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Sample records for human escherichia coli

  1. Multiplex PCR Assay for Identification of Human Diarrheagenic Escherichia coli

    OpenAIRE

    Toma, Claudia; Lu, Yan; Higa, Naomi; Nakasone, Noboru; Isabel CHINEN; Baschkier, Ariela; Rivas, Marta; Iwanaga, Masaaki

    2003-01-01

    A multiplex PCR assay for the identification of human diarrheagenic Escherichia coli was developed. The targets selected for each category were eae for enteropathogenic E. coli, stx for Shiga toxin-producing E. coli, elt and est for enterotoxigenic E. coli, ipaH for enteroinvasive E. coli, and aggR for enteroaggregative E. coli. This assay allowed the categorization of a diarrheagenic E. coli strain in a single reaction tube.

  2. Multiplex PCR Assay for Identification of Human Diarrheagenic Escherichia coli

    OpenAIRE

    2003-01-01

    A multiplex PCR assay for the identification of human diarrheagenic Escherichia coli was developed. The targets selected for each category were eae for enteropathogenic E. coli, stx for Shiga toxin-producing E. coli, elt and est for enterotoxigenic E. coli, ipaH for enteroinvasive E. coli, and aggR for enteroaggregative E. coli. This assay allowed the categorization of a diarrheagenic E. coli strain in a single reaction tube.

  3. The asymptomatic bacteriuria Escherichia coli strain 83972 outcompetes uropathogenic E. coli strains in human urine

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Ulett, G.C.; Schembri, M.A.

    2006-01-01

    Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU). In contrast to uropathogenic E. coli (UPEC), which causes symptomatic urinary tract infections (UTI), very little is known about the mechanisms by which these strains colonize the human urinary tract...

  4. Escherichia Coli

    Science.gov (United States)

    Goodsell, David S.

    2009-01-01

    Diverse biological data may be used to create illustrations of molecules in their cellular context. I describe the scientific results that support a recent textbook illustration of an "Escherichia coli cell". The image magnifies a portion of the bacterium at one million times, showing the location and form of individual macromolecules. Results…

  5. Phylogenetic analysis of Escherichia coli strains isolated from human samples

    Directory of Open Access Journals (Sweden)

    Abdollah Derakhshandeh

    2013-12-01

    Full Text Available Escherichia coli (E. coli is a normal inhabitant of the gastrointestinal tract of vertebrates, including humans. Phylogenetic analysis has shown that E. coli is composed of four main phylogenetic groups (A, B1, B2 and D. Group A and B1 are generally associated with commensals, whereas group B2 is associated with extra-intestinal pathotypes. Most enteropathogenic isolates, however, are assigned to group D. In the present study, a total of 102 E. coli strains, isolated from human samples, were used. Phylogenetic grouping was done based on the Clermont triplex PCR method using primers targeted at three genetic markers, chuA, yjaA and TspE4.C2. Group A contained the majority of the collected isolates (69 isolates, 67.64%, followed by group B2 (18 isolates, 17.64% and D (15 isolates, 14.7% and no strains were found to belong to group B1. The distribution of phylogenetic groups in our study suggests that although the majority of strains were commensals, the prevalence of enteropathogenic and extra-intestinal pathotypes was noteworthy. Therefore, the role of E. coli in human infections including diarrhea, urinary tract infections and meningitis should be considered.

  6. Curli fimbria: an Escherichia coli adhesin associated with human cystitis

    Directory of Open Access Journals (Sweden)

    Melina Aparecida Cordeiro

    2016-06-01

    Full Text Available Abstract Escherichia coli is the major causative agent of human cystitis. In this study, a preliminary molecular analysis carried out by PCR (polymerase chain reaction demonstrated that 100% of 31 E. coli strains isolated from patients with recurrent UTIs (urinary tract infections showed the presence of the curli fimbria gene (csgA. Curli fimbria is known to be associated with bacterial biofilm formation but not with the adhesion of human cystitis-associated E. coli. Therefore, this work aimed to study how curli fimbria is associated with uropathogenic E. coli (UPEC as an adhesion factor. For this purpose, the csgA gene was deleted from strain UPEC-4, which carries three adhesion factor genes (csgA, fimH and ompA. The wild-type UPEC-4 strain and its mutant (ΔcsgA were analyzed for their adhesion ability over HTB-9 (human bladder carcinoma, Vero (kidney cells of African green monkey and HUVEC (human umbilical vein cells in the presence of α-D-mannose. All the wild-type UPEC strains tested (100% were able to adhere to all three cell types, while the UPEC-4 ΔcsgA mutant lost its adherence to HTB-9 but continued to adhere to the HUVEC and Vero cells. The results suggest that curli fimbria has an important role in the adhesion processes associated with human UPEC-induced cystitis.

  7. Curli fimbria: an Escherichia coli adhesin associated with human cystitis.

    Science.gov (United States)

    Cordeiro, Melina Aparecida; Werle, Catierine Hirsch; Milanez, Guilherme Paier; Yano, Tomomasa

    2016-01-01

    Escherichia coli is the major causative agent of human cystitis. In this study, a preliminary molecular analysis carried out by PCR (polymerase chain reaction) demonstrated that 100% of 31 E. coli strains isolated from patients with recurrent UTIs (urinary tract infections) showed the presence of the curli fimbria gene (csgA). Curli fimbria is known to be associated with bacterial biofilm formation but not with the adhesion of human cystitis-associated E. coli. Therefore, this work aimed to study how curli fimbria is associated with uropathogenic E. coli (UPEC) as an adhesion factor. For this purpose, the csgA gene was deleted from strain UPEC-4, which carries three adhesion factor genes (csgA, fimH and ompA). The wild-type UPEC-4 strain and its mutant (ΔcsgA) were analyzed for their adhesion ability over HTB-9 (human bladder carcinoma), Vero (kidney cells of African green monkey) and HUVEC (human umbilical vein) cells in the presence of α-d-mannose. All the wild-type UPEC strains tested (100%) were able to adhere to all three cell types, while the UPEC-4 ΔcsgA mutant lost its adherence to HTB-9 but continued to adhere to the HUVEC and Vero cells. The results suggest that curli fimbria has an important role in the adhesion processes associated with human UPEC-induced cystitis.

  8. Measuring Escherichia coli Gene Expression during Human Urinary Tract Infections

    Science.gov (United States)

    Mobley, Harry L. T.

    2016-01-01

    Extraintestinal Escherichia coli (E. coli) evolved by acquisition of pathogenicity islands, phage, plasmids, and DNA segments by horizontal gene transfer. Strains are heterogeneous but virulent uropathogenic isolates more often have specific fimbriae, toxins, and iron receptors than commensal strains. One may ask whether it is the virulence factors alone that are required to establish infection. While these virulence factors clearly contribute strongly to pathogenesis, bacteria must survive by metabolizing nutrients available to them. By constructing mutants in all major metabolic pathways and co-challenging mice transurethrally with each mutant and the wild type strain, we identified which major metabolic pathways are required to infect the urinary tract. We must also ask what else is E. coli doing in vivo? To answer this question, we examined the transcriptome of E. coli CFT073 in the murine model of urinary tract infection (UTI) as well as for E. coli strains collected and analyzed directly from the urine of patients attending either a urology clinic or a university health clinic for symptoms of UTI. Using microarrays and RNA-seq, we measured in vivo gene expression for these uropathogenic E. coli strains, identifying genes upregulated during murine and human UTI. Our findings allow us to propose a new definition of bacterial virulence. PMID:26784237

  9. The asymptomatic bacteriuria Escherichia coli strain 83972 outcompetes uropathogenic E. coli strains in human urine

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Ulett, G.C.; Schembri, M.A.

    2006-01-01

    Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU). In contrast to uropathogenic E. coli (UPEC), which causes symptomatic urinary tract infections (UTI), very little is known about the mechanisms by which these strains colonize the human urinary tract....... The prototype ABU E. coli strain 83972 was originally isolated from a girl who had carried it asymptomatically for 3 years. Deliberate colonization of UTI-susceptible individuals with E. coli 83972 has been used successfully as an alternative approach for the treatment of patients who are refractory...... to conventional therapy. Colonization with strain 83972 appears to prevent infection with UPEC strains in such patients despite the fact that this strain is unable to express the primary adhesins involved in UTI, viz. P and type 1 fimbriae. Here we investigated the growth characteristics of E. coli 83972 in human...

  10. Evidence for a human-specific Escherichia coli clone.

    Science.gov (United States)

    Clermont, Olivier; Lescat, Mathilde; O'Brien, Claire L; Gordon, David M; Tenaillon, Olivier; Denamur, Erick

    2008-04-01

    Escherichia coli is a widespread commensal of the vertebrate intestinal tract. Until recently, no strong association between a particular clone and a given host species has been found. However, members of the B2 subgroup VIII clone with an O81 serotype appear to be human host specific. To determine the degree of host specificity exhibited by this clone, a PCR-based assay was used to screen 723 faecal and clinical isolates from humans, and 904 faecal isolates from animals. This clone was not detected among the animal isolates, but was discovered in people living in Africa, Europe and South America. The clone is rarely isolated from people suffering from intestinal or extraintestinal disease and is avirulent in a mouse model of extraintestinal infection. Fine-scale epidemiological analysis suggests that this clone is competitively dominant relative to other members of the B2 phylogenetic group and that it has increased in frequency over the past 20 years. This clone appears to be a good candidate for use as a probiotic, and may be suitable as an indicator of human faecal contamination in microbial source tracking studies.

  11. Recombinant production of human interleukin 6 in Escherichia coli.

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    Henrik Nausch

    Full Text Available In this study, we compared basic expression approaches for the efficient expression of bioactive recombinant human interleukin-6 (IL6, as an example for a difficult-to-express protein. We tested these approaches in a laboratory scale in order to pioneer the commercial production of this protein in Escherichia coli (E. coli. Among the various strategies, which were tested under Research and Development (R&D conditions, aggregation-prone IL6 was solubilized most effectively by co-expressing cytoplasmic chaperones. Expression of a Glutathion-S-Transferase (GST fusion protein was not efficient to increase IL6 solubility. Alteration of the cultivation temperature significantly increased the solubility in both cases, whereas reduced concentrations of IPTG to induce expression of the T7lac-promotor only had a positive effect on chaperone-assisted expression. The biological activity was comparable to that of commercial IL6. Targeting the expressed protein to an oxidizing environment was not effective in the generation of soluble IL6. Taken together, the presence of chaperones and a lowered cultivation temperature seem effective to isolate large quantities of soluble IL6. This approach led to in vivo soluble, functional protein fractions and reduces purification and refolding requirements caused by downstream purification procedures. The final yield of soluble recombinant protein averaged approximately 2.6 mg IL6/liter of cell culture. These findings might be beneficial for the development of the large-scale production of IL6 under the conditions of current good manufacturing practice (cGMP.

  12. Adhesion of enteropathogenic Escherichia coli to human intestinal enterocytes and cultured human intestinal mucosa.

    OpenAIRE

    1987-01-01

    The adhesion of classic enteropathogenic Escherichia coli (EPEC) strains of human origin to isolated human small intestinal enterocytes and cultured small intestinal mucosa was investigated. An adhesion assay with isolated human enterocytes prepared from duodenal biopsy samples was developed and tested with EPEC strains known to cause diarrhea in healthy adult volunteers. In the assay a mean of 53 and 55% of enterocytes had brush border-adherent E. coli E2348 (O127;H6) and E851 (O142:H6), res...

  13. Colonization of Enteroaggregative Escherichia coli and Shiga toxin-producing Escherichia coli in chickens and humans in southern Vietnam

    NARCIS (Netherlands)

    Trung, Nguyen Vinh; Nhung, Hoang Ngoc; Carrique-Mas, Juan J; Mai, Ho Huynh; Tuyen, Ha Thanh; Campbell, James; Nhung, Nguyen Thi; Van Minh, Pham; Wagenaar, Jaap A|info:eu-repo/dai/nl/126613354; Mai, Nguyen Thi Nhu; Hieu, Thai Quoc; Schultsz, Constance; Hoa, Ngo Thi

    2016-01-01

    BACKGROUND: Enteroaggregative (EAEC) and Shiga-toxin producing Escherichia coli (STEC) are a major cause of diarrhea worldwide. E. coli carrying both virulence factors characteristic for EAEC and STEC and producing extended-spectrum beta-lactamase caused severe and protracted disease during an

  14. Colonization of Enteroaggregative Escherichia coli and Shiga toxin-producing Escherichia coli in chickens and humans in southern Vietnam

    NARCIS (Netherlands)

    Trung, Nguyen Vinh; Nhung, Hoang Ngoc; Carrique-Mas, Juan J.; Mai, Ho Huynh; Tuyen, Ha Thanh; Campbell, James; Nhung, Nguyen Thi; Minh, Van Pham; Wagenaar, Jaap A.; Mai, Nguyen Thi Nhu; Hieu, Thai Quoc; Schultsz, Constance; Hoa, Ngo Thi

    2016-01-01

    Background: Enteroaggregative (EAEC) and Shiga-toxin producing Escherichia coli (STEC) are a major cause of diarrhea worldwide. E. coli carrying both virulence factors characteristic for EAEC and STEC and producing extended-spectrum beta-lactamase caused severe and protracted disease during an

  15. Colonization of Enteroaggregative Escherichia coli and Shiga toxin-producing Escherichia coli in chickens and humans in southern Vietnam

    NARCIS (Netherlands)

    Trung, Nguyen Vinh; Nhung, Hoang Ngoc; Carrique-Mas, Juan J.; Mai, Ho Huynh; Tuyen, Ha Thanh; Campbell, James; Nhung, Nguyen Thi; Minh, Van Pham; Wagenaar, Jaap A.; Mai, Nguyen Thi Nhu; Hieu, Thai Quoc; Schultsz, Constance; Hoa, Ngo Thi

    2016-01-01

    Background: Enteroaggregative (EAEC) and Shiga-toxin producing Escherichia coli (STEC) are a major cause of diarrhea worldwide. E. coli carrying both virulence factors characteristic for EAEC and STEC and producing extended-spectrum beta-lactamase caused severe and protracted disease during an ou

  16. Human Health Hazards from Antimicrobial-Resistant Escherichia coli of Animal Origin

    DEFF Research Database (Denmark)

    Hammerum, A. M.; Heuer, Ole Eske

    2009-01-01

    Because of the intensive use of antimicrobial agents in food animal production, meat is frequently contaminated with antimicrobial-resistant Escherichia coli. Humans can be colonized with E. coli of animal origin, and because of resistance to commonly used antimicrobial agents, these bacteria may...

  17. Escherichia coli lipoprotein binds human plasminogen via an intramolecular domain

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    Tammy eGonzalez

    2015-10-01

    Full Text Available Escherichia coli lipoprotein (Lpp is a major cellular component that exists in two distinct states, bound-form and free-form. Bound-form Lpp is known to interact with the periplasmic bacterial cell wall, while free-form Lpp is localized to the bacterial cell surface. A function for surface-exposed Lpp has yet to be determined. We hypothesized that the presence of C-terminal lysines in the surface-exposed region of Lpp would facilitate binding to the host zymogen plasminogen, a protease commandeered by a number of clinically important bacteria. Recombinant Lpp was synthesized and the binding of Lpp to plasminogen, the effect of various inhibitors on this binding, and the effects of various mutations of Lpp on Lpp-plasminogen interactions were examined. Additionally, the ability of Lpp-bound plasminogen to be converted to active plasmin was analyzed. We determined that Lpp binds plasminogen via an atypical domain located near the center of mature Lpp that may not be exposed on the surface of intact E. coli according to the current localization model. Finally, we found that plasminogen bound by Lpp can be converted to active plasmin. While the consequences of Lpp binding plasminogen are unclear, these results prompt further investigation of the ability of surface exposed Lpp to interact with host molecules such as extracellular matrix components and complement regulators, and the role of these interactions in infections caused by E. coli and other bacteria.

  18. Identification of factors in human urine that inhibit the binding of Escherichia coli adhesins.

    OpenAIRE

    1988-01-01

    Earlier studies on the binding of Escherichia coli adhesins to the human urinary tract have indicated that the ability to recognize binding sites on the urinary tract epithelial cells is not a characteristic for P fimbriae only, but is also shared by some other adhesins that are not associated with pyelonephritis, especially S fimbriae. In the present study we have investigated whether human urine contains inhibitors of the binding of E. coli adhesins. Normal human urine was found to inhibit ...

  19. Heterologously expressed bacterial and human multidrug resistance proteins confer cadmium resistance to Escherichia coli

    NARCIS (Netherlands)

    Achard-Joris, M; van Saparoea, HBV; Driessen, AJM; Bourdineaud, JP; Bourdineaud, Jean-Paul

    2005-01-01

    The human MDR1 gene is induced by cadmium exposure although no resistance to this metal is observed in human cells overexpressing hMDR1. To access the role of MDR proteins in cadmium resistance, human MDR1, Lactococcus lactis lmrA, and Oenococcus oeni omrA were expressed in an Escherichia coli tolC

  20. Zoonotic Escherichia coli

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    Wasteson Yngvild

    2002-03-01

    Full Text Available Escherichia coli is a normal inhabitant of the gastrointestinal tract of all warm-blooded animals, but variants of this species is also among the important etiological agents of enteritis and several extraintestinal diseases. The E. coli strains that cause diarrhoeal illness are categorised into pathogenicity groups based on virulence properties, mechanisms of pathogenicity, clinical symptoms and serology. The five main categories include enterotoxinogenic E. coli (ETEC, enteropathogenic E. coli (EPEC, enteroaggregative E. coli (EAggEC, enteroinvasive E. coli (EIEC and Shiga (Vero toxin-producing E. coli (STEC/VTEC. From a zoonotic point of view, STEC is the only E. coli pathogenicity group of major interest, as the shiga toxin-producing strains are able to cause severe disease in humans when being transmitted through the food chain from their animal reservoirs. The focus of this manuscript is therefore on STEC; pathogenicity factors, disease, the reservoirs and on-farm ecology, transmission into the food chain, growth and survival in food and in the environment, and the shiga toxin-encoding bacteriophages.

  1. Metabolic Modeling of Common Escherichia coli Strains in Human Gut Microbiome

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    Yue-Dong Gao

    2014-01-01

    Full Text Available The recent high-throughput sequencing has enabled the composition of Escherichia coli strains in the human microbial community to be profiled en masse. However, there are two challenges to address: (1 exploring the genetic differences between E. coli strains in human gut and (2 dynamic responses of E. coli to diverse stress conditions. As a result, we investigated the E. coli strains in human gut microbiome using deep sequencing data and reconstructed genome-wide metabolic networks for the three most common E. coli strains, including E. coli HS, UTI89, and CFT073. The metabolic models show obvious strain-specific characteristics, both in network contents and in behaviors. We predicted optimal biomass production for three models on four different carbon sources (acetate, ethanol, glucose, and succinate and found that these stress-associated genes were involved in host-microbial interactions and increased in human obesity. Besides, it shows that the growth rates are similar among the models, but the flux distributions are different, even in E. coli core reactions. The correlations between human diabetes-associated metabolic reactions in the E. coli models were also predicted. The study provides a systems perspective on E. coli strains in human gut microbiome and will be helpful in integrating diverse data sources in the following study.

  2. Asymptomatic bacteriuria Escherichia coli strains

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Nielsen, E.M.; Klemm, Per

    2006-01-01

    Urinary tract infections (UTIs) affect millions of people each year. Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU) in humans. Persons affected by ABU may carry a particular E. coli strain for extended periods of time without any symptoms. In contrast...

  3. Asymptomatic bacteriuria Escherichia coli strains

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Nielsen, E.M.; Klemm, Per

    2006-01-01

    Urinary tract infections (UTIs) affect millions of people each year. Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU) in humans. Persons affected by ABU may carry a particular E. coli strain for extended periods of time without any symptoms. In contrast...

  4. Expression and purification of recombinant human hemangiopoietin in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    Ren Qian; Ma Fengxia; Chen Zhong; Lu Shihong; Han Zhibo; Liu Yongiun; Xu Bin; Zhang Xiangyu; Han Zhongchao

    2008-01-01

    Objective: To express the soluble recombinant hemangiopoietin protein in E. coli BL21(DE3). Methods: Using human fetal live cDNA as a template, a partial cDNA fragment of HAPO coding N-terminal region was subcloned into plasmids pTrc99, pQE60 and pET32c to construct different recombinant prokaryotic expression systems. After selecting,the soluble rhHAPO fusion protein was expressed stably in E. coli BL21 (DE3) by vector pET32c-HAPO and further isolated by nickelnitrilotriacetic acid (NTA) affinity chromatography. After cleavage with enterokinase, the rhHAPO protein was applied to Fast Flow SP sepharose column. Results: The rhHAPO protein had a purity of more than 95% and a good bioactivity based on the cell adhesion assay in ECV304 cells. Conclusion: We have established a protein engineering system to produce rhHAPO which may provide the possibility for clinical application.

  5. Comparison of whole genome sequences from human and non-human Escherichia coli O26 strains

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    Keri N Norman

    2015-03-01

    Full Text Available Shiga toxin-producing Escherichia coli (STEC O26 is the second leading E. coli serogroup responsible for human illness outbreaks behind E. coli O157:H7. Recent outbreaks have been linked to emerging pathogenic O26:H11 strains harboring stx2 only. Cattle have been recognized as an important reservoir of O26 strains harboring stx1; however the reservoir of these emerging stx2 strains is unknown. The objective of this study was to identify nucleotide polymorphisms in human and cattle-derived strains in order to compare differences in polymorphism derived genotypes and virulence gene profiles between the two host species. Whole genome sequencing was performed on 182 epidemiologically unrelated O26 strains, including 109 human-derived strains and 73 non-human-derived strains. A panel of 289 O26 strains (241 STEC and 48 non-STEC was subsequently genotyped using a set of 283 polymorphisms identified by whole genome sequencing, resulting in 64 unique genotypes. Phylogenetic analyses identified seven clusters within the O26 strains. The seven clusters did not distinguish between isolates originating from humans or cattle; however, clusters did correspond with particular virulence gene profiles. Human and non-human-derived strains harboring stx1 clustered separately from strains harboring stx2, strains harboring eae, and non-STEC strains. Strains harboring stx2 were more closely related to non-STEC strains and strains harboring eae than to strains harboring stx1. The finding of human and cattle-derived strains with the same polymorphism derived genotypes and similar virulence gene profiles, provides evidence that similar strains are found in cattle and humans and transmission between the two species may occur.

  6. Taxonomy Icon Data: Escherichia coli [Taxonomy Icon

    Lifescience Database Archive (English)

    Full Text Available Escherichia coli Escherichia coli Escherichia_coli_L.png Escherichia_coli_NL.png Escherich...ia_coli_S.png Escherichia_coli_NS.png http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Escherichia+co...li&t=L http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Escherichia+coli&t=NL http://biosciencedbc.jp/taxono...my_icon/icon.cgi?i=Escherichia+coli&t=S http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Escherichia+coli&t=NS ...

  7. In vitro adhesion of enterotoxigenic Escherichia coli to human intestinal epithelial cells from mucosal biopsies.

    OpenAIRE

    1984-01-01

    An adhesion assay with isolated human enterocytes prepared from duodenal biopsies has been developed and tested by using human enterotoxigenic Escherichia coli expressing colonization factor antigens I and II (CFA/I and CFA/II) and type 1 fimbriae. Enterotoxigenic E. coli strains H10407 (CFA/I) and B2C (CFA/II) bound to duodenal enterocytes to a much greater extent (mean of 4.6 and 4.0 bacteria per brush border) than did strain H10407P, a CFA/I- mutant of H10407 (mean of 0.1 bacteria per brus...

  8. PATHOGENIC ESCHERICHIA COLI

    Science.gov (United States)

    Escherichia coli is a bacterial species which inhabits the gastrointestinal tract of man and warm-blooded animals. Because of the ubiquity of this bacterium in the intestinal flora, it serves as an important indicator organism of fecal contamination. E. coli, aside from serving a...

  9. Escherichia coli out in the cold: Dissemination of human-derived bacteria into the Antarctic microbiome.

    Science.gov (United States)

    Power, Michelle L; Samuel, Angelingifta; Smith, James J; Stark, Jonathon S; Gillings, Michael R; Gordon, David M

    2016-08-01

    Discharge of untreated sewage into Antarctic environments presents a risk of introducing non-native microorganisms, but until now, adverse consequences have not been conclusively identified. Here we show that sewage disposal introduces human derived Escherichia coli carrying mobile genetic elements and virulence traits with the potential to affect the diversity and evolution of native Antarctic microbial communities. We compared E. coli recovered from environmental and animal sources in Antarctica to a reference collection of E. coli from humans and non-Antarctic animals. The distribution of phylogenetic groups and frequency of 11 virulence factors amongst the Antarctic isolates were characteristic of E. coli strains more commonly associated with humans. The rapidly emerging E. coli ST131 and ST95 clones were found amongst the Antarctic isolates, and ST95 was the predominant E. coli recovered from Weddell seals. Class 1 integrons were found in 15% of the Antarctic E. coli with 4 of 5 identified gene cassette arrays containing antibiotic resistance genes matching those common in clinical contexts. Disposing untreated sewage into the Antarctic environment does disseminate non-native microorganisms, but the extent of this impact and implications for Antarctic ecosystem health are, as yet, poorly understood.

  10. Synthesis and evaluation of potential inhibitors of human and Escherichia coli histidine triad nucleotide binding proteins.

    Science.gov (United States)

    Bardaweel, Sanaa K; Ghosh, Brahma; Wagner, Carston R

    2012-01-01

    Based on recent substrate specificity studies, a series of ribonucleotide based esters and carbamates were synthesized and screened as inhibitors of the phosphoramidases and acyl-AMP hydrolases, Escherichia coli Histidine Triad Nucleotide Binding Protein (ecHinT) and human Histidine Triad Nucleotide Binding Protein 1 (hHint1). Using our established phosphoramidase assay, K(i) values were determined. All compounds exhibited non-competitive inhibition profiles. The carbamate based inhibitors were shown to successfully suppress the Hint1-associated phenotype in E. coli, suggesting that they are permeable intracellular inhibitors of ecHinT.

  11. Ultrastructural study of adhesion of enterotoxigenic Escherichia coli to erythrocytes and human intestinal epithelial cells.

    OpenAIRE

    1984-01-01

    The adhesion to erythrocytes and human intestinal epithelial cells of enterotoxigenic Escherichia coli strains H10407, B2C, and H10407P, expressing colonization factor antigen I (CFA/I), CFA/II, and type 1 fimbriae, respectively, was examined by electron microscopy. CFA and type 1 fimbriae were visualized by negative staining in thin sections after en bloc staining with ruthenium red and by immune labeling with antisera raised against purified fimbriae. By negative and ruthenium red staining,...

  12. Pathogenic potential of Escherichia coli clinical strains from orthopedic implant infections towards human osteoblastic cells

    Science.gov (United States)

    Crémet, Lise; Broquet, Alexis; Brulin, Bénédicte; Jacqueline, Cédric; Dauvergne, Sandie; Brion, Régis; Asehnoune, Karim; Corvec, Stéphane; Heymann, Dominique; Caroff, Nathalie

    2015-01-01

    Escherichia coli is one of the first causes of Gram-negative orthopedic implant infections (OII), but little is known about the pathogenicity of this species in such infections that are increasing due to the ageing of the population. We report how this pathogen interacts with human osteoblastic MG-63 cells in vitro, by comparing 20 OII E. coli strains to two Staphylococcus aureus and two Pseudomonas aeruginosa strains. LDH release assay revealed that 6/20 (30%) OII E. coli induced MG-63 cell lysis whereas none of the four control strains was cytotoxic after 4 h of coculture. This high cytotoxicity was associated with hemolytic properties and linked to hlyA gene expression. We further showed by gentamicin protection assay and confocal microscopy that the non-cytotoxic E. coli were not able to invade MG-63 cells unlike S. aureus strains (internalization rate coli versus 8.88 ± 2.31% and 4.60 ± 0.42% for both S. aureus). The non-cytotoxic E. coli also demonstrated low adherence rates (coli eliciting higher IL-6 and TNF-α mRNA expression in the osteoblastic cells. Either highly cytotoxic or slightly invasive OII E. coli do not show the same infection strategies as S. aureus towards osteoblasts. PMID:26333570

  13. Limited transmission of bla(CTX-M-9)-type-positive Escherichia coli between humans and poultry in Vietnam.

    Science.gov (United States)

    Ueda, Shuhei; Ngan, Bui Thi Kim; Huong, Bui Thi Mai; Hirai, Itaru; Tuyen, Le Danh; Yamamoto, Yoshimasa

    2015-01-01

    We examined whether Escherichia coli isolates that produce CTX-M-9-type extended-spectrum β-lactamases (ESBL) are transferred between humans and chickens in a Vietnamese community. The phylogenetic group compositions, sequence types, antimicrobial resistance profiles, the prevalence of plasmid antibiotic resistance genes, and the plasmid replicon types generally differed between the human and chicken E. coli isolates. Our results suggest that transmission of the bla(CTX-M-9)-positive E. coli between humans and poultry was limited.

  14. Dissemination of Cephalosporin Resistance Genes between Escherichia coli Strains from Farm Animals and Humans by Specific Plasmid Lineages

    NARCIS (Netherlands)

    de Been, Mark; Lanza, Val F.; de Toro, María; Scharringa, Jelle; Dohmen, Wietske|info:eu-repo/dai/nl/333690451; Du, Yu; Hu, Juan; Lei, Ying; Li, Ning; Tooming-Klunderud, Ave; Heederik, Dick J J|info:eu-repo/dai/nl/072910542; Fluit, Ad C.; Bonten, Marc J M; Willems, Rob J L; de la Cruz, Fernando; van Schaik, Willem

    2014-01-01

    Third-generation cephalosporins are a class of β-lactam antibiotics that are often used for the treatment of human infections caused by Gram-negative bacteria, especially Escherichia coli. Worryingly, the incidence of human infections caused by third-generation cephalosporin-resistant E. coli is inc

  15. Dissemination of Cephalosporin Resistance Genes between Escherichia coli Strains from Farm Animals and Humans by Specific Plasmid Lineages

    NARCIS (Netherlands)

    de Been, Mark; Lanza, Val F.; de Toro, María; Scharringa, Jelle; Dohmen, Wietske; Du, Yu; Hu, Juan; Lei, Ying; Li, Ning; Tooming-Klunderud, Ave; Heederik, Dick J J; Fluit, Ad C.; Bonten, Marc J M; Willems, Rob J L; de la Cruz, Fernando; van Schaik, Willem

    2014-01-01

    Third-generation cephalosporins are a class of β-lactam antibiotics that are often used for the treatment of human infections caused by Gram-negative bacteria, especially Escherichia coli. Worryingly, the incidence of human infections caused by third-generation cephalosporin-resistant E. coli is inc

  16. Global gene expression profiling of the asymptomatic bacteriuria Escherichia coli strain 83972 in the human urinary tract

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Klemm, Per

    2006-01-01

    Urinary tract infections (UTIs) are an important health problem worldwide, with many million cases each year. Escherichia coli is the most common organism causing UTIs in humans. The asymptomatic bacteriuria E. coli strain 83972 is an excellent colonizer of the human urinary tract, where it causes...

  17. Recombinant human lymphotoxin: Expression in Escherichia coli, purification, and some properties

    Energy Technology Data Exchange (ETDEWEB)

    Denisov, A.A.; Torskii, S.P.; Nikolaeva, O.G.; Volozhantseva, I.Y. [State Research Institute of Applied Microbiology, Obolensk (Russian Federation)

    1995-09-01

    Biosynthesis of human lymphotoxin lacking 21 amino acid residues was studied in the recombinant strain Escherichia coli SG20050/pLT21. The main content of the protein was found to be soluble and predominantly located in the cytoplasm of E. coli. Synthesis of soluble recombinant lymphotoxin was maximal under cultivation in Luria broth at 32{degrees}C for 24 h. A method for isolation and purification of the recombinant protein from E. coli was elaborated. The procedure includes gel filtration on Sephadex G-150 and ion-exchange chromatography on DEAE- and CM-Sephadex. The protein was obtained with 97-fold purification and final yield of 62%. The specific activity was 10{sup 8} units per mg protein. Some physicochemical properties of the protein were investigated. 24 refs., 5 figs., 2 tabs.

  18. Survival of Escherichia coli O26:H11 exceeds that of Escherichia coli O157:H7 as assessed by simulated human digestion of contaminated raw milk cheeses.

    Science.gov (United States)

    Miszczycha, Stéphane D; Thévenot, Jonathan; Denis, Sylvain; Callon, Cécile; Livrelli, Valérie; Alric, Monique; Montel, Marie-Christine; Blanquet-Diot, Stéphanie; Thevenot-Sergentet, Delphine

    2014-02-17

    Shiga toxin producing Escherichia coli (STEC) are an important cause of human foodborne outbreaks. The consumption of raw milk dairy products may be an important route of STEC infection. For successful foodborne transmission, STEC strains must survive stress conditions met during gastrointestinal transit in humans. The aim of this study was to evaluate the survival of two STEC strains of serotypes O157:H7 and O26:H11 during simulated human digestion in the TNO gastro-Intestinal tract Model (TIM) of contaminated uncooked pressed cheeses. The survival of cheese microflora during in vitro gastrointestinal transit was also determined for the first time. The level of STEC increased from 2 log₁₀ CFU/ml to 4 log₁₀ CFU/g during the first 24h of cheese making and remained stable at around 4 log₁₀ CFU/g during cheese ripening and conservation. During transit through the artificial stomach and duodenum, levels of STEC decreased: 0.2% of E. coli O157:H7 and 1.8% of E. coli O26:H11 were recovered at 150 min in the gastric compartment, compared with 14.3% for the transit marker. Bacterial resumption was observed in the jejunum and ileum: 35.8% of E. coli O157:H7 and 663.2% of E. coli O26:H11 were recovered at 360 min in the ileal compartment, compared with 12.6% for the transit marker. The fate of STEC was strain-dependent, the survival of E. coli O26:H11 being 13 times greater than that of E. coli O157:H7 at the end of digestion in the cumulative ileal deliveries. These data provide a better understanding of STEC behavior during gastrointestinal transit in humans after ingestion of contaminated cheese.

  19. Diarrheagenic Escherichia coli.

    Science.gov (United States)

    Gomes, Tânia A T; Elias, Waldir P; Scaletsky, Isabel C A; Guth, Beatriz E C; Rodrigues, Juliana F; Piazza, Roxane M F; Ferreira, Luís C S; Martinez, Marina B

    2016-12-01

    Most Escherichia coli strains live harmlessly in the intestines and rarely cause disease in healthy individuals. Nonetheless, a number of pathogenic strains can cause diarrhea or extraintestinal diseases both in healthy and immunocompromised individuals. Diarrheal illnesses are a severe public health problem and a major cause of morbidity and mortality in infants and young children, especially in developing countries. E. coli strains that cause diarrhea have evolved by acquiring, through horizontal gene transfer, a particular set of characteristics that have successfully persisted in the host. According to the group of virulence determinants acquired, specific combinations were formed determining the currently known E. coli pathotypes, which are collectively known as diarrheagenic E. coli. In this review, we have gathered information on current definitions, serotypes, lineages, virulence mechanisms, epidemiology, and diagnosis of the major diarrheagenic E. coli pathotypes.

  20. Expression and assembly of active human cardiac troponin in Escherichia coli.

    Science.gov (United States)

    Lassalle, Michael W

    2013-02-01

    Cardiomyopathy-related mutations in human cardiac troponin subunits, including troponin C (hcTnC), troponin I (hcTnI), and troponin T (hcTnT), are well-documented. Recently, it has been recognised that human cardiac troponin (hcTn) is a sophisticated allosteric system. Therefore, the effect of drugs on this protein complex should be studied with assembled hcTn rather than a short fragment of a subunit or the subunit itself. Here, we describe the expression and assembly of active hcTn in Escherichia coli, a novel method that is rapid and simple, and produces large amounts of functional hcTn.

  1. Recombinant expression of hydroxylated human collagen in Escherichia coli

    OpenAIRE

    Rutschmann, Christoph; Baumann, Stephan; Cabalzar, Jürg; Luther, Kelvin B.; Hennet, Thierry

    2014-01-01

    Collagen is the most abundant protein in the human body and thereby a structural protein of considerable biotechnological interest. The complex maturation process of collagen, including essential post-translational modifications such as prolyl and lysyl hydroxylation, has precluded large-scale production of recombinant collagen featuring the biophysical properties of endogenous collagen. The characterization of new prolyl and lysyl hydroxylase genes encoded by the giant virus mimivirus reveal...

  2. Expression in Escherichia coli of active human alcohol dehydrogenase lacking N-terminal acetylation.

    Science.gov (United States)

    Höög, J O; Weis, M; Zeppezauer, M; Jörnvall, H; von Bahr-Lindström, H

    1987-12-01

    Human alcohol dehydrogenase (ADH, beta beta isozyme of class I) was expressed in Escherichia coli, purified to homogeneity, and characterized regarding N-terminal processing. The expression system was obtained by ligation of a cDNA fragment corresponding to the beta-subunit of human liver alcohol dehydrogenase into the vector pKK 223-3 containing the tac promoter. The enzyme, detected by Western-blot analysis and ethanol oxidizing activity, constituted up to 3% of the total amount of protein. Recombinant ADH was separated from E. coli ADH by ion-exchange chromatography and the isolated enzyme was essentially pure as judged by SDS-polyacrylamide gel electrophoresis and sequence analysis. The N-terminal sequence was identical to that of the authentic beta-subunit except that the N-terminus was non-acetylated, indicating a correct removal of the initiator methionine, but lack of further processing.

  3. Emergence of Antimicrobial-Resistant Escherichia coli of Animal Origin Spreading in Humans

    Science.gov (United States)

    Skurnik, David; Clermont, Olivier; Guillard, Thomas; Launay, Adrien; Danilchanka, Olga; Pons, Stéphanie; Diancourt, Laure; Lebreton, François; Kadlec, Kristina; Roux, Damien; Jiang, Deming; Dion, Sara; Aschard, Hugues; Denamur, Maurice; Cywes-Bentley, Colette; Schwarz, Stefan; Tenaillon, Olivier; Andremont, Antoine; Picard, Bertrand; Mekalanos, John; Brisse, Sylvain; Denamur, Erick

    2016-01-01

    In the context of the great concern about the impact of human activities on the environment, we studied 403 commensal Escherichia coli/Escherichia clade strains isolated from several animal and human populations that have variable contacts to one another. Multilocus sequence typing (MLST) showed a decrease of diversity 1) in strains isolated from animals that had an increasing contact with humans and 2) in all strains that had increased antimicrobial resistance. A specific B1 phylogroup clonal complex (CC87, Institut Pasteur schema nomenclature) of animal origin was identified and characterized as being responsible for the increased antimicrobial resistance prevalence observed in strains from the environments with a high human-mediated antimicrobial pressure. CC87 strains have a high capacity of acquiring and disseminating resistance genes with specific metabolic and genetic determinants as demonstrated by high-throughput sequencing and phenotyping. They are good mouse gut colonizers but are not virulent. Our data confirm the predominant role of human activities in the emergence of antimicrobial resistance in the environmental bacterial strains and unveil a particular E. coli clonal complex of animal origin capable of spreading antimicrobial resistance to other members of microbial communities. PMID:26613786

  4. Characterization of pathogenic Escherichia coli isolated from humans in Austria : phenotypes, toxin gene types and epidemiology

    NARCIS (Netherlands)

    Wagner, M; Allerberger, F; Manafi, M; Lindner, G; Friedrich, A W; Sonntag, A-K; Foissy, H

    2004-01-01

    One hundred and ten clinical Escherichia coli isolates of serovar O157 (n = 102) and O26 (n = 8) were characterized for the presence of putative virulence genes by PCR. All but one of these isolates contained the eae gene. The EHEC-hly gene could be detected in all E. coli O157 and in 50% of E. coli

  5. Characterization of pathogenic Escherichia coli isolated from humans in Austria : phenotypes, toxin gene types and epidemiology

    NARCIS (Netherlands)

    Wagner, M; Allerberger, F; Manafi, M; Lindner, G; Friedrich, A W; Sonntag, A-K; Foissy, H

    2004-01-01

    One hundred and ten clinical Escherichia coli isolates of serovar O157 (n = 102) and O26 (n = 8) were characterized for the presence of putative virulence genes by PCR. All but one of these isolates contained the eae gene. The EHEC-hly gene could be detected in all E. coli O157 and in 50% of E. coli

  6. Tracking pathogen transmission at the human-wildlife interface: banded mongoose and Escherichia coli.

    Science.gov (United States)

    Pesapane, R; Ponder, M; Alexander, K A

    2013-06-01

    A primary challenge to managing emerging infectious disease is identifying pathways that allow pathogen transmission at the human-wildlife interface. Using Escherichia coli as a model organism, we evaluated fecal bacterial transmission between banded mongoose (Mungos mungo) and humans in northern Botswana. Fecal samples were collected from banded mongoose living in protected areas (n = 87, 3 troops) and surrounding villages (n = 92, 3 troops). Human fecal waste was collected from the same environment (n = 46). Isolates were evaluated for susceptibility to 10 antibiotics. Resistant E. coli isolates from mongoose were compared to human isolates using rep-PCR fingerprinting and MLST-PCR. Antimicrobial resistant isolates were identified in 57 % of the mongoose fecal samples tested (range 31-78% among troops). At least one individual mongoose fecal sample demonstrated resistance to each tested antibiotic, and multidrug resistance was highest in the protected areas (40.9%). E. coli isolated from mongoose and human sources in this study demonstrated an extremely high degree of genetic similarity on rep-PCR (AMOVA, F ST = 0.0027, p = 0.18) with a similar pattern identified on MLST-PCR. Human waste may be an important source of microbial exposure to wildlife. Evidence of high levels of antimicrobial resistance even within protected areas identifies an emerging health threat and highlights the need for improved waste management in these systems.

  7. [Optimization of fermentation of recombinant human Endostatin (rh-Endostatin) expression in Escherichia coli].

    Science.gov (United States)

    Chang, Guo-Dong; Li, Zhuang-Lin; Qin, Jia-Yang; Ma, Cui-Qing; Luo, Yong-Zhang; Xu, Ping

    2005-07-01

    The fermentation process of recombinant human Endostatin expression in Escherichia coli BL21 (DE3) was studied. The effects of factors such as concentration of IPTG, induction time, cultivation temperature and feeding strategies were investigated. Beside that, by changing the temperature to 40 degrees C after induction, the high-density cultivation finished in a much shorter period. After 9 hours cultivation, the optical density (OD) at 600 nm reached 140 and the yield of inclusion body was 3 g/L. While E. coli system was used, protein with better activity and stability was obtained. The cost was much lower and the producing process was much steadier. It will meet the demands of the industrial production.

  8. Key features of mcr-1-bearing plasmids from Escherichia coli isolated from humans and food.

    Science.gov (United States)

    Zurfluh, Katrin; Nüesch-Inderbinen, Magdalena; Klumpp, Jochen; Poirel, Laurent; Nordmann, Patrice; Stephan, Roger

    2017-01-01

    Mcr-1-harboring Enterobacteriaceae are reported worldwide since their first discovery in 2015. However, a limited number of studies are available that compared full-length plasmid sequences of human and animal origins. In this study, mcr-1-bearing plasmids from seven Escherichia coli isolates recovered from patients (n = 3), poultry meat (n = 2) and turkey meat (n = 2) in Switzerland were further analyzed and compared. Isolates were characterized by multilocus sequence typing (MLST). The mcr-1-bearing plasmids were transferred by transformation into reference strain E. coli DH5α and MCR-1-producing transformants were selected on LB-agar supplemented with 2 mg/L colistin. Purified plasmids were then sequenced and compared. MLST revealed six distinct STs, illustrating the high clonal diversity among mcr-1-positive E. coli isolates of different origins. Two different mcr-1-positive plasmids were identified from a single E. coli ST48 human isolate. All other isolates possessed a single mcr-1 harboring plasmid. Transferable IncI2 (size ca. 60-61 kb) and IncX4 (size ca. 33-35 kb) type plasmids each bearing mcr-1 were found associated with human and food isolates. None of the mcr-1-positive IncI2 and IncX4 plasmids possessed any additional resistance determinants. Surprisingly, all but one of the sequenced mcr-1-positive plasmids lacked the ISApl1 element, which is a key element mediating acquisition of mcr-1 into various plasmid backbones. There is strong evidence that the food chain may be an important transmission route for mcr-1-bearing plasmids. Our data suggest that some "epidemic" plasmids rather than specific E. coli clones might be responsible for the spread of the mcr-1 gene along the food chain.

  9. Human and Avian Extraintestinal Pathogenic Escherichia coli: Infections, Zoonotic Risks, and Antibiotic Resistance Trends

    Science.gov (United States)

    2013-01-01

    Abstract Extraintestinal pathogenic Escherichia coli (ExPEC) constitutes ongoing health concerns for women, newborns, elderly, and immunocompromised individuals due to increased numbers of urinary tract infections (UTIs), newborn meningitis, abdominal sepsis, and septicemia. E. coli remains the leading cause of UTIs, with recent investigations reporting the emergence of E. coli as the predominant cause of nosocomial and neonatal sepsis infections. This shift from the traditional Gram-positive bacterial causes of nosocomial and neonatal sepsis infections could be attributed to the use of intrapartum chemoprophylaxis against Gram-positive bacteria and the appearance of antibiotic (ATB) resistance in E. coli. While ExPEC strains cause significant healthcare concerns, these bacteria also infect chickens and cause the poultry industry economic losses due to costs of containment, mortality, and disposal of carcasses. To circumvent ExPEC-related costs, ATBs are commonly used in the poultry industry to prevent/treat microbial infections and promote growth and performance. In an unfortunate linkage, chicken products are suspected to be a source of foodborne ExPEC infections and ATB resistance in humans. Therefore, the emergence of multidrug resistance (MDR) (resistance to three or more classes of antimicrobial agents) among avian E. coli has created major economic and health concerns, affecting both human healthcare and poultry industries. Increased numbers of immunocompromised individuals, including the elderly, coupled with MDR among ExPEC strains, will continue to challenge the treatment of ExPEC infections and likely lead to increased treatment costs. With ongoing complications due to emerging ATB resistance, novel treatment strategies are necessary to control ExPEC infections. Recognizing and treating the zoonotic risk posed by ExPEC would greatly enhance food safety and positively impact human health. PMID:23962019

  10. Comparison of Escherichia coli Strains Recovered from Human Cystitis and Pyelonephritis Infections in Transurethrally Challenged Mice

    Science.gov (United States)

    Johnson, David E.; Lockatell, C. Virginia; Russell, Robert G.; Hebel, J. Richard; Island, Michael D.; Stapleton, Ann; Stamm, Walter E.; Warren, John W.

    1998-01-01

    Urinary tract infection, most frequently caused by Escherichia coli, is one of the most common bacterial infections in humans. A vast amount of literature regarding the mechanisms through which E. coli induces pyelonephritis has accumulated. Although cystitis accounts for 95% of visits to physicians for symptoms of urinary tract infections, few in vivo studies have investigated possible differences between E. coli recovered from patients with clinical symptoms of cystitis and that from patients with symptoms of pyelonephritis. Epidemiological studies indicate that cystitis-associated strains appear to differ from pyelonephritis-associated strains in elaboration of some putative virulence factors. With transurethrally challenged mice we studied possible differences using three each of the most virulent pyelonephritis and cystitis E. coli strains in our collection. The results indicate that cystitis strains colonize the bladder more rapidly than do pyelonephritis strains, while the rates of kidney colonization are similar. Cystitis strains colonize the bladder in higher numbers, induce more pronounced histologic changes in the bladder, and are more rapidly eliminated from the mouse urinary tract than pyelonephritis strains. These results provide evidence that cystitis strains differ from pyelonephritis strains in this model, that this model is useful for the study of the uropathogenicity of cystitis strains, and that it would be unwise to use pyelonephritis strains to study putative virulence factors important in the development of cystitis. PMID:9632566

  11. Human C5a anaphylatoxin: gene cloning and expression in Escherichia coli.

    Science.gov (United States)

    Bautsch, W; Emde, M; Kretzschmar, T; Köhl, J; Suckau, D; Bitter-Suermann, D

    1992-06-01

    A gene coding for the human anaphylatoxin C5a was cloned and expressed in Escherichia coli. A combination of reverse transcription of mRNA of the U937 cell line with subsequent preparative polymerase chain reaction was employed to obtain the gene. The sequence was cloned into the plasmid vector pKK 233-2 behind an ATG initiation codon under the control of a trc promotor. After purification by ion exchange chromatography and reversed phase FPLC a mixture of predominantly non-glycosylated recombinant human C5a with a beta-mercaptoethanol adduct at cysteine 27 and the N-methionyl derivative was obtained which was homogeneous on silver-stained gels, immunoreactive with C5a-specific monoclonal antibodies and functionally active in releasing myeloperoxidase from human granulocytes and ATP from guinea pig platelets. The final yield was about 0.4-0.8 mg purified recombinant C5a per liter bacterial culture.

  12. Escherichia coli RecG functionally suppresses human Bloom syndrome phenotypes

    Directory of Open Access Journals (Sweden)

    Killen Michael W

    2012-10-01

    Full Text Available Abstract Defects in the human BLM gene cause Bloom syndrome, notable for early development of tumors in a broad variety of tissues. On the basis of sequence similarity, BLM has been identified as one of the five human homologs of RecQ from Escherichia coli. Nevertheless, biochemical characterization of the BLM protein indicates far greater functional similarity to the E. coli RecG protein and there is no known RecG homolog in human cells. To explore the possibility that the shared biochemistries of BLM and RecG may represent an example of convergent evolution of cellular function where in humans BLM has evolved to fulfill the genomic stabilization role of RecG, we determined whether expression of RecG in human BLM-deficient cells could suppress established functional cellular Bloom syndrome phenotypes. We found that RecG can indeed largely suppress both the definitive elevated sister chromatid exchange phenotype and the more recently demonstrated gene cluster instability phenotype of BLM-deficient cells. In contrast, expression of RecG has no impact on either of these phenotypes in human cells with functional BLM protein. These results suggest that the combination of biochemical activities shared by RecG and BLM fill the same evolutionary niche in preserving genomic integrity without requiring exactly identical molecular mechanisms.

  13. Escherichia coli RecG functionally suppresses human Bloom syndrome phenotypes.

    Science.gov (United States)

    Killen, Michael W; Stults, Dawn M; Wilson, William A; Pierce, Andrew J

    2012-10-30

    Defects in the human BLM gene cause Bloom syndrome, notable for early development of tumors in a broad variety of tissues. On the basis of sequence similarity, BLM has been identified as one of the five human homologs of RecQ from Escherichia coli. Nevertheless, biochemical characterization of the BLM protein indicates far greater functional similarity to the E. coli RecG protein and there is no known RecG homolog in human cells. To explore the possibility that the shared biochemistries of BLM and RecG may represent an example of convergent evolution of cellular function where in humans BLM has evolved to fulfill the genomic stabilization role of RecG, we determined whether expression of RecG in human BLM-deficient cells could suppress established functional cellular Bloom syndrome phenotypes. We found that RecG can indeed largely suppress both the definitive elevated sister chromatid exchange phenotype and the more recently demonstrated gene cluster instability phenotype of BLM-deficient cells. In contrast, expression of RecG has no impact on either of these phenotypes in human cells with functional BLM protein. These results suggest that the combination of biochemical activities shared by RecG and BLM fill the same evolutionary niche in preserving genomic integrity without requiring exactly identical molecular mechanisms.

  14. Host-specific induction of Escherichia coli fitness genes during human urinary tract infection.

    Science.gov (United States)

    Subashchandrabose, Sargurunathan; Hazen, Tracy H; Brumbaugh, Ariel R; Himpsl, Stephanie D; Smith, Sara N; Ernst, Robert D; Rasko, David A; Mobley, Harry L T

    2014-12-23

    Uropathogenic Escherichia coli (UPEC) is the predominant etiological agent of uncomplicated urinary tract infection (UTI), manifested by inflammation of the urinary bladder, in humans and is a major global public health concern. Molecular pathogenesis of UPEC has been primarily examined using murine models of UTI. Translational research to develop novel therapeutics against this major pathogen, which is becoming increasingly antibiotic resistant, requires a thorough understanding of mechanisms involved in pathogenesis during human UTIs. Total RNA-sequencing (RNA-seq) and comparative transcriptional analysis of UTI samples to the UPEC isolates cultured in human urine and laboratory medium were used to identify novel fitness genes that were specifically expressed during human infection. Evidence for UPEC genes involved in ion transport, including copper efflux, nickel and potassium import systems, as key fitness factors in uropathogenesis were generated using an experimental model of UTI. Translational application of this study was investigated by targeting Cus, a bacterial copper efflux system. Copper supplementation in drinking water reduces E. coli colonization in the urinary bladder of mice. Additionally, our results suggest that anaerobic processes in UPEC are involved in promoting fitness during UTI in humans. In summary, RNA-seq was used to establish the transcriptional signature in UPEC during naturally occurring, community acquired UTI in women and multiple novel fitness genes used by UPEC during human infection were identified. The repertoire of UPEC genes involved in UTI presented here will facilitate further translational studies to develop innovative strategies against UTI caused by UPEC.

  15. One-step purification of soluble recombinant human 6-phosphogluconate dehydrogenase from Escherichia coli.

    Science.gov (United States)

    Chan, Barden; Sukhatme, Vikas P

    2013-11-01

    6-Phosphogluconate dehydrogenase (6PGD), the third enzyme in the pentose phosphate pathway, was recently identified as a novel target in human lung cancer. In this report, we present an expression and purification scheme of recombinant human 6PGD from Escherichia coli. Using a DE3 derivative strain expressing tRNAs for seven rare codons in E. coli called Rosetta2 (DE3), a large quantity of soluble human 6PGD can be expressed with an N-terminal histidine tag and purified by a one-step purification procedure to near homogeneity without denaturants or refolding. Three to seven milligrams of purified protein could be obtained from 100 ml of culture. This recombinant human 6PGD follows classic Michaelis-Menton saturation kinetics with respect to both substrates NADP(+) and 6-phosphogluconate. The respective k(cat) and K(m) were comparable to those of 6PGDs purified from mammalian tissues. Using this purified 6PGD enzyme, we devised an endpoint colorimetric assay suitable for high-throughput screening for human 6PGD inhibitors.

  16. Escherichia coli isolated from feces of brown bears (Ursus arctos) have a lower prevalence of human extraintestinal pathogenic E. coli virulence-associated genes.

    Science.gov (United States)

    Vadnov, Maruša; Barbič, Damjana; Žgur-Bertok, Darja; Erjavec, Marjanca Starčič

    2017-01-01

    Eighty-six Escherichia coli strains from feces of either wild brown bears or those living in a zoo were screened for phylogenetic groups using the revisited Clermont phylotyping method and the prevalence of 24 virulence-associated genes (VAGs) of extraintestinal pathogenic E. coli (ExPEC). Our results showed that most strains of E. coli in bears belonged to phylogenetic groups III/IV/V (29%) and B1 (26%). Only half of the tested VAGs were found in the E. coli bear strains, with fimH present in 72%, ompT in 63%, and kpsMT in 43% of the strains. When the data obtained on the fecal E. coli strains from brown bears were compared with the data obtained on 90 fecal E. coli strains from healthy humans, there were significant differences in E. coli population structures between both hosts.

  17. Comparison of ruminant and human attaching and effacing Escherichia coli (AEEC) strains.

    Science.gov (United States)

    Horcajo, Pilar; Domínguez-Bernal, Gustavo; de la Fuente, Ricardo; Ruiz-Santa-Quiteria, José A; Blanco, Jesús E; Blanco, Miguel; Mora, Azucena; Dahbi, Ghizlane; López, Cecilia; Puentes, Beatriz; Alonso, María Pilar; Blanco, Jorge; Orden, José A

    2012-03-23

    The presence of 12 genes associated with virulence in human attaching and effacing Escherichia coli (AEEC) was studied within a collection of 20 enterohemorrhagic E. coli (EHEC) and 206 atypical enteropathogenic E. coli (EPEC) isolated from ruminants. In addition, virulence genes and the clonal relationship of 49 atypical EPEC O26 strains isolated from humans and ruminants were compared to clarify whether ruminants serve as a reservoir of atypical EPEC for humans. A great diversity in the content of virulence gene was found. Thus, the espH, espG and map genes were detected in more than 85% of ruminant AEEC strains; the tccP2, espI, efa1/lifA, ehxA and paa genes were present in 50-70% of strains; and other genes such as tccP, espP, katP and toxB were detected in <25% of strains. EHEC strains contained more virulence genes than atypical EPEC strains. Our results suggest for the first time that the efa1/lifA gene is associated with diarrhea in newborn ruminants and that the AEEC strains with the H11 flagellar antigen are potentially more virulent than the non-H11 AEEC strains. Importantly, we identified a new intimin variant gene, eaeρ, in three ruminant atypical EPEC strains. The comparison of ruminant and human EPEC O26 strains showed that some ruminant strains possess virulence gene profiles and pulse-field gel electrophoresis pulsotypes similar to those of human strains. In conclusion, our data suggest that atypical EPEC is a heterogeneous group with different pathogenic potential and that ruminants could serve as a reservoir of atypical EPEC for humans.

  18. Complete solubilization and purification of recombinant human growth hormone produced in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Min-Ji Kim

    Full Text Available High-level expression of recombinant human growth hormone (hGH in Escherichia coli (E. coli leads to the formation of insoluble aggregates as inclusion bodies devoid of biological activity. Until recently, significant efforts have been made to improve the recovery of active hGH from inclusion bodies. Here, we developed an efficient procedure for the production of completely soluble hGH by minimizing the formation of inclusion bodies and optimizing protein purification conditions. Under the newly established conditions we were able to obtain most of the total hGH in the soluble fraction. We show that the soluble protein can be efficiently purified in high yield by a series of chromatographic procedures. We analyzed the resulting hGH using various analytical techniques such as reversed-phase high-performance liquid chromatography (RP-HPLC, size-exclusion chromatography (SEC, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF mass spectrometry, and circular dichroism (CD. These multiple analyses support the conclusion that we obtained highly pure hGH with the expected molecular mass and intact secondary structure. The biological activity of purified hGH was also confirmed by evaluating its growth-promoting effect using a cell proliferation assay. Taken together, we describe a straightforward strategy for the production of completely soluble and biologically active hGH in E. coli.

  19. Functional expression of a human GDP-L-fucose transporter in Escherichia coli.

    Science.gov (United States)

    Förster-Fromme, Karin; Schneider, Sarah; Sprenger, Georg A; Albermann, Christoph

    2017-02-01

    To investigate the translocation of nucleotide-activated sugars from the cytosol across a membrane into the endoplasmatic reticulum or the Golgi apparatus which is an important step in the synthesis of glycoproteins and glycolipids in eukaryotes. The heterologous expression of the recombinant and codon-adapted human GDP-L-fucose antiporter gene SLC35C1 (encoding an N-terminal OmpA-signal sequence) led to a functional transporter protein located in the cytoplasmic membrane of Escherichia coli. The in vitro transport was investigated using inverted membrane vesicles. SLC35C1 is an antiporter specific for GDP-L-fucose and depending on the concomitant reverse transport of GMP. The recombinant transporter FucT1 exhibited an activity for the transport of (3)H-GDP-L-fucose with a Vmax of 8 pmol/min mg with a Km of 4 µM. The functional expression of SLC35C1 in GDP-L-fucose overproducing E. coli led to the export of GDP-L-fucose to the culture supernatant. The export of GDP-L-fucose by E. coli provides the opportunity for the engineering of a periplasmatic fucosylation reaction in recombinant bacterial cells.

  20. Cloning and Expression of Human Keratinocyte Growth Factor in Escherichia coli for Recombinant Drug Production

    Directory of Open Access Journals (Sweden)

    Ebrahimzadeh

    2014-09-01

    Full Text Available Background Keratinocyte growth factor (KGF is a member of fibroblast growth factor (FGF family which induces proliferation and differentiation in a wide variety of epithelial tissues. KGF plays an important role in protection, repair of various types of epithelial cells, and re-epithelialization of wounds. Therefore, in patients with hematologic malignancies receiving high doses of chemotherapy and radiotherapy, treatment with KGF decreases the incidence and duration of severe oral mucositis. Objectives The aim of this study was to express the recombinant form of human keratinocyte growth factor in Escherichia coli. Materials and Methods KGF gene was amplified by PCR and cloned into the expression vector pET28a(+. The recombinant vectors were transformed into E. coli BL21(DE3 as expression host and expression of the desired protein was induced by IPTG. The expression was evaluated at RNA and protein levels by reverse transcriptase PCR (RT-PCR and SDS-PAGE analyses, respectively and the expressed protein was confirmed through western blotting. Results Cloning was confirmed by PCR and restriction digestion. RT-PCR and SDS-PAGE represented expression of KGF in E. coli. The optimized expression was achieved 16 hours after induction with 0.3 mM IPTG at 37°C in luria broth (LB containing kanamycin. The 18 kDa protein was confirmed by western blotting, using anti-His antibodies. Conclusions The result of the present study indicated that E. coli expression system was suitable for overexpression of recombinant human KGF and the expressed protein can be considered as a homemade product.

  1. Expression and purification of biologically active recombinant human paraoxonase 1 from inclusion bodies of Escherichia coli.

    Science.gov (United States)

    Bajaj, Priyanka; Tripathy, Rajan K; Aggarwal, Geetika; Pande, Abhay H

    2015-11-01

    Human PON1 (h-PON1) is a Ca(2+)-dependent serum enzyme and can hydrolyze (and inactivate) a wide range of substrates. It is a multifaceted enzyme and exhibit anti-inflammatory, anti-oxidative, anti-atherogenic, anti-diabetic, anti-microbial, and organophosphate (OP)-detoxifying properties. Thus, h-PON1 is a strong candidate for the development of therapeutic intervention against these conditions in humans. Insufficient hydrolyzing activity of native h-PON1 against desirable substrate affirms the urgent need to develop improved variant(s) of h-PON1 having enhanced activity. Production of recombinant h-PON1 (rh-PON1) using an Escherichia coli expression system is a key to develop such variant(s). However, generation of rh-PON1 using E. coli expression system has been elusive until now because of the aggregation of over-expressed rh-PON1 protein in inactive form as inclusion bodies (IBs) in the bacterial cells. In this study, we have over-expressed rh-PON1(wt) and rh-PON1(H115W;R192K) proteins as IBs in E. coli, and refolded the inactive enzymes present in the IBs to their active form using in vitro refolding. The active enzymes were isolated from the refolding mixture by ion-exchange chromatography. The catalytic properties of the refolded enzymes were similar to their soluble counterparts. Our results show that the pure and the active variant of rh-PON1 enzyme having enhanced hydrolyzing activity can be produced in large quantities using E. coli expression system. This method can be used for the industrial scale production of rh-PON1 enzymes and will aid in developing h-PON1 as a therapeutic candidate.

  2. Interactions between microsatellite instability and human gut colonization by Escherichia coli in colorectal cancer.

    Science.gov (United States)

    Gagnière, Johan; Bonnin, Virginie; Jarrousse, Anne-Sophie; Cardamone, Emilie; Agus, Allison; Uhrhammer, Nancy; Sauvanet, Pierre; Déchelotte, Pierre; Barnich, Nicolas; Bonnet, Richard; Pezet, Denis; Bonnet, Mathilde

    2017-01-16

    Recent studies suggest that colonization of colonic mucosa by pathogenic Escherichia coli (E. coli) could be involved in the development of colorectal cancer (CRC), especially through the production of genotoxins such as colibactin and/or by interfering with the DNA mismatch repair (MMR) pathway which leads to microsatellite instability (MSI). This work, performed on 88 CRC patients, revealed a significant increase in E. coli colonization in the MSI CRC phenotype. In the same way, E. coli persistence and internalization were increased in vitro in MMR-deficient cells. Moreover, we demonstrated that colibactin-producing E. coli induce inhibition of the MLH1 MMR proteins, which could lead to genomic instability. However, colibactin-producing E. coli were more frequently identified in microsatellite stable (MSS) CRC. This work suggests differences in the involvement of colibactin-producing E. coli in colorectal carcinogenesis according to the CRC phenotype. Further host/pathogens interactions studies should take into account CRC phenotypes.

  3. Identification of factors in human urine that inhibit the binding of Escherichia coli adhesins.

    Science.gov (United States)

    Parkkinen, J; Virkola, R; Korhonen, T K

    1988-10-01

    Earlier studies on the binding of Escherichia coli adhesins to the human urinary tract have indicated that the ability to recognize binding sites on the urinary tract epithelial cells is not a characteristic for P fimbriae only, but is also shared by some other adhesins that are not associated with pyelonephritis, especially S fimbriae. In the present study we have investigated whether human urine contains inhibitors of the binding of E. coli adhesins. Normal human urine was found to inhibit hemagglutination by S and type 1 fimbriae but not P fimbriae. The major inhibitor of S fimbriae in normal urine was identified as Tamm-Horsfall glycoprotein, and the interaction with S fimbriae is probably mediated by its sialyloligosaccharide chains. No significant variation was observed in the inhibitory effect of T-H glycoprotein preparations originating from different individuals. In contrast to S fimbriae, the major inhibitors of type 1 fimbriae in urine were identified as low-molecular-weight compounds. Gel filtration and ion-exchange chromatography and alpha-mannosidase treatment indicated that they were neutral alpha-mannosides, probably manno-oligosaccharides with three to five saccharides. Studies of urine samples collected from several individuals indicated the common occurrence of these inhibitory alpha-mannosides. Type 1 fimbriae bound to immobilized T-H glycoprotein, but, unlike S fimbriae, their binding was poorly inhibited by soluble T-H glycoprotein. Some urine samples were also found to contain low-molecular-weight inhibitors for the O75X adhesin of E. coli. These results emphasize that to function as a virulence factor in human urinary tract infections, an adhesin must evidently recognize such receptor structures at the infection sites that are not excreted in soluble form in urine. This prerequisite is filled by P fimbriae but not by type 1 or S fimbriae.

  4. Simplified Method to Produce Human Bioactive Leukemia Inhibitory Factor in Escherichia coli

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    Houman Kahroba

    2016-07-01

    Full Text Available Background Human leukemia inhibitory factor (hLIF is a poly functional cytokine with numerous regulatory effects on different cells. Main application of hLIF is maintaining pluripotency of embryonic stem cells. hLIF indicated effective work in implantation rate of fertilized eggs and multiple sclerosis (MS treatment. Low production of hLIF in eukaryotic cells and prokaryotic host’s problems for human protein production convinced us to develop a simple way to reach high amount of this widely used clinical and research factor. Objectives In this study we want to purify recombinant human leukemia inhibitory factor in single simple method. Materials and Methods This is an experimental study, gene expression: human LIF gene was codon optimized for expression in Escherichia coli and attached his-tag tail to make it extractable. After construction and transformation of vector to E. coli, isopropyl β-D-1-thiogalactopyranoside (IPTG used for induction. Single step immobilized metal affinity chromatography (IMAC used for purification confirmed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE and western blotting. Bioactivity of the hLIF were tested by MTT assay with TF-1 cells and CISH gene stimulation in monocyte and TF-1 by real-time PCR. Induction by 0.4 mM of IPTG in 25°C for 3 hours indicated best result for soluble expression. SPSS indicated P ˂ 0.05 that is significant for our work. Results Cloning, expression, and extraction of bio active rhLIF was successfully achieved according MTT assay and real time PCR after treatment of TF-1 and monocyte cell lines. Conclusions We developed an effective single step purification method to produce bioactive recombinant hLIF in E. coli. For the first time we used CISH gene stimulating for bioactivity test for qualifying of recombinant hLIF for application.

  5. Shiga toxin 1c-producing Escherichia coli strains : phenotypic and genetic characterization and association with human disease

    NARCIS (Netherlands)

    Friedrich, Alexander W; Borell, Julia; Bielaszewska, Martina; Fruth, Angelika; Tschäpe, Helmut; Karch, Helge

    2003-01-01

    The distribution of the stx(1c) allele among Shiga toxin (Stx)-producing Escherichia coli (STEC) and the virulence characteristics of stx(1c)-harboring STEC are unknown. In this study, we identified stx(1c) in 76 (54.3%) of 140 eae-negative, but in none of 155 eae-positive, human STEC isolates (P <

  6. Genetic Features Differentiating Bovine, Food, and Human Isolates of Shiga Toxin-Producing Escherichia coli O157 in The Netherlands

    NARCIS (Netherlands)

    Franz, E.; Hoek, van A.H.A.M.; Wal, van der F.J.; Boer, de A.G.; Zwartkruis-Nahuis, A.; Zwaluw, van der K.; Aarts, H.J.M.; Heuvelink, A.E.

    2012-01-01

    The frequency of Escherichia coli O157 genotypes among bovine, food, and human clinical isolates from The Netherlands was studied. Genotyping included the lineage-specific polymorphism assay (LSPA6), the Shiga-toxin-encoding bacteriophage insertion site assay (SBI), and PCR detection and/or subtypin

  7. Role of urinary cathelicidin LL-37 and human β-defensin 1 in uncomplicated Escherichia coli urinary tract infections

    DEFF Research Database (Denmark)

    Nielsen, Karen L; Dynesen, Pia; Larsen, Preben

    2014-01-01

    Cathelicidin (LL-37) and human β-defensin 1 (hBD-1) are important components of the innate defense in the urinary tract. The aim of this study was to characterize whether these peptides are important for developing uncomplicated Escherichia coli urinary tract infections (UTIs...

  8. Shiga toxin 1c-producing Escherichia coli strains : phenotypic and genetic characterization and association with human disease

    NARCIS (Netherlands)

    Friedrich, Alexander W; Borell, Julia; Bielaszewska, Martina; Fruth, Angelika; Tschäpe, Helmut; Karch, Helge

    2003-01-01

    The distribution of the stx(1c) allele among Shiga toxin (Stx)-producing Escherichia coli (STEC) and the virulence characteristics of stx(1c)-harboring STEC are unknown. In this study, we identified stx(1c) in 76 (54.3%) of 140 eae-negative, but in none of 155 eae-positive, human STEC isolates (P <

  9. Emergence of Escherichia coli encoding Shiga toxin 2f in human Shiga toxin-producing E-coli (STEC) infections in the Netherlands, January 2008 to December 2011

    NARCIS (Netherlands)

    Friesema, I.; van der Zwaluw, K.; Schuurman, T.; Kooistra-Smid, M.; Franz, E.; van Duynhoven, Y.; van Pelt, W.

    2014-01-01

    The Shiga toxins of Shiga toxin-producing Escherichia coli (STEC) can be divided into Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) with several sub-variants. Variant Stx(2f) is one of the latest described, but has been rarely associated with symptomatic human infections. In the enhanced STEC survei

  10. PART I. ESCHERICHIA COLI

    Directory of Open Access Journals (Sweden)

    Sanaa Mahdi Oraibi

    2016-11-01

    Full Text Available The presence of Escherichia coli in the air of facilities involved in management and composting of post-slaughter poultry wastes in selected plants of West Western Pomerania region was studied. Measurements were made on four dates in a variety of weather conditions during the year. The study was conducted at 5 objects that differ in the type of waste and the degree of preparation for composting. These were: chemical treatment and preliminary processing plant, liquid wastes reservoir, platform for preparation of materials for composting, storage of biological sediments, and composting facility. Measurement of bacteria count was carried out in accordance with the applicable procedures on selective chromogenic TBX medium. The assays revealed the presence of E. coli at all test objects, but not always on all measurement dates. It has been shown that the presence of E. coli was from 20 to 3047 CFU∙m-3 of air, although the largest quantities were most frequently detected in the air of the building for post-slaughter waste pre-treatment in chemical treatment plant.

  11. Prevalence and characterization of plasmids carrying sulfonamide resistance genes among Escherichia coli from pigs, pig carcasses and human

    DEFF Research Database (Denmark)

    Shuyu, Wu; Dalsgaard, A.; Hammerum, A. M.

    2010-01-01

    Background Sulfonamide resistance is very common in Escherichia coli. The aim of this study was to characterize plasmids carrying sulfonamide resistance genes (sul1, sul2 and sul3) in E. coli isolated from pigs and humans with a specific objective to assess the genetic diversity of plasmids...... involved in the mobility of sul genes. Methods A total of 501 E. coli isolates from pig feces, pig carcasses and human stools were tested for their susceptibility to selected antimicrobial. Multiplex PCR was conducted to detect the presence of three sul genes among the sulfonamide-resistant E. coli...... isolates. Fifty-seven sulfonamide-resistant E. coli were selected based on presence of sul resistance genes and subjected to conjugation and/or transformation experiments. S1 nuclease digestion followed by pulsed-field gel electrophoresis was used to visualize and determine the size of plasmids. Plasmids...

  12. Genes under positive selection in Escherichia coli

    DEFF Research Database (Denmark)

    Petersen, Lise; Bollback, J.P.; Dimmic, Matt

    2007-01-01

    We used a comparative genomics approach to identify genes that are under positive selection in six strains of Escherichia coli and Shigella flexneri, including five strains that are human pathogens. We find that positive selection targets a wide range of different functions in the E. coli genome...

  13. Fimbrial adhesins from extraintestinal Escherichia coli

    DEFF Research Database (Denmark)

    Klemm, Per; Hancock, Viktoria; Schembri, Mark A.

    2010-01-01

    Extraintestinal pathogenic Escherichia coli (ExPEC) represent an important subclass of E. coli that cause a wide spectrum of diseases in human and animal hosts. Fimbriae are key virulence factors of ExPEC strains. These long surface located rod-shaped organelles mediate receptor-specific attachment...

  14. Genes under positive selection in Escherichia coli

    DEFF Research Database (Denmark)

    Petersen, Lise; Bollback, J.P.; Dimmic, Matt

    2007-01-01

    We used a comparative genomics approach to identify genes that are under positive selection in six strains of Escherichia coli and Shigella flexneri, including five strains that are human pathogens. We find that positive selection targets a wide range of different functions in the E. coli genome...

  15. A novel expression system of domain I of human beta2 glycoprotein I in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Pearl Laurence H

    2006-02-01

    Full Text Available Abstract Background The antiphospholipid syndrome (APS, characterised by recurrent miscarriage and thrombosis, is a significant cause of morbidity and mortality. Domain I (DI of human beta 2 glycoprotein I (β2GPI is thought to contain crucial antibody binding epitopes for antiphospholipid antibodies (aPL, which are critical to the pathogenesis of APS. Expressing this protein in bacteria could facilitate studies investigating how this molecule interacts with aPL. Methods Using a computer programme called Juniper, sequentially overlapping primers were designed to be used in a recursive polymerase chain reaction (PCR to produce a synthetic DI gene. Specifically Juniper incorporates 'major' codons preferred by bacteria altering 41 codons out of 61. This was cloned into the expression plasmid pET(26b and expressed in BL21(DE3 Escherichia coli (E. coli. By virtue of a pelB leader sequence, periplasmic localisation of DI aided disulphide bond formation and toxicity was addressed by tightly regulating expression through the high stringency T7lac promoter. Results Purified, soluble his-tagged DI in yields of 750 μg/L bacterial culture was obtained and confirmed on Western blot. Expression using the native human cDNA sequence of DI in the same construct under identical conditions yielded significantly less DI compared to the recombinant optimised sequence. This constitutes the first description of prokaryotic expression of soluble DI of β2GPI. Binding to murine monoclonal antibodies that recognise conformationally restricted epitopes on the surface of DI and pathogenic human monoclonal IgG aPL was confirmed by direct and indirect immunoassay. Recombinant DI also bound a series of 21 polyclonal IgG samples derived from patients with APS. Conclusion By producing a synthetic gene globally optimised for expression in E. coli, tightly regulating expression and utilising periplasmic product translocation, efficient, soluble E. coli expression of the

  16. A potential camel reservoir for extended-spectrum β-lactamase-producing Escherichia coli causing human infection in Saudi Arabia.

    Science.gov (United States)

    Fadlelmula, Ali; Al-Hamam, Naser Abdallah; Al-Dughaym, Abdulla Mohamed

    2016-02-01

    The prevalence of antimicrobial resistance is continuing to increase. Consequently, efficient approaches to identify sources of resistance are required. This study aimed to compare Escherichia coli isolates from the intestinal tract of camels with isolates from human urinary tract infections (UTIs) in Al Ahsa Province, Kingdom of Saudi Arabia (KSA), for antimicrobial resistance and identification of extended-spectrum β-lactamases (ESBLs). A microbiological study was conducted on 100 samples of cecal contents from camels and 100 urine samples from female UTI patients, to isolate and confirm E. coli using the VITEK 2 Automated System. Sensitivity patterns and identification of ESBLs were analyzed using the antimicrobial susceptibility test. Molecular techniques were used to detect E. coli drug-resistant clones. The presence rate of E. coli in camels was 26.0 % (n = 26/100), and in human samples, the rate of E. coli was 33.0 % (n = 33/100). ESBLs were reported for the first time in KSA, in 26.9 % (n = 5/26) of camel samples and 36.4 % (n = 8/33) of human samples. The multi-drug resistance (MDR) index was 0.13 and 0.17, for camels and humans, respectively. Escherichia coli drug-resistant O25b:H4-sequence type 131(ST131) clone was detected in two camel and two human isolates. This study demonstrates a high presence rate of ESBL-producing E. coli (ESBL-EC) in camels for the first time in KSA. Confirmation of MDR strains and E. coli ST131 clone in human and camel isolates suggests that camels could be a potential reservoir for resistant E. coli strains contributing to the increase in antimicrobial resistance in KSA.

  17. Adherence of uropathogenic Escherichia coli to human primary epithelial cells of renal pelvis

    Institute of Scientific and Technical Information of China (English)

    CHAO GU; JIN YING CHEN; MIN HOU; JING DONG HE; JI WU CHANG

    2006-01-01

    Human primary epithelial cells of renal pelvis was established to investigate the adherence of uropathogenic Escherichia coli (UPEC) to this cell line, in which the primary cell culture was performed by using cultivation of the normal epithelium of renal pelvis in keratinocyte serum free medium (K-SFM)with epidermal growth factor (EGF) and bovine pituitary extract (BPE). Both UPEC132 obtained from urine specimen of patients with pyelonephritis and the pilus-free representative strain E. coli K-12p678-54 were used to study the adherence of these strains on human primary epithelial cells of renal pelvis.The UPEC adherence was performed with observation on the morphological changes of the adhered cells,while the adhesion rates and indices were calculated in different times of experiment. In addition, the virulence genes hly and cnf1 of UPEC132 were detected by multiplex PCR assay. In this study, the human primary epithelial cells of renal pelvis was found to exhibit the character of the transitional epithelial cells. Compared with the control group, the adhesion rates and indices began to increase from 15 min of the experiment time and reached its peak in 120 min. The adhesion rate and index of UPEC132 to human primary epithelial cells of renal pelvis were 74.4% and 34.0 respectively. Many microscopic changes in the primary cells adhered with UPEC132 could be detected, such as rounding or irregularity in shape,unevenness in staining and the cytoplasmic and nuclear changes. It suggests that human primary epithelial cells of renal pelvis can be used for the experiment on UPEC adhesion, thus providing a basis for the further study on the pathogenesis of UPEC.

  18. Global gene expression profiling of the asymptomatic bacteriuria Escherichia coli strain 83972 in the human urinary tract

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Klemm, Per

    2006-01-01

    long-term bladder colonization. The strain has been used for prophylactic purposes in patients prone to more severe and recurrent UTIs. For this study, we used DNA microarrays to monitor the expression profile of strain 83972 in the human urinary tract. Significant differences in expression levels were......Urinary tract infections (UTIs) are an important health problem worldwide, with many million cases each year. Escherichia coli is the most common organism causing UTIs in humans. The asymptomatic bacteriuria E. coli strain 83972 is an excellent colonizer of the human urinary tract, where it causes...

  19. High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor in Escherichia coli.

    Science.gov (United States)

    Ma, Yi; Yu, Jieying; Lin, Jinglian; Wu, Shaomin; Li, Shan; Wang, Jufang

    2016-01-01

    Human epidermal growth factor (hEGF) is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF in Escherichia coli has not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure using in vivo system. To tackle this problem, we fused Mxe GyrA intein (Mxe) at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO) and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H10. The fusion protein was highly expressed at the concentration of 281 mg/L and up to 59.5% of the total cellular soluble proteins. The fusion protein was purified by affinity chromatography and 29.4 mg/L of native hEGF can be released by thiol induced N-terminal cleavage without any proteases. The mitotic activity in Balb/c 3T3 cells is proliferated by commercial and recombinant hEGF measured with methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay which indicated that recombinant hEGF protein stimulates the cell proliferation similar to commercial protein. This study significantly improved the yield and reduced the cost of hEGF in the recombinant E. coli system and could be a better strategy to produce native hEGF for pharmaceutical development.

  20. Role of uropathogenic Escherichia coli OmpT in the resistance against human cathelicidin LL-37.

    Science.gov (United States)

    Brannon, John R; Thomassin, Jenny-Lee; Desloges, Isabelle; Gruenheid, Samantha; Le Moual, Hervé

    2013-08-01

    Uropathogenic Escherichia coli (UPEC) strains are among the most prevalent causative agents of urinary tract infections. To establish infection, UPEC must overcome the bactericidal action of host antimicrobial peptides. Previously, the enterohaemorrhagic E. coli outer membrane protease, OmpT, was shown to degrade and inactivate the human antimicrobial peptide LL-37. This study aims to investigate the involvement of UPEC OmpT in LL-37 degradation. An ompT deletion mutant was generated in the prototypical UPEC strain CFT073. Western blot analysis showed that the OmpT protein level is moderate in CFT073. In agreement, OmpT was shown to partially cleave LL-37. However, no difference in the minimum inhibitory concentration of LL-37 was observed between CFT073 and the ompT mutant. Plasmid complementation of ompT, which led to increased OmpT levels, resulted in complete cleavage of LL-37 and a fourfold increase in the minimum inhibitory concentration. The analysis of other UPEC isolates showed similar OmpT activity levels as CFT073. Although UPEC OmpT can cleave LL-37, we conclude that the low level of OmpT limits its contribution to LL-37 resistance. Collectively, these data suggest that UPEC OmpT is likely accompanied by other LL-37 resistance mechanisms.

  1. High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Yi Ma

    2016-01-01

    Full Text Available Human epidermal growth factor (hEGF is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF in Escherichia coli has not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure using in vivo system. To tackle this problem, we fused Mxe GyrA intein (Mxe at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H10. The fusion protein was highly expressed at the concentration of 281 mg/L and up to 59.5% of the total cellular soluble proteins. The fusion protein was purified by affinity chromatography and 29.4 mg/L of native hEGF can be released by thiol induced N-terminal cleavage without any proteases. The mitotic activity in Balb/c 3T3 cells is proliferated by commercial and recombinant hEGF measured with methylthiazolyldiphenyl-tetrazolium bromide (MTT assay which indicated that recombinant hEGF protein stimulates the cell proliferation similar to commercial protein. This study significantly improved the yield and reduced the cost of hEGF in the recombinant E. coli system and could be a better strategy to produce native hEGF for pharmaceutical development.

  2. Expression and purification of bioactive high-purity human midkine in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    Zhong-hui ZHANG; Li-juan DU; Di XIANG; Shun-ying ZHU; Ming-yuan WU; Hui-li LU; Yan YU; Wei HAN

    2009-01-01

    Midkine is a heparin-binding growth factor,which plays important roles in the regulation of cell growth and differentiation.The non-tagged recombinant human midkine (rhMK) is therefore required to facilitate its functional studies of this important growth factor.In the present work,rhMK was expressed in Escherichia coli (E.coli) BL21 (DE3).The expression of midkine was efficiently induced by isopropyl-β-D-thiogalactopyranoside (IPTG).After sonication,midkine was recovered in an insoluble form,and was dissolved in guaoidine hydrochloride buffer.Renaturation of the denatured protein was carried out in the defined protein refolding buffer,and the refolded protein was purified using S-Sepharose ion-exchange chromatography.The final preparation of the rhMK was greater than 98% pure as measured by sodium dodecylsulfate-polyacrylamid gel electrophoresis (SDS-PAGE) and reverse phase high performance liquid chromatography (RP-HPLC).The purified rhMK enhanced the proliferation of NIH3T3 cells.

  3. Replicative bypass of abasic site in Escherichia coli and human cells: similarities and differences.

    Directory of Open Access Journals (Sweden)

    Savithri Weerasooriya

    Full Text Available Abasic [apurinic/apyrimidinic (AP] sites are the most common DNA damages, opposite which dAMP is frequently inserted ('A-rule' in Escherichia coli. Nucleotide insertion opposite the AP-site in eukaryotic cells depends on the assay system and the type of cells. Accordingly, a 'C-rule', 'A-rule', or the lack of specificity has been reported. DNA sequence context also modulates nucleotide insertion opposite AP-site. Herein, we have compared replication of tetrahydrofuran (Z, a stable analog of AP-site, in E. coli and human embryonic kidney 293T cells in two different sequences. The efficiency of translesion synthesis or viability of the AP-site construct in E. coli was less than 1%, but it was 7- to 8-fold higher in the GZGTC sequence than in the GTGZC sequence. The difference in viability increased even more in pol V-deficient strains. Targeted one-base deletions occurred in 63% frequency in the GZG and 68% frequency in GZC sequence, which dropped to 49% and 21%, respectively, upon induction of SOS. The full-length products with SOS primarily involved dAMP insertion opposite the AP-site, which occurred in 49% and 71% frequency, respectively, in the GZG and GZC sequence. dAMP insertion, largely carried out by pol V, was more efficient when the AP-site was a stronger replication block. In contrast to these results in E. coli, viability was 2 to 3 orders of magnitude higher in human cells, and the 'A-rule' was more rigidly followed. The AP-site in the GZG and GZC sequences gave 76% and 89%, respectively, Z → T substitutions. In human cells, targeted one-base deletion was undetectable, and dTMP>dCMP were the next preferred nucleotides inserted opposite Z. siRNA knockdown of Rev1 or pol ζ established that both these polymerases are vital for AP-site bypass, as demonstrated by 36-67% reduction in bypass efficiency. However, neither polymerase was indispensable, suggesting roles of additional DNA polymerases in AP-site bypass in human cells.

  4. Purification and characterization of biologically active recombinant human Eppin expressed in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    ZHU Qing-yi; GU Xiao-jian; YANG Jin; WANG Jun-hong; TANG Bo; WU Hong-fei

    2008-01-01

    Background Eppin(epididymis protease inhibitor)appears to play an important role in primate fertility.However,the function of Eppin and its antibody in men and its relationship with men's infertility are poorly studied.To reveal the significance and possibility of detection of anti-Eppin antibody in clinical infertilty cases,we developed an Escherichia coli expression system for the expression of biologically actire human Eppin.Methods The human Eppin gene was cloned into PET-28a(+)vector after induction with 0.5 mmol/L isopropy-β-D-thiogalactoside(IPTG)at 26℃ for 4 hours,and the expressed fusion protein His6-Eppin was purified by Ni2+ affinity chromatography.Afterwards,six female 8-week-old Balb/c mice were immunized with purified His6-Eppin for three weeks.Their sera were collected and polyclonal antibodies against His6-Eppin were purified,all of which were further verified by Western-blot and immunofluorescence analysis.Results About 18.33 mg His6-Eppin was obtained from 1-L flask culture.The produced polyclonal antibodies against His6-Eppin recognized the Eppin protein both in human epididymis and in HEK293T cells by over-expression of the recombinant human Eppin.Conclusion The purified His6-Eppin protein has biological activity,which might be a candidate for clinical diagnosis of infertility and development of male immuno-contraceptive agents.

  5. Shiga toxin-producing Escherichia coli in humans and the food chain in Bangladesh

    NARCIS (Netherlands)

    Islam, M.A.

    2009-01-01

    Shiga toxin-producing Escherichia coli (STEC) are significant pathogenic bacteria that can cause severe gastrointestinal diseases and also the hemolytic-uremic syndrome. Domestic ruminants appear to be the main reservoirs of these organisms. Although Bangladesh is an endemic zone for diarrhea caused

  6. Distribution of intimin subtypes among Escherichia coli isolates from ruminant and human sources.

    Science.gov (United States)

    Ramachandran, Vidiya; Brett, Kim; Hornitzky, Michael A; Dowton, Mark; Bettelheim, Karl A; Walker, Mark J; Djordjevic, Steven P

    2003-11-01

    The intimin gene eae, located within the locus of enterocyte effacement pathogenicity island, distinguishes enteropathogenic Escherichia coli (EPEC) and some Shiga toxin-producing E. coli (STEC) strains from all other pathotypes of diarrheagenic E. coli. EPEC is a leading cause of infantile diarrhea in developing countries, and intimin-positive STEC isolates are typically associated with life-threatening diseases such as hemolytic-uremic syndrome and hemorrhagic colitis. Here we describe the development of a PCR-restriction fragment length polymorphism (RFLP) assay that reliably differentiates all 11 known intimin types (alpha1, alpha2, beta, gamma, kappa, epsilon, eta, iota, lambda, theta, and zeta) and three new intimin genes that show less than 95% nucleotide sequence identity with existing intimin types. We designated these new intimin genes Int- micro, Int-nu, and Int-xi. The PCR-RFLP assay was used to screen 213 eae-positive E. coli isolates derived from ovine, bovine, and human sources comprising 60 serotypes. Of these, 82 were STEC isolates, 89 were stx-negative (stx(-)) and ehxA-positive (ehxA(+)) isolates, and 42 were stx(-) and ehxA-negative isolates. Int-beta, the most commonly identified eae subtype (82 of 213 [38.5%] isolates), was associated with 21 serotypes, followed by Int-zeta (39 of 213 [18.3%] isolates; 11 serotypes), Int-theta (25 of 213 [11.7%] isolates; 15 serotypes), Int-gamma (19 of 213 [8.9%] isolates; 9 serotypes), and Int-epsilon (21 of 213 [9.9%] isolates; 5 serotypes). Intimin subtypes alpha1, alpha2, kappa, lambda, xi, micro, nu, and iota were infrequently identified; and Int-eta was not detected. Phylogenetic analyses with the Phylip package of programs clustered the intimin subtypes into nine distinct families (alpha, beta-xi, gamma, kappa, epsilon-eta-nu, iota- micro, lambda, theta, and zeta). Our data confirm that ruminants are an important source of serologically and genetically diverse intimin-containing E. coli strains.

  7. A systematic review and meta-analysis of the epidemiology of pathogenic Escherichia coli of calves and the role of calves as reservoirs for human pathogenic E. coli.

    Science.gov (United States)

    Kolenda, Rafał; Burdukiewicz, Michał; Schierack, Peter

    2015-01-01

    Escherichia coli bacteria are the most common causes of diarrhea and septicemia in calves. Moreover, calves form a major reservoir for transmission of pathogenic E. coli to humans. Systematic reviews and meta-analyses of publications on E. coli as calf pathogens and the role of calves as reservoir have not been done so far. We reviewed studies between 1951 and 2013 reporting the presence of virulence associated factors (VAFs) in calf E. coli and extracted the following information: year(s) and country of sampling, animal number, health status, isolate number, VAF prevalence, serotypes, diagnostic methods, and biological assays. The prevalence of VAFs or E. coli pathotypes was compared between healthy and diarrheic animals and was analyzed for time courses. Together, 106 papers with 25,982 E. coli isolates from 27 countries tested for VAFs were included. F5, F17, and F41 fimbriae and heat-stable enterotoxin (ST) - VAFs of enterotoxigenic E. coli (ETEC) were significantly associated with calf diarrhea. On the contrary, ETEC VAF F4 fimbriae and heat-labile enterotoxin as well as enteropathogenic (EPEC), Shiga toxin-producing (STEC), and enterohemorrhagic E. coli (EHEC) were not associated with diarrhea. The prevalence increased overtime for ST-positive isolates, but decreased for F5- and STEC-positive isolates. Our study provides useful information about the history of scientific investigations performed in this domain so far, and helps to define etiological agents of calf disease, and to evaluate calves as reservoir hosts for human pathogenic E. coli.

  8. Genetic Structure and Antimicrobial Resistance of Escherichia coli and Cryptic Clades in Birds with Diverse Human Associations

    Science.gov (United States)

    Blyton, Michaela D. J.; Pi, Hongfei; Vangchhia, Belinda; Abraham, Sam; Trott, Darren J.; Johnson, James R.

    2015-01-01

    The manner and extent to which birds associate with humans may influence the genetic attributes and antimicrobial resistance of their commensal Escherichia communities through strain transmission and altered selection pressures. In this study, we determined whether the distribution of the different Escherichia coli phylogenetic groups and cryptic clades, the occurrence of 49 virulence associated genes, and/or the prevalence of resistance to 12 antimicrobials differed between four groups of birds from Australia with contrasting types of human association. We found that birds sampled in suburban and wilderness areas had similar Escherichia communities. The Escherichia communities of backyard domestic poultry were phylogenetically distinct from the Escherichia communities sourced from all other birds, with a large proportion (46%) of poultry strains belonging to phylogenetic group A and a significant minority (17%) belonging to the cryptic clades. Wild birds sampled from veterinary and wildlife rehabilitation centers (in-care birds) carried Escherichia isolates that possessed particular virulence-associated genes more often than Escherichia isolates from birds sampled in suburban and wilderness areas. The Escherichia isolates from both the backyard poultry and in-care birds were more likely to be multidrug resistant than the Escherichia isolates from wild birds. We also detected a multidrug-resistant E. coli strain circulating in a wildlife rehabilitation center, reinforcing the importance of adequate hygiene practices when handling and caring for wildlife. We suggest that the relatively high frequency of antimicrobial resistance in the in-care birds and backyard poultry is due primarily to the use of antimicrobials in these animals, and we recommend that the treatment protocols used for these birds be reviewed. PMID:26002899

  9. Escherichia coli Uropathogenesis In Vitro

    DEFF Research Database (Denmark)

    Andersen, Thomas E; Khandige, Surabhi; Madelung, Michelle

    2012-01-01

    Uropathogenic Escherichia coli (UPEC) strains are capable of invading bladder epithelial cells (BECs) on the bladder luminal surface. Based primarily on studies in mouse models, invasion is proposed to trigger an intracellular uropathogenic cascade involving intracellular bacterial proliferation...

  10. An Escherichia coli-Based Assay to Assess the Function of Recombinant Human Hemichannels.

    Science.gov (United States)

    Krishnan, Srinivasan; Fiori, Mariana C; Whisenant, Ty E; Cortes, D Marien; Altenberg, Guillermo A; Cuello, Luis G

    2017-02-01

    Connexins form the gap junctional channels that mediate cell-to-cell communication, and also form hemichannels present at the plasma membrane. Hemichannels are permeable to small hydrophilic compounds, including molecules involved in autocrine and paracrine signaling. An abnormal hemichannel opening causes or contributes to cell damage in common human disorders (e.g., cardiac infarct, cerebrovascular accidents, deafness, skin diseases, and cataracts) and is therefore a potential pharmacological target. The discovery of useful hemichannels inhibitors has been hampered in part by the lack of suitable high-throughput functional assays. Here, we developed and characterized an assay useful to assess the function of hemichannels formed by human connexins expressed in a genetically modified Escherichia coli strain. The LB2003 cells, devoid of three key K(+) uptake transport mechanisms, cannot grow in low-[K(+)] medium, but expression of Cx26, Cx43, or Cx46 rescues their growth defect (growth complementation). We developed a protocol for a simple, inexpensive, easily scalable, reproducible, and sensitive assay that should be useful for the discovery of new and better hemichannel inhibitors based on the analysis of small-compound libraries.

  11. Human milk oligosaccharides protect bladder epithelial cells against uropathogenic Escherichia coli invasion and cytotoxicity.

    Science.gov (United States)

    Lin, Ann E; Autran, Chloe A; Espanola, Sophia D; Bode, Lars; Nizet, Victor

    2014-02-01

    The invasive pathogen uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infections (UTIs). Recurrent infection that can progress to life-threatening renal failure has remained as a serious global health concern in infants. UPEC adheres to and invades bladder epithelial cells to establish infection. Studies have detected the presence of human milk oligosaccharides (HMOs) in urine of breast-fed, but not formula-fed, neonates. We investigated the mechanisms HMOs deploy to elicit protection in human bladder epithelial cells infected with UPEC CFT073, a prototypic urosepsis-associated strain. We found a significant reduction in UPEC internalization into HMO-pretreated epithelial cells without observing any significant effect in UPEC binding to these cells. This event coincides with a rapid decrease in host cell cytotoxicity, recognized by LIVE/DEAD staining and cell detachment, but independent of caspase-mediated or mitochondrial-mediated programmed cell death pathways. Further investigation revealed HMOs, and particularly the sialic acid-containing fraction, reduced UPEC-mediated MAPK and NF-κB activation. Collectively, our results indicate that HMOs can protect bladder epithelial cells from deleterious cytotoxic and proinflammatory effects of UPEC infection, and may be one contributing mechanism underlying the epidemiological evidence of reduced UTI incidence in breast-fed infants.

  12. Efficient Expression of Bioactive Human Leptin in Escherichia coli in Soluble Fusion Form.

    Science.gov (United States)

    Li, Jian Feng; Zhang, Jie; Zhang, Zhen; Hu, Yun Long; Zhang, Shuang Quan

    2010-07-01

    Leptin, a 16 kDa nonglycosylated hormone, is produced by mature adipocytes and functions primarily in the hypothalamus to reduce food intake and body weight. To explore a new approach for high-level expression of human Leptin in Escherichia coli, the human Leptin gene, synthesized according to the published sequence, was cloned into the vector pET32a to construct a fusion expression plasmid: Trx-Leptin/pET32a. Our data showed that more than 40% of the fusion protein Trx-Leptin was expressed in soluble form. After purified by Ni-IDA affinity chromatography, cleaved by enterokinase and applied Ni-IDA affinity chromatography again, purified Leptin with homogeneity over 96% was achieved. The bio-functional experiments of purified Leptin showed a significant reduction in food intake and body weight of female mice treated with Leptin by comparing with control mice, and it indicated that the purified Leptin has full biological activity. In addition, our expression system was a very low-cost and efficient prokaryotic expression system. So taken together, our results demonstrated that our expression system of bio-active Leptin provided a new method for producing Leptin in big scale and would be widely applied in commercial Leptin producing industries.

  13. A systematic review and meta-analysis of the epidemiology of pathogenic Escherichia coli of calves and the role of calves as reservoirs for human pathogenic E. coli.

    Directory of Open Access Journals (Sweden)

    Rafał eKolenda

    2015-03-01

    Full Text Available Escherichia coli bacteria are the most common causes of diarrhea and septicemia in calves. Moreover, calves form a major reservoir for transmission of pathogenic E. coli to humans. Systematic reviews and meta-analyses of publications on Escherichia coli as calf pathogens and the role of calves as reservoir have not been done so far. We reviewed studies between 1951 and 2013 reporting the presence of virulence associated factors (VAFs in calf E. coli and extracted the following information: year(s and country of sampling, animal number, health status, isolate number, VAF prevalence, serotypes, diagnostic methods and biological assays. The prevalence of VAFs or E. coli pathotypes was compared between healthy and diarrheic animals and was analysed for time courses. Together, 106 papers with 25982 E. coli isolates from 27 countries tested for VAFs were included. F5, F17 and F41 fimbriae and heat-stable enterotoxin (ST – VAFs of enterotoxigenic E. coli (ETEC were significantly associated with calf diarrhea. On the contrary, ETEC VAF F4 fimbriae and heat-labile enterotoxin as well as enteropathogenic (EPEC, Shiga toxin-producing (STEC, and enterohaemorrhagic E. coli (EHEC were not associated with diarrhea. The prevalence increased overtime for ST-positive isolates, but decreased for F5- and STEC-positive isolates. Our study provides useful information about the history of scientific investigations performed in this domain so far, and helps to define etiological agents of calf disease, and to evaluate calves as reservoir hosts for human pathogenic E. coli.

  14. Expression of soluble, biologically active recombinant human endostatin in Escherichia coli.

    Science.gov (United States)

    Xu, Han-Mei; Zhang, Guo-Yuan; Ji, Xiao-Dan; Cao, Lin; Shu, Luan; Hua, Zi-Chun

    2005-06-01

    Endostatin, a 20kDa C-terminal fragment of collagen XVIII, is a potent anti-angiogenic protein and inhibitor of tumor growth. Recombinant endostatin was prepared from Escherichia coli deposited as insoluble, inactive inclusion bodies. In the present study, we produced soluble and biologically active recombinant human endostatin (rhEndostatin) in E. coli by employing both co-expression of the molecular chaperones and lower temperature fermentation. Two groups of chaperones Trigger factor and GroEL-GroES (GroEL/ES), DnaK-DnaJ-GrpE and GroEL/ES, were co-expressed, respectively, with rhEndostatin at different temperatures (37, 25, and 16 degrees C). It revealed that low temperature or molecular chaperones alone could enhance the production of active rhEndostatin; meanwhile, combinational employment of low temperature cultivation (16 degrees C) together with co-expression of DnaK-DnaJ-GrpE and GroEL/ES was more effective to prevent aggregation of rhEndostatin. The production of soluble rhEndostatin was about 36 mg/L, and at least 16 mg of rhEndostatin was purified from 1L flask culture. The purified rhEndostatin specifically inhibited the proliferation of endothelial cell-bovine capillary endothelial cell in a dose-dependent manner, and it showed potent anti-angiogenic capability on the chorioallantoic membrane of chick embryo in vivo. Our study provides a feasible and convenient approach to produce soluble and biologically active rhEndostatin.

  15. Surveys of human enterotoxigenic Escherichia coli from three different geographical areas for possible colonization factors.

    OpenAIRE

    1991-01-01

    Enterotoxigenic Escherichia coli (ETEC) from Burma, central Africa (Rwanda and Zaire) and Peru, were screened by enzyme-linked immunoassays for the colonization factor antigens (CFAs) and putative colonization factors (PCFs): CFA/I, CFA/II, which consists of three coli surface-associated (CS) antigens, CS1, CS2 and CS3, CFA/III, CFA/IV (CS4, CS5, CS6), CS7, PCFO9, PCFO159. H4, PCFO166, and CS17. The highest proportion of ETEC with identifiable colonization factors (71%) were found in the stra...

  16. Extraintestinal Pathogenic Escherichia coli, a Common Human Pathogen: Challenges for Vaccine Development and Progress in the Field

    OpenAIRE

    2015-01-01

    Extraintestinal pathogenic Escherichia coli (ExPEC) is the most common gram-negative bacterial pathogen in humans. ExPEC causes the vast majority of urinary tract infections (UTIs), is a leading cause of adult bacteremia, and is the second most common cause of neonatal meningitis. Increasing multidrug resistance among ExPEC strains constitutes a major obstacle to treatment and is implicated in increasing numbers of hospitalizations and deaths and increasing healthcare costs associated with Ex...

  17. Expression and Purification of Soluble Human Programmed Death-1 in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    Lihui Xu; Yi Liu; Xianhui He

    2006-01-01

    Programmed death-1 (PD-1), a member of CD28 family, is able to negatively regulate the TCR complex-initiated signaling by interacting with its cognate ligands (PD-L1 and/or PD-L2). PD-1/PD-L1 pathway plays an important role in down-regulating the effective phase of adaptive immune responses and the blockade of this pathway has been proved to enhance antiviral and antitumoral immunity, suggesting that it might be a potential target for the development of therapies to improve T cell responses in patients with virus infections or malignancies. In present study, the extracellular domain of human PD-1 with a carboxyl terminal His-tag (designated as sPD-1) was expressed as inclusion bodies in Escherichia coli. The product was on-column refolded, purified by immobilized metal affinity chromatography, and characterized by Western blotting. Furthermore, the soluble PD-1 with high purity possessed specific binding activity with its cognate ligand PD-L1, and the dissociation constant was 0.43 nmol/L as determined by Scatchard plot analysis. These results suggest that refolded sPD-1 from prokaryotic cells may be of therapeutic interest in enhancing antivirus and antitumoral immune responses.

  18. Adrenoceptor hyporeactivity is responsible for Escherichia coli endotoxin-induced acute vascular dysfunction in humans.

    Science.gov (United States)

    Pleiner, Johannes; Heere-Ress, Elisabeth; Langenberger, Herbert; Sieder, Anna E; Bayerle-Eder, Michaela; Mittermayer, Fritz; Fuchsjäger-Mayrl, Gabriele; Böhm, Johannes; Jansen, Burkhard; Wolzt, Michael

    2002-01-01

    Impaired response to catecholamines contributes to the altered hemodynamics in sepsis, which has been attributed to excessive NO formation. We have studied the systemic hemodynamic and local forearm responses and inducible NO synthase (iNOS) expression during experimental endotoxemia in humans. Escherichia coli endotoxin (lipopolysaccharide [LPS]) was administered at doses of 1 or 2 ng/kg to healthy volunteers. In 10 subjects, the systemic pressor effect of phenylephrine was assessed before and after the administration of LPS. In 9 further subjects, forearm blood flow responses to intra-arterial noradrenaline, acetylcholine, glyceryl trinitrate, and N(G)-monomethyl-L-arginine (L-NMMA) were studied at baseline and after LPS administration. Peripheral blood was collected and analyzed for iNOS mRNA and protein. Four hours after LPS, the response of systolic blood pressure (P<0.0005) and heart rate (P<0.05) to phenylephrine was significantly reduced. In the forearm, noradrenaline-induced vasoconstriction was also reduced by approximately 50% (P<0.01), but L-NMMA responsiveness was unchanged. iNOS mRNA or protein was not increased. Marked vascular adrenoceptor hyporeactivity is detectable in the absence of increased NO activity or iNOS expression in endotoxemia, arguing against major involvement of vascular iNOS activity in the acute systemic vasodilation to LPS.

  19. Transcriptomic Analysis of the Host Response and Innate Resilience to Enterotoxigenic Escherichia coli Infection in Humans.

    Science.gov (United States)

    Yang, William E; Suchindran, Sunil; Nicholson, Bradly P; McClain, Micah T; Burke, Thomas; Ginsburg, Geoffrey S; Harro, Clayton D; Chakraborty, Subhra; Sack, David A; Woods, Christopher W; Tsalik, Ephraim L

    2016-05-01

    Enterotoxigenic Escherichia coli (ETEC) is a globally prevalent cause of diarrhea. Though usually self-limited, it can be severe and debilitating. Little is known about the host transcriptional response to infection. We report the first gene expression analysis of the human host response to experimental challenge with ETEC. We challenged 30 healthy adults with an unattenuated ETEC strain, and collected serial blood samples shortly after inoculation and daily for 8 days. We performed gene expression analysis on whole peripheral blood RNA samples from subjects in whom severe symptoms developed (n = 6) and a subset of those who remained asymptomatic (n = 6) despite shedding. Compared with baseline, symptomatic subjects demonstrated significantly different expression of 406 genes highlighting increased immune response and decreased protein synthesis. Compared with asymptomatic subjects, symptomatic subjects differentially expressed 254 genes primarily associated with immune response. This comparison also revealed 29 genes differentially expressed between groups at baseline, suggesting innate resilience to infection. Drug repositioning analysis identified several drug classes with potential utility in augmenting immune response or mitigating symptoms. There are statistically significant and biologically plausible differences in host gene expression induced by ETEC infection. Differential baseline expression of some genes may indicate resilience to infection. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  20. Global gene expression profiling of asymptomatic bacteriuria Escherichia coli during biofilm growth in human urine

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Klemm, Per

    2007-01-01

    asymptomatic bacteriuria (ABU) E. coli strains 83972 and VR50 are significantly better biofilm formers in their natural growth medium, human urine, than the two uropathogenic E. coli isolates CFT073 and 536. We used DNA microarrays to monitor the expression profile during biofilm growth in urine of the two ABU...

  1. An Evidenced-Based Scale of Disease Severity following Human Challenge with Enteroxigenic Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Chad K Porter

    Full Text Available Experimental human challenge models have played a major role in enhancing our understanding of infectious diseases. Primary outcomes have typically utilized overly simplistic outcomes that fail to entirely account for complex illness syndromes. We sought to characterize clinical outcomes associated with experimental infection with enterotoxigenic Escherichia coli (ETEC and to develop a disease score.Data were obtained from prior controlled human ETEC infection studies. Correlation and univariate regression across sign and symptom severity was performed. A multiple correspondence analysis was conducted. A 3-parameter disease score with construct validity was developed in an iterative fashion, compared to standard outcome definitions and applied to prior vaccine challenge trials.Data on 264 subjects receiving seven ETEC strains at doses from 1x105 to 1x1010 cfu were used to construct a standardized dataset. The strongest observed correlation was between vomiting and nausea (r = 0.65; however, stool output was poorly correlated with subjective activity-impacting outcomes. Multiple correspondence analyses showed covariability in multiple signs and symptoms, with severity being the strongest factor corresponding across outcomes. The developed disease score performed well compared to standard outcome definitions and differentiated disease in vaccinated and unvaccinated subjects.Frequency and volumetric definitions of diarrhea severity poorly characterize ETEC disease. These data support a disease severity score accounting for stool output and other clinical signs and symptoms. Such a score could serve as the basis for better field trial outcomes and gives an additional outcome measure to help select future vaccines that warrant expanded testing in pivotal pre-licensure trials.

  2. Expression of active human sialyltransferase ST6GalNAcI in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Schwartz Marc F

    2009-09-01

    Full Text Available Abstract Background The presence of terminal, surface-exposed sialic acid moieties can greatly enhance the in vivo half-life of glycosylated biopharmaceuticals and improve their therapeutic efficacy. Complete and homogeneous sialylation of glycoproteins can be efficiently performed enzymically in vitro but this process requires large amounts of catalytically active sialyltransferases. Furthermore, standard microbial hosts used for large-scale production of recombinant enzymes can only produce small quantities of glycosyltransferases of animal origin, which lack catalytic activity. Results and conclusion In this work, we have expressed the human sialyltransferase ST6GalNAc I (ST6, an enzyme that sialylates O-linked glycoproteins, in Escherichia coli cells. We observed that wild-type bacterial cells are able to produce only very small amounts of soluble ST6 enzyme. We have found, however, that engineered bacterial strains which possess certain types of oxidative cytoplasm or which co-express the molecular chaperones/co-chaperones trigger factor, DnaK/DnaJ, GroEL/GroES, and Skp, can produce greatly enhanced amounts of soluble ST6. Furthermore, we have developed a novel high-throughput assay for the detection of sialyltransferase activity and used it to demonstrate that the bacterially expressed ST6 enzyme is active and able to transfer sialic acid onto a desialylated O-glycoprotein, bovine submaxillary mucin. To the best of our knowledge, this is the first example of expression of active human sialyltransferase in bacteria. This system may be used as a starting point for the evolution of sialyltransferases with better expression characteristics or altered donor/acceptor specificities.

  3. ESCHERICHIA COLI PATHOTYPES ASSOCIATED WITH DIARRHEA IN HUMAN AND DOMESTIC ANIMALS

    Directory of Open Access Journals (Sweden)

    I.I. Shabana

    2014-01-01

    Full Text Available Ruminants are important reservoirs for zoonotic pathogenic E. coli. The objective of this study was to characterize pathogenic E. coli isolates from cattle and sheep linked to human illness with respect to their pathotypes, serotypes and genotypes. E. coli O157:H7 isolated from cattle, sheep and human patients were compared for their genomic similarity by Pulsed-Field Gel-Electrophoresis (PFGE. PCR detection of virulence factors associated with different E. coli pathotypes (VTEC, ETEC, EPEC, EAEC and EIEC revealed that VTEC was the most prevalent pathotype (22/45; 48.9%, followed by EAEC (3/45; 6.7%, EPEC (1/45; 2.2%, a EPEC (3/45; 6.7%, ETEC (1/45; 2.2% and EIEC (1/45; 2.2%. E. coli O157:H7 represented the most prevalent VTEC serotypes (11/22; 50%. Pulsed field gel electrophoresis typing revealed exact matches between E. coli O157:H7 isolates from the human patients, sheep and cattle in the same municipality. VTEC play an important cause of diarrhea in human, sheep and cattle. The molecular relatedness between PFGE profiles of E. coli O157:H7 isolates from human, sheep and cattle supported the hypothesis that ruminants especially cattle and sheep act as reservoirs of E. coli O157:H7 for human infection.

  4. Cross feeding of glucose metabolism byproducts of Escherichia coli human gut isolates and probiotic strains affect survival of Vibrio cholerae.

    Science.gov (United States)

    Sengupta, Chirantana; Ekka, Manjula; Arora, Saurabh; Dhaware, Prashant D; Chowdhury, Rukhsana; Raychaudhuri, Saumya

    2017-01-01

    Vibrio cholerae converts glucose into either acid or the neutral end product acetoin and its survival in carbohydrate enriched media is linked to the nature of the byproducts produced. It has been demonstrated in this study that Escherichia coli strain isolated from the gut of healthy human volunteers and the commonly used probiotic E. coli Nissle strain that metabolize glucose to acidic byproducts drastically reduce the survival of V. cholerae strains irrespective of their glucose sensitivity and acetoin production status. Accordingly, E. coli glucose transport mutants that produce lower amounts of acidic metabolites had little effect on the survival of V. cholerae in cocultures. Thus, cross feeding of byproducts of glucose metabolism by heterologous bacteria modulates the survival of V. cholerae in glucose rich medium suggesting that composition of the gut microbiota could influence the outcome of V. cholerae infection especially when glucose based ORS is administered.

  5. Synthetic genes for human muscle-type adenylate kinase in Escherichia coli.

    Science.gov (United States)

    Kim, H J; Nishikawa, S; Tanaka, T; Uesugi, S; Takenaka, H; Hamada, M; Kuby, S A

    1989-01-01

    An artificial gene coding for the human muscle-type cytosolic adenylate kinase (hAK1) was chemically synthesized and directly expressed in Escherichia coli under the control of trp promoter. The DNA duplex of 596 bp was designed and constructed from 40 oligonucleotide fragments of typically 30 nucleotides in length. Twelve unique restriction sites were fairly evenly spaced in the synthetic gene to facilitate site-specific mutagenesis at any part of this recombinant protein. The genes for mutant hAK1 (Tyr 95----Phe 95, Y95F hAK1; Arg 97----Ala 97, R97A hAK1) were constructed by cassette mutagenesis and utilized restriction sites incorporated in the hAK1 gene. The recombinant hAK1 was purified to homogeneity by a two-step chromatographic procedure with a good yield, and showed the same adenylate kinase activity as that of authentic hAK1. Preliminary kinetic studies show that the enzymatic activity (Vmax app,cor/Et) of Y95F hAK1 was slightly greater than that of recombinant hAK1, whereas R97A hAK1 still possessed approximately 4% of recombinant hAK1 activity. These results suggest that the Arg-97 residue is important but not essential for catalytic activity, and that Tyr-95 can be replaced by phenylalanine without substantial effects on the enzymatic activity. Moreover, preliminary estimates of the apparent kinetic parameters suggest that these residues are not required for MgATP binding, and therefore they do not appear to be part of the MgATP binding site.

  6. Soluble Expression in Escherichia Coli and Purification of Human Carboxylesterase 1

    Institute of Scientific and Technical Information of China (English)

    WANG Lei; TONG Jin-ying; CAO Peng-rong; PENG Xiao-ning; YI Yin-sha; LV Yuan

    2014-01-01

    Objective: To achieve an optimized method for soluble expression of human carboxylesterase 1 (hCE-1) in escherichia coil and purification by Ni2+-NTA agarose affinity chromatography, to get improved protein yield and purity for further development of hepatocellular carcinoma (HCC) diagnosis ELISA kits. Methods: The best antigen epitopes of hCE1 were predicted by comparing secondary structure, flexible regions, hydrophilicity, antigenic index surface probability of residues. Afterwards, pET-42a (+) with a His-tag and a GST-tag was applied to form recombinant plasmid pET-42a (+)/hCE1, which facilitated purification when using Ni2+-NTA agarose affinity chromatography. Protein quality was measured by SDS-PAGE and BCA protein assay. Western-blot identification was also performed to ensure the correct expression of hCE1 protein. Results: The residues from 500 to 567 near C-terminal of hCE1 protein were considered the best epitopes which exhibited high hydrophilicity and high surface probability and relatively flexible secondary structure and low homology compared with hCE2 and hCE3. His-hCE1 500-567 fusion protein was achieved by IPTG-inducted expression with an expected mass of 42 kDa. After purification, the final product was specially identified, which reached over 95%purity and more than 10 mg/L of microbial culture. In Western blot, the purified fusion protein was recognized by anti-hCE1 monoclonal antibody, along with previous sequencing validation, which demonstrated the correct preparation of soluble hCE1 protein. Conclusion: This is an efficacious and affordable strategy to generate fusion hCE1 of high quality in E coli, which facilitates preparation of hCE1 monoclonal antibody and further HCC diagnosis research.

  7. Chaperone-Usher Pili Loci of Colonization Factor-Negative Human Enterotoxigenic Escherichia coli

    Science.gov (United States)

    Del Canto, Felipe; O'Ryan, Miguel; Pardo, Mirka; Torres, Alexia; Gutiérrez, Daniela; Cádiz, Leandro; Valdés, Raul; Mansilla, Aquiles; Martínez, Rodrigo; Hernández, Daniela; Caro, Benjamin; Levine, Myron M.; Rasko, David A.; Hill, Christopher M.; Pop, Mihai; Stine, O. Colin; Vidal, Roberto

    2017-01-01

    Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of diarrhea worldwide. Among the 25 different ETEC adhesins, 22 are known as “colonization factors” (CFs), of which 17 are assembled by the chaperone-usher (CU) mechanism. Currently, there is no preventive therapy against ETEC, and CFs have been proposed as components for vaccine development. However, studies of diarrhea-causing ETEC strains worldwide indicate that between 15 and 50% of these are negative for known CFs, hindering the selection of the most widespread structures and suggesting that unknown adhesins remain to be identified. Here, we report the result of a comprehensive analysis of 35 draft genomes of ETEC strains which do not carry known adhesin genes; our goal was to find new CU pili loci. The phylogenetic profiles and serogroups of these strains were highly diverse, a majority of which produced only the heat-labile toxin. We identified 10 pili loci belonging to CU families β (1 locus), γ2 (7 loci), κ (1 locus), and π (1 locus), all of which contained the required number of open reading frames (ORFs) to encode functional structures. Three loci were variants of previously-known clusters, three had been only-partially described, and four are novel loci. Intra-loci genetic variability identified would allow the synthesis of up to 14 different structures. Clusters of putative γ2-CU pili were most common (23 strains), followed by putative β-CU pili (12 strains), which have not yet been fully characterized. Overall, our findings significantly increase the number of ETEC adhesion genes associated with human infections. PMID:28111618

  8. Human Volunteers Receiving Escherichia coli Phage T4 Orally: a Safety Test of Phage Therapy

    OpenAIRE

    Bruttin, Anne; Brüssow, Harald

    2005-01-01

    Fifteen healthy adult volunteers received in their drinking water a lower Escherichia coli phage T4 dose (103 PFU/ml), a higher phage dose (105 PFU/ml), and placebo. Fecal coliphage was detected in a dose-dependent way in volunteers orally exposed to phage. All volunteers receiving the higher phage dose showed fecal phage 1 day after exposure; this prevalence was only 50% in subjects receiving the lower phage dose. No fecal phage was detectable a week after a 2-day course of oral phage applic...

  9. Drug resistance and adherence to human intestines of enteroaggregative Escherichia coli.

    Science.gov (United States)

    Yamamoto, T; Echeverria, P; Yokota, T

    1992-04-01

    Clinical isolates of enteroaggregative Escherichia coli (EAggEC) were tested for their in vitro susceptibilities to 27 antimicrobial agents. Marked drug resistance was observed with sulfamethoxazole, ampicillin, and chloramphenicol in contrast to such antimicrobial agents as cefixime, sparfloxacin, and ciprofloxacin. One of the EAggEC strains carried a plasmid that conferred on its host resistance to ampicillin, tetracycline, sulfamethoxazole, streptomycin, and spectinomycin and an ability to adhere to child ileal villi or HeLa cells in the characteristic aggregative pattern. This plasmid also mediated D-mannose-resistant hemagglutinin production and bacterial clump formation (autoagglutination). The data demonstrate appearance of marked drug resistance and an intestine-adherence and drug-resistance plasmid in the newest category of diarrheagenic E. coli.

  10. A QMRA for the Transmission of ESBL-Producing Escherichia coli and Campylobacter from Poultry Farms to Humans Through Flies.

    Science.gov (United States)

    Evers, Eric G; Blaak, Hetty; Hamidjaja, Raditijo A; de Jonge, Rob; Schets, Franciska M

    2016-02-01

    The public health significance of transmission of ESBL-producing Escherichia coli and Campylobacter from poultry farms to humans through flies was investigated using a worst-case risk model. Human exposure was modeled by the fraction of contaminated flies, the number of specific bacteria per fly, the number of flies leaving the poultry farm, and the number of positive poultry houses in the Netherlands. Simplified risk calculations for transmission through consumption of chicken fillet were used for comparison, in terms of the number of human exposures, the total human exposure, and, for Campylobacter only, the number of human cases of illness. Comparing estimates of the worst-case risk of transmission through flies with estimates of the real risk of chicken fillet consumption, the number of human exposures to ESBL-producing E. coli was higher for chicken fillet as compared with flies, but the total level of exposure was higher for flies. For Campylobacter, risk values were nearly consistently higher for transmission through flies than for chicken fillet consumption. This indicates that the public health risk of transmission of both ESBL-producing E. coli and Campylobacter to humans through flies might be of importance. It justifies further modeling of transmission through flies for which additional data (fly emigration, human exposure) are required. Similar analyses of other environmental transmission routes from poultry farms are suggested to precede further investigations into flies.

  11. [Estimation of the transfer of ESBL-producing Escherichia coli to humans in Germany].

    Science.gov (United States)

    Sharp, Hannah; Valentin, Lars; Fischer, Jennie; Guerra, Beatriz; Appel, Bernd; Käsbohrer, Annemarie

    2014-01-01

    In 2011 EFSA has evaluated the risk for the consumer caused by ESBL-/AmpC-producing bacteria in food of animal origin and in livestock animals. Human-to-human transfer in hospitals.and in the community was considered as the most relevant route of transmission for ESBL-producing E. coli. ESBL-/AmpC-producing E. coli are in Germany, as in many other Member States of the European Union, widely spread in food of animal origin and in livestock animals. In an assessment of the relevance of livestock animals as reservoir for ESBL-/AmpC-producing E. coli as well as for ESBL-coding resistance genes the heterogeneity of the resistance genes, plasmids and bacteria in animals, foods and humans needs to be considered. In this context, both, the clonal spread of bacteria as well as horizontal transfer of resistance genes, e. g. by plasmids, have to be analyzed. Whereas studies in The Netherlands identified poultry as the most relevant reservoir, the transfer of ESBL-gene carrying plasmids from pigs to the farmers was demonstrated in Denmark. First attempts to quantify the relevance of livestock animals as reservoir for ESBL-producing E. coli in Germany showed, that the proportions of the most frequent ESBL-resistance genes are quite different between animal and human derived E. coli isolates. If in addition properties of the bacterial cells, e.g. resistance to several antibiotic classes are considered, only a small proportion of human isolates showed the same patterns as animal isolates. The results achieved so far demonstrate that certain ESBL-types are prevalent in all livestock populations investigated. Currently, the majority of cases of colonizations with ESBL-producing E. coli among humans cannot be directly linked to livestock and food-producing animals as reservoirs. This reflects that transmission routes are more complex and other reservoirs and sources including human-human interactions have to be taken into consideration.

  12. Evaluation of Escherichia coli isolates from healthy chickens to determine their potential risk to poultry and human health.

    Science.gov (United States)

    Stromberg, Zachary R; Johnson, James R; Fairbrother, John M; Kilbourne, Jacquelyn; Van Goor, Angelica; Curtiss, Roy; Mellata, Melha

    2017-01-01

    Extraintestinal pathogenic Escherichia coli (ExPEC) strains are important pathogens that cause diverse diseases in humans and poultry. Some E. coli isolates from chicken feces contain ExPEC-associated virulence genes, so appear potentially pathogenic; they conceivably could be transmitted to humans through handling and/or consumption of contaminated meat. However, the actual extraintestinal virulence potential of chicken-source fecal E. coli is poorly understood. Here, we assessed whether fecal E. coli isolates from healthy production chickens could cause diseases in a chicken model of avian colibacillosis and three rodent models of ExPEC-associated human infections. From 304 E. coli isolates from chicken fecal samples, 175 E. coli isolates were screened by PCR for virulence genes associated with human-source ExPEC or avian pathogenic E. coli (APEC), an ExPEC subset that causes extraintestinal infections in poultry. Selected isolates genetically identified as ExPEC and non-ExPEC isolates were assessed in vitro for virulence-associated phenotypes, and in vivo for disease-causing ability in animal models of colibacillosis, sepsis, meningitis, and urinary tract infection. Among the study isolates, 13% (40/304) were identified as ExPEC; the majority of these were classified as APEC and uropathogenic E. coli, but none as neonatal meningitis E. coli. Multiple chicken-source fecal ExPEC isolates resembled avian and human clinical ExPEC isolates in causing one or more ExPEC-associated illnesses in experimental animal infection models. Additionally, some isolates that were classified as non-ExPEC were able to cause ExPEC-associated illnesses in animal models, and thus future studies are needed to elucidate their mechanisms of virulence. These findings show that E. coli isolates from chicken feces contain ExPEC-associated genes, exhibit ExPEC-associated in vitro phenotypes, and can cause ExPEC-associated infections in animal models, and thus may pose a health threat to

  13. Evaluation of Escherichia coli isolates from healthy chickens to determine their potential risk to poultry and human health

    Science.gov (United States)

    Johnson, James R.; Fairbrother, John M.; Kilbourne, Jacquelyn; Van Goor, Angelica; Curtiss, Roy; Mellata, Melha

    2017-01-01

    Extraintestinal pathogenic Escherichia coli (ExPEC) strains are important pathogens that cause diverse diseases in humans and poultry. Some E. coli isolates from chicken feces contain ExPEC-associated virulence genes, so appear potentially pathogenic; they conceivably could be transmitted to humans through handling and/or consumption of contaminated meat. However, the actual extraintestinal virulence potential of chicken-source fecal E. coli is poorly understood. Here, we assessed whether fecal E. coli isolates from healthy production chickens could cause diseases in a chicken model of avian colibacillosis and three rodent models of ExPEC-associated human infections. From 304 E. coli isolates from chicken fecal samples, 175 E. coli isolates were screened by PCR for virulence genes associated with human-source ExPEC or avian pathogenic E. coli (APEC), an ExPEC subset that causes extraintestinal infections in poultry. Selected isolates genetically identified as ExPEC and non-ExPEC isolates were assessed in vitro for virulence-associated phenotypes, and in vivo for disease-causing ability in animal models of colibacillosis, sepsis, meningitis, and urinary tract infection. Among the study isolates, 13% (40/304) were identified as ExPEC; the majority of these were classified as APEC and uropathogenic E. coli, but none as neonatal meningitis E. coli. Multiple chicken-source fecal ExPEC isolates resembled avian and human clinical ExPEC isolates in causing one or more ExPEC-associated illnesses in experimental animal infection models. Additionally, some isolates that were classified as non-ExPEC were able to cause ExPEC-associated illnesses in animal models, and thus future studies are needed to elucidate their mechanisms of virulence. These findings show that E. coli isolates from chicken feces contain ExPEC-associated genes, exhibit ExPEC-associated in vitro phenotypes, and can cause ExPEC-associated infections in animal models, and thus may pose a health threat to

  14. Molecular screening of virulence genes in extraintestinal pathogenic Escherichia coli isolated from human blood culture in Brazil.

    Science.gov (United States)

    Koga, Vanessa L; Tomazetto, Geizecler; Cyoia, Paula S; Neves, Meiriele S; Vidotto, Marilda C; Nakazato, Gerson; Kobayashi, Renata K T

    2014-01-01

    Extraintestinal pathogenic Escherichia coli (ExPEC) is one of the main etiological agents of bloodstream infections caused by Gram-negative bacilli. In the present study, 20 E. coli isolates from human hemocultures were characterized to identify genetic features associated with virulence (pathogenicity islands markers, phylogenetic group, virulence genes, plasmid profiles, and conjugative plasmids) and these results were compared with commensal isolates. The most prevalent pathogenicity island, in strains from hemoculture, were PAI IV536, described by many researchers as a stable island in enterobacteria. Among virulence genes, iutA gene was found more frequently and this gene enconding the aerobactin siderophore receptor. According to the phylogenetic classification, group B2 was the most commonly found. Additionally, through plasmid analysis, 14 isolates showed plasmids and 3 of these were shown to be conjugative. Although in stool samples of healthy people the presence of commensal strains is common, human intestinal tract may serve as a reservoir for ExPEC.

  15. On the trail of EHEC/EAEC--unraveling the gene regulatory networks of human pathogenic Escherichia coli bacteria.

    Science.gov (United States)

    Pauling, Josch; Röttger, Richard; Neuner, Andreas; Salgado, Heladia; Collado-Vides, Julio; Kalaghatgi, Prabhav; Azevedo, Vasco; Tauch, Andreas; Pühler, Alfred; Baumbach, Jan

    2012-07-01

    Pathogenic Escherichia coli, such as Enterohemorrhagic E. coli (EHEC) and Enteroaggregative E. coli (EAEC), are globally widespread bacteria. Some may cause the hemolytic uremic syndrome (HUS). Varying strains cause epidemics all over the world. Recently, we observed an epidemic outbreak of a multi-resistant EHEC strain in Western Europe, mainly in Germany. The Robert Koch Institute reports >4300 infections and >50 deaths (July, 2011). Farmers lost several million EUR since the origin of infection was unclear. Here, we contribute to the currently ongoing research with a computer-aided study of EHEC transcriptional regulatory interactions, a network of genetic switches that control, for instance, pathogenicity, survival and reproduction of bacterial cells. Our strategy is to utilize knowledge of gene regulatory networks from the evolutionary relative E. coli K-12, a harmless strain mainly used for wet lab studies. In order to provide high-potential candidates for human pathogenic E. coli bacteria, such as EHEC, we developed the integrated online database and an analysis platform EhecRegNet. We utilize 3489 known regulations from E. coli K-12 for predictions of yet unknown gene regulatory interactions in 16 human pathogens. For these strains we predict 40,913 regulatory interactions. EhecRegNet is based on the identification of evolutionarily conserved regulatory sites within the DNA of the harmless E. coli K-12 and the pathogens. Identifying and characterizing EHEC's genetic control mechanism network on a large scale will allow for a better understanding of its survival and infection strategies. This will support the development of urgently needed new treatments. EhecRegNet is online via http://www.ehecregnet.de.

  16. Hybrids of Shigatoxigenic and Enterotoxigenic Escherichia coli (STEC/ETEC) Among Human and Animal Isolates in Finland.

    Science.gov (United States)

    Nyholm, O; Heinikainen, S; Pelkonen, S; Hallanvuo, S; Haukka, K; Siitonen, A

    2015-11-01

    Diarrhoeagenic Escherichia coli (DEC) cause serious foodborne infections in humans. Total of 450 Shigatoxigenic E. coli (STEC) strains isolated from humans, animals and environment in Finland were examined by multiplex PCR targeting the virulence genes of various DEC pathogroups simultaneously. One per cent (3/291) of the human STEC and 14% (22/159) of the animal and environmental STEC had genes typically present in enterotoxigenic E. coli (ETEC). The strains possessed genes encoding both Shiga toxin 1 and/or 2 (stx1 and/or stx2 ) and ETEC-specific heat-stable (ST) enterotoxin Ia (estIa). The identified stx subtypes were stx1a, stx1c, stx2a, stx2d and stx2g. The three human STEC/ETEC strains were isolated from the patients with haemolytic uraemic syndrome and diarrhoea and from an asymptomatic carrier. The animal STEC/ETEC strains were isolated from cattle and moose. The human and animal STEC/ETEC strains belonged to 11 serotypes, of which O2:H27, O15:H16, O101:H-, O128:H8 and O141:H8 have previously been described to be associated with human disease. Identification of multiple virulence genes offers further information for assessing the virulence potential of STEC and other DEC. The emergence of novel hybrid pathogens should be taken into account in the patient care and epidemiological surveillance.

  17. Genetic diversity and antimicrobial resistance of Escherichia coli from human and animal sources uncovers multiple resistances from human sources.

    Science.gov (United States)

    Ibekwe, A Mark; Murinda, Shelton E; Graves, Alexandria K

    2011-01-01

    Escherichia coli are widely used as indicators of fecal contamination, and in some cases to identify host sources of fecal contamination in surface water. Prevalence, genetic diversity and antimicrobial susceptibility were determined for 600 generic E. coli isolates obtained from surface water and sediment from creeks and channels along the middle Santa Ana River (MSAR) watershed of southern California, USA, after a 12 month study. Evaluation of E. coli populations along the creeks and channels showed that E. coli were more prevalent in sediment compared to surface water. E. coli populations were not significantly different (P = 0.05) between urban runoff sources and agricultural sources, however, E. coli genotypes determined by pulsed-field gel electrophoresis (PFGE) were less diverse in the agricultural sources than in urban runoff sources. PFGE also showed that E. coli populations in surface water were more diverse than in the sediment, suggesting isolates in sediment may be dominated by clonal populations.Twenty four percent (144 isolates) of the 600 isolates exhibited resistance to more than one antimicrobial agent. Most multiple resistances were associated with inputs from urban runoff and involved the antimicrobials rifampicin, tetracycline, and erythromycin. The occurrence of a greater number of E. coli with multiple antibiotic resistances from urban runoff sources than agricultural sources in this watershed provides useful evidence in planning strategies for water quality management and public health protection.

  18. Purification and Refolding of Overexpressed Human Basic Fibroblast Growth Factor in Escherichia coli

    OpenAIRE

    2011-01-01

    International audience; This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations used to recover purified and biologically active human basic fibroblast growth factor from inclusion bodies expressed in E. coli. Insoluble overexpressed human basic fibroblast growth factor has been purified on CM Hyper Z matrix by expanded bed adsorption after isolation and solubilization in 8 M urea. The adsorption was made in expanded bed without clarifi...

  19. Prevalence and genetic relatedness of antimicrobial-resistant Escherichia coli isolated from animals, foods and humans in Iceland.

    Science.gov (United States)

    Thorsteinsdottir, T R; Haraldsson, G; Fridriksdottir, V; Kristinsson, K G; Gunnarsson, E

    2010-05-01

    The prevalence of resistant bacteria in food products in Iceland is unknown, and little is known of the prevalence in production animals. The aim of this study was to investigate the prevalence and genetic relatedness of antimicrobial-resistant Escherichia coli from healthy pigs and broiler chicken, pork, broiler meat, slaughterhouse personnel and outpatients in Iceland. A total of 419 E. coli isolates were tested for antimicrobial susceptibility using a microbroth dilution method (VetMIC), and resistant strains were compared using pulsed-field gel electrophoresis (PFGE). All samples were screened for enrofloxacin-resistant strains with selective agar plates. The resistance rates among E. coli isolates were moderate to high from caecal and meat samples of pigs (54.1% and 28%), broilers (33.6% and 52%) and slaughterhouse personnel (39.1%), whereas isolates from outpatients showed moderate resistance rates (23.1%). Of notice was resistance to quinolones (minimum inhibitory concentrations: nalidixic acid > or = 32, ciprofloxacin > or = 0.12 and enrofloxacin > or = 0.5), particularly among broiler and broiler meat isolates (18.2% and 36%), as there is no known antimicrobial selection pressure in the broiler production in Iceland. The majority (78.6%) of the resistant E. coli isolates was genotypically different, based on PFGE fingerprint analyses and clustering was limited. However, the same resistance pattern and pulsotype were found among isolates from broiler meat and a slaughterhouse worker, indicating spread of antimicrobial-resistant E. coli from animals to humans. Diverse resistance patterns and pulsotypes suggest the presence of a large population of resistant E. coli in production animals in Iceland. This study gives baseline information on the prevalence of antimicrobial-resistant E. coli from production animals, and their food products in Iceland and the moderate to high resistance rates emphasize the need for continuing surveillance. Further studies on the

  20. Neisseria meningitidis and Escherichia coli are protected from leukocyte phagocytosis by binding to erythrocyte complement receptor 1 in human blood

    DEFF Research Database (Denmark)

    Brekke, O. L.; Hellerud, B. C.; Christiansen, D.

    2011-01-01

    The initial interaction of Gram-negative bacteria with erythrocytes and its implications on leukocyte phagocytosis and oxidative burst in human whole blood were examined. Alexa-labeled Escherichia coli, wild-type H44/76 N. meningitidis and the H44/76lpxA lipopolysaccharide (LPS)-deficient mutant ......-primates and that the bacteria were mainly found in the lungs. In conclusion, complement-dependent binding of Gram-negative bacteria to erythrocyte CR1 decreases phagocytosis and oxidative burst by leukocytes in human whole blood. (C) 2011 Elsevier Ltd. All rights reserved.......The initial interaction of Gram-negative bacteria with erythrocytes and its implications on leukocyte phagocytosis and oxidative burst in human whole blood were examined. Alexa-labeled Escherichia coli, wild-type H44/76 N. meningitidis and the H44/76lpxA lipopolysaccharide (LPS)-deficient mutant...... were incubated with whole blood using lepirudin as anticoagulant which has no adverse effects on complement. Bacteria free in plasma, bound to erythrocytes or phagocytized by granulocytes and monocytes were quantified using flow cytometry. The effects of the C3 inhibitor compstatin, a C5a receptor...

  1. A Premature Termination of Human Epidermal Growth Factor Receptor Transcription in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jihene Elloumi-Mseddi

    2014-01-01

    Full Text Available Our success in producing an active epidermal growth factor receptor (EGFR tyrosine kinase in Escherichia coli encouraged us to express the full-length receptor in the same host. Despite its large size, we were successful at producing the full-length EGFR protein fused to glutathione S-transferase (GST that was detected by Western blot analysis. Moreover, we obtained a majoritarian truncated GST-EGFR form detectable by gel electrophoresis and Western blot. This truncated protein was purified and confirmed by MALDI-TOF/TOF analysis to belong to the N-terminal extracellular region of the EGFR fused to GST. Northern blot analysis showed two transcripts suggesting the occurrence of a transcriptional arrest.

  2. Global gene expression profiling of asymptomatic bacteriuria Escherichia coli during biofilm growth in human urine

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Klemm, Per

    2007-01-01

    asymptomatic bacteriuria (ABU) E. coli strains 83972 and VR50 are significantly better biofilm formers in their natural growth medium, human urine, than the two uropathogenic E. coli isolates CFT073 and 536. We used DNA microarrays to monitor the expression profile during biofilm growth in urine of the two ABU...... strains 83972 and VR50. Significant differences in expression levels were seen between the biofilm expression profiles of the two strains with the corresponding planktonic expression profiles in morpholinepropanesulfonic acid minimal laboratory medium and human urine; 417 and 355 genes were up- and down...... versions of 83972 and VR50; all mutants showed reduced biofilm formation in urine by 18 to 43% compared with the wild type (P profile of strain 83972 in the human urinary tract partially overlaps with the biofilm expression profile....

  3. Analysis of the human intestinal epithelial cell transcriptional response to Lactobacillus acidophilus, Lactobacillus salivarius, Bifidobacterium lactis and Escherichia coli

    DEFF Research Database (Denmark)

    Putaala, H; Barrangou, R; Leyer, G J

    2010-01-01

    The complex microbial population residing in the human gastrointestinal tract consists of commensal, potential pathogenic and beneficial species, which are probably perceived differently by the host and consequently could be expected to trigger specific transcriptional responses. Here, we provide...... insights into the relationship between probiotics and human intestinal epithelial cells, notably with regard to strain-specific responses, and highlight the differences between transcriptional responses to pathogenic and probiotic bacteria.......The complex microbial population residing in the human gastrointestinal tract consists of commensal, potential pathogenic and beneficial species, which are probably perceived differently by the host and consequently could be expected to trigger specific transcriptional responses. Here, we provide...... a comparative analysis of the global in vitro transcriptional response of human intestinal epithelial cells to Lactobacillus acidophilus NCFM™, Lactobacillus salivarius Ls-33, Bifidobacterium animalis subsp. lactis 420, and enterohaemorrhagic Escherichia coli O157:H7 (EHEC). Interestingly, L. salivarius Ls-33...

  4. Shiga Toxin Producing Escherichia coli.

    Science.gov (United States)

    Bryan, Allen; Youngster, Ilan; McAdam, Alexander J

    2015-06-01

    Shiga toxin-producing Escherichia coli (STEC) is among the common causes of foodborne gastroenteritis. STEC is defined by the production of specific toxins, but within this pathotype there is a diverse group of organisms. This diversity has important consequences for understanding the pathogenesis of the organism, as well as for selecting the optimum strategy for diagnostic testing in the clinical laboratory. This review includes discussions of the mechanisms of pathogenesis, the range of manifestations of infection, and the several different methods of laboratory detection of Shiga toxin-producing E coli.

  5. Escherichia coli in Europe: An Overview

    Directory of Open Access Journals (Sweden)

    Nerino Allocati

    2013-11-01

    Full Text Available Escherichia coli remains one of the most frequent causes of several common bacterial infections in humans and animals. E. coli is the prominent cause of enteritis, urinary tract infection, septicaemia and other clinical infections, such as neonatal meningitis. E. coli is also prominently associated with diarrhoea in pet and farm animals. The therapeutic treatment of E. coli infections is threatened by the emergence of antimicrobial resistance. The prevalence of multidrug-resistant E. coli strains is increasing worldwide principally due to the spread of mobile genetic elements, such as plasmids. The rise of multidrug-resistant strains of E. coli also occurs in Europe. Therefore, the spread of resistance in E. coli is an increasing public health concern in European countries. This paper summarizes the current status of E. coli strains clinically relevant in European countries. Furthermore, therapeutic interventions and strategies to prevent and control infections are presented and discussed. The article also provides an overview of the current knowledge concerning promising alternative therapies against E. coli diseases.

  6. Clonal relationship between human and avian ciprofloxacin-resistant Escherichia coli isolates in North-Eastern Algeria.

    Science.gov (United States)

    Agabou, A; Lezzar, N; Ouchenane, Z; Khemissi, S; Satta, D; Sotto, A; Lavigne, J-P; Pantel, A

    2016-02-01

    The objectives of this study were to determine rates, patterns, and mechanisms of antibiotic resistance, and to assess connections between chicken commensal, human commensal, and pathogenic ciprofloxacin-resistant Escherichia coli isolates. All E. coli isolates collected from chickens, their farmers, and patients in the Constantine region (North-east Algeria) were analyzed for bla and plasmid-mediated quinolone resistance (PMQR) gene contents, phylogroups, Rep-PCR profiles, and multilocus sequence types. A high prevalence of resistance to fluoroquinolones (51.4 % to ciprofloxacin) was recorded in avian isolates. Of these, 22.2 % carried the aac(6')-Ib-cr gene, whereas lower resistance levels to these antibiotics were recorded in chicken farmers' isolates. None of the commensal isolates harbored the qnr, qepA, or oqxAB genes. One human pathogenic isolate was ertapenem-resistant and harbored the bla OXA-48 gene, 84 showed an extended-spectrum β-lactamase phenotype, with bla CTX-M-15 gene prevalent in 87.2 % of them. Seventy isolates were resistant to fluoroquinolones, with aac(6')-Ib-cr present in 72.8 %, qnrB in 5.7 %, and qnrS in 10 %. Three Rep-PCR profiles were common to chicken commensal and human pathogenic isolates (phylogroups D and B1; ST21, ST48, and ST471 respectively); one was found in both chicken and chicken-farmer commensal strains (D; ST108), while another profile was identified in a chicken-farmer commensal strain and a human pathogenic one (B1; ST19). These findings suggest clonal and epidemiologic links between chicken and human ciprofloxacin-resistant E. coli isolates and the important role that poultry may play in the epidemiology of human E. coli infections in the Constantine region.

  7. The human homolog of Escherichia coli endonuclease V is a nucleolar protein with affinity for branched DNA structures.

    Directory of Open Access Journals (Sweden)

    Cathrine Fladeby

    Full Text Available Loss of amino groups from adenines in DNA results in the formation of hypoxanthine (Hx bases with miscoding properties. The primary enzyme in Escherichia coli for DNA repair initiation at deaminated adenine is endonuclease V (endoV, encoded by the nfi gene, which cleaves the second phosphodiester bond 3' of an Hx lesion. Endonuclease V orthologs are widespread in nature and belong to a family of highly conserved proteins. Whereas prokaryotic endoV enzymes are well characterized, the function of the eukaryotic homologs remains obscure. Here we describe the human endoV ortholog and show with bioinformatics and experimental analysis that a large number of transcript variants exist for the human endonuclease V gene (ENDOV, many of which are unlikely to be translated into functional protein. Full-length ENDOV is encoded by 8 evolutionary conserved exons covering the core region of the enzyme, in addition to one or more 3'-exons encoding an unstructured and poorly conserved C-terminus. In contrast to the E. coli enzyme, we find recombinant ENDOV neither to incise nor bind Hx-containing DNA. While both enzymes have strong affinity for several branched DNA substrates, cleavage is observed only with E. coli endoV. We find that ENDOV is localized in the cytoplasm and nucleoli of human cells. As nucleoli harbor the rRNA genes, this may suggest a role for the protein in rRNA gene transactions such as DNA replication or RNA transcription.

  8. Design of a covalently linked human interleukin-10 fusion protein and its secretory expression in Escherichia coli.

    Science.gov (United States)

    Guggenbichler, Florian; Büttner, Carolin; Rudolph, Wolfram; Zimmermann, Kurt; Gunzer, Florian; Pöhlmann, Christoph

    2016-12-01

    Wild-type human interleukin-10 (hIL-10) is a non-covalent homodimer with a short half-life, thus limiting its therapeutic applications in vivo. To avoid loss of function due to dimer dissociation, we designed a synthetic hIL-10 analog by bridging both monomers via a 15 amino acid-long peptide spacer in a C-terminal to N-terminal fashion. For secretory expression in Escherichia coli, a 1156 bp fragment was generated from template vector pAZ1 by fusion PCR encoding a T7 promoter region and the signal sequence of the E. coli outer membrane protein F fused in frame to two tandem E. coli codon-optimized mature hIL-10 genes connected via a 45 nucleotide linker sequence. The construct was cloned into pUC19 for high-level expression in E. coli BL21 (DE3). The mean concentrations of hIL-10 fusion protein in the periplasm and supernatant of E. coli at 37 °C growth temperature were 130 ± 40 and 2 ± 1 ng/ml, respectively. The molecular mass of the recombinant protein was assessed via matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis, indicating correct processing of the signaling sequence in E. coli. In vitro biological activity was shown by phosphorylation of signal transducer and activator of transcription protein 3 and suppression of tumor necrosis factor α secretion in lipopolysaccharide-stimulated macrophages.

  9. Characterization of the Shiga toxin-producing Escherichia coli O26 isolated from human in Poland between 1996 and 2014.

    Science.gov (United States)

    Januszkiewicz, A; Wołkowicz, T; Chróst, A; Szych, J

    2015-06-01

    Shiga toxin-producing Escherichia coli (STEC) O26 infections can be comparable with STEC O157 infections in severity of the acute haemolytic-uremic syndrome HUS and long-term sequelae. Among O26 STEC isolates, highly virulent clone O26:H11/H- Sequence Type 29 (ST 29) emerged in Germany in mid-1990s and spread to European countries. However, up to date, no STEC O26:H11/H- belonging to ST29 has been documented in Poland. In this study, we determined the relationship and clonal structure, stx genotypes, plasmid gene profiles and antimicrobial resistance of nine human STEC O26:H11/H- strains from human patients in Poland between 1996 and 2014. Of the 9 human STEC O26:H11/H- strains, two belonged to ST29 and were isolated from two children with HUS and renal failure with sepsis respectively. These strains showed the molecular characteristics of the emerging human-pathogenic ST29 clone (stx1-, stx2a+, eae+, ehxA+, etpD+, katP-, espP-). The remaining STEC O26:H11/H- strains examined in this study, belonged to ST21, with plasmid genes profiles frequently reported in ST21 strains in Europe. STEC O26 infections with serious human health consequences highlight the need of continuous surveillance of non-O157 STEC and implementation of the diagnostic approaches focused on their detection. Significance and impact of the study: These study provides the first data on the occurrence of emerging Shiga toxin-producing Escherichia coli O26:H11 ST 29 clone in human patients in Poland. Those strains show the molecular characteristics of highly virulent new ST29 pathotype (stx1-, stx2a+, eae+ ehxA+, etpD+, katP-, espP-). These results demonstrated prompt efforts to implement diagnostic approaches detection of those pathogen in the European countries.

  10. Dissemination of cephalosporin resistance genes between Escherichia coli strains from farm animals and humans by specific plasmid lineages.

    Directory of Open Access Journals (Sweden)

    Mark de Been

    2014-12-01

    Full Text Available Third-generation cephalosporins are a class of β-lactam antibiotics that are often used for the treatment of human infections caused by Gram-negative bacteria, especially Escherichia coli. Worryingly, the incidence of human infections caused by third-generation cephalosporin-resistant E. coli is increasing worldwide. Recent studies have suggested that these E. coli strains, and their antibiotic resistance genes, can spread from food-producing animals, via the food-chain, to humans. However, these studies used traditional typing methods, which may not have provided sufficient resolution to reliably assess the relatedness of these strains. We therefore used whole-genome sequencing (WGS to study the relatedness of cephalosporin-resistant E. coli from humans, chicken meat, poultry and pigs. One strain collection included pairs of human and poultry-associated strains that had previously been considered to be identical based on Multi-Locus Sequence Typing, plasmid typing and antibiotic resistance gene sequencing. The second collection included isolates from farmers and their pigs. WGS analysis revealed considerable heterogeneity between human and poultry-associated isolates. The most closely related pairs of strains from both sources carried 1263 Single-Nucleotide Polymorphisms (SNPs per Mbp core genome. In contrast, epidemiologically linked strains from humans and pigs differed by only 1.8 SNPs per Mbp core genome. WGS-based plasmid reconstructions revealed three distinct plasmid lineages (IncI1- and IncK-type that carried cephalosporin resistance genes of the Extended-Spectrum Beta-Lactamase (ESBL- and AmpC-types. The plasmid backbones within each lineage were virtually identical and were shared by genetically unrelated human and animal isolates. Plasmid reconstructions from short-read sequencing data were validated by long-read DNA sequencing for two strains. Our findings failed to demonstrate evidence for recent clonal transmission of

  11. Adhesion of human and animal Escherichia coli strains in association with their virulence-associated genes and phylogenetic origins.

    Science.gov (United States)

    Frömmel, Ulrike; Lehmann, Werner; Rödiger, Stefan; Böhm, Alexander; Nitschke, Jörg; Weinreich, Jörg; Groß, Julia; Roggenbuck, Dirk; Zinke, Olaf; Ansorge, Hermann; Vogel, Steffen; Klemm, Per; Wex, Thomas; Schröder, Christian; Wieler, Lothar H; Schierack, Peter

    2013-10-01

    Intestinal colonization is influenced by the ability of the bacterium to inhabit a niche, which is based on the expression of colonization factors. Escherichia coli carries a broad range of virulence-associated genes (VAGs) which contribute to intestinal (inVAGs) and extraintestinal (exVAGs) infection. Moreover, initial evidence indicates that inVAGs and exVAGs support intestinal colonization. We developed new screening tools to genotypically and phenotypically characterize E. coli isolates originating in humans, domestic pigs, and 17 wild mammal and avian species. We analyzed 317 isolates for the occurrence of 44 VAGs using a novel multiplex PCR microbead assay (MPMA) and for adhesion to four epithelial cell lines using a new adhesion assay. We correlated data for the definition of new adhesion genes. inVAGs were identified only sporadically, particularly in roe deer (Capreolus capreolus) and the European hedgehog ( Erinaceus europaeus). The prevalence of exVAGs depended on isolation from a specific host. Human uropathogenic E. coli isolates carried exVAGs with the highest prevalence, followed by badger (Meles meles) and roe deer isolates. Adhesion was found to be very diverse. Adhesion was specific to cells, host, and tissue, though it was also unspecific. Occurrence of the following VAGs was associated with a higher rate of adhesion to one or more cell lines: afa-dra, daaD, tsh, vat, ibeA, fyuA, mat, sfa-foc, malX, pic, irp2, and papC. In summary, we established new screening methods which enabled us to characterize large numbers of E. coli isolates. We defined reservoirs for potential pathogenic E. coli. We also identified a very broad range of colonization strategies and defined potential new adhesion genes.

  12. Optimization of production of recombinant human growth hormone in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Marzieh Rezaei

    2012-01-01

    Full Text Available Background: Human growth hormone (hGH is a single-chain polypeptide that participates in a wide range of biological functions such as metabolism of proteins, carbohydrates and lipids as well as in growth, development and immunity. Growth hormone deficiency in human occurs both in children and adults. The routine treatment for this condition is administration of recombinant human growth hormone (rhGH made by prokaryotes. Since nonglycosylated human growth hormone is a biologically active protein, prokaryotic expression systems are preferred for its production. Materials and Methods: Different strains of E.coli were transformed by plasmid containing human growth hormone gene and cultured in different conditions. After induction by IPTG, recombinant human growth hormone production was assessed using ELISA, dot blotting and western blotting techniques. Results: High levels of rhGH were produced using E.coli prokaryotic protein production system. Conclusion: This simple and cost effective production process could be recruited for large scale production of rhGH.

  13. [Topography and mechanisms of adhesion of uropathogenic Escherichia coli bacteria in the human kidney and renal pelvis].

    Science.gov (United States)

    Vierbuchen, M; Peters, G; Ortmann, M; Pulverer, G; Fischer, R

    1989-01-01

    The occurrence and significance of bacterial carbohydrate recognition proteins (bacterial lectins) and endogenous carbohydrate binding proteins (endogenous lectins) of human urothelium as well as kidney tubulus epithelium was analyzed with respect to the adhesion of urotoxogenic Escherichia coli bacteria. Using biotinylated neoglycoproteins, we demonstrated a wide spectrum of endogenous lectins with Galactose-, Mannose-, Fucose-, N-Acetylgalactosamine-, and N-Acetylglucosamine binding activities in the urothelium. In the kidney the distal nephron and especially the medullar collecting ducts exhibited a similar spectrum of endogenous carbohydrate binding activities as detected for the urothelium. Adhesion- as well as inhibition-experiments with selective blocking of either bacterial lectins or endogenous lectins of the target cells by different carbohydrates both reduced the bacterial adhesion. However, maximal inhibition of bacterial adhesion was achieved by simultanous blocking of microbial and target cell lectins with mannose or mannan. From these results it is reasonable to conclude that specific adhesion which may result in an organotropism (urotropism) of E. coli infection is due to a dual recognition mechanism which is accomplished by the combined interaction of the bachterial and host cell lectins with the corresponding carbohydrates of E. coli and that of the target cells respectively. Further studies showed that normal human serum possesses natural antiadhesins which are represented by the glycan parts of the serum-glycoproteins.

  14. Uncoupling of allosteric and oligomeric regulation in a functional hybrid enzyme constructed from Escherichia coli and human ribonucleotide reductase.

    Science.gov (United States)

    Fu, Yuan; Long, Marcus J C; Rigney, Mike; Parvez, Saba; Blessing, William A; Aye, Yimon

    2013-10-08

    An N-terminal-domain (NTD) and adjacent catalytic body (CB) make up subunit-α of ribonucleotide reductase (RNR), the rate-limiting enzyme for de novo dNTP biosynthesis. A strong linkage exists between ligand binding at the NTD and oligomerization-coupled RNR inhibition, inducible by both dATP and nucleotide chemotherapeutics. These observations have distinguished the NTD as an oligomeric regulation domain dictating the assembly of inactive RNR oligomers. Inactive states of RNR differ between eukaryotes and prokaryotes (α6 in human versus α4β4 in Escherichia coli , wherein β is RNR's other subunit); however, the NTD structurally interconnects individual α2 or α2 and β2 dimeric motifs within the respective α6 or α4β4 complexes. To elucidate the influence of NTD ligand binding on RNR allosteric and oligomeric regulation, we engineered a human- E. coli hybrid enzyme (HE) where human-NTD is fused to E. coli -CB. Both the NTD and the CB of the HE bind dATP. The HE specifically partners with E. coli -β to form an active holocomplex. However, although the NTD is the sole physical tether to support α2 and/or β2 associations in the dATP-bound α6 or α4β4 fully inhibited RNR complexes, the binding of dATP to the HE NTD only partially suppresses HE activity and fully precludes formation of higher-order HE oligomers. We postulate that oligomeric regulation is the ultimate mechanism for potent RNR inhibition, requiring species-specific NTD-CB interactions. Such interdomain cooperativity in RNR oligomerization is unexpected from structural studies alone or biochemical studies of point mutants.

  15. Acute Escherichia coli endotoxaemia decreases the plasma l-arginine/asymmetrical dimethylarginine ratio in humans.

    Science.gov (United States)

    Mittermayer, Friedrich; Namiranian, Khodadad; Pleiner, Johannes; Schaller, Georg; Wolzt, Michael

    2004-06-01

    Acute inflammation impairs vascular function. Based on the association between endothelial dysfunction and plasma concentrations of L-arginine and the endogenous nitric oxide synthase inhibitor ADMA (asymmetrical dimethylarginine), we hypothesized that the ratio between L-arginine and ADMA could be affected by experimental inflammation. Plasma concentrations of L-arginine, ADMA and SDMA (symmetrical dimethylarginine) were studied at baseline and 3.5 h after intravenous administration of Escherichia coli endotoxin [LPS (lipopolysaccharide), 20 units/kg of body mass; n =8] or placebo ( n =9) in healthy males. L-Arginine and dimethylarginines were quantified after solid-phase extraction by reversed-phase HPLC. Body temperature, heart rate and leucocyte count increased after LPS administration ( P <0.01 for all). LPS administration decreased plasma concentrations of L-arginine from 66 micromol/l [95% CI (confidence interval): 56, 88] at baseline to 48 micromol/l (CI: 40, 60) after 3.5 h ( P <0.02), but did not affect ADMA and SDMA concentrations. Consequently, the L-arginine/ADMA ratio declined significantly from a median of 159 (CI: 137, 193) to 135 (CI: 103, 146); a decrease of 25 (CI: -68, -13; P <0.02). L-Arginine, ADMA, SDMA and the L-arginine/ADMA ratio remained constant over time in controls. Acute inflammation reduces the L-arginine/ADMA ratio which could contribute to impaired vascular function.

  16. Effects of the Probiotic Enterococcus faecium and Pathogenic Escherichia coli Strains in a Pig and Human Epithelial Intestinal Cell Model

    Directory of Open Access Journals (Sweden)

    Ulrike Lodemann

    2015-01-01

    Full Text Available The aim of this study has been to elucidate the effect of the probiotic Enterococcus faecium NCIMB 10415 on epithelial integrity in intestinal epithelial cells and whether pre- and coincubation with this strain can reproducibly prevent damage induced by enterotoxigenic (ETEC and enteropathogenic Escherichia coli (EPEC. Porcine (IPEC-J2 and human (Caco-2 intestinal epithelial cells were incubated with bacterial strains and epithelial integrity was assessed by measuring transepithelial electrical resistance (TEER and mannitol flux rates. E. faecium alone increased TEER of Caco-2 cells without affecting mannitol fluxes whereas the E. coli strains decreased TEER and concomitantly increased mannitol flux rates in both cell lines. Preincubation with E. faecium had no effect on the TEER decrease induced by E. coli in preliminary experiments. However, in a second set of experiments using a slightly different protocol, E. faecium ameliorated the TEER decrease induced by ETEC at 4 h in IPEC-J2 and at 2, 4, and 6 h in Caco-2 cells. We conclude that E. faecium positively affected epithelial integrity in monoinfected Caco-2 cells and could ameliorate the damage on TEER induced by an ETEC strain. Reproducibility of the results is, however, limited when experiments are performed with living bacteria over longer periods.

  17. Real-time PCR for quantitative analysis of human commensal Escherichia coli populations reveals a high frequency of subdominant phylogroups.

    Science.gov (United States)

    Smati, Mounira; Clermont, Olivier; Le Gal, Frédéric; Schichmanoff, Olivier; Jauréguy, Françoise; Eddi, Alain; Denamur, Erick; Picard, Bertrand

    2013-08-01

    Escherichia coli is divided into four main phylogenetic groups, which each exhibit ecological specialization. To understand the population structure of E. coli in its primary habitat, we directly assessed the relative proportions of these phylogroups from the stools of 100 healthy human subjects using a new real-time PCR method, which allows a large number of samples to be studied. The detection threshold for our technique was 0.1% of the E. coli population, i.e., 10(5) CFU/g of feces; in other methods based on individual colony analysis, the threshold is 10%. One, two, three, or four phylogenetic groups were simultaneously found in 21%, 48%, 21%, and 8% of the subjects, respectively. Phylogroups present at a threshold of less than 10% of the population were found in 40% of the subjects, revealing high within-individual diversity. Phylogroups A and B2 were detected in 74% and 70% of the subjects, respectively; phylogroups B1 and D were detected in 36% and 32%, respectively. When phylogroup B2 was dominant, it tended not to cooccur with other phylogroups. In contrast, other phylogroups were present when phylogroup A was dominant. These data indicate a complex pattern of interactions between the members of a single species within the human gut and identify a reservoir of clones that are present at a low frequency. The presence of these minor clones could explain the fluctuation in the composition of the E. coli microbiota within single individuals that may be seen over time. They could also constitute reservoirs of virulent and/or resistant strains.

  18. The evolution of the Escherichia coli phylogeny.

    Science.gov (United States)

    Chaudhuri, Roy R; Henderson, Ian R

    2012-03-01

    Escherichia coli is familiar to biologists as a classical model system, ubiquitous in molecular biology laboratories around the world. Outside of the laboratory, E. coli strains exist as an almost universal component of the lower-gut flora of humans and animals. Although usually a commensal, E. coli has an alter ego as a pathogen, and is associated with diarrhoeal disease and extra-intestinal infections. The study of E. coli diversity predates the availability of molecular data, with strains initially distinguished by serotyping and metabolic profiling, and genomic diversity illustrated by DNA hybridisation. The quantitative study of E. coli diversity began with the application of multi-locus enzyme electrophoresis (MLEE), and has progressed with the accumulation of nucleotide sequence data, from single genes through multi-locus sequence typing (MLST) to whole genome sequencing. Phylogenetic methods have shed light on the processes of genomic evolution in this extraordinarily diverse species, and revealed the origins of pathogenic E. coli strains, including members of the phylogenetically indistinguishable "genus"Shigella. In May and June 2011, an outbreak of haemorrhagic uraemic syndrome in Germany was linked to a strain of enterohaemorrhagic E. coli (EHEC) O104:H4. Application of high-throughput sequencing technologies allowed the genome and origins of the outbreak strain to be characterised in real time as the outbreak was in progress.

  19. Escherichia coli Pathotypes Occupy Distinct Niches in the Mouse Intestine

    OpenAIRE

    Jessica P Meador; Caldwell, Matthew E.; Cohen, Paul S.; Conway, Tyrrell

    2014-01-01

    Since the first step of the infection process is colonization of the host, it is important to understand how Escherichia coli pathogens successfully colonize the intestine. We previously showed that enterohemorrhagic O157:H7 strain E. coli EDL933 colonizes a niche in the streptomycin-treated mouse intestine that is distinct from that of human commensal strains, which explains how E. coli EDL933 overcomes colonization resistance imparted by some, but not all, commensal E. coli strains. Here we...

  20. Fate of Extended-Spectrum β-Lactamase-Producing Escherichia coli from Faecal Sources in Surface Water and Probability of Human Exposure through Swimming

    NARCIS (Netherlands)

    Schijven, Jack F; Blaak, Hetty; Schets, Franciska M; de Roda Husman, Ana Maria

    2015-01-01

    The goal of this study was to determine the fate of ESBL-producing Escherichia coli (ESBL-EC) emitted from faecal sources in surface water, and the probability of human exposure through swimming. Concentrations of ESBL-EC were measured in recreational waters and in source waters, being water in ditc

  1. Characterization of epidemic IncI1-Iγ plasmids harboring ambler class A and C genes in Escherichia coli and Salmonella enterica from animals and humans

    NARCIS (Netherlands)

    Smith, Hilde; Bossers, Alex; Harders, Frank; Wu, Guanghui; Woodford, Neil; Schwarz, Stefan; Guerra, Beatriz; Rodríguez, Irene; Essen-Zandbergen, Van Alieda; Brouwer, Michael; Mevius, Dik

    2015-01-01

    The aim of the study was to identify the plasmid-encoded factors contributing to the emergence and spread of epidemic IncI1-Iγ plasmids obtained from Escherichia coli and Salmonella enterica isolates from animal and human reservoirs. For this, 251 IncI1-Iγ plasmids carrying various extended-spect

  2. Cytolethal distending toxin from Shiga toxin-producing Escherichia coli O157 causes irreversible G2/M arrest, inhibition of proliferation, and death of human endothelial cells

    NARCIS (Netherlands)

    Bielaszewska, Martina; Sinha, Bhanu; Kuczius, Thorsten; Karch, Helge

    2005-01-01

    Recently, cytolethal distending toxin V (CDT-V), a new member of the CDT family, was identified in Shiga toxin-producing Escherichia coli (STEC) O157 and particular non-O157 serotypes. Here we investigated the biological effects of CDT-V from STEC O157:H(-) (strain 493/89) on human endothelial cells

  3. Characterization of epidemic IncI1-Iγ plasmids harboring ambler class A and C genes in Escherichia coli and Salmonella enterica from animals and humans

    NARCIS (Netherlands)

    Smith, Hilde; Bossers, Alex; Harders, Frank; Wu, Guanghui; Woodford, Neil; Schwarz, Stefan; Guerra, Beatriz; Rodríguez, Irene; van Essen-Zandbergen, Alieda; Brouwer, Michael; Mevius, Dik

    2015-01-01

    The aim of the study was to identify the plasmid-encoded factors contributing to the emergence and spread of epidemic IncI1-Iγ plasmids obtained from Escherichia coli and Salmonella enterica isolates from animal and human reservoirs. For this, 251 IncI1-Iγ plasmids carrying various extended-spectrum

  4. Production in Escherichia coli of a rat chimeric proinsulin polypeptide carrying human A and B chains and its preparative chromatography.

    Science.gov (United States)

    Olmos, J; Cruz, N; Sánchez, M; López, M; Balbás, P; Gosset, G; Valle, F; Bolivar, F

    1994-11-30

    A pseudohuman proinsulin coding DNA sequence (MMRPI) carrying human A and B chains, was constructed via directed mutagenesis of a previously modified rat proinsulin cDNA (MRPI) and expressed as a tryptophan (Trp)LE-proinsulin fusion protein in Escherichia coli W3110. Expression of the hybrid gene was achieved by depletion of tryptophan from the medium. The heterologous fusion protein, accumulated as insoluble inclusion bodies within the cell, was obtained by differential centrifugation and then solubilized using formic acid. At the junction of the two peptides, a methionine residue allowed proinsulin to be released from the carrier protein by cyanogen bromide treatment. The sulfonated form of this proinsulin polypeptide was easily purified, at a preparative level, using ion exchange chromatography.

  5. The distribution of plasmids determining citrate utilization in citrate-positive variants of Escherichia coli from humans, domestic animals, feral birds and environments.

    Science.gov (United States)

    Ishiguro, N; Sato, G

    1979-10-01

    Sixty-seven isolates of citrate-positive variants of Escherichia coli were isolated from human, domestic animal, feral bird and environmental sources. With the exception of citrate utilization, all isolates were identified as typical E. coli by their biochemical reactions. The transmission of the ability to utilize citrate on Simmons' citrate agar was demonstrated in 53 (79.1%) out of the 67 citrate-positive E. coli variants obtained from various sources. Drug resistance determinants and citrate utilizing character were co-transmitted into E. coli K-12 by conjugation among citrate-positive E. coli isolates carrying R plasmids except for that isolated from horses. The other characters (haemolysin or colicin production, raffinose or sucrose fermentation) were not transmitted together with the citrate utilizing character. These facts suggested that the structural gene responsible for citrate utilizing ability in citrate-positive variants of E. coli was located on a conjugative plasmid.

  6. Cellular chain formation in Escherichia coli biofilms

    DEFF Research Database (Denmark)

    Vejborg, Rebecca Munk; Klemm, Per

    2009-01-01

    In this study we report on a novel structural phenotype in Escherichia coli biofilms: cellular chain formation. Biofilm chaining in E. coli K-12 was found to occur primarily by clonal expansion, but was not due to filamentous growth. Rather, chain formation was the result of intercellular......; type I fimbriae expression significantly reduced cellular chain formation, presumably by steric hindrance. Cellular chain formation did not appear to be specific to E coli K-12. Although many urinary tract infection (UTI) isolates were found to form rather homogeneous, flat biofilms, three isolates......, including the prototypic asymptomatic bacteriuria strain, 83972, formed highly elaborate cellular chains during biofilm growth in human urine. Combined, these results illustrate the diversity of biofilm architectures that can be observed even within a single microbial species....

  7. Serotypes, genotypes and antimicrobial resistance patterns of human diarrhoeagenic Escherichia coli isolates circulating in southeastern China.

    Science.gov (United States)

    Chen, Y; Chen, X; Zheng, S; Yu, F; Kong, H; Yang, Q; Cui, D; Chen, N; Lou, B; Li, X; Tian, L; Yang, X; Xie, G; Dong, Y; Qin, Z; Han, D; Wang, Y; Zhang, W; Tang, Y-W; Li, L

    2014-01-01

    Diarrhoeagenic Escherichia coli (DEC) infection is a major health problem in developing countries. The prevalence and characteristics of DEC have not been thoroughly investigated in China. Consecutive faecal specimens from outpatients with acute diarrhoea in nine sentinel hospitals in southeastern China were collected from July 2009 to June 2011. Bacterial and viral pathogens were detected by culture and RT-PCR, respectively. DEC isolates were further classified into five pathotypes using multiplex PCR. The O/H serotypes, sequence types (STs) and antimicrobial susceptibility profiles of the DEC isolates were determined. A total of 2466 faecal specimens were collected, from which 347 (14.1%) DEC isolates were isolated. DEC was the dominant bacterial pathogen detected. The DEC isolates included 217 EAEC, 62 ETEC, 52 EPEC, 14 STEC, one EIEC and one EAEC/ETEC. O45 (6.6%) was the predominant serotype. Genotypic analysis revealed that the major genotype was ST complex 10 (87, 25.6%). Isolates belonging to the serogroups or genotypes of O6, O25, O159, ST48, ST218, ST94 and ST1491 were highly susceptible to the majority of antimicrobials. In contrast, isolates belonging to O45, O15, O1, O169, ST38, ST226, ST69, ST31, ST93, ST394 and ST648 were highly resistant to the majority of antimicrobials. DEC accounted for the majority of bacterial pathogens causing acute diarrhoea in southeastern China, and it is therefore necessary to test for all DEC, not only the EHEC O157:H7. Some serogroups or genotypes of DEC were highly resistant to the majority of antimicrobials. DEC surveillance should be emphasized.

  8. Purification and Refolding of Overexpressed Human Basic Fibroblast Growth Factor in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Mona Alibolandi

    2011-01-01

    Full Text Available This work describes the integration of expanded bed adsorption (EBA and adsorptive protein refolding operations used to recover purified and biologically active human basic fibroblast growth factor from inclusion bodies expressed in E. coli. Insoluble overexpressed human basic fibroblast growth factor has been purified on CM Hyper Z matrix by expanded bed adsorption after isolation and solubilization in 8 M urea. The adsorption was made in expanded bed without clarification steps such as centrifugation. Column refolding was done by elimination of urea and elution with NaCl. The human basic fibroblast growth factor was obtained as a highly purified soluble monomer form with similar behavior in circular dichroism and fluorescence spectroscopy as native protein. A total of 92.52% of the available human basic fibroblast growth factor was recovered as biologically active and purified protein using the mentioned purification and refolding process. This resulted in the first procedure describing high-throughput purification and refolding of human basic fibroblast growth factor in one step and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution.

  9. Human sepsis-associated Escherichia coli (SEPEC) is able to adhere to and invade kidney epithelial cells in culture

    Energy Technology Data Exchange (ETDEWEB)

    Conceição, R.A. [Departamento de Genética, Evolução e Bioagentes, Universidade Estadual de Campinas, Campinas, SP (Brazil); Ludovico, M.S. [Departamento de Microbiologia, Universidade Estadual de Londrina, Londrina, PR (Brazil); Andrade, C.G.T.J. [Departamento de Biologia Geral, Universidade Estadual de Londrina, Londrina, PR (Brazil); Yano, T. [Departamento de Genética, Evolução e Bioagentes, Universidade Estadual de Campinas, Campinas, SP (Brazil)

    2012-04-13

    The adhesins of extraintestinal pathogenic Escherichia coli are essential for mediating direct interactions between the microbes and the host cell surfaces that they infect. Using fluorescence microscopy and gentamycin protection assays, we observed that 49 sepsis-associated E. coli (SEPEC) strains isolated from human adults adhered to and invaded Vero cells in the presence of D-mannose (100%). In addition, bacteria concentrations of approximately 2 × 10{sup 7} CFU/mL were recovered from Vero cells following an invasion assay. Furthermore, PCR analysis of adhesin genes showed that 98.0% of these SEPEC strains tested positive for fimH, 69.4% for flu, 53.1% for csgA, 38.8% for mat, and 32.7% for iha. Analysis of the invasin genes showed that 16.3% of the SEPEC strains were positive for tia, 12.3% for gimB, and 10.2% for ibeA. Therefore, these data suggest that SEPEC adhesion to cell surfaces occurs through non-fimH mechanisms. Scanning electron microscopy showed the formation of microcolonies on the Vero cell surface. SEPEC invasiveness was also confirmed by the presence of intracellular bacteria, and ultrastructural analysis using electron transmission microscopy revealed bacteria inside the Vero cells. Taken together, these results demonstrate that these SEPEC strains had the ability to adhere to and invade Vero cells. Moreover, these data support the theory that renal cells may be the predominant pathway through which SEPEC enters human blood vessels.

  10. Expression Characterization and Preparation of Human Amyloid Precursor Protein in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    XU Guang-wei; WANG Jia-peng; HUANG Xue-mei; ZHANG Ying-jiu

    2009-01-01

    To analyze whether expressed amyloid precursor protein(APP) existed in hydrophilic(cytoplasmid) or hy-drophobic(lipid bilayer) environment in E. coli and to obtain intact APP for study on its function, we investigated the expression characterization and preparation of the three intact isoforms APP770, APP751, and APP695 in E. coli. The results show that these expressed APPs existed both in hydrophilic cytoplasm region as inclusion bodies and hy-drophobic membrane region as membrane-bound state in E. coll. APPs in inclusion bodies were purified on an NTA-Ni2. agarose column after dissolving in the urea buffer and APPs in membrane-bound state were obtained by ultracentrifugation. The activity analysis indicates that APP770 and APP751 exhibited strong trypsin-inhibitory activity like the natural ones. These results indicate that E. coil cells can be used as host cells for the expression of human integral membrane protein like APP in either soluble or membrane-bound state unless the interest protein undergone post-translational modification is required.

  11. Detection of mcr-1 encoding plasmid-mediated colistin-resistant Escherichia coli isolates from human bloodstream infection and imported chicken meat, Denmark 2015.

    Science.gov (United States)

    Hasman, Henrik; Hammerum, Anette M; Hansen, Frank; Hendriksen, Rene S; Olesen, Bente; Agersø, Yvonne; Zankari, Ea; Leekitcharoenphon, Pimlapas; Stegger, Marc; Kaas, Rolf S; Cavaco, Lina M; Hansen, Dennis S; Aarestrup, Frank M; Skov, Robert L

    2015-01-01

    The plasmid-mediated colistin resistance gene, mcr-1, was detected in an Escherichia coli isolate from a Danish patient with bloodstream infection and in five E. coli isolates from imported chicken meat. One isolate from chicken meat belonged to the epidemic spreading sequence type ST131. In addition to IncI2, an incX4 replicon was found to be linked to mcr-1. This report follows a recent detection of mcr-1 in E. coli from animals, food and humans in China.

  12. Association Between Antimicrobial Resistance in Escherichia coli Isolates from Food Animals and Blood Stream Isolates from Humans in Europe: An Ecological Study

    DEFF Research Database (Denmark)

    Vieira, Antonio; Collignon, Peter; Aarestrup, Frank Møller

    2011-01-01

    Background: In addition to medical antimicrobial usage, the use of antimicrobials in food animals contributes to the occurrence of resistance among some bacterial species isolated from infections in humans. Recently, several studies have indicated that a large proportion of Escherichia coli causing...... infections in humans, especially those resistant to antimicrobials, have an animal origin.Methods: We analyzed the correlation between the prevalence of antimicrobial resistance in E. coli isolates from blood stream infections in humans and in E. coli isolates from poultry, pigs, and cattle between 2005...... and 2008 for 11 countries, using available surveillance data. We also assessed the correlation between human antimicrobial usage and the occurrence of resistance in E. coli isolates from blood stream infections.Results: Strong and significant correlations between prevalences of resistance to ampicillin (r...

  13. Effect of Culture Condition Variables on Human Endostatin Gene Expression in Escherichia coli Using Response Surface Methodology

    Science.gov (United States)

    Mohajeri, Abbas; Pilehvar-Soltanahmadi, Yones; Abdolalizadeh, Jalal; Karimi, Pouran; Zarghami, Nosratollah

    2016-01-01

    Background Recombinant human endostatin (rhES) is an angiogenesis inhibitor used as a specific drug for the treatment of non-small-cell lung cancer. As mRNA concentration affects the recombinant protein expression level, any factor affecting mRNA concentration can alter the protein expression level. Response surface methodology (RSM) based on the Box-Behnken design (BBD) is a statistical tool for experimental design and for optimizing biotechnological processes. Objectives This investigation aimed to predict and develop the optimal culture conditions for mRNA expression of the synthetic human endostatin (hES) gene in Escherichia coli BL21 (DE3). Materials and Methods The hES gene was amplified, cloned, and expressed in the E. coli expression system. Three factors, including isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, post-induction time, and cell density before induction, were selected as important factors. The mRNA expression level was determined using real-time PCR. The expression levels of hES mRNA under the different growth conditions were analyzed. SDS-PAGE and western blot analyses were carried out for further confirmation of interest-gene expression. Results A maximum rhES mRNA level of 376.16% was obtained under the following conditions: 0.6 mM IPTG, 7 hours post-induction time, and 0.9 cell density before induction. The level of rhES mRNA was significantly correlated with post-induction time, IPTG concentration, and cell density before induction (P coli.

  14. Detection of mcr-1-Carrying Escherichia coli Causing Bloodstream Infection in a New York City Hospital: Avian Origins, Human Concerns?

    Science.gov (United States)

    Macesic, Nenad; Green, Daniel; Wang, Zheng; Sullivan, Sean B; Shim, Kevin; Park, Sarah; Whittier, Susan; Furuya, E Yoko; Gomez-Simmonds, Angela; Uhlemann, Anne-Catrin

    2017-01-01

    The spread of mcr-1 in the United States remains poorly defined. mcr-1-producing Escherichia coli that also carried blaSHV-12 was detected in a hospitalized patient. No additional cases were identified during screening of 801 Gram-negative isolates. Genomic sequencing identified an IncX4 mcr-1- harboring plasmid and ST117 clonal background associated with avian pathogenic E coli.

  15. Siderophore biosynthesis coordinately modulated the virulence-associated interactive metabolome of uropathogenic Escherichia coli and human urine.

    Science.gov (United States)

    Su, Qiao; Guan, Tianbing; Lv, Haitao

    2016-04-14

    Uropathogenic Escherichia coli (UPEC) growth in women's bladders during urinary tract infection (UTI) incurs substantial chemical exchange, termed the "interactive metabolome", which primarily accounts for the metabolic costs (utilized metabolome) and metabolic donations (excreted metabolome) between UPEC and human urine. Here, we attempted to identify the individualized interactive metabolome between UPEC and human urine. We were able to distinguish UPEC from non-UPEC by employing a combination of metabolomics and genetics. Our results revealed that the interactive metabolome between UPEC and human urine was markedly different from that between non-UPEC and human urine, and that UPEC triggered much stronger perturbations in the interactive metabolome in human urine. Furthermore, siderophore biosynthesis coordinately modulated the individualized interactive metabolome, which we found to be a critical component of UPEC virulence. The individualized virulence-associated interactive metabolome contained 31 different metabolites and 17 central metabolic pathways that were annotated to host these different metabolites, including energetic metabolism, amino acid metabolism, and gut microbe metabolism. Changes in the activities of these pathways mechanistically pinpointed the virulent capability of siderophore biosynthesis. Together, our findings provide novel insights into UPEC virulence, and we propose that siderophores are potential targets for further discovery of drugs to treat UPEC-induced UTI.

  16. Robust growth of Escherichia coli.

    Science.gov (United States)

    Wang, Ping; Robert, Lydia; Pelletier, James; Dang, Wei Lien; Taddei, Francois; Wright, Andrew; Jun, Suckjoon

    2010-06-22

    The quantitative study of the cell growth has led to many fundamental insights in our understanding of a wide range of subjects, from the cell cycle to senescence. Of particular importance is the growth rate, whose constancy represents a physiological steady state of an organism. Recent studies, however, suggest that the rate of elongation during exponential growth of bacterial cells decreases cumulatively with replicative age for both asymmetrically and symmetrically dividing organisms, implying that a "steady-state" population consists of individual cells that are never in a steady state of growth. To resolve this seeming paradoxical observation, we studied the long-term growth and division patterns of Escherichia coli cells by employing a microfluidic device designed to follow steady-state growth and division of a large number of cells at a defined reproductive age. Our analysis of approximately 10(5) individual cells reveals a remarkable stability of growth whereby the mother cell inherits the same pole for hundreds of generations. We further show that death of E. coli is not purely stochastic but is the result of accumulating damages. We conclude that E. coli, unlike all other aging model systems studied to date, has a robust mechanism of growth that is decoupled from cell death.

  17. Characteristics of Quinolone Resistance in Escherichia coli Isolates from Humans, Animals, and the Environment in the Czech Republic

    Science.gov (United States)

    Röderova, Magdalena; Halova, Dana; Papousek, Ivo; Dolejska, Monika; Masarikova, Martina; Hanulik, Vojtech; Pudova, Vendula; Broz, Petr; Htoutou-Sedlakova, Miroslava; Sauer, Pavel; Bardon, Jan; Cizek, Alois; Kolar, Milan; Literak, Ivan

    2017-01-01

    Escherichia coli is a common commensal bacterial species of humans and animals that may become a troublesome pathogen causing serious diseases. The aim of this study was to characterize the quinolone resistance phenotypes and genotypes in E. coli isolates of different origin from one area of the Czech Republic. E. coli isolates were obtained from hospitalized patients and outpatients, chicken farms, retailed turkeys, rooks wintering in the area, and wastewaters. Susceptibility of the isolates grown on the MacConkey agar with ciprofloxacin (0.05 mg/L) to 23 antimicrobial agents was determined. The presence of plasmid-mediated quinolone resistance (PMQR) and ESBL genes was tested by PCR and sequencing. Specific mutations in gyrA, gyrB, parC, and parE were also examined. Multilocus sequence typing and pulsed-field gel electrophoresis were performed to assess the clonal relationship. In total, 1050 E. coli isolates were obtained, including 303 isolates from humans, 156 from chickens, 105 from turkeys, 114 from the rooks, and 372 from wastewater samples. PMQR genes were detected in 262 (25%) isolates. The highest occurrence was observed in isolates from retailed turkey (49% of the isolates were positive) and inpatients (32%). The qnrS1 gene was the most common PMQR determinant identified in 146 (56%) followed by aac(6′)-Ib-cr in 77 (29%), qnrB19 in 41 (16%), and qnrB1 in 9 (3%) isolates. All isolates with high level of ciprofloxacin resistance (>32 mg/L) carried double or triple mutations in gyrA combined with single or double mutations in parC. The most frequently identified substitutions were Ser(83)Leu; Asp(87)Asn in GyrA, together with Ser(80)Ile, or Glu(84)Val in ParC. Majority of these isolates showed resistance to beta-lactams and multiresistance phenotype was found in 95% isolates. Forty-eight different sequence types among 144 isolates analyzed were found, including five major clones ST131 (26), ST355 (19), ST48 (13), ST95 (10), and ST10 (5). No isolates

  18. Escherichia coli O157:H7.

    Science.gov (United States)

    Mead, P S; Griffin, P M

    1998-10-10

    Escherichia coli O157 was first identified as a human pathogen in 1982. One of several Shiga toxin-producing serotypes known to cause human illness, the organism probably evolved through horizontal acquisition of genes for Shiga toxins and other virulence factors. E. coli O157 is found regularly in the faeces of healthy cattle, and is transmitted to humans through contaminated food, water, and direct contact with infected people or animals. Human infection is associated with a wide range of clinical illness, including asymptomatic shedding, non-bloody diarrhoea, haemorrhagic colitis, haemolytic uraemic syndrome, and death. Since laboratory practices vary, physicians need to know whether laboratories in their area routinely test for E. coli O157 in stool specimens. Treatment with antimicrobial agents remains controversial: some studies suggest that treatment may precipitate haemolytic uraemic syndrome, and other studies suggest no effect or even a protective effect. Physicians can help to prevent E. coli O157 infections by counselling patients about the hazards of consuming undercooked ground meat or unpasteurised milk products and juices, and about the importance of handwashing to prevent the spread of diarrhoeal illness, and by informing public-health authorities when they see unusual numbers of cases of bloody diarrhoea or haemolytic uraemic syndrome.

  19. Extraintestinal Pathogenic Escherichia coli, a Common Human Pathogen: Challenges for Vaccine Development and Progress in the Field.

    Science.gov (United States)

    Poolman, Jan T; Wacker, Michael

    2016-01-01

    Extraintestinal pathogenic Escherichia coli (ExPEC) is the most common gram-negative bacterial pathogen in humans. ExPEC causes the vast majority of urinary tract infections (UTIs), is a leading cause of adult bacteremia, and is the second most common cause of neonatal meningitis. Increasing multidrug resistance among ExPEC strains constitutes a major obstacle to treatment and is implicated in increasing numbers of hospitalizations and deaths and increasing healthcare costs associated with ExPEC infections. An effective vaccine against ExPEC infection is urgently needed. The O antigen, a component of the surface lipopolysaccharide, has been identified as a promising vaccine target. With the availability of a novel bioconjugation technology it is expected that multivalent O antigen conjugate vaccines can be produced at industrial scale. Clinical proof of concept of a 4-valent O antigen conjugate vaccine is ongoing. An ExPEC vaccine effective against strains that are associated with major diseases and resistant to multiple drugs could be routinely delivered to individuals at risk of developing severe E. coli infection, such as elderly people, individuals undergoing abdominal surgery and prostatic biopsy procedures, and persons at risk of recurrent and/or complicated UTI.

  20. Soluble expression of human leukemia inhibitory factor with protein disulfide isomerase in Escherichia coli and its simple purification.

    Science.gov (United States)

    Song, Jung-A; Jung, A Song; Koo, Bon-Kyung; Chong, Seon-Ha; Kim, Kyunhoo; Choi, Dong Kyu; Thi Vu, Thu Trang; Nguyen, Minh Tan; Jeong, Boram; Ryu, Han-Bong; Kim, Injune; Jang, Yeon Jin; Robinson, Robert Charles; Choe, Han

    2013-01-01

    Human leukemia inhibitory factor (hLIF) is a multifunctional cytokine that is essential for maintaining the pluripotency of embryonic stem cells. hLIF may be also be useful in aiding fertility through its effects on increasing the implantation rate of fertilized eggs. Thus these applications in biomedical research and clinical medicine create a high demand for bioactive hLIF. However, production of active hLIF is problematic since eukaryotic cells demonstrate limited expression and prokaryotic cells produce insoluble protein. Here, we have adopted a hybrid protein disulfide isomerase design to increase the solubility of hLIF in Escherichia coli. Low temperature expression of hLIF fused to the b'a' domain of protein disulfide isomerase (PDIb'a') increased the soluble expression in comparison to controls. A simple purification protocol for bioactive hLIF was established that includes removal of the PDIb'a' domain by cleavage by TEV protease. The resulting hLIF, which contains one extra glycine residue at the N-terminus, was highly pure and demonstrated endotoxin levels below 0.05 EU/μg. The presence of an intramolecular disulfide bond was identified using mass spectroscopy. This purified hLIF effectively maintained the pluripotency of a murine embryonic stem cell line. Thus we have developed an effective method to produce a pure bioactive version of hLIF in E. coli for use in biomedical research.

  1. Crohn's disease adherent-invasive Escherichia coli colonize and induce strong gut inflammation in transgenic mice expressing human CEACAM.

    Science.gov (United States)

    Carvalho, Frédéric A; Barnich, Nicolas; Sivignon, Adeline; Darcha, Claude; Chan, Carlos H F; Stanners, Clifford P; Darfeuille-Michaud, Arlette

    2009-09-28

    Abnormal expression of CEACAM6 is observed at the apical surface of the ileal epithelium in Crohn's disease (CD) patients, and CD ileal lesions are colonized by pathogenic adherent-invasive Escherichia coli (AIEC). We investigated the ability of AIEC reference strain LF82 to colonize the intestinal mucosa and to induce inflammation in CEABAC10 transgenic mice expressing human CEACAMs. AIEC LF82 virulent bacteria, but not nonpathogenic E. coli K-12, were able to persist in the gut of CEABAC10 transgenic mice and to induce severe colitis with reduced survival rate, marked weight loss, increased rectal bleeding, presence of erosive lesions, mucosal inflammation, and increased proinflammatory cytokine expression. The colitis depended on type 1 pili expression by AIEC bacteria and on intestinal CEACAM expression because no sign of colitis was observed in transgenic mice infected with type 1 pili-negative LF82-Delta fimH isogenic mutant or in wild-type mice infected with AIEC LF82 bacteria. These findings strongly support the hypothesis that in CD patients having an abnormal intestinal expression of CEACAM6, AIEC bacteria via type 1 pili expression can colonize the intestinal mucosa and induce gut inflammation. Thus, targeting AIEC adhesion to gut mucosa represents a new strategy for clinicians to prevent and/or to treat ileal CD.

  2. Intracellular processing and presentation of T cell epitopes, expressed by recombinant Escherichia coli and Salmonella typhimurium, to human T cells

    NARCIS (Netherlands)

    G.M.G.M. Verjans (George); C.M. Janssen (Riny); F.G.C.M. Uytdehaag (Fons); C.E.M. van Doornik (C. E M); J. Tommassen (Jan)

    1995-01-01

    textabstractVaccines based on recombinant attenuated bacteria represent a potentially safe and effective immunization strategy. A carrier system was developed to analyze in vitro whether foreign T cell epitopes, inserted in the outer membrane protein PhoE of Escherichia coli and expressed by recombi

  3. Shiga toxin-producing Escherichia coli (STEC) from Humans in the Netherlands : Novel diagnostic approach, molecular characterization and phylogenetic background

    NARCIS (Netherlands)

    Ferdous, Mithila

    2017-01-01

    The shiga toxin-producing Escherichia coli (STEC) bacterium poses a serious public health risk as it can cause life threatening condition hemorrhagic colitis and hemolytic uremic syndrome besides diarrhea. STEC is associated with several food related outbreaks worldwide, including the famous one in

  4. Distribution of the urease gene cluster among and urease activities of enterohemorrhagic Escherichia coli O157 isolates from humans

    NARCIS (Netherlands)

    Friedrich, Alexander W; Köck, Robin; Bielaszewska, Martina; Zhang, Wenlan; Karch, Helge; Mathys, Werner

    2005-01-01

    Enterohemorrhagic Escherichia coli (EHEC) O157 strains belong to two closely related major groups, which are differentiated by their sorbitol fermentation phenotypes. Here we studied the conservation of urease genes and their expression in sorbitol-fermenting (SF) and non-SF EHEC O157 isolates. PCR

  5. Shiga toxin-producing Escherichia coli (STEC) from Humans in the Netherlands : Novel diagnostic approach, molecular characterization and phylogenetic background

    NARCIS (Netherlands)

    Ferdous, Mithila

    2017-01-01

    The shiga toxin-producing Escherichia coli (STEC) bacterium poses a serious public health risk as it can cause life threatening condition hemorrhagic colitis and hemolytic uremic syndrome besides diarrhea. STEC is associated with several food related outbreaks worldwide, including the famous one in

  6. Escherichia coli isolates from commercial chicken meat and eggs cause sepsis, meningitis and urinary tract infection in rodent models of human infections.

    Science.gov (United States)

    Mellata, M; Johnson, J R; Curtiss, R

    2017-07-13

    The zoonotic potential of Escherichia coli from chicken-source food products is important to define for public health purposes. Previously, genotypic and phenotypic screening of E. coli isolates from commercial chicken meat and shell eggs identified some E. coli strains that by molecular criteria resembled human-source extraintestinal pathogenic E. coli (ExPEC). Here, to clarify the zoonotic risk of such chicken-source E. coli, we compared selected E. coli isolates from chicken meat and eggs, stratified by molecularly defined ExPEC status, to human-source ExPEC and to laboratory E. coli for virulence in rodent models of sepsis, meningitis and UTI, and evaluated whether specific bacterial characteristics predict experimental virulence. Multiple chicken-source E. coli resembled human-source ExPEC in their ability to cause one or multiple different ExPEC-associated infections. Swimming ability corresponded with urovirulence, K1 capsule corresponded with ability to cause neonatal meningitis, and biofilm formation in urine corresponded with ability to cause sepsis. In contrast, molecularly defined ExPEC status and individual genotypic traits were uncorrelated with ability to cause sepsis, and neither complement sensitivity nor growth in human urine corresponded with virulence in any infection model. These findings establish that chicken-derived food products contain E. coli strains that, in rodent models of multiple human-associated ExPEC infections, are able to cause disease comparably to human-source E. coli clinical isolates, which suggests that they may pose a significant food safety threat. Further study is needed to define the level of risk they pose to human health, which if appreciable would justify efforts to monitor for and reduce or eliminate them. © 2017 Blackwell Verlag GmbH.

  7. Enteropathogenic Escherichia coli: foe or innocent bystander?

    Science.gov (United States)

    Hu, J; Torres, A G

    2015-08-01

    Enteropathogenic Escherichia coli (EPEC) remain one the most important pathogens infecting children and they are one of the main causes of persistent diarrhoea worldwide. Historically, typical EPEC (tEPEC), defined as those isolates with the attaching and effacement (A/E) genotype (eae(+)), which possess bfpA(+) and lack the stx(-) genes are found strongly associated with diarrhoeal cases. However, occurrence of atypical EPEC (aEPEC; eae(+)bfpA(-)stx(-)) in diarrhoeal and asymptomatic hosts has made investigators question the role of these pathogens in human disease. Current epidemiological data are helping to answer the question of whether EPEC is mainly a foe or an innocent bystander during infection.

  8. Refolding and Characterization of Recombinant Human GST-PD-1 Fusion Protein Expressed in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    Da-Wei LI; Jian-Feng YU; Yong-Jing CHEN; Hong-Bing MA; Zheng-Fei WANG; Yi-Bei ZHU; Xue-Guang ZHANG

    2004-01-01

    Programmed death-1 (PD-1) is a costimulatory molecule of CD28 family expressed onactivated T, B and myeloid cells. The engagement of PD-1 with its two ligands, PD-L1 and PD-L2, inhibitsproliferation of T cell and production of a series of its cytokines. The blockade of PD-1 pathway is involvedin antiviral and antitumoral immunity. In this study, human PD-1 cDNA encoding extracellular domain wasamplified and cloned into expression plasmid pGEX-5x-3. The fusion protein GST-PD-1 was effectivelyexpressed in E. coli BL21 (DE3) as inclusion bodies and a denaturation and refolding procedure was performed to obtain bioactive soluble GST-PD-1. Fusion protein of above 95% purity was acquired by a convenient two-step purification using GST affinity and size exclusion columns. Furthermore, a PD-L1-dependentin vitro bioassay method was set up to characterize GST-PD-1 bioactivity. The results suggested that GSTPD-1 could competently block the interaction between PD-L1 and PD-1 and increase the production of IL2 and IFN-γ of phytohemagglutinin-activated T cells.

  9. Refoiding and Characterization of Recombinant Human GST-PD-1 Fusion Protein Expressed in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    Da-WeiLI; Jian-FengYU; Yong-JingCHEN; Hong-BingMA; Zheng-FeiWANG; Yi-BeiZHU; Xue-GuangZHANG

    2004-01-01

    Programmed death-1 (PD-1) is a costimulatory molecule of CD28 family expressed onactivated T, B and myeloid cells. The engagement of PD-1 with its two ligands, PD-L1 and PD-L2, inhibitsproliferation of T cell and production of a series of its cytokines. The blockade of PD-1 pathway is involvedin antiviral and antitumoral immunity. In this study, human PD-1 cDNA encoding extracellular domain wasamplified and cloned into expression plasmid pGEX-Sx-3. The fusion protein GST-PD-1 was effectivelyexpressed in E. coli BL21 (DE3) as inclusion bodies and a denaturation and refolding procedure was per-formed to obtain bioactive soluble GST-PD-I. Fusion protein of above 95% purity was acquired by a conve-nient two-step purification using GST affinity and size exclusion columns. Furthermore, a PD-L1-dependentin vitro bioassay method was set up to characterize GST-PD-1 bioactivity. The results suggested that GST-PD-1 could competently block the interaction between PD-Ll and PD-l and increase the production of IL-2 and IFN-γ of phytohemagglutinin-activated T cells.

  10. Genes under positive selection in Escherichia coli

    DEFF Research Database (Denmark)

    Petersen, Lise; Bollback, Jonathan P; Dimmic, Matt

    2007-01-01

    We used a comparative genomics approach to identify genes that are under positive selection in six strains of Escherichia coli and Shigella flexneri, including five strains that are human pathogens. We find that positive selection targets a wide range of different functions in the E. coli genome......, including cell surface proteins such as beta barrel porins, presumably because of the involvement of these genes in evolutionary arms races with other bacteria, phages, and/or the host immune system. Structural mapping of positively selected sites on trans-membrane beta barrel porins reveals...... that the residues under positive selection occur almost exclusively in the extracellular region of the proteins that are enriched with sites known to be targets of phages, colicins, or the host immune system. More surprisingly, we also find a number of other categories of genes that show very strong evidence...

  11. Effects of Rhodomyrtus tomentosa Extract on Killing Activity of Human Neutrophils and Membrane Integrity of Enterohaemorrhagic Escherichia coli O157:H7.

    Science.gov (United States)

    Hmoteh, Jutharat; Syed Musthafa, Khadar; Pomwised, Rattanaruji; Voravuthikunchai, Supayang Piyawan

    2016-05-27

    Enterohaemorrhagic Escherichia coli (E. coli) O157:H7 is one of the most virulent causative agents of foodborne disease. Use of antibiotics for the treatment against E. coli O157:H7 infection leads to hemolytic uremic syndrome. The present study evaluated the potential of ethanolic leaf extract of a medicinal plant, Rhodomyrtus tomentosa in enhancing the killing activity of human neutrophils against E. coli O157:H7. In addition, the effects of the extract on membrane permeability of the organisms were studied. In the killing assay, percentage survival of the bacterial cells after being exposed to human neutrophils in the presence of various concentrations of the extract were determined. At 45 min, percentage survival of E. coli O157:H7 and E. coli ATCC 25922 after treated with neutrophils in the presence of the extract at 125-250 µg/mL was 58.48%-50.28% and 69.13%-35.35%, respectively. Furthermore, upon treatment with R. tomentosa at 250 µg/mL uptake of crystal violet by E. coli O157:H7 and E. coli ATCC 25922 was increased to 40.07% and 36.16%, respectively. Therefore, it is suggested that the extract exhibited dual effects as immunostimulant and membrane permeabilizing agent perhaps resulted in enhancing the killing activity of neutrophils against the organisms.

  12. Effects of Rhodomyrtus tomentosa Extract on Killing Activity of Human Neutrophils and Membrane Integrity of Enterohaemorrhagic Escherichia coli O157:H7

    Directory of Open Access Journals (Sweden)

    Jutharat Hmoteh

    2016-05-01

    Full Text Available Enterohaemorrhagic Escherichia coli (E. coli O157:H7 is one of the most virulent causative agents of foodborne disease. Use of antibiotics for the treatment against E. coli O157:H7 infection leads to hemolytic uremic syndrome. The present study evaluated the potential of ethanolic leaf extract of a medicinal plant, Rhodomyrtus tomentosa in enhancing the killing activity of human neutrophils against E. coli O157:H7. In addition, the effects of the extract on membrane permeability of the organisms were studied. In the killing assay, percentage survival of the bacterial cells after being exposed to human neutrophils in the presence of various concentrations of the extract were determined. At 45 min, percentage survival of E. coli O157:H7 and E. coli ATCC 25922 after treated with neutrophils in the presence of the extract at 125–250 µg/mL was 58.48%–50.28% and 69.13%–35.35%, respectively. Furthermore, upon treatment with R. tomentosa at 250 µg/mL uptake of crystal violet by E. coli O157:H7 and E. coli ATCC 25922 was increased to 40.07% and 36.16%, respectively. Therefore, it is suggested that the extract exhibited dual effects as immunostimulant and membrane permeabilizing agent perhaps resulted in enhancing the killing activity of neutrophils against the organisms.

  13. Preparation of soluble isotopically labeled human growth hormone produced in Escherichia coli.

    Science.gov (United States)

    Lee, Jin-Hee; Jeong, Ji-Seon; Kim, Sook-Kyung; Song, Jimyeong; Lee, Ji Youn; Baek, Soyun; Choi, Jun-Hyuk

    2016-11-01

    Isotopically labeled proteins have been used as internal standards for mass spectrometry (MS)-based absolute protein quantification. Although this approach can provide highly accurate analyses of proteins of interest within a complex mixture, one of the major limitations of this method is the difficulty in preparing uniformly labeled standards. Human growth hormone (hGH) is one of the most important hormones that circulate throughout the body, and its measurement is primarily of interest in the diagnosis and treatment of growth disorders. In order to provide a useful internal standard for MS-based hGH measurement, we describe an efficient strategy to produce a potentially valuable, stable isotope-labeled hGH with high purity and yield. The strategy involves the following steps: solubilization of hGH under labeling conditions, detection of stable isotope incorporation, large-scale purification, analysis of the labeled protein, and assessment of the labeling efficiency. We show that the yield of soluble hGH under selective isotopic labeling conditions can be greatly increased by optimizing protein expression and extraction. Our efficient method for generating isotopically labeled hGH does not influence the structural integrity of hGH. Finally, we assessed the efficiency of stable isotope labeling at the intact protein level, and the result was further verified by amino acid analysis. These results clearly indicate that our labeling approach allows an almost complete incorporation of (13)C6(15)N4-arginine into the hGH expressed in E.coli without detectable isotope scrambling.

  14. Enterohemorrhagic Escherichia coli induce attaching and effacing lesions and hemorrhagic colitis in human and bovine intestinal xenograft models

    Directory of Open Access Journals (Sweden)

    Lilach Golan

    2011-01-01

    Enterohemorrhagic Escherichia coli (EHEC O157:H7 is an important cause of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome in humans worldwide. The two major virulence determinants of EHEC are the Shiga toxins (Stx and the type III secretion system (T3SS, including the injected effectors. Lack of a good model system hinders the study of EHEC virulence. Here, we investigated whether bovine and human intestinal xenografts in SCID mice can be useful for studying EHEC and host tissue interactions. Fully developed, germ-free human and bovine small intestine and colon were established by subcutaneous transplantation of human and bovine fetal gut into SCID mice. Xenografts were allowed to develop for 3–4 months and thereafter were infected by direct intraluminal inoculation of Stx-negative derivatives of EHEC O157:H7, strain EDL933. The small intestine and colon xenografts closely mimicked the respective native tissues. Upon infection, EHEC induced formation of typical attaching and effacing lesions and tissue damage that resembled hemorrhagic colitis in colon xenografts. By contrast, xenografts infected with an EHEC mutant deficient in T3SS remained undamaged. Furthermore, EHEC did not attach to or damage the epithelium of small intestinal tissue, and these xenografts remained intact. EHEC damaged the colon in a T3SS-dependent manner, and this model is therefore useful for studying the molecular details of EHEC interactions with live human and bovine intestinal tissue. Furthermore, we demonstrate that Stx and gut microflora are not essential for EHEC virulence in the human gut.

  15. Phosphoramidate pronucleotides: a comparison of the phosphoramidase substrate specificity of human and Escherichia coli histidine triad nucleotide binding proteins.

    Science.gov (United States)

    Chou, Tsui-Fen; Baraniak, Janina; Kaczmarek, Renata; Zhou, Xin; Cheng, Jilin; Ghosh, Brahma; Wagner, Carston R

    2007-01-01

    To facilitate the delivery of nucleotide-based therapeutics to cells and tissues, a variety of pronucleotide approaches have been developed. Our laboratory and others have demonstrated that nucleoside phosphoramidates can be activated intracellularly to the corresponding 5'-monophosphate nucleotide and that histidine triad nucleotide binding proteins (Hints) are potentially responsible for their bioactivation. Hints are conserved and ubiquitous enzymes that hydrolyze phosphoramidate bonds between nucleoside 5'-monophosphate and an amine leaving group. On the basis of the ability of nucleosides to quench the fluorescence of covalently linked amines containing indole, a sensitive, continuous fluorescence-based assay was developed. A series of substrates linking the naturally fluorogenic indole derivatives to nucleoside 5'-monophosphates were synthesized, and their steady state kinetic parameters of hydrolysis by human Hint1 and Escherichia coli hinT were evaluated. To characterize the elemental and stereochemical effect on the reaction, two P-diastereoisomers of adenosine or guanosine phosphoramidothioates were synthesized and studied to reveal a 15-200-fold decrease in the specificity constant (kcat/Km) when the phosphoryl oxygen is replaced with sulfur. While a stereochemical preference was not observed for E. coli hinT, hHint1 exhibited a 300-fold preference for d-tryptophan phosphoramidates over l-isomers. The most efficient substrates evaluated to date are those that contain the less sterically hindering amine leaving group, tryptamine, with kcat and Km values comparable to those found for adenosine kinase. The apparent second-order rate constants (kcat/Km) for adenosine tryptamine phosphoramidate monoester were found to be 107 M-1 s-1 for hHint1 and 106 M-1 s-1 for E. coli hinT. Both the human and E. coli enzymes preferred purine over pyrimidine analogues. Consistent with observed hydrogen bonding between the 2'-OH group of adenosine monophosphate and the

  16. Overexpression and Purification of Human Calcitonin Gene Related Peptide - Receptor Component Protein (CGRP-RCP) in Escherichia coli

    Science.gov (United States)

    Tolun, Adviye A.; Dickerson, Ian M.; Malhotra, Arun

    2007-01-01

    Calcitonin gene-related peptide (CGRP) is a neuropeptide secreted by the central and peripheral nervous system nerves that has important physiological functions such as vasodilation, cardiotonic actions, metabolic and pro-inflammatory effects. The CGRP receptor is unique among G-protein coupled receptors in that a functional CGRP receptor consists of at least three proteins: Calcitonin Like Receptor (CLR), Receptor Activity Modifying Protein (RAMP1) and Receptor Component Protein (RCP). RCP is a required factor in CGRP-mediated signal transduction and it couples the CGRP receptor to the signal transduction pathway. Here we describe methods to overexpress and purify RCP for structure-function studies. Human RCP was cloned and overexpressed with a poly-histidine tag and as a Maltose Binding Protein (MBP) fusion in Escherichia coli using commercially available expression vectors. While his-tagged RCP is prone to aggregation, solubility is improved when RCP is expressed as a MBP fusion. Expression and purification procedures for these constructs are described. Results from these studies will facilitate structural analysis of human RCP, and allow further understanding of RCP function. PMID:17067815

  17. Purification and characterization of tagless recombinant human elongation factor 2 kinase (eEF-2K) expressed in Escherichia coli.

    Science.gov (United States)

    Abramczyk, Olga; Tavares, Clint D J; Devkota, Ashwini K; Ryazanov, Alexey G; Turk, Benjamin E; Riggs, Austen F; Ozpolat, Bulent; Dalby, Kevin N

    2011-10-01

    The eukaryotic elongation factor 2 kinase (eEF-2K) modulates the rate of protein synthesis by impeding the elongation phase of translation by inactivating the eukaryotic elongation factor 2 (eEF-2) via phosphorylation. eEF-2K is known to be activated by calcium and calmodulin, whereas the mTOR and MAPK pathways are suggested to negatively regulate kinase activity. Despite its pivotal role in translation regulation and potential role in tumor survival, the structure, function, and regulation of eEF-2K have not been described in detail. This deficiency may result from the difficulty of obtaining the recombinant kinase in a form suitable for biochemical analysis. Here we report the purification and characterization of recombinant human eEF-2K expressed in the Escherichia coli strain Rosetta-gami 2(DE3). Successive chromatography steps utilizing Ni-NTA affinity, anion-exchange, and gel filtration columns accomplished purification. Cleavage of the thioredoxin-His(6)-tag from the N-terminus of the expressed kinase with TEV protease yielded 9 mg of recombinant (G-D-I)-eEF-2K per liter of culture. Light scattering shows that eEF-2K is a monomer of ∼85 kDa. In vitro kinetic analysis confirmed that recombinant human eEF-2K is able to phosphorylate wheat germ eEF-2 with kinetic parameters comparable to the mammalian enzyme.

  18. High-level expression and purification of recombinant human growth hormone produced in soluble form in Escherichia coli.

    Science.gov (United States)

    Levarski, Zdenko; Šoltýsová, Andrea; Krahulec, Ján; Stuchlík, Stanislav; Turňa, Ján

    2014-08-01

    Human growth hormone (hGH) was one of the first recombinant proteins approved for the treatment of human growth disorders. Its small size (191 amino acids), possession of only 2 disulphide bonds and absence of posttranslational modifications make Escherichia coli the host of choice for its production on any scale. In this work, we have utilized an efficient T7 based expression system to produce high levels of soluble thioredoxin-hGH (Trx-hGH) fusion protein. We outline a relatively simple three step purification process employing two immobilized metal-affinity chromatography and one anion-exchange steps and removal of fusion partner by enterokinase cleavage yielding native hGH. The ability of cell populations to produce quantities of up to 1 g/L of the soluble Trx-hGH fusion protein has been tested in flask cultivations as well as in batch and fed-batch bioreactor runs. The sequence and structure of derived hGH were confirmed by mass spectrometry and circular dichroism and its native function, to induce cell proliferation, was confirmed by employing a Nb2 cell line proliferation assay.

  19. Expression of suppressor of cytokine signalling 3 (SOCS3) in human bladder epithelial cells infected with uropathogenic Escherichia coli.

    Science.gov (United States)

    Demirel, Isak; Säve, Susanne; Kruse, Robert; Persson, Katarina

    2013-02-01

    Suppressor of cytokine signalling (SOCS) proteins inhibit pro-inflammatory signalling mediated by Janus-activated kinase (JAK)-signal transducer and activator of transcription (STAT) pathways. To evade the immune response some pathogens appear to modify the host SOCS proteins. Uropathogenic Escherichia coli (UPEC) are able to subvert the host response evoked by bladder epithelial cells, but the mechanisms are not fully understood. The objective of this study was to investigate whether UPEC can modify the host SOCS and STAT3 response. Real time RT-PCR studies demonstrated an increased SOCS1 and SOCS3 expression in the isolated human bladder epithelial cell lines (RT-4 and 5637) in response to cytokines. UPEC strain IA2 increased SOCS3, but not SOCS1, mRNA levels with a peak at 6 h after infection. The increase of SOCS3 was confirmed at the protein level by Western blotting. The UPEC strain IA2 caused a time-dependent decrease in the phosphorylation of STAT3. This study demonstrates that UPEC are able to affect SOCS3 and STAT3 signalling in human uroepithelial cells. The finding that UPEC are able to induce mediators involved in suppression of host cytokine signalling may help to elucidate how UPEC may circumvent the host response during urinary tract infection.

  20. Different responses to nitrate and nitrite by the model organism Escherichia coli and the human pathogen Neisseria gonorrhoeae.

    Science.gov (United States)

    Whitehead, R N; Cole, J A

    2006-02-01

    The ability of Escherichia coli to use both nitrate and nitrite as terminal electron acceptors during anaerobic growth is mediated by the dual-acting two-component regulatory systems NarX-NarL and NarQ-NarP. In contrast, Neisseria gonorrhoeae responds only to nitrite: it expresses only NarQ-NarP. We have shown that although N. gonorrhoeae NarQ can phosphorylate E. coli NarL and NarP, the N. gonorrhoeae NarP is unable to regulate gene expression in E. coli. Mutagenesis experiments have revealed residues in E. coli NarQ that are essential for nitrate and nitrite sensing. Chimaeric proteins revealed domains of NarQ that are important for ligand sensing.

  1. A Comparative Study on the Expression, Purification and Functional Characterization of Human Adiponectin in Pichia pastoris and Escherichia coli

    Directory of Open Access Journals (Sweden)

    Hussin A. Rothan

    2012-03-01

    Full Text Available Adiponectin is one of the most bioactive substances secreted by adipose tissue and is involved in the protection against metabolic syndrome, artherosclerosis and type II diabetes. Research into the use of adiponectin as a promising drug for metabolic syndromes requires production of this hormone in high quantities considering its molecular isoforms. The objective of this study is to produce recombinant human adiponectin by Pichia pastoris (P-ADP as a cheap and convenient eukaryotic expression system for potential application in pharmaceutical therapy. For comparison, adiponectin was also expressed using the Escherichia coli (E-ADP expression system. Adiponectin was constructed by overlap-extension PCR, and cloned in standard cloning vector and hosts. Recombinant expression vectors were cloned in the P. pastoris and E. coli host strains, respectively. SDS-PAGE and western blotting were used to detect and analyse expressed recombinant protein in both systems. Adiponectin was purified by affinity chromatography and quantified using the Bradford Assay. The results of this study indicated that P-ADP quantity (0.111 mg/mL was higher than that of E-ADP (0.04 mg/mL and both were produced in soluble form. However, P-ADP was able to form high molecular weights of adiponectin molecules, whilst E-ADP was not able to form isoforms higher than trimer. In addition, P-ADP was more active in lowering blood glucose compared with E-ADP. The two types of proteins were equally efficient and significantly decreased blood triglyceride and increased high density lipoprotein. We conclude that P. pastoris is able to produce high quantity of bioactive adiponectin for potential use in treatment of metabolic syndromes.

  2. Stimulation of Escherichia coli F-18Col- Type-1 fimbriae synthesis by leuX

    DEFF Research Database (Denmark)

    Newman, Joseph V.; Burghoff, Robert L.; Pallesen, Lars

    1994-01-01

    Escherichia coli F-18, a normal human fecal isolate, is an excellent colonizer of the streptomycin-treated mouse large intestine. E. coli F-18Col-, a derivative of E. coli F-18 which no longer makes the E. coli F-18 colicin, colonizes the large intestine as well as E. coli F-18 when fed to mice a...

  3. High diversity of plasmids harbouring blaCMY-2 among clinical Escherichia coli isolates from humans and companion animals in the upper Midwestern USA

    DEFF Research Database (Denmark)

    Bortolaia, Valeria; Hansen, Katrine Hartung; Nielsen, Christine

    2014-01-01

    To determine the population structure and genetic relatedness of plasmids encoding CMY-2 β-lactamase in clinical Escherichia coli from humans and companion animals within a defined geographical area....

  4. Effects of subinhibitory amounts of ampicillin, amoxycillin and mecillinam on the adhesion of Escherichia coli bacteria to human urinary tract epithelial cells: a preliminary study.

    Science.gov (United States)

    Svanborg-Edén, C; Sandberg, T; Stenqvist, K; Ahlstedt, S

    1979-01-01

    Attachment to mucous surfaces may be a prerequisite for bacteria colonizing these surfaces or invading underlying tissues. Subinhibitory amounts of ampicillin and amoxycillin but not mecillinam decreased the attachment of Escherichia coli bacteria to human uro-epithelial cells in vitro. No significant synergistic effect on the attachment by the antibiotics was obtained. The present report indicates a new parameter for the study of antibacterial actions of drugs.

  5. Escherichia coli transcriptional regulatory network

    Directory of Open Access Journals (Sweden)

    Agustino Martinez-Antonio

    2011-06-01

    Full Text Available Escherichia coli is the most well-know bacterial model about the function of its molecular components. In this review are presented several structural and functional aspects of their transcriptional regulatory network constituted by transcription factors and target genes. The network discussed here represent to 1531 genes and 3421 regulatory interactions. This network shows a power-law distribution with a few global regulators and most of genes poorly connected. 176 of genes in the network correspond to transcription factors, which form a sub-network of seven hierarchical layers where global regulators tend to be set in superior layers while local regulators are located in the lower ones. There is a small set of proteins know as nucleoid-associated proteins, which are in a high cellular concentrations and reshape the nucleoid structure to influence the running of global transcriptional programs, to this mode of regulation is named analog regulation. Specific signal effectors assist the activity of most of transcription factors in E. coli. These effectors switch and tune the activity of transcription factors. To this type of regulation, depending of environmental signals is named the digital-precise-regulation. The integration of regulatory programs have place in the promoter region of transcription units where it is common to observe co-regulation among global and local TFs as well as of TFs sensing exogenous and endogenous conditions. The mechanistic logic to understand the harmonious operation of regulatory programs in the network should consider the globalism of TFs, their signal perceived, coregulation, genome position, and cellular concentration. Finally, duplicated TFs and their horizontal transfer influence the evolvability of members of the network. The most duplicated and transferred TFs are located in the network periphery.

  6. Structure of Escherichia coli tryptophanase.

    Science.gov (United States)

    Ku, Shao Yang; Yip, Patrick; Howell, P Lynne

    2006-07-01

    Pyridoxal 5'-phosphate (PLP) dependent tryptophanase has been isolated from Escherichia coli and its crystal structure has been determined. The structure shares the same fold with and has similar quaternary structure to Proteus vulgaris tryptophanase and tyrosine-phenol lyase, but is found in a closed conformation when compared with these two enzymes. The tryptophanase structure, solved in its apo form, does not have covalent PLP bound in the active site, but two sulfate ions. The sulfate ions occupy the phosphoryl-binding site of PLP and the binding site of the alpha-carboxyl of the natural substrate tryptophan. One of the sulfate ions makes extensive interactions with both the transferase and PLP-binding domains of the protein and appears to be responsible for holding the enzyme in its closed conformation. Based on the sulfate density and the structure of the P. vulgaris enzyme, PLP and the substrate tryptophan were modeled into the active site. The resulting model is consistent with the roles of Arg419 in orienting the substrate to PLP and acidifying the alpha-proton of the substrate for beta-elimination, Lys269 in the formation and decomposition of the PLP quinonoid intermediate, Arg230 in orienting the substrate-PLP intermediates in the optimal conformation for catalysis, and His463 and Tyr74 in determining substrate specificity and suggests that the closed conformation observed in the structure could be induced by substrate binding and that significant conformational changes occur during catalysis. A catalytic mechanism for tryptophanase is proposed. Since E. coli tryptophanase has resisted forming diffraction-quality crystals for many years, the molecular surface of tryptophanase has been analyzed in various crystal forms and it was rationalized that strong crystal contacts occur on the flat surface of the protein and that the size of crystal contact surface seems to correlate with the diffraction quality of the crystal.

  7. Structure of Escherichia Coli Tryptophanase

    Energy Technology Data Exchange (ETDEWEB)

    Ku,S.; Yip, P.; Howell, P.

    2006-01-01

    Pyridoxal 5'-phosphate (PLP) dependent tryptophanase has been isolated from Escherichia coli and its crystal structure has been determined. The structure shares the same fold with and has similar quaternary structure to Proteus vulgaris tryptophanase and tyrosine-phenol lyase, but is found in a closed conformation when compared with these two enzymes. The tryptophanase structure, solved in its apo form, does not have covalent PLP bound in the active site, but two sulfate ions. The sulfate ions occupy the phosphoryl-binding site of PLP and the binding site of the {alpha}-carboxyl of the natural substrate tryptophan. One of the sulfate ions makes extensive interactions with both the transferase and PLP-binding domains of the protein and appears to be responsible for holding the enzyme in its closed conformation. Based on the sulfate density and the structure of the P. vulgaris enzyme, PLP and the substrate tryptophan were modeled into the active site. The resulting model is consistent with the roles of Arg419 in orienting the substrate to PLP and acidifying the {alpha}-proton of the substrate for {beta}-elimination, Lys269 in the formation and decomposition of the PLP quinonoid intermediate, Arg230 in orienting the substrate-PLP intermediates in the optimal conformation for catalysis, and His463 and Tyr74 in determining substrate specificity and suggests that the closed conformation observed in the structure could be induced by substrate binding and that significant conformational changes occur during catalysis. A catalytic mechanism for tryptophanase is proposed. Since E. coli tryptophanase has resisted forming diffraction-quality crystals for many years, the molecular surface of tryptophanase has been analyzed in various crystal forms and it was rationalized that strong crystal contacts occur on the flat surface of the protein and that the size of crystal contact surface seems to correlate with the diffraction quality of the crystal.

  8. Facile purification of Escherichia coli expressed tag-free recombinant human tumor necrosis factor alpha from supernatant.

    Science.gov (United States)

    Zhang, Chun; Liu, Yongdong; Zhao, Dawei; Li, Xiunan; Yu, Rong; Su, Zhiguo

    2014-03-01

    Fusing affinity tag at N-terminus was reported to decrease the biological activity of the recombinant human tumor necrosis factor alpha. Although preparation of tag-free rhTNF-α has already been achieved, the processes were yet laborious, especially in large scale. In this paper, tag-free rhTNF-α was almost equally synthesized by Escherichia coli in both soluble and insoluble forms. A two-step ion exchange chromatography, DEAE-Sepharose combined with CM-Sepharose, was developed to purify the soluble specie from supernatant after cell lysis. Native PAGE and HP-SEC showed the rhTNF-α extracted from supernatant existed in a homogeneous form. HP-SAX and SDS-PAGE analysis demonstrated the purity of the final fraction was over 98% with a very high recovery of 75%. Circular dichroism spectrum demonstrated that β-sheet structure was dominant and fluorescence analysis suggested no dramatic exposure of aromatic amino acid residues on the protein surface. Bioassay indicated that purified rhTNF-α was biologically active with a specific activity of approximately 2.0×10(7)U/mg. All these results suggested that this two-step ion exchange chromatography is efficient for preparation of biologically active tag-free rhTNF-α from supernatant.

  9. CfaE tip mutations in enterotoxigenic Escherichia coli CFA/I fimbriae define critical human intestinal binding sites.

    Science.gov (United States)

    Baker, K K; Levine, M M; Morison, J; Phillips, A; Barry, E M

    2009-05-01

    Enterotoxigenic Escherichia coli (ETEC) use colonization factors to attach to the human intestinal mucosa, followed by enterotoxin expression that induces net secretion and diarrhoeal illness. ETEC strain H10407 expresses CFA/I fimbriae, which are composed of multiple CfaB structural subunits and a CfaE tip subunit. Currently, the contribution of these individual fimbrial subunits in intestinal binding remains incompletely defined. To identify the role of CfaE in attachment in the native ETEC background, an R181A single-amino-acid substitution was introduced by recombination into the H10407 genome. The substitution of R181A eliminated haemagglutination and binding of intestinal mucosa biopsies in in vitro organ culture assays, without loss of CFA/I fimbriae expression. Wild-type in trans plasmid-expressed cfaE restored the binding phenotype. In contrast, in trans expression of cfaE containing amino acid 181 substitutions with similar amino acids, lysine, methionine and glutamine did not restore the binding phenotype, indicating that the loss of the binding phenotype was due to localized areas of epitope disruption. R181 appears to have an irreplaceable role in the formation of a receptor-binding feature on CFA/I fimbriae. The results specifically indicate that the CfaE tip protein is a required binding factor in CFA/I-mediated ETEC colonization, making it a potentially important vaccine antigen. © 2009 Blackwell Publishing Ltd.

  10. Fosfomycin Resistance in Escherichia coli, Pennsylvania, USA.

    Science.gov (United States)

    Alrowais, Hind; McElheny, Christi L; Spychala, Caressa N; Sastry, Sangeeta; Guo, Qinglan; Butt, Adeel A; Doi, Yohei

    2015-11-01

    Fosfomycin resistance in Escherichia coli is rare in the United States. An extended-spectrum β-lactamase-producing E. coli clinical strain identified in Pennsylvania, USA, showed high-level fosfomycin resistance caused by the fosA3 gene. The IncFII plasmid carrying this gene had a structure similar to those found in China, where fosfomycin resistance is commonly described.

  11. Infectious endocarditis caused by Escherichia coli

    DEFF Research Database (Denmark)

    Lauridsen, Trine Kiilerich; Arpi, Magnus; Fritz-Hansen, Thomas

    2011-01-01

    Although Escherichia coli is among the most common causes of Gram-negative bacteraemia, infectious endocarditis (IE) due to this pathogen is rare. A 67-y-old male without a previous medical history presented with a new mitral regurgitation murmur and persisting E. coli bacteraemia in spite of broad...

  12. Prevalence of Escherichia coli O157

    NARCIS (Netherlands)

    Abdissa, Rosa; Haile, Woynshet; Fite, Akafete Teklu; Beyi, Ashenafi Feyisa; Agga, Getahun E.; Edao, Bedaso Mammo; Tadesse, Fanos; Korsa, Mesula Geloye; Beyene, Takele; Beyene, Tariku Jibat; Zutter, De Lieven; Cox, Eric; Goddeeris, Bruno Maria

    2017-01-01

    Background: There is paucity of information regarding the epidemiology of Escherichia coli O157: H7 in developing countries. In this study, we investigated the occurrence of E. coli O157: H7 associated with beef cattle at processing plants and at retail shops in Ethiopia. Methods: Various samples

  13. Escherichia coli survival in waters: Temperature dependence

    Science.gov (United States)

    Knowing the survival rates of water-borne Escherichia coli is important in evaluating microbial contamination and making appropriate management decisions. E. coli survival rates are dependent on temperature, a dependency that is routinely expressed using an analogue of the Q10 mo...

  14. Strategies for Protein Overproduction in Escherichia coli.

    Science.gov (United States)

    Mott, John E.

    1984-01-01

    Examines heterologous expression in Escherichia coli and the role of regulatory sequences which control gene expression at transcription resulting in abundant production of messenger RNA and regulatory sequences in mRNA which promote efficient translation. Also examines the role of E. coli cells in stabilizing mRNA and protein that is…

  15. Human Neutrophil’S Chemotaxis and Intracellular Killing in Response to Type 1 Piliated Uropathogenic Escherichia Coli

    Directory of Open Access Journals (Sweden)

    N Nooritalab

    2007-06-01

    Full Text Available Introduction: Uropathogenic Escherichia coli (UPEC, the commonest cause of urinary tract infections, bind to target cells and phagocytes via several distinct pairs of adhesins and receptors. In some cases bacterial binding to phagocytes ends to bacterial elimination. The survival and spread of bacteria in infected tissues are determined by the resistance of bacteria to elimination by phagocytic cells like neutrophils. The aim of this study was to determine the role of type 1 pili in interaction of UPEC with human neutrophils and its effect on bacterial killing. Methods: We used 3 clinical and 1 standard strains of type 1 piliated and 1 unpiliated standard strain of UPEC. Type 1 piliated and unpiliated strains (obtained by growth at a pilus-restrictive temperature of UPEC were used for determining the effect of this pili on migration of neutrophils towards bacteria in Boyden chamber. Also intracellular killing of bacteria by human neutrophils was estimated by counting of the number of viable bacteria in 45 minutes after incubation of piliated and unpiliated strains with purified neutrophils.The results were analyzed with t-test. Results: In chemotaxis assay, PMN migration towards piliated strains was 46-73% of that observed with FMLP, but it was 34-41% in unpiliated strains.The results obtained showed that type 1 piliated UPEC stimulated significantly greater chemotaxis than did unpiliated ones(P<0.05.In phagocytic killing assay, 40-70% of piliated strains were killed in 30 min after incubation with PMN, but the number of viable unpiliated strains was increased in this period of time .There was a significant difference between the intracellular killing of piliated and unpiliated strains with neutrophils (P<0.05. Discusion: Human granulocytes recognize type 1 piliated UPEC via α-mannose-containing structures. So the existence of this adhesin on UPEC strains can leads to increase of neutrophil chemotaxis towards bacteria, phagocytosis and

  16. Refolding and purification of recombinant human (Pro)renin receptor from Escherichia coli by ion exchange chromatography.

    Science.gov (United States)

    Wang, Fei; Guo, Jinjin; Bai, Quan; Wang, Lili

    2014-01-01

    Purification of the recombinant human renin receptor (rhRnR) is a major aspect of its biological or biophysical analysis, as well as structural research. A simple and efficient method for the refolding and purification of rhRnR expressed in Escherichia coli with weak anion-exchange chromatography (WAX) was presented in this work. The solution containing denatured rhRnR in 8.0 mol/L urea extracted from the inclusion bodies was directly injected into the WAX column. The aggregation was prevented and the soluble form of renatured rhRnR in aqueous solution was obtained after desorption from the column. Effects of the extracting solutions, the pH values and urea concentrations in the mobile phase, as well as the sample size on the refolding and purification of rhRnR were investigated, indicating that the above mentioned factors had remarkable influences on the efficiency of refolding, purification and mass recovery of rhRnR. Under the optimal conditions, rhRnR was successfully refolded and purified simultaneously by WAX in one step within only 30 min. The result was satisfactory with mass recovery of 71.8% and purity of 94.8%, which was further tested by western blotting. The specific binding of the purified rhRnR to recombinant human renin was also determined using surface plasmon resonance (SPR). The association constant of rhRnR to recombinant human renin was calculated to be 3.25 × 10(8) L/mol, which demonstrated that rhRnR was already renatured and simultaneously purified in one step using WAX. All of the above demonstrate that protein folding liquid chromatography (PFLC) should be a powerful tool for the purification and renaturation of rhRnR.

  17. ANALISIS CEMARAN BAKTERI Escherichia coli ANALISIS CEMARAN BAKTERI Escherichia coli ANALISIS CEMARAN BAKTERI Escherichia coli

    OpenAIRE

    ANGGREINI, RAHAYU

    2015-01-01

    2015 RAHAYU ANGGREINI coli Penelitian ini bertujuan untuk melakukan identifikasi cemaran bakteri E. coli O157:H7 pada daging sapi di kota Makassar. Sampel pada penelitian ini sebanyak 72 sampel Kata Kunci : Daging sapi, pasar tradisional, E. coli, E. coli O157:H7, kontaminasi bakteri, identifikasi E. coli O157:H7.

  18. ANALISIS CEMARAN BAKTERI Escherichia coli ANALISIS CEMARAN BAKTERI Escherichia coli ANALISIS CEMARAN BAKTERI Escherichia coli

    OpenAIRE

    ANGGREINI, RAHAYU

    2015-01-01

    2015 RAHAYU ANGGREINI coli Penelitian ini bertujuan untuk melakukan identifikasi cemaran bakteri E. coli O157:H7 pada daging sapi di kota Makassar. Sampel pada penelitian ini sebanyak 72 sampel Kata Kunci : Daging sapi, pasar tradisional, E. coli, E. coli O157:H7, kontaminasi bakteri, identifikasi E. coli O157:H7.

  19. The ferric yersiniabactin uptake receptor FyuA is required for efficient biofilm formation by urinary tract infectious Escherichia coli in human urine

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Ferrieres, Lionel; Klemm, Per

    2008-01-01

    Urinary tract infection (UTI) is the most common infection in patients with indwelling urinary catheters, and bacterial biofilm formation is a major problem in this type of infection. Escherichia coli is responsible for the large majority of UTIs. Free iron is strictly limited in the human urinary...... tract and there is fierce competition between the host and infectious bacteria for this essential metal. Urinary tract infectious E coli have highly efficient mechanisms of iron acquisition, one of which is the yersiniabactin system. The fyuA gene, encoding the yersiniabactin receptor, is one...... of the most upregulated genes in biofilm; it was upregulated 63-fold in the E coli UTI strain VR50. FyuA was found to be highly important for biofilm formation in iron-poor environments such as human urine. Mutants in fyuA show aberrant biofilm formation and the cells become filamentous; a VR50fyuA mutant...

  20. Escherichia coli in chronic inflammatory bowel diseases: An update on adherent invasive Escherichia coli pathogenicity

    Institute of Scientific and Technical Information of China (English)

    Margarita; Martinez-Medina; Librado; Jesus; Garcia-Gil

    2014-01-01

    Escherichia coli(E. coli), and particularly the adherent invasive E. coli(AIEC) pathotype, has been increasingly implicated in the ethiopathogenesis of Crohn’s disease(CD). E. coli strains with similar pathogenic features to AIEC have been associated with other intestinal disorders such as ulcerative colitis, colorectal cancer, and coeliac disease, but AIEC prevalence in these diseases remains largely unexplored. Since AIEC was described one decade ago, substantial progress has been made in deciphering its mechanisms of pathogenicity. However, the molecular bases that characterize the phenotypic properties of this pathotype are still not well resolved. A review of studies focused on E. coli populations in inflammatory bowel disease(IBD) is presented here and we discuss about the putative role of this species on each IBD subtype. Given the relevance of AIEC in CD pathogenesis, we present the latest research findings concerning AIEC host-microbe interactions and pathogenicity. We also review the existing data regarding the prevalence and abundance of AIEC in CD and its association with other intestinal diseases from humans and animals, in order to discuss the AIEC disease- and hostspecificity. Finally, we highlight the fact that dietarycomponents frequently found in industrialized countries may enhance AIEC colonization in the gut, which merits further investigation and the implementation of preventative measures.

  1. Differential Effects of Escherichia coli Nissle and Lactobacillus rhamnosus Strain GG on Human Rotavirus Binding, Infection, and B Cell Immunity.

    Science.gov (United States)

    Kandasamy, Sukumar; Vlasova, Anastasia N; Fischer, David; Kumar, Anand; Chattha, Kuldeep S; Rauf, Abdul; Shao, Lulu; Langel, Stephanie N; Rajashekara, Gireesh; Saif, Linda J

    2016-02-15

    Rotavirus (RV) causes significant morbidity and mortality in children worldwide. The intestinal microbiota plays an important role in modulating host-pathogen interactions, but little is known about the impact of commonly used probiotics on human RV (HRV) infection. In this study, we compared the immunomodulatory effects of Gram-positive (Lactobacillus rhamnosus strain GG [LGG]) and Gram-negative (Escherichia coli Nissle [EcN]) probiotic bacteria on virulent human rotavirus (VirHRV) infection and immunity using neonatal gnotobiotic piglets. Gnotobiotic piglets were colonized with EcN, LGG, or EcN+LGG or uncolonized and challenged with VirHRV. Mean peak virus shedding titers and mean cumulative fecal scores were significantly lower in EcN-colonized compared with LGG-colonized or uncolonized piglets. Reduced viral shedding titers were correlated with significantly reduced small intestinal HRV IgA Ab responses in EcN-colonized compared with uncolonized piglets post-VirHRV challenge. However the total IgA levels post-VirHRV challenge in the intestine and pre-VirHRV challenge in serum were significantly higher in EcN-colonized than in LGG-colonized piglets. In vitro treatment of mononuclear cells with these probiotics demonstrated that EcN, but not LGG, induced IL-6, IL-10, and IgA, with the latter partially dependent on IL-10. However, addition of exogenous recombinant porcine IL-10 + IL-6 to mononuclear cells cocultured with LGG significantly enhanced IgA responses. The greater effectiveness of EcN in moderating HRV infection may also be explained by the binding of EcN but not LGG to Wa HRV particles or HRV 2/4/6 virus-like particles but not 2/6 virus-like particles. Results suggest that EcN and LGG differentially modulate RV infection and B cell responses.

  2. Chromate Reductase YieF from Escherichia coli Enhances Hexavalent Chromium Resistance of Human HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Xuan Liu

    2015-05-01

    Full Text Available Hexavalent chromium (Cr(VI is a serious environmental pollutant and human toxicant. Mammalian cells are very sensitive to chromate as they lack efficient chromate detoxifying strategy, e.g., chromate-reducing genes that are widely present in prokaryotes. To test whether introduction of prokaryotic chromate-reducing gene into mammalian cells could render higher chromate resistance, an Escherichia coli chromate-reducing gene yieF was transfected into human HepG2 cells. The expression of yieF was measured in stably transfected cells HepG2-YieF by quantitative RT-PCR and found up-regulated by 3.89-fold upon Cr(VI induction. In chromate-reducing ability test, HepG2-YieF cells that harbored the reductase showed significantly higher reducing ability of Cr(VI than HepG2 control cells. This result was further supported by the evidence of increased Cr(VI-removing ability of crude cell extract of HepG2-YieF. Moreover, HepG2-YieF demonstrated 10% higher viability and decreased expression of GSH synthesizing enzymes under Cr(VI stress. Subcellular localization of YieF was determined by tracing GFP-YieF fusion protein that was detected in both nucleus and cytoplasm by laser confocal microscopy. Altogether, this study successfully demonstrated that the expression of a prokaryotic Cr(VI-reducing gene yieF endowed mammalian cell HepG2 with enhanced chromate resistance, which brought new insight of Cr(VI detoxification in mammalian cells.

  3. Chromate Reductase YieF from Escherichia coli Enhances Hexavalent Chromium Resistance of Human HepG2 Cells.

    Science.gov (United States)

    Liu, Xuan; Wu, Gaofeng; Zhang, Yanli; Wu, Dan; Li, Xiangkai; Liu, Pu

    2015-05-26

    Hexavalent chromium (Cr(VI)) is a serious environmental pollutant and human toxicant. Mammalian cells are very sensitive to chromate as they lack efficient chromate detoxifying strategy, e.g., chromate-reducing genes that are widely present in prokaryotes. To test whether introduction of prokaryotic chromate-reducing gene into mammalian cells could render higher chromate resistance, an Escherichia coli chromate-reducing gene yieF was transfected into human HepG2 cells. The expression of yieF was measured in stably transfected cells HepG2-YieF by quantitative RT-PCR and found up-regulated by 3.89-fold upon Cr(VI) induction. In chromate-reducing ability test, HepG2-YieF cells that harbored the reductase showed significantly higher reducing ability of Cr(VI) than HepG2 control cells. This result was further supported by the evidence of increased Cr(VI)-removing ability of crude cell extract of HepG2-YieF. Moreover, HepG2-YieF demonstrated 10% higher viability and decreased expression of GSH synthesizing enzymes under Cr(VI) stress. Subcellular localization of YieF was determined by tracing GFP-YieF fusion protein that was detected in both nucleus and cytoplasm by laser confocal microscopy. Altogether, this study successfully demonstrated that the expression of a prokaryotic Cr(VI)-reducing gene yieF endowed mammalian cell HepG2 with enhanced chromate resistance, which brought new insight of Cr(VI) detoxification in mammalian cells.

  4. Expression of Functional Human Sialyltransferases ST3Gal1 and ST6Gal1 in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Maria Elena Ortiz-Soto

    Full Text Available Sialyltransferases (STs are disulfide-containing, type II transmembrane glycoproteins that catalyze the transfer of sialic acid to proteins and lipids and participate in the synthesis of the core structure oligosaccharides of human milk. Sialic acids are found at the outermost position of glycostructures, playing a key role in health and disease. Sialylation is also essential for the production of recombinant therapeutic proteins (RTPs. Despite their importance, availability of sialyltransferases is limited due to the low levels of stable, soluble and active protein produced in bacterial expression systems, which hampers biochemical and structural studies on these enzymes and restricts biotechnological applications. We report the successful expression of active human sialyltransferases ST3Gal1 and ST6Gal1 in commercial Escherichia coli strains designed for production of disulfide-containing proteins. Fusion of hST3Gal1 with different solubility enhancers and substitution of exposed hydrophobic amino acids by negatively charged residues (supercharging-like approach were performed to promote solubility and folding. Co-expression of sialyltransferases with the chaperon/foldases sulfhydryl oxidase, protein disulfide isomerase and disulfide isomerase C was explored to improve the formation of native disulfide bonds. Active sialyltransferases fused with maltose binding protein (MBP were obtained in sufficient amounts for biochemical and structural studies when expressed under oxidative conditions and co-expression of folding factors increased the yields of active and properly folded sialyltransferases by 20%. Mutation of exposed hydrophobic amino acids increased recovery of active enzyme by 2.5-fold, yielding about 7 mg of purified protein per liter culture. Functionality of recombinant enzymes was evaluated in the synthesis of sialosides from the β-d-galactoside substrates lactose, N-acetyllactosamine and benzyl 2-acetamido-2-deoxy-3-O-(β-d-galactopyranosyl-α-d-galactopyranoside.

  5. The versatile strategies of Escherichia coli pathotypes: a mini review

    Directory of Open Access Journals (Sweden)

    C. P. Sousa

    2006-01-01

    Full Text Available The widespread species Escherichia coli includes a broad variety of different types, ranging from highly pathogenic strains to avirulent isolates. Few microorganisms are as versatile as E. coli. Pathogenic strains remain a leading cause of severe and persistent infant diarrhea in developing countries. They may be limited to colonization of a mucosal surface or can disseminate throughout the body and have been implicated in urinary tract infection, sepsis/meningitis and gastrointestinal infection. The human gastrointestinal tract is susceptible to diarrheagenic E. coli infections. Escherichia coli have effectively managed to subvert the host cytoskeleton for their own purposes causing substantial diarrheal disease, a major public health problem worldwide. This review deals with the different strategies regarding E. coli as a pathogen and the virulence traits of its pathotypes highlighting the species as a commensal, opportunistic and specialized pathogen.

  6. 21 CFR 866.3255 - Escherichia coli serological reagents.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Escherichia coli serological reagents. 866.3255... coli serological reagents. (a) Identification. Escherichia coli serological reagents are devices that consist of antigens and antisera used in serological tests to identify Escherichia coli from...

  7. Environmental Escherichia coli: Ecology and public health implications - A review

    Science.gov (United States)

    Jang, Jeonghwan; Hur, Hor-Gil; Sadowsky, Michael J.; Byappanahalli, Muruleedhara; Yan, Tao; Ishii, Satoshi

    2017-01-01

    Escherichia coli is classified as a rod-shaped, Gram-negative bacterium in the family Enterobacteriaceae. The bacterium mainly inhabits the lower intestinal tract of warm-blooded animals, including humans, and is often discharged into the environment through feces or wastewater effluent. The presence of E. coli in environmental waters has long been considered as an indicator of recent fecal pollution. However, numerous recent studies have reported that some specific strains of E. coli can survive for long periods of time, and potentially reproduce, in extra-intestinal environments. This indicates that E. coli can be integrated into indigenous microbial communities in the environment. This naturalization phenomenon calls into question the reliability of E. coli as a fecal indicator bacterium (FIB). Recently, many studies reported that E. coli populations in the environment are affected by ambient environmental conditions affecting their long-term survival. Large-scale studies of population genetics provide the diversity and complexity of E. coli strains in various environments, affected by multiple environmental factors. This review examines the current knowledge on the ecology of E. coli strains in various environments in regards to its role as a FIB and as a naturalized member of indigenous microbial communities. Special emphasis is given on the growth of pathogenic E. coli in the environment, and the population genetics of environmental members of the genus Escherichia. The impact of environmental E. coli on water quality and public health is also discussed.

  8. Characterisation of Shiga toxin-producing Escherichia coli O157 strains isolated from humans in Argentina, Australia and New Zealand

    Directory of Open Access Journals (Sweden)

    Robins-Browne Roy M

    2008-03-01

    Full Text Available Abstract Background Shiga toxin-producing Escherichia coli (STEC is an important cause of bloody diarrhoea (BD, non-bloody diarrhoea (NBD and the haemolytic uraemic syndrome (HUS. In Argentina and New Zealand, the most prevalent STEC serotype is O157:H7, which is responsible for the majority of HUS cases. In Australia, on the other hand, STEC O157:H7 is associated with a minority of HUS cases. The main aims of this study were to compare the phenotypic and genotypic characteristics of STEC O157 strains isolated between 1993 and 1996 from humans in Argentina, Australia and New Zealand, and to establish their clonal relatedness. Results Seventy-three O157 STEC strains, isolated from HUS (n = 36, BD (n = 20, NBD (n = 10, or unspecified conditions (n = 7 in Argentina, Australia and New Zealand, were analysed. The strains were confirmed to be E. coli O157 by biochemical tests and serotyping. A multiplex polymerase chain reaction (PCR was used to amplify the stx1, stx2 and rfbO157 genes and a genotyping method based on PCR-RFLP was used to determine stx1 and stx2 variants. This analysis revealed that the most frequent stx genotypes were stx2/stx2c (vh-a (91% in Argentina, stx2 (89% in New Zealand, and stx1/stx2 (30% in Australia. No stx1-postive strains were identified in Argentina or New Zealand. All strains harboured the eae gene and 72 strains produced enterohaemolysin (EHEC-Hly. The clonal relatedness of strains was investigated by phage typing and pulsed-field gel electrophoresis (PFGE. The most frequent phage types (PT identified in Argentinian, Australian, and New Zealand strains were PT49 (n = 12, PT14 (n = 9, and PT2 (n = 15, respectively. Forty-six different patterns were obtained by XbaI-PFGE; 37 strains were grouped in 10 clusters and 36 strains showed unique patterns. Most clusters could be further subdivided by BlnI-PFGE. Conclusion STEC O157 strains isolated in Argentina, Australia, and New Zealand differed from each other in terms of

  9. Effects of Escherichia Coli Subtilase Cytotoxin and Shiga Toxin 2 on Primary Cultures of Human Renal Tubular Epithelial Cells

    Science.gov (United States)

    Márquez, Laura B.; Velázquez, Natalia; Repetto, Horacio A.; Paton, Adrienne W.; Paton, James C.; Ibarra, Cristina; Silberstein, Claudia

    2014-01-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) cause post-diarrhea Hemolytic Uremic Syndrome (HUS), which is the most common cause of acute renal failure in children in many parts of the world. Several non-O157 STEC strains also produce Subtilase cytotoxin (SubAB) that may contribute to HUS pathogenesis. The aim of the present work was to examine the cytotoxic effects of SubAB on primary cultures of human cortical renal tubular epithelial cells (HRTEC) and compare its effects with those produced by Shiga toxin type 2 (Stx2), in order to evaluate their contribution to renal injury in HUS. For this purpose, cell viability, proliferation rate, and apoptosis were assayed on HRTEC incubated with SubAB and/or Stx2 toxins. SubAB significantly reduced cell viability and cell proliferation rate, as well as stimulating cell apoptosis in HRTEC cultures in a time dependent manner. However, HRTEC cultures were significantly more sensitive to the cytotoxic effects of Stx2 than those produced by SubAB. No synergism was observed when HRTEC were co-incubated with both SubAB and Stx2. When HRTEC were incubated with the inactive SubAA272B toxin, results were similar to those in untreated control cells. Similar stimulation of apoptosis was observed in Vero cells incubated with SubAB or/and Stx2, compared to HRTEC. In conclusion, primary cultures of HRTEC are significantly sensitive to the cytotoxic effects of SubAB, although, in a lesser extent compared to Stx2. PMID:24466317

  10. Effects of Escherichia coli subtilase cytotoxin and Shiga toxin 2 on primary cultures of human renal tubular epithelial cells.

    Directory of Open Access Journals (Sweden)

    Laura B Márquez

    Full Text Available Shiga toxin (Stx-producing Escherichia coli (STEC cause post-diarrhea Hemolytic Uremic Syndrome (HUS, which is the most common cause of acute renal failure in children in many parts of the world. Several non-O157 STEC strains also produce Subtilase cytotoxin (SubAB that may contribute to HUS pathogenesis. The aim of the present work was to examine the cytotoxic effects of SubAB on primary cultures of human cortical renal tubular epithelial cells (HRTEC and compare its effects with those produced by Shiga toxin type 2 (Stx2, in order to evaluate their contribution to renal injury in HUS. For this purpose, cell viability, proliferation rate, and apoptosis were assayed on HRTEC incubated with SubAB and/or Stx2 toxins. SubAB significantly reduced cell viability and cell proliferation rate, as well as stimulating cell apoptosis in HRTEC cultures in a time dependent manner. However, HRTEC cultures were significantly more sensitive to the cytotoxic effects of Stx2 than those produced by SubAB. No synergism was observed when HRTEC were co-incubated with both SubAB and Stx2. When HRTEC were incubated with the inactive SubAA272B toxin, results were similar to those in untreated control cells. Similar stimulation of apoptosis was observed in Vero cells incubated with SubAB or/and Stx2, compared to HRTEC. In conclusion, primary cultures of HRTEC are significantly sensitive to the cytotoxic effects of SubAB, although, in a lesser extent compared to Stx2.

  11. Characterization of a functional recombinant human creatine kinase-MB isoenzyme prepared by tandem affinity purification from Escherichia coli.

    Science.gov (United States)

    Zou, Lihui; Su, Wen; Wang, Meng; Huang, Wei; Zhao, Haijian; Zhang, Enyi; Jin, Junhua; Xu, Hongtao; Xiao, Fei

    2017-07-01

    Creatine kinase isoform CK-MB has been widely applied as a biomarker of myocardial injury. While a variety of methods have been used to measure CK-MB activity or mass in clinical laboratories, a CK-MB standard is needed to eliminate between-method bias. Because the in vitro expression of human creatine kinase generates three isoenzymes, CK-MM, CK-MB, and CK-BB, it is important to establish an effective method to purify the isoform CK-MB from the mixture. In this study, we aimed at using tandem affinity purification (TAP) to purify recombinant CK-MB protein and evaluate its value in clinical laboratories. After the optimized sequence coding CK-M and CK-B were synthesized, they were combined with TAP tags (6His and SBP) and inserted into a pRSFDuet vector; then, the constructed 6His-CK-M-SBP-CK-B-pRSF plasmid was transformed into Escherichia coli BL21 (DE3) for expression. After TAP, we obtained purified CK-MB protein. We also did recovery testing using the engineered CK-MB and standard CK-MB (Randox) at different concentrations, and the results suggested that the engineered CK-MB could be used as the reference material. Moreover, the stability study of recombinant CK-MB showed high stability during long-term storage at -80 °C. In conclusion, the TAP-purified recombinant CK-MB protein may be a much better and cheaper standard or reference material for clinical laboratories.

  12. Probability of recovering pathogenic Escherichia coli from foods.

    OpenAIRE

    Hill, W E; Ferreira, J. L; Payne, W L; Jones, V.M.

    1985-01-01

    The probability of recovering pathogenic Escherichia coli from food by the Bacteriological Analytical Manual method was determined by the effects of several factors: the number of strains per food, the ability of pathogenic strains to survive enrichment, and the frequency of plasmid loss during enrichment. Biochemical patterns indicated the presence of about six E. coli strains per food sample. About half of the strains isolated from humans did not survive enrichment. Among those which grew, ...

  13. Transmission of antibiotic-resistant Escherichia coli between cattle, humans and the environment in peri-urban livestock keeping communities in Morogoro, Tanzania

    DEFF Research Database (Denmark)

    Lupindu, Athumani M; Dalsgaard, Anders; Msoffe, Peter L. M.

    2015-01-01

    infrastructures (Odd Ratio=11.2, 95% CI=1.1-119.3) were associated with E. coli showing identical PFGE types within and between clusters. There is a need to improve animal husbandry and manure management practices to reduce risks of transmission of enteropathogens between livestock and humans in urban and peri......Urban and peri-urban livestock farming is expanding world-widely because of increased urbanization and demands for food of animal origin. Such farming practices pose a public health risk as livestock are reservoirs of several zoonotic pathogens. In an attempt to determine the fecal transmission...... between livestock and people, 100 household clusters keeping cattle in close proximity of humans were selected in urban and peri-urban areas of Morogoro in Tanzania. One hundred eighteen ampicillin and tetracycline resistant Escherichia coli (40 from human stool, 50 from cattle feces, 21 from soil...

  14. Differences in the antibiotic susceptibility of human Escherichia coli with poultry-associated and non-poultry-associated extended-spectrum beta-lactamases.

    Science.gov (United States)

    Platteel, T N; Leverstein-Van Hall, M A; Cohen Stuart, J W; Voets, G M; van den Munckhof, M P; Scharringa, J; van de Sande, N; Fluit, A C; Bonten, M J M

    2013-08-01

    The concurrent presence of bla CTX-M-1 and bla TEM-52 genes on similar plasmids of Escherichia coli isolated from poultry, chicken meat and humans supports the occurrence of food-borne transmission of extended-spectrum beta-lactamase (ESBL) genes. ESBL-producing E. coli (ESBL-E. coli) are most frequently detected in hospitalised patients and are known to spread in healthcare settings. We hypothesised that poultry-associated (PA) ESBL genes are predominant in the community, where acquisition is fuelled by food contamination, whereas non-PA ESBL genes are predominant in hospitals, with acquisition fuelled by cross-transmission. Then, differences in antimicrobial selective pressure in hospitals and poultry would create differences in co-resistance between PA and non-PA ESBL-E. coli. We, therefore, determined the prevalence and co-resistance of PA and non-PA ESBL-E. coli in community-acquired and nosocomial urinary tract infections in humans and bla CTX-M-1 and bla TEM-52 isolates from poultry. A total of 134 human ESBL-E. coli urine isolates were included in this study. Isolates containing bla CTX-M-1 or bla TEM-52 were considered to be PA, with the remainder being non-PA. Also, 72 poultry ESBL-E. coli were included. Minimum inhibitory concentration (MIC) values were determined by broth microdilution. The prevalence of PA ESBL genes in isolates obtained in general practice and hospitals was 28 % versus 30 % (n.s.). Human PA ESBL-E. coli were more frequently susceptible to ciprofloxacin (51 % vs. 25 %; p = 0.0056), gentamicin (86 % vs. 63 %; p = .0.0082), tobramycin (91 % vs. 34 %; p = 0.0001) and amikacin (98 % vs. 67 %; p = 0.0001) compared to human non-PA ESBL-E. coli. PA ESBL-E. coli are not more prevalent in community acquired than nosocomial urine samples, but are more often susceptible to ciprofloxacin and aminoglycosides than non-PA ESBL-E. coli. This does not support the existence of different reservoirs of ESBL genes.

  15. Occurrence and characterization of Shiga toxin-producing Escherichia coli O157:H7 and other non-sorbitol-fermenting E. coli in cattle and humans in urban areas of Morogoro, Tanzania.

    Science.gov (United States)

    Lupindu, Athumani M; Olsen, John E; Ngowi, Helena A; Msoffe, Peter L M; Mtambo, Madundo M; Scheutz, Flemming; Dalsgaard, Anders

    2014-07-01

    Escherichia coli strains such as Shiga toxin-producing E. coli (STEC), enteropathogenic E. coli, enterotoxigenic, attaching, and effacing E. coli, and enteroinvasive E. coli cause diarrhea in humans. Although other serotypes exist, the most commonly reported STEC in outbreaks is O157:H7. A cross-sectional study was conducted to isolate and characterize non-sorbitol-fermenting (NSF) E. coli O157:H7 from urban and periurban livestock settings of Morogoro, Tanzania. Human stool, cattle feces, and soil and water samples were collected. Observations and questionnaire interview studies were used to gather information about cattle and manure management practices in the study area. E. coli were isolated on sorbitol MacConkey agar and characterized by conventional biochemical tests. Out of 1049 samples, 143 (13.7%) yielded NSF E. coli. Serological and antimicrobial tests and molecular typing were performed to NSF E. coli isolates. These procedures detected 10 (7%) pathogenic E. coli including STEC (n=7), enteropathogenic E. coli (EPEC) (n=2), and attaching and effacing E. coli (A/EEC) (n=1) strains. The STEC strains had the ability to produce VT1 and different VT2 toxin subtypes that caused cytopathic effects on Vero cells. The prevalence of STEC in cattle was 1.6%, out of which 0.9% was serotype O157:H7 and the overall prevalence of diarrheagenic E. coli in cattle was 2.2%. The serotypes O157:H7, O142:H34, O113:H21, O+:H-, O+:H16, and O25:H4 were identified. One ESBL-producing isolate showed the MLST type ST131. To our knowledge, this is the first finding in Tanzania of this recently emerged worldwide pandemic clonal group, causing widespread antimicrobial-resistant infections, and adds knowledge of the geographical distribution of ST131. Cattle manure was indiscriminately deposited within residential areas, and there was direct contact between humans and cattle feces during manure handling. Cattle and manure management practices expose humans, animals, and the environment

  16. Soluble Expression and Purification of the Catalytic Domain of Human Vascular Endothelial Growth Factor Receptor 2 in Escherichia coli.

    Science.gov (United States)

    Wei, Jia; Cao, Xiaodan; Zhou, Shengmin; Chen, Chao; Yu, Haijun; Zhou, Yao; Wang, Ping

    2015-08-01

    Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis through binding to its specific receptors, which mainly occurs to VEGF receptor 2 (VEGFR-2), a kinase insert domain-containing receptor. Therefore, the disruption of VEGFR-2 signaling provides a promising therapeutic approach for the treatment of cancer by inhibiting abnormal or tumorinduced angiogenesis. To explore this potential, we expressed the catalytic domain of VEGFR- 2 (VEGFR-2-CD) as a soluble active kinase in Escherichia coli. The recombinant protein was purified and the VEGFR-2-CD activity was investigated. The obtained VEGFR-2-CD showed autophosphorylation activity and phosphate transfer activity comparable to the commercial enzyme. Furthermore, the IC50 value of known VEGFR-2 inhibitor was determined using the purified VEGFR-2-CD. These results indicated a possibility for functional and economical VEGFR-2-CD expression in E. coli to use for inhibitor screening.

  17. Overexpression of functional human oxidosqualene cyclase in Escherichia coli

    DEFF Research Database (Denmark)

    Kürten, Charlotte; Uhlén, Mathias; Syrén, Per-Olof

    2015-01-01

    The generation of multicyclic scaffolds from linear oxidosqualene by enzymatic polycyclization catalysis constitutes a cornerstone in biology for the generation of bioactive compounds. Human oxidosqualene cyclase (hOSC) is a membrane-bound triterpene cyclase that catalyzes the formation of the te......The generation of multicyclic scaffolds from linear oxidosqualene by enzymatic polycyclization catalysis constitutes a cornerstone in biology for the generation of bioactive compounds. Human oxidosqualene cyclase (hOSC) is a membrane-bound triterpene cyclase that catalyzes the formation...

  18. Recurrent Hemolytic and Uremic Syndrome Induced by Escherichia Coli

    Science.gov (United States)

    Commereuc, Morgane; Weill, Francois-Xavier; Loukiadis, Estelle; Gouali, Malika; Gleizal, Audrey; Kormann, Raphaël; Ridel, Christophe; Frémeaux-Bacchi, Véronique; Rondeau, Eric; Hertig, Alexandre

    2016-01-01

    Abstract A widespread belief is that typical hemolytic and uremic syndrome (HUS) does not recur. We report the case of a patient infected twice with raw milk taken from his own cow and containing a Shiga toxin–producing Escherichia coli O174:H21 that induced recurrent HUS causing severe renal and cerebral disorders. A genomic comparison of the human and bovine Shiga toxin–producing Escherichia coli O174:H21 isolates revealed that they were identical. Typical HUS may recur. Since milk from this animal was occasionally distributed locally, thereby posing a serious threat for the whole village, this particular cow was destroyed. PMID:26735524

  19. Efecto citotóxico en colon humano de Escherichia coli enterohemorrágico aislado de terneros con diarrea sanguinolenta Cytotoxic effect in human colon of enterohemorrhagic Escherichia coli isolated from calves with bloody diarrhea

    Directory of Open Access Journals (Sweden)

    V. Pistone Creydt

    2005-09-01

    Full Text Available Escherichia coli productor de toxina Shiga (STEC es el patógeno emergente en alimentos de mayor impacto, siendo su principal reservorio el ganado bovino. STEC puede causar diarrea, colitis hemorrágica y síndrome urémico hemolítico. El presente trabajo estudió la acción citotóxica de dos cepas de STEC aisladas de heces de terneros diarreicos en colon humano in vitro. Los fragmentos se montaron como un diafragma en una cámara de Ussing y se incubaron con las cepas patógenas. El flujo neto absortivo de agua (Jw disminuyó y la corriente de cortocircuito (Isc aumentó significativamente (P Shiga toxin-producing E. coli (STEC is one of the most important emergent pathogen in foods, being its main reservoir bovine cattle. STEC can cause diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome. The present work have studied the cytotoxic action in human colon of cultures of two STEC strains isolated from faeces of calves with bloody diarrhea. Colonic mucosa was mounted as a diaphragm in a Ussing chamber and incubated with the cultures of pathogenic strains. Net water flow (Jw decreased and the short-circuit current (Isc increased significantly (p < 0,01 compared to negative control. Tissues showed an erosion of the mucose, epithelial exfoliation, and presence of pseudo-membranes in the lumen. Mild circulatory lesions were observed in the lamina propia. A moderate neutrophils infiltration was observed in the lumen and into the epithelial cells. Colonic crypts were not disrupted. Both experimental strains caused a similar lesion on colon tissues. This is the first study that shows that cultures of STEC strains isolated from bovine cattle produce cytotoxic effects in vitro in human colon.

  20. Comparative analysis of antibiotic resistance and phylogenetic group patterns in human and porcine urinary tract infectious Escherichia coli

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Nielsen, E.M.; Krag, L.

    2009-01-01

    to relatively benign asymptomatic bacteriuria strains. Here we analyse a spectrum of porcine and human UTI E. coli strains with respect to their antibiotic resistance patterns and their phylogenetic groups, determined by multiplex PCR. The clonal profiles of the strains differed profoundly; whereas human...... strains predominantly belonged to clonal types B2 and D, these were not seen among the porcine strains, which all belonged to the E. coli clonal groups A and B1. Contrary to the human strains, the majority of the porcine strains were multidrug resistant. The distinct profiles of the porcine strains...... suggest selective pressure due to extensive antibiotic use....

  1. High Prevalence of Escherichia coli-Producing CTX-M-15 Extended-Spectrum Beta-Lactamases in Poultry and Human Clinical Isolates in Romania.

    Science.gov (United States)

    Maciuca, Iuliana E; Williams, Nicola J; Tuchilus, Cristina; Dorneanu, Olivia; Guguianu, Eleonora; Carp-Carare, Catalin; Rimbu, Cristina; Timofte, Dorina

    2015-12-01

    Use of antibiotics in food animals may contribute to development and spread of resistant organisms, particularly so in some countries. The aim of this study was two-fold; first, to establish the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in chicken production in a region within Romania. Second, to study the relatedness of ESBL-producing E. coli isolates recovered from broilers, abattoir workers where the chickens were slaughtered and from the human clinical specimens from two regional hospitals. The results indicated a very high (69%) rate of carriage of ESBL and AmpC-producing E. coli in chickens with 36% CTX-M producers. Sequencing showed that chickens in Romania have the highest worldwide prevalence (53%) of blaCTX-M-15 reported in poultry E. coli isolates. The majority (53%) of the extended-spectrum cephalosporin-resistant E. coli carried plasmid-mediated blaampC genes, mostly blaCMY-2 type, one of the highest prevalences reported in Europe. The predominant CTX-M type found in the human clinical E. coli isolates was blaCTX-M-15 and most isolates coharbored blaOXA-1, blaTEM, and aac(6')-ib-cr. The majority (60%) of the human clinical isolates belonged to the pandemic virulent clone B2-ST131. The clonal relationship between broiler and the human CTX-M-producing E. coli isolates was assessed by macrorestriction pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), which indicated strain diversity with no common STs found between human and poultry isolates. Moreover, IncI1 was the most prevalent replicon found in broiler ESBL-producing E. coli isolates and also in transconjugants, indicating that plasmids and not clonal spread may play a role in the transfer of blaCTX-M genes. This study identifies a high prevalence of ESBL-producing E. coli from broiler chickens in Romania with a high occurrence incidence of blaCTX-M-15, which reflects the main ESBL type found in human E. coli infections in this

  2. Preparative isolation by high performance liquid chromatography of human insulin B chain produced in escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Cruz, N.; Antonio, S.; De Anda, R.; Gosset, G.; Bolivar, F. (Centro de Investigacion sobre Ingenieria Genetica y Biotecnologia, Universidad Nacional Autonoma de Mexico, Apdo. Postal 510-3 Cuernavaca, Mor. 62271 (MX))

    1990-01-01

    This paper reports on a simple method developed for the analytical and preparative purification of human insulin B chain from recombinant origin. Three solvent systems: acetonitrile, isopropanol and methanol, were studied to determine their capacity to resolve the insulin B chain from a mixture of cyanogen bromide generated bacterial peptides. Using a {mu}Bondapak C18 column, it was possible to resolve the insulin B chain in all three systems. On a preparative scale, using a PrePak 500 C18 column with the isopropanol system, it was possible to purify insulin B chain and to obtain a 95% protein recovery.

  3. Molecular characterization of the Escherichia coli asymptomatic bacteriuria strain 83972

    DEFF Research Database (Denmark)

    Klemm, Per; Hancock, Viktoria; Ulett, G.C.

    2006-01-01

    Escherichia coli 83972 is a clinical asymptomatia bacteriuric isolate that is able to colonize the human urinary bladder without inducing an immune response. Here we demonstrate that one of the mechanisms by which this strain has become attenuated is through the mutation of its genes encoding type...

  4. Molecular characterization of the Escherichia coli asymptomatic bacteriuria strain 83972

    DEFF Research Database (Denmark)

    Klemm, Per; Hancock, Viktoria; Ulett, G.C.

    2006-01-01

    Escherichia coli 83972 is a clinical asymptomatia bacteriuric isolate that is able to colonize the human urinary bladder without inducing an immune response. Here we demonstrate that one of the mechanisms by which this strain has become attenuated is through the mutation of its genes encoding typ...

  5. Native valve Escherichia coli endocarditis following urosepsis.

    Science.gov (United States)

    Rangarajan, D; Ramakrishnan, S; Patro, K C; Devaraj, S; Krishnamurthy, V; Kothari, Y; Satyaki, N

    2013-05-01

    Gram-negative organisms are a rare cause of infective endocarditis. Escherichia coli, the most common cause of urinary tract infection and gram-negative septicemia involves endocardium rarely. In this case report, we describe infection of native mitral valve by E. coli following septicemia of urinary tract origin in a diabetic male; subsequently, he required prosthetic tissue valve replacement indicated by persistent sepsis and congestive cardiac failure.

  6. Comparison of multilocus sequence analysis and virulence genotyping of Escherichia coli from live birds, retail poultry meat, and human extraintestinal infection.

    Science.gov (United States)

    Danzeisen, Jessica L; Wannemuehler, Yvonne; Nolan, Lisa K; Johnson, Timothy J

    2013-03-01

    To examine the correlations between virulence genotyping and multilocus sequence analysis of Escherichia coli from poultry and humans, 88 isolates were examined. The isolates were selected from a population of over 1000 based on their assignment to nine different virulence genotyping clusters. Clustering based on multilocus sequence analysis mostly correlated with virulence genotyping, although multilocus sequence analysis demonstrated higher discriminatory ability and greater reliability related to inferred phylogenetic relationships. No distinct patterns in host source were observed using inferred phylogeny through multilocus sequence analysis, indicating that human, avian, and retail meat isolates are diverse, and some belong to multiple shared clonal complexes. Clonal complexes with host source overlap included ST95 and ST23 and additional novel groups, underscoring the diversity of avian pathogenic E. coli and the potential importance of these novel groups as avian and zoonotic pathogens.

  7. Mutation of Escherichia coli cytosine deaminase significantly enhances molecular chemotherapy of human glioma.

    Science.gov (United States)

    Kaliberov, S A; Market, J M; Gillespie, G Y; Krendelchtchikova, V; Della Manna, D; Sellers, J C; Kaliberova, L N; Black, M E; Buchsbaum, D J

    2007-07-01

    Combined treatment using adenoviral (Ad)-directed enzyme/prodrug therapy and radiation therapy has the potential to become a powerful method of cancer therapy. We have developed an Ad vector encoding a mutant bacterial cytosine deaminase (bCD) gene (AdbCD-D314A), which has a higher affinity for cytosine than wild-type bCD (bCDwt). The purpose of this study was to evaluate cytotoxicity in vitro and therapeutic efficacy in vivo of the combination of AdbCD-D314A with the prodrug 5-fluorocytosine (5-FC) and ionizing radiation against human glioma. The present study demonstrates that AdbCD-D314A infection resulted in increased 5-FC-mediated cell killing, compared with AdbCDwt. Furthermore, a significant increase in cytotoxicity following AdbCD-D314A and radiation treatment of glioma cells in vitro was demonstrated as compared to AdbCDwt. Animal studies showed significant inhibition of subcutaneous or intracranial tumor growth of D54MG glioma xenografts by the combination of AdbCD-D314A/5-FC with ionizing radiation as compared with either agent alone, and with AdbCDwt/5-FC plus radiation. The results suggest that the combination of AdbCD-D314A/5-FC with radiation produces markedly increased cytotoxic effects in cancer cells in vitro and in vivo. These data indicate that combined treatment with this novel mutant enzyme/prodrug therapy and radiotherapy provides a promising approach for cancer therapy.

  8. 77 FR 9888 - Shiga Toxin-Producing Escherichia coli

    Science.gov (United States)

    2012-02-21

    ... Food Safety and Inspection Service Shiga Toxin-Producing Escherichia coli in Certain Raw Beef Products... manufacturing trimmings for six non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45..., non-intact product, that are contaminated with Shiga toxin-producing Escherichia coli (STEC) O26,...

  9. Escherichia coli isolates from broiler chicken meat, broiler chickens, pork, and pigs share phylogroups and antimicrobial resistance with community-dwelling humans and patients with urinary tract infection.

    Science.gov (United States)

    Jakobsen, Lotte; Kurbasic, Azra; Skjøt-Rasmussen, Line; Ejrnaes, Karen; Porsbo, Lone J; Pedersen, Karl; Jensen, Lars B; Emborg, Hanne-Dorthe; Agersø, Yvonne; Olsen, Katharina E P; Aarestrup, Frank M; Frimodt-Møller, Niels; Hammerum, Anette M

    2010-05-01

    Escherichia coli is the most common cause of urinary tract infection (UTI). Phylogroup B2 and D isolates are associated with UTI. It has been proposed that E. coli causing UTI could have an animal origin. The objective of this study was to investigate the phylogroups and antimicrobial resistance, and their possible associations in E. coli isolates from patients with UTI, community-dwelling humans, broiler chicken meat, broiler chickens, pork, and pigs in Denmark. A total of 964 geographically and temporally matched E. coli isolates from UTI patients (n = 102), community-dwelling humans (n = 109), Danish (n = 197) and imported broiler chicken meat (n = 86), Danish broiler chickens (n = 138), Danish (n = 177) and imported pork (n = 10), and Danish pigs (n = 145) were tested for phylogroups (A, B1, B2, D, and nontypeable [NT] isolates) and antimicrobial susceptibility. Phylogroup A, B1, B2, D, and NT isolates were detected among all groups of isolates except for imported pork isolates. Antimicrobial resistance to three (for B2 isolates) or five antimicrobial agents (for A, B1, D, and NT isolates) was shared among isolates regardless of origin. Using cluster analysis to investigate antimicrobial resistance data, we found that UTI isolates always grouped with isolates from meat and/or animals. We detected B2 and D isolates, that are associated to UTI, among isolates from broiler chicken meat, broiler chickens, pork, and pigs. Although B2 isolates were found in low prevalences in animals and meat, these sources could still pose a risk for acquiring uropathogenic E. coli. Further, E. coli from animals and meat were very similar to UTI isolates with respect to their antimicrobial resistance phenotype. Thus, our study provides support for the hypothesis that a food animal and meat reservoir might exist for UTI-causing E. coli.

  10. No evidence for transmission of antibiotic-resistant Escherichia coli strains from humans to wild western lowland gorillas in Lopé National Park, Gabon.

    Science.gov (United States)

    Benavides, Julio Andre; Godreuil, Sylvain; Bodenham, Rebecca; Ratiarison, Sandra; Devos, Céline; Petretto, Marie-Odile; Raymond, Michel; Escobar-Páramo, Patricia

    2012-06-01

    The intensification of human activities within the habitats of wild animals is increasing the risk of interspecies disease transmission. This risk is particularly important for great apes, given their close phylogenetic relationship with humans. Areas of high human density or intense research and ecotourism activities expose apes to a high risk of disease spillover from humans. Is this risk lower in areas of low human density? We determined the prevalence of Escherichia coli antibiotic-resistant isolates in a population of the critically endangered western lowland gorilla (Gorilla gorilla gorilla) and other wild mammals in Lopé National Park (LNP), Gabon, and we tested whether the observed pattern could be explained by bacterial transmission from humans and domestic animals into wildlife populations. Our results show a high prevalence of antibiotic-resistant bacterial isolates in humans and low levels in gorillas and other wildlife. The significant differences in the genetic background of the resistant bacteria isolated from humans and gorillas suggest that transmission is low or does not occur between these two species. These findings indicate that the presence of antibiotic-resistant strains in wildlife do not imply direct bacteria transmission from humans. Thus, in areas of low human density, human-wildlife E. coli transmission seems to be low. The presence of antibiotic-resistant isolates in gorillas may be better explained by other mechanisms for resistance acquisition, such as horizontal gene exchange among bacteria or naturally acquired resistance.

  11. Detection of sul1, sul2 and sul3 in sulphonamide resistant Escherichia coli isolates obtained from healthy humans, pork and pigs in Denmark.

    Science.gov (United States)

    Hammerum, Anette M; Sandvang, Dorthe; Andersen, Sigrid R; Seyfarth, Anne Mette; Porsbo, Lone Jannok; Frimodt-Møller, Niels; Heuer, Ole E

    2006-02-01

    The occurrence of sulphonamide resistance was investigated in 998 Escherichia coli isolates, obtained from pig faeces collected at slaughter, Danish pork collected at retail outlets and from faeces from healthy persons in Denmark. In total 18% (n=35), 20% (n=38) and 26% (n=161) of the E. coli isolates obtained from humans, pork and pigs, respectively, were resistant to sulphonamide. All sulphonamide resistant E. coli isolates were investigated for the presence of sul1, sul2, sul3 and intI1 genes by PCR. The sul1 gene was detected in 40% (n=14), 29% (n=11) and 55% (n=88) of the sulphonamide resistant isolates from humans, pork and pigs, respectively. The sul2 gene was detected in 80% (n=28), 76% (n=29) and 50% (n=81) of isolates from humans, pork and pigs, respectively. None of the human isolates were PCR-positive for sul3, whereas sul3 was present in 5% of the pork isolates and 11% of the pig isolates. Of the 113 sul1 positive isolates, 97 carried the integron-associated integrase gene intI1. All 20 sul3 positive isolates were positive for intI1, and in 12 of these isolates sul3 was the only sulphonamide resistance gene detected. The origin of sul1 and sul2 found in isolates from healthy humans is speculative, but their spread from pigs to humans via the food chain is possible.

  12. Food-borne origins of Escherichia coli causing extraintestinal infections.

    Science.gov (United States)

    Manges, Amee R; Johnson, James R

    2012-09-01

    Most human extraintestinal Escherichia coli infections, including those involving antimicrobial resistant strains, are caused by the members of a limited number of distinctive E. coli lineages, termed extraintestinal pathogenic E. coli (ExPEC), that have a special ability to cause disease at extraintestinal sites when they exit their usual reservoir in the host's intestinal tract. Multiple lines of evidence suggest that many of the ExPEC strains encountered in humans with urinary tract infection, sepsis, and other extraintestinal infections, especially the most extensively antimicrobial-resistant strains, may have a food animal source, and may be transmitted to humans via the food supply. This review summarizes the evidence that food-borne organisms are a significant cause of extraintestinal E. coli infections in humans.

  13. Transfer of drug-resistance plasmids by conjugation from nosocomial strains of Serratia marcescens to Escherichia coli in biological fluids of human origin.

    Science.gov (United States)

    Mendez, F J; Mendoza, M C; Llaneza, J J; Hardisson, C

    1982-09-01

    Six independent isolates of multi-resistant Serratia marcescens associated with nosocomial infections were examined for their ability to transfer drug-resistance plasmids by conjugation to Escherichia coli in biological fluids of human origin, such as normal and pathological urine, faeces, blood plasma and ascitic fluid. Luria broth was used as a control. Positive transfer was found in all media assayed. The different patterns of linked transferable resistance found in the transconjugants corresponded to the phenotypic expression of five plasmids. The frequencies of transfer varied with plasmid types and media employed. The culture media did not affect the phenotypic expression of the plasmids.

  14. Control of Ribosome Synthesis in Escherichia coli

    DEFF Research Database (Denmark)

    Molin, Søren; Meyenburg, K. von; Måløe, O.

    1977-01-01

    The rate of ribosome synthesis and accumulation in Escherichia coli during the transition after an energy source shift-down was analyzed. The shift was imposed on cultures of stringent and relaxed strains growing in glucose minimal medium by the addition of the glucose analogue {alpha}-methylgluc...

  15. Synergistic effects in mixed Escherichia coli biofilms

    DEFF Research Database (Denmark)

    Reisner, A.; Holler, B.M.; Molin, Søren

    2006-01-01

    the pathways governing development of more complex heterogeneous communities. In this study, we established a laboratory model where biofilm-stimulating effects due to interactions between genetically diverse strains of Escherichia coli were monitored. Synergistic induction of biofilm formation resulting from...

  16. Escherichia Coli--Key to Modern Genetics.

    Science.gov (United States)

    Bregegere, Francois

    1982-01-01

    Mid-nineteenth century work by Mendel on plant hybrids and by Pasteur on fermentation gave birth by way of bacterial genetics to modern-day molecular biology. The bacterium Escherichia Coli has occupied a key position in genetic studies leading from early gene identification with DNA to current genetic engineering using recombinant DNA technology.…

  17. Progressive segregation of the Escherichia coli chromosome

    DEFF Research Database (Denmark)

    Nielsen, Henrik Jørck; Youngren, Brenda; Hansen, Flemming G.

    2006-01-01

    We have followed the fate of 14 different loci around the Escherichia coli chromosome in living cells at slow growth rate using a highly efficient labelling system and automated measurements. Loci are segregated as they are replicated, but with a marked delay. Most markers segregate in a smooth...

  18. Leaner and meaner genomes in Escherichia coli

    DEFF Research Database (Denmark)

    Ussery, David

    2006-01-01

    A 'better' Escherichia coli K-12 genome has recently been engineered in which about 15% of the genome has been removed by planned deletions. Comparison with related bacterial genomes that have undergone a natural reduction in size suggests that there is plenty of scope for yet more deletions....

  19. Compaction of isolated Escherichia coli nucleoids

    NARCIS (Netherlands)

    Wegner, Anna S.; Wintraecken, Kathelijne; Spurio, Roberto; Woldringh, Conrad L.; Vries, de Renko; Odijk, Theo

    2016-01-01

    Escherichia coli nucleoids were compacted by the inert polymer polyethylene glycol (PEG) in the presence of the H-NS protein. The protein by itself appears to have little impact on the size of the nucleoids as determined by fluorescent microscopy. However, it has a significant impact on the nucle

  20. Comparative analysis of ESBL-positive Escherichia coli isolates from animals and humans from the UK, The Netherlands and Germany

    NARCIS (Netherlands)

    Wu, G.; Day, M.J.; Mafura, T.; Nunez-Garcia, J.; Fenner, J.J.; Sharma, M.; Essen-Zandbergen, van A.; Rodriguez, I.; Dierikx, C.M.; Mevius, D.J.

    2013-01-01

    The putative virulence and antimicrobial resistance gene contents of extended spectrum ß-lactamase (ESBL)-positive E. coli (n=629) isolated between 2005 and 2009 from humans, animals and animal food products in Germany, The Netherlands and the UK were compared using a microarray approach to test the

  1. Adhesion of human and animal escherichia coli strains in association with their virulence-associated genes and phylogenetic origins

    DEFF Research Database (Denmark)

    Fr̈mmel, Ulrike; R̈diger, Stefan; B̈hm, Alexander

    2013-01-01

    ) and the European hedgehog (Erinaceus europaeus). The prevalence of exVAGs depended on isolation from a specific host. Human uropathogenic E. coli isolates carried exVAGs with the highest prevalence, followed by badger (Meles meles) and roe deer isolates. Adhesion was found to be very diverse. Adhesion was specific...

  2. A combination of PhP typing and β-d-glucuronidase gene sequence variation analysis for differentiation of Escherichia coli from humans and animals.

    Science.gov (United States)

    Masters, N; Christie, M; Katouli, M; Stratton, H

    2015-06-01

    We investigated the usefulness of the β-d-glucuronidase gene variance in Escherichia coli as a microbial source tracking tool using a novel algorithm for comparison of sequences from a prescreened set of host-specific isolates using a high-resolution PhP typing method. A total of 65 common biochemical phenotypes belonging to 318 E. coli strains isolated from humans and domestic and wild animals were analysed for nucleotide variations at 10 loci along a 518 bp fragment of the 1812 bp β-d-glucuronidase gene. Neighbour-joining analysis of loci variations revealed 86 (76.8%) human isolates and 91.2% of animal isolates were correctly identified. Pairwise hierarchical clustering improved assignment; where 92 (82.1%) human and 204 (99%) animal strains were assigned to their respective cluster. Our data show that initial typing of isolates and selection of common types from different hosts prior to analysis of the β-d-glucuronidase gene sequence improves source identification. We also concluded that numerical profiling of the nucleotide variations can be used as a valuable approach to differentiate human from animal E. coli. This study signifies the usefulness of the β-d-glucuronidase gene as a marker for differentiating human faecal pollution from animal sources.

  3. Expression and characterization of a single-chain variable fragment against human LOX-1 in Escherichia coli and Brevibacillus choshinensis.

    Science.gov (United States)

    Hu, Wei; Xiang, Jun-Yan; Kong, Ping; Liu, Ling; Xie, Qiuhong; Xiang, Hongyu

    2017-03-09

    The single-chain variable fragment (scFv) against lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a promising molecule for its potential use in the diagnosis and immunotherapy of atherosclerosis. Producing this scFv in several milligram amounts could be the starting point for further engineering and application of the scFv. In this study, the abundant expression of the anti-LOX-1 scFv was attempted using Escherichia coli (E. coli) and Brevibacillus choshinensis (B. choshinensis). The scFv had limited soluble yield in E. coli, but it was efficiently secreted by B. choshinensis. The optimized fermentation was determined using the Plackett-Burman screening design and the response surface methodology (RSM), under which the yield reached up to 1.5 g/L in a 5-L fermentor. Moreover, the properties of the scFvs obtained from the two expression systems were different. The antigen affinity, transition temperature and particle diameter size is 1.01E-07 M, 55.2 ± 0.3°C and 9.388 nm for the scFv expressed by B. choshinensis and 4.53E-07 M, 52.5 ± 0.3°C and 13.54 nm for the scFv expressed by E. coli. This study established an efficient scale-up production methodology for the anti-LOX-1 scFv, which will boost its use in LOX-1-based therapy.

  4. Characterization of IncN plasmids carrying blaCTX-M-1 and qnr genes in Escherichia coli and Salmonella from animals, the environment and humans

    DEFF Research Database (Denmark)

    Dolejska, Monika; Villa, Laura; Hasman, Henrik;

    2013-01-01

    and ST8, respectively. Related plasmids circulating in human and animal isolates were identified. Complete nucleotide sequences of the ST1 pHHA45 plasmid carrying blaCTX-M-1, isolated from E. coli from pigs in Denmark, and the ST3 pKT58A plasmid harbouring qnrS1, identified in E. coli from a water bird......Objectives The aim of the study was to characterize a collection of Escherichia coli and Salmonella harbouring qnr and blaCTX-M-1 genes on IncN plasmids isolated from humans, food-producing, companion and wild animals, and the environment from six European countries.Methods Nineteen IncN plasmids...... DNA purified from the respective E. coli transformants.Results Three types of IncN plasmids carrying blaCTX-M-1, qnrS1 and qnrB19 genes were identified in strains isolated from the Czech Republic, Poland, Slovakia, Denmark, Italy and the Netherlands, corresponding to pMLST sequence type (ST) 1, ST3...

  5. Refolding with Simultaneous Purification of Recombinant Human Granulocyte Colony-stimulating Factor from Escherichia coli Using Strong Anion Exchange Chromatography

    Institute of Scientific and Technical Information of China (English)

    Chao Zhan WANG; Jiang Feng LIU; Xin Du GENG

    2005-01-01

    The urea denatured recombinant human granulocyte colony-stimulating factor (rhGCSF) which was expressed in Escheriachia coli (E. coli) was refolded with simultaneous purification by strong anion exchange chromatography (SAX) in the presence of low concentration of urea. The effect of urea concentration on this refolding process was investigated. The obtained refolded rhG-CSF has a high specific activity of 2.3×108 U/mg, demonstrating that the proteins were completely refolded during the chromatographic process. With only one step by SAX in 40 min, purity and mass recovery of the refolded and purified rhG-CSF were 97% and43%, respectively.

  6. Prevalence of ESBLs and PMQR genes in fecal Escherichia coli isolated from the non-human primates in six zoos in China.

    Science.gov (United States)

    Wang, Yang; He, Tao; Han, Jing; Wang, Juan; Foley, Steven L; Yang, Guangyou; Wan, Shuangxiu; Shen, Jianzhong; Wu, Congming

    2012-09-14

    The aim of this study is to characterize the prevalence of extended-spectrum β-lactamases (ESBLs) and plasmid-mediated quinolone resistance (PMQR) genes in Escherichia coli from captive non-human primates. A total of 206 E. coli isolates were collected from primates in six zoos in China in 2009 and their susceptibility to 10 antimicrobials were tested by broth microdilution. The susceptibility patterns of E. coli strains varied greatly among different zoos reflecting different backgrounds of antimicrobial usage. Both the ESBL-encoding genes and the PMQR genes were detected by PCR. Of the 206 strains, 65 (32%) were confirmed as phenotypic ESBL producers with bla(CTX-M) (27%, bla(CTX-M-15), n=31, bla(CTX-M-3), n=23 and bla(CTX-M-14), n=2) mainly mediating the ESBL phenotype. qnrS1 (18%, n=36) and oqxAB (15%, n=31) were the predominant PMQR genes and the prevalence of PMQR genes was much higher among phenotypic ESBL producers than that among phenotypic non-ESBL producers from any zoo. Notably, the PMQR genes qnrS1 and oqxAB and β-lactamase genes bla(TEM-1) and bla(CTX-M-3) were found together in 23 E. coli isolates in two zoos in Shanghai. PFGE analysis of these 23 isolates demonstrated nearly identical PFGE profiles (similarity matrix >97%) indicating this specific E. coli genotype was prevalent in these two zoos. To the best of our knowledge, this is the first report of these four genes coexisting in an E. coli genotype and the first report of antimicrobial resistance profiles in E. coli isolated from primates in China. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Is Escherichia coli urinary tract infection a zoonosis?

    DEFF Research Database (Denmark)

    Jacobsen, L.; Garneau, P.; Bruant, G.

    2012-01-01

    and kidney cultures. Further, isolates with the same gene profile also yielded similar bacterial counts in urine, bladder and kidneys. This study showed a clonal link between E. coli from meat and humans, providing solid evidence that UTI is zoonosis. The close relationship between community-dwelling human......Recently, it has been suggested that the Escherichia coli causing urinary tract infection (UTI) may come from meat and animals. The purpose was to investigate if a clonal link existed between E. coli from animals, meat and UTI patients. Twenty-two geographically and temporally matched B2 E. coli...... from UTI patients, community-dwelling humans, broiler chicken meat, pork, and broiler chicken, previously identified to exhibit eight virulence genotypes by microarraydetection of approximately 300 genes, were investigated for clonal relatedness by PFGE. Nine isolates were selected and tested...

  8. High-level expression and purification of soluble bioactive recombinant human heparin-binding epidermal growth factor in Escherichia coli.

    Science.gov (United States)

    Khalili, Mostafa; Soleyman, Mohammad Reza; Baazm, Maryam; Beyer, Cordian

    2015-07-01

    Heparin-binding epidermal growth factor (HB-EGF) is a member of highly conserved superfamily of proteins that has potential mitogenic activity and stimulates differentiation and migration of various cell types. Since HB-EGF has three intra-molecular disulfide bonds, a high expression pattern of active HB-EGF in an E. coli expression system was not successfully established. The aim of this study was to increase production of soluble bioactive recombinant human HB-EGF in E. coli by modifying growth conditions and codon optimization. The open reading frame codons of human HB-EGF were optimized to achieve high level expression in E. coli. The optimized codon was amplified, cloned into plasmid pET-32a, and transformed into E. coli BL21 for further expression. The cultivation parameters (temperature and inducer) were optimized to produce a high yield of soluble HB-EGF. The fusion protein was purified by Nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. Amethylthiazole tetrazolium assay was used to evaluate the bioactivity of the produced recombinant protein. After codon optimization, the codon adaptation index (CAI) was increased from 0.255 in native gene to 0.829 using the optimized sequence. By lowering the temperature to 22°C and the inducer to 0.4 μM, we obtained 35% soluble expression of recombinant and biologically active human HB-EGF. Our data demonstrate that codon optimization increases the yield of HB-EGF in an E. coli expression system. Furthermore, the chosen modifications in cell culturing increase the solubility of recombinant human HB-EGF.

  9. Occurrence and antimicrobial resistance of pathogenic Escherichia coli and Salmonella spp. in retail raw table eggs sold for human consumption in Enugu state, Nigeria

    Science.gov (United States)

    Okorie-Kanu, O. Josephine; Ezenduka, E. Vivienne; Okorie-Kanu, C. Onwuchokwe; Ugwu, L. Chinweokwu; Nnamani, U. John

    2016-01-01

    Aim: This study was conducted to investigate the occurrence of pathogenic Escherichia coli and Salmonella species in retail raw table eggs sold for human consumption in Enugu State and to determine the resistance of these pathogens to antimicrobials commonly used in human and veterinary practices in Nigeria. Materials and Methods: A total of 340 raw table eggs comprising 68 composite samples (5 eggs per composite sample) were collected from five selected farms (13 composite samples from the farms) and 10 retail outlets (55 composite samples from the retail outlets) in the study area over a period of 4-month (March-June, 2014). The eggs were screened for pathogenic E. coli and Salmonella species following standard procedures within 24 h of sample collection. Isolates obtained were subjected to in-vitro antimicrobial susceptibility test with 15 commonly used antimicrobials using the disk diffusion method. Results: About 37 (54.4%) and 7 (10.3%) of the 68 composite samples were positive for pathogenic E. coli and Salmonella species, respectively. The shells showed significantly higher (p0.05). The organisms obtained showed a multiple drug resistance. They were completely resistant to nitrofurantoin, sulfamethoxazole/trimethoprim, penicillin G and oxacillin. In addition to these, Salmonella spp. also showed 100% resistance to tetracycline. The pathogenic E. coli isolates obtained were 100% susceptible to gentamicin, neomycin, ciprofloxacin, and amoxicillin-clavulanic acid while Salmonella spp. showed 100% susceptibility to erythromycin, neomycin, and rifampicin. Both organisms showed varying degrees of resistance to streptomycin, amoxicillin, vancomycin, and doxycycline. Conclusion: From the results of the study, it can be concluded that the raw table eggs marketed for human consumption in Enugu State, Nigeria is contaminated with pathogenic E. coli and Salmonella species that showed multiple drug resistance to antimicrobial agents commonly used in veterinary and human

  10. Occurrence and antimicrobial resistance of pathogenic Escherichia coli and Salmonella spp. in retail raw table eggs sold for human consumption in Enugu state, Nigeria

    Directory of Open Access Journals (Sweden)

    O. Josephine Okorie-Kanu

    2016-11-01

    Full Text Available Aim: This study was conducted to investigate the occurrence of pathogenic Escherichia coli and Salmonella species in retail raw table eggs sold for human consumption in Enugu State and to determine the resistance of these pathogens to antimicrobials commonly used in human and veterinary practices in Nigeria. Materials and Methods: A total of 340 raw table eggs comprising 68 composite samples (5 eggs per composite sample were collected from five selected farms (13 composite samples from the farms and 10 retail outlets (55 composite samples from the retail outlets in the study area over a period of 4-month (March-June, 2014. The eggs were screened for pathogenic E. coli and Salmonella species following standard procedures within 24 h of sample collection. Isolates obtained were subjected to in-vitro antimicrobial susceptibility test with 15 commonly used antimicrobials using the disk diffusion method. Results: About 37 (54.4% and 7 (10.3% of the 68 composite samples were positive for pathogenic E. coli and Salmonella species, respectively. The shells showed significantly higher (p0.05. The organisms obtained showed a multiple drug resistance. They were completely resistant to nitrofurantoin, sulfamethoxazole/ trimethoprim, penicillin G and oxacillin. In addition to these, Salmonella spp. also showed 100% resistance to tetracycline. The pathogenic E. coli isolates obtained were 100% susceptible to gentamicin, neomycin, ciprofloxacin, and amoxicillin-clavulanic acid while Salmonella spp. showed 100% susceptibility to erythromycin, neomycin, and rifampicin. Both organisms showed varying degrees of resistance to streptomycin, amoxicillin, vancomycin, and doxycycline. Conclusion: From the results of the study, it can be concluded that the raw table eggs marketed for human consumption in Enugu State, Nigeria is contaminated with pathogenic E. coli and Salmonella species that showed multiple drug resistance to antimicrobial agents commonly used in

  11. Pathogenomics of uropathogenic Escherichia coli

    Directory of Open Access Journals (Sweden)

    J Agarwal

    2012-01-01

    Full Text Available Subset of faecal E. coli that can enter, colonize urinary tract and cause infection are known as uropathogenic E. coli (UPEC. UPEC strains act as opportunistic intracellular pathogens taking advantage of host susceptibility using a diverse array of virulence factors. Presence of specific virulence associated genes on genomic/pathogenicity islands and involvement of horizontal gene transfer appears to account for evolution and diversity of UPEC. Recent success in large-scale genome sequencing and comparative genomics has helped in unravelling UPEC pathogenomics. Here we review recent findings regarding virulence characteristics of UPEC and mechanisms involved in pathogenesis of urinary tract infection.

  12. Prevalence of quinolone resistance mechanisms and associations to minimum inhibitory concentrations in quinolone-resistant Escherichia coli isolated from humans and swine in Denmark

    DEFF Research Database (Denmark)

    Cavaco, Lina; Frimodt-Møller, Niels; Hasman, Henrik;

    2008-01-01

    Prevalence of quinolone resistance mechanisms and associations to minimum inhibitory concentrations (MICs) of nalidixic acid (NAL) and ciprofloxacin (CIP) were investigated in 124 Escherichia coli isolated from humans (n = 85) and swine (n = 39) in Denmark. The collection included 59 high-level CIP......-resistant isolates (MIC >= 4) from human (n = 51) and pig origin (n = 8) and 65 low-level CIP-resistant isolates (MIC >= 0.125) from human (n = 34) and pig origin (n = 31). Resistance by target modification was screened by PCR amplification and sequencing, of the quinolone resistance determining regions (QRDRs......A and qnrS genes conferring quinolone resistance by target protection were detected in two human low-level CIP-resistant isolates that did not display NAL resistance. As expected, target mutation in QRDRs was the most prevalent mechanism of quinolone resistance. This mechanism was complemented by efflux...

  13. Infektionen mit darmpathogenen Escherichia coli.

    NARCIS (Netherlands)

    Friedrich, Alexander; Stein, Jürgen; Dignass, Axel

    2001-01-01

    E. coli ist ein wesentlicher Bestandteil der physiologischen Darmflora des Menschen. Die üblicherweise im Darm vorkommenden Kolibakterien sind apathogen und für den Menschen eher nützlich (Sonnenborn u. Greinwald 1990). Allerdings kennen wir bei dieser Bakterienspezies auch ein breites Spektrum von

  14. Survival of Escherichia coli in stormwater biofilters.

    Science.gov (United States)

    Chandrasena, G I; Deletic, A; McCarthy, D T

    2014-04-01

    Biofilters are widely adopted in Australia for stormwater treatment, but the reported removal of common faecal indicators (such as Escherichia coli (E. coli)) varies from net removal to net leaching. Currently, the underlying mechanisms that govern the faecal microbial removal in the biofilters are poorly understood. Therefore, it is important to study retention and subsequent survival of faecal microorganisms in the biofilters under different biofilter designs and operational characteristics. The current study investigates how E. coli survival is influenced by temperature, moisture content, sunlight exposure and presence of other microorganisms in filter media and top surface sediment. Soil samples were taken from two different biofilters to investigate E. coli survival under controlled laboratory conditions. Results revealed that the presence of other microorganisms and temperature are vital stressors which govern the survival of E. coli captured either in the top surface sediment or filter media, while sunlight exposure and moisture content are important for the survival of E. coli captured in the top surface sediment compared to that of the filter media. Moreover, increased survival was found in the filter media compared to the top sediment, and sand filter media was found be more hostile than loamy sand filter media towards E. coli survival. Results also suggest that the contribution from the tested environmental stressors on E. coli survival in biofilters will be greatly affected by the seasonality and may vary from one site to another.

  15. The StcE metalloprotease of enterohaemorrhagic Escherichia coli reduces the inner mucus layer and promotes adherence to human colonic epithelium ex vivo.

    Science.gov (United States)

    Hews, Claire L; Tran, Seav-Ly; Wegmann, Udo; Brett, Bernard; Walsham, Alistair D S; Kavanaugh, Devon; Ward, Nicole J; Juge, Nathalie; Schüller, Stephanie

    2017-01-05

    Enterohaemorrhagic Escherichia coli (EHEC) is a major foodborne pathogen and tightly adheres to human colonic epithelium by forming attaching/effacing lesions. To reach the epithelial surface, EHEC must penetrate the thick mucus layer protecting the colonic epithelium. In this study, we investigated how EHEC interacts with the intestinal mucus layer using mucin-producing LS174T colon carcinoma cells and human colonic mucosal biopsies. The level of EHEC binding and attaching/effacing lesion formation in LS174T cells was higher compared to mucin-deficient colon carcinoma cell lines, and initial adherence was independent of the presence of flagellin, Escherichia coli common pilus, or long polar fimbriae. Although EHEC infection did not affect gene expression of secreted mucins, it resulted in reduced MUC2 glycoprotein levels. This effect was dependent on the catalytic activity of the secreted metalloprotease StcE, which reduced the inner mucus layer and thereby promoted EHEC access and binding to the epithelium in vitro and ex vivo. Given the lack of efficient therapies against EHEC infection, StcE may represent a suitable target for future treatment and prevention strategies.

  16. Verotoxigenic Escherichia coli O157:H7 from Swedish cattle; isolates from prevalence studies versus strains linked to human infections - A retrospective study

    Directory of Open Access Journals (Sweden)

    Eriksson Erik

    2010-01-01

    Full Text Available Abstract Background Several cases of human infection caused by verotoxin-producing Escherichia coli (VTEC O157:H7 in Sweden have been connected with cattle farm visits. Between 1996 and 2002, 18 farms were classified as the source of human cases with isolation of EHEC (Enterohaemorrhagic Escherichia coli after VTEC O157:H7 had been isolated from cattle on those farms. Results Characterization by phage typing and molecular methods of the strains isolated from these 18 farms, including PCR for virulence genes (vtx1, vtx2 and variants thereof, eaeA and EHEC-hlyA and Pulsed-Field Gel Electrophoresis (PFGE, demonstrated a cluster of very similar strains from 16 farms. All were of phage type 4, carried the genes encoding the verotoxins VT2 and VT2c, intimin, EHEC-haemolysin and flagellin H7 as shown by PCR, and had identical or very similar PFGE patterns. When analysing strains in a prevalence study of VTEC O157:H7 from cattle at slaughter as well as from an on-farm prevalence study of dairy cattle, using the same typing methods, a rather wide variation was observed among the isolated VTEC O157:H7 strains. Conclusions In Sweden, a limited group of genetically similar and highly pathogenic VTEC O157:H7 strains seem to predominate in direct or indirect transmission from cattle to man.

  17. Detection and characterization of pCT-like plasmid vectors for blaCTX-M-14 in Escherichia coli isolates from humans, turkeys and cattle in England and Wales

    NARCIS (Netherlands)

    Stokes, M.O.; Cottel, J.L.; Piddock, L.J.; Wu, G.; Wootton, M.; Mevius, D.J.; Randall, L.P.; Teale, C.J.; Fielder, M.D.; Coldham, N.G.

    2012-01-01

    Objectives - To detect and characterize Escherichia coli strains and pCT-like plasmids implicated in the dissemination of the CTX-M-14 gene in animals and humans, in England and Wales. Methods UK CTX-M-14-producing E. coli (n¿=¿70) from cattle (n¿=¿33), turkeys (n¿=¿9), sheep (n¿=¿2) and humans (n¿=

  18. Circulation of clonal populations of fluoroquinolone-resistant CTX-M-15-producing Escherichia coli ST410 in humans and animals in Germany.

    Science.gov (United States)

    Falgenhauer, Linda; Imirzalioglu, Can; Ghosh, Hiren; Gwozdzinski, Konrad; Schmiedel, Judith; Gentil, Katrin; Bauerfeind, Rolf; Kämpfer, Peter; Seifert, Harald; Michael, Geovana Brenner; Schwarz, Stefan; Pfeifer, Yvonne; Werner, Guido; Pietsch, Michael; Roesler, Uwe; Guerra, Beatriz; Fischer, Jennie; Sharp, Hannah; Käsbohrer, Annemarie; Goesmann, Alexander; Hille, Katja; Kreienbrock, Lothar; Chakraborty, Trinad

    2016-06-01

    Multidrug-resistant Escherichia coli encoding CTX-M-type extended-spectrum β-lactamases (ESBLs) are isolated in increasing numbers from humans, companion animals and livestock, raising concern regarding the exchange and spread of isolates in these populations. In this study, whole-genome sequencing of CTX-M-15-producing E. coli isolates recently sampled from humans, companion animals, livestock and farm environments was performed. In total, 26 different sequence types (STs) were detected, of which ST410 was the most frequent and was the only ST present in all populations studied. Five clades (designated A-E) were detected within the ST410 isolates. In particular, isolates of clade B were present in all four populations and had core genomes that differed by less than 70 single nucleotide polymorphisms (SNPs). Isolates of clades B and C were also clonally marked, exhibiting identical chromosomal insertions of blaCTX-M-15 at distinct loci. These data provide strong evidence for the clonal dissemination of specific clades of CTX-M-15-producing E. coli ST410 in human and animal populations.

  19. Intramacrophage survival of uropathogenic Escherichia coli: Differences between diverse clinical isolates and between mouse and human macrophages

    DEFF Research Database (Denmark)

    Bokil, Nilesh J.; Totsika, Makrina; Carey, Alison J.;

    2011-01-01

    Uropathogenic E. coli (UPEC) are the primary cause of urinary tract infections. Recent studies have demonstrated that UPEC can invade and replicate within epithelial cells, suggesting that this bacterial pathogen may occupy an intracellular niche within the host. Given that many intracellular...... or initial uptake of bacteria. E. coli UTI89 localized to a Lamp1+ vesicular compartment within BMM. In contrast to survival within mouse BMM, intracellular bacterial loads of VR50 were low in both human monocyte-derived macrophages (HMDM) and in human T24 bladder epithelial cells. Collectively, these data......, originally isolated from patients with cystitis and asymptomatic bacteriuria respectively, showed elevated bacterial loads in BMM at 24h post-infection as compared to CFT073 and the asymptomatic bacteriuria strain 83972. These differences did not correlate with differential effects on macrophage survival...

  20. Soluble expression of active human β-defensin-3 in Escherichia coli and its effects on the growth of host cells

    Institute of Scientific and Technical Information of China (English)

    SI Li-gang; LIU Xi-cheng; LU You-yong; WANG Gen-yu; LI Wen-mei

    2007-01-01

    Background Human β-defensin-3 (HBD3) is an epithelial peptide that has been demonstrated to have a salt-insensitive broad spectrum of potent antimicrobial activity. Expressing antimicrobial peptides in Escherichia coli (E. coli) is very difficult for it can result in death of the bacterial host cells. Our aim was to establish a prokaryotic system expressing soluble HBD3 protein and demonstrate the antimicrobial activity of the expressed protein. We then studied whether the host cells would activate the suicide pathways.Methods We first cloned the complementary DNA coding for the mature chain of HBD3, inserted it into the vector PGEX-KG then transformed E. coli BL21 (DE3) with the appropriate recombinant plasmid. After induction with 0.5 mmol/L isopropyl-1-thio-β-D-galactopyranoside (IPTG) the transformed E. coli produced a recombinant glutathione S-transferase and HBD3 (GST-HBD3) fusion protein. The fusion protein was treated with thrombin to produce pure HBD3 protein then the antimicrobial activity of HBD3 was evaluated in a liquid microdilution assay.Results The fusion protein GST-HBD3 was efficiently cleaved by thrombin and yielded HBD3 that had anti-staphylococcus aureus activity with a minimal inhibitory concentration level of 12.5 μg/ml. The E. coli strain expressing the recombinant protein did not grow slower than the empty vector strain.Conclusion Active HBD3 in E. coli by expressing the recombinant protein GST-HBD3 could be produced, and suicide did not occur in the E. colistrain expressing the recombinant protein.

  1. Hydrogen production by recombinant Escherichia coli strains

    Science.gov (United States)

    Maeda, Toshinari; Sanchez‐Torres, Viviana; Wood, Thomas K.

    2012-01-01

    Summary The production of hydrogen via microbial biotechnology is an active field of research. Given its ease of manipulation, the best‐studied bacterium Escherichia coli has become a workhorse for enhanced hydrogen production through metabolic engineering, heterologous gene expression, adaptive evolution, and protein engineering. Herein, the utility of E. coli strains to produce hydrogen, via native hydrogenases or heterologous ones, is reviewed. In addition, potential strategies for increasing hydrogen production are outlined and whole‐cell systems and cell‐free systems are compared. PMID:21895995

  2. Recombinant human insulin. VIII. Isolation of fusion protein--S-sulfonate, biotechnological precursor of human insulin, from the biomass of transformed Escherichia coli cells.

    Science.gov (United States)

    Tikhonov, R V; Pechenov, S E; Belacheu, I A; Yakimov, S A; Klyushnichenko, V E; Boldireva, E F; Korobko, V G; Tunes, H; Thiemann, J E; Vilela, L; Wulfson, A N

    2001-02-01

    Various methods have been investigated for the isolation and purification of fusion proteins of precursors of human insulin in the form of S-sulfonates, from the biomass of transformed Escherichia coli cells. Fusion proteins were prepared with different sizes and structures of the leader peptide and the poly-His position (inserted for purification by metal chelate affinity chromatography). The fusion proteins contained an IgG-binding B domain of protein A from Staphylococcus aureus at the N-terminus and an Arg residue between the leader peptide of the molecule and the proinsulin sequence, for trypsin cleavage of the leader peptide. Six residues of Cys in proinsulin allow the chemical modification of the protein as a (Cys-S-SO(-)(3))(6) derivative (S-sulfonate), which increases its polyelectrolytic properties and improves the efficiency of its isolation. Various methods of oxidative sulfitolysis were compared with catalysis by sodium tetrathionate or cystine and Cu2+ or Ni2+ ions. An optimum scheme for the isolation and purification of S-sulfonated fusion proteins was developed by the combination of metal-chelating affinity and ion-exchange chromatography. Highly purified (95%) S-sulfonated fusion protein was recovered which was 85% of the fusion protein contained in the biomass of E. coli cells. Folding of fusion protein S-sulfonate occurred with high yield (up to 90-95%). We found that the fusion protein-S-sulfonate has proinsulin-like secondary structure. This structure causes highly efficient fusion protein folding. Copyright 2001 Academic Press.

  3. Evolution of the iss gene in Escherichia coli.

    Science.gov (United States)

    Johnson, Timothy J; Wannemuehler, Yvonne M; Nolan, Lisa K

    2008-04-01

    The increased serum survival gene iss has long been recognized for its role in extraintestinal pathogenic Escherichia coli (ExPEC) virulence. iss has been identified as a distinguishing trait of avian ExPEC but not of human ExPEC. This gene has been localized to large virulence plasmids and shares strong similarities with the bor gene from bacteriophage lambda. Here, we demonstrate that three alleles of iss occur among E. coli isolates that appear to have evolved from a common lambda bor precursor. In addition to the occurrence of iss on the ColV/BM virulence plasmids, at least two iss alleles occur within the E. coli chromosome. One of these alleles (designated type 3) was found to occur in the genomes of all currently sequenced ExPEC strains on a similar prophage element that also harbors the Sit iron and manganese transport system. When the prevalence of the three iss types was examined among 487 E. coli isolates, the iss type 3 gene was found to occur at a high frequency among ExPEC isolates, irrespective of the host source. The plasmid-borne iss allele (designated type 1) was highly prevalent among avian pathogenic E. coli and neonatal meningitis-associated E. coli isolates but not among uropathogenic E. coli isolates. This study demonstrates the evolution of iss in E. coli and provides an additional tool for discriminating among E. coli pathotypes through the differentiation of the three iss allele types and bor.

  4. Improving the soluble expression and purification of recombinant human stem cell factor (SCF) in endotoxin-free Escherichia coli by disulfide shuffling with persulfide.

    Science.gov (United States)

    Ueda, Takafumi; Akuta, Teruo; Kikuchi-Ueda, Takane; Imaizumi, Keitaro; Ono, Yasuo

    2016-04-01

    We here present a new method for the expression and purification of recombinant human stem cell factor (rhSCF(164)) in endotoxin-free ClearColi(®) BL21(DE3) cells harboring codon-optimized Profinity eXact™-tagged hSCF cDNA. Previously, we demonstrated that co-expression with thioredoxin increased the solubility of rhSCF in Escherichia coli BL21(DE3), and addition of l-arginine enhanced chromatography performance by removing the endotoxin-masked surface of rhSCF. Initially, we tried to express rhSCF in an endotoxin-free strain using a thioredoxin co-expression system, which resulted in significantly lower expression, possibly due to the stress imposed by overexpressed thioredoxin or antibiotics susceptibility. Therefore, we developed a new expression system without thioredoxin. External redox coupling was tested using persulfides such as glutathione persulfide or cysteine persulfide for the in vivo-folding of hSCF in the cytoplasm. Persulfides improved the protein solubility by accelerating disulfide-exchange reactions for incorrectdisulfides during folding in E. coli. Furthermore, the persulfides enhanced the expression level, likely due to upregulation of the enzymatic activity of T7 RNA polymerase. The recombinant protein was purified via affinity chromatography followed by cleavage with sodium fluoride, resulting in complete proteolytic removal of the N-terminal tag. The endotoxin-free fusion protein from ClearColi(®) BL21(DE3) could bind to the resin in the standard protocol using sodium phosphate (pH 7.2). Furthermore, purified rhSCF enhanced the proliferation and maturation of the human mast cell line LAD2. Thus, we conclude that use of the protein expression system employing E. coli by disulfide shuffling with persulfide addition could be a very useful method for efficient protein production.

  5. Genetic diversity and antibiogram profile of diarrhoeagenic Escherichia coli pathotypes isolated from human, animal, foods and associated environmental sources

    Directory of Open Access Journals (Sweden)

    Pankaj Dhaka

    2016-05-01

    Full Text Available Introduction: Infectious diarrhoea particularly due to pathogenic bacteria is a major health problem in developing countries, including India. Despite significant reports of diarrhoeagenic Escherichia coli (DEC pathotypes around the globe, studies which address genetic relatedness, antibiogram profile and their correlation with respect to their isolation from different sources are sparse. The present study determines isolation and identification of DEC pathotypes from different sources, their genetic characterisation, antibiogram profile and their correlation if any. Materials and methods: A total of 336 samples comprising diarrhoeic stool samples from infants (n=103, young animal (n=106, foods (n=68 and associated environmental sources (n=59 were collected from Bareilly region of India. All the samples were screened by using standard microbiological methods for the detection of E. coli. The identified E. coli were then confirmed as DEC pathotypes using polymerase chain reaction–based assays. Those DEC pathotypes identified as Enteroaggregative E. coli (EAEC were further confirmed using HEp-2 adherence assay. All the isolated DEC pathotypes were studied for their genetic diversity using pulsed-field gel electrophoresis (PFGE, and antimicrobial susceptibility testing was performed by using disc diffusion method as per Clinical Laboratory Standards Institute guidelines. Results and discussion: Of the four DEC pathotypes investigated, EAEC was found to be the predominant pathogen with an isolation rate of 16.5% from infants, 17.9% from young animals, 16.2% from foods and 3.4% from the associated environmental sources. These EAEC isolates, on further characterisation, revealed predominance of ‘atypical’ EAEC, with an isolation rate of 10.7% from infants, 15.1% from young animals, 16.2% from foods, and 3.4% from the associated environmental sources. On PFGE analysis, discrimination was evident within DEC pathotypes as 52 unique pulsotypes were

  6. Detection of the plasmid-mediated mcr-1 gene conferring colistin resistance in human and food isolates of Salmonella enterica and Escherichia coli in England and Wales.

    Science.gov (United States)

    Doumith, Michel; Godbole, Gauri; Ashton, Philip; Larkin, Lesley; Dallman, Tim; Day, Martin; Day, Michaela; Muller-Pebody, Berit; Ellington, Matthew J; de Pinna, Elizabeth; Johnson, Alan P; Hopkins, Katie L; Woodford, Neil

    2016-08-01

    In response to the first report of transmissible colistin resistance mediated by the mcr-1 gene in Escherichia coli and Klebsiella spp. from animals and humans in China, we sought to determine its presence in Enterobacteriaceae isolated in the UK. The PHE archive of whole-genome sequences of isolates from surveillance collections, submissions to reference services and research projects was retrospectively analysed for the presence of mcr-1 using Genefinder. The genetic environment of the gene was also analysed. Rapid screening of the genomes of ∼24 000 Salmonella enterica, E. coli, Klebsiella spp., Enterobacter spp., Campylobacter spp. and Shigella spp. isolated from food or humans identified 15 mcr-1-positive isolates. These comprised: 10 human S. enterica isolates submitted between 2012 and 2015 (8 Salmonella Typhimurium, 1 Salmonella Paratyphi B var Java and 1 Salmonella Virchow) from 10 patients; 3 isolates of E. coli from 2 patients; and 2 isolates of Salmonella Paratyphi B var Java from poultry meat imported from the EU. The mcr-1 gene was located on diverse plasmids belonging to the IncHI2, IncI2 and IncX4 replicon types and its association with ISApl1 varied. Six mcr-1-positive S. enterica isolates were from patients who had recently travelled to Asia. Analysis of WGS data allowed rapid confirmation of the presence of the plasmid-mediated colistin resistance gene mcr-1 in diverse genetic environments and plasmids. It has been present in E. coli and Salmonella spp. harboured by humans in England and Wales since at least 2012. © Crown copyright 2016.

  7. Identification and Prevalence of Escherichia coli and Escherichia coli O157: H7 in Foods

    Directory of Open Access Journals (Sweden)

    Ancuta Mihaela Rotar

    2013-11-01

    Full Text Available The objective of this study is to investigate the incidence of Escherichia coli in animal and non-animal foods, and mainly the incidence of the serotype O157: H7 producing verotoxin. The presence of common Escherichia coli and Escherichia coli O157: H7 in various foods (of animal and non animal origin was performed in Transylvania area. We analyzed a total of one hundred forty-one samples of minced meat, one hundred twenty-six samples of meat , twenty six samples of meat products, five samples of alcoholic beverages, three samples of seafood, one hundred samples of cheese from pasteurized milk, seventeen samples of butter, four samples of vegetables and one sample of milk powder, using the standard cultural method and Vidas Eco method for E. coli O157: H7 strains. E. coli was identified in 50 samples of minced meat, 55 samples of meat prepared, 4 samples of meat products, 2 samples of alcoholic beverages, 25 samples of cheese from pasteurized milk, 6 samples of butter and 1 sample of vegetables. In this study were not been identified any foods contaminated with the E. coli O157: H7 serotype. The results of this reasearch have demostrated that E. coli wich represents a hygienic indicator of recent food contamination, can be destroyed with heat treatment and hygienic handling of foods. Our country over the years has been among the few countries where the incidence of the E. coli O157: H7 serotype has been minimal.

  8. Detection of mcr-1 encoding plasmid-mediated colistin-resistant Escherichia coli isolates from human bloodstream infection and imported chicken meat, Denmark 2015

    DEFF Research Database (Denmark)

    Hasman, H.; Hammerum, A. M.; Hansen, F.

    2015-01-01

    The plasmid-mediated colistin resistance gene, mcr-1, was detected in an Escherichia coli isolate from a Danish patient with bloodstream infection and in five E. coli isolates from imported chicken meat. One isolate from chicken meat belonged to the epidemic spreading sequence type ST131. In addi...

  9. Escherichia coli Isolates from Broiler Chicken Meat, Broiler Chickens, Pork, and Pigs Share Phylogroups and Antimicrobial Resistance with Community-Dwelling Humans and Patients with Urinary Tract Infection

    DEFF Research Database (Denmark)

    Jakobsen, L.; Kurbasic, A.; Skjot-Rasmussen, L.

    2010-01-01

    Escherichia coli is the most common cause of urinary tract infection (UTI). Phylogroup B2 and D isolates are associated with UTI. It has been proposed that E. coli causing UTI could have an animal origin. The objective of this study was to investigate the phylogroups and antimicrobial resistance...

  10. Automatic tracking of Escherichia coli bacteria.

    Science.gov (United States)

    Xie, Jun; Khan, Shahid; Shah, Mubarak

    2008-01-01

    In this paper, we present an automatic method for estimating the trajectories of Escherichia coli bacteria from in vivo phase-contrast microscopy videos. To address the low-contrast boundaries in cellular images, an adaptive kernel-based technique is applied to detect cells in sequence of frames. Then a novel matching gain measure is introduced to cope with the challenges such as dramatic changes of cells' appearance and serious overlapping and occlusion. For multiple cell tracking, an optimal matching strategy is proposed to improve the handling of cell collision and broken trajectories. The results of successful tracking of Escherichia coli from various phase-contrast sequences are reported and compared with manually-determined trajectories, as well as those obtained from existing tracking methods. The stability of the algorithm with different parameter values is also analyzed and discussed.

  11. Completion of DNA replication in Escherichia coli.

    Science.gov (United States)

    Wendel, Brian M; Courcelle, Charmain T; Courcelle, Justin

    2014-11-18

    The mechanism by which cells recognize and complete replicated regions at their precise doubling point must be remarkably efficient, occurring thousands of times per cell division along the chromosomes of humans. However, this process remains poorly understood. Here we show that, in Escherichia coli, the completion of replication involves an enzymatic system that effectively counts pairs and limits cellular replication to its doubling point by allowing converging replication forks to transiently continue through the doubling point before the excess, over-replicated regions are incised, resected, and joined. Completion requires RecBCD and involves several proteins associated with repairing double-strand breaks including, ExoI, SbcDC, and RecG. However, unlike double-strand break repair, completion occurs independently of homologous recombination and RecA. In some bacterial viruses, the completion mechanism is specifically targeted for inactivation to allow over-replication to occur during lytic replication. The results suggest that a primary cause of genomic instabilities in many double-strand-break-repair mutants arises from an impaired ability to complete replication, independent from DNA damage.

  12. Mechanisms of antibiotic resistance to enrofloxacin in uropathogenic Escherichia coli in dog

    Science.gov (United States)

    Escherichia coli (E. coli) urinary tract infections (UTIs) are becoming a serious problem both for pets and humans (zoonosis) due to the close contact and to the increasing resistance to antibiotics. Canine E. coli represents a good experimental model useful to study this pathology. Moreover, as des...

  13. Detection of Escherichia Coli O157:H7 in Fecal Samples in Meat Goats

    Science.gov (United States)

    Mobley, Ray; Madden, Uford; Brooks-Walter, Alexis

    2004-01-01

    Studies have reported the isolation of Escherichia coli (E. coli)O157:H7 from pork, lamb and poultry products, and from other animals including deer, horses, dogs, birds and humans. There is limited or no information on the presence of the organism in goats. The objectives of this study were to determine if E. coli O157:H7 was naturally occurring…

  14. Multiple Antimicrobial Resistance of Escherichia coli Isolated from Chickens in Iran

    OpenAIRE

    Reza Talebiyan; Mehdi Kheradmand; Faham Khamesipour; Mohammad Rabiee-Faradonbeh

    2014-01-01

    Antimicrobial agents are used extremely in order to reduce the great losses caused by Escherichia coli infections in poultry industry. In this study, 318 pathogenic Escherichia coli (APEC) strains isolated from commercial broiler flocks with coli-septicemia were examined for antimicrobials of both veterinary and human significance by disc diffusion method. Multiple resistances to antimicrobial agents were observed in all the isolates. Resistance to the antibiotics was as follows: Tylosin (88....

  15. Escherichia coli necrotizing fasciitis in Hirschsprung's disease

    Directory of Open Access Journals (Sweden)

    Manal A. Alsaif

    2015-04-01

    Full Text Available Necrotizing fasciitis is a rare post-operative complication of Hirschsprung's disease. Very recently the only previous case of necrotizing fasciitis following a Soave procedure was reported with the etiologic agent being Pseudomonas aeruginosa. Here we are reporting the second case of necrotizing fasciitis following a Soave procedure caused by an extended spectrum beta lactamase harboring strain of Escherichia coli which is a rare pathogen in type II necrotizing fasciitis.

  16. Homology requirements for recombination in Escherichia coli.

    OpenAIRE

    Watt, V M; Ingles, C J; Urdea, M S; Rutter, W J

    1985-01-01

    The DNA sequence homology required for recombination in Escherichia coli has been determined by measuring the recombination frequency between insulin DNA in a miniplasmid pi VX and a homologous sequence in a bacteriophage lambda vector. A minimum of approximately equal to 20 base pairs in a completely homologous segment is required for significant recombination. There is an exponential increase in the frequency of recombination when the length of homologous DNA is increased from 20 base pairs...

  17. Cryopreserved, Xeno-Free Human Umbilical Cord Mesenchymal Stromal Cells Reduce Lung Injury Severity and Bacterial Burden in Rodent Escherichia coli-Induced Acute Respiratory Distress Syndrome.

    Science.gov (United States)

    Curley, Gerard F; Jerkic, Mirjana; Dixon, Steve; Hogan, Grace; Masterson, Claire; O'Toole, Daniel; Devaney, James; Laffey, John G

    2017-02-01

    Although mesenchymal stem/stromal cells represent a promising therapeutic strategy for acute respiratory distress syndrome, clinical translation faces challenges, including scarcity of bone marrow donors, and reliance on bovine serum during mesenchymal stem/stromal cell proliferation. We wished to compare mesenchymal stem/stromal cells from human umbilical cord, grown in xeno-free conditions, with mesenchymal stem/stromal cells from human bone marrow, in a rat model of Escherichia coli pneumonia. In addition, we wished to determine the potential for umbilical cord-mesenchymal stem/stromal cells to reduce E. coli-induced oxidant injury. Randomized animal study. University research laboratory. Male Sprague-Dawley rats. Acute respiratory distress syndrome was induced in rats by intratracheal instillation of E. coli (1.5-2 × 10 CFU/kg). "Series 1" compared the effects of freshly thawed cryopreserved umbilical cord-mesenchymal stem/stromal cells with bone marrow-mesenchymal stem/stromal cells on physiologic indices of lung injury, cellular infiltration, and E. coli colony counts in bronchoalveolar lavage. "Series 2" examined the effects of cryopreserved umbilical cord-mesenchymal stem/stromal cells on survival, as well as measures of injury, inflammation and oxidant stress, including production of reactive oxidative species, reactive oxidative species scavenging by superoxide dismutase-1 and superoxide dismutase-2. In "Series 1," animals subjected to E. coli pneumonia who received umbilical cord-mesenchymal stem/stromal cells had improvements in oxygenation, respiratory static compliance, and wet-to-dry ratios comparable to bone marrow-mesenchymal stem/stromal cell treatment. E. coli colony-forming units in bronchoalveolar lavage were reduced in both cell therapy groups, despite a reduction in bronchoalveolar lavage neutrophils. In series 2, umbilical cord-mesenchymal stem/stromal cells enhanced animal survival and decreased alveolar protein and proinflammatory

  18. Occurrence and characteristics of extended-spectrum β-lactamases producing Escherichia coli in foods of animal origin and human clinical samples in Chhattisgarh, India

    Directory of Open Access Journals (Sweden)

    Bhoomika

    2016-09-01

    Full Text Available Aim: To assess the prevalence of antimicrobial resistance producing extended-spectrum β-lactamases (ESBL (blaTEM, blaSHV, and blaCTX-M genes in Escherichia coli isolated from chicken meat, chevon meat, raw milk, and human urine and stool samples collected from tribal districts of Chhattisgarh, viz., Jagdalpur, Dantewada, Kondagaon, and Kanker. Materials and Methods: A total of 330 samples, comprising 98 chicken meat, 82 chevon meat, 90 raw milk, and 60 human urine and stool samples, were processed for isolation of E. coli. Isolates were confirmed biochemically and further tested against commonly used antibiotics to know their resistant pattern. The resistant isolates were tested for ESBL production by phenotypic method followed by characterization with molecular method using multiplex-polymerase chain reaction technique. Results: Overall 57.87% (191/330 samples were found positive for E. coli, which include 66.32% (65/98 chicken meat, 46.34% (38/82 chevon meat, 81.11% (73/90 raw milk, and 25% (15/60 human urine and stool samples. Isolates showed the highest resistance against cefotaxime (41.36% followed by oxytetracycline (34.03%, ampicillin (29.31%, cephalexin (24.60%, cefixime (16.75%, and ceftazidime (13.08%. Phenotypic method detected 10.99% (21/191 isolates as presumptive ESBL producers, however, molecular method detected 3.66% (7/191, 2.09% (4/191, and 0.00% (0/191 prevalence of blaTEM, blaCTX-M, and blaSHV, respectively. Conclusion: The present study indicates a high prevalence of E. coli in raw chicken meat, chevon meat, and milk due to poor hygienic practices. The antibiotic susceptibility test detected the presence of the resistance pattern against ESBL in E. coli isolated from raw chicken meat, chevon meat, milk, and also in human clinical samples is of great concern. The appearance of E. coli in the human food chain is alarming and requires adaptation of hygienic practices and stipulate use of antibiotics.

  19. Siderophore production by uropathogenic Escherichia coli

    Directory of Open Access Journals (Sweden)

    Vagrali Manjula

    2009-01-01

    Full Text Available Urinary tract infection (UTI is one of the most frequently encountered problems in ambulatory medicine. The present study was designed to determine siderophore production as the urovirulence factor of Escherichia coli isolated from the patients of UTI. A total of 160 strains of E. coli isolated from urine of patients with clinically diagnosed UTI were included in the study and 50 fecal isolates of E. coli, siderophore production was seen in 156 (97.5%. In 50 fecal isolates, siderophore production was seen in 2 (4%. Siderophore production has been shown to be more frequent in E. coli from patients with UTI, than in fecal isolates. The results suggest that siderophore production positive strains can be considered as UPEC. Thus, although a great deal has been learned regarding E. coli virulence mechanisms in UTI, much remains to be learned and the practical application of our growing understanding of E. coli virulence factors to the prevention and treatment of UTI has to be continued.

  20. Differentiation between Shigella, enteroinvasive Escherichia coli (EIEC) and noninvasive Escherichia coli.

    Science.gov (United States)

    van den Beld, M J C; Reubsaet, F A G

    2012-06-01

    Shigella causes bacillary dysentery and is classified into four species based on their antigen characteristics. This classification does not reflect genetic relatedness; in fact, Shigella species are so related to Escherichia coli , they should be classified as one distinctive species in the genus Escherichia. The differentiation of Shigella and E. coli is even more complicated with the description of enteroinvasive E. coli (EIEC). EIEC are strains that possess some of the biochemical characteristics of E. coli and have the ability to cause dysentery using the same method of invasion as Shigella does. Sequencing of multiple housekeeping genes indicates that EIEC is more related to Shigella than to non-invasive E. coli. Shigella and EIEC evolved from the same ancestor and form a single pathovar within E. coli. Shigella and EIEC could be separated from other E. coli by a PCR targeting the ipaH-gene; this is a multicopy gene exclusively found in all Shigella and EIEC. It is possible to differentiate Shigella and all E. coli, including EIEC, by using multiple tests, including ipaH-gene PCR, physiological and biochemical typing and serological typing. Based on literature study, a key is designed for daily use in diagnostic laboratories to identify Shigella and all E. coli.

  1. Interaction between Escherichia coli and lunar fines

    Science.gov (United States)

    Johansson, K. R.

    1983-01-01

    A sample of mature lunar fines (10084.151) was solubilized to a high degree (about 17 percent) by the chelating agent salicylic acid (0.01. M). The neutralized (pH adjusted to 7.0) leachate was found to inhibit the growth of Escherichia coli (ATCC 259922) in a minimial mineral salts glucose medium; however, the inhibition was somewhat less than that caused by neutralized salicylic acid alone. The presence of lunar fines in the minimal medium was highly stimulatory to growth of E. coli following an early inhibitory response. The bacterium survived less well in the lunar leachate than in distilled water, no doubt because of the salicylate. It was concluded that the sample of lunar soil tested has nutritional value to E. coli and that certain products of fermentation helped to solubilize the lunar soil.

  2. Cloning, expression, and purification of a synthetic human growth hormone in Escherichia coli using response surface methodology.

    Science.gov (United States)

    Zamani, Mozhdeh; Berenjian, Aydin; Hemmati, Shiva; Nezafat, Navid; Ghoshoon, Mohammad Bagher; Dabbagh, Fatemeh; Mohkam, Milad; Ghasemi, Younes

    2015-03-01

    The aim of this study was to achieve high-level production of the human growth hormone (hGH) in the prokaryotic expression system. In this regard, we performed cloning, expression, and purification of a synthetic hGH gene in BL21 (DE3) strain of E. coli. The hGH production was determined by SDS-PAGE and western blotting techniques, and then the protein concentration was determined by the Bradford assay. To gain insight into the effect of different nutrients on the growth of E. coli and hGH production, in a preliminary assessment nine different types of the basal medium were analyzed. The highest growth of E. coli and hGH production were observed in TB and SOB media. Accordingly, design of experiments was employed for screening the most significant nutrients, and central composite face design was applied for the optimization. The optimum medium consisted of yeast extract (10 g/L), tryptone (10 g/L), and K2HPO4 (2 g/L). The optimum hGH concentration was 391 mg/L, which was 3-fold higher than the hGH concentration in the LB basal medium (119 mg/L). This production rate is the highest hGH concentration reported in the IPTG-inducible expression systems.

  3. Human bladder uroepithelial cells synergize with monocytes to promote IL-10 synthesis and other cytokine responses to uropathogenic Escherichia coli.

    Science.gov (United States)

    Duell, Benjamin L; Carey, Alison J; Dando, Samantha J; Schembri, Mark A; Ulett, Glen C

    2013-01-01

    Urinary tract infections are a major source of morbidity for women and the elderly, with Uropathogenic Escherichia coli (UPEC) being the most prevalent causative pathogen. Studies in recent years have defined a key anti-inflammatory role for Interleukin-10 (IL-10) in urinary tract infection mediated by UPEC and other uropathogens. We investigated the nature of the IL-10-producing interactions between UPEC and host cells by utilising a novel co-culture model that incorporated lymphocytes, mononuclear and uroepithelial cells in histotypic proportions. This co-culture model demonstrated synergistic IL-10 production effects between monocytes and uroepithelial cells following infection with UPEC. Membrane inserts were used to separate the monocyte and uroepithelial cell types during infection and revealed two synergistic IL-10 production effects based on contact-dependent and soluble interactions. Analysis of a comprehensive set of immunologically relevant biomarkers in monocyte-uroepithelial cell co-cultures highlighted that multiple cytokine, chemokine and signalling factors were also produced in a synergistic or antagonistic fashion. These results demonstrate that IL-10 responses to UPEC occur via multiple interactions between several cells types, implying a complex role for infection-related IL-10 during UTI. Development and application of the co-culture model described in this study is thus useful to define the degree of contact dependency of biomarker production to UPEC, and highlights the relevance of histotypic co-cultures in studying complex host-pathogen interactions.

  4. Human bladder uroepithelial cells synergize with monocytes to promote IL-10 synthesis and other cytokine responses to uropathogenic Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Benjamin L Duell

    Full Text Available Urinary tract infections are a major source of morbidity for women and the elderly, with Uropathogenic Escherichia coli (UPEC being the most prevalent causative pathogen. Studies in recent years have defined a key anti-inflammatory role for Interleukin-10 (IL-10 in urinary tract infection mediated by UPEC and other uropathogens. We investigated the nature of the IL-10-producing interactions between UPEC and host cells by utilising a novel co-culture model that incorporated lymphocytes, mononuclear and uroepithelial cells in histotypic proportions. This co-culture model demonstrated synergistic IL-10 production effects between monocytes and uroepithelial cells following infection with UPEC. Membrane inserts were used to separate the monocyte and uroepithelial cell types during infection and revealed two synergistic IL-10 production effects based on contact-dependent and soluble interactions. Analysis of a comprehensive set of immunologically relevant biomarkers in monocyte-uroepithelial cell co-cultures highlighted that multiple cytokine, chemokine and signalling factors were also produced in a synergistic or antagonistic fashion. These results demonstrate that IL-10 responses to UPEC occur via multiple interactions between several cells types, implying a complex role for infection-related IL-10 during UTI. Development and application of the co-culture model described in this study is thus useful to define the degree of contact dependency of biomarker production to UPEC, and highlights the relevance of histotypic co-cultures in studying complex host-pathogen interactions.

  5. Effects of Escherichia Coli-derived Recombinant Human Bone Morphogenetic Protein-2 Loaded Porous Hydroxyaptite-based Ceramics on Calvarial Defect in Rabbits

    Science.gov (United States)

    Kim, Shin-Young; Lee, Youngkyun; Seo, Seung-Jun; Lim, Jae-Hong

    2017-01-01

    Background Recombinant human bone morphogenetic proteins (rhBMPs) have been widely used in regenerative therapies to promote bone formation. The production of rhBMPs using bacterial systems such as Escherichia coli (E. coli) is estimated to facilitate clinical applications by lowering the cost without compromising biological activity. In clinical practice, rhBMP-2 and osteoconductive carriers (e.g., hydroxyapatite [HA] and bovine bone xenograft) are used together. This study examined the effect of E. coli-derived rhBMP-2 combined with porous HA-based ceramics on calvarial defect in rabbits. Methods Six adult male New Zealand white rabbits were used in this study. The experimental groups were divided into the following 4 groups: untreated (NC), bovine bone graft (BO), porous HA (HA) and porous HA with rhBMP-2 (HA-BMP). Four transosseous defects of 8 mm in diameter were prepared using stainless steel trephine bur in the frontal and parietal bones. Histological and histomorphometric analyses at 4 weeks after surgery revealed significant new bone formation by porous HA alone. Results HA-BMP showed significantly higher degree of bone formation compared with BO and HA group (Pceramics can promote new bone formation. PMID:28326298

  6. Real-time toxicity and metabolic activity tracking of human cells exposed to Escherichia coli O157:H7 in a mixed consortia

    Science.gov (United States)

    Xu, Tingting; Marr, Enolia; Lam, Haylie; Ripp, Steven; Sayler, Gary

    2017-01-01

    Escherichia coli O157:H7 is a significant human pathogen that is continually responsible for sickness, and even death, on a worldwide scale. While the pathology of E. coli O157:H7 infection has been well studied, the effect of it’s multiple resulting cytotoxic mechanisms on host metabolic activity has not been well characterized. To develop a more thorough understanding of these effects, several bioluminescence assays were evaluated for their ability to track both toxicity and host metabolic activity levels in real-time. The use of continuously autobioluminescent human cells was determined to be the most favorable method for tracking these metrics, as its self-sufficient autobioluminescent phenotype was unaffected by the presence of the infecting bacteria and its signal could be measured without cellular destruction. Using this approach, it was determined that infection with as few as 10 CFU of E. coli O157:H7 could elicit cytotoxic effects. Regardless of the initial infective dose, an impact on metabolic expression was not observed until bacterial populations reached levels between 5 × 105 and 1 × 106 (R2 = 0.933), indicating that a critical bacterial infection level must be reached prior to the onset of cytotoxic effects. Supporting this hypothesis, it was found that cells displaying infection-mediated metabolic activity reductions could recover to wild type metabolic activity levels if the infecting bacteria were removed prior to cell death. These results indicate that rapid treatment of E. coli O157:H7 infection could serve to limit host metabolic impact and reduce overall host cell death. PMID:26423391

  7. antimicrobial susceptibility and plasmids from escherichia coli ...

    African Journals Online (AJOL)

    2001-10-10

    Oct 10, 2001 ... transmission to humans of E. coli containing antibiotic resistance plasmids ... resistant micro-organisms, which may in turn transfer resistance to .... cells were washed with sterile normal saline to remove leached. Я-lactamase ...

  8. ANTIMICIROBIAL SUSCEPTIBILITY PATTERNS OF Escherichia coli ...

    African Journals Online (AJOL)

    DR. AMINU

    aetiological agents of diarrhoea diseases of humans in developing ... Pathogenic organisms have developed a number of elaborate .... reported that E.coli isolated from animals haboured plasmids .... coil 0157:H7 as a model of entry of a new.

  9. Efficient Production of Hydroxylated Human-Like Collagen Via the Co-Expression of Three Key Genes in Escherichia coli Origami (DE3).

    Science.gov (United States)

    Tang, Yunping; Yang, Xiuliang; Hang, Baojian; Li, Jiangtao; Huang, Lei; Huang, Feng; Xu, Zhinan

    2016-04-01

    Mature collagen is abundant in human bodies and very valuable for a range of industrial and medical applications. The biosynthesis of mature collagen requires post-translational modifications to increase the stability of collagen triple helix structure. By co-expressing the human-like collagen (HLC) gene with human prolyl 4-hydroxylase (P4H) and D-arabinono-1, 4-lactone oxidase (ALO) in Escherichia coli, we have constructed a prokaryotic expression system to produce the hydroxylated HLC. Then, five different media, as well as the induction conditions were investigated with regard to the soluble expression of such protein. The results indicated that the highest soluble expression level of target HLC obtained in shaking flasks was 49.55 ± 0.36 mg/L, when recombinant cells were grew in MBL medium and induced by 0.1 mM IPTG at the middle stage of exponential growth phase. By adopting the glucose feeding strategy, the expression level of target HLC can be improved up to 260 mg/L in a 10 L bench-top fermentor. Further, HPLC analyses revealed that more than 10 % of proline residues in purified HLC were successfully hydroxylated. The present work has provided a solid base for the large-scale production of hydroxylated HLC in E. coli.

  10. Unexpected distribution of the fluoroquinolone-resistance gene qnrB in Escherichia coli isolates from different human and poultry origins in Ecuador.

    Science.gov (United States)

    Armas-Freire, Paulina I; Trueba, Gabriel; Proaño-Bolaños, Carolina; Levy, Karen; Zhang, Lixin; Marrs, Carl F; Cevallos, William; Eisenberg, Joseph N S

    2015-06-01

    Fluoroquinolone resistance can be conferred through chromosomal mutations or by the acquisition of plasmids carrying genes such as the quinolone resistance gene (qnr). In this study, 3,309 strains of commensal Escherichia coli were isolated in Ecuador from: (i) humans and chickens in a rural northern coastal area (n = 2368, 71.5%) and (ii) chickens from an industrial poultry operation (n = 827, 25%). In addition, 114 fluoroquinolone-resistant strains from patients with urinary tract infections who were treated at three urban hospitals in Quito, Ecuador were analyzed. All of the isolates were subjected to antibiotic susceptibility screening. Fluoroquinolone-resistant isolates (FRIs) were then screened for the presence of qnrB genes. A significantly higher phenotypic resistance to fluoroquinolones was determined in E. coli strains from chickens in both the rural area (22%) and the industrial operation (10%) than in strains isolated from humans in the rural communities (3%). However, the rates of qnrB genes in E. coli isolates from healthy humans in the rural communities (11 of 35 isolates, 31%) was higher than in chickens from either the industrial operations (3 of 81 isolates, 6%) or the rural communities (7 of 251 isolates, 2.8%). The occurrence of qnrB genes in human FRIs obtained from urban hospitals was low (1 of 114 isolates, 0.9%). These results suggested that the qnrB gene is more widely distributed in rural settings, where antibiotic usage is low, than in urban hospitals and industrial poultry operations. The role of qnrB in clinical resistance to fluoroquinolones is thus far unknown.

  11. Autodisplay of Human Hyaluronidase Hyal-1 on Escherichia coli and Identification of Plant-Derived Enzyme Inhibitors

    Directory of Open Access Journals (Sweden)

    Zoya Orlando

    2015-08-01

    Full Text Available Hyaluronan (HA is the main component of the extracellular matrix (ECM. Depending on its chain size, it is generally accepted to exert diverse effects. High molecular weight HA is anti-angiogenic, immunosuppressive and anti-inflammatory, while lower fragments are angiogenic and inflammatory. Human hyaluronidase Hyal-1 (Hyal-1 is one of the main enzymes in the metabolism of HA. This makes Hyal-1 an interesting target. Not only for functional and mechanistic studies, but also for drug development. In this work, Hyal-1 was expressed on the surface of E. coli, by applying Autodisplay, to overcome formation of inactive “inclusion bodies”. With the cells displaying Hyal-1 an activity assay was performed using “stains-all” dye. Subsequently, the inhibitory effects of four saponins and 14 plant extracts on the activity of surface displayed Hyal-1 were evaluated. The determined IC50 values were 177 µM for glycyrrhizic acid, 108 µM for gypsophila saponin 2, 371 µM for SA1657 and 296 µM for SA1641. Malvae sylvestris flos, Equiseti herba and Ononidis radix extracts showed IC50 values between 1.4 and 1.7 mg/mL. In summary, Autodisplay enabled the expression of functional human target protein Hyal-1 in E. coli and facilitated an accelerated testing of potential inhibitors.

  12. Broiler chickens, broiler chicken meat, pigs and pork as sources of ExPEC related virulence genes and resistance in Escherichia coli isolates from community-dwelling humans and UTI patients

    DEFF Research Database (Denmark)

    Jakobsen, L; Spangholm, D. J.; Pedersen, Karl

    2010-01-01

    Urinary tract infection (UTI) is one of the most common bacterial infections. UTI is primarily caused by extraintestinal pathogenic Escherichia coli (ExPEC) from the patients' own fecal flora. The ExPEC often belong to phylogroups B2 and D, the groups which include potent human ExPEC isolates cau...

  13. Prevalence and antimicrobial resistance in Escherichia coli from food and animals in Lagos, Nigeria

    Science.gov (United States)

    Background Foodborne bacteria are often associated with human infections; these infections can become more complicated to treat if the bacteria are also resistant to antimicrobials. In this study, prevalence, antimicrobial resistance, and genetic relatedness of Escherichia coli among food producing ...

  14. Efficient expression of Human papillomavirus 16 E7 oncoprotein fused to C-terminus of Tobacco mosaic virus (TMV) coat protein using molecular chaperones in Escherichia coli.

    Science.gov (United States)

    Folwarczna, Jitka; Moravec, Tomas; Plchova, Helena; Hoffmeisterova, Hana; Cerovska, Noemi

    2012-09-01

    The Human papillomavirus 16 (HPV16) E7 oncoprotein is a promising candidate for development of anti-cancer therapeutic vaccine. We have prepared the expression construct carrying mutagenized E7 oncoprotein fused to the C-terminus of Tobacco mosaic virus (TMV) coat protein via 15 amino acids β-sheet linker. The fusion protein was expressed in Escherichia coli MC 1061 cells. We have obtained high level expression, but most of the protein remained in insoluble inclusion bodies. To increase the ratio of soluble protein various molecular chaperones (TF, DnaK-DnaJ-GrpE, GroEL-GroES) were used. The immunological reactivity of expressed recombinant protein was evaluated with anti-E7 and anti-TMV antibodies. The distribution of expressed product during ultracentrifugation on sucrose gradient was studied. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Prevalence of quinolone resistance mechanisms and associations to minimum inhibitory concentrations in quinolone-resistant Escherichia coli isolated from humans and swine in Denmark.

    Science.gov (United States)

    Cavaco, Lina Maria; Frimodt-Møller, Niels; Hasman, Henrik; Guardabassi, Luca; Nielsen, Lene; Aarestrup, Frank Møller

    2008-06-01

    Prevalence of quinolone resistance mechanisms and associations to minimum inhibitory concentrations (MICs) of nalidixic acid (NAL) and ciprofloxacin (CIP) were investigated in 124 Escherichia coli isolated from humans (n=85) and swine (n=39) in Denmark. The collection included 59 high-level CIP-resistant isolates (MIC >or= 4) from human (n=51) and pig origin (n=8) and 65 low-level CIP-resistant isolates (MIC >or= 0.125) from human (n=34) and pig origin (n=31). Resistance by target modification was screened by PCR amplification and sequencing of the quinolone resistance determining regions (QRDRs) of gyrA, gyrB, parC, and parE. QRDR mutations occurred in all except two isolates (98%). All high-level CIP-resistant E. coli had one or two mutations in gyrA in combination with mutations in parC or parE. Mutations in parC and parE were only found in combination with gyrA mutations, and no mutations were observed in gyrB. Efflux pump mechanisms were detected in 10 human (11.8%) and 29 porcine (74.4%) isolates by an efflux pump inhibitor (EPI) agar dilution assay. The aac(6')-Ib-cr gene mediating resistance by enzymatic modification was found in 12 high-level CIP-resistant human isolates. The qnrA and qnrS genes conferring quinolone resistance by target protection were detected in two human low-level CIP-resistant isolates that did not display NAL resistance. As expected, target mutation in QRDRs was the most prevalent mechanism of quinolone resistance. This mechanism was complemented by efflux mechanisms in most porcine isolates. Transferable resistance by target protection or enzymatic modification was less common (10%) and restricted to human isolates.

  16. Genetic Diversity of Intimin Gene of Atypical Enteropathogenic Escherichia coli Isolated from Human, Animals and Raw Meats in China.

    Science.gov (United States)

    Xu, Yanmei; Bai, Xiangning; Zhao, Ailan; Zhang, Wang; Ba, Pengbin; Liu, Kai; Jin, Yujuan; Wang, Hong; Guo, Qiusheng; Sun, Hui; Xu, Jianguo; Xiong, Yanwen

    2016-01-01

    Atypical enteropathogenic Escherichia coli (aEPEC) is considered to be an emerging enteropathogen that is more prevalent than typical EPEC in developing and developed countries. The major adherence factor, intimin, an outer membrane protein encoded by eae, plays a pivotal role in the pathogenesis of aEPEC. This study investigated the distribution and polymorphisms of intimin subtypes of 143 aEPEC strains from diarrheal patients, healthy carriers, animals, and raw meats in China. These aEPEC strains belonged to more than 71 different serotypes, which comprised 52 O serogroups and 24 H types. Sixty-eight different eae genotypes and 19 intimin subtypes were detected. Eighteen, eight, seven, and five intimin subtypes were identified from 86 diarrheal patients, 14 healthy carriers, 19 animals, and 24 raw meats strains, respectively. Intimin β1 was the most prevalent subtype in strains from diarrheal patients (34.88%) and animals (47.37%). There was a statistically significant difference in the distribution of eae-β1 between diarrheal patients and healthy carriers (P = 0.004). Intimin-θ was more predominant among raw meat strains (50%) than among diarrheal patients strains (12.79%, P = 0.0003), healthy carrier strains (7.14%, P = 0.007), or animal strains (15.79%, P = 0.020). The two predominant subtypes (eae-β1 and eae-θ) had considerable polymorphisms with no significant differences among the four sources. PFGE analysis revealed 119 distinct patterns and the strains were clustered into 11 groups with similarity indices ranging from 63% to 100%. These results suggest that in China, aEPEC strains from different sources are highly heterogeneous. Animals and raw meats are important sources of genetically diverse intimin-harboring aEPEC, which might serve as important transmission vehicles of these bacteria.

  17. Process Control for Production of Human-like Collagen in Fed-batch Culture of Escherichia coli BL 21

    Institute of Scientific and Technical Information of China (English)

    骆艳娥; 范代娣; 马晓轩; 王德伟; 米钰; 花秀夫; 李稳宏

    2005-01-01

    Recombinant E. coli BL 21 was cultivated in high cell density to produce human-like collagen. The effects of the feeding of nitrogen source, controlled by an auto on/off-feeding mode with two different cycles of 0.5 min and 4 rain intervals, oxygen-enrichment methods and inducement strength on the cell yield and human-like collagen production were investigated. The studies showed that nitrogen source feeding in fast cycle could result in higher human-like collagen production than that in slow cycle; and the feedback regulation of glucose, increase of the pressure of fermentation bioreactor, and supply of oxygen-enriched air could all increase cell yield and human-like collagen production. The effects of inducement strength on protein expression were found important. When OD600 reached 90—100, the cultivation temperature was increased to 42℃ to begin induction for 2—3 h, and then shifted to 39℃ for 5—6 h induction, the cell density and human-like collagen production could reach 96 g·L-1 [DCW (dry cell mass)] and 19.8% (g·g-1 DCW) respectively.

  18. Lactobacillus rhamnosus and its cell-free culture supernatant differentially modulate inflammatory biomarkers in Escherichia coli-challenged human dendritic cells.

    Science.gov (United States)

    Bermudez-Brito, Miriam; Muñoz-Quezada, Sergio; Gomez-Llorente, Carolina; Romero, Fernando; Gil, Angel

    2014-05-28

    The intestinal immune system maintains a delicate balance between immunogenicity against invading pathogens and tolerance to the commensal microbiota and food antigens. Different strains of probiotics possess the ability to finely regulate the activation of dendritic cells (DC), polarising the subsequent activity of T-cells. Nevertheless, information about their underlying mechanisms of action is scarce. In the present study, we investigated the immunomodulatory effects of a potentially probiotic strain, Lactobacillus rhamnosus CNCM I-4036, and its cell-free culture supernatant (CFS) on human DC challenged with Escherichia coli. The results showed that the levels of pro-inflammatory cytokines such as IL-1β, IL-6, IL-8 and IL-12p70 were higher in the cells treated with live L. rhamnosus than in the cells treated with the CFS. In the presence of E. coli, the supernatant was more effective than the probiotic bacteria in reducing the secretion of pro-inflammatory cytokines. In addition, live L. rhamnosus potently induced the production of transforming growth factor (TGF)-β1 and TGF-β2, whereas the CFS increased the secretion of TGF-β1. However, in the presence of E. coli, both treatments restored the levels of TGF-β. The probiotic strain L. rhamnosus CNCM I-4036 and its CFS were able to activate the Toll-like receptor signalling pathway, enhancing innate immunity. The two treatments induced gene transcription of TLR-9. Live L. rhamnosus activated the expression of TLR-2 and TLR-4 genes, whereas the CFS increased the expression of TLR-1 and TLR-5 genes. In response to the stimulation with probiotic/CFS and E. coli, the expression of each gene tested was notably increased, with the exception of TNF-α and NFKBIA. In conclusion, the CFS exhibited an extraordinary ability to suppress the production of pro-inflammatory cytokines by DC, and may be used as an effective and safer alternative to live bacteria.

  19. Role of secreted glyceraldehyde-3-phosphate dehydrogenase in the infection mechanism of enterohemorrhagic and enteropathogenic Escherichia coli: interaction of the extracellular enzyme with human plasminogen and fibrinogen.

    Science.gov (United States)

    Egea, L; Aguilera, L; Giménez, R; Sorolla, M A; Aguilar, J; Badía, J; Baldoma, L

    2007-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12) is an anchorless, multifunctional protein displayed on the surface of several fungi and Gram-positive pathogens, which contributes to their adhesion and virulence. To date a role for extracellular GAPDH in the pathogenesis of Gram-negative bacteria has not been described. The aim of this study was to analyze the extracellular localization of GAPDH in enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli strains and to examine its interaction with host components that could be related to the infection mechanism. Recombinant E. coli GAPDH was purified and polyclonal antibodies were obtained. Western blotting and immunoelectron microscopy showed that GAPDH is located on the bacterial surface and released to the culture medium of EHEC and EPEC strains. GAPDH export in these Gram-negative pathogens depends on the external medium, is not mediated by vesicles and leads to an extracellular active enzyme. Non-pathogenic E. coli strains do not secrete GAPDH. Two-dimensional electrophoresis analysis showed that in E. coli GAPDH is present at least in two major forms with different isoelectric points. Of these forms, the more basic is secreted. Purified GAPDH was found to bind human plasminogen and fibrinogen in Far-Western blot and ELISA-based assays. In addition, GAPDH remained associated with colonic Caco-2 epithelial cells after adhesion of EHEC or EPEC. These observations indicate that exported GAPDH may act as a virulence factor which could contribute to EHEC and EPEC pathogenesis. This is the first description of an extracellular localization for this enzyme, with a function other than its glycolytic role in Gram-negative pathogens.

  20. Afa/Dr diffusely adhering Escherichia coli strain C1845 induces neutrophil extracellular traps that kill bacteria and damage human enterocyte-like cells.

    Science.gov (United States)

    Marin-Esteban, Viviana; Turbica, Isabelle; Dufour, Guillaume; Semiramoth, Nicolas; Gleizes, Aude; Gorges, Roseline; Beau, Isabelle; Servin, Alain L; Lievin-Le Moal, Vanessa; Sandré, Catherine; Chollet-Martin, Sylvie

    2012-05-01

    We recently documented the neutrophil response to enterovirulent diffusely adherent Escherichia coli expressing Afa/Dr fimbriae (Afa/Dr DAEC), using the human myeloid cell line PLB-985 differentiated into fully mature neutrophils. Upon activation, particularly during infections, neutrophils release neutrophil extracellular traps (NETs), composed of a nuclear DNA backbone associated with antimicrobial peptides, histones, and proteases, which entrap and kill pathogens. Here, using fluorescence microscopy and field emission scanning electron microscopy, we observed NET production by PLB-985 cells infected with the Afa/Dr wild-type (WT) E. coli strain C1845. We found that these NETs were able to capture, immobilize, and kill WT C1845 bacteria. We also developed a coculture model of human enterocyte-like Caco-2/TC7 cells and PLB-985 cells previously treated with WT C1845 and found, for the first time, that the F-actin cytoskeleton of enterocyte-like cells is damaged in the presence of bacterium-induced NETs and that this deleterious effect is prevented by inhibition of protease release. These findings provide new insights into the neutrophil response to bacterial infection via the production of bactericidal NETs and suggest that NETs may damage the intestinal epithelium, particularly in situations such as inflammatory bowel diseases.

  1. Comparative analysis of the susceptibility to biocides and heavy metals of extended-spectrum β-lactamase-producing Escherichia coli isolates of human and avian origin, Germany.

    Science.gov (United States)

    Deus, Daniela; Krischek, Carsten; Pfeifer, Yvonne; Sharifi, Ahmad Reza; Fiegen, Ulrike; Reich, Felix; Klein, Guenter; Kehrenberg, Corinna

    2017-02-08

    A total of 174 extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates collected from humans (n=140) and healthy broiler chickens (n = 34) was included in the study. The MIC values of alkyl diaminoethyl glycin hydrochloride, benzethonium chloride, benzalkonium chloride, chlorhexidine, acriflavine, copper sulfate, silver nitrate and zinc chloride were determined by the broth microdilution method. Significant differences in MIC distributions were found between human and avian isolates and between CTX-M-, SHV- and TEM-type ESBL E. coli for chlorhexidine, silver nitrate, zinc chloride and copper sulfate by statistical analysis. Isolates with reduced susceptibility were investigated for the presence and localization of tolerance-mediating genes by PCR analysis and Southern blotting. The genes emrE, mdfA, sugE(c), cueO, copA, zntA and zitB were commonly present in isolates with elevated MICs, while the genes qacE∆1, qacF, qacH, sugE(p), cusC and pcoA, were less prevalent. In several isolates, a plasmid localization of the genes qacE∆1, qacF, qacH and sugE(p) on large plasmids >20 kb was detected.

  2. Use of in vivo-induced antigen technology for identification of Escherichia coli O157:H7 proteins expressed during human infection.

    Science.gov (United States)

    John, Manohar; Kudva, Indira T; Griffin, Robert W; Dodson, Allen W; McManus, Bethany; Krastins, Bryan; Sarracino, David; Progulske-Fox, Ann; Hillman, Jeffrey D; Handfield, Martin; Tarr, Phillip I; Calderwood, Stephen B

    2005-05-01

    Using in vivo-induced antigen technology (IVIAT), a modified immunoscreening technique that circumvents the need for animal models, we directly identified immunogenic Escherichia coli O157:H7 (O157) proteins expressed either specifically during human infection but not during growth under standard laboratory conditions or at significantly higher levels in vivo than in vitro. IVIAT identified 223 O157 proteins expressed during human infection, several of which were unique to this study. These in vivo-induced (ivi) proteins, encoded by ivi genes, mapped to the backbone, O islands (OIs), and pO157. Lack of in vitro expression of O157-specific ivi proteins was confirmed by proteomic analysis of a mid-exponential-phase culture of E. coli O157 grown in LB broth. Because ivi proteins are expressed in response to specific cues during infection and might help pathogens adapt to and counter hostile in vivo environments, those identified in this study are potential targets for drug and vaccine development. Also, such proteins may be exploited as markers of O157 infection in stool specimens.

  3. Recombinant human B7-H4 expressed in Escherichia coli inhibits T lymphocyte proliferation and IL-2 secretion in vitro

    Institute of Scientific and Technical Information of China (English)

    Yi-xiang MAO; Xue-guang ZHANG; Yong-Jing CHEN; Van GE; Hong-bing MA; Jian-feng YU; Hong-ya WU; Yu-min HU; Qin WANG; Qin SHI

    2006-01-01

    Aim: To explore the biofunctions of human B7-H4 generated from prokaryotic system. Methods: The gene of human B7-H4 extracellular region (IgⅤ-like and IgC-like domains) was obtained by PCR from human cDNA FLJ22418 and then inserted into the prokaryotic expression vector pGEX-5X-3 expressing glutathione. r-transferase (GST) fusion protein. After being identified by restriction enzyme digestion and sequencing, the recombinant vector was transferred into host strain E coli BL21-RIL(DE3). A 47 kDa fusion protein (GST/hB7-H4) was induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified by standard methods reported in the prokaryotic system. The inhibitory effect of GST/hB7-H4 on proliferation of T cells was observed in vitro by CD3mAb activated T-cell cultur-ing system and [3H]-thymidine incorporation assay. The concentrations of interleukin-2 and iterferon-g in the supernatants of T cells were determined by ELISA. Results: We successfully constructed the method for high-level expression and purification of the hB7-H4 extracellular domain as GST fusion protein from E coli. The GST/hB7-H4 fusion protein produced in bacteria had obvious biological activity to inhibit T-lymphocyte proliferation and IL-2 secretion. Conclusion: The prokaryote expression system could be used to generate hB7-H4 protein with natural spatial conformations and biological functions, which provided an efficient and economical way for the preparation of this target protein.

  4. Intramammary challenge with Escherichia coli following immunization with a curli-producing Escherichia coli.

    Science.gov (United States)

    Todhunter, D A; Smith, K L; Hogan, J S; Nelson, L

    1991-03-01

    Holstein and Jersey cattle were immunized with a curli-producing strain of Escherichia coli (pCRL65/A012) or a noncurli-producing strain (pUC18/HB101) to determine differences in resistance to establishment of experimental intramammary infection. Cows (n = 6 per group) were immunized at 14 d prior to drying off, 7 d of involution, and at calving with 3 x 10(10) E. coli in Freund's Incomplete Adjuvant. At 30 d of lactation, one mammary quarter of each cow was infused with a wild strain of E. coli (727). Escherichia coli 727 was isolated from a naturally occurring intramammary infection and produced curli. All challenged quarters became infected, and all cows developed acute clinical mastitis. Geometric mean duration of intramammary infections was 6 d for both immunization groups. All infections were spontaneously eliminated within 10 d. No differences occurred between immunization groups in blood selenium and glutathione peroxidase activity, plasma selenium, number of E. coli 727 isolated from secretion after challenge, rectal temperature and SCC response, clinical status of mammary quarters, or DMI. Reduction in milk production after challenge was greater for cows immunized with E. coli pCRL65/A012. Immunization of dairy cattle with a curli-producing strain of E. coli did not protect against experimental intramammary challenge during lactation.

  5. Ethanol production by Escherichia coli KO11; Producao de etanol por Escherichia coli KO11

    Energy Technology Data Exchange (ETDEWEB)

    Lima, Katia Gianni de Carvalho [Sao Paulo Univ., SP (Brazil). Faculdade de Ciencias Farmaceuticas. Lab. de Microbiologia de Alimentos]. E-mail: gianni@usp.br; Takahashi, Caroline Maki; Alterthum, Flavio [Sao Paulo Univ., SP (Brazil). Inst. de Ciencias Biomedicas. Dept. de Microbiologia

    2002-08-01

    This paper discusses the potential use of Escherichia coli KO11 in production of ethanol, based on observation that this organism can efficiently metabolize sugar complex moistures obtained from the acid hydrolysis of lignocellulose materials such as sugar-cane bagasse, corncob, corn husk, Pinus sp and oak wood.

  6. Escherichia coli fliAZY operon.

    OpenAIRE

    Mytelka, D S; Chamberlin, M J

    1996-01-01

    We have cloned the Escherichia coli fliAZY operon, which contains the fliA gene (the alternative sigma factor sigma F) and two novel genes, fliZ and fliY. Transcriptional mapping of this operon shows two start sites, one of which is preceded by a canonical E sigma F-dependent consensus and is dependent on sigma F for expression in vivo and in vitro. We have overexpressed and purified sigma F and demonstrated that it can direct core polymerase to E sigma F-dependent promoters. FliZ and FliY ar...

  7. Global gene expression in Escherichia coli biofilms

    DEFF Research Database (Denmark)

    Schembri, Mark; Kjærgaard, K.; Klemm, Per

    2003-01-01

    in expression have no current defined function. These genes, as well as those induced by stresses relevant to biofilm growth such as oxygen and nutrient limitation, may be important factors that trigger enhanced resistance mechanisms of sessile communities to antibiotics and hydrodynamic shear forces.......It is now apparent that microorganisms undergo significant changes during the transition from planktonic to biofilm growth. These changes result in phenotypic adaptations that allow the formation of highly organized and structured sessile communities, which possess enhanced resistance...... to antimicrobial treatments and host immune defence responses. Escherichia coli has been used as a model organism to study the mechanisms of growth within adhered communities. In this study, we use DNA microarray technology to examine the global gene expression profile of E. coli during sessile growth compared...

  8. Production of recombinant avidin in Escherichia coli.

    Science.gov (United States)

    Airenne, K J; Sarkkinen, P; Punnonen, E L; Kulomaa, M S

    1994-06-24

    A recombinant avidin (re-Avd), containing amino acids (aa) 1-123 of the native chicken egg-white Avd, was produced in Escherichia coli. When cells were grown at 37 degrees C production was over 1 microgram/ml, due to altering the codon preference of the first ten codons. The re-Avd was recovered as a soluble protein from cells grown at 25 or 30 degrees C, whereas at 37 degrees C it was mostly insoluble in inclusion bodies. Our results indicated that, despite the potentially harmful biotin-binding activity of Avd, it is possible to produce biologically active Avd in E. coli which then can easily be purified by affinity chromatography on a biotin column in a single step.

  9. Escherichia coli growth under modeled reduced gravity

    Science.gov (United States)

    Baker, Paul W.; Meyer, Michelle L.; Leff, Laura G.

    2004-01-01

    Bacteria exhibit varying responses to modeled reduced gravity that can be simulated by clino-rotation. When Escherichia coli was subjected to different rotation speeds during clino-rotation, significant differences between modeled reduced gravity and normal gravity controls were observed only at higher speeds (30-50 rpm). There was no apparent affect of removing samples on the results obtained. When E. coli was grown in minimal medium (at 40 rpm), cell size was not affected by modeled reduced gravity and there were few differences in cell numbers. However, in higher nutrient conditions (i.e., dilute nutrient broth), total cell numbers were higher and cells were smaller under reduced gravity compared to normal gravity controls. Overall, the responses to modeled reduced gravity varied with nutrient conditions; larger surface to volume ratios may help compensate for the zone of nutrient depletion around the cells under modeled reduced gravity.

  10. Intramacrophage survival of uropathogenic Escherichia coli: Differences between diverse clinical isolates and between mouse and human macrophages

    KAUST Repository

    Bokil, Nilesh J.

    2011-11-01

    Uropathogenic E. coli (UPEC) are the primary cause of urinary tract infections. Recent studies have demonstrated that UPEC can invade and replicate within epithelial cells, suggesting that this bacterial pathogen may occupy an intracellular niche within the host. Given that many intracellular pathogens target macrophages, we assessed the interactions between UPEC and macrophages. Colonization of the mouse bladder by UPEC strain CFT073 resulted in increased expression of myeloid-restricted genes, consistent with the recruitment of inflammatory macrophages to the site of infection. In in vitro assays, CFT073 was able to survive within primary mouse bone marrow-derived macrophages (BMM) up to 24h post-infection. Three additional well-characterized clinical UPEC isolates associated with distinct UTI symptomatologies displayed variable long-term survival within BMM. UPEC strains UTI89 and VR50, originally isolated from patients with cystitis and asymptomatic bacteriuria respectively, showed elevated bacterial loads in BMM at 24h post-infection as compared to CFT073 and the asymptomatic bacteriuria strain 83972. These differences did not correlate with differential effects on macrophage survival or initial uptake of bacteria. E. coli UTI89 localized to a Lamp1 + vesicular compartment within BMM. In contrast to survival within mouse BMM, intracellular bacterial loads of VR50 were low in both human monocyte-derived macrophages (HMDM) and in human T24 bladder epithelial cells. Collectively, these data suggest that some UPEC isolates may subvert macrophage anti-microbial pathways, and that host species differences may impact on intracellular UPEC survival. © 2011 Elsevier GmbH.

  11. Intramacrophage survival of uropathogenic Escherichia coli: differences between diverse clinical isolates and between mouse and human macrophages.

    Science.gov (United States)

    Bokil, Nilesh J; Totsika, Makrina; Carey, Alison J; Stacey, Katryn J; Hancock, Viktoria; Saunders, Bernadette M; Ravasi, Timothy; Ulett, Glen C; Schembri, Mark A; Sweet, Matthew J

    2011-11-01

    Uropathogenic E. coli (UPEC) are the primary cause of urinary tract infections. Recent studies have demonstrated that UPEC can invade and replicate within epithelial cells, suggesting that this bacterial pathogen may occupy an intracellular niche within the host. Given that many intracellular pathogens target macrophages, we assessed the interactions between UPEC and macrophages. Colonization of the mouse bladder by UPEC strain CFT073 resulted in increased expression of myeloid-restricted genes, consistent with the recruitment of inflammatory macrophages to the site of infection. In in vitro assays, CFT073 was able to survive within primary mouse bone marrow-derived macrophages (BMM) up to 24h post-infection. Three additional well-characterized clinical UPEC isolates associated with distinct UTI symptomatologies displayed variable long-term survival within BMM. UPEC strains UTI89 and VR50, originally isolated from patients with cystitis and asymptomatic bacteriuria respectively, showed elevated bacterial loads in BMM at 24h post-infection as compared to CFT073 and the asymptomatic bacteriuria strain 83972. These differences did not correlate with differential effects on macrophage survival or initial uptake of bacteria. E. coli UTI89 localized to a Lamp1(+) vesicular compartment within BMM. In contrast to survival within mouse BMM, intracellular bacterial loads of VR50 were low in both human monocyte-derived macrophages (HMDM) and in human T24 bladder epithelial cells. Collectively, these data suggest that some UPEC isolates may subvert macrophage anti-microbial pathways, and that host species differences may impact on intracellular UPEC survival.

  12. Engineering Escherichia coli for methanol conversion.

    Science.gov (United States)

    Müller, Jonas E N; Meyer, Fabian; Litsanov, Boris; Kiefer, Patrick; Potthoff, Eva; Heux, Stéphanie; Quax, Wim J; Wendisch, Volker F; Brautaset, Trygve; Portais, Jean-Charles; Vorholt, Julia A

    2015-03-01

    Methylotrophic bacteria utilize methanol and other reduced one-carbon compounds as their sole source of carbon and energy. For this purpose, these bacteria evolved a number of specialized enzymes and pathways. Here, we used a synthetic biology approach to select and introduce a set of "methylotrophy genes" into Escherichia coli based on in silico considerations and flux balance analysis to enable methanol dissimilation and assimilation. We determined that the most promising approach allowing the utilization of methanol was the implementation of NAD-dependent methanol dehydrogenase and the establishment of the ribulose monophosphate cycle by expressing the genes for hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloisomerase (Phi). To test for the best-performing enzymes in the heterologous host, a number of enzyme candidates from different donor organisms were selected and systematically analyzed for their in vitro and in vivo activities in E. coli. Among these, Mdh2, Hps and Phi originating from Bacillus methanolicus were found to be the most effective. Labeling experiments using (13)C methanol with E. coli producing these enzymes showed up to 40% incorporation of methanol into central metabolites. The presence of the endogenous glutathione-dependent formaldehyde oxidation pathway of E. coli did not adversely affect the methanol conversion rate. Taken together, the results of this study represent a major advancement towards establishing synthetic methylotrophs by gene transfer.

  13. Escherichia coli as a bioreporter in ecotoxicology.

    Science.gov (United States)

    Robbens, Johan; Dardenne, Freddy; Devriese, Lisa; De Coen, Wim; Blust, Ronny

    2010-11-01

    Ecotoxicological assessment relies to a large extent on the information gathered with surrogate species and the extrapolation of test results across species and different levels of biological organisation. Bacteria have long been used as a bioreporter for genotoxic testing and general toxicity. Today, it is clear that bacteria have the potential for screening of other toxicological endpoints. Escherichia coli has been studied for years; in-depth knowledge of its biochemistry and genetics makes it the most proficient prokaryote for the development of new toxicological assays. Several assays have been designed with E. coli as a bioreporter, and the recent trend to develop novel, better advanced reporters makes bioreporter development one of the most dynamic in ecotoxicology. Based on in-depth knowledge of E. coli, new assays are being developed or existing ones redesigned, thanks to the availability of new reporter genes and new or improved substrates. The technological evolution towards easier and more sensitive detection of different gene products is another important aspect. Often, this requires the redesign of the bacterium to make it compatible with the novel measuring tests. Recent advances in surface chemistry and nanoelectronics open the perspective for advanced reporter based on novel measuring platforms and with an online potential. In this article, we will discuss the use of E. coli-based bioreporters in ecotoxicological applications as well as some innovative sensors awaited for the future.

  14. Transcriptional analysis of the Escherichia coli ColV-Ia plasmid pS88 during growth in human serum and urine

    Directory of Open Access Journals (Sweden)

    Lemaître Chloé

    2012-06-01

    Full Text Available Abstract Background The sequenced O45:K1:H7 Escherichia coli meningitis strain S88 harbors a large virulence plasmid. To identify possible genetic determinants of pS88 virulence, we examined the transcriptomes of 88 plasmidic ORFs corresponding to known and putative virulence genes, and 35 ORFs of unknown function. Results Quantification of plasmidic transcripts was obtained by quantitative real-time reverse transcription of extracted RNA, normalized on three housekeeping genes. The transcriptome of E. coli strain S88 grown in human serum and urine ex vivo were compared to that obtained during growth in Luria Bertani broth, with and without iron depletion. We also analyzed the transcriptome of a pS88-like plasmid recovered from a neonate with urinary tract infection. The transcriptome obtained after ex vivo growth in serum and urine was very similar to those obtained in iron-depleted LB broth. Genes encoding iron acquisition systems were strongly upregulated. ShiF and ORF 123, two ORFs encoding protein with hypothetical function and physically linked to aerobactin and salmochelin loci, respectively, were also highly expressed in iron-depleted conditions and may correspond to ancillary iron acquisition genes. Four ORFs were induced ex vivo, independently of the iron concentration. Other putative virulence genes such as iss, etsC, ompTp and hlyF were not upregulated in any of the conditions studied. Transcriptome analysis of the pS88-like plasmid recovered in vivo showed a similar pattern of induction but at much higher levels. Conclusion We identify new pS88 genes potentially involved in the growth of E. coli meningitis strain S88 in human serum and urine.

  15. Disinfectant and Antimicrobial Susceptibility Profiles of the Big Six Non-O157 Shiga Toxin-Producing Escherichia coli Strains from Food Animals and Humans.

    Science.gov (United States)

    Beier, Ross C; Franz, Eelco; Bono, James L; Mandrell, Robert E; Fratamico, Pina M; Callaway, Todd R; Andrews, Kathleen; Poole, Toni L; Crippen, Tawni L; Sheffield, Cynthia L; Anderson, Robin C; Nisbet, David J

    2016-08-01

    The disinfectant and antimicrobial susceptibility profiles of 138 non-O157 Shiga toxin-producing Escherichia coli strains (STECs) from food animals and humans were determined. Antimicrobial resistance (AMR) was moderate (39.1% of strains) in response to 15 antimicrobial agents. Animal strains had a lower AMR prevalence (35.6%) than did human strains (43.9%) but a higher prevalence of the resistance profile GEN-KAN-TET. A decreasing prevalence of AMR was found among animal strains from serogroups O45 > O145 > O121 > O111 > O26 > O103 and among human strains from serogroups O145 > O103 > O26 > O111 > O121 > O45. One animal strain from serogroups O121 and O145 and one human strain from serogroup O26 had extensive drug resistance. A high prevalence of AMR in animal O45 and O121 strains and no resistance or a low prevalence of resistance in human strains from these serogroups suggests a source other than food animals for human exposure to these strains. Among the 24 disinfectants evaluated, all strains were susceptible to triclosan. Animal strains had a higher prevalence of resistance to chlorhexidine than did human strains. Both animal and human strains had a similar low prevalence of low-level benzalkonium chloride resistance, and animal and human strains had similar susceptibility profiles for most other disinfectants. Benzyldimethylammonium chlorides and C10AC were the primary active components in disinfectants DC&R and P-128, respectively, against non-O157 STECs. A disinfectant FS512 MIC ≥ 8 μg/ml was more prevalent among animal O121 strains (61.5%) than among human O121 strains (25%), which may also suggest a source of human exposure to STEC O121 other than food animals. Bacterial inhibition was not dependent solely on pH but was correlated with the presence of dissociated organic acid species and some undissociated acids.

  16. High-level expression and efficient one-step chromatographic purification of a soluble human leukemia inhibitory factor (LIF) in Escherichia coli.

    Science.gov (United States)

    Imaizumi, Keitaro; Nishikawa, Shin-Ichi; Tarui, Hiroshi; Akuta, Teruo

    2013-07-01

    Leukemia inhibitor factor (LIF) is a three disulfide bridge-containing cytokine with numerous regulatory effects. In this report, we present the high level expression of a soluble recombinant human LIF (rhLIF) in Escherichia coli. A codon-optimized Profinity eXact™-tagged hLIF cDNA was cloned into pET3b vector, and transformed into E. coli OrigamiB(DE3) harboring the bacterial thioredoxin coexpression vector. By using an enzyme-based glucose release system (EnBase®) and high-aeration shake flask (Ultra Yield Flask™), the yield of soluble proteins was significantly improved in comparison to commonly-used 2 × YT media. The recombinant protein was purified via a single chromatographic step using an affinity tag-based protein purification system that processed by cleavage with sodium fluoride, resulting in the complete proteolytic removal the N-terminal tag. Soluble rhLIF yield was estimated to be approximately 1mg/g of wet weight cells, with above 98% purity. The rhLIF protein specifically inhibited the proliferation of the murine myeloblastic leukemia M1 cell in a dose-dependent manner, and induced Stat3 phosphorylation in mouse ES cells. This novel expression and purification protocol for the production of recombinant hLIF is a simple, suitable, and effective method.

  17. High level expression, purification and characterization of active fusion human C1q and tumor necrosis factor related protein 2 (hCTRP2) in Escherichia coli.

    Science.gov (United States)

    Li, Hongbo; Gao, Xuefei; Zhou, Yi; Li, Na; Ge, Caozuo; Hui, Xiaoyan; Wang, Yu; Xu, Aimin; Jin, Shouguang; Wu, Donghai

    2011-09-01

    C1q and tumor necrosis factor related proteins (CTRPs) are a family of adiponectin paralogues. Among them, CTRP2 is the only CTRP protein that has been shown to possess similar biological activities as adiponectin. To further explore the physiological roles of human CTRP2 and its mechanisms of action, hCTRP2 gene was expressed in Escherichia coli and Pichia pastoris, respectively. In the P. pastoris expression system, recombinant hCTRP2 could be secreted into the culture medium under induction condition, however, the resultant recombinant protein was highly unstable, resulting two main degradation products with molecular masses of approximately 20 and 26 kDa, respectively. In the E. coli expression system, a large amount of soluble thioredoxin (Trx)-hCTRP2 fusion protein could be produced, which accounts about 42% of the total soluble bacterial proteins. The recombinant Trx-hCTRP2 fusion protein was purified to an approximately 95% purity using Ni-NTA affinity chromatography and Superdex G-75 column with a yield of about 15 mg/l protein from 1l bacterial culture. The purified recombinant Trx-hCTRP2 was shown to be active under in vitro assay conditions.

  18. Distinct Renal Pathology and a Chemotactic Phenotype after Enterohemorrhagic Escherichia coli Shiga Toxins in Non-Human Primate Models of Hemolytic Uremic Syndrome

    Science.gov (United States)

    Stearns-Kurosawa, Deborah J.; Oh, Sun-Young; Cherla, Rama P.; Lee, Moo-Seung; Tesh, Vernon L.; Papin, James; Henderson, Joel; Kurosawa, Shinichiro

    2014-01-01

    Enterohemorrhagic Escherichia coli cause approximately 1.5 million infections globally with 176,000 cases occurring in the United States annually from ingesting contaminated food, most frequently E. coli O157:H7 in ground beef or fresh produce. In severe cases, the painful prodromal hemorrhagic colitis is complicated by potentially lethal hemolytic uremic syndrome (HUS), particularly in children. Bacterial Shiga-like toxins (Stx1, Stx2) are primarily responsible for HUS and the kidney and neurologic damage that ensue. Small animal models are hampered by the inability to reproduce HUS with thrombotic microangiopathy, hemolytic anemia, and acute kidney injury. Earlier, we showed that nonhuman primates (Papio) recapitulated clinical HUS after Stx challenge and that novel therapeutic intervention rescued the animals. Here, we present detailed light and electron microscopic pathology examination of the kidneys from these Stx studies. Stx1 challenge resulted in more severe glomerular endothelial injury, whereas the glomerular injury after Stx2 also included prominent mesangiolysis and an eosinophilic inflammatory infiltration. Both toxins induced glomerular platelet-rich thrombi, interstitial hemorrhage, and tubular injury. Analysis of kidney and other organs for inflammation biomarkers showed a striking chemotactic profile, with extremely high mRNA levels for IL-8, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1α and elevated urine chemokines at 48 hours after challenge. These observations give unique insight into the pathologic consequences of each toxin in a near human setting and present potential pathways for therapeutic intervention. PMID:23402998

  19. Chromatin architecture and gene expression in Escherichia coli

    DEFF Research Database (Denmark)

    Willenbrock, Hanni; Ussery, David

    2004-01-01

    Two recent genome-scale analyses underscore the importance of DNA topology and chromatin structure in regulating transcription in Escherichia coli.......Two recent genome-scale analyses underscore the importance of DNA topology and chromatin structure in regulating transcription in Escherichia coli....

  20. Escherichia coli O157 infections and unpasteurised milk

    NARCIS (Netherlands)

    Allerberger, F; Wagner, M; Schweiger, P; Rammer, H P; Resch, A; Dierich, M P; Friedrich, A W; Karch, H

    2001-01-01

    We report on two children with Escherichia coli O157 infection, one of whom developed haemolytic uraemic syndrome (HUS). Both had drunk raw cows or goats milk in the week before their illness. Molecular subtyping identified a sorbitol fermenting Escherichia coli O157:H isolate from a dairy cow. This

  1. Escherichia coli O157 infections and unpasteurised milk

    NARCIS (Netherlands)

    Allerberger, F; Wagner, M; Schweiger, P; Rammer, H P; Resch, A; Dierich, M P; Friedrich, A W; Karch, H

    2001-01-01

    We report on two children with Escherichia coli O157 infection, one of whom developed haemolytic uraemic syndrome (HUS). Both had drunk raw cows or goats milk in the week before their illness. Molecular subtyping identified a sorbitol fermenting Escherichia coli O157:H isolate from a dairy cow. This

  2. Subgrouping of ESBL-producing Escherichia coli from animal and human sources: an approach to quantify the distribution of ESBL types between different reservoirs.

    Science.gov (United States)

    Valentin, Lars; Sharp, Hannah; Hille, Katja; Seibt, Uwe; Fischer, Jennie; Pfeifer, Yvonne; Michael, Geovana Brenner; Nickel, Silke; Schmiedel, Judith; Falgenhauer, Linda; Friese, Anika; Bauerfeind, Rolf; Roesler, Uwe; Imirzalioglu, Can; Chakraborty, Trinad; Helmuth, Reiner; Valenza, Giuseppe; Werner, Guido; Schwarz, Stefan; Guerra, Beatriz; Appel, Bernd; Kreienbrock, Lothar; Käsbohrer, Annemarie

    2014-10-01

    Escherichia (E.) coli producing extended-spectrum beta-lactamases (ESBLs) are an increasing problem for public health. The success of ESBLs may be due to spread of ESBL-producing bacterial clones, transfer of ESBL gene-carrying plasmids or exchange of ESBL encoding genes on mobile elements. This makes it difficult to identify transmission routes and sources for ESBL-producing bacteria. The objectives of this study were to compare the distribution of genotypic and phenotypic properties of E. coli isolates from different animal and human sources collected in studies in the scope of the national research project RESET. ESBL-producing E. coli from two longitudinal and four cross-sectional studies in broiler, swine and cattle farms, a cross-sectional and a case-control study in humans and diagnostic isolates from humans and animals were used. In the RESET consortium, all laboratories followed harmonized methodologies for antimicrobial susceptibility testing, confirmation of the ESBL phenotype, specific PCR assays for the detection of bla(TEM), bla(CTX), and bla(SHV) genes and sequence analysis of the complete ESBL gene as well as a multiplex PCR for the detection of the four major phylogenetic groups of E. coli. Most ESBL genes were found in both, human and non-human populations but quantitative differences for distinct ESBL-types were detectable. The enzymes CTX-M-1 (63.3% of all animal isolates, 29.3% of all human isolates), CTX-M-15 (17.7% vs. 48.0%) and CTX-M-14 (5.3% vs. 8.7%) were the most common ones. More than 70% of the animal isolates and more than 50% of the human isolates contained the broadly distributed ESBL genes bla(CTX-M-1), bla(CTX-M-15), or the combinations bla(SHV-12)+bla(TEM) or bla(CTX-M-1)+bla(TEM). While the majority of animal isolates carried bla(CTX-M-1) (37.5%) or the combination bla(CTX-M-1)+bla(TEM) (25.8%), this was the case for only 16.7% and 12.6%, respectively, of the human isolates. In contrast, 28.2% of the human isolates carried bla

  3. Streamlined analysis for evaluating the use of preharvest interventions intended to prevent Escherichia coli O157:H7 illness in humans.

    Science.gov (United States)

    Withee, James; Williams, Michael; Disney, Terry; Schlosser, Wayne; Bauer, Nate; Ebel, Eric

    2009-09-01

    The U.S. Department of Agriculture Food Safety Inspection Service is responsible for ensuring the safety of meat, poultry, and egg products consumed in the United States. Here we describe a risk assessment method that provides quantitative criteria for decision makers tasked with developing food safety policies. To demonstrate the utility of this method, we apply it to a hypothetical case study on the use of an Escherichia coli O157:H7 cattle vaccine to prevent human illness caused by consuming beef. A combination of quantitative risk assessment methods and marginal economic analysis are used to describe the maximum cost per unit that would still allow the vaccine to be a cost-effective intervention as well as the minimum effectiveness it could have at a fixed cost. We create two economic production functions where the input is number of vaccinated cattle and the output is human illnesses prevented. The production functions are then used for marginal economic analysis to assess the cost/benefit ratio of using the vaccine to prevent foodborne illness. In our case study, it was determined that vaccinating the entire U.S. herd at a cost of between $2.29 and $9.14 per unit (depending on overall effectiveness of the vaccine) would be a cost-effective intervention for preventing E. coli O157:H7 illness in humans. In addition, we determined that vaccinating only a given fraction of the herd would be cost effective for vaccines that are less effective or more costly. For example, a vaccine costing $9.00 per unit that had a 100% efficacy but required 100% herd coverage for immunity would be cost effective for use in about 500,000 cattle each year-equating to an estimated 750 human illnesses prevented per annum. We believe this approach could be useful for public health policy development in a wide range of applications.

  4. Genetic characterization of atypical enteropathogenic Escherichia coli isolates from ewes' milk, sheep farm environments, and humans by multilocus sequence typing and pulsed-field gel electrophoresis.

    Science.gov (United States)

    Otero, Verónica; Rodríguez-Calleja, José-María; Otero, Andrés; García-López, María-Luisa; Santos, Jesús A

    2013-10-01

    A collection of 81 isolates of enteropathogenic Escherichia coli (EPEC) was obtained from samples of bulk tank sheep milk (62 isolates), ovine feces (4 isolates), sheep farm environment (water, 4 isolates; air, 1 isolate), and human stool samples (9 isolates). The strains were considered atypical EPEC organisms, carrying the eae gene without harboring the pEAF plasmid. Multilocus sequence typing (MLST) was carried out with seven housekeeping genes and 19 sequence types (ST) were detected, with none of them having been previously reported for atypical EPEC. The most frequent ST included 41 strains isolated from milk and human stool samples. Genetic typing by pulsed-field gel electrophoresis (PFGE) resulted in 57 patterns which grouped in 24 clusters. Comparison of strains isolated from the different samples showed phylogenetic relationships between milk and human isolates and also between milk and water isolates. The results obtained show a possible risk for humans due to the presence of atypical EPEC in ewes' milk and suggest a transmission route for this emerging pathogen through contaminated water.

  5. Production of glycoprotein vaccines in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Ihssen Julian

    2010-08-01

    Full Text Available Abstract Background Conjugate vaccines in which polysaccharide antigens are covalently linked to carrier proteins belong to the most effective and safest vaccines against bacterial pathogens. State-of-the art production of conjugate vaccines using chemical methods is a laborious, multi-step process. In vivo enzymatic coupling using the general glycosylation pathway of Campylobacter jejuni in recombinant Escherichia coli has been suggested as a simpler method for producing conjugate vaccines. In this study we describe the in vivo biosynthesis of two novel conjugate vaccine candidates against Shigella dysenteriae type 1, an important bacterial pathogen causing severe gastro-intestinal disease states mainly in developing countries. Results Two different periplasmic carrier proteins, AcrA from C. jejuni and a toxoid form of Pseudomonas aeruginosa exotoxin were glycosylated with Shigella O antigens in E. coli. Starting from shake flask cultivation in standard complex medium a lab-scale fed-batch process was developed for glycoconjugate production. It was found that efficiency of glycosylation but not carrier protein expression was highly susceptible to the physiological state at induction. After induction glycoconjugates generally appeared later than unglycosylated carrier protein, suggesting that glycosylation was the rate-limiting step for synthesis of conjugate vaccines in E. coli. Glycoconjugate synthesis, in particular expression of oligosaccharyltransferase PglB, strongly inhibited growth of E. coli cells after induction, making it necessary to separate biomass growth and recombinant protein expression phases. With a simple pulse and linear feed strategy and the use of semi-defined glycerol medium, volumetric glycoconjugate yield was increased 30 to 50-fold. Conclusions The presented data demonstrate that glycosylated proteins can be produced in recombinant E. coli at a larger scale. The described methodologies constitute an important step

  6. Influence of human urine to antimicrobial susceptibility of clinical isolates of Klebsiella pneumoniae and Escherichia coli producing β-lactamase of different types

    Directory of Open Access Journals (Sweden)

    Ž. Žagar

    2007-02-01

    Full Text Available The purpose of the study was to determine the influence of human urine on the antibiotic susceptibilities of Klebsiella pneumoniae and Escherichia coli strains producing different types of extended-spectrum β-lactamases (ESBL. The study was performed on 26 ESBL negative strains of K. pneumoniae, 80 K. pneumoniae strains producing SHV-ESBLs (52-SHV-5, 31- SHV-2 and 7- SHV-12, 94 E. coli strains harbouring TEM- ESBLs and 14 E. coli strains possessing CTX-M group 1 β-lactamases. The minimum inhibitory concentrations of amoxycillin alone and combined with clavulanate (co-amoxilcav, cephalexin, cefuroxime, ceftazidime, cefotaxime, ceftriaxone, cefepime, gentamicin and ciprofloxacin were performed in parallel in Mueller-Hinton broth and urine by broth microdilution method. With ESBL negative strains, urine increased MIC90 of amoxycillin alone and combined with clavulanate, cephalexin, cefuroxime, ceftazidime, cefotaxime, ceftriaxone, cefepime, gentamicin and ciprofloxacin. Against SHV-5 producers, an increase in MIC90 was observed with cefotaxime, cefepime and ciprofloxacin when the test was performed in urine. SHV-2 producers showed elevated MIC90 of ceftazidime, cefotaxime, ceftriaxone and cefepime in the presence of urine, in contrast to SHV-12 producers which displayed elevated MIC90 only for cefotaxime. Urine increased MIC90 of amoxycillin/clavulanate, ceftazidime and cefepime against CTX-M producers, and of amoxycillin/clavulanate, cefotaxime, ceftriaxone, cefepime and ciprofloxacin for TEM producers. According to our results the activity of antibiotics used for the treatment of urinary tract infection could be overestimated by a standard in vitro testing. However, most of antibiotics used for the treatment of urinary tract infection achieve very high concentration in urine and that could abrogate the reduction of antimicrobial activity by biological fluid.

  7. N-acylated peptides derived from human lactoferricin perturb organization of cardiolipin and phosphatidylethanolamine in cell membranes and induce defects in Escherichia coli cell division.

    Directory of Open Access Journals (Sweden)

    Dagmar Zweytick

    Full Text Available Two types of recently described antibacterial peptides derived from human lactoferricin, either nonacylated or N-acylated, were studied for their different interaction with membranes of Escherichia coli in vivo and in model systems. Electron microscopy revealed striking effects on the bacterial membrane as both peptide types induced formation of large membrane blebs. Electron and fluorescence microscopy, however demonstrated that only the N-acylated peptides partially induced the generation of oversized cells, which might reflect defects in cell-division. Further a different distribution of cardiolipin domains on the E. coli membrane was shown only in the presence of the N-acylated peptides. The lipid was distributed over the whole bacterial cell surface, whereas cardiolipin in untreated and nonacylated peptide-treated cells was mainly located at the septum and poles. Studies with bacterial membrane mimics, such as cardiolipin or phosphatidylethanolamine revealed that both types of peptides interacted with the negatively charged lipid cardiolipin. The nonacylated peptides however induced segregation of cardiolipin into peptide-enriched and peptide-poor lipid domains, while the N-acylated peptides promoted formation of many small heterogeneous domains. Only N-acylated peptides caused additional severe effects on the main phase transition of liposomes composed of pure phosphatidylethanolamine, while both peptide types inhibited the lamellar to hexagonal phase transition. Lipid mixtures of phosphatidylethanolamine and cardiolipin revealed anionic clustering by all peptide types. However additional strong perturbation of the neutral lipids was only seen with the N-acylated peptides. Nuclear magnetic resonance demonstrated different conformational arrangement of the N-acylated peptide in anionic and zwitterionic micelles revealing possible mechanistic differences in their action on different membrane lipids. We hypothesized that both peptides kill

  8. N-acylated peptides derived from human lactoferricin perturb organization of cardiolipin and phosphatidylethanolamine in cell membranes and induce defects in Escherichia coli cell division.

    Science.gov (United States)

    Zweytick, Dagmar; Japelj, Bostjan; Mileykovskaya, Eugenia; Zorko, Mateja; Dowhan, William; Blondelle, Sylvie E; Riedl, Sabrina; Jerala, Roman; Lohner, Karl

    2014-01-01

    Two types of recently described antibacterial peptides derived from human lactoferricin, either nonacylated or N-acylated, were studied for their different interaction with membranes of Escherichia coli in vivo and in model systems. Electron microscopy revealed striking effects on the bacterial membrane as both peptide types induced formation of large membrane blebs. Electron and fluorescence microscopy, however demonstrated that only the N-acylated peptides partially induced the generation of oversized cells, which might reflect defects in cell-division. Further a different distribution of cardiolipin domains on the E. coli membrane was shown only in the presence of the N-acylated peptides. The lipid was distributed over the whole bacterial cell surface, whereas cardiolipin in untreated and nonacylated peptide-treated cells was mainly located at the septum and poles. Studies with bacterial membrane mimics, such as cardiolipin or phosphatidylethanolamine revealed that both types of peptides interacted with the negatively charged lipid cardiolipin. The nonacylated peptides however induced segregation of cardiolipin into peptide-enriched and peptide-poor lipid domains, while the N-acylated peptides promoted formation of many small heterogeneous domains. Only N-acylated peptides caused additional severe effects on the main phase transition of liposomes composed of pure phosphatidylethanolamine, while both peptide types inhibited the lamellar to hexagonal phase transition. Lipid mixtures of phosphatidylethanolamine and cardiolipin revealed anionic clustering by all peptide types. However additional strong perturbation of the neutral lipids was only seen with the N-acylated peptides. Nuclear magnetic resonance demonstrated different conformational arrangement of the N-acylated peptide in anionic and zwitterionic micelles revealing possible mechanistic differences in their action on different membrane lipids. We hypothesized that both peptides kill bacteria by

  9. Prevalence of diarrheagenic Escherichia coli in suckling rabbits

    OpenAIRE

    2016-01-01

    Diarrheagenic Escherichia coli (E. coli) in suckling rabbit causes collibacillosis, which is characterized by sever yellow diarrhea, poor growth and high mortalities. This study was undertaken to investigate the prevalence of diarrheagenic E. coli in suckling rabbits in Egypt. Additionally, expression of some virulence-associated genes in the isolated E. coli serotypes were examined using the polymerase chain reaction. Finally, antibiogram of the identified E. coli serotypes was also investig...

  10. Methane production from kitchen waste using Escherichia coli.

    Science.gov (United States)

    Jayalakshmi, S; Joseph, Kurian; Sukumaran, V

    2007-04-01

    Escherichia coli (E. coli) strain isolated from biogas plant sludge was examined for its ability to enhance biogas from kitchen waste during solid phase anaerobic digestion. The laboratory experiments were conducted for total solid concentrations of 20% and 22%. Kitchen waste was characterized for physico-chemical parameters and laboratory experiments were conducted with and without E. coli strain. It was found that the reactor with E. coli produced 17% more biogas than the reactors that are operated without E. coli strain.

  11. Detection of sul1, sul2 and sul3 in sulphonamide resistant Escherichia coli isolates obtained from healthy humans, pork and pigs in Denmark

    DEFF Research Database (Denmark)

    Hammerum, Anette Marie; Sandvag, Dorthe; Andersen, Sigrid R.

    2006-01-01

    The occurrence of sulphonamide resistance was investigated in 998 Escherichia coli isolates, obtained from pig faeces collected at slaughter, Danish pork collected at retail outlets and from faeces from healthy persons in Denmark. In total 18% (n = 35), 20% (n = 38) and 26% (n = 161) of the E. co...

  12. Long term effects of Escherichia coli mastitis.

    Science.gov (United States)

    Blum, Shlomo E; Heller, Elimelech D; Leitner, Gabriel

    2014-07-01

    Escherichia coli is one of the most frequently diagnosed causes of bovine mastitis, and is typically associated with acute, clinical mastitis. The objective of the present study was to evaluate the long term effects of intramammary infections by E. coli on milk yield and quality, especially milk coagulation. Twenty-four Israeli Holstein cows diagnosed with clinical mastitis due to intramammary infection by E. coli were used in this study. Mean lactation number, days in milk (DIM) and daily milk yield (DMY) at the time of infection was 3.3 ± 1.3, 131.7 days ± 78.6 and 45.7 L ± 8.4, respectively. DMY, milk constituents, somatic cells count (SCC), differential leukocytes count and coagulation parameters were subsequently assessed. Two patterns of inflammation were identified: 'short inflammation', characterized by 15% decrease in DMY and >30 days to reach a new maximum DMY (n = 19). The estimated mean loss of marketable milk during the study was 200 L/cow for 'short inflammation' cases, and 1,500 L/cow for 'long inflammation' ones. Significant differences between 'short' and 'long inflammation' effects were found in almost all parameters studied. Long-term detrimental effects on milk quality were found regardless of clinical or bacteriological cure of affected glands.

  13. Cyclomodulins in urosepsis strains of Escherichia coli.

    Science.gov (United States)

    Dubois, Damien; Delmas, Julien; Cady, Anne; Robin, Frédéric; Sivignon, Adeline; Oswald, Eric; Bonnet, Richard

    2010-06-01

    Determinants of urosepsis in Escherichia coli remain incompletely defined. Cyclomodulins (CMs) are a growing functional family of toxins that hijack the eukaryotic cell cycle. Four cyclomodulin types are actually known in E. coli: cytotoxic necrotizing factors (CNFs), cycle-inhibiting factor (Cif), cytolethal distending toxins (CDTs), and the pks-encoded toxin. In the present study, the distribution of CM-encoding genes and the functionality of these toxins were investigated in 197 E. coli strains isolated from patients with community-acquired urosepsis (n = 146) and from uninfected subjects (n = 51). This distribution was analyzed in relation to the phylogenetic background, clinical origin, and antibiotic resistance of the strains. It emerged from this study that strains harboring the pks island and the cnf1 gene (i) were strongly associated with the B2 phylogroup (P, urosepsis origin (P, urosepsis groups, suggesting that the pks island is more important for the colonization process and the cnf1 gene for virulence. pks- or cnf1-harboring strains were significantly associated with susceptibility to antibiotics (amoxicillin, cotrimoxazole, and quinolones [P, <0.001 to 0.043]). Otherwise, only 6% and 1% of all strains harbored the cdtB and cif genes, respectively, with no particular distribution by phylogenetic background, antimicrobial susceptibility, or clinical origin.

  14. A polyclonal antibody based immunoassay detects seven subtypes of Shiga toxin 2 produced by Escherichia coli in human and environmental samples.

    Directory of Open Access Journals (Sweden)

    Xiaohua He

    Full Text Available BACKGROUND: Shiga toxin-producing Escherichia coli (STEC are frequent causes of severe human diseases ranging from diarrhea to hemolytic uremic syndrome. The existing strategy for detection of STEC relies on the unique sorbitol-negative fermentation property of the O157 strains, the most commonly identified serotype has been E. coli O157. It is becoming increasingly evident, however, that numerous non-O157 STEC serotypes also cause outbreaks and severe illnesses. It is necessary to have new methods that are capable of detecting all STEC strains. METHODS AND FINDINGS: Here we describe the development of a sandwich ELISA assay for detecting both O157 and non-O157 STECs by incorporating a novel polyclonal antibody (pAb against Stx2. The newly established immunoassay was capable of detecting Stx2a spiked in environmental samples with a limit of detection between 10 and 100 pg/mL in soil and between 100 and 500 pg/mL in feces. When applied to 36 bacterial strains isolated from human and environmental samples, this assay detected Stx2 in all strains that were confirmed to be stx2-positive by real-time PCR, demonstrating a 100% sensitivity and specificity. CONCLUSIONS: The sandwich ELISA developed in this study will enable any competent laboratory to identify and characterize Stx2-producing O157 and non-O157 strains in human and environmental samples, resulting in rapid diagnosis and patient care. The results of epitope mapping from this study will be useful for further development of a peptide-based antibody and vaccine.

  15. Expression of bioactive soluble human stem cell factor (SCF) from recombinant Escherichia coli by coproduction of thioredoxin and efficient purification using arginine in affinity chromatography.

    Science.gov (United States)

    Akuta, Teruo; Kikuchi-Ueda, Takane; Imaizumi, Keitaro; Oshikane, Hiroyuki; Nakaki, Toshio; Okada, Yoko; Sultana, Sara; Kobayashi, Kenichiro; Kiyokawa, Nobutaka; Ono, Yasuo

    2015-01-01

    Stem cell factor (SCF) known as the c-kit ligand is a two disulfide bridge-containing cytokine in the regulation of the development and function of hematopoietic cell lineages and other cells such as mast cells, germ cells, and melanocytes. The secreted soluble form of SCF exists as noncovalently associated homodimer and exerts its activity by signaling through the c-Kit receptor. In this report, we present the high level expression of a soluble recombinant human SCF (rhSCF) in Escherichia coli. A codon-optimized Profinity eXact™-tagged hSCF cDNA was cloned into pET3b vector, and transformed into E. coli BL21(DE3) harboring a bacterial thioredoxin coexpression vector. The recombinant protein was purified via an affinity chromatography processed by cleavage with sodium fluoride, resulting in the complete proteolytic removal the N-terminal tag. Although almost none of the soluble fusion protein bound to the resin in standard protocol using 0.1M sodium phosphate buffer (pH 7.2), the use of binding buffer containing 0.5M l-arginine for protein stabilization dramatically enhanced binding to resin and recovery of the protein beyond expectation. Also pretreatment by Triton X-114 for removing endotoxin was effective for affinity chromatography. In chromatography performance, l-arginine was more effective than Triton X-114 treatment. Following Mono Q anion exchange chromatography, the target protein was isolated in high purity. The rhSCF protein specifically enhanced the viability of human myeloid leukemia cell line TF-1 and the proliferation and maturation of human mast cell line LAD2 cell. This novel protocol for the production of rhSCF is a simple, suitable, and efficient method.

  16. A new approach on the purification of recombinant human soluble catechol-O-methyltransferase from an Escherichia coli extract using hydrophobic interaction chromatography.

    Science.gov (United States)

    Passarinha, L A; Bonifácio, M J; Soares-da-Silva, P; Queiroz, J A

    2008-01-11

    Catechol-O-methyltransferase (COMT) is a significant target in protein engineering due to its role not only in normal brain function but also to its possible involvement in some human disorders. In this work, a new approach was employed for the purification of recombinant human soluble COMT (hSCOMT) using hydrophobic interaction chromatography, as the main isolation method, from an Escherichia coli culture broth. A simplified overall process flow is proposed. Indeed, with an optimized heterologous expression system for recombinant hSCOMT production, such as E. coli, it was possible to produce and recover the active monomeric enzyme directly from the cell crude culture broth either by a freeze/thaw or ultrasonication lysis step. The recombinant enzyme present in the bacterial soluble fraction, exhibited similar affinity for epinephrine (K(m) 276 [215; 337] microM) and the methyl donor (S-adenosyl-L-methionine, SAMe) (K(m) 36 [30; 41]microM) as human SCOMT. After the precipitation step by 55% of ammonium sulphate, a HIC step on the butyl-sepharose resin was found to be highly effective in selectively eluting a range of contaminating key proteins present in the concentrate soluble extract. Consequently, the partially purified eluate from HIC could then be loaded and polished by gel filtration in order to increase the process efficiency. The final product appeared as a single band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The procedure resulted in a global 10.9-fold purification with a specific activity of 5500 nmol/h/mg of protein. The widespread applicability of the process, here described, to different COMT sources could make this protocol highly useful for all studies requiring purified and active COMT proteins.

  17. Genetically similar strains of Escherichia coli O157:H7 isolated from sheep, cattle and human patients

    Directory of Open Access Journals (Sweden)

    Söderlund Robert

    2012-10-01

    Full Text Available Abstract Background Comparatively little is known about the prevalence or the molecular characteristics of the zoonotic pathogen E. coli O157:H7 in the sheep reservoir. To investigate this and determine the host specificity of subclones of the bacterium, we have conducted a slaughterhouse prevalence study in sheep and compared the collected isolates to O157:H7 previously isolated from cattle and human patients. Results Verotoxin-producing O157:H7 was found in 11/597 (1.8% of samples from sheep in Swedish slaughterhouses, 9/492 faecal (1.8% and 2/105 ear samples (1.9%. All positive sheep were eaeA, hlyA, cdtV-B, vtx1, and partial sequencing of vtx2. The observed profiles were similar to those of cattle strains investigated previously. Conclusions The same pathogenic subtypes of VTEC O157:H7, including the highly virulent clade 8, appear to be present in both sheep and cattle in Sweden, suggesting strains can circulate freely between ruminant reservoirs.

  18. Enterohemorrhagic Escherichia coli infection has donor-dependent effect on human gut microbiota and may be antagonized by probiotic yeast during interaction with Peyer's patches.

    Science.gov (United States)

    Thévenot, J; Cordonnier, C; Rougeron, A; Le Goff, O; Nguyen, H T T; Denis, S; Alric, M; Livrelli, V; Blanquet-Diot, S

    2015-11-01

    Enterohemorrhagic Escherichia coli (EHEC) are major food-borne pathogens responsible for serious infections ranging from mild diarrhea to hemorrhagic colitis and life-threatening complications. Shiga toxins (Stxs) are the main virulence factor of EHEC. The antagonistic effect of a prophylactic treatment with the probiotic strain Saccharomyces cerevisiae against EHEC O157:H7 was investigated using complementary in vitro human colonic model and in vivo murine ileal loop assays. In vitro, the probiotic treatment had no effect on O157:H7 survival but favorably influenced gut microbiota activity through modulation of short-chain fatty acid production, increasing acetate production and decreasing that of butyrate. Both pathogen and probiotic strains had individual-dependent effects on human gut microbiota. For the first time, stx expression was followed in human colonic environment: at 9 and 12 h post EHEC infection, probiotic treatment significantly decreased stx mRNA levels. Besides, in murine ileal loops, the probiotic yeast specifically exerted a trophic effect on intestinal mucosa and inhibited O157:H7 interactions with Peyer's patches and subsequent hemorrhagic lesions. Taken together, the results suggest that S. cerevisiae may be useful in the fight against EHEC infection and that host associated factors such as microbiota could influence clinical evolution of EHEC infection and the effectiveness of probiotics.

  19. The pangenome structure of Escherichia coli: comparative genomic analysis of E. coli commensal and pathogenic isolates.

    Science.gov (United States)

    Rasko, David A; Rosovitz, M J; Myers, Garry S A; Mongodin, Emmanuel F; Fricke, W Florian; Gajer, Pawel; Crabtree, Jonathan; Sebaihia, Mohammed; Thomson, Nicholas R; Chaudhuri, Roy; Henderson, Ian R; Sperandio, Vanessa; Ravel, Jacques

    2008-10-01

    Whole-genome sequencing has been skewed toward bacterial pathogens as a consequence of the prioritization of medical and veterinary diseases. However, it is becoming clear that in order to accurately measure genetic variation within and between pathogenic groups, multiple isolates, as well as commensal species, must be sequenced. This study examined the pangenomic content of Escherichia coli. Six distinct E. coli pathovars can be distinguished using molecular or phenotypic markers, but only two of the six pathovars have been subjected to any genome sequencing previously. Thus, this report provides a seminal description of the genomic contents and unique features of three unsequenced pathovars, enterotoxigenic E. coli, enteropathogenic E. coli, and enteroaggregative E. coli. We also determined the first genome sequence of a human commensal E. coli isolate, E. coli HS, which will undoubtedly provide a new baseline from which workers can examine the evolution of pathogenic E. coli. Comparison of 17 E. coli genomes, 8 of which are new, resulted in identification of approximately 2,200 genes conserved in all isolates. We were also able to identify genes that were isolate and pathovar specific. Fewer pathovar-specific genes were identified than anticipated, suggesting that each isolate may have independently developed virulence capabilities. Pangenome calculations indicate that E. coli genomic diversity represents an open pangenome model containing a reservoir of more than 13,000 genes, many of which may be uncharacterized but important virulence factors. This comparative study of the species E. coli, while descriptive, should provide the basis for future functional work on this important group of pathogens.

  20. Transport of Escherichia coli in saturated porous media

    NARCIS (Netherlands)

    Foppen, J.W.A.

    2007-01-01

    Over de manier waarop de bacterie en tevens meest bekende fecale indicator soort Escherichia coli getransporteerd wordt in grondwater is relatief weinig bekend. In deze studie wordt de verwijdering van E. coli uit grondwater ten gevolge van E. coli - sediment interacties bestudeerd en modelmatig

  1. Transport of Escherichia coli in saturated porous media

    NARCIS (Netherlands)

    Foppen, J.W.A.

    2007-01-01

    Over de manier waarop de bacterie en tevens meest bekende fecale indicator soort Escherichia coli getransporteerd wordt in grondwater is relatief weinig bekend. In deze studie wordt de verwijdering van E. coli uit grondwater ten gevolge van E. coli - sediment interacties bestudeerd en modelmatig ge

  2. WGS accurately predicts antimicrobial resistance in Escherichia coli

    Science.gov (United States)

    Objectives: To determine the effectiveness of whole-genome sequencing (WGS) in identifying resistance genotypes of multidrug-resistant Escherichia coli (E. coli) and whether these correlate with observed phenotypes. Methods: Seventy-six E. coli strains were isolated from farm cattle and measured f...

  3. Transport of Escherichia coli in saturated porous media

    NARCIS (Netherlands)

    Foppen, J.W.A.

    2007-01-01

    Over de manier waarop de bacterie en tevens meest bekende fecale indicator soort Escherichia coli getransporteerd wordt in grondwater is relatief weinig bekend. In deze studie wordt de verwijdering van E. coli uit grondwater ten gevolge van E. coli - sediment interacties bestudeerd en modelmatig ge

  4. Diarrheagenic Escherichia coli Markers and Phenotypes among Fecal E. coli Isolates Collected from Nicaraguan Infants ▿

    OpenAIRE

    Reyes, Daniel; Vilchez, Samuel; Paniagua, Margarita; Colque-Navarro, Patricia; Weintraub, Andrej; Möllby, Roland; Kühn, Inger

    2010-01-01

    We analyzed the prevalence of diarrheagenic Escherichia coli (DEC) markers and common phenotypes in 2,164 E. coli isolates from 282 DEC-positive samples. Enteropathogenic E. coli (EPEC) and enteroaggregative E. coli (EAEC) were very diverse and were not correlated with diarrhea. Enterotoxigenic E. coli (ETEC) estA and enterohemorrhagic E. coli (EHEC) belonged to a few phenotypes and were significantly correlated with diarrhea.

  5. Diarrheagenic Escherichia coli Markers and Phenotypes among Fecal E. coli Isolates Collected from Nicaraguan Infants ▿

    OpenAIRE

    2010-01-01

    We analyzed the prevalence of diarrheagenic Escherichia coli (DEC) markers and common phenotypes in 2,164 E. coli isolates from 282 DEC-positive samples. Enteropathogenic E. coli (EPEC) and enteroaggregative E. coli (EAEC) were very diverse and were not correlated with diarrhea. Enterotoxigenic E. coli (ETEC) estA and enterohemorrhagic E. coli (EHEC) belonged to a few phenotypes and were significantly correlated with diarrhea.

  6. A ribosomal misincorporation of Lys for Arg in human triosephosphate isomerase expressed in Escherichia coli gives rise to two protein populations.

    Directory of Open Access Journals (Sweden)

    Beatriz Aguirre

    Full Text Available We previously observed that human homodimeric triosephosphate isomerase (HsTIM expressed in Escherichia coli and purified to apparent homogeneity exhibits two significantly different thermal transitions. A detailed exploration of the phenomenon showed that the preparations contain two proteins; one has the expected theoretical mass, while the mass of the other is 28 Da lower. The two proteins were separated by size exclusion chromatography in 3 M urea. Both proteins correspond to HsTIM as shown by Tandem Mass Spectrometry (LC/ESI-MS/MS. The two proteins were present in nearly equimolar amounts under certain growth conditions. They were catalytically active, but differed in molecular mass, thermostability, susceptibility to urea and proteinase K. An analysis of the nucleotides in the human TIM gene revealed the presence of six codons that are not commonly used in E. coli. We examined if they were related to the formation of the two proteins. We found that expression of the enzyme in a strain that contains extra copies of genes that encode for tRNAs that frequently limit translation of heterologous proteins (Arg, Ile, Leu, as well as silent mutations of two consecutive rare Arg codons (positions 98 and 99, led to the exclusive production of the more stable protein. Further analysis by LC/ESI-MS/MS showed that the 28 Da mass difference is due to the substitution of a Lys for an Arg residue at position 99. Overall, our work shows that two proteins with different biochemical and biophysical properties that coexist in the same cell environment are translated from the same nucleotide sequence frame.

  7. Initiation of Replication in Escherichia coli

    DEFF Research Database (Denmark)

    Frimodt-Møller, Jakob

    The circular chromosome of Escherichia coli is replicated by two replisomes assembled at the unique origin and moving in the opposite direction until they meet in the less well defined terminus. The key protein in initiation of replication, DnaA, facilitates the unwinding of double-stranded DNA...... to single-stranded DNA in oriC. Although DnaA is able to bind both ADP and ATP, DnaA is only active in initiation when bound to ATP. Although initiation of replication, and the regulation of this, is thoroughly investigated it is still not fully understood. The overall aim of the thesis was to investigate...... the regulation of initiation, the effect on the cell when regulation fails, and if regulation was interlinked to chromosomal organization. This thesis uncovers that there exists a subtle balance between chromosome replication and reactive oxygen species (ROS) inflicted DNA damage. Thus, failure in regulation...

  8. The eclipse period of Escherichia coli

    DEFF Research Database (Denmark)

    von Freiesleben, Ulrik; Krekling, Martin A.; Hansen, Flemming G.

    2000-01-01

    The minimal time between successive initiations on the same origin (the eclipse) in Escherichia coli was determined to be approximately 25-30 min. An inverse relationship was found between the length of the eclipse and the amount of Dam methyltransferase in the cell, indicating that the eclipse...... corresponds to the period of origin hemimethylation. The SeqA protein was absolutely required for the eclipse, and DnaA titration studies suggested that the SeqA protein prevented the binding of multiple DnaA molecules on oriC (initial complex formation). No correlation between the amount of SeqA and eclipse...... length was revealed, but increased SeqA levels affected chromosome partitioning and/or cell division. This was corroborated further by an aberrant nucleoid distribution in SeqA-deficient cells. We suggest that the SeqA protein's role in maintaining the eclipse is tied to a function in chromosome...

  9. Role of peripheral pooling in porcine Escherichia coli sepsis

    Energy Technology Data Exchange (ETDEWEB)

    Teule, G.J.; von Lingen, A.; Verwey von Vught, M.A.; Kester, A.D.; Mackaay, R.C.; Bezemer, P.D.; Heidenal, G.A.; Thijs, L.G.

    1984-01-01

    In anesthesized pigs the effects of E. coli (2 X 10(8)/kg) on hemodynamics and red cell distribution were studied. After injection of 99m-Tc red cells (15 mCi), regional radioactivity was followed during 3 hours. Gated bloodpool studies were performed to measure end-diastolic volumes (EDV). Escherichia coli E. coli was infused in 14 pigs, while 7 animals served as controls. E. coli resulted in an early increase in pulmonary arterial pressure. Systemic arterial pressure decreased gradually, while cardiac output did not change significantly. The gated studies revealed that especially left ventricular end-diastolic volume (LVEDV) declined, to 50% of the basal value. Regional radioactivity did not change over lungs, liver and abdomen. Splenic activity declined markedly. Over the hindlimb a significant increase (29 +/- 8%) was observed. It is concluded that E. coli infusion in pigs induces a hemodynamic pattern similar to human sepsis. The decrease in LVEDV is probably related to peripheral pooling and a change in right ventricle (RV) performance.

  10. Pengujian Bakteri Escherichia Coli Pada Air Sumur Di Medan Johor

    OpenAIRE

    Mahardhika, Diah

    2013-01-01

    Water is an essential material in life. Water is a means to improve public health. The spread of water borne diseases can be. Water pollution can be caused due to the entry of human and animal waste, but it can also be caused directly or through a leak or where ground soil cracks. This test aims to determine the number most likely Most Probable Number (MPN) Escherichia coli bacteria that contaminate well water located in Medan Johor still meet water quality requirements or not. The samplin...

  11. Profile of Shiga toxin-producing Escherichia coli strains isolated from dogs and cats and genetic relationships with isolates from cattle, meat and humans.

    Science.gov (United States)

    Bentancor, A; Rumi, M V; Carbonari, C; Gerhardt, E; Larzábal, M; Vilte, D A; Pistone-Creydt, V; Chinen, I; Ibarra, C; Cataldi, A; Mercado, E C

    2012-05-04

    Pets can be reservoirs of Shiga toxin-producing Escherichia coli (STEC) strains. The aim of this study was to examine nine strains belonging to several serotypes (O91:H21, O91:H16, O178:H19, O8:H19, O22:H8, O22:HNT, ONT:H8), previously recovered from cats or dogs. To this end, we assessed a set of additional virulence genes (stx(2) subtype, subAB, ehxA, eae and saa), cytotoxic activity, and genetic relationships with strains isolated from cattle, meat and humans using pulsed-field gel electrophoresis (PFGE). Most of the isolates carried the stx(2) and/or stx(2vh-b) sequences, while only the O91:H21 isolate presented the mucus-activatable stx(2d) variant, as confirmed by sequencing the genes of subunits A and B. All the strains showed cytotoxic activity in cultured cells. One of the two O178:H19, selected for its high level of cytotoxicity in Vero cells, showed the ability to cause functional alterations in the human colon mucosa in vitro. None of the strains possessed the subAB, eae or saa genes and only the strains belonging to serotype O8:H19 carried the ehxA gene. The isolates shared 90-100% similarity by PFGE to epidemiologically unrelated strains of the corresponding serotypes recovered from cattle, meat or humans. Our results demonstrate that dogs and cats may have a role in the infection of humans by STEC, probably serving as a vehicle for bovine strains in the cycle of human infection, and thus emphasize the health risks for owners and their families. Copyright © 2011 Elsevier B.V. All rights reserved.

  12. Evaluation of major types of Shiga toxin 2E-producing Escherichia coli bacteria present in food, pigs, and the environment as potential pathogens for humans.

    Science.gov (United States)

    Beutin, Lothar; Krüger, Ulrike; Krause, Gladys; Miko, Angelika; Martin, Annett; Strauch, Eckhard

    2008-08-01

    Shiga toxin 2e (Stx2e)-producing strains from food (n = 36), slaughtered pigs (n = 25), the environment (n = 21), diseased pigs (n = 19), and humans (n = 9) were investigated for production of Stx2e by enzyme-linked immunosorbent assay, for virulence markers by PCR, and for their serotypes to evaluate their role as potential human pathogens. Stx2e production was low in 64% of all 110 strains. Stx2e production was inducible by mitomycin C but differed considerably between strains. Analysis by nucleotide sequencing and transcription of stx(2e) genes in high- and low-Stx2e-producing strains showed that toxin production correlated with transcription rates of stx(2e) genes. DNA sequences specific for the int, Q, dam, and S genes of the stx(2e) bacteriophage P27 were found in 109 strains, indicating cryptic P27-like prophages, although 102 of these were not complete for all genes tested. Genes encoding intimin (eae), enterohemorrhagic Escherichia coli hemolysin (ehx), or other stx(1) or stx(2) variants were not found, whereas genes for heat-stable enterotoxins STI, STII, or EAST1 were present in 54.5% of the strains. Seven major serotypes that were associated with diseased pigs (O138:H14, O139:H1, and O141:H4) or with slaughter pigs, food, and the environment (O8:H4, O8:H9, O100:H30, and O101:H9) accounted for 60% of all Stx2e strains. The human Stx2e isolates did not belong to these major serotypes of Stx2e strains, and high production of Stx2e in human strains was not related to diarrheal disease. The results from this study and other studies do not point to Stx2e as a pathogenicity factor for diarrhea and hemolytic uremic syndrome in humans.

  13. Escherichia coli biofilms: Accepting the therapeutic challenges

    Directory of Open Access Journals (Sweden)

    Trupti Bajpai

    2016-01-01

    Full Text Available Background: Urinary tract infections (UTI′s are a major public health concern globally. Recurrent UTI′s that are predominantly caused by uropathogenic Escherichia coli′s forms biofilm that is an intracellular, structured bacterial community, enclosed in a self-produced matrix, adherent to an inert, or living surface. Biofilm physiology is characterized by increased tolerance to stress, antibiotics, and immunological defenses, which is at the origin of their resilience in most medical and industrial settings. Materials and Methods: The present prospective study was carried out from December 2013 to May 2014 in the Department of Microbiology of a Teaching Tertiary Care hospital located in central India. A total of 100 consecutive, nonrepetitive E. coli isolates were subjected to biofilm formation study by Christensen′s tube adherence method. All the isolates were also subjected to antimicrobial susceptibility testing by Kirby-Bauer disc diffusion method in accordance with the Clinical Laboratory Standard Institute 2013 guidelines. Results and Discussion: Out of the 100 E. coli isolates studied, 62 (62% were positive for biofilm formation. High percentage of resistance was detected in isolates among the male inpatient group. Overall drug resistance was found to be very high among both biofilm as well as nonbiofilm forming isolates indicating excessive drug resistance among both community and hospital organisms. Conclusion: A greater understanding of the nature of biofilm organisms in chronic UTI′s would help in the development of novel and more effective treatments for these problematic diseases.

  14. Expanding ester biosynthesis in Escherichia coli.

    Science.gov (United States)

    Rodriguez, Gabriel M; Tashiro, Yohei; Atsumi, Shota

    2014-04-01

    To expand the capabilities of whole-cell biocatalysis, we have engineered Escherichia coli to produce various esters. The alcohol O-acyltransferase (ATF) class of enzyme uses acyl-CoA units for ester formation. The release of free CoA upon esterification with an alcohol provides the free energy to facilitate ester formation. The diversity of CoA molecules found in nature in combination with various alcohol biosynthetic pathways allows for the biosynthesis of a multitude of esters. Small to medium volatile esters have extensive applications in the flavor, fragrance, cosmetic, solvent, paint and coating industries. The present work enables the production of these compounds by designing several ester pathways in E. coli. The engineered pathways generated acetate esters of ethyl, propyl, isobutyl, 2-methyl-1-butyl, 3-methyl-1-butyl and 2-phenylethyl alcohols. In particular, we achieved high-level production of isobutyl acetate from glucose (17.2 g l(-1)). This strategy was expanded to realize pathways for tetradecyl acetate and several isobutyrate esters.

  15. The crystal structure Escherichia coli Spy.

    Science.gov (United States)

    Kwon, Eunju; Kim, Dong Young; Gross, Carol A; Gross, John D; Kim, Kyeong Kyu

    2010-11-01

    Escherichia coli spheroplast protein y (EcSpy) is a small periplasmic protein that is homologous with CpxP, an inhibitor of the extracytoplasmic stress response. Stress conditions such as spheroplast formation induce the expression of Spy via the Cpx or the Bae two-component systems in E. coli, though the function of Spy is unknown. Here, we report the crystal structure of EcSpy, which reveals a long kinked hairpin-like structure of four α-helices that form an antiparallel dimer. The dimer contains a curved oval shape with a highly positively charged concave surface that may function as a ligand binding site. Sequence analysis reveals that Spy is highly conserved over the Enterobacteriaceae family. Notably, three conserved regions that contain identical residues and two LTxxQ motifs are placed at the horizontal end of the dimer structure, stabilizing the overall fold. CpxP also contains the conserved sequence motifs and has a predicted secondary structure similar to Spy, suggesting that Spy and CpxP likely share the same fold.

  16. Independence of replisomes in Escherichia coli chromosomalreplication

    Energy Technology Data Exchange (ETDEWEB)

    Breier, Adam M.; Weier, Heinz-Ulrich G.; Cozzarelli, Nicholas R.

    2005-03-13

    In Escherichia coli DNA replication is carried out by the coordinated action of the proteins within a replisome. After replication initiation, the two bidirectionally oriented replisomes from a single origin are colocalized into higher-order structures termed replication factories. The factory model postulated that the two replisomes are also functionally coupled. We tested this hypothesis by using DNA combing and whole-genome microarrays. Nascent DNA surrounding oriC in single, combed chromosomes showed instead that one replisome, usually the leftward one, was significantly ahead of the other 70% of the time. We next used microarrays to follow replication throughout the genome by measuring DNA copy number. We found in multiple E. coli strains that the replisomes are independent, with the leftward replisome ahead of the rightward one. The size of the bias was strain-specific, varying from 50 to 130 kb in the array results. When we artificially blocked one replisome, the other continued unabated, again demonstrating independence. We suggest an improved version of the factory model that retains the advantages of threading DNA through colocalized replisomes at about equal rates, but allows the cell flexibility to overcome obstacles encountered during elongation.

  17. Biosynthesis of ethylene glycol in Escherichia coli.

    Science.gov (United States)

    Liu, Huaiwei; Ramos, Kristine Rose M; Valdehuesa, Kris Niño G; Nisola, Grace M; Lee, Won-Keun; Chung, Wook-Jin

    2013-04-01

    Ethylene glycol (EG) is an important platform chemical with steadily expanding global demand. Its commercial production is currently limited to fossil resources; no biosynthesis route has been delineated. Herein, a biosynthesis route for EG production from D-xylose is reported. This route consists of four steps: D-xylose → D-xylonate → 2-dehydro-3-deoxy-D-pentonate → glycoaldehyde → EG. Respective enzymes, D-xylose dehydrogenase, D-xylonate dehydratase, 2-dehydro-3-deoxy-D-pentonate aldolase, and glycoaldehyde reductase, were assembled. The route was implemented in a metabolically engineered Escherichia coli, in which the D-xylose → D-xylulose reaction was prevented by disrupting the D-xylose isomerase gene. The most efficient construct produced 11.7 g L(-1) of EG from 40.0 g L(-1) of D-xylose. Glycolate is a carbon-competing by-product during EG production in E. coli; blockage of glycoaldehyde → glycolate reaction was also performed by disrupting the gene encoding aldehyde dehydrogenase, but from this approach, EG productivity was not improved but rather led to D-xylonate accumulation. To channel more carbon flux towards EG than the glycolate pathway, further systematic metabolic engineering and fermentation optimization studies are still required to improve EG productivity.

  18. Adhesive threads of extraintestinal pathogenic Escherichia coli

    Directory of Open Access Journals (Sweden)

    Antão Esther-Maria

    2009-12-01

    Full Text Available Abstract The ability to adhere to host surfaces is by far the most vital step in the successful colonization by microbial pathogens. Colonization begins with the attachment of the bacterium to receptors expressed by cells forming the lining of the mucosa. Long hair like extracellular appendages called fimbriae, produced by most Gram-negative pathogens, mediate specific attachment to the epithelial cell surface. Associated with the fimbriae is a protein called an adhesin, which directs high-affinity binding to specific cell surface components. In the last couple of years, an enormous amount of research has been undertaken that deals with understanding how bacterial pathogens adhere to host cells. E. coli in all probability is one of the best studied free-living organisms. A group of E. coli called Extraintestinal pathogenic E. coli (ExPEC including both human and animal pathogens like Uropathogenic E. coli (UPEC, Newborn meningitic E. coli (NMEC and Avian pathogenic E. coli (APEC, have been found to harbour many fimbriae including Type 1 fimbriae, P fimbriae, curli fibres, S fimbriae, F1C fimbriae, Dr fimbriae, afimbrial adhesins, temperature-sensitive haemagglutinin and many novel adhesin gene clusters that have not yet been characterized. Each of these adhesins is unique due to the recognition of an adhesin-specific receptor, though as a group these adhesins share common genomic organization. A newly identified putative adhesin temporarily termed ExPEC Adhesin I, encoded by gene yqi, has been recently found to play a significant role in the pathogenesis of APEC infection, thus making it an interesting candidate for future research. The aim of this review is to describe the role of ExPEC adhesins during extraintestinal infections known till date, and to suggest the idea of investigating their potential role in the colonization of the host gut which is said to be a reservoir for ExPEC.

  19. The extracellular RNA complement of Escherichia coli.

    Science.gov (United States)

    Ghosal, Anubrata; Upadhyaya, Bimal Babu; Fritz, Joëlle V; Heintz-Buschart, Anna; Desai, Mahesh S; Yusuf, Dilmurat; Huang, David; Baumuratov, Aidos; Wang, Kai; Galas, David; Wilmes, Paul

    2015-01-21

    The secretion of biomolecules into the extracellular milieu is a common and well-conserved phenomenon in biology. In bacteria, secreted biomolecules are not only involved in intra-species communication but they also play roles in inter-kingdom exchanges and pathogenicity. To date, released products, such as small molecules, DNA, peptides, and proteins, have been well studied in bacteria. However, the bacterial extracellular RNA complement has so far not been comprehensively characterized. Here, we have analyzed, using a combination of physical characterization and high-throughput sequencing, the extracellular RNA complement of both outer membrane vesicle (OMV)-associated and OMV-free RNA of the enteric Gram-negative model bacterium Escherichia coli K-12 substrain MG1655 and have compared it to its intracellular RNA complement. Our results demonstrate that a large part of the extracellular RNA complement is in the size range between 15 and 40 nucleotides and is derived from specific intracellular RNAs. Furthermore, RNA is associated with OMVs and the relative abundances of RNA biotypes in the intracellular, OMV and OMV-free fractions are distinct. Apart from rRNA fragments, a significant portion of the extracellular RNA complement is composed of specific cleavage products of functionally important structural noncoding RNAs, including tRNAs, 4.5S RNA, 6S RNA, and tmRNA. In addition, the extracellular RNA pool includes RNA biotypes from cryptic prophages, intergenic, and coding regions, of which some are so far uncharacterised, for example, transcripts mapping to the fimA-fimL and ves-spy intergenic regions. Our study provides the first detailed characterization of the extracellular RNA complement of the enteric model bacterium E. coli. Analogous to findings in eukaryotes, our results suggest the selective export of specific RNA biotypes by E. coli, which in turn indicates a potential role for extracellular bacterial RNAs in intercellular communication. © 2015 The

  20. Human body temperature (37degrees C) increases the expression of iron, carbohydrate, and amino acid utilization genes in Escherichia coli K-12.

    Science.gov (United States)

    White-Ziegler, Christine A; Malhowski, Amy J; Young, Sarah

    2007-08-01

    Using DNA microarrays, we identified 126 genes in Escherichia coli K-12 whose expression is increased at human body temperature (37 degrees C) compared to growth at 23 degrees C. Genes involved in the uptake and utilization of amino acids, carbohydrates, and iron dominated the list, supporting a model in which temperature serves as a host cue to increase expression of bacterial genes needed for growth. Using quantitative real-time PCR, we investigated the thermoregulatory response for representative genes in each of these three categories (hisJ, cysP, srlE, garP, fes, and cirA), along with the fimbrial gene papB. Increased expression at 37 degrees C compared to 23 degrees C was retained in both exponential and stationary phases for all of the genes and in most of the various media tested, supporting the relative importance of this cue in adapting to changing environments. Because iron acquisition is important for both growth and virulence, we analyzed the regulation of the iron utilization genes cirA and fes and found that growth in iron-depleted medium abrogated the thermoregulatory effect, with high-level expression at both temperatures, contrasting with papB thermoregulation, which was not greatly altered by limiting iron levels. A positive role for the environmental regulator H-NS was found for fes, cirA, hisJ, and srlE transcription, whereas it had a primarily negative effect on cysP and garP expression. Together, these studies indicate that temperature is a broadly used cue for regulating gene expression in E. coli and that H-NS regulates iron, carbohydrate, and amino acid utilization gene expression.

  1. Determination and control of low-level amino acid misincorporation in human thioredoxin protein produced in a recombinant Escherichia coli production system.

    Science.gov (United States)

    Harris, Robert P; Mattocks, Jo; Green, Philip S; Moffatt, Frank; Kilby, Peter M

    2012-08-01

    Escherichia coli is used extensively in the production of proteins within biotechnology for a number of therapeutic applications. Here, we discuss the production and overexpression of the potential biopharmaceutical human thioredoxin protein (rhTRX) within E. coli. Overexpression of foreign molecules within the cell can put an enormous amount of stress on the translation machinery. This can lead to a misfiring in the construction of a protein resulting in populations differing slightly in amino acid composition. Whilst this may still result in a population of active molecules being expressed, it does present significant problems with molecules that are destined for clinical applications. Amino acid misincorporation of this subset could potentially result in antibodies being raised to these unnatural proteins. Cross-reaction with a patient's endogenous thioredoxin could then lead to an autoimmune phenomena and serious health implications. Generally, the issue of misincorporation appears not to be a routine regulatory concern (see ICH Q6B guidelines). Therefore, amino acid misincorporation may not have been detected, much less explored in the clinic as the occurrence or absence of these random errors is not routinely reported. Using current technologies based on proteomics, the ability to find misincorporation critically depends upon the criteria for matching theoretical and experimental mass spectrometry data. Additionally, isolation and extraction of these mistranslated proteins from the production process is both difficult and expensive. Therefore, it is advantageous to find routes for removing their production during the upstream phase. In this study, we show how modern proteomic technology can be used to identify and quantify amino acid misincorporation. Using these techniques we have shown how manipulation of gene sequence and scoping of fermentation media composition can lead to the reduction and elimination of these misincorporations in rhTRX. Copyright

  2. Gene fusions with human carbonic anhydrase II for efficient expression and rapid single-step recovery of recombinant proteins: expression of the Escherichia coli F1-ATPase epsilon subunit.

    Science.gov (United States)

    Van Heeke, G; Shaw, R; Schnizer, R; Couton, J M; Schuster, S M; Wagner, F W

    1993-08-01

    A new expression vector was constructed which allows the overproduction in Escherichia coli of tripartite proteins consisting of human carbonic anhydrase isozyme II (hCAII), a peptide linker containing an enterokinase cleavage site, and a target protein of interest. Carbonic anhydrase is soluble and stable in E. coli and serves as a highly specific purification tag in the recovery of the fusion protein by a single affinity chromatography step. The enterokinase cleavage site was engineered into the construct to allow accurate and efficient release of the target protein. To demonstrate the practical value of this vector, the E. coli F1-ATPase epsilon subunit was expressed as a fusion with hCAII. After a single purification step, biologically active recombinant E. coli F1-ATPase epsilon subunit was recovered following proteolytic removal of the hCAII moiety.

  3. Persistence of Escherichia coli in batch and continuous vermicomposting systems.

    Science.gov (United States)

    Hénault-Ethier, Louise; Martin, Vincent J J; Gélinas, Yves

    2016-10-01

    Vermicomposting is a biooxidation process in which epigeicearthworms act in synergy with microbial populations to degrade organic matter. Vermicomposting does not go through a thermophilic stage as required by North American legislations for pathogen eradication. We examined the survival of a Green Fluorescent Protein (GFP) labeled Escherichia coli MG1655 as a model for the survival of pathogenic bacteria in both small-scale batch and medium-scale continuously-operated systems to discern the influence of the earthworm Eisenia fetida, nutrient content and the indigenous vermicompost microbial community on pathogen abundance. In batch systems, the microbial community had the greatest influence on the rapid decline of E. coli populations, and the effect of earthworms was only visible in microbially-impoverishedvermicomposts. No significant earthworm density-dependent relationship was observed on E. coli survival under continuous operation. E. coli numbers decreased below the US EPA compost sanitation guidelines of 10(3)Colony Forming Units (CFU)/g (dry weight) within 18-21days for both the small-scale batch and medium-scale continuous systems, but it took up to 51days without earthworms and with an impoverished microbial community to reach the legal limit. Nutrient replenishment (i.e. organic carbon) provided by continuous feed input did not appear to extend E. coli survival. In fact, longer survival of E. coli was noticed in treatments where less total and labile sugars were available, suggesting that sugars may support potentially antagonist bacteria in the vermicompost. Total N, pH and humidity did not appear to affect E. coli survival. Several opportunistic human pathogens may be found in vermicompost, and their populations are likely kept in check by antagonists.

  4. Epidemiology and clinical manifestations of enteroaggregative Escherichia coli

    DEFF Research Database (Denmark)

    Hebbelstrup Jensen, Betina; Olsen, Katharina E P; Struve, Carsten

    2014-01-01

    Enteroaggregative Escherichia coli (EAEC) represents a heterogeneous group of E. coli strains. The pathogenicity and clinical relevance of these bacteria are still controversial. In this review, we describe the clinical significance of EAEC regarding patterns of infection in humans, transmission......, reservoirs, and symptoms. Manifestations associated with EAEC infection include watery diarrhea, mucoid diarrhea, low-grade fever, nausea, tenesmus, and borborygmi. In early studies, EAEC was considered to be an opportunistic pathogen associated with diarrhea in HIV patients and in malnourished children...... in developing countries. In recent studies, associations with traveler's diarrhea, the occurrence of diarrhea cases in industrialized countries, and outbreaks of diarrhea in Europe and Asia have been reported. In the spring of 2011, a large outbreak of hemolytic-uremic syndrome (HUS) and hemorrhagic colitis...

  5. Probability of recovering pathogenic Escherichia coli from foods.

    Science.gov (United States)

    Hill, W E; Ferreira, J L; Payne, W L; Jones, V M

    1985-01-01

    The probability of recovering pathogenic Escherichia coli from food by the Bacteriological Analytical Manual method was determined by the effects of several factors: the number of strains per food, the ability of pathogenic strains to survive enrichment, and the frequency of plasmid loss during enrichment. Biochemical patterns indicated the presence of about six E. coli strains per food sample. About half of the strains isolated from humans did not survive enrichment. Among those which grew, plasmid loss, as determined by gel electrophoresis and DNA colony hybridization, ranged from 20 to 95%. The combined effects of failure to survive enrichment and plasmid loss decreased the relative numbers of these strains and reduced the chance of detecting pathogens. To counteract this tendency and obtain a 90 to 95% probability off recovering a given pathogenic strain, 40 to 50 colonies per food sample should be picked during the routine testing of foods. PMID:3893320

  6. Quinolone-resistant Escherichia coli in Poultry Farming.

    Science.gov (United States)

    Hricová, Kristýna; Röderová, Magdaléna; Pudová, Vendula; Hanulík, Vojtěch; Halová, Dana; Julínková, Pavla; Dolejská, Monika; Papoušek, Ivo; Bardoň, Jan

    2017-06-01

    Increasing bacterial resistance to quinolone antibiotics is apparent in both humans and animals. For humans, a potential source of resistant bacteria may be animals or their products entering the human food chain, for example poultry. Between July 2013 and September 2014, samples were collected and analyzed in the Moravian regions of the Czech Republic to isolate the bacterium Escherichia coli. As a result, 212 E. coli isolates were obtained comprising 126 environmental isolates from poultry houses and 86 isolates from cloacal swabs from market-weight turkeys. Subsequently, the E. coli isolates were tested for susceptibility to selected antibiotics. Resistance of the poultry isolates to quinolones ranged from 53% to 73%. Additionally, the presence of plasmid-mediated resistance genes was studied. The genes were confirmed in 58% of the tested strains. The data on resistance of isolates from poultry were compared with results of resistance tests in human isolates obtained in the same regions. The high levels of resistance determined by both phenotyping and genotyping methods and reported in the present study confirm the fact that the use of fluoroquinolones in poultry should be closely monitored. Copyright© by the National Institute of Public Health, Prague 2017.

  7. General considerations regarding the infections with the Escherichia coli pathogen

    Directory of Open Access Journals (Sweden)

    Marius Necşulescu

    2017-04-01

    Full Text Available Escherichia coli is the species of the genus Escherichia with the greatest epidemiological impact. Escherichia coli infections are found mainly in places with poor hygiene; the infants with ages between 1 and 3 years old are included in the category with the highest risk. It is a "fecal-oral" transmission mechanism as a result of consumption of contaminated food or water, or by "dirty hands". The foods most commonly implicated in the transmission of the infection are unpasteurized milk and milk products, beef, especially the one insufficiently cooked, unpasteurized fruit juice, lettuce and insufficiently washed vegetables. The disease has been reported worldwide, being described numerous episodes of infection with Escherichia coli that caused multiple illnesses and deaths. Escherichia coli has three types of antigens: antigen "O" (somatic, antigen "H" (flagella and antigen "K" (capsular. Clinical manifestations are present in the form of non-specific diarrhea, a dysentery form of enteritis, choleriform enteritis, hemorrhagic colitis and hemolytic uremic syndrome (HUS. The Escherichia coli infection diagnosis is made by identifying the etiologic agent and/or by highlighting the VTI toxin in the feces. The treatment consists in precautionary antibiotherapy, hydrodynamics and electrolyte rebalancing, blood transfusions and dialysis, if in the case of renal failure. The prevention of infections with Escherichia coli is achieved by personal hygiene, food hygiene and work hygiene.

  8. Enhancing human-like collagen accumulation by deleting the major glucose transporter ptsG in recombinant Escherichia coli BL21.

    Science.gov (United States)

    Luo, Yan'e; Zhang, Tao; Fan, Daidi; Mu, Tingzhen; Xue, Wenjiao; Hui, Junfeng; Ma, Xiaoxuan

    2014-01-01

    Collagen has been proven to be a valuable biomedical material for many medical applications. Human-like collagen (HLC) is a novel important biomedical material with diverse medical applications. In this work, recombinant Escherichia coli BL21 3.7 ∆ptsG was constructed, the characters of ptsG mutant strain were analyzed, and real-time quantitative polymerase chain reaction (PCR) was applied to investigate the effect of ptsG gene deletion on the transcriptional level of the phosphotransferase system (PTS) genes responsible for glucose transport. The HLC production and cell growth ability were 1.33- and 1.24-fold higher than those of its parent strain in the fermentation medium, respectively, and 1.16- and 1.17-fold in the modified minimal medium individually. The acetate accumulation decreased by 42%-56% compared to its parent strain in the fermentation medium, and 70%-87% in the modified minimal medium. The results of RT-qPCR showed that the transcriptional level of crr, ptsH, ptsI, and blgF in ptsG mutant all decreased dramatically, which inferred a decrease in the glucose uptake rate, but the transcriptional level of FruB and manX increased slightly, which demonstrated the activation of fructose- and mannose-specific transport pathways in the ptsG mutant. This study demonstrates that ptsG deletion is an effective strategy to reduce acetate accumulation and increase biomass and HLC production.

  9. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of CofB, the minor pilin subunit of CFA/III from human enterotoxigenic Escherichia coli.

    Science.gov (United States)

    Kawahara, Kazuki; Oki, Hiroya; Fukakusa, Shunsuke; Maruno, Takahiro; Kobayashi, Yuji; Motooka, Daisuke; Taniguchi, Tooru; Honda, Takeshi; Iida, Tetsuya; Nakamura, Shota; Ohkubo, Tadayasu

    2015-06-01

    Colonization factor antigen III (CFA/III) is one of the virulence factors of human enterotoxigenic Escherichia coli (ETEC) that forms the long, thin, proteinaceous fibres of type IV pili through assembly of its major and minor subunits CofA and CofB, respectively. The crystal structure of CofA has recently been reported; however, the lack of structural information for CofB, the largest among the known type IV pilin subunits, hampers a comprehensive understanding of CFA/III pili. In this study, constructs of wild-type CofB with an N-terminal truncation and the corresponding SeMet derivative were cloned, expressed, purified and crystallized. The crystals belonged to the rhombohedral space group R32, with unit-cell parameters a = b = 103.97, c = 364.57 Å for the wild-type construct and a = b = 103.47, c = 362.08 Å for the SeMet-derivatized form. Although the diffraction quality of these crystals was initially very poor, dehydration of the crystals substantially improved the resolution limit from ∼ 4.0 to ∼ 2.0 Å. The initial phase was solved by the single-wavelength anomalous dispersion (SAD) method using a dehydrated SeMet CofB crystal, which resulted in an interpretable electron-density map.

  10. Cloning and expression in Escherichia coli of a human gelatinase B-inhibitory single-chain immunoglobulin variable fragment (scFv).

    Science.gov (United States)

    Zhou, N; Paemen, L; Opdenakker, G; Froyen, G

    1997-09-15

    The murine monoclonal antibody REGA-3G12 selectively and specifically inhibits the activity of human gelatinase B. The cDNA fragments which encode the variable regions of the light and heavy chains were isolated by PCR-mediated cloning and sequenced. Single-chain Fv expression constructs for Escherichia coli were generated in which c-myc tag sequences were encoded. Inducible expression of the scFv and secretion to the periplasm were obtained with higher yields when the c-myc tag sequence was positioned at the amino-terminal side. The inhibitory activity of purified scFv on neutrophil gelatinase B was tested in a gelatin degradation assay and it was found to possess a similar specific activity as that of the intact monoclonal antibody and of the pepsin-clipped F(ab')2 derivative. This shows for the first time that inhibition of soluble enzymes with scFv is possible and opens new perspectives for the treatment of diseases with excessive and detrimental enzyme production in the host.

  11. Molecular characterization of a 21.4 kilobase antibiotic resistance plasmid from an α-hemolytic Escherichia coli O108:H- human clinical isolate.

    Directory of Open Access Journals (Sweden)

    Fay E Dawes

    Full Text Available This study characterizes the 21.4 kilobase plasmid pECTm80 isolated from Escherichia coli strain 80, an α hemolytic human clinical diarrhoeal isolate (serotype O108:H-. DNA sequence analysis of pECTm80 revealed it belonged to incompatibility group X1, and contained plasmid partition and toxin-antitoxin systems, an R6K-like triple origin (ori replication system, genes required for replication regulation, insertion sequences IS1R, ISEc37 and a truncated transposase gene (Tn3-like ΔtnpA of the Tn3 family, and carried a class 2 integron. The class 2 integron of pECTm80 contains an intact cassette array dfrA1-sat2, encoding resistance to trimethoprim and streptothricin, and an aadA1 gene cassette truncated by the insertion of IS1R. The complex plasmid replication system includes α, β and γ origins of replication. Pairwise BLASTn comparison of pECTm80 with plasmid pE001 reveals a conserved plasmid backbone suggestive of a common ancestral lineage. Plasmid pECTm80 is of potential clinical importance, as it carries multiple genes to ensure its stable maintenance through successive bacterial cell divisions and multiple antibiotic resistance genes.

  12. Lipopolysaccharide Phosphorylation by the WaaY Kinase Affects the Susceptibility of Escherichia coli to the Human Antimicrobial Peptide LL-37.

    Science.gov (United States)

    Bociek, Karol; Ferluga, Sara; Mardirossian, Mario; Benincasa, Monica; Tossi, Alessandro; Gennaro, Renato; Scocchi, Marco

    2015-08-07

    The human cathelicidin LL-37 is a multifunctional host defense peptide with immunomodulatory and antimicrobial roles. It kills bacteria primarily by altering membrane barrier properties, although the exact sequence of events leading to cell lysis has not yet been completely elucidated. Random insertion mutagenesis allowed isolation of Escherichia coli mutants with altered susceptibility to LL-37, pointing to factors potentially relevant to its activity. Among these, inactivation of the waaY gene, encoding a kinase responsible for heptose II phosphorylation in the LPS inner core, leads to a phenotype with decreased susceptibility to LL-37, stemming from a reduced amount of peptide binding to the surface of the cells, and a diminished capacity to lyse membranes. This points to a specific role of the LPS inner core in guiding LL-37 to the surface of Gram-negative bacteria. Although electrostatic interactions are clearly relevant, the susceptibility of the waaY mutant to other cationic helical cathelicidins was unaffected, indicating that particular structural features or LL-37 play a role in this interaction.

  13. Lipopolysaccharide Phosphorylation by the WaaY Kinase Affects the Susceptibility of Escherichia coli to the Human Antimicrobial Peptide LL-37*

    Science.gov (United States)

    Bociek, Karol; Ferluga, Sara; Mardirossian, Mario; Benincasa, Monica; Tossi, Alessandro; Gennaro, Renato; Scocchi, Marco

    2015-01-01

    The human cathelicidin LL-37 is a multifunctional host defense peptide with immunomodulatory and antimicrobial roles. It kills bacteria primarily by altering membrane barrier properties, although the exact sequence of events leading to cell lysis has not yet been completely elucidated. Random insertion mutagenesis allowed isolation of Escherichia coli mutants with altered susceptibility to LL-37, pointing to factors potentially relevant to its activity. Among these, inactivation of the waaY gene, encoding a kinase responsible for heptose II phosphorylation in the LPS inner core, leads to a phenotype with decreased susceptibility to LL-37, stemming from a reduced amount of peptide binding to the surface of the cells, and a diminished capacity to lyse membranes. This points to a specific role of the LPS inner core in guiding LL-37 to the surface of Gram-negative bacteria. Although electrostatic interactions are clearly relevant, the susceptibility of the waaY mutant to other cationic helical cathelicidins was unaffected, indicating that particular structural features or LL-37 play a role in this interaction. PMID:26100635

  14. Increased Rate of Apoptosis and Diminished Phagocytic Ability of Human Neutrophils Infected with Afa/Dr Diffusely Adhering Escherichia coli Strains

    OpenAIRE

    Brest, Patrick; Bétis, Frédéric; Çuburu, Nicolas; Selva, Eric; Herrant, Magali; Servin, Alain,; Auberger, Patrick; Hofman, Paul

    2004-01-01

    The proinflammatory effect of Afa/Dr diffusely adhering Escherichia coli (Afa/Dr DAEC) strains have been recently demonstrated in vitro by showing that polymorphonuclear leukocyte (PMN) transepithelial migration is induced after bacterial colonization of apical intestinal monolayers. The effect of Afa/Dr DAEC-PMN interaction on PMN behavior has been not investigated. Because of the putative virulence mechanism of PMN apoptosis during infectious diseases and taking into account the high level ...

  15. PARTICIPATION OF TLR4 IN ENGULFMENT OF ESCHERICHIA COLI BY HUMAN BLOOD NEUTROPHILS IN PRESENCE OF LIPOPOLYSACCHARIDES

    Directory of Open Access Journals (Sweden)

    S. V. Zubova

    2012-01-01

    Full Text Available Abstract. TLR4 is a key player in signaling system of host cells. Possible role of TLR4 is actively discussed, e.g. its significance for phagocytosis. A capacity of neutrophils to engulf FITC-labeled E. coli bacteria upon activation with LPS of different origin was studied in presence of anti-TLR4 Mab’s (HTA125 clone. It was shown that, in whole blood, TLR4 does not play any essential role in engulfment of bacteria by the neutrophils. Phagocytic activity of neutrophils in blood increases increased after their priming with E. coli endotoxins. LPS from Rb. сapsulatus did not affect phagocytosis. In presence of endotoxins, the degree of TLR4 involvement in neutrophil phagocytosis depends on LPS structure.

  16. Detection and genetic features of MCR-1-producing plasmid in human Escherichia coli infection in South Korea.

    Science.gov (United States)

    Kim, Eun Sil; Chong, Yong Pil; Park, Su-Jin; Kim, Mi-Na; Kim, Sung-Han; Lee, Sang-Oh; Choi, Sang-Ho; Woo, Jun Hee; Jeong, Jin-Yong; Kim, Yang Soo

    2017-10-01

    The plasmid-mediated colistin resistance gene, mcr-1, was identified for the first time from a hospitalized patient in South Korea. The mcr-1 gene was successfully transferred to E. coli J53 recipient and conferred resistance to colistin in the recipient. The mcr-1-harboring plasmid possessed a typical IncI2 group and did not have the mcr-1-associated ISApl1 element. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Dynamics of Escherichia coli Chromosome Segregation during Multifork Replication

    DEFF Research Database (Denmark)

    Nielsen, Henrik Jørck; Youngren, Brenda; Hansen, Flemming G.

    2007-01-01

    Slowly growing Escherichia coli cells have a simple cell cycle, with replication and progressive segregation of the chromosome completed before cell division. In rapidly growing cells, initiation of replication occurs before the previous replication rounds are complete. At cell division...

  18. The incidence and antibiotics susceptibility of Escherichia coli O157 ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-02-22

    Feb 22, 2010 ... The incidence of Escherichia coli 0157: H7 was assessed in meat samples from .... of this product resulting from contamination with STEC, a zoonotic .... Adak GK, Longs SM, O'Briens SJ (2002) Trends in indigenous forborne.

  19. mcr-1 identified in Avian Pathogenic Escherichia coli (APEC)

    National Research Council Canada - National Science Library

    Nicolle Lima Barbieri; Daniel W Nielsen; Yvonne Wannemuehler; Tia Cavender; Ashraf Hussein; Shi-gan Yan; Lisa K Nolan; Catherine M Logue

    2017-01-01

    .... Here, we examined a collection (n = 980) of Avian Pathogenic Escherichia coli (APEC) isolated from poultry with colibacillosis from the US and internationally for the presence of mcr-1 and mcr-2, genes known to encode colistin resistance...

  20. Overexpression of vsr in Escherichia coli is mutagenic.

    Science.gov (United States)

    Doiron, K M; Viau, S; Koutroumanis, M; Cupples, C G

    1996-01-01

    Overexpression of vsr in Escherichia coli stimulates transition and frameshift mutations. The pattern of mutations suggests that mutagenesis is due to saturation or inactivation of dam-directed mismatch repair. PMID:8763960

  1. Norfloxacin resistance in a clinical isolate of Escherichia coli.

    OpenAIRE

    Aoyama, H; Sato, K; Kato, T.; Hirai, K; Mitsuhashi, S.

    1987-01-01

    Analysis of DNA gyrase supercoiling and of norfloxacin uptake in Escherichia coli GN14176, a moderately norfloxacin-resistant clinical isolate, indicated that resistance was associated with both an altered drug target and a reduction in drug uptake.

  2. Identification and Characterization of a Peculiar vtx2-Converting Phage Frequently Present in Verocytotoxin-Producing Escherichia coli O157 Isolated from Human Infections

    Science.gov (United States)

    Grande, Laura; Michelacci, Valeria; Fioravanti, Rosa; Gally, David; Xu, Xuefang; La Ragione, Roberto; Anjum, Muna; Wu, Guanghui; Caprioli, Alfredo; Morabito, Stefano

    2014-01-01

    Certain verocytotoxin-producing Escherichia coli (VTEC) O157 phage types (PTs), such as PT8 and PT2, are associated with severe human infections, while others, such as PT21, seem to be restricted to cattle. In an attempt to delve into the mechanisms underlying such a differential distribution of PTs, we performed microarray comparison of human PT8 and animal PT21 VTEC O157 isolates. The main differences observed were in the vtx2-converting phages, with the PT21 strains bearing a phage identical to that present in the reference strain EDL933, BP933W, and all the PT8 isolates displaying lack of hybridization in some regions of the phage genome. We focused on the region spanning the gam and cII genes and developed a PCR tool to investigate the presence of PT8-like phages in a panel of VTEC O157 strains belonging to different PTs and determined that a vtx2 phage reacting with the primers deployed, which we named Φ8, was more frequent in VTEC O157 strains from human disease than in bovine strains. No differences were observed in the production of the VT2 mRNA when Φ8-positive strains were compared with VTEC O157 possessing BP933W. Nevertheless, we show that the gam-cII region of phage Φ8 might carry genetic determinants downregulating the transcription of the genes encoding the components of the type III secretion system borne on the locus of enterocyte effacement pathogenicity island. PMID:24799627

  3. Detection and Characterization of Shiga Toxin Producing Escherichia coli, Salmonella spp., and Yersinia Strains from Human, Animal, and Food Samples in San Luis, Argentina.

    Science.gov (United States)

    Favier, Gabriela Isabel; Lucero Estrada, Cecilia; Cortiñas, Teresa Inés; Escudero, María Esther

    2014-01-01

    Shiga toxin producing Escherichia coli (STEC), Salmonella spp., and Yersinia species was investigated in humans, animals, and foods in San Luis, Argentina. A total of 453 samples were analyzed by culture and PCR. The antimicrobial susceptibility of all the strains was studied, the genomic relationships among isolates of the same species were determined by PFGE, and the potencial virulence of Y. enterocolitica strains was analyzed. Yersinia species showed higher prevalence (9/453, 2.0%, 95% CI, 0.7-3.3%) than STEC (4/453, 0.9%, 95% CI, 0-1.8%) and Salmonella spp. (3/453, 0.7%, 95% CI, 0-1.5%). Y. enterocolitica and Y. intermedia were isolated from chicken carcasses (6/80, 7.5%, 95% CI, 1.5-13.5%) and porcine skin and bones (3/10, 30%, 95% CI, 0-65%). One STEC strain was recovered from human feces (1/70, 1.4%, 95% CI, 0-4.2%) and STEC stx1/stx2 genes were detected in bovine stools (3/129, 2.3%, 95% CI, 0-5.0%). S. Typhimurium was isolated from human feces (1/70, 1.4%, 95% CI, 0-4.2%) while one S. Newport and two S. Gaminara strains were recovered from one wild boar (1/3, 33%, 95% CI, 0-99%). The knowledge of prevalence and characteristics of these enteropathogens in our region would allow public health services to take adequate preventive measures.

  4. Shigella strains are not clones of Escherichia coli but sister species in the genus Escherichia.

    Science.gov (United States)

    Zuo, Guanghong; Xu, Zhao; Hao, Bailin

    2013-02-01

    Shigella species and Escherichia coli are closely related organisms. Early phenotyping experiments and several recent molecular studies put Shigella within the species E. coli. However, the whole-genome-based, alignment-free and parameter-free CVTree approach shows convincingly that four established Shigella species, Shigella boydii, Shigella sonnei, Shigella felxneri and Shigella dysenteriae, are distinct from E. coli strains, and form sister species to E. coli within the genus Escherichia. In view of the overall success and high resolution power of the CVTree approach, this result should be taken seriously. We hope that the present report may promote further in-depth study of the Shigella-E. coli relationship.

  5. The effects of antibiotic usage in food animals on the development of antimicrobial resistance of importance for humans in Campylobacter and Escherichia coli.

    Science.gov (United States)

    Aarestrup, F M; Wegener, H C

    1999-07-01

    Modern food animal production depends on use of large amounts of antibiotics for disease control. This provides favourable conditions for the spread and persistence of antimicrobial-resistant zoonotic bacteria such as Campylobacter and E. coli O157. The occurrence of antimicrobial resistance to antimicrobials used in human therapy is increasing in human pathogenic Campylobacter and E. coli from animals. There is an urgent need to implement strategies for prudent use of antibiotics in food animal production to prevent further increases in the occurrence of antimicrobial resistance in food-borne human pathogenic bacteria such as Campylobacter and E. coli.

  6. Identification of diarrheagenic Escherichia coli strains from avian organic fertilizers.

    Science.gov (United States)

    Puño-Sarmiento, Juan; Gazal, Luis Eduardo; Medeiros, Leonardo P; Nishio, Erick K; Kobayashi, Renata K T; Nakazato, Gerson

    2014-08-28

    The Brazilian poultry industry generates large amounts of organic waste, such as chicken litter, which is often used in agriculture. Among the bacteria present in organic fertilizer are members of the Enterobacteriaceae family. The objective of this study was to detect the presence of diarrheagenic Escherichia coli (DEC) strains in avian organic fertilizer, and assess the potential damage they can cause in humans due to antimicrobial resistance. The presence of DEC pathotypes and phylogenetic groups were detected by multiplex-PCR. Phenotypic assays, such as tests for adhesion, cytotoxicity activity, biofilm formation and especially antimicrobial susceptibility, were performed. Fifteen DEC strains from 64 E. coli were isolated. Among these, four strains were classified as enteropathogenic (EPEC; 6.2%), three strains as Shiga toxin-producing (STEC; 4.7%), 10 strains as enteroaggregative (EAEC; 12.5%), but two of these harbored the eaeA gene too. The low number of isolated strains was most likely due to the composting process, which reduces the number of microorganisms. These strains were able to adhere to HEp-2 and HeLa cells and produce Shiga-toxins and biofilms; in addition, some of the strains showed antimicrobial resistance, which indicates a risk of the transfer of resistance genes to human E. coli. These results showed that DEC strains isolated from avian organic fertilizers can cause human infections.

  7. Identification of Diarrheagenic Escherichia coli Strains from Avian Organic Fertilizers

    Directory of Open Access Journals (Sweden)

    Juan Puño-Sarmiento

    2014-08-01

    Full Text Available The Brazilian poultry industry generates large amounts of organic waste, such as chicken litter, which is often used in agriculture. Among the bacteria present in organic fertilizer are members of the Enterobacteriaceae family. The objective of this study was to detect the presence of diarrheagenic Escherichia coli (DEC strains in avian organic fertilizer, and assess the potential damage they can cause in humans due to antimicrobial resistance. The presence of DEC pathotypes and phylogenetic groups were detected by multiplex-PCR. Phenotypic assays, such as tests for adhesion, cytotoxicity activity, biofilm formation and especially antimicrobial susceptibility, were performed. Fifteen DEC strains from 64 E. coli were isolated. Among these, four strains were classified as enteropathogenic (EPEC; 6.2%, three strains as Shiga toxin-producing (STEC; 4.7%, 10 strains as enteroaggregative (EAEC; 12.5%, but two of these harbored the eaeA gene too. The low number of isolated strains was most likely due to the composting process, which reduces the number of microorganisms. These strains were able to adhere to HEp-2 and HeLa cells and produce Shiga-toxins and biofilms; in addition, some of the strains showed antimicrobial resistance, which indicates a risk of the transfer of resistance genes to human E. coli. These results showed that DEC strains isolated from avian organic fertilizers can cause human infections.

  8. Accumulation and efflux of polychlorinated biphenyls in Escherichia coli.

    Science.gov (United States)

    Geng, Shen; Fang, Jun; Turner, Kendrick B; Daunert, Sylvia; Wei, Yinan

    2012-06-01

    Polychlorinated biphenyls (PCBs) are environmental pollutants that have been associated with numerous adverse health effects in human and animals. Hydroxylated PCBs (HPCBs) are the product of the oxidative metabolism of PCBs. The presence of hydroxyl groups in HPCBs makes these compounds more hydrophilic than the parent PCBs. One of the best approaches to break down and remove these contaminants is bioremediation; an environmentally friendly process that uses microorganisms to degrade hazardous chemicals into non-toxic ones. In this study, we investigated the cellular accumulation and toxicity of selected PCBs and HPCBs in Gram-negative bacteria, using Escherichia coli as a model organism. We found that none of the five PCBs tested were toxic to E. coli, presumably due to their limited bioavailability. Nevertheless, different HPCBs tested showed different levels of toxicity. Furthermore, we demonstrated that the primary multidrug efflux system in E. coli, AcrAB-TolC, facilitated the efflux of HPCBs out of the cell. Since AcrAB-TolC is constitutively expressed in E. coli and is conserved in all sequenced Gram-negative bacterial genomes, our results suggest that the efflux activities of multidrug resistant pumps may affect the accumulation and degradation of PCBs in Gram-negative bacteria.

  9. Insights into the biology of Escherichia coli through structural proteomics.

    Science.gov (United States)

    Matte, Allan; Jia, Zongchao; Sunita, S; Sivaraman, J; Cygler, Miroslaw

    2007-09-01

    Escherichia coli has historically been an important organism for understanding a multitude of biological processes, and represents a model system as we attempt to simulate the workings of living cells. Many E. coli strains are also important human and animal pathogens for which new therapeutic strategies are required. For both reasons, a more complete and comprehensive understanding of the protein structure complement of E. coli is needed at the genome level. Here, we provide examples of insights into the mechanism and function of bacterial proteins that we have gained through the Bacterial Structural Genomics Initiative (BSGI), focused on medium-throughput structure determination of proteins from E. coli. We describe the structural characterization of several enzymes from the histidine biosynthetic pathway, the structures of three pseudouridine synthases, enzymes that synthesize one of the most abundant modified bases in RNA, as well as the combined use of protein structure and focused functional analysis to decipher functions for hypothetical proteins. Together, these results illustrate the power of structural genomics to contribute to a deeper biological understanding of bacterial processes.

  10. Genomic analysis of extra-intestinal pathogenic Escherichia coli urosepsis.

    Science.gov (United States)

    McNally, A; Alhashash, F; Collins, M; Alqasim, A; Paszckiewicz, K; Weston, V; Diggle, M

    2013-08-01

    Urosepsis is a bacteraemia infection caused by an organism previously causing an infection in the urinary tract of a patient, a diagnosis which has been classically confirmed by culture of the same species of bacteria from both blood and urine samples. Given the new insights afforded by sequencing technologies into the complicated population structures of infectious agents affecting humans, we sought to investigate urosepsis by comparing the genome sequences of blood and urine isolates of Escherichia coli from five patients with urosepsis. The results confirm the classical urosepsis hypothesis in four of the five cases, but also show the complex nature of extra-intestinal E. coli infection in the fifth case, where three distinct strains caused two distinct infections. Additionally, we show there is little to no variation in the bacterial genome as it progressed from urine to blood, and also present a minimal set of virulence genes required for bacteraemia in E. coli based on gene association. These suggest that most E. coli have the genetic propensity to cause bacteraemia.

  11. ENERGY REQUIREMENT FOR THYMINELESS DEATH IN CELLS OF ESCHERICHIA COLI.

    Science.gov (United States)

    FREIFELDER, D; MAALOE, O

    1964-10-01

    Freifelder, David (University of California, Berkeley), and Ole Maaløe. Energy requirement for thymineless death in cells of Escherichia coli. J. Bacteriol. 88:987-990. 1964.-Thymineless death in thymine-requiring Escherichia coli is arrested immediately and reversibly by nitrogenation if the bacterial population is growing in a medium containing a carbon source that can only be metabolized aerobically. The mechanism of death, therefore, involves a metabolic process.

  12. Prevalence of Virulent Escherichia coli Belonging B1 Phylogroup in Municipal Water Supply in Dhaka, Bangladesh

    DEFF Research Database (Denmark)

    Ferdous, Jannataul; Rashid, Ridwan Bin; Tulsiani, Suhella

    Escherichia coli is a commensal organism of the digestive tracts of many vertebrates, including humans. Contamination of drinking water with pathogenic E. coli is a serious public health concern. This study focused on the distribution of phylogenetic groups and virulence gene profile of E. coli i...

  13. Is Shiga Toxin-Negative Escherichia coli O157 : H7 Enteropathogenic or Enterohemorrhagic Escherichia coli? Comprehensive Molecular Analysis Using Whole-Genome Sequencing

    NARCIS (Netherlands)

    Ferdous, Mithila; Zhou, Kai; Mellmann, Alexander; Morabito, Stefano; Croughs, Peter D.; de Boer, Richard F.; Kooistra-Smid, Anna M. D.; Rossen, John W. A.; Friedrich, Alexander W.

    2015-01-01

    The ability of Escherichia coli O157:H7 to induce cellular damage leading to disease in humans is related to numerous virulence factors, most notably the stx gene, encoding Shiga toxin (Stx) and carried by a bacteriophage. Loss of the Stx-encoding bacteriophage may occur during infection or

  14. Is Shiga Toxin-Negative Escherichia coli O157 : H7 Enteropathogenic or Enterohemorrhagic Escherichia coli? Comprehensive Molecular Analysis Using Whole-Genome Sequencing

    NARCIS (Netherlands)

    Ferdous, Mithila; Zhou, Kai; Mellmann, Alexander; Morabito, Stefano; Croughs, Peter D.; de Boer, Richard F.; Kooistra-Smid, Anna M. D.; Rossen, John W. A.; Friedrich, Alexander W.

    2015-01-01

    The ability of Escherichia coli O157:H7 to induce cellular damage leading to disease in humans is related to numerous virulence factors, most notably the stx gene, encoding Shiga toxin (Stx) and carried by a bacteriophage. Loss of the Stx-encoding bacteriophage may occur during infection or culturin

  15. Nanotextile membranes for bacteria Escherichia coli capturing

    Directory of Open Access Journals (Sweden)

    Jaroslav Lev

    2010-01-01

    Full Text Available The article describes an experimental study dealing with the possibility of nanotextile materials usa­ge for microbiologically contaminated water filtration. The aim of the study is to verify filtration ability of different nanotextile materials and evaluate the possibilities of practical usage. Good detention ability of these materials in the air filtration is the presumption for nanotextile to be used for bacteria filtration from a liquid. High nanotextile porosity with the nanotextile pores dimensions smaller than a bacteria size predicates the possibility of a successful usage of these materials. For the experiment were used materials made from electrospinning nanofibres under the label PA612, PUR1, PUR2 s PUR3 on the supporting unwoven textiles (viscose and PP. As a model simulation of the microbial contamination, bacteria Escherichia coli was chosen. Contaminated water was filtered during the overpressure activity of 105Pa on the input side of the filter from the mentioned material. After three-day incubation on the nutrient medium, cultures found in the samples before and after filtration were compared. In the filtrated water, bacteria E. coli were indicated, which did not verify the theoretical presumptions about an absolut bacteria detention. However, used materials caught at least 94% of bacteria in case of material PUR1 and up to 99,996% in case of material PUR2. These results predict the possibility of producing effective nanotextile filters for microbiologically contaminated water filtration.Recommendation: For the production of materials with better filtrating qualities, experiments need to be done, enabling better understanding of the bacteria detention mechanisms on the nanotextile material, and parameters of the used materials that influence the filtrating abilities need to be verified.

  16. Longitudinal characterization of Escherichia coli in healthy captive nonhuman primates

    Directory of Open Access Journals (Sweden)

    Jonathan B Clayton

    2014-11-01

    Full Text Available The gastrointestinal (GI tracts of nonhuman primates are well known to harbor Escherichia coli, a known commensal of humans and animals. While E. coli is a normal inhabitant of the mammalian gut, it also exists in a number of pathogenic forms or pathotypes, including those with predisposition for the GI tract, as well the urogenital tract. Diarrhea in captive nonhuman primates (NHPs has long been a problem in both zoo settings and research colonies, including the Como Zoo. It is an animal welfare concern, as well as a public health concern. E. coli has not been extensively studied in correlation with diarrhea in captive primates; therefore, a study was performed during the summer of 2009 in collaboration with a zoo in Saint Paul, MN, which was experiencing an increased incidence and severity of diarrhea among their NHP collection. Fresh fecal samples were collected weekly from each member of the primate collection, between June and August of 2009, and E. coli were isolated. A total of 33 individuals were included in the study, representing eight species. E. coli isolates were examined for their genetic relatedness, phylogenetic relationships, plasmid replicon types, virulence gene profiles, and antimicrobial susceptibility profiles. A number of isolates were identified containing virulence genes commonly found in several different E. coli pathotypes, and there was evidence of clonal transmission of isolates between animals and over time. Overall, the manifestation of chronic diarrhea in the Como Zoo primate collection is a complex problem whose solution will require regular screening for microbial agents and consideration of environmental causes. This study provides some insight towards the sharing of enteric bacteria between such animals.

  17. Annual Surveillance Summary: Escherichia coli (E. coli) Infections in the Military Health System (MHS), 2016

    Science.gov (United States)

    2017-06-30

    Annual Surveillance Summary: Escherichia coli (E. coli) Infections in the Military Health System (MHS...and prevalence among all beneficiaries seeking care within the Military Health System (MHS). This report describes demographics, clinical...linked to assess descriptive and clinical factors related to E. coli. Health Level 7 (HL7)-formatted Composite Health Care System (CHCS) microbiology data

  18. Investigating the Antibacterial Effects of Plant Extracts on Pseudomonas aeruginosa and Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jahani

    2016-04-01

    Full Text Available Background Scientists are seeking an appropriate alternative method for curing infections caused by resistant bacteria, since drug resistance is continually increasing. Objectives This research aims to discover the function of some medicine plants on pestiferous Pseudomonas aeruginosa and Escherichia coli in humans. Materials and Methods Bacterial strains were obtained from a standard laboratory. The strains of Pseudomonas aeruginosa ATCC27853 and E.coli ATCC25922 bacteria were used for antimicrobial testing of the extractions. Results Our results showed that Teucrium polium extracts have the minimum density of inhibitory for Escherichia coli, 25 ppm, whereas the maximum of this is for Peganum harmala and Prangos ferulaceae with 100 ppm. The lowest minimum concentration inhibitory value of extracts P. harmala, T. polium, T. pratensis and Rumex was found in 25 ppm against P.aeruginosa. Conclusions The results of our study showed that plant extracts have good antibacterial properties against Pseudomonas aeruginosa and Escherichia coli.

  19. (ESBL)-producing Escherichia coli isolated from cl

    African Journals Online (AJOL)

    spectrum beta-lactamase (ESBL)-producing Escherichia coli isolated from clinical ... Methods: A total of 127 E. coli were collected from clinical samples in Kerman hospitals. The antibiotic ..... in Mexico and 26.1 % in Turkey [23,24]. These.

  20. Efficient expression of the yeast metallothionein gene in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Berka, T.; Shatzman, A.; Zimmerman, J.; Strickler, J.; Rosenberg, M.

    1988-01-01

    The yeast metallothionein gene CUP1 was cloned into a bacterial expression system to achieve efficient, controlled expression of the stable, unprocessed protein product. The Escherichia coli-synthesized yeast metallothionein bound copper, cadmium, zinc, indicating that the protein was functional. Furthermore, E. coli cells expressing CUP1 acquired a new, inducible ability to selectively sequester heavy metal ions from the growth medium.

  1. Antimicrobial activity of peptidomimetics against multidrug-resistant Escherichia coli

    DEFF Research Database (Denmark)

    Jahnsen, Rasmus D; Frimodt-Møller, Niels; Franzyk, Henrik

    2012-01-01

    -lactamase-producing Escherichia coli was assessed by testing an array comprising different types of cationic peptidomimetics obtained by a general monomer-based solid-phase synthesis protocol. Most of the peptidomimetics possessed high to moderate activity toward multidrug-resistant E. coli as opposed to the corresponding...

  2. Characterization of enterohemorrhagic Escherichia coli on veal hides and carcasses

    Science.gov (United States)

    Enterohemorrhagic E. coli (EHEC) are Shiga toxin–producing Escherichia coli (STEC) associated with the most severe forms of foodborne illnesses. The United States Department of Agriculture (USDA) Food Safety Inspection Service (FSIS) has identified a higher percentage of non-O157 EHEC compared to E....

  3. Escherichia coli Eyelid Abscess in a Patient with Alcoholic Cirrhosis

    Directory of Open Access Journals (Sweden)

    Matthew Stratton

    2015-01-01

    Full Text Available Escherichia coli (E. coli is a rare cause of ocular infections and has not yet been reported as a cause of an ocular abscess. We describe the case of a 47-year-old woman with a history of alcoholic cirrhosis who presented with painful left lower eyelid swelling that did not improve with oral antibiotics. The abscess was drained and cultures were positive for E. coli. Patients with cirrhosis are at increased risk for developing E. coli bacterial infections, but to our knowledge this is the first case of an E. coli eyelid abscess reported in the literature.

  4. {sup 99m}Technetium labelled Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Diniz, S.O.F.; Cardoso, V.N. [Radioisotope Laboratory, Faculty of Pharmacy/UFMG, Belo Horizonte (Brazil); Resende, B.M.; Nunan, E.A. [Biological Control Laboratory, Faculty of Pharmacy/UFMG, Belo Horizonte (Brazil); Simal, C.J.R. [Laboratory Nuclear Medicine, Faculty of Medicine/UFMG, Belo Horizonte (Brazil)

    1999-07-01

    Samples of a culture of unlabeled Escherichia coli were incubated with different concentrations of stannous chloride for various time periods. {sup 99m}Tc (26.0 MBq) was added to each preparation and the results showed a labelling yield of 98% for E. coli. Since the bacterial viability of {sup 99m}Tc-E. coli and E. coli did not show any statistical differences, these results demonstrate that labelling of E. coli with {sup 99m}Tc does not modify the bacterial viability, and the radiolabelled bacteria may be a good model to study bacterial translocation.

  5. Role of verocytotoxigenic Escherichia coli in the swine production chain

    Directory of Open Access Journals (Sweden)

    Laura Ercoli

    2015-06-01

    Full Text Available Shiga toxin-producing Escherichia coli (STEC can cause severe clinical diseases in humans, such as haemorrhagic colitis (HC and haemolytic-uremic syndrome (HUS. Although ruminants, primarily cattle, have been suggested as typical reservoirs of STEC, many food products of other origins, including pork products, have been confirmed as vehicles for STEC transmission. Only in rare cases, pork consumption is associated with severe clinical symptoms caused by high pathogenic STEC strains. However, in these outbreaks, it is unknown whether the contamination of food products occurs during swine processing or via cross-contamination from foodstuffs of different sources. In swine, STEC plays an important role in the pathogenesis of oedema disease. In particular a Shiga toxin subtype, named stx2e, it is considered as a key factor involved in the damage of swine endothelial cells. On the contrary, stx2e-producing Escherichia coli has rarely been isolated in humans, and usually only from asymptomatic carriers or from patients with mild symptoms, such as uncomplicated diarrhoea. In fact, the presence of gene stx2e, encoding for stx2e, has rarely been reported in STEC strains that cause HUS. Moreover, stx2e-producing STEC isolated from humans and pigs were found to differ in serogroup, their virulence profile and interaction with intestinal epithelial cells. Because of the limited epidemiologic data of STEC in swine and the increasing role of non-O157 STEC in human illnesses, the relationship between swine STEC and human disease needs to be further investigated.

  6. Molecular Evolutionary Relationships of Enteroinvasive Escherichia coli and Shigella spp.

    OpenAIRE

    Lan, Ruiting; Alles, M. Chehani; Donohoe, Kathy; Marina B Martinez; Reeves, Peter R.

    2004-01-01

    Enteroinvasive Escherichia coli (EIEC), a distinctive pathogenic form of E. coli causing dysentery, is similar in many properties to bacteria placed in the four species of Shigella. Shigella has been separated as a genus but in fact comprises several clones of E. coli. The evolutionary relationships of 32 EIEC strains of 12 serotypes have been determined by sequencing of four housekeeping genes and two plasmid genes which were used previously to determine the relationships of Shigella strains...

  7. Findings of Escherichia coli and Enterococcus spp. in homemade cheese

    Directory of Open Access Journals (Sweden)

    Tambur Zoran

    2007-01-01

    Full Text Available During the period from February until March 2004, 108 samples of soft cheese originating from markets of Pancevo, Subotica and Belgrade were examined. Microbiological analyses of the cheese samples to the presence of Escherichia coli was performed using methods described in the Regulations on methods for performing microbiological analyses and super analyses of consumer articles, while the presence of bacteria Enteroccocus spp. was performed on the dexter agar. From 108 samples of soft cheese from the territories of Pancevo, Belgrade and Subotica were isolated: Enterococcus spp. from 96% and Escherichia coli from 69%, cheese samples. Verocytotoxic E.coli was not isolated from any of the taken cheese samples.

  8. Impact of CD4+ T Cell Responses on Clinical Outcome following Oral Administration of Wild-Type Enterotoxigenic Escherichia coli in Humans.

    Directory of Open Access Journals (Sweden)

    Monica A McArthur

    2017-01-01

    Full Text Available Enterotoxigenic Escherichia coli (ETEC is a non-invasive enteric pathogen of considerable public health importance, being one of the most common attributable causes of diarrheal illness in infants and young children in developing countries and the most common cause of traveler's diarrhea. To enhance study-to-study consistency of our experimental challenge model of ETEC in volunteers, and to allow concomitant multi-site trials to evaluate anti-ETEC immunoprophylactic products, hundreds of vials, each containing a standardized inoculum of virulent wild-type (wt ETEC strain H10407 (serotype O78:H11 expressing colonization factor antigen I and heat-labile and heat-stable enterotoxins, were prepared under current Good Manufacturing Practices (cGMP and frozen. Following thawing, the contents of each vial can be used (diluted as necessary to prepare consistent challenge inoculum, even at different study sites. A preliminary human experimental challenge study using this cGMP inoculum was conducted on a research isolation ward and the clinical and cell-mediated immune responses evaluated. Of the 6 healthy adult volunteers challenged 83% (5/6 developed diarrhea and 50% developed moderate-to-severe diarrhea (MSD. Moderate and severe diarrhea were defined as passage of ≥ 1 liter or ≥ 3 liters of diarrheal stool respectively. We compared the CD4+ T cell responses of volunteers who developed MSD against those who did not and identified significant differences in ETEC-specific cytokine production and gut homing potential. We furthermore demonstrated that increased expression of the gut-homing molecule integrin α4β7 by peripheral T follicular helper cells (pTfh correlated with decreased stool volume and increased ETEC-specific IgA B memory cell (BM development. Collectively, despite small numbers of volunteers, our results indicate a potential role for CD4+ T cells, in particular pTfh, in modulating disease outcome following exposure to wt ETEC in a

  9. Enhancing toxic protein expression in Escherichia coli fed-batch culture using kinetic parameters: Human granulocyte-macrophage colony-stimulating factor as a model system.

    Science.gov (United States)

    Khasa, Yogender Pal; Khushoo, Amardeep; Mukherjee, Krishna Jyoti

    2013-03-01

    The kinetics of recombinant human granulocyte-macrophage colony-stimulating factor (hGM-CSF) expression was studied under the strong T7 promoter in continuous culture of Escherichia coli using complex medium to design an optimum feeding strategy for high cell density cultivation. Continuous culture studies were done at different dilution rates and the growth and product formation profiles were monitored post-induction. Recombinant protein expression was in the form of inclusion bodies with a maximum specific product formation rate (q(p)) of 63.5 mg g(-1) DCW h(-1) at a dilution rate (D) of 0.3 h(-1). The maximum volumetric product concentration achieved at this dilution rate was 474 mg l(-1), which translated a ~1.4 and ~1.75 folds increase than the values obtained at dilution rates of 0.2 h(-1) and 0.4 h(-1) respectively. The specific product yield (Y(P/x)) peaked at 138 mg g(-1) DCW, demonstrating a ~1.6 folds increase in the values obtained at other dilution rates. A drop in q(p) was observed within 5-6 h of induction at all the dilution rates, possibly due to protein toxicity and metabolic stress associated with protein expression. The data from the continuous culture studies allowed us to design an optimal feeding strategy and induction time in fed-batch cultures which resulted in a maximum product concentration of 3.95 g l(-1) with a specific hGM-CSF yield (Y(P/x)) of 107 mg g(-1) DCW.

  10. Homo-trimeric Structure of the Type IVb Minor Pilin CofB Suggests Mechanism of CFA/III Pilus Assembly in Human Enterotoxigenic Escherichia coli.

    Science.gov (United States)

    Kawahara, Kazuki; Oki, Hiroya; Fukakusa, Shunsuke; Yoshida, Takuya; Imai, Tomoya; Maruno, Takahiro; Kobayashi, Yuji; Motooka, Daisuke; Iida, Tetsuya; Ohkubo, Tadayasu; Nakamura, Shota

    2016-03-27

    In gram-negative bacteria, the assembly of type IV pilus (T4P) and the evolutionally related pseudopilus of type II secretion system involves specialized structural proteins called pilins and pseudopilins, respectively, and is dynamically regulated to promote bacterial pathogenesis. Previous studies have suggested that a structural "tip"-like hetero-complex formed through the interaction of at least three minor (pseudo) pilins plays an important role in this process, while some members of the pathogenic type IVb subfamily are known to have only one such minor pilin subunit whose function is still unknown. Here, we determined the crystal structure of the type IVb minor pilin CofB of colonization factor antigen/III from human enterotoxigenic Escherichia coli at 1.88-Å resolution. The crystal structure, in conjunction with physicochemical analysis in solution, reveals a symmetrical homo-trimeric arrangement distinct from the hetero-complexes of minor (pseudo) pilins observed in other T4P and type II secretion systems. Each CofB monomer adopts a unique three-domain architecture, in which the C-terminal β-sheet-rich lectin domain can effectively initiate trimer association of its pilin-like N-terminal domain through extensive hydrophobic interactions followed by domain swapping at the central hinge-like domain. Deletion of cofB produces a phenotype with no detectable pili formation on the cell surface, while molecular modeling indicates that the characteristic homo-trimeric structure of CofB is well situated at the pilus tip of colonization factor antigen/III formed by the major pilin CofA, suggesting a role for the minor pilin in the efficient initiation of T4P assembly. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Impact of CD4+ T Cell Responses on Clinical Outcome following Oral Administration of Wild-Type Enterotoxigenic Escherichia coli in Humans

    Science.gov (United States)

    Chen, Wilbur H.; Magder, Laurence; Levine, Myron M.; Sztein, Marcelo B.

    2017-01-01

    Enterotoxigenic Escherichia coli (ETEC) is a non-invasive enteric pathogen of considerable public health importance, being one of the most common attributable causes of diarrheal illness in infants and young children in developing countries and the most common cause of traveler’s diarrhea. To enhance study-to-study consistency of our experimental challenge model of ETEC in volunteers, and to allow concomitant multi-site trials to evaluate anti-ETEC immunoprophylactic products, hundreds of vials, each containing a standardized inoculum of virulent wild-type (wt) ETEC strain H10407 (serotype O78:H11 expressing colonization factor antigen I and heat-labile and heat-stable enterotoxins), were prepared under current Good Manufacturing Practices (cGMP) and frozen. Following thawing, the contents of each vial can be used (diluted as necessary) to prepare consistent challenge inoculum, even at different study sites. A preliminary human experimental challenge study using this cGMP inoculum was conducted on a research isolation ward and the clinical and cell-mediated immune responses evaluated. Of the 6 healthy adult volunteers challenged 83% (5/6) developed diarrhea and 50% developed moderate-to-severe diarrhea (MSD). Moderate and severe diarrhea were defined as passage of ≥ 1 liter or ≥ 3 liters of diarrheal stool respectively. We compared the CD4+ T cell responses of volunteers who developed MSD against those who did not and identified significant differences in ETEC-specific cytokine production and gut homing potential. We furthermore demonstrated that increased expression of the gut-homing molecule integrin α4β7 by peripheral T follicular helper cells (pTfh) correlated with decreased stool volume and increased ETEC-specific IgA B memory cell (BM) development. Collectively, despite small numbers of volunteers, our results indicate a potential role for CD4+ T cells, in particular pTfh, in modulating disease outcome following exposure to wt ETEC in a volunteer

  12. Free RNA polymerase in Escherichia coli.

    Science.gov (United States)

    Patrick, Michael; Dennis, Patrick P; Ehrenberg, Mans; Bremer, Hans

    2015-12-01

    The frequencies of transcription initiation of regulated and constitutive genes depend on the concentration of free RNA polymerase holoenzyme [Rf] near their promoters. Although RNA polymerase is largely confined to the nucleoid, it is difficult to determine absolute concentrations of [Rf] at particular locations within the nucleoid structure. However, relative concentrations of free RNA polymerase at different growth rates, [Rf]rel, can be estimated from the activities of constitutive promoters. Previous studies indicated that the rrnB P2 promoter is constitutive and that [Rf]rel in the vicinity of rrnB P2 increases with increasing growth rate. Recently it has become possible to directly visualize Rf in growing Escherichia coli cells. Here we examine some of the important issues relating to gene expression based on these new observations. We conclude that: (i) At a growth rate of 2 doublings/h, there are about 1000 free and 2350 non-specifically DNA-bound RNA polymerase molecules per average cell (12 and 28%, respectively, of 8400 total) which are in rapid equilibrium. (ii) The reversibility of the non-specific binding generates more than 1000 free RNA polymerase molecules every second in the immediate vicinity of the DNA. Of these, most rebind non-specifically to the DNA within a few ms; the frequency of non-specific binding is at least two orders of magnitude greater than specific binding and transcript initiation. (iii) At a given amount of RNA polymerase per cell, [Rf] and the density of non-specifically DNA-bound RNA polymerase molecules along the DNA both vary reciprocally with the amount of DNA in the cell. (iv) At 2 doublings/h an E. coli cell contains, on the average, about 1 non-specifically bound RNA polymerase per 9 kbp of DNA and 1 free RNA polymerase per 20 kbp of DNA. However some DNA regions (i.e. near active rRNA operons) may have significantly higher than average [Rf].

  13. Apramycin treatment affects selection and spread of a multidrug-resistant Escherichia coli strain able to colonize the human gut in the intestinal microbiota of pigs

    DEFF Research Database (Denmark)

    Herrero-Fresno, Ana; Zachariasen, Camilla; Hansen, Monica Hegstad;

    2016-01-01

    The effect of apramycin treatment on transfer and selection of an Escherichia coli strain (E. coli 912) in the intestine of pigs was analyzed through an in vivo experiment. The strain was sequenced and assigned to the sequence type ST101 and serotype O11. It carried resistance genes to apramycin...... of treatment, and apramycin treatment resulted in significantly higher counts compared to the non-treated group. This represents the first demonstration of how antimicrobial treatment affects spread of resistant bacteria in pig production. The use of apramycin may lead to enhanced spread of gentamicin-resistant......-treated (pen 3), along with a non-inoculated control group (pen 1). Two pigs of pen 2 and 3 were inoculated intragastrically with a rifampicin resistant variant of the strain. Apramycin treatment in pen 2 was initiated immediately after inoculation. Strain colonization was assessed in the feces from all pigs...

  14. An end-joining repair mechanism in Escherichia coli

    Science.gov (United States)

    Chayot, Romain; Montagne, Benjamin; Mazel, Didier; Ricchetti, Miria

    2010-01-01

    Bridging broken DNA ends via nonhomologous end-joining (NHEJ) contributes to the evolution and stability of eukaryote genomes. Although some bacteria possess a simplified NHEJ mechanism, the human commensal Escherichia coli is thought to rely exclusively on homology-directed mechanisms to repair DNA double-strand breaks (DSBs). We show here that laboratory and pathogenic E. coli strains possess a distinct end-joining activity that repairs DSBs and generates genome rearrangements. This mechanism, named alternative end-joining (A-EJ), does not rely on the key NHEJ proteins Ku and Ligase-D which are absent in E. coli. Differently from classical NHEJ, A-EJ is characterized by extensive end-resection largely due to RecBCD, by overwhelming usage of microhomology and extremely rare DNA synthesis. We also show that A-EJ is dependent on the essential Ligase-A and independent on Ligase-B. Importantly, mutagenic repair requires a functional Ligase-A. Although generally mutagenic, accurate A-EJ also occurs and is frequent in some pathogenic bacteria. Furthermore, we show the acquisition of an antibiotic-resistance gene via A-EJ, refuting the notion that bacteria gain exogenous sequences only by recombination-dependent mechanisms. This finding demonstrates that E. coli can integrate unrelated, nonhomologous exogenous sequences by end-joining and it provides an alternative strategy for horizontal gene transfer in the bacterial genome. Thus, A-EJ contributes to bacterial genome evolution and adaptation to environmental challenges. Interestingly, the key features of A-EJ also appear in A-NHEJ, an alternative end-joining mechanism implicated in chromosomal translocations associated with human malignancies, and we propose that this mutagenic repair might have originated in bacteria. PMID:20133858

  15. The effects of antibiotic usage in food animals on the development of antimicrobial resistance of importance for humans in Campylobacter and Escherichia coli

    DEFF Research Database (Denmark)

    Aarestrup, Frank Møller; Wegener, Henrik Caspar

    1999-01-01

    Modern food animal production depends on use of large amounts of antibiotics for disease control. This provides favourable conditions for the spread and persistence of antimicrobial-resistant zoonotic bacteria such as Campylobacter and E. coli O157. The occurrence of antimicrobial resistance...... to antimicrobials used in human therapy is increasing in human pathogenic Campylobacter and E. coli from animals. There is an urgent need to implement strategies for prudent use of antibiotics in food animal production to prevent further increases in the occurrence of antimicrobial resistance in food-borne human...

  16. Escherichia coli Heat-Stable Enterotoxin Mediates Na+/H+ Exchanger 4 Inhibition Involving cAMP in T84 Human Intestinal Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Ana R Beltrán

    Full Text Available The enterotoxigenic Escherichia coli strains lead to diarrhoea in humans due to heat-labile and heat-stable (STa enterotoxins. STa increases Cl-release in intestinal cells, including the human colonic carcinoma T84 cell line, involving increased cGMP and membrane alkalization due to reduced Na+/H+ exchangers (NHEs activity. Since NHEs modulate intracellular pH (pHi, and NHE1, NHE2, and NHE4 are expressed in T84 cells, we characterized the STa role as modulator of these exchangers. pHi was assayed by the NH4Cl pulse technique and measured by fluorescence microscopy in BCECF-preloaded cells. pHi recovery rate (dpHi/dt was determined in the absence or presence of 0.25 μmol/L STa (30 minutes, 25 μmol/L HOE-694 (concentration inhibiting NHE1 and NHE2, 500 μmol/L sodium nitroprusside (SNP, spontaneous nitric oxide donor, 100 μmol/L dibutyryl cyclic GMP (db-cGMP, 100 nmol/L H89 (protein kinase A inhibitor, or 10 μmol/L forskolin (adenylyl cyclase activator. cGMP and cAMP were measured in cell extracts by radioimmunoassay, and buffering capacity (ßi and H+ efflux (JH+ was determined. NHE4 protein abundance was determined by western blotting. STa and HOE-694 caused comparable reduction in dpHi/dt and JH+ (~63%, without altering basal pHi (range 7.144-7.172. STa did not alter ßi value in a range of 1.6 pHi units. The dpHi/dt and JH+ was almost abolished (~94% inhibition by STa + HOE-694. STa effect was unaltered by db-cGMP or SNP. However, STa and forskolin increased cAMP level. STa-decreased dpHi/dt and JH+ was mimicked by forskolin, and STa + HOE-694 effect was abolished by H89. Thus, incubation of T84 cells with STa results in reduced NHE4 activity leading to a lower capacity of pHi recovery requiring cAMP, but not cGMP. STa effect results in a causal phenomenon (STa/increased cAMP/increased PKA activity/reduced NHE4 activity ending with intracellular acidification that could have consequences in the gastrointestinal cells function promoting

  17. Escherichia coli Heat-Stable Enterotoxin Mediates Na+/H+ Exchanger 4 Inhibition Involving cAMP in T84 Human Intestinal Epithelial Cells.

    Science.gov (United States)

    Beltrán, Ana R; Carraro-Lacroix, Luciene R; Bezerra, Camila N A; Cornejo, Marcelo; Norambuena, Katrina; Toledo, Fernando; Araos, Joaquín; Pardo, Fabián; Leiva, Andrea; Sanhueza, Carlos; Malnic, Gerhard; Sobrevia, Luis; Ramírez, Marco A

    2015-01-01

    The enterotoxigenic Escherichia coli strains lead to diarrhoea in humans due to heat-labile and heat-stable (STa) enterotoxins. STa increases Cl-release in intestinal cells, including the human colonic carcinoma T84 cell line, involving increased cGMP and membrane alkalization due to reduced Na+/H+ exchangers (NHEs) activity. Since NHEs modulate intracellular pH (pHi), and NHE1, NHE2, and NHE4 are expressed in T84 cells, we characterized the STa role as modulator of these exchangers. pHi was assayed by the NH4Cl pulse technique and measured by fluorescence microscopy in BCECF-preloaded cells. pHi recovery rate (dpHi/dt) was determined in the absence or presence of 0.25 μmol/L STa (30 minutes), 25 μmol/L HOE-694 (concentration inhibiting NHE1 and NHE2), 500 μmol/L sodium nitroprusside (SNP, spontaneous nitric oxide donor), 100 μmol/L dibutyryl cyclic GMP (db-cGMP), 100 nmol/L H89 (protein kinase A inhibitor), or 10 μmol/L forskolin (adenylyl cyclase activator). cGMP and cAMP were measured in cell extracts by radioimmunoassay, and buffering capacity (ßi) and H+ efflux (JH+) was determined. NHE4 protein abundance was determined by western blotting. STa and HOE-694 caused comparable reduction in dpHi/dt and JH+ (~63%), without altering basal pHi (range 7.144-7.172). STa did not alter ßi value in a range of 1.6 pHi units. The dpHi/dt and JH+ was almost abolished (~94% inhibition) by STa + HOE-694. STa effect was unaltered by db-cGMP or SNP. However, STa and forskolin increased cAMP level. STa-decreased dpHi/dt and JH+ was mimicked by forskolin, and STa + HOE-694 effect was abolished by H89. Thus, incubation of T84 cells with STa results in reduced NHE4 activity leading to a lower capacity of pHi recovery requiring cAMP, but not cGMP. STa effect results in a causal phenomenon (STa/increased cAMP/increased PKA activity/reduced NHE4 activity) ending with intracellular acidification that could have consequences in the gastrointestinal cells function promoting human

  18. Escherichia coli O157:H7 - An Emerging Pathogen in foods of Animal Origin

    Directory of Open Access Journals (Sweden)

    Ch. Bindu Kiranmayi

    Full Text Available Escherichia coli O157:H7 is an emerging public health concern in most countries of the world. E. coli O157:H7 was known to be a human pathogen for nearly 24 years. EHEC O157 infection is estimated to be the fourth most costly food borne disease in Canada and USA, not counting the cost of possible litigation. E. coli O157:H7 and Salmonella are the leading causes of produce related outbreaks, accounting for 20 and 30% respectively. The authority of the Federal Meat Inspection Act, FSIS (Food Safety and Inspection Service declared Escherichia coli O157:H7, an adulterant in raw ground beef and enforced “zero tolerance” (USDA-FSIS, 17 December 1998. Because of the severity of these illnesses and the apparent low infective dose (less than 10 cells, Escherichia coli O157:H7 is considered one of the most serious of known food borne pathogens. Escherichia coli O157:H7 is mainly pathogenic to human but in cattle and other animals, it did not induce any clinical disease except diarrhea. So, these animals act as carriers to Escherichia coli O157:H7. The majority transmission is through eating of undercooked contaminated ground meat and consumption of raw milk, raw vegetables, fruits contaminated by water, cheese, curd and also through consumption of sprouts, lettuce and juice. The conventional isolation procedure includes growth in enrichment broth like modified EC (E. coli broth or modified tryptic soy broth (mTSB Since the infection primarily occurs via faeco-oral route, the preventive measures include food hygiene measures like proper cooking of meat, consumption of pasteurized milk, washing fruits and vegetables especially those to be eaten raw and drinking chlorine treated water and personnel hygiene measures like washing hands after toilet visits. [Veterinary World 2010; 3(8.000: 382-389

  19. Rapid Sterilization of Escherichia coli by Solution Plasma Process

    Science.gov (United States)

    Andreeva, Nina; Ishizaki, Takahiro; Baroch, Pavel; Saito, Nagahiro

    2012-12-01

    Solution plasma (SP), which is a discharge in the liquid phase, has the potential for rapid sterilization of water without chemical agents. The discharge showed a strong sterilization performance against Escherichia coli bacteria. The decimal value (D value) of the reduction time for E. coli by this system with an electrode distance of 1.0 mm was estimated to be approximately 1.0 min. Our discharge system in the liquid phase caused no physical damage to the E. coli and only a small increase in the temperature of the aqueous solution. The UV light generated by the discharge was an important factor in the sterilization of E. coli.

  20. Detection of Escherichia coli in wastewater based on enzyme immunoassay

    Institute of Scientific and Technical Information of China (English)

    XI Haiyan; CAI Qiang; HE Miao; SHI Hanchang

    2007-01-01

    This research describes a fast detection method on the basis of enzyme-linked immunosorbent assay (ELISA)for Escherichia coli in drainage of wastewater treatment plants.Optimized conditions such as the reaction format(sandwich or direct),the concentrations of diluted horseradish peroxidase (HRP)-E.coli conjugate,and anti-HPR antibody and pretreatment of E.coli were studied.Those results showed that the linear range of detection for E.coli was 10 cfu/mL-6×104 cfu/mL.Compared with conventional methods,it is a convenient and sensitive detection method with low cost.

  1. Fluorogenic assay for rapid detection of Escherichia coli in food.

    OpenAIRE

    1985-01-01

    An assay procedure to screen for Escherichia coli in foods by using 4-methylumbelliferyl-beta-D-glucuronide (MUG) incorporated into lauryl tryptose (LST) broth was evaluated. The beta-glucuronidase produced by E. coli cleaves the MUG substrate to yield a fluorescent end product. E. coli-negative samples can be identified by lack of fluorescence in LST-MUG within 24 h. MUG was not inhibitory to coliforms and E. coli. Over 1,400 food and dairy samples were tested to compare the standard three-t...

  2. Uropathogenic Escherichia coli Epigenetically Manipulate Host Cell Death Pathways.

    Science.gov (United States)

    Zhang, Zhengguo; Wang, Ming; Eisel, Florian; Tchatalbachev, Svetlin; Chakraborty, Trinad; Meinhardt, Andreas; Bhushan, Sudhanshu

    2016-04-01

    Urinary tract infections caused by uropathogenic Escherichia coli (UPEC) pathovars belong to the most frequent infections in human. It is well established that UPEC can subvert innate immune responses, but the role of UPEC in interfering with host cell death pathways is not known. Here, we show that UPEC abrogates activation of the host cell prosurvival protein kinase B signaling pathway, which results in the activation of mammalian forkhead box O (FOXO) transcription factors. Although FOXOs were localized in the nucleus and showed increased DNA-binding activity, no change in the expression levels of FOXO target genes were observed. UPEC can suppress BIM expression induced by LY249002, which results in attenuation of caspase 3 activation and blockage of apoptosis. Mechanistically, BIM expression appears to be epigenetically silenced by a decrease in histone 4 acetylation at the BIM promoter site. Taken together, these results suggest that UPEC can epigenetically silence BIM expression, a molecular switch that prevents apoptosis.

  3. Expression and purification of recombinant hemoglobin in Escherichia coli

    DEFF Research Database (Denmark)

    Natarajan, Chandrasekhar; Jiang, Xiaoben; Fago, Angela

    2011-01-01

    BACKGROUND: Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb) and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe...... a protocol for expressing Hbs with low intrinsic solubilities. Since the alpha- and beta-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression...... of wildtype and mutant Hbs of other species. METHODOLOGY/PRINCIPAL FINDINGS: As a test case for our expression system, we produced recombinant Hbs of the deer mouse (Peromyscus maniculatus), a species that has been the subject of research on mechanisms of Hb adaptation to hypoxia. By experimentally assessing...

  4. Presence of Escherichia coli O157 and O157:H7 in raw milk and Van herby cheese

    Directory of Open Access Journals (Sweden)

    Sancak Yakup Can

    2015-12-01

    Full Text Available The Shiga toxin-producing Escherichia coli (STEC strains are currently considered important emerging pathogens threatening public health. Among Shiga toxin-producing Escherichia coli, E. coli O157:H7 strains have emerged as important human pathogens. This study was conducted to determine the presence of Escherichia coli O157 and O157:H7 in raw milk samples and Van herby cheese samples. For this purpose, 100 samples of raw milk were collected and 100 samples of herby cheese sold for consumption in Van province in Turkey were obtained from grocers and markets in order to detect the presence of Escherichia coli O157 and O157:H7. The method of E. coli O157 and O157:H7 isolation proposed by the Food and Drug Administration (FDA was used. E. coli O157 in raw milk and herby cheese samples was found in 11% and 6% of samples respectively, and E. coli O157:H7 was found in 2% of herby cheese samples. No E. coli O157:H7 was detected in raw milk samples. This study showed that raw milk was contaminated with E. coli O157 and herby cheese was contaminated with both E. coli O157 and E. coli O157:H7; therefore, herby cheese poses a serious risk to public health.

  5. Ruminant Gastrointestinal Cell Proliferation and Clearance of Escherichia coli O157:H7

    OpenAIRE

    Magnuson, Bernadene A.; Davis, Margaret; Hubele, Suzanna; Austin, Paula R.; Kudva, Indira T.; Christopher J Williams; Hunt, Carl W.; Hovde, Carolyn J.

    2000-01-01

    Human infections with Escherichia coli O157:H7 cause hemorrhagic colitis that can progress to a life-threatening sequelae. The most common mode of disease transmission is ingestion of contaminated bovine food products, and it is well established that E. coli O157:H7 is a transient member of the bovine microbiota. However, the conditions that induce acquisition and subsequent clearance of this bacterium from the ruminant gastrointestinal tract (GIT) are not understood. Evidence that the rates ...

  6. Draft Genome Sequences of Escherichia coli Isolates from Wounded Military Personnel.

    Science.gov (United States)

    Arivett, Brock A; Ream, Dave C; Fiester, Steven E; Kidane, Destaalem; Actis, Luis A

    2016-08-11

    Members of the Escherichia coli bacterial family have been grouped as ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens because of their extensive drug resistance phenotypes and increasing threat to human health. The genomes of six extended-spectrum β-lactamase (ESBL)-producing E. coli strains isolated from wounded military personnel were sequenced and annotated.

  7. Enteroaggregative Escherichia coli O78:H10, the Cause of an Outbreak of Urinary Tract Infection

    OpenAIRE

    Olesen, Bente; Scheutz, Flemming; Andersen, Rebecca L.; Menard, Megan; Boisen, Nadia; Johnston, Brian; Hansen, Dennis S.; Krogfelt, Karen A.; Nataro, James P.; Johnson, James R

    2012-01-01

    In 1991, multiresistant Escherichia coli O78:H10 strains caused an outbreak of urinary tract infections in Copenhagen, Denmark. The phylogenetic origin, clonal background, and virulence characteristics of the outbreak isolates, and their relationship to nonoutbreak O78:H10 strains according to these traits and resistance profiles, are unknown. Accordingly, we extensively characterized 51 archived E. coli O78:H10 isolates (48 human isolates from seven countries, including 19 Copenhagen outbrea...

  8. Determination of metabolites of diosmetin-7-O-glucoside by a newly isolated Escherichia coli from human gut using UPLC-Q-TOF/MS.

    Science.gov (United States)

    Zhao, Min; Du, Leyue; Tao, Jinhua; Qian, Dawei; Shang, Er-xin; Jiang, Shu; Guo, Jianming; Liu, Pei; Su, Shu-lan; Duan, Jin-ao

    2014-11-26

    Different human intestinal bacteria were isolated and screened for their ability to transform diosmetin-7-O-glucoside. A Gram-negative anaerobic bacterium, strain 4, capable of metabolizing diosmetin-7-O-glucoside was newly isolated. Its 16S rRNA gene sequence displayed 99% similarity with that of Escherichia. Then strain 4 was identified as a species of the genus Escherichia and was named Escherichia sp. 4. Additionally, an ultraperformance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF/MS) technique combined with Metabolynx software method was established to screen the metabolites of diosmetin-7-O-glucoside. Comparing the retention time and MS/MS spectrum, three metabolites were detected and tentatively identified. These metabolites were acquired by four proposed metabolic pathways including dehydroxylation, deglycosylation, methylation, and acetylation. Diosmetin-7-O-glucoside was mainly bioconverted to considerable amounts of diosmetin and minor amounts of acacetin by the majority of the isolated intestinal bacteria such as Escherichia sp. 4. Subsequently, several strains could degrade acacetin to produce methylated and acetylated acacetin. The metabolites and metabolic pathways of diosmetin-7-O-glucoside by human intestinal bacterium Escherichia sp. 4 were first investigated.

  9. Both group 4 capsule and lipopolysaccharide O-antigen contribute to enteropathogenic Escherichia coli resistance to human α-defensin 5.

    Directory of Open Access Journals (Sweden)

    Jenny-Lee Thomassin

    Full Text Available Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC are food-borne pathogens that colonize the small intestine and colon, respectively. To cause disease, these pathogens must overcome the action of different host antimicrobial peptides (AMPs secreted into these distinct niches. We have shown previously that EHEC expresses high levels of the OmpT protease to inactivate the human cathelicidin LL-37, an AMP present in the colon. In this study, we investigate the mechanisms used by EPEC to resist human α-defensin 5 (HD-5, the most abundant AMP in the small intestine. Quantitative PCR was used to measure transcript levels of various EPEC surface structures. High transcript levels of gfcA, a gene required for group 4 capsule (G4C production, were observed in EPEC, but not in EHEC. The unencapsulated EPEC ∆gfcA and EHEC wild-type strains were more susceptible to HD-5 than EPEC wild-type. Since the G4C is composed of the same sugar repeats as the lipopolysaccharide O-antigen, an -antigen ligase (waaL deletion mutant was generated in EPEC to assess its role in HD-5 resistance. The ∆waaL EPEC strain was more susceptible to HD-5 than both the wild-type and ∆gfcA strains. The ∆gfcA∆waaL EPEC strain was not significantly more susceptible to HD-5 than the ∆waaL strain, suggesting that the absence of -antigen influences G4C formation. To determine whether the G4C and -antigen interact with HD-5, total polysaccharide was purified from wild-type EPEC and added to the ∆gfcA∆waaL strain in the presence of HD-5. The addition of exogenous polysaccharide protected the susceptible strain against HD-5 killing in a dose-dependent manner, suggesting that HD-5 binds to the polysaccharides present on the surface of EPEC. Altogether, these findings indicate that EPEC relies on both the G4C and the -antigen to resist the bactericidal activity of HD-5.

  10. SILAC-based comparative analysis of pathogenic Escherichia coli secretomes.

    Science.gov (United States)

    Boysen, Anders; Borch, Jonas; Krogh, Thøger Jensen; Hjernø, Karin; Møller-Jensen, Jakob

    2015-09-01

    Comparative studies of pathogenic bacteria and their non-pathogenic counterparts has led to the discovery of important virulence factors thereby generating insight into mechanisms of pathogenesis. Protein-based antigens for vaccine development are primarily selected among unique virulence-related factors produced by the pathogen of interest. However, recent work indicates that proteins that are not unique to the pathogen but instead selectively expressed compared to its non-pathogenic counterpart could also be vaccine candidates or targets for drug development. Modern methods in quantitative proteome analysis have the potential to discover both classes of proteins and hence form an important tool for discovering therapeutic targets. Adherent-invasive Escherichia coli (AIEC) and Enterotoxigenic E. coli (ETEC) are pathogenic variants of E. coli which cause intestinal disease in humans. AIEC is associated with Crohn's disease (CD), a chronic inflammatory condition of the gastrointestinal tract whereas ETEC is the major cause of human diarrhea which affects hundreds of millions annually. In spite of the disease burden associated with these pathogens, effective vaccines conferring long-term protection are still needed. In order to identify proteins with therapeutic potential, we have used mass spectrometry-based Stable Isotope Labeling with Amino acids in Cell culture (SILAC) quantitative proteomics method which allows us to compare the proteomes of pathogenic strains to commensal E. coli. In this study, we grew the pathogenic strains ETEC H10407, AIEC LF82 and the non-pathogenic reference strain E. coli K-12 MG1655 in parallel and used SILAC to compare protein levels in OMVs and culture supernatant. We have identified well-known virulence factors from both AIEC and ETEC, thus validating our experimental approach. In addition we find proteins that are not unique to the pathogenic strains but expressed at levels different from the commensal strain, including the

  11. Simplified large-scale refolding, purification, and characterization of recombinant human granulocyte-colony stimulating factor in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Chang Kyu Kim

    Full Text Available Granulocyte-colony stimulating factor (G-CSF is a pleiotropic cytokine that stimulates the development of committed hematopoietic progenitor cells and enhances the functional activity of mature cells. Here, we report a simplified method for fed-batch culture as well as the purification of recombinant human (rh G-CSF. The new system for rhG-CSF purification was performed using not only temperature shift strategy without isopropyl-l-thio-β-d-galactoside (IPTG induction but also the purification method by a single step of prep-HPLC after the pH precipitation of the refolded samples. Through these processes, the final cell density and overall yield of homogenous rhG-CSF were obtained 42.8 g as dry cell weights, 1.75 g as purified active proteins, from 1 L culture broth, respectively. The purity of rhG-CSF was finally 99% since the isoforms of rhG-CSF could be separated through the prep-HPLC step. The result of biological activity indicated that purified rhG-CSF has a similar profile to the World Health Organization (WHO 2(nd International Standard for G-CSF. Taken together, our results demonstrate that the simple purification through a single step of prep-HPLC may be valuable for the industrial-scale production of biologically active proteins.

  12. High-yield expression of human vascular endothelial growth factor VEGF(165) in Escherichia coli and purification for therapeutic applications.

    Science.gov (United States)

    Pizarro, Shelly A; Gunson, Jane; Field, Matthew J; Dinges, Rachel; Khoo, Stefanie; Dalal, Milind; Lee, Michael; Kaleas, Kimberly A; Moiseff, Kathryn; Garnick, Susan; Reilly, Dorothea E; Laird, Michael W; Schmelzer, Charles H

    2010-08-01

    Vascular endothelial growth factor (VEGF(165)) is a potent mitogen that induces angiogenesis and vascular permeability in vivo and has demonstrated potential in therapeutic applications for accelerating wound healing. An industrial production method that provides high yield as well as high purity, quality, and potency is needed. The process described in this report involves a bacterial expression system capable of producing approximately 9g of rhVEGF per liter of broth and a downstream purification process consisting of protein refolding and three chromatography steps prior to formulation of the drug substance. A high cell density (HCD) fed-batch fermentation process was used to produce rhVEGF in periplasmic inclusion bodies. The inclusion bodies are harvested from the cell lysate and subjected to a single-step protein solubilization and refolding operation to extract the rhVEGF for purification. Overall recovery yields observed during development, including refolding and chromatography, were 30+/-6%. Host cell impurities are consistently cleared below target levels at both laboratory and large-scale demonstrating process robustness. The structure of the refolded and purified rhVEGF was confirmed by mass spectrometry, N-terminal sequencing, and tryptic peptide mapping while product variants were analyzed by multiple HPLC assays. Biological activity was verified by the proliferation of human umbilical vein derived endothelial cells. Copyright 2010 Elsevier Inc. All rights reserved.

  13. Chromosomal features of Escherichia coli serotype O2:K2, an avian pathogenic E. coli

    DEFF Research Database (Denmark)

    Jørgensen, Steffen L; Kudirkiene, Egle; Li, Lili

    2017-01-01

    Escherichia coli causing infection outside the gastrointestinal system are referred to as extra-intestinal pathogenic E. coli. Avian pathogenic E. coli is a subgroup of extra-intestinal pathogenic E. coli and infections due to avian pathogenic E. coli have major impact on poultry production economy...... and welfare worldwide. An almost defining characteristic of avian pathogenic E. coli is the carriage of plasmids, which may encode virulence factors and antibiotic resistance determinates. For the same reason, plasmids of avian pathogenic E. coli have been intensively studied. However, genes encoded...... by the chromosome may also be important for disease manifestation and antimicrobial resistance. For the E. coli strain APEC_O2 the plasmids have been sequenced and analyzed in several studies, and E. coli APEC_O2 may therefore serve as a reference strain in future studies. Here we describe the chromosomal features...

  14. [Antibiotic resistance of Escherichia coli strains isolated from raw chicken meat in Senegal].

    Science.gov (United States)

    Fofana, A; Bada Alambédji, R; Seydi, M; Akakpo, A J

    2006-01-01

    Antimicrobial-resistant Escherichia coli and others pathogens bacteria can be transferred from animals to humans through consumption of contaminated food and foods products and thus present a public health risk. The increase in E. coli resistance to commonly used antimicrobials both in the public health and veterinary sectors is one of the major threats of health care worldwide. The present study was undertaken to estimate the antimicrobial resistance of E. coli isolates from raw chicken meat in Dakar. Levying of skin and muscle have been carried out on 120 chicken carcasses bought from 13 sale points and 23 flocks beetween November 2003 and April 2004. 102 Escherichia coli strains have been isolated, among which, 90 were tested for their susceptibilities to 16 selected antibiotics by agar diffusion method. All Escherichia coli strains (100%), were resistant to one or more antibiotic; 60 strains (66.66%) being resistant to more than five antibiotics. Those frequently encountererd are: ampicillin, trimethoprim, trimethoprim-sulfametoxazole, tetracycline, sulfonamides, streptomycin, nalidixic acid. Multiple resistances to 12 antibiotics were also observed. The lowest resistances were noted with gentamicin (3.33%) and neomycin (5.56%). This study showed the significance of chicken meat as source of Escherichia coli strains with a simple or multiple resistance to various antibiotics tested. Further studies are necessary in order to determine bacterium mechanisms of resistance.

  15. [Antibioresistance of Escherichia coli strains isolated from raw chicken meat in Senegal].

    Science.gov (United States)

    Fofana, A; Bada Alambedji, R; Seydi, M; Akakpo, A J

    2006-01-01

    Antimicrobial-resistant Escherichia coli and others pathogens bacteria can be transferred from animals to humans through consumption of contaminated food and foods products and thus present a public health risk. The increase in E. coli resistance to commonly used antimicrobials both in the public health and veterinary sectors is one of the major threats of health care worldwide. The present study was undertaken to estimate the antimicrobial resistance of E. coli isolates from raw chicken meat in Dakar. Levying of skin and muscle have been carried out on 120 chicken carcasses bought from 13 sale points and 23 flocks beetween November 2003 and April 2004. 102 Escherichia coli strains have been isolated, among which, 90 were tested for their susceptibilities to 16 selected antibiotics by agar diffusion method. All Escherichia coli strains (100%), were resistant to one or more antibiotic; 60 strains (66.66%) being resistant to more than five antibiotics. Those frequently encountererd are: ampicillin, trimethoprim, trimethoprim-sulfametoxazole, tetracycline, sulfonamides, streptomycin, nalidixic acid. Multiple resistances to 12 antibiotics were also observed. The lowest resistances were noted with gentamicin (3.33%) and neomycin (5.56%). This study showed the significance of chicken meat as source of Escherichia coli strains with a simple or multiple resistance to various antibiotics tested. Further studies are necessary in order to determine bacterium mechanisms of resistance.

  16. mcr-1 identified in Avian Pathogenic Escherichia coli (APEC)

    Science.gov (United States)

    Lima Barbieri, Nicolle; Nielsen, Daniel W.; Wannemuehler, Yvonne; Cavender, Tia; Hussein, Ashraf; Yan, Shi-gan; Nolan, Lisa K.; Logue, Catherine M.

    2017-01-01

    Antimicrobial resistance associated with colistin has emerged as a significant concern worldwide threatening the use of one of the most important antimicrobials for treating human disease. Here, we examined a collection (n = 980) of Avian Pathogenic Escherichia coli (APEC) isolated from poultry with colibacillosis from the US and internationally for the presence of mcr-1 and mcr-2, genes known to encode colistin resistance. Included in the analysis was an additional set of avian fecal E. coli (AFEC) (n = 220) isolates from healthy birds for comparative analysis. The mcr-1 gene was detected in a total of 12 isolates recovered from diseased production birds from China and Egypt. No mcr genes were detected in the healthy fecal isolates. The full mcr-1 gene from positive isolates was sequenced using specifically designed primers and were compared with sequences currently described in NCBI. mcr-1 positive isolates were also assessed for phenotypic colistin resistance and extended spectrum beta lactam phenotypes and genotypes. This study has identified mcr-1 in APEC isolates dating back to at least 2010 and suggests that animal husbandry practices could result in a potential source of resistance to the human food chain in countries where application of colistin in animal health is practiced. PMID:28264015

  17. mcr-1 identified in Avian Pathogenic Escherichia coli (APEC).

    Science.gov (United States)

    Lima Barbieri, Nicolle; Nielsen, Daniel W; Wannemuehler, Yvonne; Cavender, Tia; Hussein, Ashraf; Yan, Shi-Gan; Nolan, Lisa K; Logue, Catherine M

    2017-01-01

    Antimicrobial resistance associated with colistin has emerged as a significant concern worldwide threatening the use of one of the most important antimicrobials for treating human disease. Here, we examined a collection (n = 980) of Avian Pathogenic Escherichia coli (APEC) isolated from poultry with colibacillosis from the US and internationally for the presence of mcr-1 and mcr-2, genes known to encode colistin resistance. Included in the analysis was an additional set of avian fecal E. coli (AFEC) (n = 220) isolates from healthy birds for comparative analysis. The mcr-1 gene was detected in a total of 12 isolates recovered from diseased production birds from China and Egypt. No mcr genes were detected in the healthy fecal isolates. The full mcr-1 gene from positive isolates was sequenced using specifically designed primers and were compared with sequences currently described in NCBI. mcr-1 positive isolates were also assessed for phenotypic colistin resistance and extended spectrum beta lactam phenotypes and genotypes. This study has identified mcr-1 in APEC isolates dating back to at least 2010 and suggests that animal husbandry practices could result in a potential source of resistance to the human food chain in countries where application of colistin in animal health is practiced.

  18. A Murine Model for Escherichia coli Urinary Tract Infection.

    Science.gov (United States)

    Hannan, Thomas J; Hunstad, David A

    2016-01-01

    Urinary tract infections (UTI) are among the most common bacterial infections of humans. The mouse provides an excellent and tractable model system for cystitis and pyelonephritis caused by Escherichia coli and other uropathogens. Using a well-established model of experimental cystitis in which the bladders of female mice are infected via transurethral catheterization, the molecular details of the pathogenesis of bacterial cystitis have been substantially illuminated in the last decade. Uropathogenic E. coli attach to bladder epithelium (both in human and mouse) via adhesive type 1 pili, establish a replicative niche within epithelial cell cytoplasm, and form intracellular bacterial communities that are protected from antibiotic effects and immune clearance. The use of different inbred and mutant mouse strains offers the opportunity to study outcomes of infection, including resolution, formation of quiescent intracellular bacterial reservoirs, chronic bacterial cystitis, and recurrent infections. Urine, bladder, and kidney tissues can be analyzed by bacterial culture, histology, immunohistochemistry, immunofluorescent and confocal microscopy, electron microscopy, and flow cytometry, while a broad array of soluble markers (e.g., cytokines) can also be profiled in serum, urine, and tissue homogenates by ELISA, Western blotting, multiplex bead array, and other approaches. This model promises to afford continued opportunity for discovery of pathogenic mechanisms and evaluation of therapeutic and preventive strategies for acute, chronic, and recurrent UTI.

  19. Effect of cobalt on Escherichia coli metabolism and metalloporphyrin formation

    Science.gov (United States)

    Majtan, Tomas; Frerman, Frank E.

    2011-01-01

    Toxicity in Escherichia coli resulting from high concentrations of cobalt has been explained by competition of cobalt with iron in various metabolic processes including Fe–S cluster assembly, sulfur assimilation, production of free radicals and reduction of free thiol pool. Here we present another aspect of increased cobalt concentrations in the culture medium resulting in the production of cobalt protoporphyrin IX (CoPPIX), which was incorporated into heme proteins including membrane-bound cytochromes and an expressed human cystathionine beta-synthase (CBS). The presence of CoPPIX in cytochromes inhibited their electron transport capacity and resulted in a substantially decreased respiration. Bacterial cells adapted to the increased cobalt concentration by inducing a modified mixed acid fermentative pathway under aerobiosis. We capitalized on the ability of E. coli to insert cobalt into PPIX to carry out an expression of CoPPIX-substituted heme proteins. The level of CoPPIX-substitution increased with the number of passages of cells in a cobalt-containing medium. This approach is an inexpensive method to prepare cobalt-substituted heme proteins compared to in vitro enzyme reconstitution or in vivo replacement using metalloporphyrin heme analogs and seems to be especially suitable for complex heme proteins with an additional coenzyme, such as human CBS. PMID:21184140

  20. Fluorometric In Situ Monitoring of an Escherichia coli Cell Factory with Cytosolic Expression of Human Glycosyltransferase GalNAcT2: Prospects and Limitations

    Directory of Open Access Journals (Sweden)

    Karen Schwab

    2016-11-01

    Full Text Available The glycosyltransferase HisDapGalNAcT2 is the key protein of the Escherichia coli (E. coli SHuffle® T7 cell factory which was genetically engineered to allow glycosylation of a protein substrate in vivo. The specific activity of the glycosyltransferase requires time-intensive analytics, but is a critical process parameter. Therefore, it has to be monitored closely. This study evaluates fluorometric in situ monitoring as option to access this critical process parameter during complex E. coli fermentations. Partial least square regression (PLS models were built based on the fluorometric data recorded during the EnPresso® B fermentations. Capable models for the prediction of glucose and acetate concentrations were built for these fermentations with rout mean squared errors for prediction (RMSEP of 0.19 g·L−1 and 0.08 g·L−1, as well as for the prediction of the optical density (RMSEP 0.24. In situ monitoring of soluble enzyme to cell dry weight ratios (RMSEP 5.5 × 10−4 µg w/w and specific activity of the glycosyltransferase (RMSEP 33.5 pmol·min−1·µg−1 proved to be challenging, since HisDapGalNAcT2 had to be extracted from the cells and purified. However, fluorescence spectroscopy, in combination with PLS modeling, proved to be feasible for in situ monitoring of complex expression systems.

  1. Heterogeneous Genetic Location of mcr-1 in Colistin-Resistant Escherichia coli Isolated from Humans and Retail Chicken Meat in Switzerland: Emergence of mcr-1-Carrying IncK2 Plasmids.

    Science.gov (United States)

    Donà, Valentina; Bernasconi, Odette J; Pires, João; Collaud, Alexandra; Overesch, Gudrun; Ramette, Alban; Perreten, Vincent; Endimiani, Andrea

    2017-08-28

    We characterized the genetic environment of mcr-1 in colistin-resistant Escherichia coli strains isolated in Switzerland during 2014-2016 from humans (n=3) and chicken meat (n=6). Whole genome and plasmid sequencing identified mcr-1 integrated in IncX4 (of which, one carrying the mcr-1.2 variant), IncI2, IncHI2 and novel IncK2 plasmids (overall, n=7), as well as in the bacterial chromosome (n=2) in single or duplicate copies. Our study supports the easy mobilization of mcr-1 across diverse genetic locations. Copyright © 2017 American Society for Microbiology.

  2. Obscured phylogeny and possible recombinational dormancy in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Sawyer Stanley A

    2011-06-01

    Full Text Available Abstract Background Escherichia coli is one of the best studied organisms in all of biology, but its phylogenetic structure has been difficult to resolve with current data and analytical techniques. We analyzed single nucleotide polymorphisms in chromosomes of representative strains to reconstruct the topology of its emergence. Results The phylogeny of E. coli varies according to the segment of chromosome analyzed. Recombination between extant E. coli groups is largely limited to only three intergroup pairings. Conclusions Segment-dependent phylogenies most likely are legacies of a complex recombination history. However, E. coli are now in an epoch in which they no longer broadly share DNA. Using the definition of species as organisms that freely exchange genetic material, this recombinational dormancy could reflect either the end of E. coli as a species, or herald the coalescence of E. coli groups into new species.

  3. Ex vivo intestinal adhesion of Escherichia coli LF82 in Crohn's disease

    DEFF Research Database (Denmark)

    Jensen, Stina Rikke; Fink, Lisbeth Nielsen; Nielsen, Ole Haagen

    2011-01-01

    Adherent-invasive Escherichia coli (AIEC) are reported to inhabit the gut mucosa in Crohn's disease (CD), however, little is known about the importance of host factors for the interplay between AIEC and the human gut. To examine if differences in bacterial adhesion patterns are disease associated...

  4. Classification of shiga toxin-producing escherichia coli (STEC) serotypes with hyperspectral microscope imagery

    Science.gov (United States)

    Non-O157:H7 Shiga toxin-producing Escherichia coli (STEC) strains such as O26, O45, O103, O111, O121 and O145 are recognized as serious outbreak to cause human illness due to their toxicity. Since a conventional microbiological method for cell counting is laborious and time-consuming process, optica...

  5. OCCURRENCE OF ANTIBIOTIC-RESISTANT UROPATHOGENIC ESCHERICHIA COLI CLONAL GROUP A IN WASTEWATER EFFLUENTS

    Science.gov (United States)

    Isolates of Escherichia coli belonging to clonal group A (CGA), a recently described disseminated cause of drug-resistant urinary tract infections in humans, were present in four of seven sewage effluents collected from geographically dispersed areas of the United States. ...

  6. Overexpression and surface localization of the Chlamydia trachomatis major outer membrane protein in Escherichia coli

    DEFF Research Database (Denmark)

    Koehler, JF; Birkelund, Svend; Stephens, RS

    1992-01-01

    The Chlamydia trachomatis major outer membrane protein (MOMP) is the quantitatively predominant surface protein which has important functional, structural and antigenic properties. We have cloned and overexpressed the MOMP in Escherichia coli. The MOMP is surface exposed in C. trachomatis and cap...... the contribution of the MOMP variable segments to the topographical interactions which determine the antigenic structure responsible for human immune response....

  7. Evaluation of rectoanal mucosal swab sampling for molecular detection of Enterohemorrhagic Escherichia coli in beef cattle

    Science.gov (United States)

    Cattle are a primary reservoir of Enterohemorrhagic Escherichia coli (EHEC) and contaminated beef products are a source of human infections. The U.S. Department of Agriculture Food Safety and Inspection Service declared the presence of seven EHEC serogroups (O26, O45, O103, O111, O121, O145 and O157...

  8. OCCURRENCE OF ANTIBIOTIC-RESISTANT UROPATHOGENIC ESCHERICHIA COLI CLONAL GROUP A IN WASTEWATER EFFLUENTS

    Science.gov (United States)

    Isolates of Escherichia coli belonging to clonal group A (CGA), a recently described disseminated cause of drug-resistant urinary tract infections in humans, were present in four of seven sewage effluents collected from geographically dispersed areas of the United States. ...

  9. Transcriptomics and adaptive genomics of the asymptomatic bacteriuria Escherichia coli strain 83972

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Seshasayee, Aswin S.; Ussery, David

    2008-01-01

    Escherichia coli strains are the major cause of urinary tract infections in humans. Such strains can be divided into virulent, UPEC strains causing symptomatic infections, and asymptomatic, commensal-like strains causing asymptomatic bacteriuria, ABU. The best-characterized ABU strain is strain....... Strain 83972 is a deconstructed pathogen rather than a commensal strain that has acquired fitness properties....

  10. Shiga toxin, cytolethal distending toxin, and hemolysin repertoires in clinical Escherichia coli O91 isolates

    NARCIS (Netherlands)

    Bielaszewska, Martina; Stoewe, Franziska; Fruth, Angelika; Zhang, Wenlan; Prager, Rita; Brockmeyer, Jens; Mellmann, Alexander; Karch, Helge; Friedrich, Alexander W

    2009-01-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) strains of serogroup O91 are the most common human pathogenic eae-negative STEC strains. To facilitate diagnosis and subtyping of these pathogens, we genotypically and phenotypically characterized 100 clinical STEC O91 isolates. Motile strains expr

  11. Prevalence and antimicrobial resistance in Escherichia coli from food animals in Lagos, Nigeria

    Science.gov (United States)

    Foodborne bacteria are often associated with human infections; these infections can become more complicated to treat if the bacteria are also resistant to antimicrobials. In this study, prevalence, antimicrobial resistance, and genetic relatedness of Escherichia coli among food producing animals fr...

  12. Distribution of non-LEE-encoded type 3 secretion system dependent effectors in enteropathogenic Escherichia coli

    Directory of Open Access Journals (Sweden)

    Fábia A. Salvador

    2014-09-01

    Full Text Available Enteropathogenic Escherichia coli (EPEC are important human gastroenteritis agents. The prevalence of six non-LEE genes encoding type 3 translocated effectors was investigated. The nleC, cif and nleB genes were more prevalent in typical than in atypical EPEC, although a higher diversity of genes combinations was observed in atypical EPEC.

  13. 重组人尿激酶原的体外变复性研究%In vitro renaturation of recombinant human pro-urokinase expressed in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    朱慧; 刘伟; 史蔚; 薛宇鸣; 蒯乐天; 马忠

    2001-01-01

    Objective Recombinant human pro-urokinase forms insoluble inclusion body when overexpressed in Escherichia coli. It must be denatured and renatured in vitro so that it can acquire activity. This study aimed at increasing the renaturation yield of denaturant pro-urokinase. Methods We evaluated the basic renaturation conditions of pro-urokinase through qualitative and quantitative analysis of pH, temperature, denatured concentration, protein concentration, and the ratio of reduced and oxidized thiol reagents. We also compared the effects of nonspecific additives, step-wise dilution and urea gradient dialysis. Results We defined the optimal conditions of pro-urokinase renaturation with a yield of about 20%-30%. Conclusion Different recombinant denatured proteins have different renaturation conditions due to their different molecular sizes, molecular constructions, disulfide bond numbers, and hydrophobicity. The renaturation yield can be increased by optimizing the renaturation conditions of a specific protein.%目的 重组人尿激酶原在大肠杆菌中过量表达时形成不溶物包涵体,需经体外变复性后才能获得生物活性。本文旨在提高包涵体中变性尿激酶原的复性效率。 方法 通过对pH、温度、变性剂种类及浓度、蛋白浓度、以及巯基氧化还原对比率等的定性定量分析,研究重组人尿激酶原体外变复性的基本条件,并比较了添加一些非特异有效成分、脉冲稀释、梯度透析等方法对提高重组人尿激酶原体外变复性效率的作用。 结果 确定了重组人尿激酶原体外变复性的基本方法,其复性效率可达30%左右。 结论 不同的包涵体蛋白的体外变复性效率因蛋白的分子大小、二巯键数目、疏水程度等而异,对特定蛋白复性条件的优化可提高其复性效率。

  14. [Production of coenzyme Q10 by metabolically engineered Escherichia coli].

    Science.gov (United States)

    Dai, Guanping; Miao, Liangtian; Sun, Tao; Li, Qingyan; Xiao, Dongguang; Zhang, Xueli

    2015-02-01

    Coenzyme Q10 (CoQ10) is a lipophilic antioxidant that improves human immunity, delays senility and enhances the vitality of the human body and has wide applications in pharmaceutical and cosmetic industries. Microbial fermentation is a sustainable way to produce CoQ10, and attracts increased interest. In this work, the native CoQ8 synthetic pathway of Escherichia coli was replaced by the CoQ10 synthetic pathway through integrating decaprenyl diphosphate synthase gene (dps) from Rhodobacter sphaeroides into chromosome of E. coli ATCC 8739, followed by deletion of the native octaprenyl diphosphate synthase gene (ispB). The resulting strain GD-14 produced 0.68 mg/L CoQ10 with a yield of 0.54 mg/g DCW. Modulation of dxs and idi genes of the MEP pathway and ubiCA genes in combination led to 2.46-fold increase of CoQ10 production (from 0.54 to 1.87 mg/g DCW). Recruiting glucose facilitator protein of Zymomonas mobilis to replace the native phosphoenolpyruvate: carbohydrate phosphotransferase systems (PTS) further led to a 16% increase of CoQ10 yield. Finally, fed-batch fermentation of the best strain GD-51 was performed, which produced 433 mg/L CoQ10 with a yield of 11.7 mg/g DCW. To the best of our knowledge, this was the highest CoQ10 titer and yield obtained for engineered E. coli.

  15. Characterisation of commensal Escherichia coli isolated from apparently healthy cattle and their attendants in Tanzania

    DEFF Research Database (Denmark)

    Madoshi, Balichene; Kudirkiene, Egle; Mtambo, Madundo

    2016-01-01

    While pathogenic types of Escherichia coli are well characterized, relatively little is known about the commensal E. coli flora. In the current study, antimicrobial resistance in commensal E. coli and distribution of ERIC-PCR genotypes among isolates of such bacteria from cattle and cattle...... attendants on cattle farms in Tanzania were investigated. Seventeen E. coli genomes representing different ERIC-PCR types of commensal E. coli were sequenced in order to determine their possible importance as a reservoir for both antimicrobial resistance genes and virulence factors. Both human and cattle...... specific. The most frequent plasmids replicon genes found in strains from both hosts were of IncF type, which are commonly associated with carriage of antimicrobial and virulence genes. Commensal E. coli from cattle and attendants were found to share same genotypes and to carry antimicrobial resistance...

  16. Characterization of Escherichia coli isolated from migratory water fowls in Hakaluki Haor, Bangladesh

    Directory of Open Access Journals (Sweden)

    Ferdaus MohdAltaf Hossain

    2012-03-01

    Full Text Available A total of 135 fecal samples were collected to characterize the Escherichia coli isolates from the migratory waterfowls (whistling Swan harbored in the Hakaluki Haor of Bangladesh in the year of 2008 and 2009. Out of 135 fecal samples, 100 samples were distinguished as positive for isolates of Escherichia coli following cultural, biochemical and motility test. Amongst the recovered isolates only 38% were found upbeat to enterotoxin production propensity on mice inoculation test. Finally, out of 38 % enterotoxigenic E. coli (ETEC positive isolates no any isolates found to be positive for the aptitude of heat stable (ST toxin yield. So, the presence of ETEC in migratory waterfowls indicating the possibilities of them to act as vector and reservoir of E. coli to spread further infection to animals and humans. This work indicates the first time ETEC characterization from the migratory birds of Bangladesh.

  17. Arginine Catabolism and the Arginine Succinyltransferase Pathway in Escherichia coli

    OpenAIRE

    Schneider, Barbara L.; Kiupakis, Alexandros K.; Reitzer, Lawrence J.

    1998-01-01

    Arginine catabolism produces ammonia without transferring nitrogen to another compound, yet the only known pathway of arginine catabolism in Escherichia coli (through arginine decarboxylase) does not produce ammonia. Our aims were to find the ammonia-producing pathway of arginine catabolism in E. coli and to examine its function. We showed that the only previously described pathway of arginine catabolism, which does not produce ammonia, accounted for only 3% of the arginine consumed. A search...

  18. Engineered biosynthesis of bacterial aromatic polyketides in Escherichia coli

    OpenAIRE

    Zhang, Wenjun; Li, Yanran; Tang, Yi

    2008-01-01

    Bacterial aromatic polyketides are important therapeutic compounds including front line antibiotics and anticancer drugs. It is one of the last remaining major classes of natural products of which the biosynthesis has not been reconstituted in the genetically superior host Escherichia coli. Here, we demonstrate the engineered biosynthesis of bacterial aromatic polyketides in E. coli by using a dissected and reassembled fungal polyketide synthase (PKS). The minimal PKS of the megasynthase PKS4...

  19. Osmoprotection of Escherichia coli by ectoine: uptake and accumulation characteristics.

    OpenAIRE

    Jebbar, M; Talibart, R; Gloux, K; Bernard, T.; BLANCO, C.

    1992-01-01

    Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is a cyclic amino acid, identified as a compatible solute in moderately halophilic bacteria. Exogenously provided ectoine was found to stimulate growth of Escherichia coli in media of inhibitory osmotic strength. The stimulation was independent of any specific solute, electrolyte or nonelectrolyte. It is accumulated in E. coli cells proportionally to the osmotic strength of the medium, and it is not metabolized. Its osmoprotect...

  20. The Adherent/Invasive Escherichia coli Strain LF82 Invades and Persists in Human Prostate Cell Line RWPE-1, Activating a Strong Inflammatory Response

    Science.gov (United States)

    Aleandri, Marta; Marazzato, Massimiliano; Conte, Antonietta L.; Ambrosi, Cecilia; Nicoletti, Mauro; Zagaglia, Carlo; Gambara, Guido; Palombi, Fioretta; De Cesaris, Paola; Ziparo, Elio; Palamara, Anna T.; Riccioli, Anna

    2016-01-01

    Adherent/invasive Escherichia coli (AIEC) strains have recently been receiving increased attention because they are more prevalent and persistent in the intestine of Crohn's disease (CD) patients than in healthy subjects. Since AIEC strains show a high percentage of similarity to extraintestinal pathogenic E. coli (ExPEC), neonatal meningitis-associated E. coli (NMEC), and uropathogenic E. coli (UPEC) strains, here we compared AIEC strain LF82 with a UPEC isolate (strain EC73) to assess whether LF82 would be able to infect prostate cells as an extraintestinal target. The virulence phenotypes of both strains were determined by using the RWPE-1 prostate cell line. The results obtained indicated that LF82 and EC73 are able to adhere to, invade, and survive within prostate epithelial cells. Invasion was confirmed by immunofluorescence and electron microscopy. Moreover, cytochalasin D and colchicine strongly inhibited bacterial uptake of both strains, indicating the involvement of actin microfilaments and microtubules in host cell invasion. Moreover, both strains belong to phylogenetic group B2 and are strong biofilm producers. In silico analysis reveals that LF82 shares with UPEC strains several virulence factors: namely, type 1 pili, the group II capsule, the vacuolating autotransporter toxin, four iron uptake systems, and the pathogenic island (PAI). Furthermore, compared to EC73, LF82 induces in RWPE-1 cells a marked increase of phosphorylation of mitogen-activated protein kinases (MAPKs) and of NF-κB already by 5 min postinfection, thus inducing a strong inflammatory response. Our in vitro data support the hypothesis that AIEC strains might play a role in prostatitis, and, by exploiting host-cell signaling pathways controlling the innate immune response, likely facilitate bacterial multiplication and dissemination within the male genitourinary tract. PMID:27600504

  1. 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.

  2. Occurrence and characterization of verocytotoxigenic Escherichia coli (VTEC) strains from dairy farms in Trinidad.

    Science.gov (United States)

    Roopnarine, R R; Ammons, D; Rampersad, J; Adesiyun, A A

    2007-01-01

    A cross-sectional study was conducted to determine the prevalence and characteristics of verocytotoxigenic Escherichia coli (VTEC) on 25 dairy farms each located in Waller field and Carlsen field farming areas in Trinidad. On each selected farm, faecal samples were collected from milking cows, calves and humans; rectal swabs were obtained from pet farm dogs; bulk milk was sampled as well as effluent from the milking parlour. Escherichia coli was isolated from all sources on selective media using standard methods. Isolates of E. coli were subjected to slide agglutination test using E. coli O157 antiserum, vero cell cytotoxicity assay to detect verocytotoxin (VT) and heat labile toxin (LT) production, the polymerase chain reaction (PCR) to detect VT genes, and the dry spot test to screen for E. coli O157 and non-O157 strains. In addition, faecal samples from animal and human sources were tested for VT genes using PCR. Of a total of 933 E. coli isolates tested by the slide test, eight (0.9%) were positive for the O157 strain. The vero cell cytotoxicity assay detected VT-producing strains of E. coli in 16.6%, 14.6%, 3.2% and 7.1% of isolates from cows, calves, farm dogs and humans respectively (P detected amongst isolates of E. coli from calves (10.8%) and the lowest (0.0%) amongst isolates from humans and bulk milk (P detected in 62.3%, 4.9% and 1.6% respectively (P Detection of VTEC strains in milk and dairy animals poses a health risk to consumers of milk originating from these farms. In addition, the demonstration of VTEC strains in humans, VT gene in faecal samples and E. coli isolates as well as non-O157 VTEC strains of E. coli are being documented for the first time in the country.

  3. Epidemiology and Clinical Manifestations of Enteroaggregative Escherichia coli

    Science.gov (United States)

    Hebbelstrup Jensen, Betina; Olsen, Katharina E. P.; Struve, Carsten; Petersen, Andreas Munk

    2014-01-01

    SUMMARY Enteroaggregative Escherichia coli (EAEC) represents a heterogeneous group of E. coli strains. The pathogenicity and clinical relevance of these bacteria are still controversial. In this review, we describe the clinical significance of EAEC regarding patterns of infection in humans, transmission, reservoirs, and symptoms. Manifestations associated with EAEC infection include watery diarrhea, mucoid diarrhea, low-grade fever, nausea, tenesmus, and borborygmi. In early studies, EAEC was considered to be an opportunistic pathogen associated with diarrhea in HIV patients and in malnourished children in developing countries. In recent studies, associations with traveler's diarrhea, the occurrence of diarrhea cases in industrialized countries, and outbreaks of diarrhea in Europe and Asia have been reported. In the spring of 2011, a large outbreak of hemolytic-uremic syndrome (HUS) and hemorrhagic colitis occurred in Germany due to an EAEC O104:H4 strain, causing 54 deaths and 855 cases of HUS. This strain produces the potent Shiga toxin along with the aggregative fimbriae. An outbreak of urinary tract infection associated with EAEC in Copenhagen, Denmark, occurred in 1991; this involved extensive production of biofilm, an important characteristic of the pathogenicity of EAEC. However, the heterogeneity of EAEC continues to complicate diagnostics and also our understanding of pathogenicity. PMID:24982324

  4. Public Health Microbiology of Shiga Toxin-Producing Escherichia coli.

    Science.gov (United States)

    Caprioli, Alfredo; Scavia, Gaia; Morabito, Stefano

    2014-12-01

    Shiga toxin-producing Escherichia coli (STEC) strains are the only pathogenic group of E. coli that has a definite zoonotic origin, with ruminants and, in particular, cattle being recognized as the major reservoir. Most human STEC infections are food borne, but the routes of transmission include direct contact with animals and a variety of environment-related exposures. Therefore, STEC public health microbiology spans the fields of medical, veterinary, food, water, and environmental microbiology, requiring a "One Health" perspective and laboratory scientists with the ability to work effectively across disciplines. Public health microbiology laboratories play a central role in the surveillance of STEC infections, as well as in the preparedness for responding to outbreaks and in providing scientific evidence for the implementation of prevention and control measures. This article reviews (i) how the integration of surveillance of STEC infections and monitoring of these pathogens in animal reservoirs and potential food vehicles may contribute to their control; (ii) the role of reference laboratories, in both the public health and veterinary and food sectors; and (iii) the public health perspectives, including those related to regulatory issues in both the European Union and the United States.

  5. Subversion of Host Innate Immunity by Uropathogenic Escherichia coli.

    Science.gov (United States)

    Olson, Patrick D; Hunstad, David A

    2016-01-04

    Uropathogenic Escherichia coli (UPEC) cause the majority of community-onset urinary tract infections (UTI) and represent a major etiologic agent of healthcare-associated UTI. Introduction of UPEC into the mammalian urinary tract evokes a well-described inflammatory response, comprising pro-inflammatory cytokines and chemokines as well as cellular elements (neutrophils and macrophages). In human UTI, this inflammatory response contributes to symptomatology and provides means for diagnosis by standard clinical testing. Early in acute cystitis, as demonstrated in murine models, UPEC gains access to an intracellular niche that protects a population of replicating bacteria from arriving phagocytes. To ensure the establishment of this protected niche, UPEC employ multiple strategies to attenuate and delay the initiation of host inflammatory components, including epithelial secretion of chemoattractants. Recent work has also revealed novel mechanisms by which UPEC blunts neutrophil migration across infected uroepithelium. Taken together, these attributes distinguish UPEC from commensal and nonpathogenic E. coli strains. This review highlights the unique immune evasion and suppression strategies of this bacterial pathogen and offers directions for further study; molecular understanding of these mechanisms will inform the development of adjunctive, anti-virulence therapeutics for UTI.

  6. Bacteriophages with the Ability to Degrade Uropathogenic Escherichia Coli Biofilms

    Directory of Open Access Journals (Sweden)

    Amee Manges

    2012-04-01

    Full Text Available Escherichia coli-associated urinary tract infections (UTIs are among the most common bacterial infections in humans. UTIs are usually managed with antibiotic therapy, but over the years, antibiotic-resistant strains of uropathogenic E. coli (UPEC have emerged. The formation of biofilms further complicates the treatment of these infections by making them resistant to killing by the host immune system as well as by antibiotics. This has encouraged research into therapy using bacteriophages (phages as a supplement or substitute for antibiotics. In this study we characterized 253 UPEC in terms of their biofilm-forming capabilities, serotype, and antimicrobial resistance. Three phages were then isolated (vB_EcoP_ACG-C91, vB_EcoM_ACG-C40 and vB_EcoS_ACG-M12 which were able to lyse 80.5% of a subset (42 of the UPEC strains able to form biofilms. Correlation was established between phage sensitivity and specific serotypes of the UPEC strains. The phages’ genome sequences were determined and resulted in classification of vB_EcoP_ACG-C91 as a SP6likevirus, vB_EcoM_ACG-C40 as a T4likevirus and vB_EcoS_ACG-M12 as T1likevirus. We assessed the ability of the three phages to eradicate the established biofilm of one of the UPEC strains used in the study. All phages significantly reduced the biofilm within 2–12 h of incubation.

  7. Simple method for purification of enterotoxigenic Escherichia coli fimbriae.

    Science.gov (United States)

    Curtis, Brittany; Grassel, Christen; Laufer, Rachel S; Sears, Khandra T; Pasetti, Marcela F; Barry, Eileen M; Simon, Raphael

    2016-03-01

    Enterotoxigenic Escherichia coli (ETEC) are endemic pathogens in the developing world. They frequently cause illness in travelers, and are among the most prevalent causes of diarrheal disease in children. Pathogenic ETEC strains employ fimbriae as adhesion factors to bind the luminal surface of the intestinal epithelium and establish infection. Accordingly, there is marked interest in immunoprophylactic strategies targeting fimbriae to protect against ETEC infections. Multiple strategies have been reported for purification of ETEC fimbriae, however none is ideal. Purification has typically involved the use of highly virulent wild-type strains. We report here a simple and improved method to purify ETEC fimbriae, which was applied to obtain two different Class 5 fimbriae types of clinical relevance (CFA/I and CS4) expressed recombinantly in E. coli production strains. Following removal from cells by shearing, fimbriae proteins were purified by orthogonal purification steps employing ultracentrifugation, precipitation, and ion-exchange membrane chromatography. Purified fimbriae demonstrated the anticipated size and morphology by electron microscopy analysis, contained negligible levels of residual host cell proteins, nucleic acid, and endotoxin, and were recognized by convalescent human anti-sera. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. First Report of the Globally Disseminated IncX4 Plasmid Carrying the mcr-1 Gene in a Colistin-Resistant Escherichia coli Sequence Type 101 Isolate from a Human Infection in Brazil.

    Science.gov (United States)

    Fernandes, Miriam R; McCulloch, John A; Vianello, Marco A; Moura, Quézia; Pérez-Chaparro, Paula J; Esposito, Fernanda; Sartori, Luciana; Dropa, Milena; Matté, Maria H; Lira, Débora P A; Mamizuka, Elsa M; Lincopan, Nilton

    2016-10-01

    A colistin-resistant Escherichia coli strain was recovered from a patient with a diabetic foot infection in Brazil. Whole-genome analysis revealed that the E. coli isolate belonged to the widespread sequence type (ST) 101 and harbored the mcr-1 gene on an IncX4 plasmid that was highly similar to mcr-1-bearing IncX4 plasmids that were recently identified in Enterobacteriaceae from food, animal, and human samples recovered on different continents. These results suggest that self-transmissible IncX4-type plasmids may represent promiscuous plasmids contributing to the intercontinental spread of the mcr-1 gene. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  9. Detection of virulence factors of Uropathoigenic Escherichia coli isolates from infertile women high vaginal swabs

    Directory of Open Access Journals (Sweden)

    Farhad Safarpourdehkourdi

    2014-03-01

    Conclusion: The high vaginal Escherichia coli harbored certain virulence genes of uropathogenic Escherichia coli strains. The urinary tract infections should be treated well to diminish its upstream transfer into vagina. Some more investigation should be perform for identifying the epidemiological aspects of uropathogenic Escherichia coli in high vaginal part of infertile women.

  10. 76 FR 72331 - Shiga Toxin-Producing Escherichia coli in Certain Raw Beef Products

    Science.gov (United States)

    2011-11-23

    ... Toxin-Producing Escherichia coli in Certain Raw Beef Products AGENCY: Food Safety and Inspection Service... methods for controlling non-O157 Shiga toxin-producing Escherichia coli in raw, intact and non-intact beef... Escherichia coli in raw, intact and non-intact beef products and product components on or before December...

  11. [The clinical characteristics analysis of Escherichia coli bloodstream infection].

    Science.gov (United States)

    Zhang, M; Huang, J A; Chen, Y B

    2016-05-17

    To explore the clinical features of Escherichia coli bloodstream infection. The clinical data of underlying diseases, antimicrobial susceptibility, temperature at blood sampling, results of routine blood tests, venous catheterization, therapy and prognosis of Escherichia coli bloodstream infection in the First Affiliated Hospital of Soochow University from January 2007 to December 2014 were analyzed retrospectively. The pathogens were routinely isolated and identified. Susceptibilities against antimicrobial agents were determined by Kirby-Bauer methods. All patients had at least one underlying disease. Most of the basic diseases were hematological malignancies, malignant solid tumors, pneumonia and so on. Body temperature was normal in 40 patients (6.4%), fever in 587 patients (93.5%) and low temperature in 1 patient. There were 252 patients with leukopenia (40.1%), 237 patients with granulocytopenia (37.7%) and 216 patients with agranulocytosis. The resistance rate to imipenem was 3.3%, which was the lowest among the total antimicrobial susceptibilities of 628 Escherichia Coli. The extended-spectrum-β-lactamase (ESBL)-producing strains accounted for 53.8% among the total patients. The resistance rates of ESBLs-producing-Escherichia coli for the Sulfamethoxazole, Ampicillin, Gentamicin, Cefazolin, Cefuroxime, Cefotaxime, Ceftriaxone, Cefepime, Ceftazidime, Cefoperazone, Piperacillin and Ciprofloxacin were 80.2%, 100.0%, 62.4%, 99.1%, 99.1%, 98.8%, 98.2%, 48.5%, 50.6%, 95.0%, 98.2%, 79.6%, respectively, which were higher than that of non-ESBLs-producing-Escherichia coli (67.9%, 79.7%, 47.6%, 50.0%, 47.2%, 41.0%, 40.3%, 27.2%, 24.1%, 40.0%, 56.2%, 58.3%, respectively), the differences were significant statistically (χ(2)=12.33, 75.90, 13.92, 209.00, 224.94, 259.25, 256.59, 27.79, 46.19, 222.85, 165.08, 33.59, all PEscherichia coli bloodstream infection. The antimicrobial resistance rate of ESBLs-producing-Escherichia coli is higher than that of none-ESBLs-producing-Escherichia

  12. Cervical celullitis in broiler chickens for Escherichia coli/ Celulite cervical em frangos de corte causada por Escherichia coli

    Directory of Open Access Journals (Sweden)

    Ivens Gomes Guimarães

    2002-05-01

    Full Text Available In this paper was report the isolation of Escherichia coli in broiler chickens with cellulitis in the cervical region. It was carried through the isolation of E. coli of the lesion of cellulitis from broilers and carried through histopathological examination of skin that had characterized the lesion. Focal ulcerations of epidermis, fibrin in dermis and difuse infiltrated by lymphocytes and heterophils on subcutaneous tissues.Neste trabalho, relata-se o isolamento de Escherichia coli em frangos de corte apresentando lesão de celulite na região cervical. Foi realizado o isolamento de E. coli da lesão de celulite e realizado exames histopatológicos que caracterizaram a lesão. Na epiderme foram verificadas lesões ulcerativas, presença de fibrina na derme e infiltração difusa de linfócitos e heterófilos no tecido subcutâneo.

  13. Immunomodulatory intervention with interferon-γ in Escherichia coli pyelonephritis.

    Science.gov (United States)

    Katsaris, Matthew P; Adamis, Theodoros; Pistiki, Aikaterini; Carrer, Dionyssia-Pinelopi; Galani, Irene; Sabracos, Labros; Droggiti, Dionyssia-Irini; Georgitsi, Marianna; Damoraki, Georgia; Giamarellos-Bourboulis, Evangelos J; Chrisofos, Michael

    2014-08-01

    We investigated the efficacy of recombinant human interferon-γ in experimental pyelonephritis due to Escherichia coli. Pyelonephritis was induced by intrapelvic inoculation of bacteria after ureteral ligation in 38 rabbits assigned to 1 of 3 groups, including group 1-16 controls, group 2-14 rabbits treated with intravenous recombinant human interferon-γ and group 3-8 rabbits treated with intravenous recombinant human interferon-γ plus amikacin. Bacterial counts, cytokines and malondialdehyde were measured in blood. Peripheral blood mononuclear cells were isolated to measure TNFα transcripts, cytokine stimulation and apoptosis. Survival was recorded, and the tissue bacterial load and myeloperoxidase activity were measured after sacrifice. The mortality rate in groups 1, 2 and 3 was 66.7%, 25% and 12.5%, respectively. The circulating bacterial count and tissue bacterial load were less in group 2 than in group 1. Circulating malondialdehyde negatively correlated with the bacterial load of the spleen. Although the number of TNFα transcripts in circulating peripheral blood mononuclear cells did not differ, peripheral blood mononuclear cells isolated from group 2 at 48 hours produced much greater concentrations of tumor necrosis factor-α after stimulation with Pam3Cys. In parallel, the apoptosis rate of circulating monocytes was increased in group 2 at 48 hours. Lung myeloperoxidase activity at 24 hours, serving as indirect evidence of neutrophil infiltration, was decreased in group 2. Recombinant human interferon-γ administration prolonged survival in rabbits with experimental E. coli urosepsis. Its action was probably related to increased bacterial phagocytosis after modulation of oxidant status and reversal of monocyte immunoparalysis. Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  14. Data for the co-expression and purification of human recombinant CaMKK2 in complex with calmodulin in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Lisa Gerner

    2016-09-01

    Full Text Available Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2 has been implicated in a range of conditions and pathologies from prostate to hepatic cancer. Here, we describe the expression in Escherichia coli and the purification protocol for the following constructs: full-length CaMKK2 in complex with CaM, CaMKK2 ‘apo’, CaMKK2 (165-501 in complex with CaM, and the CaMKK2 F267G mutant. The protocols described have been optimized for maximum yield and purity with minimal purification steps required and the proteins subsequently used to develop a fluorescence-based assay for drug binding to the kinase, “Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2” [1].

  15. YeeO from Escherichia coli exports flavins.

    Science.gov (United States)

    McAnulty, Michael J; Wood, Thomas K

    2014-01-01

    Multidrug and toxic compound extrusion (MATE) proteins help maintain cellular homeostasis by secreting metabolic wastes. Flavins may occur as cellular waste products, with their production and secretion providing potential benefit for industrial applications related to biofuel cells. Here we find that MATE protein YeeO from Escherichia coli exports both flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Significant amounts of flavins were trapped intracellularly when YeeO was produced indicating transport limits secretion of flavins. Wild-type E. coli secreted 3 flavins (riboflavin, FMN, and FAD), so E. coli likely produces additional flavin transporters.

  16. Escherichia coli O26 IN RAW BUFFALO MILK: PRELIMINARY RESULTS

    Directory of Open Access Journals (Sweden)

    A. Rella

    2013-02-01

    Full Text Available Escherichia coli O26 is considered to be one of the most important food-borne pathogen. In this study, 120 buffalo milk samples collected in Lazio and in Apulia regions were tested for the presence of E. coli O26. One buffalo milk sample (0,8% tested positive for E. coli O26; the isolate was positive at the verocytotoxicity test and it showed resistance properties to different antimicrobial classes. These preliminary results highlight the need to monitor the foods of animal origin used for production and eaten by a wide range of persons, respect VTEC organism.

  17. Lack of the RNA chaperone hfq attenuates pathogenicity of several Escherichia coli pathotypes towards Caenorhabditis elegans

    DEFF Research Database (Denmark)

    Bojer, Martin Saxtorph; Jakobsen, Henrik; Struve, Carsten;

    2012-01-01

    as a model for virulence characterization and screening for novel antimicrobial entities. Several E. coli human pathotypes are also pathogenic towards C. elegans, and we show here that lack of the RNA chaperone Hfq significantly reduces pathogenicity of VTEC, EAEC, and UPEC in the nematode model. Thus, Hfq...... is intrinsically essential to pathogenic E. coli for survival and virulence exerted in the C. elegans host.......Escherichia coli is an important agent of Gram-negative bacterial infections worldwide, being one of the leading causes of diarrhoea and urinary tract infections. Strategies to understand pathogenesis and develop therapeutic compounds include the use of the nematode Caenorhabditis elegans...

  18. Development of a Vaccine Against Experimental Cholera and Escherichia coli Diarrheal Disease

    OpenAIRE

    Rappaport, R. S.; Bonde, G.

    1981-01-01

    The results of the present investigation indicate a simple approach to the development of a single-vaccine formula which may ultimately be used to confer protection against both cholera and certain types of Escherichia coli diarrheal disease in humans and domestic animals. The design of the vaccine is based on the well-documented ability of cholera antitoxin to neutralize both cholera and heat-labile E. coli enterotoxins (CT and LT, respectively) and on the ability of killed E. coli to enhanc...

  19. Contribution of siderophore systems to growth and urinary tract colonization of asymptomatic bacteriuria Escherichia coli

    DEFF Research Database (Denmark)

    Watts, Rebecca E; Totsika, Makrina; Challinor, Victoria L

    2012-01-01

    The molecular mechanisms that define asymptomatic bacteriuria (ABU) Escherichia coli colonization of the human urinary tract remain to be properly elucidated. Here, we utilize ABU E. coli strain 83972 as a model to dissect the contribution of siderophores to iron acquisition, growth, fitness......, and colonization of the urinary tract. We show that E. coli 83972 produces enterobactin, salmochelin, aerobactin, and yersiniabactin and examine the role of these systems using mutants defective in siderophore biosynthesis and uptake. Enterobactin and aerobactin contributed most to total siderophore activity...

  20. Shiga toxin-producing Escherichia coli and rectoanal junction persistence in ruminants: a study of bacterial-epithelial interactions.

    Science.gov (United States)

    Escherichia coli O157:H7 (O157) was the first Shiga toxin-producing E. coli serotype to be associated with bloody diarrhea or hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) in humans. It has since been implicated in several outbreaks in the U.S. and globally. Non-O157 STEC have not bee...

  1. Synergistic effects in mixed Escherichia coli biofilms

    DEFF Research Database (Denmark)

    Reisner, A.; Holler, B.M.; Molin, Søren

    2006-01-01

    the strongest effects was most often linked to conjugative transmission of natural plasmids carried by the E. coli isolates (70%). Thus, the capacity of an isolate to promote the biofilm through cocultivation was (i) transferable to the K-12 strain, (ii) was linked with the acquisition of conjugation genes...... promotion in this system is not dependent on the laboratory strain and that the described model system could provide relevant insights on mechanisms of biofilm development in natural E. coli populations....

  2. Antibiotic treatment of verocytotoxin-producing Escherichia coli (VTEC) infection

    DEFF Research Database (Denmark)

    Agger, Morten; Scheutz, Flemming; Villumsen, Steen;

    2015-01-01

    OBJECTIVES: A consensus has existed on not to treat verocytotoxin-producing Escherichia coli (VTEC)-infected individuals with antibiotics because of possible subsequent increased risk of developing haemolytic uraemic syndrome (HUS). The aim of this systematic review is to clarify the risk...

  3. Escherichia coli mutants with a temperature-sensitive alcohol dehydrogenase.

    OpenAIRE

    Lorowitz, W; Clark, D.

    1982-01-01

    Mutants of Escherichia coli resistant to allyl alcohol were selected. Such mutants were found to lack alcohol dehydrogenase. In addition, mutants with temperature-sensitive alcohol dehydrogenase activity were obtained. These mutations, designated adhE, are all located at the previously described adh regulatory locus. Most adhE mutants were also defective in acetaldehyde dehydrogenase activity.

  4. New types of Escherichia coli recombination-deficient mutants.

    Science.gov (United States)

    Freifelder, D

    1976-11-01

    A set of Escherichia coli mutants deficient in intramolecular recombination and different from those previously found is described. All have temperature-sensitive lethal mutations. The mutants have been characterized with respect to the following properties: the Pap phenotype, deoxyribonucleic acid synthesis, sensitivity to ultraviolet light, ability to support the growth of phage lambda, filament formation, and mutation frequency.

  5. Escherichia coli and virus isolated from ''sticky kits''

    DEFF Research Database (Denmark)

    Jørgensen, M.; Scheutz, F.; Strandbygaard, Bertel

    1996-01-01

    A total of 121 Escherichia coli strains isolated from 3-week-old mink kits were serotyped and examined for virulence factors. 56 strains were isolated from healthy kits while 65 were from ''sticky kits''. Among these, 34 different serotypes were detected. No difference in serotypes or the presenc...

  6. armA and aminoglycoside resistance in Escherichia coli.

    Science.gov (United States)

    González-Zorn, Bruno; Teshager, Tirushet; Casas, María; Porrero, María C; Moreno, Miguel A; Courvalin, Patrice; Domínguez, Lucas

    2005-06-01

    We report armA in an Escherichia coli pig isolate from Spain. The resistance gene was borne by self-transferable IncN plasmid pMUR050. Molecular analysis of the plasmid and of the armA locus confirmed the spread of this resistance determinant.

  7. armA and Aminoglycoside Resistance in Escherichia coli

    OpenAIRE

    González-Zorn, Bruno; Teshager, Tirushet; Casas, María; Porrero, María C.; Moreno, Miguel A.; Courvalin, Patrice; Domínguez, Lucas

    2005-01-01

    We report armA in an Escherichia coli pig isolate from Spain. The resistance gene was borne by self-transferable IncN plasmid pMUR050. Molecular analysis of the plasmid and of the armA locus confirmed the spread of this resistance determinant.

  8. Combating enteropathogenic Escherichia coli (EPEC) infections: the way forward

    OpenAIRE

    Michael S Donnenberg; Finlay, B. Brett

    2013-01-01

    Enteropathogenic Escherichia coli (EPEC) strains continue to cause severe and sometimes fatal infantile diarrhea, particularly in Africa. Increased efforts at diagnosis, defining the clinical spectrum of disease, understanding pathogenic mechanisms, and delineating immune responses are desperately needed to develop new strategies to combat EPEC.

  9. Characterization of Escherichia coli nucleoids released by osmotic shock.

    NARCIS (Netherlands)

    Wegner, A.S.; Alexeeva, S.; Odijk, T.; Woldringh, C.L.

    2012-01-01

    Nucleoids were isolated by osmotic shock from Escherichia coli spheroplasts at relatively low salt concentrations and in the absence of detergents. Sucrose-protected cells, made osmotically sensitive by growth in the presence of ampicillin or by digestion with low lysozyme concentrations (50-5 μg/ml

  10. Characterization of Escherichia coli nucleoids released by osmotic shock

    NARCIS (Netherlands)

    Wegner, S.; Alexeeva, S.V.; Odijk, T.; Woldringh, C.L.

    2012-01-01

    Nucleoids were isolated by osmotic shock from Escherichia coli spheroplasts at relatively low salt concentrations and in the absence of detergents. Sucrose-protected cells, made osmotically sensitive by growth in the presence of ampicillin or by digestion with low lysozyme concentrations (50–5 µg/ml

  11. Immunologic Control of Diarrheal Disease Due to Enterotoxigenic Escherichia coli

    Science.gov (United States)

    1984-01-01

    Classical Enteropathogenic (Serotyped) Escherichia coli Strains of Proven Pathogenicity. Infect. Immun. 38:798-801, 1982. 8. Levine, M.M. Vacunas Contra...Microbiol., 18:808-815, 1983. 8 15. Levine, M.M., Lanata, C. Progresos en Vacunas Contra Diarrea Bacteriana. Adelantos Microbiol. Enferm. Inf., 2:67-117

  12. Sequencing of Escherichia coli that cause persistent and transient Mastitis

    Science.gov (United States)

    The genomes of two strains of Escherichia coli that cause bovine mastitis were sequenced. These strains are known to be associated with persistent and transient mastitis: strain ECA-B causes a transient infection, and ECC-M leads to a persistent infection....

  13. Comparative Genomics of Escherichia coli Strains Causing Urinary Tract Infections

    DEFF Research Database (Denmark)

    Vejborg, Rebecca Munk; Hancock, Viktoria; Schembri, Mark A.

    2011-01-01

    The virulence determinants of uropathogenic Escherichia coli have been studied extensively over the years, but relatively little is known about what differentiates isolates causing various types of urinary tract infections. In this study, we compared the genomic profiles of 45 strains from a range...

  14. Inactivation of Escherichia coli by titanium dioxide photocatalytic oxidation.

    Science.gov (United States)

    Titanium dioxide in the anatase crystalline form was used as a photocatalyst to generate hydroxyl radicals in a flowthrough water reactor. Experiments were performed on pure cultures of Escherichia coli in dechlorinated tap water and a surface water sample to evaluate the disinfe...

  15. Chromosomal replication incompatibility in Dam methyltransferase deficient Escherichia coli cells

    DEFF Research Database (Denmark)

    Freiesleben, Ulrik Von

    1996-01-01

    Dam methyltransferase deficient Escherichia coli cells containing minichromosomes were constructed. Free plasmid DNA could not be detected in these cells and the minichromosomes were found to be integrated in multiple copies in the origin of replication (oriC) region of the host chromosome...

  16. FimH-mediated autoaggregation of Escherichia coli

    DEFF Research Database (Denmark)

    Schembri, Mark; Christiansen, G.; Klemm, Per

    2001-01-01

    Autoaggregation is a phenomenon thought to contribute to colonization of mammalian hosts by pathogenic bacteria. Type 1 fimbriae are surface organelles of Escherichia coli that mediate D-mannose-sensitive binding to various host surfaces. This binding is conferred by the minor fimbrial component...

  17. DNA supercoiling depends on the phosphorylation potential in Escherichia coli

    DEFF Research Database (Denmark)

    Van Workum, M.; van Dooren, S.J.M; Oldenburg, N

    1996-01-01

    ATP/ADP ratios were varied in different ways and the degree of negative supercoiling was determined in Escherichia coli. Independent of whether the ATP/ADP ratio was reduced by a shift to anaerobic conditions, by addition of protonophore (dinitrophenol) or by potassium cyanide addition, DNA...

  18. Novel Aggregative Adherence Fimbria Variant of Enteroaggregative Escherichia coli

    DEFF Research Database (Denmark)

    Jønsson, Rie; Struve, Carsten; Boisen, Nadia

    2015-01-01

    Enteroaggregative Escherichia coli (EAEC) organisms belong to a diarrheagenic pathotype known to cause diarrhea and can be characterized by distinct aggregative adherence (AA) in a stacked-brick pattern to cultured epithelial cells. In this study, we investigated 118 EAEC strains isolated from...

  19. Peptide nucleic acid (PNA) antisense effects in Escherichia coli

    DEFF Research Database (Denmark)

    Good, L; Nielsen, P E

    1999-01-01

    Antisense peptide nucleic acid (PNA) can be used to control cell growth, gene expression and growth phenotypes in the bacteria Escherichia coli. PNAs targeted to the RNA components of the ribosome can inhibit translation and cell growth, and PNAs targeted to mRNA can limit gene expression with gene...

  20. Suppressors of DnaAATP imposed overinitiation in Escherichia coli

    DEFF Research Database (Denmark)

    Charbon, Godefroid; Riber, Leise; Cohen, Malene

    2011-01-01

    Chromosome replication in Escherichia coli is limited by the supply of DnaA associated with ATP. Cells deficient in RIDA (Regulatory Inactivation of DnaA) due to a deletion of the hda gene accumulate suppressor mutations (hsm) to counteract the overinitiation caused by an elevated DnaAATP level...