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Sample records for heat-labile enterotoxin lt-iia

  1. Heat-Labile Enterotoxin: Beyond G M1 Binding

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    Benjamin Mudrak

    2010-06-01

    Full Text Available Enterotoxigenic Escherichia coli (ETEC is a significant source of morbidity and mortality worldwide. One major virulence factor released by ETEC is the heat-labile enterotoxin LT, which is structurally and functionally similar to cholera toxin. LT consists of five B subunits carrying a single catalytically active A subunit. LTB binds the monosialoganglioside GM1, the toxin’s host receptor, but interactions with A-type blood sugars and E. coli lipopolysaccharide have also been identified within the past decade. Here, we review the regulation, assembly, and binding properties of the LT B-subunit pentamer and discuss the possible roles of its numerous molecular interactions.

  2. Type II heat-labile enterotoxins from 50 diverse Escherichia coli isolates belong almost exclusively to the LT-IIc family and may be prophage encoded.

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    Michael G Jobling

    Full Text Available Some enterotoxigenic Escherichia coli (ETEC produce a type II heat-labile enterotoxin (LT-II that activates adenylate cyclase in susceptible cells but is not neutralized by antisera against cholera toxin or type I heat-labile enterotoxin (LT-I. LT-I variants encoded by plasmids in ETEC from humans and pigs have amino acid sequences that are ≥ 95% identical. In contrast, LT-II toxins are chromosomally encoded and are much more diverse. Early studies characterized LT-IIa and LT-IIb variants, but a novel LT-IIc was reported recently. Here we characterized the LT-II encoding loci from 48 additional ETEC isolates. Two encoded LT-IIa, none encoded LT-IIb, and 46 encoded highly related variants of LT-IIc. Phylogenetic analysis indicated that the predicted LT-IIc toxins encoded by these loci could be assigned to 6 subgroups. The loci corresponding to individual toxins within each subgroup had DNA sequences that were more than 99% identical. The LT-IIc subgroups appear to have arisen by multiple recombinational events between progenitor loci encoding LT-IIc1- and LT-IIc3-like variants. All loci from representative isolates encoding the LT-IIa, LT-IIb, and each subgroup of LT-IIc enterotoxins are preceded by highly-related genes that are between 80 and 93% identical to predicted phage lysozyme genes. DNA sequences immediately following the B genes differ considerably between toxin subgroups, but all are most closely related to genomic sequences found in predicted prophages. Together these data suggest that the LT-II loci are inserted into lambdoid type prophages that may or may not be infectious. These findings raise the possibility that production of LT-II enterotoxins by ETEC may be determined by phage conversion and may be activated by induction of prophage, in a manner similar to control of production of Shiga-like toxins by converting phages in isolates of enterohemmorhagic E. coli.

  3. GM1 erythroimmunoassay for detection and titration of Escherichia coli heat-labile enterotoxin.

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    Germani, Y; Bégaud, E; Guesdon, J L; Moreau, J P

    1986-01-01

    A GM1 ganglioside erythroimmunoassay for the detection of heat-labile Escherichia coli enterotoxin (LT) was developed for use in poorly equipped laboratories in developing countries. This assay is based on the immunological similarity between Vibrio cholerae toxin and LT and uses cholera toxin antiserum and sheep anti-rabbit immunoglobulin covalently coupled to sheep erythrocytes as conjugate. This assay has the following advantages over other currently available techniques: the reagents it u...

  4. Parenteral adjuvant potential of recombinant B subunit of Escherichia coli heat-labile enterotoxin

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    Carlos Eduardo Pouey da Cunha

    Full Text Available BACKGROUND The B subunit of Escherichia coli heat-labile enterotoxin (LTB is a potent mucosal immune adjuvant. However, there is little information about LTB's potential as a parenteral adjuvant. OBJECTIVES We aimed at evaluating and better understanding rLTB's potential as a parenteral adjuvant using the fused R1 repeat of Mycoplasma hyopneumoniae P97 adhesin as an antigen to characterise the humoral immune response induced by this construct and comparing it to that generated when aluminium hydroxide is used as adjuvant instead. METHODS BALB/c mice were immunised intraperitoneally with either rLTBR1 or recombinant R1 adsorbed onto aluminium hydroxide. The levels of systemic anti-rR1 antibodies (total Ig, IgG1, IgG2a, and IgA were assessed by enzyme-linked immunosorbent assay (ELISA. The ratio of IgG1 and IgG2a was used to characterise a Th1, Th2, or mixed Th1/Th2 immune response. FINDINGS Western blot confirmed rR1, either alone or fused to LTB, remained antigenic; anti-cholera toxin ELISA confirmed that LTB retained its activity when expressed in a heterologous system. Mice immunised with the rLTBR1 fusion protein produced approximately twice as much anti-rR1 immunoglobulins as mice vaccinated with rR1 adsorbed onto aluminium hydroxide. Animals vaccinated with either rLTBR1 or rR1 adsorbed onto aluminium hydroxide presented a mixed Th1/Th2 immune response. We speculate this might be a result of rR1 immune modulation rather than adjuvant modulation. Mice immunised with rLTBR1 produced approximately 1.5-fold more serum IgA than animals immunised with rR1 and aluminium hydroxide. MAIN CONCLUSIONS The results suggest that rLTB is a more powerful parenteral adjuvant than aluminium hydroxide when administered intraperitoneally as it induced higher antibody titres. Therefore, we recommend that rLTB be considered an alternative adjuvant, even if different administration routes are employed.

  5. [Escherichia coli heat-labile enterotoxin B subunit enhances the immune response against canine parvovirus VP2 in mice immunized by VP2 DNA vaccine].

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    Han, Dongmei; Zhong, Fei; Li, Xiujin; Wang, Wei; Wang, Xingxing; Pan, Sumin

    2011-01-01

    To investigate the effect of Escherichia coli heat-labile enterotoxin (LT) B subunit (LTB) gene on canine parvovirus (CPV) VP2 gene vaccine. The LTB gene was amplified by PCR from genomic DNA of E. coli 44815 strain. The VP2-70 fragment (210 bp) encoding major epitope of VP2 (70 amino acids) was amplified by PCR from a plasmid encoding VP2 gene. VP2-70 and LTB genes were inserted into the eukaryotic vector to construct VP2-70 gene,LTB gene and VP2-70-LTB fused gene vectors. The mice were immunized with VP2-70 vector, VP2-70-LTB fused vector, or VP2-70 vector plus LTB vector, respectively. The antibody titers at the different time were measured by using ELISA method. The spleen lymphocyte proliferation activity was analyzed by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The sequence of VP2-70 and LTB genes was identified. The recombinant VP2-70 and LTB proteins could be expressed in HEK293T cells in a secretory manner. The mice immunized with VP2-70 vector, VP2-70-LTB vector or VP2-70 vector plus LTB vector could generate the specific antibody against VP2 protein. The antibody titer immunized with VP2-70-LTB vector reached 1:5120 at 35 d post immunization, significantly higher than that of other two groups (P vaccine in mice.

  6. Immunogenicity of recombinant Lactobacillus casei-expressing F4 (K88) fimbrial adhesin FaeG in conjunction with a heat-labile enterotoxin A (LTAK63) and heat-labile enterotoxin B (LTB) of enterotoxigenic Escherichia coli as an oral adjuvant in mice.

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    Yu, M; Qi, R; Chen, C; Yin, J; Ma, S; Shi, W; Wu, Y; Ge, J; Jiang, Y; Tang, L; Xu, Y; Li, Y

    2017-02-01

    The aims of this study were to develop an effective oral vaccine against enterotoxigenic Escherichia coli (ETEC) infection and to design new and more versatile mucosal adjuvants. Genetically engineered Lactobacillus casei strains expressing F4 (K88) fimbrial adhesin FaeG (rLpPG-2-FaeG) and either co-expressing heat-labile enterotoxin A (LTA) subunit with an amino acid mutation associated with reduced virulence (LTAK63) and a heat-labile enterotoxin B (LTB) subunit of E. coli (rLpPG-2-LTAK63-co-LTB) or fused-expressing LTAK63 and LTB (rLpPG-2-LTAK63-fu-LTB) were constructed. The immunogenicity of rLpPG-2-FaeG in conjunction with rLpPG-2-LTAK63-co-LTB or rLpPG-2-LTAK63-fu-LTB as an orally administered mucosal adjuvant in mice was evaluated. Results showed that the levels of FaeG-specific serum IgG and mucosal sIgA, as well as the proliferation of lymphocytes, were significantly higher in mice orally co-administered rLpPG-2-FaeG and rLpPG-2-LTAK63-fu-LTB compared with those administered rLpPG-2-FaeG alone, and were lower than those co-administered rLpPG-2-FaeG and rLpPG-2-LTAK63-co-LTB. Moreover, effective protection was observed after challenge with F4+ ETEC strain CVCC 230 in mice co-administered rLpPG-2-FaeG and rLpPG-2-LTAK63-co-LTB or rLpPG-2-FaeG and rLpPG-2-LTAK63-fu-LTB group compared with those that received rLpPG-2-FaeG alone. rLpPG-2-FaeG showed greater immunogenicity in combination with LTAK63 and LTB as molecular adjuvants. Recombinant Lactobacillus provides a promising platform for the development of vaccines against F4+ ETEC infection. © 2016 The Society for Applied Microbiology.

  7. Incorporation of membrane-anchored flagellin or Escherichia coli heat-labile enterotoxin B subunit enhances the immunogenicity of rabies virus-like particles in mice and dogs

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    Yinglin eQi

    2015-03-01

    Full Text Available Rabies remains an important worldwide public health threat, so safe, effective and affordable vaccines are still being sought. Virus-like particle (VLP-based vaccines targeting various viral pathogens have been successfully produced, licensed and commercialized. Here, we designed and constructed two chimeric rabies virus-like particles (cRVLPs containing rabies virus (RABV glycoprotein (G, matrix (M protein, and membrane-anchored flagellin (EVLP-F or Escherichia coli heat-labile enterotoxin B subunit (EVLP-L as molecular adjuvants to enhance the immune response against rabies. The immunogenicity and potential of cRVLPs as novel rabies vaccine were evaluated by intramuscular vaccination in mouse and dog models. Mouse studies demonstrated that both EVLP-F and EVLP-L induced faster and larger virus-neutralizing antibodies (VNA responses and elicited greater numbers of CD4+ and CD8+ T cells secreting IFN-γ or IL-4 compared with a standard rabies VLP (sRVLP containing only G and M. Moreover, cRVLPs recruited and/or activated more B cells and dendritic cells in inguinal lymph nodes. EVLP-F induced a strong, specific IgG2a response but not an IgG1 response, suggesting the activation of Th1 class immunity; in contrast, Th2 class immunity was observed with EVLP-L. The significantly enhanced humoral and cellular immune responses induced by cRVLPs provided complete protection against lethal challenge with RABV. Most importantly, dogs vaccinated with EVLP-F or EVLP-L exhibited increased VNA titers in sera and enhanced IFN-γ and IL-4 secretion from peripheral blood mononuclear cells. Taken together, these results illustrate that when incorporated into sRVLP, membrane-anchored flagellin and LTB possess strong adjuvant activity. EVLP-F and EVLP-L induce significantly enhanced RABV-specific humoral and cellular immune responses in both mouse and dog. Therefore, these cRVLPs may be developed as safe and more efficacious rabies vaccine candidate for animals.

  8. Influence of Host Interleukin-10 Polymorphisms on Development of Traveler's Diarrhea Due to Heat-Labile Enterotoxin-Producing Escherichia coli in Travelers from the United States Who Are Visiting Mexico▿

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    Flores, Jose; DuPont, Herbert L.; Lee, Stephanie A.; Belkind-Gerson, Jaime; Paredes, Mercedes; Mohamed, Jamal A.; Armitige, Lisa Y.; Guo, Dong-Chuan; Okhuysen, Pablo C.

    2008-01-01

    Up to 60% of U.S. visitors to Mexico develop traveler's diarrhea (TD), mostly due to enterotoxigenic Escherichia coli (ETEC) strains that produce heat-labile (LT) and/or heat-stable (ST) enterotoxins. Distinct single-nucleotide polymorphisms (SNPs) within the interleukin-10 (IL-10) promoter have been associated with high, intermediate, or low production of IL-10. We conducted a prospective study to investigate the association of SNPs in the IL-10 promoter and the occurrence of TD in ETEC LT-exposed travelers. Sera from U.S. travelers to Mexico collected on arrival and departure were studied for ETEC LT seroconversion by using cholera toxin as the antigen. Pyrosequencing was performed to genotype IL-10 SNPs. Stools from subjects who developed diarrhea were also studied for other enteropathogens. One hundred twenty-one of 569 (21.3%) travelers seroconverted to ETEC LT, and among them 75 (62%) developed diarrhea. Symptomatic seroconversion was more commonly seen in subjects who carried a genotype producing high levels of IL-10; it was seen in 83% of subjects with the GG genotype versus 54% of subjects with the AA genotype at IL-10 gene position −1082 (P, 0.02), in 71% of those with the CC genotype versus 33% of those with the TT genotype at position −819 (P, 0.005), and in 71% of those with the CC genotype versus 38% of those with the AA genotype at position −592 (P, 0.02). Travelers with the GCC haplotype were more likely to have symptomatic seroconversion than those with the ATA haplotype (71% versus 38%; P, 0.002). Travelers genetically predisposed to produce high levels of IL-10 were more likely to experience symptomatic ETEC TD. PMID:18579697

  9. Influence of host interleukin-10 polymorphisms on development of traveler's diarrhea due to heat-labile enterotoxin-producing Escherichia coli in travelers from the United States who are visiting Mexico.

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    Flores, Jose; DuPont, Herbert L; Lee, Stephanie A; Belkind-Gerson, Jaime; Paredes, Mercedes; Mohamed, Jamal A; Armitige, Lisa Y; Guo, Dong-Chuan; Okhuysen, Pablo C

    2008-08-01

    Up to 60% of U.S. visitors to Mexico develop traveler's diarrhea (TD), mostly due to enterotoxigenic Escherichia coli (ETEC) strains that produce heat-labile (LT) and/or heat-stable (ST) enterotoxins. Distinct single-nucleotide polymorphisms (SNPs) within the interleukin-10 (IL-10) promoter have been associated with high, intermediate, or low production of IL-10. We conducted a prospective study to investigate the association of SNPs in the IL-10 promoter and the occurrence of TD in ETEC LT-exposed travelers. Sera from U.S. travelers to Mexico collected on arrival and departure were studied for ETEC LT seroconversion by using cholera toxin as the antigen. Pyrosequencing was performed to genotype IL-10 SNPs. Stools from subjects who developed diarrhea were also studied for other enteropathogens. One hundred twenty-one of 569 (21.3%) travelers seroconverted to ETEC LT, and among them 75 (62%) developed diarrhea. Symptomatic seroconversion was more commonly seen in subjects who carried a genotype producing high levels of IL-10; it was seen in 83% of subjects with the GG genotype versus 54% of subjects with the AA genotype at IL-10 gene position -1082 (P, 0.02), in 71% of those with the CC genotype versus 33% of those with the TT genotype at position -819 (P, 0.005), and in 71% of those with the CC genotype versus 38% of those with the AA genotype at position -592 (P, 0.02). Travelers with the GCC haplotype were more likely to have symptomatic seroconversion than those with the ATA haplotype (71% versus 38%; P, 0.002). Travelers genetically predisposed to produce high levels of IL-10 were more likely to experience symptomatic ETEC TD.

  10. Attenuated Escherichia coli strains expressing the colonization factor antigen I (CFA/I) and a detoxified heat-labile enterotoxin (LThK63) enhance clearance of ETEC from the lungs of mice and protect mice from intestinal ETEC colonization and LT-induced fluid accumulation.

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    Byrd, Wyatt; Boedeker, Edgar C

    2013-03-15

    Although enterotoxigenic Escherichia coli (ETEC) infections are important causes of infantile and traveler's diarrhea there is no licensed vaccine available for those at-risk. Our goal is to develop a safe, live attenuated ETEC vaccine. We used an attenuated E. coli strain (O157:H7, Δ-intimin, Stx1-neg, Stx2-neg) as a vector (ZCR533) to prepare two vaccine strains, one strain expressing colonization factor antigen I (ZCR533-CFA/I) and one strain expressing CFA/I and a detoxified heat-labile enterotoxin (ZCR533-CFA/I+LThK63) to deliver ETEC antigens to mucosal sites in BALB/c mice. Following intranasal and intragastric immunization with the vaccine strains, serum IgG and IgA antibodies were measured to the CFA/I antigen, however, only serum IgG antibodies were detected to the heat-labile enterotoxin. Intranasal administration of the vaccine strains induced respiratory and intestinal antibody responses to the CFA/I and LT antigens, while intragastric administration induced only intestinal antibody responses with no respiratory antibodies detected to the CFA/I and LT antigens. Mice immunized intranasally with the vaccine strains showed enhanced clearance of wild-type (wt) ETEC bacteria from the lungs. Mice immunized intranasally and intragastrically with the vaccine strains were protected from intestinal colonization following oral challenge with ETEC wt bacteria. Mice immunized intragastrically with the ZCR533-CFA/I+LThK63 vaccine strain had less fluid accumulate in their intestine following challenge with ETEC wt bacteria or with purified LT as compared to the sham mice indicating that the immunized mice were protected from LT-induced intestinal fluid accumulation. Thus, mice intragastrically immunized with the ZCR533-CFA/I+LThK63 vaccine strain were able to effectively neutralize the activity of the LT enterotoxin. However, no difference in intestinal fluid accumulation was detected in the mice immunized intranasally with the vaccine strain as compared to the sham

  11. Inhibition of Binding of the AB5-Type Enterotoxins LT-I and Cholera Toxin to Ganglioside GM1 by Galactose-Rich Dietary Components

    NARCIS (Netherlands)

    Becker, P.M.; Widjaja-Greefkes, H.C.A.; Wikselaar, van P.G.

    2010-01-01

    Cholera, travelers' diarrhea, or colibacillosis in pigs can possibly be prevented or attenuated by dietary provision of competitive inhibitors that react with the GM1-binding sites of the enterotoxins cholera toxin (CT), human Escherichia coli heat-labile enterotoxin of serogroup I (LTh-I), and

  12. Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli.

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    Christiane Y Ozaki

    Full Text Available Diarrhea is a prevalent pathological condition frequently associated to the colonization of the small intestine by enterotoxigenic Escherichia coli (ETEC strains, known to be endemic in developing countries. These strains can produce two enterotoxins associated with the manifestation of clinical symptoms that can be used to detect these pathogens. Although several detection tests have been developed, minimally equipped laboratories are still in need of simple and cost-effective methods. With the aim to contribute to the development of such diagnostic approaches, we describe here two mouse hybridoma-derived single chain fragment variable (scFv that were produced in E. coli against enterotoxins of ETEC strains.Recombinant scFv were developed against ETEC heat-labile toxin (LT and heat-stable toxin (ST, from previously isolated hybridoma clones. This work reports their design, construction, molecular and functional characterization against LT and ST toxins. Both antibody fragments were able to recognize the cell-interacting toxins by immunofluorescence, the purified toxins by ELISA and also LT-, ST- and LT/ST-producing ETEC strains.The developed recombinant scFvs against LT and ST constitute promising starting point for simple and cost-effective ETEC diagnosis.

  13. Alkaline pH Is a signal for optimal production and secretion of the heat labile toxin, LT in enterotoxigenic Escherichia coli (ETEC.

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    Lucia Gonzales

    Full Text Available Enterotoxigenic Escherichia coli (ETEC cause secretory diarrhea in children and travelers to endemic areas. ETEC spreads through the fecal-oral route. After ingestion, ETEC passes through the stomach and duodenum before it colonizes the lower part of the small intestine, exposing bacteria to a wide range of pH and environmental conditions. This study aimed to determine the impact of external pH and activity of the Cyclic AMP receptor protein (CRP on the regulation of production and secretion of heat labile (LT enterotoxin. ETEC strain E2863wt and its isogenic mutant E2863ΔCRP were grown in LBK media buffered to pH 5, 7 and 9. GM1 ELISA, cDNA and cAMP analyses were carried out on bacterial pellet and supernatant samples derived from 3 and 5 hours growth and from overnight cultures. We confirm that CRP is a repressor of LT transcription and production as has been shown before but we show for the first time that CRP is a positive regulator of LT secretion both in vitro and in vivo. LT secretion increased at neutral to alkaline pH compared to acidic pH 5 where secretion was completely inhibited. At pH 9 secretion of LT was optimal resulting in 600 percent increase of secreted LT compared to unbuffered LBK media. This effect was not due to membrane leakage since the bacteria were viable at pH 9. The results indicate that the transition to the alkaline duodenum and/or exposure to high pH close to the epithelium as well as activation of the global transcription factor CRP are signals that induce secretion of the LT toxin in ETEC.

  14. Toxicity and immunogenicity of Enterotoxigenic Escherichia coli heat-labile and heat-stable toxoid fusion 3xSTa(A14Q-LT(S63K/R192G/L211A in a murine model.

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    Chengxian Zhang

    Full Text Available Diarrhea is the second leading cause of death to young children. Enterotoxigenic Escherichia coli (ETEC are the most common bacteria causing diarrhea. Adhesins and enterotoxins are the virulence determinants in ETEC diarrhea. Adhesins mediate bacterial attachment and colonization, and enterotoxins including heat-labile (LT and heat-stable type Ib toxin (STa disrupt fluid homeostasis in host cells that leads to fluid hyper-secretion and diarrhea. Thus, adhesins and enterotoxins have been primarily targeted in ETEC vaccine development. A recent study reported toxoid fusions with STa toxoid (STa(P13F fused at the N- or C-terminus, or inside the A subunit of LT(R192G elicited neutralizing antitoxin antibodies, and suggested application of toxoid fusions in ETEC vaccine development (Liu et al., Infect. Immun. 79:4002-4009, 2011. In this study, we generated a different STa toxoid (STa(A14Q and a triple-mutant LT toxoid (LT(S63K/R192G/L211A, tmLT, constructed a toxoid fusion (3xSTa(A14Q-tmLT that carried 3 copies of STa(A14Q for further facilitation of anti-STa immunogenicity, and assessed antigen safety and immunogenicity in a murine model to explore its potential for ETEC vaccine development. Mice immunized with this fusion antigen showed no adverse effects, and developed antitoxin antibodies particularly through the IP route. Anti-LT antibodies were detected and were shown neutralizing against CT in vitro. Anti-STa antibodies were also detected in the immunized mice, and serum from the IP immunized mice neutralized STa toxin in vitro. Data from this study indicated that toxoid fusion 3xSTa(A14Q-tmLT is safe and can induce neutralizing antitoxin antibodies, and provided helpful information for vaccine development against ETEC diarrhea.

  15. Induction of heat-labile sites in DNA of mammalian cells by the antitumor alkylating drug CC-1065

    International Nuclear Information System (INIS)

    Zsido, T.J.; Woynarowski, J.M.; Baker, R.M.; Gawron, L.S.; Beerman, T.A.

    1991-01-01

    CC-1065 is a very potent antitumor antibiotic capable of covalent and noncovalent binding to the minor groove of naked DNA. Upon thermal treatment, covalent adducts formed between CC-1065 and DNA generate strand break. The authors have shown that this molecular damage can be detected following CC-1065 treatment of mammalian whole cells. Using alkaline sucrose gradient analysis, They observe thermally induced breakage of [ 14 C]thymidine-prelabeled DNA from drug-treated African green monkey kidney BSC-1 cells. Very little damage to cellular DNA by CC-1065 can be detected without first heating the drug-treated samples. CC-1065 can also generate heat-labile sites within DNA during cell lysis and heating, subsequent to the exposure of cells to drug, suggesting that a pool of free and noncovalently bound drug is available for posttreatment adduct formation. This effect was controlled for by mixing [ 3 H]thymidine-labeled untreated cells with the [ 14 C]thymidine-labeled drug-treated samples. The lowest drug dose at which heat-labile sites were detected was 3 nM CC-1065 (3 single-stranded breaks/10 6 base pairs). This concentration reduced survival of BSC-1 cells to 0.1% in cytotoxicity assays. The generation of CC-1065-induced lesions in cellular DNA is time dependent (the frequency of lesions caused by a 60 nM treatment reaching a plateau at 2 h) and is not readily reversible. The results of this study demonstrate that CC-1065 does generate heat-labile sites with the cellular DNA of intact cells and suggest that a mechanism of cytotoxic action of CC-1065 involves formation of covalent adducts to DNA

  16. Repair of radiation-induced heat-labile sites is independent of DNA-PKcs, XRCC1 or PARP

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    Stenerl& #246; w, Bo; Karlsson, Karin H.; Radulescu, Irina; Rydberg, Bjorn; Stenerlow, Bo

    2008-04-29

    Ionizing radiation induces a variety of different DNA lesions: in addition to the most critical DNA damage, the DSB, numerous base alterations, SSBs and other modifications of the DNA double-helix are formed. When several non-DSB lesions are clustered within a short distance along DNA, or close to a DSB, they may interfere with the repair of DSBs and affect the measurement of DSB induction and repair. We have previously shown that a substantial fraction of DSBs measured by pulsed-field gel electrophoresis (PFGE) are in fact due to heat-labile sites (HLS) within clustered lesions, thus reflecting an artifact of preparation of genomic DNA at elevated temperature. To further characterize the influence of HLS on DSB induction and repair, four human cell lines (GM5758, GM7166, M059K, U-1810) with apparently normal DSB rejoining were tested for bi-phasic rejoining after gamma irradiation. When heat-released DSBs were excluded from the measurements the fraction of fast rejoining decreased to less than 50% of the total. However, neither the half-times of the fast (t{sub 1/2} = 7-8 min) or slow (t{sub 1/2} = 2.5 h) DSB rejoining were changed significantly. At t=0 the heat-released DSBs accounted for almost 40% of the DSBs, corresponding to 10 extra DSB/cell/Gy in the initial DSB yield. These heat-released DSBs were repaired within 60-90 min in all tested cells, including M059K cells treated with wortmannin or DNA-PKcs defect M059J cells. Furthermore, cells lacking XRCC1 or Poly(ADP-ribose) polymerase-1 (PARP-1) rejoined both total DSBs and heat-released DSBs similar to normal cells. In summary, the presence of heat-labile sites have a substantial impact on DSB induction yields and DSB rejoining rates measured by pulsed-field gel electrophoresis, and HLS repair is independent of DNA-PKcs, XRCC1 and PARP.

  17. Allele Variants of Enterotoxigenic Escherichia coli Heat-Labile Toxin Are Globally Transmitted and Associated with Colonization Factors

    KAUST Repository

    Joffré, Enrique

    2015-01-15

    Enterotoxigenic Escherichia coli (ETEC) is a significant cause of morbidity and mortality in the developing world. ETEC-mediated diarrhea is orchestrated by heat-labile toxin (LT) and heat-stable toxins (STp and STh), acting in concert with a repertoire of more than 25 colonization factors (CFs). LT, the major virulence factor, induces fluid secretion after delivery of a monomeric ADP-ribosylase (LTA) and its pentameric carrier B subunit (LTB). A study of ETEC isolates from humans in Brazil reported the existence of natural LT variants. In the present study, analysis of predicted amino acid sequences showed that the LT amino acid polymorphisms are associated with a geographically and temporally diverse set of 192 clinical ETEC strains and identified 12 novel LT variants. Twenty distinct LT amino acid variants were observed in the globally distributed strains, and phylogenetic analysis showed these to be associated with different CF profiles. Notably, the most prevalent LT1 allele variants were correlated with major ETEC lineages expressing CS1 + CS3 or CS2 + CS3, and the most prevalent LT2 allele variants were correlated with major ETEC lineages expressing CS5 + CS6 or CFA/I. LTB allele variants generally exhibited more-stringent amino acid sequence conservation (2 substitutions identified) than LTA allele variants (22 substitutions identified). The functional impact of LT1 and LT2 polymorphisms on virulence was investigated by measuring total-toxin production, secretion, and stability using GM1-enzyme-linked immunosorbent assays (GM1-ELISA) and in silico protein modeling. Our data show that LT2 strains produce 5-fold more toxin than LT1 strains (P < 0.001), which may suggest greater virulence potential for this genetic variant. Our data suggest that functionally distinct LT-CF variants with increased fitness have persisted during the evolution of ETEC and have spread globally.

  18. Allele Variants of Enterotoxigenic Escherichia coli Heat-Labile Toxin Are Globally Transmitted and Associated with Colonization Factors

    KAUST Repository

    Joffré , Enrique; von Mentzer, Astrid; Abd El Ghany, Moataz; Oezguen, Numan; Savidge, Tor; Dougan, Gordon; Svennerholm, Ann-Mari; Sjö ling, Å sa

    2015-01-01

    Enterotoxigenic Escherichia coli (ETEC) is a significant cause of morbidity and mortality in the developing world. ETEC-mediated diarrhea is orchestrated by heat-labile toxin (LT) and heat-stable toxins (STp and STh), acting in concert with a repertoire of more than 25 colonization factors (CFs). LT, the major virulence factor, induces fluid secretion after delivery of a monomeric ADP-ribosylase (LTA) and its pentameric carrier B subunit (LTB). A study of ETEC isolates from humans in Brazil reported the existence of natural LT variants. In the present study, analysis of predicted amino acid sequences showed that the LT amino acid polymorphisms are associated with a geographically and temporally diverse set of 192 clinical ETEC strains and identified 12 novel LT variants. Twenty distinct LT amino acid variants were observed in the globally distributed strains, and phylogenetic analysis showed these to be associated with different CF profiles. Notably, the most prevalent LT1 allele variants were correlated with major ETEC lineages expressing CS1 + CS3 or CS2 + CS3, and the most prevalent LT2 allele variants were correlated with major ETEC lineages expressing CS5 + CS6 or CFA/I. LTB allele variants generally exhibited more-stringent amino acid sequence conservation (2 substitutions identified) than LTA allele variants (22 substitutions identified). The functional impact of LT1 and LT2 polymorphisms on virulence was investigated by measuring total-toxin production, secretion, and stability using GM1-enzyme-linked immunosorbent assays (GM1-ELISA) and in silico protein modeling. Our data show that LT2 strains produce 5-fold more toxin than LT1 strains (P < 0.001), which may suggest greater virulence potential for this genetic variant. Our data suggest that functionally distinct LT-CF variants with increased fitness have persisted during the evolution of ETEC and have spread globally.

  19. Molecular Characterization of Enterotoxin-Producing Escherichia coli Collected in 2011-2012, Russia.

    Directory of Open Access Journals (Sweden)

    Nikolay N Kartsev

    Full Text Available Enterotoxin-producing Escherichia coli (ETEC are one of the main causative agents of diarrhea in children especially in developing countries and travel diarrhoea in adults. Pathogenic properties of ETEC associated with their ability to produce a heat-stable (ST and/or heat-labile (LT enterotoxins, as well as adhesins providing bacterial adhesion to intestinal epithelial cells. This study presents the molecular characterization of the ETEC isolates collected from the Central and Far-Eastern regions of Russia in 2011-2012. It was shown that all ETEC under study (n=18 had the heat-labile enterotoxin-coding operon elt, and had no the genes of the heat-stable enterotoxin operon est. DNA sequencing revealed two types of nucleotide exchanges in the eltB gene coding subunit B of LT in isolates collected from Cherepovets city (Central region, Russia and Vladivostok city (Far-East region, Russia. Only one ETEC strain carried genes cfaA, cfaB, cfaC and cfaD coding adhesion factor CFA/I. Expression of LT in four ETEC isolates in the agglutination reaction was detected using a latex test-system. The isolates were assigned to serogroups O142 (n = 6, О6 (n = 4, О25 (n = 5, О26 (n = 2, and O115 (n = 1. Genotyping showed that they belonged to an earlier described sequence-type ST4 (n = 3 as well as to 11 novel sequence-types ST1043, ST1312, ST3697, ST3707, ST3708, ST3709, ST3710, ST3755, ST3756, ST3757 and ST4509. The ETEC isolates displayed different levels of antimicrobial resistance. Eight isolates were resistant to only one drug, three isolates-to two drugs, one isolate-to three drugs, two isolates-to four antibacterials, and only one isolate to each of the five, six and ten antibacterials simultaneously. Genetic determinants of the resistance to beta-lactams and other classes of antibacterials on the ETEC genomes were identified. There are blaTEM (n = 10, blaCTX-M-15 (n = 1, class 1 integron (n = 3 carrying resistance cassettes to aminoglycosides and

  20. Molecular Characterization of Enterotoxin-Producing Escherichia coli Collected in 2011-2012, Russia.

    Science.gov (United States)

    Kartsev, Nikolay N; Fursova, Nadezhda K; Pachkunov, Dmitry M; Bannov, Vasiliy A; Eruslanov, Boris V; Svetoch, Edward A; Dyatlov, Ivan A

    2015-01-01

    Enterotoxin-producing Escherichia coli (ETEC) are one of the main causative agents of diarrhea in children especially in developing countries and travel diarrhoea in adults. Pathogenic properties of ETEC associated with their ability to produce a heat-stable (ST) and/or heat-labile (LT) enterotoxins, as well as adhesins providing bacterial adhesion to intestinal epithelial cells. This study presents the molecular characterization of the ETEC isolates collected from the Central and Far-Eastern regions of Russia in 2011-2012. It was shown that all ETEC under study (n=18) had the heat-labile enterotoxin-coding operon elt, and had no the genes of the heat-stable enterotoxin operon est. DNA sequencing revealed two types of nucleotide exchanges in the eltB gene coding subunit B of LT in isolates collected from Cherepovets city (Central region, Russia) and Vladivostok city (Far-East region, Russia). Only one ETEC strain carried genes cfaA, cfaB, cfaC and cfaD coding adhesion factor CFA/I. Expression of LT in four ETEC isolates in the agglutination reaction was detected using a latex test-system. The isolates were assigned to serogroups O142 (n = 6), О6 (n = 4), О25 (n = 5), О26 (n = 2), and O115 (n = 1). Genotyping showed that they belonged to an earlier described sequence-type ST4 (n = 3) as well as to 11 novel sequence-types ST1043, ST1312, ST3697, ST3707, ST3708, ST3709, ST3710, ST3755, ST3756, ST3757 and ST4509. The ETEC isolates displayed different levels of antimicrobial resistance. Eight isolates were resistant to only one drug, three isolates-to two drugs, one isolate-to three drugs, two isolates-to four antibacterials, and only one isolate to each of the five, six and ten antibacterials simultaneously. Genetic determinants of the resistance to beta-lactams and other classes of antibacterials on the ETEC genomes were identified. There are blaTEM (n = 10), blaCTX-M-15 (n = 1), class 1 integron (n = 3) carrying resistance cassettes to aminoglycosides and

  1. Prolonged protection against Intranasal challenge with influenza virus following systemic immunization or combinations of mucosal and systemic immunizations with a heat-labile toxin mutant.

    Science.gov (United States)

    Zhou, Fengmin; Goodsell, Amanda; Uematsu, Yasushi; Vajdy, Michael

    2009-04-01

    Seasonal influenza virus infections cause considerable morbidity and mortality in the world, and there is a serious threat of a pandemic influenza with the potential to cause millions of deaths. Therefore, practical influenza vaccines and vaccination strategies that can confer protection against intranasal infection with influenza viruses are needed. In this study, we demonstrate that using LTK63, a nontoxic mutant of the heat-labile toxin from Escherichia coli, as an adjuvant for both mucosal and systemic immunizations, systemic (intramuscular) immunization or combinations of mucosal (intranasal) and intramuscular immunizations protected mice against intranasal challenge with a lethal dose of live influenza virus at 3.5 months after the second immunization.

  2. Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfite restriction analysis

    International Nuclear Information System (INIS)

    Watanabe, Kousuke; Emoto, Noriko; Sunohara, Mitsuhiro; Kawakami, Masanori; Kage, Hidenori; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

    2010-01-01

    Research highlights: → Incubating PCR products at a high temperature causes smears in gel electrophoresis. → Smears interfere with the interpretation of methylation analysis using COBRA. → Treatment with exonuclease I and heat-labile alkaline phosphatase eliminates smears. → The elimination of smears improves the visibility of COBRA. -- Abstract: DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonuclease I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 o C or 65 o C, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis.

  3. An Enterotoxin-Like Binary Protein from Pseudomonas protegens with Potent Nematicidal Activity.

    Science.gov (United States)

    Wei, Jun-Zhi; Siehl, Daniel L; Hou, Zhenglin; Rosen, Barbara; Oral, Jarred; Taylor, Christopher G; Wu, Gusui

    2017-10-01

    Soil microbes are a major food source for free-living soil nematodes. It is known that certain soil bacteria have evolved systems to combat predation. We identified the nematode-antagonistic Pseudomonas protegens strain 15G2 from screening of microbes. Through protein purification we identified a binary protein, designated Pp-ANP, which is responsible for the nematicidal activity. This binary protein inhibits Caenorhabditis elegans growth and development by arresting larvae at the L1 stage and killing older-staged worms. The two subunits, Pp-ANP1a and Pp-ANP2a, are active when reconstituted from separate expression in Escherichia coli The binary toxin also shows strong nematicidal activity against three other free-living nematodes ( Pristionchus pacificus , Panagrellus redivivus , and Acrobeloides sp.), but we did not find any activity against insects and fungi under test conditions, indicating specificity for nematodes. Pp-ANP1a has no significant identity to any known proteins, while Pp-ANP2a shows ∼30% identity to E. coli heat-labile enterotoxin (LT) subunit A and cholera toxin (CT) subunit A. Protein modeling indicates that Pp-ANP2a is structurally similar to CT/LT and likely acts as an ADP-ribosyltransferase. Despite the similarity, Pp-ANP shows several characteristics distinct from CT/LT toxins. Our results indicate that Pp-ANP is a new enterotoxin-like binary toxin with potent and specific activity to nematodes. The potency and specificity of Pp-ANP suggest applications in controlling parasitic nematodes and open an avenue for further research on its mechanism of action and role in bacterium-nematode interaction. IMPORTANCE This study reports the discovery of a new enterotoxin-like binary protein, Pp-ANP, from a Pseudomonas protegens strain. Pp-ANP shows strong nematicidal activity against Caenorhabditis elegans larvae and older-staged worms. It also shows strong activity on other free-living nematodes ( Pristionchus pacificus , Panagrellus redivivus , and

  4. The detection of K88, K99 fimbrial antigen and enterotoxin genes of Escherichia coli isolated from piglets and calves with diarrhoea in Indonesia

    Directory of Open Access Journals (Sweden)

    Supar

    1996-03-01

    Full Text Available Enterotoxigenic Escherichia coli (ETEC strains cause diarrhoeal disease in piglets and calves in Indonesia. These strains possess two virulence factors namely attachment and enterotoxin antigens . These factors could be detected phenotypically and genetically. Haemolytic Escherichia coli (E coli isolates possessing K88 fimbrial antigen associated with 0-group 108 and 149. They were positive for K88 gene and demonstrated their ability to produce heat labile enterotoxin (LT and genetically were all positive for LT gene . Seventeen isolates ofE coli K88 which associated with 0-group 149 were positive forSTb gene, other O-serotypes were negative . Ten isolates of Ecoli K88 which associated with 0-group 108 possessed K88, K99, LT and STa genes, but negative for STb gene . However, phenotypically the K99 antigen and STa toxin were not expressed under laboratory conditions, the reason was not well understood . E. coli K99 strains isolated from calves wit h diarrhoea were all associated with 0-group 9 and produced STa toxin when tested by suckling mousse bioassay. The E. coli K99 calf isolates were all hybridized with K99 and STa gene only . It is likely that K99 gene is associated with STa gene . The DNA hybridization technique is more convenience to be used for confirmation diagnosis of colibacillosis, however, not all veterinary laboratories could perform these tests .

  5. Genetic fusions of a CFA/I/II/IV MEFA (multiepitope fusion antigen) and a toxoid fusion of heat-stable toxin (STa) and heat-labile toxin (LT) of enterotoxigenic Escherichia coli (ETEC) retain broad anti-CFA and antitoxin antigenicity.

    Science.gov (United States)

    Ruan, Xiaosai; Sack, David A; Zhang, Weiping

    2015-01-01

    Immunological heterogeneity has long been the major challenge in developing broadly effective vaccines to protect humans and animals against bacterial and viral infections. Enterotoxigenic Escherichia coli (ETEC) strains, the leading bacterial cause of diarrhea in humans, express at least 23 immunologically different colonization factor antigens (CFAs) and two distinct enterotoxins [heat-labile toxin (LT) and heat-stable toxin type Ib (STa or hSTa)]. ETEC strains expressing any one or two CFAs and either toxin cause diarrhea, therefore vaccines inducing broad immunity against a majority of CFAs, if not all, and both toxins are expected to be effective against ETEC. In this study, we applied the multiepitope fusion antigen (MEFA) strategy to construct ETEC antigens and examined antigens for broad anti-CFA and antitoxin immunogenicity. CFA MEFA CFA/I/II/IV [CVI 2014, 21(2):243-9], which carried epitopes of seven CFAs [CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5, CS6)] expressed by the most prevalent and virulent ETEC strains, was genetically fused to LT-STa toxoid fusion monomer 3xSTaA14Q-dmLT or 3xSTaN12S-dmLT [IAI 2014, 82(5):1823-32] for CFA/I/II/IV-STaA14Q-dmLT and CFA/I/II/IV-STaN12S-dmLT MEFAs. Mice intraperitoneally immunized with either CFA/I/II/IV-STa-toxoid-dmLT MEFA developed antibodies specific to seven CFAs and both toxins, at levels equivalent or comparable to those induced from co-administration of the CFA/I/II/IV MEFA and toxoid fusion 3xSTaN12S-dmLT. Moreover, induced antibodies showed in vitro adherence inhibition activities against ETEC or E. coli strains expressing these seven CFAs and neutralization activities against both toxins. These results indicated CFA/I/II/IV-STa-toxoid-dmLT MEFA or CFA/I/II/IV MEFA combined with 3xSTaN12S-dmLT induced broadly protective anti-CFA and antitoxin immunity, and suggested their potential application in broadly effective ETEC vaccine development. This MEFA strategy may be generally used in multivalent

  6. Genetic fusions of a CFA/I/II/IV MEFA (multiepitope fusion antigen and a toxoid fusion of heat-stable toxin (STa and heat-labile toxin (LT of enterotoxigenic Escherichia coli (ETEC retain broad anti-CFA and antitoxin antigenicity.

    Directory of Open Access Journals (Sweden)

    Xiaosai Ruan

    Full Text Available Immunological heterogeneity has long been the major challenge in developing broadly effective vaccines to protect humans and animals against bacterial and viral infections. Enterotoxigenic Escherichia coli (ETEC strains, the leading bacterial cause of diarrhea in humans, express at least 23 immunologically different colonization factor antigens (CFAs and two distinct enterotoxins [heat-labile toxin (LT and heat-stable toxin type Ib (STa or hSTa]. ETEC strains expressing any one or two CFAs and either toxin cause diarrhea, therefore vaccines inducing broad immunity against a majority of CFAs, if not all, and both toxins are expected to be effective against ETEC. In this study, we applied the multiepitope fusion antigen (MEFA strategy to construct ETEC antigens and examined antigens for broad anti-CFA and antitoxin immunogenicity. CFA MEFA CFA/I/II/IV [CVI 2014, 21(2:243-9], which carried epitopes of seven CFAs [CFA/I, CFA/II (CS1, CS2, CS3, CFA/IV (CS4, CS5, CS6] expressed by the most prevalent and virulent ETEC strains, was genetically fused to LT-STa toxoid fusion monomer 3xSTaA14Q-dmLT or 3xSTaN12S-dmLT [IAI 2014, 82(5:1823-32] for CFA/I/II/IV-STaA14Q-dmLT and CFA/I/II/IV-STaN12S-dmLT MEFAs. Mice intraperitoneally immunized with either CFA/I/II/IV-STa-toxoid-dmLT MEFA developed antibodies specific to seven CFAs and both toxins, at levels equivalent or comparable to those induced from co-administration of the CFA/I/II/IV MEFA and toxoid fusion 3xSTaN12S-dmLT. Moreover, induced antibodies showed in vitro adherence inhibition activities against ETEC or E. coli strains expressing these seven CFAs and neutralization activities against both toxins. These results indicated CFA/I/II/IV-STa-toxoid-dmLT MEFA or CFA/I/II/IV MEFA combined with 3xSTaN12S-dmLT induced broadly protective anti-CFA and antitoxin immunity, and suggested their potential application in broadly effective ETEC vaccine development. This MEFA strategy may be generally used in

  7. Rotavirus 2/6 Viruslike Particles Administered Intranasally with Cholera Toxin, Escherichia coli Heat-Labile Toxin (LT), and LT-R192G Induce Protection from Rotavirus Challenge

    OpenAIRE

    O’Neal, Christine M.; Clements, John D.; Estes, Mary K.; Conner, Margaret E.

    1998-01-01

    We have shown that rotavirus 2/6 viruslike particles composed of proteins VP2 and VP6 (2/6-VLPs) administered to mice intranasally with cholera toxin (CT) induced protection from rotavirus challenge, as measured by virus shedding. Since it is unclear if CT will be approved for human use, we evaluated the adjuvanticity of Escherichia coli heat-labile toxin (LT) and LT-R192G. Mice were inoculated intranasally with 10 μg of 2/6-VLPs combined with CT, LT, or LT-R192G. All three adjuvants induced ...

  8. Double antibody solid-phase radioimmunoassay for staphylococcal enterotoxin A

    International Nuclear Information System (INIS)

    Lindroth, S.; Niskanen, A.

    1977-01-01

    A double antibody solid-phase (DASP) radioimmunoassay for staphylococcal enterotoxin A is described. In the assay the antigen-antibody complex is precipitated by anti-rabbit serum which is adsorbed onto a solid carrier (cellulose). The method is sensitive to 200 pg of enterotoxin. It was possible to detect a little as 2-5 ng of enterotoxin A/ml food extract from minced meat and sausage. Enterotoxins B and C were not found to inhibit the uptake of labled enterotoxin A at a level which might distort the results of the enterotoxin A assay. The DASP technique is sensitive, rapid, and easy to perform and thus compares favorably with other radioimmunoassays for enterotoxin. (orig.) [de

  9. Staphylococcal aureus Enterotoxin C and Enterotoxin-Like L Associated with Post-partum Mastitis

    DEFF Research Database (Denmark)

    Franck, Kristina T; Gumpert, Heidi; Olesen, Bente

    2017-01-01

    between isolates from the two outbreaks revealed a S. aureus pathogenicity island containing enterotoxin C and enterotoxin-like L only in isolates from outbreak 2. Enterotoxin C and enterotoxin-like L carrying S. aureus are associated with bovine mastitis and our findings indicate that these may also...... PVL and ACME negative. In outbreak 1, the isolates harbored SCCmec IVa and in outbreak 2 SCCmec V. The clinical presentation differed between the two outbreaks, as none of five MRSA positive mothers in outbreak 1 had mastitis vs. five of six MRSA positive mothers in outbreak 2 (p ....02). To investigate if whole-genome sequencing could identify virulence genes associated with mastitis, t015:ST45 isolates from Denmark (N = 101) were whole-genome sequenced. Sequence analysis confirmed two separate outbreaks with no sign of sustained spread into the community. Analysis of the accessory genome...

  10. Staphylococcus aureus enterotoxins A- and B

    DEFF Research Database (Denmark)

    Danielsen, E Michael; Hansen, Gert H; Karlsdóttir, Edda

    2013-01-01

    Enterotoxins of Staphylococcus aureus are among the most common causes of food poisoning. Acting as superantigens they intoxicate the organism by causing a massive uncontrolled T cell activation that ultimately may lead to toxic shock and death. In contrast to our detailed knowledge regarding...

  11. Basis of Virulence in Enterotoxin-Mediated Staphylococcal Food Poisoning

    Directory of Open Access Journals (Sweden)

    Emilie L. Fisher

    2018-03-01

    Full Text Available The Staphylococcus aureus enterotoxins are a superfamily of secreted virulence factors that share structural and functional similarities and possess potent superantigenic activity causing disruptions in adaptive immunity. The enterotoxins can be separated into two groups; the classical (SEA-SEE and the newer (SEG-SElY and counting enterotoxin groups. Many members from both these groups contribute to the pathogenesis of several serious human diseases, including toxic shock syndrome, pneumonia, and sepsis-related infections. Additionally, many members demonstrate emetic activity and are frequently responsible for food poisoning outbreaks. Due to their robust tolerance to denaturing, the enterotoxins retain activity in food contaminated previously with S. aureus. The genes encoding the enterotoxins are found mostly on a variety of different mobile genetic elements. Therefore, the presence of enterotoxins can vary widely among different S. aureus isolates. Additionally, the enterotoxins are regulated by multiple, and often overlapping, regulatory pathways, which are influenced by environmental factors. In this review, we also will focus on the newer enterotoxins (SEG-SElY, which matter for the role of S. aureus as an enteropathogen, and summarize our current knowledge on their prevalence in recent food poisoning outbreaks. Finally, we will review the current literature regarding the key elements that govern the complex regulation of enterotoxins, the molecular mechanisms underlying their enterotoxigenic, superantigenic, and immunomodulatory functions, and discuss how these activities may collectively contribute to the overall manifestation of staphylococcal food poisoning.

  12. Rotavirus 2/6 Viruslike Particles Administered Intranasally with Cholera Toxin, Escherichia coli Heat-Labile Toxin (LT), and LT-R192G Induce Protection from Rotavirus Challenge

    Science.gov (United States)

    O’Neal, Christine M.; Clements, John D.; Estes, Mary K.; Conner, Margaret E.

    1998-01-01

    We have shown that rotavirus 2/6 viruslike particles composed of proteins VP2 and VP6 (2/6-VLPs) administered to mice intranasally with cholera toxin (CT) induced protection from rotavirus challenge, as measured by virus shedding. Since it is unclear if CT will be approved for human use, we evaluated the adjuvanticity of Escherichia coli heat-labile toxin (LT) and LT-R192G. Mice were inoculated intranasally with 10 μg of 2/6-VLPs combined with CT, LT, or LT-R192G. All three adjuvants induced equivalent geometric mean titers of rotavirus-specific serum antibody and intestinal immunoglobulin G (IgG). Mice inoculated with 2/6-VLPs with LT produced significantly higher titers of intestinal IgA than mice given CT as the adjuvant. All mice inoculated with 2/6-VLPs mixed with LT and LT-R192G were totally protected (100%) from rotavirus challenge, while mice inoculated with 2/6-VLPs mixed with CT showed a mean 91% protection from challenge. The availability of a safe, effective mucosal adjuvant such as LT-R192G will increase the practicality of administering recombinant vaccines mucosally. PMID:9525668

  13. A modified multiscale peak alignment method combined with trilinear decomposition to study the volatile/heat-labile components in Ligusticum chuanxiong Hort - Cyperus rotundus rhizomes by HS-SPME-GC/MS.

    Science.gov (United States)

    He, Min; Yan, Pan; Yang, Zhi-Yu; Zhang, Zhi-Min; Yang, Tian-Biao; Hong, Liang

    2018-03-15

    Head Space/Solid Phase Micro-Extraction (HS-SPME) coupled with Gas Chromatography/Mass Spectrometer (GC/MS) was used to determine the volatile/heat-labile components in Ligusticum chuanxiong Hort - Cyperus rotundus rhizomes. Facing co-eluting peaks in k samples, a trilinear structure was reconstructed to obtain the second-order advantage. The retention time (RT) shift with multi-channel detection signals for different samples has been vital in maintaining the trilinear structure, thus a modified multiscale peak alignment (mMSPA) method was proposed in this paper. The peak position and peak width of representative ion profile were firstly detected by mMSPA using Continuous Wavelet Transform with Haar wavelet as the mother wavelet (Haar CWT). Then, the raw shift was confirmed by Fast Fourier Transform (FFT) cross correlation calculation. To obtain the optimal shift, Haar CWT was again used to detect the subtle deviations and be amalgamated in calculation. Here, to ensure there is no peaks shape alternation, the alignment was performed in local domains of data matrices, and all data points in the peak zone were moved via linear interpolation in non-peak parts. Finally, chemical components of interest in Ligusticum chuanxiong Hort - Cyperus rotundus rhizomes were analyzed by HS-SPME-GCMS and mMSPA-alternating trilinear decomposition (ATLD) resolution. As a result, the concentration variation between herbs and their pharmaceutical products can provide a scientific basic for the quality standard establishment of traditional Chinese medicines. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. Production of staphylococcal enterotoxin A in cream-filled cake.

    Science.gov (United States)

    Anunciaçao, L L; Linardi, W R; do Carmo, L S; Bergdoll, M S

    1995-07-01

    Cakes were baked with normal ingredients and filled with cream, inoculated with different size enterotoxigenic-staphylococcal inocula. Samples of the cakes were incubated at room temperature and put in the refrigerator. Samples of cake and filling were taken at different times and analyzed for staphylococcal count and presence of enterotoxin. The smaller the inoculum, the longer the time required for sufficient growth (10(6)) to occur for production of detectable enterotoxin. Enterotoxin added to the cake dough before baking (210 degrees C, 45 min) did not survive the baking. The presence of enterotoxin in the contaminated cream filling indicated this as the cause of staphylococcal food poisoning from cream-filled cakes. Refrigeration of the cakes prevented the growth of the staphylococci.

  15. Safety and efficacy of E coli enterotoxin adjuvant for urease-based rectal immunization against Helicobacter pylori.

    Science.gov (United States)

    Sougioultzis, Stavros; Lee, Cynthia K; Alsahli, Mazen; Banerjee, Subhas; Cadoz, Michel; Schrader, Robert; Guy, Bruno; Bedford, Philip; Monath, Thomas P; Kelly, Ciaran P; Michetti, Pierre

    2002-12-13

    Low dose E. coli heat-labile enterotoxin (LT), delivered orally or enterically, has been used as an adjuvant for Helicobacter pylori (H. pylori) urease in healthy adults. In this study we aim to test the safety and adjuvant efficacy of LT delivered rectally together with recombinant H. pylori urease. Eighteen healthy adults without present or past H. pylori infection were enrolled in a double blind, randomized, ascending dose study to receive either urease (60 mg), or urease (60 mg) + LT (5 or 25 microg). The immunization preparation was administered per rectum on days 0, 14 and 28. Serum, stool and saliva anti-urease and anti-LT IgG and IgA antibodies (Abs) were measured and urease-specific and LT-specific antigen secreting cells (ASCs) were counted in peripheral blood at baseline and 7 (ASC counts) or 14 days (antibody levels) after each dosing. Peripheral blood lymphoproliferation assays were also performed at baseline and at the end of the study. Rectally delivered urease and LT were well tolerated. Among the 12 subjects assigned to urease+LT, 2 (16.7%) developed anti-urease IgG Abs, 1 (8.3%) developed anti-urease IgA Abs, and 3 (25%) showed urease-specific IgA(+) ASCs. Immune responses to LT were more vigorous, especially in subjects exposed to 5 microg LT. In the urease+ 5 microg LT group, anti-LT IgG and IgA Abs developed in 60 and 80% of the subjects, respectively, while LT-specific IgG(+) and IgA(+) ASCs were detected in all subjects. The magnitude of the anti-LT response was much higher than the response to urease. No IgA anti-urease or anti-LT Abs were detected in stool or saliva and lymphocyte proliferative responses to urease were unsatisfactory. In conclusion, rectal delivery of 5 microg LT is safe and induces vigorous systemic anti-LT immune responses. Further studies are needed to determine if LT can be an effective adjuvant for rectally delivered antigens.

  16. Intranasal Administration of 2/6-Rotavirus-Like Particles with Mutant Escherichia coli Heat-Labile Toxin (LT-R192G) Induces Antibody-Secreting Cell Responses but Not Protective Immunity in Gnotobiotic Pigs

    Science.gov (United States)

    Yuan, Lijuan; Geyer, Annelise; Hodgins, Douglas C.; Fan, Zhiqian; Qian, Yuan; Chang, Kyeong-Ok; Crawford, Sue E.; Parreño, Viviana; Ward, Lucy A.; Estes, Mary K.; Conner, Margaret E.; Saif, Linda J.

    2000-01-01

    We investigated the immunogenicity of recombinant double-layered rotavirus-like particle (2/6-VLPs) vaccines derived from simian SA11 or human (VP6) Wa and bovine RF (VP2) rotavirus strains. The 2/6-VLPs were administered to gnotobiotic pigs intranasally (i.n.) with a mutant Escherichia coli heat-labile toxin, LT-R192G (mLT), as mucosal adjuvant. Pigs were challenged with virulent Wa (P1A[8],G1) human rotavirus at postinoculation day (PID) 21 (two-dose VLP regimen) or 28 (three-dose VLP regimen). In vivo antigen-activated antibody-secreting cells (ASC) (effector B cells) and in vitro antigen-reactivated ASC (derived from memory B cells) from intestinal and systemic lymphoid tissues (duodenum, ileum, mesenteric lymph nodes [MLN], spleen, peripheral blood lymphocytes [PBL], and bone marrow lymphocytes) collected at selected times were quantitated by enzyme-linked immunospot assays. Rotavirus-specific immunoglobulin M (IgM), IgA, and IgG ASC and memory B-cell responses were detected by PID 21 or 28 in intestinal and systemic lymphoid tissues after i.n. inoculation with two or three doses of 2/6-VLPs with or without mLT. Greater mean numbers of virus-specific ASC and memory B cells in all tissues prechallenge were induced in pigs inoculated with two doses of SA11 2/6-VLPs plus mLT compared to SA11 2/6-VLPs without mLT. After challenge, anamnestic IgA and IgG ASC and memory B-cell responses were detected in intestinal lymphoid tissues of all VLP-inoculated groups, but serum virus-neutralizing antibody titers were not significantly enhanced compared to the challenged controls. Pigs inoculated with Wa-RF 2/6-VLPs (with or without mLT) developed higher anamnestic IgA and IgG ASC responses in ileum after challenge compared to pigs inoculated with SA11 2/6-VLPs (with or without mLT). Three doses of SA 11 2/6-VLP plus mLT induced the highest mean numbers of IgG memory B cells in MLN, spleen, and PBL among all groups postchallenge. However, no significant protection against

  17. Production of enterotoxin by Salmonella exposed to X rays

    International Nuclear Information System (INIS)

    Szulc, M.; Pliszka, A.; Peconek, J.

    1982-01-01

    The influence of irradiation on enterotoxin production by Salmonella enteritidis and S. typhimurium was evaluated using skin test elaborated by Tschaepe and others. The strains examined appeared to be equally sensitive to X rays. A dose of 100 Gy decreased the number of bacterial cells at approx. 10 D 10 and 200 Gy at 2-3 D 10 . Irradiation with 100 and 200 Gy of X rays did not influence enterotoxin production by microorganisms which were resistant to the process of irradiation. (author)

  18. Prevalence of enterotoxigenic Bacillus Cereus and Its enterotoxins ...

    African Journals Online (AJOL)

    Objectives: To determine the prevalence of enterotoxigenic Bacillus cereus (B. cereus) and enterotoxins in milk and milk products. Design: A random sampling of milk products was carried out. Setting: Market milk and milk products were collected from retail shops in Nairobi and analysed for contamination with ...

  19. Prevalence, antibiotic-resistance properties and enterotoxin gene ...

    African Journals Online (AJOL)

    Prevalence, antibiotic-resistance properties and enterotoxin gene profile of Bacillus cereus strains isolated from milk-based baby foods. ... Conclusion: Considerable prevalence of resistant and toxigenic B. cereus and high consumption of milk-based infant foods in Iran, represent an important public health issue which ...

  20. Construction of PVX virus-expression vector to express enterotoxin ...

    African Journals Online (AJOL)

    Potato X potyvirus (PVX)-based vector has been comprehensively applied in transient expression system. In order to produce the heterologous proteins more quickly and stably, the ClaI and NotI enzyme sites were introduced into the Enterotoxin fusion gene LTB-ST by polymerase chain reaction (PCR) and the LTB-ST ...

  1. Evaluation of an alternative extraction procedure for enterotoxin determination in dairy products.

    Science.gov (United States)

    Meyrand, A; Atrache, V; Bavai, C; Montet, M P; Vernozy-Rozand, C

    1999-06-01

    A concentration protocol based on trichloroacetic acid precipitation was evaluated and compared with the reference method using dialysis concentration. Different quantities of purified staphylococcal enterotoxins were added to pasteurized Camembert-type cheeses. Detection of enterotoxins in these cheeses was performed using an automated detection system. Raw goat milk Camembert-type cheeses involved in a staphylococcal food poisoning were also tested. Both enterotoxin extraction methods allowed detection of the lowest enterotoxin concentration level used in this study (0.5 ng g-1). Compared with the dialysis concentration method, TCA precipitation of staphylococcal enterotoxins was 'user-friendly' and less time-consuming. These results suggest that TCA precipitation is a rapid (1 h), simple and reliable method of extracting enterotoxin from food which gives excellent recovery from dairy products.

  2. Stimulation of Clostridium perfringens enterotoxin formation by caffeine and theobromine.

    Science.gov (United States)

    Labbe, R G; Nolan, L L

    1981-01-01

    In the presence of 100 micrograms of caffeine per ml or 200 micrograms of theobromine per ml, sporulation of Clostridium perfringens NCTC 8679 rose from less than 1 to 80 or 85%. Enterotoxin concentration increased from undetectable levels to 450 micrograms/mg of cell extract protein. Heat-resistant spore levels increased from less than 1,000 to between 1 X 10(7) and 2 X 10(7)/ml. These effects were partially reversible by the addition of adenosine or thymidine. In the case of NCTC 8238, caffeine and theobromine caused a three- to fourfold increase in the percentages of cells possessing refractile spores and a similar increase in enterotoxin concentration. Heat-resistant spore levels, however, were unaffected. Inosine was ineffective in promoting sporulation in NCTC 8679. PMID:6271685

  3. Monkey Feeding Assay for Testing Emetic Activity of Staphylococcal Enterotoxin.

    Science.gov (United States)

    Seo, Keun Seok

    2016-01-01

    Staphylococcal enterotoxins (SEs) are unique bacterial toxins that cause gastrointestinal toxicity as well as superantigenic activity. Since systemic administration of SEs induces superantigenic activity leading to toxic shock syndrome that may mimic enterotoxic activity of SEs such as vomiting and diarrhea, oral administration of SEs in the monkey feeding assay is considered as a standard method to evaluate emetic activity of SEs. This chapter summarizes and discusses practical considerations of the monkey feeding assay used in studies characterizing classical and newly identified SEs.

  4. The prevalence of methicillin-resistant Staph. aureus strains producing enterotoxin A and B

    Directory of Open Access Journals (Sweden)

    Safiyeh Abbasi

    2016-02-01

    Full Text Available Background: Staphylococcus aureus is a gram positive coccus which is able to cause different kinds of infection in certain condition. The function of this bacteria is to provide the conditions for the invasion of it to the host with the secretion of different sorts of toxins such as Staphylococcus aureus enterotoxin, including important virulence factors that super antigens are all factors digestive inconvenience. Staphylococcus aureus enterotoxin-secreting toxins such conditions provides invasion of host genes. There are different types of SE, but type A enterotoxin (SEA and type B enterotoxin (SEB are the most important types. Therefore, in this study, the prevalence of Staphylococcus aureus toxin-producing enterotoxin genes (SEB, SEA in clinical strains isolated from patients in teaching hospitals of Shahrekord city, Iran, were studied. Methods: This cross-sectional and descriptive study, which was conducted from May 2014 to December 2014. A hundred and ten isolates of Staphylococcus aureus from patients collected over a period of 8 months and were first identified using standard biochemical methods and laboratory. Using standard methods and laboratory tests were identified and compared with the antibiotic oxacillin minimum inhibitory concentration were determined by broth micro dilution, and then they were assessed by polymerase chain reaction (PCR technique. Results: The results indicated that, 110 samples of dairy products infected by Staphylococcus aureus were detected. Two cases (1.8% of these infected samples were carrying both enterotoxin A and enterotoxin B genes. The frequencies of enterotoxin A genes were twenty-six cases (23/6% and The frequencies of enterotoxin B genes were two cases (1/8%, respectively. Conclusion: The detection of enterotoxin A and enterotoxin B genes, shows the most important role they have in bringing about superinfection. The detection of enterotoxin A and B genes, shows the most important role they have in

  5. Cloning the enterotoxin gene from Clostridium perfringens type A

    OpenAIRE

    Iwanejko, Lesley Ann.

    1991-01-01

    A C. perfringens type A genomic library was constructed in E. coli by banking overlapping 6-10 kbp Hind III fragments of chromosomal DNA from the enterotoxin (CPE) positive strain NCTC 8239 into the pUC derived vector pHG165. The library was screened by colony hybridization with a degenerate 26 bp oligonucleotide probe, derived from the amino acid sequence CPE9_17A. complex mixture of plasmid DNA was isolated from the only hybridization positive clone. A second round of screening picked out a...

  6. Growth and enterotoxin production of Bacillus cereus in cow, goat, and sheep milk

    Directory of Open Access Journals (Sweden)

    Lenka Necidová

    2014-01-01

    Full Text Available The aim of this study was to compare Bacillus cereus growth rates and diarrhoeal enterotoxin production in raw and pasteurized goat, sheep, and cow milk in terms of storage conditions. Milk samples were inoculated with B. cereus (CCM 2010, which produces diarrhoeal enterotoxins. Enterotoxin production was tested by ELISA (Enzyme-Linked Immunosorbent Assay, and the count of B. cereus was determined by the plate method. With raw cow milk, B. cereus growth and enterotoxin production can be completely suppressed; in raw goat and sheep milk, enterotoxin was produced at 22 °C. In pasteurized cow, goat, and sheep milk, the B. cereus count increased under all storage conditions, with more rapid growth being observed at 15 °C (sheep milk and 22 °C (cow and goat milk. Enterotoxin presence was detected at 15 °C and 22 °C, and with pasteurized cow milk also at 8 °C. Our model experiments have determined that B. cereus multiplication and subsequent enterotoxin production depend on storage temperature and milk type.

  7. Sulfasalazine Attenuates Staphylococcal Enterotoxin B-Induced Immune Responses

    Directory of Open Access Journals (Sweden)

    Teresa Krakauer

    2015-02-01

    Full Text Available Staphylococcal enterotoxin B (SEB and related exotoxins are important virulence factors produced by Staphylococcus aureus as they cause human diseases such as food poisoning and toxic shock. These toxins bind directly to cells of the immune system resulting in hyperactivation of both T lymphocytes and monocytes/macrophages. The excessive release of proinflammatory cytokines from these cells mediates the toxic effects of SEB. This study examined the inhibitory activities of an anti-inflammatory drug, sulfasalazine, on SEB-stimulated human peripheral blood mononuclear cells (PBMC. Sulfasalazine dose-dependently inhibited tumor necrosis factor α, interleukin 1 (IL-1 β, IL-2, IL-6, interferon γ (IFNγ, and various chemotactic cytokines from SEB-stimulated human PBMC. Sulfasalazine also potently blocked SEB-induced T cell proliferation and NFκB activation. These results suggest that sulfasalazine might be useful in mitigating the toxic effects of SEB by blocking SEB-induced host inflammatory cascade and signaling pathways.

  8. The staphylococcal enterotoxin (SE) family: SEB and siblings.

    Science.gov (United States)

    Krakauer, Teresa; Stiles, Bradley G

    2013-11-15

    Staphylococcus aureus plays an important role in numerous human cases of food poisoning, soft tissue, and bone infections, as well as potentially lethal toxic shock. This common bacterium synthesizes various virulence factors that include staphylococcal enterotoxins (SEs). These protein toxins bind directly to major histocompatibility complex class II on antigen-presenting cells and specific Vβ regions of T-cell receptors, resulting in potentially life-threatening stimulation of the immune system. Picomolar concentrations of SEs ultimately elicit proinflammatory cytokines that can induce fever, hypotension, multi-organ failure, and lethal shock. Various in vitro and in vivo models have provided important tools for studying the biological effects of, as well as potential vaccines/therapeutics against, the SEs. This review succinctly presents known physical and biological properties of the SEs, including various intervention strategies. In particular, SEB will often be portrayed as per biodefense concerns dating back to the 1960s.

  9. Effect of Sodium Chloride and pH on Enterotoxin C Production

    Science.gov (United States)

    Genigeorgis, Constantin; Foda, Mohamed S.; Mantis, Antony; Sadler, Walter W.

    1971-01-01

    Growth and production of enterotoxin C by Staphylococcus aureus strain 137 in 3% + 3% protein hydrolysate powder N-Z Amine NAK broths with 0 to 12% NaCl and an initial pH of 4.00 to 9.83 were studied during an 8-day incubation period at 37 C. Growth was initiated at pH values as low as 4.00 and as high as 9.83 at 0% salt level as long as the inoculum contained at least 108 cells per ml. Rate of growth decreased as the NaCl concentration was increased gradually to 12%. Enterotoxin C was produced in broths inoculated with 108 cells per ml and above and having initial pH ranges of 4.00 to 9.83, 4.40 to 9.43, 4.50 to 8.55 and respective NaCl concentrations of 0, 4, and 8%. In the presence of 10% NaCl, the pH range supporting enterotoxin C production was 5.45 to 7.30 for an inoculum level of 108 cells per ml and 6.38 to 7.30 for 3.6 × 106 cells per ml. In repeated experiments in which the inoculum contained 108 cells per ml, we failed to demonstrate enterotoxin C production in broths with 12% NaCl and a pH range of 4.50 to 8.55 and concentrated up to 14 times. The effect of NaCl on enterotoxin C production followed the same pattern as its effect on enterotoxin B production. As the concentration of NaCl increased from 0 to 10%, yields of enterotoxin B and C decreased to undetectable amounts. PMID:5574320

  10. Purification of heat labile toxin from Bordetella pertussis vaccine ...

    African Journals Online (AJOL)

    K.C. Shivanandappa

    2015-06-23

    Jun 23, 2015 ... heat inactivation at 56 °C for 30 min during Wcpv prepara- tion. Therefore, the ... The B2 culture medium was prepared with following composi- tions: Bacto casamino ... 15min at the ambient temperature (+18 to 22 °C). The.

  11. Purification of heat labile toxin from Bordetella pertussis vaccine ...

    African Journals Online (AJOL)

    . pertussis vaccine strain 134 by employing indigenous technology and examining the immuno-biochemical aspects of the purified protein. Materials and methods: Shaker cultivation of B. pertussis strain 134, sterility, opacity confirmation, TCA ...

  12. Staphylococcus aureus growth and enterotoxin production in different types of milk

    Directory of Open Access Journals (Sweden)

    Bohdana Janštová, Jr.

    2012-01-01

    Full Text Available The aim of our study was to assess Staphylococcus aureus growth and the time of first detection of staphylococcal enterotoxins type A, B and C (SEA, SEB, SEC in different type of milk, depending on the strain and storage conditions. Raw, pasteurized, and UHT milk were inoculated with three strains of S. aureus, and growth patterns were determined by the plate method in accordance with EN ISO 6888-1. Baird-Parker agar medium was used for the detection of S. aureus and the Enzyme Linked Fluorescent Assay (ELFA used with a miniVIDAS analyzer tested the production of staphylococcal enterotoxins. The results of model experiments showed the dependence of the growth rate and subsequent production of staphylococcal enterotoxins on incubation (storage temperature, S. aureus strain, and type of milk. A significant finding was that the growth of S. aureus and production of enterotoxins in raw milk was inhibited by natural microflora, and production of enterotoxins was therefore not detected in raw milk within 102 hours of storage either at 15 °C or 22 °C. The highest risk of SEs production is associated with secondary contamination of pasteurized and UHT milk when stored at room temperature, where production was first detected after 12 hours of incubation.

  13. Staphylococcus aureus Enterotoxin A Gene Isolated From Raw Red Meat and Poultry in Tehran, Iran

    Directory of Open Access Journals (Sweden)

    Mohammad Hossein Sarrafzadeh Zargar

    2014-07-01

    Full Text Available Background: Staphylococcus aureus is the most prevalent infectious agent of food materials. Enterotoxin producing types of S. aureus cause well-known food-borne disease. Staphylococcal Enterotoxin A (SEA is the most important agent of gastroenteritis. Objectives: The present study aimed to screen the raw meat samples collected from different regions of Tehran for S. aureus infection and type of encoding enterotoxin. Materials and Methods: Hundred and eighty six meat samples were collected randomly from city dealers and transferred to laboratory within screw cap containers. The samples were first cultured according to the standard bacteriological methods and then S. aureus isolates were identified using standard bacteriological tests. The isolates were subjected to Polymerase Chain Reaction (PCR to detect gene encoding SEA. Results: Staphylococcus aureus isolated from 29 (15.6% meat samples including beef 14.8%, raw lamb 15%, raw chicken 15.7% and raw turkey 16.6%. Using special primer sets proved that the species isolated from five samples (two raw chicken, two raw beef and one raw turkey encoded enterotoxin A. Conclusions: Although staphylococcal contamination within food material is more or less a routine, but detection of enterotoxin encoding species from raw meat samples is alarming for health authorities. These data highlight the importance of periodic surveillance of raw meat distributed among ordinary consumers.

  14. Sub-inhibitory stress with essential oil affects enterotoxins production and essential oil susceptibility in Staphylococcus aureus.

    Science.gov (United States)

    Turchi, Barbara; Mancini, Simone; Pistelli, Luisa; Najar, Basma; Cerri, Domenico; Fratini, Filippo

    2018-03-01

    Fourteen wild strains of Staphylococcus aureus positive for gene sea were tested for enterotoxins production and the minimum inhibitory concentration of Leptospermum scoparium, Origanum majorana, Origanum vulgare, Satureja montana and Thymus vulgaris essential oils (EOs) were determined. After this trial, bacteria stressed with sub-inhibitory concentration of each EO were tested for enterotoxins production by an immunoenzymatic assay and resistance to the same EO. Oregano oil exhibited the highest antibacterial activity followed by manuka and thyme oils. After the exposure to a sub-inhibitory concentration of EOs, strains displayed an increased sensitivity in more than 95% of the cases. After treatment with oregano and marjoram EOs, few strains showed a modified enterotoxins production, while 43% of the strains were no longer able to produce enterotoxins after treatment with manuka EO. The results obtained in this study highlight that exposure to sub-inhibitory concentration of EO modifies strains enterotoxins production and EOs susceptibility profile.

  15. Receptors and cGMP signalling mechanism for E. coli enterotoxin in opossum kidney

    International Nuclear Information System (INIS)

    Forte, L.R.; Krause, W.J.; Freeman, R.H.

    1988-01-01

    Receptors for the heat-stable enterotoxin produced by Escherichia coli were found in the kidney and intestine of the North American opossum and in cultured renal cell lines. The enterotoxin markedly increased guanosine 3',5'-cyclic monophosphate (cGMP) production in slices of kidney cortex and medulla, in suspensions of intestinal mucosa, and in the opossum kidney (OK) and rat kangaroo kidney (PtK-2) cell lines. In contrast, atrial natriuretic factor elicited much smaller increases in cGMP levels of kidney, intestine, or cultured kidney cell lines. The enterotoxin receptors in OK cells had a molecular mass of approximately 120 kDa when measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of receptors crosslinked with 125 I-enterotoxin. The occurrence of receptors for the E. coli peptide in OK implies that these receptors may be involved in the regulation of renal tubular function in the opossum. E. coli enterotoxin caused a much larger increase in urine cGMP excretion than did atrial natriuretic factor when these peptides were injected intravenously into opossums. However, atrial natriuretic factor elicited a marked diuresis, natriuresis, and increased urinary excretion of calcium, phosphate, potassium, and magnesium. In contrast, the enterotoxin did not acutely influence OK fluid and electrolyte excretion. Thus the substantial increase in cGMP synthesis produced by the bacterial peptide in OK cortex and medulla in vitro and the increased renal excretion of cGMP in vivo were not associated with changes in electrolyte or water excretion. Whether cGMP represents a second messenger molecule in the kidney is an interesting question that was raised but not answered in this series of experiments

  16. Organ Culture as a Model System for Studies on Enterotoxin Interactions with the Intestinal Epithelium

    DEFF Research Database (Denmark)

    Lorenzen, Ulver Spangsberg; Hansen, Gert H; Danielsen, E Michael

    2015-01-01

    Studies on bacterial enterotoxin-epithelium interactions require model systems capable of mimicking the events occurring at the molecular and cellular levels during intoxication. In this chapter, we describe organ culture as an often neglected alternative to whole-animal experiments or enterocyte......-like cell lines. Like cell culture, organ culture is versatile and suitable for studying rapidly occurring events, such as enterotoxin binding and uptake. In addition, it is advantageous in offering an epithelium with more authentic permeability/barrier properties than any cell line, as well...

  17. Bystander Host Cell Killing Effects of Clostridium perfringens Enterotoxin

    Directory of Open Access Journals (Sweden)

    Archana Shrestha

    2016-12-01

    Full Text Available Clostridium perfringens enterotoxin (CPE binds to claudin receptors, e.g., claudin-4, and then forms a pore that triggers cell death. Pure cultures of host cells that do not express claudin receptors, e.g., fibroblasts, are unaffected by pathophysiologically relevant CPE concentrations in vitro. However, both CPE-insensitive and CPE-sensitive host cells are present in vivo. Therefore, this study tested whether CPE treatment might affect fibroblasts when cocultured with CPE-sensitive claudin-4 fibroblast transfectants or Caco-2 cells. Under these conditions, immunofluorescence microscopy detected increased death of fibroblasts. This cytotoxic effect involved release of a toxic factor from the dying CPE-sensitive cells, since it could be reproduced using culture supernatants from CPE-treated sensitive cells. Supernatants from CPE-treated sensitive cells, particularly Caco-2 cells, were found to contain high levels of membrane vesicles, often containing a CPE species. However, most cytotoxic activity remained in those supernatants even after membrane vesicle depletion, and CPE was not detected in fibroblasts treated with supernatants from CPE-treated sensitive cells. Instead, characterization studies suggest that a major cytotoxic factor present in supernatants from CPE-treated sensitive cells may be a 10- to 30-kDa host serine protease or require the action of that host serine protease. Induction of caspase-3-mediated apoptosis was found to be important for triggering release of the cytotoxic factor(s from CPE-treated sensitive host cells. Furthermore, the cytotoxic factor(s in these supernatants was shown to induce a caspase-3-mediated killing of fibroblasts. This bystander killing effect due to release of cytotoxic factors from CPE-treated sensitive cells could contribute to CPE-mediated disease.

  18. Effects of Bile Acids and Nisin on the Production of Enterotoxin by Clostridium perfringens in a Nutrient-Rich Medium

    Directory of Open Access Journals (Sweden)

    Miseon Park

    2018-01-01

    Full Text Available Clostridium perfringens is the second most common cause of bacterial foodborne illness in the United States, with nearly a million cases each year. C. perfringens enterotoxin (CPE, produced during sporulation, damages intestinal epithelial cells by pore formation, which results in watery diarrhea. The effects of low concentrations of nisin and bile acids on sporulation and toxin production were investigated in C. perfringens SM101, which carries an enterotoxin gene on the chromosome, in a nutrient-rich medium. Bile acids and nisin increased production of enterotoxin in cultures; bile acids had the highest effect. Both compounds stimulated the transcription of enterotoxin and sporulation-related genes and production of spores during the early growth phase. They also delayed spore outgrowth and nisin was more inhibitory. Bile acids and nisin enhanced enterotoxin production in some but not all other C. perfringens isolates tested. Low concentrations of bile acids and nisin may act as a stress signal for the initiation of sporulation and the early transcription of sporulation-related genes in some strains of C. perfringens, which may result in increased strain-specific production of enterotoxin in those strains. This is the first report showing that nisin and bile acids stimulated the transcription of enterotoxin and sporulation-related genes in a nutrient-rich bacterial culture medium.

  19. Effects of Bile Acids and Nisin on the Production of Enterotoxin by Clostridium perfringens in a Nutrient-Rich Medium.

    Science.gov (United States)

    Park, Miseon; Rafii, Fatemeh

    2018-01-01

    Clostridium perfringens is the second most common cause of bacterial foodborne illness in the United States, with nearly a million cases each year. C. perfringens enterotoxin (CPE), produced during sporulation, damages intestinal epithelial cells by pore formation, which results in watery diarrhea. The effects of low concentrations of nisin and bile acids on sporulation and toxin production were investigated in C. perfringens SM101, which carries an enterotoxin gene on the chromosome, in a nutrient-rich medium. Bile acids and nisin increased production of enterotoxin in cultures; bile acids had the highest effect. Both compounds stimulated the transcription of enterotoxin and sporulation-related genes and production of spores during the early growth phase. They also delayed spore outgrowth and nisin was more inhibitory. Bile acids and nisin enhanced enterotoxin production in some but not all other C. perfringens isolates tested. Low concentrations of bile acids and nisin may act as a stress signal for the initiation of sporulation and the early transcription of sporulation-related genes in some strains of C. perfringens , which may result in increased strain-specific production of enterotoxin in those strains. This is the first report showing that nisin and bile acids stimulated the transcription of enterotoxin and sporulation-related genes in a nutrient-rich bacterial culture medium.

  20. Use of inactivated E.Coli enterotoxins to enhance respiratory mucosal adjuvanticity during vaccination in swine

    Science.gov (United States)

    In order to augment responses to respiratory vaccines in swine, various adjuvants were intranasally co-administered with an antigen to pigs. Detoxified E. coli enterotoxins LTK63 and LTR72 enhanced mucosal and systemic immunity to the model peptide, exhibiting their efficacy as mucosal adjuvants for...

  1. Production of enterotoxins of Staphylococcus spp. isolated from samples of sheep milk

    Directory of Open Access Journals (Sweden)

    František Zigo

    2014-02-01

    Full Text Available In our study was followed occurrence of mastitis in herd of 430 sheep of breed zoslachtena valaska with hand milking technology examined two times during one lactation season. Individual examination consisted from clinical examination of udder and microbiological examination of milk samples. By PCR was determined presence of genes coding production of enterotoxins, and by ELISA methods production individual types of enterotoxins. From individual forms of mastitis were frequently detected subacute (6.7%, subclinical (5.7% and acute (2.9%. The coagulase-negative staphylococci (CNS were identified in 102 (65.4% from all 156 positive isolates. The CNS and S. aureus caused subacute (5.1%, subclinical (3.9% and acute (2.4% forms of mastitis. The most frequently isolated were S. epidermidis, followed by S. chromogenes and S. xylosus from ewes with subacute and subclinical mastitis. From acute and chronical forms of mastitis were  predominantly isolated S. aureus, S. uberis and S. epidermidis. The production of staphylococcal enterotoxins (SE - SEA, SEB, SEC, SED and the presence of genes sec (3, sea (2, seb (2 and sed (2 were determined in S. aureus, S. epidermidis, S. schleiferi and S. chromogenes, respectively. The results suggested on the high occurrence (12.4% of subacute and subclinical forms. Confirmed production of enterotoxins and presence of genes coding their production present a risk for human health and decreased a quality of milk and products from sheep´s milk.

  2. Lung and pharyngeal abscess caused by enterotoxin G- and I-producing Staphylococcus aureus.

    Science.gov (United States)

    Barnett, S Y; Hattotuwa, K L; Teare, L

    2012-05-01

    We report a particularly serious case of extensive meticillin sensitive Staphylococcal lung and pharyngeal abscess. Our patient had no significant risk factors for severe infection. The detection of enterotoxin G and I here suggest that when present together, these toxins work synergistically to produce a more virulent strain of Staphylococcus aureus. Copyright © 2011. Published by Elsevier Ltd.

  3. Lab-on-a-chip for label free biological semiconductor analysis of Staphylococcal Enterotoxin B

    NARCIS (Netherlands)

    Yang, Minghui; Sun, Steven; Bruck, Hugh Alan; Kostov, Yordan; Rasooly, Avraham

    2010-01-01

    We describe a new lab-on-a-chip (LOC) which utilizes a biological semiconductor (BSC) transducer for label free analysis of Staphylococcal Enterotoxin B (SEB) (or other biological interactions) directly and electronically. BSCs are new transducers based on electrical percolation through a

  4. Inhibitory effect of totarol on exotoxin proteins hemolysin and enterotoxins secreted by Staphylococcus aureus.

    Science.gov (United States)

    Shi, Ce; Zhao, Xingchen; Li, Wenli; Meng, Rizeng; Liu, Zonghui; Liu, Mingyuan; Guo, Na; Yu, Lu

    2015-10-01

    Staphylococcus aureus (S. aureus) causes a wide variety of infections, which are of major concern worldwide. S. aureus produces multiple virulence factors, resulting in food infection and poisoning. These virulence factors include hyaluronidases, proteases, coagulases, lipases, deoxyribonucleases and enterotoxins. Among the extracellular proteins produced by S. aureus that contribute to pathogenicity, the exotoxins α-hemolysin, staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin B (SEB) are thought to be of major significance. Totarol, a plant extract, has been revealed to inhibit the proliferation of several pathogens effectively. However, there are no reports on the effects of totarol on the production of α-hemolysin, SEA or SEB secreted by S. aureus. The aim of this study was to evaluate the effects of totarol on these three exotoxins. Hemolysis assay, western blotting and real-time reverse transcriptase-PCR assay were performed to identify the influence of graded subinhibitory concentrations of totarol on the production of α-hemolysin and the two major enterotoxins, SEA and SEB, by S. aureus in a dose-dependent manner. Moreover, an enzyme linked immunosorbent assay showed that the TNF-α production of RAW264.7 cells stimulated by S. aureus supernatants was inhibited by subinhibitory concentrations of totarol. Form the data, we propose that totarol could potentially be used as a promising natural compound in the food and pharmaceutical industries.

  5. Rauvolfia grandiflora (Apocynaceae extract interferes with staphylococcal density, enterotoxin production and antimicrobial activity

    Directory of Open Access Journals (Sweden)

    Lanamar de Almeida Carlos

    2010-10-01

    Full Text Available Staphylococci bacteria are involved in many human and animal infections and development of alternative antimicrobial drugs against pathogenic bacteria is of great interest to the pharmaceutical industry. This study investigated the in vitro effect of Rauvolfia grandiflora methanol extract (root bark fraction (RGE on the density of ATCC strains of Staphylococcus aureus and Staphylococcus epidermidis, and a clinical enterotoxin-producer, S. aureus bovine strain. The alkaloid, isoreserpiline, obtained from dichloromethane extract of R. grandiflora was ineffective against the strains tested. After incubation of staphylococci strains in the presence of 1.2 mg.mL-1 RGE, a significant inhibition of cell growth was observed using both spectrophotometry and ELISA assays. Twelve drugs were evaluated for their antimicrobial effects on culture RGE-treated cells using the disk diffusion method. Penicillin resistant strains became sensitive to the drug after RGE treatment. Furthermore, enterotoxin production by RGE-treated S. aureus was evaluated using a standardized ELISA method. Although staphylococcal LSA 88 bovine strain cells remained viable after exposure to the extract, enterotoxin production was precluded in 20% after RGE treatment. Significant interference in staphylococci cell density, drug sensitivity and enterotoxin secretion was observed after treatment. The study highlights the necessity to find new methods of disease prevention and new antibiotic therapies against staphylococcal infections.

  6. Rauvolfia grandiflora (apocynaceae) extract interferes with staphylococcal density, enterotoxin production and antimicrobial activity.

    Science.gov (United States)

    de Almeida Carlos, Lanamar; da Silva Amaral, Kenas Aguiar; Curcino Vieira, Ivo José; Mathias, Leda; Braz-Filho, Raimundo; Silva Samarão, Solange; Vieira-da-Motta, Olney

    2010-07-01

    Staphylococci bacteria are involved in many human and animal infections and development of alternative antimicrobial drugs against pathogenic bacteria is of great interest to the pharmaceutical industry. This study investigated the in vitro effect of Rauvolfia grandiflora methanol extract (root bark fraction) (RGE) on the density of ATCC strains of Staphylococcus aureus and Staphylococcus epidermidis, and a clinical enterotoxin-producer, S. aureus bovine strain. The alkaloid, isoreserpiline, obtained from dichloromethane extract of R. grandiflora was ineffective against the strains tested. After incubation of staphylococci strains in the presence of 1.2 μg.mL(-1) RGE, a significant inhibition of cell growth was observed using both spectrophotometry and ELISA assays. Twelve drugs were evaluated for their antimicrobial effects on culture RGE-treated cells using the disk diffusion method. Penicillin resistant strains became sensitive to the drug after RGE treatment. Furthermore, enterotoxin production by RGE-treated S. aureus was evaluated using a standardized ELISA method. Although staphylococcal LSA 88 bovine strain cells remained viable after exposure to the extract, enterotoxin production was precluded in 20% after RGE treatment. Significant interference in staphylococci cell density, drug sensitivity and enterotoxin secretion was observed after treatment. The study highlights the necessity to find new methods of disease prevention and new antibiotic therapies against staphylococcal infections.

  7. Expression of Staphylococcal Enterotoxins under Stress Encountered during Food Production and Preservation.

    Science.gov (United States)

    Schelin, Jenny; Susilo, Yusak Budi; Johler, Sophia

    2017-12-15

    Staphylococcal food poisoning (SFP) is the most prevalent cause of food-borne intoxications worldwide. Consumption of enterotoxins preformed in food causes violent vomiting and can be fatal in children and the elderly. While being repressed by competing bacteria in most matrices, Staphylococcus aureus benefits from crucial competitive advantages in foods with high osmolarity or low pH. During recent years, the long-standing belief in the feasibility of assessing SFP risk based on colony-forming units of S. aureus present in food products has been disproven. Instead, researchers and food business operators are acutely aware of the imminent threat arising from unforeseeable enterotoxin production under stress conditions. This paradigm shift led to a variety of new publications enabling an improved understanding of enterotoxin expression under stress conditions encountered in food. The wealth of data provided by these studies is extremely diverse, as it is based on different methodological approaches, staphylococcal strains, stressors, and enterotoxins. Therefore, in this review, we aggregated and critically evaluated the complex findings of these studies, to provide readers with a current overview of the state of research in the field.

  8. The olive compound 4-hydroxytyrosol inactivates Staphyloccoccus aureus bacteria and Staphylococcal enterotoxin A (SEA)

    Science.gov (United States)

    The foodborne pathogen Staphylococcus aureus produces the virulent staphylococcal enterotoxin A (SEA), a single chain protein which consists of 233 amino acid residues with a molecular weight of 27,078 Da. SEA is a superantigen that is reported to contribute to animal (mastitis) and human (emesis, ...

  9. Cloning and Expression of Nano Body Gene against Enterotoxin B of Staphylococcus Aureus

    Directory of Open Access Journals (Sweden)

    Zahra Tavassoli

    2017-02-01

    Full Text Available Background & Objectives: Staphylococcus aureus bacteria causes many different diseases by secretion of various enterotoxins. Therefore, it is necessary to develop ways that facilitate the detection of enterotoxins. Nowadays, immunochemical methods which are based on monoclonal antibody technology are used. The heavy chain antibodies that are called VHH or Nano body were found in blood serum of the Camelidae family. The unique properties of this antibody such as their binding to small molecules like toxins make them attractive candidates for the development of immunodiagnostic tests. The present study was done to achieve a VHH molecules against Staphylococcus enterotoxin B. Materials & Methods: Freighting phage library for isolate private Nano bodies against enterotoxin B was done in previous works. Next, pCANTAB 5E vector that consists VHH, extracted from E.coli bacteria strain xl1blue, and after doing PCR process with relative primers, sub cloning in pET21a(+ as an expression vector with cut sites NdeI and XhoI was done. Transformation in E.coli bacteria strain BL21(DE3 was done. Then, the cells effected with IPTG and producing time, and other terms were optimized. Finally, the expression of the protein with SDS-PAGE and western blot techniques was evaluated. Result: For proving cloning of nano body gene in pET21a (+ vector, nucleotide sequence of gene was analyzed, and transforming to E.coli bacteria strain BL21(DE3 was successful. After inspiration, active protein in cell was seen by SDS-PAGE technique and proved by western blot. Conclusion: cloning, sub cloning, and nonabody expression were surveyed in this research. Production of this protein can help to develop new therapeutic methods and produce vaccine against enterotoxin B of Staphylococcus aureus

  10. Antidiarrhoeal activity of different plants used in traditional medicine

    African Journals Online (AJOL)

    SERVER

    2007-12-28

    Dec 28, 2007 ... components such as artificial sweeteners and lactose and the result is the .... decrease in severe diarrhoea of rats treated with castor oil. This extract also ..... heat-labile enterotoxin and ganglioside interaction. J. Ethnopharma-.

  11. Enhancement of Intranasal Vaccination in Mice with Deglycosylated Chain A Ricin by LTR72, a Novel Mucosal Adjuvant

    National Research Council Canada - National Science Library

    Kende, Meir; Del Giudice, Giuseppe; Rivera, Noelia; Hewetson, John

    2006-01-01

    .... However, in the presence of 4, 2, or 1 microg of the mucosal adjuvant LTR72, a mutant of the heat-labile enterotoxin of Escherichia coli, the low antibody response and protection were substantially enhanced...

  12. Enhancement of Intranasal Vaccination in Mice with Deglycosylated Chain A Ricin by LTR72, a Novel Mucosal Adjuvant

    National Research Council Canada - National Science Library

    Kende, Meir; Del Giudice, Giuseppe; Rivera, Noelia; Hewetson, John

    2006-01-01

    .... However, in the presence of 4, 2, or 1 micro-gram of the mucosal adjuvant LTR72, a mutant of the heat-labile enterotoxin of Escherichia coli, the low antibody response and protection were substantially enhanced...

  13. [Enterotoxin genes occurance among S. aureus strains isolated from inpatients and carriers].

    Science.gov (United States)

    Lawrynowicz-Paciorek, Maja; Kochman, Maria; Piekarska, Katarzyna; Wyrebiak, Agata; Potracka, Ewa; Leniak-Chmiel, Urszula; Magdziak, Agnieszka

    2006-01-01

    We examined 44 inpatients and 66 carriers Staphylococcus aureus strains, isolated in years 2002-2005, for the presence of 18 enterotoxin genes (se/sel) (by PCR), the ability for A-D enterotoxin production (by SET-RPLA) and antibiotic resistance distribution (by disc diffusion method). se/sel genes were detected in 90,9% of all strains, sea (70,5%) and selk and selq (52,3%) - among inpatients strains and egc (65,2%) - among carriers strains were the most frequently se/sel genes found. Positive results of SET-RPLA were consistent with PCR results. There was no correlation observed between antibiotic resistance and se/sel genes distribution among tested S. aureus strains.

  14. Prevalence of Staphylococcal Enterotoxins in Ready-to-Eat Foods Sold in Istanbul.

    Science.gov (United States)

    Ulusoy, Beyza H; Çakmak Sancar, Burcu; Öztürk, Muhsin

    2017-10-01

    The aim of this study was to investigate the prevalence of staphylococcal enterotoxins (SEs) in ready-to-eat (RTE) foods sold in Istanbul, Turkey. A total of 5,241 samples were randomly collected from various caterers, hotels, and restaurants from 2014 to 2016. The samples were classified into four groups: (i) various cooked RTE meat and vegetable meals, (ii) various RTE salads, charcuterie, and cold appetizers, (iii) various cooked RTE bakery products (pasta, pastries, pizza, pita, ravioli, etc.), and (iv) any cooked RTE sweets and desserts (pudding, custard, cream, ashura, etc.). The samples were examined for the presence of SEs by 3M Tecra Staph Enterotoxin Visual Immunoassay method, which is a manual enzyme-linked immunosorbent assay method. Among all samples, only 1 (0.019%) RTE meal (vegetable meal with meat) was found to be contaminated with SEs, a good result in terms of staphylococcal food poisoning risk and public health.

  15. The influence of radiation on the enterotoxin and thermoresistant deoxyribo-nuclease production by Staphylococcus sp

    International Nuclear Information System (INIS)

    Szulc, M.; Pliszka, A.; Peconek, J.

    1980-01-01

    Six strains of Staph. aureus present in the environment with and without protein were exposed to X rays in the doses range from 1 to 400 Gy. The production of enterotoxin and thermoresistant deoxyribonuclease by enterotoxic strains No 262 and 100 was determined. All the strains showed similar radiosensitivity to X rays. About 1 per cent of protein in the environment exposed to radiation increased the resistance of Staphylococcus strains. The doses of radiation from 1 to 400 Gy did not influence the enterotoxin A and B production by bacteria multiplied from those which had survived the radiation. The irradiation of the strains present in the environment without protein brought about a temporary inhibition of thermoresistant deoxyribonuclease production. (author)

  16. Cloning in Escherichia coli of the enterotoxin gene from Clostridium perfringens type A.

    Science.gov (United States)

    Iwanejko, L A; Routledge, M N; Stewart, G S

    1989-04-01

    A 26 bp DNA probe has been constructed with minimal degeneracy to the protein sequence for Clostridium perfringens enterotoxin. The probe has been hybridized against a 6-10 kb chromosomal bank from C. perfringens 8239, prepared as a HindIII partial digest in pHG165. From this survey a clone has been identified containing a 6.8 kb DNA insert with strong hybridization to the probe. Direct plasmid sequencing has identified a translational reading frame within this clone which correlates with the known protein sequence for the type A enterotoxin. DNA sequences 5' to this open reading frame and containing the putative transcriptional control regions show areas of significant homology with regions upstream from the ATG codon of the tetanus toxin gene.

  17. Selection and characterization of DNA aptamers against Staphylococcus aureus enterotoxin C1.

    Science.gov (United States)

    Huang, Yukun; Chen, Xiujuan; Duan, Nuo; Wu, Shijia; Wang, Zhouping; Wei, Xinlin; Wang, Yuanfeng

    2015-01-01

    Enterotoxins from pathogenic bacteria are known as the main reason that can cause the bacterial foodborne diseases. In this study, aptamers that bound to Staphylococcus aureus enterotoxin C1 (SEC1) with high affinity and selectivity were generated in vitro by twelve rounds of selection based on magnetic separation technology, with a low-level dissociation constant (Kd) value of 65.14 ± 11.64 nmol/L of aptamer C10. Aptamer-based quantification of SEC1 in the food sample by a graphene oxide (GO)-based method was implemented to investigate the potential of the aptamer against SEC1 with a limit of detection of 6 ng/mL. On the basis of this work, biosensors using the selected SEC1 aptamers as new molecular recognition elements could be applied for innovative determinations of SEC1. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Estrogen inhibits chloride secretion caused by cholera and Escherichia coli enterotoxins in female rat distal colon.

    LENUS (Irish Health Repository)

    Alzamora, Rodrigo

    2011-05-08

    Excessive Cl(-) secretion is the driving force for secretory diarrhea. 17β-Estradiol has been shown to inhibit Cl(-) secretion in rat distal colon through a nongenomic pathway. We examined whether 17β-estradiol inhibits Cl(-) secretion in an animal model of secretory diarrhea and the downstream effectors involved. The effect of 17β-estradiol on cholera toxin and heat-stable enterotoxin induced Cl(-) secretion in rat colonic mucosal sheets was studied by current-voltage clamping. Selective permeabilization of apical or basolateral membranes with amphotericin B or nystatin was used to isolate basolateral K(+) channel and apical Cl(-) channel activity, respectively. 17β-Estradiol dose-dependently inhibited secretory responses to both toxins with IC(50) values of approximately 1nM. This effect was female-gender specific, with no inhibition observed in male tissues. 17β-Estradiol responses were insensitive to the pure anti-estrogen ICI 182,720. 17β-Estradiol exerted its effects downstream of enterotoxin-induced production of second messengers (cAMP and cGMP) but was dependent on PKCδ activation. In nystatin-permeabilized tissues, apical Cl(-) currents were unaffected by 17β-estradiol treatment while basolateral K(+) current was profoundly inhibited by the hormone. This current was sensitive to the specific KCNQ1 channel inhibitors chromanol 293B and HMR-1556. In conclusion, 17β-estradiol inhibits enterotoxin-induced Cl(-) secretion via a PKCδ-dependent mechanism involving inhibition of basolateral KCNQ1 channels. These data elucidate mechanisms of 17β-estradiol inhibition of Cl(-) secretion induced by enterotoxins in intestinal epithelia, which may be relevant for the treatment of diarrheal diseases.

  19. Estrogen inhibits chloride secretion caused by cholera and Escherichia coli enterotoxins in female rat distal colon.

    LENUS (Irish Health Repository)

    Alzamora, Rodrigo

    2012-02-01

    Excessive Cl(-) secretion is the driving force for secretory diarrhea. 17beta-Estradiol has been shown to inhibit Cl(-) secretion in rat distal colon through a nongenomic pathway. We examined whether 17beta-estradiol inhibits Cl(-) secretion in an animal model of secretory diarrhea and the downstream effectors involved. The effect of 17beta-estradiol on cholera toxin and heat-stable enterotoxin induced Cl(-) secretion in rat colonic mucosal sheets was studied by current-voltage clamping. Selective permeabilization of apical or basolateral membranes with amphotericin B or nystatin was used to isolate basolateral K(+) channel and apical Cl(-) channel activity, respectively. 17beta-Estradiol dose-dependently inhibited secretory responses to both toxins with IC(50) values of approximately 1nM. This effect was female-gender specific, with no inhibition observed in male tissues. 17beta-Estradiol responses were insensitive to the pure anti-estrogen ICI 182,720. 17beta-Estradiol exerted its effects downstream of enterotoxin-induced production of second messengers (cAMP and cGMP) but was dependent on PKCdelta activation. In nystatin-permeabilized tissues, apical Cl(-) currents were unaffected by 17beta-estradiol treatment while basolateral K(+) current was profoundly inhibited by the hormone. This current was sensitive to the specific KCNQ1 channel inhibitors chromanol 293B and HMR-1556. In conclusion, 17beta-estradiol inhibits enterotoxin-induced Cl(-) secretion via a PKCdelta-dependent mechanism involving inhibition of basolateral KCNQ1 channels. These data elucidate mechanisms of 17beta-estradiol inhibition of Cl(-) secretion induced by enterotoxins in intestinal epithelia, which may be relevant for the treatment of diarrheal diseases.

  20. PCR detection of staphylococcal enterotoxin genes in Staphylococcus aureus strains isolated from raw and pasteurized milk.

    Science.gov (United States)

    Rall, V L M; Vieira, F P; Rall, R; Vieitis, R L; Fernandes, A; Candeias, J M G; Cardoso, K F G; Araújo, J P

    2008-12-10

    Milk is considered a nutritious food because it contains several important nutrients including proteins and vitamins. Conversely, it can be a vehicle for several pathogenic bacteria such as Staphylococcus aureus. This study aimed to analyze the frequency of genes encoding the staphylococcal enterotoxins (SEs) SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI and SEJ in S. aureus strains isolated from raw or pasteurized bovine milk. S. aureus was found in 38 (70.4%) out of 54 raw milk samples at concentrations of up to 8.9 x 10(5) CFU/ml. This microorganism was present in eight samples of pasteurized milk before the expiration date and in 11 samples analyzed on the expiration date. Of the 57 strains studied, 68.4% were positive for one or more genes encoding the enterotoxins, and 12 different genotypes were identified. The gene coding for enterotoxin A, sea, was the most frequent (16 strains, 41%), followed by sec (8 strains, 20.5%), sed (5 strains, 12.8%), seb (3 strains, 7.7%) and see (2 strains, 5.1%). Among the genes encoding the other enterotoxins, seg was the most frequently observed (11 strains, 28.2%), followed by sei (10 strains) and seh and sej (3 strains each). With the recent identification of new SEs, the perceived frequency of enterotoxigenic strains has increased, suggesting that the pathogenic potential of staphylococci may be higher than previously thought; however, further studies are required to assess the expression of these new SEs by S. aureus, and their impact in foodborne disease. The quality of Brazilian milk is still low, and efforts from the government and the entire productive chain are required to attain consumer safety.

  1. Antibiotic resistance and enterotoxin genes in Staphylococcus sp. isolates from polluted water in Southern Brazil

    OpenAIRE

    BASSO, ANA P.; MARTINS, PAULA D.; NACHTIGALL, GISELE; SAND, SUELI VAN DER; MOURA, TIANE M. DE; FRAZZON, ANA PAULA G.

    2014-01-01

    The aim of this study was to evaluate the species distribution, antibiotic-resistance profile and presence of enterotoxin (SE) genes in staphylococci isolated from the Dilúvio stream in South Brazil. Eighty-eight staphylococci were identified, 93.18% were identified as coagulase-negative (CNS) and 6.82% coagulase-positive (CPS). Fourteen Staphylococcus species were detected and the most frequently were Staphylococcus cohnii (30.48%) and S. haemolyticus (21.95%). Resistance to erythromycin was...

  2. Estrogen inhibits chloride secretion caused by cholera and Escherichia coli enterotoxins in female rat distal colon.

    OpenAIRE

    Alzamora, Rodrigo; O'Mahony, Fiona; Harvey, Brian J

    2011-01-01

    Excessive Cl(-) secretion is the driving force for secretory diarrhea. 17β-Estradiol has been shown to inhibit Cl(-) secretion in rat distal colon through a nongenomic pathway. We examined whether 17β-estradiol inhibits Cl(-) secretion in an animal model of secretory diarrhea and the downstream effectors involved. The effect of 17β-estradiol on cholera toxin and heat-stable enterotoxin induced Cl(-) secretion in rat colonic mucosal sheets was studied by current-voltage clamping. Selective per...

  3. A Single-Stranded DNA Aptamer That Selectively Binds to Staphylococcus aureus Enterotoxin B

    OpenAIRE

    DeGrasse, Jeffrey A.

    2012-01-01

    The bacterium Staphylococcus aureus is a common foodborne pathogen capable of secreting a cocktail of small, stable, and strain-specific, staphylococcal enterotoxins (SEs). Staphylococcal food poisoning (SFP) results when improperly handled food contaminated with SEs is consumed. Gastrointestinal symptoms of SFP include emesis, diarrhea and severe abdominal pain, which manifest within hours of ingesting contaminated food. Immuno-affinity based methods directly detect, identify, and quantify s...

  4. Effects of frozen storage on survival of Staphylococcus aureus and enterotoxin production in precooked tuna meat.

    Science.gov (United States)

    Wu, Xulei; Su, Yi-Cheng

    2014-08-01

    This study investigated the survival of Staphylococcus aureus in precooked tuna meat for producing canned products during frozen storage (-20 ± 2 °C) as well as its growth and enterotoxin production at 35 to 37 °C after the storage. Samples (50 ± 5 g) of precooked albacore (loin, chunk, and flake) and skipjack (chunk and flake) tuna were inoculated with 5 enterotoxin-producing strains of S. aureus at a level of approximately 3.5 log CFU/g and individually packed in a vacuum bag after 3 h incubation at 35 to 37 °C. Vacuum-packed samples were stored in a freezer (-20 ± 2 °C) for 4 wk. The frozen samples were then thawed in 37 °C circulating water for 2 h and incubated at 35 to 37 °C for 22 h. Populations of S. aureus in all precooked tuna samples decreased slightly (canned tuna within 6 to 8 h of thawing to avoid product spoilage and potential enterotoxin production by S. aureus in contaminated precooked tuna meat. © 2014 Institute of Food Technologists®

  5. Staphylococcal superantigens; toxic shock syndrome toxin-1 and enterotoxins in pediatric otitis media effusion: a brief report

    Directory of Open Access Journals (Sweden)

    Noorbakhsh S

    2013-03-01

    Full Text Available Background: Staphylococcal superantigens (SAg's may have some role in otitis media with effusion (OME. The aim of this study was the search of staphylococcal SAg's in middle ear effusion of children with OME.  Methods: This cross sectional-analytic study was done in ENT & pediatric wards upon 64 children with otitis media with effusion (OME between 1-15 years, (mean age=7.42+4 years of Rasoul Akram University Hospital, Tehran, Iran in 2009-2011. Fifty six percent (36 of cases were male, 43.8% (28 were female. Staphylococcal SAg's; Toxic Shock Syndrome Toxin-1 (TSST-1, Staphylococcal enterotoxin A, B, C, D (Enzyme immune assay, AB Cam, USA were detected in middle ear effusion samples after conventional culture.Results: None type of SAg's found in 39% of OME cases, enterotoxin B found in: 22%; enterotoxin A: 17%, enterotoxin C: 15.6%, enterotoxin D: 12.5%, Toxic Shock Syndrome Toxin-1 (TSST-1: 7.8% Mean age of cases with positive TSST-1, enterotoxin A, B, C, and D was: 1, 5, 8.6, 9.6 and 9.6 years respectively. Positive TSST had no agreement with positive enterotoxin A and C but had weak agreement with type B and D. Mean age of cases with positive TSST was one years which had significant difference with (7.9 years in cases with negative TSST test (P<0.0001.Conclusion: At least one or more type of staphylococcal toxins had found in middle ear effusion of 70% of OME cases with negative culture for Staphylococcus aureus. Even in culture negative cases, staphylococcal toxins might have some immunologic role in middle ear effusion forming. Finding the SAg's (at least one type are important for treatment of immunosuppressive or corticosteroid in cases with resistant OME.

  6. Effect of low-dose gamma irradiation on enterotoxin-positive strains of Clostridium perfringens Type A

    International Nuclear Information System (INIS)

    Barnhart, H.M. Jr.

    1976-01-01

    The effect of low-dose gamma irradiation on selected enterotoxin producing strains of Clostridium perfringens Type A was studied. The radioresistance of three strains NCTC-8239, NCTC-10239 and NCTC-8798 in 0.1 percent peptone water, beef gravy and ground beef was determined for both vegetative cells and spores. D 10 values were approximately 30 Krad in 0.1 percent peptone water and 175 Krad in beef menstruums. D 10 values for spores were approximately 250 Krad in 0.1 percent peptone water and 335 Krad in beef. Low-level irradiation induced a 2 hr lag for cell recovery at 37 0 C following irradiation though this was strain dependent. Heat resistance of vegetative cells decreased following irradiation, although one strain was stimulated in growth response and unaltered in its heat resistance. Spore activation and germination were not affected by low-level irradiation. Spores were not significantly inactivated at this level. Irradiation had no effect on subsequent survival of vegetative cells stored at cold temperatures. Enterotoxin production by irradiated cultures was not affected by the irradiation treatment. A method for quantitating C. perfringens enterotoxin using crossed-immunoelectrophoresis was developed. It was found that this technique could detect at least .05 g of enterotoxin, could utilize crude enterotoxin preparations and was more sensitive than other methods based on biological activity

  7. A complex array of Hpr consensus DNA recognition sequences proximal to the enterotoxin gene in Clostridium perfringens type A.

    Science.gov (United States)

    Brynestad, S; Iwanejko, L A; Stewart, G S; Granum, P E

    1994-01-01

    Enterotoxin production in Clostridium perfringens is both strain dependent and sporulation associated. Underlying these phenotypic observations must lie a genetic and molecular explanation and the principal keys will be held within the DNA sequence both upstream and downstream of the structural gene cpe. In accordance with the above we have sequenced 4.1 kbp of DNA upstream of cpe in the type strain NCTC 8239. A region of DNA extending up to 1.5 kb 5' to cpe is conserved in all enterotoxin-positive strains. This region contains a putative ORF with substantial homology to an ORF in the Salmonella typhimurium IS200 insertion element and, in addition, contains multiple perfect consensus DNA-binding sequences for the Bacillus subtilis transition state regulator Hpr. The detailed structural elements revealed by the sequence analysis are presented and used to develop a new perspective on the molecular basis of enterotoxin production in this important food-poisoning bacterium.

  8. How do the rotavirus NSP4 and bacterial enterotoxins lead differently to diarrhea?

    Directory of Open Access Journals (Sweden)

    Vasseur Monique

    2007-03-01

    Full Text Available Abstract Rotavirus is the major cause of infantile gastroenteritis and each year causes 611 000 deaths worldwide. The virus infects the mature enterocytes of the villus tip of the small intestine and induces a watery diarrhea. Diarrhea can occur with no visible tissue damage and, conversely, the histological lesions can be asymptomatic. Rotavirus impairs activities of intestinal disaccharidases and Na+-solute symports coupled with water transport. Maldigestion of carbohydrates and their accumulation in the intestinal lumen as well as malabsorption of nutrients and a concomitant inhibition of water reabsorption can lead to a malabsorption component of diarrhea. Since the discovery of the NSP4 enterotoxin, diverse hypotheses have been proposed in favor of an additional secretion component in the pathogenesis of diarrhea. Rotavirus induces a moderate net chloride secretion at the onset of diarrhea, but the mechanisms appear to be quite different from those used by bacterial enterotoxins that cause pure secretory diarrhea. Rotavirus failed to stimulate Cl- secretion in crypt, whereas it stimulated Cl- reabsorption in villi, questioning, therefore, the origin of net Cl- secretion. A solution to this riddle was that intestinal villi do in fact secrete chloride as a result of rotavirus infection. Also, the overall chloride secretory response is regulated by a phospholipase C-dependent calcium signaling pathway induced by NSP4. However, the overall response is weak, suggesting that NSP4 may exert both secretory and subsequent anti-secretory actions, as did carbachol, hence limiting Cl- secretion. All these characteristics provide the means to make the necessary functional distinction between viral NSP4 and bacterial enterotoxins.

  9. Uncoupling of T Cell Receptor Zeta Chain Function during the Induction of Anergy by the Superantigen, Staphylococcal Enterotoxin A

    Directory of Open Access Journals (Sweden)

    William D. Cornwell

    2010-06-01

    Full Text Available Staphylococcus aureus enterotoxins have immunomodulatory properties. In this study, we show that Staphylococcal enterotoxin A (SEA induces a strong proliferative response in a murine T cell clone independent of MHC class II bearing cells. SEA stimulation also induces a state of hypo-responsiveness (anergy. We characterized the components of the T cell receptor (TCR during induction of anergy by SEA. Most interestingly, TCR zeta chain phosphorylation was absent under SEA anergizing conditions, which suggests an uncoupling of zeta chain function. We characterize here a model system for studying anergy in the absence of confounding costimulatory signals.

  10. Suppression of colorectal tumorigenesis by recombinant Bacteroides fragilis enterotoxin-2 in vivo

    OpenAIRE

    Lv, You; Ye, Tao; Wang, Hui-Peng; Zhao, Jia-Ying; Chen, Wen-Jie; Wang, Xin; Shen, Chen-Xia; Wu, Yi-Bin; Cai, Yuan-Kun

    2017-01-01

    AIM To evaluate the impact of recombinant Bacteroides fragilis enterotoxin-2 (BFT-2, or Fragilysin) on colorectal tumorigenesis in mice induced by azoxymethane/dextran sulfate sodium (AOM/DSS). METHODS Recombinant proBFT-2 was expressed in Escherichia coli strain Rosetta (DE3) and BFT-2 was obtained and tested for its biological activity via colorectal adenocarcinoma cell strains SW-480. Seventy C57BL/6J mice were randomly divided into a blank (BC; n = 10), model (AD; n = 20), model + low-dos...

  11. Plant-Derived Polyphenols Interact with Staphylococcal Enterotoxin A and Inhibit Toxin Activity

    OpenAIRE

    Shimamura, Yuko; Aoki, Natsumi; Sugiyama, Yuka; Tanaka, Takashi; Murata, Masatsune; Masuda, Shuichi

    2016-01-01

    This study was performed to investigate the inhibitory effects of 16 different plant-derived polyphenols on the toxicity of staphylococcal enterotoxin A (SEA). Plant-derived polyphenols were incubated with the cultured Staphylococcus aureus C-29 to investigate the effects of these samples on SEA produced from C-29 using Western blot analysis. Twelve polyphenols (0.1-0.5 mg/mL) inhibited the interaction between the anti-SEA antibody and SEA. We examined whether the polyphenols could directly i...

  12. Cloning Sequencing and Structural Manipulation of the Enterotoxin D and E Genes from Staphylococcus aureus

    Science.gov (United States)

    1990-07-01

    Iandolo, J.J. and R.K. Tweten. 1988. Purification of staphylococcal enterotoxin. In. Methods of Enzymology, (N.O. Kaplan , S. Harshman, Eds.) volume 165...N C V 9 L G 0 K 1 S P A K I C T S N 0 149 "T7AACTGG&&C&hICG&clhht:7caflCATGTAATAa~aI ACca CAACaCAA 942 L K D G D K L Z L I G T P r D H K V N 0 H L..L

  13. Elevated Enterotoxin A Expression and Formation in Staphylococcus aureus and Its Association with Prophage Induction

    OpenAIRE

    Cao, Rong; Zeaki, Nikoleta; Wallin-Carlquist, Nina; Skandamis, Panagiotis N.; Schelin, Jenny; Rådström, Peter

    2012-01-01

    Staphylococcus aureus strains producing the bacteriophage-encoded staphylococcal enterotoxin A (SEA) were divided into two groups, high- and low-SEA-producing strains, based on the amount of SEA produced. After growth under favorable conditions in batch cultures, 10 of the 21 strains tested produced more than 1,000 ng/ml SEA, and 9 strains produced less than 10 ng/ml SEA; two enterotoxigenic strains, MRSA252 and Newman, produced intermediate levels of SEA (around 450 ng/ml). The differences i...

  14. Characterization of Enterotoxigenic Bacillus cereus sensu lato and Staphylococcus aureus Isolates and Associated Enterotoxin Production Dynamics in Milk or Meat-Based Broth.

    Science.gov (United States)

    Walker-York-Moore, Laura; Moore, Sean C; Fox, Edward M

    2017-07-15

    Bacillus cereus sensu lato species, as well as Staphylococcus aureus , are important pathogenic bacteria which can cause foodborne illness through the production of enterotoxins. This study characterised enterotoxin genes of these species and examined growth and enterotoxin production dynamics of isolates when grown in milk or meat-based broth. All B. cereus s. l. isolates harboured nheA , hblA and entFM toxin genes, with lower prevalence of bceT and hlyII . When grown at 16 °C, toxin production by individual B. cereus s. l. isolates varied depending on the food matrix; toxin was detected at cell densities below 5 log 10 (CFU/mL). At 16 °C no staphylococcal enterotoxin C (SEC) production was detected by S. aureus isolates, although low levels of SED production was noted. At 30 °C all S. aureus isolates produced detectable enterotoxin in the simulated meat matrix, whereas SEC production was significantly reduced in milk. Relative to B. cereus s. l. toxin production, S. aureus typically required reaching higher cell numbers to produce detectable levels of enterotoxin. Phylogenetic analysis of the sec and sel genes suggested population evolution which correlated with animal host adaptation, with subgroups of bovine isolates or caprine/ovine isolates noted, which were distinct from human isolates. Taken together, this study highlights the marked differences in the production of enterotoxins both associated with different growth matrices themselves, but also in the behaviour of individual strains when exposed to different food matrices.

  15. Role of Enterotoxin-Producing Staphylococci in Zoonotic Infections in Iran

    Directory of Open Access Journals (Sweden)

    Abdolmajid Ghasemian

    2018-05-01

    Full Text Available Context: Staphylococci spp, are enterotoxin-producing zoonotic agents causing a variety of infections such as mastitis in animals and wound bite infections in humans. This review was conducted to determine the prevalence of Staphylococci infections especially to uncover enterotoxin-producing species in Iran. Evidence acquisition: for this review, words of "Staphylococcus", "zoonotic", "prevalence", "animals", "human" and "Iran" were searched in the internet engines such as Google Scholar, PubMed, Google, Science Direct and so on. Patients with no history of contact with animals were also included in the study for comparison aims. Both veterinary and human coagulase positive isolates were included. Data was analyzed with Graph Pad Prism 6, meta-analysis section. A total of 20 previous studies (450 clinical samples were found. S. intermedius was the predominant isolate identified in veterinary sources. Other coagulase positive spp such as S. hycus and S. simulans were isolated with lower prevalence, but S. delphini has not been detected. Conclusion: S. intermedius was the most isolate identified in veterinary sources with potential of causing infections in humans. Other coagulase positive spp such as S. hycus and S. simulans were isolated with lower prevalence, but S. delphini was not detected.

  16. Detection of enterotoxins and genotyping of Staphylococcus aureus strains isolated from Isfahan Educational Hospital, Iran

    Directory of Open Access Journals (Sweden)

    Seyed Asghar Havaei

    2017-10-01

    Full Text Available Background and aims: Staphylococcus aureus is known as one of the most important nosocomial pathogens, which may lead to several infections. The aim of this study was determining the enterotoxins A, C, and TSST-1 and molecular characterization of S. aureus strains with PFGE and MLST typing methods. Materials and methods: In the present study during the sixmonths sampling, fifty S. aureus strains were isolated from patients admitted to Al-Zahra university hospital. Antimicrobial susceptibility testing, Multiplex PCR for detection of enterotoxin A, C and TSST-1, pulse field gel electrophoresis (PFGE and multilocus sequence typing (MLST were used for molecular typing. Results: In antibiogram the highest and lowest percentage of resistance was belonged to tetracycline and rifampin respectively. Multiplex PCR indicated that 30% of the strains harbored sea and 34% harbored sec genes. However, only 4% of our collected isolates had tsst gene. In PFGE method analysis on all S. aureus strains, a total of 19 different patterns were identified. Nine various sequence types in 27 selected S. aureus isolates were identified by MLST. Conclusions: Present study indicates a possible higher variability among our S. aureus strains by two different molecular typing methods; nevertheless four main common types (CT1, CT7, CT9, and CT11 with at least one toxin genes were determined.

  17. Antibiotic resistance and enterotoxin genes in Staphylococcus sp. isolates from polluted water in Southern Brazil.

    Science.gov (United States)

    Basso, Ana P; Martins, Paula D; Nachtigall, Gisele; Van Der Sand, Sueli; De Moura, Tiane M; Frazzon, Ana Paula G

    2014-12-01

    The aim of this study was to evaluate the species distribution, antibiotic-resistance profile and presence of enterotoxin (SE) genes in staphylococci isolated from the Dilúvio stream in South Brazil. Eighty-eight staphylococci were identified, 93.18% were identified as coagulase-negative (CNS) and 6.82% coagulase-positive (CPS). Fourteen Staphylococcus species were detected and the most frequently were Staphylococcus cohnii (30.48%) and S. haemolyticus (21.95%). Resistance to erythromycin was verified in 37.50% of the strains, followed by 27.27% to penicillin, 12.50% to clindamycin, 6.81% to trimethoprim-sulfamethoxazole, 5.68% to chloramphenicol and 2.27% to norfloxacin. None of the investigated strains showed gentamicin and ciprofloxacin resistance. The strains were tested for the presence of sea, seb, sec, sed and see genes by PCR and only CNS strains (43.18%) showed positive results to one or more SE genes. The scientific importance of our results is due to the lack of data about these topics in polluted waters in Brazil. In conclusion, polluted waters from the Dilúvio stream may constitute a reservoir for disseminating antibiotic-resistance and enterotoxin into the community. In addition, the detection of staphylococci in the polluted waters of the Dilúvio stream indicated a situation of environmental contamination and poor sanitation conditions.

  18. Antibiotic resistance and enterotoxin genes in Staphylococcus sp. isolates from polluted water in Southern Brazil

    Directory of Open Access Journals (Sweden)

    ANA P. BASSO

    2014-12-01

    Full Text Available The aim of this study was to evaluate the species distribution, antibiotic-resistance profile and presence of enterotoxin (SE genes in staphylococci isolated from the Dilúvio stream in South Brazil. Eighty-eight staphylococci were identified, 93.18% were identified as coagulase-negative (CNS and 6.82% coagulase-positive (CPS. Fourteen Staphylococcus species were detected and the most frequently were Staphylococcus cohnii (30.48% and S. haemolyticus (21.95%. Resistance to erythromycin was verified in 37.50% of the strains, followed by 27.27% to penicillin, 12.50% to clindamycin, 6.81% to trimethoprim-sulfamethoxazole, 5.68% to chloramphenicol and 2.27% to norfloxacin. None of the investigated strains showed gentamicin and ciprofloxacin resistance. The strains were tested for the presence of sea, seb, sec, sed and see genes by PCR and only CNS strains (43.18% showed positive results to one or more SE genes. The scientific importance of our results is due to the lack of data about these topics in polluted waters in Brazil. In conclusion, polluted waters from the Dilúvio stream may constitute a reservoir for disseminating antibiotic-resistance and enterotoxin into the community. In addition, the detection of staphylococci in the polluted waters of the Dilúvio stream indicated a situation of environmental contamination and poor sanitation conditions.

  19. Staphylococcal enterotoxins in the Etiopathogenesis of Mucosal Autoimmunity within the Gastrointestinal Tract

    Directory of Open Access Journals (Sweden)

    MaryAnn Principato

    2014-04-01

    Full Text Available The staphylococcal enterotoxins (SEs are the products of Staphylococcus aureus and are recognized as the causative agents of classical food poisoning in humans following the consumption of contaminated food. While illness evoked by ingestion of the SE or its producer organism in tainted food are often self-limited, our current understanding regarding the evolution of S. aureus provokes the utmost concern. The organism and its associated toxins, has been implicated in a wide variety of disease states including infections of the skin, heart, sinuses, inflammatory gastrointestinal disease, toxic shock, and Sudden Infant Death Syndrome. The intricate relationship between the various subsets of immunocompetent T cells and accessory cells and the ingested material found within the gastrointestinal tract present daunting challenges to the maintenance of immunologic homeostasis. Dysregulation of the intricate balances within this environment has the potential for extreme consequences within the host, some of which are long-lived. The focus of this review is to evaluate the relevance of staphylococcal enterotoxin in the context of mucosal immunity, and the underlying mechanisms that contribute to the pathogenesis of gastrointestinal autoimmune disease.

  20. Contamination of beef products with staphylococcal classical enterotoxins in Egypt and Saudi Arabia

    Directory of Open Access Journals (Sweden)

    Shawish, Reyad R.

    2016-04-01

    Full Text Available Food-borne pathogens are of high concern for public health and food safety. food poisoning is one of the most economically devastating types of food poisoning globally. The purpose of this study was to detect staphylococcal classical enterotoxins (SEs in processed beef from Kingdom of Saudi Arabia (KSA and Egypt. In the present investigation a total of 250 random processed meat samples (50 each of minced meat, beef burger, beef sausage, beef kofta and beef luncheon were collected from different super markets in the study area. Using conventional cultural methods, samples were cultured for isolation and identification of . Multiplex PCR was used to detect SEs of the classical type SEA, SEB, SEC and SED from isolates.The percentage presence of in minced meat, beef burger, beef sausage, beef kofta and beef luncheon was 38%, 22%, 30%, 32% and 12%, respectively. Multiplex PCR indicated that all examined samples contain different types of classical staphylococcal enterotoxins and only minced meat samples contained all four types of toxins. Multiplex PCR is efficient in detection of SEs from food and may be used in tracing of toxins to promote food hygiene. Implications of contamination of processed meat to food hygiene in the study area are highlighted.

  1. A probability model for enterotoxin production of Bacillus cereus as a function of pH and temperature

    Science.gov (United States)

    Bacillus cereus is frequently isolated from a variety of foods including vegetables, dairy products, meat, and other raw and processed foods. The bacterium is capable of producing enterotoxin and emetic toxin that can cause severe nausea, vomiting and diarrhea. The objectives of this study were to a...

  2. Claudins Overexpression in Ovarian Cancer: Potential Targets for Clostridium Perfringens Enterotoxin (CPE Based Diagnosis and Therapy

    Directory of Open Access Journals (Sweden)

    Diana P. English

    2013-05-01

    Full Text Available Claudins are a family of tight junction proteins regulating paracellular permeability and cell polarity with different patterns of expression in benign and malignant human tissues. There are approximately 27 members of the claudin family identified to date with varying cell and tissue-specific expression. Claudins-3, -4 and -7 represent the most highly differentially expressed claudins in ovarian cancer. While their exact role in ovarian tumors is still being elucidated, these proteins are thought to be critical for ovarian cancer cell invasion/dissemination and resistance to chemotherapy. Claudin-3 and claudin-4 are the natural receptors for the Clostridium perfringens enterotoxin (CPE, a potent cytolytic toxin. These surface proteins may therefore represent attractive targets for the detection and treatment of chemotherapy-resistant ovarian cancer and other aggressive solid tumors overexpressing claudin-3 and -4 using CPE-based theranostic agents.

  3. Association and dissociation of Escherichia coli heat-stable enterotoxin from rat brush border membrane receptors

    International Nuclear Information System (INIS)

    Cohen, M.B.; Thompson, M.R.; Overmann, G.J.; Giannella, R.A.

    1987-01-01

    Escherichia coli heat-stable enterotoxin (ST) binds to receptors on rat intestinal cells and brush border membranes (BBM). We devised experiments to examine the reversibility of ST binding. We found that both 125 I-labeled ST and native ST were spontaneously dissociable from the BBM receptor. Radiolabeled ST bound to BBM was also dissociated by the addition of avid goat anti-ST antiserum. Furthermore, using a computer program for analysis of ligand binding, we calculated an apparent Ka of 10(8) liters/mol from competitive inhibition and saturation-binding data. This is significantly lower than the value previously reported by others. Our findings, of a lower Ka and a reversible ST-binding process, suggest that a therapeutic strategy of removing bound ST from its receptor or competing with the enterocyte receptor for unbound ST might be successful in terminating ST-induced secretion

  4. Staphylococcal enterotoxin C2 promotes osteogenesis and suppresses osteoclastogenesis of human mesenchymal stem cells.

    Science.gov (United States)

    Fu, Wei-ming; Zhu, Xiao; Wang, Hua; Wei-Mao Wang; Chen, Ju-yu; Liang, Yan; Zhang, Jin-fang; Kung, Hsiang-fu

    2014-03-10

    As a super-antigen, staphylococcal enterotoxin C2 (SEC2) stimulates the release of massive inflammatory cytokines such as interferon-gamma (IFN-γ), interleukin-1 (IL-1) and interleukin-2 (IL-2) which are documented to implicate osteoblast differentiation. In the present study, SEC2 was found to significantly improve the osteoblast differentiation by up-regulating BMP2 and Runx2/Cbfa1 expression. Interferon (IFN)-inducible gene IFI16, a co-activator of Runx2/Cbfa1, was also activated by SEC2 in the osteoblast differentiation. In addition, exogenous introduction of SEC2 stimulated OPG expression and suppressed RANKL, suggesting suppression of osteoclastogenesis in hMSCs. Therefore, our results displayed that SEC2 plays an important role in the commitment of MSC to the osteoblast and it might be a potential new therapeutic candidate for bone regeneration. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Identification and Characterization of a New Enterotoxin Produced by Clostridium perfringens Isolated from Food Poisoning Outbreaks.

    Directory of Open Access Journals (Sweden)

    Daisuke Irikura

    Full Text Available There is a strain of Clostridium perfringens, W5052, which does not produce a known enterotoxin. We herein report that the strain W5052 expressed a homologue of the iota-like toxin components sa and sb of C. spiroforme, named Clostridium perfringens iota-like enterotoxin, CPILE-a and CPILE-b, respectively, based on the results of a genome sequencing analysis and a systematic protein screening. In the nicotinamide glyco-hydrolase (NADase assay the hydrolysis activity was dose-dependently increased by the concentration of rCPILE-a, as judged by the mass spectrometry analysis. In addition, the actin monomer of the lysates of Vero and L929 cells were radiolabeled in the presence of [32P]NAD and rCPILE-a. These findings indicated that CPILE-a possesses ADP-ribosylation activity. The culture supernatant of W5052 facilitated the rounding and killing of Vero and L929 cells, but the rCPILE-a or a non-proteolyzed rCPILE-b did not. However, a trypsin-treated rCPILE-b did. Moreover, a mixture of rCPILE-a and the trypsin-treated rCPILE-b enhanced the cell rounding and killing activities, compared with that induced by the trypsin-treated rCPILE-b alone. The injection of the mixture of rCPILE-a and the trypsin-treated rCPILE-b into an ileum loop of rabbits evoked the swelling of the loop and accumulation of the fluid dose-dependently, suggesting that CPILE possesses enterotoxic activity. The evidence presented in this communication will facilitate the epidemiological, etiological, and toxicological studies of C. perfringens food poisoning, and also stimulate studies on the transfer of the toxins' gene(s among the Genus Clostridium.

  6. Comparative Effects of Food Preservatives on the Production of Staphylococcal Enterotoxin I from Staphylococcus aureus Isolate

    Directory of Open Access Journals (Sweden)

    Yanying Zhao

    2017-01-01

    Full Text Available Staphylococcal enterotoxin I (SEI is associated with staphylococcal food poisoning, but little is known about different food preservatives on the production of SEI. In this study, the effect of different food preservatives (sodium nitrite, polylysine, chitosan, and tea catechin on the bacteria growth, sei gene expression, and extracellular SEI production of Staphylococcus aureus isolate H4 was detected in tryptone soya broth (TSB culture. Our results showed that all of these preservatives depressed S. aureus H4 growth and the order of inhibitory effect was 0.8 g/L tea catechin > 6 g/L chitosan > 0.25 g/L polylysine > 0.4 g/L tea catechin > 0.15 g/L sodium nitrite. Furthermore, 0.25 g/L polylysine or 0.15 g/L sodium nitrite did not significantly alter sei gene transcription, while 6 g/L chitosan obviously increased the relative mRNA level of sei gene expression. 0.4 g/L tea catechin remarkably inhibited sei gene transcription. In addition, 0.15 g/L sodium nitrite and 6 g/L chitosan significantly enhanced SEI secretion. 0.25 g/L polylysine, especially 0.4 g/L tea catechin, sharply inhibited the level of SEI secretion. The results indicated that tea catechin not only suppressed Staphylococcus aureus growth, but also inhibited SEI production and secretion, suggesting that tea catechin may be better than sodium nitrite, polylysine, or chitosan for keeping the food from the contamination of SEI. These investigations would be useful for food industry to provide safer food products due to S. aureus enterotoxins-related control strategy.

  7. Studies on the mechanism of action of enterotoxin-induced fluid secretion in the gut

    International Nuclear Information System (INIS)

    Schirgi-Degen, A.

    1992-12-01

    The mechanism of action of Clostridium difficile enterotoxin A (CA), of Escherichia coli enterotoxin (STa) and of cholera toxin (CT), which are known to cause severe diarrhea, were studied in a preparation of ligated jejunal loops of anesthetized rats in vivo. The toxins were administered intraluminally. Pharmacological agents, which were tested for their potency to influence toxin-related effects, were administered subcutaneously. Net fluid transport was determined gravimetrically, prostaglandin (PG) E 2 -output into the lumen, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) contents in the mucosa were measured by radioimmunoassay, serotonin-(5-HT)-output into the lumen was determined by high performance liquid chromatography. The histopathological effects of CA and CT were examined by light- and scanning electron microscopy. All three toxins caused net fluid secretion (FS). 5-HT 2 -(ketanserin) and 5-HT 3 -receptor antagonists (tropisetron, ondansetron, granisetron) dose-dependently reduced or abolished CT- and STa-induced net FS, CA-induced net FS was not influenced. Indomethacin reduced CA-, CT- and STa-induced net FS. Elevation of PGE 2 -output occurred after exposure to CA and CT and was reduced by indomethacin. CA caused severe histopathological lesions and also CT time-dependently caused morphological changes, which may take part in the secretory response. It is concluded that 5-HT, using both 5-HT 2 - and 5-HT 3 -receptors, mediates CT- and STa, but not CA-induced FS. PGE 2 is involved in FS caused by all three toxins. CAMP and cGMP are presumedly no causative mediators of toxin-induced FS

  8. Detection of Staphylococcus aureus enterotoxins in sheep cheese and dairy desserts by multiplex PCR technique.

    Science.gov (United States)

    Ertas, Nurhan; Gonulalan, Zafer; Yildirim, Yeliz; Kum, Erhan

    2010-08-15

    The aim of this study was to investigate the presence of Staphylococcus aureus (S. aureus) and staphylococcal enterotoxins (SEs) genes in sheep cheese and dairy dessert samples by multiplex PCR (mPCR) technique. A total of 150 samples were analyzed consisting of 50 dairy dessert samples and 100 sheep cheese. Coagulase positive staphylococci (CPS) were found in 86 (57.3%) out of 150 analyzed samples. S. aureus were isolated from 60 (60%), 26 (52%) of sheep cheese and from of dairy desserts, respectively. Five suspected colonies were tested from each sheep cheese and dairy dessert samples for phenotypic and genotypic characterizations. A total of 430 isolates from the 86 positive samples were investigated in this study. Eighty (18.6%) isolates were characterized as S. aureus. The enterotoxin genes (sea, seb, sec, sed) were found in 13 (3.02%) out of 80 isolates. From cheese isolates, sea, seb and sed were detected in 5 (1.6%), 2 (0.6%), 1 (0.3%), respectively. From dairy dessert isolates, sea, sec and sed were detected in 3 (2.3%), 1 (0.76%), 1 (0.76%), respectively. The presence of SEs was identified in 12 (2.8%) out of 80 isolates by using ELISA technique. It was determined that these SEs had a distribution of 7 (1.6%) SEA, 2 (0.46%) SEB, 1 (0.23%) SEC, and 2 (0.46%) SED. SEs were found in 7 (2.3%) cheese and 5 (3.8%) dairy dessert isolates. In conclusion, S.aureus and their SEs were found to be present in sheep cheese and dairy desserts in this study. It is emphasized that the presence of S. aureus and their SEs genes in sheep cheese and dairy desserts may be regarded as a potential risk for human health. Copyright 2010 Elsevier B.V. All rights reserved.

  9. Short communication: Pasteurization as a means of inactivating staphylococcal enterotoxins A, B, and C in milk.

    Science.gov (United States)

    Necidova, Lenka; Bogdanovicova, Katerina; Harustiakova, Danka; Bartova, Katerina

    2016-11-01

    Our aim was to assess the effect of pasteurization temperature on inactivation of staphylococcal enterotoxins (SE). Milk samples were inoculated with log 4.38 to 5.18cfu/mL of 40 different Staphylococcus aureus strains having the ability to produce types A, B, or C SE and incubated at 37°C for 24h to develop SE. This incubation was followed by heat treatment for 15 s at 72, 85, and 92°C. Samples were analyzed for Staph. aureus count by plate method and, specifically, for SE presence. An enzyme-linked immunofluorescent assay on a MiniVIDAS analyzer (bioMérieux, Marcy l'Étoile, France) was used to detect SE, which were determined semiquantitatively based on test values. The Staph. aureus count in milk before pasteurization did not affect the amount of SE. Before pasteurization, SEB was detected in the lowest amount compared with other SE types. Staphylococcal enterotoxins were markedly reduced with pasteurization and inactivated at pasteurization temperatures to an extent depending on the amount in the sample before pasteurization. After pasteurization at 72°C, SE were detected in 87.5% of samples (35/40), after pasteurization at 85°C in 52.5% of samples (21/40), and after pasteurization at 92°C in 45.0% of samples (18/40). We determined that SE may still persist in milk even when Staph. aureus bacteria are inactivated through pasteurization. Although pasteurization may partially inactivate SE in milk, a key measure in the prevention of staphylococcal enterotoxicosis linked to pasteurized milk consumption is to avoid any cold chain disruption during milk production and processing. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  10. A single-stranded DNA aptamer that selectively binds to Staphylococcus aureus enterotoxin B.

    Science.gov (United States)

    DeGrasse, Jeffrey A

    2012-01-01

    The bacterium Staphylococcus aureus is a common foodborne pathogen capable of secreting a cocktail of small, stable, and strain-specific, staphylococcal enterotoxins (SEs). Staphylococcal food poisoning (SFP) results when improperly handled food contaminated with SEs is consumed. Gastrointestinal symptoms of SFP include emesis, diarrhea and severe abdominal pain, which manifest within hours of ingesting contaminated food. Immuno-affinity based methods directly detect, identify, and quantify several SEs within a food or clinical sample. However, the success of these assays depends upon the availability of a monoclonal antibody, the development of which is non-trivial and costly. The current scope of the available immuno-affinity based methods is limited to the classical SEs and does not encompass all of the known or emergent SEs. In contrast to antibodies, aptamers are short nucleic acids that exhibit high affinity and specificity for their targets without the high-costs and ethical concerns of animal husbandry. Further, researchers may choose to freely distribute aptamers and develop assays without the proprietary issues that increase the per-sample cost of immuno-affinity assays. This study describes a novel aptamer, selected in vitro, with affinity to staphylococcal enterotoxin B (SEB) that may be used in lieu of antibodies in SE detection assays. The aptamer, designated APT(SEB1), successfully isolates SEB from a complex mixture of SEs with extremely high discrimination. This work sets the foundation for future aptamer and assay development towards the entire family of SEs. The rapid, robust, and low-cost identification and quantification of all of the SEs in S. aureus contaminated food is essential for food safety and epidemiological efforts. An in vitro generated library of SE aptamers could potentially allow for the comprehensive and cost-effective analysis of food samples that immuno-affinity assays currently cannot provide.

  11. A single-stranded DNA aptamer that selectively binds to Staphylococcus aureus enterotoxin B.

    Directory of Open Access Journals (Sweden)

    Jeffrey A DeGrasse

    Full Text Available The bacterium Staphylococcus aureus is a common foodborne pathogen capable of secreting a cocktail of small, stable, and strain-specific, staphylococcal enterotoxins (SEs. Staphylococcal food poisoning (SFP results when improperly handled food contaminated with SEs is consumed. Gastrointestinal symptoms of SFP include emesis, diarrhea and severe abdominal pain, which manifest within hours of ingesting contaminated food. Immuno-affinity based methods directly detect, identify, and quantify several SEs within a food or clinical sample. However, the success of these assays depends upon the availability of a monoclonal antibody, the development of which is non-trivial and costly. The current scope of the available immuno-affinity based methods is limited to the classical SEs and does not encompass all of the known or emergent SEs. In contrast to antibodies, aptamers are short nucleic acids that exhibit high affinity and specificity for their targets without the high-costs and ethical concerns of animal husbandry. Further, researchers may choose to freely distribute aptamers and develop assays without the proprietary issues that increase the per-sample cost of immuno-affinity assays. This study describes a novel aptamer, selected in vitro, with affinity to staphylococcal enterotoxin B (SEB that may be used in lieu of antibodies in SE detection assays. The aptamer, designated APT(SEB1, successfully isolates SEB from a complex mixture of SEs with extremely high discrimination. This work sets the foundation for future aptamer and assay development towards the entire family of SEs. The rapid, robust, and low-cost identification and quantification of all of the SEs in S. aureus contaminated food is essential for food safety and epidemiological efforts. An in vitro generated library of SE aptamers could potentially allow for the comprehensive and cost-effective analysis of food samples that immuno-affinity assays currently cannot provide.

  12. Passive therapy with humanized anti-staphylococcal enterotoxin B antibodies attenuates systemic inflammatory response and protects from lethal pneumonia caused by staphylococcal enterotoxin B-producing Staphylococcus aureus.

    Science.gov (United States)

    Karau, Melissa J; Tilahun, Mulualem E; Krogman, Ashton; Osborne, Barbara A; Goldsby, Richard A; David, Chella S; Mandrekar, Jayawant N; Patel, Robin; Rajagopalan, Govindarajan

    2017-10-03

    Drugs such as linezolid that inhibit bacterial protein synthesis may be beneficial in treating infections caused by toxigenic Staphylococcus aureus. As protein synthesis inhibitors have no effect on preformed toxins, neutralization of pathogenic exotoxins with anti-toxin antibodies may be beneficial in conjunction with antibacterial therapy. Herein, we evaluated the efficacy of human-mouse chimeric high-affinity neutralizing anti-staphylococcal enterotoxin B (SEB) antibodies in the treatment of experimental pneumonia caused by SEB-producing S. aureus. Since HLA class II transgenic mice mount a stronger systemic immune response following challenge with SEB and are more susceptible to SEB-induced lethal toxic shock than conventional mice strains, HLA-DR3 transgenic mice were used. Lethal pneumonia caused by SEB-producing S. aureus in HLA-DR3 transgenic mice was characterized by robust T cell activation and elevated systemic levels of several pro-inflammatory cytokines and chemokines. Prophylactic administration of a single dose of linezolid 30 min prior to the onset of infection attenuated the systemic inflammatory response and protected from mortality whereas linezolid administered 60 min after the onset of infection failed to confer significant protection. Human-mouse chimeric high-affinity neutralizing anti-SEB antibodies alone, but not polyclonal human IgG, mitigated this response and protected from death when administered immediately after initiation of infection. Further, anti-SEB antibodies as well as intact polyclonal human IgG, but not its Fab or Fc fragments, protected from lethal pneumonia when followed with linezolid therapy 60 min later. In conclusion, neutralization of superantigens with high-affinity antibodies may have beneficial effects in pneumonia.

  13. Inhibitory effects of food additives derived from polyphenols on staphylococcal enterotoxin A production and biofilm formation by Staphylococcus aureus.

    Science.gov (United States)

    Shimamura, Yuko; Hirai, Chikako; Sugiyama, Yuka; Shibata, Masaharu; Ozaki, Junya; Murata, Masatsune; Ohashi, Norio; Masuda, Shuichi

    2017-12-01

    In this study, we examined the inhibitory effects of 14 food additives derived from polyphenol samples on staphylococcal enterotoxin A (SEA) production and biofilm formation by Staphylococcus aureus. Tannic acid AL (TA), Purephenon 50 W (PP) and Polyphenon 70A (POP) at 0.25 mg/mL and Gravinol®-N (GN), Blackcurrant polyphenol AC10 (BP), and Resveratrol-P5 (RT) at 1.0 mg/mL significantly decreased SEA production by S. aureus C-29 (p Food additives derived from polyphenols have viability to be used as a means to inhibit the enterotoxin production and control the biofilm formation of foodborne pathogens.

  14. Effects of nisin and temperature on survival, growth, and enterotoxin production characteristics of psychrotrophic Bacillus cereus in beef gravy.

    OpenAIRE

    Beuchat, L R; Clavero, M R; Jaquette, C B

    1997-01-01

    The presence of psychrotrophic enterotoxigenic Bacillus cereus in ready-to-serve meats and meat products that have not been subjected to sterilization treatment is a public health concern. A study was undertaken to determine the survival, growth, and diarrheal enterotoxin production characteristics of four strains of psychrotrophic B. cereus in brain heart infusion (BHI) broth and beef gravy as affected by temperature and supplementation with nisin. A portion of unheated vegetative cells from...

  15. Characterization of Staphylococcus aureus strains and evidence for the involvement of non-classical enterotoxin genes in food poisoning outbreaks.

    Science.gov (United States)

    Ciupescu, Laurentiu-Mihai; Auvray, Frederic; Nicorescu, Isabela Madalina; Meheut, Thomas; Ciupescu, Veronica; Lardeux, Anne-Laure; Tanasuica, Rodica; Hennekinne, Jacques-Antoine

    2018-06-05

    To an increasing extent, molecular and genetic characterization is now used to investigate foodborne outbreaks. The aim of this study was to seek molecular links among coagulase-positive staphylococci (CPS) isolated from three recent food poisoning outbreaks in Romania using polymerase chain reaction and pulsed-field gel electrophoresis (PFGE) techniques. Nineteen CPS isolates were identified as Staphylococcus aureus by detection of the 23S rDNA gene. Among them, 15 carried at least one staphylococcal enterotoxin-encoding gene (se). The Calarași outbreak strains grouped in pulsotype 2 and were sed/sej/ser-positive, whereas the Arad outbreak strains clustered in pulsotype 17 and were either sed/seg/sei/sej/ser- or seg/sei-positive. The Pitești outbreak strains clustered in pulsotype 1 and, surprisingly, possessed only one enterotoxin gene, i.e. seh. Similar to other European countries, the seh gene has been identified with increasing frequency in Romanian outbreaks; this highlights the importance of considering the application of methods recommended for staphylococcal enterotoxin regulation in Europe.

  16. Effects of nisin and temperature on survival, growth, and enterotoxin production characteristics of psychrotrophic Bacillus cereus in beef gravy.

    Science.gov (United States)

    Beuchat, L R; Clavero, M R; Jaquette, C B

    1997-05-01

    The presence of psychrotrophic enterotoxigenic Bacillus cereus in ready-to-serve meats and meat products that have not been subjected to sterilization treatment is a public health concern. A study was undertaken to determine the survival, growth, and diarrheal enterotoxin production characteristics of four strains of psychrotrophic B. cereus in brain heart infusion (BHI) broth and beef gravy as affected by temperature and supplementation with nisin. A portion of unheated vegetative cells from 24-h BHI broth cultures was sensitive to nisin as evidenced by an inability to form colonies on BHI agar containing 10 micrograms of nisin/ml. Heat-stressed cells exhibited increased sensitivity to nisin. At concentrations as low as 1 microgram/ml, nisin was lethal to B. cereus, the effect being more pronounced in BHI broth than in beef gravy. The inhibitory effect of nisin (1 microgram/ml) was greater on vegetative cells than on spores inoculated into beef gravy and was more pronounced at 8 degrees C than at 15 degrees C. Nisin, at a concentration of 5 or 50 micrograms/ml, inhibited growth in gravy inoculated with vegetative cells and stored at 8 or 15 degrees C, respectively, for 14 days. Growth of vegetative cells and spores of B. cereus after an initial period of inhibition is attributed to loss of activity of nisin. One of two test strains produced diarrheal enterotoxin in gravy stored at 8 or 15 degrees C within 9 or 3 days, respectively. Enterotoxin production was inhibited in gravy supplemented with 1 microgram of nisin/ml and stored at 8 degrees C for 14 days; 5 micrograms of nisin/ml was required for inhibition at 15 degrees C. Enterotoxin was not detected in gravy in which less than 5.85 log10 CFU of B. cereus/ml had grown. Results indicate that as little as 1 microgram of nisin/ml may be effective in inhibiting or retarding growth of and diarrheal enterotoxin production by vegetative cells and spores of psychrotrophic B. cereus in beef gravy at 8 degrees C, a

  17. CodY Promotes Sporulation and Enterotoxin Production by Clostridium perfringens Type A Strain SM101.

    Science.gov (United States)

    Li, Jihong; Freedman, John C; Evans, Daniel R; McClane, Bruce A

    2017-03-01

    Clostridium perfringens type D strains cause enterotoxemia and enteritis in livestock via epsilon toxin production. In type D strain CN3718, CodY was previously shown to increase the level of epsilon toxin production and repress sporulation. C. perfringens type A strains producing C. perfringens enterotoxin (CPE) cause human food poisoning and antibiotic-associated diarrhea. Sporulation is critical for C. perfringens type A food poisoning since spores contribute to transmission and resistance in the harsh food environment and sporulation is essential for CPE production. Therefore, the current study asked whether CodY also regulates sporulation and CPE production in SM101, a derivative of C. perfringens type A food-poisoning strain NCTC8798. An isogenic codY -null mutant of SM101 showed decreased levels of spore formation, along with lower levels of CPE production. A complemented strain recovered wild-type levels of both sporulation and CPE production. When this result was coupled with the earlier results obtained with CN3718, it became apparent that CodY regulation of sporulation varies among different C. perfringens strains. Results from quantitative reverse transcriptase PCR analysis clearly demonstrated that, during sporulation, codY transcript levels remained high in SM101 but rapidly declined in CN3718. In addition, abrB gene expression patterns varied significantly between codY -null mutants of SM101 and CN3718. Compared to the levels in their wild-type parents, the level of abrB gene expression decreased in the CN3718 codY -null mutant strain but significantly increased in the SM101 codY -null mutant strain, demonstrating CodY-dependent regulation differences in abrB expression between these two strains. This difference appears to be important since overexpression of the abrB gene in SM101 reduced the levels of sporulation and enterotoxin production, supporting the involvement of AbrB repression in regulating C. perfringens sporulation. Copyright © 2017

  18. The Isolation and Detection of Staphylococcus aureus Enterotoxins A-E and TSST-1 Genes from Different Sources by PCR Method

    Directory of Open Access Journals (Sweden)

    Norouzi J

    2012-09-01

    Full Text Available Background and Objectives: Enterotoxins and toxic shock syndrome toxin–1 (TSST-1 are an important virulence factor of S. aureus. The purpose of this study was to analyze the presence of S. aureus enterotoxin (sea-see and tst genes in the samples that collected from different sources with PCR method. Methods: During 5 months from 150 collected samples, 80 strains were identified as S. aureus. PCR reaction was used for investigation on the presence of genes for staphylococcal enterotoxins (A-E and toxic shock syndrome toxin-1 (TSST-1. Results: Fifty three samples (66.25% out of 80 samples were positive for one or more ETs and TSST-1 genes. of these positive strains, 17 (32.07% were positive for sea 39 (73.58% for seb, 30 (56.6% for sec, 2 (3.7% for sed, 21 (39.62% for see, and 14 (26.41% for tst. Enterotoxins and tst in 40 samples (75.47% observed.Conclusion: In this study, high prevalence of S. aureus, its enterotoxin, and tst genes were observed in clinical samples, food samples, and healthy people. This fact emphasizes on the role of human as original source and carrier of S. aureus. Also, use of PCR reaction for detection of these genes in S. aureus that isolated from various sources is recommended.

  19. [Poisoning by enterotoxin from Staphylococcus aureus associated with mocha pastry. Microbiology and epidemiology].

    Science.gov (United States)

    Escartín, E F; Saldaña-Lozano, J; Montiel-Falcón, A

    1998-01-01

    A brief description of a foodborne outbreak due to S. aureus enterotoxin associated with the consumption of mocha cake in the city of Guadalajara is presented. The cake was prepared in a bakery and affected nearly 100 persons. S. aureus was isolated from the nose and skin of one of the pastry cooks. A S. aureus strain isolated from the cake involved in the outbreak was not only unable to grow in the mocha cream, but it actually decreased in numbers by 2 log after 72 h of storage at 30 degrees C. The pH of mocha cream ranged from 6.2 to 6.6, and water activity from 0.833 to 0.859, with a media of 0.841. In preparing mocha cake at the shop, one half of the dough used to be sprayed with a sucrose solution in water (20% w/v); mocha cream was spread on the other half of the dough before overlapping the two halves. When mocha cake was prepared in this manner, and stored at 30 degrees C, S. aureus increased in number by more than 4 log after 48 h. S. aureus did not grow in the cake stored at 4-7 degrees C. Contributory factors in this outbreak were an increase of water activity in the interphase of the mocha and the cake dough, storage of the cake in an unrefrigerated area, and an unusually high ambient temperature (28-32 degrees C) at that time.

  20. Formation of cereulide and enterotoxins by Bacillus cereus in fermented African locust beans

    DEFF Research Database (Denmark)

    Thorsen, Line; Azokpota, Paulin; Munk Hansen, Bjarne

    2011-01-01

    Afitin, iru and sonru are three spontaneously fermented African locust bean Benin condiments. The fermentation processes are exothermic, with temperatures mostly being above 40 °C. A total of 19 predominant Bacillus cereus isolates from afitin, iru and sonru, were investigated. The enterotoxin...... genes nhe (A, B, C) were present in all 19 isolates, the hbl (A, C, D) in one (afitin), and the cytK gene in three isolates (afitin). Levels of cytotoxicity to Vero cells and NheA production in BHI-broth was within the range of known diarrheal outbreak strains. Autoclaved cooked African locust beans...... inoculated with emetic (cereulide producing) B. cereus Ba18H2/RIF supported growth at 25, 30 and 40 °C with highly different maximum cereulide productions of 6 ± 5, 97 ± 3 and 0.04 ± 0.02 μg/g beans, respectively (48 h). For non-autoclaved cooked beans inoculated with 2, 4 and 6 log10 B. cereus Ba18H2/RIF...

  1. Colorimetric determination of staphylococcal enterotoxin B via DNAzyme-guided growth of gold nanoparticles

    International Nuclear Information System (INIS)

    Zhou, Dandan; Chen, Hui; Xie, Guoming; Cao, Xianqing; Chen, Xueping; Zhang, Xing

    2016-01-01

    The authors describe a colorimetric method for the determination of the staphylococcal enterotoxin B (SEB) that also allows for visual readout. The assay is based on the growth of gold nanoparticles (AuNPs) mediated by a hemin/G-quadruplex DNAzyme which generates a color change from red to blue in the presence of SEB. The method is enzyme-free and does not require a label. The kinetics of the formation of the AuNPs is controlled by the hemin/G-quadruplex DNAzyme and this is key to the signal generation mechanism. In the presence of SEB, the reactions between aptamer and target modulated the amount of single probe G strands that form DNAzyme capable of consuming hydrogen peroxide. The growth process of AuNPs is influenced by the resulting concentration of H 2 O 2 and leads to the color change. Under optimal conditions, a linear relationship exists between absorbance and SEB concentration in the range from 0.1 to 500 pg·mL -1 which covers the clinically relevant range. In case of visual detection, the lower limit of detection is 1 pg·mL −1 . The assay described here is sensitive, comparably inexpensive and can detect SEB rapidly without the need for sophisticated equipment. In our perception, the method has a wide scope in that it may be adapted to various nucleic acids, proteins and other biomolecules if respective aptamers are available. (author)

  2. From Escherichia coli heat-stable enterotoxin to mammalian endogenous guanylin hormones

    International Nuclear Information System (INIS)

    Lima, A.A.M.; Fonteles, M.C.

    2014-01-01

    The isolation of heat-stable enterotoxin (STa) from Escherichia coli and cholera toxin from Vibrio cholerae has increased our knowledge of specific mechanisms of action that could be used as pharmacological tools to understand the guanylyl cyclase-C and the adenylyl cyclase enzymatic systems. These discoveries have also been instrumental in increasing our understanding of the basic mechanisms that control the electrolyte and water balance in the gut, kidney, and urinary tracts under normal conditions and in disease. Herein, we review the evolution of genes of the guanylin family and STa genes from bacteria to fish and mammals. We also describe new developments and perspectives regarding these novel bacterial compounds and peptide hormones that act in electrolyte and water balance. The available data point toward new therapeutic perspectives for pathological features such as functional gastrointestinal disorders associated with constipation, colorectal cancer, cystic fibrosis, asthma, hypertension, gastrointestinal barrier function damage associated with enteropathy, enteric infection, malnutrition, satiety, food preferences, obesity, metabolic syndrome, and effects on behavior and brain disorders such as attention deficit, hyperactivity disorder, and schizophrenia

  3. Colorimetric DNAzyme Biosensor for Convenience Detection of Enterotoxin B Harboring Staphylococcus aureus from Food Samples.

    Science.gov (United States)

    Mondal, Bhairab; N, Bhavanashri; Ramlal, Shylaja; Kingston, Joseph

    2018-02-14

    In the present study, a colorimetric DNAzymes biosensor strategy was devised in combination with immunomagnetic separation for rapid and easy detection of enterotoxin B harboring Staphylococcus aureus from food and clinical samples. The method employs immunocapture of S. aureus and amplification of seb gene by DNAzyme complementary sequence integrated forward primer and with specific reverse primer. The DNAzyme sequence integrated dsDNA PCR products when treated with hemin and TMB (3,3',5,5'-tetramethylbenzidine) in the presence of H 2 O 2 produce colorimetric signal. A linear relationship of optical signal with the initial template of seb was obtained which could be monitored by visually or spectrophotrometrically for qualitative and quantitative detection. The limit of detection for the assay was approximately 10 2 CFU/mL of seb gene harboring target. This method is convenient compared to gel based and ELISA systems. Further, spiking studies and analysis on natural samples emphasized the robustness and applicability of developed method. Altogether, the established assay could be a reliable alternative, low-cost, viable detection tool for the routine investigation of seb from food and clinical sources.

  4. Plant-Derived Polyphenols Interact with Staphylococcal Enterotoxin A and Inhibit Toxin Activity.

    Science.gov (United States)

    Shimamura, Yuko; Aoki, Natsumi; Sugiyama, Yuka; Tanaka, Takashi; Murata, Masatsune; Masuda, Shuichi

    2016-01-01

    This study was performed to investigate the inhibitory effects of 16 different plant-derived polyphenols on the toxicity of staphylococcal enterotoxin A (SEA). Plant-derived polyphenols were incubated with the cultured Staphylococcus aureus C-29 to investigate the effects of these samples on SEA produced from C-29 using Western blot analysis. Twelve polyphenols (0.1-0.5 mg/mL) inhibited the interaction between the anti-SEA antibody and SEA. We examined whether the polyphenols could directly interact with SEA after incubation of these test samples with SEA. As a result, 8 polyphenols (0.25 mg/mL) significantly decreased SEA protein levels. In addition, the polyphenols that interacted with SEA inactivated the toxin activity of splenocyte proliferation induced by SEA. Polyphenols that exerted inhibitory effects on SEA toxic activity had a tendency to interact with SEA. In particular, polyphenol compounds with 1 or 2 hexahydroxydiphenoyl groups and/or a galloyl group, such as eugeniin, castalagin, punicalagin, pedunculagin, corilagin and geraniin, strongly interacted with SEA and inhibited toxin activity at a low concentration. These polyphenols may be used to prevent S. aureus infection and staphylococcal food poisoning.

  5. Plant-Derived Polyphenols Interact with Staphylococcal Enterotoxin A and Inhibit Toxin Activity.

    Directory of Open Access Journals (Sweden)

    Yuko Shimamura

    Full Text Available This study was performed to investigate the inhibitory effects of 16 different plant-derived polyphenols on the toxicity of staphylococcal enterotoxin A (SEA. Plant-derived polyphenols were incubated with the cultured Staphylococcus aureus C-29 to investigate the effects of these samples on SEA produced from C-29 using Western blot analysis. Twelve polyphenols (0.1-0.5 mg/mL inhibited the interaction between the anti-SEA antibody and SEA. We examined whether the polyphenols could directly interact with SEA after incubation of these test samples with SEA. As a result, 8 polyphenols (0.25 mg/mL significantly decreased SEA protein levels. In addition, the polyphenols that interacted with SEA inactivated the toxin activity of splenocyte proliferation induced by SEA. Polyphenols that exerted inhibitory effects on SEA toxic activity had a tendency to interact with SEA. In particular, polyphenol compounds with 1 or 2 hexahydroxydiphenoyl groups and/or a galloyl group, such as eugeniin, castalagin, punicalagin, pedunculagin, corilagin and geraniin, strongly interacted with SEA and inhibited toxin activity at a low concentration. These polyphenols may be used to prevent S. aureus infection and staphylococcal food poisoning.

  6. From Escherichia coli heat-stable enterotoxin to mammalian endogenous guanylin hormones

    Energy Technology Data Exchange (ETDEWEB)

    Lima, A.A.M. [Universidade Federal do Ceará, Departamento de Fisiologia e Farmacologia, Escola de Medicina, Instituto de Biomedicina, Unidade de Pesquisas Clínicas, Fortaleza, CE, Brasil, Unidade de Pesquisas Clínicas, Instituto de Biomedicina, Departamento de Fisiologia e Farmacologia, Escola de Medicina, Universidade Federal do Ceará, Fortaleza, CE (Brazil); Fonteles, M.C. [Universidade Federal do Ceará, Departamento de Fisiologia e Farmacologia, Escola de Medicina, Instituto de Biomedicina, Unidade de Pesquisas Clínicas, Fortaleza, CE, Brasil, Unidade de Pesquisas Clínicas, Instituto de Biomedicina, Departamento de Fisiologia e Farmacologia, Escola de Medicina, Universidade Federal do Ceará, Fortaleza, CE (Brazil); Universidade Estadual do Ceará, Instituto de Ciências Biomédicas, Fortaleza, CE, Brasil, Instituto de Ciências Biomédicas, Universidade Estadual do Ceará, Fortaleza, CE (Brazil)

    2014-03-03

    The isolation of heat-stable enterotoxin (STa) from Escherichia coli and cholera toxin from Vibrio cholerae has increased our knowledge of specific mechanisms of action that could be used as pharmacological tools to understand the guanylyl cyclase-C and the adenylyl cyclase enzymatic systems. These discoveries have also been instrumental in increasing our understanding of the basic mechanisms that control the electrolyte and water balance in the gut, kidney, and urinary tracts under normal conditions and in disease. Herein, we review the evolution of genes of the guanylin family and STa genes from bacteria to fish and mammals. We also describe new developments and perspectives regarding these novel bacterial compounds and peptide hormones that act in electrolyte and water balance. The available data point toward new therapeutic perspectives for pathological features such as functional gastrointestinal disorders associated with constipation, colorectal cancer, cystic fibrosis, asthma, hypertension, gastrointestinal barrier function damage associated with enteropathy, enteric infection, malnutrition, satiety, food preferences, obesity, metabolic syndrome, and effects on behavior and brain disorders such as attention deficit, hyperactivity disorder, and schizophrenia.

  7. Concerted action of sphingomyelinase and non-hemolytic enterotoxin in pathogenic Bacillus cereus.

    Directory of Open Access Journals (Sweden)

    Viktoria M Doll

    Full Text Available Bacillus cereus causes food poisoning and serious non-gastrointestinal-tract infections. Non-hemolytic enterotoxin (Nhe, which is present in most B. cereus strains, is considered to be one of the main virulence factors. However, a B. cereus ΔnheBC mutant strain lacking Nhe is still cytotoxic to intestinal epithelial cells. In a screen for additional cytotoxic factors using an in vitro model for polarized colon epithelial cells we identified B. cereus sphingomyelinase (SMase as a strong inducer of epithelial cell death. Using single and double deletion mutants of sph, the gene encoding for SMase, and nheBC in B. cereus we demonstrated that SMase is an important factor for B. cereus cytotoxicity in vitro and pathogenicity in vivo. SMase substantially complemented Nhe induced cytotoxicity in vitro. In addition, SMase but not Nhe contributed significantly to the mortality rate of larvae in vivo in the insect model Galleria mellonella. Our study suggests that the role of B. cereus SMase as a secreted virulence factor for in vivo pathogenesis has been underestimated and that Nhe and SMase complement each other significantly to cause full B. cereus virulence hence disease formation.

  8. Inhibition of biological activity of staphylococcal enterotoxin A (SEA) by apple juice and apple polyphenols.

    Science.gov (United States)

    Rasooly, Reuven; Do, Paula M; Friedman, Mendel

    2010-05-12

    The foodborne pathogen Staphylococcus aureus produces the virulent staphylococcal enterotoxin A (SEA), a single-chain protein that consists of 233 amino acid residues with a molecular weight of 27 078 Da. SEA is a superantigen that is reported to contribute to animal (mastitis) and human (emesis, diarrhea, atopic dermatitis, arthritis, and toxic shock) syndromes. Changes of the native structural integrity may inactivate the toxin by preventing molecular interaction with cell membrane receptor sites of their host cells. In the present study, we evaluated the ability of one commercial and two freshly prepared apple juices and a commercial apple polyphenol preparation (Apple Poly) to inhibit the biological activity of SEA. Dilutions of freshly prepared apple juices and Apple Poly inhibited the biological activity of SEA without any significant cytotoxic effect on the spleen cells. Additional studies with antibody-coated immunomagnetic beads bearing specific antibodies against the toxin revealed that SEA added to apple juice appears to be largely irreversibly bound to the juice constituents. The results suggest that food-compatible and safe anti-toxin phenolic compounds can be used to inactivate SEA in vitro and possibly also in vivo, even after induction of T-cell proliferation by long-term exposure to SEA. The significance of the results for microbial food safety and human health is discussed.

  9. Evaluation of Handheld Assays for the Detection of Ricin and Staphylococcal Enterotoxin B in Disinfected Waters

    Directory of Open Access Journals (Sweden)

    Mary Margaret Wade

    2011-01-01

    Full Text Available Development of a rapid field test is needed capable of determining if field supplies of water are safe to drink by the warfighter during a military operation. The present study sought to assess the effectiveness of handheld assays (HHAs in detecting ricin and Staphylococcal Enterotoxin B (SEB in water. Performance of HHAs was evaluated in formulated tap water with and without chlorine, reverse osmosis water (RO with chlorine, and RO with bromine. Each matrix was prepared, spiked with ricin or SEB at multiple concentrations, and then loaded onto HHAs. HHAs were allowed to develop and then read visually. Limits of detection (LOD were determined for all HHAs in each water type. Both ricin and SEB were detected by HHAs in formulated tap water at or below the suggested health effect levels of 455 ng/mL and 4.55 ng/mL, respectively. However, in brominated or chlorinated waters, LODs for SEB increased to approximately 2,500 ng/mL. LODs for ricin increased in chlorinated water, but still remained below the suggested health effect level. In brominated water, the LOD for ricin increased to approximately 2,500 ng/mL. In conclusion, the HHAs tested were less effective at detecting ricin and SEB in disinfected water, as currently configured.

  10. Selection of staphylococcal enterotoxin B (SEB)-binding peptide using phage display technology

    International Nuclear Information System (INIS)

    Soykut, Esra Acar; Dudak, Fahriye Ceyda; Boyaci, Ismail Hakki

    2008-01-01

    In this study, peptides were selected to recognize staphylococcal enterotoxin B (SEB) which cause food intoxication and can be used as a biological war agent. By using commercial M13 phage library, single plaque isolation of 38 phages was done and binding affinities were investigated with phage-ELISA. The specificities of the selected phage clones showing high affinity to SEB were checked by using different protein molecules which can be found in food samples. Furthermore, the affinities of three selected phage clones were determined by using surface plasmon resonance (SPR) sensors. Sequence analysis was realized for three peptides showing high binding affinity to SEB and WWRPLTPESPPA, MNLHDYHRLFWY, and QHPQINQTLYRM amino acid sequences were obtained. The peptide sequence with highest affinity to SEB was synthesized with solid phase peptide synthesis technique and thermodynamic constants of the peptide-SEB interaction were determined by using isothermal titration calorimetry (ITC) and compared with those of antibody-SEB interaction. The binding constant of the peptide was determined as 4.2 ± 0.7 x 10 5 M -1 which indicates a strong binding close to that of antibody

  11. Evaluation of Potential Effects of NaCl and Sorbic Acid on Staphylococcal Enterotoxin A Formation

    Directory of Open Access Journals (Sweden)

    Nikoleta Zeaki

    2015-09-01

    Full Text Available The prophage-encoded staphylococcal enterotoxin A (SEA is recognized as the main cause of staphylococcal food poisoning (SFP, a common foodborne intoxication disease, caused by Staphylococcus aureus. Studies on the production of SEA suggest that activation of the SOS response and subsequent prophage induction affect the regulation of the sea gene and the SEA produced, increasing the risk for SFP. The present study aims to evaluate the effect of NaCl and sorbic acid, in concentrations relevant to food production, on SOS response activation, prophage induction and SEA production. The impact of stress was initially evaluated on steady state cells for a homogenous cell response. NaCl 2% was found to activate the SOS response, i.e., recA expression, and trigger prophage induction, in a similar way as the phage-inducer mitomycin C. In contrast, sorbic acid decreased the pH of the culture to a level where prophage induction was probably suppressed, even when combined with NaCl stress. The impact of previous physiological state of the bacteria was also addressed on cells pre-exposed to NaCl, and was found to potentially affect cell response upon exposure to further stress. The results obtained highlight the possible SFP-related risks arising from the use of preservatives during food processing.

  12. Inhibition of the superantigenic activities of Staphylococcal enterotoxin A by an aptamer antagonist.

    Science.gov (United States)

    Wang, Kaiyu; Wu, Dong; Chen, Zhuang; Zhang, Xianhui; Yang, Xiangyue; Yang, Chaoyong James; Lan, Xiaopeng

    2016-09-01

    Staphylococcal enterotoxin A (SEA) is an important component of Staphylococcus aureus pathogenesis. SEA induces T lymphocytes activation and proliferation, resulting in the release of a large number of inflammatory cytokines. Blocking the toxic cascade triggered by SEA may be an effective strategy for the treatment of SEA-induced diseases. Through a systematic evolution of ligands by exponential enrichment process, we obtained an aptamer (S3) that could bind SEA with both high affinity and specificity, with a Kd value 36.93 ± 7.29 nM (n = 3). This aptamer antagonist effectively inhibited SEA-mediated human peripheral blood mononuclear cells proliferation and inflammatory cytokines (IFN-γ, TNF-α, IL-2 and IL-6) secretion. Moreover, PEGylated S3 significantly reduced mortality in murine lethal toxic shock models established by lipopolysaccharide-potentiated SEA. Therefore, this novel aptamer antagonist has the potential to become a new strategy for treating S. aureus infections and SEA-induced diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Lactobacillus zeae protects Caenorhabditis elegans from enterotoxigenic Escherichia coli-caused death by inhibiting enterotoxin gene expression of the pathogen.

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    Mengzhou Zhou

    Full Text Available BACKGROUND: The nematode Caenorhabditis elegans has become increasingly used for screening antimicrobials and probiotics for pathogen control. It also provides a useful tool for studying microbe-host interactions. This study has established a C. elegans life-span assay to preselect probiotic bacteria for controlling K88(+ enterotoxigenic Escherichia coli (ETEC, a pathogen causing pig diarrhea, and has determined a potential mechanism underlying the protection provided by Lactobacillus. METHODOLOGY/PRINCIPAL FINDINGS: Life-span of C. elegans was used to measure the response of worms to ETEC infection and protection provided by lactic acid-producing bacteria (LAB. Among 13 LAB isolates that varied in their ability to protect C. elegans from death induced by ETEC strain JG280, Lactobacillus zeae LB1 offered the highest level of protection (86%. The treatment with Lactobacillus did not reduce ETEC JG280 colonization in the nematode intestine. Feeding E. coli strain JFF4 (K88(+ but lacking enterotoxin genes of estA, estB, and elt did not cause death of worms. There was a significant increase in gene expression of estA, estB, and elt during ETEC JG280 infection, which was remarkably inhibited by isolate LB1. The clone with either estA or estB expressed in E. coli DH5α was as effective as ETEC JG280 in killing the nematode. However, the elt clone killed only approximately 40% of worms. The killing by the clones could also be prevented by isolate LB1. The same isolate only partially inhibited the gene expression of enterotoxins in both ETEC JG280 and E. coli DH5α in-vitro. CONCLUSIONS/SIGNIFICANCE: The established life-span assay can be used for studies of probiotics to control ETEC (for effective selection and mechanistic studies. Heat-stable enterotoxins appeared to be the main factors responsible for the death of C. elegans. Inhibition of ETEC enterotoxin production, rather than interference of its intestinal colonization, appears to be the

  14. A regenerable piezoelectric immunosensor on the basis of electropolymerized polypyrrole for highly selective detection of Staphylococcal Enterotoxin A in foodstuffs

    International Nuclear Information System (INIS)

    Karaseva, Nadezhda; Ermolaeva, Tatyana

    2015-01-01

    Piezoelectric immunosensors have been developed for the determination of trace concentrations of Staphylococcal enterotoxin A (SEA) in foodstuff. Antibodies against SEA were covalently immobilized on an electropolymerized polypyrrole substrate on a gold electrode which warrants increased stability of the biolayer and sensitive detection. The calibration plot is linear in the 1–80 ng∙mL -1 SEA concentration range, the limit of detection is 0.4 ng∙mL -1 , and the time of analysis is <30 min. The sensor was successfully applied to quantify SEA in chicken meat and milk. (author)

  15. Nitrate salts suppress sporulation and production of enterotoxin in Clostridium perfringens strain NCTC8239.

    Science.gov (United States)

    Yasugi, Mayo; Otsuka, Keisuke; Miyake, Masami

    2016-10-01

    Clostridium perfringens type A is a common source of food-borne illness in humans. Ingested vegetative cells sporulate in the small intestinal tract and in the process produce C. perfringens enterotoxin (CPE). Although sporulation plays a critical role in the pathogenesis of food-borne illness, the molecules triggering/inhibiting sporulation are still largely unknown. It has previously been reported by our group that sporulation is induced in C. perfringens strain NCTC8239 co-cultured with Caco-2 cells in Dulbecco's Modified Eagle Medium (DMEM). In contrast, an equivalent amount of spores was not observed when bacteria were co-cultured in Roswell Park Memorial Institute-1640 medium (RPMI). In the present study it was found that, when these two media are mixed, RPMI inhibits sporulation and CPE production induced in DMEM. When a component of RPMI was added to DMEM, it was found that calcium nitrate (Ca[NO 3 ] 2 ) significantly inhibits sporulation and CPE production. The number of spores increased when Ca(NO 3 ) 2 -deficient RPMI was used. The other nitrate salts significantly suppressed sporulation, whereas the calcium salts used did not. qPCR revealed that nitrate salts increased expression of bacterial nitrate/nitrite reductase. Furthermore, it was found that nitrite and nitric oxide suppress sporulation. In the sporulation stages, Ca(NO 3 ) 2 down-regulated the genes controlled by Spo0A, a master regulator of sporulation, but not spo0A itself. Collectively, these results indicate that nitrate salts suppress sporulation and CPE production by down-regulating Spo0A-regulated genes in C. perfringens strain NCTC8239. Nitrate reduction may be associated with inhibition of sporulation. © 2016 The Societies and John Wiley & Sons Australia, Ltd.

  16. Suppression of colorectal tumorigenesis by recombinant Bacteroides fragilis enterotoxin-2 in vivo.

    Science.gov (United States)

    Lv, You; Ye, Tao; Wang, Hui-Peng; Zhao, Jia-Ying; Chen, Wen-Jie; Wang, Xin; Shen, Chen-Xia; Wu, Yi-Bin; Cai, Yuan-Kun

    2017-01-28

    To evaluate the impact of recombinant Bacteroides fragilis enterotoxin-2 (BFT-2, or Fragilysin) on colorectal tumorigenesis in mice induced by azoxymethane/dextran sulfate sodium (AOM/DSS). Recombinant proBFT-2 was expressed in Escherichia coli strain Rosetta (DE3) and BFT-2 was obtained and tested for its biological activity via colorectal adenocarcinoma cell strains SW-480. Seventy C57BL/6J mice were randomly divided into a blank (BC; n = 10), model (AD; n = 20), model + low-dose toxin (ADLT; n = 20, 10 μg), and a model + high-dose toxin (ADHT; n = 20, 20 μg) group. Mice weight, tumor formation and pathology were analyzed. Immunohistochemistry determined Ki-67 and Caspase-3 expression in normal and tumor tissues of colorectal mucosa. Recombinant BFT-2 was successfully obtained, along with its biological activity. The most obvious weight loss occurred in the AD group compared with the ADLT group (21.82 ± 0.68 vs 23.23 ± 0.91, P ADHT group (21.82 ± 0.68 vs 23.57 ± 1.06, P ADHT groups (19.75 ± 3.30 vs 6.50 ± 1.73, P ADHT group. The incidence of colorectal adenocarcinoma in both the ADHT group and the ADHT group was reduced compared to that in the AD group ( P ADHT group was 50% and 40%, respectively, both of which were lower than that found in the AD group (94.44%, P ADHT group was 45% and 55%, both of which were higher than that found in the BC group (16.67%, P < 0.05, P < 0.05). Oral administration with lower-dose biologically active recombinant BFT-2 inhibited colorectal tumorigenesis in mice.

  17. Modeling the effects of a Staphylococcal Enterotoxin B (SEB on the apoptosis pathway

    Directory of Open Access Journals (Sweden)

    Hammamieh Rasha

    2006-05-01

    Full Text Available Abstract Background The lack of detailed understanding of the mechanism of action of many biowarfare agents poses an immediate challenge to biodefense efforts. Many potential bioweapons have been shown to affect the cellular pathways controlling apoptosis 1234. For example, pathogen-produced exotoxins such as Staphylococcal Enterotoxin B (SEB and Anthrax Lethal Factor (LF have been shown to disrupt the Fas-mediated apoptotic pathway 24. To evaluate how these agents affect these pathways it is first necessary to understand the dynamics of a normally functioning apoptosis network. This can then serve as a baseline against which a pathogen perturbed system can be compared. Such comparisons can expose both the proteins most susceptible to alteration by the agent as well as the most critical reaction rates to better instill control on a biological network. Results We explore this through the modeling and simulation of the Fas-mediated apoptotic pathway under normal and SEB influenced conditions. We stimulated human Jurkat cells with an anti-Fas antibody in the presence and absence of SEB and determined the relative levels of seven proteins involved in the core pathway at five time points following exposure. These levels were used to impute relative rate constants and build a quantitative model consisting of a series of ordinary differential equations (ODEs that simulate the network under both normal and pathogen-influenced conditions. Experimental results show that cells exposed to SEB exhibit an increase in the rate of executioner caspase expression (and subsequently apoptosis of 1 hour 43 minutes (± 14 minutes, as compared to cells undergoing normal cell death. Conclusion Our model accurately reflects these results and reveals intervention points that can be altered to restore SEB-influenced system dynamics back to levels within the range of normal conditions.

  18. Effect of different ionizing radiation dose rates on the Staphylococcal enterotoxin in mechanically deboned chicken meat

    International Nuclear Information System (INIS)

    Azevedo, Heliana de; Brito, Poliana de Paula; Fukuma, Henrique Takuji; Roque, Claudio Vitor; Custodio, Wilson; Kodama, Yasko; Miya, Norma Terugo Nago; Pereira, Jose Luiz

    2009-01-01

    Samples weighing 50g each were prepared from allotments of back with skin MDCM, to the EEB contamination or not (control). Each sample of MDCM contaminated or not with EEB was conditioned in low density polyethylene bag, frozen (-18 ± 1 deg C) for one night in a tunnel and irradiated with gamma rays from 60 Co source in this state with doses of 0.0 kGy (control), 1.5 kGy (5.7 kGy.h -1 - higher dose rate, 1.8 kGy.h -1 - intermediary dose rate and 0.6 kGy.h -1 - lower dose rate) and 3.0 kGy (8.4 kGy.h - '1 - higher dose rate, 2.4 kGy.h -1 - intermediary dose rate and 0.6 kGy.h -1 - lower dose rate). Irradiated or non irradiated MDCM samples were processed to the EEB extraction, according to the VIDAS Staph enterotoxin II kit (bioMerieux) manufacturer protocol. The calculation to determinate the MDCM EEB recovery after the sample (control or irradiated) processing were carried out applying the principle of mass balance, along the whole process. Described experiment was performed in triplicate. Results showed that the irradiation process was effective to remove the MDCM EEB, to both 1.5 kGy and 3.0 kGy. According to the expected, doses of 3.0 kGy showed the highest values of MDCM EEB removal. Regarding the effect of dose rate of radiation on the removal of EEB of the MDCM, it could be observed only for samples irradiated with 1.5 kGy radiation dose; in these processing conditions, the highest value of EEB removal was obtained for samples processed with low radiation dose rate. (author)

  19. Novel Tissue Level Effects of the Staphylococcus aureus Enterotoxin Gene Cluster Are Essential for Infective Endocarditis.

    Science.gov (United States)

    Stach, Christopher S; Vu, Bao G; Merriman, Joseph A; Herrera, Alfa; Cahill, Michael P; Schlievert, Patrick M; Salgado-Pabón, Wilmara

    2016-01-01

    Superantigens are indispensable virulence factors for Staphylococcus aureus in disease causation. Superantigens stimulate massive immune cell activation, leading to toxic shock syndrome (TSS) and contributing to other illnesses. However, superantigens differ in their capacities to induce body-wide effects. For many, their production, at least as tested in vitro, is not high enough to reach the circulation, or the proteins are not efficient in crossing epithelial and endothelial barriers, thus remaining within tissues or localized on mucosal surfaces where they exert only local effects. In this study, we address the role of TSS toxin-1 (TSST-1) and most importantly the enterotoxin gene cluster (egc) in infective endocarditis and sepsis, gaining insights into the body-wide versus local effects of superantigens. We examined S. aureus TSST-1 gene (tstH) and egc deletion strains in the rabbit model of infective endocarditis and sepsis. Importantly, we also assessed the ability of commercial human intravenous immunoglobulin (IVIG) plus vancomycin to alter the course of infective endocarditis and sepsis. TSST-1 contributed to infective endocarditis vegetations and lethal sepsis, while superantigens of the egc, a cluster with uncharacterized functions in S. aureus infections, promoted vegetation formation in infective endocarditis. IVIG plus vancomycin prevented lethality and stroke development in infective endocarditis and sepsis. Our studies support the local tissue effects of egc superantigens for establishment and progression of infective endocarditis providing evidence for their role in life-threatening illnesses. In contrast, TSST-1 contributes to both infective endocarditis and lethal sepsis. IVIG may be a useful adjunct therapy for infective endocarditis and sepsis.

  20. Rhinosinusitis derived Staphylococcal enterotoxin B possibly associates with pathogenesis of ulcerative colitis

    Directory of Open Access Journals (Sweden)

    An Zi-Yuan

    2005-09-01

    Full Text Available Abstract Background During clinical practice, we noticed that some patients with both ulcerative colitis (UC and chronic rhinosinusitis (CRS showed amelioration of UC after treatment of CRS. This study was designed to identify a possible association between CRS and UC. Methods Thirty-two patients with both CRS and UC received treatment with functional endoscopic sinus surgery (FESS for CRS. Clinical symptom scores for CRS and UC, as well as serum levels of anti-Staphylococcal enterotoxin B (SEB were evaluated at week 0 and week 12. Sinus wash fluid SEB content was measured with enzyme-linked immunosorbent assay (ELISA. The surgically removed tissues were cultured to identify growth of Staphylococcus. aureus (S. aureus. Immunohistochemistry was employed to identify anti-SEB positive cells in the colonic mucosa. Colonic biopsies were obtained and incubated with SEB. Mast cell activation in the colonic mucosa in response to incubation with SEB was observed with electron microscopy and immunoassay. Results The clinical symptom scores of CRS and UC severe scores (UCSS were significantly reduced in the UC-CRS patients after FESS. The number of cultured S. aureus colonies from the surgically removed sinus mucosa significantly correlated with the decrease in UCSS. High levels of SEB were detected in the sinus wash fluids of the patients with UC-CRS. Histamine and tryptase release was significantly higher in the culture supernate in the patients with UC-CRS than the patients with UC-only and normal controls. Anti-SEB positive cells were located in the colonic mucosa. Conclusion The pathogenesis of UC in some patients may be associated with their pre-existing CRS by a mechanism of swallowing sinusitis-derived SEB. We speculate that SEB initiates inappropriate immune reactions and inflammation in the colonic mucosa that further progresses to UC.

  1. Evaluation of the adjuvant effect of Escherichia coli heat-labile ...

    African Journals Online (AJOL)

    Data on IgG antibody isotype profiles showed that IgG 1 and IgG 2a were predominant in mice immunized with rMVNP + LT or LTK63 whereas IgG 1 predominated when rMVNP was given on its own implying that LT and LTK63 induce both Th1 and Th2-type immune responses. These results highlight the great potential of ...

  2. Peracetic Acid: A Practical Agent for Sterilizing Heat-Labile Polymeric Tissue-Engineering Scaffolds

    Science.gov (United States)

    Yoganarasimha, Suyog; Trahan, William R.; Best, Al M.; Bowlin, Gary L.; Kitten, Todd O.; Moon, Peter C.

    2014-01-01

    Advanced biomaterials and sophisticated processing technologies aim at fabricating tissue-engineering scaffolds that can predictably interact within a biological environment at the cellular level. Sterilization of such scaffolds is at the core of patient safety and is an important regulatory issue that needs to be addressed before clinical translation. In addition, it is crucial that meticulously engineered micro- and nano- structures are preserved after sterilization. Conventional sterilization methods involving heat, steam, and radiation are not compatible with engineered polymeric systems because of scaffold degradation and loss of architecture. Using electrospun scaffolds made from polycaprolactone, a low melting polymer, and employing spores of Bacillus atrophaeus as biological indicators, we compared ethylene oxide, autoclaving and 80% ethanol to a known chemical sterilant, peracetic acid (PAA), for their ability to sterilize as well as their effects on scaffold properties. PAA diluted in 20% ethanol to 1000 ppm or above sterilized electrospun scaffolds in 15 min at room temperature while maintaining nano-architecture and mechanical properties. Scaffolds treated with PAA at 5000 ppm were rendered hydrophilic, with contact angles reduced to 0°. Therefore, PAA can provide economical, rapid, and effective sterilization of heat-sensitive polymeric electrospun scaffolds that are used in tissue engineering. PMID:24341350

  3. Inhibition of Cronobacter sakazakii by heat labile bacteriocins produced by probiotic LAB isolated from healthy infants.

    Science.gov (United States)

    Awaisheh, Saddam S; Al-Nabulsi, Anas A; Osaili, Tareq M; Ibrahim, Salam; Holley, Richard

    2013-09-01

    Cronobacter sakazakii is an opportunistic pathogen that can cause bacteremia, meningitis, and necrotizing enterocolitis, most often in neonates with case-fatality rates that may reach 80%. The antimicrobial activity of lactic acid bacteria against a wide range of foodborne pathogens is well-established in different types of food products. The objective of the current study was to investigate the antibacterial activity of Lactobacillus acidophilus and L. casei isolated from feces of healthy infants against different strains of C. sakazakii in agar and a rehydrated infant milk formula (RIMF) model. The inhibition zones of C. sakazakii around L. acidophilus or L. casei ranged from 22 to 32 mm on eMan Rogosa Sharpe (MRS) agar under aerobic conditions, while a slight reduction in antibacterial activity was noted on modified MRS (0.2% glucose) under anaerobic conditions. It was observed that pH-neutralized cell-free supernatant (CFS) of L. acidophilus or L. casei was inhibitory against tested C. sakazakii strains. The inhibition zones of neutralized CFS were lower than the antibacterial activities of live cultures. The antibacterial activity of CFS was abolished when CFS from L. acidophilus or L. casei was heated at 60 or 80 °C for either 10 min or 2 h, or treated with trypsin or pepsin. This was considered strong evidence that the inhibition was due to the production of bacteriocins by L. casei and L. acidophilus. Both the CFS and active growing cells of L. casei and L. acidophilus were able to reduce the viability of C. sakazakii in the RIMF model. The results may extend the use of natural antimicrobials instead of conventional preservation methods to improve the safety of RIMF. © 2013 Institute of Food Technologists®

  4. Detergent inhibited, heat labile nucleoside triphosphatase in cores of avian myeloblastosis virus

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank

    1978-01-01

    Endogenous DNA synthesis was studied in isolated core particles of avian myeloblastosis virus. It was found that cores contained an enzymatic activity which rapidly converted the added nucleoside triphosphates to diphosphates (but not further) at 0 degrees C, thus inhibiting DNA synthesis...

  5. Comparison of Genotypes and Enterotoxin Genes Between Staphylococcus aureus Isolates from Blood and Nasal Colonizers in a Korean Hospital

    Science.gov (United States)

    Peck, Kyong Ran; Baek, Jin Yang; Song, Jae-Hoon

    2009-01-01

    In this study, we investigated the genetic background of 70 Staphylococcus aureus isolates (36 methicillin-resistant S. aureus [MRSA] and 34 methicillin-susceptible S. aureus [MSSA]) obtained from blood at a Korean tertiary-care hospital, using spa typing, multilocus sequence typing, and SCCmec typing. In addition, the prevalence of enterotoxin (sea, seb, sec, sed, see, seg, seh, sei, and sek), tst, and pvl genes among the samples was assessed via polymerase chain reaction, and the results were compared with those of 95 isolates of S. aureus obtained from nasal swabs. All MRSA isolates from blood, except one, belonged to three major clones: sequence type (ST)5-MRSA-II, ST72-MRSA-II (or IVA), and ST239-MRSA-III, among which ST5-MRSA-II was the predominant clone. The prevalence of enterotoxin genes in the S. aureus isolates obtained from blood differed significantly from those from the nasal swabs for the sea, seb, sec, and seh gene. In particular, the seb and sec genes were detected exclusively in the MRSA isolates of ST5 or spa-CC002, thereby suggesting the co-adaptation of virulence genes with the genetic background and their contribution to biological fitness. PMID:19654937

  6. Sensitive chemiluminescence immunoassay for staphylococcal enterotoxin C1 based on the use of dye-encapsulated mesoporous silica nanoparticles

    International Nuclear Information System (INIS)

    Tao, Liang; Zhang, Chunmei; Sun, Yuanjie; Jin, Boquan; Yang, Kun; Li, Xiaohua; Zhang, Zhujun; Zhang, Jinpeng; Yan, Kuocheng

    2016-01-01

    A chemiluminescent immunoassay for the staphylococcal enterotoxin C1 (SEC1) based on the use of dye-encapsulated mesoporous silica nanoparticles (m-SiNPs) as a label is described. The dyes are retained in the m-SiNPs via strong hydrophobic interactions. The assay comprises the following steps: (a) Microplates coated with antibody against SEC1 are filled with sample upon which the SEC antigen will be bound to the surface; (b) following a washing step, secondary antibody linked to m-SiNPs (that were covalently labeled with rhodamine 6G and fluorescein) were added to form the sandwich complex; (c) after another washing step, bis(2,4,6-trichlorophenyl) oxalate, H_2O_2 and imidazole are added to generate chemiluminescence whose intensity is proportional to the number of m-SiNPs and thus to the number of antigen (SEC) molecules. It is found that the use of functionalized m-SiNPs strongly amplifies the signal. Enterotoxin SEC1 can be detected by this method in the 0.025 to 2 ng⋅mL"-"1 concentration range, the detection limit is 19 pg⋅mL"-"1 (at 3σ), and the relative standard deviation (for 11 parallel measurements at a 1 ng⋅mL"-"1 level) is 4.6 %. The use of an automated chemiluminescence analyzer further improves detection. (author)

  7. Magnetic Bead and Fluorescent Silica Nanoparticles Based Optical Immunodetection of Staphylococcal Enterotoxin B (SEB in Bottled Water

    Directory of Open Access Journals (Sweden)

    Shiva K. RASTOGI

    2009-10-01

    Full Text Available Staphylococcal enterotoxins (SEs are a major cause of food-borne diseases, most commonly SEs assayed immunologically with ELISA. An immunoassay based on fluorescein dye doped silica dioxide nanoparticles (F-SiNPs and magnetic bead (MB is described here for the detection of staphylococcal enterotoxin B (SEB. F-SiNPs have unique optical properties which make them attractive for biosensing. The water-in-oil (W/O reverse microemulsion method was used for the synthesis of F-SiNPs (~ 95 nm of diameter. The F-SiNPs were characterized using SEM, TEM and FTIR spectroscopy. The detection of SEB is preformed in PBS buffer, and bottled drinking water using sandwich immunoassay format. Target analytes were captured using MBs modified with the antigen-specific “capture” antibody, and detected using F-SiNP labeled secondary antigen-specific antibody. We report a limit of detection down to 1 ng/mL SEB spiked sample in less than 2 hr assay time using fluorocount method. This study demonstrates the bio warfare agent SEB capture by magnetic beads and detection using F-SiNPs.

  8. Presence of Classical Enterotoxin Genes, agr Typing, Antimicrobial Resistance, and Genetic Diversity of Staphylococcus aureus from Milk of Cows with Mastitis in Southern Brazil.

    Science.gov (United States)

    Kroning, Isabela S; Iglesias, Mariana A; Mendonça, Karla S; Lopes, Graciela V; Silva, Wladimir P

    2018-05-01

    Staphylococcus aureus is a common causative agent of bovine mastitis in dairy cows and commonly associated with foodborne disease outbreaks. The aim of this study was to evaluate the presence of enterotoxin genes, agr typing, antimicrobial resistance, and genetic diversity of S. aureus isolated from milk of cows with mastitis in dairy farms from southern Brazil. Results showed that 7 (22.6%) of 31 S. aureus isolates were positive for enterotoxin genes. Specifically, the genes encoding for enterotoxins A ( n = 4), C ( n = 2), and B ( n = 1) were detected. Isolates belonging to the agr group III (10 of 31, 32.2%) and agr group I (7 of 31, 22.5%) were the most common. To our knowledge, this is the first report of both agr I and III in the same S. aureus isolate from milk of cows with bovine mastitis. The antimicrobial resistance test showed that 54% of the isolates were multiresistant to antimicrobial agents. The macrorestriction analysis produced 16 different major SmaI pulsed-field gel electrophoresis patterns, with up to two subpatterns. Moreover, the presence of some S. aureus clones in a distinct area was observed. Although this study characterized a limited number of S. aureus isolates, the presence of classical enterotoxin genes and resistance to multiple antimicrobial agents reinforces the importance of this microorganism to animal and human health. In addition, similar genetic profiles have been identified in distinct geographic areas, suggesting clonal dissemination of S. aureus in dairy herds from southern Brazil.

  9. Detection of coding genes for enterotoxins in Bacillus cereus by PCR and their products by BCET-RPLA and ELISA Assay

    Directory of Open Access Journals (Sweden)

    Marcela Vyletělová

    2010-01-01

    Full Text Available Determination of enterotoxin production, diarrhoeal and emetic gene identification was studied in 41 Bacillus cereus strains isolated from raw cows’ and raw goats’ milk, pasteurized milk, dairy products during technological processing and from dairy plant equipment. Presence of enterotoxins was detected by BCET-RPLA (HBL and ELISA immunoassay (NHE. Gene identification (nheA, nheB, nheC, hblA, hblC, hblD, bceT, cytK-1, cytK-2, entFM and ces was achieved by means of PCR. Enterotoxin HBL was detected in 32 strains, enterotoxin NHE in all 41 strains. Presence of all three genes nheA, nheB and nheC was confirmed in 40 strains and genes hblA, hblC and hblD in 29 strains. Comparison of used methods was as follow: 1 BCET-RPLA (which detects L2 component and PCR (positive or negative all three hblA, hblC and hblD gene detection were identical in 30 (73%; 2 ELISA (NheA and PCR (all three nheC, nheB and nheA gene expression were identical in 40 (98% cases isolated strains.

  10. Comparison of toxicity neutralization-, ELISA- and PCR tests for typing of Clostridium perfringens and detection of the enterotoxin gene by PCR

    DEFF Research Database (Denmark)

    Møller, Kristian; Ahrens, Peter

    1996-01-01

    A polymerase chain reaction (PCR) was developed for the specific amplification of a part of each of the five Clostridium perfringens toxin genes: alpha (alpha), beta (beta), epsilon (epsilon), iota (iota), and enterotoxin (CPE). While the toxicity neutralization test (TNT) only showed limited...

  11. Enterotoxin Producing Ability And Antimicrobial Susceptibility Of Coagulase-Negative Staphylococci Isolated From Goat Milk Cheese And Salted Yoghurt In Turkey

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    S. Pehlivanlar Onen

    2017-10-01

    Full Text Available The aim of this study was to determine enterotoxin producing ability and antimicrobial susceptibility of coagulase negative staphylococci CNS in goats bulk milk cheese and salted yoghurt. CNS strains were identified by using GP card in VITEK 2 system. The presence of enterotoxins was determined by enzyme immunoassay test by using RIDASCREEN test kit. Antibiotic susceptibility in CNS strains was detected by using AST-P640 card in VITEK 2 system. A total of 100 CNS strains were isolated in 22 55 bulk milk samples and in 23 57.5 cheese samples. Staphylococcus spp. could not be isolated from salted yoghurt samples. The most encountered species were S. caprae 51.9 S. chromogenes 11.5 and S. xylosus 9.6 from milk samples and S. saprophyticus 60.4 S. xylosus 12.5 and S. haemolyticus 8.3 from cheese samples. Four CNS strains 4 isolated from samples were capable of producing enterotoxin. While all isolates were resistant to at least one antibiotic 74 of CNS strains showed resistance to two or more antibiotics. Enterotoxin production ability and high antibiotic resistance of the CNS strains isolated from goat bulk milk and cheese can lead to a risk for public health.

  12. Noncontiguous Finished Genome Sequence of Staphylococcus aureus KLT6, a Staphylococcal Enterotoxin B-Positive Strain Involved in a Food Poisoning Outbreak in Switzerland

    Science.gov (United States)

    Tobes, Raquel; Manrique, Marina; Brozynska, Marta; Stephan, Roger; Pareja, Eduardo

    2013-01-01

    We present the first complete genome sequence of a Staphylococcus aureus strain assigned to clonal complex 12. The strain was isolated in a food poisoning outbreak due to contaminated potato salad in Switzerland in 2009, and it produces staphylococcal enterotoxin B. PMID:23704175

  13. Growth and enterotoxin production of Staphylococcus aureus during the manufacture and ripening of Camembert-type cheeses from raw goats' milk.

    Science.gov (United States)

    Meyrand, A; Boutrand-Loei, S; Ray-Gueniot, S; Mazuy, C; Gaspard, C E; Jaubert, G; Perrin, G; Lapeyre, C; Vernozy-Rozand, C

    1998-09-01

    Tests were carried out to determine the effect of manufacturing procedures for a Camembert-type cheese from raw goats' milk on the growth and survival of Staphylococcus aureus organisms added to milk at the start of the process, and to study the possible presence of staphylococcal enterotoxin A in these cheeses. The initial staphylococcal counts were, respectively, 2, 3, 4, 5 and 6 log cfu ml-1. Cheese was prepared following the industrial specifications and ripened for 41 d. Detection of enterotoxins was done by the Vidas SET test and by an indirect double-sandwich ELISA technique using antienterotoxin monoclonal antibodies. Generally, numbers of microbes increased at a similar rate during manufacture in all cheeses until salting. During the ripening period, the aerobic plate count population and Staph. aureus levels remained stable and high. There was an approximately 1 log reduction of Staph. aureus in cheeses made with an initial inoculum of Staph. aureus greater than 10(3) cfu ml-1 at the end of the ripening period (41 d) compared with the count at 22 h. The level of staphylococcal enterotoxin A recovered varied from 1 to 3.2 ng g-1 of cheese made with an initial population of 10(3)-10(6) cfu ml-1. No trace of enterotoxin A was detected in cheeses made with the lowest Staph. aureus inoculum used in this study.

  14. Detection of enterotoxins produced by B. cereus through PCR analysis of ground and roasted coffee samples in Rio de Janeiro, Brazil

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    Cyllene de Matos Ornelas da Cunha Corrêa de Souza

    2011-06-01

    Full Text Available Coffee is one of the most appreciated drinks in the world. Coffee ground is obtained from the fruit of a small plant that belongs to the genus Coffea. Coffea arabica and Coffea canephora robusta are the two most commercially important species. They are more commonly known as arabica and robusta, respectively. Two-thirds of Coffea arabica plants are grown in South and Central America, and Eastern Africa - the place of origin for this coffee species. Contamination by microorganisms has been a major matter affecting coffee quality in Brazil, mainly due to the harvesting method adopted. Brazilian harvests are based on fruits collected from the ground mixed with those that fall on collection cloths. As the Bacillus cereus bacterium frequently uses the soil as its environmental reservoir, it is easily capable of becoming a contaminant. This study aimed to evaluate the contamination and potential of B. cereus enterotoxin genes encoding the HBL and NHE complexes, which were observed in strains of ground and roasted coffee samples sold in Rio de Janeiro. The PCR (Polymerase Chain Reaction results revealed high potential of enterotoxin production in the samples. The method described by Speck (1984 was used for the isolation of contaminants. The investigation of the potential production of enterotoxins through isolates of the microorganism was performed using the B. cereus enterotoxin Reverse Passive Latex Agglutination test-kit (BCET-RPLA, Oxoid, according to the manufacturer's instructions. The potential of enterotoxin production was investigated using polymerase chain reaction (PCR methods for hblA, hblD and hblC genes (encoding hemolysin HBL and for nheA, nheB and nheC genes (encoding non-hemolytic enterotoxin - NHE. Of all the 17 strains, 100% were positive for at least 1 enterotoxin gene; 52.9% (9/17 were positive for the 3 genes encoding the HBL complex; 35.3% (6/17 were positive for the three NHE encoding genes; and 29.4% (5/17 were positive for

  15. Acetic acid increases the phage-encoded enterotoxin A expression in Staphylococcus aureus

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    da Silva Ayla

    2010-05-01

    Full Text Available Abstract Background The effects of acetic acid, a common food preservative, on the bacteriophage-encoded enterotoxin A (SEA expression and production in Staphylococcus aureus was investigated in pH-controlled batch cultures carried out at pH 7.0, 6.5, 6.0, 5.5, 5.0, and 4.5. Also, genomic analysis of S. aureus strains carrying sea was performed to map differences within the gene and in the temperate phage carrying sea. Results The sea expression profile was similar from pH 7.0 to 5.5, with the relative expression peaking in the transition between exponential and stationary growth phase and falling during stationary phase. The levels of sea mRNA were below the detection limit at pH 5.0 and 4.5, confirmed by very low SEA levels at these pH values. The level of relative sea expression at pH 6.0 and 5.5 were nine and four times higher, respectively, in the transitional phase than in the exponential growth phase, compared to pH 7.0 and pH 6.5, where only a slight increase in relative expression in the transitional phase was observed. Furthermore, the increase in sea expression levels at pH 6.0 and 5.5 were observed to be linked to increased intracellular sea gene copy numbers and extracellular sea-containing phage copy numbers. The extracellular SEA levels increased over time, with highest levels produced at pH 6.0 in the four growth phases investigated. Using mitomycin C, it was verified that SEA was at least partially produced as a consequence of prophage induction of the sea-phage in the three S. aureus strains tested. Finally, genetic analysis of six S. aureus strains carrying the sea gene showed specific sea phage-groups and two versions of the sea gene that may explain the different sea expression and production levels observed in this study. Conclusions Our findings suggest that the increased sea expression in S. aureus caused by acetic acid induced the sea-encoding prophage, linking SEA production to the lifecycle of the phage.

  16. Staphylococcal enterotoxin-specific IgE antibodies in atopic dermatitis.

    Science.gov (United States)

    Ide, Fumihito; Matsubara, Tomoyo; Kaneko, Miho; Ichiyama, Takashi; Mukouyama, Tokuko; Furukawa, Susumu

    2004-06-01

    The authors clarified the clinical significance of the measurement of serum concentrations of specific IgE antibodies to staphylococcal enterotoxin (SE) A- and SEB in atopic dermatitis (AD). The serum concentrations of SEA- and SEB-specific IgE antibodies in 140 pediatric patients with AD were measured with an immuno CAP -radioallergosorbent test system (RAST). To check the cross-reaction of specific IgE antibodies to SEA/SEB and other allergens, the CAP RAST fluorescent enzyme immunoassay inhibition test was performed. Forty-seven patients (33.6%) tested positive for either SEA- or SEB-specific IgE antibodies. School children showed higher positive rates of SEA/SEB-specific IgE antibodies than infants or young children. The patients with severe AD and those with exacerbation of symptoms in summer, had higher positive rates of SEA/SEB-specific IgE antibodies than patients with mild AD or those with exacerbation in winter. In addition, the positive rates of specific IgE antibodies to both dog-dander and cat-dander were higher in patients with positive SEA/SEB-specific IgE antibodies than in patients with negative ones. No cross-reactions occurred among specific IgE antibodies to SEA/SEB and dog/cat dander with one patient's serum, which had positive IgE-specific antibodies against cat/dog dander and SEA/SEB. The positive rate of SEA/SEB-specific IgE antibodies in the patients with dogs and/or cats as pets was 48.4%, which was higher than in those with no pets. Atopic dermatitis patients who exhibit high positive rates of SEA/SEB-specific IgE antibodies were found to be school children, severe cases, cases with high serum concentrations of total IgE, cases with exacerbation in summer, and cases with dogs and/or cats as pets. The measurement of serum concentrations of specific IgE antibodies to SEA and SEB, thus has some value for evaluating AD patients.

  17. Staphylococcal enterotoxin A regulates bone marrow granulocyte trafficking during pulmonary inflammatory disease in mice

    Energy Technology Data Exchange (ETDEWEB)

    Takeshita, W.M.; Gushiken, V.O.; Ferreira-Duarte, A.P.; Pinheiro-Torres, A.S.; Roncalho-Buck, I.A. [Department of Biology and Physiology, Faculty of Medicine of Jundiai (FMJ), Jundiai, SP (Brazil); Squebola-Cola, D.M.; Mello, G.C.; Anhê, G.F.; Antunes, E. [Department of Pharmacology, Faculty of Medical Sciences, University of Campinas (UNICAMP), Campinas, SP (Brazil); DeSouza, I.A., E-mail: ivanidesouza@uol.com.br [Department of Biology and Physiology, Faculty of Medicine of Jundiai (FMJ), Jundiai, SP (Brazil)

    2015-09-15

    Pulmonary neutrophil infiltration produced by Staphylococcal enterotoxin A (SEA) airway exposure is accompanied by marked granulocyte accumulation in bone marrow (BM). Therefore, the aim of this study was to investigate the mechanisms of BM cell accumulation, and trafficking to circulating blood and lung tissue after SEA airway exposure. Male BALB/C mice were intranasally exposed to SEA (1 μg), and at 4, 12 and 24 h thereafter, BM, circulating blood, bronchoalveolar lavage (BAL) fluid and lung tissue were collected. Adhesion of BM granulocytes and flow cytometry for MAC-1, LFA1-α and VLA-4 and cytokine and/or chemokine levels were assayed after SEA-airway exposure. Prior exposure to SEA promoted a marked PMN influx to BAL and lung tissue, which was accompanied by increased counts of immature and/or mature neutrophils and eosinophils in BM, along with blood neutrophilia. Airway exposure to SEA enhanced BM neutrophil MAC-1 expression, and adhesion to VCAM-1 and/or ICAM-1-coated plates. Elevated levels of GM-CSF, G-CSF, INF-γ, TNF-α, KC/CXCL-1 and SDF-1α were detected in BM after SEA exposure. SEA exposure increased production of eosinopoietic cytokines (eotaxin and IL-5) and BM eosinophil VLA-4 expression, but it failed to affect eosinophil adhesion to VCAM-1 and ICAM-1. In conclusion, BM neutrophil accumulation after SEA exposure takes place by integrated action of cytokines and/or chemokines, enhancing the adhesive responses of BM neutrophils and its trafficking to lung tissues, leading to acute lung injury. BM eosinophil accumulation in SEA-induced acute lung injury may occur via increased eosinopoietic cytokines and VLA-4 expression. - Highlights: • Airway exposure to SEA causes acute lung inflammation. • SEA induces accumulation of bone marrow (BM) in immature and mature neutrophils. • SEA increases BM granulocyte or BM PMN adhesion to ICAM-1 and VCAM-1, and MAC-1 expression. • SEA induces BM elevations of CXCL-1, INF-γ, TNF-α, GM-CSF, G-CSF and

  18. Staphylococcal enterotoxin A regulates bone marrow granulocyte trafficking during pulmonary inflammatory disease in mice

    International Nuclear Information System (INIS)

    Takeshita, W.M.; Gushiken, V.O.; Ferreira-Duarte, A.P.; Pinheiro-Torres, A.S.; Roncalho-Buck, I.A.; Squebola-Cola, D.M.; Mello, G.C.; Anhê, G.F.; Antunes, E.; DeSouza, I.A.

    2015-01-01

    Pulmonary neutrophil infiltration produced by Staphylococcal enterotoxin A (SEA) airway exposure is accompanied by marked granulocyte accumulation in bone marrow (BM). Therefore, the aim of this study was to investigate the mechanisms of BM cell accumulation, and trafficking to circulating blood and lung tissue after SEA airway exposure. Male BALB/C mice were intranasally exposed to SEA (1 μg), and at 4, 12 and 24 h thereafter, BM, circulating blood, bronchoalveolar lavage (BAL) fluid and lung tissue were collected. Adhesion of BM granulocytes and flow cytometry for MAC-1, LFA1-α and VLA-4 and cytokine and/or chemokine levels were assayed after SEA-airway exposure. Prior exposure to SEA promoted a marked PMN influx to BAL and lung tissue, which was accompanied by increased counts of immature and/or mature neutrophils and eosinophils in BM, along with blood neutrophilia. Airway exposure to SEA enhanced BM neutrophil MAC-1 expression, and adhesion to VCAM-1 and/or ICAM-1-coated plates. Elevated levels of GM-CSF, G-CSF, INF-γ, TNF-α, KC/CXCL-1 and SDF-1α were detected in BM after SEA exposure. SEA exposure increased production of eosinopoietic cytokines (eotaxin and IL-5) and BM eosinophil VLA-4 expression, but it failed to affect eosinophil adhesion to VCAM-1 and ICAM-1. In conclusion, BM neutrophil accumulation after SEA exposure takes place by integrated action of cytokines and/or chemokines, enhancing the adhesive responses of BM neutrophils and its trafficking to lung tissues, leading to acute lung injury. BM eosinophil accumulation in SEA-induced acute lung injury may occur via increased eosinopoietic cytokines and VLA-4 expression. - Highlights: • Airway exposure to SEA causes acute lung inflammation. • SEA induces accumulation of bone marrow (BM) in immature and mature neutrophils. • SEA increases BM granulocyte or BM PMN adhesion to ICAM-1 and VCAM-1, and MAC-1 expression. • SEA induces BM elevations of CXCL-1, INF-γ, TNF-α, GM-CSF, G-CSF and

  19. Structure of Staphylococcal Enterotoxin E in Complex with TCR Defines the Role of TCR Loop Positioning in Superantigen Recognition.

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    Karin E J Rödström

    Full Text Available T cells are crucial players in cell-mediated immunity. The specificity of their receptor, the T cell receptor (TCR, is central for the immune system to distinguish foreign from host antigens. Superantigens are bacterial toxins capable of inducing a toxic immune response by cross-linking the TCR and the major histocompatibility complex (MHC class II and circumventing the antigen specificity. Here, we present the structure of staphylococcal enterotoxin E (SEE in complex with a human T cell receptor, as well as the unligated T cell receptor structure. There are clear structural changes in the TCR loops upon superantigen binding. In particular, the HV4 loop moves to circumvent steric clashes upon complex formation. In addition, a predicted ternary model of SEE in complex with both TCR and MHC class II displays intermolecular contacts between the TCR α-chain and the MHC, suggesting that the TCR α-chain is of importance for complex formation.

  20. Staphylococcus aureus and staphylococcal enterotoxin A in breaded chicken products: detection and behavior during the cooking process.

    Science.gov (United States)

    Pepe, Olimpia; Blaiotta, Giuseppe; Bucci, Francesca; Anastasio, Marilena; Aponte, Maria; Villani, Francesco

    2006-11-01

    In this study we examined the presence of Staphylococcus aureus and staphylococcal enterotoxin A (SEA) in 20 industrial breaded chicken products obtained from different retail butchers and supermarket stores in Italy. The levels of contamination in the products analyzed were quite low, although the pH values and water activities (a(w)) in the samples considered were in ranges favorable for S. aureus growth. As demonstrated by phenotypic and molecular characterization, in spite of the high percentage of coagulase-positive Staphylococcus strains, only three strains could be referred to the species S. aureus. Moreover, all the strains were negative in PCR assays targeting staphylococcal enterotoxin genes (seA to seE, seG to seJ, and seM to seO), as well as the toxic shock syndrome toxin 1 gene, and no SEA was detected in the retail breaded chicken samples analyzed by a reversed passive latex agglutination assay or by Western blotting. Hence, we evaluated the thermal resistance of two strains of SEA-producing S. aureus in a laboratory-scale preparation of precooked breaded chicken cutlets. The heat treatment employed in the manufacture determined the inactivation of S. aureus cells, but the preformed SEA remained active during product storage at 4 degrees C. The presence of the staphylococci and, in particular, of S. aureus in the retail breaded chicken products analyzed is a potential health risk for consumers since the pH and a(w) values of these kinds of products are favorable for S. aureus growth. The thermal process used during their manufacture can limit staphylococcal contamination but cannot eliminate preformed toxins.

  1. Comparative Genomics and Identification of an Enterotoxin-Bearing Pathogenicity Island, SEPI-1/SECI-1, in Staphylococcus epidermidis Pathogenic Strains.

    Science.gov (United States)

    Argemi, Xavier; Nanoukon, Chimène; Affolabi, Dissou; Keller, Daniel; Hansmann, Yves; Riegel, Philippe; Baba-Moussa, Lamine; Prévost, Gilles

    2018-02-25

    Staphylococcus epidermidis is a leading cause of nosocomial infections, majorly resistant to beta-lactam antibiotics, and may transfer several mobile genetic elements among the members of its own species, as well as to Staphylococcus aureus ; however, a genetic exchange from S. aureus to S. epidermidis remains controversial. We recently identified two pathogenic clinical strains of S. epidermidis that produce a staphylococcal enterotoxin C3-like (SEC) similar to that by S. aureus pathogenicity islands. This study aimed to determine the genetic environment of the SEC-coding sequence and to identify the mobile genetic elements. Whole-genome sequencing and annotation of the S. epidermidis strains were performed using Illumina technology and a bioinformatics pipeline for assembly, which provided evidence that the SEC-coding sequences were located in a composite pathogenicity island that was previously described in the S. epidermidis strain FRI909, called SePI-1/SeCI-1, with 83.8-89.7% nucleotide similarity. Various other plasmids were identified, particularly p_3_95 and p_4_95, which carry antibiotic resistance genes ( hsrA and dfrG , respectively), and share homologies with SAP085A and pUSA04-2-SUR11, two plasmids described in S. aureus . Eventually, one complete prophage was identified, ΦSE90, sharing 30 out of 52 coding sequences with the Acinetobacter phage vB_AbaM_IME200. Thus, the SePI-1/SeCI-1 pathogenicity island was identified in two pathogenic strains of S. epidermidis that produced a SEC enterotoxin causing septic shock. These findings suggest the existence of in vivo genetic exchange from S. aureus to S. epidermidis .

  2. Comparative Genomics and Identification of an Enterotoxin-Bearing Pathogenicity Island, SEPI-1/SECI-1, in Staphylococcus epidermidis Pathogenic Strains

    Directory of Open Access Journals (Sweden)

    Xavier Argemi

    2018-02-01

    Full Text Available Staphylococcus epidermidis is a leading cause of nosocomial infections, majorly resistant to beta-lactam antibiotics, and may transfer several mobile genetic elements among the members of its own species, as well as to Staphylococcus aureus; however, a genetic exchange from S. aureus to S. epidermidis remains controversial. We recently identified two pathogenic clinical strains of S. epidermidis that produce a staphylococcal enterotoxin C3-like (SEC similar to that by S. aureus pathogenicity islands. This study aimed to determine the genetic environment of the SEC-coding sequence and to identify the mobile genetic elements. Whole-genome sequencing and annotation of the S. epidermidis strains were performed using Illumina technology and a bioinformatics pipeline for assembly, which provided evidence that the SEC-coding sequences were located in a composite pathogenicity island that was previously described in the S. epidermidis strain FRI909, called SePI-1/SeCI-1, with 83.8–89.7% nucleotide similarity. Various other plasmids were identified, particularly p_3_95 and p_4_95, which carry antibiotic resistance genes (hsrA and dfrG, respectively, and share homologies with SAP085A and pUSA04-2-SUR11, two plasmids described in S. aureus. Eventually, one complete prophage was identified, ΦSE90, sharing 30 out of 52 coding sequences with the Acinetobacter phage vB_AbaM_IME200. Thus, the SePI-1/SeCI-1 pathogenicity island was identified in two pathogenic strains of S. epidermidis that produced a SEC enterotoxin causing septic shock. These findings suggest the existence of in vivo genetic exchange from S. aureus to S. epidermidis.

  3. Subinhibitory concentrations of thymol reduce enterotoxins A and B and alpha-hemolysin production in Staphylococcus aureus isolates.

    Directory of Open Access Journals (Sweden)

    Jiazhang Qiu

    Full Text Available BACKGROUND: Targeting bacterial virulence factors is now gaining interest as an alternative strategy to develop new types of anti-infective agents. It has been shown that thymol, when used at low concentrations, can inhibit the TSST-1 secretion in Staphylococcus aureus. However, there are no data on the effect of thymol on the production of other exotoxins (e.g., alpha-hemolysin and enterotoxins by S. aureus. METHODOLOGY/PRINCIPAL FINDINGS: Secretion of alpha-hemolysin, SEA and SEB in both methicillin-sensitive and methicillin-resistant S. aureus isolates cultured with graded subinhibitory concentrations of thymol was detected by immunoblot analysis. Hemolysin and tumor necrosis factor (TNF release assays were performed to elucidate the biological relevance of changes in alpha-hemolysin, SEA and SEB secretion induced by thymol. In addition, the influence of thymol on the transcription of hla, sea, and seb (the genes encoding alpha-hemolysin, SEA and SEB, respectively was analyzed by quantitative RT-PCR. Thymol inhibited transcription of hla, sea and seb in S. aureus, resulting in a reduction of alpha-hemolysin, SEA and SEB secretion and, thus, a reduction in hemolytic and TNF-inducing activities. CONCLUSIONS/SIGNIFICANCE: Subinhibitory concentrations of thymol decreased the production of alpha-hemolysin, SEA and SEB in both MSSA and MRSA in a dose-dependent manner. These data suggest that thymol may be useful for the treatment of S. aureus infections when used in combination with beta-lactams and glycopeptide antibiotics, which induce expression of alpha-hemolysin and enterotoxins at subinhibitory concentrations. Furthermore, the structure of thymol may potentially be used as a basic structure for development of drugs aimed against these bacterial virulence factors.

  4. Possible Application of Biotechnology to the Development of Biological Agents by Potential Enemies

    Science.gov (United States)

    1987-06-01

    7 beta chains Shigella dysenteriae type 1 plus other Inhibits 60S ribosome in eukaryotes -22- E. coli enterotoxin (heat labile) 2 subunits...toxin and those from E. colt because they do not act directly on intestinal cells. The same clinical response is obtained in monkeys following...there arc few good models in which to stud> the toxicity of the enterotoxins. Monkeys show many of the symptoms of toxic shock syndrome, however

  5. Nanodisc-based Co-immunoprecipitation for Mass Spectrometric Identification of Membrane-interacting Proteins

    DEFF Research Database (Denmark)

    Borch-Jensen, Jonas; Roepstorff, Peter; Møller-Jensen, Jakob

    2011-01-01

    enterotoxigenic Escherischia coli, GM1-nanodiscs were employed for co-immunoprecipitation. The B subunit of heat labile enterotoxin was identified as a specific interaction partner by mass spectrometry, thus demonstrating that nanodisc technology is useful for highly specific detection and identification...

  6. Coupling immunomagnetic separation on magnetic beads with matrix-assisted laser desorption ionization-time of flight mass spectrometry for detection of Staphylococcal Enterotoxin B

    Czech Academy of Sciences Publication Activity Database

    Schlosser, G.; Kačer, P.; Kuzma, Marek; Szilágyi, Z.; Sorrentino, Z.; Manzo, C.; Pizzano, R.; Malorni, L.; Poscfalvi, G.

    2007-01-01

    Roč. 73, č. 21 (2007), s. 6945-6952 ISSN 0099-2240 Grant - others:IT(IT) project SIFORTI (MIUR-CNR, Legge 449/97); XE(XE) 94.05.09.103, ARINCO 94.IT.16.028 Institutional research plan: CEZ:AV0Z50200510 Keywords : mass spectrometry * staphylococcal enterotoxin b * bacterial protein toxins Subject RIV: EE - Microbiology, Virology Impact factor: 4.004, year: 2007

  7. Prevalence of enterotoxin-encoding genes and antimicrobial resistance in coagulase-negative and coagulase-positive Staphylococcus isolates from black pudding

    Directory of Open Access Journals (Sweden)

    Tiane Martin de Moura

    2012-10-01

    Full Text Available INTRODUCTION: Staphylococcal species are pathogens that are responsible for outbreaks of foodborne diseases. The aim of this study was to investigate the prevalence of enterotoxin-genes and the antimicrobial resistance profile in staphylococcus coagulase-negative (CoNS and coagulasepositive (CoPS isolates from black pudding in southern Brazil. METHODS: Two hundred typical and atypical colonies from Baird-Parker agar were inoculated on mannitol salt agar. Eighty-two mannitol-positive staphylococci were submitted to conventional biochemical tests and antimicrobial susceptibility profiling. The presence of coagulase (coa and enterotoxin (se genes was investigated by polymerase chain reaction. RESULTS: The isolates were divided into 2 groups: 75.6% (62/82 were CoNS and 24.4% (20/82 were CoPS. The biochemical tests identified 9 species, of which Staphylococcus saprophyticus (37.8% and Staphylococcus carnosus (15.9% were the most prevalent. Antimicrobial susceptibility tests showed resistance phenotypes to antibiotics widely administered in humans, such as gentamicin, tetracycline, chloramphenicol, and erythromycin. The coa gene was detected in 19.5% (16/82 of the strains and 4 polymorphic DNA fragments were observed. Five CoNS isolates carrying the coa gene were submitted for 16S rRNA sequencing and 3 showed similarity with CoNS. Forty strains were positive for at least 1 enterotoxin-encoding gene, the genes most frequently detected were sea (28.6% and seb (27.5%. CONCLUSIONS: The presence of antimicrobial resistant and enterotoxin-encoding genes in staphylococci isolates from black pudding indicated that this fermented food may represent a potential health risk, since staphylococci present in food could cause foodborne diseases or be a possible route for the transfer of antimicrobial resistance to humans.

  8. Influence of holding temperature on the growth and survival of Salmonella spp. and Staphylococcus aureus and the production of staphylococcal enterotoxin in egg products.

    Science.gov (United States)

    Yang, S E; Yu, R C; Chou, C C

    2001-01-22

    In this study, growth and survival of Salmonella spp. and Staphylococcus aureus in steamed egg and scrambled egg held at 5, 18, 22, 37, 55 and 60 degrees C are investigated. The production of staphylococcal enterotoxin in steamed egg is also examined. Results reveal that Salmonella spp. and Staph. aureus in the egg products multiply best at 37 degrees C, followed closely by 22 and 18 degrees C. Neither pathogen showed growth in the egg products held at 5 degrees C. Initial inoculation dose, holding temperature and holding time affected the population of both organisms found in the egg products. Staphylococcal enterotoxin A (SEA) and B (SEB) are detected only in the egg products held at 37 or 22 degrees C. After holding at 37 degrees C for 36 h, scrambled egg inoculated with ca. 5.0 log cfu/g Staph. aureus contains the highest levels of SEA (> 64 ng/g) and SEB (> 64 ng/g). Although Salmonella spp. and Staph. aureus grow better in steamed eggs than in scrambled eggs, production of staphylococcal enterotoxin, in general, was higher in scrambled eggs than in steamed eggs. On the other hand, a repaid destruction of the test organisms in steamed eggs held at 60 degrees C was observed. Holding the steamed eggs at 60 degrees C, Salmonella spp. and Staph. aureus with an initial population of ca. 5.9 and 5.6 log cfu/g, respectively, reduced to a non-detectable level in 1 h.

  9. Prevalence and Genetic Characteristics of Staphylococcus aureus and Staphylococcus argenteus Isolates Harboring Panton-Valentine Leukocidin, Enterotoxins, and TSST-1 Genes from Food Handlers in Myanmar.

    Science.gov (United States)

    Aung, Meiji Soe; San, Thida; Aye, Mya Mya; Mya, San; Maw, Win Win; Zan, Khin Nyein; Htut, Wut Hmone Win; Kawaguchiya, Mitsuyo; Urushibara, Noriko; Kobayashi, Nobumichi

    2017-08-04

    Asymptomatic carriers of toxigenic Staphylococcus aureus are potential source of diseases, including food poisoning. Toxigenic potential and genetic traits of colonizing S. aureus were investigated for 563 healthy food handlers in Myanmar. Carriage of S. aureus was found in 110 individuals (19.5%), and a total of 144 S. aureus isolates were recovered from nasal cavities (110 isolates) and hands (34 isolates). Panton-Valentine leucocidin genes ( pvl ) were detected in 18 isolates (12.5%), among which 11 isolates were classified into coa -VIa, agr type III, and ST1930 (CC96) that had been also detected in pvl -positive clinical isolates in Myanmar. A pvl -positive, ST2250 nasal isolate was identified as S. argenteus , a novel coagulase-positive staphylococcus species. Toxic shock syndrome toxin-1 (TSST-1) gene was detected in five pvl -negative isolates. All of the 144 isolates harbored at least one of the 21 enterotoxin(-like) gene(s). The most prevalent enterotoxin(-like) gene was selw (98%), followed by selx (97%), sei (28%), sely (28%), sem (26%), sel (24%), and sea and sec (22% each). Considerable genetic diversity with five groups was detected for selw . The present study revealed the relatively high rate of pvl , as well as the wide distribution of enterotoxin(-like) genes among colonizing S. aureus in Myanmar.

  10. An alternative explanation for the occurrence of short circuit current increases in the small intestine following challenge by bacterial enterotoxins.

    Science.gov (United States)

    Lucas, M L

    2013-10-01

    Secretory diarrhoeal disease due to enterotoxins is thought to arise from the enhancement to pathologically high rates of normally occurring chloride ion and therefore fluid secretion from enterocytes. In support of this concept, many enterotoxins increase intestinal short-circuit current, regarded now as faithfully reflecting the increased chloride ion secretion. Contradicting this assumption, STa reduces absorption but does not cause secretion in vivo although short-circuit current is increased in vitro. There is therefore a mismatch between an assumed enterocyte mediated secretory event that should but does not cause net fluid secretion and an undoubtedly increased short-circuit current. It is proposed here that short-circuit current increases are not themselves secretory events but result from interrupted fluid absorption. A noteworthy feature of compounds that inhibit the increase in short-circuit current is that the majority are vasoactive, neuroactive or both. In general, vasodilator substances increase current. An alternative hypothesis for the origin of short-circuit current increases is that these result from reflex induction of electrogenic fluid absorption. This reflex enhances a compensatory response that is also present at a cellular level. An intestinal reflex is therefore proposed by which decreases in interstitial and intravascular volume or pressure within the intestine initiate an electrogenic fluid absorption mechanism that compensates for the loss of electrically neutral fluid absorption. This hypothesis would explain the apparently complex pharmacology of short-circuit current increases since many depressor substances have receptors in common with enterocytes and enteric nerves. The proposed alternative view of the origin of short-circuit current increases assumes that these do not represent chloride secretion from the enterocytes. This view may therefore aid the successful development of anti-diarrhoeal drugs to overcome a major cause of

  11. Attenuation of massive cytokine response to the staphylococcal enterotoxin B superantigen by the innate immunomodulatory protein lactoferrin

    Science.gov (United States)

    Hayworth, J L; Kasper, K J; Leon-Ponte, M; Herfst, C A; Yue, D; Brintnell, W C; Mazzuca, D M; Heinrichs, D E; Cairns, E; Madrenas, J; Hoskin, D W; McCormick, J K; Haeryfar, S M M

    2009-01-01

    Staphylococcal enterotoxin B (SEB) is a pyrogenic exotoxin and a potent superantigen which causes massive T cell activation and cytokine secretion, leading to profound immunosuppression and morbidity. The inhibition of SEB-induced responses is thus considered a goal in the management of certain types of staphylococcal infections. Lactoferrin (LF) is a multi-functional glycoprotein with both bacteriostatic and bactericidal activities. In addition, LF is known to have potent immunomodulatory properties. Given the anti-microbial and anti-inflammatory properties of this protein, we hypothesized that LF can modulate T cell responses to SEB. Here, we report that bovine LF (bLF) was indeed able to attenuate SEB-induced proliferation, interleukin-2 production and CD25 expression by human leucocyte antigen (HLA)-DR4 transgenic mouse T cells. This inhibition was not due to bLF's iron-binding capacity, and could be mimicked by the bLF-derived peptide lactoferricin. Cytokine secretion by an engineered SEB-responsive human Jurkat T cell line and by peripheral blood mononuclear cells from healthy donors was also inhibited by bLF. These findings reveal a previously unrecognized property of LF in modulation of SEB-triggered immune activation and suggest a therapeutic potential for this naturally occurring protein during toxic shock syndrome. PMID:19659771

  12. Ultrasensitive electrochemical immunoassay of staphylococcal enterotoxin B in food using enzyme-nanosilica-doped carbon nanotubes for signal amplification.

    Science.gov (United States)

    Tang, Dianping; Tang, Juan; Su, Biling; Chen, Guonan

    2010-10-27

    A new sandwich-type electrochemical immunoassay for ultrasensitive detection of staphylococcal enterotoxin B (SEB) in food was developed using horseradish peroxidase-nanosilica-doped multiwalled carbon nanotubes (HRPSiCNTs) for signal amplification. Rabbit polyclonal anti-SEB antibodies immobilized on the screen-printed carbon electrode (SPCE) and covalently bound to the HRPSiCNTs were used as capture antibodies and detection antibodies, respectively. In the presence of SEB analyte, the sandwich-type immunocomplex could be formed between the immobilized anti-SEB on the SPCE and anti-SEB-labeled HRPSiCNTs, and the carried HRP could catalyze the electrochemical reduction of H2O2 with the help of thionine. The high content of HRP in the HRPSiCNTs could greatly amplify the electrochemical signal. Under optimal conditions, the reduction current increased with the increase of SEB in the sample, and exhibited a dynamic range of 0.05-15 ng/mL with a low detection limit (LOD) of 10 pg/mL SEB (at 3σ). Intra- and interassay coefficients of variation were below 10%. In addition, the assay was evaluated with SEB spiked samples including watermelon juice, soymilk, apple juice, and pork food, receiving excellent correlation with results from commercially available enzyme-linked immunosorbent assay (ELISA).

  13. Prophage-Encoded Staphylococcal Enterotoxin A: Regulation of Production in Staphylococcus aureus Strains Representing Different Sea Regions

    Directory of Open Access Journals (Sweden)

    Nikoleta Zeaki

    2015-12-01

    Full Text Available The present study investigates the nature of the link between the staphylococcal enterotoxin A (SEA gene and the lifecycle of Siphoviridae bacteriophages, including the origin of strain variation regarding SEA production after prophage induction. Five strains representing three different genetic lines of the sea region were studied under optimal and prophage-induced growth conditions and the Siphoviridae lifecycle was followed through the phage replicative form copies and transcripts of the lysogenic repressor, cro. The role of SOS response on prophage induction was addressed through recA transcription in a recA-disruption mutant. Prophage induction was found to increase the abundance of the phage replicative form, the sea gene copies and transcripts and enhance SEA production. Sequence analysis of the sea regions revealed that observed strain variances were related to strain capacity for prophage induction, rather than sequence differences in the sea region. The impact of SOS response activation on the phage lifecycle was demonstrated by the absence of phage replicative form copies in the recA-disruption mutant after prophage induction. From this study it emerges that all aspects of SEA-producing strain, the Siphoviridae phage and the food environment must be considered when evaluating SEA-related hazards.

  14. Immunochemical and PCR analysis of Staphylococcus aureus Enterotoxin B (SEB in milk and fruit juices collected in Lahore, Pakistan

    Directory of Open Access Journals (Sweden)

    Dar Nadia

    2013-01-01

    Full Text Available Enterotoxins secreted by S. aureus are known as a food-poisoning agent that is associated with various gastro-intestinal pathological conditions. In this study, a one-step immunodetection method was devised for routine checking of SEB in milk and fruit juices available locally. Antibodies against recombinant SEB were raised, purified, and cross reactivity was checked against clinically important bacteria (Shigella flexneri, Streptococci, Salmonella typhi, Klebsiella and Bacillus subtilis. Purified anti-SEB antibodies were conjugated with gold nanoparticles (Ab-GNPs for direct detection of SEB in samples. SEB (33%, 4.76% and 15% was found in non-sterilized milk (118, sterilized milk (42 and juices (60, respectively. Coagulase, MSA tests and PCR amplification of 725 bp of the SEB gene confirmed the presence of S. aureus in the collected samples positive for SEB. Immunoassay is easy, reliable and less time consuming and will be helpful to detect the SEB in food samples at local level.

  15. Effect of different ionizing radiation doses and dose rates, using Cobalt-60 and electrons beam sources, on the staphylococcal enterotoxin inoculated in mechanically deboned chicken meat

    Energy Technology Data Exchange (ETDEWEB)

    Pomarico Neto, Walter; Brito, Poliana de Paula; Azevedo, Heliana de; Roque, Claudio Vitor; Fukuma, Henrique Takuji, E-mail: pbrito@cnen.gov.br, E-mail: hazevedo@cnen.gov.br, E-mail: cvroque@cnen.gov.br, E-mail: htfukuma@cnen.gov.br [Brazilian Nuclear Energy Commission (LAPOC/CNEN), Pocos de Caldas, MG (Brazil); Kodama, Yasko, E-mail: ykodama@ipen.br [Nuclear and Energy Research Institute (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Miya, Norma Terugo Nago; Pereira, Jose Luiz, E-mail: miya@fea.unicamp.br, E-mail: pereira@fea.unicamp.br [Campinas State University (UNICAMP), SP (Brazil). Dept. of Food Sciences

    2011-07-01

    The purpose of food irradiation is the destruction of present pathogenic microorganisms and the increase of shelf life of foods. To achieve this process, the source of cobalt-60 and the electron accelerator can be used. The mechanically deboned chicken meat (MDCM) is used for the production of traditional meat products, and it may come to present pathogenic microorganisms such as staphylococcus aureus, a bacterium that produces enterotoxin, which causes food poisoning. The objective of this study is to analyze the effect of ionizing irradiation with different doses and dose rates, deriving from different radiation sources, on staphylococcal enterotoxin type B (SEB) in the MDCM. 50 g samples of MDCM were prepared in a batch of 6 kg of MDCM. The samples were contaminated, with the exception of the control, with SEB in amounts of about 100 ng. Then they were conditioned in a transparent bag made of low density polyethylene, frozen at -18{+-}1 deg C overnight and irradiated in these conditions with doses of 0.0 kGy (control), 1.5 kGy and 3.0 kGy, and with three different dose rates, both in the Cobalt-60 and the electron accelerator. The experiments were conducted in quintuplicate. The SEB extraction from the MDCM was performed according to the protocol recommended by the manufacturer of the kit VIDAS Staph Enterotoxin II (bioMerrieux). The principle of mass balance was used to determine the actual amount of SEB removed by irradiation. The treatment that presented the best results was the one with a dose of 1.5 kGy, high dose rate of the electron accelerator. (author)

  16. Effect of different ionizing radiation doses and dose rates, using Cobalt-60 and electrons beam sources, on the staphylococcal enterotoxin inoculated in mechanically deboned chicken meat

    International Nuclear Information System (INIS)

    Pomarico Neto, Walter; Brito, Poliana de Paula; Azevedo, Heliana de; Roque, Claudio Vitor; Fukuma, Henrique Takuji; Kodama, Yasko; Miya, Norma Terugo Nago; Pereira, Jose Luiz

    2011-01-01

    The purpose of food irradiation is the destruction of present pathogenic microorganisms and the increase of shelf life of foods. To achieve this process, the source of cobalt-60 and the electron accelerator can be used. The mechanically deboned chicken meat (MDCM) is used for the production of traditional meat products, and it may come to present pathogenic microorganisms such as staphylococcus aureus, a bacterium that produces enterotoxin, which causes food poisoning. The objective of this study is to analyze the effect of ionizing irradiation with different doses and dose rates, deriving from different radiation sources, on staphylococcal enterotoxin type B (SEB) in the MDCM. 50 g samples of MDCM were prepared in a batch of 6 kg of MDCM. The samples were contaminated, with the exception of the control, with SEB in amounts of about 100 ng. Then they were conditioned in a transparent bag made of low density polyethylene, frozen at -18±1 deg C overnight and irradiated in these conditions with doses of 0.0 kGy (control), 1.5 kGy and 3.0 kGy, and with three different dose rates, both in the Cobalt-60 and the electron accelerator. The experiments were conducted in quintuplicate. The SEB extraction from the MDCM was performed according to the protocol recommended by the manufacturer of the kit VIDAS Staph Enterotoxin II (bioMerrieux). The principle of mass balance was used to determine the actual amount of SEB removed by irradiation. The treatment that presented the best results was the one with a dose of 1.5 kGy, high dose rate of the electron accelerator. (author)

  17. Multiple-Strain Approach and Probabilistic Modeling of Consumer Habits in Quantitative Microbial Risk Assessment: A Quantitative Assessment of Exposure to Staphylococcal Enterotoxin A in Raw Milk.

    Science.gov (United States)

    Crotta, Matteo; Rizzi, Rita; Varisco, Giorgio; Daminelli, Paolo; Cunico, Elena Cosciani; Luini, Mario; Graber, Hans Ulrich; Paterlini, Franco; Guitian, Javier

    2016-03-01

    Quantitative microbial risk assessment (QMRA) models are extensively applied to inform management of a broad range of food safety risks. Inevitably, QMRA modeling involves an element of simplification of the biological process of interest. Two features that are frequently simplified or disregarded are the pathogenicity of multiple strains of a single pathogen and consumer behavior at the household level. In this study, we developed a QMRA model with a multiple-strain approach and a consumer phase module (CPM) based on uncertainty distributions fitted from field data. We modeled exposure to staphylococcal enterotoxin A in raw milk in Lombardy; a specific enterotoxin production module was thus included. The model is adaptable and could be used to assess the risk related to other pathogens in raw milk as well as other staphylococcal enterotoxins. The multiplestrain approach, implemented as a multinomial process, allowed the inclusion of variability and uncertainty with regard to pathogenicity at the bacterial level. Data from 301 questionnaires submitted to raw milk consumers were used to obtain uncertainty distributions for the CPM. The distributions were modeled to be easily updatable with further data or evidence. The sources of uncertainty due to the multiple-strain approach and the CPM were identified, and their impact on the output was assessed by comparing specific scenarios to the baseline. When the distributions reflecting the uncertainty in consumer behavior were fixed to the 95th percentile, the risk of exposure increased up to 160 times. This reflects the importance of taking into consideration the diversity of consumers' habits at the household level and the impact that the lack of knowledge about variables in the CPM can have on the final QMRA estimates. The multiple-strain approach lends itself to use in other food matrices besides raw milk and allows the model to better capture the complexity of the real world and to be capable of geographical

  18. Identification of novel Clostridium perfringens type E strains that carry an iota toxin plasmid with a functional enterotoxin gene.

    Directory of Open Access Journals (Sweden)

    Kazuaki Miyamoto

    Full Text Available Clostridium perfringens enterotoxin (CPE is a major virulence factor for human gastrointestinal diseases, such as food poisoning and antibiotic associated diarrhea. The CPE-encoding gene (cpe can be chromosomal or plasmid-borne. Recent development of conventional PCR cpe-genotyping assays makes it possible to identify cpe location (chromosomal or plasmid in type A isolates. Initial studies for developing cpe genotyping assays indicated that all cpe-positive strains isolated from sickened patients were typable by cpe-genotypes, but surveys of C. perfringens environmental strains or strains from feces of healthy people suggested that this assay might not be useful for some cpe-carrying type A isolates. In the current study, a pulsed-field gel electrophoresis Southern blot assay showed that four cpe-genotype untypable isolates carried their cpe gene on a plasmid of ∼65 kb. Complete sequence analysis of the ∼65 kb variant cpe-carrying plasmid revealed no intact IS elements and a disrupted cytosine methyltransferase (dcm gene. More importantly, this plasmid contains a conjugative transfer region, a variant cpe gene and variant iota toxin genes. The toxin genes encoded by this plasmid are expressed based upon the results of RT-PCR assays. The ∼65 kb plasmid is closely related to the pCPF4969 cpe plasmid of type A isolates. MLST analyses indicated these isolates belong to a unique cluster of C. perfringens. Overall, these isolates carrying a variant functional cpe gene and iota toxin genes represent unique type E strains.

  19. Review Over a 3-Year Period of European Union Proficiency Tests for Detection of Staphylococcal Enterotoxins in Food Matrices.

    Science.gov (United States)

    Nia, Yacine; Mutel, Isabelle; Assere, Adrien; Lombard, Bertrand; Auvray, Frederic; Hennekinne, Jacques-Antoine

    2016-04-13

    Staphylococcal food poisoning outbreaks are a major cause of foodborne illnesses in Europe and their notifications have been mandatory since 2005. Even though the European regulation on microbiological criteria for food defines a criterion on staphylococcal enterotoxin (SE) only in cheese and dairy products, European Food Safety Authority (EFSA) data reported that various types of food matrices are involved in staphylococcal food poisoning outbreaks. The European Screening Method (ESM) of European Union Reference Laboratory for Coagulase Positive Staphylococci (EURL CPS) was validated in 2011 for SE detection in food matrices and is currently the official method used for screening purposes in Europe. In this context, EURLCPS is annually organizing Inter-Laboratory Proficiency Testing Trials (ILPT) to evaluate the competency of the European countries' National Reference Laboratories (NRLs) to analyse SE content in food matrices. A total of 31 NRLs representing 93% of European countries participated in these ILPTs. Eight food matrices were used for ILPT over the period 2013-2015, including cheese, freeze-dried cheese, tuna, mackerel, roasted chicken, ready-to-eat food, milk, and pastry. Food samples were spiked with four SE types (i.e., SEA, SEC, SED, and SEE) at various concentrations. Homogeneity and stability studies showed that ILPT samples were both homogeneous and stable. The analysis of results obtained by participants for a total of 155 blank and 620 contaminated samples allowed for evaluation of trueness (>98%) and specificity (100%) of ESM. Further to the validation study of ESM carried out in 2011, these three ILPTs allowed for the assessment of the proficiency of the NRL network and the performance of ESM on a large variety of food matrices and samples. The ILPT design presented here will be helpful for the organization of ILPT on SE detection by NRLs or other expert laboratories.

  20. Review Over a 3-Year Period of European Union Proficiency Tests for Detection of Staphylococcal Enterotoxins in Food Matrices

    Directory of Open Access Journals (Sweden)

    Yacine Nia

    2016-04-01

    Full Text Available Staphylococcal food poisoning outbreaks are a major cause of foodborne illnesses in Europe and their notifications have been mandatory since 2005. Even though the European regulation on microbiological criteria for food defines a criterion on staphylococcal enterotoxin (SE only in cheese and dairy products, European Food Safety Authority (EFSA data reported that various types of food matrices are involved in staphylococcal food poisoning outbreaks. The European Screening Method (ESM of European Union Reference Laboratory for Coagulase Positive Staphylococci (EURL CPS was validated in 2011 for SE detection in food matrices and is currently the official method used for screening purposes in Europe. In this context, EURLCPS is annually organizing Inter-Laboratory Proficiency Testing Trials (ILPT to evaluate the competency of the European countries’ National Reference Laboratories (NRLs to analyse SE content in food matrices. A total of 31 NRLs representing 93% of European countries participated in these ILPTs. Eight food matrices were used for ILPT over the period 2013–2015, including cheese, freeze-dried cheese, tuna, mackerel, roasted chicken, ready-to-eat food, milk, and pastry. Food samples were spiked with four SE types (i.e., SEA, SEC, SED, and SEE at various concentrations. Homogeneity and stability studies showed that ILPT samples were both homogeneous and stable. The analysis of results obtained by participants for a total of 155 blank and 620 contaminated samples allowed for evaluation of trueness (>98% and specificity (100% of ESM. Further to the validation study of ESM carried out in 2011, these three ILPTs allowed for the assessment of the proficiency of the NRL network and the performance of ESM on a large variety of food matrices and samples. The ILPT design presented here will be helpful for the organization of ILPT on SE detection by NRLs or other expert laboratories.

  1. Influence of carvacrol and thymol on the physiological attributes, enterotoxin production and surface characteristics of Staphylococcus aureus strains isolated from foods

    Directory of Open Access Journals (Sweden)

    E.L. Souza

    2013-01-01

    Full Text Available This study evaluated the influence of the phenolic compounds carvacrol (CAR and thymol (THY on some physiological characteristics and on the modulation of the secretion of some staphylococcal virulence factors, that is, coagulase and enterotoxin. This study also investigated possible mechanisms for the establishment of the anti-staphylococcal activity of these compounds. Sublethal concentrations (0.3 and 0.15 µL/mL of CAR and THY inhibited the activity of the enzymes coagulase and lipase and led to a decrease in salt tolerance. At the tested sublethal concentrations, both CAR and THY led to a total suppression of enterotoxin production. The loss of a 260-nm-absorbing material and an efflux of potassium ions occurred immediately after the addition of CAR and THY at 0.6 and 1.2 µL/mL and increased up to 120 min of exposure. Electron microscopy of cells exposed to CAR and THY (0.6 µL/mL revealed that individual cells appeared to be deformed, with projections of cellular material. The observations of leakage of cellular material and an altered cell surface suggest that gross damage to a cell's cytoplasmic membrane, which results in a disruption in protein secretion, could be responsible for the anti-staphylococcal properties of CAR and THY.

  2. Structure of the superantigen staphylococcal enterotoxin B in complex with TCR and peptide-MHC demonstrates absence of TCR-peptide contacts.

    Science.gov (United States)

    Rödström, Karin E J; Elbing, Karin; Lindkvist-Petersson, Karin

    2014-08-15

    Superantigens are immune-stimulatory toxins produced by Staphylococcus aureus, which are able to interact with host immune receptors to induce a massive release of cytokines, causing toxic shock syndrome and possibly death. In this article, we present the x-ray structure of staphylococcal enterotoxin B (SEB) in complex with its receptors, the TCR and MHC class II, forming a ternary complex. The structure, in combination with functional analyses, clearly shows how SEB adopts a wedge-like position when binding to the β-chain of TCR, allowing for an interaction between the α-chain of TCR and MHC. Furthermore, the binding mode also circumvents contact between TCR and the peptide presented by MHC, which enables SEB to initiate a peptide-independent activation of T cells. Copyright © 2014 by The American Association of Immunologists, Inc.

  3. Antimicrobial resistance and detection of the mecA gene besides enterotoxin-encoding genes among coagulase-negative Staphylococci isolated from clam meat of Anomalocardia brasiliana.

    Science.gov (United States)

    Batista, Jacqueline Ellen Camelo; Ferreira, Ewerton Lucena; Nascimento, Danielle Cristina de Oliveira; Ventura, Roberta Ferreira; de Oliveira, Wagner Luis Mendes; Leal, Nilma Cintra; Lima-Filho, José Vitor

    2013-12-01

    The marine clam Anomalocardia brasiliana is a candidate as a sentinel animal to monitor the contamination levels of coliforms in shellfish-harvesting areas of Brazil's northeastern region. The aim of the present study was to search enterotoxin-encoding genes plus the mecA gene among coagulase-negative staphylococci (CNS) isolates from shellfish meats of A. brasiliana. The specimen clam (n=48; 40 clams per sample) was collected during low tide in the bay area of Mangue Seco from April through June 2009, and random samples of chilled and frozen shelled clam meat (n=33; 250 g per sample) were obtained from retail shops from January through March 2012. Seventy-nine CNS isolates were identified, including Staphylococcus xylosus, S. cohnii spp. urealyticus, S. sciuri, and S. lentus. A high percentage of isolates resistant to erythromycin (58.5%), penicillin (51.2%), and tetracycline (43.9%), and the fluoroquinolones levofloxacin (39%) and ciprofloxacin (34.1%) were recorded from those environmental samples. Isolates from retail shops were particularly resistant to oxacillin (55.3%) and penicillin (36.8%). All CNS resistant to oxacillin and/or cefoxitin were positive for the presence of the mecA gene, but phenotypically susceptible to vancomycin. Also, the enterotoxin-encoding genes seg and seh were detected through multiplex-polymerase chain reaction in 77.7% and 88.8% of the isolates from environmental samples, versus 90.5% and 100% of the isolates from retail shops, respectively. The data reveal the risk to public health due to consuming raw or undercooked shellfish containing enterotoxigenic plus methicillin-resistant CNS.

  4. The fecal presence of enterotoxin and F4 genes as an indicator of efficacy of treatment with colistin sulfate in pigs.

    Science.gov (United States)

    Rhouma, Mohamed; Fairbrother, John Morris; Thériault, William; Beaudry, Francis; Bergeron, Nadia; Laurent-Lewandowski, Sylvette; Letellier, Ann

    2017-01-05

    Enterotoxigenic Escherichia coli (ETEC) strains producing multiple enterotoxins are important causes of post-weaning diarrhea (PWD) in pigs. The aim of the present study was to investigate the fecal presence of ETEC enterotoxin as well as F4 and F18 genes as an indicator of colistin sulfate (CS) efficacy for treatment of PWD in pigs. Forty-eight piglets were weaned at the age of 21 days, and were divided into four groups: challenged treated, challenged untreated, unchallenged treated, and unchallenged untreated. Challenge was performed using 10 9  CFU of an ETEC: F4 strain, and treatment was conducted using oral CS at the dose of 50,000 IU/kg. The fecal presence of genes encoding for STa, STb, LT, F4 and F18 was detected using PCR. The PCR amplification of ETEC virulence genes showed that nearly 100% of pigs excreted genes encoding for STa and STb toxins in the feces before the challenge. These genes, in the absence of the gene encoding F4, were considered as a marker for F4-negative ETEC. One day after ETEC: F4 oral challenge pigs in the two challenged groups excreted the genes encoding LT and F4 in the feces. These genes were considered as a marker for F4-positive ETEC. However, the gene encoding F18 was not detected in any fecal samples of the 4 groups throughout the experiment. After only 3 days of successive oral treatment with CS, a significant reduction in both the F4-positive and negative ETEC populations was observed in the challenged treated group compared to the challenged untreated group (p F4-positive and F4-negative ETEC in pigs. However, CS clinical efficiency was correlated with non-detection of F4-positive ETEC in the feces. Furthermore the fecal presence of F4-negative ETEC was not associated with clinical symptoms of post-weaning diarrhea in pigs.

  5. Molecular and epidemiological characterization of staphylococcal foodborne outbreak of Staphylococcus aureus harboring seg, sei, sem, sen, seo, and selu genes without production of classical enterotoxins.

    Science.gov (United States)

    Umeda, Kaoru; Nakamura, Hiromi; Yamamoto, Kaori; Nishina, Nobuko; Yasufuku, Kiyoshi; Hirai, Yuki; Hirayama, Teruo; Goto, Kaoru; Hase, Atsushi; Ogasawara, Jun

    2017-09-01

    Staphylococcal food poisoning is the result of consumption of food contaminated with staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus. To date, 23 SEs and SE-like enterotoxins (SEls) have been described in the literature. They are divided into classical SEs (SEA-SEE) and new SE/SEls (SEG-SElX). Some have proved to be foodborne-inducible, but others remain unidentified. In May 2016, at an elderly group home in Osaka city, Japan, an outbreak from foodborne pathogens occurred among lunch party participants. Within 2h 30min to 4h 40min, 15 of 53 participants presented gastrointestinal symptoms of vomiting, diarrhea, and nausea. A subsequent laboratory investigation detected S. aureus from most stool samples from patients, several left-over food items, a kitchen swab, and hand swabs from two food handlers. Classical SEs was not detected from S. aureus isolates or left-over food items. From examination for the presence of SE/SEl genes of 20 kinds by PCR, seg, sei, sem, sen, seo, and selu genes were detected in almost all isolates. These isolates exhibited identical or closely related types by coagulase type (type VII), Sma I digested pulsed-field gel electrophoresis analysis and multi-locus sequence typing (MLST-CC45 lineage). These results suggest that the foodborne outbreak was caused by S. aureus harboring seg, sei, sem, sen, seo, and selu genes without production of classical SEs. Additionally, some S. aureus isolates from human nasal swabs and healthy human feces harboring seg, sei, sem, sen, seo, and selu genes without production of classical SEs were classified into CC45 lineage using MLST. These findings suggest new SE/SEls as a potential cause of foodborne outbreaks. Copyright © 2017. Published by Elsevier B.V.

  6. Positive Regulation of Staphylococcal Enterotoxin H by Rot (Repressor of Toxin) Protein and Its Importance in Clonal Complex 81 Subtype 1 Lineage-Related Food Poisoning.

    Science.gov (United States)

    Sato'o, Yusuke; Hisatsune, Junzo; Nagasako, Yuria; Ono, Hisaya K; Omoe, Katsuhiko; Sugai, Motoyuki

    2015-11-01

    We previously demonstrated the clonal complex 81 (CC81) subtype 1 lineage is the major staphylococcal food poisoning (SFP)-associated lineage in Japan (Y. Sato'o et al., J Clin Microbiol 52:2637-2640, 2014, http://dx.doi.org/10.1128/JCM.00661-14). Strains of this lineage produce staphylococcal enterotoxin H (SEH) in addition to SEA. However, an evaluation of the risk for the recently reported SEH has not been sufficiently conducted. We first searched for staphylococcal enterotoxin (SE) genes and SE proteins in milk samples that caused a large SFP outbreak in Japan. Only SEA and SEH were detected, while there were several SE genes detected in the samples. We next designed an experimental model using a meat product to assess the productivity of SEs and found that only SEA and SEH were detectably produced in situ. Therefore, we investigated the regulation of SEH production using a CC81 subtype 1 isolate. Through mutant analysis of global regulators, we found the repressor of toxin (Rot) functioned oppositely as a stimulator of SEH production. SEA production was not affected by Rot. seh mRNA expression correlated with rot both in media and on the meat product, and the Rot protein was shown to directly bind to the seh promoter. The seh promoter sequence was predicted to form a loop structure and to hide the RNA polymerase binding sequences. We propose Rot binds to the promoter sequence of seh and unfolds the secondary structure that may lead the RNA polymerase to bind the promoter, and then seh mRNA transcription begins. This alternative Rot regulation for SEH may contribute to sufficient toxin production by the CC81 subtype 1 lineage in foods to induce SFP. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  7. [The staphylococcal enterotoxin burden determines the ultrastructure of ciliated epithelia and inflammatory changes in maxillary sinus mucosa of rabbits].

    Science.gov (United States)

    Wei, Hongqi; Zhu, Zhengwen; Cao, Zhongsheng; Liu, Zhiyong; Wu, Xiaofan; Yuan, Hui

    2014-12-01

    To investigate the ultrastructure of ciliated epithelia and inflammatory changes upon repeated exposure to staphylococcal enterotoxin A (SEA) of different concentrations in the maxillary sinus mucosa of rabbits. The rabbits were randomly divided into 2 groups (24 rabbits per group): low-dose SEA group and high-dose SEA group. The low-dose SEA group and high-dose SEA group received daily injections of 0.6 ng of SEA (2 ml) and 60 ng of SEA (2 ml) into the left maxillary sinus of rabbits for 28 days, respectively. Concurrent treatment of the right maxillary sinus with normal saline was used as control. Six rabbits chosen randomly in two groups were examined by computed tomography (CT) scans and then sacrificed to obtain the sinus mucosa from the two-side of maxillary sinuses for histological assessment on days 3, 7, 14 and 28. To characterize the inflammatory changes of the sinus mucosa examined using light microscope, hematoxylin and eosin (HE) and toluidine blue staining was performed. Scanning and transmission electron microscopy were performed to observe ultrastructure of ciliated epithelia in the maxillary sinus mucosa. SPSS 13.0 software was used to analyze the data. On days 14 and 28, CT images showed opacification of the left maxillary sinus in the high-dose SEA group. The percentage of epithelial disruption was (22.73 ± 5.72) % and (30.79 ± 4.30)% in the high-dose SEA group respectively, and were significantly greater than those in the low-dose SEA group (5.12% ± 1.98% and 5.38% ± 1.64%, q value was 10.079 and 19.132) and control group (4.08% ± 1.29% and 4.81% ± 1.62%, q value was 11.016 and 19.592, respectively, all P microscope, loss of cilia was observed, a few compound cilia and cytoplasmic protrusion were found, an obvious stretching of the endoplasmic reticulum and an obvious turgescence of the mitochondria was also observed. However, in the low-dose SEA group on days 14 and 28, CT scan of the left maxillary sinus showed transparency; light

  8. Genotypic and Phenotypic Characterization of Enterotoxigenic Escherichia coli Strains Isolated from Peruvian Children

    Science.gov (United States)

    2010-09-01

    adhesion, ETEC elabo- rates one or both of two enterotoxins: heat-labile toxin (LT), a protein multimer which shares many features with cholera toxin...pathogens (Shigella, Salmonella. Vibrio , Campylo/Jacter, Gi- ardia Iamblia, Cryptosporidium, and rotavirus) by conventional methods. Five lactose...studies performed in developing coun- tries: 38% in children ɚ years of age in Nicaragua (19), 33% in children ə year of age in Mexico (6), and 18% in

  9. Simulation of carbohydrate-protein interactions : Computer-aided design of a second generation GM1 mimic

    NARCIS (Netherlands)

    Bernardi, A; Galgano, M; Belvisi, L; Colombo, G

    The oligosaccharide of ganglioside GM1 [Gal beta1-3GalNAc beta1-4(NeuAc alpha2-3)Gal beta1-4Glc beta1-1Cer] is the cellular target of two bacterial enterotoxins: the cholera toxin (CT) and the heat-labile toxin of E.coli (LT). We recently reported that the pseudosaccharide 2[Gal beta1-3GalNAc

  10. Assessment of Enterotoxin Production and Cross-Contamination of Staphylococcus aureus between Food Processing Materials and Ready-To-Eat Cooked Fish Paste.

    Science.gov (United States)

    Tango, Charles Nkufi; Hong, Sung-Sam; Wang, Jun; Oh, Deog-Hwan

    2015-12-01

    This study evaluated Staphylococcus aureus growth and subsequent staphylococcal enterotoxin A production in tryptone soy broth and on ready-to-eat cooked fish paste at 12 to 37 °C, as well as cross-contamination between stainless steel, polyethylene, and latex glove at room temperature. A model was developed using Barany and Roberts's growth model, which satisfactorily described the suitable growth of S. aureus with R(2)-adj from 0.94 to 0.99. Except at 12 °C, S. aureus cells in TSB presented a lag time lower (14.64 to 1.65 h), grew faster (0.08 to 0.31 log CFU/h) and produced SEA at lower cell density levels (5.65 to 6.44 log CFU/mL) compare to those inoculated on cooked fish paste with data of 16.920 to 1.985 h, 0.02 to 0.23 log CFU/h, and 6.19 to 7.11 log CFU/g, respectively. Staphylococcal enterotoxin type A (SEA) visual immunoassay test showed that primary SEA detection varied considerably among different storage temperature degrees and media. For example, it occurred only during exponential phase at 30 and 37 °C in TSB, but in cooked fish paste it took place at late exponential phase of S. aureus growth at 20 and 25 °C. The SEA detection test was negative on presence of S. aureus on cooked fish paste stored at 12 and 15 °C, although cell density reached level of 6.12 log CFU/g at 15 °C. Cross-contamination expressed as transfer rate of S. aureus from polyethylene surface to cooked fish paste surface was slower than that observed with steel surface to cooked fish paste under same conditions. These results provide helpful information for controlling S. aureus growth, SEA production and cross-contamination during processing of cooked fish paste. © 2015 Institute of Food Technologists®

  11. Characteristics of enterotoxin distribution, hemolysis, lecithinase, and starch hydrolysis of Bacillus cereus isolated from infant formulas and ready-to-eat foods.

    Science.gov (United States)

    Hwang, Ji-Yeon; Park, Jong-Hyun

    2015-03-01

    Bacillus cereus is a ubiquitous environmental microbe implicated as a main cause of food poisoning with various symptoms, depending on the strain type and the isolation source. In this study, the potential virulence factors and biochemical properties of B. cereus isolated from infant formulas and ready-to-eat (RTE) foods were analyzed and compared. A total of 347 B. cereus strains were isolated and identified from 687 infant food formulas and RTE food samples. All the isolates had one or more enterotoxin genes, and one-half of the strains had all 3 enterotoxin genes (hbl, nhe, and cytK) that are involved in food poisoning in humans. Here, all the 3 genes were detected in 50% of the B. cereus isolates from RTE foods and only 14% of the isolates were identified from infant formulas. The latter harbored low cytK and bceT, and very low hbl genes. Most B. cereus isolates possessed the hemolysis gene, but not the ces gene. The infant formula isolates showed stronger hemolysis activity than the other isolates. In addition, 26% of the total isolates showed low lecithinase activities and 10% showed high lecithinase activities. A greater number of isolates from the infant formula showed high lecithinase activity than those from the RTE foods. Approximately 83% of the isolates were positive and 17% were negative for starch hydrolysis. Over 90% of the RTE food isolates and only 35% of the infant formula isolates were positive for starch hydrolysis. However, all the strains possessed nhe, but their harboring patterns of hbl and cytK were significantly different. Most starch-hydrolyzing strains possessed hbl, but only 23% nonstarch-hydrolyzing isolates possessed this gene. Moreover, very low nonstarch hydrolyzing strains harbored cytK. Most nonstarch-hydrolyzing isolates showed high lecithinase and strong hemolysis activities, and very low hbl and cytK harboring. In summary, most infant formula isolates showed stronger hemolysis and higher lecithinase activities with lower

  12. A highly sensitive fluorescence resonance energy transfer aptasensor for staphylococcal enterotoxin B detection based on exonuclease-catalyzed target recycling strategy

    International Nuclear Information System (INIS)

    Wu, Shijia; Duan, Nuo; Ma, Xiaoyuan; Xia, Yu; Wang, Hongxin; Wang, Zhouping

    2013-01-01

    Graphical abstract: -- Highlights: •An ultrasensitive FRET aptasensor was developed for staphylococcal enterotoxin B determination. •SEB was recognized by SEB aptamer with high affinity and specificity. •The Mn 2+ doped NaYF 4 :Yb/Er UCNPs used as donor to quencher dye (BHQ 3 ) in new FRET. •The fluorescence intensity was prominently amplified using an exonuclease-catalyzed target recycling strategy. -- Abstract: An ultrasensitive fluorescence resonance energy transfer (FRET) bioassay was developed to detect staphylococcal enterotoxin B (SEB), a low molecular exotoxin, using an aptamer-affinity method coupled with upconversion nanoparticles (UCNPs)-sensing, and the fluorescence intensity was prominently enhanced using an exonuclease-catalyzed target recycling strategy. To construct this aptasensor, both fluorescence donor probes (complementary DNA 1 –UCNPs) and fluorescence quencher probes (complementary DNA 2 –Black Hole Quencher 3 (BHQ 3 )) were hybridized to an SEB aptamer, and double-strand oligonucleotides were fabricated, which quenched the fluorescence of the UCNPs via FRET. The formation of an aptamer–SEB complex in the presence of the SEB analyte resulted in not only the dissociation of aptamer from the double-strand DNA but also both the disruption of the FRET system and the restoration of the UCNPs fluorescence. In addition, the SEB was liberated from the aptamer–SEB complex using exonuclease I, an exonuclease specific to single-stranded DNA, for analyte recycling by selectively digesting a particular DNA (SEB aptamer). Based on this exonuclease-catalyzed target recycling strategy, an amplified fluorescence intensity could be produced using different SEB concentrations. Using optimized experimental conditions produced an ultrasensitive aptasensor for the detection of SEB, with a wide linear range of 0.001–1 ng mL −1 and a lower detection limit (LOD) of 0.3 pg mL −1 SEB (at 3σ). The fabricated aptasensor was used to measure SEB in a

  13. The three-dimensional crystal structure of cholera toxin

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Rong-Guang; Westbrook, M.L.; Nance, S.; Spangler, B.D. [Argonne National Lab., IL (United States); Scott, D.L. [Yale Univ., New Haven, CT (United States). Dept. of Molecular Biophysics and Biochemistry; Westbrook, E.M. [Northwestern Univ., Evanston, IL (United States)

    1996-02-01

    The clinical manifestations of cholera are largely attributable to the actions of a secreted hexameric AB{sub 5} enterotoxin (choleragen). We have solved the three-dimensional structure of choleragen at 2.5 {Angstrom} resolution and compared the refined coordinates with those of choleragenoid (isolated B pentamer) and the heat-labile enterotoxin from Escherichia coli (LT). The crystalline coordinates provide a detailed view of the stereochemistry implicated in binding to GM1 gangliosides and in carrying out ADP-ribosylation. The A2 chain of choleragen, in contrast to that of LT, is a nearly continuous {alpha}-helix with an interpretable carboxyl tail.

  14. Distinct cytokine profiles of circulating mononuclear cells stimulated with Staphylococcus aureus enterotoxin A in vitro during early and late episodes of chronic osteomyelitis

    Directory of Open Access Journals (Sweden)

    Gabriella Freitas Ferreira

    2012-05-01

    Full Text Available We investigated the cytokine profile of peripheral mononuclear cells from chronic osteomyelitis (OST patients following in vitro stimulation with staphylococcal enterotoxin A (SEA. We demonstrate that stimulation with SEA induced prominent lymphocyte proliferation and high levels of tumour necrosis factor (TNF-α, interleukin (IL-4 and IL-10 secretion in both OST and non-infected individuals (NI. Even though stimulation with SEA had no impact on IL-6 production in either patient group, the baseline level of IL-6 production by cells from OST patients was always significantly less than that produced by cells from NI. After classifying the osteomyelitic episodes based on the time after the last reactivation event as "early" (1-4 months or "late" osteomyelitis (5-12 months, we found that increased levels of TNF-α and IL-4 in combination with decreased levels of IL-6 were observed in the early episodes. By contrast, increased levels of IL-10, IL-2 and IL-6 were hallmarks of late episodes. Our data demonstrate that early osteomyelitic episodes are accompanied by an increased frequency of "high producers" of TNF-α and IL-4, whereas late events are characterised by increased frequencies of "high producers" of IL-10, IL-6 and IL-2. These findings demonstrate the distinct cytokine profiles in chronic osteomyelitis, with a distinct regulation of IL-6 production during early and late episodes.

  15. Frequency of enterotoxins, toxic shock syndrome toxin-1, and biofilm formation genes in Staphylococcus aureus isolates from cows with mastitis in the Northeast of Brazil.

    Science.gov (United States)

    Costa, F N; Belo, N O; Costa, E A; Andrade, G I; Pereira, L S; Carvalho, I A; Santos, R L

    2018-06-01

    Staphylococcus aureus is among the microorganisms more frequently associated with subclinical bovine mastitis. S. aureus may produce several virulence factors. This study aimed at determining the frequency of virulence factors such as enterotoxins, toxic shock syndrome toxin 1, and ica adhesion genes. In addition, we assessed antimicrobial drug resistance in S. aureus isolated from clinical and subclinical cases of mastitis. A total of 88 cows with clinical or subclinical mastitis were sampled, resulting in 38 S. aureus isolates, from which 25 (65.78%) carried toxin genes, including seb, sec, sed, tst, and icaD adhesion gene. These S. aureus isolates belong to 21 ribotypes and three S. aureus strains belonged to the same ribotype producing ica adhesion gene. Approximately 90% of S. aureus strains obtained in our study demonstrated multiple resistance to different antimicrobial agents. The most efficacious antimicrobial agents against the isolates were gentamicin, amoxicillin, and norfloxacin. Gentamicin was the most efficacious agent inhibiting 78.95% of the S. aureus isolates. The least efficacious were penicillin, streptomycin, and ampicillin. Our results can help in understanding the relationship between virulence factors and subclinical mastitis caused by S. aureus. Further research about diversity of S. aureus isolates and genes responsible for the pathogenicity of subclinical mastitis is essential.

  16. Involvement of protein kinase C in the mechanism of action of Escherichia coli heat-stable enterotoxin (STa) in a human colonic carcinoma cell line, COLO-205

    International Nuclear Information System (INIS)

    Gupta, Dyuti Datta; Saha, Subhrajit; Chakrabarti, Manoj K.

    2005-01-01

    The present study was undertaken to determine the involvement of calcium-protein kinase C pathway in the mechanism of action of Escherichia coli heat stable enterotoxin (STa) apart from STa-induced activation of guanylate cyclase in human colonic carcinoma cell line COLO-205, which was used as a model cultured cell line to study the mechanism of action of E. coli STa. In response to E. coli STa, protein kinase C (PKC) activity was increased in a time-dependent manner with its physical translocation from cytosol to membrane. Inhibition of the PKC activity in membrane fraction and inhibition of its physical translocation in response to IP 3 -mediated calcium release inhibitor dantrolene suggested the involvement of intracellular store depletion in the regulation of PKC activity. Among different PKC isoforms, predominant involvement of calcium-dependent protein kinase C (PKCα) was specified using isotype-specific pseudosubstrate, which showed pronounce enzyme activity. Inhibition of enzyme activity by PKCα-specific inhibitor Goe6976 and immunoblott study employing isotype-specific antibody further demonstrated the involvement of calcium-dependent isoform of PKC in the mechanism of action of E. coli STa. Moreover, inhibition of guanylate cyclase activity by PKCα-specific inhibitor Goe6976 suggested the involvement of PKCα in the regulation of guanylate cyclase activity

  17. Chimeric anti-staphylococcal enterotoxin B antibodies and lovastatin act synergistically to provide in vivo protection against lethal doses of SEB.

    Directory of Open Access Journals (Sweden)

    Mulualem E Tilahun

    Full Text Available Staphylococcal enterotoxin B (SEB is one of a family of toxins secreted by Staphylococcus aureus that act as superantigens, activating a large fraction of the T-cell population and inducing production of high levels of inflammatory cytokines that can cause toxic shock syndrome (TSS and death. Extracellular engagement of the TCR of T-cells and class II MHC of antigen presenting cells by SEB triggers the activation of many intracellular signaling processes. We engineered chimeric antibodies to block the extracellular engagement of cellular receptors by SEB and used a statin to inhibit intracellular signaling. Chimeric human-mouse antibodies directed against different neutralizing epitopes of SEB synergistically inhibited its activation of human T-cells in vitro. In the in vivo model of lethal toxic shock syndrome (TSS in HLA-DR3 transgenic mice, two of these antibodies conferred significant partial protection when administered individually, but offered complete protection in a synergistic manner when given together. Similarly, in vivo, lovastatin alone conferred only partial protection from TSS similar to single anti-SEB antibodies. However, used in combination with one chimeric neutralizing anti-SEB antibody, lovastatin provided complete protection against lethal TSS in HLA-DR3 transgenic mice. These experiments demonstrate that in vivo protection against lethal doses of SEB can be achieved by a statin of proven clinical safety and chimeric human-mouse antibodies, agents now widely used and known to be of low immunogenicity in human hosts.

  18. Effects of tetrodotoxin and ion replacements on the short-circuit current induced by Escherichiacoli heat stable enterotoxin across small intestine of the gerbil (Gerbillus cheesmani

    Directory of Open Access Journals (Sweden)

    Fawzia Yaqoub Al-Balool

    2004-03-01

    Full Text Available The effects of mucosally added Escherichia coli heat stable enterotoxin (STa 30 ng ml-1 on the basal short-circuit current (Isc in µA cm-2 across stripped and unstripped sheets of jejuna and ilea taken from fed, starved (4 days, water ad lib and undernourished (50% control food intake for 21 days gerbil (Gerbillus cheesmani were investigated. The effect of neurotoxin tetrodotoxin (TTX 10 µM and the effects of replacing chloride by gluconate or the effects of removing bicarbonate from bathing buffers on the maximum increase in Isc induced by STa were also investigated. The maximum increase in Isc which resulted from the addition of STa were significantly higher in jejuna and ilea taken from starved and undernourished gerbils when compared with the fed control both using stripped and unstripped sheets. In the two regions of the small intestine taken from fed and starved animals TTX reduced the maximum increase in Isc induced by STa across unstripped sheets only. Moreover in jejuna and ilea taken from undernourished gerbils TTX reduced significantly the maximum increase in Isc induced by STa across stripped and unstripped sheets. Replacing chloride by gluconate decreased the maximum increase in Isc induced by STa across jejuna and ilea taken from undernourished gerbils only. Removing bicarbonates from bathing buffer decreased the maximum increase in Isc across the jejuna and ilea taken from starved and undernourished gerbils.

  19. Detection of target staphylococcal enterotoxin B antigen in orange juice and popular carbonated beverages using antibody-dependent antigen-capture assays.

    Science.gov (United States)

    Principato, MaryAnn; Njoroge, Joyce M; Perlloni, Andrei; O' Donnell, Michael; Boyle, Thomas; Jones, Robert L

    2010-10-01

    There is a critical need for qualitative and quantitative methodologies that provide the rapid and accurate detection of food contaminants in complex food matrices. However, the sensitivity of the assay can be affected when antigen-capture is applied to certain foods or beverages that are extremely acidic. This study was undertaken to assess the effects of orange juice and popular carbonated soft drink upon the fidelity of antibody-based antigen-capture assays and to develop simple approaches that could rescue assay performance without the introduction of additional or extensive extraction procedures. We examined the effects of orange juice and a variety of popular carbonated soft drink beverages upon a quantitative Interleukin-2 (IL-2) enzyme-linked immunosorbent assay (ELISA) assay system and a lateral flow device (LFD) adapted for the detection of staphylococcal enterotoxin B (SEB) in foods. Alterations in the performance and sensitivity of the assay were directly attributable to the food matrix, and alterations in pH were especially critical. The results demonstrate that approaches such as an alteration of pH and the use of milk as a blocking agent, either singly or in combination, will partially rescue ELISA performance. The same approaches permit lateral flow to efficiently detect antigen. Practical Application: The authors present ways to rescue an ELISA assay compromised by acidity in beverages and show that either the alteration of pH, or the use of milk as a blocking agent are not always capable of restoring the assay to its intended efficiency. However, the same methods, when employed with lateral flow technology, are rapid and extremely successful.

  20. The immunogenic SigA enterotoxin of Shigella flexneri 2a binds to HEp-2 cells and induces fodrin redistribution in intoxicated epithelial cells.

    Directory of Open Access Journals (Sweden)

    Keith Al-Hasani

    Full Text Available BACKGROUND: We have previously shown that the enterotoxin SigA which resides on the she pathogenicity island (PAI of S. flexneri 2a is an autonomously secreted serine protease capable of degrading casein. We have also demonstrated that SigA is cytopathic for HEp-2 cells and plays a role in the intestinal fluid accumulation associated with S. flexneri infections. METHODS/PRINCIPAL FINDINGS: In this work we show that SigA binds specifically to HEp-2 cells and degrades recombinant human alphaII spectrin (alpha-fodrin in vitro, suggesting that the cytotoxic and enterotoxic effects mediated by SigA are likely associated with the degradation of epithelial fodrin. Consistent with our data, this study also demonstrates that SigA cleaves intracellular fodrin in situ, causing its redistribution within cells. These results strongly implicate SigA in altering the cytoskeleton during the pathogenesis of shigellosis. On the basis of these findings, cleavage of fodrin is a novel mechanism of cellular intoxication for a Shigella toxin. Furthermore, information regarding immunogenicity to SigA in infected patients is lacking. We studied the immune response of SigA from day 28 post-challenge serum of one volunteer from S. flexneri 2a challenge studies. Our results demonstrate that SigA is immunogenic following infection with S. flexneri 2a. CONCLUSIONS: This work shows that SigA binds to epithelial HEp-2 cells as well as being able to induce fodrin degradation in vitro and in situ, further extending its documented role in the pathogenesis of Shigella infections.

  1. Staphylococcal enterotoxin-like X (SElX is a unique superantigen with functional features of two major families of staphylococcal virulence factors.

    Directory of Open Access Journals (Sweden)

    Ries J Langley

    2017-09-01

    Full Text Available Staphylococcus aureus is an opportunistic pathogen that produces many virulence factors. Two major families of which are the staphylococcal superantigens (SAgs and the Staphylococcal Superantigen-Like (SSL exoproteins. The former are immunomodulatory toxins that induce a Vβ-specific activation of T cells, while the latter are immune evasion molecules that interfere with a wide range of innate immune defences. The superantigenic properties of Staphylococcal enterotoxin-like X (SElX have recently been established. We now reveal that SElX also possesses functional characteristics of the SSLs. A region of SElX displays high homology to the sialyl-lactosamine (sLacNac-specific binding site present in a sub-family of SSLs. By analysing the interaction of SElX with sLacNac-containing glycans we show that SElX has an equivalent specificity and host cell binding range to the SSLs. Mutation of key amino acids in this conserved region affects the ability of SElX to bind to cells of myeloid origin and significantly reduces its ability to protect S. aureus from destruction in a whole blood killing (WBK assay. Like the SSLs, SElX is up-regulated early during infection and is under the control of the S. aureus exotoxin expression (Sae two component gene regulatory system. Additionally, the structure of SElX in complex with the sLacNac-containing tetrasaccharide sialyl Lewis X (sLeX reveals that SElX is a unique single-domain SAg. In summary, SElX is an 'SSL-like' SAg.

  2. Isolation of a new ssDNA aptamer against staphylococcal enterotoxin B based on CNBr-activated sepharose-4B affinity chromatography.

    Science.gov (United States)

    Hedayati Ch, Mojtaba; Amani, Jafar; Sedighian, Hamid; Amin, Mohsen; Salimian, Jafar; Halabian, Raheleh; Imani Fooladi, Abbas Ali

    2016-09-01

    Staphylococcus aureus are potent human pathogens possessing arsenal of virulence factors. Staphylococcal food poisoning (SFP) and respiratory infections mediated by staphylococcal enterotoxin B (SEB) are common clinical manifestations. Many diagnostic techniques are based on serological detection and quantification of SEB in different food and clinical samples. Aptamers are known as new therapeutic and detection tools which are available in different ssDNA, dsDNA and protein structures. In this study, we used a new set of ssDNA aptamers against SEB. The methods used included preparation of a dsDNA library using standard SEB protein as the target analyte, affinity chromatography matrix in microfuge tubes, SELEX procedures to isolate specific ssDNA-aptamer as an affinity ligand, aptamer purification using ethanol precipitation method, affinity binding assay using ELISA, aptamer cloning and specificity test. Among 12 readable sequences, three of them were selected as the most appropriate aptamer because of their affinity and specificity to SEB. This study presents a new set of ssDNA aptamer with favorable selectivity to SEB through 12 rounds of SELEX. Selected aptamers were used to detect SEB in infected serum samples. Results showed that SEB c1 aptamer (2 µg SEB/100 nM aptamer) had favorable specificity to SEB (kd  = 2.3 × 10(-11) ). In conclusion, aptamers can be considered as useful tools for detecting and evaluating SEB. The results showed that affinity chromatography was an affordable assay with acceptable accuracy to isolate sensitive and selective novel aptamers. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  3. Up-regulation of granzyme B and perforin by staphylococcal enterotoxin C2 mutant induces enhanced cytotoxicity in Hepa1–6 cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Guojun [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China); University of Chinese Academy of Sciences, Beijing (China); Xu, Mingkai, E-mail: mkxu@iae.ac.cn [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China); Zhang, Huiwen [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China); Song, Yubo [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China); University of Chinese Academy of Sciences, Beijing (China); Wang, Jian; Zhang, Chenggang [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China)

    2016-12-15

    Staphylococcal enterotoxin C2 (SEC2), a member of bacterial superantigen, is one of the most potent known activators of T lymphocytes. With this property, SEC2 has already been used in clinic as a tumor immunotherapy agent in China. To increase the antitumor activity, a SEC2 mutant named ST-4 (GKVTG102-106WWH) with amino acid substitutions in T cell receptor (TCR)-binding domain was generated by site-directed mutagenesis, and the molecular mechanism of the enhanced antitumor activity was investigated. Results showed that ST-4 could activate much more Vβ 8.2 and 8.3 T cells and NK cells compared with SEC2, and exhibited significantly enhanced immunocyte stimulation and antitumor activity in vitro. The synthetic peptide sequencing the residues of mutant TCR-binding domain could competitively inhibit the immunocyte stimulation activity of ST-4. Most importantly, ST-4 up-regulated granzyme B and perforin at both mRNA and protein levels. We also found that expression of proapoptotic proteins cytochrome c, BAX and activation of caspase-3, 9 was up-regulated, and antiapoptotic protein Bcl-xL was down-regulated in the treatment with either ST-4 or SEC2. When granzyme B inhibitor or perforin inhibitor is presented, tumor cell viability was significantly rescued. Taken together, we demonstrate that increased ST-4-TCR recognition contributed to massive T cells and NK cells activation. These activated cells released up-regulated granzyme B and perforin, which induced the enhanced tumor cells apoptosis by mitochondrial apoptotic pathway, and ultimately led to enhanced tumor cell growth inhibition. ST-4 may be a promising candidate for antitumor clinic usage in future. - Highlights: • We obtained a SEC2 mutant ST-4 with enhanced superantigen and antitumor activity. • Increased ST-4-TCR recognition contributed to massive T cells and NK cells activation. • Up-regulated GzmB and PRF1 in T cell by ST-4 induced enhanced tumor cells apoptosis. • Enhanced tumor cell apoptosis

  4. Natural indoles, indole-3-carbinol and 3,3′-diindolymethane, inhibit T cell activation by staphylococcal enterotoxin B through epigenetic regulation involving HDAC expression

    International Nuclear Information System (INIS)

    Busbee, Philip B.; Nagarkatti, Mitzi; Nagarkatti, Prakash S.

    2014-01-01

    Staphylococcal enterotoxin B (SEB) is a potent exotoxin produced by the Staphylococcus aureus. This toxin is classified as a superantigen because of its ability to directly bind with MHC-II class molecules followed by activation of a large proportion of T cells bearing specific Vβ-T cell receptors. Commonly associated with classic food poisoning, SEB has also been shown to induce toxic shock syndrome, and is also considered to be a potential biological warfare agent because it is easily aerosolized. In the present study, we assessed the ability of indole-3-carbinol (I3C) and one of its byproducts, 3,3′-diindolylmethane (DIM), found in cruciferous vegetables, to counteract the effects of SEB-induced activation of T cells in mice. Both I3C and DIM were found to decrease the activation, proliferation, and cytokine production by SEB-activated Vβ8 + T cells in vitro and in vivo. Interestingly, inhibitors of histone deacetylase class I (HDAC-I), but not class II (HDAC-II), showed significant decrease in SEB-induced T cell activation and cytokine production, thereby suggesting that epigenetic modulation plays a critical role in the regulation of SEB-induced inflammation. In addition, I3C and DIM caused a decrease in HDAC-I but not HDAC-II in SEB-activated T cells, thereby suggesting that I3C and DIM may inhibit SEB-mediated T cell activation by acting as HDAC-I inhibitors. These studies not only suggest for the first time that plant-derived indoles are potent suppressors of SEB-induced T cell activation and cytokine storm but also that they may mediate these effects by acting as HDAC inhibitors. - Highlights: • I3C and DIM reduce SEB-induced T cell activation and inflammatory cytokines. • Inhibiting class I HDACs reduces T cell activation and inflammatory cytokines. • Inhibiting class II HDACs increases T cell activation and inflammatory cytokines. • I3C and DIM selectively reduce mRNA expression of class I HDACs. • Novel use and mechanism to counteract SEB

  5. Natural indoles, indole-3-carbinol and 3,3′-diindolymethane, inhibit T cell activation by staphylococcal enterotoxin B through epigenetic regulation involving HDAC expression

    Energy Technology Data Exchange (ETDEWEB)

    Busbee, Philip B.; Nagarkatti, Mitzi; Nagarkatti, Prakash S., E-mail: prakash@mailbox.sc.edu

    2014-01-01

    Staphylococcal enterotoxin B (SEB) is a potent exotoxin produced by the Staphylococcus aureus. This toxin is classified as a superantigen because of its ability to directly bind with MHC-II class molecules followed by activation of a large proportion of T cells bearing specific Vβ-T cell receptors. Commonly associated with classic food poisoning, SEB has also been shown to induce toxic shock syndrome, and is also considered to be a potential biological warfare agent because it is easily aerosolized. In the present study, we assessed the ability of indole-3-carbinol (I3C) and one of its byproducts, 3,3′-diindolylmethane (DIM), found in cruciferous vegetables, to counteract the effects of SEB-induced activation of T cells in mice. Both I3C and DIM were found to decrease the activation, proliferation, and cytokine production by SEB-activated Vβ8{sup +} T cells in vitro and in vivo. Interestingly, inhibitors of histone deacetylase class I (HDAC-I), but not class II (HDAC-II), showed significant decrease in SEB-induced T cell activation and cytokine production, thereby suggesting that epigenetic modulation plays a critical role in the regulation of SEB-induced inflammation. In addition, I3C and DIM caused a decrease in HDAC-I but not HDAC-II in SEB-activated T cells, thereby suggesting that I3C and DIM may inhibit SEB-mediated T cell activation by acting as HDAC-I inhibitors. These studies not only suggest for the first time that plant-derived indoles are potent suppressors of SEB-induced T cell activation and cytokine storm but also that they may mediate these effects by acting as HDAC inhibitors. - Highlights: • I3C and DIM reduce SEB-induced T cell activation and inflammatory cytokines. • Inhibiting class I HDACs reduces T cell activation and inflammatory cytokines. • Inhibiting class II HDACs increases T cell activation and inflammatory cytokines. • I3C and DIM selectively reduce mRNA expression of class I HDACs. • Novel use and mechanism to counteract

  6. Staphylococcal Entertotoxins of the Enterotoxin Gene Cluster (egcSEs Induce Nitrous Oxide- and Cytokine Dependent Tumor Cell Apoptosis in a Broad Panel of Human Tumor Cells

    Directory of Open Access Journals (Sweden)

    David eTerman

    2013-08-01

    Full Text Available The egcSEs comprise five genetically linked staphylococcal enterotoxins, SEG, SEI, SElM, SElN and SElO and two pseudotoxins which constitute an operon present in up to 80% of Staphylococcus aureus isolates. A preparation containing theses proteins was recently used to treat advanced lung cancer with pleural effusion. We investigated the hypothesis that egcSEs induce nitrous oxide (NO and associated cytokine production and that these agents may be involved in tumoricidal effects against a broad panel of clinically relevant human tumor cells. Preliminary studies showed that egcSEs and SEA activated T cells (range: 11-25% in a concentration dependent manner. Peripheral blood mononuclear cells (PBMCs stimulated with equimolar quantities of egcSEs expressed NO synthase and generated robust levels of nitrite (range: 200-250 µM, a breakdown product of NO; this reaction was inhibited by NG-monomethyl-L-arginine (L-NMMA (0.3 mM, an NO synthase antagonist. Cell free supernatants (CSFs of all egcSE-stimulated PBMCs were also equally effective in inducing concentration dependent tumor cell apoptosis in a broad panel of human tumor cells. The latter effect was due in part to the generation of NO and TNF-α since it was significantly abolished by L-NMMA, anti-TNF-α antibodies respectively and a combination thereof. A hierarchy of tumor cell sensitivity to these CFSs was as follows: lung carcinoma>osteogenic sarcoma>melanoma>breast carcinoma>neuroblastoma. Notably, SEG induced robust activation of NO/TNFα-dependent tumor cell apoptosis comparable to the other egcSEs and SEA despite TNF-α and IFN-γ levels that were 2 and 8 fold lower respectively than the other egcSEs and SEA. Thus, egcSEs produced by S. aureus induce NO synthase and the increased NO formation together with TNF-α appear to contribute to egcSE-mediated apoptosis against a broad panel of human tumor cells.

  7. Production of Escherichia coli heat labile toxin (LT) B subunit in soybean seed and analysis of its immunogenicity as an oral vaccine

    Czech Academy of Sciences Publication Activity Database

    Moravec, Tomáš; Schmidt, M.A.; Herman, E.M.; Woodford-Thomas, T.

    2007-01-01

    Roč. 25, - (2007), s. 1647-1657 ISSN 0264-410X Grant - others:Marie Curie Fellowship(XE) MOIF CT 2005-008692 Institutional research plan: CEZ:AV0Z50380511 Keywords : Microbial toxin * Plant-based vaccines * Transgenic soybean seed Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.377, year: 2007

  8. The yield of DNA double strand breaks determined after exclusion of those forming from heat-labile lesions predicts tumor cell radiosensitivity to killing.

    Science.gov (United States)

    Cheng, Yanlei; Li, Fanghua; Mladenov, Emil; Iliakis, George

    2015-09-01

    The radiosensitivity to killing of tumor cells and in-field normal tissue are key determinants of radiotherapy response. In vitro radiosensitivity of tumor- and normal-tissue-derived cells often predicts radiation response, but high determination cost in time and resources compromise utility as routine response-predictor. Efforts to use induction or repair of DNA double-strand-breaks (DSBs) as surrogate-predictors of cell radiosensitivity to killing have met with limited success. Here, we re-visit this issue encouraged by our recent observations that ionizing radiation (IR) induces not only promptly-forming DSBs (prDSBs), but also DSBs developing after irradiation from the conversion to breaks of thermally-labile sugar-lesions (tlDSBs). We employ pulsed-field gel-electrophoresis and flow-cytometry protocols to measure total DSBs (tDSB=prDSB+tlDSBs) and prDSBs, as well as γH2AX and parameters of chromatin structure. We report a fully unexpected and in many ways unprecedented correlation between yield of prDSBs and radiosensitivity to killing in a battery of ten tumor cell lines that is not matched by yields of tDSBs or γH2AX, and cannot be explained by simple parameters of chromatin structure. We propose the introduction of prDSBs-yield as a novel and powerful surrogate-predictor of cell radiosensitivity to killing with potential for clinical application. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  9. Treponema pallidum 3-Phosphoglycerate Mutase Is a Heat-Labile Enzyme That May Limit the Maximum Growth Temperature for the Spirochete

    Science.gov (United States)

    Benoit, Stéphane; Posey, James E.; Chenoweth, Matthew R.; Gherardini, Frank C.

    2001-01-01

    In the causative agent of syphilis, Treponema pallidum, the gene encoding 3-phosphoglycerate mutase, gpm, is part of a six-gene operon (tro operon) that is regulated by the Mn-dependent repressor TroR. Since substrate-level phosphorylation via the Embden-Meyerhof pathway is the principal way to generate ATP in T. pallidum and Gpm is a key enzyme in this pathway, Mn could exert a regulatory effect on central metabolism in this bacterium. To study this, T. pallidum gpm was cloned, Gpm was purified from Escherichia coli, and antiserum against the recombinant protein was raised. Immunoblots indicated that Gpm was expressed in freshly extracted infective T. pallidum. Enzyme assays indicated that Gpm did not require Mn2+ while 2,3-diphosphoglycerate (DPG) was required for maximum activity. Consistent with these observations, Mn did not copurify with Gpm. The purified Gpm was stable for more than 4 h at 25°C, retained only 50% activity after incubation for 20 min at 34°C or 10 min at 37°C, and was completely inactive after 10 min at 42°C. The temperature effect was attenuated when 1 mM DPG was added to the assay mixture. The recombinant Gpm from pSLB2 complemented E. coli strain PL225 (gpm) and restored growth on minimal glucose medium in a temperature-dependent manner. Increasing the temperature of cultures of E. coli PL225 harboring pSLB2 from 34 to 42°C resulted in a 7- to 11-h period in which no growth occurred (compared to wild-type E. coli). These data suggest that biochemical properties of Gpm could be one contributing factor to the heat sensitivity of T. pallidum. PMID:11466272

  10. The influence of headspace and dissolved oxygen level on growth and haemolytic BL enterotoxin production of a psychrotolerant Bacillus weihenstephanensis isolate on potato based ready-to-eat food products.

    Science.gov (United States)

    Samapundo, S; Everaert, H; Wandutu, J N; Rajkovic, A; Uyttendaele, M; Devlieghere, F

    2011-04-01

    The major objective of this study was to determine the influence of the initial headspace and dissolved O(2) level and vacuum packaging on growth and diarrhoeal enterotoxin production by Bacillus weihenstephanensis on potato based ready-to-eat food products. In general, the lower the initial headspace or dissolved O(2) level the slower the maximum growth rate (μ(max), log(10) CFU g(-1) d(-1)), the longer the lag phase duration (λ, d) and the smaller the maximum population density (N(max), log(10) CFU g(-1)) became. The slowest μ(max), the longest λ and the smallest N(max) were generally found for growth under vacuum packaging. This implies shorter shelf-lives will occur at higher initial headspace or dissolved O(2) levels as the growth of B. weihenstephanensis to the infective dose of 10(5) CFU g(-1) in such atmospheres takes a shorter time. Significant consumption of dissolved O(2) only occurred when growth shifted from the lag to the exponential phase and growth generally transitioned from the exponential to the stationary phase when the dissolved O(2) levels fell below ca. 75 ppb. Diarrhoeal enterotoxin production (determined via detection of the L2 component of haemolytic BL) was similar for growth under initial headspace O(2) levels of 1-20.9%, and was only reduced when growth took place under vacuum packaging. The reduction in L2 production when growth took place under vacuum was most probably related to the low final cell densities observed under this condition. Both growth and L2 production were inhibited over a 32-day incubation period at 7 °C by 40% CO(2) irrespective of the headspace or dissolved O(2) levels. The results illustrate the importance of residual O(2) and CO(2) on the shelf-stability and safety of modified atmosphere packaged potato based ready-to-eat food products with regards to B. weihenstephanensis. Copyright © 2010 Elsevier Ltd. All rights reserved.

  11. Claudin-4 Overexpression in Epithelial Ovarian Cancer Is Associated with Hypomethylation and Is a Potential Target for Modulation of Tight Junction Barrier Function Using a C-Terminal Fragment of Clostridium perfringens Enterotoxin

    Directory of Open Access Journals (Sweden)

    Babak Litkouhi

    2007-04-01

    Full Text Available BACKGROUND: Claudin-4, a tight junction (TJ protein and receptor for the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE, is overexpressed in epithelial ovarian cancer (EOC. Previous research suggests DNA methylation is a mechanism for claudin-4 overexpression in cancer and that C-CPE acts as an absorption-enhancing agent in claudin-4expressing cells. We sought to correlate claudin-4 overexpression in EOC with clinical outcomes and TJ barrier function, investigate DNA methylation as a mechanism for overexpression, and evaluate the effect of C-CPE on the TJ. METHODS: Claudin-4 expression in EOC was quantified and correlated with clinical outcomes. Claudin-4 methylation status was determined, and claudin-4-negative cell lines were treated with a demethylating agent. Electric cell-substrate impedance sensing was used to calculate junctional (paracellular resistance (Rb in EOC cells after claudin-4 silencing and after C-CPE treatment. RESULTS: Claudin4 overexpression in EOC does not correlate with survival or other clinical endpoints and is associated with hypomethylation. Claudin-4 overexpression correlates with Rb and C-CPE treatment of EOC cells significantly decreased Rb in a dose- and claudin-4-dependent noncytotoxic manner. CONCLUSIONS: C-CPE treatment of EOC cells leads to altered TJ function. Further research is needed to determine the potential clinical applications of C-CPE in EOC drug delivery strategies.

  12. Experimental investigation into the effects of irradiation with mixed neutron-gamma-rays on the immune system as demonstrated by the model of immunity to Staphylococcal Enterotoxin B in rabbits

    International Nuclear Information System (INIS)

    Kraus, W.

    1984-01-01

    Using the model of immunisation of rabbits with Staphylococcal Enterotoxin B the influence of neutron radiation on the antibody response was investigated. Four groups were formed, which differed by the moment of irradiation with regard to the moment of immunisation and re-immunisation, and were irradiated with neutron-rays of 200 rad and 300 rad doses. Both doses were each given before and after vaccination, respectively before and after re-vaccination. The antibody response has been evaluated by the Radio-Immuno-Assay (RIA): Neutron-radiation given 24 hours before vaccination effects a stronger immune suppression than given 24 hours after vaccination. By pre-antigenic radiation a secondary immune response is more suppressed than by radiation following the antigen; the recovery of lymphoid tissue, however, takes place in a shorter time than in case of radiation before or after a primary vaccination. The secondary antibody response to a re-vaccination is not influenced by a subsequent neutron-radiation. The formation of secondary antibodies is not as radiosensitive as the formation of primary ones. By a neutron-radiation preformed antibodies are hardly damaged. (orig./MG) [de

  13. Organization and ELISA-Based Results of the First Proficiency Testing to Evaluate the Ability of European Union Laboratories to Detect Staphylococcal Enterotoxin Type B (SEB in Buffer and Milk

    Directory of Open Access Journals (Sweden)

    Yacine Nia

    2016-09-01

    Full Text Available The aim of this work was to organize the first proficiency test (PT dedicated to staphylococcal enterotoxin B (SEB detection in milk and buffer solutions. This paper describes the organization of the PT trial according to EN ISO 17043 requirements. Characterization of the SEB stock solution was performed using SDS-PAGE and SE-specific ELISA, and amino acid analysis was used to assign its protein concentration. The solution was then used to prepare six PT materials (four milk and two buffer batches at a ng/g toxin level, which included one blank and one SEA-containing milk as specificity control. Suitable material homogeneity and stability were assessed using screening and quantitative ELISAs. Among the methods used by the participants, ELISA-based methods demonstrated their efficiency for the detection of SEB in both simple and complex matrices. The results serve as a basis for further improving the detection capabilities in expert laboratories and can therefore be considered as a contribution to biopreparedness.

  14. Bacteroides fragilis Enterotoxin Induces Formation of Autophagosomes in Endothelial Cells but Interferes with Fusion with Lysosomes for Complete Autophagic Flux through a Mitogen-Activated Protein Kinase-, AP-1-, and C/EBP Homologous Protein-Dependent Pathway.

    Science.gov (United States)

    Ko, Su Hyuk; Jeon, Jong Ik; Myung, Hyun Soo; Kim, Young-Jeon; Kim, Jung Mogg

    2017-10-01

    Bacteroides fragilis enterotoxin (BFT), a virulence factor of enterotoxigenic B. fragilis (ETBF), plays an essential role in mucosal inflammation. Although autophagy contributes to the pathogenesis of diverse infectious diseases, little is known about autophagy in ETBF infection. This study was conducted to investigate the role of BFT in the autophagic process in endothelial cells (ECs). Stimulation of human umbilical vein ECs (HUVECs) with BFT increased light chain 3 protein II (LC3-II) conversion from LC3-I and protein expression of p62, Atg5, and Atg12. In addition, BFT-exposed ECs showed increased indices of autophagosomal fusion with lysosomes such as LC3-lysosome-associated protein 2 (LAMP2) colocalization and the percentage of red vesicles monitored by the expression of dual-tagged LC3B. BFT also upregulated expression of C/EBP homologous protein (CHOP), and inhibition of CHOP significantly increased indices of autophagosomal fusion with lysosomes. BFT activated an AP-1 transcription factor, in which suppression of AP-1 activity significantly downregulated CHOP and augmented autophagosomal fusion with lysosomes. Furthermore, suppression of Jun N-terminal protein kinase (JNK) mitogen-activated protein kinase (MAPK) significantly inhibited the AP-1 and CHOP signals, leading to an increase in autophagosomal fusion with lysosomes in BFT-stimulated ECs. These results suggest that BFT induced accumulation of autophagosomes in ECs, but activation of a signaling pathway involving JNK, AP-1, and CHOP may interfere with complete autophagy. Copyright © 2017 American Society for Microbiology.

  15. Produção de enterotoxinas e da toxina da síndrome do choque tóxico por cepas de Staphylococcus aureus isoladas na mastite bovina Production of enterotoxins and toxic shock syndrome toxin by Staphylococcus aureus strains isolated from bovine mastitis

    Directory of Open Access Journals (Sweden)

    A. Nader Filho

    2007-10-01

    Full Text Available A total of 72 strains of Staphylococcus aureus were examined for the production of staphylococcal enterotoxins (SE A, B, C, D and toxic shock syndrome toxin (TSST-1. The strains were isolated from milk samples from cows with mastitis in dairy herds of São Paulo State, Brazil. Off 72 isolates, 38 (52.8% produced SEA, 38 (52.8% SEB, 32 (44.4% SED, 28 (38.9% SEC and 27 (37.5% TSST-1. From the 72 strains, 66 (91.7% produced, at least, one or more toxin, including TSST-1.

  16. Natural indoles, indole-3-carbinol (I3C and 3,3'-diindolylmethane (DIM, attenuate staphylococcal enterotoxin B-mediated liver injury by downregulating miR-31 expression and promoting caspase-2-mediated apoptosis.

    Directory of Open Access Journals (Sweden)

    Philip B Busbee

    Full Text Available Staphylococcal enterotoxin B (SEB is a potent superantigen capable of inducing inflammation characterized by robust immune cell activation and proinflammatory cytokine release. Exposure to SEB can result in food poisoning as well as fatal conditions such as toxic shock syndrome. In the current study, we investigated the effect of natural indoles including indole-3-carbinol (I3C and 3,3'-diindolylmethane (DIM on SEB-mediated liver injury. Injection of SEB into D-galactosamine-sensitized female C57BL/6 mice resulted in liver injury as indicated by an increase in enzyme aspartate transaminase (AST levels, induction of inflammatory cytokines, and massive infiltration of immune cells into the liver. Administration of I3C and DIM (40 mg/kg, by intraperitonal injection, attenuated SEB-induced acute liver injury, as evidenced by decrease in AST levels, inflammatory cytokines and cellular infiltration in the liver. I3C and DIM triggered apoptosis in SEB-activated T cells primarily through activation of the intrinsic mitochondrial pathway. In addition, inhibitor studies involving caspases revealed that I3C and DIM-mediated apoptosis in these activated cells was dependent on caspase-2 but independent of caspase-8, 9 and 3. In addition, I3C and DIM caused a decrease in Bcl-2 expression. Both compounds also down-regulated miR-31, which directly targets caspase-2 and influences apoptosis in SEB-activated cells. Our data demonstrate for the first time that indoles can effectively suppress acute hepatic inflammation caused by SEB and that this may be mediated by decreased expression of miR-31 and consequent caspase-2-dependent apoptosis in T cells.

  17. Perfil de contaminação por Staphylococcus e suas enterotoxinas e monitorização das condições de higiene em uma linha de produção de queijo de coalho Contamination profile for staphylococci and its enterotoxins and monitorization of the conditions of hygiene in a ‘coalho' cheese production line

    Directory of Open Access Journals (Sweden)

    Maria de Fatima Borges

    2008-08-01

    Full Text Available Avaliou-se o perfil de contaminação por Staphylococcus e suas enterotoxinas e monitorou-se as condições de higiene em uma linha de produção de queijo de coalho por meio da técnica de bioluminescência (ATP. A população de Staphylococcus sp. variou de This research aimed to evaluate the contamination by staphylococci and its enterotoxins as well as to monitor the conditions of hygiene from a coalho cheese production line, using ATP bioluminescence assay. Staphylococcus sp. population varied from <1CFU mL-1, in pasteurized milk to 1.5 x 107CFU mL-1, in raw milk, whereas coagulase-positive staphylococci count ranged from <1CFU mL-1, in pasteurized milk to 5.0 x 106CFU mL-1 in raw milk. Coagulase-positive staphylococci were detected in 100% (25/25 of the raw milk samples and in 8% (2/25 of cheese samples. Twelve Staphylococcus species were identified within the selected 68 isolates, being nine negative and three positive for coagulase. Raw milk samples showed a high rate of coagulase-positive, being S. aureus the most common, whereas other product samples and equipment surfaces, pieces of furniture, utensils and manipulator gloves samples presented a high frequency of coagulase-negative and low frequency of coagulase-positive. Staphylococcal enterotoxin was detected in 20% of the raw milk samples and therefore in pasteurized milk, curd and cheese. ATP measurement permitted to assess the effectiveness of the surfaces cleaning, being considered adequate in 62.1% (36/95, "alert state" in 23.2% (22/95 and inadequate in 14.7% (14/95 of surfaces evaluated. Detection of staphylococci species with enterotoxigenic potential as well as enterotoxin presence reveal dissemination of contamination at the "coalho" cheese production line, possibly due inappropriate Good Manufacturing Practices (GMP from the initial milking step until the final cheese production.

  18. ANIMAL ENTEROTOXIGENIC ESCHERICHIA COLI

    Science.gov (United States)

    Dubreuil, J. Daniel; Isaacson, Richard E.; Schifferli, Dieter M.

    2016-01-01

    Enterotoxigenic Escherichia coli (ETEC) is the most common cause of E. coli diarrhea in farm animals. ETEC are characterized by the ability to produce two types of virulence factors; adhesins that promote binding to specific enterocyte receptors for intestinal colonization and enterotoxins responsible for fluid secretion. The best-characterized adhesins are expressed in the context of fimbriae, such as the F4 (also designated K88), F5 (K99), F6 (987P), F17 and F18 fimbriae. Once established in the animal small intestine, ETEC produces enterotoxin(s) that lead to diarrhea. The enterotoxins belong to two major classes; heat-labile toxin that consist of one active and five binding subunits (LT), and heat-stable toxins that are small polypeptides (STa, STb, and EAST1). This chapter describes the disease and pathogenesis of animal ETEC, the corresponding virulence genes and protein products of these bacteria, their regulation and targets in animal hosts, as well as mechanisms of action. Furthermore, vaccines, inhibitors, probiotics and the identification of potential new targets identified by genomics are presented in the context of animal ETEC. PMID:27735786

  19. Histopathological Changes Induced By Staphylococcal Enterotoxin ...

    African Journals Online (AJOL)

    In this study, six Staphylococcus aureus strains isolated from contaminated yoghurt were evaluated for enterotoxigenicity. Two o the strains were enterotoxigenic and caused fluid accumulation in rabbit ileal loops. Fluid aspirated from the loops was bloody and histopathological changes in sections collected from rabbit ...

  20. Development and accuracy of quantitative real-time polymerase chain reaction assays for detection and quantification of enterotoxigenic Escherichia coli (ETEC) heat labile and heat stable toxin genes in travelers' diarrhea samples.

    Science.gov (United States)

    Youmans, Bonnie P; Ajami, Nadim J; Jiang, Zhi-Dong; Petrosino, Joseph F; DuPont, Herbert L; Highlander, Sarah K

    2014-01-01

    Enterotoxigenic Escherichia coli (ETEC), the leading bacterial pathogen of travelers' diarrhea, is routinely detected by an established DNA hybridization protocol that is neither sensitive nor quantitative. Quantitative real-time polymerase chain reaction (qPCR) assays that detect the ETEC toxin genes eltA, sta1, and sta2 in clinical stool samples were developed and tested using donor stool inoculated with known quantities of ETEC bacteria. The sensitivity of the qPCR assays is 89%, compared with 22% for the DNA hybridization assay, and the limits of detection are 10,000-fold lower than the DNA hybridization assays performed in parallel. Ninety-three clinical stool samples, previously characterized by DNA hybridization, were tested using the new ETEC qPCR assays. Discordant toxin profiles were observed for 22 samples, notably, four samples originally typed as ETEC negative were ETEC positive. The qPCR assays are unique in their sensitivity and ability to quantify the three toxin genes in clinical stool samples.

  1. PCR detection and serotyping of enterotoxigenic and shigatoxigenic Escherichia coli isolates obtained from chicken meat in Mumbai, India

    Directory of Open Access Journals (Sweden)

    R. J. Zende,

    2013-08-01

    Full Text Available Aim: Present study was undertaken to find out the frequency of few virulent genes and prevalence of related strains of Escherichia coli isolated from chicken meat obtained from chicken retail shops by Polymerase Chain Reaction (PCR.Materials and Methods: 66 samples of freshly slaughtered chicken meat were collected from 22 identified retail shops located at Mumbai city, randomly. Processed meat samples were cultured in EMB agar and presumptive colonies were confirmed by various biochemical tests. PCR method was accustomed for identification of the genes coding for heat-stable enterotoxin a (STa, heat labile enterotoxin (LT, shiga-like toxins 1 and 2 (SLT1 and SLT2. E. coli isolates were sent to National Salmonella and Escherichia Centre, CRI, Kasauli, HP, India for serotyping.Results: 11 (16.67% E. coli strains were isolated from 66 chicken meat samples. 3 (27.27% out of 11 harbored the gene for SLT2, and 2 (18.18% for STa. None of the strain contains SLT1 and LT genes. Serotypes detected were rough, O2, O20, O22, O102 each for one isolate and 6 isolates were untypable (UT.Conclusion: The results concluded that chicken meat samples analysed harbored genes for shiga like toxins and enterotoxins and different serotypes of E. coli. These findings indicating that regular monitoring of chicken meat is essential for this pathogen to prevent potential public health problems.

  2. Automated 5 ' nuclease assay for detection of virulence factors in porcine Escherichia coli

    DEFF Research Database (Denmark)

    Frydendahl, K.; Imberechts, H.; Lehmann, S.

    2001-01-01

    (STa, STb, EAST1) and heat labile LT) enterotoxins and the verocytotoxin variant 2e (VT2e). To correctly identify false negative results, an endogenous internal control targeting the E. coil 16S rRNA gene was incorporated in each test tube. The assay was evaluated using a collection of E. coil...... reference strains which have previously been examined with phenotypical assays or DNA hybridization. Furthermore, the assay was evaluated by testing porcine E. coil field strains, previously characterized. The 5' nuclease assay correctly detected the presence of virulence genes in all reference strains....... When testing field strains there was generally excellent agreement with results obtained by laboratories in Belgium and Germany. In conclusion, the 5' nuclease assay developed is a fast and specific tool for detection of E. coli virulence genes in the veterinary diagnostic laboratory....

  3. Production of vaccines for treatment of infectious diseases by transgenic plants

    Directory of Open Access Journals (Sweden)

    Kristina LEDL

    2016-04-01

    Full Text Available Since the first pathogen antigen was expressed in transgenic plants with the aim of producing edible vaccine in early 1990s, transgenic plants have become a well-established expression system for production of alternative vaccines against various human and animal infectious diseases. The main focus of plant expression systems in the last five years has been on improving expression of well-studied antigens such as porcine reproductive and respiratory syndrome (PRRSV, bovine viral diarrhea disease virus (BVDV, footh and mouth disease virus (FMDV, hepatitis B surface antigen (HBsAg, rabies G protein, rotavirus, Newcastle disease virus (NDV, Norwalk virus capsid protein (NVCP, avian influenza virus H5N1, Escherichia coli heat-labile enterotoxin subunit B (LT-B, cholera toxin B (CT-B, human immunodeficiency virus (HIV, artherosclerosis, ebola and anthrax. Significant increases in expression have been obtained using improved expression vectors, different plant species and transformation methods.

  4. Presença de Staphylococcus spp. produtores de enterotoxinas e da toxina da síndrome do choque tóxico em manipuladores de queijo de cabra Presence of Staphylococcus strains producer of enterotoxins and toxic shock toxin syndrome isolated from goat's cheese handlers

    Directory of Open Access Journals (Sweden)

    L.S. Rapini

    2005-12-01

    Full Text Available Agruparam-se 167 cepas de Staphylococcus, isoladas de diferentes sítios (fossas nasais, orofaringe, palma das mãos e leitos subungueais, de dois manipuladores de queijos de leite de cabra, em pools (45, de acordo com a similaridade da espécie e o local de origem, e testou-se o seu potencial de produção de enterotoxinas (SE A, B, C e D, e da toxina da síndrome do choque tóxico (TSST-1. Constatou-se que 62,2% dos pools foram capazes de produzir, de forma individual ou associada, SEA (33,3%; SEB (46,7%; SEC (8,9%; SED (4,4% e TSST-1 (4,4%. Dos pools enterotoxigênicos, 96,4% corresponderam a espécies coagulase negativa (Staphylococcus epidermidis e Staphylococcus cohnii. A capacidade de produção de SE e de TSST-1 pelas cepas de Staphylococcus spp. isoladas dos manipuladores, além de reafirmar sua importância na transmissão de intoxicações de origem alimentar, enfatiza a necessidade imprescindível da adoção de condutas higiênicas e sanitárias durante a manipulação do alimento.A total of 167 strains of Staphylococcus isolated from nasal cavities, oropharynx, palm of hands and subunguial of two goat's cheese handlers were collected. The strains were pooled (45 according to the species similarity and place of origin and tested for the production of enterotoxins (SE A, B, C, D and toxic shock toxin syndrome (TSST-1. It was observed that 62.2% of the pools presented the capacity to produce, individually or in association, SEA (33.3%; SEB (46.7%; SEC (8.9%; SED (4.4% and TSST-1 (4.4%. From the enterotoxigenic pools, 96.4% corresponded to species negative coagulase (Staphylococcus epidermidis and Staphylococcus cohnii. The capacity to produce SE and TSST-1 by Staphylococcus spp. strains isolated from food handlers reaffirms its hole on the transmission of food poisoning, beyond emphasizing the indispensable necessity of the adoption of satisfactory hygienic and sanitary procedures during the food manufacture.

  5. The 2.3 {angstrom} crystal structure of cholera toxin B subunit pentamer: Choleragenoid

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Rong-Guang; Westbrook, M.L. [Argonne National Lab., IL (United States); Maulik, P.R.; Reed, R.A.; Shipley, G. [Boston Univ., MA (United States). School of Medicine; Westbrook, E.M. [Argonne National Lab., IL (United States)]|[Northwestern Univ., Evanston, IL (United States); Scott, D.L.; Otwinowski, Z. [Yale Univ., New Haven, CT (United States)

    1996-02-01

    Cholera toxin, a heterohexameric AB{sub 5} enterotoxin released by Vibrio cholera, induces a profuse secretory diarrhea in susceptible hosts. Choleragenoid, the B subunit pentamer of cholera toxin, directs the enzymatic A subunit to its target by binding to GM{sub 1} gangliosides exposed on the luminal surface of intestinal epithelial cells. We have solved the crystal structure of choleragenoid at 2.3 {Angstrom} resolution by combining single isomorphous replacement with non-crystallographic symmetry averaging. The structure of the B subunits, and their pentameric arrangement, closely resembles that reported for the intact holotoxin (choleragen), the heat-labile enterotoxin from E. coli, and for a choleragenoid-GM{sub 1} pentasaccharide complex. In the absence of the A subunit the central cavity of the B pentamer is a highly solvated channel. The binding of the A subunit or the receptor pentasaccharide to choleragenoid has only a modest effect on the local stereochemistry and does not perceptibly alter the subunit interface.

  6. Contagem de Staphylococcus sp. e detecção de enterotoxinas estafilocócicas e toxina da síndrome do choque tóxico em amostras de leite cru refrigerado Staphylococcus sp. counting and detection of staphylococcal enterotoxins and toxic shock toxin syndrome from cooled raw milk

    Directory of Open Access Journals (Sweden)

    H.C. Lamaita

    2005-10-01

    Full Text Available Analisaram-se 80 amostras de leite cru refrigerado a 4°C e estocado por 48 horas em tanques refrigeradores de propriedades rurais do estado de Minas Gerais quanto à contagem e identificação de Staphylococcus sp. e detecção de enterotoxinas estafilocócicas (SE e da toxina da síndrome do choque tóxico (TSST-1. Staphylococcus sp. foi detectado em 100% das amostras de leite de tanque refrigerador em contagens que variaram de 1,0 × 10(5 a 2,5 × 10(7 UFC/ml (média = 5,60 log UFC/ml; s = 0,53 e CV = 9,5%. Isolaram-se e identificaram-se 436 estirpes como: S. aureus, S hyicus, S. epidermidis, S. intermedius, S. cohnii, S. sciuri, S. schleirferi e S. delphini. As estirpes de mesmo perfil bioquímico, oriundas da mesma amostra, foram agrupadas (pools e induzidas a produzir SE e TSST-1. A detecção dessas enterotoxinas foi feita pelo método optimum sensitivity plate, usando-se técnica de celofane sobre ágar. Identificou-se a produção de SEA, SEB, SEC, SED e de TSST-1 em percentuais variados. Dos 138 pools preparados, 91 produziram, pelo menos, uma toxina isoladamente ou em associação a outras toxinas. Dos pools enterotoxigênicos, 24,6% eram coagulase positiva e 41,3%, coagulase negativa. A confirmação de estirpes enterotoxigênicas de Staphylococcus coagulase negativa isoladas de amostras de leite é importante em relação à saúde pública.In order to count and identify Staphylococcus sp., the detection of the Staphylococcal enterotoxins (SE and toxic shock toxin syndrome (TSST-1, 80 raw milk samples cooled at 4°C and stored in bulk tanks for 48 hours in different farms from Minas Gerais State were analyzed. Staphylococcus sp. was observed in all samples and the counts varied from 1.0 × 10(5 to 2.5 × 10(7 CFU/ml (mean = 5.60 log CFU/ml; sd = 0.53 and CV = 9.46 %. A total of 436 strains of Staphylococcus were isolated and identified as S. aureus, S hyicus, S. epidermidis, S. intermedius, S. cohnii, S. sciuri, S. schleirferi and S

  7. Staphylococcal enterotoxins stimulate lymphoma-associated immune dysregulation

    DEFF Research Database (Denmark)

    Krejsgaard, Thorbjørn Frej; Willerslev-Olsen, Andreas; Lindahl, Lise Maria

    2014-01-01

    a cascade of events involving cell-cell and asymmetric cytokine interactions between malignant and benign T cells, which stimulated the malignant T cells to express high levels of IL-10. Much evidence supports that malignant activation of the Stat3/IL-10 axis plays a key role in driving the immune...... dysregulation and severe immunodeficiency that characteristically develops in CTCL patients. The present findings thereby establish a novel link between SEs and immune dysregulation in CTCL strengthening the rationale for antibiotic treatment of colonized patients with severe or progressive disease....

  8. Prevalence, antibiotic-resistance properties and enterotoxin gene ...

    African Journals Online (AJOL)

    Methods: Three-hundred milk-based infant foods were collected and immediately transferred to the laboratory. ... and entFM (55.5 %) were the most commonly detected enterotoxigenic genes. Bacteria showed the ... conditions like pasteurization and sterilization. [2,3]. ... spaghetti, noodle, milk powder and infant formula.

  9. Prevalence, antibiotic-resistance properties and enterotoxin gene ...

    African Journals Online (AJOL)

    milk-based infant foods in Iran, represent an important public health issue which should be considered ... Keywords: Prevalence, Bacillus cereus, Antibiotic resistance, Enterotoxigenic genes, Milk-based infant food. Tropical Journal of Pharmaceutical Research is indexed by Science ..... and cereals collected in Korea.

  10. Clonal relatedness of enterotoxigenic Escherichia coli (ETEC) strains expressing LT and CS17 isolated from children with diarrhoea in La Paz, Bolivia.

    Science.gov (United States)

    Rodas, Claudia; Klena, John D; Nicklasson, Matilda; Iniguez, Volga; Sjöling, Asa

    2011-01-01

    Enterotoxigenic Escherichia coli (ETEC) is a major cause of traveller's and infantile diarrhoea in the developing world. ETEC produces two toxins, a heat-stable toxin (known as ST) and a heat-labile toxin (LT) and colonization factors that help the bacteria to attach to epithelial cells. In this study, we characterized a subset of ETEC clinical isolates recovered from Bolivian children under 5 years of age using a combination of multilocus sequence typing (MLST) analysis, virulence typing, serotyping and antimicrobial resistance test patterns in order to determine the genetic background of ETEC strains circulating in Bolivia. We found that strains expressing the heat-labile (LT) enterotoxin and colonization factor CS17 were common and belonged to several MLST sequence types but mainly to sequence type-423 and sequence type-443 (Achtman scheme). To further study the LT/CS17 strains we analysed the nucleotide sequence of the CS17 operon and compared the structure to LT/CS17 ETEC isolates from Bangladesh. Sequence analysis confirmed that all sequence type-423 strains from Bolivia had a single nucleotide polymorphism; SNP(bol) in the CS17 operon that was also found in some other MLST sequence types from Bolivia but not in strains recovered from Bangladeshi children. The dominant ETEC clone in Bolivia (sequence type-423/SNP(bol)) was found to persist over multiple years and was associated with severe diarrhoea but these strains were variable with respect to antimicrobial resistance patterns. The results showed that although the LT/CS17 phenotype is common among ETEC strains in Bolivia, multiple clones, as determined by unique MLST sequence types, populate this phenotype. Our data also appear to suggest that acquisition and loss of antimicrobial resistance in LT-expressing CS17 ETEC clones is more dynamic than acquisition or loss of virulence factors.

  11. Clonal Relatedness of Enterotoxigenic Escherichia coli (ETEC) Strains Expressing LT and CS17 Isolated from Children with Diarrhoea in La Paz, Bolivia

    Science.gov (United States)

    Rodas, Claudia; Klena, John D.; Nicklasson, Matilda; Iniguez, Volga; Sjöling, Åsa

    2011-01-01

    Background Enterotoxigenic Escherichia coli (ETEC) is a major cause of traveller's and infantile diarrhoea in the developing world. ETEC produces two toxins, a heat-stable toxin (known as ST) and a heat-labile toxin (LT) and colonization factors that help the bacteria to attach to epithelial cells. Methodology/Principal Findings In this study, we characterized a subset of ETEC clinical isolates recovered from Bolivian children under 5 years of age using a combination of multilocus sequence typing (MLST) analysis, virulence typing, serotyping and antimicrobial resistance test patterns in order to determine the genetic background of ETEC strains circulating in Bolivia. We found that strains expressing the heat-labile (LT) enterotoxin and colonization factor CS17 were common and belonged to several MLST sequence types but mainly to sequence type-423 and sequence type-443 (Achtman scheme). To further study the LT/CS17 strains we analysed the nucleotide sequence of the CS17 operon and compared the structure to LT/CS17 ETEC isolates from Bangladesh. Sequence analysis confirmed that all sequence type-423 strains from Bolivia had a single nucleotide polymorphism; SNPbol in the CS17 operon that was also found in some other MLST sequence types from Bolivia but not in strains recovered from Bangladeshi children. The dominant ETEC clone in Bolivia (sequence type-423/SNPbol) was found to persist over multiple years and was associated with severe diarrhoea but these strains were variable with respect to antimicrobial resistance patterns. Conclusion/Significance The results showed that although the LT/CS17 phenotype is common among ETEC strains in Bolivia, multiple clones, as determined by unique MLST sequence types, populate this phenotype. Our data also appear to suggest that acquisition and loss of antimicrobial resistance in LT-expressing CS17 ETEC clones is more dynamic than acquisition or loss of virulence factors. PMID:22140423

  12. Genotypic and Phenotypic Characterization of Enterotoxigenic Escherichia coli Strains Isolated from Peruvian Children ▿

    Science.gov (United States)

    Rivera, F. P.; Ochoa, T. J.; Maves, R. C.; Bernal, M.; Medina, A. M.; Meza, R.; Barletta, F.; Mercado, E.; Ecker, L.; Gil, A. I.; Hall, E. R.; Huicho, L.; Lanata, C. F.

    2010-01-01

    Enterotoxigenic Escherichia coli (ETEC) is a major cause of childhood diarrhea. The present study sought to determine the prevalence and distribution of toxin types, colonization factors (CFs), and antimicrobial susceptibility of ETEC strains isolated from Peruvian children. We analyzed ETEC strains isolated from Peruvian children between 2 and 24 months of age in a passive surveillance study. Five E. coli colonies per patient were studied by multiplex real-time PCR to identify ETEC virulence factors. ETEC-associated toxins were confirmed using a GM1-based enzyme-linked immunosorbent assay. Confirmed strains were tested for CFs by dot blot assay using 21 monoclonal antibodies. We analyzed 1,129 samples from children with diarrhea and 744 control children and found ETEC in 5.3% and 4.3%, respectively. ETEC was more frequently isolated from children >12 months of age than from children <12 months of age (P < 0.001). Fifty-two percent of ETEC isolates from children with diarrhea and 72% of isolates from controls were heat-labile enterotoxin (LT) positive and heat-stable enterotoxin (ST) negative; 25% and 19%, respectively, were LT negative and ST positive; and 23% and 9%, respectively, were LT positive and ST positive. CFs were identified in 64% of diarrheal samples and 37% of control samples (P < 0.05). The most common CFs were CS6 (14% and 7%, respectively), CS12 (12% and 4%, respectively), and CS1 (9% and 4%, respectively). ST-producing ETEC strains caused more severe diarrhea than non-ST-producing ETEC strains. The strains were most frequently resistant to ampicillin (71%) and co-trimoxazole (61%). ETEC was thus found to be more prevalent in older infants. LT was the most common toxin type; 64% of strains had an identified CF. These data are relevant in estimating the burden of disease due to ETEC and the potential coverage of children in Peru by investigational vaccines. PMID:20631096

  13. Molecular characterization and antibiotic resistance of enterotoxigenic and entero-aggregative Escherichia coli isolated from raw milk and unpasteurized cheeses

    Directory of Open Access Journals (Sweden)

    Mojtaba Bonyadian

    2014-04-01

    Full Text Available The aim of this study was to determine the occurrence of enterotoxigenic and enteroaggregative Escherichia coli strains and antibiotic resistance of the isolates in raw milk and unpasteurized cheese. Out of 200 samples of raw milk and 50 samples of unpasteurized cheeses, 96 and 24 strains of E. coli were isolated, respectively. Polymerase chain reaction (PCR was used to detect the genes encoding heat-stable enterotoxin a (STa, heat-stable enterotoxin b (STb, heat labile toxin (LT and enteroaggregative heat-stable toxin1 (EAST1. Twelve out of 120 (10.00% isolates harbored the gene for EAST1, 2(1.66% isolates were detected as producing STb and LT toxins and 12 (10.00% strains contained STb and EAST1 genes. None of the strains contain the STa gene. All of the strains were tested for antibiotic resistance by disk diffusion method. Disks included: ciprofloxacin (CFN, trimetoprim-sulfamethoxazole (TSX, oxytetracycline (OTC, gentamicin (GMN, cephalexin (CPN, nalidixic acid (NDA and nitrofurantoin (NFN, ampicillin (AMP, neomycin (NEO and streptomycin (STM. Among 120 isolated strains of E. coli, the resistance to each antibiotics were as follows: OTC100%, CPN 86.00%, NDA 56.00%, NFN 42.00%, GMN 30.00%, TSX 28.00%, CFN 20%, AM 23.40% and STM 4.25%. None of the isolates were resistant to NEO. The present data indicate that different resistant E. coli pathogens may be found in raw milk and unpasteurized cheese. It poses an infection risk for human and transferring the resistant factors to microflora of the consumers gut.

  14. The Proteome of Biologically Active Membrane Vesicles from Piscirickettsia salmonis LF-89 Type Strain Identifies Plasmid-Encoded Putative Toxins

    Directory of Open Access Journals (Sweden)

    Cristian Oliver

    2017-09-01

    Full Text Available Piscirickettsia salmonis is the predominant bacterial pathogen affecting the Chilean salmonid industry. This bacterium is the etiological agent of piscirickettsiosis, a significant fish disease. Membrane vesicles (MVs released by P. salmonis deliver several virulence factors to host cells. To improve on existing knowledge for the pathogenicity-associated functions of P. salmonis MVs, we studied the proteome of purified MVs from the P. salmonis LF-89 type strain using multidimensional protein identification technology. Initially, the cytotoxicity of different MV concentration purified from P. salmonis LF-89 was confirmed in an in vivo adult zebrafish infection model. The cumulative mortality of zebrafish injected with MVs showed a dose-dependent pattern. Analyses identified 452 proteins of different subcellular origins; most of them were associated with the cytoplasmic compartment and were mainly related to key functions for pathogen survival. Interestingly, previously unidentified putative virulence-related proteins were identified in P. salmonis MVs, such as outer membrane porin F and hemolysin. Additionally, five amino acid sequences corresponding to the Bordetella pertussis toxin subunit 1 and two amino acid sequences corresponding to the heat-labile enterotoxin alpha chain of Escherichia coli were located in the P. salmonis MV proteome. Curiously, these putative toxins were located in a plasmid region of P. salmonis LF-89. Based on the identified proteins, we propose that the protein composition of P. salmonis LF-89 MVs could reflect total protein characteristics of this P. salmonis type strain.

  15. Algevir: An Expression System for Microalgae Based on Viral Vectors

    Directory of Open Access Journals (Sweden)

    Bernardo Bañuelos-Hernández

    2017-06-01

    Full Text Available The use of recombinant algae for the production of valuable compounds is opening promising biotechnological applications. However, the development of efficient expression approaches is still needed to expand the exploitation of microalgae in biotechnology. Herein, the concept of using viral expression vectors in microalgae was explored for the first time. An inducible geminiviral vector leading to Rep-mediated replication of the expression cassette allowed the production of antigenic proteins at high levels. This system, called Algevir, allows the production of complex viral proteins (GP1 from Zaire ebolavirus and bacterial toxin subunits (B subunit of the heat-labile Escherichia coli enterotoxin, which retained their antigenic activity. The highest achieved yield was 1.25 mg/g fresh biomass (6 mg/L of culture, which was attained 3 days after transformation. The Algevir system allows for a fast and efficient production of recombinant proteins, overcoming the difficulties imposed by the low yields and unstable expression patterns frequently observed in stably transformed microalgae at the nuclear level; as well as the toxicity of some target proteins.

  16. Cholera toxin B subunits assemble into pentamers--proposition of a fly-casting mechanism.

    Directory of Open Access Journals (Sweden)

    Jihad Zrimi

    Full Text Available The cholera toxin B pentamer (CtxB(5, which belongs to the AB(5 toxin family, is used as a model study for protein assembly. The effect of the pH on the reassembly of the toxin was investigated using immunochemical, electrophoretic and spectroscopic methods. Three pH-dependent steps were identified during the toxin reassembly: (i acquisition of a fully assembly-competent fold by the CtxB monomer, (ii association of CtxB monomer into oligomers, (iii acquisition of the native fold by the CtxB pentamer. The results show that CtxB(5 and the related heat labile enterotoxin LTB(5 have distinct mechanisms of assembly despite sharing high sequence identity (84% and almost identical atomic structures. The difference can be pinpointed to four histidines which are spread along the protein sequence and may act together. Thus, most of the toxin B amino acids appear negligible for the assembly, raising the possibility that assembly is driven by a small network of amino acids instead of involving all of them.

  17. Bystander protein protects potential vaccine-targeting ligands against intestinal proteolysis.

    Science.gov (United States)

    Reuter, Fabian; Bade, Steffen; Hirst, Timothy R; Frey, Andreas

    2009-07-20

    Endowing mucosal vaccines with ligands that target antigen to mucosal lymphoid tissues may improve immunization efficacy provided that the ligands withstand the proteolytic environment of the gastro-intestinal tract until they reach their destination. Our aim was to investigate whether and how three renowned ligands - Ulex europaeus agglutinin I and the B subunits of cholera toxin and E. coli heat-labile enterotoxin - master this challenge. We assessed the digestive power of natural murine intestinal fluid (natIF) using assays for trypsin, chymotrypsin and pancreatic elastase along with a test for nonspecific proteolysis. The natIF was compared with simulated murine intestinal fluid (simIF) that resembled the trypsin, chymotrypsin and elastase activities of its natural counterpart but lacked or contained albumins as additional protease substrates. The ligands were exposed to the digestive fluids and degradation was determined. The studies revealed that (i) the three pancreatic endoproteases constitute only one third of the total protease activity of natIF and (ii) the ligands resist proteolysis in natIF and protein-enriched simIF over 3 h but (iii) are partially destroyed in simIF that lacks additional protease substrate. We assume that the proteins of natIF are preferred substrates for the intestinal proteases and thus can protect vaccine-targeting ligands from destruction.

  18. [A study on the construction, expression and immunosterility of Lagurus laguru zona pellucida 3 DNA vaccine pVAX1-sig-LTB-lZP3-C3d3].

    Science.gov (United States)

    Li, Chen-Chen; Yu, Ji-Yun; Jiang, Min; Tu, Yi-Xian; Ma, Xiao-Lin; Zhang, Fu-Chun

    2011-09-01

    To enhance the immunocontraceptive effect of Lagurus lagurus zona pellucida 3 DNA vaccine, and to achieve the prospect of application through the pVAX1-sig-LTB-lZP3-C3d3 different immunity pathway. Two adjuvant molecules were constructed into the recombinant plasmid pVAX1-sig-LTB-lZP3-C3d3 as DNA vaccine which contains Escherichia coli heat-labile enterotoxin B subunit and the molecular adjuvant 3 copies of C3d. The results of RT-PCR and western blot showed that the DNA vaccine was expressed in mRNA and protein level. The female C57BL/6 mice were immunized by three ways: intramuscular injection, intranasal or oral route.Antibody levels and types were detected by ELISA. ELISA results showed that recombinant plasmid pVAX1-sig-LTB-lZP3-C3d3 immunization induced specific IgG, IgA levels were significantly different comparing with control (Psig-LTB-lZP3-C3d3 can induce the specific immune response efficiently and enhance the immunocontraceptive effects.

  19. Algevir: An Expression System for Microalgae Based on Viral Vectors

    Science.gov (United States)

    Bañuelos-Hernández, Bernardo; Monreal-Escalante, Elizabeth; González-Ortega, Omar; Angulo, Carlos; Rosales-Mendoza, Sergio

    2017-01-01

    The use of recombinant algae for the production of valuable compounds is opening promising biotechnological applications. However, the development of efficient expression approaches is still needed to expand the exploitation of microalgae in biotechnology. Herein, the concept of using viral expression vectors in microalgae was explored for the first time. An inducible geminiviral vector leading to Rep-mediated replication of the expression cassette allowed the production of antigenic proteins at high levels. This system, called Algevir, allows the production of complex viral proteins (GP1 from Zaire ebolavirus) and bacterial toxin subunits (B subunit of the heat-labile Escherichia coli enterotoxin), which retained their antigenic activity. The highest achieved yield was 1.25 mg/g fresh biomass (6 mg/L of culture), which was attained 3 days after transformation. The Algevir system allows for a fast and efficient production of recombinant proteins, overcoming the difficulties imposed by the low yields and unstable expression patterns frequently observed in stably transformed microalgae at the nuclear level; as well as the toxicity of some target proteins. PMID:28713333

  20. Immune responses of a chimaeric protein vaccine containing Mycoplasma hyopneumoniae antigens and LTB against experimental M. hyopneumoniae infection in pigs.

    Science.gov (United States)

    Marchioro, Silvana B; Sácristan, Rubén Del Pozo; Michiels, Annelies; Haesebrouck, Freddy; Conceição, Fabricio R; Dellagostin, Odir A; Maes, Dominiek

    2014-08-06

    A recombinant chimaeric protein containing three Mycoplasma hyopneumoniae antigens (C-terminal portion of P97, heat shock protein P42, and NrdF) fused to an adjuvant, the B subunit of heat-labile enterotoxin of Escherichia coli (LTB), was used to immunize pigs against enzootic pneumonia. The systemic and local immune responses, as well as the efficacy of the chimaeric protein in inducing protection against experimental M. hyopneumoniae infection were evaluated. In total, 60 male piglets, purchased from a M. hyopneumoniae-free herd, at 4 weeks of age were randomly allocated to six different experimental groups of 10 animals each: recombinant chimaeric protein by intramuscular (IM) (1) or intranasal (IN) (2) administration, commercial bacterin by IM administration (3), and the adjuvant LTB by IM (4, control group A) or IN (5, control group B) administration. All groups were immunized at 24 and 38 days of age and challenged at 52 days of age. The sixth group that was not challenged was used as the negative control (IN [n=5] or IM [n=5] administration of the LTB adjuvant). Compared with the non-challenged group, administration of the chimaeric protein induced significant (Phyopneumoniae infection in pigs. This lack of effectiveness points towards the need for further studies to improve the efficacy of this subunit-based vaccine approach. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. An adenovirus vectored mucosal adjuvant augments protection of mice immunized intranasally with an adenovirus-vectored foot-and-mouth disease virus subunit vaccine.

    Science.gov (United States)

    Alejo, Diana M; Moraes, Mauro P; Liao, Xiaofen; Dias, Camila C; Tulman, Edan R; Diaz-San Segundo, Fayna; Rood, Debra; Grubman, Marvin J; Silbart, Lawrence K

    2013-04-26

    Foot-and-mouth disease virus (FMDV) is a highly contagious pathogen that causes severe morbidity and economic losses to the livestock industry in many countries. The oral and respiratory mucosae are the main ports of entry of FMDV, so the stimulation of local immunity in these tissues may help prevent initial infection and viral spread. E. coli heat-labile enterotoxin (LT) has been described as one of the few molecules that have adjuvant activity at mucosal surfaces. The objective of this study was to evaluate the efficacy of replication-defective adenovirus 5 (Ad5) vectors encoding either of two LT-based mucosal adjuvants, LTB or LTR72. These vectored adjuvants were delivered intranasally to mice concurrent with an Ad5-FMDV vaccine (Ad5-A24) to assess their ability to augment mucosal and systemic humoral immune responses to Ad5-A24 and protection against FMDV. Mice receiving Ad5-A24 plus Ad5-LTR72 had higher levels of mucosal and systemic neutralizing antibodies than those receiving Ad5-A24 alone or Ad5-A24 plus Ad5-LTB. The vaccine plus Ad5-LTR72 group also demonstrated 100% survival after intradermal challenge with a lethal dose of homologous FMDV serotype A24. These results suggest that Ad5-LTR72 could be used as an important tool to enhance mucosal and systemic immunity against FMDV and potentially other pathogens with a common route of entry. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Immunogenicity of nuclear-encoded LTB:ST fusion protein from Escherichia coli expressed in tobacco plants.

    Science.gov (United States)

    Rosales-Mendoza, Sergio; Soria-Guerra, Ruth E; Moreno-Fierros, Leticia; Govea-Alonso, Dania O; Herrera-Díaz, Areli; Korban, Schuyler S; Alpuche-Solís, Ángel G

    2011-06-01

    Enterotoxigenic Escherichia coli (ETEC) is one of the main causative agents of diarrhea in infants and for travelers. Inclusion of a heat-stable (ST) toxin into vaccine formulations is mandatory as most ETEC strains can produce both heat-labile (LT) and ST enterotoxins. In this study, a genetic fusion gene encoding for an LTB:ST protein has been constructed and transferred into tobacco via Agrobacterium tumefaciens-mediated transformation. Transgenic tobacco plants carrying the LTB:ST gene are then subjected to GM1-ELISA revealing that the LTB:ST has assembled into pentamers and displays antigenic determinants from both LTB and ST. Protein accumulation of up to 0.05% total soluble protein is detected. Subsequently, mucosal and systemic humoral responses are elicited in mice orally dosed with transgenic tobacco leaves. This has suggested that the plant-derived LTB:ST is immunogenic via the oral route. These findings are critical for the development of a plant-based vaccine capable of eliciting broader protection against ETEC and targeting both LTB and ST. Features of this platform in comparison to transplastomic approaches are discussed.

  3. The Proteome of Biologically Active Membrane Vesicles from Piscirickettsia salmonis LF-89 Type Strain Identifies Plasmid-Encoded Putative Toxins.

    Science.gov (United States)

    Oliver, Cristian; Hernández, Mauricio A; Tandberg, Julia I; Valenzuela, Karla N; Lagos, Leidy X; Haro, Ronie E; Sánchez, Patricio; Ruiz, Pamela A; Sanhueza-Oyarzún, Constanza; Cortés, Marcos A; Villar, María T; Artigues, Antonio; Winther-Larsen, Hanne C; Avendaño-Herrera, Ruben; Yáñez, Alejandro J

    2017-01-01

    Piscirickettsia salmonis is the predominant bacterial pathogen affecting the Chilean salmonid industry. This bacterium is the etiological agent of piscirickettsiosis, a significant fish disease. Membrane vesicles (MVs) released by P. salmonis deliver several virulence factors to host cells. To improve on existing knowledge for the pathogenicity-associated functions of P. salmonis MVs, we studied the proteome of purified MVs from the P. salmonis LF-89 type strain using multidimensional protein identification technology. Initially, the cytotoxicity of different MV concentration purified from P. salmonis LF-89 was confirmed in an in vivo adult zebrafish infection model. The cumulative mortality of zebrafish injected with MVs showed a dose-dependent pattern. Analyses identified 452 proteins of different subcellular origins; most of them were associated with the cytoplasmic compartment and were mainly related to key functions for pathogen survival. Interestingly, previously unidentified putative virulence-related proteins were identified in P. salmonis MVs, such as outer membrane porin F and hemolysin. Additionally, five amino acid sequences corresponding to the Bordetella pertussis toxin subunit 1 and two amino acid sequences corresponding to the heat-labile enterotoxin alpha chain of Escherichia coli were located in the P. salmonis MV proteome. Curiously, these putative toxins were located in a plasmid region of P. salmonis LF-89. Based on the identified proteins, we propose that the protein composition of P. salmonis LF-89 MVs could reflect total protein characteristics of this P. salmonis type strain.

  4. Species Distribution and Prevalence of Putative Virulence Factors in Mesophilic Aeromonas spp. Isolated from Fresh Retail Sushi

    Directory of Open Access Journals (Sweden)

    Sunniva Hoel

    2017-05-01

    Full Text Available Aeromonas spp. are ubiquitous bacteria that have received increasing attention as human pathogens because of their widespread occurrence in food, especially seafood and vegetables. The aim of this work was to assess the species identity and phylogenetic relationship of 118 Aeromonas strains isolated from fresh retail sushi from three producers, and to characterize the isolates with respect to genetic and phenotypic virulence factors. We also evaluate the potential hazard associated with their presence in ready-to-eat seafood not subjected to heat treatment. Mesophilic Aeromonas salmonicida was most prevalent (74%, followed by A. bestiarum (9%, A. dhakensis (5%, A. caviae (5%, A. media (4%, A. hydrophila (2%, and A. piscicola (1%. All isolates were considered potentially pathogenic due to the high prevalence of genes encoding hemolysin (hlyA (99%, aerolysin (aerA (98%, cytotoxic enterotoxin (act (86%, heat-labile cytotonic enterotoxin (alt (99%, and heat-stable cytotonic enterotoxin (ast (31%. The shiga-like toxins 1 and 2 (stx-1 and stx-2 were not detected. Moreover, there was heterogeneity in toxin gene distribution among the isolates, and the combination of act/alt/hlyA/aerA was most commonly detected (63%. β-hemolysis was species-dependent and observed in 91% of the isolates. All A. media and A. caviae strains were non-hemolytic. For isolates belonging to this group, lack of hemolysis was possibly related to the absence of the act gene. Swimming motility, linked to adhesion and host invasion, occurred in 65% of the isolates. Partial sequencing of the gyrB gene demonstrated its suitability as a genetic marker for Aeromonas species identification and for assessment of the phylogenetic relationship between the isolates. The gyrB sequence divergence within a given species ranged from 1.3 to 2.9%. A. bestiarum, A. salmonicida, and A. piscicola were the most closely related species; their sequences differed by 2.7–3.4%. The average gyrB sequence

  5. Pilot Scale Production and Testing of a Recombinant Staphylococcal Enterotoxin (SEB) Triple Mutant

    Science.gov (United States)

    2017-09-01

    Polymerase Chain Reaction . J. Food Prot. 2000, 63, (4), 479–488. 6. Ulrich, R.G.; Olson, M.A.; Bavari, S. Development of Engineered Vaccines...TR-1471 9. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) Joint Program Executive Office for Chemical and Biological Defense, Joint Project...distribution unlimited. 13. SUPPLEMENTARY NOTES 14. ABSTRACT: The use of chemically or thermally inactivated biothreat toxins in detection

  6. Stress-caused Anergy of Leukocytes towards Staphylococcal enterotoxin B and Exposure Transcriptome Signatures

    Science.gov (United States)

    2015-05-28

    concerted immune depletion was highly pronounced in the case of genes involved in synthesis /secretion of chemokines and antigen presentation. (c) The 21...biosynthetic process 48545 8.03E-03 12 Response to steroid hormone stimulus 16126 9.50E-03 5 Sterol biosynthetic process 6950 1.01E-02 44 Response to

  7. Molecular analysis of the interaction between Clostridium perfringens Enterotoxin and Claudins

    OpenAIRE

    Protze, Jonas

    2015-01-01

    Claudins are essential constituents of Tight Junctions (TJs) and responsible for maintenance of these cell-cell contacts. Binding of Clostridium perfringens Enterotoxin’s C-terminal domain (cCPE) to the extracellular loop 2 (EZS2) of claudins, especially Cld3, Cld4 and Cld6-Cld9 causes a reversible opening of TJs. Thus, a structure-function analysis of this system is relevant for biomedical application, since cCPE could be used to enhance paracellular drug uptake. Furthermore cCPE respectivel...

  8. Genetic mapping of the regulator gene determining enterotoxin synthesis in Vibrio cholerae

    International Nuclear Information System (INIS)

    Smirnova, N.I.; Livanova, L.F.; Shaginyan, I.A.; Motin, V.L.

    1986-01-01

    Data on the genetic mapping of mutation tox-7 (the mutation affecting the synthesis of the cholera toxin) were obtained by conjugation crosses between the atoxigenic donor strain Vibrio cholerae Eltor and the toxigenic recipient strain V. cholera classica. The molecular and genetic analysis of the Tox - recombinants indicated that, when the synthesis of the cholera toxin is disrupted in these strains, the tox-7 mutation (which impairs the regulator gene tox) is gained. Close linkage between the tox-7 and pur-63 mutations was established (during the selection procedure there was 81.1% combined transfer with respect to marker pur-63 situated in the donor strain chromosome more proximal than mutation tox-7). The markers were localized in the following order in the region under investigation: asp-cys-nal-pur-61-trp-his-pur-63-tox-7-ile

  9. Staphylococcus enterotoxin A modulates interleukin 15-induced signaling and mitogenesis in human T cells

    DEFF Research Database (Denmark)

    Gerwien, J; Kaltoft, K; Nielsen, M

    1998-01-01

    the anti-mitogenic effect of SEA on cytokine-induced proliferation and the pro-mitogenic effect of PMA. In contrast, inhibitors of PP1, PP2A, protein kinase C (PKC), phosphatidyl-inositol-3-kinase (PI-3K) and mammalian target of rapamycin (mTOR) are unable to inhibit the effects of SEA. In a SEA "non...

  10. Inhibition of Biological Activity of Staphylococcal Enterotoxin A (SEA) by Apple Juice and Apple Polyphenols

    Science.gov (United States)

    The foodborne pathogen Staphylococcus aureus produces the virulent staphylococcal entertoxin A (SEA), a single-chain protein that consists of 233 amino acid residues with a molecular weight of 27 078 Da. SEA is a superantigen that is reported to contribute to animal (mastitis) and human (emesis, dia...

  11. Bacillus anthracis Edema Toxin Inhibits Staphylococcus aureus Enterotoxin B Effects in Vitro: A Potential Protein Therapeutic?

    Science.gov (United States)

    2005-10-01

    5). Inherent characteristics of edema toxin and other procaryotic adenylate cyclases from Bordetella pertussis, Pseudomonas aeruginosa, and Yersinia...by mouse peritoneal macrophages: the role of cellular cyclic AMP. Immunology 64:719–724. 12. Krakauer, T. 1999. Induction of CC chemokines in human

  12. Side-by-side comparison of gene-based smallpox vaccine with MVA in nonhuman primates.

    Science.gov (United States)

    Golden, Joseph W; Josleyn, Matthew; Mucker, Eric M; Hung, Chien-Fu; Loudon, Peter T; Wu, T C; Hooper, Jay W

    2012-01-01

    Orthopoxviruses remain a threat as biological weapons and zoonoses. The licensed live-virus vaccine is associated with serious health risks, making its general usage unacceptable. Attenuated vaccines are being developed as alternatives, the most advanced of which is modified-vaccinia virus Ankara (MVA). We previously developed a gene-based vaccine, termed 4pox, which targets four orthopoxvirus antigens, A33, B5, A27 and L1. This vaccine protects mice and non-human primates from lethal orthopoxvirus disease. Here, we investigated the capacity of the molecular adjuvants GM-CSF and Escherichia coli heat-labile enterotoxin (LT) to enhance the efficacy of the 4pox gene-based vaccine. Both adjuvants significantly increased protective antibody responses in mice. We directly compared the 4pox plus LT vaccine against MVA in a monkeypox virus (MPXV) nonhuman primate (NHP) challenge model. NHPs were vaccinated twice with MVA by intramuscular injection or the 4pox/LT vaccine delivered using a disposable gene gun device. As a positive control, one NHP was vaccinated with ACAM2000. NHPs vaccinated with each vaccine developed anti-orthopoxvirus antibody responses, including those against the 4pox antigens. After MPXV intravenous challenge, all control NHPs developed severe disease, while the ACAM2000 vaccinated animal was well protected. All NHPs vaccinated with MVA were protected from lethality, but three of five developed severe disease and all animals shed virus. All five NHPs vaccinated with 4pox/LT survived and only one developed severe disease. None of the 4pox/LT-vaccinated animals shed virus. Our findings show, for the first time, that a subunit orthopoxvirus vaccine delivered by the same schedule can provide a degree of protection at least as high as that of MVA.

  13. Intradermal delivery of Shigella IpaB and IpaD type III secretion proteins: kinetics of cell recruitment and antigen uptake, mucosal and systemic immunity, and protection across serotypes.

    Science.gov (United States)

    Heine, Shannon J; Diaz-McNair, Jovita; Andar, Abhay U; Drachenberg, Cinthia B; van de Verg, Lillian; Walker, Richard; Picking, Wendy L; Pasetti, Marcela F

    2014-02-15

    Shigella is one of the leading pathogens contributing to the vast pediatric diarrheal disease burden in low-income countries. No licensed vaccine is available, and the existing candidates are only partially effective and serotype specific. Shigella type III secretion system proteins IpaB and IpaD, which are conserved across Shigella spp., are candidates for a broadly protective, subunit-based vaccine. In this study, we investigated the immunogenicity and protective efficacy of IpaB and IpaD administered intradermally (i.d.) with a double-mutant of the Escherichia coli heat-labile enterotoxin (dmLT) adjuvant using microneedles. Different dosage levels of IpaB and IpaD, with or without dmLT, were tested in mice. Vaccine delivery into the dermis, recruitment of neutrophils, macrophages, dendritic cells, and Langerhans cells, and colocalization of vaccine Ag within skin-activated APC were demonstrated through histology and immunofluorescence microscopy. Ag-loaded neutrophils, macrophages, dendritic cells, and Langerhans cells remained in the tissue at least 1 wk. IpaB, IpaD, and dmLT-specific serum IgG- and IgG-secreting cells were produced following i.d. immunization. The protective efficacy was 70% against Shigella flexneri and 50% against Shigella sonnei. Similar results were obtained when the vaccine was administered intranasally, with the i.d. route requiring 25-40 times lower doses. Distinctively, IgG was detected in mucosal secretions; secretory IgA, as well as mucosal and systemic IgA Ab-secreting cells, were seemingly absent. Vaccine-induced T cells produced IFN-γ, IL-2, TNF-α, IL-17, IL-4, IL-5, and IL-10. These results demonstrate the potential of i.d. vaccination with IpaB and IpaD to prevent Shigella infection and support further studies in humans.

  14. The oral, live attenuated enterotoxigenic Escherichia coli vaccine ACE527 reduces the incidence and severity of diarrhea in a human challenge model of diarrheal disease.

    Science.gov (United States)

    Darsley, Michael J; Chakraborty, Subhra; DeNearing, Barbara; Sack, David A; Feller, Andrea; Buchwaldt, Charlotte; Bourgeois, A Louis; Walker, Richard; Harro, Clayton D

    2012-12-01

    An oral, live attenuated, three-strain recombinant bacterial vaccine, ACE527, was demonstrated to generate strong immune responses to colonization factor and toxin antigens of enterotoxigenic Escherichia coli (ETEC) in human volunteers. The vaccine was safe and well tolerated at doses of up to 10(11) CFU, administered in each of two doses given 21 days apart. These observations have now been extended in a phase 2b study with a total of 70 subjects. Fifty-six of these subjects were challenged 28 days after the second dose of vaccine with the highly virulent ETEC strain H10407 to obtain preliminary indicators of efficacy against disease and to support further development of the vaccine for both travelers and infants in countries where ETEC is endemic. The vaccine had a significant impact on intestinal colonization by the challenge strain, as measured by quantitative fecal culture 2 days after challenge, demonstrating the induction of a functional immune response to the CFA/I antigen. The incidence and severity of diarrhea were also reduced in vaccinees as measured by a number of secondary and ad hoc endpoints, although the 27% reduction seen in the primary endpoint, moderate to severe diarrhea, was not statistically significant. Together, these observations support the hypothesis that the ACE527 vaccine has a dual mode of action, targeting both colonization factors and the heat-labile enterotoxin (LT), and suggest that it should be further developed for more advanced trials to evaluate its impact on the burden of ETEC disease in field settings.

  15. Production and evaluation of a recombinant chimeric vaccine against clostridium botulinum neurotoxin types C and D.

    Directory of Open Access Journals (Sweden)

    Luciana A F Gil

    Full Text Available Bovine botulism is a fatal disease that is caused by botulinum neurotoxins (BoNTs produced by Clostridium botulinum serotypes C and D and that causes great economic losses, with nearly 100% lethality during outbreaks. It has also been considered a potential source of human food-borne illness in many countries. Vaccination has been reported to be the most effective way to control bovine botulism. However, the commercially available toxoid-based vaccines are difficult and hazardous to produce. Neutralizing antibodies targeted against the C-terminal fragment of the BoNT heavy chain (HC are known to confer efficient protection against lethal doses of BoNTs. In this study, a novel recombinant chimera, consisting of Escherichia coli heat-labile enterotoxin B subunit (LTB, a strong adjuvant of the humoral immune response, fused to the HC of BoNT serotypes C and D, was produced in E. coli. Mice vaccinated with the chimera containing LTB and an equivalent molar ratio of the chimera without LTB plus aluminum hydroxide (Al(OH3 developed 2 IU/mL of antitoxins for both serotypes. Guinea pigs immunized with the recombinant chimera with LTB plus Al(OH3 developed a protective immune response against both BoNT/C (5 IU/mL and BoNT/D (10 IU/mL, as determined by a mouse neutralization bioassay with pooled sera. The results achieved with guinea pig sera fulfilled the requirements of commercial vaccines for prevention of botulism, as determined by the Brazilian Ministry of Agriculture, Livestock and Food, Supply. The presence of LTB was essential for the development of a strong humoral immune response, as it acted in synergism with Al(OH3. Thus, the vaccine described in this study is a strong candidate for the control of botulism in cattle.

  16. Side-by-side comparison of gene-based smallpox vaccine with MVA in nonhuman primates.

    Directory of Open Access Journals (Sweden)

    Joseph W Golden

    Full Text Available Orthopoxviruses remain a threat as biological weapons and zoonoses. The licensed live-virus vaccine is associated with serious health risks, making its general usage unacceptable. Attenuated vaccines are being developed as alternatives, the most advanced of which is modified-vaccinia virus Ankara (MVA. We previously developed a gene-based vaccine, termed 4pox, which targets four orthopoxvirus antigens, A33, B5, A27 and L1. This vaccine protects mice and non-human primates from lethal orthopoxvirus disease. Here, we investigated the capacity of the molecular adjuvants GM-CSF and Escherichia coli heat-labile enterotoxin (LT to enhance the efficacy of the 4pox gene-based vaccine. Both adjuvants significantly increased protective antibody responses in mice. We directly compared the 4pox plus LT vaccine against MVA in a monkeypox virus (MPXV nonhuman primate (NHP challenge model. NHPs were vaccinated twice with MVA by intramuscular injection or the 4pox/LT vaccine delivered using a disposable gene gun device. As a positive control, one NHP was vaccinated with ACAM2000. NHPs vaccinated with each vaccine developed anti-orthopoxvirus antibody responses, including those against the 4pox antigens. After MPXV intravenous challenge, all control NHPs developed severe disease, while the ACAM2000 vaccinated animal was well protected. All NHPs vaccinated with MVA were protected from lethality, but three of five developed severe disease and all animals shed virus. All five NHPs vaccinated with 4pox/LT survived and only one developed severe disease. None of the 4pox/LT-vaccinated animals shed virus. Our findings show, for the first time, that a subunit orthopoxvirus vaccine delivered by the same schedule can provide a degree of protection at least as high as that of MVA.

  17. Comparative Genomics and Characterization of Hybrid Shigatoxigenic and Enterotoxigenic Escherichia coli (STEC/ETEC) Strains.

    Science.gov (United States)

    Nyholm, Outi; Halkilahti, Jani; Wiklund, Gudrun; Okeke, Uche; Paulin, Lars; Auvinen, Petri; Haukka, Kaisa; Siitonen, Anja

    2015-01-01

    Shigatoxigenic Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC) cause serious foodborne infections in humans. These two pathogroups are defined based on the pathogroup-associated virulence genes: stx encoding Shiga toxin (Stx) for STEC and elt encoding heat-labile and/or est encoding heat-stable enterotoxin (ST) for ETEC. The study investigated the genomics of STEC/ETEC hybrid strains to determine their phylogenetic position among E. coli and to define the virulence genes they harbor. The whole genomes of three STEC/ETEC strains possessing both stx and est genes were sequenced using PacBio RS sequencer. Two of the strains were isolated from the patients, one with hemolytic uremic syndrome, and one with diarrhea. The third strain was of bovine origin. Core genome analysis of the shared chromosomal genes and comparison with E. coli and Shigella spp. reference genomes was performed to determine the phylogenetic position of the STEC/ETEC strains. In addition, a set of virulence genes and ETEC colonization factors were extracted from the genomes. The production of Stx and ST were studied. The human STEC/ETEC strains clustered with strains representing ETEC, STEC, enteroaggregative E. coli, and commensal and laboratory-adapted E. coli. However, the bovine STEC/ETEC strain formed a remote cluster with two STECs of bovine origin. All three STEC/ETEC strains harbored several other virulence genes, apart from stx and est, and lacked ETEC colonization factors. Two STEC/ETEC strains produced both toxins and one strain Stx only. This study shows that pathogroup-associated virulence genes of different E. coli can co-exist in strains originating from different phylogenetic lineages. The possibility of virulence genes to be associated with several E. coli pathogroups should be taken into account in strain typing and in epidemiological surveillance. Development of novel hybrid E. coli strains may cause a new public health risk, which challenges the traditional diagnostics

  18. Studies on the antidiarrhoeal activity of Aegle marmelos unripe fruit: Validating its traditional usage

    Directory of Open Access Journals (Sweden)

    Antia Noshir

    2009-11-01

    Full Text Available Abstract Background Aegle marmelos (L. Correa has been widely used in indigenous systems of Indian medicine due to its various medicinal properties. However, despite its traditional usage as an anti-diarrhoeal there is limited information regarding its mode of action in infectious forms of diarrhoea. Hence, we evaluated the hot aqueous extract (decoction of dried unripe fruit pulp of A. marmelos for its antimicrobial activity and effect on various aspects of pathogenicity of infectious diarrhoea. Methods The decoction was assessed for its antibacterial, antigiardial and antirotaviral activities. The effect of the decoction on adherence of enteropathogenic Escherichia coli and invasion of enteroinvasive E. coli and Shigella flexneri to HEp-2 cells were assessed as a measure of its effect on colonization. The effect of the decoction on production of E. coli heat labile toxin (LT and cholera toxin (CT and their binding to ganglioside monosialic acid receptor (GM1 were assessed by GM1-enzyme linked immuno sorbent assay whereas its effect on production and action of E. coli heat stable toxin (ST was assessed by suckling mouse assay. Results The decoction showed cidal activity against Giardia and rotavirus whereas viability of none of the six bacterial strains tested was affected. It significantly reduced bacterial adherence to and invasion of HEp-2 cells. The extract also affected production of CT and binding of both LT and CT to GM1. However, it had no effect on ST. Conclusion The decoction of the unripe fruit pulp of A. marmelos, despite having limited antimicrobial activity, affected the bacterial colonization to gut epithelium and production and action of certain enterotoxins. These observations suggest the varied possible modes of action of A. marmelos in infectious forms of diarrhoea thereby validating its mention in the ancient Indian texts and continued use by local communities for the treatment of diarrhoeal diseases.

  19. LTB-R1: an alternative to swine mycoplasmal pneumoniae control LTB-R1: uma alternativa para o controle da pneumonia micoplásmica suína

    Directory of Open Access Journals (Sweden)

    Fabrício Rochedo Conceição

    2003-11-01

    Full Text Available Mycoplasmal pneumoniae is the main respiratory disease in swine. The most efficient way to control it is through the use of vaccines (bacterins, whose production cost is high. The objective of this work was to develop a new alternative for controlling Swine Mycoplasmal Pneumoniae, based on a recombinant subunit vaccine containing the R1 region of P97 adhesin of Mycoplasma hyopneumoniae fused to the B subunit of the heat-labile enterotoxin of Escherichia coli (rLTB-R1. In this work we report the amplification of the genes, genetic fusion between LTB and R1 coding sequences, cloning, construction of the expression vector, as well as expression and purification of rLTB-R1 in E. coli.A Pneumonia Micoplásmica é a doença respiratória mais importante dos suínos. A forma mais eficaz de controlá-la é mediante a utilização de vacinas (bacterinas, cujo custo de produção é elevado. Uma nova alternativa para o controle desta doença, baseada em uma vacina de subunidade recombinante contendo a região R1 da adesina P97 de Mycoplasma hyopneumoniae fusionada a subunidade B da enterotoxina termolábel de Escherichia coli (rLTB-R1, foi o alvo deste trabalho. Nele abordou-se a amplificação dos genes, a fusão genética entre as seqüências codificadoras para LTB e R1, a clonagem, a construção do vetor de expressão, assim como a expressão em E. coli e purificação da rLTBR1.

  20. Prophylactic Administration of Bacterially Derived Immunomodulators Improves the Outcome of Influenza Virus Infection in a Murine Model▿

    Science.gov (United States)

    Norton, Elizabeth B.; Clements, John D.; Voss, Thomas G.; Cárdenas-Freytag, Lucia

    2010-01-01

    Prophylactic or therapeutic immunomodulation is an antigen-independent strategy that induces nonspecific immune system activation, thereby enhancing host defense to disease. In this study, we investigated the effect of prophylactic immunomodulation on the outcome of influenza virus infection using three bacterially derived immune-enhancing agents known for promoting distinct immunological profiles. BALB/c mice were treated nasally with either cholera toxin (CT), a mutant form of the CT-related Escherichia coli heat-labile enterotoxin designated LT(R192G), or CpG oligodeoxynucleotide. Mice were subsequently challenged with a lethal dose of influenza A/PR/8/34 virus 24 h after the last immunomodulation treatment and either monitored for survival or sacrificed postchallenge for viral and immunological analysis. Treatment with the three immunomodulators prevented or delayed mortality and weight loss, but only CT and LT(R192G) significantly reduced initial lung viral loads as measured by plaque assay. Analysis performed 4 days postinfection indicated that prophylactic treatments with CT, LT(R192G), or CpG resulted in significantly increased numbers of CD4 T cells, B cells, and dendritic cells and altered costimulatory marker expression in the airways of infected mice, coinciding with reduced expression of pulmonary chemokines and the appearance of inducible bronchus-associated lymphoid tissue-like structures in the lungs. Collectively, these results suggest that, despite different immunomodulatory mechanisms, CT, LT(R192G), and CpG induce an initial inflammatory process and enhance the immune response to primary influenza virus challenge while preventing potentially damaging chemokine expression. These studies provide insight into the immunological parameters and immune modulation strategies that have the potential to enhance the nonspecific host response to influenza virus infection. PMID:20053748

  1. Newer insights into the mechanism of action of Psidium guajava L. leaves in infectious diarrhoea.

    Science.gov (United States)

    Birdi, Tannaz; Daswani, Poonam; Brijesh, S; Tetali, Pundarikakshudu; Natu, Arvind; Antia, Noshir

    2010-06-28

    Psidium guajava L., Myrtaceae, is used widely in traditional medicine for the treatment of diarrhoea, dysentery, gastroenteritis, stomachaches, and indigestion. However, the effect of the leaf extract of P. guajava on the pathogenesis of infectious diarrhoea has not been studied. The present study evaluates the effect of a hot aqueous extract (decoction) of dried leaves of P. guajava on parameters associated with pathogenicity of infectious diarrhoea. The aim was to understand its possible mechanism(s) of action in controlling infectious diarrhoea and compare it with quercetin, one of the most reported active constituents of P. guajava with antidiarrhoeal activity. The crude decoction and quercetin were studied for their antibacterial activity and effect on virulence features of common diarrhoeal pathogens viz. colonization of epithelial cells and production and action of enterotoxins. Colonization as measured by adherence of enteropathogenic Escherichia coli (EPEC) and invasion of enteroinvasive E. coli (EIEC) and Shigella flexneri was assessed using HEp-2 cell line. The production of E. coli heat labile toxin (LT) and cholera toxin (CT) and their binding to ganglioside monosialic acid (GM1) were studied by GM1-ELISA whereas the production and action of E. coli heat stable toxin (ST) was assessed by suckling mouse assay. The decoction of P. guajava showed antibacterial activity towards S. flexneri and Vibrio cholerae. It decreased production of both LT and CT and their binding to GM1. However, it had no effect on production and action of ST. The decoction also inhibited the adherence of EPEC and invasion by both EIEC and S. flexneri to HEp-2 cells. Quercetin, on the other hand, had no antibacterial activity at the concentrations used nor did it affect any of the enterotoxins. Although it did not affect adherence of EPEC, it inhibited the invasion of both EIEC and S. flexneri to HEp-2 cells. Collectively, the results indicate that the decoction of P. guajava leaves

  2. Newer insights into the mechanism of action of Psidium guajava L. leaves in infectious diarrhoea

    Directory of Open Access Journals (Sweden)

    Natu Arvind

    2010-06-01

    Full Text Available Abstract Background Psidium guajava L., Myrtaceae, is used widely in traditional medicine for the treatment of diarrhoea, dysentery, gastroenteritis, stomachaches, and indigestion. However, the effect of the leaf extract of P. guajava on the pathogenesis of infectious diarrhoea has not been studied. The present study evaluates the effect of a hot aqueous extract (decoction of dried leaves of P. guajava on parameters associated with pathogenicity of infectious diarrhoea. The aim was to understand its possible mechanism(s of action in controlling infectious diarrhoea and compare it with quercetin, one of the most reported active constituents of P. guajava with antidiarrhoeal activity. Methods The crude decoction and quercetin were studied for their antibacterial activity and effect on virulence features of common diarrhoeal pathogens viz. colonization of epithelial cells and production and action of enterotoxins. Colonization as measured by adherence of enteropathogenic Escherichia coli (EPEC and invasion of enteroinvasive E. coli (EIEC and Shigella flexneri was assessed using HEp-2 cell line. The production of E. coli heat labile toxin (LT and cholera toxin (CT and their binding to ganglioside monosialic acid (GM1 were studied by GM1-ELISA whereas the production and action of E. coli heat stable toxin (ST was assessed by suckling mouse assay. Results The decoction of P. guajava showed antibacterial activity towards S. flexneri and Vibrio cholerae. It decreased production of both LT and CT and their binding to GM1. However, it had no effect on production and action of ST. The decoction also inhibited the adherence of EPEC and invasion by both EIEC and S. flexneri to HEp-2 cells. Quercetin, on the other hand, had no antibacterial activity at the concentrations used nor did it affect any of the enterotoxins. Although it did not affect adherence of EPEC, it inhibited the invasion of both EIEC and S. flexneri to HEp-2 cells. Conclusion Collectively

  3. Liquid Food Concentration

    Indian Academy of Sciences (India)

    First page Back Continue Last page Graphics. For ease of transport & storage. For ease of transport & storage. Heat labile liquids. Natural Colors; Fruit Juices. Thermal Evaporation. Thermal deterioration; Loss of volatiles; High energy consumption.

  4. Detection of Staphylococcus Aureus Enterotoxin A and B Genes with PCR-EIA and a Hand-Held Electrochemical Sensor

    National Research Council Canada - National Science Library

    Aitichou, Mohamed; Henkins, Robert; Sultana, Afroz M; Ulrich, Robert G; Ibrahim, M. S

    2004-01-01

    ... S. aureus DNA, and genomic DNA from Alcaligens, Bacillus, Bacteroides, Bordetella, Burkholderia, Clostridium, Comanonas, Enterobacter, Enterococcus, Escherichia, Francisella, Haemophilus, Klebsiella...

  5. Monoclonal antibodies protect from Staphylococcal Enterotoxin K (SEK) induced toxic shock and sepsis by USA300 Staphylococcus aureus.

    Science.gov (United States)

    Aguilar, Jorge L; Varshney, Avanish K; Pechuan, Ximo; Dutta, Kaushik; Nosanchuk, Joshua D; Fries, Bettina C

    2017-08-18

    Staphylococcus aureus is a leading infectious cause of life-threatening disease in humans, yet there is currently no vaccine to combat this bacterium. The pathogenesis of S. aureus is mediated by a diverse array of protein toxins including a large family of secreted pyrogenic superantigens. Neutralization of superantigens, including SEB and TSST-1, has proven to be protective in several animal models of toxic shock and sepsis. We demonstrate, for the first time, that a far more prevalent staphylococcal superantigen, SEK, can also induce lethal shock in mice. Additionally, we describe monoclonal antibodies (mAbs) that inhibit SEK-induced mitogenicity as well as protect against SEK-induced lethality, and enhance survival from S. aureus septicemia in murine models. MAb-4G3 (IgG2b), mAb-5G2 (IgG1), and mAb-9H2 (IgG1), all inhibit SEK-induced proliferation and cytokine production of human immune cells. We then demonstrate that passive immunization with a combination of mAb-4G3 and mAb-5G4, 2 mAbs that do not compete for epitope(s) on SEK, significantly enhance survival in a murine model of SEK-induced toxic shock (p = 0.006). In the setting of sepsis, passive immunization with this combination of mAbs also significantly enhances survival in mice after challenge with CA-MRSA strain USA300 (p = 0.03). Furthermore, septic mice that received mAb treatment in conjunction with vancomycin exhibit less morbidity than mice treated with vancomycin alone. Taken together, these findings suggest that the contribution of SEK to S. aureus pathogenesis may be greater than previously appreciated, and that adjunctive therapy with passive immunotherapy against SEs may be beneficial.

  6. Food compounds inhibit Staphylococcus aureus bacteria and the toxicity of Staphylococcus Enterotoxin A (SEA) associated with atopic dermatitis

    Science.gov (United States)

    Atopic dermatitis or eczema is characterized by skin rashes and itching is an inflammatory disease that affects 10-20% of children and 1-3% of adults. Staphylococcus aureus bacteria are present on the skin of nearly all patients with atopic dermatitis. Antibiotics that suppress colonization of S. au...

  7. Staphylococcal enterotoxin-A directly stimulates signal transduction and interferon-gamma production in psoriatic T-cell lines

    DEFF Research Database (Denmark)

    Nielsen, M B; Odum, N; Gerwien, J

    1998-01-01

    class II. Here we address the question of whether SEA can directly activate psoriatic T cells in the absence of professional antigen-presenting cells. We show that SEA induces i) tyrosine phosphorylation of several proteins, ii) downregulation of the T-cell receptor (TCR), and iii) production......-mediated proliferation. In contrast, SEA with a mutation in the MHC class II alpha-binding site induces IFN-gamma and a qualitatively changed tyrosine phosphorylation profile. Both mutations delete the co-stimulatory effect on cytokine-mediated proliferation. This suggests that both MHC class II binding sites...

  8. Dietary n-3 PUFAs augment caspase 8 activation in Staphylococcal aureus enterotoxin B stimulated T-cells

    Energy Technology Data Exchange (ETDEWEB)

    Gill, R. [Department of Immunology and Microbiology, Wayne State University, Detroit, MI (United States); Jen, K.L. [Department of Nutrition and Food Science, Wayne State University, Detroit, MI (United States); Center for Urban Responses to Environmental Stressors (CURES), Wayne State University, Detroit, MI (United States); McCabe, M.J.J. [Department of Environmental Medicine, University of Rochester, Rochester, NY (United States); Rosenspire, A., E-mail: arosenspire@wayne.edu [Department of Immunology and Microbiology, Wayne State University, Detroit, MI (United States); Center for Urban Responses to Environmental Stressors (CURES), Wayne State University, Detroit, MI (United States)

    2016-10-15

    Epidemiological studies have linked consumption of n-3 PUFAs with a variety of beneficial health benefits, particularly with respect to putative anti-inflammatory effects. Unfortunately, many of these results remain somewhat controversial because in most instances there has not been a linkage to specific molecular mechanisms. For instance, dietary exposure to low levels of mercury has been shown to be damaging to neural development, but concomitant ingestion of n-3 PUFAs as occurs during consumption of fish, has been shown to counteract the detrimental effects. As the mechanisms mediating the neurotoxicity of environmental mercury are not fully delineated, it is difficult to conceptualize a testable molecular mechanism explaining how n-3 PUFAs negate its neurotoxic effects. However, environmental exposure to mercury also has been linked to increased autoimmunity. By way of a molecular understanding of this immuno-toxic association, disruption of CD95 signaling is well established as a triggering factor for autoimmunity, and we have previously shown that environmentally relevant in vitro and dietary exposures to mercury interfere with CD95 signaling. In particular we have shown that activation of caspase 8, as well as downstream activation of caspase 3, in response to CD95 agonist stimulation is depressed by mercury. More recently we have shown in vitro that the n-3 PUFA docosahexaenoic acid counteracts the negative effect of mercury on CD95 signaling by restoring caspase activity. We hypothesized that concomitant ingestion of n-3 PUFAs with mercury might be protective from the immuno-toxic effects of mercury, as it is with mercury's neuro-toxic effects, and in the case of immuno-toxicity this would be related to restoration of CD95 signal strength. We now show that dietary ingestion of n-3 PUFAs generally promotes CD95 signaling by upregulating caspase 8 activation. Apart from accounting for the ability of n-3 PUFAs to specifically counteract autoimmune sequelae of mercury exposure, this novel finding for the first time suggests a testable molecular mechanism explaining the overall anti-inflammatory properties of n-3 PUFAs. - Highlights: • Dietary n-3 PUFAs counter Hg{sup 2+} immunotoxicity • Hg{sup 2+} interference with SEB-mediated signal transduction is ameliorated by n-3 PUFA rich diets. • Dietary n-3 PUFAs augment SEB-mediated activation of caspase 8 in vivo.

  9. Dietary n-3 PUFAs augment caspase 8 activation in Staphylococcal aureus enterotoxin B stimulated T-cells

    International Nuclear Information System (INIS)

    Gill, R.; Jen, K.L.; McCabe, M.J.J.; Rosenspire, A.

    2016-01-01

    Epidemiological studies have linked consumption of n-3 PUFAs with a variety of beneficial health benefits, particularly with respect to putative anti-inflammatory effects. Unfortunately, many of these results remain somewhat controversial because in most instances there has not been a linkage to specific molecular mechanisms. For instance, dietary exposure to low levels of mercury has been shown to be damaging to neural development, but concomitant ingestion of n-3 PUFAs as occurs during consumption of fish, has been shown to counteract the detrimental effects. As the mechanisms mediating the neurotoxicity of environmental mercury are not fully delineated, it is difficult to conceptualize a testable molecular mechanism explaining how n-3 PUFAs negate its neurotoxic effects. However, environmental exposure to mercury also has been linked to increased autoimmunity. By way of a molecular understanding of this immuno-toxic association, disruption of CD95 signaling is well established as a triggering factor for autoimmunity, and we have previously shown that environmentally relevant in vitro and dietary exposures to mercury interfere with CD95 signaling. In particular we have shown that activation of caspase 8, as well as downstream activation of caspase 3, in response to CD95 agonist stimulation is depressed by mercury. More recently we have shown in vitro that the n-3 PUFA docosahexaenoic acid counteracts the negative effect of mercury on CD95 signaling by restoring caspase activity. We hypothesized that concomitant ingestion of n-3 PUFAs with mercury might be protective from the immuno-toxic effects of mercury, as it is with mercury's neuro-toxic effects, and in the case of immuno-toxicity this would be related to restoration of CD95 signal strength. We now show that dietary ingestion of n-3 PUFAs generally promotes CD95 signaling by upregulating caspase 8 activation. Apart from accounting for the ability of n-3 PUFAs to specifically counteract autoimmune sequelae of mercury exposure, this novel finding for the first time suggests a testable molecular mechanism explaining the overall anti-inflammatory properties of n-3 PUFAs. - Highlights: • Dietary n-3 PUFAs counter Hg 2+ immunotoxicity • Hg 2+ interference with SEB-mediated signal transduction is ameliorated by n-3 PUFA rich diets. • Dietary n-3 PUFAs augment SEB-mediated activation of caspase 8 in vivo

  10. Staphylococcal Enterotoxins, Toxic Shock Syndrome Toxin-I, and Streptococcal Pyrogenic Exotoxins: Some Basic Biology of Bacterial Superantigens

    National Research Council Canada - National Science Library

    Krakauer, Teresa

    2003-01-01

    Staphylococcus aureus and Streptococcus pyogenes are facultative gram-positive cocci that play an important role in a myriad of human illnesses, including food poisoning, skin infections, pharyngitis...

  11. Staphylococcus aureus enterotoxin A (SEA) stimulates STAT3 activation and IL-17 expression in cutaneous T-cell lymphoma

    DEFF Research Database (Denmark)

    Willerslev-Olsen, Andreas; Krejsgaard, Thorbjørn; Lindahl, Lise Maria

    2016-01-01

    cells. The response is induced via IL-2 receptor common g chain cytokines and a Janus kinase 3 (JAK3)-dependent pathway in malignant T cells, and blocked by tofacitinib, a clinical-grade JAK3 inhibitor. In conclusion, we demonstrate that SEA induces cell cross talk-dependent activation of STAT3...

  12. Association between enteropathogens and malnutrition in children aged 6-23 mo in Bangladesh: a case-control study.

    Science.gov (United States)

    Platts-Mills, James A; Taniuchi, Mami; Uddin, Md Jashim; Sobuz, Shihab Uddin; Mahfuz, Mustafa; Gaffar, Sm Abdul; Mondal, Dinesh; Hossain, Md Iqbal; Islam, M Munirul; Ahmed, Am Shamsir; Petri, William A; Haque, Rashidul; Houpt, Eric R; Ahmed, Tahmeed

    2017-05-01

    Background: Early exposure to enteropathogens has been associated with malnutrition in children in low-resource settings. However, the contribution of individual enteropathogens remains poorly defined. Molecular diagnostics offer an increase in sensitivity for detecting enteropathogens but have not been comprehensively applied to studies of malnutrition. Objective: We sought to identify enteropathogens associated with malnutrition in Bangladesh. Design: Malnourished children [weight-for-age z score (WAZ) children (WAZ >-1) of the same age and from the same community were enrolled as controls. Stools were collected at enrollment and, for cases, after a 5-mo nutritional intervention. Enrollment and follow-up stools were tested by quantitative polymerase chain reaction for 32 enteropathogens with the use of a custom-developed TaqMan Array Card. Results: Enteropathogen testing was performed on 486 cases and 442 controls upon enrollment and 365 cases at follow-up. At enrollment, the detection of enteroaggregative Escherichia coli (OR: 1.39; 95% CI: 1.05, 1.83), Campylobacter spp. (OR: 1.46; 95% CI: 1.11, 1.91), heat-labile enterotoxin-producing E. coli (OR: 1.55; 95% CI: 1.04, 2.33), Shigella /enteroinvasive E. coli (OR: 1.65; 95% CI: 1.10, 2.46), norovirus genogroup I (OR: 1.66; 95% CI: 1.23, 2.25), and Giardia (OR: 1.73; 95% CI: 1.20, 2.49) were associated with malnourished cases, and the total burden of these pathogens remained associated with malnutrition after adjusting for sociodemographic factors. The number of these pathogens at follow-up was negatively associated with the change in WAZ during the intervention (-0.10 change in WAZ per pathogen detected; 95% CI: -0.14, -0.06), whereas the number at enrollment was positively associated with the change in WAZ (0.05 change in WAZ per pathogen detected; 95% CI: 0.00, 0.10). Conclusions: A subset of enteropathogens was associated with malnutrition in this setting. Broad interventions designed to reduce the burden of

  13. In-silico design, expression, and purification of novel chimeric Escherichia coli O157:H7 OmpA fused to LTB protein in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Aytak Novinrooz

    Full Text Available E. coli O157:H7, one of the major EHEC serotypes, is capable of developing bloody diarrhea, hemorrhagic colitis (HC, and fatal hemolytic uremic syndrome (HUS and is accompanied by high annual economic loss worldwide. Due to the increased risk of HC and HUS development following antibiotic therapy, the prevention of infections caused by this pathogen is considered to be one of the most effective ways of avoiding the consequences of this infection. The main aim of the present study was to design, express, and purify a novel chimeric protein to develope human vaccine candidate against E. coli O157:H7 containing loop 2-4 of E. coli O157:H7, outer membrane protein A (OmpA, and B subunit of E. coli heat labile enterotoxin (LTB which are connected by a flexible peptide linker. Several online databases and bioinformatics software were utilized to choose the peptide linker among 537 analyzed linkers, design the chimeric protein, and optimize the codon of the relative gene encoding this protein. Subsequently, the recombinant gene encoding OmpA-LTB was synthesized and cloned into pET-24a (+ expression vector and transferred to E. coli BL21(DE3 cells. The expression of OmpA-LTB chimeric protein was then carried out by induction of cultured E. coli Bl21 (DE3 cells with 1mM isopropyl-β-D-thiogalactopyranoside (IPTG. The purification of OmpA-LTB was then performed by nickel affinity chromatography. Expression and purification were analyzed by sodium dodecyl sulphate poly acrylamide gel electrophoresis. Moreover, the identity of the expressed protein was analyzed by western blotting. SDS-PAGE and western immunoblotting confirmed the successful expression of a 27 KDa recombinant protein after 24 hours at 37°C post-IPTG induction. OmpA-LTB was then successfully purified, using nickel affinity chromatography under denaturing conditions. The yield of purification was 12 mg per liter of culture media. Ultimately, we constructed the successful design and efficient

  14. Lactobacillus plantarum BSGP201683 Isolated from Giant Panda Feces Attenuated Inflammation and Improved Gut Microflora in Mice Challenged with Enterotoxigenic Escherichia coli

    Directory of Open Access Journals (Sweden)

    Qian Liu

    2017-09-01

    Full Text Available In this work, we searched for an effective probiotic that can help control intestinal infection, particularly enterotoxigenic Escherichia coli K88 (ETEC invasion, in giant panda (Ailuropoda melanoleuca. As a potential probiotic strain, Lactobacillus plantarum BSGP201683 (L. plantarum G83 was isolated from the feces of giant panda and proven beneficial in vitro. This study was aimed to evaluate the protective effect of L. plantarum G83 in mice challenged with ETEC. The mice were orally administered with 0.2 mL of PBS containing L. plantarum G83 at 0 colony-forming units (cfu mL−1 (control; negative control, ETEC group, 5.0 × 108 cfu mL−1 (LDLP, 5.0 × 109 cfu mL−1 (MDLP, and 5.0 × 1010 cfu mL−1 (HDLP for 14 consecutive days. At day 15, the mice (LDLP, MDLP, HDLP, and ETEC groups were challenged with ETEC and assessed at 0, 24, and 144 h. Animal health status; chemical and biological intestinal barriers; and body weight were measured. Results showed that L. plantarum G83 supplementation protected the mouse gut mainly by attenuating inflammation and improving the gut microflora. Most indices significantly changed at 24 h after challenge compared to those at 0 and 144 h. All treatment groups showed inhibited plasma diamine oxidase activity and D-lactate concentration. Tight-junction protein expression was down-regulated, and interleukin (IL-1β, IL-6, IL-8, TLR4, and MyD88 levels were up-regulated in the jejunum in the LDLP and MDLP groups. The number of the Enterobacteriaceae family and the heat-labile enterotoxin (LT gene decreased (P < 0.05 in the colons in the LDLP and MDLP groups. All data indicated that L. plantarum G83 could attenuate acute intestinal inflammation caused by ETEC infection, and the low and intermediate doses were superior to the high dose. These findings suggested that L. plantarum G83 may serve as a protective probiotic for intestinal disease and merits further investigation.

  15. Lactobacillus plantarum BSGP201683 Isolated from Giant Panda Feces Attenuated Inflammation and Improved Gut Microflora in Mice Challenged with Enterotoxigenic Escherichia coli.

    Science.gov (United States)

    Liu, Qian; Ni, Xueqin; Wang, Qiang; Peng, Zhirong; Niu, Lili; Wang, Hengsong; Zhou, Yi; Sun, Hao; Pan, Kangcheng; Jing, Bo; Zeng, Dong

    2017-01-01

    In this work, we searched for an effective probiotic that can help control intestinal infection, particularly enterotoxigenic Escherichia coli K88 (ETEC) invasion, in giant panda ( Ailuropoda melanoleuca ). As a potential probiotic strain, Lactobacillus plantarum BSGP201683 ( L. plantarum G83) was isolated from the feces of giant panda and proven beneficial in vitro . This study was aimed to evaluate the protective effect of L. plantarum G83 in mice challenged with ETEC. The mice were orally administered with 0.2 mL of PBS containing L. plantarum G83 at 0 colony-forming units (cfu) mL -1 (control; negative control, ETEC group), 5.0 × 10 8 cfu mL -1 (LDLP), 5.0 × 10 9 cfu mL -1 (MDLP), and 5.0 × 10 10 cfu mL -1 (HDLP) for 14 consecutive days. At day 15, the mice (LDLP, MDLP, HDLP, and ETEC groups) were challenged with ETEC and assessed at 0, 24, and 144 h. Animal health status; chemical and biological intestinal barriers; and body weight were measured. Results showed that L. plantarum G83 supplementation protected the mouse gut mainly by attenuating inflammation and improving the gut microflora. Most indices significantly changed at 24 h after challenge compared to those at 0 and 144 h. All treatment groups showed inhibited plasma diamine oxidase activity and D -lactate concentration. Tight-junction protein expression was down-regulated, and interleukin (IL)-1β, IL-6, IL-8, TLR4, and MyD88 levels were up-regulated in the jejunum in the LDLP and MDLP groups. The number of the Enterobacteriaceae family and the heat-labile enterotoxin (LT) gene decreased ( P < 0.05) in the colons in the LDLP and MDLP groups. All data indicated that L. plantarum G83 could attenuate acute intestinal inflammation caused by ETEC infection, and the low and intermediate doses were superior to the high dose. These findings suggested that L. plantarum G83 may serve as a protective probiotic for intestinal disease and merits further investigation.

  16. Comparative Genomics and Characterization of Hybrid Shigatoxigenic and Enterotoxigenic Escherichia coli (STEC/ETEC Strains.

    Directory of Open Access Journals (Sweden)

    Outi Nyholm

    Full Text Available Shigatoxigenic Escherichia coli (STEC and enterotoxigenic E. coli (ETEC cause serious foodborne infections in humans. These two pathogroups are defined based on the pathogroup-associated virulence genes: stx encoding Shiga toxin (Stx for STEC and elt encoding heat-labile and/or est encoding heat-stable enterotoxin (ST for ETEC. The study investigated the genomics of STEC/ETEC hybrid strains to determine their phylogenetic position among E. coli and to define the virulence genes they harbor.The whole genomes of three STEC/ETEC strains possessing both stx and est genes were sequenced using PacBio RS sequencer. Two of the strains were isolated from the patients, one with hemolytic uremic syndrome, and one with diarrhea. The third strain was of bovine origin. Core genome analysis of the shared chromosomal genes and comparison with E. coli and Shigella spp. reference genomes was performed to determine the phylogenetic position of the STEC/ETEC strains. In addition, a set of virulence genes and ETEC colonization factors were extracted from the genomes. The production of Stx and ST were studied.The human STEC/ETEC strains clustered with strains representing ETEC, STEC, enteroaggregative E. coli, and commensal and laboratory-adapted E. coli. However, the bovine STEC/ETEC strain formed a remote cluster with two STECs of bovine origin. All three STEC/ETEC strains harbored several other virulence genes, apart from stx and est, and lacked ETEC colonization factors. Two STEC/ETEC strains produced both toxins and one strain Stx only.This study shows that pathogroup-associated virulence genes of different E. coli can co-exist in strains originating from different phylogenetic lineages. The possibility of virulence genes to be associated with several E. coli pathogroups should be taken into account in strain typing and in epidemiological surveillance. Development of novel hybrid E. coli strains may cause a new public health risk, which challenges the

  17. Subunit Vaccine Preparation of Bovine Rotavirus and Its Efficacy in Mice.

    Science.gov (United States)

    Suocheng, Wei; Tuanjie, Che; Changjun, Song; Fengling, Tian; Zhongren, Ma

    2015-09-01

    Rotaviruses (RV) are important viral diarrheal agents in calves. Vaccination is an optimum measure to prevent bovine rotaviruses (BRV) infection. However, little research on BRV VP7 vaccine has been done and currently there is no BRV vaccine. To prepare a subunit vaccine of BRV and investigate its efficacy. Total RNA was extracted from MA104 cells infected with bovine rotavirus (BRV) strain GSB01. BRV VP7 gene was amplified using real time fluorescence quantitative PCR (qPCR). The pEASY-T3-VP7 plasmid was digested using HindⅢ and BamHI restriction endonucleases, then recombined into the prokaryotic expression vector pET32a. The pET32a-VP7 and pET32a-VP7-LTB (heat-labile enterotoxin B subunit) were transformed into BL21 (DE3) competent cells of Escherichia coli, respectively, and induced with IPTG, then analyzed using SDS-PAGE. Sixty mice were randomly divided into three groups (n=20). Group A mice was used as His-tag control and mice in group B and C were inoculated with pET32a-VP7 and pET32a-VP7-LTB, respectively. VP7 IgG antibody titers and protection efficiency of pET32a-VP7-LTB were further determined in neonatal mice challenged with GSB01 BRV strain. SDS-PAGE analysis showed that the pET32a-VP7 was highly expressed in the BL21 (DE3) cells. PET32a-VP7 and pET32a-VP7-LTB protein could promote VP7 IgG antibody titer(8.33×103 vs. 17.26×103)in mice. Immunization protection ratios of pET32a-VP7 and pET32a-VP7-LTB proteins in the neonatal mice were 86.4% and 91.7%, respectively. The fusion protein of pET32a-VP7-LTB had excellent immunogenicity and protected mice from BRV infection. Our findings can be used for further developing of a high-efficiency subunit vaccine of BRV.

  18. Ant-infecting Ophiocordyceps genomes reveal a high diversity of potential behavioral manipulation genes and a possible major role for enterotoxins

    NARCIS (Netherlands)

    De Bekker, Charissa; Ohm, Robin A.; Evans, Harry C.; Brachmann, Andreas; Hughes, David P

    2017-01-01

    Much can be gained from revealing the mechanisms fungal entomopathogens employ. Especially intriguing are fungal parasites that manipulate insect behavior because, presumably, they secrete a wealth of bioactive compounds. To gain more insight into their strategies, we compared the genomes of five

  19. Spreading of genes encoding enterotoxins, haemolysins, adhesin and biofilm among methicillin resistant Staphylococcus aureus strains with staphylococcal cassette chromosome mec type IIIA isolated from burn patients.

    Science.gov (United States)

    Motallebi, Mitra; Jabalameli, Fereshteh; Asadollahi, Kheirollah; Taherikalani, Morovat; Emaneini, Mohammad

    2016-08-01

    The emergence of antibiotic-resistant Staphylococcus aureus in particular methicillin-resistant S. aureus (MRSA) is an important concern in burn medical centers either in Iran or worldwide. A total of 128 S. aureus isolates were collected from wound infection of burn patients during June 2013 to June 2014. Multiplex-polymerase chain reaction (MPCR) assay was performed for the characterization of the staphylococcal cassette chromosome mec (SCCmec). Genes encoding virulence factors and biofilm were targeted by PCR. Of 128 S. aureus isolates, 77 (60.1%) isolates were MRSA. Fifty four (70.1%) isolates were identified as SCCmec type IIIA. The most frequently detected toxin genes among MRSA isolates with SCCmec type IIIA were sea (64.1%) and hla (51.8%). The rate of coexistence of sea with hla and sea with hla and hlb was 37% and12.9%, respectively. The sec, eta, tst, pvl, hla and hlb genes were not detected in any of the MRSA isolates. The most prevalent genes encoding biofilm was eno, found in 61.1% of isolates, followed by fib and icaA found in 48.1% and 38.8% of the isolates, respectively. The rate of coexistence of fib + eno + icaA + icaD and fib + eno was 20.3% and 9.2%, respectively. The ebps gene was not detected in any of the isolates. In conclusion, our study indicated that the sea, hla, fib and icaA were most frequent genes encoding virulence factors among MRSA with SCCmec type IIIA isolated from burn wound infection. Moreover, the results of this study shows that the rate of coexistence of genes encoding different virulence factor were high. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Persistent production of TH2-type cytokines and polyclonal B cell activation after chronic administration of staphylococcal enterotoxin B in mice

    NARCIS (Netherlands)

    Florquin, S.; Amraoui, Z.; Goldman, M.

    1996-01-01

    In order to study the immunopathological consequences of repeated exposure to bacterial superantigens, we evaluated the production of cytokines, the profile of serum immunoglobulins and the tissue damage in BALB/c mice injected twice a week for 3 weeks with 50 micrograms of staphylococcal

  1. X-ray crystal structure of the passenger domain of plasmid encoded toxin(Pet), an autotransporter enterotoxin from enteroaggregative Escherichia coli (EAEC)

    Energy Technology Data Exchange (ETDEWEB)

    Domingo Meza-Aguilar, J. [Departamento de Salud Pública Facultad de Medicina UNAM, Ciudad Universitaria Coyoacán 04510, D.F. (Mexico); Laboratorio de Patogenicidad Bacteriana, Unidad de Hemato Oncología e Investigación, Hospital Infantil de México Federico Gómez 06720, D.F. (Mexico); Fromme, Petra [Department of Chemistry and Biochemistry, Arizona State University, Physical Sciences BLDG D-102, Tempe, AZ 85287 (United States); Torres-Larios, Alfredo [Instituto de Fisiología Celular UNAM, Ciudad Universitaria Coyoacán 04510, D.F. (Mexico); Mendoza-Hernández, Guillermo [Instituto de Química UNAM, Ciudad Universitaria Coyoacán 04510, D.F (Mexico); Hernandez-Chiñas, Ulises [Departamento de Salud Pública Facultad de Medicina UNAM, Ciudad Universitaria Coyoacán 04510, D.F. (Mexico); Laboratorio de Patogenicidad Bacteriana, Unidad de Hemato Oncología e Investigación, Hospital Infantil de México Federico Gómez 06720, D.F. (Mexico); Arreguin-Espinosa de los Monteros, Roberto A. [Instituto de Química UNAM, Ciudad Universitaria Coyoacán 04510, D.F (Mexico); and others

    2014-03-07

    Highlights: • X-ray crystal structure of the passenger domain of Plasmid encoded toxin at 2.3 Å. • Structural differences between Pet passenger domain and EspP protein are described. • High flexibility of the C-terminal beta helix is structurally assigned. - Abstract: Autotransporters (ATs) represent a superfamily of proteins produced by a variety of pathogenic bacteria, which include the pathogenic groups of Escherichia coli (E. coli) associated with gastrointestinal and urinary tract infections. We present the first X-ray structure of the passenger domain from the Plasmid-encoded toxin (Pet) a 100 kDa protein at 2.3 Å resolution which is a cause of acute diarrhea in both developing and industrialized countries. Pet is a cytoskeleton-altering toxin that induces loss of actin stress fibers. While Pet (pdb code: 4OM9) shows only a sequence identity of 50% compared to the closest related protein sequence, extracellular serine protease plasmid (EspP) the structural features of both proteins are conserved. A closer structural look reveals that Pet contains a β-pleaded sheet at the sequence region of residues 181–190, the corresponding structural domain in EspP consists of a coiled loop. Secondary, the Pet passenger domain features a more pronounced beta sheet between residues 135 and 143 compared to the structure of EspP.

  2. Cloning and Expression of Fusion Genes of Domain A-1 Protective Antigen of Bacillus Anthracis and Shigella Enterotoxin B Subunit (Stxb In E. Coil

    Directory of Open Access Journals (Sweden)

    AH ahmadi

    2015-02-01

    Conclusion: The findings of the current study revealed that this antigen can be raised as an anti-cancer and recombinant vaccine candidate against types of Shigella, Escherichia coli and Bacillus anthracis which can be due to such factors as identification of antigen(PA by antibody PA20, its apoptosis induction properties, property of immunogenicity, adjuvant and delivery of STxB protein and high expression levels of Gb3 in human cancer cells.

  3. Therapeutic Inhibition of Pro-Inflammatory Signaling and Toxicity to Staphylococcal Enterotoxin B by a Synthetic Dimeric BB-Loop Mimetic of MyD88

    Science.gov (United States)

    2012-07-27

    56 stock solution and 75 ml of the 16 dilution buffer was mixed with 25 ml of the supernatant, incubated for 30 min at 65 oC to inactivate endogenous ...shock by staphylococcal pyrogenic exotoxin type C. Infect Immun 36: 123–128. Meier survival analysis and log-rank tests were performed to compare

  4. Antimicrobial activities of the bacteriocin-like substances produced ...

    African Journals Online (AJOL)

    Administrator

    2011-09-07

    Sep 7, 2011 ... bacteriocins (subclass IIc); the large (>30 kDa) heat-labile non-lantibiotics (class III) and ... LAB from cow and goat milk and dairy products were ... (Oxoid, Hapshire, UK) was used to determine hydrolysis of esculin and growth ...

  5. In vivo and in vitro antibacterial activity of conglutinin, a mammalian plasma lectin

    DEFF Research Database (Denmark)

    Friis-Christiansen, P; Thiel, S; Svehag, S E

    1990-01-01

    of BALB/c mice with Salmonella typhimurium is mediated by conglutinin. Conglutinin also demonstrated antibacterial activity against E. coli and S. typhimurium in vitro. The expression of this activity required the presence of heat-labile serum factors and peritoneal exudate or spleen cells...

  6. Alexandria Journal of Medicine - Vol 52, No 2 (2016)

    African Journals Online (AJOL)

    Purification of heat labile toxin from Bordetella pertussis vaccine strain 134 employed indigenous technology · EMAIL FREE FULL TEXT EMAIL FREE FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT. KC Shivanandappa, S Jagannathan, S Pandiyarajan, P Tamilvanan, T Umadevi, Jeeva Kalaiselvan, ...

  7. Developing novel immunogens for a safe and effective Alzheimer's disease vaccine.

    Science.gov (United States)

    Lemere, Cynthia A

    2009-01-01

    , a safe and effective active Abeta vaccine would be more cost-effective and more readily available to a larger AD population. We have developed several novel short Abeta immunogens that target the Abeta N-terminus containing a strong B cell epitope while avoiding the Abeta mid-region and C-terminus containing T cell epitopes. These immunogens include dendrimeric Abeta1-15 (16 copies of Abeta1-15 on a lysine antigen tree), 2xAbeta1-15 (a tandem repeat of two lysine-linked Abeta1-15 peptides), and 2xAbeta1-15 with the addition of a three amino acid RGD motif (R-2xAbeta1-15). Intranasal immunization with our short Abeta fragment immunogens and a mucosal adjuvant, mutant Escherichia coli heat-labile enterotoxin LT(R192G), resulted in reduced cerebral Abeta levels, plaque deposition, and gliosis, as well as increased plasma Abeta levels and improved cognition in a transgenic mouse model of AD. Preclinical trials in nonhuman primates, and human clinical trials using similar Abeta immunogens, are now underway. Abeta immunotherapy looks promising but must be made safer and more effective at generating antibody titers in the elderly. It is hoped that these novel immunogens will enhance Abeta antibody generation across a broad population and avoid the adverse events seen in the earlier clinical trial.

  8. Enterotoxins and emetic toxins production by Bacillus cereus and other species of Bacillus isolated from Soumbala and Bikalga, African alkaline fermentedfood condiments

    DEFF Research Database (Denmark)

    Ouoba, Labia Irene I.; Thorsen, Line; Varnam, Alan H.

    2008-01-01

    The ability of various species of Bacillus from fermented seeds of Parkia biglobosa known as African locust bean(Soumbala) and fermented seeds of Hibiscus sabdariffa (Bikalga) was investigated. The study included screening of the isolates by haemolysis on blood agar, detection of toxins in broth...

  9. Detection of B. fragilis group and diversity of bft enterotoxin and antibiotic resistance markers cepA, cfiA and nim among intestinal Bacteroides fragilis strains in patients with inflammatory bowel disease.

    Science.gov (United States)

    Rashidan, Marjan; Azimirad, Masoumeh; Alebouyeh, Masoud; Ghobakhlou, Mehdi; Asadzadeh Aghdaei, Hamid; Zali, Mohammad Reza

    2018-04-01

    We compared frequency of the members of B. fragilis group in 100 and 20 colon biopsy specimens of inflammatory bowel disease (IBD) and non-IBD patients. Agar dilution and PCR were orderly used to detect minimal inhibitory concentration of ampicillin, imipenem, and metronidazole, and carriage of related resistance genes cepA, cfi, and nim. B. fragilis group was detected in 38% of IBD (UC: 36/89; CD:1/11) and 25% (5/20) of non-IBD patients. While B. vulgatus (UC: 20/36, CD: 1/2, control: 1/6); B. fragilis (UC: 18/36, CD: 1/2, control: 5/6); B. ovatus (UC: 2/36); B. caccae (UC: 1/36); and B. eggerthii (UC: 1/36) were characterized, colonization of B. thetaiotamicron, B. merdae, B. distasonis, B. stercoris and B. dorei species was not detected in these specimens. Co-existence of B. fragilis + B. vulgatus (5 patients) and B. vulgatus + B. caccae (1 patient) was detected just in UC patients. bft was detected among 31.5% (6/19) of B. fragilis strains in the IBD and 40% (2/5) in the non-IBD groups. Nearly, 73.6% of the strains from the patient group and 80% in control group harbored cepA; 31.5% and 20% in the patients and control groups harbored cfiA, and none of them harbored nim determinant. Co-occurrence of the cepA and cfiA was orderly detected in 10.5% (2/19) and 20% (1/5) of the strains in these groups. The resistance rates were detected as 95.8% (23/24 (to ampicillin (MIC range of ≤0.5-≥16 μg/ml), 0% to metronidazole and 29.1% to imipenem (7/24, MIC range ≤4-32 μg/ml). Nearly 25% (6/24) of the strains were resistant to ampicillin and imipenem, simultaneously. No statistically significant difference was detected between the IBD and control groups for drug resistance phenotypes. Statistical analysis showed significant associations between resistance to ampicillin or imipenem and carriage of cepA or cfiA, respectively (p value = 0.0007). PCR results on the extracted plasmids confirmed their roles in carriage of cfiA and cepA. These data provide guide for antibiotic therapy and highlights wide distribution of β-lactam resistant B. fragilis strains in patients with IBD and non-IBD intestinal disorders. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. Anaerobic Aryl Reductive Dehalogenation of Halobenzoates by Cell Extracts of “Desulfomonile tiedjei”

    OpenAIRE

    DeWeerd, Kim A.; Suflita, Joseph M.

    1990-01-01

    We studied the transformation of halogenated benzoates by cell extracts of a dehalogenating anaerobe, “Desulfomonile tiedjei.” We found that cell extracts possessed aryl reductive dehalogenation activity. The activity was heat labile and dependent on the addition of reduced methyl viologen, but not on that of reduced NAD, NADP, flavin mononucleotide, flavin adenine dinucleotide, desulfoviridin, cytochrome c3, or benzyl viologen. Dehalogenation activity in extracts was stimulated by formate, C...

  11. Segregation of Tay-Sachs and Sandhoff alleles in a non-Jewish family.

    OpenAIRE

    Lane, A B; Young, E; Jenkins, T

    1980-01-01

    A non-Jewish family is presented in which the genes for Tay-Sachs disease and Sandhoff disease are segregating. Individuals heterozygous for both alleles have low serum and white cell total hexosaminidase levels together with a proportion of heat-labile hexosaminidase A (HEX A) which falls in the normal range. The individuals would not be detected as carriers of Tay-Sachs disease or Sandhoff disease in a population screening program.

  12. Segregation of Tay-Sachs and Sandhoff alleles in a non-Jewish family.

    Science.gov (United States)

    Lane, A B; Young, E; Jenkins, T

    1980-01-01

    A non-Jewish family is presented in which the genes for Tay-Sachs disease and Sandhoff disease are segregating. Individuals heterozygous for both alleles have low serum and white cell total hexosaminidase levels together with a proportion of heat-labile hexosaminidase A (HEX A) which falls in the normal range. The individuals would not be detected as carriers of Tay-Sachs disease or Sandhoff disease in a population screening program. PMID:7446530

  13. 40 CFR 725.421 - Introduced genetic material.

    Science.gov (United States)

    2010-07-01

    ... fever toxins, pyrogenic exotoxins) Yersinia enterocolitica Heat-stable enterotoxins (ST) ... Neurotoxin Staphylococcus aureus Alpha toxin (alpha lysin) Yersinia pestis Murine toxin Snake toxins Bungarus...

  14. Food-Borne Outbreak Investigation and Molecular Typing: High Diversity of Staphylococcus aureus Strains and Importance of Toxin Detection

    Science.gov (United States)

    Denayer, Sarah; Nia, Yacine; Botteldoorn, Nadine

    2017-01-01

    Staphylococcus aureus is an important aetiological agent of food intoxications in the European Union as it can cause gastro-enteritis through the production of various staphylococcal enterotoxins (SEs) in foods. Reported enterotoxin dose levels causing food-borne illness are scarce and varying. Three food poisoning outbreaks due to enterotoxin-producing S. aureus strains which occurred in 2013 in Belgium are described. The outbreaks occurred in an elderly home, at a barbecue event and in a kindergarten and involved 28, 18, and six cases, respectively. Various food leftovers contained coagulase positive staphylococci (CPS). Low levels of staphylococcal enterotoxins ranging between 0.015 ng/g and 0.019 ng/g for enterotoxin A (SEA), and corresponding to 0.132 ng/g for SEC were quantified in the food leftovers for two of the reported outbreaks. Molecular typing of human and food isolates using pulsed-field gel electrophoresis (PFGE) and enterotoxin gene typing, confirmed the link between patients and the suspected foodstuffs. This also demonstrated the high diversity of CPS isolates both in the cases and in healthy persons carrying enterotoxin genes encoding emetic SEs for which no detection methods currently exist. For one outbreak, the investigation pointed out to the food handler who transmitted the outbreak strain to the food. Tools to improve staphylococcal food poisoning (SFP) investigations are presented. PMID:29261162

  15. Food-Borne Outbreak Investigation and Molecular Typing: High Diversity of Staphylococcus aureus Strains and Importance of Toxin Detection.

    Science.gov (United States)

    Denayer, Sarah; Delbrassinne, Laurence; Nia, Yacine; Botteldoorn, Nadine

    2017-12-20

    Staphylococcus aureus is an important aetiological agent of food intoxications in the European Union as it can cause gastro-enteritis through the production of various staphylococcal enterotoxins (SEs) in foods. Reported enterotoxin dose levels causing food-borne illness are scarce and varying. Three food poisoning outbreaks due to enterotoxin-producing S. aureus strains which occurred in 2013 in Belgium are described. The outbreaks occurred in an elderly home, at a barbecue event and in a kindergarten and involved 28, 18, and six cases, respectively. Various food leftovers contained coagulase positive staphylococci (CPS). Low levels of staphylococcal enterotoxins ranging between 0.015 ng/g and 0.019 ng/g for enterotoxin A (SEA), and corresponding to 0.132 ng/g for SEC were quantified in the food leftovers for two of the reported outbreaks. Molecular typing of human and food isolates using pulsed-field gel electrophoresis (PFGE) and enterotoxin gene typing, confirmed the link between patients and the suspected foodstuffs. This also demonstrated the high diversity of CPS isolates both in the cases and in healthy persons carrying enterotoxin genes encoding emetic SEs for which no detection methods currently exist. For one outbreak, the investigation pointed out to the food handler who transmitted the outbreak strain to the food. Tools to improve staphylococcal food poisoning (SFP) investigations are presented.

  16. Synergistic effects of heat and irradiation treatment (thermoradiation) in the sterilization of medical products

    International Nuclear Information System (INIS)

    Trauth, C.A. Jr.; Sivinski, H.D.

    1975-01-01

    This paper describes a generic class of sterilization processes is which properly chosen combinations of radiation and heat synergistically inactivate many bacteria and viruses. Treatments with optimal combinations are shown to offer the possibility of using lower total doses and lower temperatures than would be required separately for sterilization. This results from easier elimination of heat-labile, radioresistant organisms and radiolabile, heat-resistant organisms, and from synergistic inactivation of organisms which are both radioresistant and heat resistant. These processes depend upon temperature, dose-rate, and time in fairly complex ways; therefore, an analytical framework in which they can be defined is also presented. (author)

  17. Oxidation of indole-3-acetic acid to oxindole-3-acetic acid by an enzyme preparation from Zea mays

    Science.gov (United States)

    Reinecke, D. M.; Bandurski, R. S.

    1988-01-01

    Indole-3-acetic acid is oxidized to oxindole-3-acetic acid by Zea mays tissue extracts. Shoot, root, and endosperm tissues have enzyme activities of 1 to 10 picomoles per hour per milligram protein. The enzyme is heat labile, is soluble, and requires oxygen for activity. Cofactors of mixed function oxygenase, peroxidase, and intermolecular dioxygenase are not stimulatory to enzymic activity. A heat-stable, detergent-extractable component from corn enhances enzyme activity 6- to 10-fold. This is the first demonstration of the in vitro enzymic oxidation of indole-3-acetic acid to oxindole-3-acetic acid in higher plants.

  18. INFECTIOUS MYXOMATOSIS OF RABBITS

    Science.gov (United States)

    Smadel, Joseph E.; Ward, S. M.; Rivers, Thomas M.

    1940-01-01

    A second soluble antigen, separable from the virus, occurs in extracts of infected skin and in the serum of rabbits acutely ill with infectious myxomatosis. Like the first antigen (A), the second (B) is heat labile and has certain characteristics of a globulin. The two antigens precipitate in different concentrations of ammonium sulfate and can be separated by this method. Neither of the antigens after being heated at 56°C. precipitates in the presence of specific antibody but each is capable of inhibiting the activity of its antibody. PMID:19871012

  19. Development and fabrication of heat-sterilizable inhalation therapy equipment

    Science.gov (United States)

    Irons, A. S.

    1974-01-01

    The development of a completely heat sterilizable intermittent positive pressure breathing (IPPB) ventilator in an effort to reduce the number of hospital acquired infections is reported. After appropriate changes in materials and design were made, six prototype units were fabricated and were successfully field tested in local hospitals. Most components of the modified ventilators are compatible with existing machines. In all but a few instances, such as installation of bacteria-retentive filters and a modified venturi, the change over from non-heat-sterilizable to sterilizable units was accomplished by replacement of heat labile materials with heat stable materials.

  20. THE EFFECT OF COOKING AND STORAGE ON FLORFENICOL AND FLORFENICOL AMINE RESIDUES IN EGGS

    Directory of Open Access Journals (Sweden)

    Ayhan Filazi

    2015-09-01

    Full Text Available The aim of this study was to evaluate the effects of storage conditions (room temperature, refrigerator and cooking methods (frying, boiling on florfenicol (FF and florfenicol amine (FFA residue levels in eggs. Without any significant difference between storage conditions at 20˚C and +4˚C, residue levels decreased within days, but were still present on day 28. Frying and boiling for 1 and 5 min yielded similar results to the storage conditions just described; there was a significant decrease in residue levels, but still not enough for decomposing. These findings indicate that FF and FFA residues are heat-labile.

  1. Partiel fingeramputation efter fjæsingstik og andengradsfrobrænding

    DEFF Research Database (Denmark)

    Josiassen, Michael; Partoft, Søren; Leicht, Pernille

    2017-01-01

    The greater weever (Trachinus draco) is the only venomous fish in Denmark, and if stung by the weever the venom may cause symptoms such as pain, headache, nausea, dizziness and in severe cases systemic allergic reaction or tissue necrosis. The venom is heat-labile and should therefore be treated ...... with warm water to the afflicted area. We report a case of a patient who was stung in the second finger and because of the treatment, he got a second-degree burn, that may have enhanced the effect of the venom, thus resulting in partial finger amputation....

  2. Aeromonas caviae strain induces Th1 cytokine response in mouse intestinal tract

    Science.gov (United States)

    Aeromonas caviae has been associated with human gastrointestinal disease. Strains of this species typically lack virulence factors (VFs) such as enterotoxins and hemolysins that are produced by other human pathogens of the Aeromonas genus,. Microarray profiling of...

  3. Real-Time Fluorogenic Polymerase Chain Reaction (PCR) Assays to Nucleic Acid-Based Detection of Simulants and Biothreat Agents

    National Research Council Canada - National Science Library

    Khan, Akbar S; O'Connell, Kevin P; Bucher, Jennifer R; Anderson, Patricia E; Cao, Cheng J; Gostomski, Mark V; Valdes, James J

    2003-01-01

    .... We describe here assays for the detection of the gene encoding staphylococcal enterotoxin A (SEA), a toxin produced by the bacterium Staphylococcus aureus and an agent responsible for a significant fraction of food poisoning incidents worldwide...

  4. Marine toxins and their toxicological significance: An overview

    Digital Repository Service at National Institute of Oceanography (India)

    Sarkar, A.

    , Hemolysins-1 and hemolysin-2, saxitoxin, neosaxitoxin, gonyautoxin, tetrodotoxin, ptychodiscus brevis toxin and theonellamide F. According to their mode of action, these toxins are classified into different categories such as cytotoxin, enterotoxin...

  5. Polyclonal antibodies production against Staphylococcus aureus ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-02-01

    Feb 1, 2010 ... ... and the enterotoxins staphylococcies dried dosage remains the unique means to define the food innocuousness. The standard methods of cultivation used to detect .... A second sample of pasteurized and packaged milk.

  6. 21 CFR 866.3700 - Staphylococcus aureus serological reagents.

    Science.gov (United States)

    2010-04-01

    ... affecting the intestine) producing staphylococci from cultured isolates. The identification aids in the... while growing in meat, dairy, or bakery products. After ingestion, this enterotoxin is absorbed in the...

  7. Distribusi Gen Enterotoksin Staphylococcus aureus dari Susu Segar dan Pangan Asal Hewan

    Directory of Open Access Journals (Sweden)

    Siti Isrina Oktavia Salasia

    2009-09-01

    Full Text Available Staphylococcus aureus is a potential pathogen causing disease in human and animals due to severalvirulence factors. Staphylococcal enterotoxins which is responsible for foodborne disease is considered tobe one of the important virulence factor for the bacteria. The research was conducted to identify variousenterotoxin genes of S. aureus. Twenty three S. aureus isolates from milk cows (12 isolates and foodanimal products (11 isolates were used to study various enterotoxins genes (sea, seb, sec, sed, see, seg, seh,sei, and sej. The enterotoxins genes of S. aureus were investigated by using polymerase chain reaction(PCR with specific primers. There were 3 isolates (13.04% negative for staphylococcal enterotoxin genes.Twenty isolates (86.96% harboured for one or more staphylococcal enterotoxin genes such as: sec (6 isolates/26.09%, see, seh for 1 isolate (4.35%, combination of 2 genes se (b,i, se (c,g, se (g,i for 1 isolate of each(4.35%, se(c,e for 2 isolates (8.70%, se(b,c for 4 isolates (17.39%. Staphylococcal enterotoxin could bedetected in 3 combination genes of se(b,c,i, se(c,e,i, se(c,g,i for 1 isolate of each (4.35%.

  8. Detection of enterotoxin A and cytotoxin B, and isolation of Clostridium difficile in piglets in Minas Gerais, Brazil Detecção da enterotoxina A e citotoxina B e isolamento de Clostridium difficile em leitões em Minas Gerais, Brasil

    Directory of Open Access Journals (Sweden)

    Rodrigo Otávio Silveira Silva

    2011-08-01

    Full Text Available Clostridium difficile has emerged as a major cause of neonatal colitis in piglets, displacing classic bacterial pathogens. However, there is no information regarding the distribution of this microorganism in pig farms in Brazil. In the present study, the presence of toxins A/B and of C. difficile strains in stool samples from 60 diarrheic or non-diarrheic newborn piglets (one to seven days old, from 15 different farms, was studied. The presence of toxins A/B was detected by ELISA and PCR was used to identify toxin A, toxin B and binary toxin gene in each isolated strain. C. difficile A/B toxins were detected in ten samples (16.7%. Of these, seven were from diarrheic and three were from non-diarrheic piglets. C. difficile was recovered from 12 out of 60 (20% fecal samples. Of those, three strains were non-toxigenic (A-B- and nine were toxigenic. Of the nine toxigenic strains, four were A+B+ strains and five were A-B+ strains. The presence of binary toxin observed in the present study was much higher (50% than in previously reported studies. All three non-toxigenic strains were isolated from otherwise healthy piglets. The results suggest the occurrence of neonatal diarrhea by C. difficile in farms in Brazil.Clostridium difficile tem sido relatado como o principal causador de colite neonatal em suínos. Apesar da crescente importância deste agente, não há dados sobre infecções causadas por C. difficile em suínos no Brasil. O objetivo do presente estudo foi detectar as toxinas A/B e isolar C. difficile a partir de 60 amostras de fezes de leitões diarreicos ou apararentemente saudáveis, com no máximo sete dias de vida, e oriundos de 15 granjas diferentes. As toxinas A/B foram detectadas por ELISA e uma PCR multiplex foi utilizada para detecção dos genes responsáveis pela codificação das toxinas A, B e toxina binária. As toxinas A/B de C. difficile foram detectadas em dez amostras de fezes (16.7%. Dessas, sete eram de animais diarreicos e três de leitões aparentemente saudáveis. Foi possível isolar C. difficile em 12 das 60 (20% amostras trabalhadas. Dessas, três estirpes eram não-toxigênicas (A-B- e nove eram toxigênicas, sendo quatro caracterizadas como A+B+ e cinco como A-B+. O gene responsável pela codificação da toxina binária foi encontrado em 50% das estirpes isoladas, proporção superior ao relatado em estudos anteriores. Todas as estirpes não toxigênicas foram isoladas de animais não diarreicos. Os resultados encontrados sugerem a ocorrência de diarreia por C. difficile em granjas no Brasil.

  9. Heat-stable proteins and abscisic acid action in barley aleurone cells

    International Nuclear Information System (INIS)

    Jacobsen, J.V.; Shaw, D.C.

    1989-01-01

    [ 35 S]Methionine labeling experiments showed that abscisic acid (ABA) induced the synthesis of at least 25 polypeptides in mature barley (Hordeum vulgare) aleurone cells. The polypeptides were not secreted. Whereas most of the proteins extracted from aleurone cells were coagulated by heating to 100 degree C for 10 minutes, most of the ABA-induced polypeptides remained in solution (heat-stable). ABA had little effect on the spectrum of polypeptides that were synthesized and secreted by aleurone cells, and most of these secreted polypeptides were also heat-stable. Coomassie blue staining of sodium dodecyl sulfate polyacrylamide gels indicated that ABA-induced polypeptides already occurred in high amounts in mature aleurone layers having accumulated during grain development. About 60% of the total protein extracted from mature aleurone was heat stable. Amino acid analyses of total preparations of heat-stable and heat-labile proteins showed that, compared to heat-labile proteins, heat-stable intracellular proteins were characterized by higher glutamic acid/glutamine (Glx) and glycine levels and lower levels of neutral amino acids. Secreted heat-stable proteins were rich in Glx and proline. The possibilities that the accumulation of the heat-stable polypeptides during grain development is controlled by ABA and that the function of these polypeptides is related to their abundance and extraordinary heat stability are considered

  10. Randomised, double-blind, safety and efficacy of a killed oral vaccine for enterotoxigenic E. Coli diarrhoea of travellers to Guatemala and Mexico.

    Science.gov (United States)

    Sack, David A; Shimko, Janet; Torres, Olga; Bourgeois, August L; Francia, Domingo Sanchez; Gustafsson, Björn; Kärnell, Anders; Nyquist, Iréne; Svennerholm, Ann-Mari

    2007-05-30

    We tested the efficacy of a killed oral vaccine for enterotoxigenic Escherichia coli (ETEC) diarrhoea to determine if two doses of vaccine with colonization factor antigens (CF) and cholera B subunit would protect against ETEC diarrhoea of travellers. Six hundred seventy-two healthy travellers going to Mexico or Guatemala were studied in a prospective, randomised, placebo-controlled trial. The primary outcome was a vaccine preventable outcome (VPO), defined as an episode of ETEC diarrhoea with an ETEC organism producing heat labile toxin (LT) or CF homologous with the vaccine, without other known causes. The vaccine was safe and stimulated anti-heat labile toxin antibodies. There was a significant decrease in more severe VPO episodes (PE=77%, p=0.039) as defined by symptoms that interfered with daily activities or more than five loose stools in a day, although the total number of VPO events did not differ significantly in the vaccine and placebo groups. We conclude that the new oral ETEC vaccine reduces the rate of more severe episodes of traveller's diarrhoea (TD) due to VPO-ETEC, but it did not reduce the overall rate of ETEC diarrhoea or of travellers' diarrhoea due to other causes.

  11. Potential applications for Annona squamosa leaf extract in the treatment and prevention of foodborne bacterial disease.

    Science.gov (United States)

    Dholvitayakhun, Achara; Trachoo, Nathanon; Sakee, Uthai; Cushnie, T P Tim

    2013-03-01

    Foodborne disease is a major public health problem. The present study examined Annona squamosa leaves, which are traditionally used to treat diarrhea and other infections, for their potential to be used in modern food safety or medicine. Active constituents were partially purified by ethanol extraction and column chromatography. MICs of the extract were 62.5 to 125 microg/mL against Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus, and 250 microg/mL against Campylobacter jejuni. In time-kill assays, 500 microg/mL of the extract reduced colony forming unit numbers of C. jejuni almost 10 000-fold within 12 hours. Similar decreases were seen against B. cereus, but over a longer time-frame. LC-MS analysis indicated the presence of reticuline and oxophoebine. Assessment of stability by MIC assay showed activity was heat-labile, with loss of activity greatest following high temperature treatments. Activity was relatively stable at refrigeration temperature. These results indicate A. squamosa has broad-spectrum but heat-labile activity against foodborne bacterial pathogens, and bactericidal activity against B. cereus and C. jejuni. This bactericidal activity is not sufficiently rapid for A. squamosa to be used as a food sanitizer, but the extract could potentially be developed as an additive for refrigerated foods, or a modern treatment for foodborne illness.

  12. A comparison of the toluene distillation and vacuum/heat methods for extracting soil water for stable isotopic analysis

    Science.gov (United States)

    Ingraham, Neil L.; Shadel, Craig

    1992-12-01

    Hanford Loam, from Richland, Washington, was used as a test soil to determine the precision, accuracy and nature of two methods to extract soil water for stable isotopic analysis: azeotropic distillation using toluene, and simple heating under vacuum. The soil was oven dried, rehydrated with water of known stable isotopic compositions, and the introduced water was then extracted. Compared with the introduced water, initial aliquots of evolved water taken during a toluene extraction were as much as 30 ‰ more depleted in D and 2.7 ‰ more depleted in 18O, whereas final aliquots were as much as 40 ‰ more enriched in D and 14.3 ‰ more enriched in 18O. Initial aliquots collected during the vacuum/heat extraction were as much as 64 ‰ more depleted in D and 8.4 ‰ more depleted in 18O than was the introduced water, whereas the final aliquots were as much as 139 ‰ more enriched in D, and 20.8 ‰ more enriched in 18O. Neither method appears quantitative; however, the difference in stable isotopic composition between the first and last aliquots of water extracted by the toluene method is less than that from the vacuum/heat method. This is attributed to the smaller fractionation factors involved with the higher average temperatures of distillation of the toluene. The average stable isotopic compositions of the extracted water varied from that of the introduced water by up to 1.4 ‰ in δD and 4.2 ‰ in δ18O with the toluene method, and by 11.0 ‰ in δD and 1.8 ‰ in δ18O for the vacuum/heat method. The lack of accuracy of the extraction methods is thought to be due to isotopic fractionation associated with water being weakly bound (not released below 110°C) in the soil. The isotopic effect of this heat-labile water is larger at low water contents (3.6 and 5.2% water by weight) as the water bound in the soil is a commensurately larger fraction of the total. With larger soilwater contents the small volume of water bound with an associated fractionation is

  13. Effects of vitamin D supplementation in pregnancy.

    Science.gov (United States)

    Marya, R K; Rathee, S; Lata, V; Mudgil, S

    1981-01-01

    Serum calcium, inorganic phosphate and heat-labile alkaline phosphatase (HLAP) have been estimated in maternal and cord sera of 120 pregnant women at labour. 75 women who did not take any vitamin D supplements during pregnancy showed statistically significant hypocalcaemia, hypophosphataemia and elevation of HLAP. Hypocalcaemia and hypophosphataemia were present in cord blood, too. 25 women who had received 1,200 U vitamin D/day throughout the 3rd trimester, showed significantly lower HLAP levels and increased fetal birth weight but there was no other improvement in maternal or cord blood chemistry. Administration of vitamin D in two large doses of 600,000 U each in the 7th and 8th months of pregnancy in 20 women proved more efficacious. Statistically significant improvement was observed in all the three biochemical parameters in maternal as well as cord sera. Fetal birth weight was also significantly greater with this mode of therapy.

  14. Immunoglobulin E antibodies to pollens augmented in dogs by virus vaccines.

    Science.gov (United States)

    Frick, O L; Brooks, D L

    1983-03-01

    An inbred "atopic dog colony" was established to study the effect of viruses on immunoregulation of immunoglobulin (Ig) E antibodies. Dogs were selected for high skin reactivity to grass and weed pollens. Their offspring were inoculated with pollen extracts in alum immediately after routine vaccinations (attenuated live-virus vaccines for canine distemper and infectious canine hepatitis, and a killed bacterin for Leptospira). Heat labile antipollen IgE antibodies were measured by passive cutaneous anaphylaxis. Pups vaccinated for canine distemper before being given pollen extracts had many more IgE antibodies than did their control littermates who were not vaccinated until after the last pollen extract injection. This may be a natural example of the "allergic break-through phenomenon."

  15. Pilot study of whole-blood gamma interferon response to the Vibrio cholerae toxin B subunit and resistance to enterotoxigenic Escherichia coli-associated diarrhea.

    Science.gov (United States)

    Flores, Jose; DuPont, Herbert L; Paredes-Paredes, Mercedes; Aguirre-Garcia, M Magdalena; Rojas, Araceli; Gonzalez, Alexei; Okhuysen, Pablo C

    2010-05-01

    Enterotoxigenic Escherichia coli (ETEC), which produces heat-labile toxin (LT), is a common cause of travelers' diarrhea (TD). The B subunit of ETEC LT is immunologically related to the B subunit of Vibrio cholerae toxin (CT). In this pilot study we evaluated the whole-blood gamma interferon response to CT B in 17 U.S. adults traveling to Mexico. Only one of nine subjects who demonstrated a cellular immune response as determined by whole-blood gamma interferon production to CT B on arrival to Mexico developed diarrhea, whereas five of eight without a cellular response developed diarrhea. Markers of the cellular immune response to ETEC LT could help in identifying individuals immune to ETEC LT, and these markers deserve additional study.

  16. Suppression of linalool acetate production in Lavandula x intermedia.

    Science.gov (United States)

    Desautels, Amy; Biswas, Kamal; Lane, Alexander; Boeckelmann, Astrid; Mahmoud, Soheil S

    2009-11-01

    Linalool acetate, one of the major constituent of several essential oils, is heat-labile and decomposes upon exposure to the high injector temperature during gas chromatography. Here we report the development of an improved method for detection of this compound by gas chromatography mass spectrometry (GCMS) using cold on-column injection of the sample. By using this sensitive method, it has been demonstrated that a lavandin (L. x intermedia) mutant accumulates trace quantities of linalool acetate and camphor and higher amounts of cineole and borneol compared to its parent. This plant, which very likely carries a point mutation in one or more of the genes involved in essential oil production, provides a unique tool for investigating regulation of essential oil biogenesis in plants.

  17. Waste resources utilization program. Interim report, June 30, 1976

    International Nuclear Information System (INIS)

    1976-07-01

    This is an interim report on the effects of the combined use of heat and ionizing radiation (thermoradiation) as a treatment for ridding sewage sludge of pathogenic organisms as well as its effect on the physical-chemical properties. This activity couples two major environmental problems, disposition of human and of nuclear waste, in an attempt to provide a framework in which both will become useful resources. This combined treatment might be chosen to inactivate both heat labile (but possibly radiation resistant) and radiation labile (but possibly heat resistant) organisms. The cost-effective analyses of such a treatment are being examined. Sludge treated with thermoradiation offers considerable potential for use as a fertilizer in agriculture or a soil conditioner for land reclamation free of the potential health hazards associated with conventional methods of land disposal. Treated sludge may also provide a low-cost substitute for high-nutritional components in ruminant diets

  18. Importance in dairy technology of bacteriocins produced by dairy starter cultures

    Directory of Open Access Journals (Sweden)

    Bedia Şimşek

    2002-03-01

    Full Text Available Bacteriocins produced by Lactic acid bacteria (LAB and propionic acid bacteria (PAB are heterogeneous group of peptide inhibitors which include lantibiotics (class I, e. g. nisin, small heat-stable peptides (class II, e. g. pediocin PA-1 and large heat-labile proteins (class III, e. g. helveticin J. Many bacteriocins belonging to the first two groups can be successfully used to inhibit undesirable microorganisms in foods, but only nisin is produced industrially and is used as a food preservative. LAB and PAB develops easily in milk and milk products. LAB and PAB growth in dairy products can cause microbial interference to spoilage and pathogenic bacteria through several metabolits, specially bacteriocins. The review deals with the description of milk-borne bacteriocins and their application in milk and milk products either to extend the shelf life or to inhibit milk pathogens.

  19. TOPICAL REVIEW: Protein stability and enzyme activity at extreme biological temperatures

    Science.gov (United States)

    Feller, Georges

    2010-08-01

    Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 °C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins.

  20. Protein stability and enzyme activity at extreme biological temperatures

    International Nuclear Information System (INIS)

    Feller, Georges

    2010-01-01

    Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 0 C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins. (topical review)

  1. Tomato contact dermatitis

    DEFF Research Database (Denmark)

    Paulsen, Evy; Christensen, Lars P; Andersen, Klaus Ejner

    2012-01-01

    The tomato plant (Solanum lycopersicum) is an important crop worldwide. Whereas immediate-type reactions to tomato fruits are well known, contact dermatitis caused by tomatoes or tomato plants is rarely reported. The aims of this study were to present new data on contact sensitization to tomato...... plants and review the literature on contact dermatitis caused by both plants and fruits. An ether extract of tomato plants made as the original oleoresin plant extracts, was used in aimed patch testing, and between 2005 and 2011. 8 of 93 patients (9%) tested positive to the oleoresin extracts....... This prevalence is in accordance with the older literature that reports tomato plants as occasional sensitizers. The same applies to tomato fruits, which, in addition, may cause protein contact dermatitis. The allergens of the plant are unknown, but both heat-stable and heat-labile constituents seem...

  2. Mechanisms of pancreatic beta-cell growth and regeneration

    DEFF Research Database (Denmark)

    Nielsen, Jens Høiriis

    1989-01-01

    Information about the mechanism of beta-cell growth and regeneration may be obtained by studies of insulinoma cells. In the present study the growth and function of the rat insulinoma cell lines RINm5F and 5AH were evaluated by addition of serum, hormones, and growth factors. It was found...... of insulin mRNA content showed that the insulinoma cells only contained about 2% of that of normal rat beta-cells. These results are discussed in relation to the role of growth factors, oncogenes, and differentiation in the growth and regeneration of beta-cells....... that transferrin is the only obligatory factor whereas growth hormone, epidermal growth factor, fibroblast growth factor, and TRH had modulating effects. A heat-labile heparin binding serum factor which stimulated thymidine incorporation but not cell proliferation was demonstrated in human serum. Measurements...

  3. Efficient loading of primary alcohols onto a solid phase using a trityl bromide linker

    DEFF Research Database (Denmark)

    Crestey, François; Ottesen, Lars Korsgaard; Jaroszewski, Jerzy Witold

    2008-01-01

    The Letter describes an improved, rapid and mild strategy for the loading of primary alcohols onto a polystyrene trityl resin via a highly reactive trityl bromide linker. This protocol facilitates an efficient resin loading even of acid-sensitive or heat-labile alcohols, which otherwise require...... expensive or non-commercial resin types. Secondary alcohols were only attached in moderate to low yields, while attempts to load a tertiary alcohol expectedly failed. Importantly, selective attachment of diols via a primary alcohol group in the presence of more hindered alcohol groups proved possible....... The effects of activation time and reagent excess as well as alcohol structure were investigated. This improved method provides a convenient access to O-linked resin-bound N-Fmoc-protected amino alcohols that may be employed in SPS of peptides with C-terminal alcohol functionalities. In the case...

  4. Importance of Photobacterium phosphoreum in relation to spoilage of modified atmosphere-packed fish products

    DEFF Research Database (Denmark)

    Dalgaard, Paw; Mejlholm, Ole; Christiansen, T.J.

    1997-01-01

    Occurrence and growth of Photobacterium phosphoreum were studied in 20 experiments with fresh fish from Denmark, Iceland and Greece. The organism was detected in all marine fish species but not in fish from fresh water. Growth of P. phosphoreum to high levels (>10(7) cfu g(-1)) was observed in most...... products and the organism is likely to be of importance for spoilage of several modified atmosphere-packed (MAP) marine fish species when stored at chill temperatures. Some microbiological methods recommended for control of fish products by national and international authorities are inappropriate...... for detection of psychrotolerant and heat-labile micro-organisms like P. phosphoreum. These methods have been used in many previous studies of MAP fish and this could explain why, contrary to the findings in the present study, P. phosphoreum in general was not detected previously in spoiled MAP fish....

  5. Chromosomal and plasmid-encoded factors of Shigella flexneri induce secretogenic activity ex vivo.

    Directory of Open Access Journals (Sweden)

    Christina S Faherty

    Full Text Available Shigella flexneri is a Gram-negative, facultative intracellular pathogen that causes millions of cases of watery or bloody diarrhea annually, resulting in significant global mortality. Watery diarrhea is thought to arise in the jejunum, and subsequent bloody diarrhea occurs as a result of invasion of the colonic epithelium. Previous literature has demonstrated that Shigella encodes enterotoxins, both chromosomally and on the 220 kilobase virulence plasmid. The ShigellaEnterotoxins 1 and 2 (ShET1 and ShET2 have been shown to increase water accumulation in the rabbit ileal loop model. In addition, these toxins increase the short circuit current in rabbit tissue mounted in Ussing chambers, which is a model for the ion exchange that occurs during watery diarrhea. In this study, we sought to validate the use of mouse jejunum in Ussing chamber as an alternative, more versatile model to study bacterial pathogenesis. In the process, we also identified enterotoxins in addition to ShET1 and ShET2 encoded by S. flexneri. Through analysis of proteins secreted from wildtype bacteria and various deletion mutants, we have identified four factors responsible for enterotoxin activity: ShET1 and Pic, which are encoded on the chromosome; ShET2 (encoded by sen or ospD3, which requires the type-III secretion system for secretion; and SepA, an additional factor encoded on the virulence plasmid. The use of mouse jejunum serves as a reliable and reproducible model to identify the enterotoxins elaborated by enteric bacteria. Moreover, the identification of all Shigella proteins responsible for enterotoxin activity is vital to our understanding of Shigella pathogenicity and to our success in developing safe and effective vaccine candidates.

  6. Oxidation of the diphosphoinositol polyphosphate phosphohydrolase-like Nudix hydrolase Aps from Drosophila melanogaster induces thermolability--A possible regulatory switch?

    Science.gov (United States)

    Winward, Lucinda; Whitfield, William G F; McLennan, Alexander G; Safrany, Stephen T

    2010-07-01

    Unlike mammalian cells, Drosophila melanogaster contains only a single member of the diphosphoinositol polyphosphate phosphohydrolase subfamily of the Nudix hydrolases, suggesting that functional specialisation has not occurred in this organism. In order to evaluate its function, Aps was cloned and characterized. It hydrolyses a range of (di)nucleoside polyphosphates, the most efficient being guanosine 5'-tetraphosphate (K(m)=11 microM, k(cat)=0.79 s(-1)). However, it shows a 5-fold preference for the hydrolysis of diphosphoinositol pentakisphosphate (PP-InsP(5), K(m)=0.07 microM, k(cat)=0.024 s(-1)). Assayed at 26 degrees C, Aps had an alkaline pH optimum and required a divalent ion: Mg(2+) (10-20 mM) or Mn(2+) (1 mM) were preferred for nucleotide hydrolysis and Mg(2+) (0.5-1 mM) or Co(2+) (1-100 microM) for PP-InsP(5) hydrolysis. GFP-fusions showed that Aps was predominantly cytoplasmic, with some nuclear localization. In the absence of dithiothreitol Aps was heat labile, rapidly losing activity even at 36 degrees C, while in the presence of dithiothreitol, Aps was heat stable, surviving for 5 min at 76 degrees C. Heat lability was restored by H(2)O(2) and mass spectrometric analysis suggested that this was due to reversible dimerisation involving two inter-molecular disulphides between Cys23 and Cys25. Aps expression was highest in embryos and declined throughout development. The ratio of PP-InsP(5) to inositol hexakisphosphate also decreased throughout development, with the highest level of PP-InsP(5) found in embryos. These data suggest that the redox state of Aps may play a role in controlling its activity by altering its stability, something that could be important for regulating PP-InsP(5) during development. Copyright 2010 Elsevier Ltd. All rights reserved.

  7. Isolation of Bacillus cereus Group from the Fecal Material of Endangered Wood Turtles.

    Science.gov (United States)

    Nfor, Nancy Ngvumbo; Lapin, Carly N; McLaughlin, Richard William

    2015-10-01

    Members of the Bacillus cereus group are opportunistic human pathogens. They can be found in a broad range of foods. Diarrheal food poisoning and/or emetic type syndromes can result from eating contaminated food. In this study, seven B. cereus group members were isolated from the fecal material of Wood Turtles (Glyptemys insculpta). The isolates were then assessed for the presence of enterotoxin genes (nheA, entFM, hblC, and cytK) using PCR. The most prevalent is the nonhemolytic enterotoxin gene which was found in all seven isolates.

  8. Inhibition of Clostridium difficile toxin A and B by 1,2-cyclohexanedione modification of an arginine residue.

    Science.gov (United States)

    Balfanz, J; Rautenberg, P

    1989-12-29

    Toxin A (enterotoxin) and toxin B (cytotoxin) of Clostridium difficile were both inactivated by the arginine specific reagent 1,2-cyclohexanedione. Molecular stability during the inactivation process was demonstrated by SDS-PAGE analysis showing the same migration rates for modified and unmodified forms of the 230 kDa toxin A and of the 250 kDa toxin B. Cytotoxicity of both toxins as well as mouse lethality of the enterotoxin were drastically decreased as a result of the arginine modification. The reaction followed pseudo-first-order kinetics. Analysis of the data suggested that modification of a single arginine residue was sufficient to abolish the activity of both toxins.

  9. Molecular Typing of Staphylococcus Aureus Isolate Responsible for Staphylococcal Poisoning Incident in Homemade Food.

    Science.gov (United States)

    Macori, Guerrino; Bellio, Alberto; Bianchi, Daniela Manila; Gallina, Silvia; Adriano, Daniela; Zuccon, Fabio; Chiesa, Francesco; Acutis, Pier Luigi; Casalinuovo, Francesco; Decastelli, Lucia

    2016-04-19

    In October 2012, two persons fell ill with symptoms consistent with staphylococcal food poisoning after eating home-canned tuna fish and tomatoes. Laboratory investigation detected the enterotoxins in the home-canned tuna and molecular analysis of the isolated Staphylococcus aureus confirmed it carried toxin genes. Qualitative enzyme-linked immunosorbent assay and enzime linked fluorescent assay methods and quantitative assay identified the enterotoxins in the food leftovers, specifically staphylococcal enterotoxins type A (SEA) and D (SED), respectively 0.49 and 2.04 ng/g. The laboratory results are discussed considering the relation to the fish in oil, survival and heat resistance of S. aureus , and presumptive microbial contamination due to improper handling during home-canning procedures. This is the first reported cluster of foodborne illnesses due to staphylococcal enterotoxins in tuna in Italy. In this study, we reported cases described and analysed for their spa -type. Showing a high heterogeneity of isolates, spa -type t 13252 is correlated in a node of the minimum spanning tree and it has never been reported as responsible for foodborne outbreak. This case underlines the importance of risk communication and dissemination of home-canning guidelines to reduce the incidence of foodborne outbreaks caused by homemade conserves.

  10. Molecular typing of Staphylococcus aureus isolate responsible for staphylococcal poisoning incident in homemade food

    Directory of Open Access Journals (Sweden)

    Guerrino Macori

    2016-06-01

    Full Text Available In October 2012, two persons fell ill with symptoms consistent with staphylococcal food poisoning after eating home-canned tuna fish and tomatoes. Laboratory investigation detected the enterotoxins in the home-canned tuna and molecular analysis of the isolated Staphylococcus aureus confirmed it carried toxin genes. Qualitative enzyme-linked immunosorbent assay and enzime linked fluorescent assay methods and quantitative assay identified the enterotoxins in the food leftovers, specifically staphylococcal enterotoxins type A (SEA and D (SED, respectively 0.49 and 2.04 ng/g. The laboratory results are discussed considering the relation to the fish in oil, survival and heat resistance of S. aureus, and presumptive microbial contamination due to improper handling during home-canning procedures. This is the first reported cluster of foodborne illnesses due to staphylococcal enterotoxins in tuna in Italy. In this study, we reported cases described and analysed for their spa-type. Showing a high heterogeneity of isolates, spa-type t13252 is correlated in a node of the minimum spanning tree and it has never been reported as responsible for foodborne outbreak. This case underlines the importance of risk communication and dissemination of home-canning guidelines to reduce the incidence of foodborne outbreaks caused by homemade conserves.

  11. Optimizing Two-Color Semiconductor Nanocrystal Immunoassays in Single Well Microtiter Plate Formats

    Directory of Open Access Journals (Sweden)

    W. Russ Algar

    2011-08-01

    Full Text Available The simultaneous detection of two analytes, chicken IgY (IgG and Staphylococcal enterotoxin B (SEB, in the single well of a 96-well plate is demonstrated using luminescent semiconductor quantum dot nanocrystal (NC tracers. The NC-labeled antibodies were prepared via sulfhydryl-reactive chemistry using a facile protocol that took

  12. The N-Terminal Flanking Region of the Invariant Chain Peptide Augments the Immunogenicity of a Cryptic “Self” Epitope from a Tumor-Associated Antigen

    NARCIS (Netherlands)

    Hess, A.D.; Thoburn, C.; Chen, W.; Miura, Y.; Wall, E. van der

    2001-01-01

    The N-terminal flanking region of the invariant chain peptide termed CLIP appears to have superagonistic properties interacting with the T cell receptor and the MHC class II molecule at or near the binding site for the bacterial superantigen Staphylococcal enterotoxin B (SEB). The present studies

  13. Food irradiation and bacterial toxins

    International Nuclear Information System (INIS)

    Tranter, H.S.; Modi, N.K.; Hambleton, P.; Melling, J.; Rose, S.; Stringer, M.F.

    1987-01-01

    The authors' findings indicate that irradiation confers no advantage over heat processing in respect of bacterial toxins (clostridium botulinum, neurotoxin A and staphylococcal enterotoxin A). It follows that irradiation at doses less than the ACINF recommended upper limit of 10 kGy could not be used to improve the ambient temperature shelf life on non-acid foods. (author)

  14. DNA microarray genotyping and virulence and antimicrobial resistance gene profiling of methicillin-resistant Staphylococcus aureus bloodstream isolates from renal patients.

    LENUS (Irish Health Repository)

    McNicholas, Sinead

    2012-02-01

    Thirty-six methicillin-resistant Staphylococcus aureus (MRSA) bloodstream isolates from renal patients were genetically characterized by DNA microarray analysis and spa typing. The isolates were highly clonal, belonging mainly to ST22-MRSA-IV. The immune evasion and enterotoxin gene clusters were found in 29\\/36 (80%) and 33\\/36 (92%) isolates, respectively.

  15. Fate and effect of ingested Bacillus cereus spores and vegetative cells in the intestinal tract of human-flora-associated rats

    DEFF Research Database (Denmark)

    Wilcks, Andrea; Hansen, Bjarne Munk; Hendriksen, Niels Bohse

    2006-01-01

    The fate and effect of Bacillus cereus F4433/73R in the intestine of human-flora-associated rats was studied using bacteriological culturing techniques and PCR-denaturing gradient gel electrophoresis in combination with cell assays and immunoassays for detection of enterotoxins. In faecal samples...

  16. Occurrence of Natural Bacillus thuringiensis Contaminants and Residues of Bacillus thuringiensis-Based Insecticides on Fresh Fruits and Vegetables

    Science.gov (United States)

    Frederiksen, Kristine; Rosenquist, Hanne; Jørgensen, Kirsten; Wilcks, Andrea

    2006-01-01

    A total of 128 Bacillus cereus-like strains isolated from fresh fruits and vegetables for sale in retail shops in Denmark were characterized. Of these strains, 39% (50/128) were classified as Bacillus thuringiensis on the basis of their content of cry genes determined by PCR or crystal proteins visualized by microscopy. Random amplified polymorphic DNA analysis and plasmid profiling indicated that 23 of the 50 B. thuringiensis strains were of the same subtype as B. thuringiensis strains used as commercial bioinsecticides. Fourteen isolates were indistinguishable from B. thuringiensis subsp. kurstaki HD1 present in the products Dipel, Biobit, and Foray, and nine isolates grouped with B. thuringiensis subsp. aizawai present in Turex. The commercial strains were primarily isolated from samples of tomatoes, cucumbers, and peppers. A multiplex PCR method was developed to simultaneously detect all three genes in the enterotoxin hemolysin BL (HBL) and the nonhemolytic enterotoxin (NHE), respectively. This revealed that the frequency of these enterotoxin genes was higher among the strains indistinguishable from the commercial strains than among the other B. thuringiensis and B. cereus-like strains isolated from fruits and vegetables. The same was seen for a third enterotoxin, CytK. In conclusion, the present study strongly indicates that residues of B. thuringiensis-based insecticides can be found on fresh fruits and vegetables and that these are potentially enterotoxigenic. PMID:16672488

  17. Identification of Staphylococcus spp. isolated during the ripening process of a traditional minas cheese

    Directory of Open Access Journals (Sweden)

    B.M. Borelli

    2011-04-01

    Full Text Available The population dynamics of Staphylococcus spp. was studied during the ripening of Canastra Minas cheese at three farms located in the State of Minas Gerais, Brazil. The presence of coagulase (coa, thermonuclease (nuc, and enterotoxin (sea, seb, sec, and sed genes was investigated in Staphylococcus strains isolated during the 60-day cheese-ripening period. The presence of the staphylococcal enterotoxins A, C, and D was also investigated in the cheese samples. Cheese samples that were matured for 0, 7, 15, 30, and 45 days presented staphylococci counts from 10³ to 10(8cfu/g. All isolates considered coagulase-positive by physiological tests had the coa gene. However, no association was observed between the results obtained with biochemical tests and those obtained by PCR using gene-specific primers for coagulase-negative strains. Coagulase and thermonuclease genes occurred simultaneously in 41.3% of Staphylococcus spp. tested. None of the investigated Staphylococcus strains expressed enterotoxins SEA, SEB, SEC, and SED. Enterotoxins A, C, and D were not detected in any of the cheese samples.

  18. Laser Induced Breakdown Spectroscopy (LIBS)

    Science.gov (United States)

    2010-03-31

    Radium 226 Page 34 Nitrogen mustard (HN) Lassa Polonium 210 Phosgene oxime (CX) Staphylococcal Enterotoxin B (SEB) ( Food Poisoning...sample preparation. Diverse solid or liquid samples including environmental, biological, geological, industrial, forensic, food , thin coatings...Thallium 201 Hydrogen cyanide (AC) FUNGI Thorium 232 Cyanogen chloride (CK) Aflatoxin Lead 210 Arsine (SA) Bacillus Sodium 22

  19. DNA microarray genotyping and virulence and antimicrobial resistance gene profiling of methicillin-resistant Staphylococcus aureus bloodstream isolates from renal patients.

    LENUS (Irish Health Repository)

    McNicholas, Sinead

    2011-12-01

    Thirty-six methicillin-resistant Staphylococcus aureus (MRSA) bloodstream isolates from renal patients were genetically characterized by DNA microarray analysis and spa typing. The isolates were highly clonal, belonging mainly to ST22-MRSA-IV. The immune evasion and enterotoxin gene clusters were found in 29\\/36 (80%) and 33\\/36 (92%) isolates, respectively.

  20. Multilocus Sequence Typing And Antibiotic Resistance Of Staphylococcus Aureus Isolated From The Brazilian Dairy Industry

    DEFF Research Database (Denmark)

    Dittmann, Karen Kiesbye; Chaul, Luiza; Lee, Sarah

    2015-01-01

    Staphylococcus aureus is a common cause of food poisoning due to enterotoxin production. This is particularly an issue in the dairy industry, where S. aureus can contaminate the product e.g. from raw milk or the handlers. In Brazil, soft cheese is mainly produced in small dairy plants where good ...

  1. TCR-Vß8 as alternative to animal testing for quantifying active SEE

    Science.gov (United States)

    Staphylococcal food poisoning is a result of ingestion of Staphylococcal enterotoxins (SEs) produced by the bacterium Staphylococcus aureus. SEs cause gastroenteritis and also cause activation of T cells and massive cytokine release. A current method for the detection of active SEs relies on its eme...

  2. A Study To Assess the Numbers and Prevalence of Bacillus cereus and Its Toxins in Pasteurized Fluid Milk.

    Science.gov (United States)

    Saleh-Lakha, Saleema; Leon-Velarde, Carlos G; Chen, Shu; Lee, Susan; Shannon, Kelly; Fabri, Martha; Downing, Gavin; Keown, Bruce

    2017-07-01

    Bacillus cereus is a pathogenic adulterant of raw milk and can persist as spores and grow in pasteurized milk. The objective of this study was to determine the prevalence of B. cereus and its enterotoxins in pasteurized milk at its best-before date when stored at 4, 7, and 10°C. More than 5.5% of moderately temperature-abused products (stored at 7°C) were found to contain >10 5 CFU/mL B. cereus , and about 4% of them contained enterotoxins at a level that may result in foodborne illness; in addition, more than 31% of the products contained >10 5 CFU/mL B. cereus and associated enterotoxins when stored at 10°C. Results from a growth kinetic study demonstrated that enterotoxin production by B. cereus in pasteurized milk can occur in as short as 7 to 8 days of storage at 7°C. The higher B. cereus counts were associated with products containing higher butterfat content or with those produced using the conventional high-temperature, short-time pasteurization process. Traditional indicators, aerobic colony counts and psychrotrophic counts, were found to have no correlation with level of B. cereus in milk. The characterization of 17 representative B. cereus isolates from pasteurized milk revealed five toxigenic gene patterns, with all the strains carrying genes encoding for diarrheal toxins but not for an emetic toxin, and with one strain containing all four diarrheal enterotoxin genes (nheA, entFM, hblC, and cytK). The results of this study demonstrate the risks associated even with moderately temperature-abused pasteurized milk and the necessity of a controlled cold chain throughout the shelf life of fluid milk to enhance product safety and minimize foodborne illness.

  3. Prevalence of toxin genes among the clinical isolates of Staphylococcus aureus and its clinical impact

    Directory of Open Access Journals (Sweden)

    Divya Deodhar

    2015-01-01

    Full Text Available Introduction: Staphylococcus aureus (S. aureus causes a variety of infections, ranging from a mild skin infection to blood stream infections and deep seated infections. As Stapylococcus aureus bacteremia (SAB has the tendency to cause endovascular and metastatic infections, complications can occur at almost all sites of the body. Hence, SAB is associated with increased morbidity and mortality in spite of appropriate antimicrobial treatment. The virulence in S. aureus is determined by the presence of adhesins and toxins, which behave like superantigens (SAgs and leads to a massive release of proinflammatory cytokines causing overwhelming inflammatory response leading to endothelial leakage, hemodynamic shock, multiorgan failure, and possibly death. Materials and Methods: One year prospective study conducted in a tertiary care hospital in southern part of India included all patients with SAB. Clinical details were filled according to. All isolates were subjected to polymerase chain reaction (PCR for enterotoxin profiling. Results: A total of 101 patients of SAB were identified which comprises of 61 (60.4% patients with methicillin-susceptible S. aureus (MSSA and 40 (39.6% patients with methicillin-resistant S. aureus (MRSA. Most common predictors of mortality were prior hospitalization and antibiotic intake, severe organ dysfunction, shock, tachycardia, and leukocytosis. Two-third of the isolates had at least one enterotoxin, most prevalent was sea; 28% and 27% (P - value = 0.001 MSSA isolates had seg and sei; whereas, 38.6% (P - value < 0.001 of MRSA isolates were found to have sea. The most common enterotoxin associated with mortality was sei, which comprised of 38% of all mortality. Conclusion: In SAB, the significant predictors of mortality were prior hospitalization and antibiotic intake, presence of multiorgan dysfunction, and shock. Although overall significance between the enterotoxin and shock could not be demonstrated, it successfully

  4. Prevalence of Toxin Genes among the Clinical Isolates of Staphylococcus aureus and its Clinical Impact.

    Science.gov (United States)

    Deodhar, Divya; Varghese, George; Balaji, Veeraraghavan; John, James; Rebekah, Grace; Janardhanan, Jeshina; Jeyaraman, Ranjith; Jasmine, Sudha; Mathews, Prasad

    2015-01-01

    Staphylococcus aureus (S. aureus) causes a variety of infections, ranging from a mild skin infection to blood stream infections and deep seated infections. As Stapylococcus aureus bacteremia (SAB) has the tendency to cause endovascular and metastatic infections, complications can occur at almost all sites of the body. Hence, SAB is associated with increased morbidity and mortality in spite of appropriate antimicrobial treatment. The virulence in S. aureus is determined by the presence of adhesins and toxins, which behave like superantigens (SAgs) and leads to a massive release of proinflammatory cytokines causing overwhelming inflammatory response leading to endothelial leakage, hemodynamic shock, multiorgan failure, and possibly death. One year prospective study conducted in a tertiary care hospital in southern part of India included all patients with SAB. Clinical details were filled according to. All isolates were subjected to polymerase chain reaction (PCR) for enterotoxin profiling. A total of 101 patients of SAB were identified which comprises of 61 (60.4%) patients with methicillin-susceptible S. aureus (MSSA) and 40 (39.6%) patients with methicillin-resistant S. aureus (MRSA). Most common predictors of mortality were prior hospitalization and antibiotic intake, severe organ dysfunction, shock, tachycardia, and leukocytosis. Two-third of the isolates had at least one enterotoxin, most prevalent was sea; 28% and 27% (P - value = 0.001) MSSA isolates had seg and sei; whereas, 38.6% (P - value < 0.001) of MRSA isolates were found to have sea. The most common enterotoxin associated with mortality was sei, which comprised of 38% of all mortality. In SAB, the significant predictors of mortality were prior hospitalization and antibiotic intake, presence of multiorgan dysfunction, and shock. Although overall significance between the enterotoxin and shock could not be demonstrated, it successfully demonstrated the difference of enterotoxin between MSSA and MRSA.

  5. Genome sequencing and molecular characterisation of Staphylococcus aureus ST772-MRSA-V, "Bengal Bay Clone".

    Science.gov (United States)

    Monecke, Stefan; Baier, Vico; Coombs, Geoffrey W; Slickers, Peter; Ziegler, Albrecht; Ehricht, Ralf

    2013-12-20

    The PVL-positive ST772-MRSA-V is an emerging community-associated (CA-) MRSA clone that has been named Bengal Bay Clone since most patients have epidemiological connections to the Indian subcontinent. It is found increasingly common in other areas of the world. One isolate of ST772-MRSA-V was sequenced using the Illumina Genome Analyzer System. After initial assembling the multiple sequence contigs were analysed using different in-house annotation scripts. Results were compared to microarray hybridisation results of clinical isolates of ST772-MRSA-V, of related strains and to another ST772-MRSA-V genome sequence. According to MLST e-burst analysis, ST772-MRSA-V belongs to Clonal Complex (CC)1, differing from ST1 only in one MLST allele (pta-22). However, there are several additional differences including agr alleles (group II rather than III), capsule type (5 rather than 8), the presence of the egc enterotoxin gene cluster and of the enterotoxin homologue ORF CM14 as well as the absence of the enterotoxin H gene seh. Enterotoxin genes sec and sel are present. ST772-MRSA-V harbours the genes encoding enterotoxin A (sea) and PVL (lukS/F-PV). Both are located on the same prophage. ST772-MRSA-V may have emerged from the same lineage as globally spread CC1 and CC5 strains. It has acquired a variety of virulence factors, and for a CA-MRSA strain it has an unusually high number of genes associated with antibiotic resistance.

  6. Networked T cell death following macrophage infection by Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Stephen H-F Macdonald

    Full Text Available BACKGROUND: Depletion of T cells following infection by Mycobacterium tuberculosis (Mtb impairs disease resolution, and interferes with clinical test performance that relies on cell-mediated immunity. A number of mechanisms contribute to this T cell suppression, such as activation-induced death and trafficking of T cells out of the peripheral circulation and into the diseased lungs. The extent to which Mtb infection of human macrophages affects T cell viability however, is not well characterised. METHODOLOGY/PRINCIPAL FINDINGS: We found that lymphopenia (<1.5 × 10(9 cells/l was prevalent among culture-positive tuberculosis patients, and lymphocyte counts significantly improved post-therapy. We previously reported that Mtb-infected human macrophages resulted in death of infected and uninfected bystander macrophages. In the current study, we sought to examine the influence of infected human alveolar macrophages on T cells. We infected primary human alveolar macrophages (the primary host cell for Mtb or PMA-differentiated THP-1 cells with Mtb H37Ra, then prepared cell-free supernatants. The supernatants of Mtb-infected macrophages caused dose-dependent, caspase-dependent, T cell apoptosis. This toxic effect of infected macrophage secreted factors did not require TNF-α or Fas. The supernatant cytotoxic signal(s were heat-labile and greater than 50 kDa in molecular size. Although ESAT-6 was toxic to T cells, other Mtb-secreted factors tested did not influence T cell viability; nor did macrophage-free Mtb bacilli or broth from Mtb cultures. Furthermore, supernatants from Mycobacterium bovis Bacille de Calmette et Guerin (BCG- infected macrophages also elicited T cell death suggesting that ESAT-6 itself, although cytotoxic, was not the principal mediator of T cell death in our system. CONCLUSIONS: Mtb-Infected macrophages secrete heat-labile factors that are toxic to T cells, and may contribute to the immunosuppression seen in tuberculosis as well as

  7. Adaptation in Bacillus cereus: from stress to disease

    Directory of Open Access Journals (Sweden)

    Catherine Duport

    2016-10-01

    Full Text Available Bacillus cereus is a foodborne pathogen that causes diarrheal disease in humans. After ingestion B. cereus experiences in the human gastro-intestinal tract abiotic physical variables encountered in food, such as acidic pH in the stomach and changing oxygen conditions in the human intestine. B. cereus responds to environmental changing conditions (stress by reversibly adjusting its physiology to maximize resource utilization while maintaining structural and genetic integrity by repairing and minimizing damage to cellular infrastructure. As reviewed in this article, B. cereus adapts to acidic pH and changing oxygen conditions through diverse regulatory mechanisms and then exploits its metabolic flexibility to grow and produce enterotoxins. We then focus on the intricate link between metabolism, redox homeostasis and enterotoxins, which are recognized as important contributors of food-borne disease.

  8. Superantigen and HLA-DR ligation induce phospholipase-C gamma 1 activation in class II+ T cells

    DEFF Research Database (Denmark)

    Kanner, S B; Odum, Niels; Grosmaire, L

    1992-01-01

    Bacterial enterotoxin superantigens bind directly to HLA class II molecules (HLA-DR) expressed on both APC and activated human T cells, and simultaneously bind to certain V beta chains of the TCR. In this report, we compared early T cell signaling events in human alloantigen-stimulated T cells when...... activated by HLA-DR ligation through antibody cross-linking or by direct enterotoxin superantigen binding. Both types of stimuli induced tyrosine phosphorylation of phosphatidylinositol-specific phospholipase C gamma 1 (PLC gamma 1) and an increase in intracellular calcium concentration; however......, superantigen-induced signaling was stronger than class II ligation alone. Antibody-mediated ligation of HLA-DR with CD3 resulted in augmented PLC gamma 1 activation and increased calcium mobilization, consistent with a mechanism of superantigen activity through a combination of class II and CD3/Ti signals...

  9. Incidence of enterotoxigenic staphylococci and their toxins in foods.

    Science.gov (United States)

    Soriano, J M; Font, G; Rico, H; Moltó, J C; Mañes, J

    2002-05-01

    Of 504 food samples collected from cafeterias, 19 (3.8%) yielded strains of enterotoxigenic staphylococci, and 10 (52.6%), 4 (21.1%), 3 (15.8%), and 2 (10.5%) of these strains produced enterotoxins C (SEC), D (SED), B (SEB), and A (SEA), respectively. Moreover, SEA, SEB, and SEC were isolated from three hamburger samples. Of 181 food samples collected from four restaurants before the implementation of the hazard analysis and critical control point (HACCP) system, 7 (3.9%) were found to contain enterotoxigenic strains, and SED, SEC, and SEA were produced by 4 (57.1%), 2 (28.6%), and 1 (14.3%) of these strains, respectively. One meatball sample with SEC was detected in a restaurant. After the implementation of the HACCP system in four restaurants, neither enterotoxigenic staphylococci nor enterotoxins were detected in 196 studied samples.

  10. Studies on the irradiation of toxins of Clostridium botulinum and Staphylococcus aureus

    International Nuclear Information System (INIS)

    Rose, S.A.; Bailey, N.E.; Stringer, M.F.; Modi, N.K.; Tranter, H.S.; Hambleton, P.

    1989-01-01

    The effects of irradiation of Clostridium botulinum neutotoxin type A (BNTA) and staphylococcal enterotoxin A (SEA) in gelatin phosphate buffer and cooked mince beef slurries were investigated. Estimation of toxins by immunoassays showed that in buffer, toxins were destroyed by irradiation at 8.0 kGy; in mince slurries however, 45% of BTNA and 27-34% of SEA remained after this level of irradiation. At 23.7 kGy, over twice the dose of irradiation proposed for legal acceptance in the UK, 15% of BNTA and 16-26% of SEA still remained. Increasing concentrations of mince conferred increased protection against the effect of irradiation on both toxins. The biological activity of BNTA was more sensitive to irradiation than the immunological activity. Staphylococcal enterotoxin was more resistant to irradiation than BNTA. Irradiation should therefore only be used in conjunction with good manufacturing practices to prevent microbial proliferation and toxin production prior to irradiation. (author)

  11. Studies on the irradiation of toxins of Clostridium botulinum and Staphylococcus aureus

    Energy Technology Data Exchange (ETDEWEB)

    Rose, S A; Bailey, N E; Stringer, M F [Campden Food and Drink Research Association, Chipping Campden (UK); Modi, N K; Tranter, H S [Porton International plc., London (UK); Hambleton, P [Centre for Applied Microbiological Research, Porton Down (UK)

    1988-10-01

    The effects of irradiation of Clostridium botulinum neurotoxin type A (BNTA) and staphylococcal enterotoxin A (SEA) in gelatin phosphate buffer and cooked mince beef slurries were investigated. Estimation of toxins by immunoassays showed that in buffer, toxins were destroyed by irradiation at 8.0 kGy; in mince slurries however, 45% of BTNA and 27-34% of SEA remained after this level of irradiation. At 23.7 kGy, over twice the dose of irradiation proposed for legal acceptance in the UK, 15% of BNTA and 16-26% of SEA still remained. Increasing concentrations of mince conferred increased protection against the effect of irradiation on both toxins. The biological activity of BNTA was more sensitive to irradiation than the immunological activity. Staphylococcal enterotoxin was more resistant to irradiation than BNTA. Irradiation should therefore only be used in conjunction with good manufacturing practices to prevent microbial proliferation and toxin production prior to irradiation. (author).

  12. Geographical variation in the presence of genes encoding superantigenic exotoxins and beta-hemolysin among Staphylococcus aureus isolated from bovine mastitis in Europe and USA

    DEFF Research Database (Denmark)

    Larsen, H. D.; Aarestrup, Frank Møller; Jensen, N. E.

    2002-01-01

    The object was to examine the geographical variation in the presence of superantigenic exotoxins and beta-hemolysin among epidemiologically independent Staphyirrcoccus aureus isolates from bovine mastitis. A total of 462 S. aureus isolates from nine European countries and USA were examined...... for the individual exotoxins. The genes encoding enterotoxin C, TSST-1, and enterotoxin D were the most common superantigens. The present and earlier studies demonstrate that the superantigenic exotoxins that were investigated in this study, do not play a role in the pathogenesis of bovine S. aureus mastitis...... regions in the beta-hemolysin encoding gene of the Norwegian isolates is suggested, and should be investigated further. The consistent presence of beta-hemolysin suggests that this factor, or a co-existing gene correlated to beta-hemolysin, may be an active virulence factor in the pathogenesis of bovine S...

  13. Studies on the irradiation of toxins of Clostridium botulinum and Staphylococcus aureus

    International Nuclear Information System (INIS)

    Rose, S.A.; Bailey, N.E.; Stringer, M.F.; Modi, N.K.; Tranter, H.S.; Hambleton, P.

    1988-01-01

    The effects of irradiation of Clostridium botulinum neurotoxin type A (BNTA) and staphylococcal enterotoxin A (SEA) in gelatin phosphate buffer and cooked mince beef slurries were investigated. Estimation of toxins by immunoassays showed that in buffer, toxins were destroyed by irradiation at 8.0 kGy; in mince slurries however, 45% of BTNA and 27-34% of SEA remained after this level of irradiation. At 23.7 kGy, over twice the dose of irradiation proposed for legal acceptance in the UK, 15% of BNTA and 16-26% of SEA still remained. Increasing concentrations of mince conferred increased protection against the effect of irradiation on both toxins. The biological activity of BNTA was more sensitive to irradiation than the immunological activity. Staphylococcal enterotoxin was more resistant to irradiation than BNTA. Irradiation should therefore only be used in conjunction with good manufacturing practices to prevent microbial proliferation and toxin production prior to irradiation. (author)

  14. Studies on the irradiation of toxins of Clostridium botulinum and Staphylococcus aureus

    Energy Technology Data Exchange (ETDEWEB)

    Rose, S.A.; Bailey, N.E.; Stringer, M.F. (Campden Food and Drink Research Association, Chipping Campden (UK)); Modi, N.K.; Tranter, H.S. (Centre for Applied Micobiological Research, Porton Down, (UK)); Hambleton, P. (Porton International plc, London (UK))

    1989-09-01

    The effects of irradiation of Clostridium botulinum neutotoxin type A (BNTA) and staphylococcal enterotoxin A (SEA) in gelatin phosphate buffer and cooked mince beef slurries were investigated. Estimation of toxins by immunoassays showed that in buffer, toxins were destroyed by irradiation at 8.0 kGy; in mince slurries however, 45% of BTNA and 27-34% of SEA remained after this level of irradiation. At 23.7 kGy, over twice the dose of irradiation proposed for legal acceptance in the UK, 15% of BNTA and 16-26% of SEA still remained. Increasing concentrations of mince conferred increased protection against the effect of irradiation on both toxins. The biological activity of BNTA was more sensitive to irradiation than the immunological activity. Staphylococcal enterotoxin was more resistant to irradiation than BNTA. Irradiation should therefore only be used in conjunction with good manufacturing practices to prevent microbial proliferation and toxin production prior to irradiation. (author).

  15. Selection of bacteriocin producer strains of lactic acid bacteria from a dairy environment.

    Science.gov (United States)

    Lasagno, M; Beoleito, V; Sesma, F; Raya, R; Font de Valdez, G; Eraso, A

    2002-01-01

    Two strains showing bacteriocin production were selected from a total of 206 lactic acid bacteria isolated from samples of milk, milk serum, whey and homemade cheeses in Southern Cordoba, Argentina. This property was detected by means of well diffusion assays. The strains were identified as Enterococcus hirae and Enterococcus durans. The protein nature of those substances was proved by showing their sensitivity to type IV and XXV proteases, papaine, trypsin, pepsin and K proteinase. The bacteriocins inhibited the growth of Listeria monocytogenes, Bacillus cereus, Clostridium perfringes and two strains of Staphylococcus aureus, an A-enterotoxin and a B-enterotoxin producers. All of these bacteria are common pathogens usually associated with food borne diseases (ETA). These lactic acid bacteria or their bacteriocins could be suitable candidates for food preservation and specially useful in the our regional dairy industry.

  16. Clostridium perfringens in London, July 2009: two weddings and an outbreak.

    OpenAIRE

    Eriksen, J.; Zenner, D.; Anderson, S. R.; Grant, K.; Kumar, D.

    2010-01-01

    : Food poisoning outbreaks caused by Clostridium perfringens enterotoxin occur occasionally in Europe but have become less common in recent years. This paper presents the microbiological and epidemiological results of a large C. perfringens outbreak occurring simultaneously at two weddings that used the same caterer. The outbreak involved several London locations and required coordination across multiple agencies. A case-control study (n=134) was carried out to analyse possible associations b...

  17. Detection of the enterotoxigenic genes (sei,sej) in Staphylococcus aureus isolates from bovine mastitis milk in the West Azerbaijan of Iran

    OpenAIRE

    Ahmady, Malahat; Kazemi, Sahar

    2012-01-01

    Staphylococcus aureus is a major causative pathogen of clinical and subclinical mastitis of dairy domestic ruminants. This organism produces a variety of extracellular toxins and virulence factors such as enterotoxin SEI and SEJ that contribute to its pathogenic potential. In this study 25 S. aureus isolates obtained from four dairy herds of Urmia region which is located in West Azerbaijan province in Iran. The tested isolates were identified on the basis of the cultural and biochemical prope...

  18. Housefly (Musca domestica as Carrier of Enterotoxigenic Staphylococcus aureus in Broiler Farms in Iran: Is it Important for Public Health?

    Directory of Open Access Journals (Sweden)

    Mehdi Rashki Ghalehnoo

    2015-08-01

    Full Text Available Background: During the last decade, the prevalence of insect borne diseases due to contaminated food as well as the outbreaks of diseases due to enterotoxin-producing Staphylococcus aureus (ETSA strains has increased. Objectives: This study was conducted to determine the prevalence rate, enterotoxigenecity, and antimicrobial resistance of S. aureus isolated from M. domestica collected from the residential areas of nine districts broiler farms in Zabol, Iran. Materials and Methods: The flies were captured with a sterile nylon net and washed twice with distilled water. The contents were streaked onto selective media and S. aureus was identified using the conventional biochemical tests. Antimicrobial susceptibility testing was performed using CLSI guideline. Staphylococcal enterotoxins (SEs, SEA, SEB, SEC and SED, was detected by the reverse passive latex agglutination (PRLA method. Coagulase typing was performed with coagulase typing kit. Results: A total of 87 (17% S. aureus was isolated from 450 samples. The ability to synthesize staphylococcal enterotoxins (SEs was determined in 62 of 87 (71% isolates. SE type B was the most common enterotoxin found in the isolated S. aureus (45%, followed by SE type A (26%, SE type C (5%, SE type D (8%, SEA + SEC (7% and SEA + SEB (7%. Among the antibiotic tested, Penicillin was the most resistant antibiotic tested. Sixty seven percent of isolates belonged to coagulase type III, VI, VII, VIII, IV, and V. Conclusions: Our results suggest that S. aureus, including ETSA, is being carried by house flies such as M. domestica and may contribute to the spread of pathogenic isolates, with an impact on public health.

  19. Bacillus cereus and related species.

    OpenAIRE

    Drobniewski, F A

    1993-01-01

    Bacillus cereus is a gram-positive aerobic or facultatively anaerobic spore-forming rod. It is a cause of food poisoning, which is frequently associated with the consumption of rice-based dishes. The organism produces an emetic or diarrheal syndrome induced by an emetic toxin and enterotoxin, respectively. Other toxins are produced during growth, including phospholipases, proteases, and hemolysins, one of which, cereolysin, is a thiol-activated hemolysin. These toxins may contribute to the pa...

  20. Volunteer Challenge With Enterotoxigenic Escherichia coli That Express Intestinal Colonization Factor Fimbriae CS17 and CS19

    Science.gov (United States)

    2011-07-01

    Serotype was determined by classic serological methods at the Universidad Nacional Aut6noma de Mexico [UNAMl. H- indrcates non-motility. b CF...Levine MM, Merson MM. Serologic differentiation between antitoxin responses to infection with Vibrio cholerae and enterotoxin-producing Escherichia coli...prototype cholera B subunit-colonization factor antigen cnterotoxigenic Escherichia coli vaccine. Vaccine 1993; 1[:929-34. 15. Levine MM, Nalin DR

  1. Molecular characterization and antimicrobial susceptibility of Staphylococcus aureus from small-scale dairy systems in the highlands of Central México

    OpenAIRE

    Salgado-Ruiz, T. B.; Rodríguez González, Ana; Gutiérrez, Diana; Martínez Fernández, Beatriz; García Suárez, María Pilar; Arriaga-Jordán, C. M.

    2015-01-01

    Staphylococcus aureus is involved in human and animal infections. Because of mastitis in dairy cows, milk can be contaminated by enterotoxin-producing strains, which constitutes a food poisoning risk. Animal handlers can be asymptomatic carriers, becoming an additional source of contamination. This research aims to improve our understanding of Staphylococcus aureus in small-scale dairy systems in central Mexico. Samples were taken in 12 dairy farms and included composite milk (from the four t...

  2. Occurrence of Salmonella, Listeria monocytogenes, and enterotoxigenic Staphylococcus in goat milk from small and medium-sized farms located in Minas Gerais State, Brazil.

    Science.gov (United States)

    Cavicchioli, V Q; Scatamburlo, T M; Yamazi, A K; Pieri, F A; Nero, L A

    2015-12-01

    Consumption of goat milk has been increasing due to its nutritional characteristics and health benefits. Therefore, assessment of the presence of foodborne pathogens in this product is critical to ensure its safety to consumers. The present study aimed to identify common foodborne pathogens in raw goat milk. Fifty-three samples of raw goat milk from 11 farms were collected and cultured for the presence of Salmonella spp. and Listeria monocytogenes, as well as for enumeration and isolation of coagulase-positive and coagulase-negative Staphylococcus (CPS and CNS, respectively). All samples tested negative for Salmonella spp. and L. monocytogenes. The CPS counts in raw goat milk samples were predominantly less than 2 log cfu/mL (n=39), and CNS counts were predominantly higher than 3 log cfu/mL (n=42). Based on Staphylococcus counts, 51 isolates were selected (CPS=26; CNS=25) and tested by PCR for the presence of classic enterotoxin-encoding genes (sea, seb, sec, sed, and see). Only 3 isolates (CPS=2, CNS=1) were negative for all enterotoxin-encoding genes tested, and the genotype sec and see was the most frequent (n=16), followed by sea, sec, and see (n=13) and sec (n=13); sed was not detected in any isolate. The frequencies of enterotoxin-encoding genes for CPS and CNS were similar, demonstrating the equivalence of both groups in harboring these virulent markers. These results suggest that Salmonella and L. monocytogenes are not frequent contaminants of raw goat milk, but that Staphylococcus spp. that are capable of producing enterotoxins are prevalent; therefore, consumers of raw goat milk and products made from raw milk are at risk. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  3. Enterotoxigenic coagulase positive Staphylococcus in milk and milk products, lben and jben, in northern Morocco.

    Science.gov (United States)

    Bendahou, Abdrezzak; Abid, Mohammed; Bouteldoun, Nadine; Catelejine, Dierick; Lebbadi, Mariam

    2009-04-30

    The aim of this research was to determine the prevalence of enterotoxin genes (sea-seo) in Coagulase Positive Staphylococcus (CPS) isolated from unpasteurized milk and milk products. These results were compared with the results obtained by using the detection kit SET-RPLA for the specific detection of staphylococcal enterotoxins (SEA-SED). Eighty-one samples of milk and milk products were analyzed for the presence of Staphylococcus strains. Forty-six coagulase positive Staphylococcus isolates were tested for the production of staphylococcal enterotoxins (SEA-SED) by using the reversed passive latex agglutination method. The strains were also tested for the presence of se genes (sea-seo) by polymerase chain reaction. One or more classical enterotoxin products (SEA-SED) were observed in 39% of the strains tested, while se genes were detected in 56.5%. SEA and sea were most commonly detected. For newly discovered se genes among CPS isolates tested in this study, except the seh gene which was revealed in four isolates (8.7 %), none of the strains harbored any of the other se genes (see, seg, sei, sej, sek, sel, sem, seo and sen). The finding of a pathogen such as staphylococci-producing SEs and containing se genes in milk and milk products in northern Morocco may indicate a problem for public health in this region. The presence of enterotoxigenic strains in food does not always necessarily mean that the toxin will be produced. For that reason, the combination of both methods (RPLA and PCR) is a guarantee for success in diagnostic analysis tests.

  4. Bacillus cereus Fnr binds a [4Fe-4S] cluster and forms a ternary complex with ResD and PlcR

    Directory of Open Access Journals (Sweden)

    Esbelin Julia

    2012-06-01

    Full Text Available Abstract Background Bacillus cereus is a facultative anaerobe that causes diarrheal disease in humans. Diarrheal syndrome may result from the secretion of various virulence factors including hemolysin BL and nonhemolytic enterotoxin Nhe. Expression of genes encoding Hbl and Nhe is regulated by the two redox systems, ResDE and Fnr, and the virulence regulator PlcR. B. cereus Fnr is a member of the Crp/Fnr family of iron-sulfur (Fe-S proteins. Only its apo-form has so far been studied. A major goal in deciphering the Fnr-dependent regulation of enterotoxin genes is thus to obtain and characterize holoFnr. Results Fnr has been subjected to in vitro Fe-S cluster reconstitution under anoxic conditions. UV-visible and EPR spectroscopic analyses together with the chemical estimation of the iron content indicated that Fnr binds one [4Fe-4S]2+ cluster per monomer. Atmospheric O2 causes disassembly of the Fe-S cluster, which exhibited a half-life of 15 min in air. Holo- and apoFnr have similar affinities for the nhe and hbl promoter regions, while holoFnr has a higher affinity for fnr promoter region than apoFnr. Both the apo- and holo-form of Fnr interact with ResD and PlcR to form a ternary complex. Conclusions Overall, this work shows that incorporation of the [4Fe-4S]2+ cluster is not required for DNA binding of Fnr to promoter regions of hbl and nhe enterotoxin genes or for the formation of a ternary complex with ResD and PlcR. This points to some new unusual properties of Fnr that may have physiological relevance in the redox regulation of enterotoxin gene regulation.

  5. Evaluación de estrategias de control frente a biopelículas de "Staphylococcus aureus" potencialmente presentes en la industria pesquera

    OpenAIRE

    Vázquez Sánchez, Daniel

    2014-01-01

    Staphylococcus aureus is one of the major bacterial agents causing foodborne diseases in humans worldwide, due to the ingestion of food containing staphylococcal enterotoxins. Spain is one of the largest producers and consumers of fishery products in the European Union. However, S. aureus is repeatedly detected in these products as a consequence of cross-contamination from food handlers and food contact surfaces. Biofilm formation also provides S. aureus a high tolerance to biocides allowing ...

  6. Enhancement of antibiotic activity by Cordia verbenacea DC

    OpenAIRE

    Matias, Edinardo F.F.; Santos, Karla K. A.; Almeida, Thiago S.; Costa, José G.M. da; Coutinho, Henrique D.M.

    2010-01-01

    Escherichia coli is known to produce enterotoxins whose properties and its role in diarrheal disease has been extensively investigated. Some species of Staphylococcus are often recognized as etiological agents of many animal and human opportunistic infections. This study is the first test of change in resistance of antibiotic activity by Cordia verbenacea DC. against multiresistant strains of Escherichia coli and Staphylococcus aureus. In this study, the hexane and methanol extract of Cordia ...

  7. Detection of the enterotoxigenic genes (sei,sej) in Staphylococcus aureus isolates from bovine mastitis milk in the West Azerbaijan of Iran.

    Science.gov (United States)

    Ahmady, Malahat; Kazemi, Sahar

    2013-07-01

    Staphylococcus aureus is a major causative pathogen of clinical and subclinical mastitis of dairy domestic ruminants. This organism produces a variety of extracellular toxins and virulence factors such as enterotoxin SEI and SEJ that contribute to its pathogenic potential. In this study 25 S. aureus isolates obtained from four dairy herds of Urmia region which is located in West Azerbaijan province in Iran. The tested isolates were identified on the basis of the cultural and biochemical properties, as well as amplification of the aroA gene which is specific for S. aureus . All isolates were also analyzed for the presence of the SEI ( sei ) and SEJ ( sej ) encoding genes using polymerase chain reaction (PCR). Seven positive isolates were detected for sei , but sej gene was not detected in any of the total number of 25 isolates. The present study revealed that the PCR amplification of the aroA gene could be used as a powerful tool for identification of S. aureus from the cases of bovine mastitis. Results of the present study also showed that the strains of S. aureus which cause mastitis can potentially produce enterotoxin SEI. Overall, our results suggest that it is of special importance to follow the presence of enterotoxin-producing S. aureus in other dairy products, especially for protecting the consumers from staphylococcal food poisoning.

  8. Prevalence of foodborne pathogens in grilled chicken from street vendors and retail outlets in Reynosa, Tamaulipas, Mexico.

    Science.gov (United States)

    Díaz-López, A; Cantú-Ramírez, R C; Garza-González, E; Ruiz-Tolentino, L; Tellez-Luis, S J; Rivera, G; Bocanegra-García, V

    2011-08-01

    We analyzed a total of 70 grilled chicken samples bought randomly from street vendors and retail outlets in the city of Reynosa, Mexico, to determine the prevalence of Escherichia coli (Shiga toxin producing and enterotoxin producing), Salmonella spp., Staphylococcus aureus, Listeria spp., and Campylobacter spp. using microbiological methods and PCR detection of bacterial sequences. Of the 70 samples, 27 (38.5%) were from retail outlets and 43 (61.4%) from street vendors. All specimens were negative by both microbiological and molecular methods for Listeria monocytogenes, Shiga toxin 2 of Shiga toxin-producing E. coli, lt of enterotoxin-producing E. coli, and st enterotoxin, and all were negative for Salmonella spp. and Campylobacter jejuni by PCR. Of the samples studied, 49 (70%) had undetectable levels of the foodborne pathogens studied with the methods used. In the remaining 21 (30%) specimens, at least one pathogen was isolated or detected, with E. coli being the pathogen most frequently isolated and with two samples bearing the hlyA gene. We found no statistical difference in bacterial prevalence between retail and street vendor samples. The presence of pathogens in grilled chicken is an important public health risk because of the great demand for and daily consumption of this product in this region.

  9. Manufacturing of a novel double-function ssDNA aptamer for sensitive diagnosis and efficient neutralization of SEA.

    Science.gov (United States)

    Sedighian, Hamid; Halabian, Raheleh; Amani, Jafar; Heiat, Mohammad; Taheri, Ramezan Ali; Imani Fooladi, Abbas Ali

    2018-05-01

    Staphylococcal enterotoxin A (SEA) is an enterotoxin produced mainly by Staphylococcus aureus. In recent years, it has become the most prevalent compound for staphylococcal food poisoning (SFP) around the world. In this study, we isolate new dual-function single-stranded DNA (ssDNA) aptamers by using some new methods, such as the Taguchi method, by focusing on the detection and neutralization of SEA enterotoxin in food and clinical samples. For the asymmetric polymerase chain reaction (PCR) optimization of each round of systematic evolution of ligands by exponential enrichment (SELEX), we use Taguchi L9 orthogonal arrays, and the aptamer mobility shift assay (AMSA) is used for initial evaluation of the protein-DNA interactions on the last SELEX round. In our investigation the dissociation constant (K D ) value and the limit of detection (LOD) of the candidate aptamer were found to be 8.5 ± 0.91 of nM and 5 ng/ml using surface plasmon resonance (SPR). In the current study, the Taguchi and mobility shift assay methods were innovatively harnessed to improve the selection process and evaluate the protein-aptamer interactions. To the best of our knowledge, this is the first report on employing these two methods in aptamer technology especially against bacterial toxin. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. KARAKTERISASI ENZIM B-GLUKOSIDASE VANILI [Characterization of B-glukosidase Enzyme from Vanilla Bean

    Directory of Open Access Journals (Sweden)

    Dwi setyaningsih 1

    2007-12-01

    Full Text Available The Indonesian natural vanilla is know for having a unigue woody, smooky, and phenolic flavor. Development of the aroma and flavor vanilla was formed by the action of a hydrolytic enzyme B-glucosidase on glucovanillin. The objective of this research was to characterize vanilla B-glucosidase. The vanilla B-glucosidase activity was increased by detergent. The enzyme was found as heat labile. Scalding should be conducted at 400C for 2-3 minutes. The result from B-glucosidase activity in each part of vanilla and microscopic analisis of vanilla bean slice showed that the highest B-glucosidase activity and vanillin concentrations were found in the seed funicles and placental tissue the of vanilla bean. The activity of vanilla B-glucosidase was optimum at pH 6,0, and temperature of 400C, found as and activation energy was 5,78 kcal/mole. After 44 minutes incubation time at 400C. The activity was reduced down to 10%. The apparent of moleculer weight was 100-400 kDa according to gel setration (Sephacryl S-300 analysis.

  11. Outbreaks of cholera-like diarrhoea caused by enterotoxigenic Escherichia coli in the Brazilian Amazon Rainforest.

    Science.gov (United States)

    Vicente, Ana C P; Teixeira, Luiz F M; Iniguez-Rojas, L; Luna, M G; Silva, L; Andrade, J R C; Guth, B E C

    2005-09-01

    The relationship between enteropathogens and severe diarrhoea in the Brazilian Amazon is poorly understood. In 1998, outbreaks of acute diarrhoea clinically diagnosed as cholera occurred in two small villages localized far from the main cholera route in the Brazilian rainforest. PCR was performed on some enteropathogens and heat-labile (LT) and/or heat-stable (STh) toxin genes, the virulence determinants of enterotoxigenic Escherichia coli (ETEC), were detected. Further characterization of ETEC isolates revealed the presence of two clones, one from each outbreak. One presenting serotype O167:H5 harboured LT-I and STh toxin genes and expressed the CS5CS6 colonization factor. The other, a non-typeable serotype, was positive for the LT-I gene and expressed the CS7 colonization factor. The current study demonstrates the importance of molecular diagnosis in regions such as the Amazon basin, where the enormous distances and local support conditions make standard laboratory diagnosis difficult. Here we also show that the mis-identified cholera cases were in fact associated with ETEC strains. This is the first report of ETEC, molecularly characterized as the aetiological agent of severe diarrhoea in children and adults in the Brazilian Amazon Rainforest.

  12. The epidemiology, evaluation, and management of stingray injuries.

    Science.gov (United States)

    Diaz, James H

    2007-01-01

    A descriptive analysis and review of the world's salient scientific literature on stingray injuries was conducted in light of recent high-profile cases of fatal and near-fatal thoracic stingray injuries to guide clinicians in evaluating and managing stingray injuries. Data was extracted from observational and longitudinal studies over the period, 1950-2006, to permit (1) a stratification of stingray injuries as bites, penetrating lacerations with and without envenoming, and combinations of deeply penetrating and envenoming wounds; and (2) an assessment of new management strategies for thoracoabdominal penetrating trauma and non-healing, necrotic stingray wounds. Unlike their Chondrichthyes classmates, the sharks, stingrays are docile and non-aggressive; and will not attack with their spined tails, unless provoked. Although some occupations are predisposed to stingray injuries, most stingray injuries can be avoided by observing seafloors and adopting simple practices when wading, swimming, diving, or fishing in temperate oceans and some tropical freshwater river systems. All stingray injuries should be managed initially with wound irrigation to dislodge retained spine fragments and envenoming tissues and warm water immersion to inactivate heat-labile toxins.

  13. Pulmonary biochemistry

    International Nuclear Information System (INIS)

    Mavis, R.D.

    1986-01-01

    Regeneration of vitamin E from its intermediate oxidation product, the chromanoxyl radical, was studied as a heat-labile, vitamin E-dependent antioxidant activity of glutathione in delaying peroxidation of a microsomal fraction of liver and lung in vitro. The characteristics of this system are consistent with an enzyme-catalyzed reaction between glutathione and the chromanoxyl radical. We have developed a method of labeling the vitamin E of microsomes with tracer amounts of tritiated α-tocopherol. The oxidation of tocopherol can thus be monitored coincidentally with the peroxidation of fatty acids. Fatty acid peroxidation is monitored by spectrophotometrically measuring malondialdehyde and oxidation of vitamin E is monitored by extracting and chromatographing the radiolabelled vitamin E in a system which separates -tocopherol from its oxidation products. We have found that the presence of glutathione in this system retards both peroxidation of membrane phospholipids and oxidation of membrane-associated α-tocopherol, with a time sequence that is consistent with regeneration of vitamin E by reduction of the chromanoxyl radical by glutathione

  14. Glutathione delays varies as-tocopherol oxidation and subsequent lipid peroxidation in rat liver microsomes

    International Nuclear Information System (INIS)

    Robey, S.; Mavis, R.

    1986-01-01

    A method has been developed for in vitro trace radiolabeling of rat liver microsomes with 3 H-α-tocopherol (αT*) which allows virtually complete oxidation of the αT* under oxidizing conditions. The supernatant of a 16,000 xg centrifugation of homogenized rat liver, containing the cytosolic rat liver vitamin E (VE) transfer protein, was incubated with an ethanolic solution of αT* for 10 minutes at 37 0 C. Labeled microsomes were collected in the washed 100,000 xg pellet. Microsomes were then incubated with 30 μM Fe 2+ in an NADPH-generating system, and both production of malondialdehyde (MDA) (a product of lipid peroxidation) and oxidation of αT* were monitored over a time course in the presence and absence of glutathione (GSH). The results indicate virtually complete oxidation of αT* precedes significant membrane lipid peroxidation, and that addition of 5 mM GSH delays both αT* oxidation and subsequent MDA production. This suggests that the previously observed VE-dependent heat labile inhibition of microsomal lipid peroxidation by GSH involves maintaining membrane levels of α-tocopherol

  15. Identification of a haemolysin-like peptide with antibacterial activity using the draft genome sequence of Staphylococcus epidermidis strain A487.

    Science.gov (United States)

    Al-Mahrous, Mohammed M; Jack, Ralph W; Sandiford, Stephanie K; Tagg, John R; Beatson, Scott A; Upton, Mathew

    2011-08-01

    Our interest in Staphylococcus epidermidis strain A487 was prompted by the unusual nature of its inhibitory activity in screening tests against methicillin-resistant Staphylococcus aureus isolates. The inhibitory activity was detected in deferred antagonism tests only if the agar plate was preheated for at least 35 min at ≥ 55 °C before inoculation of the indicator bacteria, this phenomenon indicating possible involvement of a heat-labile immunity agent or protease. The inhibitor was purified to homogeneity by ammonium sulphate precipitation, followed by cation-exchange and reversed-phase chromatography. Tandem MS revealed a novel peptide of molecular weight 2588.4 Da. The draft genome sequence of strain A487 was determined using 454 GS FLX technology, allowing the identification of the structural gene (hlp) encoding the mature peptide MQFITDLIKKAVDFFKGLFGNK. The deduced amino acid sequence of peptide 487 exhibited 70.8% similarity to that of a putative haemolysin from Staphylococcus cohnii. Analysis of the genome of strain A487 showed several additional inhibitor-encoding genes, including hld, the determinant for staphylococcal δ-lysin. This work indicates that potentially useful inhibitors could be overlooked in agar-based inhibitor screening programmes lacking a heat pretreatment step and also highlights the utility of draft genome sequence examination in antibacterial agent discovery. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  16. Toxic, antimicrobial and hemagglutinating activities of the purple fluid of the sea hare Aplysia dactylomela Rang, 1828

    Directory of Open Access Journals (Sweden)

    Melo V.M.M.

    1998-01-01

    Full Text Available The antimicrobial, hemagglutinating and toxic activities of the purple fluid of the sea hare Aplysia dactylomela are described. Intact or dialyzed purple fluid inhibited the growth of species of Gram-positive and Gram-negative bacteria and the action was not bactericidal but bacteriostatic. The active factor or factors were heat labile and sensitive to extreme pH values. The fluid preferentially agglutinated rabbit erythrocytes and, to a lesser extent, human blood cells, and this activity was inhibited by the glycoprotein fetuin, a fact suggesting the presence of a lectin. The fluid was also toxic to brine shrimp nauplii (LD50 141.25 µg protein/ml and to mice injected intraperitoneally (LD50 201.8 ± 8.6 mg protein/kg, in a dose-dependent fashion. These toxic activities were abolished when the fluid was heated. Taken together, the data suggest that the activities of the purple fluid are due primarily to substance(s of a protein nature which may be involved in the chemical defense mechanism of this sea hare.

  17. Comparative genomics and transcriptomics of Escherichia coli isolates carrying virulence factors of both enteropathogenic and enterotoxigenic E. coli.

    Science.gov (United States)

    Hazen, Tracy H; Michalski, Jane; Luo, Qingwei; Shetty, Amol C; Daugherty, Sean C; Fleckenstein, James M; Rasko, David A

    2017-06-14

    Escherichia coli that are capable of causing human disease are often classified into pathogenic variants (pathovars) based on their virulence gene content. However, disease-associated hybrid E. coli, containing unique combinations of multiple canonical virulence factors have also been described. Such was the case of the E. coli O104:H4 outbreak in 2011, which caused significant morbidity and mortality. Among the pathovars of diarrheagenic E. coli that cause significant human disease are the enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC). In the current study we use comparative genomics, transcriptomics, and functional studies to characterize isolates that contain virulence factors of both EPEC and ETEC. Based on phylogenomic analysis, these hybrid isolates are more genomically-related to EPEC, but appear to have acquired ETEC virulence genes. Global transcriptional analysis using RNA sequencing, demonstrated that the EPEC and ETEC virulence genes of these hybrid isolates were differentially-expressed under virulence-inducing laboratory conditions, similar to reference isolates. Immunoblot assays further verified that the virulence gene products were produced and that the T3SS effector EspB of EPEC, and heat-labile toxin of ETEC were secreted. These findings document the existence and virulence potential of an E. coli pathovar hybrid that blurs the distinction between E. coli pathovars.

  18. Culicoides antigen extract stimulates equine blood mononuclear (BMN) cell proliferation and the release of eosinophil adherence-inducing factor(s).

    Science.gov (United States)

    Mckelvie, J; Foster, A P; Hamblin, A S; Cunningham, F M

    2001-04-01

    Intradermal injection of a Culicoides antigen extract (CAgX) induces T lymphocyte and eosinophil accumulation in the skin of horses with sweet itch. Blood mononuclear (BMN) cells from normal ponies proliferate when stimulated by mitogen (phytohaemagglutinin, PHA) or antigen (tetanus toxoid, TT) and, as shown here, release soluble factor(s) that induce eosinophil adherence. CAgX also caused concentration dependent proliferation of BMN cells from sweet itch and normal ponies [stimulation index: 29 (13) and 17 (7) for BMN cells from sweet itch and normal ponies, respectively during the active phase of disease; 4 microg protein ml(-1)CAgX; 168 h]. A heat labile factor(s) which caused eosinophil adherence was also released [sweet itch ponies: 6.0 (1.6) per cent adherence versus 1.3 (0.4) per cent; normal ponies: 6.6 (0.5) per cent adherence versus 0.9 (0.1) per cent for supernatants from CAgX (4 microg protein ml(-1); 48 hours) stimulated versus unstimulated BMN cells, respectively]. These results suggest that soluble proteins released from T lymphocytes could affect eosinophil function in the lesional skin of sweet itch horses. Copyright 2001 Harcourt Publishers Ltd.

  19. Studies on allergoids from naturally occurring allergens. III. Preparation of ragweed pollen allergoids by aldehyde modification in two steps.

    Science.gov (United States)

    Marsh, D G; Norman, P S; Roebber, M; Lichtenstein, L M

    1981-12-01

    We have devised a new process for modifying heat-labile allergens, which employs a sequential "two-step" incubation at temperatures of 10 degree C and 30 degree to 32 degree C. This process was found to produce effective ragweed "allergoids" with low allergenicity and good immunogenicity, which makes them useful for the therapy of allergic humans. Modification with formaldehyde produced derivatives ("formallergoids") that were about 10-fold less allergenic in allergic humans (as measured by leukocyte histamine-release assay), and similarly or more immunogenic in guinea pigs, than glutaraldehyde-modified allergens ("glutarallergoids"). Further analysis by RAST inhibition showed that a ragweed formallergoid was sixfold less reactive than a glutarallergoid with a pool of human IgE antibodies. However, the formallergoid had retained the ability to induce a wide array of antibodies against native ragweed antigens, since rabbit anti-formallergoid serum was able to recognize at least 12 different ragweed antigens, including AgE. Gel-filtration experiments showed that both the formallergoid and glutarallergoid materials contained polymers having apparent molecular weights distributed around 260,000 and 230,000 daltons, respectively (approximate range 30,000 to 900,000 daltons). Our studies provide the immunochemical basis for the use of these allergoids in the therapy of allergic humans.

  20. Phenotypic characterization of adenovirus type 12 temperature-sensitive mutants in productive infection and transformation.

    Science.gov (United States)

    Hama, S; Kimura, G

    1980-01-01

    Eleven temperature-sensitive mutants of adenovirus type 12, capable of forming plaques in human cells at 33 C but not at 39.5 C, were isolated from a stock of a wild-type strain after treatment with either nitrous acid or hydroxylamine. Complementation tests in doubly infected human cells permitted a tentative assignment of eight of these mutants to six complementation groups. Temperature-shift experiments revealed that one mutant is affected early and most of the other mutants are affected late. Only the early mutant, H12ts505, was temperature sensitive in viral DNA replication. Infectious virions of all the mutants except H12ts505 and two of the late mutants produced at 33 C, appeared to be more heat labile than those of the wild type. Only H12ts505 was temperature sensitive for the establishment of transformation of rat 3Y1 cells. One of the late mutants (H12ts504) had an increased transforming ability at the permissive temperature. Results of temperature-shift transformation experiments suggest that a viral function affected in H12ts505 is required for "initiation" of transformation. Some of the growth properties of H12ts505-transformed cells were also temperature dependent, suggesting that a functional expression of a gene mutation in H12ts505 is required to maintain at least some aspects of the transformed state.

  1. Multiple loci affecting photoreactivation in Escherichia coli

    International Nuclear Information System (INIS)

    Sutherland, B.M.; Hausrath, S.G.

    1979-01-01

    Sutherland et al. mapped a phr gene in Escherichia coli at 17 min and found that induction of an E. coli stain lysogenic for a lambda phage carrying this gene increased photoreactivating enzyme levels 2,000-fold. Recently, Smith and Youngs and Sancar and Rupert located a phr gene at 15.9 min. We have therefore investigated the properties of photoreactivating enzyme and cellular photoreactivation in cells containing deletions of the gene at 17 min. Cells with this deletion photoreactivated ultraviolet-induced killing at a rate 20% of normal; they also contained approximately 20% of the normal photoreactivating enzyme level. The residual enzyme in these cells was characterized to determine whether the reduced cellular photoreactivation rate and photoreactivating enzyme levels resulted from reduced numbers of normal enzymes or from an altered enzyme. Photoreactivating enzymes from strains carrying a deletion of the region at 17 min has an apparent K/sub m/ about two- to threefold higher than normal enzyme and showed markedly increased heat lability. The gene at 17 min thus contains information determining the function of the E. coli photoreactivating enzyme rather than the quantity of the enzyme. It is proposed that the gene at 17 min be termed phrA and that located at 15.9 min be termed phrB

  2. Cytotoxic Activity of Coagulase-Negative Staphylococci in Bovine Mastitis

    Science.gov (United States)

    Zhang, Songlin; Maddox, Carol W.

    2000-01-01

    Secreted toxins play important roles in the pathogenesis of bacterial infections. In this study, we examined the presence of secreted cytotoxic factors of coagulase-negative staphylococci (CoNS) from bovine clinical and subclinical mastitis. A 34- to 36-kDa protein with cell-rounding cytotoxic activity was found in many CoNS strains, especially in Staphylococcus chromogenes strains. The protein caused cell detachment and cell rounding in several cell lines, including HEp-2, Int 407, CHO-K1, and Y-1 cells. Native protein recovered from nondenatured polyacrylamide gel electrophoresis showed both cytotoxic activity and casein hydrolysis activity. The purified protein had a pH optimal at 7.2 to 7.5 and a pI of 5.1 and was heat labile. The proteolytic activity could be inhibited by zinc and metal specific inhibitors such as 1,10-phenanthroline and EDTA, indicating that it is a metalloprotease. Protein mass analysis and peptide sequencing indicated that the protein is a novel metalloprotease. Different bacterial strains expressed variable levels of 34- to 36-kDa protease, which may provide an indication of strain virulence. PMID:10678913

  3. Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis) 1

    Science.gov (United States)

    Samac, Deborah; Storey, Richard

    1981-01-01

    Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling. Jojoba cotyledonary extracts were found to inhibit the enzymic activity of trypsin, chymotrypsin, and pepsin but not the protease from Aspergillus saotoi. The heat-labile trypsin inhibitor substance(s) was found in commercially processed jojoba seed meal and the albumin fraction of seed proteins. Trypsin inhibitor activity decreased with germination. PMID:16662104

  4. Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis).

    Science.gov (United States)

    Samac, D; Storey, R

    1981-12-01

    Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling.Jojoba cotyledonary extracts were found to inhibit the enzymic activity of trypsin, chymotrypsin, and pepsin but not the protease from Aspergillus saotoi. The heat-labile trypsin inhibitor substance(s) was found in commercially processed jojoba seed meal and the albumin fraction of seed proteins. Trypsin inhibitor activity decreased with germination.

  5. Decreased uv mutagenesis in cdc8, a DNA replication mutant of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Prakash, L.; Hinkle, D.; Prakash, S.

    1978-01-01

    A DNA replication mutant of yeast, cdc8, was found to decrease uv-induced reversion of lys2-1, arg4-17, tryl and ural. This effect was observed with all three alleles of cdc8 tested. Survival curves obtained following uv irradiation in cdc8 rad double mutants show that cdc8 is epistatic to rad6, as well as to rad1; cdc8 rad51 double mutants seem to be more sensitive than the single mutants. Since uv-induced reversion in cdc8 rad1 and cdc8 rad51 double mutants is like that of the cdc8 single mutants, we conclude that CDC8 plays a direct role in error-prone repair. To test whether CDC8 codes for a DNA polymerase, we have purified both DNA polymerase I and DNA polymerase II from cdc8 and CDC+ cells. The purified DNA polymerases from cdc8 were no more heat labile than those from CDC+, suggesting that CDC8 is not a structural gene for either enzyme

  6. The stability of complement-mediated bactericidal activity in human serum against Salmonella.

    Directory of Open Access Journals (Sweden)

    Colette M O'Shaughnessy

    Full Text Available The complement cascade includes heat-labile proteins and care is required when handling serum in order to preserve its functional integrity. We have previously used a whole human serum bactericidal assay to show that antibody and an intact complement system are required in blood for killing of invasive isolates of Salmonella. The aim of the present study was to evaluate the conditions under which human serum can be stored and manipulated while maintaining complement integrity. Serum bactericidal activity against Salmonella was maintained for a minimum of 35 days when stored at 4°C, eight days at 22°C and 54 hours at 37°C. Up to three freeze-thaw cycles had no effect on the persistence of bactericidal activity and hemolytic complement assays confirmed no effect on complement function. Delay in the separation of serum for up to four days from clotted blood stored at 22°C did not affect bactericidal activity. Dilution of serum resulted in an increased rate of loss of bactericidal activity and so serum should be stored undiluted. These findings indicate that the current guidelines concerning manipulation and storage of human serum to preserve complement integrity and function leave a large margin for safety with regards to bactericidal activity against Salmonella. The study provides a scheme for determining the requirements for serum handling in relation to functional activity of complement in other systems.

  7. An exceptionally cold-adapted alpha-amylase from a metagenomic library of a cold and alkaline environment

    DEFF Research Database (Denmark)

    Vester, Jan Kjølhede; Glaring, Mikkel Andreas; Stougaard, Peter

    2015-01-01

    A cold-active α-amylase, AmyI3C6, identified by a functional metagenomics approach was expressed in Escherichia coli and purified to homogeneity. Sequence analysis showed that the AmyI3C6 amylase was similar to α-amylases from the class Clostridia and revealed classical characteristics of cold......-adapted enzymes, as did comparison of the kinetic parameters Km and kcat to a mesophilic α-amylase. AmyI3C6 was shown to be heat-labile. Temperature optimum was at 10-15 °C, and more than 70 % of the relative activity was retained at 1 °C. The pH optimum of AmyI3C6 was at pH 8-9, and the enzyme displayed activity...... in two commercial detergents tested, suggesting that the AmyI3C6 α-amylase may be useful as a detergent enzyme in environmentally friendly, low-temperature laundry processes....

  8. Bacteriocin Isolated From Halomon sp.: A Bacterial Ding Protein?

    International Nuclear Information System (INIS)

    Atirah Azemin; Klappa, P.; Mohd Shahir Shamsir Omar

    2015-01-01

    A marine halophile, Halomonas sp. strain M3 was isolated from Straits of Johor, Malaysia and produce bacteriocin CC that acts as bacteriostatic agent. Characterisation of the bacterium showed that optimal growth and bacteriocin production is at ambient temperature, pH of 8-8.5 in nutrient broth medium supplemented with 2.9 % w/v NaCI to mimic saltwater conditions. The stability studies indicated that bacteriocin CC is heat-labile (35-50 degree Celsius) and was stable over 2 years when stored in 0.02 M Tris- HCI with 30-60 % glycerol at 4 degree Celsius. A loss of activity was detected after proteolytic enzymes treatment, indicating the proteinaceous nature of the antimicrobial compound. The amino acid sequence of bacteriocin CC was obtained by Edman degradation and MALDI-TOF analysis, showed the characteristic sequence of a DING protein (D-I-N-G-G-G-A-T-L-P-Q-A-LY- Q) in size 38.9-kDa at pI of 6.8. These proteins constitute a conserved and widely distributed set of proteins found in all kingdoms with ligand-binding activities and hydrolytic enzyme, suggesting a possible role in cell signalling and bio mineralization in DING isolates. Intriguingly, DING proteins also have been involved as an anti-tumour agent in humans. Thus, bacteriocin CC as DING protein family members should be further studied to investigate its potential as a novel antimicrobial agent. (author)

  9. Studies on the interaction between the Ehrlich ascites tumor cell and its fluid environment

    International Nuclear Information System (INIS)

    Magnani, B.

    1984-01-01

    In this dissertation, the glycolytic nature of the Ehrlich ascites tumor (EAT) cell is disclosed both in vivo and in vitro by experiments challenging it with glucose. It is demonstrated that EAT cells can cause the extracellular pH to drop to values sufficiently acidic so as to inhibit EAT glycolysis. However, the extracellular fluid or the Ascites Supernatant Fluid (ASF) reduced the extent to which the pH dropped during EAT cell glycolysis. A comparison of the activities of the sera from tumor-bearing mice and normal mice revealed that the serumfrom the tumor-bearing mice reduced the pH fall generated by the EAT cell in the same way as did ASF; normal mouse serum had no such effect. The metabolic pathways utilized during glucose catabolism were examined by radio-respirometry and the results demonstrated that the high percentage of the glucose conversion to lactate occurred because of partial blockade of the TCA cycle. The databolism of glutamine, glutamic acid, asparagine, aspartic acid, and alanine was enhanced by ASF as determined by measuring 14 CO 2 from 14 C-labelled amino acids, with glutamine catabolism enhanced about three-fold. Fractionation experiments revealed that ASF contained a factor(s) responsible for this enhancement that had a molecular weight greater than 300,000 daltons and was heat-labile

  10. Comparative analysis of antimicrobial resistance in enterotoxigenic Escherichia coli isolates from two paediatric cohort studies in Lima, Peru.

    Science.gov (United States)

    Medina, Anicia M; Rivera, Fulton P; Pons, Maria J; Riveros, Maribel; Gomes, Cláudia; Bernal, María; Meza, Rina; Maves, Ryan C; Huicho, Luis; Chea-Woo, Elsa; Lanata, Claudio F; Gil, Ana I; Ochoa, Theresa J; Ruiz, Joaquim

    2015-08-01

    Antibiotic resistance is increasing worldwide, being of special concern in low- and middle-income countries. The aim of this study was to determine the antimicrobial susceptibility and mechanisms of resistance in 205 enterotoxigenic Escherichia coli (ETEC) isolates from two cohort studies in children Peru. ETEC were identified by an in-house multiplex real-time PCR. Susceptibility to 13 antimicrobial agents was tested by disk diffusion; mechanisms of resistance were evaluated by PCR. ETEC isolates were resistant to ampicillin (64%), cotrimoxazole (52%), tetracycline (37%); 39% of the isolates were multidrug-resistant. Heat-stable toxin producing (ETEC-st) (48%) and heat-labile toxin producing ETEC (ETEC-lt) (40%) had higher rates of multidrug resistance than isolates producing both toxins (ETEC-lt-st) (21%), pPeru. However, further development of resistance should be closely monitored. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  11. Effect of temperature stress on polyphenol oxidase activity in grains of some wheat cultivars

    International Nuclear Information System (INIS)

    Kayani, W.K.

    2011-01-01

    Color is a key quality trait of wheat-based products and polyphenol oxidase (PPO) is implicated to play a significant role in their undesirable darkening. Polyphenol oxidase catalyzes the oxidation of phenols to quinines, which auto oxidize and polymerize with amino acid of cellular proteins resulting brown and black pigmentation propounding reduced nutritional values. In present study, the PPO activity in 50 different Pakistani wheat cultivars was investigated and grouped into three categories viz; low, medium and high PPO activity cultivars. PPO is a heat labile enzyme. To investigate effect of heat stress, nine cultivars from each category were chosen for treatment at 30, 40 and 50 deg. C for 30, 60, and 120 minutes each. A substantial change was experienced in PPO activity as compared to room temperature. Two wheat cultivar Wafaq-2001 and AS-2002 showed a compromising attitude of minimum PPO activity at 30 deg. C for a period of 30 and 60 minutes of incubation. In general, an incubation of 30 deg. C or 60 deg. C (low or high) for a period of 30 minutes can be recommended for suppressing PPO activity. (author)

  12. Utilization of bacteriocin-producing bacteria in dairy products

    Directory of Open Access Journals (Sweden)

    Matěj Patrovský

    2016-07-01

    Full Text Available Lactic acid bacteria have been used since ancient times for food preparation and for bio-conservation by fermentation. Selected strains are capable of producing antimicrobial peptides - bacteriocins, which can be natural preservatives, especially in products with short shelf lives. The present study is focused on inhibitory effects of the bacteriocin-producing bacteria strains Enterococcus faecium, Pediococccus acidilactici and Lactobacillus plantarum against Listeria innocua as an indicator microorganism. Freeze-dried preparations of bacterial strains producing particular bacteriocins were tested by agar well-diffusion assay and by the traditional spread plate method. Plantaricin exhibited the highest anti-listerial effect among the tested bacteriocins. Pediocin also demonstrated a distinct inhibitory effect, but enterocin appeared to be heat labile and its efficiency was also suppressed under cold storage conditions. Plantaricin reduced Listeria innocua counts by 1 log in dairy spread made from cheese and quark. The formation of bacteriocins by various Lactobacillus plantarum strains were substantially influenced by the cultivation conditions of the mother culture and by the microbial preparation process before freeze-drying. Bacteriocins introduced into foodstuffs via protective cultures in situ offer new perspectives on enhancing food quality and safety.

  13. Cell wall glycans and soluble factors determine the interactions between the hyphae of Candida albicans and Pseudomonas aeruginosa.

    Science.gov (United States)

    Brand, Alexandra; Barnes, Julia D; Mackenzie, Kevin S; Odds, Frank C; Gow, Neil A R

    2008-10-01

    The fungus, Candida albicans, and the bacterium, Pseudomonas aeruginosa, are opportunistic human pathogens that have been coisolated from diverse body sites. Pseudomonas aeruginosa suppresses C. albicans proliferation in vitro and potentially in vivo but it is the C. albicans hyphae that are killed while yeast cells are not. We show that hyphal killing involves both contact-mediated and soluble factors. Bacterial culture filtrates contained heat-labile soluble factors that killed C. albicans hyphae. In cocultures, localized points of hyphal lysis were observed, suggesting that adhesion and subsequent bacteria-mediated cell wall lysis is involved in the killing of C. albicans hyphae. The glycosylation status of the C. albicans cell wall affected the rate of contact-dependent killing because mutants with severely truncated O-linked, but not N-linked, glycans were hypersensitive to Pseudomonas-mediated killing. Deletion of HWP1, ALS3 or HYR1, which encode major hypha-associated cell wall proteins, had no effect on fungal susceptibility.

  14. Radioimmunoassay and heat denaturation enzyme assay for the detection of Tay-Sachs heterozygotes during pregnancy

    International Nuclear Information System (INIS)

    Nguyen, C.; Gold, R.J.M.; Mahuran, D.; Lowden, J.A.

    1981-01-01

    Tay-Sachs disease results from a loss of activity of hexosaminidase A (HEX A) in body tissues and fluids. Heterozygotes for the disease are usually identified by their relatively low ratio of heat-labile HEX A to total hexosaminidase. During pregnancy an intermediate isoenzyme (HEX I) increases in activity in serum and obscures the heterozygote status. HEX I does not increase in leucocytes, tears and other body tissues but because of technical difficulties in these assays the authors examined the feasibility of using a radioimmunoassay for HEX A. By univariate analysis, the heat denaturation assay gave a lower cost of misclassification for non-pregnant normals while RIA did so for pregnant normals. A combination of both tests led to reduced cost of misclassification compared to either alone. Bayesian analysis of bivariate gaussian density functions for heat denaturation and for radioimmunoassays of HEX isoenzymes was employed to calculate misclassification frequencies. Among the parameters examined, HEX A measured by RIA and % HEX A by heat-denaturation assay were the two having the best discriminatory power. (Auth.)

  15. Cellular protein receptors of maculosin, a host specific phytotoxin of spotted knapweed (Centaurea maculosa L.).

    Science.gov (United States)

    Park, S H; Strobel, G A

    1994-01-05

    Maculosin (the diketopiperazine, cyclo (L-Pro-L-Tyr)) is a host specific phytotoxin produced by Alternaria alternata on spotted knapweed (Centaurea maculosa L.). Receptors for this phytotoxin have been isolated from spotted knapweed. Knapweed leaves possess most of the maculosin-binding activity in the cytosolic fraction. However, activity was also observed in the whole membrane fraction of the leaf. The binding component of the cytosolic fraction was identified as a protein(s) because of its heat-lability and sensitivity to proteases. A 16-fold purification of a toxin-binding protein was carried out by ammonium sulfate fractionation, and Sephadex G-200, and maculosin-affinity column chromatography. The affinity column was prepared with epoxy activated Sepharose 6B to which the phenolic group of maculosin was attached. The receptor was estimated to contain more than one binding protein by native and SDS-PAGE. At least one of the maculosin-binding proteins was identified as ribulose-1,5-biphosphate carboxylase (RuBPcase).

  16. Hemolytic activity of Fusobacterium necrophorum culture supernatants due to presence of phospholipase A and lysophospholipase.

    Science.gov (United States)

    Abe, P M; Kendall, C J; Stauffer, L R; Holland, J W

    1979-01-01

    Culture supernatants of Fusobacterium necrophorum demonstrated hemolytic activity. The hemolysin(s), which was partially purified by ammonium sulfate precipitation, was temperature-dependent and heat labile. The spectrum of hemolytic activity against various erythrocytes included rabbit, human, and dog erythrocytes. Goats, sheep, and bovine erythrocytes showed only trace hemolysis. According to results of thin-layer chromatography, the hemolysin hydrolyzed rabbit erythrocyte phosphatidyl choline, phosphatidyl ethanolamine, lysophosphatidyl choline, and bovine phosphatidyl choline. Hydrolysis of egg yolk phosphatidyl choline, bovine phosphatidyl ethanolamine, cholesterol, 1,2-dipalmitin, 1,3-dipalmitin, sphingomyelin, or triolein was not detected by thin layer chromatography. A more sensitive procedure utilizing gas-liquid chromatography revealed that, of the substrates tested, the following were bein hydrolyzed: bovine and egg yolk phosphatidyl choline, lysophosphatidyl choline, alpha-palmito-beta-eleoyl-L-alpha lecithin and alpha-oleoyl-betal-palmitoyl-L-alpha lecithin. Substrates which were weakly hydrolyzed were bovine phosphatidyl ethanolamine, DL-alpha-hosphatidyl ethanolamine dipalmitoyl, 1,2-dipalmitin, 1,3-dipalmitin, and triolein.

  17. Environmental fate of ceftiofur sodium, a cephalosporin antibiotic. Role of animal excreta in its decomposition

    International Nuclear Information System (INIS)

    Gilbertson, T.J.; Hornish, R.E.; Jaglan, P.S.; Koshy, K.T.; Nappier, J.L.; Stahl, G.L.; Cazers, A.R.; Nappier, J.M.; Kubicek, M.F.; Hoffman, G.A.; Hamlow, P.J.

    1990-01-01

    The degradation of ceftiofur sodium, a wide-spectrum cephalosporin antibiotic, was studied in the urine and feces of cattle, in three soils, and in buffers of pH 5, 7, and 9. Photodegradation was also studied. Fortification of cattle feces with [ 14 C]ceftiofur showed that it was quickly degraded to microbiologically inactive metabolites. Sterilization of the cattle feces inhibited the degradation of ceftiofur, which suggests that microorganisms or heat-labile substances may be responsible for the degradation. The t 1/2 values of aerobic degradation of ceftiofur sodium in California, Florida, and Wisconsin soil were found to be 22.2, 49.0, and 41.4 days, respectively. Hydrolysis of ceftiofur, as measured by either HPLC or microbiological methods, was accelerated by increasing pH. The t 1/2 values at pH 5, 7, and 9 were 100.3, 8.0, and 4.2 days, respectively, at 22 degree C and dramatically increased at 47 degree C. The photodegradation of ceftiofur, as determined by HPLC and a microbiological method, showed that after initial degradation for several days the rate of degradation was minimal, probably due to a protective film formed from degradation products. A major role for feces in the degradation of ceftiofur was observed, although other pathways of degradation such as soil, light, and water were also important

  18. Lipopolysaccharide (LPS) stimulates fresh human monocytes to lyse actinomycin D-treated WEHI-164 target cells via increased secretion of a monokine similar to tumor necrosis factor

    International Nuclear Information System (INIS)

    Chen, A.R.; McKinnon, K.P.; Koren, H.S.

    1985-01-01

    The effects of lipopolysaccharide (LPS) on tumoricidal activity of human monocytes freshly isolated from peripheral blood were studied. Actinomycin D-treated WEHI-164 cells were used as targets because they are NK insensitive and are lysed rapidly by monocytes in 6-hr 51 Cr-release assays. Monocytes exhibited significant spontaneous activity without endotoxin. Monocytes either pretreated for 1 hr with LPS or assayed in the presence of LPS exhibited 100- to 1000-fold increased cytolytic activity. Cytolytic activity was heat labile and trypsin sensitive, and was recovered from Sepharose S-200 columns in a single peak with an apparent m.w. between 25,000 and 40,000. Actinomycin D or cycloheximide treatment of monocytes before the addition of LPS inhibited cytolytic monokine production. Cytolytic monokine activity was practically neutralized by specific rabbit antisera to human tumor necrosis factor (TNF). It was concluded that, although fresh human monocytes exhibit spontaneous tumoricidal activity, LPS is a potent activating agent. Its stimulatory effects depend on new transcription and translation and are mediated by enhanced secretion of a cytolytic monokine similar to TNF

  19. Free terminal amines in DNA-binding peptides alter the product distribution from guanine radicals produced by single electron oxidation.

    Science.gov (United States)

    Konigsfeld, Katie M; Lee, Melissa; Urata, Sarah M; Aguilera, Joe A; Milligan, Jamie R

    2012-03-01

    Electron deficient guanine radical species are major intermediates produced in DNA by the direct effect of ionizing irradiation. There is evidence that they react with amine groups in closely bound ligands to form covalent crosslinks. Crosslink formation is very poorly characterized in terms of quantitative rate and yield data. We sought to address this issue by using oligo-arginine ligands to model the close association of DNA and its binding proteins in chromatin. Guanine radicals were prepared in plasmid DNA by single electron oxidation. The product distribution derived from them was assayed by strand break formation after four different post-irradiation incubations. We compared the yields of DNA damage produced in the presence of four ligands in which neither, one, or both of the amino and carboxylate termini were blocked with amides. Free carboxylate groups were unreactive. Significantly higher yields of heat labile sites were observed when the amino terminus was unblocked. The rate of the reaction was characterized by diluting the unblocked amino group with its amide blocked derivative. These observations provide a means to develop quantitative estimates for the yields in which these labile sites are formed in chromatin by exposure to ionizing irradiation.

  20. Characterization of phospholipid composition and its control in the plasma membrane of developing soybean root

    International Nuclear Information System (INIS)

    Whitman, C.E.

    1985-01-01

    The phospholipid composition of plasma membrane enriched fractions from developing soybean root and several mechanisms which may regulate it have been examined. Plasma membrane vesicles were isolated from meristematic and mature sections of four-day-old dark grown soybean roots (Glycine max [L.] Merr. Cult. Wells II). Analysis of lipid extracts revealed two major phospholipid classes: phosphatidylcholine and phosphatidylethanolamine. Minor phospholipid classes were phosphatidylinositol, phosphatidylserine, phosphatidylgylcerol and diphosphatidylgylcerol. Phospholipid composition was similar at each developmental stage. Fatty acids of phosphatidylcholine and phosphatidylethanolamine were 16:0, 18:0, 18:2, and 18:3. Fatty acid composition varied with both phospholipid class and the developmental stage of the root. The degradation of phosphatidylcholine by endogenous phospholipase D during membrane isolation indicated that this enzyme might be involved in phospholipid turnover within the membrane. Phospholipase D activity was heat labile and increasing the pH of the enzyme assay from 5.3 to 7.8 resulted in 90% inhibition of activity. The turnover of fatty acids within the phospholipids of the plasma membrane was studied. Mature root sections were incubated with [1- 14 C] acetate, 1 mM Na acetate and 50 μg/ml chloramphenicol. Membrane lipid extracts analyzed for phospholipid class and acyl chain composition revealed that the long incubation times did not alter the phospholipid composition of the plasma membrane enriched fraction

  1. Competition between Burkholderia pseudomallei and B. thailandensis.

    Science.gov (United States)

    Ngamdee, Wikanda; Tandhavanant, Sarunporn; Wikraiphat, Chanthiwa; Reamtong, Onrapak; Wuthiekanun, Vanaporn; Salje, Jeanne; Low, David A; Peacock, Sharon J; Chantratita, Narisara

    2015-03-03

    Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis, an often fatal disease in tropical countries. Burkholderia thailandensis is a non-virulent but closely related species. Both species are soil saprophytes but are almost never isolated together. We identified two mechanisms by which B. pseudomallei affects the growth of B. thailandensis. First, we found that six different isolates of B. pseudomallei inhibited the growth of B. thailandensis on LB agar plates. Second, our results indicated that 55% of isolated strains of B. pseudomallei produced a secreted compound that inhibited the motility but not the viability of B. thailandensis. Analysis showed that the active compound was a pH-sensitive and heat-labile compound, likely a protein, which may affect flagella processing or facilitate their degradation. Analysis of bacterial sequence types (STs) demonstrated an association between this and motility inhibition. The active compound was produced from B. pseudomallei during the stationary growth phase. Taken together, our results indicate that B. pseudomallei inhibits both the growth and motility of its close relative B. thailandensis. The latter phenomenon appears to occur via a previously unreported mechanism involving flagellar processing or degradation.

  2. Assay for plasma somatomedin: (/sup 3/H)thymidine incorporation by isolated rabbit chondrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Ashton, I K; Francis, M J.O. [Nuffield Orthopaedic Centre, Oxford (UK)

    1977-01-01

    The incorporation of (/sup 3/H)thymidine by rabbit chondrocytes in vitro has been developed as a sensitive assay for plasma somatomedin. A concentration of normal plasma of 2.5% enhanced (/sup 3/H)thymidine incorporation by 5- to 20-fold compared with basal levels in the absence of plasma. The mean potency of plasma from normal adult men was 0.96 +-0.1 u./ml (mean +- S.D.) and from acromegalic patients 1.9 +- 0.4 u./ml. The apparent potency of hypopituitary plasma alone increased on heating which suggested the presence of heat-labile inhibitors of somatomedin activity. The potency of heated hypopituitary plasma (0.6 +- 0.09 u./ml) remained significantly lower (P < 0.01) than normal plasma. Human growth hormone (0.1 to 20 ..mu..u/ml), bovine growth hormone (0.5 to 20 ..mu..u/ml), insulin (0.5 to 5 ..mu..u/ml) and glucose (0.3 to 2 mmol/l) had no direct effect on the incorporation of (/sup 3/H)thymidine. Chondrocytes which had been previously stored frozen also showed a response to plasma somatomedin.

  3. Purification and characterization of factors produced by Aspergillus fumigatus which affect human ciliated respiratory epithelium.

    Science.gov (United States)

    Amitani, R; Taylor, G; Elezis, E N; Llewellyn-Jones, C; Mitchell, J; Kuze, F; Cole, P J; Wilson, R

    1995-09-01

    The mechanisms by which Aspergillus fumigatus colonizes the respiratory mucosa are unknown. Culture filtrates of eight of nine clinical isolates of A. fumigatus slowed ciliary beat frequency and damaged human respiratory epithelium in vitro. These changes appeared to occur concurrently. Culture filtrates of two clinical isolates of Candida albicans had no effect on ciliated epithelium. We have purified and characterized cilioinhibitory factors of a clinical isolate of A. fumigatus. The cilioinhibitory activity was heat labile, reduced by dialysis, and partially extractable into chloroform. The activity was associated with both high- and low-molecular-weight factors, as determined by gel filtration on Sephadex G-50. A low-molecular-weight cilioinhibitory factor was further purified by reverse-phase high-performance liquid chromatography and shown by mass spectrometry to be gliotoxin, a known metabolite of A. fumigatus. Gliotoxin significantly slowed ciliary beat frequency in association with epithelial damage at concentrations above 0.2 microgram/ml; other Aspergillus toxins, i.e., fumagillin and helvolic acid, were also cilioinhibitory but at much higher concentrations. High-molecular-weight (> or = 35,000 and 25,000) cilioinhibitory materials had neither elastolytic nor proteolytic activity and remain to be identified. Thus, A. fumigatus produces a number of biologically active substances which slow ciliary beating and damage epithelium and which may influence colonization of the airways.

  4. Methicillin-resistant Staphylococcus aureus: a controversial food-borne pathogen.

    Science.gov (United States)

    Sergelidis, D; Angelidis, A S

    2017-06-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of severe healthcare-associated (HA) infections. Although during the last decade the incidence of HA invasive infections has dropped, the incidence of community-associated MRSA (CA-MRSA) infections has risen among the general population. Moreover, CA-MRSA, livestock-associated MRSA (LA-MRSA) and HA-MRSA (HA-MRSA) can be found in foods intended for human consumption. Several studies from different geographical areas have reported the presence of enterotoxin genes in several MRSA food isolates. Molecular typing studies have revealed genetic relatedness of these enterotoxigenic isolates with isolates incriminated in human infections. The contamination sources for foods, especially animal-origin foods, may be livestock as well as humans involved in animal husbandry and food-processing. Under favourable environmental conditions for growth and enterotoxin production, enterotoxigenic S. aureus isolates present in foods can cause staphylococcal food poisoning (SFP), irrespective of the contamination origin. Owing to the typically moderate clinical manifestations of SFP, the S. aureus strains responsible for SFP (cases or outbreaks) are frequently either not identified or not further characterized. Antimicrobial susceptibility testing is rarely performed, because administration of antimicrobial therapy is not required in the vast majority of cases. Staphylococcal food poisoning is the result of consumption of foods with preformed enterotoxins. Hence, similar to methicillin-sensitive enterotoxigenic S. aureus, enterotoxigenic MRSA can also act as food-borne pathogens upon favourable conditions for growth and enterotoxin production. The severity of the intoxication is not related to the antimicrobial resistance profile of the causative S. aureus strain and therefore MRSA food-borne outbreaks are not expected to be more severe. This review evaluates the potential of methicillin-resistant Staphylococcus

  5. Prevalence and molecular characteristics of Staphylococcus aureus, including methicillin resistant strains, isolated from bulk can milk and raw milk products in pastoral communities of South-West Uganda.

    Science.gov (United States)

    Asiimwe, Benon B; Baldan, Rossella; Trovato, Alberto; Cirillo, Daniela M

    2017-06-13

    Staphylococcus aureus strains are now regarded as zoonotic agents. In pastoral settings where human-animal interaction is intimate, multi-drug resistant microorganisms have become an emerging zoonotic issue of public health concern. The study of S. aureus prevalence, antimicrobial resistance and clonal lineages in humans, animals and food in African settings has great relevance, taking into consideration the high diversity of ethnicities, cultures and food habits that determine the lifestyle of the people. Little is known about milk carriage of methicillin resistant S. aureus strains (MRSA) and their virulence factors in Uganda. Here, we present the prevalence of MRSA in bulk can milk and raw milk products in pastoral communities of south-west Uganda. We also present PFGE profiles, spa-types, as well as frequency of enterotoxins genes. S. aureus was identified by the coagulase test, susceptibility testing by the Kirby-Bauer disc diffusion and E-test methods and MRSA by detection of the mecA gene and SCCmec types. The presence of Panton - Valentine Leucocidin (PVL) genes and staphylococcal enterotoxins was determined by PCR, while genotyping was by PFGE and spa typing. S. aureus were isolated from 30/148 (20.3%) milk and 11/91(12%) sour milk samples. mecA gene carriage, hence MRSA, was detected in 23/41 (56.1%) of the isolates, with 21 of the 23 (91.3%) being SCCmec type V; while up to 30/41 (73.2%) of the isolates were resistant to tetracycline. Only five isolates carried the PVL virulence gene, while PFGE typing revealed ten clusters (ranging from two seven isolates each) that comprised 83% of the sample, and only eight isolates with unique pulsotypes. The largest PFGE profile (E) consisted of seven isolates while t7753, t1398, and t2112 were the most common spa-types. Thirty seven of the 41 strains (90.2%) showed at least one of the eight enterotoxin genes tested, with sem 29 (70.7%), sei 25 (61%) and seg 21 (51.2%) being the most frequently observed genes. This

  6. A 5 year longitudinal study of water quality for final rinsing in the single chamber washer-disinfector with a reverse osmosis plant.

    Science.gov (United States)

    Uetera, Yushi; Kishii, Kozue; Yasuhara, Hiroshi; Kumada, Naohito; Moriya, Kyoji; Saito, Ryoichi; Okazaki, Mitsuhiro; Misawa, Yoshiki; Kawamura, Kunio

    2013-01-01

    This report deals with the construction and management of the reverse osmosis (RO) water system for final rinsing of surgical instruments in the washer-disinfector. Numerous operational challenges were encountered in our RO water system and these were analyzed utilizing the Ishikawa Fishbone diagram. The aim was to find potential problems and promote preventive system management for RO water. It was found that the measures that existed were inappropriate for preventing contamination in the heat-labile RO water system. The storage tank was found to be significantly contaminated and had to be replaced with a new one equipped with a sampling port and water drainage system. Additional filters and an UV treatment lamp were installed. The whole system disinfection started 1.5 years later using a peracetic acid-based compound after confirming the material compatibility. Operator errors were found when a new water engineer took over the duty from his predecessor. It was also found that there were some deficiencies in the standard operating procedures (SOPs), and that on-the-job training was not enough. The water engineer failed to disinfect the sampling port and water drainage system. The RO membrane had been used for 4 years, even though the SOP standard specified changing it as every 3 years. Various bacteria, such as Rothia mucilaginosa, were cultured from the RO water sampled from the equipment. Because Rothia mucilaginosa is a resident in the oral cavity and upper respiratory tract, it is believed that the bacteria were introduced into the system by the maintenance personnel or working environment. Therefore, the presence of R. mucilaginosa implied the failure of sanitary maintenance procedures. This study suggests that water systems should be designed based on the plans for profound system maintenance. It also suggests that SOP and on-the job training are essential to avoid any operator errors. These results must be carefully considered when either constructing new

  7. Palmito de pupunha (Bactris gasipaes Kunth. composição mineral e cinética de enzimas oxidativas Heart of palm (Bactris gasipaes Kunth.: mineral composition and kinetics of oxidative enzymes

    Directory of Open Access Journals (Sweden)

    Nathália Ottoboni Galdino

    2008-09-01

    Full Text Available A análise da presença de enzimas oxidativas como a peroxidase (POD e a polifenoloxidase (PPO e o controle da atividade destas enzimas são importantes na preservação e no processamento de alimentos. Este trabalho teve por objetivo determinar a atividade enzimática da polifenoloxidase (PPO e da peroxidase (POD do palmito de pupunha, bem como avaliar o comportamento destas enzimas frente ao tratamento térmico e assim calcular a cinética de inativação térmica das mesmas para suas porções termorresistente e termolábil. Para a extração de peroxidase (POD e polifenoloxidase (PPO de palmito, utilizou-se solução tampão fosfato de sódio 100 mM com diferentes pHs (5,5; 6,0; 6,5 e 7,0. O melhor pH de extração da POD foi 5,5 e da PPO, 6,5. Estes extratos foram tratados em diferentes temperaturas (65, 70, 75 e 80 °C por períodos de 1 a 10 minutos. A POD e a PPO sofreram um decréscimo de 70 e 80%, respectivamente, em relação às suas atividades iniciais. As energias de ativação, nas temperaturas estudadas, para a porção termolábil e termorresistente da peroxidase foram 154,0 e 153,0 kJ.mol-1, respectivamente, enquanto que para a polifenoloxidase foram 26,3 e 27,0 kJ.mol-1, respectivamente. Resultados apresentaram valores que estão dentro da faixa de energia de ativação reportada para o processo de inativação térmica de enzimas.Analysis of oxidative enzymes such as peroxidase (POD and polyphenoloxidase (PPO and the control of the activity of these enzymes are important in food preservation and also in food processing. The aim of this work was to determine polyphenoloxidase (PPO and peroxidase (POD enzymatic activity in heart of palm, as well as to evaluate enzyme behavior during thermal treatment, determining the kinetics of thermal inactivation of the heat resistant and heat labile portions. For the extraction of peroxidase (POD and polyphenoloxidase (PPO from the heart of palm solution, 100 mM sodium phosphate buffer with

  8. The odyssey of a young gene: structure-function studies in human glutamate dehydrogenases reveal evolutionary-acquired complex allosteric regulation mechanisms.

    Science.gov (United States)

    Zaganas, Ioannis V; Kanavouras, Konstantinos; Borompokas, Nikolas; Arianoglou, Giovanna; Dimovasili, Christina; Latsoudis, Helen; Vlassi, Metaxia; Mastorodemos, Vasileios

    2014-01-01

    Mammalian glutamate dehydrogenase (GDH) catalyzes the reversible inter-conversion of glutamate to α-ketoglutarate and ammonia, interconnecting carbon skeleton and nitrogen metabolism. In addition, it functions as an energy switch by its ability to fuel the Krebs cycle depending on the energy status of the cell. As GDH lies at the intersection of several metabolic pathways, its activity is tightly regulated by several allosteric compounds that are metabolic intermediates. In contrast to other mammals that have a single GDH-encoding gene, humans and great apes possess two isoforms of GDH (hGDH1 and hGDH2, encoded by the GLUD1 and GLUD2 genes, respectively) with distinct regulation pattern, but remarkable sequence similarity (they differ, in their mature form, in only 15 of their 505 amino-acids). The GLUD2 gene is considered a very young gene, emerging from the GLUD1 gene through retro-position only recently (<23 million years ago). The new hGDH2 iso-enzyme, through random mutations and natural selection, is thought to have conferred an evolutionary advantage that helped its persistence through primate evolution. The properties of the two highly homologous human GDHs have been studied using purified recombinant hGDH1 and hGDH2 proteins obtained by expression of the corresponding cDNAs in Sf21 cells. According to these studies, in contrast to hGDH1 that maintains basal activity at 35-40 % of its maximal, hGDH2 displays low basal activity that is highly responsive to activation by rising levels of ADP and/or L-leucine which can also act synergistically. While hGDH1 is inhibited potently by GTP, hGDH2 shows remarkable GTP resistance. Furthermore, the two iso-enzymes are differentially inhibited by estrogens, polyamines and neuroleptics, and also differ in heat-lability. To elucidate the molecular mechanisms that underlie these different regulation patterns of the two iso-enzymes (and consequently the evolutionary adaptation of hGDH2 to a new functional role), we have

  9. Staphylococcal poisoning foodborne outbreak: epidemiological investigation and strain genotyping.

    Science.gov (United States)

    Gallina, S; Bianchi, D M; Bellio, A; Nogarol, C; Macori, G; Zaccaria, T; Biorci, F; Carraro, E; Decastelli, L

    2013-12-01

    In June 2011, an outbreak of Staphylococcus aureus enterotoxin food poisoning gastroenteritis occurred in Turin, Italy, following a catered dinner party at a private home. Within a few hours, 26 of the 47 guests experienced gastrointestinal illness, and 9 were hospitalized. A retrospective cohort study using a standardized questionnaire was carried out, and the risk ratios for each food item were calculated. The analysis indicated consumption of seafood salad as the most probable cause of the outbreak (risk ratio = 11.72; 95 % confidence interval, 1.75 to 78.54). Biological samples were collected from four of the hospitalized guests (stool and vomit), nasal mucosa swabs from three food handlers employed with the caterer, and available food residuals. All stool and vomit samples tested positive for enterotoxigenic S. aureus. As residues of the seafood salad were no longer available for sampling, suspected contamination could not be verified. However, no other food was found contaminated by S. aureus or its enterotoxins. All isolates from the biological samples were characterized at the genomic level by means of two multiplex PCR protocols to determine the presence of genes encoding staphylococcal enterotoxins, pulsed-field gel electrophoresis and staphylococcal protein A gene (spa) typing to describe their genetic profiles. All the isolates presented genes encoding SEA and SEI; the pulsed-field gel electrophoresis genetic profiles revealed the same pulsotype in the microorganism isolated from the hospitalized guests as in one of the isolates from a food handler's nasal mucosa, and the spa typing analysis reported two closely related spa types (t701 and t267), implicating the food handler as the most likely outbreak source.

  10. Outbreak of Staphylococcal food poisoning due to SEA-producing Staphylococcus aureus.

    Science.gov (United States)

    Johler, Sophia; Tichaczek-Dischinger, Petra S; Rau, Jörg; Sihto, Henna-Maria; Lehner, Angelika; Adam, Maja; Stephan, Roger

    2013-09-01

    In 2008, 150 people gathered for a wedding celebration in Baden-Württemberg, Germany. Three hours after ingestion of a variety of foods including pancakes filled with minced chicken, several guests exhibited symptoms of acute gastroenteritis such as vomiting, diarrhea, fever, and ague. Twelve guests were reported to have fallen ill, with nine of these seeking medical care in hospitals. At least four patients were admitted to the hospital and received inpatient treatment, among them a 2-year-old child and a woman in the 4th month of pregnancy. Within 24 h of the event, an investigative team collected a variety of samples including refrigerated leftovers, food in the storage unit of the caterer, nasal swabs of the caterer, as well as 21 environmental swabs. Five stool samples from patients were provided by the hospitals. Staphylococcus aureus isolates were gathered from eight samples, among them nasal swabs of the caterer, food samples, and one stool sample. Fourier transform-infrared spectroscopy was used for species identification and for primary clustering of the isolates in a similarity tree. The isolates were further characterized by spa typing and pulsed-field gel electrophoresis, and a DNA microarray was used to determine the presence/absence of genes involved in virulence and antimicrobial resistance. We were able to match an enterotoxigenic strain from the stool sample of a patient to isolates of the same strain obtained from food and the nasal cavity of a food handler. The strain produced the enterotoxin SEA and the toxic shock syndrome toxin-1, and was also found to exhibit the genes encoding enterotoxins SEG and SEI, as well as the enterotoxin gene cluster egc. This is one of only a few studies that were able to link a staphylococcal food poisoning outbreak to its source.

  11. Identification of virulence factors in 16S-23S rRNA intergenic spacer genotyped Staphylococcus aureus isolated from water buffaloes and small ruminants.

    Science.gov (United States)

    Cremonesi, P; Zottola, T; Locatelli, C; Pollera, C; Castiglioni, B; Scaccabarozzi, L; Moroni, P

    2013-01-01

    Staphylococcus aureus is an important human and animal pathogen, and is regarded as an important cause of intramammary infection (IMI) in ruminants. Staphylococcus aureus genetic variability and virulence factors have been well studied in veterinary medicine, especially in cows as support for control and management of IMI. The aim of the present study was to genotype 71 Staph. aureus isolates from the bulk tank and foremilk of water buffaloes (n=40) and from udder tissue (n=7) and foremilk (n=24) from small ruminants. The method used was previously applied to bovine Staph. aureus and is based on the amplification of the 16S-23S rRNA intergenic spacer region. The technique applied was able to identify different Staph. aureus genotypes isolated from dairy species other than the bovine species, and cluster the genotypes according to species and herds. Virulence gene distribution was consistent with genotype differentiation. The isolates were also characterized through determination of the presence of 19 virulence-associated genes by specific PCR. Enterotoxins A, C, D, G, I, J, and L were associated with Staph. aureus isolates from buffaloes, whereas enterotoxins C and L were linked to small ruminants. Genes coding for methicillin resistance, Panton-Valentine leukocidin, exfoliative toxins A and B, and enterotoxins B, E, and H were undetected. These findings indicate that RNA template-specific PCR is a valid technique for typing Staph. aureus from buffaloes and small ruminants and is a useful tool for understanding udder infection epidemiology. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  12. Subinhibitory concentrations of perilla oil affect the expression of secreted virulence factor genes in Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Jiazhang Qiu

    Full Text Available BACKGROUND: The pathogenicity of staphylococcus aureus is dependent largely upon its ability to secrete a number of virulence factors, therefore, anti-virulence strategy to combat S. aureus-mediated infections is now gaining great interest. It is widely recognized that some plant essential oils could affect the production of staphylococcal exotoxins when used at subinhibitory concentrations. Perilla [Perilla frutescens (L. Britton], a natural medicine found in eastern Asia, is primarily used as both a medicinal and culinary herb. Its essential oil (perilla oil has been previously demonstrated to be active against S. aureus. However, there are no data on the influence of perilla oil on the production of S. aureus exotoxins. METHODOLOGY/PRINCIPAL FINDINGS: A broth microdilution method was used to determine the minimum inhibitory concentrations (MICs of perilla oil against S. aureus strains. Hemolysis, tumour necrosis factor (TNF release, Western blot, and real-time RT-PCR assays were performed to evaluate the effects of subinhibitory concentrations of perilla oil on exotoxins production in S. aureus. The data presented here show that perilla oil dose-dependently decreased the production of α-toxin, enterotoxins A and B (the major staphylococcal enterotoxins, and toxic shock syndrome toxin 1 (TSST-1 in both methicillin-sensitive S. aureus (MSSA and methicillin-resistant S. aureus (MRSA. CONCLUSIONS/SIGNIFICANCE: The production of α-toxin, SEA, SEB, and TSST-1 in S. aureus was decreased by perilla oil. These data suggest that perilla oil may be useful for the treatment of S. aureus infections when used in combination with β-lactam antibiotics, which can increase exotoxins production by S. aureus at subinhibitory concentrations. Furthermore, perilla oil could be rationally applied in food systems as a novel food preservative both to inhibit the growth of S. aureus and to repress the production of exotoxins, particularly staphylococcal enterotoxins.

  13. Virulence Factors of Staphylococcus aureus Isolates in an Iranian Referral Children's Hospital.

    Science.gov (United States)

    Sabouni, Farah; Mahmoudi, Shima; Bahador, Abbas; Pourakbari, Babak; Sadeghi, Reihaneh Hosseinpour; Ashtiani, Mohammad Taghi Haghi; Nikmanesh, Bahram; Mamishi, Setareh

    2014-04-01

    The clinical importance of Staphylococcus aureus (S. aureus) is attributed to notable virulence factors, surface proteins, toxins, and enzymes as well as the rapid development of drug resistance. The aim of this study was to compare the occurrence of virulence factors produced by S. aureus strains isolated from children in an Iranian referral children's hospital. The presence of genes encoding for the enterotoxins A (sea), B (seb), C (sec), D (sed), TSST-1 (tsst), exfoliative toxin A (eta), and exfoliative toxin B (etb) were detected by Multiplex polymerase chain reaction (PCR) using specific primers. In addition, the standardized Kirby-Bauer disc-diffusion method was performed on Mueller-Hinton agar. In total, 133 S. aureus isolates were obtained from different patients. Of these S. aureus isolates, 64 (48%) were methicillin-resistant S. aureus (MRSA), and all of these tested positive for the mecA gene. Regarding the classical enterotoxin genes, sea gene (40.6%) was the most prevalent followed by seb (19.6%), tsst (12.8%), eta (11.3%), etb (9%), sed (4.5%), and sec (3%). Among methicillin-susceptible S. aureus (MSSA) isolates, seb and tsst were the more prevalent toxins in comparison with MRSA isolates (p  0.05). In our study enterotoxin A was produced by 40.6% of the isolates (48% from MRSA and 33% from MSSA isolates) which was higher than in previous reports. According to our results, strict hygiene and preventative measures during food processing are highly recommended.

  14. Characterisation of MRSA from Malta and the description of a Maltese epidemic MRSA strain.

    LENUS (Irish Health Repository)

    Scicluna, E A

    2010-02-01

    Malta has one of the highest prevalence rates of methicillin-resistant Staphylococcus aureus (MRSA) in Europe. However, only limited typing data are currently available. In order to address this situation, 45 MRSA isolates from the Mater Dei Hospital in Msida, Malta, were characterised using DNA microarrays. The most common strain was ST22-MRSA-IV (UK-EMRSA-15, 30 isolates). Sporadic strains included ST36-MRSA-II (UK-EMRSA-16, two isolates), PVL-positive ST80-MRSA-IV (European Clone, one isolate), ST228-MRSA-I (Italian Clone\\/South German Epidemic Strain, one isolate) and ST239-MRSA-III (Vienna\\/Hungarian\\/Brazilian Epidemic Strain, one isolate). Ten MRSA isolates belonged to a clonal complex (CC) 5\\/ST149, spa type t002 strain. This strain harboured an SCCmec IV element (mecA, delta mecR, ugpQ, dcs, ccrA2 and ccrB2), as well as novel alleles of ccrA\\/B and the fusidic acid resistance element Q6GD50 (previously described in the sequenced strain MSSA476, BX571857.1:SAS0043). It also carried the gene for enterotoxin A (sea) and the egc enterotoxin locus, as well as (in nine out of ten isolates) genes encoding the toxic shock syndrome toxin (tst1) and enterotoxins C and L (sec, sel). While the presence of the other MRSA strains suggests foreign importation due to travel between Malta and other European countries, the CC5\\/t002 strain appears, so far, to be restricted to Malta.

  15. Modulation of Interleukin-8 and staphylococcal flora by Avène hydrotherapy in patients suffering from chronic inflammatory dermatoses.

    Science.gov (United States)

    Casas, C; Ribet, V; Alvarez-Georges, S; Sibaud, V; Guerrero, D; Schmitt, A-M; Redoulès, D

    2011-02-01

    A number of studies argue in favour of an important role of microbial colonization, in particular of Staphylococcus aureus, in triggering atopic dermatitis (AD) flare-up and psoriasis, in particular through the superantigenic properties of toxins generated by S. aureus. The aim of this study was to assess the efficacy of a 3-week Avène hydrotherapy on the skin surface of patients suffering from psoriasis or atopic dermatitis. Skin samples were taken from healthy subjects or atopic (n = 18) or psoriatic patients (n = 39) undergoing hydrotherapy at Avène at the beginning (D0) and the end of treatment (D18). The severity of the dermatosis was evaluated according to SCORing Atopic Dermatitis (SCORAD) or Psoriasis Area Severity Index (PASI) scores at D0 and D18. Marker of inflammation interleukin 8 (IL-8), S. aureus colonization (protein A) and enterotoxins were assessed in skin samples using RT-PCR. At D0, significant differences were observed between healthy subjects and atopic or psoriatic patients in all the parameters evaluated (IL-8, protein A). At the end of the hydrotherapy, a significant decrease in SCORAD was associated with a significant reduction of IL-8, S. aureus colonization and enterotoxin D in patients with atopic dermatitis. Similarly, a significant decrease in PASI was associated with a significant reduction of IL-8, S. aureus colonization and enterotoxin N in patients with psoriasis. This study demonstrates the positive effects of Avène hydrotherapy on the skin of patients suffering from chronic dermatosis, with decreased inflammation and reduced colonization by S. aureus. © 2010 The Authors. JEADV © 2010 European Academy of Dermatology and Venereology.

  16. Effect of High Pressure and Heat on Bacterial Toxins

    Directory of Open Access Journals (Sweden)

    Dirk Margosch

    2005-01-01

    Full Text Available Even though the inactivation of microorganisms by high pressure treatment is a subject of intense investigations, the effect of high pressure on bacterial toxins has not been studied so far. In this study, the influence of combined pressure/temperature treatment (0.1 to 800 MPa and 5 to 121 °C on bacterial enterotoxins was determined. Therefore, heat-stable enterotoxin (STa of cholera toxin (CT from Vibrio cholerae, staphylococcal enterotoxins A-E, haemolysin BL (HBL from Bacillus cereus, and Escherichia coli (STa were subjected to different treatment schemes. Structural alterations were monitored in enzyme immunoassays (EIAs. Cytotoxicity of the pressure treated supernatant of toxigenic B. cereus DSM 4384 was investigated with Vero cells. High pressure of 200 to 800 MPa at 5 °C leads to a slight increase of the reactivity of the STa of E. coli. However, reactivity decreased at 800 MPa and 80 °C to (66±21 % after 30 min and to (44±0.3 % after 128 min. At ambient pressure no decrease in EIA reactivity could be observed after 128 min. Pressurization (0.1 to 800 MPa of heat stable monomeric staphylococcal toxins at 5 and 20 °C showed no effect. A combined heat (80 °C and pressure (0.1 to 800 MPa treatment lead to a decrease in the immuno-reactivity to 20 % of its maximum. For cholera toxin a significant loss in latex agglutination was observable only at 80 °C and 800 MPa for holding times higher than 20 min. Interestingly, the immuno-reactivity of B. cereus HBL toxin increased with the increase of pressure (182 % at 800 MPa, 30 °C, and high pressure showed only minor effects on cytotoxicity to Vero cells. Our results indicate that pressurization can increase inactivation observed by heat treatment, and combined treatments may be effective at lower temperatures and/or shorter incubation time.

  17. Enterotoxigenicity and Antimicrobial Resistance of Staphylococcus aureus Isolated from Retail Food in China

    Science.gov (United States)

    Wang, Wei; Baloch, Zulqarnain; Jiang, Tao; Zhang, Cunshan; Peng, Zixin; Li, Fengqin; Fanning, Séamus; Ma, Aiguo; Xu, Jin

    2017-01-01

    Staphylococcus aureus is one of the most common causes of zoonotic agent in the world, which are attributable to the contamination of food with enterotoxins. In this study, a total of 1,150 S. aureus isolates were cultured from 27,000 retail foods items from 203 cities of 24 provinces in China in 2015 and were test for antimicrobial susceptibility. Additionally, the role of the genes responsible for the staphylococcal enterotoxins (SEA to SEE), methicillin resistance (mecA) and the toxigenic capabilities were also assessed. The results showed that 4.3% retail foods were contaminated with S. aureus, and 7.9% retail foods isolates were mecA positive. Some 97.6% of S. aureus isolates were resistant to at least one antimicrobial compound, and 57.5% of these were multi drug resistant (MDR). Resistance to penicillin (83.7%, 963/1,150), was common, followed by linezolid (67.7%, 778/1,150) and erythromycin (52.1%, 599/1,150). The isolates cultured from raw meats showed high levels of resistant to tetracycline (42.8%), ciprofloxacin (17.4%), and chloramphenicol (12.0%) and expressed a MDR phenotype (62.4%). A total of 29.7% S. aureus isolates harbored the classical SEs genes (sea, seb, sec, and sed). The sea and seb genes were the most frequent SEs genes detected. Of note, 22% of the SEs genes positive S. aureus harbored two or three SEs genes, and 16 isolates were confirmed with the capacity to simultaneously produce two or three enterotoxin types. Moreover, nearly 50% of the MRSA isolates were positive for at least one SE gene in this study. Therefore, it is important to monitor the antimicrobial susceptibility and enterotoxigenicity of MDR S. aureus and MRSA in the food chain and to use these data to develop food safety measures, designed to reduce the contamination and transmission of this bacterium. PMID:29209290

  18. Detection and characterisation of Complement protein activity in bovine milk by bactericidal sequestration assay.

    Science.gov (United States)

    Maye, Susan; Stanton, Catherine; Fitzgerald, Gerald F; Kelly, Philip M

    2015-08-01

    While the Complement protein system in human milk is well characterised, there is little information on its presence and activity in bovine milk. Complement forms part of the innate immune system, hence the importance of its contribution during milk ingestion to the overall defences of the neonate. A bactericidal sequestration assay, featuring a Complement sensitive strain, Escherichia coli 0111, originally used to characterise Complement activity in human milk was successfully applied to freshly drawn bovine milk samples, thus, providing an opportunity to compare Complement activities in both human and bovine milks. Although not identical in response, the levels of Complement activity in bovine milk were found to be closely comparable with that of human milk. Differential counts of Esch. coli 0111 after 2 h incubation were 6.20 and 6.06 log CFU/ml, for raw bovine and human milks, respectively - the lower value representing a stronger Complement response. Exposing bovine milk to a range of thermal treatments e.g. 42, 45, 65, 72, 85 or 95 °C for 10 min, progressively inhibited Complement activity by increasing temperature, thus confirming the heat labile nature of this immune protein system. Low level Complement activity was found, however, in 65 and 72 °C heat treated samples and in retailed pasteurised milk which highlights the outer limit to which high temperature, short time (HTST) industrial thermal processes should be applied if retention of activity is a priority. Concentration of Complement in the fat phase was evident following cream separation, and this was also reflected in the further loss of activity recorded in low fat variants of retailed pasteurised milk. Laboratory-based churning of the cream during simulated buttermaking generated an aqueous (buttermilk) phase with higher levels of Complement activity than the fat phase, thus pointing to a likely association with the milk fat globule membrane (MFGM) layer.

  19. Molecular Structural Basis for the Cold Adaptedness of the Psychrophilic β-Glucosidase BglU in Micrococcus antarcticus.

    Science.gov (United States)

    Miao, Li-Li; Hou, Yan-Jie; Fan, Hong-Xia; Qu, Jie; Qi, Chao; Liu, Ying; Li, De-Feng; Liu, Zhi-Pei

    2016-01-22

    Psychrophilic enzymes play crucial roles in cold adaptation of microbes and provide useful models for studies of protein evolution, folding, and dynamic properties. We examined the crystal structure (2.2-Å resolution) of the psychrophilic β-glucosidase BglU, a member of the glycosyl hydrolase 1 (GH1) enzyme family found in the cold-adapted bacterium Micrococcus antarcticus. Structural comparison and sequence alignment between BglU and its mesophilic and thermophilic counterpart enzymes (BglB and GlyTn, respectively) revealed two notable features distinct to BglU: (i) a unique long-loop L3 (35 versus 7 amino acids in others) involved in substrate binding and (ii) a unique amino acid, His299 (Tyr in others), involved in the stabilization of an ordered water molecule chain. Shortening of loop L3 to 25 amino acids reduced low-temperature catalytic activity, substrate-binding ability, the optimal temperature, and the melting temperature (Tm). Mutation of His299 to Tyr increased the optimal temperature, the Tm, and the catalytic activity. Conversely, mutation of Tyr301 to His in BglB caused a reduction in catalytic activity, thermostability, and the optimal temperature (45 to 35°C). Loop L3 shortening and H299Y substitution jointly restored enzyme activity to the level of BglU, but at moderate temperatures. Our findings indicate that loop L3 controls the level of catalytic activity at low temperatures, residue His299 is responsible for thermolability (particularly heat lability of the active center), and long-loop L3 and His299 are jointly responsible for the psychrophilic properties. The described structural basis for the cold adaptedness of BglU will be helpful for structure-based engineering of new cold-adapted enzymes and for the production of mutants useful in a variety of industrial processes at different temperatures. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  20. Specific amino acids responsible for the cold adaptedness of Micrococcus antarcticus β-glucosidase BglU.

    Science.gov (United States)

    Miao, Li-Li; Fan, Hong-Xia; Qu, Jie; Liu, Ying; Liu, Zhi-Pei

    2017-03-01

    Psychrophilic enzymes display efficient activity at moderate or low temperatures (4-25 °C) and are therefore of great interest in biotechnological industries. We previously examined the crystal structure of BglU, a psychrophilic β-glucosidase from the bacterium Micrococcus antarcticus, at 2.2 Å resolution. In structural comparison and sequence alignment with mesophilic (BglB) and thermophilic (GlyTn) counterpart enzymes, BglU showed much lower contents of Pro residue and of charged amino acids (particularly positively charged) on the accessible surface area. In the present study, we investigated the roles of specific amino acid residues in the cold adaptedness of BglU. Mutagenesis assays showed that the mutations G261R and Q448P increased optimal temperature (from 25 to 40-45 °C) at the expense of low-temperature activity, but had no notable effects on maximal activity or heat lability. Mutations A368P, T383P, and A389E significantly increased optimal temperature (from 25 to 35-40 °C) and maximal activity (~1.5-fold relative to BglU). Thermostability of A368P and A389E increased slightly at 30 °C. Mutations K163P, N228P, and H301A greatly reduced enzymatic activity-almost completely in the case of H301A. Low contents of Pro, Arg, and Glu are important factors contributing to BglU's psychrophilic properties. Our findings will be useful in structure-based engineering of psychrophilic enzymes and in production of mutants suitable for a variety of industrial processes (e.g., food production, sewage treatment) at cold or moderate temperatures.

  1. Antigenic and anticorpal diagnosis of invasive aspergillosis and candidosis in immunocompromised host

    Directory of Open Access Journals (Sweden)

    Marco Machetti

    2009-03-01

    Full Text Available Invasive aspergillosis (IA and candidemia are an increasing cause of morbidity and mortality in immunocompromised patients, but diagnostic procedures are often hampered by critical patients conditions. In recent years, non-culture methods have reached a standardization level suitable to be marketed and widely employed. A method for a panfungal antigenic diagnosis, is the detection in serum of (1→3-ß-D-Glucan (BDG, a polisaccaridic component of fungal cell wall. Best results can be obtained in Aspergillus, Candida and Fusarium infections, while the test performs poorly with Cryptococcus and in zygomycosis. The use of this method are limited by the high costs and by the need of disposable materials certified as glucan-free, in order to avoid false positive results. Galactomannan (GM antigen detection is a method for non-invasive diagnosis of IA. The assay, in latex agglutination (LA and enzymeimmunoassay (EIA format, detect GM in serum, whose presence correlates with IA. Sensitivity and specificity strongly fluctuate (50-100% and 81-98% respectively depending on the time of sampling, the positivity cut-off employed, the concomitant administration of antifungal drugs and of some antibiotics. Nevertheless the method is a very useful and widely employed tool for the diagnosis of IA. For the diagnosis of candidemia, two Candida antigens may be detected in serum: the 56°C heat-labile antigen in LA format, and the mannan antigen, in LA and EIA format. Both the methods perform quite well, but sensitivities and specificities are not so good to allow a routinely and useful use in clinical practice.

  2. Neonatal plasma polarizes TLR4-mediated cytokine responses towards low IL-12p70 and high IL-10 production via distinct factors.

    Directory of Open Access Journals (Sweden)

    Mirjam E Belderbos

    Full Text Available Human neonates are highly susceptible to infection, which may be due in part to impaired innate immune function. Neonatal Toll-like receptor (TLR responses are biased against the generation of pro-inflammatory/Th1-polarizing cytokines, yet the underlying mechanisms are incompletely defined. Here, we demonstrate that neonatal plasma polarizes TLR4-mediated cytokine production. When exposed to cord blood plasma, mononuclear cells (MCs produced significantly lower TLR4-mediated IL-12p70 and higher IL-10 compared to MC exposed to adult plasma. Suppression by neonatal plasma of TLR4-mediated IL-12p70 production, but not induction of TLR4-mediated IL-10 production, was maintained up to the age of 1 month. Cord blood plasma conferred a similar pattern of MC cytokine responses to TLR3 and TLR8 agonists, demonstrating activity towards both MyD88-dependent and MyD88-independent agonists. The factor causing increased TLR4-mediated IL-10 production by cord blood plasma was heat-labile, lost after protein depletion and independent of lipoprotein binding protein (LBP or soluble CD14 (sCD14. The factor causing inhibition of TLR4-mediated IL-12p70 production by cord blood plasma was resistant to heat inactivation or protein depletion and was independent of IL-10, vitamin D and prostaglandin E2. In conclusion, human neonatal plasma contains at least two distinct factors that suppress TLR4-mediated IL-12p70 production or induce IL-10 or production. Further identification of these factors will provide insight into the ontogeny of innate immune development and might identify novel targets for the prevention and treatment of neonatal infection.

  3. Neonatal Plasma Polarizes TLR4-Mediated Cytokine Responses towards Low IL-12p70 and High IL-10 Production via Distinct Factors

    Science.gov (United States)

    Belderbos, Mirjam E.; Levy, Ofer; Stalpers, Femke; Kimpen, Jan L.; Meyaard, Linde; Bont, Louis

    2012-01-01

    Human neonates are highly susceptible to infection, which may be due in part to impaired innate immune function. Neonatal Toll-like receptor (TLR) responses are biased against the generation of pro-inflammatory/Th1-polarizing cytokines, yet the underlying mechanisms are incompletely defined. Here, we demonstrate that neonatal plasma polarizes TLR4-mediated cytokine production. When exposed to cord blood plasma, mononuclear cells (MCs) produced significantly lower TLR4-mediated IL-12p70 and higher IL-10 compared to MC exposed to adult plasma. Suppression by neonatal plasma of TLR4-mediated IL-12p70 production, but not induction of TLR4-mediated IL-10 production, was maintained up to the age of 1 month. Cord blood plasma conferred a similar pattern of MC cytokine responses to TLR3 and TLR8 agonists, demonstrating activity towards both MyD88-dependent and MyD88-independent agonists. The factor causing increased TLR4-mediated IL-10 production by cord blood plasma was heat-labile, lost after protein depletion and independent of lipoprotein binding protein (LBP) or soluble CD14 (sCD14). The factor causing inhibition of TLR4-mediated IL-12p70 production by cord blood plasma was resistant to heat inactivation or protein depletion and was independent of IL-10, vitamin D and prostaglandin E2. In conclusion, human neonatal plasma contains at least two distinct factors that suppress TLR4-mediated IL-12p70 production or induce IL-10 or production. Further identification of these factors will provide insight into the ontogeny of innate immune development and might identify novel targets for the prevention and treatment of neonatal infection. PMID:22442690

  4. The role of complement in CD4⁺ T cell homeostasis and effector functions.

    Science.gov (United States)

    Kolev, Martin; Le Friec, Gaëlle; Kemper, Claudia

    2013-02-01

    The complement system is among the evolutionary oldest 'players' of the immune system. It was discovered in 1896 by Jules Bordet as a heat-labile fraction of the serum responsible for the opsonisation and subsequent killing of bacteria. The decades between the 1920s and 1990s then marked the discovery and biochemical characterization of the proteins comprising the complement system. Today, complement is defined as a complex system consisting of more than 30 membrane-bound and soluble plasma proteins, which are activated in a cascade-like manner, very similarly to the caspase proteases and blood coagulation systems. Complement is engrained in the immunologist's mind as a serum-effective, quintessential part of innate immunity, vitally required for the detection and removal of pathogens or other dangerous entities. Three decades ago, this rather confined definition was challenged and then refined when it was shown that complement participates vitally in the induction and regulation of B cell responses, thus adaptive immunity. Similarly, research work published in more recent years supports an equally important role for the complement system in shaping T cell responses. Today, we are again facing paradigm shifts in the field: complement is actively involved in the negative control of T cell effector immune responses, and thus, by definition in immune homeostasis. Further, while serum complement activity is without doubt fundamental in the defence against invading pathogens, local immune cell-derived production of complement emerges as key mediator of complement's impact on adaptive immune responses. And finally, the impact of complement on metabolic pathways and the crosstalk between complement and other immune effector systems is likely more extensive than previously anticipated and is fertile ground for future discoveries. In this review, we will discuss these emerging new roles of complement, with a focus on Th1 cell biology. Copyright © 2013 Elsevier Ltd. All

  5. Production, secretion, and stability of human secreted alkaline phosphatase in tobacco NT1 cell suspension cultures.

    Science.gov (United States)

    Becerra-Arteaga, Alejandro; Mason, Hugh S; Shuler, Michael L

    2006-01-01

    Tobacco NT1 cell suspension cultures secreting active human secreted alkaline phosphatase (SEAP) were generated for the first time as a model system to study recombinant protein production, secretion, and stability in plant cell cultures. The SEAP gene encodes a secreted form of the human placental alkaline phosphatase (PLAP). During batch culture, the highest level of active SEAP in the culture medium (0.4 U/mL, corresponding to approximately 27 mg/L) was observed at the end of the exponential growth phase. Although the level of active SEAP decreased during the stationary phase, the activity loss did not appear to be due to SEAP degradation (based on Western blots) but due to SEAP denaturation. The protein-stabilizing agents polyvinylpirrolidone (PVP) and bacitracin were added extracellularly to test for their ability to reduce the loss of SEAP activity during the stationary phase. Bacitracin (100 mg/L) was the most effective treatment at sustaining activity levels for up to 17 days post-subculture. Commercially available human placental alkaline phosphatase (PLAP) was used to probe the mechanism of SEAP deactivation. Experiments with PLAP in sterile and conditioned medium corroborated the denaturation of SEAP by factors generated by cell growth and not due to simple proteolysis. We also show for the first time that the factors promoting activity loss are heat labile at 95 degrees C but not at 70 degrees C, and they are not inactivated after a 5 day incubation period under normal culture conditions (27 degrees C). In addition, there were no significant changes in pH or redox potential when comparing sterile and cell-free conditioned medium during PLAP incubation, indicating that these factors were unimportant.

  6. Radiation sterilization: an industrial process

    International Nuclear Information System (INIS)

    Ley, F.J.

    1975-01-01

    A new dimension has been added to the use of radiation in the medical field with the introduction of radiation as a sterilizing agent. Its use in diagnosis through radioactive tracers or X-rays and in therapy administered with the most sophisticated of electrical machines or radioisotope units, is familiar in the hospital world, being well established therein. In contrast, the application of radiation sterilization is in industry where the installation of large radiation sources is already commonplace in many countries. The beginnings in the early 1950's centered on the Van de Graaff machine and linear accelerators and the pioneering efforts of Ethicon Inc. here in the United States must be recognized. However, although sterilization with electron beams is still current practice in a number of plants, the use of gamma rays from cobalt-60 is preferred. The first steps in this direction were taken by the U.K.A.E.A. which, in common with similar organizations elsewhere, was attempting to exploit the tremendous potential for cobalt-60 production arising through the rapid construction of nuclear reactors. The first full-scale commercial gamma plant was commissioned in the U.K. in 1960. It reached a loading of 500,000 curies before its demolition after twelve years of operation. The process gained rapid acceptance within industry and approval by health authorities because it provided a ''cold'' sterilization method combining the property of lethal effect with penetration. Its immediate impact occurred in the introduction of disposable products making it possible, for example, to use heat-labile plastics and new packaging materials and package designs. Certainly, the technique has proved complementary to sterilization methods based on heat and to the use of chemical agents, in particular ethylene oxide gas

  7. Study the possibility of use of extracts of the mushroom Boletus edulis in the production of functional dairy products

    Directory of Open Access Journals (Sweden)

    L. I. Aleksandrova

    2016-01-01

    Full Text Available Fruiting bodies of mushrooms are rich in proteins. The most rich in proteins is Boletus edulis (white mushroom, and total protein contains all the essential amino acids. The low bioavailability of proteins Boletus edulis stimulates the search for ways to improve. One way to improve the bioavailability of nutrients of Boletus edulis is an extraction. This article presents the results of studies of extracts of white mushroom (Boletus edulis, obtained using different methods of processing the chopped dry fruiting body. The effect of physical and biotechnological parameters on the efficiency of the extraction of proteins was studied. The spectral characteristics and protein content of the obtained extracts were determined. It is shown that the ultrasonic extraction activation leads to the most efficient protein extraction(an increase of 9.5%. It is also significantly affected by the duration of extraction. The use of the enzyme preparation in the investigated ratios do not have a positive impact, however, enzymatic treatment in conjunction with ultrasound treatment increased the efficiency of 13.5%, showing the complex diffusion processes in the fruiting bodies of mushrooms. Increasing the length of the extraction, despite the high content of protein in the extract, is impractical because of the risk of bacterial contamination of high-nutritive extract. Increasing the temperature of the process has no significant effect on the protein content, but it reduces the value of the extract, due to the destruction of heat-labile components, which include vitamins and secondary metabolites. The extract application rate for curd product was determined. For the test samples organoleptic characteristics and titratable acidity were determined. The most appropriate dose of the extract in the manufacture of making curd product is 15% by weight of the finished product.

  8. Cataracts induced by microwave and ionizing radiation

    International Nuclear Information System (INIS)

    Lipman, R.M.; Tripathi, B.J.; Tripathi, R.C.

    1988-01-01

    Microwaves most commonly cause anterior and/or posterior subcapsular lenticular opacities in experimental animals and, as shown in epidemiologic studies and case reports, in human subjects. The formation of cataracts seems to be related directly to the power of the microwave and the duration of exposure. The mechanism of cataractogenesis includes deformation of heat-labile enzymes, such as glutathione peroxide, that ordinarily protect lens cell proteins and membrane lipids from oxidative damage. Oxidation of protein sulfhydryl groups and the formation of high-molecular-weight aggregates cause local variations in the orderly structure of the lens cells. An alternative mechanism is thermoelastic expansion through which pressure waves in the aqueous humor cause direct physical damage to the lens cells. Cataracts induced by ionizing radiation (e.g., X-rays and gamma rays) usually are observed in the posterior region of the lens, often in the form of a posterior subcapsular cataract. Increasing the dose of ionizing radiation causes increasing opacification of the lens, which appears after a decreasing latency period. Like cataract formation by microwaves, cataractogenesis induced by ionizing radiation is associated with damage to the lens cell membrane. Another possible mechanism is damage to lens cell DNA, with decreases in the production of protective enzymes and in sulfur-sulfur bond formation, and with altered protein concentrations. Until further definitive conclusions about the mechanisms of microwaves and ionizing radiation induced cataracts are reached, and alternative protective measures are found, one can only recommend mechanical shielding from these radiations to minimize the possibility of development of radiation-induced cataracts. 74 references

  9. High-affinity binding of [3H]estradiol-17 beta by an estrogen receptor in the liver of the turtle

    International Nuclear Information System (INIS)

    Ho, S.M.; Fehrer, S.; Yu, M.; Liang, L.C.; Press, D.

    1988-01-01

    Specific [3H]estradiol-17 beta ([3H]E2) binding activity (EBA) with characteristics of an estrogen receptor (ER) was demonstrated in cytosols and nuclear extracts of the female turtle, Chrysemys picta. Three different receptor assays (dextran-coated charcoal assay, hydroxylapatite batch procedure, and DNA-cellulose chromatography) were evaluated in terms of their applicability in analyzing large numbers of samples. For the measurement of cytosolic EBA, the hydroxylapatite batch procedure was found to be the most reliable assay. On the other hand, the dextran-coated charcoal assay was found to be the most appropriate method for the measurement of nuclear EBA. Turtle hepatic EBA binds [3H]E2 with high affinity (cytosolic, 17.4 +/- 2.8 X 10(9) M-1; nuclear, 17.7 +/- 1.9 X 10(9) M-1), limited capacity (cytosolic, 133.7 +/- 4.6 fmol/g tissue; nuclear, 81.1 +/- 9.0 fmol/g tissue), and strict steroid specificity. The EBA bound natural estrogens (E2, estrone, estriol) as well as the nonsteroidal estrogen, diethylstilbestrol, but exhibited little affinity for androgens, progesterone, or corticosterone. The turtle hepatic EBA resembled mammalian and avian ERs in terms of binding characteristics; however, unlike mammalian and avian ERs it was shown to be heat-labile. Incubation at 30 degrees caused rapid loss of [3H]E2 binding activity in both cytosolic and nuclear fractions. The exchange between [3H]E2 and the endogenously bound estrogen was slow at 4 and 15 degrees, but the exchange process was facilitated in the presence of the chaotropic salt, NaSCN. Establishment of quantitation methods for both cytosolic and nuclear forms of EBA will enable future investigation of the mechanism and regulation of estrogen action in the liver of this turtle species

  10. The excretory-secretory products of Echinococcus granulosus protoscoleces directly regulate the differentiation of B10, B17 and Th17 cells.

    Science.gov (United States)

    Pan, Wei; Hao, Wen-Ting; Shen, Yu-Juan; Li, Xiang-Yang; Wang, Yan-Juan; Sun, Fen-Fen; Yin, Jian-Hai; Zhang, Jing; Tang, Ren-Xian; Cao, Jian-Ping; Zheng, Kui-Yang

    2017-07-21

    Excretory-secretory products (ESPs) released by helminths are well-known to regulate T cell responses in the host. However, their direct influence in the differentiation of naïve T cells, and especially B cells, remains largely unknown. This study investigated the effects of Echinococcus granulosus protoscoleces ESPs (EgPSC-ESPs) on the differentiation of IL-10-producing B cells (B10), IL-17A-producing B cells (B17) and Th17 cells. BALB/c mice injected with EgPSC were used to evaluate the in vivo profiles of B10, B17 and Th17 cells. In vitro purified CD19 + B and naïve CD4 + T cells were cultured in the presence of native, heat-inactivated or periodate-treated EgPSC-ESPs, and the differentiation of these cell subsets were compared. In contrast to the control group, infected mice showed higher frequencies of B10, B17 and Th17 cells, and higher levels of IL-10 and IL-17A in the sera. Interestingly, B17 cells were first identified to express CD19 + CD1d high . In vitro, B cells cultured with native ESPs exhibited a higher percentage of B10 cells but lower percentage of B17 and Th17 cells compared to the PBS group. Moreover, the relative expression of IL-10 and IL-17A mRNA were consistent with the altered frequencies. However, ESPs subjected to heat-inactivation or periodate treatment exhibited an inverse effect on the induction of these cell subsets. Our findings indicate that ESPs released by EgPSC can directly regulate the differentiation of B10, B17 and Th17 cells, which appear to be heat-labile and carbohydrate-dependent.

  11. Analysis of transgenic wheat (Triticum aestivum L.) harboring a maize (Zea mays L.) gene for plastid EF-Tu: segregation pattern, expression and effects of the transgene.

    Science.gov (United States)

    Fu, Jianming; Ristic, Zoran

    2010-06-01

    We previously reported that transgenic wheat (Triticum aestivum L.) carrying a maize (Zea mays L.) gene (Zmeftu1) for chloroplast protein synthesis elongation factor, EF-Tu, displays reduced thermal aggregation of leaf proteins, reduced injury to photosynthetic membranes (thylakoids), and enhanced rate of CO(2) fixation following exposure to heat stress (18 h at 45 degrees C) [Fu et al. in Plant Mol Biol 68:277-288, 2008]. In the current study, we investigated the segregation pattern and expression of the transgene Zmeftu1 and determined the grain yield of transgenic plants after exposure to a brief heat stress (18 h at 45 degrees C). We also assessed thermal aggregation of soluble leaf proteins in transgenic plants, testing the hypothesis that increased levels of EF-Tu will lead to a non-specific protection of leaf proteins against thermal aggregation. The transgenic wheat displayed a single-gene pattern of segregation of Zmeftu1. Zmeftu1 was expressed, and the transgenic plants synthesized and accumulated three anti-EF-Tu cross-reacting polypeptides of similar molecular mass but different pI, suggesting the possibility of posttranslational modification of this protein. The transgenic plants also showed better grain yield after exposure to heat stress compared with their non-transgenic counterparts. Soluble leaf proteins of various molecular masses displayed lower thermal aggregation in transgenic than in non-transgenic wheat. The results suggest that overexpression of chloroplast EF-Tu can be beneficial to wheat tolerance to heat stress. Moreover, the results also support the hypothesis that EF-Tu contributes to heat tolerance by acting as a molecular chaperone and protecting heat-labile proteins from thermal aggregation in a non-specific manner.

  12. Differing mechanisms of simple nitrile formation on glucosinolate degradation in Lepidium sativum and Nasturtium officinale seeds.

    Science.gov (United States)

    Williams, David J; Critchley, Christa; Pun, Sharon; Chaliha, Mridusmita; O'Hare, Timothy J

    2009-01-01

    Glucosinolates are sulphur-containing glycosides found in brassicaceous plants that can be hydrolysed enzymatically by plant myrosinase or non-enzymatically to form primarily isothiocyanates and/or simple nitriles. From a human health perspective, isothiocyanates are quite important because they are major inducers of carcinogen-detoxifying enzymes. Two of the most potent inducers are benzyl isothiocyanate (BITC) present in garden cress (Lepidium sativum), and phenylethyl isothiocyanate (PEITC) present in watercress (Nasturtium officinale). Previous studies on these salad crops have indicated that significant amounts of simple nitriles are produced at the expense of the isothiocyanates. These studies also suggested that nitrile formation may occur by different pathways: (1) under the control of specifier protein in garden cress and (2) by an unspecified, non-enzymatic path in watercress. In an effort to understand more about the mechanisms involved in simple nitrile formation in these species, we analysed their seeds for specifier protein and myrosinase activities, endogenous iron content and glucosinolate degradation products after addition of different iron species, specific chelators and various heat treatments. We confirmed that simple nitrile formation was predominantly under specifier protein control (thiocyanate-forming protein) in garden cress seeds. Limited thermal degradation of the major glucosinolate, glucotropaeolin (benzyl glucosinolate), occurred when seed material was heated to >120 degrees C. In the watercress seeds, however, we show for the first time that gluconasturtiin (phenylethyl glucosinolate) undergoes a non-enzymatic, iron-dependent degradation to a simple nitrile. On heating the seeds to 120 degrees C or greater, thermal degradation of this heat-labile glucosinolate increased simple nitrile levels many fold.

  13. Cell-to-cell transformation in Escherichia coli: a novel type of natural transformation involving cell-derived DNA and a putative promoting pheromone.

    Directory of Open Access Journals (Sweden)

    Rika Etchuuya

    Full Text Available Escherichia coli is not assumed to be naturally transformable. However, several recent reports have shown that E. coli can express modest genetic competence in certain conditions that may arise in its environment. We have shown previously that spontaneous lateral transfer of non-conjugative plasmids occurs in a colony biofilm of mixed E. coli strains (a set of a donor strain harbouring a plasmid and a plasmid-free recipient strain. In this study, with high-frequency combinations of strains and a plasmid, we constructed the same lateral plasmid transfer system in liquid culture. Using this system, we demonstrated that this lateral plasmid transfer was DNase-sensitive, indicating that it is a kind of transformation in which DNase-accessible extracellular naked DNA is essential. However, this transformation did not occur with purified plasmid DNA and required a direct supply of plasmid from co-existing donor cells. Based on this feature, we have termed this transformation type as 'cell-to-cell transformation'. Analyses using medium conditioned with the high-frequency strain revealed that this strain released a certain factor(s that promoted cell-to-cell transformation and arrested growth of the other strains. This factor is heat-labile and protease-sensitive, and its roughly estimated molecular mass was between ∼9 kDa and ∼30 kDa, indicating that it is a polypeptide factor. Interestingly, this factor was effective even when the conditioned medium was diluted 10(-5-10(-6, suggesting that it acts like a pheromone with high bioactivity. Based on these results, we propose that cell-to-cell transformation is a novel natural transformation mechanism in E. coli that requires cell-derived DNA and is promoted by a peptide pheromone. This is the first evidence that suggests the existence of a peptide pheromone-regulated transformation mechanism in E. coli and in Gram-negative bacteria.

  14. Sublingual immunization with the phosphate-binding-protein (PstS) reduces oral colonization by Streptococcus mutans.

    Science.gov (United States)

    Ferreira, E L; Batista, M T; Cavalcante, R C M; Pegos, V R; Passos, H M; Silva, D A; Balan, A; Ferreira, L C S; Ferreira, R C C

    2016-10-01

    Bacterial ATP-binding cassette (ABC) transporters play a crucial role in the physiology and pathogenicity of different bacterial species. Components of ABC transporters have also been tested as target antigens for the development of vaccines against different bacterial species, such as those belonging to the Streptococcus genus. Streptococcus mutans is the etiological agent of dental caries, and previous studies have demonstrated that deletion of the gene encoding PstS, the substrate-binding component of the phosphate uptake system (Pst), reduced the adherence of the bacteria to abiotic surfaces. In the current study, we generated a recombinant form of the S. mutans PstS protein (rPstS) with preserved structural features, and we evaluated the induction of antibody responses in mice after sublingual mucosal immunization with a formulation containing the recombinant protein and an adjuvant derived from the heat-labile toxin from enterotoxigenic Escherichia coli strains. Mice immunized with rPstS exhibited systemic and secreted antibody responses, measured by the number of immunoglobulin A-secreting cells in draining lymph nodes. Serum antibodies raised in mice immunized with rPstS interfered with the adhesion of bacteria to the oral cavity of naive mice challenged with S. mutans. Similarly, mice actively immunized with rPstS were partially protected from oral colonization after challenge with the S. mutans NG8 strain. Therefore, our results indicate that S. mutans PstS is a potential target antigen capable of inducing specific and protective antibody responses after sublingual administration. Overall, these observations raise interesting perspectives for the development of vaccines to prevent dental caries. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. Evaluation of immunogenicity and protective efficacy of orally delivered Shigella type III secretion system proteins IpaB and IpaD.

    Science.gov (United States)

    Heine, Shannon J; Diaz-McNair, Jovita; Martinez-Becerra, Francisco J; Choudhari, Shyamal P; Clements, John D; Picking, Wendy L; Pasetti, Marcela F

    2013-06-19

    Shigella spp. are food- and water-borne pathogens that cause shigellosis, a severe diarrheal and dysenteric disease that is associated with a high morbidity and mortality in resource-poor countries. No licensed vaccine is available to prevent shigellosis. We have recently demonstrated that Shigella invasion plasmid antigens (Ipas), IpaB and IpaD, which are components of the bacterial type III secretion system (TTSS), can prevent infection in a mouse model of intranasal immunization and lethal pulmonary challenge. Because they are conserved across Shigella spp. and highly immunogenic, these proteins are excellent candidates for a cross-protective vaccine. Ideally, such a vaccine could be administered to humans orally to induce mucosal and systemic immunity. In this study, we investigated the immunogenicity and protective efficacy of Shigella IpaB and IpaD administered orally with a double mutant of the Escherichia coli heat labile toxin (dmLT) as a mucosal adjuvant. We characterized the immune responses induced by oral vs. intranasal immunization and the protective efficacy using a mouse pulmonary infection model. Serum IgG and fecal IgA against IpaB were induced after oral immunization. These responses, however, were lower than those obtained after intranasal immunization despite a 100-fold dosage increase. The level of protection induced by oral immunization with IpaB and IpaD was 40%, while intranasal immunization resulted in 90% protective efficacy. IpaB- and IpaD-specific IgA antibody-secreting cells in the lungs and spleen and T-cell-derived IL-2, IL-5, IL-17 and IL-10 were associated with protection. These results demonstrate the immunogenicity of orally administered IpaB and IpaD and support further studies in humans. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Development and preclinical evaluation of safety and immunogenicity of an oral ETEC vaccine containing inactivated E. coli bacteria overexpressing colonization factors CFA/I, CS3, CS5 and CS6 combined with a hybrid LT/CT B subunit antigen, administered alone and together with dmLT adjuvant.

    Science.gov (United States)

    Holmgren, J; Bourgeois, L; Carlin, N; Clements, J; Gustafsson, B; Lundgren, A; Nygren, E; Tobias, J; Walker, R; Svennerholm, A-M

    2013-05-07

    A first-generation oral inactivated whole-cell enterotoxigenic Escherichia coli (ETEC) vaccine, comprising formalin-killed ETEC bacteria expressing different colonization factor (CF) antigens combined with cholera toxin B subunit (CTB), when tested in phase III studies did not significantly reduce overall (generally mild) ETEC diarrhea in travelers or children although it reduced more severe ETEC diarrhea in travelers by almost 80%. We have now developed a novel more immunogenic ETEC vaccine based on recombinant non-toxigenic E. coli strains engineered to express increased amounts of CF antigens, including CS6 as well as an ETEC-based B subunit protein (LCTBA), and the optional combination with a nontoxic double-mutant heat-labile toxin (LT) molecule (dmLT) as an adjuvant. Two test vaccines were prepared under GMP: (1) A prototype E. coli CFA/I-only formalin-killed whole-cell+LCTBA vaccine, and (2) A "complete" inactivated multivalent ETEC-CF (CFA/I, CS3, CS5 and CS6 antigens) whole-cell+LCTBA vaccine. These vaccines, when given intragastrically alone or together with dmLT in mice, were well tolerated and induced strong intestinal-mucosal IgA antibody responses as well as serum IgG and IgA responses to each of the vaccine CF antigens as well as to LT B subunit (LTB). Both mucosal and serum responses were further enhanced (adjuvanted) when the vaccines were co-administered with dmLT. We conclude that the new multivalent oral ETEC vaccine, both alone and especially in combination with the dmLT adjuvant, shows great promise for further testing in humans. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Human platelet lysate supplementation of mesenchymal stromal cell delivery: issues of xenogenicity and species variability.

    Science.gov (United States)

    Allen, Ashley B; Butts, Emily B; Copland, Ian B; Stevens, Hazel Y; Guldberg, Robert E

    2017-10-01

    Immunogenicity of fetal bovine serum (FBS) poses a problem for its use in the propagation of autologous mesenchymal stromal cells (MSCs) for cell therapy. Human platelet lysate (hPL), an enriched growth factor solution containing mitogenic and angiogenic cues, has potential utility in replacing FBS for human MSC (hMSC) delivery strategies. Despite its potentiation of hMSC number in vitro, little is known concerning its capacity to supplement implanted hMSC-seeded constructs and promote tissue regeneration in vivo. In this study, we tested the effects of incorporating hPL in cell-seeded constructs implanted subcutaneously into immunocompromised rats, investigated in vitro interactions between hPL and rat MSCs (rMSCs) and determined interspecies variability in the PL product [hPL vs rat PL (rPL)] and its effect on cultured MSCs (hPL/hMSCs vs rPL/rMSCs). The overarching aim was to determine the utility of hPL to foster MSC survival in preclinical rodent models. Exposure to hPL-supplemented media resulted in rMSC death, by a process attributable to heat-labile proteins, but not membrane attack complex formation. In the in vitro syngeneic model, the rodent product proved fundamentally distinct from the human product, with rPL having substantially lower growth factor content than hPL. Moreover, contrary to the positive effects of hPL on hMSC expansion, rPL did not reduce rMSC doubling time for the serum concentrations examined. When tested in vivo, hPL did not improve cell survival within hydrogel constructs through 2 weeks postimplantation. In summary, this study highlights the many facets of xenogenicity and interspecies variability that must be considered in the preclinical evaluation of hPL. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  18. Characterization of Ice Nucleating Particles at the Western US Coast

    Science.gov (United States)

    Rocci, K.; McCluskey, C. S.; Hill, T. C. J.; DeMott, P. J.; Kreidenweis, S. M.

    2015-12-01

    In temperate climates, ice nucleating particles (INPs) are vital for precipitation initiation. Because INPs may affect precipitation efficiency, and thereby the supply of water resources, it is paramount to have a clear understanding of both natural and anthropogenic sources of INPs. This is especially important to understand in California where drought continues to be a major problem. The CalWater 2015 field campaign, which took place in California from January 15 - March 9, 2015, included comprehensive characterizations of aerosols and their ice nucleating ability via ground-, air-, and ship-based measurements. As part of this campaign, we characterized and analyzed the intra-air mass differences of INPs at a coastal site (Bodega Bay) using immersion freezing measurements of particles collected on filters. Aerosol filters collected throughout the campaign were characterized by their loading and dominant type using meteorology, aerosol size distributions, aerosol composition, and trace gas concentration data. Samples contained a variety of aerosol influences, including biomass burning, nitrogen pollution, sulfur pollution, and sea spray. This study had a particular focus on the INP activity spectra of sea spray aerosol (SSA). We used the online aerosol data to infer variations in SSA types and heat-treated specific samples to look for the presence of heat-labile biological INPs. Furthermore, we ran the NOAA HYSPLIT model to obtain back trajectories for samples dominated by SSA. We found that air masses dominated by distinct terrestrial source types are not well distinguished by their INP number concentrations. However, we did see significantly higher (up to 5000-fold) INP number concentrations in SSA samples taken at the coast compared with number concentrations in samples obtained over open ocean. This difference could be attributable to differences in overall aerosol abundance, which will be evaluated in future studies. Overall, our findings suggest that an

  19. Involvement of DNA-PK and ATM in radiation- and heat-induced DNA damage recognition and apoptotic cell death

    International Nuclear Information System (INIS)

    Tomita, Masanori

    2010-01-01

    Exposure to ionizing radiation and hyperthermia results in important biological consequences, e.g. cell death, chromosomal aberrations, mutations, and DNA strand breaks. There is good evidence that the nucleus, specifically cellular DNA, is the principal target for radiation-induced cell lethality. DNA double-strand breaks (DSBs) are considered to be the most serious type of DNA damage induced by ionizing radiation. On the other hand, verifiable mechanisms which can lead to heat-induced cell death are damage to the plasma membrane and/or inactivation of heat-labile proteins caused by protein denaturation and subsequent aggregation. Recently, several reports have suggested that DSBs can be induced after hyperthermia because heat-induced phosphorylated histone H2AX (γ-H2AX) foci formation can be observed in several mammalian cell lines. In mammalian cells, DSBs are repaired primarily through two distinct and complementary mechanisms: non-homologous end joining (NHEJ), and homologous recombination (HR) or homology-directed repair (HDR). DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) are key players in the initiation of DSB repair and phosphorylate and/or activate many substrates, including themselves. These phosphorylated substrates have important roles in the functioning of cell cycle checkpoints and in cell death, as well as in DSB repair. Apoptotic cell death is a crucial cell suicide mechanism during development and in the defense of homeostasis. If DSBs are unrepaired or misrepaired, apoptosis is a very important system which can protect an organism against carcinogenesis. This paper reviews recently obtained results and current topics concerning the role of DNA-PK and ATM in heat- or radiation-induced apoptotic cell death. (author)

  20. Mycolytic enzymes produced by Streptomyces violaceusniger and their role in antagonism towards wood-rotting fungi.

    Science.gov (United States)

    Nagpure, Anand; Choudhary, Bharti; Gupta, Rajinder K

    2014-05-01

    Extracellular mycolytic enzymes produced under submerged fermentation by the fungal antagonist Streptomyces violaceusniger MTCC 3959 were characterized. This streptomycete produced higher amounts of extracellular chitinase and protease during late exponential phase, whereas β-1,3-glucanase production was at peak in mid-stationary phase. Cell-free culture filtrate (CCF) exhibited a broad range of antifungal activity against both white rot and brown rot fungi. The inhibitory activity was completely lost after treatment with proteinase K and heat, indicating that extracellular antifungal metabolites are heat labile and proteinaceous in nature. Optimum pH and temperature for enzyme activity were: 9.0 and 60 °C for chitinase; 6.0 and 60 °C for β-1,3-glucanase; and 9.0 and 70 °C for protease. Mycolytic enzymes were moderately thermostable, and had a wide pH stability range extending from pH 5.0 to 10.0. The zymogram analysis of CCF revealed five chitinase isoenzymes with an apparent molecular weight of 20.8, 33.3, 45.6, 67.4, and 114.8 kDa, one β-1,3-glucanase appeared as a single band of ∼131.8 kDa and four protease isoenzymes with approximate molecular weights of 22.8, 62.52, 74.64, and 120.5 kDa. S. violaceusniger MTCC 3959 produced mycolytic enzymes that can be effectively used for suppression of phytopathogenic basidiomycetes. It has the potential to be an effective biofungicide. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Microbiological quality of milk in Tanzania: from Maasai stable to African consumer table.

    Science.gov (United States)

    Schoder, Dagmar; Maichin, Andreas; Lema, Benedict; Laffa, John

    2013-11-01

    In Tanzania, pastoralists such as the Maasai and small urban farmers are responsible for the country's milk production, and 95% of the national milk supply is sold without regulation. This study was conducted using hygiene checklists and milk sampling to investigate milk quality and safety at various steps throughout the milk production chain. In regions of Dar es Salaam and Lake Victoria, 196 milk samples were collected: 109 samples of raw milk, 41 samples of packed or open served heat-treated products, and 46 samples of fermented products. Samples were taken from (i) the production level (pastoralists and urban farmers), (ii) the collection level (middlemen and depots), (iii) processors (dairies), and (iv) retailers (kiosks). Samples were analyzed for hygiene criteria (total bacteria, total coliforms, Escherichia coli, and coagulase-positive staphylococci) and foodborne pathogens such as Salmonella, enterohemorrhagic E. coli O157:H7, and Listeria monocytogenes. Adequate heating of milk for drinking was determined via heat labile alkaline phosphatase and lactoperoxidase analysis. Total bacterial counts indicated that only 67% (73 of 109) of raw milk samples and 46% (19 of 41) of heat-treated samples met national Tanzanian standards. Bulk milk samples taken from the traditional milking vessels of Maasai pastoralists had the lowest total bacterial counts: ≥ 1 × 10(2) CFU/ml. Foodborne pathogens such as E. coli O157:H7 and Salmonella were isolated from 10.1% (11 of 109) of raw milk samples but were not detected in heat-treated or fermented products, and 83% of heat-treated milk samples were lactoperoxidase negative, indicating overpasteurization. Coliforms were detected in 41% (17 of 41) of processed milk samples, thus indicating a high rate of recontamination. A progressive decrease in microbial quality along the milk production chain was attributed to departures from traditional methods, inadequate milk containers, long transport distances, lack of cooling, and

  2. Pseudomonas fluorescens strain CL145A - a biopesticide for the control of zebra and quagga mussels (Bivalvia: Dreissenidae).

    Science.gov (United States)

    Molloy, Daniel P; Mayer, Denise A; Gaylo, Michael J; Morse, John T; Presti, Kathleen T; Sawyko, Paul M; Karatayev, Alexander Y; Burlakova, Lyubov E; Laruelle, Franck; Nishikawa, Kimi C; Griffin, Barbara H

    2013-05-01

    Zebra mussels (Dreissena polymorpha) and quagga mussels (Dreissena rostriformis bugensis) are the "poster children" of high-impact aquatic invasive species. In an effort to develop an effective and environmentally acceptable method to control their fouling of raw-water conduits, we have investigated the potential use of bacteria and their natural metabolic products as selective biological control agents. An outcome of this effort was the discovery of Pseudomonas fluorescens strain CL145A - an environmental isolate that kills these dreissenid mussels by intoxication (i.e., not infection). In the present paper, we use molecular methods to reconfirm that CL145A is a strain of the species P. fluorescens, and provide a phylogenetic analysis of the strain in relation to other Pseudomonas spp. We also provide evidence that the natural product lethal to dreissenids is associated with the cell wall of P. fluorescens CL145A, is a heat-labile secondary metabolite, and has degradable toxicity within 24 h when applied to water. CL145A appears to be an unusual strain of P. fluorescens since it was the only one among the ten strains tested to cause high mussel mortality. Pipe trials conducted under once-through conditions indicated: (1) P. fluorescens CL145A cells were efficacious against both zebra and quagga mussels, with high mortalities achieved against both species, and (2) as long as the total quantity of bacterial cells applied during the entire treatment period was the same, similar mussel mortality could be achieved in treatments lasting 1.5-12.0 h, with longer treatment durations achieving lower mortalities. The efficacy data presented herein, in combination with prior demonstration of its low risk of non-target impact, indicate that P. fluorescens CL145A cells have significant promise as an effective and environmentally safe control agent against these invasive mussels. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Adjuvant-Mediated Epitope Specificity and Enhanced Neutralizing Activity of Antibodies Targeting Dengue Virus Envelope Protein

    Directory of Open Access Journals (Sweden)

    Denicar Lina Nascimento Fabris Maeda

    2017-09-01

    Full Text Available The heat-labile toxins (LT produced by enterotoxigenic Escherichia coli display adjuvant effects to coadministered antigens, leading to enhanced production of serum antibodies. Despite extensive knowledge of the adjuvant properties of LT derivatives, including in vitro-generated non-toxic mutant forms, little is known about the capacity of these adjuvants to modulate the epitope specificity of antibodies directed against antigens. This study characterizes the role of LT and its non-toxic B subunit (LTB in the modulation of antibody responses to a coadministered antigen, the dengue virus (DENV envelope glycoprotein domain III (EDIII, which binds to surface receptors and mediates virus entry into host cells. In contrast to non-adjuvanted or alum-adjuvanted formulations, antibodies induced in mice immunized with LT or LTB showed enhanced virus-neutralization effects that were not ascribed to a subclass shift or antigen affinity. Nonetheless, immunosignature analyses revealed that purified LT-adjuvanted EDIII-specific antibodies display distinct epitope-binding patterns with regard to antibodies raised in mice immunized with EDIII or the alum-adjuvanted vaccine. Notably, the analyses led to the identification of a specific EDIII epitope located in the EF to FG loop, which is involved in the entry of DENV into eukaryotic cells. The present results demonstrate that LT and LTB modulate the epitope specificity of antibodies generated after immunization with coadministered antigens that, in the case of EDIII, was associated with the induction of neutralizing antibody responses. These results open perspectives for the more rational development of vaccines with enhanced protective effects against DENV infections.

  4. Distinct pathogenesis and host responses during infection of C. elegans by P. aeruginosa and S. aureus.

    Science.gov (United States)

    Irazoqui, Javier E; Troemel, Emily R; Feinbaum, Rhonda L; Luhachack, Lyly G; Cezairliyan, Brent O; Ausubel, Frederick M

    2010-07-01

    The genetically tractable model host Caenorhabditis elegans provides a valuable tool to dissect host-microbe interactions in vivo. Pseudomonas aeruginosa and Staphylococcus aureus utilize virulence factors involved in human disease to infect and kill C. elegans. Despite much progress, virtually nothing is known regarding the cytopathology of infection and the proximate causes of nematode death. Using light and electron microscopy, we found that P. aeruginosa infection entails intestinal distention, accumulation of an unidentified extracellular matrix and P. aeruginosa-synthesized outer membrane vesicles in the gut lumen and on the apical surface of intestinal cells, the appearance of abnormal autophagosomes inside intestinal cells, and P. aeruginosa intracellular invasion of C. elegans. Importantly, heat-killed P. aeruginosa fails to elicit a significant host response, suggesting that the C. elegans response to P. aeruginosa is activated either by heat-labile signals or pathogen-induced damage. In contrast, S. aureus infection causes enterocyte effacement, intestinal epithelium destruction, and complete degradation of internal organs. S. aureus activates a strong transcriptional response in C. elegans intestinal epithelial cells, which aids host survival during infection and shares elements with human innate responses. The C. elegans genes induced in response to S. aureus are mostly distinct from those induced by P. aeruginosa. In contrast to P. aeruginosa, heat-killed S. aureus activates a similar response as live S. aureus, which appears to be independent of the single C. elegans Toll-Like Receptor (TLR) protein. These data suggest that the host response to S. aureus is possibly mediated by pathogen-associated molecular patterns (PAMPs). Because our data suggest that neither the P. aeruginosa nor the S. aureus-triggered response requires canonical TLR signaling, they imply the existence of unidentified mechanisms for pathogen detection in C. elegans, with

  5. Distinct pathogenesis and host responses during infection of C. elegans by P. aeruginosa and S. aureus.

    Directory of Open Access Journals (Sweden)

    Javier E Irazoqui

    2010-07-01

    Full Text Available The genetically tractable model host Caenorhabditis elegans provides a valuable tool to dissect host-microbe interactions in vivo. Pseudomonas aeruginosa and Staphylococcus aureus utilize virulence factors involved in human disease to infect and kill C. elegans. Despite much progress, virtually nothing is known regarding the cytopathology of infection and the proximate causes of nematode death. Using light and electron microscopy, we found that P. aeruginosa infection entails intestinal distention, accumulation of an unidentified extracellular matrix and P. aeruginosa-synthesized outer membrane vesicles in the gut lumen and on the apical surface of intestinal cells, the appearance of abnormal autophagosomes inside intestinal cells, and P. aeruginosa intracellular invasion of C. elegans. Importantly, heat-killed P. aeruginosa fails to elicit a significant host response, suggesting that the C. elegans response to P. aeruginosa is activated either by heat-labile signals or pathogen-induced damage. In contrast, S. aureus infection causes enterocyte effacement, intestinal epithelium destruction, and complete degradation of internal organs. S. aureus activates a strong transcriptional response in C. elegans intestinal epithelial cells, which aids host survival during infection and shares elements with human innate responses. The C. elegans genes induced in response to S. aureus are mostly distinct from those induced by P. aeruginosa. In contrast to P. aeruginosa, heat-killed S. aureus activates a similar response as live S. aureus, which appears to be independent of the single C. elegans Toll-Like Receptor (TLR protein. These data suggest that the host response to S. aureus is possibly mediated by pathogen-associated molecular patterns (PAMPs. Because our data suggest that neither the P. aeruginosa nor the S. aureus-triggered response requires canonical TLR signaling, they imply the existence of unidentified mechanisms for pathogen detection in C

  6. SANITARY EVALUATION OF MILK PRODUCTS IN MOUNTAIN DAIRIES

    Directory of Open Access Journals (Sweden)

    R. Mioni

    2010-03-01

    Full Text Available between 2006 and 2007 881 samples of “malga” (little mountain dairies milk products were analysed to estimate their hygienic characteristics. Several samples showed high counts for Escherichia coli and coagulase-positive staphylococci, while Salmonella spp. and Campylobacter spp. were absent in all of the samples; 0,9% of cheese samples, 4,1% of butter samples and 4,7% of “ricotta” samples were positive for Listeria monocytogenes, so as 14,7% of cheese samples for staphylococcal enterotoxins.

  7. [Carriage of Staphylococcus aureus among food service workers].

    Science.gov (United States)

    Alarcón-Lavín, María Paula; Oyarzo, Carolina; Escudero, Carlos; Cerda-Leal, Fabiola; Valenzuela, Francisco J

    2017-12-01

    Background Staphylococcus aureus produces 11 serotypes of endotoxins that may cause food poisoning. Aim To determine the prevalence of type A enterotoxigenic Staphylococcus aureus carriage among food service workers in Chillan, Chile. Material and Methods Pharyngeal swabs were obtained from 100 food service workers and were cultured in Agar plates. After identifying the presence of Staphylococcus aureus, DNA was extracted to identify type A toxin by conventional PCR. Results Thirty eight percent of samples were colonized with Staphylococcus aureus. Among these, 26% were toxin A producers. Conclusions Half of the sampled workers carried Staphylococcus aureus and a quarter of these produced type A enterotoxin.

  8. Staphylococcus aureus in Some Brazilian Dairy Industries: Changes of Contamination and Diversity

    DEFF Research Database (Denmark)

    Dittmann, Karen Kiesbye; Chaul, Luiza T.; Lee, Sarah H. I.

    2017-01-01

    Staphylococcus aureus, a major food-poisoning pathogen, is a common contaminant in dairy industries worldwide, including in Brazil. We determined the occurrence of S. aureus in five dairies in Brazil over 8 months. Of 421 samples, 31 (7.4%) were positive for S. aureus and prevalence varied from 0.......8% of strains being sensitive to all antibiotic classes and no Methicillin-resistant S. aureus (MRSA) strains were found. The enterotoxin-encoding genes involved in food-poisoning, e.g., sea, sed, see, and seg were targeted by PCR. The two toxin-encoding genes, sed and see, were not detected. Only three strains...... contamination....

  9. The Role of Intestinal Bacteria in Acute Diarrheal Diseases

    Science.gov (United States)

    1981-01-01

    another enteric pathogen, Vibrio cholerae , is present in high number in the cral cavity during acute and convalescent periods. Also, the buccal cells...h42 LT/ST + - - 2 K32&c4 AD) Kenya ... . .- r K32ScT All Kenya - LT - - - NT TD46ZcT ADY Mexfco 06:HT6 LT/ST - - - Z13 TD260cl AD Mexico 06:H16 LT...2 TD514c! AD Mexico .. . . NT TD472cI A Mexico LT/ST - - NT TD514cZ AD Mexico - NT "TABLE 1 CONTINUED 2 1 Enterotoxins MR-HA Pili 3 Human Strains

  10. Investigating potential exogenous tumor initiating and promoting factors for Cutaneous T-Cell Lymphomas (CTCL), a rare skin malignancy

    DEFF Research Database (Denmark)

    Litvinov, Ivan V.; Shtreis, Anna; Kobayashi, Kenneth

    2016-01-01

    -Cell lymphotropic virus type 1 (HTLV1), Epstein-Barr virus (EBV), and herpes simplex virus (HSV). In this report, we review recent evidence evaluating the involvement of these agents in cancer initiation/progression. Most importantly, recent molecular experimental evidence documented for the first time that S....... aureus can activate oncogenic STAT3 signaling in malignant T cells. Specifically, S. aureus Enterotoxin type A (SEA) was recently shown to trigger non-malignant infiltrating T cells to release IL-2 and other cytokines. These signals upon binging to their cognate receptors on malignant T cells...

  11. Effect of atopic skin stressors on natural moisturizing factors and cytokines in healthy adult epidermis

    DEFF Research Database (Denmark)

    Engebretsen, K A; Kezic, S; Jakasa, I

    2018-01-01

    : To explore the effect of selected exogenous skin stressors on NMF and skin cytokines levels in healthy adult epidermis. MATERIAL AND METHODS: 40 healthy volunteers (18-49 years) were exposed to hard, soft, and chlorinated water, 0.5% SLS, house dust mite, cat allergen, staphylococcal enterotoxin B (SEB...... of various skin cytokines in healthy individuals. Our data highlight environmental factors that might play a role in AD pathophysiology, but needs confirmation in AD patients. This article is protected by copyright. All rights reserved....

  12. The application of alkaline and acidic electrolyzed water in the sterilization of chicken breasts and beef liver

    OpenAIRE

    Shimamura, Yuko; Shinke, Momoka; Hiraishi, Miki; Tsuchiya, Yusuke; Masuda, Shuichi

    2015-01-01

    Abstract The sterilization effect of a combination treatment with alkaline electrolyzed water (AlEW) and strong acidic electrolyzed water (StAEW) on fresh chicken breasts and beef liver was evaluated. Samples (1, 5, and 10?g) were inoculated with Salmonella Enteritidis NBRC3313, Escherichia coli ATCC 10798, Staphylococcus aureus FDA209P, and S.?aureus C?29 [staphylococcal enterotoxin A (SEA) productive strain] and subjected to a dipping combination treatment (4?C and 25?C for 3?min) with AlEW...

  13. Viability of Staphylococcus xylosus isolated from artisanal sausages for application as starter cultures in meat products.

    Science.gov (United States)

    Fiorentini, Angela Maria; Sawitzki, Maristela Cortez; Bertol, Teresinha Marisa; Sant'anna, Ernani S

    2009-01-01

    Viability of Staphylococcus xylosus isolated from artisanal sausages for application as starter cultures in meat products Viability of Staphylococcus xylosus strains AD1 and U5 isolated from natural fermented sausages was investigated as starter cultures in fermented sausages produced in the South Region of Brazil. The study demonstrated that the Staphylococcus xylosus strains AD1 and U5 showed significant growth during fermentation, stability over freeze-dried process, negative reaction for staphylococcal enterotoxins and viability for using as a single-strain culture or associated with lactic acid bacteria for production of fermented sausages.

  14. Portación y caracterización de Staphylococcus aureus en manipuladores de alimentos Carriage and characterization of Staphylococcus aureus in food handlers

    Directory of Open Access Journals (Sweden)

    Graciela B Jordá

    2012-06-01

    Full Text Available Staphylococcus aureus es una causa de intoxicaciones alimentarias por su capacidad de producir enterotoxinas. Los manipuladores de alimentos que portan S. aureus productores de enterotoxinas pueden provocar intoxicaciones alimentarias. Se estudiaron muestras tomadas de fosas nasales de 88 manipuladores de alimentos en la provincia de Misiones. El 37,5 % de los individuos analizados eran portadores de S. aureus. Mediante técnicas de amplificación (PCR, se detectaron genes que codifican la producción de enterotoxinas en 13 de los 33 aislamientos obtenidos (39,4 % y en el 14,7 % de los manipuladores. De estos aislamientos, 10 portaban el gen sea y 3 el gen sec. El estudio de sensibilidad a los antibióticos mostró un 100 % de sensibilidad a teicoplanina, gentamiclna y rifampicina; 2 aislamientos fueron resistentes a clindamicina y a eritromicina y 4 resultaron resistentes a la meticilina. Estos resultados son un alerta e indicarían la necesidad de desarrollar medidas racionales para reducir el riesgo potencial de intoxicaciones alimentarias.Staphylococcus aureus causes food poisoning due to its ability to produce enterotoxins. Food handlers carrying enterotoxin-producing S. aureus can contaminate food, thus leading to food poisoning. Samples were obtained from 88 food handlers in the Province of Misiones, Argentina. S. aureus was isolated from nasal swaps and PCR amplification was performed for genes encoding staphylococcal enterotoxins. A total of 37.5 % food handlers were positive for S. aureus. Expression of enterotoxin genes was found in 13 of the 33 (39.4 % S. aureus isolates studied, accounting for 14.7 % of food handlers. Gene sea was detected in 10 isolates followed by gene sec in 3 isolates. All isolates were susceptible to teicoplanin, gentamicin and rifampicin. Four isolates were resistant to methicillin whereas 2 isolates were resistant to clindamycin and erythromycin. These results constitute a critical alert and indicate the need

  15. Prevalence, virulence factor genes and antibiotic resistance of Bacillus cereus sensu lato isolated from dairy farms and traditional dairy products

    DEFF Research Database (Denmark)

    Owusu-Kwarteng, James; Wuni, Alhassan; Akabanda, Fortune

    2017-01-01

    of B. cereus sensu lato isolated from cattle grazing soils and dairy products in Ghana. A total of 114 samples made up of 25 soil collected from cattle grazing farm land, 30 raw milk, 28 nunu (yoghurt-like product) and 31 woagashie (West African soft cheese). Ninety-six B. cereus sensu lato isolates......%), oxacillin (92%), penicillin (100%), amoxicillin (100%), and cefepime (100%) but susceptible to other antibiotics tested. Conclusions: Bacillus cereus s. l. is prevalent in soil, raw milk and dairy products in Ghana. However, loads are at levels considered to be safe for consumption. Various enterotoxin...

  16. Serologic test systems development. Progress report, July 1, 1976--September 30, 1977

    Energy Technology Data Exchange (ETDEWEB)

    Saunders, G.C.; Clinard, E.H.; Bartlett, M.L.; Petersen, P.M.; Sanders, W.M.; Payne, R.J.; Martinez, E.

    1978-01-01

    Work has continued on the development and application of the Enzyme-Labeled Antibody (ELA) test to the USDA needs. Results on trichinosis, brucellosis, and staphylococcal enterotoxin A detection are very encouraging. A field test for trichinosis detection is being worked out in cooperation with Food Safety and Quality Service personnel. Work is in progress with the Technicon Instrument Corporation to develop a modification of their equipment to automatically process samples by the ELA procedure. An automated ELA readout instrument for 96-well trays has been completed and is being checked out.

  17. Clostridium perfringens Sporulation and Sporulation-Associated Toxin Production

    Science.gov (United States)

    Li, Jihong; Paredes-Sabja, Daniel; Sarker, Mahfuzur R.; McClane, Bruce A.

    2015-01-01

    The ability of Clostridium perfringens to form spores plays a key role during the transmission of this Gram-positive bacterium to cause disease. Of particular note, the spores produced by food poisoning strains are often exceptionally resistant to food environment stresses such as heat, cold and preservatives, which likely facilitates their survival in temperature-abused foods. The exceptional resistance properties of spores made by most type A food poisoning strains and some type C foodborne disease strains involves their production of a variant small acid soluble protein-4 that binds more tightly to spore DNA compared to the small acid soluble protein-4 made by most other C. perfringens strains. Sporulation and germination by C. perfringens and Bacillus spp. share both similarities and differences. Finally, sporulation is essential for production of C. perfringens enterotoxin, which is responsible for the symptoms of C. perfringens type A food poisoning, the second most common bacterial foodborne disease in the USA. During this foodborne disease, C. perfringens is ingested with food and then, using sporulation-specific alternate sigma factors, this bacterium sporulates and produces the enterotoxin in the intestines. PMID:27337447

  18. Clostridium perfringens in London, July 2009: two weddings and an outbreak.

    Science.gov (United States)

    Eriksen, J; Zenner, D; Anderson, S R; Grant, K; Kumar, D

    2010-06-24

    Food poisoning outbreaks caused by Clostridium perfringens enterotoxin occur occasionally in Europe but have become less common in recent years. This paper presents the microbiological and epidemiological results of a large C. perfringens outbreak occurring simultaneously at two weddings that used the same caterer. The outbreak involved several London locations and required coordination across multiple agencies. A case-control study (n=134) was carried out to analyse possible associations between the food consumed and becoming ill. Food, environmental and stool samples were tested for common causative agents, including enterotoxigenic C. perfringens. The clinical presentation and the epidemiological findings were compatible with C. perfringens food poisoning and C. perfringens enterotoxin was detected in stool samples from two cases. The case-control study found statistically significant associations between becoming ill and eating either a specific chicken or lamb dish prepared by the same food handler of the implicated catering company. A rapid outbreak investigation with preliminary real-time results and the successful collaboration between the agencies and the caterer led to timely identification and rectification of the failures in the food handling practices.

  19. Prevalence of enteropathogens and their antibiotic sensitivity pattern in puppies with hemorrhagic gastroenteritis

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    A. Kokila Priya

    2017-08-01

    Full Text Available Aim: Hemorrhagic gastroenteritis (HGE ranging from mild to severe forms is commonly encountered in puppies. The aim of the study was to identify the prevalence of common enteropathogens and the antibiotic sensitivity pattern in puppies reported with HGE. Materials and Methods: The canine HGE activity index, with little modification, was adopted to identify Grade III/ severely affected puppies below 6 months of age. Fecal polymerase chain reaction (PCR assay was employed to screen and compare the enteropathogens in puppies with hemorrhagic diarrhea and healthy control. Results: Canine parvovirus 2b was identified in 90.3% of the diarrheic and 10% of the non-diarrheic healthy puppies. Clostridium difficile was identified in all the diarrheic puppies and in 80% of the healthy puppies. Among the diarrheic puppies, 17.7% were positive for Clostridium perfringens enterotoxin, 9.7% were positive for C. perfringens alpha toxin, 6.4% were positive for Escherichia coli shiga toxin, 6.4% were positive for E. coli enterotoxin (LT, and 3.2% were positive for canine distemper virus. Whereas, none of the healthy puppies were positive for these bacteria and toxins. Fecal antibiotic sensitivity test pattern revealed gentamicin to be sensitive in 95% of the cases, azithromycin in 50%, enrofloxacin in 25%, cefotaxime in 20%, and tetracycline in 5% of the cases. Conclusion: Parvoviral enteritis is predominant among puppies. Yet, bacteria and their toxins also play an important role in HGE. Gentamicin has higher sensitivity against the enteropathogens associated with the condition.

  20. Evaluation of PCR and DNA hybridization protocols for detection of viable enterotoxigenic Clostridium perfringens in irradiated beef

    International Nuclear Information System (INIS)

    Baez, L.A.; Juneja, V.K.; Thayer, D.W.; Sackitey, S.

    1997-01-01

    The sensitivity of DNA hybridization and polymerase chain reaction (PCR), was evaluated in irradiated cooked and raw beef samples. A membrane-based colony hybridization assay and a PCR protocol, both with specificity for the enterotoxin A gene of Clostridium perfringens, were compared with viable plate counts. The results of the colony hybridization procedure were in agreement with viable plate counts for detection and enumeration of enterotoxigenic C. perfringens. The PCR procedure combined a 4 h enrichment followed by a nucleic acid extraction step and assessed the amplification of 183 and 750 base pair enterotoxin gene targets. Detection of C. perfringens by PCR did not show a reliable correlation with viable plate counts or the colony hybridization assay. C. perfringens killed by irradiation were not detected by the plate count or colony hybridization methods; however, killed cells were detected with the PCR technique. By relying on the growth of viable cells for detection and/or enumeration, the colony hybridization and plate count methods provided a direct correlation with the presence of viable bacteria

  1. Microbiological quality and safety of Skipjack tuna loins (Katsuwonus Pelamis intented for canning

    Directory of Open Access Journals (Sweden)

    Francesco Casalinuovo

    2018-03-01

    Full Text Available Tuna is a food widely consumed fresh and canned as well. After catching and filleting, a pre-cooking step is normally followed by freezing and shipping to canning industry as loins. The aim of this paper was to assess the microbiological quality and safety of tuna loins (50 samples imported by an Italian canned tuna producer from two different FAO fishing areas. Total bacterial count (TBC, Coliforms, Enterobacteriaceae, Escherichia coli, Listeria monocytogenes, Salmonella, Staphylococcus aureus, Vibrio parahaemolyticus, Vibrio cholera, pH measurement, S. aureus enterotoxin and histamine levels were evaluated. Significant differences were evidenced for TBC, Enterobacteriaceae, E. coli, Coliforms and pH between fishing areas. Staphylococcus aureus was detected in all the samples tested at 1 to 3 Log cfu/g; Staphylococcus warneri, S. saprophyticus, S. epidermidis, S. hominis, S. intermedius, S. vitulinus and S. sciuri were also identified. Listeria monocytogenes, Salmonella, Vibrio parahaemolyticus, Vibrio cholera and Staphylococcus aureus enterotoxin were not detected. Histamine ranged between 0 to 5 mg/kg. The tuna loins analysed in this paper have to be considered safe; staphylococci presence represents a possible concern and has to been taken in account when risk analysis is carried out.

  2. Prevalence of Staphylococcus aureus Isolated From Various Foods of Animal Origin in Kırşehir, Turkey and Their Enterotoxigenicity

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    M. Dilek Avşaroğlu

    2016-12-01

    Full Text Available The aim of this study was to detect Staphylococcus aureus contamination to different types of animal origin foods collected in the Kırşehir province of Turkey and to examine their enterotoxin production ability. Out of 120 food samples 38 suspected colonies were obtained and 23 of them were identified as S. aureus by biochemical and molecular analyses. Other species detected were S. chromogenes, S. cohnii ssp. cohnii, S. hominis, S. lentus, S. warneri, and S. xylosus. The isolates were also analysed with regard to carry mecA gene. None of them was found to have mecA gene indicating susceptibility to methicillin. To determine the enterotoxigenic ability of the isolates phenotypically, reversed-passive-latex-agglutination test against SEA-SED was used. Six out of 23 S. aureus isolates were determined to produce SEA, SEC and SED. Three of them had only one enterotoxin production, whereas others had SEA and SED production together. The results of phenotypic analyses were confirmed by PCR based examination. None of the coagulase-negative staphylococci were found to be enterotoxigenic by both phenotypical and PCR-based analyses. In conclusion, enterotoxigenic S. aureus is a risk in foods of animal origin in Kırşehir and its counties.

  3. Control of Virulence Gene Expression by the Master Regulator, CfaD, in the Prototypical Enterotoxigenic Escherichia coli Strain, H10407

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    Carla Hodson

    2017-08-01

    Full Text Available Enterotoxigenic Escherichia coli (ETEC is the most common bacterial cause of diarrhea in children in developing countries, as well as in travelers to these countries. To cause disease, ETEC needs to produce a series of virulence proteins including enterotoxins, colonization factors and secretion pathways, which enable this pathogen to colonize the human small intestine and deliver enterotoxins to epithelial cells. Previously, a number of studies have demonstrated that CfaD, an AraC-like transcriptional regulator, plays a key role in virulence gene expression by ETEC. In this study, we carried out a transcriptomic analysis of ETEC strain, H10407, grown under different conditions, and determined the complete set of genes that are regulated by CfaD. In this way, we identified a number of new target genes, including rnr-1, rnr-2, etpBAC, agn43, flu, traM and ETEC_3214, whose expression is strongly activated by CfaD. Using promoter-lacZ reporters, primer extension and electrophoretic mobility shift assays, we characterized the CfaD-mediated activation of several selected target promoters. We also showed that the gut-associated environmental signal, sodium bicarbonate, stimulates CfaD-mediated upregulation of its virulence target operons. Finally, we screened a commercial small molecule library and identified a compound (CH-1 that specifically inhibited the regulatory function of CfaD, and by 2-D analoging, we identified a second inhibitor (CH-2 with greater potency.

  4. GENOTYPING OF CLOSTRIDIUM PERFRINGENS FROM FRESH WATER FISH AND FISH PICKLES

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    Adarsh Jain

    2012-08-01

    Full Text Available This study aims to evaluate the genotypes of Clostridium perfringens in fish and fish based products from Tamil Nadu and Kerala states of India. A total of 301 samples consisting intestinal contents of freshwater fish (234 from various dams, freshwater lakes, ponds, retail shops and markets and fish pickles (67 obtained from randomly selected retail shops and supermarkets were investigated. Bacterial isolations, identifications and phenotypic characterization of virulence factors were carried out as per standard microbiological procedures. Genotyping of the C. perfringens isolates were done by amplifying four major lethal toxin genes namely- alpha toxin gene (cpa, beta toxin gene (cpb, epsilon toxin gene (etx, iota toxin gene (iA in a Thermal Cycler. Isolates were also screened for the presence of enterotoxin gene (cpe and beta2 toxin gene (cpb2 by single step PCR. Biochemical tests and phenotypic determination of virulence factors tentatively identified 82 (27.24% isolates of C. perfringens. In PCR assay, all 82 (100% isolates harbored cpa toxin genes of C. perfringens, however, 65 (79.26% isolates also carried additional cpb2 toxin genes. None of the isolates were found positive for beta, epsilon, iota and enterotoxin genes. Genotyping of the 82 isolates by PCR revealed that all the isolated bacteria were belonged to C. perfringens type A and both cpa and cpb2 toxin genes were prevalent among the isolates of C. perfringens type A, impending the risk of pathogenicity to human via freshwater fish and fish pickles.

  5. Oral Administration of Astrovirus Capsid Protein Is Sufficient To Induce Acute Diarrhea In Vivo

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    Victoria A. Meliopoulos

    2016-11-01

    Full Text Available The disease mechanisms associated with the onset of astrovirus diarrhea are unknown. Unlike other enteric virus infections, astrovirus infection is not associated with an inflammatory response or cellular damage. In vitro studies in differentiated Caco-2 cells demonstrated that human astrovirus serotype 1 (HAstV-1 capsid protein alone disrupts the actin cytoskeleton and tight junction complex, leading to increased epithelial barrier permeability. In this study, we show that oral administration of purified recombinant turkey astrovirus 2 (TAstV-2 capsid protein results in acute diarrhea in a dose- and time-dependent manner in turkey poults. Similarly to that induced by infectious virus, TAstV-2 capsid-induced diarrhea was independent of inflammation or histological changes but was associated with increased intestinal barrier permeability, as well as redistribution of sodium hydrogen exchanger 3 (NHE3 from the membrane to the cytoplasm of the intestinal epithelium. Unlike other viral enterotoxins that have been identified, astrovirus capsid induces diarrhea after oral administration, reproducing the natural route of infection and demonstrating that ingestion of intact noninfectious capsid protein may be sufficient to provoke acute diarrhea. Based on these data, we hypothesize that the astrovirus capsid acts like an enterotoxin and induces intestinal epithelial barrier dysfunction.

  6. Safety Evaluation of the Coagulase-Negative Staphylococci Microbiota of Salami: Superantigenic Toxin Production and Antimicrobial Resistance

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    Raquel Soares Casaes Nunes

    2015-01-01

    Full Text Available The risks of contracting staphylococci food poisoning by the consumption of improperly manufactured salami and the possibility of this food being reservoirs for antibiotic resistance were evaluated. Nineteen coagulase-negative staphylococci (CNS strains were found in commercial and artisanal salami. The species in commercial salami were S. saprophyticus, S. sciuri, S. xylosus, and S. carnosus. Artisanal salami showed S. succinus, S. epidermidis, and S. hominis but no S. carnosus. Phylogenetic analyses grouped the strains into three major staphylococcal species groups, comprised of 4 refined clusters with similarities superior to 90%. Fifteen strains harbored multiple enterotoxin genes, with high incidence of seb/sec and sea, 57% and 50%, respectively, intermediate incidence of sed/seh/selm and sei/seln/tst-H, 33% and 27%, correspondingly, and low incidence of see/selj/selo and seg, of respectively 13% and 1%. Real time RT-PCR and enzyme-linked-immunosorbent assays confirmed the enterotoxigenicity of the strains, which expressed and produced enterotoxins in vitro. The CNS strains showed multiresistance to several antimicrobials of therapeutic importance in both human and veterinarian medicine, such as β-lactams, vancomycin, and linezolid. The effective control of undue staphylococci in fermented meat products should be adopted to prevent or limit the risk of food poisoning and the spread of antimicrobial-resistant strains.

  7. Determination of haemolytic and non haemolytic genes profiles of Bacillus cereus strains isolated from food samples by polymerase chain reaction (pcr) technique

    Science.gov (United States)

    Jawad, Nisreen; Ahemd, Asmat; Abdullah, Aminah

    2018-04-01

    The aim of this study was to investigate the presence of Bacillus cereus and detection of enterotoxigenic genes in food samples by utilizing a Polymerase Chain Reaction technique (PCR). In this study the providence of B. cereus was carried out to food samples. The B. cereus isolates were investigated for enterotoxigenic gene. The cooked seafood, and raw milk samples were purchased from several restaurants and market in the area of (Bangi, Kajang, Serdang and UKM) Selangor, Malaysia. A total of 60 samples have been analyzed. B. cereus contamination has been formed between 1.4×105 - 3×105 cfu/mL of cooked seafood and raw milk samples. Five colonies have been detected as B. cereus using biochemical test. All B. cereus isolates named BC1 to BC27, were characterized for haemolytic enterotoxin (HBL) complex encoding genes (hblA), non-haemolytic enterotoxin encoding gene (NheA). 10 isolates have been reported to be positive towards hblA and 12 isolates were positive towards NheA. The presence of B. cereus and their enterotoxigenic genes in cooked seafood and raw milk from to food samples obtained may pose a potential risk for public health.

  8. Study of immunoglobulin G thin layers obtained by the Langmuir-Blodgett method: application to immunosensors.

    Science.gov (United States)

    Barraud, A; Perrot, H; Billard, V; Martelet, C; Therasse, J

    1993-01-01

    Nowadays, immunosensors play a leading part in the field of bioanalytical chemistry research. As with any biosensor, they need appropriate transducers and a suitable technique to immobilize the active biocomponents. In this study, two transduction modes were chosen: mass effects (quartz microbalance measurements) and geometric and dielectric effects (capacitance measurements). The Langmuir-Blodgett (LB) method appears to be quite suitable for generating biospecific surfaces. This work has focused on the detection of staphylococcal enterotoxin B, the corresponding antibody being immobilized at the surface of fatty acids by a variant of the LB method. The composition of the film and the nature of antibody-fatty acid interactions were studied by means of the two transducers mentioned above. FTIR (Fourier transform infra-red) spectroscopy and protein diagnostic assay. Influence of several parameters (pH, ionic strength, transfer pressure, antibody concentration in the subphase) was investigated. The immobilization rate reached its maximum when experimental conditions allowed optimal electrostatic interactions. In this case, the quartz crystal microbalance response, in air, reached 55 Hz per monolayer of immobilized immunoglobulin G and the equivalent capacitance variation, measured in liquid media, was around 300 pF cm-2. Activity of the biospecific LB films, when binding enterotoxin, was checked by the classical ELISA (enzyme immuno-linked assay) technique.

  9. Potential enterotoxicity and antimicrobial resistance pattern of Aeromonas species isolated from pet turtles and their environment.

    Science.gov (United States)

    Wimalasena, S H M P; Shin, Gee-Wook; Hossain, Sabrina; Heo, Gang-Joon

    2017-05-23

    To investigate the potential enterotoxicity and antimicrobial resistance of aeromonads from pet turtles as a risk for human infection, one hundred and two Aeromonas spp. were isolated from the feces, skin and rearing environments of pet turtles and identified by biochemical and gyrB sequence analyses. Aeromonas enteropelogenes was the predominant species among the isolates (52.9%) followed by A. hydrophila (32.4%), A. dharkensis (5.9%), A. veronii (4.9%) and A. caviae (3.9%). Their potential enterotoxicities were evaluated by PCR assays for detecting genes encoding cytotoxic enterotoxin (act) and two cytotonic enterotoxins (alt and ast). 75.8% of A. hydrophila isolates exhibited the act + /alt + /ast + genotype, whereas 94.4% of A. enteropelogenes isolates were determined to be act - /alt - /ast - . In an antimicrobial susceptibility test, most isolates were susceptible to all tested antibiotics except amoxicillin, ampicillin, cephalothin, chloramphenicol and tetracycline. Non-susceptible isolates to penicillins (ampicillin and amoxicillin) and fluoroquinolones (ciprofloxacin and norfloxacin) were frequently observed among the A. enteropelogenes isolates. Few isolates were resistant to imipenem, amikacin, ceftriaxone and cefotaxime. Collectively, these results suggest that pet turtles may pose a public health risk of infection by enterotoxigenic and antimicrobial resistant Aeromonas strains.

  10. Prevalence and antimicrobial resistance of Bacillus cereus isolated from beef products in Egypt

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    Reyad Shawish

    2017-12-01

    Full Text Available Foodborne pathogens have the main concern in public health and food safety. Bacillus cereus food poisoning is one of the most important foodborne pathogens worldwide. In the present study, a total of 200 random beef product samples were collected from different supermarkets located at Menofia and Cairo governorates were examined for the presence of B. cereus. In addition, the presence of some virulence encoding genes was evaluated using Multiplex PCR. Finally, the antibiogram testing was conveyed to illustrate the resistance pattern of the confirmed B. cereus. The data showed that B. cereus was recovered from 22.5%, 30%, 25%, 37.5% and 15% of the minced meat, burger, sausage, kofta, and luncheon respectively. Among the 20 examined isolates 18/20 (90% were harbor hblC enterotoxin encoding gene compared with 20/20 (100 were have cytK enterotoxin encoding gene. The isolated strains of B. cereus were resistant to penicillin G and sensitive to oxacillin, clindamycin, vancomycin, erythromycin, gentamicin, ciprofloxacin, and ceftriaxone. In all, the obtained data showed the importance of emerging B. cereus in disease control and prevention programs, and in regular clinical and food quality control laboratories in Egypt.

  11. Antibiotic resistance and molecular analysis of Staphylococcus aureus isolated from cow's milk and dairy products in northeast Brazil.

    Science.gov (United States)

    Silveira-Filho, Vladimir M; Luz, Isabelle S; Campos, Ana Paula F; Silva, Wellington M; Barros, Maria Paloma S; Medeiros, Elizabeth S; Freitas, Manuela F L; Mota, Rinaldo A; Sena, Maria J; Leal-Balbino, Tereza C

    2014-04-01

    This work aimed to assess the clonal distribution among 94 strains of Staphylococcus aureus isolated from cow's milk, raw cheese, and a milking machine in 12 dairy farms in northeast Brazil, by analyzing different typing methods and detecting resistance and toxigenic profiles. For the first time, isolates of this region were assessed simultaneously by the polymorphism of the 3'-end coa gene and 16S-23S rDNA, pulsed-field gel electrophoresis, antibiotic resistance phenotyping, and toxigenic arsenal. Although pulsed-field gel electrophoresis patterns showed a wider variation (discriminatory index 0.83) than the PCR-based methods, the internal transcribed spacer-PCR proved to be a useful and inexpensive procedure for conducting epidemiological surveys of S. aureus on a regional scale. Each dairy farm had its own resistance profile, and in two herds, 63% of the strains were multiresistant, probably due to the indiscriminate use of antibiotics in bovine mastitis treatment. No methicillin-resistant S. aureus strains were detected in this study; however, 93.6% of S. aureus strains harbored variable profiles of staphylococcal enterotoxin genes seg, seh, sei, and sej. Transcriptional analysis revealed that 53.3% of staphylococcal enterotoxin genes actually transcribed, pointing out the food poisoning risk of these dairy products to consumers in the region. Based on the detection of the most prevalent clones in a herd or region, appropriate antibiotic therapy and specific immunization can be used for the treatment and control of staphylococcal mastitis.

  12. Modeling the level of contamination of Staphylococcus aureus in ready-to-eat kimbab in Korea.

    Science.gov (United States)

    Bahk, Gyung-Jin; Hong, Chong-Hae; Oh, Deog-Hwan; Ha, Sang-Do; Park, Ki-Hwan; Todd, Ewen C D

    2006-06-01

    The risk of Staphylococcus aureus in ready-to-eat kimbab (rice rolled in laver) sold in Korea was evaluated by a mathematical modeling approach. Four nodes were constructed from preparation at retail to consumption. A predictive microbial growth model and survey data were combined with probabilistic modeling to simulate the level of S. aureus in a single kimbab at the time of consumption. We estimated the mean level of S. aureus to be 2.92 log CFU/g for a typical kimbab (150 to 200 g each) at the time of consumption. Our model also showed that 29.73% of the kimbabs had > or = 100,000 S. aureus CFU/g, which poses some risk of illness, since some level of enterotoxin would be expected from toxigenic strains. However, because of the lack of dose-response models for staphylococcal enterotoxin, the final level of S. aureus in the kimbabs could not be used to estimate how many people would become ill from eating them. Correlation sensitivity results showed that consumer eating patterns and initial contamination levels at retail stores were the most significant risk factors for illness and that temperature control under 10 degrees C was a critical control point in kimbab retail establishments to prevent the growth of S. aureus.

  13. Effect of packaging conditions on the growth of micro-organisms and the quality characteristics of fresh mushrooms (Agaricus bisporus) stored at inadequate temperatures.

    Science.gov (United States)

    González-Fandos, E; Giménez, M; Olarte, C; Sanz, S; Simón, A

    2000-10-01

    Mushrooms were packed in two polymeric films (perforated and non-perforated PVC) and stored at 17 degrees C and 25 degrees C. The carbon dioxide and oxygen content inside the packages, aerobic mesophiles, Pseudomonas spp., faecal coliforms, Escherichia coli, anaerobic spores and major sensory factors (colour, texture, development stage and presence of moulds) were determined. The non-perforated packages had the highest contents of CO2 (6-7%), the lowest contents of O2 (0.013-0.17%) and the most desirable quality parameters (texture, development stage and absence of moulds). Pseudomonas spp. counts were around 1 logarithmic unit lower in mushrooms packaged in non-perforated film as the O2 concentrations were lower than in perforated film. The mushrooms themselves were inoculated with an enterotoxin A-producing strain of Staphylococcus aureus, packaged in overwrapped trays and stored at 17 and 25 degrees C. Staphylococcus aureus did not grow in the samples stored at 17 degrees C. Only slight growth was observed in mushrooms packaged with non-perforated film after 1 day at 25 degrees C. No enterotoxin was detected in any package. Faecal coliform counts were <2 log cfu g(-1). Escherichia coli was not isolated in any of the samples. At 25 degrees C, counts of anaerobic spores of around 2 log cfu g(-1) were detected in those mushrooms packaged in non-perforated film.

  14. Putative new heat-stable cytotoxic and enterotoxic factors in culture supernatant of Escherichia coli isolated from drinking water

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    DA Ribeiro

    2011-01-01

    Full Text Available Enteric infections caused by the ingestion of contaminated water, especially by Escherichia coli, are important to define the virulence properties of these bacteria. Due to frequent infantile diarrhea in the city of Ouro Preto, Minas Gerais state, Brazil, the phenotypic and genotypic diarrheagenic properties of E. coli isolated from drinking water were studied. The culture supernatants of 39 (40% among a total of 97 E. coli isolates from drinking water were positive by suckling mouse assay and induced cytotoxic effects on Vero cells. The enterotoxic and cytotoxic activities were present in the fraction with less than 10 kDa and were not lost when heated up to 60°C and 100°C for 30 minutes. PCR assays showed that among these 39 Vero cytotoxigenic E. coli, four (10.2% were positive for ST II (estB and two (5% positive for αHly (hlyA. Gene amplification of SLT (stx 1, stx 2, ST I (estA, LT (eltI, eltII, EAST1 (astA, EHly (enhly and plasmid-encoded enterotoxin (pet were not observed. This heat-stable cytotoxic enterotoxin of E. coli is probably a new putative diarrheagenic virulence factor, as a toxin presenting these characteristics has not yet been described.

  15. Investigating an outbreak of staphylococcal food poisoning among travellers across two Australian states

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    Stephanie Fletcher

    2015-05-01

    Full Text Available Introduction: Staphylococcus aureus is a common cause of staphylococcal food poisoning in Australia with several outbreaks associated with foods prepared by commercial caterers. Laboratory testing on cases of gastrointestinal illness caused by enterotoxin-producing S. aureus is not routinely done as this condition is self-limiting. Hence outbreaks of such illness may go undetected. Methods: A retrospective cohort study was conducted among a group of tourists who were hospitalized in Sydney shortly after flying from Queensland. The group had consumed food prepared by a restaurant on the Gold Coast before transit. Laboratory analyses on stool specimens were conducted in Sydney. An environmental assessment of the restaurant in the Gold Coast was conducted, and environmental specimens were assessed for contamination. Results: Epidemiological investigations linked the outbreak to a restaurant in the Gold Coast where the suspected food was produced. Stool samples from two of the hospitalized cases were confirmed to have enterotoxin-producing S. aureus, and several environmental samples were found to be contaminated with S. aureus as well. Investigations suggested that absence of hand washing and other unhygienic food handling at the implicated restaurant was the likely cause of this outbreak. Conclusion: Food poisoning due to toxin-mediated S. aureus is frequently undetected and underreported. Public health units should consider toxin-producing pathogens such as S. aureus when investigating outbreaks where vomiting is the predominant symptom and occurs rapidly after consuming food.

  16. Use of the Real Time xCelligence System for Purposes of Medical Microbiology.

    Science.gov (United States)

    Junka, Adam Feliks; Janczura, Adriana; Smutnicka, Danuta; Mączyńska, Beata; Anna, Secewicz; Nowicka, Joanna; Bartoszewicz, Marzenna; Gościniak, Grażyna

    2012-09-28

    Roche's xCelligence impedance-measuring instrument is one of a few commercially available systems of such type. According to the best knowledge of authors, instrument was tested so far only for eukaryotic cell research. The aim of this work was to estimate xCELLigence suitability for the microbiological tests, including (i) measurement of morphological changes in eukaryotic cells as a result of bacterial toxin activity, (ii) measurement of bacterial biofilm formation and (iii) impact of antiseptics on the biofilm structure. To test the infuence of bacterial LT enterotoxin on eukaryotic cell lines, Chinese Hamster Ovary (CHO) cell line and reference strain Escherichia coli ATTC 35401 were used. To investigate Roche's instrument ability to measure biofilm formation and impact of antiseptics on its development, Staphylococcus aureus ATTC6538 reference strain was used. The data generated during the experiments indicate excellent ability of xCelligence instrument to detect cytopathic effect caused by bacterial LT endotoxin and to detect staphylococcal biofilm formation. However, interpretation of the results obtained during real-time measurement of antiseptic's bactericidal activity against staphylococcal biofilm, caused many difficulties. xCelligence instrument can be used for real-time monitoring of morphological changes in CHO cells treated with bacterial LT enterotoxin and for real-time measurement of staphylococcal biofilm formation in vitro. Further investigation is necessary to confirm suitability of system to analyze antiseptic's antimicrobial activity against biofilm in vitro.

  17. Prevalence and Toxin Characteristics of Bacillus thuringiensis Isolated from Organic Vegetables.

    Science.gov (United States)

    Kim, Jung-Beom; Choi, Ok-Kyung; Kwon, Sun-Mok; Cho, Seung-Hak; Park, Byung-Jae; Jin, Na Young; Yu, Yong Man; Oh, Deog-Hwan

    2017-08-28

    The prevalence and toxin characteristics of Bacillus thuringiensis isolated from 39 organic vegetables were investigated. B. thuringiensis was detected in 30 out of the 39 organic vegetables (76.9%) with a mean value of 2.60 log CFU/g. Twenty-five out of the 30 B. thuringiensis isolates (83.3%) showed insecticidal toxicity against Spodoptera exigua . The hblCDA, nheABC , and entFM genes were found to be the major toxin genes, but the ces gene was not detected in any of the tested B. thuringiensis isolates. The hemolysin BL enterotoxin was detected in all 30 B. thuringiensis isolates (100%). The non-hemolytic enterotoxin complex was found in 27 out of 30 B. thuringiensis isolates (90.0%). The B. thuringiensis tested in this study had similar toxin gene characteristics to B. cereus , which possessed more than one toxin gene. B. thuringiensis could have the potential risk of foodborne illness based on the toxin genes and toxin-producing ability.

  18. A Chimeric protein of CFA/I, CS6 subunits and LTB/STa toxoid protects immunized mice against enterotoxigenic Escherichia coli.

    Science.gov (United States)

    Zeinalzadeh, Narges; Salmanian, Ali Hatef; Goujani, Goli; Amani, Jafar; Ahangari, Ghasem; Akhavian, Asal; Jafari, Mahyat

    2017-07-01

    Enterotoxigenic Escherichia Coli (ETEC) strains are the commonest bacteria causing diarrhea in children in developing countries and travelers to these areas. Colonization factors (CFs) and enterotoxins are the main virulence determinants in ETEC pathogenesis. Heterogeneity of CFs is commonly considered the bottleneck to developing an effective vaccine. It is believed that broad spectrum protection against ETEC would be achieved by induced anti-CF and anti-enterotoxin immunity simultaneously. Here, a fusion antigen strategy was used to construct a quadrivalent recombinant protein called 3CL and composed of CfaB, a structural subunit of CFA/I, and CS6 structural subunits, LTB and STa toxoid of ETEC. Its anti-CF and antitoxin immunogenicity was then assessed. To achieve high-level expression, the 3CL gene was synthesized using E. coli codon bias. Female BALB/C mice were immunized with purified recombinant 3CL. Immunized mice developed antibodies that were capable of detecting each recombinant subunit in addition to native CS6 protein and also protected the mice against ETEC challenge. Moreover, sera from immunized mice also neutralized STa toxin in a suckling mouse assay. These results indicate that 3CL can induce anti-CF and neutralizing antitoxin antibodies along with introducing CFA/I as a platform for epitope insertion. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

  19. Enterotoxigenic Genes in strains of Staphylococcus spp., isolated from cheese made in Pamplona-Colombia

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    Fanny Herrera A.

    2015-01-01

    Full Text Available Objective. To determine the incidence of coagulase-positive strains of enterotoxigenic Staphylococcus in doble crema (double cream cheese samples produced in Pamplona. Materials and methods. Bacterial isolation was performed following the routine method for coagulase positive Staphylococcus provided by the Colombian Technical Standard 4779, by using Baird Parker medium with confirmation of typical colonies by performing the coagulase test. Detection of genes for principal enterotoxins was done by PCR. Results. The prevalence of coagulase positive Staphylococcus in cheese samples was 31%, with 27% of the samples failing to meet the requirements of the NTC 750. In 24.6% of the studied isolates, genes for enterotoxin production were detected. The presence, in the isolated strains, of genes for SEB, SEA and SED was 18.5%, 4.6% and 3.0%, respectively. Conclusions. The significant presence of enterotoxigenic genes found in the isolates obtained from samples of double cream cheese made in Pamplona, suggests an important hazard to the health of consumers.

  20. Poor survival of Methicillin-resistant Staphylococcus aureus on inanimate objects in the public spaces.

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    Domon, Hisanori; Uehara, Yoshio; Oda, Masataka; Seo, Hiromi; Kubota, Noriko; Terao, Yutaka

    2016-02-01

    We investigated the prevalence of Staphylococcus aureus on shopping baskets in Osaka Prefecture, Japan. Multilocus sequence typing was performed to determine the genotypes of S. aureus isolates, and then a polymerase chain reaction method was used to detect staphylococcal enterotoxins and antibiotic resistance genes. In addition, desiccation tolerance of S. aureus isolates was evaluated in vitro. Forty-six (6.2%) S. aureus isolates were collected from 740 shopping baskets, though only one MRSA strain was identified. In multilocus sequence typing findings, ten sequence types and 24 singletons were classified, which were divided into ten clonal complexes and six singletons. The most frequent staphylococcal enterotoxin gene was seg (30.4%). Our in vitro findings demonstrated that 70% of the S. aureus isolates, including the MRSA strain, became undetectable at 12 h after desiccation at an appropriate cell density, while the others remained viable for up to 24 h. Thus, it is difficult for MRSA organisms to survive on dry surfaces found in public areas. We speculated that inanimate objects in the community are unlikely to be a potential source for transmission of MRSA and that S. aureus on such objects outside of hospital settings is not a public health threat. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  1. Investigating an outbreak of staphylococcal food poisoning among travellers across two Australian states.

    Science.gov (United States)

    Fletcher, Stephanie; Boonwaat, Leng; Moore, Terry; Chavada, Ruchir; Conaty, Stephen

    2015-01-01

    Staphylococcus aureus is a common cause of staphylococcal food poisoning in Australia with several outbreaks associated with foods prepared by commercial caterers. Laboratory testing on cases of gastrointestinal illness caused by enterotoxin-producing S. aureus is not routinely done as this condition is self-limiting. Hence outbreaks of such illness may go undetected. A retrospective cohort study was conducted among a group of tourists who were hospitalized in Sydney shortly after flying from Queensland. The group had consumed food prepared by a restaurant on the Gold Coast before transit. Laboratory analyses on stool specimens were conducted in Sydney. An environmental assessment of the restaurant in the Gold Coast was conducted, and environmental specimens were assessed for contamination. Epidemiological investigations linked the outbreak to a restaurant in the Gold Coast where the suspected food was produced. Stool samples from two of the hospitalized cases were confirmed to have enterotoxin-producing S. aureus, and several environmental samples were found to be contaminated with S. aureus as well. Investigations suggested that absence of hand washing and other unhygienic food handling at the implicated restaurant was the likely cause of this outbreak. Food poisoning due to toxin-mediated S. aureus is frequently undetected and underreported. Public health units should consider toxin-producing pathogens such as S. aureus when investigating outbreaks where vomiting is the predominant symptom and occurs rapidly after consuming food.

  2. EPIDEMIOLOGIA MOLECULAR APLICADA AO MONITORAMENTO DE ESTIRPES DE Staphylococcus aureus NA PRODUÇÃO DE QUEIJO MINAS FRESCAL

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    Maria Izabel Merino de Medeiros

    2013-03-01

    Full Text Available We studied the molecular epidemiology of Staphylococcus aureus strains potentially toxigenic, isolated from the production process of Minas frescal cheese in a small dairy plant in the state of São Paulo. For this, samples were taken during the period from June 2008 to July 2009. Samples were collected from the surface of the receiving and storage tanks of raw milk, the surface of the balance tank of pasteurized milk, the water supply system, the pipes and equipments, the hands of the handler and from the packaged cheese, totaling 140 samples. The colonies isolated on Baird-Parker Agar confirmed as Gram positive and positive for catalase, coagulase and acetoin production, were submitted to extraction of bacterial DNA using the Invitek - Uniscience® kit. Confirmation of the isolated species and enterotoxins SEA, SEB, SEC, SED and TSST-1 toxin was carried out through the amplification of specific fragments of chromosomal DNA. Among the 74 strains of isolated coagulase-positive staphylococci, only 41 (55.4% strains were confirmed as Staphylococcus aureus, of which 25 (61.0% were positive to the presence of staphylococcal toxins. The most frequently identified enterotoxin was SEA. The toxigenic strains of Staphylococcus aureus were more frequently isolated from hands of the handler (16.0%, raw milk receiving tank (12.0%, pasteurized milk for cheese making (12.0% and fresh white cheese ready for consumption (12.0%.

  3. Genospecies and virulence factors of Aeromonas species in different sources in a North African country

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    Khalifa Sifaw Ghenghesh

    2014-09-01

    Full Text Available Introduction: Aeromonads of medical importance have been reported from numerous clinical, food, and water sources, but identification of genospecies and virulence factors of Aeromonas species from countries in North Africa and the Middle East are few. Methods: In total 99 Aeromonas species isolates from different sources (diarrheal children [n=23], non-diarrheal children [n=16], untreated drinking water from wells [n=32], and chicken carcasses [n=28] in Tripoli, Libya, were included in the present investigation. Genus identification was confirmed by biochemical analysis, and genospecies were determined using a combination of 16S rDNA variable region and gyrB sequence analysis. Polymerase chain reaction (PCR was used to detect genes encoding toxins from 52 of the isolates. Results: We identified 44 isolates (44% as A. hydrophila (3 [3.0%] subspecies anaerogenes, 23 [23%] subspecies dhakensis, and 18 [18%] subspecies ranae; 27 isolates (27% as A. veronii; 23 isolates (23% as A. caviae; and 5 isolates (5.0% as other genospecies. The genes encoding aerolysin (aer, cytolytic enterotoxin (act, and A. hydrophila isolate SSU enterotoxin (ast were detected in 45 (87%, 4 (7.7%, and 9 (17% of the 52 isolates tested, respectively. The gene encoding an extracellular lipase (alt was not detected. Conclusion: The majority of aeromonads from Libya fall within three genospecies (i.e. A. hydrophila, A. veronii, and A. caviae, and genes coding for toxin production are common among them.

  4. Assessment of hygienic quality of some types of cheese sampled from retail outlets.

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    Prencipe, Vincenza; Migliorati, Giacomo; Matteucci, Osvaldo; Calistri, Paolo; Di Giannatale, Elisabetta

    2010-01-01

    The authors evaluated the prevalence of Listeria monocytogenes, Escherichia coli O157:H7, Salmonella spp. and staphylococcal enterotoxin, in 2,132 samples selected from six types of cheese on the basis of recorded consumption in Italy in 2004. In L. monocytogenes-positive samples the precise level of contamination was determined. To define the physical-chemical characteristics of the selected natural cheeses, the pH values, water activity and sodium chloride content were determined. The results suggest that blue and soft cheeses (Brie, Camembert, Gorgonzola and Taleggio) are more likely to be contaminated with L. monocytogenes. The mean prevalence of L. monocytogenes in the six types of cheese was 2.4% (from 0.2% in Asiago and Crescenza to 6.5% in Taleggio), with contamination levels of up to 460 MPN/g. No presence of Salmonella spp. and E. coli O157 was found in any sample. Staphylococcus enterotoxin was found in 0.6% of the samples examined. Physical and chemical parameter values confirmed that all types of cheese are considered capable of supporting the growth of L. monocytogenes. The study confirmed the need to apply effective control at production and sales levels to reduce the probability of contamination by L. monocytogenes. This micro-organism can attain high levels of contamination in food products, such as cheeses that have a long shelf-life when associated with difficulties of maintaining appropriate storage temperatures in both sales points and in the home.

  5. Assessment of hygienic quality of some types of cheese sampled from retail outlets

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    Elisabetta Di Giannatale

    2010-06-01

    Full Text Available The authors evaluated the prevalence of Listeria monocytogenes, Escherichia coli O157:H7, Salmonella spp. and Staphylococcus enterotoxin, in 2 132 samples selected from six types of cheese on the basis of recorded consumption in Italy in 2004. In L. monocytogenes-positive samples the precise level of contamination was determined. To define the physical-chemical characteristics of the selected natural cheeses, the pH values, water activity and sodium chloride content were determined. The results suggest that blue and soft cheeses (Brie, Camembert, Gorgonzola and Taleggio are more likely to be contaminated with L. monocytogenes. The mean prevalence of L. monocytogenes in the six types of cheese was 2.4% (from 0.2% in Asiago and Crescenza to 6.5% in Taleggio, with contamination levels of up to 460 MPN/g. No presence of Salmonella spp. and E. coli O157 was found in any sample. Staphylococcus enterotoxin was found in 0.6% of the samples examined. Physical and chemical parameter values confirmed that all types of cheese are considered capable of supporting the growth of L. monocytogenes. The study confirmed the need to apply effective control at production and sales levels to reduce the probability of contamination by L. monocytogenes. This micro-organism can attain high levels of contamination in food products, such as cheeses that have a long shelf-life when associated with difficulties of maintaining appropriate storage temperatures in both sales points and in the home.

  6. Effect of food-related stress conditions and loss of agr and sigB on seb promoter activity in S. aureus.

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    Sihto, Henna-Maria; Stephan, Roger; Engl, Christoph; Chen, John; Johler, Sophia

    2017-08-01

    Staphylococcal enterotoxin B (SEB) causes staphylococcal food poisoning and is produced in up to ten times higher quantities than other major enterotoxins. While Staphylococcus aureus growth is often repressed by competing flora, the organism exhibits a decisive growth advantage under some stress conditions. So far, data on the influence of food-related stressors and regulatory mutations on seb expression is limited and largely based on laboratory strains, which were later reported to harbor mutations. Therefore, the aim of this study was to investigate the influence of stress and regulatory mutations on seb promoter activity. To this end, transcriptional fusions were created in two strains, USA300 and HG003, carrying different seb upstream sequences fused to a blaZ reporter. NaCl, nitrite, and glucose stress led to significantly decreased seb promoter activity, while lactic acid stress resulted in significantly increased seb promoter activity. Loss of agr decreased seb promoter activity and loss of sigB increased promoter activity, with the magnitude of change depending on the strain. These results demonstrate that mild stress conditions encountered during food production and preservation can induce significant changes in seb promoter activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Partial Sequencing of 16S rRNA Gene of Selected Staphylococcus aureus Isolates and its Antibiotic Resistance

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    Harsi Dewantari Kusumaningrum

    2016-08-01

    Full Text Available The choice of primer used in 16S rRNA sequencing for identification of Staphylococcus species found in food is important. This study aimed to characterize Staphylococcus aureus isolates by partial sequencing based on 16S rRNA gene employing primers 16sF, 63F or 1387R. The isolates were isolated from milk, egg dishes and chicken dishes and selected based on the presence of sea gene that responsible for formation of enterotoxin-A. Antibiotic susceptibility of the isolates towards six antibiotics was also tested. The use of 16sF resulted generally in higher identity percentage and query coverage compared to the sequencing by 63F or 1387R. BLAST results of all isolates, sequenced by 16sF, showed 99% homology to complete genome of four S. aureus strains, with different characteristics on enterotoxin production and antibiotic resistance. Considering that all isolates were carrying sea gene, indicated by the occurence of 120 bp amplicon after PCR amplification using primer SEA1/SEA2,  the isolates were most in agreeing to S. aureus subsp. aureus ST288. This study indicated that 4 out of 8 selected isolates were resistant towards streptomycin. The 16S rRNA gene sequencing using 16sF is useful for identification of S. aureus. However, additional analysis such as PCR employing specific gene target, should give a valuable supplementary information, when specific characteristic is expected.

  8. Antimicrobial resistance levels amongst staphylococci isolated from clinical cases of bovine mastitis in Kosovo.

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    Mehmeti, Ibrahim; Behluli, Behlul; Mestani, Mergim; Ademi, Arsim; Nes, Ingolf F; Diep, Dzung B

    2016-10-31

    Mastitis is one of the most frequent and costly disease in cattle. We studied milk samples from cattle with mastitis from farms in Kosovo to identify mastitis-causing pathogens and possible effective antibiotics. Our ultimate goal is to help implement adequate antibiotic management and treatment practices in Kosovo METHODOLOGY: A total of 152 milk samples were collected from cows with clinical mastitis from different farms in Kosovo. After identification of microorganisms, antibiotic susceptibility and the occurrence of enterotoxins was investigated. Staphylococci were found in 89 samples, of which 58 were coagulase negative and 31 coagulase positive. S. aureus was isolated from 27 samples, S. epidermidis from 25, and S. chromogenes from 15, while other species of staphylococci were isolated from the remaining 22 isolates. Interestingly, the bacterial diversity was different between cows in different periods of lactation and among different breeds. Most of the isolates (76/89) were resistant to two or more antibiotics. The highest resistance was to penicillin and ampicillin (> 65%), followed by tetracycline, oxacillin, streptomycin, chloramphenicol (> 23%), and less than 3% to erythromycin. Of the 89 isolates, 40 produced enterotoxins that were most frequently typed as A and C. We detected human bacterial pathogens in the cultures of milk samples from cows with mastitis. The isolates demonstrated resistance to two or more antibiotics, some of which are frequently used to treat animal and human infections. We recommend increased control and more stringent use of antibiotics in veterinary as well as human medicine.

  9. Balneotherapy of Psoriasis

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    Golušin Zoran

    2014-09-01

    Full Text Available Application of different kinds of mineral waters and peloids on the skin exerts mechanical, thermal and chemical effects. Significant reduction of inflammation and increased differentiation of keratinocytes may explain why balneotherapy has positive clinical effects in psoriatic patients. In vitro models have shown that thermal water stimulates interleukin-2 production after cell stimulation by staphylococcal enterotoxin B, and reduces interleukin-4 secretion. After balneotherapy, a significant decrease in Psoriasis Area Severity Index (PASI, associated with a significant reduction of interleukin-8, Staphylococcus aureus colonization and enterotoxin N, have been reported in patients with psoriasis. Mineral water was found to have inhibitory in vitro effects on substance P, TNF-α release and antigen-induced cell degranulation. Immunomodulatory effects of water depend on its content. Sulfur waters have beneficial anti-inflammatory, keratolytic, and antipruriginous effects and also possess antibacterial and antifungal properties. The effectiveness of balneotherapy in the treatment of psoriasis has been reported in many studies conducted all over the world. The majority of studies were conducted at the Dead Sea coast. Investigations showed that balneotherapy factors are important therapeutic factors in the treatment of psoriatic patients. The first and only comparable study of this kind in Serbia, was conducted in Prolom Spa with satisfactory therapeutic results.

  10. Isolation and identification of Staphylococcus sp. in powdered infant milk

    Science.gov (United States)

    Palilu, Prayolga Toban; Budiarso, Tri Yahya

    2017-05-01

    Staphylococcus sp. is one of the most dangerous bacteria that could cause food poisoning. It is a pathogenic bacterium which is able to produce enterotoxin in foods. Milk is an ideal growth medium for Staphylococcus sp., that may cause problem if it is to be consumed, especially by infant. It is the objective of this research to detect the presence of Staphylococcus sp. in powdered infant milk. As many as 14 samples obtained from market were used as samples for bacterial isolation. The isolation were done by employing enrichment step on BHI-broth, continued with Baird-Parker Agar which will produce a typical colony. It is then picked and grown on Mannitol Salt Agar, and gram staining, coagulase assay, and fermentation tests. The confirmation step was done by using API-Staph which gives the identification of Staphylococcus hemoliticus, Staphylococcus aureus and Staphylococcus epidermidis, with a percentage of identity ranging from 65.9-97.7%. Two isolates with the highest identification similarity values were then picked for molecular detection. A PCR primer pair targeting gene coding for enterotoxin A was used, and it gives positive result for the two isolates being tested. It is then concluded that the two isolates belong to Staphylococcus sp., and further research need to be done to correctly identify these isolates.

  11. THE STUDY OF ENTEROTOXIGENICITY OF THE BIOTYPE 1A YERSINIA ENTEROCOLITICA

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    E. A. Bogumilchik

    2011-01-01

    Full Text Available Abstract. The representatives of Y. enterocolitica biotype 1А which are considered as nonpathogenic microorganisms were tested for production of the thermostable enterotoxin YST B (Yersinia Stable Toxin. This toxin is characterized by strong toxic action and it can bring on diarrhea in human and animals. The chromosome gene of thermostable enterotoxin ystB was detected by PCR in 87.1% out of 116 studied strains of different origin and territorial isolation. To determine toxin production in vitro the studied strains cultivated in various conditions: in 26°C and 37°С in usual culture medium and in 37°С in the medium corresponded to the content of intestine. In part of the studied strains the toxin production was revealed on the model of newborn mice in both temperature regimes of cultivation 26°С and 37°С. The study of toxin production in representatives of Y. enterocolitica biotype 1А showed their possible role as etiological agents of diarrhea.

  12. Enterotoxigenic gene profiles of Bacillus cereus and Bacillus megaterium isolates recovered from honey Búsqueda de factores de virulencia en cepas de Bacillus cereus y de Bacillus megaterium aisladas de miel

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    A. C. López

    2010-09-01

    Full Text Available One hundred and thirty two Bacillus cereus and 52 Bacillus megaterium isolates from honeys were evaluated for the presence of genes encoding enterotoxin HBL, enterotoxin-T, cytotoxin K and the NHE complex, respectively. The relationship between hemolytic and coagulase activity and its correlation with the presence of the four mentioned enterotoxins was determined by principal component analysis (PCA. PCA in B. cereus revealed a positive correlation among free coagulase, hemolysis and the presence of genes hblA, hblB, hblC, hblD (HBL complex and bceT (enterotoxin-T, but no correlation with the clumping factor (bound coagulase and the presence of sequences of the NHE complex. On the other hand, PCA in B. megaterium showed a high positive correlation between coagulase (bound and free and the haemolytic activity but no correlation in relation to the presence of genes of the HBL complex, cytotoxin K, enterotoxin T and the NHE complex. To our knowledge, this is the first report of the detection of cytotoxin K and of the NHE complex genes in B. megaterium. The relationship between the coagulase activity and the presence of virulence factors has not been described before in the genus Bacillus, being this work the first report of this correlation. Interestingly, the presence of the cytK gene was almost independent of the presence of the rest of virulence factors herein analyzed both in B. cereus and B. megaterium populations. Our results suggest that honey could be a possible vehicle for foodborne illness due to the presence of toxigenic B. cereus and B. megaterium strains containing different virulence factors.Se evaluaron 132 aislamientos de Bacillus cereus y 52 de Bacillus megaterium provenientes de mieles de distintos orígenes geográficos para investigar la presencia de secuencias de ADN relacionadas con genes de virulencia y su posible correlación con la actividad hemolítica y coagulasa. Con respecto a los genes de virulencia, se analizaron por

  13. Transcriptomic and metabolic responses of Staphylococcus aureus in mixed culture with Lactobacillus plantarum, Streptococcus thermophilus and Enterococcus durans in milk.

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    Zdenkova, Kamila; Alibayov, Babek; Karamonova, Ludmila; Purkrtova, Sabina; Karpiskova, Renata; Demnerova, Katerina

    2016-09-01

    Staphylococcus aureus is a major food-borne pathogen due to the production of enterotoxin and is particularly prevalent in contaminated milk and dairy products. The lactic acid bacteria (LAB) are widely used as biocontrol agents in fermented foods which can inhibit pathogenic flora. In our work, we investigated the influence of three strains of LAB (Lactobacillus plantarum, Streptococcus thermophilus and Enterococcus durans) on the relative expression of three enterotoxin genes (sea, sec, sell) and eight virulence and/or regulatory genes (sarA, saeS, codY, srrA, rot, hld/RNAIII, agrA/RNAII, sigB) in two S. aureus strains (MW2 and Sa1612) in TSB and reduced-fat milk (1.5 %) at 30 °C over a 24-h period. The tested LAB and S. aureus strains proved to be mutually non-competitive or only slightly competitive during co-cultivation. In addition, under the above-mentioned conditions, differential gene expression between the S. aureus MW2 and Sa1612 strains was well documented. S. aureus growth was changed in mixed culture with LAB; however, its effect on the repression of sea and sec expression correlated with production of these virulence factors. In comparison, the presence of LAB strains generally inhibited the expression of sec, sell, sarA, seaS, agrA/RNAII and hld/RNAIII genes. The effect of LAB strains presence on the expression of sea, codY, srrA, rot and sigB genes was medium, time, LAB and S. aureus strain specific. SEA and SEC production was significantly reduced in milk compared to TSB in pure culture. After the 24-h cultivation, S. aureus MW2 and Sa1612 SEC production was 187 and 331 times lower in milk compared to TSB, respectively (0.07 and 0.39 ng/mL in milk, versus 13.1 and 129.2 ng/mL in TSB, respectively). At the same time S. aureus MW2 and Sa1612 SEA production was 77 and 68 times lower in milk compared to TSB, respectively (0.99 and 0.17 ng/mL in milk, versus 76.4 and 11.5 ng/mL in TSB, respectively). This study has revealed new insights into the

  14. Toxin production ability of Bacillus cereus strains from food product of Ukraine

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    I. Pylypenko

    2017-10-01

    Full Text Available Potential pathogens of foodborne toxic infections – bacterial contaminants Bacillus cereus isolated from plant raw materials and food products from the Ukrainian region were investigated. When determining of the proportion of isolated bacilli from the plant samples, it was established that the epidemiologically significant microorganisms of Bacillus cereus as agents of food poisoning are the second largest. The average value of contaminated samples of Ukrainian plant raw materials and processed products with Bacillus cereus is 36,2 %. The ability of Bacillus cereus strains identified by a complex of morphological, tinctorial, cultural and biochemical properties, to produce specific emetic and enterotoxins was studied. Molecular genetic diagnosis and detection of the toxin-producing ability of isolated 42 Bacillus cereus strains showed both the possibility of their rapid identification and the presence of specific toxicity genes. Multiplex polymerase chain reaction (PCR was carried out with specific primers to detect toxicity determined of various bacilli genes: nheA, hblD, cytK, cesВ. The distribution of toxigenic genes is significantly different among the Bacillus cereus isolates from various sources. The nheA, hblD and cytK enterotoxin genes were detected in 100, 83,3 and 61,9 % of the investigated strains of Bacillus cereus, respectively. The cesB gene encoding emetic toxin was detected in 4,8 % of  strains. Molecular-genetic PCR-method confirmed that all the isolated strains belong to the Bacillus cereus group, and the ability to produce toxins can be attributed to five groups. The main toxins that produce the investigated Bacillus cereus strains were nhe and hbl enterotoxins encoded by the corresponding genes of nheA and hblD. The enterotoxic type of Bacillus cereus was predominant in Ukrainian region.  Studies of domestic plant food raw materials and products have confirmed the need to improve microbiological control of product safety

  15. Shark complement: an assessment.

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    Smith, S L

    1998-12-01

    The classical (CCP) and alternative (ACP) pathways of complement activation have been established for the nurse shark (Ginglymostoma cirratum). The isolation of a cDNA clone encoding a mannan-binding protein-associated serine protease (MASP)-1-like protein from the Japanese dogfish (Triakis scyllia) suggests the presence of a lectin pathway. The CCP consists of six functionally distinct components: C1n, C2n, C3n, C4n, C8n and C9n, and is activated by immune complexes in the presence of Ca++ and Mg++ ions. The ACP is antibody independent, requiring Mg++ ions and a heat-labile 90 kDa factor B-like protein for activity. Proteins considered homologues of C1q, C3 and C4 (C2n) of the mammalian complement system have been isolated from nurse shark serum. Shark C1q is composed of at least two chain types each showing 50% identity to human C1q chains A and B. Partial sequence of the globular domain of one of the chains shows it to be C1q-like rather than like mannan-binding protein. N-terminal amino acid sequences of the alpha and beta chain of shark C3 and C4 molecules show significant identity with corresponding human C3 and C4 chains. A sequence representing shark C4 gamma chain, shows little similarity to human C4 gamma chain. The terminal shark components C8n and C9n are functional analogues of mammalian C8 and C9. Anaphylatoxin activity has been demonstrated in activated shark serum, and porcine C5a desArg induces shark leucocyte chemotaxis. The deduced amino acid sequence of a partial C3 cDNA clone from the nurse shark shows 50%, 30% and 24% homology with the corresponding region of mammalian C3, C4 and alpha 2-macroglobulin. Deduced amino acid sequence data from partial Bf/C2 cDNA clones, two from the nurse shark and one from the Japanese dogfish, suggest that at least one species of elasmobranch has two distinct Bf/C2 genes.

  16. Prevalence of adhesin and toxin genes in E. coli strains isolated from diarrheic and non-diarrheic pigs from smallholder herds in northern and eastern Uganda.

    Science.gov (United States)

    Ikwap, Kokas; Larsson, Jenny; Jacobson, Magdalena; Owiny, David Okello; Nasinyama, George William; Nabukenya, Immaculate; Mattsson, Sigbrit; Aspan, Anna; Erume, Joseph

    2016-08-05

    Enterotoxigenic E. coli (ETEC) significantly contribute to diarrhea in piglets and