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Sample records for pseudomallei selective agar

  1. Comparison of four selective media for the isolation of Burkholderia mallei and Burkholderia pseudomallei.

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    Glass, Mindy B; Beesley, Cari A; Wilkins, Patricia P; Hoffmaster, Alex R

    2009-06-01

    Currently there are no commercially available selective media indicated for the isolation of Burkholderia mallei and Burkholderia pseudomallei. Ashdown's agar, a custom selective medium for isolation of B. pseudomallei, is well described in the literature but unavailable commercially. Three commercially available media, Burkholderia cepacia selective agar (BCSA), oxidative-fermentative-polymyxin B-bacitracin-lactose (OFPBL) agar, and Pseudomonas cepacia (PC) agar are recommended for isolation of B. cepacia from respiratory secretions of cystic fibrosis patients. We evaluated the sensitivity and selectivity of these four media using 20 B. mallei, 20 B. pseudomallei, 20 Burkholderia spp., and 15 diagnostically challenging organisms. Ashdown's agar was the most sensitive medium for the isolation of B. pseudomallei, but it was unable to support growth of B. mallei. Pseudomonas cepacia agar was highly sensitive and selective for both organisms. In non-endemic areas, we suggest the use of the commercially available PC agar for the isolation of B. mallei and B. pseudomallei.

  2. An objective approach for Burkholderia pseudomallei strain selection as challenge material for medical countermeasures efficacy testing

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    Kristopher E. Van Zandt

    2012-09-01

    Full Text Available Burkholderia pseudomallei is the causative agent of melioidosis, a rare disease of biodefense concern with high mortality and extreme difficulty in treatment. No human vaccines are available that protect against B. pseudomallei infection, and with the current limitations of antibiotic treatment, the development of new preventative and therapeutic interventions is crucial. Although clinical trials could be used to test the efficacy of new medical countermeasures (MCMs, the high mortality rates associated with melioidosis raises significant ethical issues concerning treating individuals with new compounds with unknown efficacies. The US Food and Drug Administration (FDA has formulated a set of guidelines for the licensure of new MCMs to treat diseases in which it would be unethical to test the efficacy of these drugs in humans. The FDA Animal Rule 21 CFR 314 calls for consistent, well-characterized B. pseudomallei strains to be used as challenge material in animal models. In order to facilitate the efficacy testing of new MCMs for melioidosis using animal models, we intend to develop a well-characterized panel of strains for use. This panel will comprise of strains that were isolated from human cases, have a low passage history, are virulent in animal models, and are well characterized phenotypically and genotypically. We have reviewed published and unpublished data on various B. pseudomallei strains to establish an objective method for selecting the strains to be included in the panel of B. pseudomallei strains with attention to five categories: animal infection models, genetic characterization, clinical and passage history, and availability of the strain to the research community. We identified 109 strains with data in at least one of the five categories, scored each strain based on the gathered data and identified 6 strains as candidate for a B. pseudomallei strain panel.

  3. An allelic exchange system for compliant genetic manipulation of the select agents Burkholderia pseudomallei and Burkholderia mallei.

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    Hamad, Mohamad A; Zajdowicz, Sheryl L; Holmes, Randall K; Voskuil, Martin I

    2009-02-01

    Burkholderia pseudomallei and B. mallei are Gram-negative bacterial pathogens that cause melioidosis in humans and glanders in horses, respectively. Both bacteria are classified as category B select agents in the United States. Due to strict select-agent regulations, the number of antibiotic selection markers approved for use in these bacteria is greatly limited. Approved markers for B. pseudomallei include genes encoding resistance to kanamycin (Km), gentamicin (Gm), and zeocin (Zeo); however, wild type B. pseudomallei is intrinsically resistant to these antibiotics. Selection markers for B. mallei are limited to Km and Zeo resistance genes. Additionally, there are few well developed counter-selection markers for use in Burkholderia. The use of SacB as a counter-selection method has been of limited success due to the presence of endogenous sacBC genes in the genomes of B. pseudomallei and B. mallei. These impediments have greatly hampered the genetic manipulation of B. pseudomallei and B. mallei and currently few reliable tools for the genetic manipulation of Burkholderia exist. To expand the repertoire of genetic tools for use in Burkholderia, we developed the suicide plasmid pMo130, which allows for the compliant genetic manipulation of the select agents B. pseudomallei and B. mallei using allelic exchange. pMo130 harbors an aphA gene which allows for Km selection, the reporter gene xylE, which allows for reliable visual detection of Burkholderia transformants, and carries a modified sacB gene that allows for the resolution of co-integrants. We employed this system to generate multiple unmarked and in-frame mutants in B. pseudomallei, and one mutant in B. mallei. This vector significantly expands the number of available tools that are select-agent compliant for the genetic manipulation of B. pseudomallei and B. mallei.

  4. Selected elements in fly agaric Amanita muscaria.

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    Falandysz, J; Kunito, T; Kubota, R; Lipka, K; Mazur, A; Falandysz, Justyna J; Tanabe, S

    2007-09-01

    Concentrations of Ag, Al, Ba, Ca, Cd, Co, Cu, Cr, Cs, Fe, Ga, Hg, K, Mg, Mn, Mo, Na, Pb, Rb, Se, Sb, Sr, V, Tl and Zn have been determined in the whole fruiting bodies, as well as separately in caps and stalks, of fly agaric collected from three geographically distant sites in northern part of Poland. The elements were determined using ICP-MS, ICP-OES, HG-AAS and CV-AAS, respectively. For elements such as Al, Ba, Cr, Fe, Ga, Mo, Mn, Pb, Sb, Sr, Tl, and V concentrations were similar in the caps and stalks, respectively, and for K, Zn, Ag, Ca, Cd, Cu, Hg, Mg, Rb and Se were greater in the caps, while for Co, Cs and Na in the stalks. For Ag, Al, Ba, Ca, Cd, Co, Cr, Cs, Fe, Ga, Hg, Mn, Mo, Pb, Rb, Sb, Sr, Tl and V concentration in the caps showed spatial variations (P<0.05), while for Cu, K, Mg, Na, Se and Zn was independent of the site. The elements such as K with median or mean in the caps between 37,000 and 43,000 microg/g.dm and Mg with 920 and 1,100 microg/g dm were most abundant. Next, within median values range from approximately 100 to 500 microg/g dm were such as Ca, Fe and Al, and in descending order they followed by Rb (100-400 microg/g dm); V, Na, Zn (50-200 microg/g dm); Cu, Mn (10-50 microg/g dm); Cd (10-20 microg/g dm); Se (5 microg/g dm); Ba (<1-3); Cr, Ag, Pb, Sr (<1-2 microg/g dm); Cs, Co, Hg (<1-1 microg/g dm); Ga (<0.5), Sb, Mo and Tl (<0.1 microg/g dm).

  5. Mupirocin-mucin agar for selective enumeration of Bifidobacterium bifidum.

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    Pechar, Radko; Rada, Vojtech; Parafati, Lucia; Musilova, Sarka; Bunesova, Vera; Vlkova, Eva; Killer, Jiri; Mrazek, Jakub; Kmet, Vladimir; Svejstil, Roman

    2014-11-17

    Bifidobacterium bifidum is a bacterial species exclusively found in the human intestinal tract. This species is becoming increasingly popular as a probiotic organism added to lyophilized products. In this study, porcine mucin was used as the sole carbon source for the selective enumeration of B. bifidum in probiotic food additives. Thirty-six bifidobacterial strains were cultivated in broth with mucin. Only 13 strains of B. bifidum utilized the mucin to produce acids. B. bifidum was selectively enumerated in eight probiotic food supplements using agar (MM agar) containing mupirocin (100 mg/L) and mucin (20 g/L) as the sole carbon source. MM agar was fully selective if the B. bifidum species was presented together with Bifidobacterium animalis subsp. lactis, Bifidobacterium breve, and Bifidobacterium longum subsp. longum species and with lactic acid bacteria (lactobacilli, streptococci). Isolated strains of B. bifidum were identified using biochemical, PCR, MALDI-TOF procedures and 16S rRNA gene sequencing. The novel selective medium was also suitable for the isolation of B. bifidum strains from human fecal samples. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Detection of Burkholderia pseudomallei in Sputum using Selective Enrichment Broth and Ashdown's Medium at Kampong Cham Provincial Hospital, Cambodia.

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    Nhem, Somary; Letchford, Joanne; Meas, Chea; Thann, Sovanndeth; McLaughlin, James C; Baron, Ellen Jo; West, T Eoin

    2014-01-01

    Melioidosis, infection caused by Burkholderia pseudomallei, is increasingly reported in Cambodia. We hypothesized that implementation of an enhanced sputum testing protocol in a provincial hospital diagnostic microbiology laboratory would increase detection of B. pseudomallei. We tested 241 sputum specimens that were deemed acceptable for culture, comparing culture in selective enrichment broth followed by sub-culture on Ashdown's medium to standard culture methods. Two specimens (0.8%) were positive for B. pseudomallei using the enhanced protocol whereas one specimen (0.4%) was positive using standard methods. Given the low numbers of positive specimens, we could not conclusively determine the utility of the enhanced sputum testing protocol. However, the ramifications of identification of  B. pseudomallei are substantial, and the benefit of the enhanced testing protocol may be more apparent in patients selected based on risk factors and clinical presentation. Promoting clinician awareness of the infection and encouraging utilization of diagnostic microbiology services are also likely to be important factors in facilitating identification of melioidosis.

  7. Alanine racemase mutants of Burkholderia pseudomallei and Burkholderia mallei and use of alanine racemase as a non-antibiotic-based selectable marker.

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    Sheryl L W Zajdowicz

    Full Text Available Burkholderia pseudomallei and Burkholderia mallei are category B select agents and must be studied under BSL3 containment in the United States. They are typically resistant to multiple antibiotics, and the antibiotics used to treat B. pseudomallei or B. mallei infections may not be used as selective agents with the corresponding Burkholderia species. Here, we investigated alanine racemase deficient mutants of B. pseudomallei and B. mallei for development of non-antibiotic-based genetic selection methods and for attenuation of virulence. The genome of B. pseudomallei K96243 has two annotated alanine racemase genes (bpsl2179 and bpss0711, and B. mallei ATCC 23344 has one (bma1575. Each of these genes encodes a functional enzyme that can complement the alanine racemase deficiency of Escherichia coli strain ALA1. Herein, we show that B. pseudomallei with in-frame deletions in both bpsl2179 and bpss0711, or B. mallei with an in-frame deletion in bma1575, requires exogenous D-alanine for growth. Introduction of bpsl2179 on a multicopy plasmid into alanine racemase deficient variants of either Burkholderia species eliminated the requirement for D-alanine. During log phase growth without D-alanine, the viable counts of alanine racemase deficient mutants of B. pseudomallei and B. mallei decreased within 2 hours by about 1000-fold and 10-fold, respectively, and no viable bacteria were present at 24 hours. We constructed several genetic tools with bpsl2179 as a selectable genetic marker, and we used them without any antibiotic selection to construct an in-frame ΔflgK mutant in the alanine racemase deficient variant of B. pseudomallei K96243. In murine peritoneal macrophages, wild type B. mallei ATCC 23344 was killed much more rapidly than wild type B. pseudomallei K96243. In addition, the alanine racemase deficient mutant of B. pseudomallei K96243 exhibited attenuation versus its isogenic parental strain with respect to growth and survival in murine

  8. Alanine racemase mutants of Burkholderia pseudomallei and Burkholderia mallei and use of alanine racemase as a non-antibiotic-based selectable marker.

    Science.gov (United States)

    Zajdowicz, Sheryl L W; Jones-Carson, Jessica; Vazquez-Torres, Andres; Jobling, Michael G; Gill, Ronald E; Holmes, Randall K

    2011-01-01

    Burkholderia pseudomallei and Burkholderia mallei are category B select agents and must be studied under BSL3 containment in the United States. They are typically resistant to multiple antibiotics, and the antibiotics used to treat B. pseudomallei or B. mallei infections may not be used as selective agents with the corresponding Burkholderia species. Here, we investigated alanine racemase deficient mutants of B. pseudomallei and B. mallei for development of non-antibiotic-based genetic selection methods and for attenuation of virulence. The genome of B. pseudomallei K96243 has two annotated alanine racemase genes (bpsl2179 and bpss0711), and B. mallei ATCC 23344 has one (bma1575). Each of these genes encodes a functional enzyme that can complement the alanine racemase deficiency of Escherichia coli strain ALA1. Herein, we show that B. pseudomallei with in-frame deletions in both bpsl2179 and bpss0711, or B. mallei with an in-frame deletion in bma1575, requires exogenous D-alanine for growth. Introduction of bpsl2179 on a multicopy plasmid into alanine racemase deficient variants of either Burkholderia species eliminated the requirement for D-alanine. During log phase growth without D-alanine, the viable counts of alanine racemase deficient mutants of B. pseudomallei and B. mallei decreased within 2 hours by about 1000-fold and 10-fold, respectively, and no viable bacteria were present at 24 hours. We constructed several genetic tools with bpsl2179 as a selectable genetic marker, and we used them without any antibiotic selection to construct an in-frame ΔflgK mutant in the alanine racemase deficient variant of B. pseudomallei K96243. In murine peritoneal macrophages, wild type B. mallei ATCC 23344 was killed much more rapidly than wild type B. pseudomallei K96243. In addition, the alanine racemase deficient mutant of B. pseudomallei K96243 exhibited attenuation versus its isogenic parental strain with respect to growth and survival in murine peritoneal macrophages.

  9. Comparison of Four Selective Agars for the Isolation of Pseudomonads

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    Hart, A.; Kite, Atricia E.

    1977-01-01

    Significant differences were found between Dettol (chloroxylenol) agar, nalidixic acid-cetrimide agar, and two other cetrimide-containing agars used for the isolation of small numbers of pseudomonads. PMID:879777

  10. Development of a selective agar plate for the detection of Campylobacter spp. in fresh produce.

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    Yoo, Jin-Hee; Choi, Na-Young; Bae, Young-Min; Lee, Jung-Su; Lee, Sun-Young

    2014-10-17

    This study was conducted to develop a selective medium for the detection of Campylobacter spp. in fresh produce. Campylobacter spp. (n=4), non-Campylobacter (showing positive results on Campylobacter selective agar) strains (n=49) isolated from fresh produce, indicator bacteria (n=13), and spoilage bacteria isolated from fresh produce (n=15) were plated on four Campylobacter selective media. Bolton agar and modified charcoal cefoperazone deoxycholate agar (mCCDA) exhibited higher sensitivity for Campylobacter spp. than did Preston agar and Hunt agar, although certain non-Campylobacter strains isolated from fresh produce by using a selective agar isolation method, were still able to grow on Bolton agar and mCCDA. To inhibit the growth of non-Campylobacter strains, Bolton agar and mCCDA were supplemented with 5 antibiotics (rifampicin, polymyxin B, sodium metabisulfite, sodium pyruvate, ferrous sulfate) and the growth of Campylobacter spp. (n=7) and non-Campylobacter strains (n=44) was evaluated. Although Bolton agar supplemented with rifampicin (BR agar) exhibited a higher selectivity for Campylobacter spp. than did mCCDA supplemented with antibiotics, certain non-Campylobacter strains were still able to grow on BR agar (18.8%). When BR agar with various concentrations of sulfamethoxazole-trimethoprim were tested with Campylobacter spp. (n=8) and non-Campylobacter (n=7), sulfamethoxazole-trimethoprim was inhibitory against 3 of 7 non-Campylobacter strains. Finally, we validated the use of BR agar containing 50mg/L sulfamethoxazole (BRS agar) or 0.5mg/L ciprofloxacin (BRCS agar) and other selective agars for the detection of Campylobacter spp. in chicken and fresh produce. All chicken samples were positive for Campylobacter spp. when tested on mCCDA, BR agar, and BRS agar. In fresh produce samples, BRS agar exhibited the highest selectivity for Campylobacter spp., demonstrating its suitability for the detection of Campylobacter spp. in fresh produce. Copyright

  11. Utility of a Lateral Flow Immunoassay (LFI to Detect Burkholderia pseudomallei in Soil Samples.

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    Patpong Rongkard

    2016-12-01

    Full Text Available Culture is the gold standard for the detection of environmental B. pseudomallei. In general, soil specimens are cultured in enrichment broth for 2 days, and then the culture broth is streaked on an agar plate and incubated further for 7 days. However, identifying B. pseudomallei on the agar plates among other soil microbes requires expertise and experience. Here, we evaluate a lateral flow immunoassay (LFI developed to detect B. pseudomallei capsular polysaccharide (CPS in clinical samples as a tool to detect B. pseudomallei in environmental samples.First, we determined the limit of detection (LOD of LFI for enrichment broth of the soil specimens. Soil specimens (10 grams/specimen culture negative for B. pseudomallei were spiked with B. pseudomallei ranging from 10 to 105 CFU, and incubated in 10 ml of enrichment broth in air at 40°C. Then, on day 2, 4 and 7 of incubation, 50 μL of the upper layer of the broth were tested on the LFI, and colony counts to determine quantity of B. pseudomallei in the broth were performed. We found that all five soil specimens inoculated at 10 CFU were negative by LFI on day 2, but four of those five specimens were LFI positive on day 7. The LOD of the LFI was estimated to be roughly 3.8x106 CFU/ml, and culture broth on day 7 was selected as the optimal sample for LFI testing. Second, we evaluated the utility of the LFI by testing 105 soil samples from Northeast Thailand. All samples were also tested by standard culture and quantitative PCR (qPCR targeting orf2. Of 105 soil samples, 35 (33% were LFI positive, 25 (24% were culture positive for B. pseudomallei, and 79 (75% were qPCR positive. Of 11 LFI positive but standard culture negative specimens, six were confirmed by having the enrichment broth on day 7 culture positive for B. pseudomallei, and an additional three by qPCR. The LFI had 97% (30/31 sensitivity to detect soil specimens culture positive for B. pseudomallei.The LFI can be used to detect B

  12. Accurate and rapid identification of the Burkholderia pseudomallei near-neighbour, Burkholderia ubonensis, using real-time PCR.

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    Price, Erin P; Sarovich, Derek S; Webb, Jessica R; Ginther, Jennifer L; Mayo, Mark; Cook, James M; Seymour, Meagan L; Kaestli, Mirjam; Theobald, Vanessa; Hall, Carina M; Busch, Joseph D; Foster, Jeffrey T; Keim, Paul; Wagner, David M; Tuanyok, Apichai; Pearson, Talima; Currie, Bart J

    2013-01-01

    Burkholderia ubonensis is an environmental bacterium belonging to the Burkholderia cepacia complex (Bcc), a group of genetically related organisms that are associated with opportunistic but generally nonfatal infections in healthy individuals. In contrast, the near-neighbour species Burkholderia pseudomallei causes melioidosis, a disease that can be fatal in up to 95% of cases if left untreated. B. ubonensis is frequently misidentified as B. pseudomallei from soil samples using selective culturing on Ashdown's medium, reflecting both the shared environmental niche and morphological similarities of these species. Additionally, B. ubonensis shows potential as an important biocontrol agent in B. pseudomallei-endemic regions as certain strains possess antagonistic properties towards B. pseudomallei. Current methods for characterising B. ubonensis are laborious, time-consuming and costly, and as such this bacterium remains poorly studied. The aim of our study was to develop a rapid and inexpensive real-time PCR-based assay specific for B. ubonensis. We demonstrate that a novel B. ubonensis-specific assay, Bu550, accurately differentiates B. ubonensis from B. pseudomallei and other species that grow on selective Ashdown's agar. We anticipate that Bu550 will catalyse research on B. ubonensis by enabling rapid identification of this organism from Ashdown's-positive colonies that are not B. pseudomallei.

  13. Accurate and rapid identification of the Burkholderia pseudomallei near-neighbour, Burkholderia ubonensis, using real-time PCR.

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    Erin P Price

    Full Text Available Burkholderia ubonensis is an environmental bacterium belonging to the Burkholderia cepacia complex (Bcc, a group of genetically related organisms that are associated with opportunistic but generally nonfatal infections in healthy individuals. In contrast, the near-neighbour species Burkholderia pseudomallei causes melioidosis, a disease that can be fatal in up to 95% of cases if left untreated. B. ubonensis is frequently misidentified as B. pseudomallei from soil samples using selective culturing on Ashdown's medium, reflecting both the shared environmental niche and morphological similarities of these species. Additionally, B. ubonensis shows potential as an important biocontrol agent in B. pseudomallei-endemic regions as certain strains possess antagonistic properties towards B. pseudomallei. Current methods for characterising B. ubonensis are laborious, time-consuming and costly, and as such this bacterium remains poorly studied. The aim of our study was to develop a rapid and inexpensive real-time PCR-based assay specific for B. ubonensis. We demonstrate that a novel B. ubonensis-specific assay, Bu550, accurately differentiates B. ubonensis from B. pseudomallei and other species that grow on selective Ashdown's agar. We anticipate that Bu550 will catalyse research on B. ubonensis by enabling rapid identification of this organism from Ashdown's-positive colonies that are not B. pseudomallei.

  14. How selective are agar cultures for malignant transformation?

    NARCIS (Netherlands)

    Klein, J.C.

    1981-01-01

    In vitro assays that permit cloning of tumour cells in soft agar have been improved during the last 5 years. Two of them are claimed to be useful as test systems for the screening of new anticancer drugs and even for drug sensitivity testing of individual human tumours in devising individualized

  15. Detection of Burkholderia pseudomallei in Sputum using Selective Enrichment Broth and Ashdown’s Medium at Kampong Cham Provincial Hospital, Cambodia [v1; ref status: indexed, http://f1000r.es/4w7

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    Somary Nhem

    2014-12-01

    Full Text Available Melioidosis infection, caused by Burkholderia pseudomallei, is increasingly reported in Cambodia. We hypothesized that implementation of an enhanced sputum testing protocol in a provincial hospital diagnostic microbiology laboratory would increase detection of B. pseudomallei. We tested 241 sputum specimens that were deemed acceptable for culture, comparing culture in selective enrichment broth followed by sub-culture on Ashdown’s medium to standard culture methods. Two specimens (0.8% were positive for B. pseudomallei using the enhanced protocol whereas one specimen (0.4% was positive using standard methods. These findings demonstrate that B. pseudomallei is rarely detected in sputum at this hospital. The low frequency of B. pseudomallei in sputum specimens precludes drawing any conclusions about the relative benefits of an enhanced sputum testing protocol at this site. Promoting clinician awareness of the infection and encouraging utilization of diagnostic microbiology services are likely to be important factors in facilitating identification of melioidosis.

  16. Comparison of selective agars recommended by method ISO 11290-1 and chromogenic agars for the isolation of Listeria sp. in refrigerated sausages

    OpenAIRE

    Thalyta Marina Benetti; Cristina Leise Bastos Monteiro; Márcia Regina Beux; Wanda Moscalewski Abrahão

    2012-01-01

    The aim of this study was to determine the prevalence of Listeria sp. in refrigerated sausages, and to compare the performance of the selective plating media employed in the ISO 11290-1 method (PALCAM and Oxford agars) with chromogenic agars (Chromogenic Listeria agars CM 1080 (OCLA) and CM 1084). The prevalence of Listeria sp. detected was 52.9%, comprising 13.7% L. monocytogenes strains. The efficacy of the four agars for the isolation of L. monocytogenes proved to be satisfactory. Despite ...

  17. [THE APPLICATION OF SELECTIVE CHROMOGENIC AGAR FOR DETECTING ENTEROBACTERIA WITH PRODUCTION OF BETA-LACTAMASES].

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    Korobova, A G; Frolova, L N; Kliasova, G A

    2015-11-01

    The detection of enterobacteria with production of beta-lactamases of extended spectrum in selective chromogenic agar was analyzed The results ofdetection of beta-lactamases of extended spectrum was compared with "double disc" technique. The smears from mucous membrane of guttur and rectum from patients were analyzed in parallel on solid growth agar (Endo or Mac Conkey) and on selective agar CHROMagartm ESBL (CHROMagar France). The production of beta-lactamases of extended spectrum was confirmed using "double discs" technique. To exclude hyper-production of ampC beta-lactamases E-test was applied containing cefotetan and cefotetan with cloxacillin. The sampling consisted of 1552 samples from patients. The study permitted to isolate 1243 strains of enterobacteria on agar Endo or Mac Conkey and 409 strains of enterobacteria on selective agar CHROMagartm ESBL (Escherichia coli n = 226, Klebsiella pneumoniae n = 105, enterobacter spp. n = 35, Citrobacter spp. n = 21, others n = 22). The application of "double discs" technique confirmed production of beta-lactamases of extended spectrum in 386 (94%) out of 409 strains isolated on agar CHROMagartm ESBL. In 23 (6%) of strains no confirmation was established and hyper-production of ampC of beta-lactamases was established 15 out of total. Additionally, 8 were sensitive to cephalosporin of third generation. All enterobacteria isolated on agar Endo or Mac Conkey also were tested by "double discs" technique. Overall, 394 strains of enterobacteria with production of beta-lactamases of extended spectrum were obtained. On all agars (agar Endo or Mac Conkey and CHROMagartm ESBL)--263 (67%) strains; only on CHROMagartm ESBL--123 (31%) and only on agar Endo or Mac Conkey--8 (2%) (p enterobacteria producing beta-lactamases of extended spectrum were submitted to clinic in 18-24 hours after arrival ofsamplesfrom patients in laboratory. The CHR OMagartm ESBL has higher sensitivity and specificity to detect enterobacteria with production

  18. Competition between Burkholderia pseudomallei and B. thailandensis.

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    Ngamdee, Wikanda; Tandhavanant, Sarunporn; Wikraiphat, Chanthiwa; Reamtong, Onrapak; Wuthiekanun, Vanaporn; Salje, Jeanne; Low, David A; Peacock, Sharon J; Chantratita, Narisara

    2015-03-03

    Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis, an often fatal disease in tropical countries. Burkholderia thailandensis is a non-virulent but closely related species. Both species are soil saprophytes but are almost never isolated together. We identified two mechanisms by which B. pseudomallei affects the growth of B. thailandensis. First, we found that six different isolates of B. pseudomallei inhibited the growth of B. thailandensis on LB agar plates. Second, our results indicated that 55% of isolated strains of B. pseudomallei produced a secreted compound that inhibited the motility but not the viability of B. thailandensis. Analysis showed that the active compound was a pH-sensitive and heat-labile compound, likely a protein, which may affect flagella processing or facilitate their degradation. Analysis of bacterial sequence types (STs) demonstrated an association between this and motility inhibition. The active compound was produced from B. pseudomallei during the stationary growth phase. Taken together, our results indicate that B. pseudomallei inhibits both the growth and motility of its close relative B. thailandensis. The latter phenomenon appears to occur via a previously unreported mechanism involving flagellar processing or degradation.

  19. Comparison of ChromID agar and Clostridium difficile selective agar for effective isolation of C. difficile from stool specimens.

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    Shin, Bo-Moon; Lee, Eun Joo

    2014-01-01

    ChromID Clostridium difficile agar (IDCd; bioMérieux SA, France) is a recently developed chromogenic medium for rapid and specific isolation of C. difficile. We compared the performance of IDCd with that of Clostridium difficile Selective Agar (CDSA). A total of 530 fresh stool specimens were collected from patients with clinical signs compatible with C. difficile infection, and cultures for C. difficile were performed on IDCd and CDSA. C. difficile colonies were identified by spore staining, odor, use of an ANI identification test kit (bioMérieux SA), and multiplex PCR for tcdA, tcdB, and tpi. The concordance rate between IDCd and CDSA was 90.6% (480/530). The positivity rates on IDCd on days 1 and 2 (55.6% and 85.0%, respectively) were significantly higher than those on CDSA (19.4% and 75.6%, respectively) (P<0.001 for day 1 and P=0.02 for day 2), but the detection rates on IDCd and CDSA on day 3 were not different (89.4% vs. 82.8%, P=0.0914). On day 3, the recovery rates for non-C. difficile isolates on IDCd and CDSA were 30.2% (160/530) and 22.1% (117/530), respectively (P=0.0075). Clostridium spp. other than C. difficile were the most prevalent non-C. difficile isolates on both media. The culture positivity rates on IDCd and CDSA were not different on day 3 but IDCd may allow for rapid and sensitive detection of C. difficile within 2 days of cultivation.

  20. A genomic survey of positive selection in Burkholderia pseudomallei provides insights into the evolution of accidental virulence.

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    Tannistha Nandi

    2010-04-01

    Full Text Available Certain environmental microorganisms can cause severe human infections, even in the absence of an obvious requirement for transition through an animal host for replication ("accidental virulence". To understand this process, we compared eleven isolate genomes of Burkholderia pseudomallei (Bp, a tropical soil microbe and causative agent of the human and animal disease melioidosis. We found evidence for the existence of several new genes in the Bp reference genome, identifying 282 novel genes supported by at least two independent lines of supporting evidence (mRNA transcripts, database homologs, and presence of ribosomal binding sites and 81 novel genes supported by all three lines. Within the Bp core genome, 211 genes exhibited significant levels of positive selection (4.5%, distributed across many cellular pathways including carbohydrate and secondary metabolism. Functional experiments revealed that certain positively selected genes might enhance mammalian virulence by interacting with host cellular pathways or utilizing host nutrients. Evolutionary modifications improving Bp environmental fitness may thus have indirectly facilitated the ability of Bp to colonize and survive in mammalian hosts. These findings improve our understanding of the pathogenesis of melioidosis, and establish Bp as a model system for studying the genetics of accidental virulence.

  1. Evaluation of CP Chromo Select Agar for the enumeration of Clostridium perfringens from water.

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    Manafi, Mammad; Waldherr, Kerstin; Kundi, Michael

    2013-10-01

    The European Directive on drinking water quality has included mCP agar as the reference method for recovering Clostridium perfringens from drinking waters. In the present study, three media (mCP, TSCF and CP Chromo Select Agar) were evaluated for recovery of C. perfringens in different surface water samples. Out of 139 water samples, using a membrane filtration technique, 131 samples (94.2%) were found to be presumptively positive for C. perfringens in at least one of the culture media. Green colored colonies on CP Chromo Select Agar (CCP agar) were counted as presumptive C. perfringens isolates. Out of 483 green colonies on CCP agar, 96.3% (465 strains, indole negative) were identified as C. perfringens, and 15 strains (3.1%) were indole positive and were identified as Clostridium sordellii, Clostridium bifermentans or Clostridium tetani. Only 3 strains (0.6%) gave false positive results and were identified as Clostridium fallax, Clostridium botulinum, and Clostridium tertium. Variance analysis of the data obtained shows statistically no significant differences in the counts obtained between media employed in this work. The mCP method is very onerous for routine screening and bacterial colonies could not be used for further biochemical testing. The colonies on CCP and TSCF were easy to count and subculture for confirmation tests. TSCF detects sulfite-reducing clostridia, including species other than C. perfringens, and in some cases excessive blackening of the agar frustrated counting of the colonies. If the contamination was too high, TSCF did not consistently produce black colonies and as a consequence, the colonies were white and gave false negative results. On the other hand, the identification of typical and atypical colonies isolated from all media demonstrated that CCP agar was the most useful medium for C. perfringens recovery in water samples. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Detection of bacterioplankton in immersed cadavers using selective agar plates.

    Science.gov (United States)

    Kakizaki, Eiji; Kozawa, Shuji; Tashiro, Noriko; Sakai, Masahiro; Yukawa, Nobuhiro

    2009-04-01

    We measured bacterioplankton in blood from cadavers retrieved from the sea (n=12), near estuaries (n=4), rivers (fresh water, n=8) and from bathtubs (n=4) as well as from non-drowned victims (n=10) discovered near aquatic environments. Blood from 11 victims drowned in seawater developed bioluminescent and/or blue colonies (oxidase test positive) on selective media containing 2-4% NaCl. Homology analyses of the 16S rRNA gene showed that all of them were marine bacteria (genera: Photobacterium, Vibrio, Shewanella, Psychrobacter). Blood from all victims drowned in rivers generated blue colonies on plates containing 3%, but not 4% NaCl. Homology analyses showed that the blue colonies were generated from bacteria that inhabit fresh water (Aeromonas). None of the blood samples from victims that drowned in bathtubs generated bioluminescent and blue colonies. However, all cadavers contained bacteria that produced unstained colonies (Staphylococcus, Bacillus, Enterobacter, Escherichia, etc.). Among non-drowned victims, blood from two gave rise to blue colonies on plates containing < or =3% NaCl (Pseudomonas). Of the cadavers found near estuaries, bioluminescent and blue colonies developed from two of them on media containing 2-4% NaCl (Photobacterium, Vibrio, Listonella), but not from two others on plates containing 4% NaCl (at < or =3%; blue colonies, Aeromonas; unstained colonies, Citrobacter, Vagococcus, Proteus, Enterobacter). These results suggested that the presence of numerous bacterioplankton in immersed cadavers could support a conclusion of death by drowning.

  3. Selection of Brain Metastasis-Initiating Breast Cancer Cells Determined by Growth on Hard Agar

    Science.gov (United States)

    Guo, Lixia; Fan, Dominic; Zhang, Fahao; Price, Janet E.; Lee, Ju-Seog; Marchetti, Dario; Fidler, Isaiah J.; Langley, Robert R.

    2011-01-01

    An approach that facilitates rapid isolation and characterization of tumor cells with enhanced metastatic potential is highly desirable. Here, we demonstrate that plating GI-101A human breast cancer cells on hard (0.9%) agar selects for the subpopulation of metastasis-initiating cells. The agar-selected cells, designated GI-AGR, were homogeneous for CD44+ and CD133+ and five times more invasive than the parental GI-101A cells. Moreover, mice injected with GI-AGR cells had significantly more experimental brain metastases and shorter overall survival than did mice injected with GI-101A cells. Comparative gene expression analysis revealed that GI-AGR cells were markedly distinct from the parental cells but shared an overlapping pattern of gene expression with the GI-101A subline GI-BRN, which was generated by repeated in vivo recycling of GI-101A cells in an experimental brain metastasis model. Data mining on 216 genes shared between GI-AGR and GI-BRN breast cancer cells suggested that the molecular phenotype of these cells is consistent with that of cancer stem cells and the aggressive basal subtype of breast cancer. Collectively, these results demonstrate that analysis of cell growth in a hard agar assay is a powerful tool for selecting metastasis-initiating cells in a heterogeneous population of breast cancer cells, and that such selected cells have properties similar to those of tumor cells that are selected based on their potential to form metastases in mice. PMID:21514446

  4. Growth of non-Campylobacter, oxidase-positive bacteria on selective Campylobacter agar.

    OpenAIRE

    Moskowitz, L B; Chester, B

    1982-01-01

    A total of 67 oxidase-positive, gram-negative bacteria were tested for growth on selective Campylobacter agar (Blaser formulation, BBL Microbiology Systems, Cockeysville, Md.) at 42 degrees C under microaerophilic conditions. Although the growth of most of these bacteria was prevented, all strains of Achromobacter xylosoxidans, Pseudomonas aeruginosa, Pseudomonas putrefaciens, Pseudomonas alcaligenes, and Pseudomonas pseudoalcaligenes grew as well as Campylobacter fetus subsp. jejuni.

  5. Evaluation of cephamycins as supplements to selective agar for detecting Campylobacter spp. in chicken carcass rinses.

    Science.gov (United States)

    Chon, Jung-Whan; Kim, Young-Ji; Kim, Hong-Seok; Kim, Dong-Hyeon; Kim, Hyunsook; Song, Kwang-Young; Sung, Kidon; Seo, Kun-Ho

    2016-04-16

    Although cefoperazone is the most commonly used antibiotic in Campylobacter-selective media, the distribution of cefoperazone-resistant bacteria such as extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli is increasing. Here we evaluated the potential of cephamycins for use as supplements to improve modified charcoal-cefoperazone-deoxycholate agar (mCCDA) by replacing cefoperazone with the same concentrations (32 mg/L) of cefotetan (modified charcoal-cefotetan-deoxycholate agar, mCCtDA) and cefoxitin (modified charcoal-cefoxitin-deoxycholate agar, mCCxDA). In chicken carcass rinse samples, the number of mCCDA plates detecting for Campylobacter (18/70, 26%) was significantly lower than that of mCCtDA (42/70, 60%) or mCCxDA plates (40/70, 57%). The number of mCCDA plates (70/70, 100%) that were contaminated with non-Campylobacter species was significantly higher than that of mCCtDA (20/70, 29%) or mCCxDA plates (21/70, 30%). The most common competing species identified using mCCDA was ESBL-producing E. coli, while Pseudomonas species frequently appeared on mCCtDA and mCCxDA. Copyright © 2016. Published by Elsevier B.V.

  6. Screening and expression of selected taxonomically conserved and unique hypothetical proteins in Burkholderia pseudomallei K96243

    Science.gov (United States)

    Akhir, Nor Azurah Mat; Nadzirin, Nurul; Mohamed, Rahmah; Firdaus-Raih, Mohd

    2015-09-01

    Hypothetical proteins of bacterial pathogens represent a large numbers of novel biological mechanisms which could belong to essential pathways in the bacteria. They lack functional characterizations mainly due to the inability of sequence homology based methods to detect functional relationships in the absence of detectable sequence similarity. The dataset derived from this study showed 550 candidates conserved in genomes that has pathogenicity information and only present in the Burkholderiales order. The dataset has been narrowed down to taxonomic clusters. Ten proteins were selected for ORF amplification, seven of them were successfully amplified, and only four proteins were successfully expressed. These proteins will be great candidates in determining the true function via structural biology.

  7. Novel pan-genomic analysis approach in target selection for multiplex PCR identification and detection of Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia cepacia complex species: a proof-of-concept study.

    Science.gov (United States)

    Ho, Chi-Chun; Lau, Candy C Y; Martelli, Paolo; Chan, San-Yuen; Tse, Cindy W S; Wu, Alan K L; Yuen, Kwok-Yung; Lau, Susanna K P; Woo, Patrick C Y

    2011-03-01

    Burkholderia pseudomallei, Burkholderia thailandensis, and the Burkholderia cepacia complex differ greatly in pathogenicity and epidemiology. Yet, they are occasionally misidentified by biochemical profiling, and even 16S rRNA gene sequencing may not offer adequate discrimination between certain species groups. Using the 23 B. pseudomallei, four B. thailandensis, and 16 B. cepacia complex genome sequences available, we identified gene targets specific to each of them (a Tat domain protein, a 70-kDa protein, and a 12-kDa protein for B. pseudomallei, B. thailandensis, and the B. cepacia complex, respectively), with an in-house developed algorithm. Using these targets, we designed a robust multiplex PCR assay useful for their identification and detection from soil and simulated sputum samples. For all 43 B. pseudomallei, seven B. thailandensis, and 20 B. cepacia complex (B. multivorans, n = 6; B. cenocepacia, n = 3; B. cepacia, n = 4; B. arboris, n = 2; B. contaminans, B. anthina, and B. pyrrocinia, n = 1 each; other unnamed members, n = 2) isolates, the assay produced specific products of predicted size without false positives or negatives. Of the 60 soil samples screened, 19 (31.6%) and 29 (48.3%) were positive for B. pseudomallei and the B. cepacia complex, respectively, and in four (6.7%) soil samples, the organisms were codetected. DNA sequencing confirmed that all PCR products originated from their targeted loci. This novel pan-genomic analysis approach in target selection is simple, computationally efficient, and potentially applicable to any species that harbors species-specific genes. A multiplex PCR assay for rapid and accurate identification and detection of B. pseudomallei, B. thailandensis, and the B. cepacia complex was developed and verified.

  8. Novel Pan-Genomic Analysis Approach in Target Selection for Multiplex PCR Identification and Detection of Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia cepacia Complex Species: a Proof-of-Concept Study▿

    Science.gov (United States)

    Ho, Chi-Chun; Lau, Candy C. Y.; Martelli, Paolo; Chan, San-Yuen; Tse, Cindy W. S.; Wu, Alan K. L.; Yuen, Kwok-Yung; Lau, Susanna K. P.; Woo, Patrick C. Y.

    2011-01-01

    Burkholderia pseudomallei, Burkholderia thailandensis, and the Burkholderia cepacia complex differ greatly in pathogenicity and epidemiology. Yet, they are occasionally misidentified by biochemical profiling, and even 16S rRNA gene sequencing may not offer adequate discrimination between certain species groups. Using the 23 B. pseudomallei, four B. thailandensis, and 16 B. cepacia complex genome sequences available, we identified gene targets specific to each of them (a Tat domain protein, a 70-kDa protein, and a 12-kDa protein for B. pseudomallei, B. thailandensis, and the B. cepacia complex, respectively), with an in-house developed algorithm. Using these targets, we designed a robust multiplex PCR assay useful for their identification and detection from soil and simulated sputum samples. For all 43 B. pseudomallei, seven B. thailandensis, and 20 B. cepacia complex (B. multivorans, n = 6; B. cenocepacia, n = 3; B. cepacia, n = 4; B. arboris, n = 2; B. contaminans, B. anthina, and B. pyrrocinia, n = 1 each; other unnamed members, n = 2) isolates, the assay produced specific products of predicted size without false positives or negatives. Of the 60 soil samples screened, 19 (31.6%) and 29 (48.3%) were positive for B. pseudomallei and the B. cepacia complex, respectively, and in four (6.7%) soil samples, the organisms were codetected. DNA sequencing confirmed that all PCR products originated from their targeted loci. This novel pan-genomic analysis approach in target selection is simple, computationally efficient, and potentially applicable to any species that harbors species-specific genes. A multiplex PCR assay for rapid and accurate identification and detection of B. pseudomallei, B. thailandensis, and the B. cepacia complex was developed and verified. PMID:21177905

  9. Comparison of selective agars recommended by method ISO 11290-1 and chromogenic agars for the isolation of Listeria sp. in refrigerated sausages

    Directory of Open Access Journals (Sweden)

    Thalyta Marina Benetti

    2012-12-01

    Full Text Available The aim of this study was to determine the prevalence of Listeria sp. in refrigerated sausages, and to compare the performance of the selective plating media employed in the ISO 11290-1 method (PALCAM and Oxford agars with chromogenic agars (Chromogenic Listeria agars CM 1080 (OCLA and CM 1084. The prevalence of Listeria sp. detected was 52.9%, comprising 13.7% L. monocytogenes strains. The efficacy of the four agars for the isolation of L. monocytogenes proved to be satisfactory. Despite differences in composition of the chromogenic media assessed, these disparities did not affect concordance among results. However, PALCAM agar was shown to suppress other microorganisms more effectively, being more applicable for detecting Listeria strains present in lower quantities. Based on these results, the use of PALCAM agar, in combination with a chromogenic media, is recommended for enhanced isolation of atypical Listeria sp. strains in meat products.Este estudo teve como objetivo a análise da prevalência de Listeria sp. em linguiças resfriadas e a comparação dos meios seletivos utilizados no plaqueamento do método ISO 11290-1 (Ágar PALCAM e Ágar Oxford, e ágares cromogênicos (Ágares Listeria Cromogênico CM 1080 (OCLA e CM 1084 (ISO. A frequência de Listeria sp. foi de 52,9%, sendo que destas, 13,7% corresponderam à L. monocytogenes. A eficácia dos quatro ágares para o isolamento de L. monocytogenes demonstrou-se satisfatória. Apesar de haver algumas diferenças nas composições dos meios cromogênicos analisados, estas não pareceram influenciar nas concordâncias entre os resultados expressos. Contudo, o ágar PALCAM mostrou-se mais eficaz na supressão de outros micro-organismos, aumentando, assim, a possibilidade de detecção de espécies de Listeria presentes em número reduzido. Através deste trabalho sugere-se a utilização do ágar PALCAM associado a um meio cromogênico para aumentar a chance de isolamento de cepas at

  10. Detection of Burkholderia pseudomallei in Sputum using Selective Enrichment Broth and Ashdown’s Medium at Kampong Cham Provincial Hospital, Cambodia [v2; ref status: indexed, http://f1000r.es/5e9

    Directory of Open Access Journals (Sweden)

    Somary Nhem

    2015-05-01

    Full Text Available Melioidosis, infection caused by Burkholderia pseudomallei, is increasingly reported in Cambodia. We hypothesized that implementation of an enhanced sputum testing protocol in a provincial hospital diagnostic microbiology laboratory would increase detection of B. pseudomallei. We tested 241 sputum specimens that were deemed acceptable for culture, comparing culture in selective enrichment broth followed by sub-culture on Ashdown’s medium to standard culture methods. Two specimens (0.8% were positive for B. pseudomallei using the enhanced protocol whereas one specimen (0.4% was positive using standard methods. Given the low numbers of positive specimens, we could not conclusively determine the utility of the enhanced sputum testing protocol. However, the ramifications of identification of B. pseudomallei are substantial, and the benefit of the enhanced testing protocol may be more apparent in patients selected based on risk factors and clinical presentation. Promoting clinician awareness of the infection and encouraging utilization of diagnostic microbiology services are also likely to be important factors in facilitating identification of melioidosis.

  11. Evaluation of commercial selective agars in screening for methicillin-resistant Staphylococcus aureus.

    Science.gov (United States)

    Yang, Hee-Young; Suh, Jin-Tae; Lee, Hee-Joo

    2010-01-01

    To evaluate methicillin-resistant S. aureus (MRSA) detection methods, we compared (a) mannitol salt agar with cefoxitin (MSA-FX), (b) MRSASelect agar (Bio-Rad), (c) MRSA ID (bioMerieuex), and (d) CHROMagar MRSA (BD Diagnostics) as selective media for culturing nasal swab specimens collected from intensive care unit (ICU) patients and healthcare personnel. A total of 99 (17.1%) cases of MRSA were recovered from 578 specimens. Four (5.5%) cases were identified from healthcare personnel and 95 (18.8%) were from ICU patients. The sensitivity of MSA-FX, MRSASelect, MRSA ID, and CHROMagar MRSA was 83.8, 87.9, 80.8, and 84.8% after 18 hr; 92.9, 94.9, 90.9, and 91.9% after 24 hr; and 96.0, 100, 99.0, and 99.0% after 48 hr, respectively. The specificity of MSA-FX, MRSASelect, MRSA ID, and CHROMagar MRSA was 97.7, 99.0, 98.7. and 99.8% after 18 hr; 97.1, 98.5, 98.1, and 99.5% after 24 hr; and 95.2, 97.7, 97.9, and 99.0% after 48 hr, respectively. In conclusion, all four media showed good results after the 24 hr readings, but MRSA ID and CHROMagar MRSA media required readings at 48 hr due to increased sensitivity at this time point.

  12. Performance of chromID Clostridium difficile agar compared with BBL C. difficile selective agar for detection of C. difficile in stool specimens.

    Science.gov (United States)

    Han, Sang Bong; Chang, Jiyoung; Shin, Sang Hyun; Park, Kang Gyun; Lee, Gun Dong; Park, Yong Gyu; Park, Yeon-Joon

    2014-09-01

    We evaluated the performance of a new chromogenic medium for detection of Clostridium difficile, chromID C. difficile agar (CDIF; bioMérieux, France), by comparison with BBL C. difficile Selective Agar (CDSA; Becton Dickinson and Company, USA). After heat pre-treatment (80℃, 5 min), 185 diarrheal stool samples were inoculated onto the two media types and incubated anaerobically for 24 hr and 48 hr for CDIF and for 48 hr and 72 hr for CDSA. All typical colonies on each medium were examined by Gram staining, and the gram-positive rods confirmed to contain the tpi gene by PCR were identified as C. difficile. C. difficile was recovered from 36 samples by using a combination of the two media. The sensitivity with CDIF 48 hr was highest (100%) and was significantly higher than that with CDIF 24 hr (58.3%; P<0.001), because samples with a low burden of C. difficile tended to require prolonged incubation up to 48 hr (P<0.001). The specificity of CDIF 24 hr and CDIF 48 hr (99.3% and 90.6%, respectively) was significantly higher than that of CDSA 48 hr and CDSA 72 hr (72.5% and 67.1%, respectively; P<0.001). CDIF was effective for detecting C. difficile in heat-pretreated stool specimens, thus reducing unnecessary testing for toxin production in non-C. difficile isolates and turnaround time.

  13. Recent Advances in Burkholderia mallei and B. pseudomallei Research.

    Science.gov (United States)

    Hatcher, Christopher L; Muruato, Laura A; Torres, Alfredo G

    2015-06-01

    Burkholderia mallei and Burkholderia pseudomallei are Gram-negative organisms, which are etiological agents of glanders and melioidosis, respectively. Although only B. pseudomallei is responsible for a significant number of human cases, both organisms are classified as Tier 1 Select Agents and their diseases lack effective diagnosis and treatment. Despite a recent resurgence in research pertaining to these organisms, there are still a number of knowledge gaps. This article summarizes the latest research progress in the fields of B. mallei and B. pseudomallei pathogenesis, vaccines, and diagnostics.

  14. Volatile-sulfur-compound profile distinguishes Burkholderia pseudomallei from Burkholderia thailandensis.

    Science.gov (United States)

    Inglis, Timothy J J; Hahne, Dorothee R; Merritt, Adam J; Clarke, Michael W

    2015-03-01

    Solid-phase microextraction gas chromatography-mass spectrometry (SPME-GCMS) was used to show that dimethyl sulfide produced by Burkholderia pseudomallei is responsible for its unusual truffle-like smell and distinguishes the species from Burkholderia thailandensis. SPME-GCMS can be safely used to detect dimethyl sulfide produced by agar-grown B. pseudomallei. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  15. [Selective-differential nutrient medium "Shewanella IRHLS agar" for isolation of Shewanella genus bacteria].

    Science.gov (United States)

    Sivolodsky, E P

    2015-01-01

    Development of a selective-differential nutrient medium for isolation of Shewanella genus bacteria. 73 strains of Shewanella bacteria (S. algae--3, S. baltica--26, S. putrefaciens--44) and 80 strains of 22 other bacteria genera were used. Shewanella species were identified by methods and criteria proposed by Nozue H. et al., 1992; Khashe S. et al., 1998. Nutrient media "Shewanella IRHLS Agar" for shewanella isolation was developed. Medium selective factors: irgazan DP-300 (I). 0.14-0.2 g/l and rifampicin (R) 0.0005-0.001 g/l. Shevanella colonies were detected by the production of hydrogen sulfide (H), lipase presence (L), lack of sorbitol fermentation (S). The medium suppressed the growth of hydrogen sulfide producers (Salmonella, Proteus) and blocked hydrogen sulfide production by Citrobacter. Growth of Escherichia, Enterobacter, Klebsiella, Shigella, Staphylococcus, Bacillus was also suppressed, Analytical sensitivity of the medium was 1-2 CFU/ml for Shewanella and Stenotrophomonas, Aerombnas, Serratia genera bacteria. 72 strains of Shewanella were isolated from water of Neva river in this medium, 91.7 ± 3.2% of those produced H2S. 1 strain of S. algae was isolated from clinical material. The developed media allows to use it in a complex for Stenotrophomo- nas sp., Aeromonas sp., Serratia sp., Citrobactersp. and Shewanella bacteria isolation.

  16. Evaluation of four selective agars and two enrichment broths in screening for methicillin-resistant Staphylococcus aureus.

    Science.gov (United States)

    Böcher, S; Smyth, R; Kahlmeter, G; Kerremans, J; Vos, M C; Skov, R

    2008-09-01

    To evaluate methicillin-resistant Staphylococcus aureus detection, we tested in vitro four selective agars and two enrichment broths apart and in combination. Tryptone soya broth with salt, aztreonam, and cefoxitin appeared to be the most sensitive medium. This broth was superior to a phenol red mannitol broth with aztreonam and ceftizoxime.

  17. Pseudomonas pseudomallei, a common pathogen in Thailand that is resistant to the bactericidal effects of many antibiotics.

    Science.gov (United States)

    Sookpranee, T; Sookpranee, M; Mellencamp, M A; Preheim, L C

    1991-03-01

    The purpose of this investigation was to identify newer antimicrobial agents that may be useful in the therapy of melioidosis. The in vitro susceptibilities of 199 clinical isolates of Pseudomonas pseudomallei to 22 antibiotics were determined by standard disk diffusion, and those to 13 antibiotics were determined by agar dilution. Over 90% of the isolates were susceptible to imipenem, piperacillin-tazobactam, piperacillin, ceftazidime, ticarcillin-clavulanate, ampicillin-sulbactam, and carumonam by both methods. Standard disk diffusion yielded unacceptably high false-susceptibility results with aztreonam, ciprofloxacin, and temafloxacin. Piperacillin, ceftazidime, imipenem, and ciprofloxacin were not bactericidal for three selected P. pseudomallei strains as determined by time-kill curve methods. Furthermore, addition of ciprofloxacin to piperacillin, ceftazidime, or imipenem did not enhance bactericidal activity. One hundred ninety-four strains showed weak beta-lactamase production that did not increase upon incubation with cefoxitin. These findings suggest that several newer antimicrobial agents may prove useful in the treatment of melioidosis. However, results of susceptibility studies involving P. pseudomallei and newer agents must be interpreted with caution.

  18. Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei.

    Science.gov (United States)

    Su, Shengchang; Bangar, Hansraj; Saldanha, Roland; Pemberton, Adin; Aronow, Bruce; Dean, Gary E; Lamkin, Thomas J; Hassett, Daniel J

    2014-10-01

    Here, we constructed stable, chromosomal, constitutively expressed, green and red fluorescent protein (GFP and RFP) as reporters in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei. Using bioinformatic approaches and other experimental analyses, we identified P0253 and P1 as potent promoters that drive the optimal expression of fluorescent reporters in single copy in B. anthracis and Burkholderia spp. as well as their surrogate strains, respectively. In comparison, Y. pestis and its surrogate strain need two chromosomal copies of cysZK promoter (P2cysZK) for optimal fluorescence. The P0253-, P2cysZK-, and P1-driven GFP and RFP fusions were first cloned into the vectors pRP1028, pUC18R6KT-mini-Tn7T-Km, pmini-Tn7-gat, or their derivatives. The resultant constructs were delivered into the respective surrogates and subsequently into the select agent strains. The chromosomal GFP- and RFP-tagged strains exhibited bright fluorescence at an exposure time of less than 200 msec and displayed the same virulence traits as their wild-type parental strains. The utility of the tagged strains was proven by the macrophage infection assays and lactate dehydrogenase release analysis. Such strains will be extremely useful in high-throughput screens for novel compounds that could either kill these organisms, or interfere with critical virulence processes in these important bioweapon agents and during infection of alveolar macrophages. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  19. PCR-based Methodologies Used to Detect and Differentiate the Burkholderia pseudomallei complex: B. pseudomallei, B. mallei, and B. thailandensis.

    Science.gov (United States)

    Lowe, Woan; March, Jordon K; Bunnell, Annette J; O'Neill, Kim L; Robison, Richard A

    2014-01-01

    Methods for the rapid detection and differentiation of the Burkholderia pseudomallei complex comprising B. pseudomallei, B. mallei, and B. thailandensis, have been the topic of recent research due to the high degree of phenotypic and genotypic similarities of these species. B. pseudomallei and B. mallei are recognized by the CDC as tier 1 select agents. The high mortality rates of glanders and melioidosis, their potential use as bioweapons, and their low infectious dose, necessitate the need for rapid and accurate detection methods. Although B. thailandensis is generally avirulent in mammals, this species displays very similar phenotypic characteristics to that of B. pseudomallei. Optimal identification of these species remains problematic, due to the difficulty in developing a sensitive, selective, and accurate assay. The development of PCR technologies has revolutionized diagnostic testing and these detection methods have become popular due to their speed, sensitivity, and accuracy. The purpose of this review is to provide a comprehensive overview and evaluation of the advancements in PCR-based detection and differentiation methodologies for the B. pseudomallei complex, and examine their potential uses in diagnostic and environmental testing.

  20. Comparison of culture screening protocols for methicillin-resistant Staphylococcus aureus (MRSA) using a chromogenic agar (MRSA-Select).

    Science.gov (United States)

    Lee, Seungok; Park, Yeon-Joon; Yoo, Jin-Hong; Kahng, Jimin; Jeong, In-Hee; Kwon, Young-Mi; Han, Kyungja

    2008-01-01

    To compare the culture screening protocols for methicillin-resistant Staphylococcus aureus (MRSA), a total of 300 duplicate nasal swabs (233 initial cultures and 67 weekly follow-up cultures) were collected consecutively from 233 patients in the Intensive Care Unit (ICU). One swab was plated directly on MRSA-Select agar (D-MRSA-Select) and observed at 24 hr. The duplicate swab was incubated in tryptic soy broth (TSB) with 6.5% NaCl for 24 hr, and then subcultured on MRSA-Select (B-MRSA-Select), BAP (B-BAP), and mannitol salt agar with 4 mg/L oxacillin (B-MSA(OXA)), and observed at 24 hr. MRSA was detected in 13.7% (32/233) of the initial and 22.4% (15/67) of the follow-up specimens. A patient was classified as MRSA-positive if any of the media grew colonies that were tested and confirmed to be MRSA. In the initial screening samples, the sensitivities of D-MRSA-Select, B-MRSA-Select, B-BAP, and B-MSA(OXA) were 78.1%, 84.4%, 78.1%, and 65.6%, respectively, and the specificities were 100%, 98.0%, 83.1%, and 93.5%, respectively. The sensitivities of all but the B-MRSA-Select protocol were significantly lower (p Salt-containing enrichment broth in conjunction with MRSA-Select (B-MRSA-Select) provides a promising way to increase sensitivity in initial and follow-up screening for MRSA.

  1. Evaluation of a latex agglutination assay for the identification of Burkholderia pseudomallei and Burkholderia mallei.

    Science.gov (United States)

    Duval, Brea D; Elrod, Mindy G; Gee, Jay E; Chantratita, Narisara; Tandhavanant, Sarunporn; Limmathurotsakul, Direk; Hoffmaster, Alex R

    2014-06-01

    Cases of melioidosis and glanders are rare in the United States, but the etiologic agents of each disease (Burkholderia pseudomallei and Burkholderia mallei, respectively) are classified as Tier 1 select agents because of concerns about their potential use as bioterrorism agents. A rapid, highly sensitive, and portable assay for clinical laboratories and field use is required. Our laboratory has further evaluated a latex agglutination assay for its ability to identify B. pseudomallei and B. mallei isolates. This assay uses a monoclonal antibody that specifically recognizes the capsular polysaccharide produced by B. pseudomallei and B. mallei, but is absent in closely related Burkholderia species. A total of 110 B. pseudomallei and B. mallei were tested, and 36 closely related Burkholderia species. The latex agglutination assay was positive for 109 of 110 (99.1% sensitivity) B. pseudomallei and B. mallei isolates tested. © The American Society of Tropical Medicine and Hygiene.

  2. Antimicrobial activity of Tachyplesin 1 against Burkholderia pseudomallei: an in vitro and in silico approach

    Directory of Open Access Journals (Sweden)

    Lyn-Fay Lee

    2016-10-01

    Full Text Available Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to many conventional antibiotics. Therefore, alternative antimicrobial agents such as antimicrobial peptides (AMPs are extensively studied to combat this issue. Our study aims to identify and understand the mode of action of the potential AMP(s that are effective against B. pseudomallei in both planktonic and biofilm state as well as to predict the possible binding targets on using in vitro and in silico approaches. In the in vitro study, 11 AMPs were tested against 100 B. pseudomallei isolates for planktonic cell susceptibility, where LL-37, and PG1, demonstrated 100.0% susceptibility and TP1 demonstrated 83% susceptibility. Since the B. pseudomallei activity was reported on LL-37 and PG1, TP1 was selected for further investigation. TP1 inhibited B. pseudomallei cells at 61.69 μM, and membrane blebbing was observed using scanning electron microscopy. Moreover, TP1 inhibited B. pseudomallei cell growth, reaching bactericidal endpoint within 2 h post exposure as compared to ceftazidime (CAZ (8 h. Furthermore, TP1 was shown to suppress the growth of B. pseudomallei cells in biofilm state at concentrations above 221 μM. However, TP1 was cytotoxic to the mammalian cell lines tested. In the in silico study, molecular docking revealed that TP1 demonstrated a strong interaction to the common peptide or inhibitor binding targets for lipopolysaccharide of Escherichia coli, as well as autolysin, pneumolysin, and pneumococcal surface protein A (PspA of Streptococcus pneumoniae. Homology modelled B. pseudomallei PspA protein (YDP also showed a favourable binding with a strong electrostatic contribution and nine hydrogen bonds. In conclusion, TP1 demonstrated a good potential as an anti-B. pseudomallei agent.

  3. Selective growth of Staphylococcus aureus from flushed dairy manure wastewater using acriflavine-supplemented mannitol salt agar.

    Science.gov (United States)

    Davis, J A; Farrah, S R; Wilkie, A C

    2006-06-01

    To investigate the use of mannitol salt agar (MSA) supplemented with acriflavine for selective growth and quantification of Staphylococcus aureus from flushed dairy manure wastewater (FDMW). Minimal inhibitory concentrations of acriflavine in MSA were determined by comparing the growth of S. aureus subsp. aureus (ATCC 33591) and Staphylococcus epidermidis (ATCC 155) in pure culture. Acriflavine concentrations of 1.3, 1.4 and 1.5 mg l(-1) reduced CFU of S. epidermidis by 43%, 55% and 87%, respectively, while CFU of S. aureus subsp. aureus were only reduced by 15%, 20% and 26% at the respective concentrations of acriflavine. MSA supplemented with 1.5 mg l(-1) acriflavine was tested for selective growth of indigenous S. aureus from three grab samples of FDMW. Acriflavine concentrations of 1.5 mg l(-1) reduced background flora without significantly reducing (P < 0.05) indigenous S. aureus counts. Acriflavine-supplemented MSA provides an effective media for selective growth and quantification of indigenous S. aureus from FDMW in the presence of high levels of background microflora. S. aureus is implicated for mastitis infections in dairy cows. Therefore, a reliable means for monitoring and detecting the organism in FDMW provides a tool for measuring the effectiveness of treatment for reducing S. aureus levels and implementing flushwater recycling without affecting herd health.

  4. Enumeration of sublethally injured Escherichia coli O157:H7 ATCC 43895 and Escherichia coli strain B-41560 using selective agar overlays versus commercial methods.

    Science.gov (United States)

    Smith, Amanda R; Ellison, Alysha L; Robinson, Amanda L; Drake, Maryanne; McDowell, Susan A; Mitchell, James K; Gerard, Patrick D; Heckler, Rachel A; McKillip, John L

    2013-04-01

    Quality control procedures during food processing may involve direct inoculation of food samples onto appropriate selective media for subsequent enumeration. However, sublethally injured bacteria often fail to grow, enabling them to evade detection and intervention measures and ultimately threaten the health of consumers. This study compares traditional selective and nonselective agar-based overlays versus two commercial systems (Petrifilm and Easygel) for recovery of injured E. coli B-41560 and O157:H7 strains. Bacteria were propagated in tryptic soy broth (TSB), ground beef slurry, and infant milk formula to a density of 10(6) to 10(8) CFU/ml and then were stressed for 6 min either in lactic acid (pH 4.5) or heat shocked for 3 min at 60°C. Samples were pour plated in basal layers of either tryptic soy agar (TSA), sorbitol MacConkey agar (SMAC), or violet red bile agar (VRB) and were resuscitated for 4 h prior to addition of agar overlays. Other stressed bacteria were plated directly onto Petrifilm and Easygel. Results indicate that selective and nonselective agar overlays recovered significantly higher numbers (greater than 1 log) of acid- and heat-injured E. coli O157:H7 from TSB, ground beef, and infant milk formula compared with direct plating onto selective media, Petrifilm, or Easygel, while no significant differences among these media combinations were observed for stressed E. coli B-41560. Nonstressed bacteria from TSB and ground beef were also recovered at densities significantly higher in nonselective TSA-TSA and in VRB-VRB and SMAC-SMAC compared with Petrifilm and Easygel. These data underscore the need to implement food safety measures that address sublethally injured pathogens such as E. coli O157:H7 in order to avoid underestimation of true densities for target pathogens.

  5. Burkholderia pseudomallei: Its Detection in Soil and Seroprevalence in Bangladesh.

    Science.gov (United States)

    Jilani, Md Shariful Alam; Robayet, Jamshedul Alam Mohammad; Mohiuddin, Md; Hasan, Md Rokib; Ahsan, Chowdhury Rafiqul; Haq, Jalaluddin Ashraful

    2016-01-01

    Melioidosis, caused by Burkholderia pseudomallei, is an endemic disease in Bangladesh. No systematic study has yet been done to detect the environmental source of the organism and its true extent in Bangladesh. The present study attempted to isolate B. pseudomallei in soil samples and to determine its seroprevalence in several districts in Bangladesh. Soil samples were collected from rural areas of four districts of Bangladesh from where culture confirmed melioidosis cases were detected earlier. Multiple soil samples, collected from 5-7 sampling points of 3-5 sites of each district, were cultured in Ashdown selective media. Suspected colonies of B. pseudomallei were identified by biochemical and serological test, and by polymerase chain reaction (PCR) using 16s rRNA specific primers. Blood samples were collected from 940 healthy individuals of four districts to determine anti- B. pseudomallei IgG antibody levels by indirect enzyme linked immunosorbent assay (ELISA) using sonicated crude antigen. Out of 179 soil samples, B. pseudomallei was isolated from two samples of Gazipur district which is located 58 km north of capital Dhaka city. Both the isolates were phenotypically identical, arabinose negative and showed specific 550bp band in PCR. Out of 940 blood samples, anti- B. pseudomallei IgG antibody, higher than the cut-off value (>0.8), was detected in 21.5% individuals. Seropositivity rate was 22.6%-30.8% in three districts from where melioidosis cases were detected earlier, compared to 9.8% in a district where no melioidosis case was either detected or reported (p 50 years respectively. The seropositivity rates were 26.0% and 20.6% in male and female respectively, while it was 20-27% among different occupational groups. No significant association was observed with gender (χ2 = 3.441, p = 0.064) or any occupational group (χ2 = 3.835, p = 0.280). This is the first study demonstrating the presence of B. pseudomallei in the environmental (soil) samples of

  6. Antimicrobial susceptibility testing of Helicobacter pylori to selected agents by agar dilution method in Shiraz-Iran

    Directory of Open Access Journals (Sweden)

    Kohanteb J

    2007-01-01

    Full Text Available Purpose: To assess the pattern of antimicrobial susceptibility profile of Helicobacter pylori isolates from patients with gastritis, duodenal ulcer (DU and gastroesophageal reflux disease (GERD residing in Shiraz, Iran. Methods: One hundred and six H. pylori isolates from patients with gastritis, DU and GERD undergoing endoscopy at our university hospitals and clinics were analysed for their antimicrobial susceptibility to metronidazole, clarithromycin, amoxicillin, co-amoxiclav, tetracycline, ciprofloxacin and furazolidone. The minimum inhibitory concentrations were determined by agar dilution method. Results: Overall H. pylori resistance rate was 72.6% to metronidazole, 9.4% to clarithromycin and furazolidone, 20.8% to amoxicillin and 4.7% to tetracycline and ciprofloxacin. No resistance to co-amoxiclav was detected among H. pylori isolates. No significant differences between antimicrobial resistance and clinical outcome were detected. Conclusions: With regard to the increasing resistance of H. pylori isolates to various antibiotics, susceptibility testing of H. pylori isolates prior to the treatment of infection must be performed to achieve better eradication and to reduce the risk of selection of H. pylori resistant strains.

  7. Defense mechanisms of hepatocytes against Burkholderia pseudomallei

    Directory of Open Access Journals (Sweden)

    Antje eBast

    2012-01-01

    Full Text Available The gram-negative facultative intracellular rod Burkholderia pseudomallei causes melioidosis, an infectious disease with a wide range of clinical presentations. Among the observed visceral abscesses, the liver is commonly affected. However, neither this organotropism of B. pseudomallei nor local hepatic defense mechanisms have been thoroughly investigated so far. Own previous studies using electron microscopy of the murine liver after systemic infection of mice indicated that hepatocytes might be capable of killing B. pseudomallei. Therefore, the aim of this study was to further elucidate the interaction of B. pseudomallei with these cells and to analyse the role of hepatocytes in anti-B. pseudomallei host defense. In vitro studies using the human hepatocyte cell line HepG2 revealed that B. pseudomallei can invade these cells. Subsequently, B. pseudomallei is able to escape from the vacuole, to replicate within the cytosol of HepG2 cells involving its type 3 and type 6 secretion systems, and to induce actin tail formation. Furthermore, stimulation of HepG2 cells showed that IFNgamma can restrict growth of B. pseudomallei in the early and late phase of infection whereas the combination of IFNgamma, IL-1beta and TNFalpha is required for the maximal antibacterial activity. This anti-B. pseudomallei defense of HepG2 cells did not seem to be mediated by iNOS-derived nitric oxide or NADPH oxidase-derived superoxide. In summary, this is the first study describing B. pseudomallei intracellular life cycle characteristics in hepatocytes and showing that IFNgamma-mediated, but nitric oxide- and reactive oxygen species-independent, effector mechanisms are important in anti-B. pseudomallei host defense of hepatocytes.

  8. Randomized Soil Survey of the Distribution of Burkholderia pseudomallei in Rice Fields in Laos ▿ †

    Science.gov (United States)

    Rattanavong, Sayaphet; Wuthiekanun, Vanaporn; Langla, Sayan; Amornchai, Premjit; Sirisouk, Joy; Phetsouvanh, Rattanaphone; Moore, Catrin E.; Peacock, Sharon J.; Buisson, Yves; Newton, Paul N.

    2011-01-01

    Melioidosis is a major cause of morbidity and mortality in Southeast Asia, where the causative organism (Burkholderia pseudomallei) is present in the soil. In the Lao People's Democratic Republic (Laos), B. pseudomallei is a significant cause of sepsis around the capital, Vientiane, and has been isolated in soil near the city, adjacent to the Mekong River. We explored whether B. pseudomallei occurs in Lao soil distant from the Mekong River, drawing three axes across northwest, northeast, and southern Laos to create nine sampling areas in six provinces. Within each sampling area, a random rice field site containing a grid of 100 sampling points each 5 m apart was selected. Soil was obtained from a depth of 30 cm and cultured for B. pseudomallei. Four of nine sites (44%) were positive for B. pseudomallei, including all three sites in Saravane Province, southern Laos. The highest isolation frequency was in east Saravane, where 94% of soil samples were B. pseudomallei positive with a geometric mean concentration of 464 CFU/g soil (95% confidence interval, 372 to 579 CFU/g soil; range, 25 to 10,850 CFU/g soil). At one site in northwest Laos (Luangnamtha), only one sample (1%) was positive for B. pseudomallei, at a concentration of 80 CFU/g soil. Therefore, B. pseudomallei occurs in Lao soils beyond the immediate vicinity of the Mekong River, alerting physicians to the likelihood of melioidosis in these areas. Further studies are needed to investigate potential climatic, soil, and biological determinants of this heterogeneity. PMID:21075883

  9. Burkholderia pseudomallei Data Gap Analysis

    Science.gov (United States)

    2015-11-01

    was clinically isolated by Alfred Whitmore in Burma (Myanmar). Figure 1-1 illustrates the prevalence of melioidosis worldwide. Since the addition of B...probability of survival. Animals used in this research include mice, hamsters, rats, ferrets, guinea pigs , rabbits, pigs , and non-human primates...and approved vaccine there is no protective countermeasure against B. pseudomallei . Prophylaxis using antibiotics has been investigated in mice

  10. Novel pan-genomic analysis approach in target selection for multiplex PCR identification and detection of Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia cepacia complex species: A proof-of-concept study

    OpenAIRE

    Ho, Chi-Chun; Lau, Candy C. Y.; Martelli, Paolo; Chan, San-Yuen; Tse, Cindy W. S.; Wu, Alan K.L.; Yuen, Kwok-yung; Lau, Susanna K.P.; Patrick C Y Woo

    2011-01-01

    Burkholderia pseudomallei, Burkholderia thailandensis, and the Burkholderia cepacia complex differ greatly in pathogenicity and epidemiology. Yet, they are occasionally misidentified by biochemical profiling, and even 16S rRNA gene sequencing may not offer adequate discrimination between certain species groups. Using the 23 B. pseudomallei, four B. thailandensis, and 16 B. cepacia complex genome sequences available, we identified gene targets specific to each of them (a Tat domain protein, a ...

  11. Pathogenesis of Burkholderia pseudomallei and Burkholderia mallei.

    Science.gov (United States)

    Larsen, Joseph C; Johnson, Nathan H

    2009-06-01

    Burkholderia pseudomallei and mallei are biological agents of military significance. There has been significant research in recent years to develop medical countermeasures for these organisms. This review summarizes work which details aspects of the pathogenesis of B. pseudomallei and mallei and discusses key scientific questions and directions for future research.

  12. Development of mouse hybridomas for production of monoclonal antibodies specific to Burkholderia mallei and Burkholderia pseudomallei.

    Science.gov (United States)

    Feng, Shaw-Huey; Tsai, Shien; Rodriguez, Jose; Newsome, Tamara; Emanuel, Peter; Lo, Shyh-Ching

    2006-08-01

    Burkholderia mallei and B. pseudomallei are designated category B biothreat agents on the "select agents" list established by the NIH and CDC. Development of monoclonal antibodies (MAbs) that could effectively differentiate these two closely related species of bacteria and other non-pathogenic Burkholderia bacteria is urgently needed. Splenocytes from mice immunized with various antigen preparations from either B. mallei (American Type Culture Collection [ATCC] 23344) or B. pseudomallei (ATCC 23343) were used for production of hybridomas. Using a three-step cross-screening protocol, a total of 10 hybridomas were selected that produced MAbs which specifically recognized B. mallei 23344 but did not bind B. pseudomallei, Pseudomonas aeruginasa, or any of the other nine Burkholderia species tested. All 10 MAbs targeted to the lipopolysaccharide (LPS) molecules of B. mallei and reacted strongly with 12 out of 15 different strains of B. mallei tested. A total of 14 hybridomas that produced MAbs reacting with B. pseudomallei 23343, but not with B. mallei, P. aeruginasa, or any other nine non-pathogenic Burkholderia species were also selected. All 14 MAbs appeared to react with a proteinase K-sensitive 200-kDa band by immunoblotting analysis. Surprisingly, these 14 MAbs that were raised against the ATCC 23343 strain failed to react to any of the other 13 different strains of B. pseudomallei examined. In conclusion, our B. mallei-specific MAbs can effectively recognize 80% of the different B. mallei strains tested, and all the B. pseudomallei-specific MAbs appeared to react with a unique antigen present only in the ATCC 23343 strain, but not in any other strains of B. pseudomallei tested.

  13. Development and validation of Burkholderia pseudomallei-specific real-time PCR assays for clinical, environmental or forensic detection applications.

    Directory of Open Access Journals (Sweden)

    Erin P Price

    Full Text Available The bacterium Burkholderia pseudomallei causes melioidosis, a rare but serious illness that can be fatal if untreated or misdiagnosed. Species-specific PCR assays provide a technically simple method for differentiating B. pseudomallei from near-neighbor species. However, substantial genetic diversity and high levels of recombination within this species reduce the likelihood that molecular signatures will differentiate all B. pseudomallei from other Burkholderiaceae. Currently available molecular assays for B. pseudomallei detection lack rigorous validation across large in silico datasets and isolate collections to test for specificity, and none have been subjected to stringent quality control criteria (accuracy, precision, selectivity, limit of quantitation (LoQ, limit of detection (LoD, linearity, ruggedness and robustness to determine their suitability for environmental, clinical or forensic investigations. In this study, we developed two novel B. pseudomallei specific assays, 122018 and 266152, using a dual-probe approach to differentiate B. pseudomallei from B. thailandensis, B. oklahomensis and B. thailandensis-like species; other species failed to amplify. Species specificity was validated across a large DNA panel (>2,300 samples comprising Burkholderia spp. and non-Burkholderia bacterial and fungal species of clinical and environmental relevance. Comparison of assay specificity to two previously published B. pseudomallei-specific assays, BurkDiff and TTS1, demonstrated comparable performance of all assays, providing between 99.7 and 100% specificity against our isolate panel. Last, we subjected 122018 and 266152 to rigorous quality control analyses, thus providing quantitative limits of assay performance. Using B. pseudomallei as a model, our study provides a framework for comprehensive quantitative validation of molecular assays and provides additional, highly validated B. pseudomallei assays for the scientific research community.

  14. Plating in Top Agar

    OpenAIRE

    sprotocols

    2014-01-01

    1. Warm plates to room temperature before use. Cold plates causes the top agar to solidify irregularly. DO not warm plates to 37° as the top agar will take forever to solidify. - Prepare top agar as the appropriate liquid medium with 0.7% agar. Keeping 100 mL bottles is convenient. For phages, use λ top agar, which is less rich and yields bigger plaques. - Melt top agar in the microwave completely. Allow the agar to boil after liquification; incompletely melted agar looks liquid, but is...

  15. Development of vaccines against burkholderia pseudomallei

    National Research Council Canada - National Science Library

    Patel, Natasha; Conejero, Laura; De Reynal, Melanie; Easton, Anna; Bancroft, Gregory J; Titball, Richard W

    2011-01-01

    Burkholderia pseudomallei is a Gram-negative bacterium which is the causative agent of melioidosis, a disease which carries a high mortality and morbidity rate in endemic areas of South East Asia and Northern Australia...

  16. Burkholderia pseudomallei transcriptional adaptation in macrophages

    Directory of Open Access Journals (Sweden)

    Chieng Sylvia

    2012-07-01

    Full Text Available Abstract Background Burkholderia pseudomallei is a facultative intracellular pathogen of phagocytic and non-phagocytic cells. How the bacterium interacts with host macrophage cells is still not well understood and is critical to appreciate the strategies used by this bacterium to survive and how intracellular survival leads to disease manifestation. Results Here we report the expression profile of intracellular B. pseudomallei following infection of human macrophage-like U937 cells. During intracellular growth over the 6 h infection period, approximately 22 % of the B. pseudomallei genome showed significant transcriptional adaptation. B. pseudomallei adapted rapidly to the intracellular environment by down-regulating numerous genes involved in metabolism, cell envelope, motility, replication, amino acid and ion transport system and regulatory function pathways. Reduced expression in catabolic and housekeeping genes suggested lower energy requirement and growth arrest during macrophage infection, while expression of genes encoding anaerobic metabolism functions were up regulated. However, whilst the type VI secretion system was up regulated, expression of many known virulence factors was not significantly modulated over the 6hours of infection. Conclusions The transcriptome profile described here provides the first comprehensive view of how B. pseudomallei survives within host cells and will help identify potential virulence factors and proteins that are important for the survival and growth of B. pseudomallei within human cells.

  17. Multitarget Quantitative PCR Improves Detection and Predicts Cultivability of the Pathogen Burkholderia pseudomallei.

    Science.gov (United States)

    Göhler, Andre; Trung, Trinh Thanh; Hopf, Verena; Kohler, Christian; Hartleib, Jörg; Wuthiekanun, Vanaporn; Peacock, Sharon J; Limmathurotsakul, Direk; Tuanyok, Apichai; Steinmetz, Ivo

    2017-04-15

    Burkholderia pseudomallei is present in the environment in many parts of the world and causes the often-fatal disease melioidosis. The sensitive detection and quantification of B. pseudomallei in the environment are a prerequisite for assessing the risk of infection. We recently reported the direct detection of B. pseudomallei in soil samples using a quantitative PCR (qPCR) targeting a single type three secretion system 1 (TTSS1) gene. Here, we extend the qPCR-based analysis of B. pseudomallei in soil by validating novel qPCR gene targets selected from a comparative genomic analysis. Two hundred soil samples from two rice paddies in northeast Thailand were evaluated, of which 47% (94/200) were B. pseudomallei culture positive. The TTSS1 qPCR and two novel qPCR assays that targeted open reading frames (ORFs) BPSS0087 and BPSS0745 exhibited detection rates of 76.5% (153/200), 34.5% (69/200), and 74.5% (150/200), respectively. The combination of TTSS1 and BPSS0745 qPCR increased the detection rate to 90% (180/200). Combining the results of the three qPCR assays and the BPSS1187 nested PCR previously published, all 200 samples were positive by at least one PCR assay. Samples positive by either TTSS1 (n = 153) or BPSS0745 (n = 150) qPCR were more likely to be direct-culture positive, with odds ratios of 4.0 (95% confidence interval [CI], 1.7 to 9.5; P rate in soil samples and predicted culture positivity. This approach has the potential for use as a sensitive environmental screening method for B. pseudomalleiIMPORTANCE The worldwide environmental distribution of the soil bacterium Burkholderia pseudomallei remains to be determined. So far, most environmental studies have relied on culture-based approaches to detect this pathogen. Since current culture methods are laborious, are time consuming, and have limited sensitivity, culture-independent and more sensitive methods are needed. In this study, we show that a B. pseudomallei-specific qPCR approach can detect

  18. [Evaluation of the usefulness of selective chromogenic agar medium (chromID VRE) and multiplex PCR method for the detection of vancomycin-resistant enterococci].

    Science.gov (United States)

    Kim, Do-Hoon; Lee, Jae-Hee; Ha, Jung-Sook; Ryoo, Nam-Hee; Jeon, Dong-Seok; Kim, Jae-Ryong

    2010-12-01

    Accurate and early detection of vancomycin-resistant enterococci (VRE) is critical for controlling nosocomial infection. In this study, we evaluated the usefulness of a selective chromogenic agar medium and of multiplex PCR for detection of VRE, and both these techniques were compared with the conventional culture method for VRE detection. We performed the following 3 methods for detecting VRE infection in stool specimens: the routine culture method, culturing in selective chromogenic agar medium (chromID VRE, bioMérieux, France), and multiplex PCR using the Seeplex® VRE ACE Detection kit (Seegene Inc., Korea) with additional PCR for vanC genes. We isolated 109 VRE strains from 100 stool specimens by the routine culture method. In chromID VRE, all the isolates showed purple colonies, including Enterococcus gallinarum and E. raffinosus, which were later identified using the Vitek card. All VRE isolates were identified by the multiplex PCR method; 100 were vanA-positive E. faecium, 8 were vanA- and vanC-1-positive E. gallinarum, and 1 was vanA-positive E. raffinosus. For VRE surveillance, culturing the isolates in chromID VRE after broth enrichment appears to be an accurate, rapid, and easy method for routine screening test. Multiplex PCR is relatively expensive and needs skilled techniques for detecting VRE, but it can be an auxiliary tool for rapid detection of genotype during a VRE outbreak.

  19. Detection of Burkholderia pseudomallei O-antigen serotypes in near-neighbor species

    Directory of Open Access Journals (Sweden)

    Stone Joshua K

    2012-11-01

    Full Text Available Abstract Background Burkholderia pseudomallei is the etiological agent of melioidosis and a CDC category B select agent with no available effective vaccine. Previous immunizations in mice have utilized the lipopolysaccharide (LPS as a potential vaccine target because it is known as one of the most important antigenic epitopes in B. pseudomallei. Complicating this strategy are the four different B. pseudomallei LPS O-antigen types: A, B, B2, and rough. Sero-crossreactivity is common among O-antigens of Burkholderia species. Here, we identified the presence of multiple B. pseudomallei O-antigen types and sero-crossreactivity in its near-neighbor species. Results PCR screening of O-antigen biosynthesis genes, phenotypic characterization using SDS-PAGE, and immunoblot analysis showed that majority of B. mallei and B. thailandensis strains contained the typical O-antigen type A. In contrast, most of B. ubonensis and B. thailandensis-like strains expressed the atypical O-antigen types B and B2, respectively. Most B. oklahomensis strains expressed a distinct and non-seroreactive O-antigen type, except strain E0147 which expressed O-antigen type A. O-antigen type B2 was also detected in B. thailandensis 82172, B. ubonensis MSMB108, and Burkholderia sp. MSMB175. Interestingly, B. thailandensis-like MSMB43 contained a novel serotype B positive O-antigen. Conclusions This study expands the number of species which express B. pseudomallei O-antigen types. Further work is required to elucidate the full structures and how closely these are to the B. pseudomallei O-antigens, which will ultimately determine the efficacy of the near-neighbor B serotypes for vaccine development.

  20. 21 CFR 582.7115 - Agar-agar.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Agar-agar. 582.7115 Section 582.7115 Food and..., FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Stabilizers § 582.7115 Agar-agar. (a) Product. Agar-agar. (b) Conditions of use. This substance is generally recognized as safe when used in...

  1. Screening for potential anti-infective agents towards Burkholderia pseudomallei infection

    Science.gov (United States)

    Eng, Su Anne; Nathan, Sheila

    2014-09-01

    The established treatment for melioidosis is antibiotic therapy. However, a constant threat to this form of treatment is resistance development of the causative agent, Burkholderia pseudomallei, towards antibiotics. One option to circumvent this threat of antibiotic resistance is to search for new alternative anti-infectives which target the host innate immune system and/or bacterial virulence. In this study, 29 synthetic compounds were evaluated for their potential to increase the lifespan of an infected host. The nematode Caenorhabditis elegans was adopted as the infection model as its innate immune pathways are homologous to humans. Screens were performed in a liquid-based survival assay containing infected worms exposed to individual compounds and survival of untreated and compound-treated worms were compared. A primary screen identified nine synthetic compounds that extended the lifespan of B. pseudomallei-infected worms. Subsequently, a disc diffusion test was performed on these selected compounds to delineate compounds into those that enhanced the survival of worms via antimicrobial activity i.e. reducing the number of infecting bacteria, or into those that did not target pathogen viability. Out of the nine hits selected, two demonstrated antimicrobial effects on B. pseudomallei. Therefore, the findings from this study suggest that the other seven identified compounds are potential anti-infectives which could protect a host against B. pseudomallei infection without developing the risk of drug resistance.

  2. φX216, a P2-like bacteriophage with broad Burkholderia pseudomallei and B. mallei strain infectivity.

    Science.gov (United States)

    Kvitko, Brian H; Cox, Christopher R; DeShazer, David; Johnson, Shannon L; Voorhees, Kent J; Schweizer, Herbert P

    2012-12-07

    Burkholderia pseudomallei and B. mallei are closely related Category B Select Agents of bioterrorism and the causative agents of the diseases melioidosis and glanders, respectively. Rapid phage-based diagnostic tools would greatly benefit early recognition and treatment of these diseases. There is extensive strain-to-strain variation in B. pseudomallei genome content due in part to the presence or absence of integrated prophages. Several phages have previously been isolated from B. pseudomallei lysogens, for example φK96243, φ1026b and φ52237. We have isolated a P2-like bacteriophage, φX216, which infects 78% of all B. pseudomallei strains tested. φX216 also infects B. mallei, but not other Burkholderia species, including the closely related B. thailandensis and B. oklahomensis. The nature of the φX216 host receptor remains unclear but evidence indicates that in B. mallei φX216 uses lipopolysaccharide O-antigen but a different receptor in B. pseudomallei. The 37,637 bp genome of φX216 encodes 47 predicted open reading frames and shares 99.8% pairwise identity and an identical strain host range with bacteriophage φ52237. Closely related P2-like prophages appear to be widely distributed among B. pseudomallei strains but both φX216 and φ52237 readily infect prophage carrying strains. The broad strain infectivity and high specificity for B. pseudomallei and B. mallei indicate that φX216 will provide a good platform for the development of phage-based diagnostics for these bacteria.

  3. φX216, a P2-like bacteriophage with broad Burkholderia pseudomallei and B. mallei strain infectivity

    Directory of Open Access Journals (Sweden)

    Kvitko Brian H

    2012-12-01

    Full Text Available Abstract Background Burkholderia pseudomallei and B. mallei are closely related Category B Select Agents of bioterrorism and the causative agents of the diseases melioidosis and glanders, respectively. Rapid phage-based diagnostic tools would greatly benefit early recognition and treatment of these diseases. There is extensive strain-to-strain variation in B. pseudomallei genome content due in part to the presence or absence of integrated prophages. Several phages have previously been isolated from B. pseudomallei lysogens, for example φK96243, φ1026b and φ52237. Results We have isolated a P2-like bacteriophage, φX216, which infects 78% of all B. pseudomallei strains tested. φX216 also infects B. mallei, but not other Burkholderia species, including the closely related B. thailandensis and B. oklahomensis. The nature of the φX216 host receptor remains unclear but evidence indicates that in B. mallei φX216 uses lipopolysaccharide O-antigen but a different receptor in B. pseudomallei. The 37,637 bp genome of φX216 encodes 47 predicted open reading frames and shares 99.8% pairwise identity and an identical strain host range with bacteriophage φ52237. Closely related P2-like prophages appear to be widely distributed among B. pseudomallei strains but both φX216 and φ52237 readily infect prophage carrying strains. Conclusions The broad strain infectivity and high specificity for B. pseudomallei and B. mallei indicate that φX216 will provide a good platform for the development of phage-based diagnostics for these bacteria.

  4. Paravertebral Abscess Caused by Bukholderia Pseudomallei in

    Directory of Open Access Journals (Sweden)

    S Ahmad

    2009-05-01

    Full Text Available A 53-year-old Malay man was admitted with intestinal obstruction, fever and lower limb weakness. Initial clinical impression was myelitis causing paralytic ilues and paraperesis. Blood culture showed Burkholderia pseudomallei infection and subsequent MRI showed paravertebral abscess. This case highlights a rare manifestation of melioidosis involving the spine and difficulties in establishing the diagnosis.

  5. Incubation of premise plumbing water samples on Buffered Charcoal Yeast Extract agar at elevated temperature and pH selects for Legionella pneumophila.

    Science.gov (United States)

    Veenendaal, Harm R; Brouwer-Hanzens, Anke J; van der Kooij, Dick

    2017-10-15

    Worldwide, over 90% of the notified cases of Legionnaires' disease are caused by Legionella pneumophila. However, the standard culture medium for the detection of Legionella in environmental water samples, Buffered Charcoal Yeast Extract (BCYE) agar of pH 6.9 ± 0.4 with or without antimicrobial agents incubated at 36 ± 1 °C, supports the growth of a large diversity of Legionella species. BCYE agar of elevated pH or/and incubation at elevated temperature gave strongly reduced recoveries of most of 26 L. non-pneumophila spp. tested, but not of L. pneumophila. BCYE agar of pH 7.3 ± 0.1, incubated at 40 ± 0.5 °C (BCYE pH 7.3/40 °C) was tested for selective enumeration of L. pneumophila. Of the L. non-pneumophila spp. tested, only L. adelaidensis and L. londiniensis multiplied under these conditions. The colony counts on BCYE pH 7.3/40 °C of a L. pneumophila serogroup 1 strain cultured in tap water did not differ significantly from those on BCYE pH 6.9/36 °C when directly plated and after membrane filtration and showed repeatability's of 13-14%. By using membrane filtration L. pneumophila was detected in 58 (54%) of 107 Legionella-positive water samples from premise plumbing systems under one or both of these culture conditions. The L. pneumophila colony counts (log-transformed) on BCYE pH 7.3/40 °C were strongly related (r2 = 0.87) to those on BCYE pH 6.9/36 °C, but differed significantly (p < 0.05) by a mean of - 0.12 ± 0.30 logs. L. non-pneumophila spp. were detected only on BCYE pH 6.9/36 °C in 49 (46%) of the samples. Hence, BCYE pH 7.3/40 °C can facilitate the enumeration of L. pneumophila and their isolation from premise plumbing systems with culturable L. non-pneumophila spp., some of which, e.g. L. anisa, can be present in high numbers. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. The Identification and Differentiation between Burkholderia mallei and Burkholderia pseudomallei Using One Gene Pyrosequencing.

    Science.gov (United States)

    Gilling, Damian H; Luna, Vicki Ann; Pflugradt, Cori

    2014-01-01

    The etiologic agents for melioidosis and glanders, Burkholderia mallei and Burkholderia pseudomallei respectively, are genetically similar making identification and differentiation from other Burkholderia species and each other challenging. We used pyrosequencing to determine the presence or absence of an insertion sequence IS407A within the flagellin P (fliP) gene and to exploit the difference in orientation of this gene in the two species. Oligonucleotide primers were designed to selectively target the IS407A-fliP interface in B. mallei and the fliP gene specifically at the insertion point in B. pseudomallei. We then examined DNA from ten B. mallei, ten B. pseudomallei, 14 B. cepacia, eight other Burkholderia spp., and 17 other bacteria. Resultant pyrograms encompassed the target sequence that contained either the fliP gene with the IS407A interruption or the fully intact fliP gene with 100% sensitivity and 100% specificity. These pyrosequencing assays based upon a single gene enable investigators to reliably identify the two species. The information obtained by these assays provides more knowledge of the genomic reduction that created the new species B. mallei from B. pseudomallei and may point to new targets that can be exploited in the future.

  7. Survival of Burkholderia pseudomallei in Water

    Directory of Open Access Journals (Sweden)

    Woods Donald E

    2008-05-01

    Full Text Available Abstract Background The ability of Burkholderia pseudomallei to survive in water likely contributes to its environmental persistence in endemic regions. To determine the physiological adaptations which allow B. pseudomallei to survive in aqueous environments, we performed microarray analyses of B. pseudomallei cultures transferred from Luria broth (LB to distilled water. Findings Increased expression of a gene encoding for a putative membrane protein (BPSL0721 was confirmed using a lux-based transcriptional reporter system, and maximal expression was noted at approximately 6 hrs after shifting cells from LB to water. A BPSL0721 deficient mutant of B. pseudomallei was able to survive in water for at least 90 days indicating that although involved, BPSL0721 was not essential for survival. BPSL2961, a gene encoding a putative phosphatidylglycerol phosphatase (PGP, was also induced when cells were shifted to water. This gene is likely involved in cell membrane biosynthesis. We were unable to construct a PGP mutant suggesting that the gene is not only involved in survival in water but is essential for cell viability. We also examined mutants of polyhydroxybutyrate synthase (phbC, lipopolysaccharide (LPS oligosaccharide and capsule synthesis, and these mutations did not affect survival in water. LPS mutants lacking outer core were found to lose viability in water by 200 days indicating that an intact LPS core provides an outer membrane architecture which allows prolonged survival in water. Conclusion The results from these studies suggest that B. pseudomallei survival in water is a complex process that requires an LPS molecule which contains an intact core region.

  8. Antimicrobial Carbohydrate Vaccines: Development of Burkholderia pseudomallei immunogens

    OpenAIRE

    Donaldson, Matthew

    2013-01-01

    The potential bio-terror threat posed by Burkholderia pseudomallei highlights the need for an effective vaccine. Immunisation and challenge experiments in mice have demonstrated that the capsular polysaccharide (CPS-1) of B. pseudomallei, which is composed of β-1,3-linked 6-deoxy-D-manno-heptopyranose residues, is a promising candidate for vaccine development. This thesis set out to explore routes to potential vaccine candidates for Burkholderia pseudomallei infection based on ...

  9. 21 CFR 184.1115 - Agar-agar.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Agar-agar. 184.1115 Section 184.1115 Food and Drugs... Substances Affirmed as GRAS § 184.1115 Agar-agar. (a) Agar-agar (CAS Reg. No. PM 9002-18-0) is a dried... ingredient is used in food in accordance with § 184.1(b)(2) under the following conditions: Maximum Usage...

  10. Evaluation of resistance in a selected field strain of Haemonchus contortus to ivermectin and moxidectin using the Larval Migration on Agar Test

    Directory of Open Access Journals (Sweden)

    Fernanda S. Fortes

    2013-02-01

    Full Text Available Haemonchus contortus is one of the most common and economically significant causes of disease in small ruminants worldwide, and the control programs of parasitic nematodes - including H. contortus - rely mostly on the use of anthelmintic drugs. The consequence of the use of this, as the sole sanitary strategy to avoid parasite infections, was the reduction of the efficacy of all chemotherapeutic products with a heavy selection for resistance. The widespread of anthelmintic resistance and the difficulty of its early diagnosis has been a major concern for the sustainable parasite management on farms. The objective of this research was to determine and compare the ivermectin (IVM and moxidectin (MOX effect in a selected field strain of H. contortus with a known resistance status, using the in vitro larval migration on agar test (LMAT. Third stage larvae of the selected isolate were obtained from faecal cultures of experimentally infected sheep and incubated in eleven increasing diluted concentrations of IVM and MOX (6, 12, 24, 48, 96, 192, 384, 768, 1536, 3072 and 6144µg/mL. The dose-response sigmoidal curves were obtained using the R² value of >0.90 and the lethal concentration (LC50 dose for the tested anthelmintic drugs using a four-parameter logistic model. The LC50 value for MOX was significantly lower than IVM (1.253µg/mL and 91.06µg/mL, identifying the H. contortus isolate as considerably less susceptible to IVM compared to MOX. Furthermore, the LMAT showed a high consistency (p<0.0001 and provided to be a useful diagnostic tool for monitoring the resistance status of IVM and MOX in H. contortus field isolate, as well as it may be used for official routine drug monitoring programs under the Ministry of Agriculture (MAPA guidance.

  11. Phylogeography of Burkholderia pseudomallei Isolates, Western Hemisphere.

    Science.gov (United States)

    Gee, Jay E; Gulvik, Christopher A; Elrod, Mindy G; Batra, Dhwani; Rowe, Lori A; Sheth, Mili; Hoffmaster, Alex R

    2017-07-01

    The bacterium Burkholderia pseudomallei causes melioidosis, which is mainly associated with tropical areas. We analyzed single-nucleotide polymorphisms (SNPs) among genome sequences from isolates of B. pseudomallei that originated in the Western Hemisphere by comparing them with genome sequences of isolates that originated in the Eastern Hemisphere. Analysis indicated that isolates from the Western Hemisphere form a distinct clade, which supports the hypothesis that these isolates were derived from a constricted seeding event from Africa. Subclades have been resolved that are associated with specific regions within the Western Hemisphere and suggest that isolates might be correlated geographically with cases of melioidosis. One isolate associated with a former World War II prisoner of war was believed to represent illness 62 years after exposure in Southeast Asia. However, analysis suggested the isolate originated in Central or South America.

  12. Burkholderia pseudomallei in Unchlorinated Domestic Bore Water, Tropical Northern Australia

    Science.gov (United States)

    Mayo, Mark; Kaestli, Mirjam; Harrington, Glenda; Cheng, Allen C.; Ward, Linda; Karp, Danuta; Jolly, Peter; Godoy, Daniel; Spratt, Brian G.

    2011-01-01

    To determine whether unchlorinated bore water in northern Australia contained Burkholderia pseudomallei organisms, we sampled 55 bores; 18 (33%) were culture positive. Multilocus sequence typing identified 15 sequence types. The B. pseudomallei sequence type from 1 water sample matched a clinical isolate from a resident with melioidosis on the same property. PMID:21762588

  13. Persistent gastric colonization with Burkholderia pseudomallei and dissemination from the gastrointestinal tract following mucosal inoculation of mice.

    Directory of Open Access Journals (Sweden)

    Andrew Goodyear

    Full Text Available Melioidosis is a disease of humans caused by opportunistic infection with the soil and water bacterium Burkholderia pseudomallei. Melioidosis can manifest as an acute, overwhelming infection or as a chronic, recurrent infection. At present, it is not clear where B. pseudomallei resides in the mammalian host during the chronic, recurrent phase of infection. To address this question, we developed a mouse low-dose mucosal challenge model of chronic B. pseudomallei infection and investigated sites of bacterial persistence over 60 days. Sensitive culture techniques and selective media were used to quantitate bacterial burden in major organs, including the gastrointestinal (GI tract. We found that the GI tract was the primary site of bacterial persistence during the chronic infection phase, and was the only site from which the organism could be consistently cultured during a 60-day infection period. The organism could be repeatedly recovered from all levels of the GI tract, and chronic infection was accompanied by sustained low-level fecal shedding. The stomach was identified as the primary site of GI colonization as determined by fluorescent in situ hybridization. Organisms in the stomach were associated with the gastric mucosal surface, and the propensity to colonize the gastric mucosa was observed with 4 different B. pseudomallei isolates. In contrast, B. pseudomallei organisms were present at low numbers within luminal contents in the small and large intestine and cecum relative to the stomach. Notably, inflammatory lesions were not detected in any GI tissue examined in chronically-infected mice. Only low-dose oral or intranasal inoculation led to GI colonization and development of chronic infection of the spleen and liver. Thus, we concluded that in a mouse model of melioidosis B. pseudomallei preferentially colonizes the stomach following oral inoculation, and that the chronically colonized GI tract likely serves as a reservoir for dissemination

  14. A genetic programming approach for Burkholderia Pseudomallei diagnostic pattern discovery

    Science.gov (United States)

    Yang, Zheng Rong; Lertmemongkolchai, Ganjana; Tan, Gladys; Felgner, Philip L.; Titball, Richard

    2009-01-01

    Motivation: Finding diagnostic patterns for fighting diseases like Burkholderia pseudomallei using biomarkers involves two key issues. First, exhausting all subsets of testable biomarkers (antigens in this context) to find a best one is computationally infeasible. Therefore, a proper optimization approach like evolutionary computation should be investigated. Second, a properly selected function of the antigens as the diagnostic pattern which is commonly unknown is a key to the diagnostic accuracy and the diagnostic effectiveness in clinical use. Results: A conversion function is proposed to convert serum tests of antigens on patients to binary values based on which Boolean functions as the diagnostic patterns are developed. A genetic programming approach is designed for optimizing the diagnostic patterns in terms of their accuracy and effectiveness. During optimization, it is aimed to maximize the coverage (the rate of positive response to antigens) in the infected patients and minimize the coverage in the non-infected patients while maintaining the fewest number of testable antigens used in the Boolean functions as possible. The final coverage in the infected patients is 96.55% using 17 of 215 (7.4%) antigens with zero coverage in the non-infected patients. Among these 17 antigens, BPSL2697 is the most frequently selected one for the diagnosis of Burkholderia Pseudomallei. The approach has been evaluated using both the cross-validation and the Jack–knife simulation methods with the prediction accuracy as 93% and 92%, respectively. A novel approach is also proposed in this study to evaluate a model with binary data using ROC analysis. Contact: z.r.yang@ex.ac.uk PMID:19561021

  15. Development of Burkholderia mallei and pseudomallei vaccines

    Directory of Open Access Journals (Sweden)

    Ediane Batista Silva

    2013-03-01

    Full Text Available B. mallei and B. pseudomallei are Gram-negative bacteria that cause glanders and melioidosis, respectively. Inhalational infection with either organism can result in severe and rapidly fatal pneumonia. Inoculation by the oral and cutaneous routes can also produce infection. chronic infection develops after recovery from acute infection with both agents, and control of infection with antibiotics requires prolonged treatment. Symptoms for both meliodosis and glanders are non-specific, making diagnosis difficult.B. pseudomallei can be located in the environment, but in the host, B. mallei and B. psedomallei are intracellular organisms. Thefection results in similar immune responses to both agents. Effective early innate immune responses are critical to controlling the early phase of the infection. Innate immune signaling molecules such as TLR, NOD, MyD88 and pro-inflammatory cytokines such as IFN- and TNF-α play key roles in regulating control of infection. Neutrophils and monocytes are critical cells in the early infection for these microorganisms. Both monocytes and macrophages are necessary for limiting dissemination of B. pseudomallei. In contrast, the role of adaptive immune responses in controlling Burkholderia infection is less well understood. However, T cell responses are critical for vaccine protection from Burkholderia infection. At present, effective vaccines for prevention of glanders or meliodosis have not been developed, although recently progress of Burkholderia vaccines has received renewed attention.This review will summarize current and past approaches to develop Burkholderia mallei and pseudomalllei vaccines, with emphasis on immune mechanisms of protection and the challenges facing the field. At present, immunization with live attenuated bacteria provides the most effective and durable immunity, and it is important therefore to understand the immune correlates of protection induced by live attenuated vaccines.

  16. Capsule influences the deposition of critical complement C3 levels required for the killing of Burkholderia pseudomallei via NADPH-oxidase induction by human neutrophils.

    Directory of Open Access Journals (Sweden)

    Michael E Woodman

    Full Text Available Burkholderia pseudomallei is the causative agent of melioidosis and is a major mediator of sepsis in its endemic areas. Because of the low LD(50 via aerosols and resistance to multiple antibiotics, it is considered a Tier 1 select agent by the CDC and APHIS. B. pseudomallei is an encapsulated bacterium that can infect, multiply, and persist within a variety of host cell types. In vivo studies suggest that macrophages and neutrophils are important for controlling B. pseudomallei infections, however few details are known regarding how neutrophils respond to these bacteria. Our goal is to describe the capacity of human neutrophils to control highly virulent B. pseudomallei compared to the relatively avirulent, acapsular B. thailandensis using in vitro analyses. B. thailandensis was more readily phagocytosed than B. pseudomallei, but both displayed similar rates of persistence within neutrophils, indicating they possess similar inherent abilities to escape neutrophil clearance. Serum opsonization studies showed that both were resistant to direct killing by complement, although B. thailandensis acquired significantly more C3 on its surface than B. pseudomallei, whose polysaccharide capsule significantly decreased the levels of complement deposition on the bacterial surface. Both Burkholderia species showed significantly enhanced uptake and killing by neutrophils after critical levels of C3 were deposited. Serum-opsonized Burkholderia induced a significant respiratory burst by neutrophils compared to unopsonized bacteria, and neutrophil killing was prevented by inhibiting NADPH-oxidase. In summary, neutrophils can efficiently kill B. pseudomallei and B. thailandensis that possess a critical threshold of complement deposition, and the relative differences in their ability to resist surface opsonization may contribute to the distinct virulence phenotypes observed in vivo.

  17. Strategies for intracellular survival of Burkholderia pseudomallei

    Directory of Open Access Journals (Sweden)

    Ben eAdler

    2011-08-01

    Full Text Available Burkholderia pseudomallei is the causative agent of melioidosis, a disease with high mortality that is prevalent in tropical regions of the world. A key component of the pathogenesis of melioidosis is the ability of B. pseudomallei to enter, survive and replicate within mammalian host cells. For non-phagocytic cells, bacterial adhesins have been identified both on the bacterial surface and associated with Type 4 pili. Cell invasion involves components of one or more of the three Type 3 Secretion System clusters, which also mediate, at least in part, the escape of bacteria from the endosome into the cytoplasm, where bacteria move by actin-based motility. The mechanism of actin-based motility is not clearly understood, but appears to differ from characterised mechanisms in other bacterial species. A small proportion of intracellular bacteria is targeted by host cell autophagy, involving direct recruitment of LC3 to endosomes rather than through uptake by canonical autophagosomes. However, the majority of bacterial cells are able to circumvent autophagy and other intracellular defence mechanisms such as the induction of iNOS, and then replicate in the cytoplasm and spread to adjacent cells via membrane fusion, resulting in the formation of multi-nucleated giant cells. A potential role for host cell ubiquitin in the autophagic response to bacterial infection has recently been proposed.

  18. Brain abscess caused by Burkholderia pseudomallei

    Energy Technology Data Exchange (ETDEWEB)

    Padigione, A.; Spelman, D.; Ferris, N. [Alfred Hospital, Prahran, VIC (Australia)

    1997-10-01

    Full text: Melioidosis, or infection with Burkholderia pseudomallei, is an important human disease in South East Asia and Northern Australia. Neurological manifestations are well recognized amongst its protean presentations, but direct focal central nervous system infection is infrequently described with only 9 adult and 5 paediatric cases reported in the English language literature. A case of brain abscess due to Burkholderia pseudomallei occurring in a 20 year old Dutch visitor to Australia which progressed despite antibiotic treatment is described. A review of the clinical manifestations, Magnetic Resonance (MR) appearance, diagnosis and treatment of melioidosis is presented, highlighting that: (i) physicians outside endernic areas should consider melioidosis in any patient with an appropriate travel history, (ii) MR imaging is more sensitive then CT in diagnosing early brain infection, especially of the brainstem; (iii) Bacterial culture, the mainstay of diagnosis, has many shortcomings; (iv)In vitro antibiotic sensitivity testing may not translate into clinical efficacy; and (v) Steroids appear to have little role, even in severe disease.

  19. Burkholderia pseudomallei Genotype Distribution in the Northern Territory, Australia.

    Science.gov (United States)

    Chapple, Stephanie N J; Price, Erin P; Sarovich, Derek S; McRobb, Evan; Mayo, Mark; Kaestli, Mirjam; Spratt, Brian G; Currie, Bart J

    2016-01-01

    Melioidosis is a tropical disease of high mortality caused by the environmental bacterium, Burkholderia pseudomallei. We have collected clinical isolates from the highly endemic Northern Territory of Australia routinely since 1989, and animal and environmental B. pseudomallei isolates since 1991. Here we provide a complete record of all B. pseudomallei multilocus sequence types (STs) found in the Northern Territory to date, and distribution maps of the eight most common environmental STs. We observed surprisingly restricted geographic distributions of STs, which is contrary to previous reports suggesting widespread environmental dissemination of this bacterium. Our data suggest that B. pseudomallei from soil and water does not frequently disperse long distances following severe weather events or by migration of infected animals. © The American Society of Tropical Medicine and Hygiene.

  20. Promethazine improves antibiotic efficacy and disrupts biofilms of Burkholderia pseudomallei.

    Science.gov (United States)

    Sidrim, José Júlio Costa; Vasconcelos, David Caldas; Riello, Giovanna Barbosa; Guedes, Glaucia Morgana de Melo; Serpa, Rosana; Bandeira, Tereza de Jesus Pinheiro Gomes; Monteiro, André Jalles; Cordeiro, Rossana de Aguiar; Castelo-Branco, Débora de Souza Collares Maia; Rocha, Marcos Fábio Gadelha; Brilhante, Raimunda Sâmia Nogueira

    2017-01-01

    Efflux pumps are important defense mechanisms against antimicrobial drugs and maintenance of Burkholderia pseudomallei biofilms. This study evaluated the effect of the efflux pump inhibitor promethazine on the structure and antimicrobial susceptibility of B. pseudomallei biofilms. Susceptibility of planktonic cells and biofilms to promethazine alone and combined with antimicrobials was assessed by the broth microdilution test and biofilm metabolic activity was determined with resazurin. The effect of promethazine on 48 h-grown biofilms was also evaluated through confocal and electronic microscopy. The minimum inhibitory concentration (MIC) of promethazine was 780 mg l(-1), while the minimum biofilm elimination concentration (MBEC) was 780-3,120 mg l(-1). Promethazine reduced the MIC values for erythromycin, trimethoprim/sulfamethoxazole, gentamicin and ciprofloxacin and reduced the MBEC values for all tested drugs (pbiofilm structure of B. pseudomallei, even at subinhibitory concentrations, possibly facilitating antibiotic penetration. Promethazine improves antibiotics efficacy against B. pseudomallei biofilms, by disrupting biofilm structure.

  1. The Madagascar hissing cockroach as a novel surrogate host for Burkholderia pseudomallei, B. mallei and B. thailandensis

    Directory of Open Access Journals (Sweden)

    Fisher Nathan A

    2012-06-01

    Full Text Available Abstract Background Burkholderia pseudomallei and Burkholderia mallei are gram-negative pathogens responsible for the diseases melioidosis and glanders, respectively. Both species cause disease in humans and animals and have been designated as category B select agents by the Centers for Disease Control and Prevention (CDC. Burkholderia thailandensis is a closely related bacterium that is generally considered avirulent for humans. While it can cause disease in rodents, the B. thailandensis 50% lethal dose (LD50 is typically ≥ 104-fold higher than the B. pseudomallei and B. mallei LD50 in mammalian models of infection. Here we describe an alternative to mammalian hosts in the study of virulence and host-pathogen interactions of these Burkholderia species. Results Madagascar hissing cockroaches (MH cockroaches possess a number of qualities that make them desirable for use as a surrogate host, including ease of breeding, ease of handling, a competent innate immune system, and the ability to survive at 37°C. MH cockroaches were highly susceptible to infection with B. pseudomallei, B. mallei and B. thailandensis and the LD50 was 50 for Escherichia coli in MH cockroaches was >105 cfu. B. pseudomallei, B. mallei, and B. thailandensis cluster 1 type VI secretion system (T6SS-1 mutants were all attenuated in MH cockroaches, which is consistent with previous virulence studies conducted in rodents. B. pseudomallei mutants deficient in the other five T6SS gene clusters, T6SS-2 through T6SS-6, were virulent in both MH cockroaches and hamsters. Hemocytes obtained from MH cockroaches infected with B. pseudomallei harbored numerous intracellular bacteria, suggesting that this facultative intracellular pathogen can survive and replicate inside of MH cockroach phagocytic cells. The hemolymph extracted from these MH cockroaches also contained multinuclear giant cells (MNGCs with intracellular B. pseudomallei, which indicates that infected hemocytes can

  2. The Madagascar hissing cockroach as a novel surrogate host for Burkholderia pseudomallei, B. mallei and B. thailandensis.

    Science.gov (United States)

    Fisher, Nathan A; Ribot, Wilson J; Applefeld, Willard; DeShazer, David

    2012-06-22

    Burkholderia pseudomallei and Burkholderia mallei are gram-negative pathogens responsible for the diseases melioidosis and glanders, respectively. Both species cause disease in humans and animals and have been designated as category B select agents by the Centers for Disease Control and Prevention (CDC). Burkholderia thailandensis is a closely related bacterium that is generally considered avirulent for humans. While it can cause disease in rodents, the B. thailandensis 50% lethal dose (LD50) is typically ≥ 104-fold higher than the B. pseudomallei and B. mallei LD50 in mammalian models of infection. Here we describe an alternative to mammalian hosts in the study of virulence and host-pathogen interactions of these Burkholderia species. Madagascar hissing cockroaches (MH cockroaches) possess a number of qualities that make them desirable for use as a surrogate host, including ease of breeding, ease of handling, a competent innate immune system, and the ability to survive at 37°C. MH cockroaches were highly susceptible to infection with B. pseudomallei, B. mallei and B. thailandensis and the LD50 was 105 cfu. B. pseudomallei, B. mallei, and B. thailandensis cluster 1 type VI secretion system (T6SS-1) mutants were all attenuated in MH cockroaches, which is consistent with previous virulence studies conducted in rodents. B. pseudomallei mutants deficient in the other five T6SS gene clusters, T6SS-2 through T6SS-6, were virulent in both MH cockroaches and hamsters. Hemocytes obtained from MH cockroaches infected with B. pseudomallei harbored numerous intracellular bacteria, suggesting that this facultative intracellular pathogen can survive and replicate inside of MH cockroach phagocytic cells. The hemolymph extracted from these MH cockroaches also contained multinuclear giant cells (MNGCs) with intracellular B. pseudomallei, which indicates that infected hemocytes can fuse while flowing through the insect's open circulatory system in vivo. The results

  3. Development of Burkholderia mallei and pseudomallei vaccines.

    Science.gov (United States)

    Silva, Ediane B; Dow, Steven W

    2013-01-01

    Burkholderia mallei and Burkholderia pseudomallei are Gram-negative bacteria that cause glanders and melioidosis, respectively. Inhalational infection with either organism can result in severe and rapidly fatal pneumonia. Inoculation by the oral and cutaneous routes can also produce infection. Chronic infection may develop after recovery from acute infection with both agents, and control of infection with antibiotics requires prolonged treatment. Symptoms for both meliodosis and glanders are non-specific, making diagnosis difficult. B. pseudomallei can be located in the environment, but in the host, B. mallei and B. psedomallei are intracellular organisms, and infection results in similar immune responses to both agents. Effective early innate immune responses are critical to controlling the early phase of the infection. Innate immune signaling molecules such as TLR, NOD, MyD88, and pro-inflammatory cytokines such as IFN-γ and TNF-α play key roles in regulating control of infection. Neutrophils and monocytes are critical cells in the early infection for both microorganisms. Both monocytes and macrophages are necessary for limiting dissemination of B. pseudomallei. In contrast, the role of adaptive immune responses in controlling Burkholderia infection is less well understood. However, T cell responses are critical for vaccine protection from Burkholderia infection. At present, effective vaccines for prevention of glanders or meliodosis have not been developed, although recently development of Burkholderia vaccines has received renewed attention. This review will summarize current and past approaches to develop B. mallei and B. pseudomalllei vaccines, with emphasis on immune mechanisms of protection and the challenges facing the field. At present, immunization with live attenuated bacteria provides the most effective and durable immunity, and it is important therefore to understand the immune correlates of protection induced by live attenuated vaccines. Subunit

  4. Development of Burkholderia mallei and pseudomallei vaccines

    Science.gov (United States)

    Silva, Ediane B.; Dow, Steven W.

    2013-01-01

    Burkholderia mallei and Burkholderia pseudomallei are Gram-negative bacteria that cause glanders and melioidosis, respectively. Inhalational infection with either organism can result in severe and rapidly fatal pneumonia. Inoculation by the oral and cutaneous routes can also produce infection. Chronic infection may develop after recovery from acute infection with both agents, and control of infection with antibiotics requires prolonged treatment. Symptoms for both meliodosis and glanders are non-specific, making diagnosis difficult. B. pseudomallei can be located in the environment, but in the host, B. mallei and B. psedomallei are intracellular organisms, and infection results in similar immune responses to both agents. Effective early innate immune responses are critical to controlling the early phase of the infection. Innate immune signaling molecules such as TLR, NOD, MyD88, and pro-inflammatory cytokines such as IFN-γ and TNF-α play key roles in regulating control of infection. Neutrophils and monocytes are critical cells in the early infection for both microorganisms. Both monocytes and macrophages are necessary for limiting dissemination of B. pseudomallei. In contrast, the role of adaptive immune responses in controlling Burkholderia infection is less well understood. However, T cell responses are critical for vaccine protection from Burkholderia infection. At present, effective vaccines for prevention of glanders or meliodosis have not been developed, although recently development of Burkholderia vaccines has received renewed attention. This review will summarize current and past approaches to develop B. mallei and B. pseudomalllei vaccines, with emphasis on immune mechanisms of protection and the challenges facing the field. At present, immunization with live attenuated bacteria provides the most effective and durable immunity, and it is important therefore to understand the immune correlates of protection induced by live attenuated vaccines. Subunit

  5. Evaluation of a biodegradable microparticulate polymer as a carrier for Burkholderia pseudomallei subunit vaccines in a mouse model of melioidosis.

    Science.gov (United States)

    Schully, K L; Bell, M G; Prouty, A M; Gallovic, M D; Gautam, S; Peine, K J; Sharma, S; Bachelder, E M; Pesce, J T; Elberson, M A; Ainslie, K M; Keane-Myers, A

    2015-11-30

    Melioidosis, a potentially lethal disease of humans and animals, is caused by the soil-dwelling bacterium Burkholderia pseudomallei. Due to B. pseudomallei's classification as a Tier 1 Select Agent, there is substantial interest in the development of an effective vaccine. Yet, despite decades of research, no effective target, adjuvant or delivery vehicle capable of inducing protective immunity against B. pseudomallei infection has been identified. We propose a microparticulate delivery vehicle comprised of the novel polymer acetalated dextran (Ac-DEX). Ac-DEX is an acid-sensitive biodegradable carrier that can be fabricated into microparticles (MPs) that are relatively stable at pH 7.4, but rapidly degrade after phagocytosis by antigen presenting cells where the pH can drop to 5.0. As compared to other biomaterials, this acid sensitivity has been shown to enhance cross presentation of subunit antigens. To evaluate this platform as a delivery system for a melioidosis vaccine, BALB/c mice were vaccinated with Ac-DEX MPs separately encapsulating B. pseudomallei whole cell lysate and the toll-like receptor (TLR) 7/8 agonist resiquimod. This vaccine elicited a robust antibody response that included both Th1 and Th2 immunity. Following lethal intraperitoneal challenge with B. pseudomallei 1026b, vaccinated mice demonstrated a significant delay to time of death compared to untreated mice. The formulation, however, demonstrated incomplete protection indicating that lysate protein offers limited value as an antigen. Nevertheless, our Ac-DEX MPs may offer an effective delivery vehicle for a subunit B. psuedomallei vaccine. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Improved isolation of Vibro vulnificus from seawater and sediment with cellobiose-colistin agar

    DEFF Research Database (Denmark)

    Høi, L.; Dalsgaard, Inger; Dalsgaard, A.

    1998-01-01

    An improved selective medium, cellobiose-colistin (CC) agar, gave a significantly higher (P agar, In a total of 446 alkaline peptone water preenrichments amended...... with polymyxin B, V. vulnificus was isolated from 154 preenrichments (35%) with mCPC agar and from 179 preenrichments (40%) with CC agar. CC agar gave a higher plating efficiency of V. vulnificus cells than did cellobiose-polymyxin B-colistin (CPC) agar, mCPC agar, or thiosulfate-citrate-bile salts-sucrose (TCBS......) agar; the only significant difference was observed with TCBS agar, which gave much lower plating efficiencies than the other selective media. Determination of MICs demonstrated that the concentrations of colistin and polymyxin B in CPC agar inhibit growth of a proportion of V. vulnificus strains....

  7. Versatile dual-technology system for markerless allele replacement in Burkholderia pseudomallei.

    Science.gov (United States)

    López, Carolina M; Rholl, Drew A; Trunck, Lily A; Schweizer, Herbert P

    2009-10-01

    Burkholderia pseudomallei is the etiologic agent of melioidosis, a rare but serious tropical disease. In the United States, genetic research with this select agent bacterium is strictly regulated. Although several select agent compliant methods have been developed for allelic replacement, all of them suffer from some drawbacks, such as a need for specific host backgrounds or use of minimal media. Here we describe a versatile select agent compliant allele replacement system for B. pseudomallei based on a mobilizable vector, pEXKm5, which contains (i) a multiple cloning site within a lacZalpha gene for facile cloning of recombinant DNA fragments, (ii) a constitutively expressed gusA indicator gene for visual detection of merodiploid formation and resolution, and (iii) elements required for resolution of merodiploids using either I-SceI homing endonuclease-stimulated recombination or sacB-based counterselection. The homing endonuclease-based allele replacement system is completed by pBADSce, which contains an araC-P(BAD)-I-sceI expression cassette for arabinose-inducible I-SceI expression and a temperature-sensitive pRO1600 replicon for facile plasmid curing. Complementing these systems is the improved Deltaasd Escherichia coli mobilizer strain RHO3. This strain is susceptible to commonly used antibiotics and allows nutritional counterselection on rich media because of its diaminopimelic acid auxotrophy. The versatility of the I-SceI- and sacB-based methods afforded by pEXKm5 in conjunction with E. coli RHO3 was demonstrated by isolation of diverse deletion mutants in several clinical, environmental, and laboratory B. pseudomallei strains. Finally, sacB-based counterselection was employed to isolate a defined chromosomal fabD(Ts) allele that causes synthesis of a temperature-sensitive FabD, an essential fatty acid biosynthesis enzyme.

  8. Global and regional dissemination and evolution of Burkholderia pseudomallei

    Science.gov (United States)

    Chewapreecha, Claire; Holden, Matthew T. G.; Vehkala, Minna; Välimäki, Niko; Yang, Zhirong; Harris, Simon R; Mather, Alison E.; Tuanyok, Apichai; De Smet, Birgit; Le Hello, Simon; Bizet, Chantal; Mayo, Mark; Wuthiekanun, Vanaporn; Limmathurotsakul, Direk; Phetsouvanh, Rattanaphone; Spratt, Brian G; Corander, Jukka; Keim, Paul; Dougan, Gordon; Dance, David A. B.; Currie, Bart J; Parkhill, Julian; Peacock, Sharon J.

    2017-01-01

    The environmental bacterium Burkholderia pseudomallei causes an estimated 165,000 cases of human melioidosis per year worldwide, and is also classified as a biothreat agent. We used whole genome sequences of 469 B. pseudomallei isolates from 30 countries collected over 79 years to explore its geographic transmission. Our data point to Australia as an early reservoir, with transmission to Southeast Asia followed by onward transmission to South Asia, and East Asia. Repeated reintroduction was observed within the Malay Peninsula, and between countries bordered by the Mekong river. Our data support an African origin of the Central and South American isolates with introduction of B. pseudomallei into the Americas between 1650 and 1850, providing a temporal link with the slave trade. We also identified geographically distinct genes/variants in Australasian or Southeast Asian isolates alone, with virulence-associated genes being among those overrepresented. This provides a potential explanation for clinical manifestations of melioidosis that are geographically restricted. PMID:28112723

  9. Comparison of inhibitory mold agar to Sabouraud dextrose agar as a primary medium for isolation of fungi.

    Science.gov (United States)

    Scognamiglio, Theresa; Zinchuk, Riva; Gumpeni, Pramod; Larone, Davise H

    2010-05-01

    Clinical specimens cultured on two selective fungal media, inhibitory mold agar (IMA) and Sabouraud dextrose agar (SDA), were compared with respect to recovery of fungi. Of the 840 fungal isolates recovered, 69.3% grew on both IMA and SDA; 24.9% grew only on IMA; and 5.8% grew only on SDA, showing that IMA is superior (P=0.003).

  10. Automatic agar tray inoculation device

    Science.gov (United States)

    Wilkins, J. R.; Mills, S. M.

    1972-01-01

    Automatic agar tray inoculation device is simple in design and foolproof in operation. It employs either conventional inoculating loop or cotton swab for uniform inoculation of agar media, and it allows technician to carry on with other activities while tray is being inoculated.

  11. Development of ceftazidime resistance in an acute Burkholderia pseudomallei infection

    Directory of Open Access Journals (Sweden)

    Sarovich DS

    2012-08-01

    Full Text Available Derek S Sarovich,1,2,* Erin P Price,1,2,* Direk Limmathurotsakul,3 James M Cook,1 Alex T Von Schulze,1 Spenser R Wolken,1 Paul Keim,1 Sharon J Peacock,3,4 Talima Pearson1 1Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, AZ, USA; 2Tropical and Emerging Infectious Diseases Division, Menzies School of Health Research, Darwin, Australia; 3Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; 4Department of Medicine, University of Cambridge, Cambridge, United Kingdom*These authors contributed equally to this workAbstract: Burkholderia pseudomallei, a bacterium that causes the disease melioidosis, is intrinsically resistant to many antibiotics. First-line antibiotic therapy for treating melioidosis is usually the synthetic β-lactam, ceftazidime (CAZ, as almost all B. pseudomallei strains are susceptible to this drug. However, acquired CAZ resistance can develop in vivo during treatment with CAZ, which can lead to mortality if therapy is not switched to a different drug in a timely manner. Serial B. pseudomallei isolates obtained from an acute Thai melioidosis patient infected by a CAZ susceptible strain, who ultimately succumbed to infection despite being on CAZ therapy for the duration of their infection, were analyzed. Isolates that developed CAZ resistance due to a proline to serine change at position 167 in the β-lactamase PenA were identified. Importantly, these CAZ resistant isolates remained sensitive to the alternative melioidosis treatments; namely, amoxicillin-clavulanate, imipenem, and meropenem. Lastly, real-time polymerase chain reaction-based assays capable of rapidly identifying CAZ resistance in B. pseudomallei isolates at the position 167 mutation site were developed. The ability to rapidly identify the emergence of CAZ resistant B. pseudomallei populations in melioidosis patients will allow timely alterations in treatment strategies

  12. Burkholderia pseudomallei musculoskeletal infections (melioidosis in India

    Directory of Open Access Journals (Sweden)

    Pandey Vivek

    2010-01-01

    Full Text Available Melioidosis, an infection due to gram negative Burkholderia pseudomallei, is an important cause of sepsis in east Asia especially Thailand and northern Australia. It usually causes abscesses in lung, liver, spleen, skeletal muscle and parotids especially in patients with diabetes, chronic renal failure and thalassemia. Musculoskeletal melioidosis is not common in India even though sporadic cases have been reported mostly involving soft tissues. During a two-year-period, we had five patients with musculoskeletal melioidosis. All patients presented with multifocal osteomyelitis, recurrent osteomyelitis or septic arthritis. One patient died early because of septicemia and multi-organ failure. All patients were diagnosed on the basis of positive pus culture. All patients were treated by surgical debridement followed by a combination of antibiotics; (ceftazidime, amoxy-clavulanic acid, co-trimoxazole and doxycycline for six months except for one who died due to fulminant septicemia. All other patients recovered completely with no recurrences. With increasing awareness and better diagnostic facilities, probably musculoskeletal melioidosis will be increasingly diagnosed in future.

  13. Molecular insights into Burkholderia pseudomallei and Burkholderia mallei pathogenesis.

    Science.gov (United States)

    Galyov, Edouard E; Brett, Paul J; DeShazer, David

    2010-01-01

    Burkholderia pseudomallei and Burkholderia mallei are closely related gram-negative bacteria that can cause serious diseases in humans and animals. This review summarizes the current and rapidly expanding knowledge on the specific virulence factors employed by these pathogens and their roles in the pathogenesis of melioidosis and glanders. In particular, the contributions of recently identified virulence factors are described in the context of the intracellular lifestyle of these pathogens. Throughout this review, unique and shared virulence features of B. pseudomallei and B. mallei are discussed.

  14. Burkholderia pseudomallei resistance to antibiotics in biofilm-induced conditions is related to efflux pumps.

    Science.gov (United States)

    Sirijant, Nopphasul; Sermswan, Rasana W; Wongratanacheewin, Surasakdi

    2016-11-01

    Burkholderia pseudomallei, the causative agent of melioidosis, has been found to increase its resistance to antibiotics when growing as a biofilm. The resistance is related to several mechanisms. One of the possible mechanisms is the efflux pump. Using bioinformatics analysis, it was found that BPSL1661, BPSL1664 and BPSL1665 were orthologous genes of the efflux transporter encoding genes for biofilm-related antibiotic resistance, PA1874-PA1877 genes in Pseudomonas aeruginosa strain PAO1. Expression of selected encoding genes for the efflux transporter system during biofilm formation were investigated. Real-time reverse transcriptase PCR expression of amrB, cytoplasmic membrane protein of AmrAB-OprA efflux transporter encoding gene, was slightly increased, while BPSL1665 was significantly increased during growth of bacteria in biofilm formation. Minimum biofilm inhibition concentration and minimum biofilm eradication concentration (MBEC) of ceftazidime (CTZ), doxycycline (DOX) and imipenem were found to be 2- to 1024-times increased when compared to their MICs for of planktonic cells. Inhibition of the efflux transporter by adding phenylalanine arginine β-napthylamide (PAβN), a universal efflux inhibitor, decreased 2 to 16 times as much as MBEC in B. pseudomallei biofilms with CTZ and DOX. When the intracellular accumulation of antibiotics was tested to reveal the pump inhibition, only the concentrations of CTZ and DOX increased in PAβN treated biofilm. Taken together, these results indicated that BPSL1665, a putative precursor of the efflux pump gene, might be related to the adaptation of B. pseudomallei in biofilm conditions. Inhibition of efflux pumps may lead to a decrease of resistance to CTZ and DOX in biofilm cells.

  15. Characterization of BcaA, a putative classical autotransporter protein in Burkholderia pseudomallei.

    Science.gov (United States)

    Campos, Cristine G; Borst, Luke; Cotter, Peggy A

    2013-04-01

    Burkholderia pseudomallei is a tier 1 select agent, and the causative agent of melioidosis, a disease with effects ranging from chronic abscesses to fulminant pneumonia and septic shock, which can be rapidly fatal. Autotransporters (ATs) are outer membrane proteins belonging to the type V secretion system family, and many have been shown to play crucial roles in pathogenesis. The open reading frame Bp1026b_II1054 (bcaA) in B. pseudomallei strain 1026b is predicted to encode a classical autotransporter protein with an approximately 80-kDa passenger domain that contains a subtilisin-related domain. Immediately 3' to bcaA is Bp11026_II1055 (bcaB), which encodes a putative prolyl 4-hydroxylase. To investigate the role of these genes in pathogenesis, large in-frame deletion mutations of bcaA and bcaB were constructed in strain Bp340, an efflux pump mutant derivative of the melioidosis clinical isolate 1026b. Comparison of Bp340ΔbcaA and Bp340ΔbcaB mutants to wild-type B. pseudomallei in vitro demonstrated similar levels of adherence to A549 lung epithelial cells, but the mutant strains were defective in their ability to invade these cells and to form plaques. In a BALB/c mouse model of intranasal infection, similar bacterial burdens were observed after 48 h in the lungs and liver of mice infected with Bp340ΔbcaA, Bp340ΔbcaB, and wild-type bacteria. However, significantly fewer bacteria were recovered from the spleen of Bp340ΔbcaA-infected mice, supporting the idea of a role for this AT in dissemination or in survival in the passage from the site of infection to the spleen.

  16. Release Control of Dye from Agar Ball

    OpenAIRE

    板屋, 智之; 山村, 俊貴; 唐澤, 有太朗

    2013-01-01

    Agar is a special product of Nagano prefecture. To utilize agar gel as adsorbing or releasing material of dyes or drugs, spherical agar gel “agar ball” was prepared by dropping aqueous agar solution into salad oil. And releasing behavior of a dye (rhodamine B) from agar ball was studied. The dye is released easily from agar ball, but the release can be controlled by hybiridazation of agar and galatin. In addition, it was found that agar ball could extract the dye from oil phase containing the...

  17. What drives the occurrence of the melioidosis bacterium Burkholderia pseudomallei in domestic gardens?

    Science.gov (United States)

    Kaestli, Mirjam; Harrington, Glenda; Mayo, Mark; Chatfield, Mark D; Harrington, Ian; Hill, Audrey; Munksgaard, Niels; Gibb, Karen; Currie, Bart J

    2015-03-01

    Melioidosis is an often fatal infectious disease affecting humans and animals in tropical regions and is caused by the saprophytic environmental bacterium Burkholderia pseudomallei. Domestic gardens are not only a common source of exposure to soil and thus to B. pseudomallei, but they also have been found to contain more B. pseudomallei than other environments. In this study we addressed whether anthropogenic manipulations common to gardens such as irrigation or fertilizers change the occurrence of B. pseudomallei. We conducted a soil microcosm experiment with a range of fertilizers and soil types as well as a longitudinal interventional study over three years on an experimental fertilized field site in an area naturally positive for B. pseudomallei. Irrigation was the only consistent treatment to increase B. pseudomallei occurrence over time. The effects of fertilizers upon these bacteria depended on soil texture, physicochemical soil properties and biotic factors. Nitrates and urea increased B. pseudomallei load in sand while phosphates had a positive effect in clay. The high buffering and cation exchange capacities of organic material found in a commercial potting mix led to a marked increase in soil salinity with no survival of B. pseudomallei after four weeks in the potting mix sampled. Imported grasses were also associated with B. pseudomallei occurrence in a multivariate model. With increasing population density in endemic areas these findings inform the identification of areas in the anthropogenic environment with increased risk of exposure to B. pseudomallei.

  18. Molecular Characterization of Putative Virulence Determinants in Burkholderia pseudomallei

    Directory of Open Access Journals (Sweden)

    Suat Moi Puah

    2014-01-01

    Full Text Available The Gram-negative saprophyte Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease which is endemic in Southeast Asia and northern Australia. This bacterium possesses many virulence factors which are thought to contribute to its survival and pathogenicity. Using a virulent clinical isolate of B. pseudomallei and an attenuated strain of the same B. pseudomallei isolate, 6 genes BPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131, and BURPS1710A_1419 were identified earlier by PCR-based subtractive hybridization. These genes were extensively characterized at the molecular level, together with an additional gene BPSL3147 that had been identified by other investigators. Through a reverse genetic approach, single-gene knockout mutants were successfully constructed by using site-specific insertion mutagenesis and were confirmed by PCR. BPSL2033::Km and BURPS1710A_1419::Km mutants showed reduced rates of survival inside macrophage RAW 264.7 cells and also low levels of virulence in the nematode infection model. BPSL2033::Km demonstrated weak statistical significance (P=0.049 at 8 hours after infection in macrophage infection study but this was not seen in BURPS1710A_1419::Km. Nevertheless, complemented strains of both genes were able to partially restore the gene defects in both in vitro and in vivo studies, thus suggesting that they individually play a minor role in the virulence of B. pseudomallei.

  19. A Burkholderia pseudomallei Infection Imported from Eritrea to Israel

    OpenAIRE

    Almog, Yaniv; Yagel, Yael; Geffen, Yuval; Yagupsky, Pablo

    2016-01-01

    Although it has been predicted that melioidosis is probably endemic in the Horn of Africa, no confirmed cases have ever been detected in the region. We have recently isolated Burkholderia pseudomallei from an Eritrean patient in Israel. The isolate was assigned a novel multilocus sequence type (ST-1479). The observation has important epidemiological implications in an era of massive human migration.

  20. Genetic diversity and microevolution of Burkholderia pseudomallei in the environment.

    Directory of Open Access Journals (Sweden)

    Narisara Chantratita

    2008-02-01

    Full Text Available The soil dwelling Gram-negative pathogen Burkholderia pseudomallei is the cause of melioidosis. The diversity and population structure of this organism in the environment is poorly defined.We undertook a study of B. pseudomallei in soil sampled from 100 equally spaced points within 237.5 m(2 of disused land in northeast Thailand. B. pseudomallei was present on direct culture of 77/100 sampling points. Genotyping of 200 primary plate colonies from three independent sampling points was performed using a combination of pulsed field gel electrophoresis (PFGE and multilocus sequence typing (MLST. Twelve PFGE types and nine sequence types (STs were identified, the majority of which were present at only a single sampling point. Two sampling points contained four STs and the third point contained three STs. Although the distance between the three sampling points was low (7.6, 7.9, and 13.3 meters, respectively, only two STs were present in more than one sampling point. Each of the three samples was characterized by the localized expansion of a single B. pseudomallei clone (corresponding to STs 185, 163, and 93. Comparison of PFGE and MLST results demonstrated that two STs contained strains with variable PFGE banding pattern types, indicating geographic structuring even within a single MLST-defined clone.We discuss the implications of this extreme structuring of genotype and genotypic frequency in terms of micro-evolutionary dynamics and ecology, and how our results may inform future sampling strategies.

  1. In vitro susceptibility of Burkholderia pseudomallei to antimicrobial peptides

    NARCIS (Netherlands)

    Kanthawong, S.; Nazmi, K.; Wongratanacheewin, S.; Bolscher, J.G.M.; Wuthiekanun, V.; Taweechaisupapong, S.

    2009-01-01

    Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to many antibiotics, resulting in high mortality rates of 19% in Australia and even 50% in Thailand. Antimicrobial peptides (AMPs) possess potent broad-spectrum bactericidal activities and are regarded as

  2. Ultrastructural effects and antibiofilm activity of LFchimera against Burkholderia pseudomallei

    NARCIS (Netherlands)

    Puknun, A.; Kanthawong, S.; Anutrakunchai, C.; Nazmi, K.; Tigchelaar, W.; Hoeben, K.A.; Veerman, E.C.I.; Bolscher, J.G.M.; Taweechaisupong, S.

    2016-01-01

    Lactoferrin chimera (LFchimera), a hybrid peptide containing the two antimicrobial stretches of the innate immunity factor bovine lactoferrin, viz. LFampin265-284 and LFcin17-30, has strikingly high antimicrobial activity against the category B pathogen Burkholderia pseudomallei. The action

  3. Burkholderia pseudomallei genome plasticity associated with genomic island variation

    Directory of Open Access Journals (Sweden)

    Currie Bart J

    2008-04-01

    Full Text Available Abstract Background Burkholderia pseudomallei is a soil-dwelling saprophyte and the cause of melioidosis. Horizontal gene transfer contributes to the genetic diversity of this pathogen and may be an important determinant of virulence potential. The genome contains genomic island (GI regions that encode a broad array of functions. Although there is some evidence for the variable distribution of genomic islands in B. pseudomallei isolates, little is known about the extent of variation between related strains or their association with disease or environmental survival. Results Five islands from B. pseudomallei strain K96243 were chosen as representatives of different types of genomic islands present in this strain, and their presence investigated in other B. pseudomallei. In silico analysis of 10 B. pseudomallei genome sequences provided evidence for the variable presence of these regions, together with micro-evolutionary changes that generate GI diversity. The diversity of GIs in 186 isolates from NE Thailand (83 environmental and 103 clinical isolates was investigated using multiplex PCR screening. The proportion of all isolates positive by PCR ranged from 12% for a prophage-like island (GI 9, to 76% for a metabolic island (GI 16. The presence of each of the five GIs did not differ between environmental and disease-associated isolates (p > 0.05 for all five islands. The cumulative number of GIs per isolate for the 186 isolates ranged from 0 to 5 (median 2, IQR 1 to 3. The distribution of cumulative GI number did not differ between environmental and disease-associated isolates (p = 0.27. The presence of GIs was defined for the three largest clones in this collection (each defined as a single sequence type, ST, by multilocus sequence typing; these were ST 70 (n = 15 isolates, ST 54 (n = 11, and ST 167 (n = 9. The rapid loss and/or acquisition of gene islands was observed within individual clones. Comparisons were drawn between isolates obtained

  4. Construction and molecular characterization of mouse single-chain variable fragment antibodies against Burkholderia mallei and Burkholderia pseudomallei.

    Science.gov (United States)

    Kim, Ho San; Tsai, Shien; Zou, Nianxiang; Lo, Shyh-Ching; Wear, Douglas J; Izadjoo, Mina J

    2011-02-28

    We have selected two lipopolysaccharide (LPS) specific Burkholderia mallei mouse monoclonal antibodies (mAbs) and four anti-capsular B. pseudomallei-specific mAbs to generate mouse single-chain variable fragment (scFv) antibodies. This selection was made through extensive in vitro and in vivo assay from our library of mAbs against B. mallei and B. pseudomallei. We initially generated the mouse immunoglobulin variable heavy chain (VH) and light chain (VL) regions from each of these six selected mAbs using a phage display scFv technology. We determined the coding sequences of the VH and VL regions and successfully constructed two B. mallei-specific scFv phage antibodies consisting of two different VH (VH1 and VH2) and one Vλ1 families. Four scFvs constructed against B. pseudomallei had two VH (VH1 and VH6) and two VL (Vκ4/5 and Vκ21) families. All of six scFv antibodies constructed demonstrated good binding activity without any rounds of biopanning against B. mallei (M5D and M18F were 0.425 and 0.480 at OD405nm) and B. pseudomallei (P1E7, P2I67, P7C6, and P7F4 were 0.523, 0.859, 0.775, and 0.449 at OD405nm) by ELISA, respectively. A comparison of the immunoglobulin gene segments revealed that the gene sequences in complementarity-determining regions (CDRs) of three out of four B. pseudomallei-specific scFvs are highly conserved. We determined that the two B. mallei-specific scFvs have different CDRs in the VH, but the amino acid sequences of CDRs in the VL are conserved. This high sequence homology found in CDRs of VH or VL of these mAbs contributes to our better understanding and determination of binding to the specific antigenic epitope(s). The scFv phage display technology may be a valuable tool to develop and engineer mAbs with improved antigen-binding affinity. Copyright © 2010 Elsevier B.V. All rights reserved.

  5. Polar lipids of Burkholderia pseudomallei induce different host immune responses.

    Directory of Open Access Journals (Sweden)

    Mercedes Gonzalez-Juarrero

    Full Text Available Melioidosis is a disease in tropical and subtropical regions of the world that is caused by Burkholderia pseudomallei. In endemic regions the disease occurs primarily in humans and goats. In the present study, we used the goat as a model to dissect the polar lipids of B. pseudomallei to identify lipid molecules that could be used for adjuvants/vaccines or as diagnostic tools. We showed that the lipidome of B. pseudomallei and its fractions contain several polar lipids with the capacity to elicit different immune responses in goats, namely rhamnolipids and ornithine lipids which induced IFN-γ, whereas phospholipids and an undefined polar lipid induced strong IL-10 secretion in CD4(+ T cells. Autologous T cells co-cultured with caprine dendritic cells (cDCs and polar lipids of B. pseudomallei proliferated and up-regulated the expression of CD25 (IL-2 receptor molecules. Furthermore, we demonstrated that polar lipids were able to up-regulate CD1w2 antigen expression in cDCs derived from peripheral blood monocytes. Interestingly, the same polar lipids had only little effect on the expression of MHC class II DR antigens in the same caprine dendritic cells. Finally, antibody blocking of the CD1w2 molecules on cDCs resulted in decreased expression for IFN-γ by CD4(+ T cells. Altogether, these results showed that polar lipids of B. pseudomallei are recognized by the caprine immune system and that their recognition is primarily mediated by the CD1 antigen cluster.

  6. Polar Lipids of Burkholderia pseudomallei Induce Different Host Immune Responses

    Science.gov (United States)

    Gonzalez-Juarrero, Mercedes; Mima, Naoko; Trunck, Lily A.; Schweizer, Herbert P.; Bowen, Richard A.; Dascher, Kyle; Mwangi, Waithaka; Eckstein, Torsten M.

    2013-01-01

    Melioidosis is a disease in tropical and subtropical regions of the world that is caused by Burkholderia pseudomallei. In endemic regions the disease occurs primarily in humans and goats. In the present study, we used the goat as a model to dissect the polar lipids of B. pseudomallei to identify lipid molecules that could be used for adjuvants/vaccines or as diagnostic tools. We showed that the lipidome of B. pseudomallei and its fractions contain several polar lipids with the capacity to elicit different immune responses in goats, namely rhamnolipids and ornithine lipids which induced IFN-γ, whereas phospholipids and an undefined polar lipid induced strong IL-10 secretion in CD4+ T cells. Autologous T cells co-cultured with caprine dendritic cells (cDCs) and polar lipids of B. pseudomallei proliferated and up-regulated the expression of CD25 (IL-2 receptor) molecules. Furthermore, we demonstrated that polar lipids were able to up-regulate CD1w2 antigen expression in cDCs derived from peripheral blood monocytes. Interestingly, the same polar lipids had only little effect on the expression of MHC class II DR antigens in the same caprine dendritic cells. Finally, antibody blocking of the CD1w2 molecules on cDCs resulted in decreased expression for IFN-γ by CD4+ T cells. Altogether, these results showed that polar lipids of B. pseudomallei are recognized by the caprine immune system and that their recognition is primarily mediated by the CD1 antigen cluster. PMID:24260378

  7. Outer membrane proteome of Burkholderia pseudomallei and Burkholderia mallei from diverse growth conditions.

    Science.gov (United States)

    Schell, Mark A; Zhao, Peng; Wells, Lance

    2011-05-06

    Burkholderia mallei and Burkholderia pseudomallei are closely related, aerosol-infective human pathogens that cause life-threatening diseases. Biochemical analyses requiring large-scale growth and manipulation at biosafety level 3 under select agent regulations are cumbersome and hazardous. We developed a simple, safe, and rapid method to prepare highly purified outer membrane (OM) fragments from these pathogens. Shotgun proteomic analyses of OMs by trypsin shaving and mass spectrometry identified >155 proteins, the majority of which are clearly outer membrane proteins (OMPs). These included: 13 porins, 4 secretins for virulence factor export, 11 efflux pumps, multiple components of a Type VI secreton, metal transport receptors, polysaccharide exporters, and hypothetical OMPs of unknown function. We also identified 20 OMPs in each pathogen that are abundant under a wide variety of conditions, including in serum and with macrophages, suggesting these are fundamental for growth and survival and may represent prime drug or vaccine targets. Comparison of the OM proteomes of B. mallei and B. pseudomallei showed many similarities but also revealed a few differences, perhaps reflecting evolution of B. mallei away from environmental survival toward host-adaptation.

  8. Fastidious anaerobe agar compared with Wilkins-Chalgren agar, brain heart infusion agar, and brucella agar for susceptibility testing of Fusobacterium species.

    OpenAIRE

    Brazier, J S; Goldstein, E J; Citron, D M; Ostovari, M I

    1990-01-01

    Fastidious anaerobe agar supported the growth of 82 strains of fusobacteria better than brain heart infusion agar, brucella agar, and Wilkins-Chalgren agar. Fastidious anaerobe agar showed less hazing and fewer tailing endpoints with beta-lactam antibiotics. Whole-blood supplementation improved the performance of all media. Wilkins-Chalgren agar without blood failed to support the growth of 17% of the strains. All Fusobacterium ulcerans strains were resistant to clindamycin.

  9. DBSecSys 2.0: a database of Burkholderia mallei and Burkholderia pseudomallei secretion systems

    OpenAIRE

    Memi?evi?, Vesna; Kumar, Kamal; Zavaljevski, Nela; DeShazer, David; Wallqvist, Anders; Reifman, Jaques

    2016-01-01

    Background Burkholderia mallei and B. pseudomallei are the causative agents of glanders and melioidosis, respectively, diseases with high morbidity and mortality rates. B. mallei and B. pseudomallei are closely related genetically; B. mallei evolved from an ancestral strain of B. pseudomallei by genome reduction and adaptation to an obligate intracellular lifestyle. Although these two bacteria cause different diseases, they share multiple virulence factors, including bacterial secretion syste...

  10. Rapid identification of Burkholderia mallei and Burkholderia pseudomallei by intact cell Matrix-assisted Laser Desorption/Ionisation mass spectrometric typing

    Science.gov (United States)

    2012-01-01

    nucleic amplification methods. Our spectra demonstrated a higher homogeneity in B. mallei than in B. pseudomallei isolates. As expected for closely related species, the identification process with MALDI Biotyper software (Bruker Daltonik GmbH, Bremen, Germany) requires the careful selection of spectra from reference strains. When a dedicated reference set is used and spectra of high quality are acquired, it is possible to distinguish both species unambiguously. The need for a careful curation of reference spectra databases is stressed. PMID:23046611

  11. Rapid identification of Burkholderia mallei and Burkholderia pseudomallei by intact cell Matrix-assisted Laser Desorption/Ionisation mass spectrometric typing.

    Science.gov (United States)

    Karger, Axel; Stock, Rüdiger; Ziller, Mario; Elschner, Mandy C; Bettin, Barbara; Melzer, Falk; Maier, Thomas; Kostrzewa, Markus; Scholz, Holger C; Neubauer, Heinrich; Tomaso, Herbert

    2012-10-10

    spectra demonstrated a higher homogeneity in B. mallei than in B. pseudomallei isolates. As expected for closely related species, the identification process with MALDI Biotyper software (Bruker Daltonik GmbH, Bremen, Germany) requires the careful selection of spectra from reference strains. When a dedicated reference set is used and spectra of high quality are acquired, it is possible to distinguish both species unambiguously. The need for a careful curation of reference spectra databases is stressed.

  12. Rapid identification of Burkholderia mallei and Burkholderia pseudomallei by intact cell Matrix-assisted Laser Desorption/Ionisation mass spectrometric typing

    Directory of Open Access Journals (Sweden)

    Karger Axel

    2012-10-01

    analysis being faster than nucleic amplification methods. Our spectra demonstrated a higher homogeneity in B. mallei than in B. pseudomallei isolates. As expected for closely related species, the identification process with MALDI Biotyper software (Bruker Daltonik GmbH, Bremen, Germany requires the careful selection of spectra from reference strains. When a dedicated reference set is used and spectra of high quality are acquired, it is possible to distinguish both species unambiguously. The need for a careful curation of reference spectra databases is stressed.

  13. 48 CFR 401.371 - AGAR Advisories.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false AGAR Advisories. 401.371... ACQUISITION REGULATION SYSTEM Agency Acquisition Regulations 401.371 AGAR Advisories. The SPE may issue AGAR Advisories, consistent with the policies of the FAR and the AGAR, for the following purposes: (a) To...

  14. A Burkholderia pseudomallei Infection Imported from Eritrea to Israel.

    Science.gov (United States)

    Almog, Yaniv; Yagel, Yael; Geffen, Yuval; Yagupsky, Pablo

    2016-11-02

    Although it has been predicted that melioidosis is probably endemic in the Horn of Africa, no confirmed cases have ever been detected in the region. We have recently isolated Burkholderia pseudomallei from an Eritrean patient in Israel. The isolate was assigned a novel multilocus sequence type (ST-1479). The observation has important epidemiological implications in an era of massive human migration. © The American Society of Tropical Medicine and Hygiene.

  15. Pyogenic Liver Abscess Caused by Burkhoderia pseudomallei in Taiwan

    Directory of Open Access Journals (Sweden)

    Yu-Lin Lee

    2006-01-01

    Full Text Available Pyogenic liver abscess in Taiwan is a well-known disease entity, commonly associated with a single pathogen, Klebsiella pneumoniae. Melioidosis is an endemic disease in Taiwan that can manifest as multiple abscesses in sites including the liver. We report three cases of liver abscesses caused by Burkholderia pseudomallei. The first patient was a 54-year-old diabetic woman, who presented with liver abscess and a left subphrenic abscess resulting from a ruptured splenic abscess, co-infected with K. pneumoniae and B. pseudomallei. The second patient, a 58-year-old diabetic man, developed bacteremic pneumonia over the left lower lung due to B. pseudomallei with acute respiratory distress syndrome, and relapsed 5 months later with bacteremic abscesses of the liver, spleen, prostate and osteomyelitis, due to lack of compliance with prescribed antibiotic therapy. The third patient was a 61-year-old diabetic man with a history of travel to Thailand, who presented with jaundice and fever of unknown origin. Liver and splenic abscesses due to B. pseudomallei were diagnosed. A high clinical alertness to patients' travel history, underlying diseases, and the presence of concomitant splenic abscess is essential to early detection of the great mimicker, melioidosis. The treatment of choice is intravenous ceftazidime for at least 14 days or more. An adequate duration of maintenance oral therapy, with amoxicillin-clavulanate or trimethoprim-sulfamethoxazole for 12-20 weeks, is necessary to prevent relapse. Liver abscess in Taiwan is most commonly due to K. pneumoniae, but clinicians should keep in mind that this may be a presenting feature of melioidosis.

  16. Antimicrobial and antibiofilm activity of LL-37 and its truncated variants against Burkholderia pseudomallei

    NARCIS (Netherlands)

    Kanthawong, S.; Bolscher, J.G.M.; Veerman, E.C.I.; van Marle, J.; de Soet, H.J.J.; Nazmi, K.; Wongratanacheewin, S.; Taweechaisupapong, S.

    2012-01-01

    The Gram-negative bacterium Burkholderia pseudomallei is the aetiological agent of melioidosis, which is an endemic disease in tropical areas of Southeast Asia and Northern Australia. Burkholderia pseudomallei has intrinsic resistance to a number of commonly used antibiotics and has also been

  17. Molecular Procedure for Rapid Detection of Burkholderia mallei and Burkholderia pseudomallei

    OpenAIRE

    Bauernfeind, Adolf; Roller, Carsten; Meyer, Detlef; Jungwirth, Renate; Schneider, Ines

    1998-01-01

    A PCR procedure for the discrimination of Burkholderia mallei and Burkholderia pseudomallei was developed. It is based on the nucleotide difference T 2143 C (T versus C at position 2143) between B. mallei and B. pseudomallei detected in the 23S rDNA sequences. In comparison with conventional methods the procedure allows more rapid identification at reduced risk for infection of laboratory personnel.

  18. Assessing the potential for Burkholderia pseudomallei in the southeastern United States

    Science.gov (United States)

    Burkholderia pseudomallei, the causative agent of melioidosis, is an underreported zoonosis in many countries where environmental conditions may be favorable for B. pseudomallei. This soil saprophyte is most often detected in tropical areas such as Southeast Asia and Northern Australia where the cas...

  19. Survival and Intra-Nuclear Trafficking of Burkholderia pseudomallei: Strategies of Evasion from Immune Surveillance?

    Directory of Open Access Journals (Sweden)

    Jamuna Vadivelu

    2017-01-01

    Full Text Available During infection, successful bacterial clearance is achieved via the host immune system acting in conjunction with appropriate antibiotic therapy. However, it still remains a tip of the iceberg as to where persistent pathogens namely, Burkholderia pseudomallei (B. pseudomallei reside/hide to escape from host immune sensors and antimicrobial pressure.We used transmission electron microscopy (TEM to investigate post-mortem tissue sections of patients with clinical melioidosis to identify the localisation of a recently identified gut microbiome, B. pseudomallei within host cells. The intranuclear presence of B. pseudomallei was confirmed using transmission electron microscopy (TEM of experimentally infected guinea pig spleen tissues and Live Z-stack, and ImageJ analysis of fluorescence microscopy analysis of in vitro infection of A549 human lung epithelial cells.TEM investigations revealed intranuclear localization of B. pseudomallei in cells of infected human lung and guinea pig spleen tissues. We also found that B. pseudomallei induced actin polymerization following infection of A549 human lung epithelial cells. Infected A549 lung epithelial cells using 3D-Laser scanning confocal microscopy (LSCM and immunofluorescence microscopy confirmed the intranuclear localization of B. pseudomallei.B. pseudomallei was found within the nuclear compartment of host cells. The nucleus may play a role as an occult or transient niche for persistence of intracellular pathogens, potentially leading to recurrrent episodes or recrudescence of infection.

  20. Improvement of Karmali agar by addition of polymyxin B for the detection of Campylobacter jejuni and C. coli in whole-chicken carcass rinse.

    Science.gov (United States)

    Chon, Jung-Whan; Kim, Hyunsook; Yim, Jin-Hyeok; Song, Kwang-Young; Moon, Jin-San; Kim, Young-Jo; Seo, Kun-Ho

    2013-05-01

    The Karmali agar was modified by supplementation with a high concentration of polymyxin B. The goal of the study was to evaluate the effect of a high concentration of polymyxin B on the ability and selectivity of the modified Karmali agar to isolate Campylobacter jejuni and Campylobacter coli from whole chicken carcass rinse. A total of 80 whole chickens were rinsed with 400 mL of buffer peptone water. The rinsed samples were incubated with 2× blood-free modified Bolton enrichment broth for 48 h, and then streaked onto unmodified Karmali agar and modified Karmali agar supplemented with 100000 IU/L polymixin B (P-Karmali agar). The suspected colonies were finally confirmed by colony PCR. The P-Karmali agar exhibited a significantly better (P agar (P-Karmali agar, 73.8%; unmodified Karmali agar, 33.8%). Moreover, the selectivity of the P-Karmali agar was also better (P agar when comparing the number of contaminated plates (P-Karmali agar, 68.8%; unmodified Karmali agar, 87.5%) and growth index of competing flora (P-Karmali agar, 1.4; unmodified Karmali agar, 2.7). The improved selective agar excluded competing flora resistant to antibiotic agents in unmodified Karmali agar, increasing isolation rate and selectivity for C. jejuni and C. coli. © 2013 Institute of Food Technologists®

  1. Pengaruh Iradiasi Gamma Pada Sifat Fisiko-Kimia Agar- Agar Dalam Bentuk Padat

    OpenAIRE

    Erizal, Erizal; A., Basril; Sudirman, Sudirman; W., Thamrin; Budianto, Emil

    2011-01-01

    Dalam kerangka aplikasi radiasi pada pengembangan bahan polimer yang berasal dari tumbuhan laut, telah dievaluasi karakter fisiko-kimia agar-agar dalam bentuk padat (serbuk dan film) akibat pengaruh iradiasi gamma pada rentang dosis 0 kGy sampai 50 kGy. Viscometer, Instron dan FT-IR digunakan untuk memonitor Perubahan struktur dan sifat mekanik agar-agar hasil iradiasi dan kontrol. Hasil menunjukkan meningkatnya dosis iradiasi menyebabkan viskositas intrinsik dan tegangan putus agar-agar menu...

  2. Novo meio seletivo-indicador para detecção de Aeromonas e Plesiomonas: ágar UNISC New selective indicator medium for detection of Aeromonas and Plesiomonas: UNISC agar

    Directory of Open Access Journals (Sweden)

    Marion Pereira da Rocha

    2008-08-01

    Full Text Available Avaliou-se um novo meio seletivo-indicador (ágar UNISC para o isolamento de enteropatógenos clássicos e Aeromonas e Plesiomonas shigelloides. A capacidade de fermentação da xilose é indicada pela coloração amarela (fermentadores ou azul (não fermentadores que, aliada à prova da oxidase, constitui-se em indicador para a detecção de Aeromonas spp e Plesiomonas shigelloides. A produtividade e seletividade, avaliadas pelos índice de contagem absoluta e índice de contagem relativa indicam-no como uma alternativa aos coprocultivos clássicos porque permite, num só meio, o isolamento de Escherichia coli, Shigella spp, Salmonella spp, bem como, Aeromonas spp e Plesiomonas shigelloides, favorecendo o diagnóstico laboratorial das gastroenterites.We evaluated a new selective indicator medium (UNISC Agar for isolation of classical enteropathogens, Aeromonas spp and Plesiomonas shigelloides. The xylose fermentation capacity is indicated by a yellow color (fermenting agents or blue (no fermenting agent. This, together with the oxidase test, establishes it as an indicator for detecting Aeromonas and Plesiomonas shigelloides. Its productivity and selectivity, as assessed using the absolute count index and relative count index, indicate it as an alternative to the classical feces culturing media. This is because, in a single medium, it enables isolation of Escherichia coli, Shigella spp and Salmonella spp, in addition to Aeromonas and Plesiomonas shigelloides, thereby favoring the laboratory diagnosis of gastroenteritis.

  3. Evaluation of eight agar media for the isolation of shiga toxin-Producing Escherichia coli.

    Science.gov (United States)

    Gill, Alexander; Huszczynski, George; Gauthier, Martine; Blais, Burton

    2014-01-01

    The growth characteristics of 96 shiga toxin-producing Escherichia coli (STEC) strains representing 36 different O-types (including priority O types O26, O45, O103, O111, O121, O145 and O157) on commercial and in-house agar media were studied. The ability of the strains to grow on agar media with varying selective supplement formulations was evaluated using MacConkey Agar (MAC); Rainbow® Agar O157 (RBA); Rainbow® Agar O157 with manufacturer-recommended selective supplements (RBA-NT); Rainbow® Agar O157 with USDA-recommended selective supplements (RBA-USDA); CHROMagar STEC™ (CH STEC); Tryptone Bile agar containing cefixime and tellurite (TBA-CT); Tryptone Bile agar containing cefixime, tellurite, eosin and methylene blue (TBA-EM); and VTEC agar. All of the strains were able to grow on MAC, RBA and VTEC agar, whereas a number of strains (including some non-O157 priority O types) were unable to grow on the highly selective media CH STEC, RBA-NT, RBA-USDA, TBA-EM and TBA-CT. Only RBA-NT and CH STEC exhibited significant inhibition of background flora from ground beef enrichment. Significant inhibition of background flora from beef trim enrichment was observed with RBA-NT, RBA-USDA, CH STEC, TBA-EM and VTEC agar. With exception of E. coli O157, several different colony morphologies were observed on the differential plating media among strains of the same O type, indicating that this colony morphology is not a reliable means of identifying target STEC. These results suggest that an approach to maximize the recovery of target STEC from beef enrichment cultures is dual plating on lesser (RBA, MAC, VTEC agar) and more highly (RBA-NT, CH STEC) selective agars. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  4. Detection of extracellular proteases from microorganisms on agar plates

    Directory of Open Access Journals (Sweden)

    Alane Beatriz Vermelho

    1996-12-01

    Full Text Available We present herein an improved assay for detecting the presence of extracellular proteases from microorganisms on agar plates. Using different substrates (gelatin, BSA, hemoglobin incorporated into the agar and varying the culture medium composition, we were able to detect proteolytic activities from Pseudomonas aeruginosa, Micrococcus luteus and Serratia marcescens as well as the influence that these components displayed in the expression of these enzymes. For all microorganisms tested we found that in agar-BHI or yeast extract medium containing gelatin the sensitivity of proteinase detection was considerably greater than in BSA-agar or hemoglobin-agar. However, when BSA or hemoglobin were added to the culture medium, there was an increase in growth along with a marked reduction in the amount of proteinase production. In the case of M. luteus the incorporation of glycerol in BHI or yeast extract gelatin-agar induced protease liberation. Our results indicate that the technique described here is of value for detecting extracellular proteases directly in the culture medium, by means of a qualitative assay, simple, inexpensive, straight forward method to assess the presence of the proteolytic activity of a given microorganism colony with great freedom in substrate selection.

  5. Improvement of Karmali Agar by Supplementation with Tazobactam for Detecting Campylobacter in Raw Poultry.

    Science.gov (United States)

    Kim, Young-Ji; Whan, Chon-Jung; Kim, Hong-Seok; Kim, Kwang-Yeop; Yim, Jin-Hyeok; Cho, Seung-Hak; Seo, Kun-Ho

    2016-11-01

    In this study, Karmali agar was modified by adding tazobactam (T-Karmali agar) to suppress the growth of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli , which frequently contaminates raw poultry meat. By inoculating 30 Campylobacter spp. strains and 25 ESBL-producing E. coli strains onto Karmali agar and T-Karmali agar containing various concentrations of the antibacterial agent, we determined the optimum concentration of tazobactam to be 4 mg/liter. The Campylobacter spp. isolation rate on T-Karmali agar (13.3%) was higher than that on Karmali agar (8.3%), although the difference was not significant (P > 0.05). However, T-Karmali agar showed a significantly greater selectivity than Karmali agar, as evaluated by comparing the numbers of contaminated agar plates (20.8 versus 82.5%; P agar were identified as ESBL-producing E. coli . Thus, T-Karmali agar might be effective for determining the real prevalence of Campylobacter in raw poultry and, especially, contamination with ESBL-producing E. coli .

  6. Land use and soil type determine the presence of the pathogen Burkholderia pseudomallei in tropical rivers.

    Science.gov (United States)

    Ribolzi, Olivier; Rochelle-Newall, Emma; Dittrich, Sabine; Auda, Yves; Newton, Paul N; Rattanavong, Sayaphet; Knappik, Michael; Soulileuth, Bounsamai; Sengtaheuanghoung, Oloth; Dance, David A B; Pierret, Alain

    2016-04-01

    Burkholderia pseudomallei is the bacterium that causes melioidosis in humans. While B. pseudomallei is known to be endemic in South East Asia (SEA), the occurrence of the disease in other parts of the tropics points towards a potentially large global distribution. We investigated the environmental factors that influence the presence (and absence) of B. pseudomallei in a tropical watershed in SEA. Our main objective was to determine whether there is a link between the presence of the organism in the hydrographic network and the upstream soil and land-use type. The presence of B. pseudomallei was determined using a specific quantitative real-time PCR assay following enrichment culture. Land use, soil, geomorphology, and environmental data were then analyzed using partial least squares discriminant analysis (PLSDA) to compare the B. pseudomallei positive and negative sites. Soil type in the surrounding catchment and turbidity had a strong positive influence on the presence (acrisols and luvisols) or absence (ferralsols) of B. pseudomallei. Given the strong apparent links between soil characteristics, water turbidity, and the presence/absence of B. pseudomallei, actions to raise public awareness about factors increasing the risk of exposure should be undertaken in order to reduce the incidence of melioidosis in regions of endemicity.

  7. Burkholderia pseudomallei Differentially Regulates Host Innate Immune Response Genes for Intracellular Survival in Lung Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Kumutha Malar Vellasamy

    2016-07-01

    Full Text Available Burkholderia pseudomallei, the causative agent of melioidosis poses a serious threat to humankind. B. pseudomallei secretes numerous virulence proteins that alter host cell functions to escape from intracellular immune sensors. However, the events underlying disease pathogenesis are poorly understood.We determined the ability of B. pseudomallei to invade and survive intracellularly in A549 human lung epithelial cells, and also investigated the early transcriptional responses using an Illumina HumanHT-12 v4 microarray platform, after three hours of exposure to live B. pseudomallei (BCMS and its secreted proteins (CCMS.We found that the ability of B. pseudomallei to invade and survive intracellularly correlated with increase of multiplicity of infection and duration of contact. Activation of host carbohydrate metabolism and apoptosis as well as suppression of amino acid metabolism and innate immune responses both by live bacteria and its secreted proteins were evident. These early events might be linked to initial activation of host genes directed towards bacterial dissemination from lungs to target organs (via proposed in vivo mechanisms or to escape potential sensing by macrophages.Understanding the early responses of A549 cells toward B. pseudomallei infection provide preliminary insights into the likely pathogenesis mechanisms underlying melioidosis, and could contribute to development of novel intervention strategies to combat B. pseudomallei infections.

  8. Post-exposure therapeutic efficacy of COX-2 inhibition against Burkholderia pseudomallei.

    Directory of Open Access Journals (Sweden)

    Saja Asakrah

    Full Text Available Burkholderia pseudomallei is a Gram-negative, facultative intracellular bacillus and the etiologic agent of melioidosis, a severe disease in Southeast Asia and Northern Australia. Like other multidrug-resistant pathogens, the inherent antibiotic resistance of B. pseudomallei impedes treatment and highlights the need for alternative therapeutic strategies that can circumvent antimicrobial resistance mechanisms. In this work, we demonstrate that host prostaglandin E2 (PGE2 production plays a regulatory role in the pathogenesis of B. pseudomallei. PGE2 promotes B. pseudomallei intracellular survival within macrophages and bacterial virulence in a mouse model of pneumonic melioidosis. PGE2-mediated immunosuppression of macrophage bactericidal effector functions is associated with increased arginase 2 (Arg2 expression and decreased nitric oxide (NO production. Treatment with a commercially-available COX-2 inhibitor suppresses the growth of B. pseudomallei in macrophages and affords significant protection against rapidly lethal pneumonic melioidosis when administered post-exposure to B. pseudomallei-infected mice. COX-2 inhibition may represent a novel immunotherapeutic strategy to control infection with B. pseudomallei and other intracellular pathogens.

  9. Ceftibuten-containing agar plate for detecting group B streptococci with reduced penicillin susceptibility (PRGBS).

    Science.gov (United States)

    Kamiya, Chitose; Kimura, Kouji; Doyama, Yo; Miyazaki, Akira; Morimoto, Makiko; Banno, Hirotsugu; Nagano, Noriyuki; Jin, Wanchun; Wachino, Jun-ichi; Yamada, Keiko; Arakawa, Yoshichika

    2015-08-01

    Penicillins remain first-line agents for treatment of group B Streptococcus (Streptococcus agalactiae; GBS) infections; however, several reports have confirmed the existence of GBS with reduced penicillin susceptibility (PRGBS). Because no selective agar plates for detection of PRGBS are available to date, in this investigation, we developed the selective agar plate for detection of PRGBS. We used 19 genetically well-confirmed PRGBS isolates and 38 penicillin-susceptible GBS isolates identified in Japan. For preparation of trial PRGBS-selective agar plates, we added 1 of antimicrobial agents (among oxacillin, ceftizoxime, and ceftibuten) to a well-established GBS-selective agar plate. Among 12 trial PRGBS-selective agar plates, Muller-Hinton agar containing 128 μg/mL ceftibuten with 5% sheep blood, 8 μg/mL gentamicin, and 12 μg/mL nalidixic acid was the most appropriate selective agar for PRGBS, showing 100% sensitivity and 81.6% specificity. In cases of potential nosocomial spread of PRGBS, the selective agar plate could be useful and reliable. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Nitric oxide from IFNγ-primed macrophages modulates the antimicrobial activity of β-lactams against the intracellular pathogens Burkholderia pseudomallei and Nontyphoidal Salmonella.

    Directory of Open Access Journals (Sweden)

    Jessica Jones-Carson

    2014-08-01

    Full Text Available Our investigations show that nonlethal concentrations of nitric oxide (NO abrogate the antibiotic activity of β-lactam antibiotics against Burkholderia pseudomallei, Escherichia coli and nontyphoidal Salmonella enterica serovar Typhimurium. NO protects B. pseudomallei already exposed to β-lactams, suggesting that this diatomic radical tolerizes bacteria against the antimicrobial activity of this important class of antibiotics. The concentrations of NO that elicit antibiotic tolerance repress consumption of oxygen (O2, while stimulating hydrogen peroxide (H2O2 synthesis. Transposon insertions in genes encoding cytochrome c oxidase-related functions and molybdenum assimilation confer B. pseudomallei a selective advantage against the antimicrobial activity of the β-lactam antibiotic imipenem. Cumulatively, these data support a model by which NO induces antibiotic tolerance through the inhibition of the electron transport chain, rather than by potentiating antioxidant defenses as previously proposed. Accordingly, pharmacological inhibition of terminal oxidases and nitrate reductases tolerizes aerobic and anaerobic bacteria to β-lactams. The degree of NO-induced β-lactam antibiotic tolerance seems to be inversely proportional to the proton motive force (PMF, and thus the dissipation of ΔH+ and ΔΨ electrochemical gradients of the PMF prevents β-lactam-mediated killing. According to this model, NO generated by IFNγ-primed macrophages protects intracellular Salmonella against imipenem. On the other hand, sublethal concentrations of imipenem potentiate the killing of B. pseudomallei by NO generated enzymatically from IFNγ-primed macrophages. Our investigations indicate that NO modulates the antimicrobial activity of β-lactam antibiotics.

  11. Application of lipovitellin-salt-mannitol agar for screening, isolation, and presumptive identification of Staphylococcus aureus in a teaching hospital.

    OpenAIRE

    Merlino, J; Gill, R; Robertson, G J

    1996-01-01

    Lipovitellin-salt-mannitol (LSM) plate medium was examined for its ability to directly isolate, recover, and presumptively identify Staphylococcus aureus from 418 clinical specimens. The criteria for medium evaluation included colony morphology reactions, selectivity, and ease of isolation. For 298 specimens used for screening, LSM agar medium was compared with the other conventional media used, mannitol salt agar (MSA), 5% horse blood agar (HBA), and phenolphthalein phosphate agar (PPA), to ...

  12. [Evaluation of a new medium, eggplant (Solanum melongena) agar as a screening medium for Cryptococcus neoformans in environmental samples].

    Science.gov (United States)

    Sengul, Mustafa; Ergin, Cağrı; Kartal, Tuğba

    2014-04-01

    Cryptococcus neofomans is an encapsulated yeast-like fungus that causes life-threatening infections, especially in immunosuppresive patients. C.neoformans infection is believed to be acquired via inhalation of aerosolized particles from the environment. Avian guano, decaying tree hollows and soil are the related known environmental niches. Brown pigmented yeast growth from the precursors in growth media is an important step for the identification and isolation of C.neoformans. Seeds of plants in nature are preferred owing to easy accessibility and low costs for the preparation of such media. Guizotia abysinicca (Niger seed) as Staib agar, Helianthus annus (Sunflower) as Pal's medium, Brassica nigra (Mustard) agar, tobacco agar, Mucuna pruriens (Velvet bean) seed agar, Perilla frutescens (Beefsteak plant) seed agar, Rubus fruticosus (Blackberry) agar and ground red hot pepper agar are pigment-based selective media for the differentiation of C.neoformans. The aim of this study was to observe the pigment production of C.neoformans in a new medium based on eggplant (Solanum melongena) and also to compare its performance with the simplified Staib, Pal's and tobacco agar for isolation from the environment. Three different eggplant-based medium (S.melongena Melanzaza viserba, S.melongena Pinstripe F1 and S.ovigerum Ivory F1) were included in the study. Pigment-forming eggplant medium, simplified Staib agar, Pal's agar and tobacco agar were used for the cultivation of the environmental swabbed samples from 19 Eucalyptus camaldulensis trunk hollows in continuous colonization region. While pigment formation were observed with S.melongena Melanzaza viserba and S.melongena Pinstripe F1 containing media, S.ovigerum Ivory F1 medium was found to be non-reactive. In colonization area (Gökova-Akyaka, Turkey), 11 (57.9%) out of 19 E.camaldulensis samples were positive with simplified Staib agar, Pal's agar and eggplant agar while 10 (52.6%) of them are positive with tobacco agar. C

  13. In Vitro and In Vivo Studies of Monoclonal Antibodies with Prominent Bactericidal Activity against Burkholderia pseudomallei and Burkholderia mallei▿

    Science.gov (United States)

    Zhang, Shimin; Feng, Shaw-Huey; Li, Bingjie; Kim, Hyung-Yong; Rodriguez, Joe; Tsai, Shien; Lo, Shyh-Ching

    2011-01-01

    Our laboratory has developed more than a hundred mouse monoclonal antibodies (MAbs) against Burkholderia pseudomallei and Burkholderia mallei. These antibodies have been categorized into different groups based on their specificities and the biochemical natures of their target antigens. The current study first examined the bactericidal activities of a number of these MAbs by an in vitro opsonic assay. Then, the in vivo protective efficacy of selected MAbs was evaluated using BALB/c mice challenged intranasally with a lethal dose of the bacteria. The opsonic assay using dimethyl sulfoxide-treated human HL-60 cells as phagocytes revealed that 19 out of 47 tested MAbs (40%) have prominent bactericidal activities against B. pseudomallei and/or B. mallei. Interestingly, all MAbs with strong opsonic activities are those with specificity against either the capsular polysaccharides (PS) or the lipopolysaccharides (LPS) of the bacteria. On the other hand, none of the MAbs reacting to bacterial proteins or glycoproteins showed prominent bactericidal activity. Further study revealed that the antigenic epitopes on either the capsular PS or LPS molecules were readily available for binding in intact bacteria, while the epitopes on proteins/glycoproteins were less accessible to the MAbs. Our in vivo study showed that four MAbs reactive to either the capsular PS or LPS were highly effective in protecting mice against lethal bacterial challenge. The result is compatible with that of our in vitro study. The MAbs with the highest protective efficacy are those reactive to either the capsular PS or LPS of the Burkholderia bacteria. PMID:21450976

  14. In Vitro and In Vivo studies of monoclonal antibodies with prominent bactericidal activity against Burkholderia pseudomallei and Burkholderia mallei.

    Science.gov (United States)

    Zhang, Shimin; Feng, Shaw-Huey; Li, Bingjie; Kim, Hyung-Yong; Rodriguez, Joe; Tsai, Shien; Lo, Shyh-Ching

    2011-05-01

    Our laboratory has developed more than a hundred mouse monoclonal antibodies (MAbs) against Burkholderia pseudomallei and Burkholderia mallei. These antibodies have been categorized into different groups based on their specificities and the biochemical natures of their target antigens. The current study first examined the bactericidal activities of a number of these MAbs by an in vitro opsonic assay. Then, the in vivo protective efficacy of selected MAbs was evaluated using BALB/c mice challenged intranasally with a lethal dose of the bacteria. The opsonic assay using dimethyl sulfoxide-treated human HL-60 cells as phagocytes revealed that 19 out of 47 tested MAbs (40%) have prominent bactericidal activities against B. pseudomallei and/or B. mallei. Interestingly, all MAbs with strong opsonic activities are those with specificity against either the capsular polysaccharides (PS) or the lipopolysaccharides (LPS) of the bacteria. On the other hand, none of the MAbs reacting to bacterial proteins or glycoproteins showed prominent bactericidal activity. Further study revealed that the antigenic epitopes on either the capsular PS or LPS molecules were readily available for binding in intact bacteria, while the epitopes on proteins/glycoproteins were less accessible to the MAbs. Our in vivo study showed that four MAbs reactive to either the capsular PS or LPS were highly effective in protecting mice against lethal bacterial challenge. The result is compatible with that of our in vitro study. The MAbs with the highest protective efficacy are those reactive to either the capsular PS or LPS of the Burkholderia bacteria.

  15. Crystal formation in furunculosis agar

    Science.gov (United States)

    Bullock, G.L.; Ross, A.J.

    1964-01-01

    SINCE ITS INTRODUCTION SOME MONTHS AGO, FURUNCULOSIS AGAR has been employed in the diagnosis of suspect furunculosis and also as a general purpose medium. During our work with this medium we have noticed discrete "colonies," of crystalline material, which very closely resemble microbial colonies. These crystal colonies are compact and appear on both the surface and subsurface; they occur in inoculated slants and plates incubated for long periods (2 to 3 weeks), as well as in uninoculated stored medium. As the crystal colonies could be confusing to workers using this medium, we decided to attempt to identify them and also to determine whether storage conditions and different lots of medium affect crystal formation.

  16. Rheological properties of agar and carrageenan from Ghanaian red seaweeds

    DEFF Research Database (Denmark)

    Rhein-Knudsen, Nanna; Ale, Marcel Tutor; Ajalloueian, Fatemeh

    2017-01-01

    Red seaweeds contain unique galactose-rich hydrocolloids, carrageenans and agar, which find use as gelling agents in high value applications. This study examined the chemical and rheological properties of hydrocolloids from selected wild red seaweed species collected in Ghana: Hypnea musciformis...... and Cryptonemia crenulata, expected to hold carrageenan, contained 21–26% by weight of galactose. A commercial Kappaphycus alvarezii carrageenan sample had 30% galactose residues by weight. Hydropuntia dentata, expected to contain agar, contained 15% by weight of galactose-monomers. Fourier transform infrared...... spectroscopy (FTIR) analysis on the hydrocolloids extracted from H. musciformis (and K. alvarezii) indicated κ-carrageenan, C. crenulata hydrocolloids were mainly ι-carrageenan, and the H. dentata hydrocolloids were agar. Gelling temperatures ranged from 32 to 36 °C for the κ-carrageenan hydrocolloid samples...

  17. Detection and differentiation of Burkholderia pseudomallei, Burkholderia mallei and Burkholderia thailandensis by multiplex PCR.

    Science.gov (United States)

    Lee, May-Ann; Wang, Dongling; Yap, Eu Hian

    2005-03-01

    Burkholderia pseudomallei, a Gram-negative bacterium that causes melioidosis may be differentiated from closely related species of Burkholderia mallei that causes glanders and non-pathogenic species of Burkholderia thailandensis by multiplex PCR. The multiplex PCR consists of primers that flank a 10-bp repetitive element in B. pseudomallei and B. mallei amplifying PCR fragment of varying sizes between 400-700 bp, a unique sequence in B. thailandensis amplifying a PCR fragment of 308 bp and the metalloprotease gene amplifying a PCR fragment of 245 bp in B. pseudomallei and B. thailandensis. The multiplex PCR not only can differentiate the three Burkholderia species but can also be used for epidemiological typing of B. pseudomallei and B. mallei strains.

  18. DBSecSys 2.0: a database of Burkholderia mallei and Burkholderia pseudomallei secretion systems.

    Science.gov (United States)

    Memišević, Vesna; Kumar, Kamal; Zavaljevski, Nela; DeShazer, David; Wallqvist, Anders; Reifman, Jaques

    2016-09-20

    Burkholderia mallei and B. pseudomallei are the causative agents of glanders and melioidosis, respectively, diseases with high morbidity and mortality rates. B. mallei and B. pseudomallei are closely related genetically; B. mallei evolved from an ancestral strain of B. pseudomallei by genome reduction and adaptation to an obligate intracellular lifestyle. Although these two bacteria cause different diseases, they share multiple virulence factors, including bacterial secretion systems, which represent key components of bacterial pathogenicity. Despite recent progress, the secretion system proteins for B. mallei and B. pseudomallei, their pathogenic mechanisms of action, and host factors are not well characterized. We previously developed a manually curated database, DBSecSys, of bacterial secretion system proteins for B. mallei. Here, we report an expansion of the database with corresponding information about B. pseudomallei. DBSecSys 2.0 contains comprehensive literature-based and computationally derived information about B. mallei ATCC 23344 and literature-based and computationally derived information about B. pseudomallei K96243. The database contains updated information for 163 B. mallei proteins from the previous database and 61 additional B. mallei proteins, and new information for 281 B. pseudomallei proteins associated with 5 secretion systems, their 1,633 human- and murine-interacting targets, and 2,400 host-B. mallei interactions and 2,286 host-B. pseudomallei interactions. The database also includes information about 13 pathogenic mechanisms of action for B. mallei and B. pseudomallei secretion system proteins inferred from the available literature or computationally. Additionally, DBSecSys 2.0 provides details about 82 virulence attenuation experiments for 52 B. mallei secretion system proteins and 98 virulence attenuation experiments for 61 B. pseudomallei secretion system proteins. We updated the Web interface and data access layer to speed-up users

  19. Molecular Procedure for Rapid Detection of Burkholderia mallei and Burkholderia pseudomallei

    Science.gov (United States)

    Bauernfeind, Adolf; Roller, Carsten; Meyer, Detlef; Jungwirth, Renate; Schneider, Ines

    1998-01-01

    A PCR procedure for the discrimination of Burkholderia mallei and Burkholderia pseudomallei was developed. It is based on the nucleotide difference T 2143 C (T versus C at position 2143) between B. mallei and B. pseudomallei detected in the 23S rDNA sequences. In comparison with conventional methods the procedure allows more rapid identification at reduced risk for infection of laboratory personnel. PMID:9705426

  20. Autotransporters and Their Role in the Virulence of Burkholderia pseudomallei and Burkholderia mallei

    OpenAIRE

    Natalie eLazar Adler; Joanne eStevens; Mark eStevens; Edouard eGalyov

    2011-01-01

    Burkholderia pseudomallei and Burkholderia mallei are closely related Gram-negative bacteria responsible for the infectious diseases melioidosis and glanders, respectively. Autotransporters (ATs) comprise a large and diverse family of secreted and outer membrane proteins that includes virulence-associated invasins, adhesins, proteases and actin-nucleating factors. The B. pseudomallei K96243 genome contains eleven predicted ATs, eight of which share homologues in the B. mallei ATCC 23344 genom...

  1. Burkholderia pseudomallei kills the nematode Caenorhabditis elegans using an endotoxin-mediated paralysis.

    Science.gov (United States)

    O'Quinn, A L; Wiegand, E M; Jeddeloh, J A

    2001-06-01

    We investigated a non-mammalian host model system for fitness in genetic screening for virulence-attenuating mutations in the potential biowarfare agents Burkholderia pseudomallei and Burkholderia mallei. We determined that B. pseudomallei is able to cause 'disease-like' symptoms and kill the nematode Caenorhabditis elegans. Analysis of killing in the surrogate disease model with B. pseudomallei mutants indicated that killing did not require lipopolysaccharide (LPS) O-antigen, aminoglycoside/macrolide efflux pumping, type II pathway-secreted exoenzymes or motility. Burkholderia thailandensis and some strains of Burkholderia cepacia also killed nematodes. Manipulation of the nematode host genotype suggests that the neuromuscular intoxication caused by both B. pseudomallei and B. thailandensis acts in part through a disruption of normal Ca2+ signal transduction. Both species produce a UV-sensitive, gamma-irradiation-resistant, limited diffusion, paralytic agent as part of their nematode pathogenic mechanism. The results of this investigation suggest that killing by B. pseudomallei is an active process in C. elegans, and that the C. elegans model might be useful for the identification of vertebrate animal virulence factors in B. pseudomallei.

  2. Development of novel agar media for isolating guaiacol producing Alicyclobacillus spp.

    Science.gov (United States)

    Chang, S S; Park, S H; Kang, D H

    2013-06-03

    The purpose of this study is to develop a selective and differential medium (SK2 agar) for isolating guaiacol producing Alicyclobacillus. Forty-one selected dyes and vanillic acid were incorporated in SK agar for screening selective and differential agents. Two guaiacol producing (1016, 1101) and two non-guaiacol producing (19220, C-GD 1-1) Alicyclobacillus isolates were streaked onto media and color differentiation of the isolates was assessed. Among 41 tested dyes, Chrome Azurol S (CAS) allowed color differentiation of the two types of Alicyclobacillus. Colonies of guaiacol producing Alicyclobacillus isolates appeared as dark purple to royal blue color with yellow background, whereas non-guaiacol producing Alicyclobacillus isolates produced cream colored colonies with yellow background. Vanillic acid not only served as a precursor for guaiacol formation but also inhibited non-guaiacol producing Alicyclobacillus. Non-guaiacol producing isolates did not grow on SK agar containing more than 70 ppm vanillic acid, whereas the recovery of guaiacol producing isolates was unaffected. When compared with other Alicyclobacillus isolation media, not only was SK2 agar capable of selectively recovering guaiacol-producing Alicyclobacillus, the degree of growth was also approximately equal if not better than orange serum agar, potato dextrose agar, and K agar. The development of SK2 agar provides the fruit juice industry with an inexpensive, simple to use alternative for the detection of guaiacol producing Alicyclobacillus. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Tandem repeat regions within the Burkholderia pseudomallei genome and their application for high resolution genotyping

    Directory of Open Access Journals (Sweden)

    Harvey Steven P

    2007-03-01

    Full Text Available Abstract Background The facultative, intracellular bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals. We identified and categorized tandem repeat arrays and their distribution throughout the genome of B. pseudomallei strain K96243 in order to develop a genetic typing method for B. pseudomallei. We then screened 104 of the potentially polymorphic loci across a diverse panel of 31 isolates including B. pseudomallei, B. mallei and B. thailandensis in order to identify loci with varying degrees of polymorphism. A subset of these tandem repeat arrays were subsequently developed into a multiple-locus VNTR analysis to examine 66 B. pseudomallei and 21 B. mallei isolates from around the world, as well as 95 lineages from a serial transfer experiment encompassing ~18,000 generations. Results B. pseudomallei contains a preponderance of tandem repeat loci throughout its genome, many of which are duplicated elsewhere in the genome. The majority of these loci are composed of repeat motif lengths of 6 to 9 bp with 4 to 10 repeat units and are predominately located in intergenic regions of the genome. Across geographically diverse B. pseudomallei and B.mallei isolates, the 32 VNTR loci displayed between 7 and 28 alleles, with Nei's diversity values ranging from 0.47 and 0.94. Mutation rates for these loci are comparable (>10-5 per locus per generation to that of the most diverse tandemly repeated regions found in other less diverse bacteria. Conclusion The frequency, location and duplicate nature of tandemly repeated regions within the B. pseudomallei genome indicate that these tandem repeat regions may play a role in generating and maintaining adaptive genomic variation. Multiple-locus VNTR analysis revealed extensive diversity within the global isolate set containing B. pseudomallei and B. mallei, and it detected genotypic differences within clonal lineages of both species that were

  4. Survey of innate immune responses to Burkholderia pseudomallei in human blood identifies a central role for lipopolysaccharide.

    Directory of Open Access Journals (Sweden)

    Narisara Chantratita

    Full Text Available B. pseudomallei is a gram-negative bacterium that causes the tropical infection melioidosis. In northeast Thailand, mortality from melioidosis approaches 40%. As exemplified by the lipopolysaccharide-Toll-like receptor 4 interaction, innate immune responses to invading bacteria are precipitated by activation of host pathogen recognition receptors by pathogen associated molecular patterns. Human melioidosis is characterized by up-regulation of pathogen recognition receptors and pro-inflammatory cytokine release. In contrast to many gram-negative pathogens, however, the lipopolysaccharide of B. pseudomallei is considered only weakly inflammatory. We conducted a study in 300 healthy Thai subjects to investigate the ex vivo human blood response to various bacterial pathogen associated molecular patterns, including lipopolysaccharide from several bacteria, and to two heat-killed B. pseudomallei isolates. We measured cytokine levels after stimulation of fresh whole blood with a panel of stimuli. We found that age, sex, and white blood cell count modulate the innate immune response to B. pseudomallei. We further observed that, in comparison to other stimuli, the innate immune response to B. pseudomallei is most highly correlated with the response to lipopolysaccharide. The magnitude of cytokine responses induced by B. pseudomallei lipopolysaccharide was significantly greater than those induced by lipopolysaccharide from Escherichia coli and comparable to many responses induced by lipopolysaccharide from Salmonella minnesota despite lower amounts of lipid A in the B. pseudomallei lipopolysaccharide preparation. In human monocytes stimulated with B. pseudomallei, addition of polymyxin B or a TLR4/MD-2 neutralizing antibody inhibited the majority of TNF-α production. Challenging existing views, our data indicate that the innate immune response to B. pseudomallei in human blood is largely driven by lipopolysaccharide, and that the response to B

  5. Agar agar-stabilized milled zerovalent iron particles for in situ groundwater remediation

    Energy Technology Data Exchange (ETDEWEB)

    Velimirovic, Milica; Schmid, Doris; Wagner, Stephan; Micić, Vesna; Kammer, Frank von der; Hofmann, Thilo, E-mail: thilo.hofmann@univie.ac.at

    2016-09-01

    Submicron-scale milled zerovalent iron (milled ZVI) particles produced by grinding macroscopic raw materials could provide a cost-effective alternative to nanoscale zerovalent iron (nZVI) particles for in situ degradation of chlorinated aliphatic hydrocarbons in groundwater. However, the aggregation and settling of bare milled ZVI particles from suspension presents a significant obstacle to their in situ application for groundwater remediation. In our investigations we reduced the rapid aggregation and settling rate of bare milled ZVI particles from suspension by stabilization with a “green” agar agar polymer. The transport potential of stabilized milled ZVI particle suspensions in a diverse array of natural heterogeneous porous media was evaluated in a series of well-controlled laboratory column experiments. The impact of agar agar on trichloroethene (TCE) removal by milled ZVI particles was assessed in laboratory-scale batch reactors. The use of agar agar significantly enhanced the transport of milled ZVI particles in all of the investigated porous media. Reactivity tests showed that the agar agar-stabilized milled ZVI particles were reactive towards TCE, but that their reactivity was an order of magnitude less than that of bare, non-stabilized milled ZVI particles. Our results suggest that milled ZVI particles could be used as an alternative to nZVI particles as their potential for emplacement into contaminated zone, their reactivity, and expected longevity are beneficial for in situ groundwater remediation. - Highlights: • Rapid aggregation and sedimentation were observed in bare milled ZVI particles. • Agar agar improved the stability of milled ZVI particle suspensions. • Agar agar enhanced the transport of milled ZVI particles in heterogeneous sands. • Agar agar reduced the reactivity of milled ZVI particles towards TCE.

  6. Comparative in vivo and in vitro analyses of putative virulence factors of Burkholderia pseudomallei using lipopolysaccharide, capsule and flagellin mutants

    NARCIS (Netherlands)

    Wikraiphat, C.; Charoensap, J.; Utaisincharoen, P.; Wongratanacheewin, S.; Taweechaisupapong, S.; Woods, D.E.; Bolscher, J.G.M.; Sirisinha, S.

    2009-01-01

    Burkholderia pseudomallei is a gram-negative bacillus that is the causative agent of melioidosis. We evaluated host-pathogen interaction at different levels using three separate B. pseudomallei mutants generated by insertional inactivation. One of these mutants is defective in the production of the

  7. Identification of Burkholderia mallei and Burkholderia pseudomallei adhesins for human respiratory epithelial cells.

    Science.gov (United States)

    Balder, Rachel; Lipski, Serena; Lazarus, John J; Grose, William; Wooten, Ronald M; Hogan, Robert J; Woods, Donald E; Lafontaine, Eric R

    2010-09-28

    Burkholderia pseudomallei and Burkholderia mallei cause the diseases melioidosis and glanders, respectively. A well-studied aspect of pathogenesis by these closely-related bacteria is their ability to invade and multiply within eukaryotic cells. In contrast, the means by which B. pseudomallei and B. mallei adhere to cells are poorly defined. The purpose of this study was to identify adherence factors expressed by these organisms. Comparative sequence analyses identified a gene product in the published genome of B. mallei strain ATCC23344 (locus # BMAA0649) that resembles the well-characterized Yersinia enterocolitica autotransporter adhesin YadA. The gene encoding this B. mallei protein, designated boaA, was expressed in Escherichia coli and shown to significantly increase adherence to human epithelial cell lines, specifically HEp2 (laryngeal cells) and A549 (type II pneumocytes), as well as to cultures of normal human bronchial epithelium (NHBE). Consistent with these findings, disruption of the boaA gene in B. mallei ATCC23344 reduced adherence to all three cell types by ~50%. The genomes of the B. pseudomallei strains K96243 and DD503 were also found to contain boaA and inactivation of the gene in DD503 considerably decreased binding to monolayers of HEp2 and A549 cells and to NHBE cultures.A second YadA-like gene product highly similar to BoaA (65% identity) was identified in the published genomic sequence of B. pseudomallei strain K96243 (locus # BPSL1705). The gene specifying this protein, termed boaB, appears to be B. pseudomallei-specific. Quantitative attachment assays demonstrated that recombinant E. coli expressing BoaB displayed greater binding to A549 pneumocytes, HEp2 cells and NHBE cultures. Moreover, a boaB mutant of B. pseudomallei DD503 showed decreased adherence to these respiratory cells. Additionally, a B. pseudomallei strain lacking expression of both boaA and boaB was impaired in its ability to thrive inside J774A.1 murine macrophages

  8. Identification of Burkholderia mallei and Burkholderia pseudomallei adhesins for human respiratory epithelial cells

    Directory of Open Access Journals (Sweden)

    Hogan Robert J

    2010-09-01

    Full Text Available Abstract Background Burkholderia pseudomallei and Burkholderia mallei cause the diseases melioidosis and glanders, respectively. A well-studied aspect of pathogenesis by these closely-related bacteria is their ability to invade and multiply within eukaryotic cells. In contrast, the means by which B. pseudomallei and B. mallei adhere to cells are poorly defined. The purpose of this study was to identify adherence factors expressed by these organisms. Results Comparative sequence analyses identified a gene product in the published genome of B. mallei strain ATCC23344 (locus # BMAA0649 that resembles the well-characterized Yersinia enterocolitica autotransporter adhesin YadA. The gene encoding this B. mallei protein, designated boaA, was expressed in Escherichia coli and shown to significantly increase adherence to human epithelial cell lines, specifically HEp2 (laryngeal cells and A549 (type II pneumocytes, as well as to cultures of normal human bronchial epithelium (NHBE. Consistent with these findings, disruption of the boaA gene in B. mallei ATCC23344 reduced adherence to all three cell types by ~50%. The genomes of the B. pseudomallei strains K96243 and DD503 were also found to contain boaA and inactivation of the gene in DD503 considerably decreased binding to monolayers of HEp2 and A549 cells and to NHBE cultures. A second YadA-like gene product highly similar to BoaA (65% identity was identified in the published genomic sequence of B. pseudomallei strain K96243 (locus # BPSL1705. The gene specifying this protein, termed boaB, appears to be B. pseudomallei-specific. Quantitative attachment assays demonstrated that recombinant E. coli expressing BoaB displayed greater binding to A549 pneumocytes, HEp2 cells and NHBE cultures. Moreover, a boaB mutant of B. pseudomallei DD503 showed decreased adherence to these respiratory cells. Additionally, a B. pseudomallei strain lacking expression of both boaA and boaB was impaired in its ability to

  9. Protective response to subunit vaccination against intranasal Burkholderia mallei and B. pseudomallei challenge.

    Science.gov (United States)

    Whitlock, Gregory C; Deeraksa, Arpaporn; Qazi, Omar; Judy, Barbara M; Taylor, Katherine; Propst, Katie L; Duffy, Angie J; Johnson, Kate; Kitto, G Barrie; Brown, Katherine A; Dow, Steven W; Torres, Alfredo G; Estes, D Mark

    2010-01-01

    Burkholderia mallei and B. pseudomallei are Gram-negative pathogenic bacteria, responsible for the diseases glanders and melioidosis, respectively. Furthermore, there is currently no vaccine available against these Burkholderia species. In this study, we aimed to identify protective proteins against these pathogens. Immunization with recombinant B. mallei Hcp1 (type VI secreted/structural protein), BimA (autotransporter protein), BopA (type III secreted protein), and B. pseudomallei LolC (ABC transporter protein) generated significant protection against lethal inhaled B. mallei ATCC23344 and B. pseudomallei 1026b challenge. Immunization with BopA elicited the greatest protective activity, resulting in 100% and 60% survival against B. mallei and B. pseudomallei challenge, respectively. Moreover, sera from recovered mice demonstrated reactivity with the recombinant proteins. Dendritic cells stimulated with each of the different recombinant proteins showed distinct cytokine patterns. In addition, T cells from immunized mice produced IFN-γ following in vitro re-stimulation. These results indicated therefore that it was possible to elicit cross-protective immunity against both B. mallei and B. pseudomallei by vaccinating animals with one or more novel recombinant proteins identified in B. mallei.

  10. Autotransporters and Their Role in the Virulence of Burkholderia pseudomallei and Burkholderia mallei.

    Science.gov (United States)

    Lazar Adler, Natalie R; Stevens, Joanne M; Stevens, Mark P; Galyov, Edouard E

    2011-01-01

    Burkholderia pseudomallei and Burkholderia mallei are closely related Gram-negative bacteria responsible for the infectious diseases melioidosis and glanders, respectively. Autotransporters (ATs) comprise a large and diverse family of secreted and outer membrane proteins that includes virulence-associated invasins, adhesins, proteases, and actin-nucleating factors. The B. pseudomallei K96243 genome contains 11 predicted ATs, eight of which share homologs in the B. mallei ATCC 23344 genome. This review distils key findings from in silico, in vitro, and in vivo studies on the ATs of B. pseudomallei and B. mallei. To date, the best characterized of the predicted ATs of B. pseudomallei and B. mallei is BimA, a predicted trimeric AT mediating actin-based motility which varies in sequence and mode of action between Burkholderia species. Of the remaining eight predicted B. pseudomallei trimeric autotransporters, five of which are also present in B. mallei, two (BoaA and BoaB), have been implicated in bacterial adhesion to epithelial cells. Several predicted Burkholderia ATs are recognized by human humoral and cell-mediated immunity, indicating that they are expressed during infection and may be useful for diagnosis and vaccine-mediated protection. Further studies on the mode of secretion and functions of Burkholderia ATs will facilitate the rational design of control strategies.

  11. Autotransporters and their role in the virulence of Burkholderia pseudomallei and Burkholderia mallei.

    Directory of Open Access Journals (Sweden)

    Natalie eLazar Adler

    2011-07-01

    Full Text Available Burkholderia pseudomallei and Burkholderia mallei are closely related Gram-negative bacteria responsible for the infectious diseases melioidosis and glanders, respectively. Autotransporters (ATs comprise a large and diverse family of secreted and outer membrane proteins that includes virulence-associated invasins, adhesins, proteases and actin-nucleating factors. The B. pseudomallei K96243 genome contains eleven predicted ATs, eight of which share homologues in the B. mallei ATCC 23344 genome. This review distils key findings from in silico, in vitro and in vivo studies on the ATs of B. pseudomallei and B. mallei. To date, the best characterized of the predicted ATs of B. pseudomallei and B. mallei is BimA, a predicted trimeric AT mediating actin-based motility which varies in sequence and mode of action between Burkholderia species. Of the remaining eight predicted B. pseudomallei trimeric autotransporters, five of which are also present in B. mallei, two (BoaA and BoaB, have been implicated in bacterial adhesion to epithelial cells. Several predicted Burkholderia ATs are recognized by human humoral and cell-mediated immunity, indicating that they are expressed during infection and may be useful for diagnosis and vaccine-mediated protection. Further studies on the mode of secretion and functions of Burkholderia ATs will facilitate the rational design of control strategies.

  12. Agar from some Hawaiian red algae

    Energy Technology Data Exchange (ETDEWEB)

    Santos, G.A.; Doty, M.S.

    1983-08-01

    From describing the agars of Gelidiella acerosa Forskk., Gelidium pluma Loomis, G. pusillum (Stackh.) Lejolis, Gracilaria abbotiana Hoyle, G. bursapastoris (Gmelin) Silva, G. canaliculata (Kutzing) Sonder, G. coronopifolia J.Ag., G. epihippisora Hoyle, Pterocladia caerulescens (Kutzing) Santelices and P. capillacea (Gmelin) Born. and Thur. as found in Hawaiian samples of these species, it is concluded that the species of Gelidium and especially Pterocladia and Gelidiella may merit more consideration for usage due to their agar gel strengths. The nature of the gel from Gracilaria abbottiana suggests the generic status might well be reexamined. The agars from the Gelidiella and the other Gracilaria species should be studied further for their prospective values to the food industry other than gel strength. Mixtures of the agars from G. bursapastoris and G. coronopifolia would merit attention for the taste texture of their mixtures. (Refs. 18).

  13. Acetamide Agar for Differentiation of Nonfermentative Bacteria

    Science.gov (United States)

    Oberhofer, Thomas R.; Rowen, Joyce W.

    1974-01-01

    An acetamide agar medium is described for use in the differentiation of nonfermentative gram-negative bacteria. With few exceptions, indicator reactions were rapid, intense, and clear-cut. PMID:4417708

  14. Characterization of physicochemical properties of carboxymethyl agar.

    Science.gov (United States)

    Cao, Mingzhao; Liu, Xin; Luan, Jimei; Zhang, Xiaodong

    2014-10-13

    A series of carboxymethyl agars (CMAs) with different degree of substitution (DS) were prepared, and their properties were determined and analyzed. The results showed that with the increase of DS, the dissolving temperature, the gelling temperature, the gel melting temperature, the gel strength, the gel hardness, the gel fracturability, and the solution apparent viscosity of CMA all decreased, except that its gel cohesiveness and gel springiness increased. The variation process of agar molecules in solution from coil to helix could be observed by measuring the optical rotation of the solution at such a low concentration, at which even the solution could not form a gel. The gel skeleton microstructures of both agar and CMA were of porous network structure, and the pore size of CMA became smaller and denser with the increase of its DS. After carboxymethylation, the agar hygroscopicity was improved, but its thermal stability was lowered. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Revised structures for the predominant O-polysaccharides expressed by Burkholderia pseudomallei and Burkholderia mallei.

    Science.gov (United States)

    Heiss, Christian; Burtnick, Mary N; Roberts, Rosemary A; Black, Ian; Azadi, Parastoo; Brett, Paul J

    2013-11-15

    O-Polysaccharides (OPS) were isolated from purified Burkholderia pseudomallei and Burkholderia mallei lipopolysaccharides by mild-acid hydrolysis and gel-permeation chromatography. 1-D and 2-D (1)H and (13)C NMR spectroscopy experiments revealed that the OPS antigens were unbranched heteropolymers with the following structures: Collectively, our results demonstrate that the predominant OPS antigens expressed by B. pseudomallei and B. mallei isolates are structurally more complex than previously described and provide evidence that different capping residues are used by these closely related pathogens to terminate chain elongation. Additionally, they confirm that Burkholderia thailandensis and B. pseudomallei express OPS antigens that are essentially identical to one another. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Clear distinction between Burkholderia mallei and Burkholderia pseudomallei using fluorescent motB primers.

    Science.gov (United States)

    Schmoock, Gernot; Elschner, Mandy; Sprague, Lisa D

    2015-03-07

    A frame-shift mutation in the flagellum motor gene motB coding for the chemotaxis MotB protein of Burkholderia mallei has been utilized to design a conventional duplex PCR assay with fluorescent labelled primers. Species specificity was tested with a panel of 13 Burkholderia type strains. A total of 41 B. mallei field strains, 36 B. pseudomallei field strains, and 1 B. thailandensis field strain from different geographic regions were tested and correctly identified. Testing of 55 non-Burkholderia bacterial species revealed 100% specificity of the assay. The minimum detection limit was 1 pg DNA or 160 GE for B. mallei and 130 GE for B. pseudomallei, respectively. This assay enables the clear distinction between B. mallei and B. pseudomallei/B. thailandensis.

  17. Phenotypic and functional characterization of human memory T cell responses to Burkholderia pseudomallei.

    Directory of Open Access Journals (Sweden)

    Patcharaporn Tippayawat

    Full Text Available Infection with the Gram-negative bacterium Burkholderia pseudomallei is an important cause of community-acquired lethal sepsis in endemic regions in southeast Asia and northern Australia and is increasingly reported in other tropical areas. In animal models, production of interferon-gamma (IFN-gamma is critical for resistance, but in humans the characteristics of IFN-gamma production and the bacterial antigens that are recognized by the cell-mediated immune response have not been defined.Peripheral blood from 133 healthy individuals who lived in the endemic area and had no history of melioidosis, 60 patients who had recovered from melioidosis, and 31 other patient control subjects were stimulated by whole bacteria or purified bacterial proteins in vitro, and IFN-gamma responses were analyzed by ELISPOT and flow cytometry.B. pseudomallei was a potent activator of human peripheral blood NK cells for innate production of IFN-gamma. In addition, healthy individuals with serological evidence of exposure to B. pseudomallei and patients recovered from active melioidosis developed CD4(+ (and CD8(+ T cells that recognized whole bacteria and purified proteins LolC, OppA, and PotF, members of the B. pseudomallei ABC transporter family. This response was primarily mediated by terminally differentiated T cells of the effector-memory (T(EMRA phenotype and correlated with the titer of anti-B. pseudomallei antibodies in the serum.Individuals living in a melioidosis-endemic region show clear evidence of T cell priming for the ability to make IFN-gamma that correlates with their serological status. The ability to detect T cell responses to defined B. pseudomallei proteins in large numbers of individuals now provides the opportunity to screen candidate antigens for inclusion in protein or polysaccharide-conjugate subunit vaccines against this important but neglected disease.

  18. Phenotypic and functional characterization of human memory T cell responses to Burkholderia pseudomallei.

    Science.gov (United States)

    Tippayawat, Patcharaporn; Saenwongsa, Wipawee; Mahawantung, Jirawan; Suwannasaen, Duangchan; Chetchotisakd, Ploenchan; Limmathurotsakul, Direk; Peacock, Sharon J; Felgner, Philip L; Atkins, Helen S; Titball, Richard W; Bancroft, Gregory J; Lertmemongkolchai, Ganjana

    2009-01-01

    Infection with the Gram-negative bacterium Burkholderia pseudomallei is an important cause of community-acquired lethal sepsis in endemic regions in southeast Asia and northern Australia and is increasingly reported in other tropical areas. In animal models, production of interferon-gamma (IFN-gamma) is critical for resistance, but in humans the characteristics of IFN-gamma production and the bacterial antigens that are recognized by the cell-mediated immune response have not been defined. Peripheral blood from 133 healthy individuals who lived in the endemic area and had no history of melioidosis, 60 patients who had recovered from melioidosis, and 31 other patient control subjects were stimulated by whole bacteria or purified bacterial proteins in vitro, and IFN-gamma responses were analyzed by ELISPOT and flow cytometry. B. pseudomallei was a potent activator of human peripheral blood NK cells for innate production of IFN-gamma. In addition, healthy individuals with serological evidence of exposure to B. pseudomallei and patients recovered from active melioidosis developed CD4(+) (and CD8(+)) T cells that recognized whole bacteria and purified proteins LolC, OppA, and PotF, members of the B. pseudomallei ABC transporter family. This response was primarily mediated by terminally differentiated T cells of the effector-memory (T(EMRA)) phenotype and correlated with the titer of anti-B. pseudomallei antibodies in the serum. Individuals living in a melioidosis-endemic region show clear evidence of T cell priming for the ability to make IFN-gamma that correlates with their serological status. The ability to detect T cell responses to defined B. pseudomallei proteins in large numbers of individuals now provides the opportunity to screen candidate antigens for inclusion in protein or polysaccharide-conjugate subunit vaccines against this important but neglected disease.

  19. Burkholderia pseudomallei Colony Morphotypes Show a Synchronized Metabolic Pattern after Acute Infection.

    Science.gov (United States)

    Gierok, Philipp; Kohler, Christian; Steinmetz, Ivo; Lalk, Michael

    2016-03-01

    Burkholderia pseudomallei is a water and soil bacterium and the causative agent of melioidosis. A characteristic feature of this bacterium is the formation of different colony morphologies which can be isolated from environmental samples as well as from clinical samples, but can also be induced in vitro. Previous studies indicate that morphotypes can differ in a number of characteristics such as resistance to oxidative stress, cellular adhesion and intracellular replication. Yet the metabolic features of B. pseudomallei and its different morphotypes have not been examined in detail so far. Therefore, this study aimed to characterize the exometabolome of B. pseudomallei morphotypes and the impact of acute infection on their metabolic characteristics. We applied nuclear magnetic resonance spectroscopy (1H-NMR) in a metabolic footprint approach to compare nutrition uptake and metabolite secretion of starvation induced morphotypes of the B. pseudomallei strains K96243 and E8. We observed gluconate production and uptake in all morphotype cultures. Our study also revealed that among all morphotypes amino acids could be classified with regard to their fast and slow consumption. In addition to these shared metabolic features, the morphotypes varied highly in amino acid uptake profiles, secretion of branched chain amino acid metabolites and carbon utilization. After intracellular passage in vitro or murine acute infection in vivo, we observed a switch of the various morphotypes towards a single morphotype and a synchronization of nutrient uptake and metabolite secretion. To our knowledge, this study provides first insights into the basic metabolism of B. pseudomallei and its colony morphotypes. Furthermore, our data suggest, that acute infection leads to the synchronization of B. pseudomallei colony morphology and metabolism through yet unknown host signals and bacterial mechanisms.

  20. The in vitro tolerant persister population in Burkholderia pseudomallei is altered by environmental factors

    Directory of Open Access Journals (Sweden)

    William Charles Nierman

    2015-12-01

    Full Text Available Bacterial persistence due to antibiotic tolerance is a critical aspect of antibiotic treatment failure, disease latency, and chronic or reemergent infections. The levels of persisters is especially notable for the opportunistic Gram-negative pathogens from the Burkholderia and Pseudomonas genera. We examined the rate of drug tolerant persisters in Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia cepacia complex organisms, and Pseudomonas aeruginosa at mid-log growth in LB broth culture. We found that a fraction of the antibiotic-sensitive cells from every species were tolerant to a 24 hour high-dose antibiotic challenge. All tested Burkholderia strains demonstrated a drug tolerant persister population at a rate that was at least 100 – 500 times higher than P. aeruginosa. When challenged with a 10X minimum inhibitory concentration 24 hour exposure to five different antibiotics with different modes of action we found that in B. pseudomallei Bp82 the same fraction of persisters in the bacterial population was revealed when using 4 of them. This observation suggests that our assay is detecting a single homogeneous persister population. Persistence in B. pseudomallei Bp82 was highly dependent on growth stage, with a surprisingly high persister fraction of >64% of the late stationary phase cells being antibiotic tolerant. Adaptation of B. pseudomallei to distilled water storage resulted in a population of drug tolerant cells up to 100% of the non-drug-challenged viable cell count. Cultivation of B. pseudomallei with a sub-inhibitory concentration of several antibiotics resulted in altered persister fractions within the population relative to cultures lacking the antibiotic. Our study provides insight into the sensitivity of the persister fraction within the population of B. pseudomallei due to environmental variables and suggests a lack of diversity within the persister population.

  1. Antibiotic susceptibility testing (agar disk diffusion and agar dilution) of clinical isolates of Corynebacterium jeikeium.

    Science.gov (United States)

    Traub, W H; Geipel, U; Leonhard, B; Bauer, D

    1998-01-01

    Thirty-three clinical isolates of Corynebacterium jeikeium were examined for susceptibility to 27 antimicrobial drugs with the agar dilution test. Sheep-blood-supplemented Mueller-Hinton agar performed better than Wilkins-Chalgren agar. Disk susceptibility (Bauer-Kirby) tests were carried out in parallel with 24 of the chemotherapeutic agents. All isolates were susceptible to teicoplanin and vancomycin. All isolates resisted fosfomycin, mupirocin, and trimethoprim-sulfamethoxazole. The isolates varied in susceptibility to ciprofloxacin, doxycycline, fusidic acid, ofloxacin, and tetracycline; most were susceptible to rifampin. Surprisingly few discrepancies between agar dilution and disk diffusion tests were encountered when utilizing NCCLS interpretive criteria currently valid for enterococcal isolates.

  2. The Efficiency of UVC Radiation in the Inactivation of Listeria monocytogenes on Beef-Agar Food Models

    OpenAIRE

    Hamidi-Oskouei, Amir M.; James, Christian; James, Stephen

    2015-01-01

    The aim of this study is to evaluate the eff ect of meat content and surface smoothness on the deactivation of Listeria monocytogenes in beef-agar food models achieved by shortwave ultraviolet (UVC) light. Food models with various meat contents were made using chopped beef slices and agar solution. Prepared models together with a Listeria selective agar (LSA) plate and a slice of cooked beef were inoculated with L. monocytogenes and then exposed to UVC light. Population of Listeria reduced to...

  3. The Efficiency of UVC Radiation in the Inactivation of
Listeria monocytogenes on Beef-Agar Food Models

    OpenAIRE

    Christian James; Stephen James; Hamidi-Oskouei, Amir M.

    2015-01-01

    The aim of this study is to evaluate the eff ect of meat content and surface smoothness on the deactivation of Listeria monocytogenes in beef-agar food models achieved by shortwave ultraviolet (UVC) light. Food models with various meat contents were made using chopped beef slices and agar solution. Prepared models together with a Listeria selective agar (LSA) plate and a slice of cooked beef were inoculated with L. monocytogenes and then exposed to UVC light. Population of Listeria reduced to...

  4. Two new media Pinus halepensis seed agar and blackberry agar for rapid identification of Cryptococcus neoformans.

    Science.gov (United States)

    Mseddi, Fatma; Sellami, Amira; Sellami, Hayet; Cheikhrouhou, Fatma; Makni, Fattouma; Ayadi, Ali

    2011-07-01

    Cryptococcus neoformans is an encapsulated yeast-like fungus that causes life-threatening infections, particularly in immunocompromised patients. The formation of brown pigment on many media described in the literature, such as that in Niger seed (Guizotia abyssinica) agar, has been used to identify C. neoformans. The present study compares melanin production by clinical and environmental isolates of C. neoformans and other medically important yeast on two new media, Pinus halepensis seed (PHS) agar and blackberry (BlaB) agar, and the classic medium Niger seed agar. Results obtained after the culture of 46 strains of C. neoformans, for 4, 24 and 48 h at 37 °C on these three media, showed that at 24 h, 100% of strains were pigmented on BlaB agar, 91.3% on PHS agar but only 34.8% on Niger seed agar. In conclusion, PHS and BlaB agar are two interesting new media for the rapid identification of C. neoformans isolates. © 2010 Blackwell Verlag GmbH.

  5. STABILITAS ANTOSIANIN JANTUNG PISANG KEPOK (Musa paradisiaca L TERHADAP CAHAYA SEBAGAI PEWARNA AGAR-AGAR

    Directory of Open Access Journals (Sweden)

    Lydia Ninan Lestario

    2015-02-01

    jantung pisang kepok yang disinari dengan intensitas 780-2.214 lux selama 10 jam masih disukai panelis, sedangkan yang disinari dengan intensitas 10.340 sudah tidak disukai panelis. Kata kunci: Antosianin, jantung pisang, agar-agar, intensitas cahaya, laju degradasi warna

  6. Cassava starch as an alternative to agar-agar in microbiological media

    African Journals Online (AJOL)

    ... in pour plates and slants. 10% concentration produced setting usually associated with solid nutrient media within 30 min. Cassava starch powder can therefore be a potential solidifying agent in microbiological nutrient media as an alternative to agar-agar. African Journal of Biotechnology Vol. 4 (6), pp. 573-574, 2005 ...

  7. Pigments of fly agaric (Amanita muscaria).

    Science.gov (United States)

    Stintzing, Florian; Schliemann, Willibald

    2007-01-01

    The complex pigment pattern of fly agaric (Amanita muscaria) cap skins has been studied by LC-DAD and mass spectrometry. Among the betaxanthins the corresponding derivatives of serine, threonine, ethanolamine, alanine, Dopa, phenylalanine and tryptophan are reported for the first time to contribute to the pigment pattern of fly agarics. Betalamic acid, the chromophoric precursor of betaxanthins and betacyanins, muscaflavin and seco-dopas were also detected. Furthermore, the red-purple muscapurpurin and the red muscarubrin were tentatively assigned while further six betacyanin-like components could not be structurally allocated. Stability studies indicated a high susceptibility of pigment extracts to degradation which led to rapid colour loss thus rendering a complete characterization of betacyanin-like compounds impossible at present. Taking into account these difficulties the presented results may be a starting point for a comprehensive characterization of the pigment composition of fly agarics.

  8. Extraction of agar from Gelidium sesquipedale (Rhodopyta) and surface characterization of agar based films.

    Science.gov (United States)

    Guerrero, P; Etxabide, A; Leceta, I; Peñalba, M; de la Caba, K

    2014-01-01

    The chemical structure of the agar obtained from Gelidium sesquipedale (Rhodophyta) has been determined by (13)C nuclear magnetic resonance ((13)C NMR) and Fourier transform infrared spectroscopy (FTIR). Agar (AG) films with different amounts of soy protein isolate (SPI) were prepared using a thermo-moulding method, and transparent and hydrophobic films were obtained and characterized. FTIR analysis provided a detailed description of the binding groups present in the films, such as carboxylic, hydroxyl and sulfonate groups, while the surface composition was examined using X-ray photoelectron spectroscopy (XPS). The changes observed by FTIR and XPS spectra suggested interactions between functional groups of agar and SPI. This is a novel approach to the characterization of agar-based films and provides knowledge about the compatibility of agar and soy protein for further investigation of the functional properties of biodegradable films based on these biopolymers. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. BurkDiff: a real-time PCR allelic discrimination assay for Burkholderia pseudomallei and B. mallei.

    Directory of Open Access Journals (Sweden)

    Jolene R Bowers

    Full Text Available A real-time PCR assay, BurkDiff, was designed to target a unique conserved region in the B. pseudomallei and B. mallei genomes containing a SNP that differentiates the two species. Sensitivity and specificity were assessed by screening BurkDiff across 469 isolates of B. pseudomallei, 49 isolates of B. mallei, and 390 isolates of clinically relevant non-target species. Concordance of results with traditional speciation methods and no cross-reactivity to non-target species show BurkDiff is a robust, highly validated assay for the detection and differentiation of B. pseudomallei and B. mallei.

  10. BurkDiff: a real-time PCR allelic discrimination assay for Burkholderia pseudomallei and B. mallei.

    Science.gov (United States)

    Bowers, Jolene R; Engelthaler, David M; Ginther, Jennifer L; Pearson, Talima; Peacock, Sharon J; Tuanyok, Apichai; Wagner, David M; Currie, Bart J; Keim, Paul S

    2010-11-12

    A real-time PCR assay, BurkDiff, was designed to target a unique conserved region in the B. pseudomallei and B. mallei genomes containing a SNP that differentiates the two species. Sensitivity and specificity were assessed by screening BurkDiff across 469 isolates of B. pseudomallei, 49 isolates of B. mallei, and 390 isolates of clinically relevant non-target species. Concordance of results with traditional speciation methods and no cross-reactivity to non-target species show BurkDiff is a robust, highly validated assay for the detection and differentiation of B. pseudomallei and B. mallei.

  11. Genomic Diversity of Burkholderia pseudomallei Clinical Isolates: Subtractive Hybridization Reveals a Burkholderia mallei-Specific Propage in B. pseudomallei 1026b

    Science.gov (United States)

    2004-06-01

    Kitajima. 2003. Comparative analysis of the complete genome sequence of Pierce’s disease and citrus varigated chlorosis strains of Xylella...currently valid OMB control number. 1. REPORT DATE 8 MAR 2004 2. REPORT TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Genomic ...the disease melioidosis and is a category B biological threat agent. The genomic sequence of B. pseudomallei K96243 was recently determined, but

  12. Genomic Diversity of Burkholderia pseudomallei Clinical Isolates: Subtractive Hybridization Reveals a Burkholderia mallei-Specific Prophage in B. pseudomallei 1026b

    Science.gov (United States)

    2004-06-01

    J. P. Kitajima. 2003. Comparative analysis of the complete genome sequence of Pierce’s disease and citrus varigated chlorosis strains of Xylella...JOURNAL OF BACTERIOLOGY, June 2004, p. 3938–3950 Vol. 186, No. 12 0021-9193/04/$08.000 DOI: 10.1128/JB.186.12.3938–3950.2004 Genomic Diversity of... genomic sequence of B. pseudomallei K96243 was recently determined, but little is known about the overall genetic diversity of this species

  13. NAS agar is more suitable than McKay agar for primary culture of Streptococcus milleri group (SMG) fastidious bacteria, S. intermedius in particular.

    Science.gov (United States)

    Raclavsky, Vladislav; Novotny, Radko; Stary, Lubomir; Navratilova, Lucie; Zatloukal, Jaromir; Jakubec, Petr; Zapalka, Martin; Kopriva, Frantisek; Kolek, Vitezslav

    2017-01-01

    Streptococcus milleri group (SMG) is a group of three streptococcal species (S. anginosus, intermedius and constellatus) that act as opportunist pathogens, among others in cystic fibrosis. Due to their fastidious character, they are both difficult to cultivate and to differentiate from less pathogenic streptococcal species, therefore being most probably underdiagnosed. Semi-selective McKay agar and NAS agar were developed to facilitate SMG recovery from clinical samples; however, direct comparison of recovery rates has not been published yet. We tested the performance of both media on 123 patient samples and demonstrated general superiority of NAS agar for SMG recovery during primary cultivation convincingly. This observation was also confirmed by quantitative drop tests during subculture. Despite the undisputed overall superiority of NAS agar over McKay agar, a smaller fraction of strains grew better on McKay agar. Inter-strain differences were the most probable explanation. Therefore, when economic conditions are not limiting and maximum recovery rate is desirable, both plates are advised to be used in parallel for primary cultivation of clinical samples.

  14. Molecular Characterization of Genetic Loci Required for Secretion of Exoproducts in Burkholderia pseudomallei

    OpenAIRE

    DeShazer, David; Brett, Paul J.; Burtnick, Mary N; Woods, Donald E.

    1999-01-01

    Previous studies have demonstrated that Burkholderia pseudomallei secretes protease, lipase, and phospholipase C (PLC) into the extracellular milieu, but their mechanisms of secretion and roles in pathogenesis have not been elucidated. In this study, we isolated and characterized 29 transposon mutants unable to secrete protease, lipase, and PLC.

  15. Rapid DNA vaccination against Burkholderia pseudomallei flagellin by tattoo or intranasal application

    NARCIS (Netherlands)

    Lankelma, Jacqueline M.; Wagemakers, Alex; Birnie, Emma; Haak, Bastiaan W.; Trentelman, Jos J. A.; Weehuizen, Tassili A. F.; Ersöz, Jasmin; Roelofs, Joris J. T. H.; Hovius, Joppe W.; Wiersinga, W. Joost; Bins, Adriaan D.

    2017-01-01

    Melioidosis is a severe infectious disease with a high mortality that is endemic in South-East Asia and Northern Australia. The causative pathogen, Burkholderia pseudomallei, is listed as potential bioterror weapon due to its high virulence and potential for easy dissemination. Currently, there is

  16. Antimicrobial susceptibility patterns of Burkholderia pseudomallei among melioidosis cases in Kedah, Malaysia.

    Science.gov (United States)

    Hassan, Muhammad R A; Vijayalakshmi, Natesan; Pani, Subhada Prasad; Peng, Ng P; Mehenderkar, Ranjith; Voralu, Kirtanaa; Michael, Edwin

    2014-05-01

    Burkholderia pseudomallei, the causative agent of melioidosis is an important cause of morbidity and mortality particularly among diabetics. We evaluated 228 isolates of B. pseudomallei for antimicrobial sensitivity during 2005-2010 using the disc diffusion technique, of which 144 were obtained from blood culture. More than 90% of the strains were susceptible to cefoperazone, ceftazidime, chloramphenicol and imipenem. Eighty-two percent of the isolates were susceptible to tetracycline and amoxicillin/clavulanate. The susceptibilities to ciprofloxacin was 78% and to trimethoprim-sulfamethoxezole was 47%. The susceptibilities to aminoglycoside antibiotics were low (21% to gentamicin and 6% to amikacin). The susceptibilities were similar between isolates from females and males, bacteremic and abacteremic cases, diabetics and non-diabetics, pneumonia and non-pneumonia cases and between those who died and those who survived. Our findings show antibiotic susceptibility patterns are not a major factor in determining outcomes of B. pseudomallei infection. Monitoring the drug susceptibilities among B. pseudomallei isolates needs to be conducted regularly to guide empiric therapy for melioidosis, as it causes high mortality, especially among diabetic cases.

  17. Antimicrobial Susceptibility and Genetic Characterisation of Burkholderia pseudomallei Isolated from Malaysian Patients

    Directory of Open Access Journals (Sweden)

    Yalda Khosravi

    2014-01-01

    Full Text Available Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to many antibiotics. Ceftazidime (CAZ, the synthetic β-lactam, is normally used as the first-line antibiotic therapy for treatment of melioidosis. However, acquired CAZ resistance can develop in vivo during treatment with CAZ, leading to mortality if therapy is not switched to a different antibiotic(s in a timely manner. In this study, susceptibilities of 81 B. pseudomallei isolates to nine different antimicrobial agents were determined using the disk diffusion method, broth microdilution test and Etest. Highest percentage of susceptibility was demonstrated to CAZ, amoxicillin/clavulanic acid, meropenem, imipenem, and trimethoprim/sulfamethoxazole. Although these drugs demonstrated the highest percentage of susceptibility in B. pseudomallei, the overall results underline the importance of the emergence of resistance in this organism. PCR results showed that, of the 81 B. pseudomallei, six multidrug resistant (MDR isolates carried bpeB, amrB, and BPSS1119 and penA genes. Genotyping of the isolates using random amplified polymorphic DNA analysis showed six different PCR fingerprinting patterns generated from the six MDR isolates clusters (A and eight PCR fingerprinting patterns generated for the remaining 75 non-MDR isolates clusters (B.

  18. A heterodimer comprised of two bovine lactoferrin antimicrobial peptides exhibits powerful bactericidal activity against Burkholderia pseudomallei

    NARCIS (Netherlands)

    Puknun, A.; Bolscher, J.G.M.; Nazmi, K.; Veerman, E.C.I.; Tungpradabkul, S.; Wongratanacheewin, S.; Kanthawong, S.; Taweechaisupapong, S.

    2013-01-01

    Melioidosis is a severe infectious disease that is endemic in Southeast Asia and Northern Australia. Burkholderia pseudomallei, the causative agent of this disease, has developed resistance to an increasing list of antibiotics, demanding a search for novel agents. Lactoferricin and lactoferrampin

  19. Osteopontin Impairs Host Defense during Established Gram-Negative Sepsis Caused by Burkholderia pseudomallei (Melioidosis)

    NARCIS (Netherlands)

    van der Windt, G.J.W.; Wiersinga, W.J.; Wieland, C.W.; Tjia, I.C.S.I.; Day, N.P.; Peacock, S.J.; Florquin, S.; van der Poll, T.

    2010-01-01

    Background: Melioidosis, caused by infection with Burkholderia (B.) pseudomallei, is a severe illness that is endemic in Southeast Asia. Osteopontin (OPN) is a phosphorylated glycoprotein that is involved in several immune responses including induction of T-helper 1 cytokines and recruitment of

  20. Molecular identification and typing of Burkholderia pseudomallei and Burkholderia mallei: when is enough enough?

    Science.gov (United States)

    Antonov, Valery A; Tkachenko, Galina A; Altukhova, Viktoriya V; Savchenko, Sergey S; Zinchenko, Olga V; Viktorov, Dmitry V; Zamaraev, Valery S; Ilyukhin, Vladimir I; Alekseev, Vladimir V

    2008-12-01

    Burkholderia mallei and B. pseudomallei are highly pathogenic microorganisms for both humans and animals. Moreover, they are regarded as potential agents of bioterrorism. Thus, rapid and unequivocal detection and identification of these dangerous pathogens is critical. In the present study, we describe the use of an optimized protocol for the early diagnosis of experimental glanders and melioidosis and for the rapid differentiation and typing of Burkholderia strains. This experience with PCR-based identification methods indicates that single PCR targets (23S and 16S rRNA genes, 16S-23S intergenic region, fliC and type III secretion gene cluster) should be used with caution for identification of B. mallei and B. pseudomallei, and need to be used alongside molecular methods such as gene sequencing. Several molecular typing procedures have been used to identify genetically related B. pseudomallei and B. mallei isolates, including ribotyping, pulsed-field gel electrophoresis and multilocus sequence typing. However, these methods are time consuming and technically challenging for many laboratories. RAPD, variable amplicon typing scheme, Rep-PCR, BOX-PCR and multiple-locus variable-number tandem repeat analysis have been recommended by us for the rapid differentiation of B. mallei and B. pseudomallei strains.

  1. Revised structures for the predominant O-polysaccharides expressed by Burkholderia pseudomallei and Burkholderia mallei

    OpenAIRE

    Heiss, Christian; Burtnick, Mary N.; Rosemary A Roberts; Black, Ian; Azadi, Parastoo; Brett, Paul J

    2013-01-01

    O-Polysaccharides (OPS) were isolated from purified Burkholderia pseudomallei and Burkholderia mallei lipopolysaccharides by mild-acid hydrolysis and gel-permeation chromatography. 1-D and 2-D 1H and 13C NMR spectroscopy experiments revealed that the OPS antigens were unbranched heteropolymers with the following structures:

  2. Oropharyngeal aspiration of Burkholderia mallei and Burkholderia pseudomallei in BALB/c mice.

    Directory of Open Access Journals (Sweden)

    Kevin L Schully

    Full Text Available Burkholderia mallei and Burkholderia pseudomallei are potentially lethal pathogens categorized as biothreat agents due, in part, to their ability to be disseminated via aerosol. There are no protective vaccines against these pathogens and treatment options are limited and cumbersome. Since disease severity is greatest when these agents are inhaled, efforts to develop pre- or post-exposure prophylaxis focus largely on inhalation models of infection. Here, we demonstrate a non-invasive and technically simple method for affecting the inhalational challenge of BALB/c mice with B. pseudomallei and B. mallei. In this model, two investigators utilized common laboratory tools such as forceps and a micropipette to conduct and characterize an effective and reproducible inhalational challenge of BALB/c mice with B. mallei and B. pseudomallei. Challenge by oropharyngeal aspiration resulted in acute disease. Additionally, 50% endpoints for B. pseudomallei K96243 and B. mallei ATCC 23344 were nearly identical to published aerosol challenge methods. Furthermore, the pathogens disseminated to all major organs typically targeted by these agents where they proliferated. The pro-inflammatory cytokine production in the proximal and peripheral fluids demonstrated a rapid and robust immune response comparable to previously described murine and human studies. These observations demonstrate that OA is a viable alternative to aerosol exposure.

  3. Oropharyngeal aspiration of Burkholderia mallei and Burkholderia pseudomallei in BALB/c mice.

    Science.gov (United States)

    Schully, Kevin L; Bell, Matthew G; Ward, Jerrold M; Keane-Myers, Andrea M

    2014-01-01

    Burkholderia mallei and Burkholderia pseudomallei are potentially lethal pathogens categorized as biothreat agents due, in part, to their ability to be disseminated via aerosol. There are no protective vaccines against these pathogens and treatment options are limited and cumbersome. Since disease severity is greatest when these agents are inhaled, efforts to develop pre- or post-exposure prophylaxis focus largely on inhalation models of infection. Here, we demonstrate a non-invasive and technically simple method for affecting the inhalational challenge of BALB/c mice with B. pseudomallei and B. mallei. In this model, two investigators utilized common laboratory tools such as forceps and a micropipette to conduct and characterize an effective and reproducible inhalational challenge of BALB/c mice with B. mallei and B. pseudomallei. Challenge by oropharyngeal aspiration resulted in acute disease. Additionally, 50% endpoints for B. pseudomallei K96243 and B. mallei ATCC 23344 were nearly identical to published aerosol challenge methods. Furthermore, the pathogens disseminated to all major organs typically targeted by these agents where they proliferated. The pro-inflammatory cytokine production in the proximal and peripheral fluids demonstrated a rapid and robust immune response comparable to previously described murine and human studies. These observations demonstrate that OA is a viable alternative to aerosol exposure.

  4. Genotypic and phenotypic diversity in Enterococcus faecalis: is agar invasion a pathogenicity score?

    Science.gov (United States)

    Cafini, F; Gómez-Aguado, F; Corcuera, M T; Ramos, C; Bas, P; Collado, L; Gómez-Lus, M L; Prieto, J

    2015-04-01

    The main objective of the present study is to analyze different genotypic and phenotypic traits related to virulence in Enterococcus faecalis, as well as evaluated the agar invasion phenotype in a collection of isolates with different clinical origins. Seventy-nine E. faecalis isolates, with invasive and non-invasive clinical origins, have been used in this work. Presence of cytolysin activator (cylA), gelatinase (gelE), surface protein (esp), aggregation substance (asa1), endocarditis antigen (efaA), and collagen-binding protein (ace) have been analyzed by PCR. Phenotypic characterization included gelatinase activity, haemolysin production, biofilm formation and agar invasion. All the isolates tested harboured at least one of the virulence determinants. The 95.5% of isolates from haematologic samples were positive for agar invasion test, significantly higher than isolates from non-invasive diseases. A significant reduction in relative invasion area was observed in three selected agar-invasive strains after 15 serial passages. It has been observed a significant high prevalence of agar-invasion positive isolates among strains belonged to haematological samples. Agar invasiveness is reduced after adaptation of clinical isolates to laboratory conditions, showing that agar invasion phenotype can be modulate by culture conditions as other virulence factors observed in different bacterial species.

  5. Secondary metabolites from Bacillus amyloliquefaciens isolated from soil can kill Burkholderia pseudomallei.

    Science.gov (United States)

    Boottanun, Patcharaporn; Potisap, Chotima; Hurdle, Julian G; Sermswan, Rasana W

    2017-12-01

    Bacillus species are Gram-positive bacteria found in abundance in nature and their secondary metabolites were found to possess various potential activities, notably antimicrobial. In this study, Bacillus amyloliquefaciens N2-4 and N3-8 were isolated from soil and their metabolites could kill Burkholderia pseudomallei, a Gram-negative pathogenic bacterium also found in soil in its endemic areas. Moreover, the metabolites were able to kill drug resistant isolates of B. pseudomallei and also inhibit other pathogenic bacteria such as Staphylococcus aureus, Escherichia coli and Acinetobacter baumannii but not the non-pathogenic Burkholderia thailandensis, which is closely related to B. pseudomallei. Since the antimicrobial activity of N3-8 was not partially decreased or abolished when treated with proteolytic enzymes or autoclaved, but N2-4 was, these two strains should have produced different compounds. The N3-8 metabolites with antimicrobial activity consisted of both protein and non-protein compounds. The inhibition spectrum of the precipitated proteins compared to the culture supernatant indicated a possible synergistic effect of the non-protein and peptide compounds of N3-8 isolates against other pathogens. When either N2-4 or N3-8 isolates was co-cultured with B. pseudomallei the numbers of the bacteria decreased by 5 log10 within 72 h. Further purification and characterization of the metabolites is required for future use of the bacteria or their metabolites as biological controls of B. pseudomallei in the environment or for development as new drugs for problematic pathogenic bacteria.

  6. Avaliação da eficiência de três ágares seletivos no isolamento de Listeria monocytogenes Evaluation of the efficiency of three selective agars for L. monocytogenes isolation

    Directory of Open Access Journals (Sweden)

    Denize Aparecida Rodrigues

    2003-12-01

    Full Text Available A variedade de protocolos existentes para a pesquisa de Listeria sp em alimentos e outras amostras é muito grande, o que dificulta a escolha daquele que possa apresentar melhores resultados. Os protocolos recomendados nas metodologias tradicionais indicam vários caldos de pré-enriquecimento e enriquecimento seletivo, assim como meios de isolamento seletivo. O presente estudo teve por finalidade comparar a eficiência dos ágares cloreto de lítio-feniletanol-moxalactam (LPM, PALCAM (PAL e do ágar hemolítico ceftazidima-cloreto de lítio (HCLA no isolamento de L. monocytogenes a partir de diferentes tipos de amostras colhidas em uma linha de processamento de "nuggets" congelados de frango. Um total de 413 amostras foi examinado. As amostras foram primeiramente enriquecidas em caldo Half Fraser, seguido do enriquecimento em caldo BLEB. Alíquotas deste enriquecimento foram semeadas por esgotamento em placas contendo os meios LPM, PAL e HCLA. Colônias típicas foram selecionadas e submetidas à identificação bioquímica. O meio HCLA permitiu o isolamento de L. monocytogenes em 60,1% do total de amostras examinadas, enquanto PAL e LPM permitiram o isolamento em aproximadamente 47,9% das amostras. O desempenho do HCLA foi estatisticamente diferente dos demais meios (pThere is a variety of protocols for Listeria spp detection in foods and other sources. Therefore, the decision on which method to be used becomes very difficult. Conventional isolation methodologies indicate different pre-enrichment and enrichment broths as well as different isolation media. The comparison between the efficiency of PALCAM (PAL, lithium chloride-phenylethanol-moxalactam (LPM and haemolitic ceftazidime-lithium chloride (HCLA agars was carried out. A total of 413 samples from different sources, collected from a chicken nuggets processing plant, was pre-enriched in Half Fraser broth followed by enrichment in BLEB. They were streaked onto LPM, PAL and HCLA plates and

  7. The Madagascar hissing cockroach as a novel surrogate host for Burkholderia pseudomallei, B. mallei and B. thailandensis

    National Research Council Canada - National Science Library

    Fisher, Nathan A; Ribot, Wilson J; Applefeld, Willard; DeShazer, David

    2012-01-01

    .... pseudomallei and B. mallei LD50 in mammalian models of infection. Here we describe an alternative to mammalian hosts in the study of virulence and host-pathogen interactions of these Burkholderia species...

  8. Rapid identification of Burkholderia pseudomallei and Burkholderia mallei by fluorescence in situ hybridization (FISH) from culture and paraffin-embedded tissue samples.

    Science.gov (United States)

    Hagen, Ralf M; Frickmann, Hagen; Elschner, Mandy; Melzer, Falk; Neubauer, Heinrich; Gauthier, Yves P; Racz, Paul; Poppert, Sven

    2011-11-01

    We evaluated newly developed probes for rapid identification of Burkholderia (B.) pseudomallei and B. mallei and differentiation from B. thailandensis by fluorescence in situ hybridization (FISH). FISH correctly identified 100% of the tested B. pseudomallei (11), B. mallei (11), and B. thailandensis (1) strains, excluded 100% of all tested negative controls (61), and allowed demonstration of B. pseudomallei infection in a paraffin-embedded spleen tissue sample of an experimentally infected mouse. Copyright © 2011 Elsevier GmbH. All rights reserved.

  9. Activation of NADPH oxidase is essential, but not sufficient, in controlling intracellular multiplication of Burkholderia pseudomallei in primary human monocytes.

    Science.gov (United States)

    Wikraiphat, Chanthiwa; Pudla, Matsayapan; Baral, Pankaj; Kitthawee, Sangvorn; Utaisincharoen, Pongsak

    2014-06-01

    Burkholderia pseudomallei is a Gram-negative intracellular bacterium and the causative agent of melioidosis. Innate immune mechanisms against this pathogen, which might contribute to outcomes of melioidosis, are little known. We demonstrated here that B. pseudomallei could activate NADPH oxidase in primary human monocytes as judged by production of reactive oxygen species (ROS) and p40(phox) phosphorylation after infection. However, as similar to other intracellular bacteria, this bacterium was able to resist and multiply inside monocytes despite being able to activate NADPH oxidase. In the presence of NADPH oxidase inhibitor, diphenyleneiodonium or apocynin, intracellular multiplication of B. pseudomallei was significantly increased, suggesting that NADPH oxidase-mediated ROS production is essential in suppressing intracellular multiplication of B. pseudomallei. Additionally, interferon-γ (IFN-γ)-mediated intracellular killing of B. pseudomallei requires NADPH oxidase activity, even though ROS level was not detected at higher levels in IFN-γ-treated infected monocytes. Altogether, these results imply that the activation of NADPH plays an essential role in suppressing intracellular multiplication of B. pseudomallei in human monocytes, although this enzyme is not sufficient to stop intracellular multiplication. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  10. Application of lipovitellin-salt-mannitol agar for screening, isolation, and presumptive identification of Staphylococcus aureus in a teaching hospital.

    Science.gov (United States)

    Merlino, J; Gill, R; Robertson, G J

    1996-12-01

    Lipovitellin-salt-mannitol (LSM) plate medium was examined for its ability to directly isolate, recover, and presumptively identify Staphylococcus aureus from 418 clinical specimens. The criteria for medium evaluation included colony morphology reactions, selectivity, and ease of isolation. For 298 specimens used for screening, LSM agar medium was compared with the other conventional media used, mannitol salt agar (MSA), 5% horse blood agar (HBA), and phenolphthalein phosphate agar (PPA), to detect and recover S. aureus and methicillin-resistant S. aureus. The results indicated that LSM agar is more effective than MSA, HBA, or PPA for the recovery and isolation of S. aureus and methicillin-resistant S. aureus. On a replicator multipoint inoculation system, we compared the reactions on LSM agar, MSA, and DNase agar of 227 different strains of staphylococci, which included 178 different strains of S. aureus and 49 different strains of coagulase-negative staphylococci isolated from clinical specimens. By using the lipovitellin precipitation activity and mannitol fermentation characteristics, LSM agar gave a 100% correlation in presumptively identifying S. aureus. LSM agar may be an alternative plate medium for large hospital extensive screening for the detection and isolation of S. aureus.

  11. DISCRIMINATION OF Burkholderia mallei/pseudomallei FROM Burkholderia thailandensis BY SEQUENCE COMPARISON OF A FRAGMENT OF THE RIBOSOMAL PROTEIN S21 (RPSU) GENE.

    Science.gov (United States)

    Frickmann, H; Chantratita, N; Gauthier, Y P; Neubauer, H; Hagen, R M

    2012-06-13

    Discrimination of Burkholderia (B.) pseudomallei and B. mallei from environmental B. thailandensis is challenging. We describe a discrimination method based on sequence comparison of the ribosomal protein S21 (rpsU) gene.The rpsU gene was sequenced in ten B. pseudomallei, six B. mallei, one B. thailandensis reference strains, six isolates of B. pseudomallei, and 37 of B. thailandensis. Further rpsU sequences of six B. pseudomallei, three B. mallei, and one B. thailandensis were identified via NCBI GenBank. Three to four variable base-positions were identified within a 120-base-pair fragment, allowing discrimination of the B. pseudomallei/mallei-cluster from B. thailandensis, whose sequences clustered identically. All B. mallei and three B. pseudomallei sequences were identical, while 17/22 B. pseudomallei strains differed in one nucleotide (78A>C). Sequences of the rpsU fragment of 'out-stander' reference strains of B. cepacia, B. gladioli, B. plantarii, and B. vietnamensis clustered differently.Sequence comparison of the described rpsU gene fragment can be used as a supplementary diagnostic procedure for the discrimination of B. mallei/pseudomallei from B. thailandensis as well as from other species of the genus Burkholderia, keeping in mind that it does not allow for a differentiation between B. mallei and B. pseudomallei.

  12. Diversity of 16S-23S rDNA internal transcribed spacer (ITS reveals phylogenetic relationships in Burkholderia pseudomallei and its near-neighbors.

    Directory of Open Access Journals (Sweden)

    Andrew P Liguori

    Full Text Available Length polymorphisms within the 16S-23S ribosomal DNA internal transcribed spacer (ITS have been described as stable genetic markers for studying bacterial phylogenetics. In this study, we used these genetic markers to investigate phylogenetic relationships in Burkholderia pseudomallei and its near-relative species. B. pseudomallei is known as one of the most genetically recombined bacterial species. In silico analysis of multiple B. pseudomallei genomes revealed approximately four homologous rRNA operons and ITS length polymorphisms therein. We characterized ITS distribution using PCR and analyzed via a high-throughput capillary electrophoresis in 1,191 B. pseudomallei strains. Three major ITS types were identified, two of which were commonly found in most B. pseudomallei strains from the endemic areas, whereas the third one was significantly correlated with worldwide sporadic strains. Interestingly, mixtures of the two common ITS types were observed within the same strains, and at a greater incidence in Thailand than Australia suggesting that genetic recombination causes the ITS variation within species, with greater recombination frequency in Thailand. In addition, the B. mallei ITS type was common to B. pseudomallei, providing further support that B. mallei is a clone of B. pseudomallei. Other B. pseudomallei near-neighbors possessed unique and monomorphic ITS types. Our data shed light on evolutionary patterns of B. pseudomallei and its near relative species.

  13. Adhesion and Disintegration Phenomena on Fractal Agar Gel Surfaces.

    Science.gov (United States)

    Kudo, Ayano; Sato, Marika; Sawaguchi, Haruna; Hotta, Jun-Ichi; Mayama, Hiroyuki; Nonomura, Yoshimune

    2016-11-01

    In the present study, mechanical phenomena on fractal agar gel were analyzed to understand the interfacial properties of hydrophilic biosurfaces. The evaluation of adhesion strength between the fractal agar gel surfaces showed that the fractal structure inhibits the adhesion between the agar gel surfaces. In addition, when the disintegration behavior of an agar gel block was observed between fractal agar gel substrates, the rough structure prevented the sliding of an agar gel block. These findings are useful for understanding the biological significance of rough structure on the biological surfaces.

  14. Effect of gamma irradiation on Burkholderia thailandensis (Burkholderia pseudomallei surrogate) survival under combinations of pH and NaCl

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Yohan; Kim, Jae-Hun; Byun, Myung-Woo [Team for Radiation Food Science and Biotechnology, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup, Jeollabuk 580-185 (Korea, Republic of); Choi, Kyoung-Hee [Department of Oral Microbiology, College of Dentistry, Wonkwang University, Iksan, Jeollabuk 570-749 (Korea, Republic of); Lee, Ju-Woon, E-mail: sjwlee@kaeri.re.k [Team for Radiation Food Science and Biotechnology, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup, Jeollabuk 580-185 (Korea, Republic of)

    2010-04-15

    This study evaluated the effect of gamma irradiation on Burkholderia thailandensis (Burkholderia pseudomallei surrogate; potential bioterrorism agent) survival under different levels of NaCl and pH. B. thailandensis in Luria Bertani broth supplemented with NaCl (0-3%), and pH-adjusted to 4-7 was treated with gamma irradiation (0-0.5 kGy). Surviving cell counts of bacteria were then enumerated on tryptic soy agar. Data for the cell counts were also used to calculate D{sub 10} values (the dose required to reduce 1 log CFU/mL of B. thailandensis). Cell counts of B. thailandensis were decreased (P<0.05) as irradiation dose increased, and no differences (P>=0.05) in cell counts of the bacteria were observed among different levels of NaCl and pH. D{sub 10} values ranged from 0.04 to 0.07 kGy, regardless of NaCl and pH level. These results indicate that low doses of gamma irradiation should be a useful treatment in decreasing the potential bioterrorism bacteria, which may possibly infect humans through foods.

  15. Effect of gamma irradiation on Burkholderia thailandensis ( Burkholderia pseudomallei surrogate) survival under combinations of pH and NaCl

    Science.gov (United States)

    Yoon, Yohan; Kim, Jae-Hun; Byun, Myung-Woo; Choi, Kyoung-Hee; Lee, Ju-Woon

    2010-04-01

    This study evaluated the effect of gamma irradiation on Burkholderia thailandensis ( Burkholderia pseudomallei surrogate; potential bioterrorism agent) survival under different levels of NaCl and pH. B. thailandensis in Luria Bertani broth supplemented with NaCl (0-3%), and pH-adjusted to 4-7 was treated with gamma irradiation (0-0.5 kGy). Surviving cell counts of bacteria were then enumerated on tryptic soy agar. Data for the cell counts were also used to calculate D10 values (the dose required to reduce 1 log CFU/mL of B. thailandensis). Cell counts of B. thailandensis were decreased ( P<0.05) as irradiation dose increased, and no differences ( P≥0.05) in cell counts of the bacteria were observed among different levels of NaCl and pH. D10 values ranged from 0.04 to 0.07 kGy, regardless of NaCl and pH level. These results indicate that low doses of gamma irradiation should be a useful treatment in decreasing the potential bioterrorism bacteria, which may possibly infect humans through foods.

  16. Two-Phase Bactericidal Mechanism of Silver Nanoparticles against Burkholderia pseudomallei.

    Directory of Open Access Journals (Sweden)

    Pawinee Siritongsuk

    Full Text Available Silver nanoparticles (AgNPs have a strong antimicrobial activity against a variety of pathogenic bacteria. The killing mechanism of AgNPs involves direct physical membrane destruction and subsequent molecular damage from both AgNPs and released Ag+. Burkholderia pseudomallei is the causative agent of melioidosis, an endemic infectious disease primarily found in northern Australia and Southeast Asia. B. pseudomallei is intrinsically resistant to most common antibiotics. In this study, the antimicrobial activity and mechanism of AgNPs (10-20 nm against B. pseudomallei were investigated. The MIC and MBC for nine B. pseudomallei strains ranged from 32-48 μg/mL and 96-128 μg/mL, respectively. Concentrations of AgNPs less than 256 μg/mL were not toxic to human red blood cells. AgNPs exhibited a two-phase mechanism: cell death induction and ROS induction. The first phase was a rapid killing step within 5 min, causing the direct damage of the cytoplasmic membrane of the bacterial cells, as observed by a time-kill assay and fluorescence microscopy. During the period of 5-30 min, the cell surface charge was rapidly neutralized from -8.73 and -7.74 to 2.85 and 2.94 mV in two isolates of B. pseudomallei, as revealed by zeta potential measurement. Energy-dispersive X-ray (EDX spectroscopy showed the silver element deposited on the bacterial membrane, and TEM micrographs of the AgNP-treated B. pseudomallei cells showed severe membrane damage and cytosolic leakage at 1/5 MIC and cell bursting at MBC. During the killing effect the released Ag+ from AgNPs was only 3.9% from the starting AgNPs concentration as observed with ICP-OES experiment. In the second phase, the ROS induction occurred 1-4 hr after the AgNP treatment. Altogether, we provide direct kinetic evidence of the AgNPs killing mechanism, by which cell death is separable from the ROS induction and AgNPs mainly contributes in the killing action. AgNPs may be considered a potential candidate to

  17. Automatic surface inoculation of agar trays.

    Science.gov (United States)

    Wilkins, J. R.; Mills, S. M.; Boykin, E. H.

    1972-01-01

    Description of a machine and technique for the automatic inoculation of a plastic tray containing agar media with a culture, using either a conventional inoculation loop or a cotton swab. The design of the machine is simple, it is easy to use, and it relieves the operator from the manual task of streaking cultures. The described technique makes possible the visualization of the overall qualitative and, to some extent, quantitative relationships of various bacterial types in a sample tested.

  18. 21 CFR 866.4600 - Ouchterlony agar plate.

    Science.gov (United States)

    2010-04-01

    ...) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunology Laboratory Equipment and Reagents § 866.4600 Ouchterlony agar plate. (a) Identification. An ouchterlony agar plate for clinical use is a device...

  19. Workshop on treatment of and postexposure prophylaxis for Burkholderia pseudomallei and B. mallei Infection, 2010.

    Science.gov (United States)

    Lipsitz, Rebecca; Garges, Susan; Aurigemma, Rosemarie; Baccam, Prasith; Blaney, David D; Cheng, Allen C; Currie, Bart J; Dance, David; Gee, Jay E; Larsen, Joseph; Limmathurotsakul, Direk; Morrow, Meredith G; Norton, Robert; O'Mara, Elizabeth; Peacock, Sharon J; Pesik, Nicki; Rogers, L Paige; Schweizer, Herbert P; Steinmetz, Ivo; Tan, Gladys; Tan, Patrick; Wiersinga, W Joost; Wuthiekanun, Vanaporn; Smith, Theresa L

    2012-12-01

    The US Public Health Emergency Medical Countermeasures Enterprise convened subject matter experts at the 2010 HHS Burkholderia Workshop to develop consensus recommendations for postexposure prophylaxis against and treatment for Burkholderia pseudomallei and B. mallei infections, which cause melioidosis and glanders, respectively. Drugs recommended by consensus of the participants are ceftazidime or meropenem for initial intensive therapy, and trimethoprim/sulfamethoxazole or amoxicillin/clavulanic acid for eradication therapy. For postexposure prophylaxis, recommended drugs are trimethoprim/sulfamethoxazole or co-amoxiclav. To improve the timely diagnosis of melioidosis and glanders, further development and wide distribution of rapid diagnostic assays were also recommended. Standardized animal models and B. pseudomallei strains are needed for further development of therapeutic options. Training for laboratory technicians and physicians would facilitate better diagnosis and treatment options.

  20. Agar alternatives for micropropagation of African violet ( Saintpaulia ...

    African Journals Online (AJOL)

    Agar is one of the most popular solidifying agents in plant tissue culture. High price of pure grade agar and fear of over exploitation of its resources caused searching for low cost alternatives. In this study, liquid medium with cotton substratum and different combinations of starch, semolina, potato powder and agar in two ...

  1. Gari agar as culture media for mycological studies | Okorondu ...

    African Journals Online (AJOL)

    Gari agar was prepared by weighing 28 g of Gari, 14 g of agar powder and 8 g of Hibiscus rabdariffa powder to 1 L of sterile water. A conventional media, Sabouraud Dextrose Agar (SDA) was prepared as control according to manufacturer's procedure. Aliquot of appropriate dilutions of 1 g of agricultural soil was inoculated ...

  2. Evaluation of mannitol salt agar with oxacillin as a screening medium for methicillin-resistant Staphylococcus aureus.

    Science.gov (United States)

    Lally, R T; Ederer, M N; Woolfrey, B F

    1985-10-01

    We evaluated the use of mannitol salt agar with oxacillin for use as a primary screening medium for the simultaneous detection and identification of methicillin-resistant Staphylococcus aureus in clinical surveillance specimens. Oxacillin agar dilution susceptibility tests with mannitol salt agar and Mueller-Hinton agar were performed in parallel with disk-agar diffusion testing on 95 oxacillin-susceptible and 105 oxacillin-resistant S. aureus stock isolates. MICs were found to be comparable, showing distinct separation of susceptible and resistant isolates into two groups with MICs of less than or equal to 2 and greater than or equal to 32 micrograms/ml, respectively. In accord with these findings, 4 micrograms of oxacillin per ml was selected for use in the screening medium. For performance evaluation, mannitol salt agar with 4 micrograms of oxacillin per ml was compared with mannitol salt agar without oxacillin by performing parallel screening tests on 153 clinical surveillance specimens. For detection of methicillin-resistant S. aureus, mannitol salt agar with 4 micrograms of oxacillin per ml was as sensitive as mannitol salt agar without oxacillin and required significantly fewer confirmatory tests. For primary identification of methicillin-resistant S. aureus, mannitol salt agar with 4 micrograms of oxacillin per ml was 6.4% false-positive and 1.1% false-negative, with a 93.6% positive predictive value. These findings indicate that mannitol salt agar with 4 micrograms of oxacillin per ml can be used as a reliable and cost-effective screening medium for the simultaneous detection and identification of methicillin-resistant S. aureus in clinical surveillance specimens.

  3. Altered Proteome of Burkholderia pseudomallei Colony Variants Induced by Exposure to Human Lung Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Anis Rageh Al-Maleki

    Full Text Available Burkholderia pseudomallei primary diagnostic cultures demonstrate colony morphology variation associated with expression of virulence and adaptation proteins. This study aims to examine the ability of B. pseudomallei colony variants (wild type [WT] and small colony variant [SCV] to survive and replicate intracellularly in A549 cells and to identify the alterations in the protein expression of these variants, post-exposure to the A549 cells. Intracellular survival and cytotoxicity assays were performed followed by proteomics analysis using two-dimensional gel electrophoresis. B. pseudomallei SCV survive longer than the WT. During post-exposure, among 259 and 260 protein spots of SCV and WT, respectively, 19 were differentially expressed. Among SCV post-exposure up-regulated proteins, glyceraldehyde 3-phosphate dehydrogenase, fructose-bisphosphate aldolase (CbbA and betaine aldehyde dehydrogenase were associated with adhesion and virulence. Among the down-regulated proteins, enolase (Eno is implicated in adhesion and virulence. Additionally, post-exposure expression profiles of both variants were compared with pre-exposure. In WT pre- vs post-exposure, 36 proteins were differentially expressed. Of the up-regulated proteins, translocator protein, Eno, nucleoside diphosphate kinase (Ndk, ferritin Dps-family DNA binding protein and peptidyl-prolyl cis-trans isomerase B were implicated in invasion and virulence. In SCV pre- vs post-exposure, 27 proteins were differentially expressed. Among the up-regulated proteins, flagellin, Eno, CbbA, Ndk and phenylacetate-coenzyme A ligase have similarly been implicated in adhesion, invasion. Protein profiles differences post-exposure provide insights into association between morphotypic and phenotypic characteristics of colony variants, strengthening the role of B. pseudomallei morphotypes in pathogenesis of melioidosis.

  4. Roles and Interactions of Burkholderia pseudomallei BpsIR Quorum-Sensing System Determinants▿

    OpenAIRE

    Kiratisin, Pattarachai; Sanmee, Sittinee

    2008-01-01

    The Burkholderia pseudomallei quorum-sensing system (QSS), designated BpsIR, is encoded by five bpsR genes and three bpsI genes. This study investigated the roles and interactions of the QSS determinants in terms of gene regulation and protein interaction. We report two novel findings, that BpsR can function as an activator and a repressor for bpsI expression and that BpsR may form homodimers and heterodimers.

  5. The BpsIR Quorum-Sensing System of Burkholderia pseudomallei

    OpenAIRE

    Song, Yan; Xie, Chao; Ong, Yong-Mei; Gan, Yunn-Hwen; Chua, Kim-Lee

    2005-01-01

    BpsIR, a LuxIR quorum-sensing homolog, is required for optimal expression of virulence and secretion of exoproducts in Burkholderia pseudomallei. Cell density-dependent expression of bpsI and bpsR, the positive regulation of bpsIR expression by BpsR, and the synthesis of N-octanoyl-homoserine lactone (C8HSL) by BpsI are described in this report.

  6. Rapid DNA vaccination against Burkholderia pseudomallei flagellin by tattoo or intranasal application.

    Science.gov (United States)

    Lankelma, Jacqueline M; Wagemakers, Alex; Birnie, Emma; Haak, Bastiaan W; Trentelman, Jos J A; Weehuizen, Tassili A F; Ersöz, Jasmin; Roelofs, Joris J T H; Hovius, Joppe W; Wiersinga, W Joost; Bins, Adriaan D

    2017-11-17

    Melioidosis is a severe infectious disease with a high mortality that is endemic in South-East Asia and Northern Australia. The causative pathogen, Burkholderia pseudomallei, is listed as potential bioterror weapon due to its high virulence and potential for easy dissemination. Currently, there is no licensed vaccine for prevention of melioidosis. Here, we explore the use of rapid plasmid DNA vaccination against B. pseudomallei flagellin for protection against respiratory challenge. We tested three flagellin DNA vaccines with different subcellular targeting designs. C57BL/6 mice were vaccinated via skin tattoo on day 0, 3 and 6 before intranasal challenge with B. pseudomallei on day 21. Next, the most effective construct was used as single vaccination on day 0 by tattoo or intranasal formulation. Mice were sacrificed 72 hours post-challenge to assess bacterial loads, cytokine responses, inflammation and microscopic lesions. A construct encoding a cellular secretion signal resulted in the most effective protection against melioidosis via tattooing, with a 10-fold reduction in bacterial loads in lungs and distant organs compared to the empty vector. Strikingly, a single intranasal administration of the same vaccine resulted in >1000-fold lower bacterial loads and increased survival. Pro-inflammatory cytokine responses were significantly diminished and strong reductions in markers for distant organ damage were observed. A rapid vaccination scheme using flagellin DNA tattoo provides significant protection against intranasal challenge with B. pseudomallei, markedly improved by a single administration via airway mucosa. Hence intranasal vaccination with flagellin-encoding DNA may be applicable when acute mass vaccination is indicated and warrants further testing.

  7. The effect of environmental conditions on biofilm formation of Burkholderia pseudomallei clinical isolates.

    Directory of Open Access Journals (Sweden)

    Nur Siti K Ramli

    Full Text Available Burkholderia pseudomallei, a Gram-negative saprophytic bacterium, is the causative agent of the potentially fatal melioidosis disease in humans. In this study, environmental parameters including temperature, nutrient content, pH and the presence of glucose were shown to play a role in in vitro biofilm formation by 28 B. pseudomallei clinical isolates, including four isolates with large colony variants (LCVs and small colony variants (SCVs morphotypes. Enhanced biofilm formation was observed when the isolates were tested in LB medium, at 30 °C, at pH 7.2, and in the presence of as little as 2 mM glucose respectively. It was also shown that all SVCs displayed significantly greater capacity to form biofilms than the corresponding LCVs when cultured in LB at 37 °C. In addition, octanoyl-homoserine lactone (C(8-HSL, a quorum sensing molecule, was identified by mass spectrometry analysis in bacterial isolates referred to as LCV CTH, LCV VIT, SCV TOM, SCV CTH, 1 and 3, and the presence of other AHL's with higher masses; decanoyl-homoserine lactone (C(10-HSL and dodecanoyl-homoserine lactone (C(12-HSL were also found in all tested strain in this study. Last but not least, we had successfully acquired two Bacillus sp. soil isolates, termed KW and SA respectively, which possessed strong AHLs degradation activity. Biofilm formation of B. pseudomallei isolates was significantly decreased after treated with culture supernatants of KW and SA strains, demonstrating that AHLs may play a role in B. pseudomallei biofilm formation.

  8. Characterization of an autotransporter adhesin protein shared by Burkholderia mallei and Burkholderia pseudomallei.

    Science.gov (United States)

    Lafontaine, Eric R; Balder, Rachel; Michel, Frank; Hogan, Robert J

    2014-04-14

    Autotransporters form a large family of outer membrane proteins specifying diverse biological traits of Gram-negative bacteria. In this study, we report the identification and characterization of a novel autotransporter gene product of Burkholderia mallei (locus tag BMA1027 in strain ATCC 23344). Database searches identified the gene in at least seven B. mallei isolates and the encoded proteins were found to be 84% identical. Inactivation of the gene encoding the autotransporter in the genome of strain ATCC 23344 substantially reduces adherence to monolayers of HEp-2 laryngeal cells and A549 type II pneumocytes, as well as to cultures of normal human bronchial epithelium (NHBE). Consistent with these findings, expression of the autotransporter on the surface of recombinant E. coli bacteria increases adherence to these cell types by 5-7 fold. The gene specifying the autotransporter was identified in the genome of 29 B. pseudomallei isolates and disruption of the gene in strain DD503 reduced adherence to NHBE cultures by 61%. Unlike B. mallei, the mutation did not impair binding of B. pseudomallei to A549 or HEp-2 cells. Analysis of sera from mice infected via the aerosol route with B. mallei and B. pseudomallei revealed that animals inoculated with as few as 10 organisms produce antibodies against the autotransporter, therefore indicating expression in vivo. Our data demonstrate that we have identified an autotransporter protein common to the pathogenic species B. mallei and B. pseudomallei which mediates adherence to respiratory epithelial cells and is expressed in vivo during the course of aerosol infection.

  9. Gene Expression Profile of Human Cytokines in Response to B.pseudomallei Infection

    Science.gov (United States)

    2017-04-19

    responses 81 to an infection (6). Activation of leukocytes and cytokine networks are prominent 82 features of inflammation and the septic response (7...and of leukocyte Interleukin 8 mRNA 539 levels during septicemic and localized pseudomonas pseudomallei infection. 540 Infection and Immunity 60...which is chemotactic for T cells, eosnophils and basophils, plays an active role in recruiting leukocytes into inflammatory sites IFNγ Interferon

  10. In vitro antimicrobial activity of natural toxins and animal venoms tested against Burkholderia pseudomallei

    Directory of Open Access Journals (Sweden)

    Chow Vincent TK

    2006-06-01

    Full Text Available Abstract Background Burkholderia pseudomallei are the causative agent of melioidosis. Increasing resistance of the disease to antibiotics is a severe problem in treatment regime and has led to intensification of the search for new drugs. Antimicrobial peptides are the most ubiquitous in nature as part of the innate immune system and host defense mechanism. Methods Here, we investigated a group of venoms (snakes, scorpions and honey bee venoms for antimicrobial properties against two strains of Gram-negative bacteria Burkholderia pseudomallei by using disc-diffusion assay for in vitro susceptibility testing. The antibacterial activities of the venoms were compared with that of the isolated L-amino acid oxidase (LAAO and phospholipase A2 (PLA2s enzymes. MICs were determined using broth dilution method. Bacterial growth was assessed by measurement of optical density at the lowest dilutions (MIC 0.25 mg/ml. The cell viability was measured using tetrazolium salts (XTT based cytotoxic assay. Results The studied venoms showed high antimicrobial activity. The venoms of C. adamanteus, Daboia russelli russelli, A. halys, P. australis, B. candidus and P. guttata were equally as effective as Chloramphenicol and Ceftazidime (30 μg/disc. Among those tested, phospholipase A2 enzymes (crotoxin B and daboiatoxin showed the most potent antibacterial activity against Gram-negative (TES bacteria. Naturally occurring venom peptides and phospholipase A2 proved to possess highly potent antimicrobial activity against Burkholderia pseudomallei. The XTT-assay results showed that the cell survival decreased with increasing concentrations (0.05–10 mg/mL of Crotalus adamanteus venom, with no effect on the cell viability evident at 0.5 mg/mL. Conclusion This antibacterial profile of snake venoms reported herein will be useful in the search for potential antibacterial agents against drug resistant microorganisms like B. pseudomallei.

  11. In vitro antimicrobial activity of natural toxins and animal venoms tested against Burkholderia pseudomallei

    Science.gov (United States)

    Perumal Samy, R; Pachiappan, A; Gopalakrishnakone, P; Thwin, Maung M; Hian, Yap E; Chow, Vincent TK; Bow, Ho; Weng, Joseph T

    2006-01-01

    Background Burkholderia pseudomallei are the causative agent of melioidosis. Increasing resistance of the disease to antibiotics is a severe problem in treatment regime and has led to intensification of the search for new drugs. Antimicrobial peptides are the most ubiquitous in nature as part of the innate immune system and host defense mechanism. Methods Here, we investigated a group of venoms (snakes, scorpions and honey bee venoms) for antimicrobial properties against two strains of Gram-negative bacteria Burkholderia pseudomallei by using disc-diffusion assay for in vitro susceptibility testing. The antibacterial activities of the venoms were compared with that of the isolated L-amino acid oxidase (LAAO) and phospholipase A2 (PLA2s) enzymes. MICs were determined using broth dilution method. Bacterial growth was assessed by measurement of optical density at the lowest dilutions (MIC 0.25 mg/ml). The cell viability was measured using tetrazolium salts (XTT) based cytotoxic assay. Results The studied venoms showed high antimicrobial activity. The venoms of C. adamanteus, Daboia russelli russelli, A. halys, P. australis, B. candidus and P. guttata were equally as effective as Chloramphenicol and Ceftazidime (30 μg/disc). Among those tested, phospholipase A2 enzymes (crotoxin B and daboiatoxin) showed the most potent antibacterial activity against Gram-negative (TES) bacteria. Naturally occurring venom peptides and phospholipase A2 proved to possess highly potent antimicrobial activity against Burkholderia pseudomallei. The XTT-assay results showed that the cell survival decreased with increasing concentrations (0.05–10 mg/mL) of Crotalus adamanteus venom, with no effect on the cell viability evident at 0.5 mg/mL. Conclusion This antibacterial profile of snake venoms reported herein will be useful in the search for potential antibacterial agents against drug resistant microorganisms like B. pseudomallei. PMID:16784542

  12. Burkholderia pseudomallei known siderophores and hemin uptake are dispensable for lethal murine melioidosis.

    Directory of Open Access Journals (Sweden)

    Brian H Kvitko

    Full Text Available Burkholderia pseudomallei is a mostly saprophytic bacterium, but can infect humans where it causes the difficult-to-manage disease melioidosis. Even with proper diagnosis and prompt therapeutic interventions mortality rates still range from >20% in Northern Australia to over 40% in Thailand. Surprisingly little is yet known about how B. pseudomallei infects, invades and survives within its hosts, and virtually nothing is known about the contribution of critical nutrients such as iron to the bacterium's pathogenesis. It was previously assumed that B. pseudomallei used iron-acquisition systems commonly found in other bacteria, for example siderophores. However, our previous discovery of a clinical isolate carrying a large chromosomal deletion missing the entire malleobactin gene cluster encoding the bacterium's major high-affinity siderophore while still being fully virulent in a murine melioidosis model suggested that other iron-acquisition systems might make contributions to virulence. Here, we deleted the major siderophore malleobactin (mba and pyochelin (pch gene clusters in strain 1710b and revealed a residual siderophore activity which was unrelated to other known Burkholderia siderophores such as cepabactin and cepaciachelin, and not due to increased secretion of chelators such as citrate. Deletion of the two hemin uptake loci, hmu and hem, showed that Hmu is required for utilization of hemin and hemoglobin and that Hem cannot complement a Hmu deficiency. Prolonged incubation of a hmu hem mutant in hemoglobin-containing minimal medium yielded variants able to utilize hemoglobin and hemin suggesting alternate pathways for utilization of these two host iron sources. Lactoferrin utilization was dependent on malleobactin, but not pyochelin synthesis and/or uptake. A mba pch hmu hem quadruple mutant could use ferritin as an iron source and upon intranasal infection was lethal in an acute murine melioidosis model. These data suggest that B

  13. Nasal Acai Polysaccharides Potentiate Innate Immunity to Protect against Pulmonary Francisella tularensis and Burkholderia pseudomallei Infections

    OpenAIRE

    Skyberg, Jerod A.; Rollins, MaryClare F.; Holderness, Jeff S.; Marlenee, Nicole L.; Schepetkin, Igor A.; Goodyear, Andrew; Dow, Steven W.; Jutila, Mark A.; Pascual, David W.

    2012-01-01

    Pulmonary Francisella tularensis and Burkholderia pseudomallei infections are highly lethal in untreated patients, and current antibiotic regimens are not always effective. Activating the innate immune system provides an alternative means of treating infection and can also complement antibiotic therapies. Several natural agonists were screened for their ability to enhance host resistance to infection, and polysaccharides derived from the Acai berry (Acai PS) were found to have potent abilitie...

  14. Molecular phylogeny of Burkholderia pseudomallei from a remote region of Papua New Guinea.

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    Anthony Baker

    Full Text Available BACKGROUND: The island of New Guinea is located midway between the world's two major melioidosis endemic regions of Australia and Southeast Asia. Previous studies in Papua New Guinea have demonstrated autochthonous melioidosis in Balimo, Western province. In contrast to other regions of endemicity, isolates recovered from both environmental and clinical sources demonstrate narrow genetic diversity over large spatial and temporal scales. METHODOLOGY/PRINCIPAL FINDINGS: We employed molecular typing techniques to determine the phylogenetic relationships of these isolates to each other and to others worldwide to aid in understanding the origins of the Papua New Guinean isolates. Multi-locus sequence typing of the 39 isolates resolved three unique sequence types. Phylogenetic reconstruction and Structure analysis determined that all isolates were genetically closer to those from Australia than those from Southeast Asia. Gene cluster analysis however, identified a Yersinia-like fimbrial gene cluster predominantly found among Burkholderia pseudomallei derived from Southeast Asia. Higher resolution VNTR typing and phylogenetic reconstruction of the Balimo isolates resolved 24 genotypes with long branch lengths. These findings are congruent with long term persistence in the region and a high level of environmental stability. CONCLUSIONS/SIGNIFICANCE: Given that anthropogenic influence has been hypothesized as a mechanism for the dispersal of B. pseudomallei, these findings correlate with limited movement of the indigenous people in the region. The palaeogeographical and anthropogenic history of Australasia and the results from this study indicate that New Guinea is an important region for the further study of B. pseudomallei origins and dissemination.

  15. Genomic islands as a marker to differentiate between clinical and environmental Burkholderia pseudomallei.

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    Thanatchaporn Bartpho

    Full Text Available Burkholderia pseudomallei, as a saprophytic bacterium that can cause a severe sepsis disease named melioidosis, has preserved several extra genes in its genome for survival. The sequenced genome of the organism showed high diversity contributed mainly from genomic islands (GIs. Comparative genome hybridization (CGH of 3 clinical and 2 environmental isolates, using whole genome microarrays based on B. pseudomallei K96243 genes, revealed a difference in the presence of genomic islands between clinical and environmental isolates. The largest GI, GI8, of B. pseudomallei was observed as a 2 sub-GI named GIs8.1 and 8.2 with distinguishable %GC content and unequal presence in the genome. GIs8.1, 8.2 and 15 were found to be more common in clinical isolates. A new GI, GI16c, was detected on chromosome 2. Presences of GIs8.1, 8.2, 15 and 16c were evaluated in 70 environmental and 64 clinical isolates using PCR assays. A combination of GIs8.1 and 16c (positivity of either GI was detected in 70% of clinical isolates and 11.4% of environmental isolates (P0.05. Some virulence genes located in the absent GIs and the difference of GIs seems to contribute less to bacterial virulence. The PCR detection of 2 GIs could be used as a cost effective and rapid tool to detect potentially virulent isolates that were contaminated in soil.

  16. Production of a Recombinant Vaccine Candidate against Burkholderia pseudomallei Exploiting the Bacterial N-Glycosylation Machinery

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    Fatima eGarcia-Quintanilla

    2014-07-01

    Full Text Available Vaccines developing immune responses towards surface carbohydrates conjugated to proteins are effective in preventing infection and death by bacterial pathogens. Traditional production of these vaccines utilizes complex synthetic chemistry to acquire and conjugate the glycan to a protein. However, glycoproteins produced by bacterial protein glycosylation systems are significantly easier to produce, and could possible be used as vaccine candidates. In this work we functionally expressed the B. pseudomallei O polysaccharide (OPS II, the C. jejuni oligosaccharyltransferase (OTase, and a suitable glycoprotein (AcrA in a designer E. coli strain with a higher efficiency for production of glycoconjugates. We were able to produce and purify the OPS II-AcrA glycoconjugate, and MS analysis confirmed correct glycan was produced and attached. We observed the attachment of the O-acetylated deoxyhexose directly to the acceptor protein, which expands the range of substrates utilized by the OTase PglB. Injection of the glycoprotein into mice generated an IgG immune response against B. pseudomallei, and this response was partially protective against an intranasal challenge. Our experiments show that bacterial engineered glycoconjugates can be utilized as vaccine candidates against B. pseudomallei. Additionally, our new E. coli strain SDB1 is more efficient in glycoprotein production, and could have additional applications in the future.

  17. Polysaccharide specific monoclonal antibodies provide passive protection against intranasal challenge with Burkholderia pseudomallei.

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    David P AuCoin

    Full Text Available Burkholderia pseudomallei is a Gram-negative bacillus that is the causative agent of melioidosis. The bacterium is inherently resistant to many antibiotics and mortality rates remain high in endemic areas. The lipopolysaccharide (LPS and capsular polysaccharide (CPS are two surface-associated antigens that contribute to pathogenesis. We previously developed two monoclonal antibodies (mAbs specific to the CPS and LPS; the CPS mAb was shown to identify antigen in serum and urine from melioidosis patients. The goal of this study was to determine if passive immunization with CPS and LPS mAbs alone and in combination would protect mice from a lethal challenge with B. pseudomallei. Intranasal (i.n. challenge experiments were performed with B. pseudomallei strains 1026b and K96423. Both mAbs provided significant protection when administered alone. A combination of mAbs was protective when low doses were administered. In addition, combination therapy provided a significant reduction in spleen colony forming units (cfu compared to results when either the CPS or LPS mAbs were administered alone.

  18. Burkholderia pseudomallei and Burkholderia mallei vaccines: Are we close to clinical trials?

    Science.gov (United States)

    Titball, Richard W; Burtnick, Mary N; Bancroft, Gregory J; Brett, Paul

    2017-10-20

    B. pseudomallei is the cause of melioidosis, a serious an often fatal disease of humans and animals. The closely related bacterium B. mallei, which cases glanders, is considered to be a clonal derivative of B. pseudomallei. Both B. pseudomallei and B. mallei were evaluated by the United States and the former USSR as potential bioweapons. Much of the effort to devise biodefence vaccines in the past decade has been directed towards the identification and formulation of sub-unit vaccines which could protect against both melioidosis and glanders. A wide range of proteins and polysaccharides have been identified which protective immunity in mice. In this review we highlight the significant progress that has been made in developing glycoconjugates as sub-unit vaccines. We also consider some of the important the criteria for licensing, including the suitability of the "animal rule" for assessing vaccine efficacy, the protection required from a vaccine and the how correlates of protection will be identified. Vaccines developed for biodefence purposes could also be used in regions of the world where naturally occurring disease is endemic. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Multiplex qPCR for reliable detection and differentiation of Burkholderia mallei and Burkholderia pseudomallei

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    Janse Ingmar

    2013-02-01

    Full Text Available Abstract Background Burkholderia mallei and B. pseudomallei are two closely related species of highly virulent bacteria that can be difficult to detect. Pathogenic Burkholderia are endemic in many regions worldwide and cases of infection, sometimes brought by travelers from unsuspected regions, also occur elsewhere. Rapid, sensitive methods for identification of B. mallei and B. pseudomallei are urgently needed in the interests of patient treatment and epidemiological surveillance. Methods Signature sequences for sensitive, specific detection of pathogenic Burkholderia based on published genomes were identified and a qPCR assay was designed and validated. Results A single-reaction quadruplex qPCR assay for the detection of pathogenic Burkholderia, which includes a marker for internal control of DNA extraction and amplification, was developed. The assay permits differentiation of B. mallei and B. pseudomallei strains, and probit analysis showed a very low detection limit. Use of a multicopy signature sequence permits detection of less than 1 genome equivalent per reaction. Conclusions The new assay permits rapid detection of pathogenic Burkholderia and combines enhanced sensitivity, species differentiation, and inclusion of an internal control for both DNA extraction and PCR amplification.

  20. Multiplex qPCR for reliable detection and differentiation of Burkholderia mallei and Burkholderia pseudomallei.

    Science.gov (United States)

    Janse, Ingmar; Hamidjaja, Raditijo A; Hendriks, Amber C A; van Rotterdam, Bart J

    2013-02-14

    Burkholderia mallei and B. pseudomallei are two closely related species of highly virulent bacteria that can be difficult to detect. Pathogenic Burkholderia are endemic in many regions worldwide and cases of infection, sometimes brought by travelers from unsuspected regions, also occur elsewhere. Rapid, sensitive methods for identification of B. mallei and B. pseudomallei are urgently needed in the interests of patient treatment and epidemiological surveillance. Signature sequences for sensitive, specific detection of pathogenic Burkholderia based on published genomes were identified and a qPCR assay was designed and validated. A single-reaction quadruplex qPCR assay for the detection of pathogenic Burkholderia, which includes a marker for internal control of DNA extraction and amplification, was developed. The assay permits differentiation of B. mallei and B. pseudomallei strains, and probit analysis showed a very low detection limit. Use of a multicopy signature sequence permits detection of less than 1 genome equivalent per reaction. The new assay permits rapid detection of pathogenic Burkholderia and combines enhanced sensitivity, species differentiation, and inclusion of an internal control for both DNA extraction and PCR amplification.

  1. ATP-binding cassette systems in Burkholderia pseudomallei and Burkholderia mallei

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    Titball Richard W

    2007-03-01

    Full Text Available Abstract Background ATP binding cassette (ABC systems are responsible for the import and export of a wide variety of molecules across cell membranes and comprise one of largest protein superfamilies found in prokarya, eukarya and archea. ABC systems play important roles in bacterial lifestyle, virulence and survival. In this study, an inventory of the ABC systems of Burkholderia pseudomallei strain K96243 and Burkholderia mallei strain ATCC 23344 has been compiled using bioinformatic techniques. Results The ABC systems in the genomes of B. pseudomallei and B. mallei have been reannotated and subsequently compared. Differences in the number and types of encoded ABC systems in belonging to these organisms have been identified. For example, ABC systems involved in iron acquisition appear to be correlated with differences in genome size and lifestyles between these two closely related organisms. Conclusion The availability of complete inventories of the ABC systems in B. pseudomallei and B. mallei has enabled a more detailed comparison of the encoded proteins in this family. This has resulted in the identification of ABC systems which may play key roles in the different lifestyles and pathogenic properties of these two bacteria. This information has the potential to be exploited for improved clinical identification of these organisms as well as in the development of new vaccines and therapeutics targeted against the diseases caused by these organisms.

  2. Membrane-active mechanism of LFchimera against Burkholderia pseudomallei and Burkholderia thailandensis.

    Science.gov (United States)

    Kanthawong, Sakawrat; Puknun, Aekkalak; Bolscher, Jan G M; Nazmi, Kamran; van Marle, Jan; de Soet, Johannes J; Veerman, Enno C I; Wongratanacheewin, Surasakdi; Taweechaisupapong, Suwimol

    2014-10-01

    LFchimera, a construct combining two antimicrobial domains of bovine lactoferrin, lactoferrampin265-284 and lactoferricin17-30, possesses strong bactericidal activity. As yet, no experimental evidence was presented to evaluate the mechanisms of LFchimera against Burkholderia isolates. In this study we analyzed the killing activity of LFchimera on the category B pathogen Burkholderia pseudomallei in comparison to the lesser virulent Burkholderia thailandensis often used as a model for the highly virulent B. pseudomallei. Killing kinetics showed that B. thailandensis E264 was more susceptible for LFchimera than B. pseudomallei 1026b. Interestingly the bactericidal activity of LFchimera appeared highly pH dependent; B. thailandensis killing was completely abolished at and below pH 6.4. FITC-labeled LFchimera caused a rapid accumulation within 15 min in the cytoplasm of both bacterial species. Moreover, freeze-fracture electron microscopy demonstrated extreme effects on the membrane morphology of both bacterial species within 1 h of incubation, accompanied by altered membrane permeability monitored as leakage of nucleotides. These data indicate that the mechanism of action of LFchimera is similar for both species and encompasses disruption of the plasma membrane and subsequently leakage of intracellular nucleotides leading to cell dead.

  3. Characteristic features and dye degrading capability of agar-agar gel immobilized manganese peroxidase.

    Science.gov (United States)

    Bilal, Muhammad; Asgher, Muhammad; Shahid, Muhammad; Bhatti, Haq Nawaz

    2016-05-01

    Immobilization of enzymes has been regarded as an efficient approach to develop biocatalyst with improved activity and stability characteristics under reaction conditions. In the present study, purified manganese peroxidase (MnP) from Ganoderma lucidum IBL-05 was immobilized in agar-agar support using entrapment technique. Maximum immobilization yield was accomplished at 4.0% agar-agar gel. The immobilized MnP exhibited better resistance to changes in pH and temperature than the free enzyme, with optimal conditions being pH 6.0 and 50 °C. The kinetic parameters Km and Kcat/Km for free and entrapped MnP were calculated to be 65.6 mM and 6.99 M(-1) s(-1), and 82 mM and 8.15 M(-1) s(-1), respectively. Thermo-stability was significantly improved after immobilization. After 120 h, the insolubilized MnP retained its activity up to 71.9% and 60.3% at 30 °C and 40 °C, respectively. It showed activity until 10th cycle and retained 74.3% residual activity after 3th cycle. The effects of H2O2, ionic strength and potential inhibitors on activity of free and immobilized enzyme were investigated. Moreover, the decolorization of three structurally different dyes was monitored in order to assess the degrading capability of the entrapped MnP. The decolorization efficiencies for all the tested dyes were 78.6-84.7% after 12h. The studies concluded that the toxicity of dyes aqueous solutions was significantly reduced after treatment. The remarkable catalytic, thermo-stability and re-cycling features of the agar-agar immobilized MnP display a high potential for biotechnological applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Differential MicroRNA Analyses of Burkholderia pseudomallei- and Francisella tularensis-Exposed hPBMCs Reveal Potential Biomarkers

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    Regina Z. Cer

    2017-01-01

    Full Text Available Increasing evidence that microRNAs (miRNAs play important roles in the immune response against infectious agents suggests that miRNA might be exploitable as signatures of exposure to specific infectious agents. In order to identify potential early miRNA biomarkers of bacterial infections, human peripheral blood mononuclear cells (hPBMCs were exposed to two select agents, Burkholderia pseudomallei K96243 and Francisella tularensis SHU S4, as well as to the nonpathogenic control Escherichia coli DH5α. RNA samples were harvested at three early time points, 30, 60, and 120 minutes postexposure, then sequenced. RNAseq analyses identified 87 miRNAs to be differentially expressed (DE in a linear fashion. Of these, 31 miRNAs were tested using the miScript miRNA qPCR assay. Through RNAseq identification and qPCR validation, we identified differentially expressed miRNA species that may be involved in the early response to bacterial infections. Based upon its upregulation at early time points postexposure in two different individuals, hsa-mir-30c-5p is a miRNA species that could be studied further as a potential biomarker for exposure to these gram-negative intracellular pathogens. Gene ontology functional analyses demonstrated that programmed cell death is the first ranking biological process associated with miRNAs that are upregulated in F. tularensis-exposed hPBMCs.

  5. Population-Sequencing as a Biomarker of Burkholderia mallei and Burkholderia pseudomallei Evolution through Microbial Forensic Analysis

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    John P. Jakupciak

    2013-01-01

    Full Text Available Large-scale genomics projects are identifying biomarkers to detect human disease. B. pseudomallei and B. mallei are two closely related select agents that cause melioidosis and glanders. Accurate characterization of metagenomic samples is dependent on accurate measurements of genetic variation between isolates with resolution down to strain level. Often single biomarker sensitivity is augmented by use of multiple or panels of biomarkers. In parallel with single biomarker validation, advances in DNA sequencing enable analysis of entire genomes in a single run: population-sequencing. Potentially, direct sequencing could be used to analyze an entire genome to serve as the biomarker for genome identification. However, genome variation and population diversity complicate use of direct sequencing, as well as differences caused by sample preparation protocols including sequencing artifacts and mistakes. As part of a Department of Homeland Security program in bacterial forensics, we examined how to implement whole genome sequencing (WGS analysis as a judicially defensible forensic method for attributing microbial sample relatedness; and also to determine the strengths and limitations of whole genome sequence analysis in a forensics context. Herein, we demonstrate use of sequencing to provide genetic characterization of populations: direct sequencing of populations.

  6. Evaluation of Molecular Methods To Improve the Detection of Burkholderia pseudomallei in Soil and Water Samples from Laos.

    Science.gov (United States)

    Knappik, Michael; Dance, David A B; Rattanavong, Sayaphet; Pierret, Alain; Ribolzi, Olivier; Davong, Viengmon; Silisouk, Joy; Vongsouvath, Manivanh; Newton, Paul N; Dittrich, Sabine

    2015-06-01

    Burkholderia pseudomallei is the cause of melioidosis, a severe and potentially fatal disease of humans and animals. It is endemic in northern Australia and Southeast Asia and is found in soil and surface water. The environmental distribution of B. pseudomallei worldwide and within countries where it is endemic, such as the Lao People's Democratic Republic (Laos), remains unclear. However, this knowledge is important to our understanding of the ecology and epidemiology of B. pseudomallei and to facilitate public health interventions. Sensitive and specific methods to detect B. pseudomallei in environmental samples are therefore needed. The aim of this study was to compare molecular and culture-based methods for the detection of B. pseudomallei in soil and surface water in order to identify the optimal approach for future environmental studies in Laos. Molecular detection by quantitative real-time PCR (qPCR) was attempted after DNA extraction directly from soil or water samples or after an overnight enrichment step. The positivity rates obtained by qPCR were compared to those obtained by different culture techniques. The rate of detection from soil samples by qPCR following culture enrichment was significantly higher (84/100) than that by individual culture methods and all culture methods combined (44/100; P Lao environmental samples and is recommended as the preferred method for future surveys. Copyright © 2015, Knappik et al.

  7. Biological treatment of textile dyes by agar-agar immobilized consortium in a packed bed reactor.

    Science.gov (United States)

    Patel, Yogesh; Gupte, Akshaya

    2015-03-01

    The decolorization of Acid Maroon V was investigated using bacterial consortium EDPA containing Enterobacter dissolvens AGYP1 and Pseudomonas aeruginosa AGYP2 immobilized in different entrapment matrices. The consortium displayed 96% removal of dye (100 mg/l) within 6 h when immobilized in agar-agar. Under optimum concentrations of agar-agar (3.0% w/v) and cell biomass (0.9 g% w/v), the consortium displayed decolorization for 18 successive batches of Acid Maroon V and also decolorized 14 other different textile dyes. A packed bed reactor under batch mode showed 89% decolorization of dye after 56 repetitive cycles. Under continuous flow mode, maximum color removal was achieved with bed length of 36 cm, hydraulic retention time of 2.66 h, and dye concentration of 100 mg/l. Additionally, the reactor decolorized relatively higher concentrations (100-2000 mg/l) of dye. The synthetic dye wastewater containing five textile dyes was decolorized 92% with 62% COD reduction using an immobilized consortium.

  8. Formulation of Hydrocolloid-Agar, Sucrose, and Acidulant on Jam Leather Product Development

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    Wahyu Ramadhan

    2017-04-01

    Full Text Available Tallying agar powder as a texturizer in guava single sheet jam instigate the product more convenience to consumed. The aims of this research were to determine the best concentration of sucrose, citric acid and agar powder to form a good quality guava jam slice. The research method are optimization and formulationof sucrose, citric acid and agar-agar on making guava jam single sheets product. Physochemical and sensory tests were performed to reveal the best formulation of guava jam slice and the Bayes method used to determine the optimization of the selected formula. Based on the results of formulation and analysis, itwas obtained that  the guava jam slice with Acidulant concentration (0.02%, 0.04%, 0.06%, sucrose (70%, 80%, 90%, 100% and agar powder (0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2% had pH 3.63-3.90, sugar content 34.68 g/100 g – 35.76 g/100 g, color intensity L*, a*, b* with ΔE* value was 37,88-53,97, fiber content 1.01%-1.59%, and water activity 0.852-0.893. Rheology properties for texture profile (hardness, cohesiveness, springiness, adhesive force, and gumminess also showed significant value with agar powder formulation. Based on the Bayes test and hedonic test, it was found that the best formula was for guava jam slices with the addition of 90% sucrose, citric acid 0.04% and agar powder 0.9%. From the best formula, it was found the shelf life prediction model of Arrhenius formula was ln k = 20.222-6660.6(1/T and the nutrition facts contribute total energy 45 kcal, fat 0%, carbohydrate 9%, protein 2% and dietary fiber 3%.

  9. Cleaning plaster surfaces with agar-agar gels: evaluation of the technique

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    Sonia Tortajada Hernando

    2013-07-01

    Full Text Available Abstract: Cleaning plaster surfaces represent a challenge for conservators It should only be performed following fully tested methods that guarantee the conservation of such fragile material. The goal of this work is to establishing a suitable cleaning method for this type of artworks from the tested concentrations and time of applications, using agar gels on plaster supports. Morphological, porosity and weight variations have been studied. Confocal and stereomicroscopy have been used as analytical techniques, as well as the measurement of water vapor permeability and weight have been taken on the samples. La limpieza de superficies de yeso-escayola con geles de agar-agar: evaluación de la técnica Resumen: La limpieza segura y eficiente de las superficies de yeso constituye un reto y una responsabilidad para el conservador-restaurador, y debe llevarse a cabo siguiendo métodos testados que garanticen su correcta conservación. La intención de este trabajo es determinar, a partir de las concentraciones y tiempos de aplicación ensayados, cuáles serían los parámetros óptimos para la ejecución de una limpieza eficaz e inocua empleando geles de agar-agar sobre soportes de yeso. Se han comprobado las posibles variaciones morfológicas de la superficie, las variaciones de la porosidad y del peso, así como la presencia de residuos, para lo cual se ha empleado la microscopía confocal, microscopía binocular, la medida de la permeabilidad al vapor de agua y la medida del peso de las muestras.

  10. Improving agar electrospinnability with choline-based deep eutectic solvents.

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    Sousa, Ana M M; Souza, Hiléia K S; Uknalis, Joseph; Liu, Shih-Chuan; Gonçalves, Maria P; Liu, LinShu

    2015-09-01

    Very recently our group has produced novel agar-based fibers by an electrospinning technique using water as solvent and polyvinyl alcohol (PVA) as co-blending polymer. Here, we tested the deep eutectic solvent (DES), (2-hydroxyethyl)trimethylammonium chloride/urea prepared at 1:2 molar ratio, as an alternative solvent medium for agar electrospinning. The electrospun materials were collected with an ethanol bath adapted to a previous electrospinning set-up. One weight percent agar-in-DES showed improved viscoelasticity and hence, spinnability, when compared to 1 wt% agar-in-water and pure agar nanofibers were successfully electrospun if working above the temperature of sol-gel transition (∼80 °C). By changing the solvent medium we decreased the PVA concentration (5 wt% starting solution) and successfully produced composite fibers with high agar contents (50/50 agar/PVA). Best composite fibers were formed with the 50/50 and 30/70 agar/PVA solutions. These fibers were mechanically resistant, showed tailorable surface roughness and diverse size distributions, with most of the diameters falling in the sub-micron range. Both nano and micro forms of agar fibers (used separately or combined) may have potential for the design of new and highly functional agar-based materials. Published by Elsevier B.V.

  11. Liver abscess caused by Burkholderia pseudomallei in a young man: A case report and review of literature

    Science.gov (United States)

    Pal, Partha; Ray, Sayantan; Moulick, Avijit; Dey, Subhasis; Jana, Anirban; Banerjee, Kokila

    2014-01-01

    Pyogenic liver abscess is a common entity in Indian subcontinent and is mostly caused by gram negative bacteria. Melioidosis is not commonly seen in India and only a few cases are reported. It can give rise to multiple abscesses at different sites including liver. We report a case of isolated liver abscess caused by Burkholderia pseudomallei (B. pseudomallei) in a 29-year-old recently diagnosed diabetic, immunocompetent male. Diagnosis was made by imaging and culture of pus aspirated from the abscess and he was treated with percutaneous pigtail catheter drainage followed by antibiotics (meropenem and trimethoprim-sulphmethoxazole). Melioidosis is an emerging infection in India and has high mortality rate, so early diagnosis and prompt management is warranted which requires clinical vigilance and an intensive microbiological workup. Clinicians should be aware of isolated liver abscess caused by B. pseudomallei in appropriate clinical settings. PMID:25325075

  12. On some agarics occurring in carr forests

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    Anna Bujakiewicz

    2013-12-01

    Full Text Available Remarks on ecology of several small agarics growing in carr forests of Querco-Ulmetum minoris and Astrantio-Fraxinetum type are presented and discussed. They all occupy special microhabitat (bare ground, belong to certain functional group (saprotrophs and seem to be connected with carr forest habitat. The base rich or neutral soil humus, generally not favoured by fungi, together with extreme moisture and fast decomposition of litter causes that some humicolous macrofungi in carr forests form mostly small and short-living fruit bodies of hygrophyte character. They represent several genera: Conocybe, Coprinus, Cystolepiota, Entoloma, Flammulaster, Lepiota, Melanophyllum and Pholiotina of the order Agaricales. New localities are given, ecological amplitude is analised and preliminary mycosynusial consideration is presented.

  13. Cell aggregation on agar as an indicator for cell-matrix adhesion: effects of opioids.

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    Debruyne, Delphine; Mareel, Marc; Vanhoecke, Barbara; Bracke, Marc

    2009-09-01

    The slow aggregation assay is generally used to study the functionality of cell-cell adhesion complexes. Single cells are seeded on a semisolid agar substrate in a 96-well plate and the cells spontaneously aggregate. We used HEK FLAG-MOP cells that stably overexpress the mu opioid receptor and the mu-opioid-receptor-selective agonists DAMGO and morphine to study whether other factors than functionality of cell-cell adhesions complexes can contribute to changes in the pattern of slow aggregation on agar. HEK FLAG-MOP cells formed small compact aggregates. In the presence of DAMGO and morphine, larger and fewer aggregates were formed in comparison to the vehicle control. These aggregates were localized in the center of the agar surface, whereas in the vehicle control they were dispersed over the substrate. However, in suspension culture on a Gyrotory shaker, no stimulation of aggregation was observed by DAMGO and morphine, showing that opioids do not affect affinity. A dissociation experiment revealed that HEK FLAG-MOP aggregates formed in the absence or presence of opioids are resistant to de-adhesion. We demonstrated that the larger aggregates are neither the result of cell growth stimulation by DAMGO and morphine. Since manipulations of the substrate such as increasing the agar concentration or mixing agar with agarose induced the same changes in the pattern of slow aggregation as treatment with opioids, we suggest that cell-substrate adhesion may be involved in opioid-stimulated aggregation.

  14. McKay agar enables routine quantification of the 'Streptococcus milleri' group in cystic fibrosis patients.

    Science.gov (United States)

    Sibley, Christopher D; Grinwis, Margot E; Field, Tyler R; Parkins, Michael D; Norgaard, Jens C; Gregson, Daniel B; Rabin, Harvey R; Surette, Michael G

    2010-05-01

    The 'Streptococcus milleri' group (SMG) has recently been recognized as a contributor to bronchopulmonary disease in cystic fibrosis (CF). Routine detection and quantification is limited by current CF microbiology protocols. McKay agar was developed previously for the semi-selective isolation of this group. Here, McKay agar was validated against a panel of clinical SMG isolates, which revealed improved SMG recovery compared with Columbia blood agar. The effectiveness of this medium was evaluated by appending it to the standard CF sputum microbiology protocols in a clinical laboratory for a 6-month period. All unique colony types were isolated and identified by 16S rRNA gene sequencing. Whilst a wide variety of organisms were isolated, members of the SMG were the most prevalent bacteria cultured, and McKay agar allowed routine quantification of the SMG from 10(3) to >10(8) c.f.u. ml(-1) directly from sputum. All members of the SMG were detected [Streptococcus anginosus (40.7 %), Streptococcus intermedius (34.3 %) and Streptococcus constellatus (25 %)] with an overall prevalence rate of 40.6 % in our adult CF population. Without exception, samples where SMG isolates were cultured at 10(7) c.f.u. ml(-1) or greater were associated with pulmonary exacerbations. This study demonstrates that McKay agar can be used routinely to quantify the SMG from complex clinical samples.

  15. Long-term biological hydrogen production by agar immobilized Rhodobacter capsulatus in a sequential batch photobioreactor.

    Science.gov (United States)

    Elkahlout, Kamal; Alipour, Siamak; Eroglu, Inci; Gunduz, Ufuk; Yucel, Meral

    2017-04-01

    In this study, agar immobilization technique was employed for biological hydrogen production using Rhodobacter capsulatus DSM 1710 (wild type) and YO3 (hup-mutant) strains in sequential batch process. Different agar and glutamate concentrations were tested with defined nutrient medium. Agar concentration 4% (w/v) and 4 mM glutamate were selected for bacterial immobilization in terms of rate and longevity of hydrogen production. Acetate concentration was increased from 40 to 60-100 and 60 mM gave best results with both bacterial strains immobilized in 4% (w/v) agar. Cell concentration was increased from 2.5 to 5 mg dcw mL -1 agar and it was found that increasing cell concentration of wild-type strain caused decrease in yield and productivity while these parameters improved by increasing cell concentration of mutant strain. Also, the hydrogen production time has extended from 17 days up to 60 days according to the process conditions and parameters. Hydrogen production by immobilized photosynthetic bacteria is a convenient technology for hydrogen production as it enables to produce hydrogen with high organic acid concentrations comparing to suspended cultures. Besides, immobilization increases the stability of the system and allowed sequential batch operation for long-term application.

  16. Comparative evaluation of chromogenic agar CM1046 and mFC agar for detection of E. coli and thermotolerant coliform bacteria from water samples.

    Science.gov (United States)

    Wohlsen, T D

    2011-08-01

    The equivalence of Oxoid (CM 1046) Brilliance((TM)) E. coli/coliform selective agar to mFC agar, as used in the Australian/New Zealand Standard Method to detect thermotolerant coliforms and Escherichia coli in water samples, was assessed. A total of 244 water samples were analysed in parallel over a 5-month period. Sewage effluent samples (n = 131, sites = 43), freshwater (n = 62, sites = 18) and marine/brackish water samples (n = 51, sites = 23) were analysed. The Wilcoxon matched-pairs signed-ranks test showed a varying degree of statistical difference between the two methods. All matrices had a higher recovery in the trial method. Enterococci faecalis, Aeromonas spp. and Vibrio spp. did not grow on the CM1046 agar, and Pseudomonas aeruginosa and Enterobacter aerogenes were inhibited. The use of CM 1046 for the detection and enumeration of E. coli and thermotolerant coliforms in water samples is a suitable alternative to the AS/NZS Standard Method. The use of CM1046 agar was less labour intensive and time consuming, as no secondary confirmation steps were required. Confirmed results could be reported within 24 h of sample analysis, as compared to 48 h with the reference method. Public health concerns can be addressed in a more efficient manner. © 2011 Unitywater. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

  17. Burkholderia pseudomallei Rapidly Infects the Brain Stem and Spinal Cord via the Trigeminal Nerve after Intranasal Inoculation.

    Science.gov (United States)

    St John, James A; Walkden, Heidi; Nazareth, Lynn; Beagley, Kenneth W; Ulett, Glen C; Batzloff, Michael R; Beacham, Ifor R; Ekberg, Jenny A K

    2016-09-01

    Infection with Burkholderia pseudomallei causes melioidosis, a disease with a high mortality rate (20% in Australia and 40% in Southeast Asia). Neurological melioidosis is particularly prevalent in northern Australian patients and involves brain stem infection, which can progress to the spinal cord; however, the route by which the bacteria invade the central nervous system (CNS) is unknown. We have previously demonstrated that B. pseudomallei can infect the olfactory and trigeminal nerves within the nasal cavity following intranasal inoculation. As the trigeminal nerve projects into the brain stem, we investigated whether the bacteria could continue along this nerve to penetrate the CNS. After intranasal inoculation of mice, B. pseudomallei caused low-level localized infection within the nasal cavity epithelium, prior to invasion of the trigeminal nerve in small numbers. B. pseudomallei rapidly invaded the trigeminal nerve and crossed the astrocytic barrier to enter the brain stem within 24 h and then rapidly progressed over 2,000 μm into the spinal cord. To rule out that the bacteria used a hematogenous route, we used a capsule-deficient mutant of B. pseudomallei that does not survive in the blood and found that it also entered the CNS via the trigeminal nerve. This suggests that the primary route of entry is via the nerves that innervate the nasal cavity. We found that actin-mediated motility could facilitate initial infection of the olfactory epithelium. Thus, we have demonstrated that B. pseudomallei can rapidly infect the brain and spinal cord via the trigeminal nerve branches that innervate the nasal cavity. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  18. The Burkholderia pseudomallei type III secretion system and BopA are required for evasion of LC3-associated phagocytosis.

    Directory of Open Access Journals (Sweden)

    Lan Gong

    Full Text Available Burkholderia pseudomallei is the causative agent of melioidosis, a fatal infectious disease endemic in tropical regions worldwide, and especially prevalent in southeast Asia and northern Australia. This intracellular pathogen can escape from phagosomes into the host cytoplasm, where it replicates and infects adjacent cells. We previously demonstrated that, in response to B. pseudomallei infection of macrophage cell line RAW 264.7, a subset of bacteria co-localized with the autophagy marker protein, microtubule-associated protein light chain 3 (LC3, implicating autophagy in host cell defence against infection. Recent reports have suggested that LC3 can be recruited to both phagosomes and autophagosomes, thereby raising questions regarding the identity of the LC3-positive compartments in which invading bacteria reside and the mechanism of the autophagic response to B. pseudomallei infection. Electron microscopy analysis of infected cells demonstrated that the invading bacteria were either free in the cytosol, or sequestered in single-membrane phagosomes rather than double-membrane autophagosomes, suggesting that LC3 is recruited to B. pseudomallei-containing phagosomes. Partial or complete loss of function of type III secretion system cluster 3 (TTSS3 in mutants lacking the BopA (effector or BipD (translocator proteins respectively, resulted in delayed or no escape from phagosomes. Consistent with these observations, bopA and bipD mutants both showed a higher level of co-localization with LC3 and the lysosomal marker LAMP1, and impaired survival in RAW264.7 cells, suggesting enhanced killing in phagolysosomes. We conclude that LC3 recruitment to phagosomes stimulates killing of B. pseudomallei trapped in phagosomes. Furthermore, BopA plays an important role in efficient escape of B. pseudomallei from phagosomes.

  19. Comparison of Six Chromogenic Agar Media for the Isolation of a Broad Variety of Non-O157 Shigatoxin-Producing Escherichia coli (STEC) Serogroups.

    Science.gov (United States)

    Verhaegen, Bavo; De Reu, Koen; Heyndrickx, Marc; De Zutter, Lieven

    2015-06-17

    The isolation of non-O157 STEC from food samples has proved to be challenging. The selection of a suitable selective isolation agar remains problematic. The purpose of this study was to qualitatively and quantitatively evaluate six chromogenic agar media for the isolation of STEC: Tryptone Bile X-glucuronide agar (TBX), Rainbow® Agar O157 (RB), Rapid E. coli O157:H7 (RE), Modified MacConkey Agar (mMac), CHROMagarTM STEC (Chr ST) and chromIDTM EHEC (Chr ID). During this study, 45 E. coli strains were used, including 39 STEC strains belonging to 16 different O serogroups and 6 non-STEC E. coli. All E. coli strains were able to grow on TBX and RB, whereas one STEC strain was unable to grow on Chr ID and a number of other STEC strains did not grow on mMac, CHROMagar STEC and Rapid E. coli O157:H7. However, only the latter three agars were selective enough to completely inhibit the growth of the non-STEC E. coli. Our conclusion was that paired use of a more selective agar such as CHROMagar STEC together with a less selective agar like TBX or Chr ID might be the best solution for isolating non-O157 STEC from food.

  20. Chocolate agar, a differential medium for gram-positive cocci.

    OpenAIRE

    Gunn, B A

    1984-01-01

    Reactions incurred on chocolate agar by gram-positive cocci were correlated with species identity. Darkening and clearing of the medium was usually associated with the species Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus simulans, and Streptococcus faecalis. Yellowing of chocolate agar was associated with alpha-hemolytic species of Streptococcus. The study demonstrated that reactions occurring on chocolate agar are useful in identifying gram-positive cocci.

  1. Acanthamoeba on Sabouraud's agar from a patient with keratitis

    OpenAIRE

    Baradkar, Vasant; Samal, Badhuli; Swapna A Mali; Kulkarni, Ketaki; Shastri, Jayanthi

    2011-01-01

    A 25-year-old transgender patient came with complaints of watery discharge, red eye and photophobia in the left eye since 2 days. The patient had a history of wearing colored contact lenses since 4 years and cleaning the lens with tap water. Culture of lenses on Mac Conkey and blood agar yielded Klebsiella pneumoniae and Pseudomonas aeruginosa. Sabouroud's agar showed yeast cells and double-walled cysts of Acanthamoeba species. On further incubation of Sabouroud's agar, the cysts transformed ...

  2. Improved Agar Diffusion Method for Detecting Residual Antimicrobial Agents

    National Research Council Canada - National Science Library

    Tsai C-E; Kondo F

    2001-01-01

    The improved agar diffusion method for determination of residual antimicrobial agents was investigated, and the sensitivities of various combinations of test organisms and assay media were determined...

  3. Acanthamoeba on Sabouraud's agar from a patient with keratitis.

    Science.gov (United States)

    Baradkar, Vasant; Samal, Badhuli; Mali, Swapna A; Kulkarni, Ketaki; Shastri, Jayanthi

    2011-07-01

    A 25-year-old transgender patient came with complaints of watery discharge, red eye and photophobia in the left eye since 2 days. The patient had a history of wearing colored contact lenses since 4 years and cleaning the lens with tap water. Culture of lenses on Mac Conkey and blood agar yielded Klebsiella pneumoniae and Pseudomonas aeruginosa. Sabouroud's agar showed yeast cells and double-walled cysts of Acanthamoeba species. On further incubation of Sabouroud's agar, the cysts transformed to trophozoites. Parallel results were obtained on tap water agar. The previous therapy of moxifloxacin was changed to local Neosporin application.

  4. A case report of Tubo-ovarian abscess caused by Burkholderia pseudomallei.

    Science.gov (United States)

    Nernsai, Pattaranit; Sophonsritsuk, Areepan; Lertvikool, Srithean; Jinawath, Artit; Chitasombat, Maria Nina

    2018-02-08

    Melioidosis, the disease caused by Burkholderia pseudomallei is endemic in the Northeastern part of Thailand, South-East Asia, and Northern Australia. The pelvic involvement of disease is rare even in an endemic area. Therefore, we describe in this report the clinical presentation, management, and outcome of the patient with primary tubo-ovarian abscess due to melioidosis. A 31-year-old Thai cassava farmer woman presented with fever and abdominal pain at left lower quadrant for one month. She also had pain, swelling, and redness of the genitalia without any ulcer. She had odorless whitish vaginal discharge. The pelvic examination revealed excitation pain on the left side of her cervix. Transvaginal ultrasonography revealed a large left tubo-ovarian abscess size 9.4 × 4.8 cm located at anterior of the uterus. Urgent exploratory laparotomy revealed left hydrosalpinx with a large amount of pus. The pus culture grew Burkholderia pseudomallei. The computer tomography of the abdomen revealed multiple hepatosplenic abscesses. The patient underwent left salpingo-oophorectomy and pus drainage. The pathological examination of excised left adnexa revealed chronic and acute suppurative inflammation with necrotic tissue. She was given intravenous ceftazidime for one month, and her clinical symptom improved. She was diagnosed with type 2 diabetes mellitus at this visit and treated with insulin injection. She continued to take oral co-trimoxazole for 20 weeks. The final diagnosis was disseminated melioidosis with left tubo-ovarian abscess and hepatosplenic abscesses in a newly diagnosed morbidly obese diabetic patient. Burkholderia pseudomallei should be considered as the causative organism of gynecologic infection among patient with risk factor resided in an endemic area who do not respond to standard antibiotics. The pus culture from the site of infection is the only diagnostic method of pelvic melioidosis, appropriate antibiotics, and adequate surgical drainage were the

  5. Identification of Burkholderia pseudomallei Near-Neighbor Species in the Northern Territory of Australia

    Science.gov (United States)

    Ginther, Jennifer L.; Mayo, Mark; Warrington, Stephanie D.; Kaestli, Mirjam; Mullins, Travis; Wagner, David M.; Currie, Bart J.; Tuanyok, Apichai; Keim, Paul

    2015-01-01

    Identification and characterization of near-neighbor species are critical to the development of robust molecular diagnostic tools for biothreat agents. One such agent, Burkholderia pseudomallei, a soil bacterium and the causative agent of melioidosis, is lacking in this area because of its genomic diversity and widespread geographic distribution. The Burkholderia genus contains over 60 species and occupies a large range of environments including soil, plants, rhizospheres, water, animals and humans. The identification of novel species in new locations necessitates the need to identify the true global distribution of Burkholderia species, especially the members that are closely related to B. pseudomallei. In our current study, we used the Burkholderia-specific recA sequencing assay to analyze environmental samples from the Darwin region in the Northern Territory of Australia where melioidosis is endemic. Burkholderia recA PCR negative samples were further characterized using 16s rRNA sequencing for species identification. Phylogenetic analysis demonstrated that over 70% of the bacterial isolates were identified as B. ubonensis indicating that this species is common in the soil where B. pseudomallei is endemic. Bayesian phylogenetic analysis reveals many novel branches within the B. cepacia complex, one novel B. oklahomensis-like species, and one novel branch containing one isolate that is distinct from all other samples on the phylogenetic tree. During the analysis with recA sequencing, we discovered 2 single nucleotide polymorphisms in the reverse priming region of B. oklahomensis. A degenerate primer was developed and is proposed for future use. We conclude that the recA sequencing technique is an effective tool to classify Burkholderia and identify soil organisms in a melioidosis endemic area. PMID:26121041

  6. Growing Burkholderia pseudomallei in biofilm stimulating conditions significantly induces antimicrobial resistance.

    Directory of Open Access Journals (Sweden)

    Chakrit Sawasdidoln

    Full Text Available BACKGROUND: Burkholderia pseudomallei, a gram-negative bacterium that causes melioidosis, was reported to produce biofilm. As the disease causes high relapse rate when compared to other bacterial infections, it therefore might be due to the reactivation of the biofilm forming bacteria which also provided resistance to antimicrobial agents. However, the mechanism on how biofilm can provide tolerance to antimicrobials is still unclear. METHODOLOGY/PRINCIPAL FINDINGS: The change in resistance of B. pseudomallei to doxycycline, ceftazidime, imipenem, and trimethoprim/sulfamethoxazole during biofilm formation were measured as minimum biofilm elimination concentration (MBEC in 50 soil and clinical isolates and also in capsule, flagellin, LPS and biofilm mutants. Almost all planktonic isolates were susceptible to all agents studied. In contrast, when they were grown in the condition that induced biofilm formation, they were markedly resistant to all antimicrobial agents even though the amount of biofilm production was not the same. The capsule and O-side chains of LPS mutants had no effect on biofilm formation whereas the flagellin-defective mutant markedly reduced in biofilm production. No alteration of LPS profiles was observed when susceptible form was changed to resistance. The higher amount of N-acyl homoserine lactones (AHLs was detected in the high biofilm-producing isolates. Interestingly, the biofilm mutant which produced a very low amount of biofilm and was sensitive to antimicrobial agents significantly resisted those agents when grown in biofilm inducing condition. CONCLUSIONS/SIGNIFICANCE: The possible drug resistance mechanism of biofilm mutants and other isolates is not by having biofilm but rather from some factors that up-regulated when biofilm formation genes were stimulated. The understanding of genes related to this situation may lead us to prevent B. pseudomallei biofilms leading to the relapse of melioidosis.

  7. Raman spectroscopic detection and identification of Burkholderia mallei and Burkholderia pseudomallei in feedstuff.

    Science.gov (United States)

    Stöckel, Stephan; Meisel, Susann; Elschner, Mandy; Melzer, Falk; Rösch, Petra; Popp, Jürgen

    2015-01-01

    Burkholderia mallei (the etiologic agent of glanders in equines and rarely humans) and Burkholderia pseudomallei, causing melioidosis in humans and animals, are designated category B biothreat agents. The intrinsically high resistance of both agents to many antibiotics, their potential use as bioweapons, and their low infectious dose, necessitate the need for rapid and accurate detection methods. Current methods to identify these organisms may require up to 1 week, as they rely on phenotypic characteristics and an extensive set of biochemical reactions. In this study, Raman microspectroscopy, a cultivation-independent typing technique for single bacterial cells with the potential for being a rapid point-of-care analysis system, is evaluated to identify and differentiate B. mallei and B. pseudomallei within hours. Here, not only broth-cultured microbes but also bacteria isolated out of pelleted animal feedstuff were taken into account. A database of Raman spectra allowed a calculation of classification functions, which were trained to differentiate Raman spectra of not only both pathogens but also of five further Burkholderia spp. and four species of the closely related genus Pseudomonas. The developed two-stage classification system comprising two support vector machine (SVM) classifiers was then challenged by a test set of 11 samples to simulate the case of a real-world-scenario, when "unknown samples" are to be identified. In the end, all test set samples were identified correctly, even if the contained bacterial strains were not incorporated in the database before or were isolated out of animal feedstuff. Specifically, the five test samples bearing B. mallei and B. pseudomallei were correctly identified on species level with accuracies between 93.9 and 98.7%. The sample analysis itself requires no biomass enrichment step prior to the analysis and can be performed under biosafety level 1 (BSL 1) conditions after inactivating the bacteria with formaldehyde.

  8. Identification of Burkholderia pseudomallei Near-Neighbor Species in the Northern Territory of Australia.

    Directory of Open Access Journals (Sweden)

    Jennifer L Ginther

    Full Text Available Identification and characterization of near-neighbor species are critical to the development of robust molecular diagnostic tools for biothreat agents. One such agent, Burkholderia pseudomallei, a soil bacterium and the causative agent of melioidosis, is lacking in this area because of its genomic diversity and widespread geographic distribution. The Burkholderia genus contains over 60 species and occupies a large range of environments including soil, plants, rhizospheres, water, animals and humans. The identification of novel species in new locations necessitates the need to identify the true global distribution of Burkholderia species, especially the members that are closely related to B. pseudomallei. In our current study, we used the Burkholderia-specific recA sequencing assay to analyze environmental samples from the Darwin region in the Northern Territory of Australia where melioidosis is endemic. Burkholderia recA PCR negative samples were further characterized using 16s rRNA sequencing for species identification. Phylogenetic analysis demonstrated that over 70% of the bacterial isolates were identified as B. ubonensis indicating that this species is common in the soil where B. pseudomallei is endemic. Bayesian phylogenetic analysis reveals many novel branches within the B. cepacia complex, one novel B. oklahomensis-like species, and one novel branch containing one isolate that is distinct from all other samples on the phylogenetic tree. During the analysis with recA sequencing, we discovered 2 single nucleotide polymorphisms in the reverse priming region of B. oklahomensis. A degenerate primer was developed and is proposed for future use. We conclude that the recA sequencing technique is an effective tool to classify Burkholderia and identify soil organisms in a melioidosis endemic area.

  9. Estudio comparativo del agar Iso-Sensitest y el agar Mueller-Hinton

    Directory of Open Access Journals (Sweden)

    Margaret Ordoñez Smith de Danies

    1993-12-01

    Full Text Available Se estudiaron 710 cepas bacterianas provenientes de diferentes muestras para comparar las zonas de inhibición de los diámetros obtenidos en agar Iso-Sensitest y el agar Mueller Hinton. Se realizaron bajo la técnica de difusión en disco de la NCCLS (Comité Nacional para los Estándares de Laboratorio Clínico. Estadísticamente, al analizar el chi-cuadrado de las muestras estudiadas, se observó una confiabilidad del 99%, por lo tanto no hay diferencia entre los dos medios de cultivo. En el agar Iso-Sensitest se obtuvo una mejor nitidez con los diámetros de los halos de inhibición, se encontró un 62,0% de mejor lectura o mayor visibilidad en los diámetros de las zonas de inhibición con los cultivos de Enterococcus sp., un 21,4% con el Staphylococcus aureus y 20,0% en el caso de la Providencia stuartii.

  10. Structural analysis of capsular polysaccharides expressed by Burkholderia mallei and Burkholderia pseudomallei.

    Science.gov (United States)

    Heiss, Christian; Burtnick, Mary N; Wang, Zhirui; Azadi, Parastoo; Brett, Paul J

    2012-02-15

    Capsular polysaccharides (CPSs) were isolated from O-polysaccharide deficient strains of Burkholderia mallei and Burkholderia pseudomallei using a modified hot phenol/water extraction procedure. Glycosyl composition, methylation, MALDI-TOF MS analyses as well as (1)H NMR spectroscopy including COSY, TOCSY, NOESY, HMBC and HSQC experiments identified the presence of two distinct CPS antigens in the samples exhibiting the following structures: This study confirms the ability of B. mallei to express a 6-deoxy-heptan CPS and represents the first report of a mannan CPS being expressed by these bacterial pathogens. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. Identification and cloning of four riboswitches from Burkholderia pseudomallei strain K96243

    Science.gov (United States)

    Munyati-Othman, Noor; Fatah, Ahmad Luqman Abdul; Piji, Mohd Al Akmarul Fizree Bin Md; Ramlan, Effirul Ikhwan; Raih, Mohd Firdaus

    2015-09-01

    Structured RNAs referred as riboswitches have been predicted to be present in the genome sequence of Burkholderia pseudomallei strain K96243. Four of the riboswitches were identified and analyzed through BLASTN, Rfam search and multiple sequence alignment. The RNA aptamers belong to the following riboswitch classifications: glycine riboswitch, cobalamin riboswitch, S-adenosyl-(L)-homocysteine (SAH) riboswitch and flavin mononucleotide (FMN) riboswitch. The conserved nucleotides for each aptamer were identified and were marked on the secondary structure generated by RNAfold. These riboswitches were successfully amplified and cloned for further study.

  12. EFFICIENCY OF THE ENRICHMENT BROTHES (LEB1 AND LEB2 AND SELECTIVE AGARS (LPM AND LSAB-CAN TO ISOLATE BACTERIA OF THE GENUS Listeria IN MEAT AND RESIDUAL WATER OF WASHING CARCASS EFICIÊNCIA DOS CALDOS DE ENRIQUECIMENTO (LEB1 E LEB2 E DOS ÁGARES SELETIVOS (LPM E LSAB-CAN NO ISOLAMENTO DE BACTÉRIAS DO GÊNERO Listeria EM CARNE BOVINA E ÁGUA RESIDUÁRIA DE LAVAGEM DE CARCAÇA

    Directory of Open Access Journals (Sweden)

    Albenones José de Mesquita

    2007-09-01

    Full Text Available

    In this paper it was observed the efficiency of enrichment brothes for Listeria (LEB1 and LEB2, associated to the passage of the culture through a 0.25% potassium hidroxide solution, verifying that the secondary enrichment broth (LEB2 and LEB2KOH was better than the primary enrichment broth (LEB1KOH for this purpose. On the other hand, lithium chloride-phenylethanol-moxalactam agar and the selective Listeria agar base, supplemented with cicloheximide, acriflavine and nalidixic acid (LSAIB-CAN showed equivalency, at the statistic point of view (p=0.l442, in relation to the number of positive samples for Listeria spp., although the LSAB-CAN agar had given the greatest number of bacteria isolated from this genus.

    KEY-WORDS: Listeria; enrichment broth; selective agar.

    No presente trabalho verificou-se a eficiência dos caldos de enriquecimento para Listeria (LEB1 e LEB 2 associados à passagem da cultura por uma solução de hidróxido de potássio a 0,25%, constatando-se que o caldo de enriquecimento secundário (LEB2 e LEB2KOH foi superior ao caldo de enriquecimento primário (LEB1KOH. Por outro lado, o ágar cloreto de lítio-feniletanol-moxalactam (LPM e o ágar base seletivo para Listeria, suplementado com cicloheximida, acriflavina e ácido nalidíxico (LSAB-CAN equivaleram-se estatisticamente (p= 0,l442 em relação ao número de amostras positivas para Listeria, embora o ágar LSAB-CAN tenha proporcionado um maior número de isolamentos da bactéria.

    PALAVRAS-CHAVE: Caldo de enriquecimento; ágar seletivo; Listeria.

  13. Anticlostridial agent 8-hydroxyquinoline improves the isolation of faecal bifidobacteria on modified Wilkins-Chalgren agar with mupirocin.

    Science.gov (United States)

    Novakova, J; Vlkova, E; Salmonova, H; Pechar, R; Rada, V; Kokoska, L

    2016-04-01

    The need for suitable selective cultivation media for the isolation of Bifidobacterium spp. continues to be a real concern in the field of intestinal microbiology. Isolation of bifidobacteria from human and animal faecal samples using selective agar plating may be problematic especially in samples with increased clostridial counts than bifidobacterial counts. Due to the absence of anticlostridial agents in existing selective media, clostridia can displace bifidobacteria resulting in incorrect estimation of their counts. Therefore, we supplemented the existing selective medium 'modified Wilkins Chalgren agar with mupirocin' (MWM) with 90 mg l(-1) of 8-hydroxyquinoline (8HQ), which was recently proved to act selectively against clostridia. The newly composed 'modified Wilkins-Chalgren agar with 8HQ' (MWMQ) was tested on pure bifidobacterial and clostridial strains, their mixtures, and using faecal samples of mammalian origin; its selectivity was evaluated by genus-specific identification of isolates. The results demonstrated that the presence of 8HQ in this agar eliminated the growth of nonbifidobacterial strains on MWMQ compared to that on MWM, whereas the recovery of bifidobacterial counts was at satisfactory levels. In conclusion, MWMQ could be recommended for bifidobacterial isolation from human and animal faeces especially when bifidobacteria are not numerically dominant and there are chances of clostridial contamination. Routine isolation of bifidobacteria from mammalian faeces does not use a reliable selective agar with an anticlostridial agent. Overgrowth of clostridia may result in incorrect estimation of bifidobacterial counts. Thus, in order to improve the selectivity of existing media for bifidobacterial isolation, we chose the modified Wilkins-Chalgren agar with mupirocin and supplemented it with 8-hydroxyquinoline (8HQ), a molecule that shows anticlostridial activity without affecting the growth of bifidobacteria. This newly composed medium showed

  14. Activity staining method of chitinase on chitin agar plate through ...

    African Journals Online (AJOL)

    A method for detection of chitinase activity on chitin agar plate after polyacrylamide gel electrophoresis is described. Different staining dyes such as calcofluor white M2R, fluorescein isothiocyanate, rhodamine B, ruthenium red and congo red were separately incorporated in chitin agar plates. After running polyacrylamide ...

  15. Assessment of Native Agar Gels Extracted from Gracilaria debilis ...

    African Journals Online (AJOL)

    Native agar gels extracted from Gracilaria debilis and G. salicornia harvested during the rainy and dry seasons, were assessed for culturing the microorganisms Micrococcus luteus, Saccharomyces cerevisiae and Pleurotus flabellatus. Agars extracted from plants harvested during the rainy season were suitable for culturing ...

  16. Palm kernel agar: An alternative culture medium for rapid detection ...

    African Journals Online (AJOL)

    Palm kernel agar: An alternative culture medium for rapid detection of aflatoxins in agricultural commodities. ... a pink background and blue or blue green fluorescence of palm kernel agar Under long wave UV light (366nm) as against the white background of DCA, which often interferes with fluorescence with corresponding ...

  17. Passive Protection of Diabetic Rats with Antisera Specific for the Polysaccharide Portion of the Lipopolysaccharide Isolated from Pseudomonas pseudomallei

    Directory of Open Access Journals (Sweden)

    Larry E Bryan

    1994-01-01

    Full Text Available Polyclonal and monoclonal antisera raised to tetanus toxoid-conjugated polysaccharide of lipopolysaccharide (lps and purified lps of Pseudomonas pseudomallei that reacted with a collection of 41 strains of this bacterium from 23 patients are described. The common antigen recognized by these sera was within the polysaccharide component of the lps of the cells. The sera were specific for P pseudomallei in that none of 37 strains of other bacteria, including 20 Gram-negative and three Gram-positive species, were recognized, although cross-reaction occurred using the anticonjugate serum with some strains of Pseudomonas cepacia serotype A, a closely related bacterium. Passive protection studies using a diabetic rat model of P pseudomallei infection showed that partially purified rabbit polyclonal and mouse monoclonal antisera were protective when the median lethal dose was raised by four to five orders of magnitude. The wide distribution of the polysaccharide antigen among isolates of P pseudomallei used in this study and the protective role of antibody to the conjugated polysaccharide antigen suggest potential as a vaccine.

  18. Development of a multiplex PCR assay for rapid identification of Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex.

    Science.gov (United States)

    Koh, Seng Fook; Tay, Sun Tee; Sermswan, Rasana; Wongratanacheewin, Surasakdi; Chua, Kek Heng; Puthucheary, Savithri D

    2012-09-01

    We have developed a multiplex PCR assay for rapid identification and differentiation of cultures for Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex. The assay is valuable for use in clinical and veterinary laboratories, and in a deployable laboratory during outbreaks. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Relationship between antigenicity and pathogenicity for Burkholderia pseudomallei and Burkholderia mallei revealed by a large panel of mouse MAbs.

    Science.gov (United States)

    Zou, Nianxiang; Tsai, Shien; Feng, Shaw-Huey; Newsome, Tamara; Kim, Hyung-Yong; Li, Bingjie; Zhang, Shimin; Lo, Shyh-Ching

    2008-08-01

    Burkholderia pseudomallei and Burkholderia mallei are two closely related gram-negative bacterial species classified by the CDC as category B biowarfare agents. To develop monoclonal antibodies (MAbs) that can recognize as many different strains and/or clinical isolates of these two pathogens as possible, we immunized mice with heat-killed bacterial whole cells and membrane preparations from multiple strains and/or clinical isolates of B. pseudomallei and B. mallei. More than 100 different hybridoma clones that produced MAbs strongly reacting to B. pseudomallei and/or B. mallei have been developed. These MAbs were categorized into eight different groups according to their reaction specificity against different species of Burkholderia bacteria as well as the different nature of target antigens (LPS, capsule polysaccharides, proteins, and glycoproteins) on the bacteria they recognized. Characterization of this large panel of MAbs has demonstrated an interesting pattern of various antigenic epitopes shared by the different species of Burkholderia bacteria. More importantly, this study has revealed a pathogenicity-linked antigen epitope(s) on capsule-like polysaccharides found only in the pathogenic species of Burkholderia bacteria and a Burkholderia-specific antigen epitope(s) that did not exist in other gram-negative bacterial species. Our MAbs should prove to be highly valuable in the development of detection, diagnosis, and therapeutic applications against B. mallei and B. pseudomallei infections.

  20. Cross-species comparison of the Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia mallei quorum-sensing regulons.

    Science.gov (United States)

    Majerczyk, Charlotte D; Brittnacher, Mitchell J; Jacobs, Michael A; Armour, Christopher D; Radey, Matthew C; Bunt, Richard; Hayden, Hillary S; Bydalek, Ryland; Greenberg, E Peter

    2014-11-01

    Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia mallei (the Bptm group) are close relatives with very different lifestyles: B. pseudomallei is an opportunistic pathogen, B. thailandensis is a nonpathogenic saprophyte, and B. mallei is a host-restricted pathogen. The acyl-homoserine lactone quorum-sensing (QS) systems of these three species show a high level of conservation. We used transcriptome sequencing (RNA-seq) to define the quorum-sensing regulon in each species, and we performed a cross-species analysis of the QS-controlled orthologs. Our analysis revealed a core set of QS-regulated genes in all three species, as well as QS-controlled factors shared by only two species or unique to a given species. This global survey of the QS regulons of B. pseudomallei, B. thailandensis, and B. mallei serves as a platform for predicting which QS-controlled processes might be important in different bacterial niches and contribute to the pathogenesis of B. pseudomallei and B. mallei. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  1. Effects of Colonization of the Roots of Domestic Rice (Oryza sativa L. cv. Amaroo) by Burkholderia pseudomallei.

    Science.gov (United States)

    Prasertsincharoen, Noppadol; Constantinoiu, Constantin; Gardiner, Christopher; Warner, Jeffrey; Elliman, Jennifer

    2015-07-01

    Burkholderia pseudomallei is a saprophytic bacterium that causes melioidosis and is often isolated from rice fields in Southeast Asia, where the infection incidence is high among rice field workers. The aim of this study was to investigate the relationship between this bacterium and rice through growth experiments where the effect of colonization of domestic rice (Oryza sativa L. cv Amaroo) roots by B. pseudomallei could be observed. When B. pseudomallei was exposed to surface-sterilized seeds, the growth of both the root and the aerosphere was retarded compared to that in controls. The organism was found to localize in the root hairs and endodermis of the plant. A biofilm formed around the root and root structures that were colonized. Growth experiments with a wild rice species (Oryza meridionalis) produced similar retardation of growth, while another domestic cultivar (O. sativa L. cv Koshihikari) did not show retarded growth. Here we report B. pseudomallei infection and inhibition of O. sativa L. cv Amaroo, which might provide insights into plant interactions with this important human pathogen. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  2. Effects of Colonization of the Roots of Domestic Rice (Oryza sativa L. cv. Amaroo) by Burkholderia pseudomallei

    Science.gov (United States)

    Constantinoiu, Constantin; Gardiner, Christopher; Warner, Jeffrey

    2015-01-01

    Burkholderia pseudomallei is a saprophytic bacterium that causes melioidosis and is often isolated from rice fields in Southeast Asia, where the infection incidence is high among rice field workers. The aim of this study was to investigate the relationship between this bacterium and rice through growth experiments where the effect of colonization of domestic rice (Oryza sativa L. cv Amaroo) roots by B. pseudomallei could be observed. When B. pseudomallei was exposed to surface-sterilized seeds, the growth of both the root and the aerosphere was retarded compared to that in controls. The organism was found to localize in the root hairs and endodermis of the plant. A biofilm formed around the root and root structures that were colonized. Growth experiments with a wild rice species (Oryza meridionalis) produced similar retardation of growth, while another domestic cultivar (O. sativa L. cv Koshihikari) did not show retarded growth. Here we report B. pseudomallei infection and inhibition of O. sativa L. cv Amaroo, which might provide insights into plant interactions with this important human pathogen. PMID:25911477

  3. Biosynthesis of agar in red seaweeds: A review.

    Science.gov (United States)

    Lee, Wei-Kang; Lim, Yi-Yi; Leow, Adam Thean-Chor; Namasivayam, Parameswari; Ong Abdullah, Janna; Ho, Chai-Ling

    2017-05-15

    Agar is a jelly-like biopolymer synthesized by many red seaweeds as their major cell wall component. Due to its excellent rheological properties, it has been exploited commercially for applications in food, cosmetic, pharmaceutical, biomedical and biotechnology industries. Despite its multiple uses, the biosynthesis of this phycocolloid is not fully understood. The current knowledge on agar biosynthesis is inferred from plant biochemistry and putative pathways for ulvan and alginate biosynthesis in green and brown seaweeds, respectively. In this review, the gaps in our current knowledge on agar biosynthetic pathway are discussed, focusing on the biosynthesis of agar precursors, elongation of agar polysaccharide chain and side chain modification. The development of molecular markers for the screening of desired seaweeds for industrial exploitation is also discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Improved Detection of Strongyloides stercoralis in Modified Agar Plate Cultures.

    Science.gov (United States)

    Pocaterra, Leonor A; Ferrara, Giuseppe; Peñaranda, Rosaura; Rojas, Elsy; Pérez-Chacón, Gladymar; Hernán, Aurora; Certad, Gabriela; Goldstein, Carlos; Núñez, Luz

    2017-04-01

    AbstractA modification of Koga agar plate culture was performed, consisting of a 2 × 2-cm cellophane paper centered on the agar plate to prevent bacterial contamination of the agar and daily dish examinations (days 2-5). Between January 2000 and July 2005, we examined 1,708 infection-suspected patients, of which 147 (8.6%) harbored S. stercoralis. Single modified agar plate cultures exhibited superior sensitivity (93.2%), compared with different three-sample screening methods (sensitivity-Baermann: 76.6%, formalin-ethyl acetate: 22%, and direct smear: 15.3%). Agar plate cultures stand out as helpful alternatives for improved detection and therapy monitoring in poor countries and endemic areas. Combined with Baermann methods, they provide increased probability for S. stercoralis detection.

  5. Modified Sabouraud dextrose agar for isolation and identification of dermatophytes

    Directory of Open Access Journals (Sweden)

    Bulajić Nina M.

    2005-01-01

    Full Text Available The most common causative agents of dermatomycoses are fungi belonging to genders Trichophyton, Microsporum and Epidermophyton. Media mainly used for isolation of dermatophytes are mycobiotic agar, dermatophyte test medium Sabouraud agar (original formula or modification by Emmons with or without antibiotics and cycloheximide. Peptones are the most important components of the media, which enable adequate reproductivity in identification of dermatophytes. Standard medium for isolation of dermatophytes is not produced in our country. The aim of the study was to create an optimal easily accessible and economic medium which enables isolation and identification of dermatophytes according to criteria for morphological diagnosis provided by identification guides. We examined 57 strains of Trichophyton, 24 of Microsporum and 5 of Epidermophyton floccosum (E. floccosum. Each strain was seeded on Sabouraud dextrose agar (Torlak Serbia and Montenegro, Sabouraud maltose agar (Torlak, two experimental modified Sabouraud dextrose agar media marked as SA-2 and SA-3 (Torlak Sabouraud-Chloramp- henicole agar (Biomerieux, France Sabouraud-Chloramphenicole agar (Himedia, India, Glucose-peptone agar (Himedia, India and Sabouraud Emmons dextrose Agar with Chloramphenicole and Cycloheximide (Biolife, Italy. Colony morphology of Trichophyton mentagrophytes (T. mentagrophytes was uni- form on all the media, while morphology of Trichophyton rubrum (T. rubrum and Microsporum canis (M. canis depended more on the media type. Colonies of E. floccosum were typical and uniform on all the media, as were the control species of Trichophyton schoenleinii (T. schoenleinii and Trichophyton soudanense (T. soudanense. Experimental modified Sabouraud dextrose agar (Torlak marked as SA-3 demonstrated the best results in identification of dermatophytes in this study.

  6. Osteopontin impairs host defense during established gram-negative sepsis caused by Burkholderia pseudomallei (melioidosis.

    Directory of Open Access Journals (Sweden)

    Gerritje J W van der Windt

    2010-08-01

    Full Text Available Melioidosis, caused by infection with Burkholderia (B. pseudomallei, is a severe illness that is endemic in Southeast Asia. Osteopontin (OPN is a phosphorylated glycoprotein that is involved in several immune responses including induction of T-helper 1 cytokines and recruitment of inflammatory cells.OPN levels were determined in plasma from 33 melioidosis patients and 31 healthy controls, and in wild-type (WT mice intranasally infected with B. pseudomallei. OPN function was studied in experimental murine melioidosis using WT and OPN knockout (KO mice. Plasma OPN levels were elevated in patients with severe melioidosis, even more so in patients who went on to die. In patients who recovered plasma OPN concentrations had decreased after treatment. In experimental melioidosis in mice plasma and pulmonary OPN levels were also increased. Whereas WT and OPN KO mice were indistinguishable during the first 24 hours after infection, after 72 hours OPN KO mice demonstrated reduced bacterial numbers in their lungs, diminished pulmonary tissue injury, especially due to less necrosis, and decreased neutrophil infiltration. Moreover, OPN KO mice displayed a delayed mortality as compared to WT mice. OPN deficiency did not influence the induction of proinflammatory cytokines.These data suggest that sustained production of OPN impairs host defense during established septic melioidosis.

  7. Variable virulence factors in Burkholderia pseudomallei (melioidosis associated with human disease.

    Directory of Open Access Journals (Sweden)

    Derek S Sarovich

    Full Text Available Burkholderia pseudomallei is a Gram-negative environmental bacterium that causes melioidosis, a potentially life-threatening infectious disease affecting mammals, including humans. Melioidosis symptoms are both protean and diverse, ranging from mild, localized skin infections to more severe and often fatal presentations including pneumonia, septic shock with multiple internal abscesses and occasionally neurological involvement. Several ubiquitous virulence determinants in B. pseudomallei have already been discovered. However, the molecular basis for differential pathogenesis has, until now, remained elusive. Using clinical data from 556 Australian melioidosis cases spanning more than 20 years, we identified a Burkholderia mallei-like actin polymerization bimA(Bm gene that is strongly associated with neurological disease. We also report that a filamentous hemagglutinin gene, fhaB3, is associated with positive blood cultures but is negatively correlated with localized skin lesions without sepsis. We show, for the first time, that variably present virulence factors play an important role in the pathogenesis of melioidosis. Collectively, our study provides a framework for assessing other non-ubiquitous bacterial virulence factors and their association with disease, such as candidate loci identified from large-scale microbial genome-wide association studies.

  8. DNA microarray-based detection and identification of Burkholderia mallei, Burkholderia pseudomallei and Burkholderia spp.

    Science.gov (United States)

    Schmoock, Gernot; Ehricht, Ralf; Melzer, Falk; Rassbach, Astrid; Scholz, Holger C; Neubauer, Heinrich; Sachse, Konrad; Mota, Rinaldo Aparecido; Saqib, Muhammad; Elschner, Mandy

    2009-01-01

    We developed a rapid oligonucleotide microarray assay based on genetic markers for the accurate identification and differentiation of Burkholderia (B.) mallei and Burkholderia pseudomallei, the agents of glanders and melioidosis, respectively. These two agents were clearly identified using at least 4 independent genetic markers including 16S rRNA gene, fliC, motB and also by novel species-specific target genes, identified by in silico sequence analysis. Specific hybridization signal profiles allowed the detection and differentiation of up to 10 further Burkholderia spp., including the closely related species Burkholderia thailandensis and Burkholderia-like agents, such as Burkholderia cepacia, Burkholderia cenocepacia, Burkholderia vietnamiensis, Burkholderia ambifaria, and Burkholderia gladioli, which are often associated with cystic fibrosis (CF) lung disease. The assay was developed using the easy-to-handle and economical ArrayTube (AT) platform. A representative strain panel comprising 44 B. mallei, 32 B. pseudomallei isolates, and various Burkholderia type strains were examined to validate the test. Assay specificity was determined by examination of 40 non-Burkholderia strains.

  9. Variable virulence factors in Burkholderia pseudomallei (melioidosis) associated with human disease.

    Science.gov (United States)

    Sarovich, Derek S; Price, Erin P; Webb, Jessica R; Ward, Linda M; Voutsinos, Marcos Y; Tuanyok, Apichai; Mayo, Mark; Kaestli, Mirjam; Currie, Bart J

    2014-01-01

    Burkholderia pseudomallei is a Gram-negative environmental bacterium that causes melioidosis, a potentially life-threatening infectious disease affecting mammals, including humans. Melioidosis symptoms are both protean and diverse, ranging from mild, localized skin infections to more severe and often fatal presentations including pneumonia, septic shock with multiple internal abscesses and occasionally neurological involvement. Several ubiquitous virulence determinants in B. pseudomallei have already been discovered. However, the molecular basis for differential pathogenesis has, until now, remained elusive. Using clinical data from 556 Australian melioidosis cases spanning more than 20 years, we identified a Burkholderia mallei-like actin polymerization bimA(Bm) gene that is strongly associated with neurological disease. We also report that a filamentous hemagglutinin gene, fhaB3, is associated with positive blood cultures but is negatively correlated with localized skin lesions without sepsis. We show, for the first time, that variably present virulence factors play an important role in the pathogenesis of melioidosis. Collectively, our study provides a framework for assessing other non-ubiquitous bacterial virulence factors and their association with disease, such as candidate loci identified from large-scale microbial genome-wide association studies.

  10. Evaluation of mannitol salt agar for detection of oxacillin resistance in Staphylococcus aureus by disk diffusion and agar screening.

    Science.gov (United States)

    Kampf, G; Lecke, C; Cimbal, A K; Weist, K; Rüden, H

    1998-08-01

    Mannitol salt agar was evaluated for detection of oxacillin resistance in 136 Staphylococcus aureus isolates. All mecA-positive isolates (n = 54) were correctly categorized as oxacillin resistant by the disk diffusion test (1-microgram disk; zone diameter, Agar screening (2 micrograms of oxacillin per ml) revealed a sensitivity of 98.1% and a specificity of 95.1%.

  11. Comparison of DNA extraction kits for detection of Burkholderia pseudomallei in spiked human whole blood using real-time PCR.

    Directory of Open Access Journals (Sweden)

    Nicole L Podnecky

    Full Text Available Burkholderia pseudomallei, the etiologic agent of melioidosis, is endemic in northern Australia and Southeast Asia and can cause severe septicemia that may lead to death in 20% to 50% of cases. Rapid detection of B. pseudomallei infection is crucial for timely treatment of septic patients. This study evaluated seven commercially available DNA extraction kits to determine the relative recovery of B. pseudomallei DNA from spiked EDTA-containing human whole blood. The evaluation included three manual kits: the QIAamp DNA Mini kit, the QIAamp DNA Blood Mini kit, and the High Pure PCR Template Preparation kit; and four automated systems: the MagNAPure LC using the DNA Isolation Kit I, the MagNAPure Compact using the Nucleic Acid Isolation Kit I, and the QIAcube using the QIAamp DNA Mini kit and the QIAamp DNA Blood Mini kit. Detection of B. pseudomallei DNA extracted by each kit was performed using the B. pseudomallei specific type III secretion real-time PCR (TTS1 assay. Crossing threshold (C T values were used to compare the limit of detection and reproducibility of each kit. This study also compared the DNA concentrations and DNA purity yielded for each kit. The following kits consistently yielded DNA that produced a detectable signal from blood spiked with 5.5×10(4 colony forming units per mL: the High Pure PCR Template Preparation, QIAamp DNA Mini, MagNA Pure Compact, and the QIAcube running the QIAamp DNA Mini and QIAamp DNA Blood Mini kits. The High Pure PCR Template Preparation kit yielded the lowest limit of detection with spiked blood, but when this kit was used with blood from patients with confirmed cases of melioidosis, the bacteria was not reliably detected indicating blood may not be an optimal specimen.

  12. Discrimination of Burkholderia mallei/pseudomallei from Burkholderia thailandensis by sequence comparison of a fragment of the ribosomal protein S21 (rpsU) gene

    OpenAIRE

    Frickmann, H.; Chantratita, N; Gauthier, Y. P.; Neubauer, H.; Hagen, R. M.

    2012-01-01

    Discrimination of Burkholderia (B.) pseudomallei and B. mallei from environmental B. thailandensis is challenging. We describe a discrimination method based on sequence comparison of the ribosomal protein S21 (rpsU) gene.

  13. Increased Neurotropic Threat from Burkholderia pseudomallei Strains with a B. mallei-like Variation in the bimA Motility Gene, Australia.

    Science.gov (United States)

    Morris, Jodie L; Fane, Anne; Sarovich, Derek S; Price, Erin P; Rush, Catherine M; Govan, Brenda L; Parker, Elizabeth; Mayo, Mark; Currie, Bart J; Ketheesan, Natkunam

    2017-05-01

    Neurologic melioidosis is a serious, potentially fatal form of Burkholderia pseudomallei infection. Recently, we reported that a subset of clinical isolates of B. pseudomallei from Australia have heightened virulence and potential for dissemination to the central nervous system. In this study, we demonstrate that this subset has a B. mallei-like sequence variation of the actin-based motility gene, bimA. Compared with B. pseudomallei isolates having typical bimA alleles, isolates that contain the B. mallei-like variation demonstrate increased persistence in phagocytic cells and increased virulence with rapid systemic dissemination and replication within multiple tissues, including the brain and spinal cord, in an experimental model. These findings highlight the implications of bimA variation on disease progression of B. pseudomallei infection and have considerable clinical and public health implications with respect to the degree of neurotropic threat posed to human health.

  14. Cost-effectiveness of blood agar for isolation of mycobacteria.

    Directory of Open Access Journals (Sweden)

    Michel Drancourt

    Full Text Available BACKGROUND: Mycobacterium species are grown using specific media that increase laboratory cost, thus hampering their diffusion in resource-limited countries. Preliminary data suggested that versatile blood agar may be also used for mycobacterial culture. METHODOLOGY: We examined the growth of 41 different Mycobacterium species on 5% blood agar. Over a 24-month period we analysed isolation of mycobacteria after parallel inoculation of clinical specimens into both a reference automated system (BACTEC 9000 MB broth and 5% blood agar slant tubes, after NaOH decontamination, and compared the cost of performing 1,000 analyses using these two techniques. CONCLUSIONS: Mycobacterium reference species cultured on blood agar, with the exception of Mycobacterium ulcerans. Inoculation of 1,634 specimens yielded 95 Mycobacterium isolates. Blood agar performed significantly more efficiently than BACTEC 9000 MB broth (94 vs 88 isolates, P = 0.03. Decontamination of Candida albicans in 5 specimens by addition of amphotericin B in blood agar yielded one more M. tuberculosis isolate that could not be isolated in BACTEC broth. Uneven distribution of time to culture positivity for M. tuberculosis had a median (range of 19+/-5 days using blood agar and 26+/-6 days using BACTEC 9000 MB broth. Cost for 1,000 analyses in France was estimated to be of 1,913 euros using the blood agar method and 8,990 euros using the BACTEC 9000 MB method. Blood agar should be regarded as a first-line medium for culturing Mycobacterium species. It saves time, is cost-effective, is more sensitive than, and at least as rapid as the automated method. This is of particular importance for resource-limited countries in which the prevalence of tuberculosis is high.

  15. Cost-Effectiveness of Blood Agar for Isolation of Mycobacteria

    Science.gov (United States)

    Drancourt, Michel; Raoult, Didier

    2007-01-01

    Background Mycobacterium species are grown using specific media that increase laboratory cost, thus hampering their diffusion in resource-limited countries. Preliminary data suggested that versatile blood agar may be also used for mycobacterial culture. Methodology We examined the growth of 41 different Mycobacterium species on 5% blood agar. Over a 24-month period we analysed isolation of mycobacteria after parallel inoculation of clinical specimens into both a reference automated system (BACTEC 9000 MB broth) and 5% blood agar slant tubes, after NaOH decontamination, and compared the cost of performing 1,000 analyses using these two techniques. Conclusions Mycobacterium reference species cultured on blood agar, with the exception of Mycobacterium ulcerans. Inoculation of 1,634 specimens yielded 95 Mycobacterium isolates. Blood agar performed significantly more efficiently than BACTEC 9000 MB broth (94 vs 88 isolates, P = 0.03). Decontamination of Candida albicans in 5 specimens by addition of amphotericin B in blood agar yielded one more M. tuberculosis isolate that could not be isolated in BACTEC broth. Uneven distribution of time to culture positivity for M. tuberculosis had a median (range) of 19±5 days using blood agar and 26±6 days using BACTEC 9000 MB broth. Cost for 1,000 analyses in France was estimated to be of 1,913 euros using the blood agar method and 8,990 euros using the BACTEC 9000 MB method. Blood agar should be regarded as a first-line medium for culturing Mycobacterium species. It saves time, is cost-effective, is more sensitive than, and at least as rapid as the automated method. This is of particular importance for resource-limited countries in which the prevalence of tuberculosis is high. PMID:18060087

  16. Genome-scale analysis of the genes that contribute to Burkholderia pseudomallei biofilm formation identifies a crucial exopolysaccharide biosynthesis gene cluster.

    Directory of Open Access Journals (Sweden)

    Grace I Borlee

    2017-06-01

    Full Text Available Burkholderia pseudomallei, the causative agent of melioidosis, is an important public health threat due to limited therapeutic options for treatment. Efforts to improve therapeutics for B. pseudomallei infections are dependent on the need to understand the role of B. pseudomallei biofilm formation and its contribution to antibiotic tolerance and persistence as these are bacterial traits that prevent effective therapy. In order to reveal the genes that regulate and/or contribute to B. pseudomallei 1026b biofilm formation, we screened a sequence defined two-allele transposon library and identified 118 transposon insertion mutants that were deficient in biofilm formation. These mutants include transposon insertions in genes predicted to encode flagella, fimbriae, transcriptional regulators, polysaccharides, and hypothetical proteins. Polysaccharides are key constituents of biofilms and B. pseudomallei has the capacity to produce a diversity of polysaccharides, thus there is a critical need to link these biosynthetic genes with the polysaccharides they produce to better understand their biological role during infection. An allelic exchange deletion mutant of the entire B. pseudomallei biofilm-associated exopolysaccharide biosynthetic cluster was decreased in biofilm formation and produced a smooth colony morphology suggestive of the loss of exopolysaccharide production. Conversely, deletion of the previously defined capsule I polysaccharide biosynthesis gene cluster increased biofilm formation. Bioinformatics analyses combined with immunoblot analysis and glycosyl composition studies of the partially purified exopolysaccharide indicate that the biofilm-associated exopolysaccharide is neither cepacian nor the previously described acidic exopolysaccharide. The biofilm-associated exopolysaccharide described here is also specific to the B. pseudomallei complex of bacteria. Since this novel exopolysaccharide biosynthesis cluster is retained in B. mallei

  17. The Efficiency of UVC Radiation in the Inactivation of
Listeria monocytogenes on Beef-Agar Food Models.

    Science.gov (United States)

    Hamidi-Oskouei, Amir M; James, Christian; James, Stephen

    2015-06-01

    The aim of this study is to evaluate the effect of meat content and surface smoothness on the deactivation of Listeria monocytogenes in beef-agar food models achieved by shortwave ultraviolet (UVC) light. Food models with various meat contents were made using chopped beef slices and agar solution. Prepared models together with a Listeria selective agar (LSA) plate and a slice of cooked beef were inoculated with L. monocytogenes and then exposed to UVC light. Population of Listeria reduced to below the level of detection on the LSA plates. As the content of beef in the beef-agar models increased, more L. monocytogenes cells survived. Survival was greatest on the treated cooked slice of beef. To better understand the effect of surface irregularities, a white light interferometer was used to analyse the surface smoothness of beef-agar media and LSA plates. No correlation was observed between the surface roughness of seven out of nine types of produced beef-agar media and the degree of inactivation resulting from UVC radiation at the given dose, whereas, less bacterial cells were killed as beef content of the food models increased. The findings of the current study show that the chemical composition of the treated sample also plays an important role in pathogen resistance and survival, meaning that two samples with similar surface irregularities but different chemical composition might produce very different inactivation results when exposed to UVC light.

  18. The Efficiency of UVC Radiation in the Inactivation of Listeria monocytogenes on Beef-Agar Food Models

    Directory of Open Access Journals (Sweden)

    Christian James

    2015-01-01

    Full Text Available The aim of this study is to evaluate the eff ect of meat content and surface smoothness on the deactivation of Listeria monocytogenes in beef-agar food models achieved by shortwave ultraviolet (UVC light. Food models with various meat contents were made using chopped beef slices and agar solution. Prepared models together with a Listeria selective agar (LSA plate and a slice of cooked beef were inoculated with L. monocytogenes and then exposed to UVC light. Population of Listeria reduced to below the level of detection on the LSA plates. As the content of beef in the beef-agar models increased, more L. monocytogenes cells survived. Survival was greatest on the treated cooked slice of beef. To bett er understand the effect of surface irregularities, a white light interferometer was used to analyse the surface smoothness of beef-agar media and LSA plates. No correlation was observed between the surface roughness of seven out of nine types of produced beef-agar media and the degree of inactivation resulting from UVC radiation at the given dose, whereas, less bacterial cells were killed as beef content of the food models increased. The findings of the current study show that the chemical composition of the treated sample also plays an important role in pathogen resistance and survival, meaning that two samples with similar surface irregularities but diff erent chemical composition might produce very diff erent inactivation results when exposed to UVC light.

  19. Comparison of manual mycobacteria growth indicator tube and epsilometer test with agar proportion method for susceptibility testing of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    N Karabulut

    2014-01-01

    Full Text Available Background and Objectives: Antimycobacterial susceptibility tests take weeks, and delayed therapy can lead to spread of Mycobacterium tuberculosis. Therefore, rapid, accurate and cost-effective methods are required for proper therapy selection. In this study, the Mycobacteria growth indicator tube (MGIT and epsilometer test (Etest methods were compared to the agar proportion method for susceptibility testing of Mycobacterium tuberculosis. Materials and Methods: The susceptibility tests against isoniazid (INH, rifampin (RIF, streptomycin (STM and ethambutol (ETM of 51 M. tuberculosis complex isolates were analyzed by the MGIT, Etest and agar proportion methods. Results: The concordance between MGIT/Etest and agar proportion methods was 98% for INH and 100% for RIF, STM, ETM. There were not statistically significant differences in results of the susceptibility tests between MGIT/Etest and the reference agar proportion method. Conclusion: The results have shown that MGIT and Etest methods can be used instead of the agar proportion method, because these two methods are more rapid and easier than the agar proportion method.

  20. Mannitol Salt Agar-Cefoxitin Combination as a Screening Medium for Methicillin-Resistant Staphylococcus aureus

    Science.gov (United States)

    Smyth, R. W.; Kahlmeter, Gunnar

    2005-01-01

    In disk diffusion tests, cefoxitin is now considered a better indicator than oxacillin for the presence of the mecA gene in Staphylococcus aureus. A logical extension of this work is the incorporation of cefoxitin into media selective for methicillin-resistant Staphylococcus aureus (MRSA). This paper describes the development and subsequent testing of mannitol salt agar containing 4 mg/liter cefoxitin with a unique collection of well-characterized MRSA strains, including low-level methicillin-resistant strains and an equal number of known mecA-negative S. aureus strains. The agar supported the growth of 96.6% of the mecA-positive strains in the collection and inhibited the growth of 100% of the mecA-negative strains. These results suggest that selective media based on cefoxitin are superior to those based on oxacillin for the detection of MRSA. PMID:16081913

  1. History and environmental education- A review of agar's thesis with ...

    African Journals Online (AJOL)

    History has a major contribution to make to the understanding of the present day environment, and consequently a significcnt role to play in environmental education. Agar's (1973) arguments for this are reviewed in the context of contemporary Southern Africa.

  2. Suitability of various plant derived gelling agents as agar substitute ...

    African Journals Online (AJOL)

    Suitability of various plant derived gelling agents as agar substitute in microbiological growth media. Abdul Mateen, Shaukat Hussain, Shams Ur Rehman, Basharat Mahmood, Muhammad Aslam Khan, Abdul Rashid, Majid Sohail, Muhammad Farooq, S Jawad Ahmed Shah ...

  3. AGAR PENULISAN RESEP TETAP UP TO DATE

    Directory of Open Access Journals (Sweden)

    Rahmatini Rahmatini

    2009-09-01

    Full Text Available AbstrakResep adalah suatu permintaan tertulis dari dokter, dokter gigi atau dokter hewan kepada apoteker untuk membuatkan obat dalam bentuk sediaan tertentu dan menyerahkannya kepada pasien. Resep merupakan perwujudan akhir dari kompetensi, pengetahuan dan keahlian dokter dalam menerapkan pengetahuannya dalam bidang farmakologi dan terapi.Penulisan resep harus ditulis dengan jelas sehingga dapat dibaca oleh petugas di apotek. Resep yang ditulis dengan tidak jelas akan menimbulkan terjadinya kesalahan saat peracikan / penyiapan obat dan penggunaan obat yang diresepkan.Ilmu pengetahuan tentang obat selalu berubah, obat – obat baru selalu muncul di pasaran.Secara umum, seorang dokter harus mengikuti perkembangan dalam terapi obat. Bila muncul efek samping akibat obat yang seharusnya diketahui dan dapat dicegah oleh dokter, maka dokter akan berhadapan dengan hukum.Agar penulisan resep tetap up to date, seorang dokter harus mengumpulkan berbagai informasi yang tersedia. Sumber informasi yang dapat digunakan adalah : Buku acuan, Kompendium obat, Daftar Obat Esensial Nasional (DOEN dan Pedoman terapi, Buletin obat, Jurnal Kedokteran, Pusat informasi obat,Informasi melalui komputer, Sumber informasi dari industri farmasi, dan informasi lisan.Bandingkan kelebihan dan kekurangan berbagai sumber informasi. Tugas seorang dokter adalah melakukan cara terbaik untuk tetap up to date dengan mendaftar sumber informasi yang dapat dimanfaatkan. Carilah sedikitnya satu dari yang berikut ini : (1 jurnal kedokteran: (2 buletin obat; (3 buku acuan farmakologi atau acuan klinis; (4 komisi terapi maupun konsultan, atau lulusan pasca sarjana farmakologi. Dengan bekal pengetahuan dan kemampuan untuk melakukan penilaian secara kritis setiap bentuk informasi, diharapkan dokter tetap up to date dalam menulis resepKata kunci : Resep – up to- date.Abstract Prescription is a written request from a doctor, dentist or veterinarian to the pharmacist to make a particular drug

  4. Inflammasome-dependent pyroptosis and IL-18 protect against Burkholderia pseudomallei lung infection while IL-1β is deleterious.

    Directory of Open Access Journals (Sweden)

    Ivonne Ceballos-Olvera

    2011-12-01

    Full Text Available Burkholderia pseudomallei is a Gram-negative bacterium that infects macrophages and other cell types and causes melioidosis. The interaction of B. pseudomallei with the inflammasome and the role of pyroptosis, IL-1β, and IL-18 during melioidosis have not been investigated in detail. Here we show that the Nod-like receptors (NLR NLRP3 and NLRC4 differentially regulate pyroptosis and production of IL-1β and IL-18 and are critical for inflammasome-mediated resistance to melioidosis. In vitro production of IL-1β by macrophages or dendritic cells infected with B. pseudomallei was dependent on NLRC4 and NLRP3 while pyroptosis required only NLRC4. Mice deficient in the inflammasome components ASC, caspase-1, NLRC4, and NLRP3, were dramatically more susceptible to lung infection with B. pseudomallei than WT mice. The heightened susceptibility of Nlrp3⁻/⁻ mice was due to decreased production of IL-18 and IL-1β. In contrast, Nlrc4⁻/⁻ mice produced IL-1β and IL-18 in higher amount than WT mice and their high susceptibility was due to decreased pyroptosis and consequently higher bacterial burdens. Analyses of IL-18-deficient mice revealed that IL-18 is essential for survival primarily because of its ability to induce IFNγ production. In contrast, studies using IL-1RI-deficient mice or WT mice treated with either IL-1β or IL-1 receptor agonist revealed that IL-1β has deleterious effects during melioidosis. The detrimental role of IL-1β appeared to be due, in part, to excessive recruitment of neutrophils to the lung. Because neutrophils do not express NLRC4 and therefore fail to undergo pyroptosis, they may be permissive to B. pseudomallei intracellular growth. Administration of neutrophil-recruitment inhibitors IL-1ra or the CXCR2 neutrophil chemokine receptor antagonist antileukinate protected Nlrc4⁻/⁻ mice from lethal doses of B. pseudomallei and decreased systemic dissemination of bacteria. Thus, the NLRP3 and NLRC4 inflammasomes have

  5. Thermal characterization of magnetically aligned carbonyl iron/agar composites.

    Science.gov (United States)

    Diaz-Bleis, D; Vales-Pinzón, C; Freile-Pelegrín, Y; Alvarado-Gil, J J

    2014-01-01

    Composites of magnetic particles into polymeric matrices have received increasing research interest due to their capacity to respond to external magnetic or electromagnetic fields. In this study, agar from Gelidium robustum has been chosen as natural biocompatible polymer to build the matrix of the magnetic carbonyl iron particles (CIP) for their uses in biomedical fields. Heat transfer behavior of the CIP-agar composites containing different concentrations (5, 10, 15, 20, 25 and 30% w/w) of magnetically aligned and non-aligned CIP in the agar matrix was studied using photothermal radiometry (PTR) in the back-propagation emission configuration. The morphology of the CIP-agar composites with aligned and non-aligned CIP under magnetic field was also evaluated by scanning electron microscopy (SEM). The results revealed a dominant effect of CIP concentration over the alignment patterns induced by the magnetic field, which agrees with the behavior of the thermal diffusivity and thermal conductivity. Agar served as a perfect matrix to be used with CIP, and CIP-agar composites magnetically aligned at 20% CIP concentration can be considered as promising 'smart' material for hyperthermia treatments in the biomedical field. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Cost-effective nanoporous Agar-Agar polymer/Nickel powder composite particle for effective bio-products adsorption by expanded bed chromatography.

    Science.gov (United States)

    Asgari, Setareh; Jahanshahi, Mohsen; Rahimpour, Ahmad

    2014-09-26

    In the present work a novel kind of dense nanoporous composite matrix for expanded bed application has been successfully first prepared with Nickel powder as a densifier and was covered with Agar-Agar layer as a skeleton, through the method of water-in-oil emulsification. Agar-Agar is a porous and inexpensive polymer. In order to fabricate cost-effective adsorbent with favorable qualities Agar-Agar polymer was used. Thereafter, the customized composite particle was modified by pseudo-affinity dye-ligand, Reactive Blue 4 (RB4), aimed at preparing a pseudo-affinity adsorbent (RB4-Agar-Ni) for bioprodut adsorption from aqueous solution. Bovine Serum Albumin (BSA) was selected as a model protein to investigate the adsorption behavior in batchwise and expanded bed chromatography, and the obtained results were evaluated with that of Streamline™ (Amersham-Pharmacia Biotech, Sweden). Spherical appearance and porous structure of composite particles were observed by the optical microscope (OM) and scanning electronic microscope (SEM). The results suggested that the matrices followed the logarithmic normal size distribution with the range of 65-300 μm and average diameter of 126.81-151.47 μm, proper wet density of 1.64-2.78 g/ml, water content of 62.74-34%, porosity of 98-90% and pore size of about 38-130 nm. For better comprehension of the impact of solid phase properties on the performance of the expanded bed, the expansion and hydrodynamic properties of a composite matrix with a series of densities was evaluated and estimated by the retention time distribution method (RTD) in an expanded bed and was compared with that of other matrices. According to obtained results the expansion factors under the same fluid velocity decreased by increasing the matrix density. Moreover, the axial dispersion coefficient (Dax) is the most appropriate parameter for evaluating the stability of expanded bed, on various operating conditions, such as different flow velocity, bed expansion

  7. An ensemble of structures of Burkholderia pseudomallei 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase

    Energy Technology Data Exchange (ETDEWEB)

    Davies, Douglas R.; Staker, Bart L.; Abendroth, Jan A.; Edwards, Thomas E.; Hartley, Robert; Leonard, Jess; Kim, Hidong; Rychel, Amanda L.; Hewitt, Stephen N.; Myler, Peter J.; Stewart, Lance J. (UWASH); (Emerald)

    2011-12-07

    Burkholderia pseudomallei is a soil-dwelling bacterium endemic to Southeast Asia and Northern Australia. Burkholderia is responsible for melioidosis, a serious infection of the skin. The enzyme 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (PGAM) catalyzes the interconversion of 3-phosphoglycerate and 2-phosphoglycerate, a key step in the glycolytic pathway. As such it is an extensively studied enzyme and X-ray crystal structures of PGAM enzymes from multiple species have been elucidated. Vanadate is a phosphate mimic that is a powerful tool for studying enzymatic mechanisms in phosphoryl-transfer enzymes such as phosphoglycerate mutase. However, to date no X-ray crystal structures of phosphoglycerate mutase have been solved with vanadate acting as a substrate mimic. Here, two vanadate complexes together with an ensemble of substrate and fragment-bound structures that provide a comprehensive picture of the function of the Burkholderia enzyme are reported.

  8. Interim report on updated microarray probes for the LLNL Burkholderia pseudomallei SNP array

    Energy Technology Data Exchange (ETDEWEB)

    Gardner, S; Jaing, C

    2012-03-27

    The overall goal of this project is to forensically characterize 100 unknown Burkholderia isolates in the US-Australia collaboration. We will identify genome-wide single nucleotide polymorphisms (SNPs) from B. pseudomallei and near neighbor species including B. mallei, B. thailandensis and B. oklahomensis. We will design microarray probes to detect these SNP markers and analyze 100 Burkholderia genomic DNAs extracted from environmental, clinical and near neighbor isolates from Australian collaborators on the Burkholderia SNP microarray. We will analyze the microarray genotyping results to characterize the genetic diversity of these new isolates and triage the samples for whole genome sequencing. In this interim report, we described the SNP analysis and the microarray probe design for the Burkholderia SNP microarray.

  9. Identification of the conserved hypothetical protein BPSL0317 in Burkholderia pseudomallei K96243

    Science.gov (United States)

    Yusoff, Nur Syamimi; Damiri, Nadzirah; Firdaus-Raih, Mohd

    2014-09-01

    Burkholderia pseudomallei K96243 is the causative agent of melioidosis, a disease which is endemic in Northern Australia and Southeastern Asia. The genome encodes several essential proteins including those currently annotated as hypothetical proteins. We studied the conservation and the essentiality of expressed hypothetical proteins in normal and different stress conditions. Based on the comparative genomics, we identified a hypothetical protein, BPSL0317, a potential essential gene that is being expressed in all normal and stress conditions. BPSL0317 is also phylogenetically conserved in the Burkholderiales order suggesting that this protein is crucial for survival among the order's members. BPSL0317 therefore has a potential to be a candidate antimicrobial drug target for this group of bacteria.

  10. In vivo Distribution and Clearance of Purified Capsular Polysaccharide from Burkholderia pseudomallei in a Murine Model.

    Directory of Open Access Journals (Sweden)

    Teerapat Nualnoi

    2016-12-01

    Full Text Available Burkholderia pseudomallei is the causative agent of melioidosis, a severe infection prominent in northern Australia and Southeast Asia. The "gold standard" for melioidosis diagnosis is bacterial isolation, which takes several days to complete. The resulting delay in diagnosis leads to delayed treatments, which could result in death. In an attempt to develop better methods for early diagnosis of melioidosis, B. pseudomallei capsular polysaccharide (CPS was identified as an important diagnostic biomarker. A rapid lateral flow immunoassay utilizing CPS-specific monoclonal antibody was developed and tested in endemic regions worldwide. However, the in vivo fate and clearance of CPS has never been thoroughly investigated. Here, we injected mice with purified CPS intravenously and determined CPS concentrations in serum, urine, and major organs at various intervals. The results indicate that CPS is predominantly eliminated through urine and no CPS accumulation occurs in the major organs. Immunoblot analysis demonstrated that intact CPS was excreted through urine. To understand how a large molecule like CPS was eliminated without degradation, a 3-dimenational structure of CPS was modeled. The predicted CPS structure has a rod-like shape with a small diameter that could allow it to flow through the glomerulus of the kidney. CPS clearance was determined using exponential decay models and the corrected Akaike Information Criterion. The results show that CPS has a relatively short serum half-life of 2.9 to 4.4 hours. Therefore, the presence of CPS in the serum and/or urine suggests active melioidosis infection and provides a marker to monitor treatment of melioidosis.

  11. Pangenome Analysis of Burkholderia pseudomallei: Genome Evolution Preserves Gene Order despite High Recombination Rates.

    Directory of Open Access Journals (Sweden)

    Senanu M Spring-Pearson

    Full Text Available The pangenomic diversity in Burkholderia pseudomallei is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of B. pseudomallei and demonstrate that the pangenome is 'open', with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order.

  12. Activation of MAPK/ERK signaling by Burkholderia pseudomallei cycle inhibiting factor (Cif).

    Science.gov (United States)

    Ng, Mei Ying; Wang, Mei; Casey, Patrick J; Gan, Yunn-Hwen; Hagen, Thilo

    2017-01-01

    Cycle inhibiting factors (Cifs) are virulence proteins secreted by the type III secretion system of some Gram-negative pathogenic bacteria including Burkholderia pseudomallei. Cif is known to function to deamidate Nedd8, leading to inhibition of Cullin E3 ubiquitin ligases (CRL) and consequently induction of cell cycle arrest. Here we show that Cif can function as a potent activator of MAPK/ERK signaling without significant activation of other signaling pathways downstream of receptor tyrosine kinases. Importantly, we found that the ability of Cif to activate ERK is dependent on its deamidase activity, but independent of Cullin E3 ligase inhibition. This suggests that apart from Nedd8, other cellular targets of Cif-dependent deamidation exist. We provide evidence that the mechanism involved in Cif-mediated ERK activation is dependent on recruitment of the Grb2-SOS1 complex to the plasma membrane. Further investigation revealed that Cif appears to modify the phosphorylation status of SOS1 in a region containing the CDC25-H and proline-rich domains. It is known that prolonged Cullin E3 ligase inhibition leads to cellular apoptosis. Therefore, we hypothesize that ERK activation is an important mechanism to counter the pro-apoptotic effects of Cif. Indeed, we show that Cif dependent ERK activation promotes phosphorylation of the proapoptotic protein Bim, thereby potentially conferring a pro-survival signal. In summary, we identified a novel deamidation-dependent mechanism of action of the B. pseudomallei virulence factor Cif/CHBP to activate MAPK/ERK signaling. Our study demonstrates that bacterial proteins such as Cif can serve as useful molecular tools to uncover novel aspects of mammalian signaling pathways.

  13. Activation of MAPK/ERK signaling by Burkholderia pseudomallei cycle inhibiting factor (Cif.

    Directory of Open Access Journals (Sweden)

    Mei Ying Ng

    Full Text Available Cycle inhibiting factors (Cifs are virulence proteins secreted by the type III secretion system of some Gram-negative pathogenic bacteria including Burkholderia pseudomallei. Cif is known to function to deamidate Nedd8, leading to inhibition of Cullin E3 ubiquitin ligases (CRL and consequently induction of cell cycle arrest. Here we show that Cif can function as a potent activator of MAPK/ERK signaling without significant activation of other signaling pathways downstream of receptor tyrosine kinases. Importantly, we found that the ability of Cif to activate ERK is dependent on its deamidase activity, but independent of Cullin E3 ligase inhibition. This suggests that apart from Nedd8, other cellular targets of Cif-dependent deamidation exist. We provide evidence that the mechanism involved in Cif-mediated ERK activation is dependent on recruitment of the Grb2-SOS1 complex to the plasma membrane. Further investigation revealed that Cif appears to modify the phosphorylation status of SOS1 in a region containing the CDC25-H and proline-rich domains. It is known that prolonged Cullin E3 ligase inhibition leads to cellular apoptosis. Therefore, we hypothesize that ERK activation is an important mechanism to counter the pro-apoptotic effects of Cif. Indeed, we show that Cif dependent ERK activation promotes phosphorylation of the proapoptotic protein Bim, thereby potentially conferring a pro-survival signal. In summary, we identified a novel deamidation-dependent mechanism of action of the B. pseudomallei virulence factor Cif/CHBP to activate MAPK/ERK signaling. Our study demonstrates that bacterial proteins such as Cif can serve as useful molecular tools to uncover novel aspects of mammalian signaling pathways.

  14. Egg yolk emulsion agar, a new medium for the cultivation of Helicobacter pylori.

    OpenAIRE

    Westblom, T U; Madan, E; Midkiff, B R

    1991-01-01

    We developed a new agar, egg yolk emulsion (EYE) agar, for cultivation of Helicobacter pylori. EYE agar contains Columbia agar base (Oxoid), 10% EYE (Oxoid), 1% IsoVitaleX (BBL), and 40 mg of Triphenyleteraxolium chloride (Sigma) per liter. We compared EYE agar with the following agars: (i) brain heart infusion agar-7% horse blood-1% IsoVitaleX (GDW agar; C. S. Goodwin, E. D. Blincow, J. R. Warren, T. E. Waters, C. R. Sanderson, and L. Easton, J. Clin. Pathol. 38:1127-1131, 1985), (ii) brain ...

  15. A comparison of EF-18 agar and modified brilliant green agar with lutensit for isolation of Salmonella from poultry samples

    DEFF Research Database (Denmark)

    Petersen, Line Hedegård

    1997-01-01

    -Vassiliadis broth, and 3) Plating onto EF-18 agar and BGA/L simultaneously. From a total of 1101 samples, Salmonella was isolated from 158, 157 of which were faecal samples. Thirtyone of these isolates were recovered on one medium only, 18 could not be found on BGA/L and 13 could not be found on EF-18 agar....... The relative specificity and sensitivity of each plating agar was determined by enumeration of false-positive and false-negative reactions. EF-18 agar compared favourably with BGA/L, displaying a sensitivity of 0.92 as opposed to 0.89 for BGA/L, calculated for the ''fecal samples'' group only. The calculated...... specificities for each group of samples were likewise considerably higher for EF-18 agar (0.75-0.91) than for BGA/L (0.35-0.55). Though EF-18 agar is slightly more expensive than BGA/L, the routine use of the former may result in a considerable reduction in overall laboratory costs due to its superior...

  16. Immobilization of pectin degrading enzyme from Bacillus licheniformis KIBGE IB-21 using agar-agar as a support.

    Science.gov (United States)

    Rehman, Haneef Ur; Aman, Afsheen; Zohra, Raheela Rahmat; Qader, Shah Ali Ul

    2014-02-15

    Pectinase from Bacillus licheniformis KIBGE IB-21 was immobilized in agar-agar matrix using entrapment technique. Effect of different concentrations of agar-agar on pectinase immobilization was investigated and it was found that maximum immobilization was achieved at 3.0% agar-agar with 80% enzyme activity. After immobilization, the optimum temperature of enzyme increased from 45 to 50 °C and reaction time from 5 to 10 minutes as compared to free enzyme. Due to the limited diffusion of high molecular weight substrate, K(m) of immobilized enzyme slightly increased from 1.017 to 1.055 mg ml(-1), while Vmax decreased from 23,800 to 19,392 μM min(-1) as compared to free enzyme. After 120 h entrapped pectinase retained their activity up to 82% and 71% at 30 °C and 40 °C, respectively. The entrapped pectinase showed activity until 10th cycle and maintain 69.21% activity even after third cycle. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Production and characterization of chimeric monoclonal antibodies against Burkholderia pseudomallei and B. mallei using the DHFR expression system.

    Directory of Open Access Journals (Sweden)

    Hyung-Yong Kim

    Full Text Available Burkholderia pseudomallei (BP and B. mallei (BM are closely related gram-negative, facultative anaerobic bacteria which cause life-threatening melioidosis in human and glanders in horse, respectively. Our laboratory has previously generated and characterized more than 100 mouse monoclonal antibodies (MAbs against BP and BM, according to in vitro and in vivo assay. In this study, 3 MAbs (BP7 10B11, BP7 2C6, and BP1 7F7 were selected to develop into chimeric mouse-human monoclonal antibodies (cMAbs against BP and/or BM. For the stable production of cMAbs, we constructed 4 major different vector systems with a dihydrofolate reductase (DHFR amplification marker, and optimized transfection/selection conditions in mammalian host cells with the single-gene and/or double-gene expression system. These 3 cMAbs were stably produced by the DHFR double mutant Chinese hamster ovarian (CHO-DG44 cells. By ELISA and Western blot analysis using whole bacterial antigens treated by heat (65°C/90 min, sodium periodate, and proteinase K, the cMAb BP7 10B11 (cMAb CK1 reacted with glycoproteins (34, 38, 48 kDa in BP; 28, 38, 48 kDa in BM. The cMAb BP7 2C6 (cMAb CK2 recognized surface-capsule antigens with molecular sizes of 38 to 52 kDa, and 200 kDa in BM. The cMAb CK2 was weakly reactive to 14∼28, 200 kDa antigens in BP. The cMAb BP1 7F7 (cMAb CK3 reacted with lipopolysaccharides (38∼52 kDa in BP; 38∼60 kDa in B. thailandensis. Western blot results with the outer surface antigens of the 3 Burkholderia species were consistent with results with the whole Burkholderia cell antigens, suggesting that these immunodominant antigens reacting with the 3 cMAbs were primarily present on the outer surface of the Burkholderia species. These 3 cMAbs would be useful for analyzing the role of the major outer surface antigens in Burkholderia infection.

  18. Production and characterization of chimeric monoclonal antibodies against Burkholderia pseudomallei and B. mallei using the DHFR expression system.

    Science.gov (United States)

    Kim, Hyung-Yong; Tsai, Shien; Lo, Shyh-Ching; Wear, Douglas J; Izadjoo, Mina J

    2011-05-09

    Burkholderia pseudomallei (BP) and B. mallei (BM) are closely related gram-negative, facultative anaerobic bacteria which cause life-threatening melioidosis in human and glanders in horse, respectively. Our laboratory has previously generated and characterized more than 100 mouse monoclonal antibodies (MAbs) against BP and BM, according to in vitro and in vivo assay. In this study, 3 MAbs (BP7 10B11, BP7 2C6, and BP1 7F7) were selected to develop into chimeric mouse-human monoclonal antibodies (cMAbs) against BP and/or BM. For the stable production of cMAbs, we constructed 4 major different vector systems with a dihydrofolate reductase (DHFR) amplification marker, and optimized transfection/selection conditions in mammalian host cells with the single-gene and/or double-gene expression system. These 3 cMAbs were stably produced by the DHFR double mutant Chinese hamster ovarian (CHO)-DG44 cells. By ELISA and Western blot analysis using whole bacterial antigens treated by heat (65°C/90 min), sodium periodate, and proteinase K, the cMAb BP7 10B11 (cMAb CK1) reacted with glycoproteins (34, 38, 48 kDa in BP; 28, 38, 48 kDa in BM). The cMAb BP7 2C6 (cMAb CK2) recognized surface-capsule antigens with molecular sizes of 38 to 52 kDa, and 200 kDa in BM. The cMAb CK2 was weakly reactive to 14∼28, 200 kDa antigens in BP. The cMAb BP1 7F7 (cMAb CK3) reacted with lipopolysaccharides (38∼52 kDa in BP; 38∼60 kDa in B. thailandensis). Western blot results with the outer surface antigens of the 3 Burkholderia species were consistent with results with the whole Burkholderia cell antigens, suggesting that these immunodominant antigens reacting with the 3 cMAbs were primarily present on the outer surface of the Burkholderia species. These 3 cMAbs would be useful for analyzing the role of the major outer surface antigens in Burkholderia infection.

  19. Characterization of a laboratory-generated variant of BPS beta-lactamase from Burkholderia pseudomallei that hydrolyses ceftazidime.

    Science.gov (United States)

    Ho, P L; Cheung, Terence K M; Yam, W C; Yuen, K Y

    2002-11-01

    Burkholderia pseudomallei produces an Ambler class A beta-lactamase, known as BPS-1. The beta-lactamase gene from a laboratory-derived, ceftazidime-resistant strain of B. pseudomallei (LH-1-2) was cloned and expressed in Escherichia coli. The beta-lactamase, named BPS-1m, had an identical isoelectric focusing point (pI 7.7) to that of BPS-1, but differed in having a stronger hydrolytic activity against ceftazidime. Susceptibility testing showed that BPS-1m when expressed in E. coli conferred resistance to ceftazidime (MIC >or= 32 mg/L). The amino acid sequence of BPS-1m differed from that of BPS-1 by a Pro-to-Ser change at position 167 in the omega loop.

  20. Prevalence and Identification of Burkholderia pseudomallei and Near-Neighbor Species in the Malabar Coastal Region of India

    Science.gov (United States)

    Peddayelachagiri, Bhavani V.; Paul, Soumya; Nagaraj, Sowmya; Gogoi, Madhurjya; Sripathy, Murali H.; Batra, Harsh V.

    2016-01-01

    Accurate identification of pathogens with biowarfare importance requires detection tools that specifically differentiate them from near-neighbor species. Burkholderia pseudomallei, the causative agent of a fatal disease melioidosis, is one such biothreat agent whose differentiation from its near-neighbor species is always a challenge. This is because of its phenotypic similarity with other Burkholderia species which have a wide spread geographical distribution with shared environmental niches. Melioidosis is a major public health concern in endemic regions including Southeast Asia and northern Australia. In India, the disease is still considered to be emerging. Prevalence surveys of this saprophytic bacterium in environment are under-reported in the country. A major challenge in this case is the specific identification and differentiation of B. pseudomallei from the growing list of species of Burkholderia genus. The objectives of this study included examining the prevalence of B. pseudomallei and near-neighbor species in coastal region of South India and development of a novel detection tool for specific identification and differentiation of Burkholderia species. Briefly, we analyzed soil and water samples collected from Malabar coastal region of Kerala, South India for prevalence of B. pseudomallei. The presumptive Burkholderia isolates were identified using recA PCR assay. The recA PCR assay identified 22 of the total 40 presumptive isolates as Burkholderia strains (22.72% and 77.27% B. pseudomallei and non-pseudomallei Burkholderia respectively). In order to identify each isolate screened, we performed recA and 16S rDNA sequencing. This two genes sequencing revealed that the presumptive isolates included B. pseudomallei, non-pseudomallei Burkholderia as well as non-Burkholderia strains. Furthermore, a gene termed D-beta hydroxybutyrate dehydrogenase (bdha) was studied both in silico and in vitro for accurate detection of Burkholderia genus. The optimized bdha

  1. Prevalence and Identification of Burkholderia pseudomallei and Near-Neighbor Species in the Malabar Coastal Region of India.

    Directory of Open Access Journals (Sweden)

    Bhavani V Peddayelachagiri

    2016-09-01

    Full Text Available Accurate identification of pathogens with biowarfare importance requires detection tools that specifically differentiate them from near-neighbor species. Burkholderia pseudomallei, the causative agent of a fatal disease melioidosis, is one such biothreat agent whose differentiation from its near-neighbor species is always a challenge. This is because of its phenotypic similarity with other Burkholderia species which have a wide spread geographical distribution with shared environmental niches. Melioidosis is a major public health concern in endemic regions including Southeast Asia and northern Australia. In India, the disease is still considered to be emerging. Prevalence surveys of this saprophytic bacterium in environment are under-reported in the country. A major challenge in this case is the specific identification and differentiation of B. pseudomallei from the growing list of species of Burkholderia genus. The objectives of this study included examining the prevalence of B. pseudomallei and near-neighbor species in coastal region of South India and development of a novel detection tool for specific identification and differentiation of Burkholderia species. Briefly, we analyzed soil and water samples collected from Malabar coastal region of Kerala, South India for prevalence of B. pseudomallei. The presumptive Burkholderia isolates were identified using recA PCR assay. The recA PCR assay identified 22 of the total 40 presumptive isolates as Burkholderia strains (22.72% and 77.27% B. pseudomallei and non-pseudomallei Burkholderia respectively. In order to identify each isolate screened, we performed recA and 16S rDNA sequencing. This two genes sequencing revealed that the presumptive isolates included B. pseudomallei, non-pseudomallei Burkholderia as well as non-Burkholderia strains. Furthermore, a gene termed D-beta hydroxybutyrate dehydrogenase (bdha was studied both in silico and in vitro for accurate detection of Burkholderia genus. The

  2. Application of solid-phase extraction to agar-supported fermentation.

    Science.gov (United States)

    Le Goff, Géraldine; Adelin, Emilie; Cortial, Sylvie; Servy, Claudine; Ouazzani, Jamal

    2013-09-01

    Agar-supported fermentation (Ag-SF), a variant of solid-state fermentation, has recently been improved by the development of a dedicated 2 m(2) scale pilot facility, Platotex. We investigated the application of solid-phase extraction (SPE) to Ag-SF in order to increase yields and minimize the contamination of the extracts with agar constituents. The selection of the appropriate resin was conducted on liquid-state fermentation and Diaion HP-20 exhibited the highest recovery yield and selectivity for the metabolites of the model fungal strains Phomopsis sp. and Fusarium sp. SPE applied to Ag-SF resulted in a particular compartmentalization of the culture. The mycelium that requires oxygen to grow migrates to the top layer and formed a thick biofilm. The resin beads intercalate between the agar surface and the mycelium layer, and trap directly the compounds secreted by the mycelium through a "solid-solid extraction" (SSE) process. The resin/mycelium layer is easily recovered by scraping the surface and the target metabolites extracted by methanol. Ag-SF associated to SSE represents an ideal compromise for the production of bioactive secondary metabolites with limited economic and environmental impact.

  3. Crystallization and preliminary X-ray diffraction analysis of BipD, a virulence factor from Burkholderia pseudomallei

    Energy Technology Data Exchange (ETDEWEB)

    Knight, M. J.; Ruaux, A.; Mikolajek, H.; Erskine, P. T.; Gill, R.; Wood, S. P. [School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX (United Kingdom); Wood, M. [Institute of Animal Health, Division of Environmental Microbiology, Institute for Animal Health, Compton Laboratory, Berkshire RG20 7NN (United Kingdom); Cooper, J. B., E-mail: j.b.cooper@soton.ac.uk [School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX (United Kingdom)

    2006-08-01

    BipD is likely to be a component of a type-III protein secretion system (TTSS) in B. pseudomallei. Native and selenomethionyl-BipD proteins have been expressed and crystals have been obtained which diffract to 2.1 Å. Burkholderia pseudomallei, the causative agent of melioidosis, possesses a protein-secretion apparatus that is similar to those found in Salmonella and Shigella. A major function of these secretion systems is to secrete virulence-associated proteins into target cells of the host organism. The BipD gene of B. pseudomallei encodes a secreted virulence factor that is similar in sequence and most likely functionally analogous to IpaD from Shigella and SipD from Salmonella. Thus, the BipD protein is likely to be a component of a type III protein-secretion system (TTSS) in B. pseudomallei. Proteins in the same class as BipD, such as IpaD and SipD, are thought to act as extracellular chaperones to help the hydrophobic translocator proteins enter the target cell membrane, where they form a pore and might even link the translocon pore with the secretion needle. There is evidence that the translocator proteins also bind an integrin which stimulates actin-mediated insertion of the bacterium into the host-cell membrane. Native BipD has been crystallized in a monoclinic crystal form that diffracts X-rays to 2.5 Å resolution. BipD protein which incorporates selenomethionine (SeMet-BipD) has also been expressed and forms crystals which diffract to a higher resolution of 2.1 Å.

  4. Diverse Burkholderia Species Isolated from Soils in the Southern United States with No Evidence of B. pseudomallei.

    Science.gov (United States)

    Hall, Carina M; Busch, Joseph D; Shippy, Kenzie; Allender, Christopher J; Kaestli, Mirjam; Mayo, Mark; Sahl, Jason W; Schupp, James M; Colman, Rebecca E; Keim, Paul; Currie, Bart J; Wagner, David M

    2015-01-01

    The global distribution of the soil-dwelling bacterium Burkholderia pseudomallei, causative agent of melioidosis, is poorly understood. We used established culturing methods developed for B. pseudomallei to isolate Burkholderia species from soil collected at 18 sampling sites in three states in the southern United States (Arizona (n = 4), Florida (n = 7), and Louisiana (n = 7)). Using multi-locus sequence typing (MLST) of seven genes, we identified 35 Burkholderia isolates from these soil samples. All species belonged to the B. cepacia complex (Bcc), including B. cenocepacia, B. cepacia, B. contaminans, B. diffusa, B. metallica, B. seminalis, B. vietnamiensis and two unnamed members of the Bcc. The MLST analysis provided a high level of resolution among and within these species. Despite previous clinical cases within the U.S. involving B. pseudomallei and its close phylogenetic relatives, we did not isolate any of these taxa. The Bcc contains a number of opportunistic pathogens that cause infections in cystic fibrosis patients. Interestingly, we found that B. vietnamiensis was present in soil from all three states, suggesting it may be a common component in southern U.S. soils. Most of the Burkholderia isolates collected in this study were from Florida (30/35; 86%), which may be due to the combination of relatively moist, sandy, and acidic soils found there compared to the other two states. We also investigated one MLST gene, recA, for its ability to identify species within Burkholderia. A 365bp fragment of recA recovered nearly the same species-level identification as MLST, thus demonstrating its cost effective utility when conducting environmental surveys for Burkholderia. Although we did not find B. pseudomallei, our findings document that other diverse Burkholderia species are present in soils in the southern United States.

  5. Preparation of Burkholderia pseudomallei Polysaccharide-CRM197 Conjugate, a Potential Vaccine Candidate for Glanders and Melioidosis

    Science.gov (United States)

    2005-10-01

    Glanders Glanders is an infectious disease that is caused by the bacterium Burkholderia mallei The types of infection include localized, pus- forming...Glanders Burkholderia mallei and B. pseudomallei are the causative agents for glanders and melioidosis, respectively Both of these organisms have... virulence factor : – Dave DeShazer prepared a capsule mutant (DD3008) and demonstrated that the mouse aerosol LD50 was at least 103 times greater than the

  6. Burkholderia humptydooensis sp. nov., A Burkholderia thailandensis-Like Species and the Fifth Member of the pseudomallei Complex

    Science.gov (United States)

    2016-06-02

    2012). The type strain, MSMB43T, has been previously referred to as B. 312 Page 14 of 23 thailandensis-like species in multiple studies (Currie...closely related species were used to reconstruct the phylogenetic relationships. 339 Genomes from this study in bold and assembly numbers in...The In Vitro Antibiotic Susceptibility of Malaysian 379 Isolates of Burkholderia pseudomallei. Int J Microbiol, 2013, 121845. 380 BARNES, J. L

  7. Light transfer in agar immobilized microalgae cell cultures

    Science.gov (United States)

    Kandilian, Razmig; Jesus, Bruno; Legrand, Jack; Pilon, Laurent; Pruvost, Jérémy

    2017-09-01

    This paper experimentally and theoretically investigates light transfer in agar-immobilized cell cultures. Certain biotechnological applications such as production of metabolites secreted by photosynthetic microorganisms require cells to be immobilized in biopolymers to minimize contamination and to facilitate metabolite recovery. In such applications, light absorption by cells is one of the most important parameters affecting cell growth or metabolite productivity. Modeling light transfer therein can aid design and optimize immobilized-cell reactors. In this study, Parachlorella kessleri cells with areal biomass concentrations ranging from 0.36 to 16.9 g/m2 were immobilized in 2.6 mm thick agar gels. The average absorption and scattering cross-sections as well as the scattering phase function of P. kessleri cells were measured. Then, the absorption and transport scattering coefficients of the agar gel were determined using an inverse method based on the modified two-flux approximation. The forward model was used to predict the normal-hemispherical transmittance and reflectance of the immobilized-cell films accounting for absorption and scattering by both microalgae and the agar gel. Good agreement was found between the measured and predicted normal-hemispherical transmittance and reflectance provided absorption and scattering by agar were taken into account. Moreover, good agreement was found between experimentally measured and predicted mean rate of photon absorption. Finally, optimal areal biomass concentration was determined to achieve complete absorption of the incident radiation.

  8. Isolation and characterization of agar-digesting Vibrio species from the rotten thallus of Gracilariopsis heteroclada Zhang et Xia.

    Science.gov (United States)

    Martinez, Joval N; Padilla, Philip Ian P

    2016-08-01

    Gracilariopsis heteroclada Zhang et Xia (Gracilariaceae, Rhodophyta) is one of the most studied marine seaweeds due to its economic importance. This has been cultivated extensively on commercial scale in the Philippines and other Asian countries. However, sustainable production of G. heteroclada in the Philippines could not be maximized due to the occurrence of rotten thallus disease. Thus, isolation and characterization of agar-digesting bacteria from the rotten thalli of G. heteroclada was conducted. A total of seven representative bacterial isolates were randomly selected based on their ability to digest agar as evidenced by the formation of depressions around the bacterial colonies on nutrient agar plates supplemented with 1.5% NaCl and liquefaction of agar. Gram-staining and biochemical characterization revealed that isolates tested were gram-negative rods and taxonomically identified as Vibrio parahaemolyticus (86-99.5%) and Vibrio alginolyticus (94.2-97.7%), respectively. It is yet to be confirmed whether these agar-digesting vibrios are involved in the induction and development of rotten thallus disease in G. heteroclada in concomitance with other opportunistic bacterial pathogens coupled with adverse environmental conditions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Fly agaric (Amanita muscaria) poisoning, case report and review.

    Science.gov (United States)

    Satora, Leszek; Pach, Dorota; Butryn, Beata; Hydzik, Piotr; Balicka-Slusarczyk, Barbara

    2005-06-01

    Gathering and eating mushrooms and other plants containing psychoactive substances has become increasingly popular among young people experimenting with drugs. Dried fly agaric Amanita muscaria fruiting bodies were eaten by five young persons (18-21 years of age) at a party in order to evoke hallucinations. Visual and auditory hallucinations occurred in four of them, whereas a 18-year-old girl lost consciousness. The following morning, she went to the Clinic of Toxicology. Due to the fact that not all the active substances present in the fly agaric have been identified, and some of them have an effect after a period of latency, the patient was admitted for several days of observation during which check-up examinations were performed. After four days without any problems, she was discharged. The poisoning regressed with no organ complications. The remaining persons who had eaten the fly agaric were free from any complaints.

  10. Burkholderia pseudomallei Evades Nramp1 (Slc11a1- and NADPH Oxidase-Mediated Killing in Macrophages and Exhibits Nramp1-Dependent Virulence Gene Expression

    Directory of Open Access Journals (Sweden)

    Veerachat Muangsombut

    2017-08-01

    Full Text Available Bacterial survival in macrophages can be affected by the natural resistance-associated macrophage protein 1 (Nramp1; also known as solute carrier family 11 member a1 or Slc11a1 which localizes to phagosome membranes and transports divalent cations, including iron. Little is known about the role of Nramp1 in Burkholderia infection, in particular whether this differs for pathogenic species like Burkholderia pseudomallei causing melioidosis or non-pathogenic species like Burkholderia thailandensis. Here we show that transfected macrophages stably expressing wild-type Nramp1 (Nramp1+ control the net replication of B. thailandensis, but not B. pseudomallei. Control of B. thailandensis was associated with increased cytokine responses, and could be abrogated by blocking NADPH oxidase-mediated production of reactive oxygen species but not by blocking generation of reactive nitrogen species. The inability of Nramp1+ macrophages to control B. pseudomallei was associated with rapid escape of bacteria from phagosomes, as indicated by decreased co-localization with LAMP1 compared to B. thailandensis. A B. pseudomallei bipB mutant impaired in escape from phagosomes was controlled to a greater extent than the parent strain in Nramp1+ macrophages, but was also attenuated in Nramp1− cells. Consistent with reduced escape from phagosomes, B. thailandensis formed fewer multinucleated giant cells in Nramp1+ macrophages at later time points compared to B. pseudomallei. B. pseudomallei exhibited elevated transcription of virulence-associated genes of Type VI Secretion System cluster 1 (T6SS-1, the Bsa Type III Secretion System (T3SS-3 and the bimA gene required for actin-based motility in Nramp1+ macrophages. Nramp1+ macrophages were found to contain decreased iron levels that may impact on expression of such genes. Our data show that B. pseudomallei is able to evade Nramp1- and NADPH oxidase-mediated killing in macrophages and that expression of virulence

  11. Tellurite resistance profiles and performance of different chromogenic agars for detection of non-O157 Shiga toxin-producing Escherichia coli.

    Science.gov (United States)

    Fan, Ruyue; Bai, Xiangning; Fu, Shanshan; Xu, Yanmei; Sun, Hui; Wang, Hong; Xiong, Yanwen

    2018-02-02

    Shiga toxin-producing Escherichia coli (STEC) are globally important food-borne pathogens. The isolation of non-O157 STEC is a significant public health challenge due to the dramatic diversity of their phenotypes and genotypes. In the present study, 476 non-O157 STEC strains representing 95 different O-serogroups were used to evaluate tellurite resistance and the performance of 12 different chromogenic agars. Of 476 strains, only 108 (22.7%) strains showed the minimal inhibitory concentration (MIC) values for potassium tellurite being higher than 4μg/ml, and 96 (20.2%) strains harbored intact ter genes cluster. The presence of ter genes was significantly correlated with tellurite resistance. Six commercial chromogenic agars (TBX, MAC, SMAC, Rainbow® Agar O157, CHROMagar™ ECC, and Fluorocult O157) supported the growth of all strains. However, CT-SMAC, CHROMagar™ O157, and CHROMagar™ STEC agars exhibited 12.2%, 31.1%, and 38.0% of growth inhibition, respectively. Furthermore, 4.6%, 33.2%, and 45.0% of strains were inhibited on RBA-USDA, RBA-NT, and BCM O157 agar media. Variations in tellurite resistance and colony appearance might result in discrepant performance of non-O157 STEC recovery from different chromogenic agars. Using inclusive agars or less selective agar in combination with highly selective agar should be suggested to recover most non-O157 STEC strains, which would increase the probability of recovering STECs from complex background microflora. Copyright © 2018 Elsevier B.V. All rights reserved.

  12. Toll-like receptor 2 impairs host defense in gram-negative sepsis caused by Burkholderia pseudomallei (Melioidosis.

    Directory of Open Access Journals (Sweden)

    W Joost Wiersinga

    2007-07-01

    Full Text Available Toll-like receptors (TLRs are essential in host defense against pathogens by virtue of their capacity to detect microbes and initiate the immune response. TLR2 is seen as the most important receptor for gram-positive bacteria, while TLR4 is regarded as the gram-negative TLR. Melioidosis is a severe infection caused by the gram-negative bacterium, Burkholderia pseudomallei, that is endemic in Southeast Asia. We aimed to characterize the expression and function of TLRs in septic melioidosis.Patient studies: 34 patients with melioidosis demonstrated increased expression of CD14, TLR1, TLR2, and TLR4 on the cell surfaces of monocytes and granulocytes, and increased CD14, TLR1, TLR2, TLR4, LY96 (also known as MD-2, TLR5, and TLR10 mRNA levels in purified monocytes and granulocytes when compared with healthy controls. In vitro experiments: Whole-blood and alveolar macrophages obtained from TLR2 and TLR4 knockout (KO mice were less responsive to B. pseudomallei in vitro, whereas in the reverse experiment, transfection of HEK293 cells with either TLR2 or TLR4 rendered these cells responsive to this bacterium. In addition, the lipopolysaccharide (LPS of B. pseudomallei signals through TLR2 and not through TLR4. Mouse studies: Surprisingly, TLR4 KO mice were indistinguishable from wild-type mice with respect to bacterial outgrowth and survival in experimentally induced melioidosis. In contrast, TLR2 KO mice displayed a markedly improved host defenses as reflected by a strong survival advantage together with decreased bacterial loads, reduced lung inflammation, and less distant-organ injury.Patients with melioidosis displayed an up-regulation of multiple TLRs in peripheral blood monocytes and granulocytes. Although both TLR2 and TLR4 contribute to cellular responsiveness to B. pseudomallei in vitro, TLR2 detects the LPS of B. pseudomallei, and only TLR2 impacts on the immune response of the intact host in vivo. Inhibition of TLR2 may be a novel treatment

  13. Effects of season on the yield and quality of agar from Gelidium ...

    African Journals Online (AJOL)

    ... of 11.11 mPa.s (± 1.50 mPa.s.). In general, the physicochemical characteristics of the agar of G.sesquipedale are similar to those of the commercial agar, which leads to the conclusion that this alga can be a source of production of agar. Key words: Agarophytes, seaweed, agar, 3,6-anhydrogalactose, sulphate, pyruvate.

  14. Effect of BiTek agar on lysostaphin susceptibility of staphylococci.

    OpenAIRE

    Langlois, B E; Dawson, K.; Akers, K

    1990-01-01

    Staphylococci which were considered to be lysostaphin susceptible on P agar containing Bacto-Agar showed different degrees of resistance to lysostaphin when tested on P agar made with BiTek agar. As a result, lysostaphin-susceptible strains were misidentified as lysostaphin-resistant strains.

  15. Comparison between the antimicrobial susceptibility of Burkholderia pseudomallei to trimethoprim-sulfamethoxazole by standard disk diffusion method and by minimal inhibitory concentration determination.

    Science.gov (United States)

    Lumbiganon, P; Tattawasatra, U; Chetchotisakd, P; Wongratanacheewin, S; Thinkhamrop, B

    2000-08-01

    Melioidosis, an infection caused by Burkholderia pseudomallei, usually occurs in immunocompromised patients and requires prolonged antibiotic therapy. Previously, oral trimethoprim-sulfamethoxazole (TM/SM), an inexpensive and effective drug has been used as a maintenance therapy. The susceptibility of B. pseudomallei to TM/SM by the standard disk diffusion method is very low. However, some patients who were treated with TM/SM as a maintenance therapy despite the in vitro resistance showed good clinical responses. There were no data comparing the susceptibility of B. pseudomallei by the standard disk diffusion method with other quantitative susceptibility tests. The objective of this study was to determine the agreement between the antimicrobial susceptibility of B. pseudomallei to TM/SM by standard disk diffusion and minimal inhibitory concentration determination (MIC). We performed the susceptibility test of 144 strains of B. pseudomallei to TM/SM by both the standard disk diffusion and microbroth dilution MIC. The sensitivity results were 53.5 per cent and 84.0 per cent respectively. The agreement between the 2 tests was very poor (Kappa = 0.14; 95% CI = -0.01 to 0.29). The false resistant rate by the standard disk diffusion test was 67.9 per cent. Further in vitro susceptibility and clinical study are needed to define the interpretive criteria that correlate with clinical response.

  16. Role of Burkholderia pseudomallei Sigma N2 in Amino Acids Utilization and in Regulation of Catalase E Expression at the Transcriptional Level

    Directory of Open Access Journals (Sweden)

    Duong Thi Hong Diep

    2015-01-01

    Full Text Available Burkholderia pseudomallei is the causative agent of melioidosis. The complete genome sequences of this pathogen have been revealed, which explain some pathogenic mechanisms. In various hostile conditions, for example, during nitrogen and amino acid starvation, bacteria can utilize alternative sigma factors such as RpoS and RpoN to modulate genes expression for their adaptation and survival. In this study, we demonstrate that mutagenesis of rpoN2, which lies on chromosome 2 of B. pseudomallei and encodes a homologue of the sigma factor RpoN, did not alter nitrogen and amino acid utilization of the bacterium. However, introduction of B. pseudomallei rpoN2 into E. coli strain deficient for rpoN restored the ability to utilize amino acids. Moreover, comparative partial proteomic analysis of the B. pseudomallei wild type and its rpoN2 isogenic mutant was performed to elucidate its amino acids utilization property which was comparable to its function found in the complementation assay. By contrast, the rpoN2 mutant exhibited decreased katE expression at the transcriptional and translational levels. Our finding indicates that B. pseudomallei RpoN2 is involved in a specific function in the regulation of catalase E expression.

  17. Predicting the growth situation of Pseudomonas aeruginosa on agar plates and meat stuffs using gas sensors

    Science.gov (United States)

    Gu, Xinzhe; Sun, Ye; Tu, Kang; Dong, Qingli; Pan, Leiqing

    2016-12-01

    A rapid method of predicting the growing situation of Pseudomonas aeruginosa is presented. Gas sensors were used to acquire volatile compounds generated by P. aeruginosa on agar plates and meat stuffs. Then, optimal sensors were selected to simulate P. aeruginosa growth using modified Logistic and Gompertz equations by odor changes. The results showed that the responses of S8 or S10 yielded high coefficients of determination (R2) of 0.89-0.99 and low root mean square errors (RMSE) of 0.06-0.17 for P. aeruginosa growth, fitting the models on the agar plate. The responses of S9, S4 and the first principal component of 10 sensors fit well with the growth of P. aeruginosa inoculated in meat stored at 4 °C and 20 °C, with R2 of 0.73-0.96 and RMSE of 0.25-1.38. The correlation coefficients between the fitting models, as measured by electronic nose responses, and the colony counts of P. aeruginosa were high, ranging from 0.882 to 0.996 for both plate and meat samples. Also, gas chromatography-mass spectrometry results indicated the presence of specific volatiles of P. aeruginosa on agar plates. This work demonstrated an acceptable feasibility of using gas sensors—a rapid, easy and nondestructive method for predicting P. aeruginosa growth.

  18. Alternative plasticizers for the production of thermo-compressed agar films.

    Science.gov (United States)

    Sousa, Ana M M; Souza, Hiléia K S; Liu, LinShu; Gonçalves, Maria P

    2015-05-01

    Agar films were produced by thermo-compression using choline chloride (ChCl) as a plasticizer with urea. The three solid components were mixed together with the salt and urea (minor components) added to agar (main component) according to a fixed mass ratio of, respectively, 1.16:1:5. A central composite rotatable design (CCRD) with three parameters, 2(3), was used to evaluate the effects of temperature (X1; °C), time (X2; min) and applied load (X3; kN) of heat-pressing on the maximum tensile strength (TS) of the films (Y; MPa). Mixtures of urea and agar prepared at a mass ratio of 1:5 did not form homogeneous films suggesting the important plasticizing role of the salt. Heat-pressing the mixtures at more draconian conditions led to much darker and opaque films, with better mechanical resistance (higher values of TS). The most resistant film (∼ 15 MPa) was obtained at 140°C, 20 min and 176 kN. Selected films, including the optimal, showed similar water sorption profiles and close values of water vapor permeability (∼ 2.5-3.7 × 10(-9)gm(-1)s(-1)Pa(-1)). The fracture behavior and mechanical properties of the films were greatly affected by additional water plasticization when the films were stored at different conditions of relative humidity. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Comparison of eight different agars for the recovery of clinically relevant non-O157 Shiga toxin-producing Escherichia coli from baby spinach, cilantro, alfalfa sprouts and raw milk.

    Science.gov (United States)

    Kase, Julie A; Maounounen-Laasri, Anna; Son, Insook; Lin, Andrew; Hammack, Thomas S

    2015-04-01

    The FDA Bacteriological Analytical Manual (BAM) Chapter 4a recommends several agars for isolating non-O157 Shiga toxin-producing Escherichia coli (STEC); not all have been thoroughly tested for recovering STECs from food. Using E. coli strains representing ten clinically relevant O serogroups (O26, O45, O91, O103, O104, O111, O113, O121, O128, O145) in artificially-contaminated fresh produce--bagged baby spinach, alfalfa sprouts, cilantro, and raw milk--we evaluated the performance of 8 different agars. Performance was highly dependent upon strain used and the presence of inhibitors, but not necessarily dependent on food matrix. Tellurite resistant-negative strains, O91:-, O103:H6, O104:H21, O113:H21, and O128, grew poorly on CHROMagar STEC, Rainbow agar O157, and a modified Rainbow O157 (mRB) agar. Although adding washed sheep's blood to CHROMagar STEC and mRB agars improved overall performance; however, this also reversed the inhibition of non-target bacteria provided by original formulations. Variable colony coloration made selecting colonies from Rainbow agar O157 and mRB agars difficult. Study results support a strategy using inclusive agars (e.g. L-EMB, SHIBAM) in combination with selective agars (R & F E. coli O157:H7, CHROMagar STEC) to allow for recovery of the most STECs while increasing the probability of recovering STEC in high bacterial count matrices. Published by Elsevier Ltd.

  20. Assessment of Native Agar Gels Extracted from Gracilaria debilis ...

    African Journals Online (AJOL)

    PhD thesis. University of Dar es Salaam. Buriyo, A.S. & Kivaisi, A.K. (2003) Standing stock, agar yield and properties of Gracilaria salicornia harvested along the Tanzanian coast. Western. Indian Ocean J. Mar. Sci. 2: 171-178. Buriyo, A. S., Oliveira, E. C., Mtolera, M. S. P. &. Kivaisi, A. K. (2004). Taxonomic challenges and.

  1. Modified agar diffusion bioassay for better quantification of Nisaplin

    National Research Council Canada - National Science Library

    Lalpuria, M; Karwa, V; Anantheswaran, R.C; Floros, J.D

    2013-01-01

    To investigate the effect of different well sizes and pre-diffusion times at 4 °C, on the sensitivity, accuracy and precision of nisin quantification by agar diffusion bioassay. Nisin solution (0.625-125 μg ml(-1...

  2. Internal structure and thermo-viscoelastic properties of agar ionogels.

    Science.gov (United States)

    Sharma, Anshu; Rawat, Kamla; Solanki, Pratima R; Aswal, V K; Kohlbrecher, J; Bohidar, H B

    2015-12-10

    Ionic liquids (IL) can alter the physical properties of agar hydrogels. Rheology studies show that gels with wide range of storage moduli (gel strength) G0 values ranging from 1 to 20 KPa could be made in imidazolium based IL solutions where the IL concentration may not exceed 5% (w/v). Gelation and gel melting temperatures (tgel and Tm) could be altered by as much as ≈ 10 °C. Small angle neutron scattering studies revealed the presence of fibre bundles of agar double helices having typical length of 120 nm that increased to ≈ 180 nm under favorable conditions. These structures gain flexibility from the cladding of the agar bundles by IL molecules which in turn caused partial charge neutralization of its surface. Raman spectroscopy revealed differential hydration of these bundles. It was found that IL molecules with longer alkyl chain (more hydrophobic) altered the gel homogeneity, and changed its thermal and mechanical properties significantly. Therefore, customization of agar hydrogels in green solvent medium (IL solutions) widens the scope of its application potential that may include sensing. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Burkholderia pseudomallei Capsule Exacerbates Respiratory Melioidosis but Does Not Afford Protection against Antimicrobial Signaling or Bacterial Killing in Human Olfactory Ensheathing Cells.

    Science.gov (United States)

    Dando, Samantha J; Ipe, Deepak S; Batzloff, Michael; Sullivan, Matthew J; Crossman, David K; Crowley, Michael; Strong, Emily; Kyan, Stephanie; Leclercq, Sophie Y; Ekberg, Jenny A K; St John, James; Beacham, Ifor R; Ulett, Glen C

    2016-07-01

    Melioidosis, caused by the bacterium Burkholderia pseudomallei, is an often severe infection that regularly involves respiratory disease following inhalation exposure. Intranasal (i.n.) inoculation of mice represents an experimental approach used to study the contributions of bacterial capsular polysaccharide I (CPS I) to virulence during acute disease. We used aerosol delivery of B. pseudomallei to establish respiratory infection in mice and studied CPS I in the context of innate immune responses. CPS I improved B. pseudomallei survival in vivo and triggered multiple cytokine responses, neutrophil infiltration, and acute inflammatory histopathology in the spleen, liver, nasal-associated lymphoid tissue, and olfactory mucosa (OM). To further explore the role of the OM response to B. pseudomallei infection, we infected human olfactory ensheathing cells (OECs) in vitro and measured bacterial invasion and the cytokine responses induced following infection. Human OECs killed >90% of the B. pseudomallei in a CPS I-independent manner and exhibited an antibacterial cytokine response comprising granulocyte colony-stimulating factor, tumor necrosis factor alpha, and several regulatory cytokines. In-depth genome-wide transcriptomic profiling of the OEC response by RNA-Seq revealed a network of signaling pathways activated in OECs following infection involving a novel group of 378 genes that encode biological pathways controlling cellular movement, inflammation, immunological disease, and molecular transport. This represents the first antimicrobial program to be described in human OECs and establishes the extensive transcriptional defense network accessible in these cells. Collectively, these findings show a role for CPS I in B. pseudomallei survival in vivo following inhalation infection and the antibacterial signaling network that exists in human OM and OECs. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  4. Hair sheep blood, citrated or defibrinated, fulfills all requirements of blood agar for diagnostic microbiology laboratory tests.

    Directory of Open Access Journals (Sweden)

    Ellen Yeh

    Full Text Available BACKGROUND: Blood agar is used for the identification and antibiotic susceptibility testing of many bacterial pathogens. In the developing world, microbiologists use human blood agar because of the high cost and inhospitable conditions for raising wool sheep or horses to supply blood. Many pathogens either fail to grow entirely or exhibit morphologies and hemolytic patterns on human blood agar that confound colony recognition. Furthermore, human blood can be hazardous to handle due to HIV and hepatitis. This study investigated whether blood from hair sheep, a hardy, low-maintenance variety of sheep adapted for hot climates, was suitable for routine clinical microbiology studies. METHODS AND FINDINGS: Hair sheep blood obtained by jugular venipuncture was anticoagulated by either manual defibrination or collection in human blood bank bags containing citrate-phosphate-dextrose. Trypticase soy 5% blood agar was made from both forms of hair sheep blood and commercial defibrinated wool sheep blood. Growth characteristics, colony morphologies, and hemolytic patterns of selected human pathogens, including several streptococcal species, were evaluated. Specialized identification tests, including CAMP test, reverse CAMP test, and satellite colony formation with Haemophilus influenzae and Abiotrophia defectiva were also performed. Mueller-Hinton blood agar plates prepared from the three blood types were compared in antibiotic susceptibility tests by disk diffusion and E-test. CONCLUSIONS: The results of all studies showed that blood agar prepared from citrated hair sheep blood is suitable for microbiological tests used in routine identification and susceptibility profiling of human pathogens. The validation of citrated hair sheep blood eliminates the labor-intensive and equipment-requiring process of manual defibrination. Use of hair sheep blood, in lieu of human blood currently used by many developing world laboratories and as an alternative to cost

  5. Leveraging structure determination with fragment screening for infectious disease drug targets: MECP synthase from Burkholderia pseudomallei

    Energy Technology Data Exchange (ETDEWEB)

    Begley, Darren W.; Hartley, Robert C.; Davies, Douglas R.; Edwards, Thomas E.; Leonard, Jess T.; Abendroth, Jan; Burris, Courtney A.; Bhandari, Janhavi; Myler, Peter J.; Staker, Bart L.; Stewart, Lance J. (UWASH); (Emerald)

    2011-09-28

    As part of the Seattle Structural Genomics Center for Infectious Disease, we seek to enhance structural genomics with ligand-bound structure data which can serve as a blueprint for structure-based drug design. We have adapted fragment-based screening methods to our structural genomics pipeline to generate multiple ligand-bound structures of high priority drug targets from pathogenic organisms. In this study, we report fragment screening methods and structure determination results for 2C-methyl-D-erythritol-2,4-cyclo-diphosphate (MECP) synthase from Burkholderia pseudomallei, the gram-negative bacterium which causes melioidosis. Screening by nuclear magnetic resonance spectroscopy as well as crystal soaking followed by X-ray diffraction led to the identification of several small molecules which bind this enzyme in a critical metabolic pathway. A series of complex structures obtained with screening hits reveal distinct binding pockets and a range of small molecules which form complexes with the target. Additional soaks with these compounds further demonstrate a subset of fragments to only bind the protein when present in specific combinations. This ensemble of fragment-bound complexes illuminates several characteristics of MECP synthase, including a previously unknown binding surface external to the catalytic active site. These ligand-bound structures now serve to guide medicinal chemists and structural biologists in rational design of novel inhibitors for this enzyme.

  6. Deciphering minimal antigenic epitopes associated with Burkholderia pseudomallei and Burkholderia mallei lipopolysaccharide O-antigens.

    Science.gov (United States)

    Tamigney Kenfack, Marielle; Mazur, Marcelina; Nualnoi, Teerapat; Shaffer, Teresa L; Ngassimou, Abba; Blériot, Yves; Marrot, Jérôme; Marchetti, Roberta; Sintiprungrat, Kitisak; Chantratita, Narisara; Silipo, Alba; Molinaro, Antonio; AuCoin, David P; Burtnick, Mary N; Brett, Paul J; Gauthier, Charles

    2017-07-24

    Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm), the etiologic agents of melioidosis and glanders, respectively, cause severe disease in both humans and animals. Studies have highlighted the importance of Bp and Bm lipopolysaccharides (LPS) as vaccine candidates. Here we describe the synthesis of seven oligosaccharides as the minimal structures featuring all of the reported acetylation/methylation patterns associated with Bp and Bm LPS O-antigens (OAgs). Our approach is based on the conversion of an L-rhamnose into a 6-deoxy-L-talose residue at a late stage of the synthetic sequence. Using biochemical and biophysical methods, we demonstrate the binding of several Bp and Bm LPS-specific monoclonal antibodies with terminal OAg residues. Mice immunized with terminal disaccharide-CRM197 constructs produced high-titer antibody responses that crossreacted with Bm-like OAgs. Collectively, these studies serve as foundation for the development of novel therapeutics, diagnostics, and vaccine candidates to combat diseases caused by Bp and Bm.Melioidosis and glanders are multifaceted infections caused by gram-negative bacteria. Here, the authors synthesize a series of oligosaccharides that mimic the lipopolysaccharides present on the pathogens' surface and use them to develop novel glycoconjugates for vaccine development.

  7. Functional characterisation of Burkholderia pseudomallei biotin protein ligase: A toolkit for anti-melioidosis drug development.

    Science.gov (United States)

    Bond, Thomas E H; Sorenson, Alanna E; Schaeffer, Patrick M

    2017-06-01

    Burkholderia pseudomallei (Bp) is the causative agent of melioidosis. The bacterium is responsible for 20% of community-acquired sepsis cases and 40% of sepsis-related mortalities in northeast Thailand, and is intrinsically resistant to aminoglycosides, macrolides, rifamycins, cephalosporins, and nonureidopenicillins. There is no vaccine and its diagnosis is problematic. Biotin protein ligase (BirA) which is essential for fatty acid synthesis has been proposed as a drug target in bacteria. Very few bacterial BirA have been characterized, and a better understanding of these enzymes is necessary to further assess their value as drug targets. BirA within the Burkholderia genus have not yet been investigated. We present for the first time the cloning, expression, purification and functional characterisation of the putative Bp BirA and orthologous B. thailandensis (Bt) biotin carboxyl carrier protein (BCCP) substrate. A GFP-tagged Bp BirA was produced and applied for the development of a high-throughput (HT) assay based on our differential scanning fluorimetry of GFP-tagged proteins (DSF-GTP) principle as well as an electrophoretic mobility shift assay. Our biochemical data in combination with the new HT DSF-GTP and biotinylation activity assay could facilitate future drug screening efforts against this drug-resistant organism. Copyright © 2017 Elsevier GmbH. All rights reserved.

  8. Crystal structures of Cif from bacterial pathogens Photorhabdus luminescens and Burkholderia pseudomallei.

    Science.gov (United States)

    Crow, Allister; Race, Paul R; Jubelin, Grégory; Varela Chavez, Carolina; Escoubas, Jean-Michel; Oswald, Eric; Banfield, Mark J

    2009-01-01

    A pre-requisite for bacterial pathogenesis is the successful interaction of a pathogen with a host. One mechanism used by a broad range of Gram negative bacterial pathogens is to deliver effector proteins directly into host cells through a dedicated type III secretion system where they modulate host cell function. The cycle inhibiting factor (Cif) family of effector proteins, identified in a growing number of pathogens that harbour functional type III secretion systems and have a wide host range, arrest the eukaryotic cell cycle. Here, the crystal structures of Cifs from the insect pathogen/nematode symbiont Photorhabdus luminescens (a gamma-proteobacterium) and human pathogen Burkholderia pseudomallei (a beta-proteobacterium) are presented. Both of these proteins adopt an overall fold similar to the papain sub-family of cysteine proteases, as originally identified in the structure of a truncated form of Cif from Enteropathogenic E. coli (EPEC), despite sharing only limited sequence identity. The structure of an N-terminal region, referred to here as the 'tail-domain' (absent in the EPEC Cif structure), suggests a surface likely to be involved in host-cell substrate recognition. The conformation of the Cys-His-Gln catalytic triad is retained, and the essential cysteine is exposed to solvent and addressable by small molecule reagents. These structures and biochemical work contribute to the rapidly expanding literature on Cifs, and direct further studies to better understand the molecular details of the activity of these proteins.

  9. Evaluation of Mannitol Salt Agar for Detection of Oxacillin Resistance in Staphylococcus aureus by Disk Diffusion and Agar Screening

    OpenAIRE

    Kampf, Günter; Lecke, Christoph; Cimbal, Ann-Katrin; Weist, Klaus; Rüden, Henning

    1998-01-01

    Mannitol salt agar was evaluated for detection of oxacillin resistance in 136 Staphylococcus aureus isolates. All mecA-positive isolates (n = 54) were correctly categorized as oxacillin resistant by the disk diffusion test (1-μg disk; zone diameter,

  10. Combination cellulose plate (non-agar solid support) and agar plate method improves isolation of fungi from soil.

    Science.gov (United States)

    Nonaka, Kenichi; Todaka, Nemuri; Ōmura, Satoshi; Masuma, Rokuro

    2014-11-01

    This is the first report describing the improved isolation of common filamentous fungi via a method combining cellulose plate and agar plate system. A cellulose plate is a porous plate made of nanofibrous crystaline cellulose. Isolating fungi from soils using these types of media separately resulted in the number of fungal colonies appearing on cellulose plates being lower than that on agar plates. However, the number of actual fungal species isolated using cellulose plates alone was more or less the same as that found using agar plates. Significantly, the diversity of isolates using a combination of the two media was greater than using each media individually. As a result, numerous new or rare fungal species with potential, including previously proposed new species, were isolated successfully in this way. All fungal colonies, including the Penicillium species, that appeared on the cellulose plate penetrated in potato dextrose were either white or yellow. Cultivation on cellulose plates with added copper ion overcomes the change in coloration, the colonies appearing as they do following cultivation on potato dextrose agar.

  11. Actin-Binding Proteins from Burkholderia mallei and Burkholderia thailandensis Can Functionally Compensate for the Actin-Based Motility Defect of a Burkholderia pseudomallei bimA Mutant

    OpenAIRE

    Stevens, J M; Ulrich, R L; Taylor, L A; Wood, M W; DeShazer, D; Stevens, M P; Galyov, E E

    2005-01-01

    Recently we identified a bacterial factor (BimA) required for actin-based motility of Burkholderia pseudomallei. Here we report that Burkholderia mallei and Burkholderia thailandensis are capable of actin-based motility in J774.2 cells and that BimA homologs of these bacteria can restore the actin-based motility defect of a B. pseudomallei bimA mutant. While the BimA homologs differ in their amino-terminal sequence, they interact directly with actin in vitro and vary in their ability to bind ...

  12. Discrimination of Pathogenic vs. Nonpathogenic Francisella tularensis and Burkholderia pseudomallei Using Proteomics Mass Spectrometry

    Science.gov (United States)

    2011-03-01

    F. tularensis LVS and B. thailandensis E264. Working cultures were prepared by streaking cells from cryopreserved stocks onto chocolate agar (CA...Escherichia coli and Salmonella : cellular and molecular biology, 2nd ed. ASM Press, Washington, DC. 3. Haake, D.A. 2000. Spirochaetal lipoproteins and

  13. Pathogenesis of percutaneous infection of goats with Burkholderia pseudomallei: clinical, pathologic, and immunological responses in chronic melioidosis

    Science.gov (United States)

    Soffler, Carl; Bosco-Lauth, Angela M; Aboellail, Tawfik A; Marolf, Angela J; Bowen, Richard A

    2014-01-01

    Melioidosis is a severe suppurative to granulomatous infection caused by Burkholderia pseudomallei. The disease is endemic to South-East Asia and Northern Australasia and is also of interest as a potential biological weapon. Natural infection can occur by percutaneous inoculation, inhalation or ingestion, but the relative importance of each route is unknown. Experimental infection models using mice have shown inhalation to be the most lethal route of exposure, but few studies have examined the pathogenesis of percutaneous infection despite its presumptive importance in natural disease. Caprine models are useful in the study of melioidosis because goats are susceptible to natural infection by B. pseudomallei, display similar epizootiology/epidemiology to that of humans within the endemic range and develop similar pathologic lesions. Percutaneous inoculation with 104 CFU of B. pseudomallei produced disease in all experimental animals with rapid dissemination to the lungs, spleen and kidneys. Initial fever was brief, but temperatures did not return to pre-infection levels until day 18, concurrent with a dramatic lymphocytosis and the transition to chronic disease. Distribution and appearance of gross pathologic and radiographic lesions in goats were similar to caprine aerosol infection and to reported human disease. The similarities seen despite different routes of infection suggest that host or bacterial factors may be more important than the route of infection in disease pathogenesis. The nature of melioidosis in goats makes it amenable for modelling additional risk factors to produce acute clinical disease, which is important to the study of human melioidosis. PMID:24571408

  14. BipC, a Predicted Burkholderia pseudomallei Type 3 Secretion System Translocator Protein with Actin Binding Activity

    Directory of Open Access Journals (Sweden)

    Charles W. Vander Broek

    2017-07-01

    Full Text Available Burkholderia pseudomallei is an intracellular bacterial pathogen and the causative agent of melioidosis, a severe disease of humans and animals. Like other clinically important Gram-negative bacteria, fundamental to B. pseudomallei pathogenesis is the Bsa Type III Secretion System. The Bsa system injects bacterial effector proteins into the cytoplasm of target host cells subverting cellular pathways for the benefit of the bacteria. It is required for invasion of non-phagocytic host cells, escape from the endocytic compartment into the host cell cytoplasm, and for virulence in murine models of melioidosis. We have recently described the repertoire of effector proteins secreted by the B. pseudomallei Bsa system, however the functions of many of these effector proteins remain an enigma. One such protein is BipC, a homolog of the translocator/effector proteins SipC and IpaC from Salmonella spp. and Shigella flexneri respectively. SipC and IpaC each have separate and distinct roles acting both as translocators, involved in creating a pore in the eukaryotic cell membrane through which effector proteins can transit, and as effectors by interacting with and polymerizing host cell actin. In this study, pull-down assays demonstrate an interaction between BipC and actin. Furthermore, we show that BipC directly interacts with actin, preferentially with actin polymers (F-actin and has the ability to polymerize actin in a similar manner as that described for SipC. Yet unlike SipC, BipC does not stabilize F-actin filaments, indicating a functionally distinct interaction with actin. Expression of Myc-tagged BipC in HeLa cells induces the formation of pseudopodia similar to that seen for IpaC. This study explores the effector function of BipC and reveals that actin interaction is conserved within the BipC/SipC/IpaC family of translocator/effector proteins.

  15. Performance validation of chromogenic coliform agar for the enumeration of Escherichia coli and coliform bacteria.

    Science.gov (United States)

    Lange, B; Strathmann, M; Oßmer, R

    2013-12-01

    The performance of chromogenic coliform agar (CCA) for the enumeration of Escherichia coli and coliform bacteria was validated according to ENV ISO 13843 using pure cultures and naturally contaminated water samples. The results indicate that for the detection of E. coli and coliform bacteria, respectively, the method is sensitive (94 and 91%), specific (97 and 94%), selective (selectivity -0·78 and -0·32) and efficient (96 and 92%). Relative recovery of E. coli and coliform bacteria on CCA in comparison with tryptone soy agar (TSA) was good (104 and 94% in mean, >80 and >70% in all cases), and repeatability and reproducibility were sufficient. The linear working range was defined for 10-100 total target colonies per 47-mm membrane filter. A high precision of the method was confirmed by low overdispersion in comparison with Poisson distribution. The robustness of the method with respect to the variable incubation time of 21 ± 3 h was found to be low, because an incidental increase in presumptive colonies especially between 18 and 21 h was observed. In conclusion, the CCA method was proved as a reliable method for the quantification of E. coli and coliform bacteria. The international standard for the detection and enumeration of E. coli and coliform bacteria by membrane filtration (ISO 9308-1) is currently under revision and will be published in 2014. In the new standard, lactose-triphenyl tetrazolium chloride (TTC) agar will be replaced by a CCA. A performance validation of this revised method according to ENV ISO 13843 is presented in this study to determine fundamental data on its applicability and to provide reference data for secondary validation by users of this method. © 2013 The Society for Applied Microbiology.

  16. Corn and potato starch as an agar alternative for Solanum ...

    African Journals Online (AJOL)

    Potato single nodes were subcultured onto fresh MS medium gelled with 0, 1 and 2 g/l of agar + 40, 50 or 60 g/l of commercial corn and potato starch (CS or PS, respectively). After 4 weeks of culture, the pH of medium supplemented with 50 or 60 g/l of CS or 60 g/l of PS was significantly decreased to 3.91 - 4.00.

  17. Water vapor adsorption isotherms of agar-based nanocomposite films.

    Science.gov (United States)

    Rhim, Jong-Whan

    2011-10-01

    Adsorption isotherms of agar and agar/clay nanocomposite films prepared with different types of nanoclays, that is, a natural montmorillonite (Cloisite Na(+) ) and 2 organically modified montmorillonites (Cloisite 30B and Cloisite 20A), were determined at 3 different temperatures (10, 25, and 40 °C). The water vapor adsorption behavior of the nanocomposite films was found to be greatly influenced with the type of clay. The Guggenheim-Anderson-de Boer (GAB) isotherm model parameters were estimated by using both polynomial regression and nonlinear regression methods and it was found that the GAB model fitted adequately for describing experimental adsorption isotherm data for the film samples. The monolayer moisture content (m(o) ) of the film samples was also greatly affected by the type of nanoclay used, that is, m(o) of nanocomposite films was significantly lower than that of the neat agar film. Nanocomposite films prepared with hydrophobic nanoclays (Cloisite 30B and Cloisite 20A) exhibited lower m(o) values than those prepared with hydrophilic nanoclay (Cloisite Na(+) ). © 2011 Institute of Food Technologists®

  18. Synergistic enzymatic saccharification and fermentation of agar for biohydrogen production.

    Science.gov (United States)

    Wu, Yi-Rui; Zhang, Mingming; Zhong, Mingqi; Hu, Zhong

    2017-10-01

    Nowadays, marine biomass is gradually considered as another utilizable material for the sustainable bioenergy development. In the present study, galactose, the main component of agar polysaccharide, was utilized for the biohydrogen production by Enterobacter sp. CN1. The highest hydrogen yield of 303.2mL/g was obtained in the cultivation media containing 5.87g/L of galactose, together with initial pH of 7.3 and incubation temperature of 36°C, after the response surface methodology (RSM) analysis. After the saccharification process by the agarase (AgaXa) and neoagarobiose hydrolase (NH852), the agar hydrolysate obtained was further applied to generate biohydrogen by strain CN1. Under the synergistic enzymatic saccharification and fermentation process, the production of biohydrogen was obtained to be 5047±228mL/L from 50g/L of agar, resulting in 3.86-fold higher than the control without enzymatic pretreatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Use of electron beam on aflatoxins degradation in coconut agar

    Energy Technology Data Exchange (ETDEWEB)

    Rogovschi, Vladimir D.; Nunes, Thaise C.F.; Villavicencio, Anna L.C.H. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)], e-mail: vrogovschi@ipen.br; Aquino, Simone; Goncalez, Edlayne [Instituto Biologico (IB-SP), Sao Paulo, SP (Brazil); Correa, Benedito [Universidade de Sao Paulo (USP), SP (Brazil). Inst. de Ciencias Biomedicas

    2009-07-01

    The fungi Aspergillus flavus are capable of producing toxic metabolites, such as aflatoxin, that is one of the most important human carcinogens, according to the 'International Agency for Research on Cancer'. The aim of this study was to compare the effect of electron beam irradiation on degradation of aflatoxin B1 present in laboratorial residues with a dose of 0 kGy and 5.0 kGy. The fungi were cultivated in potato dextrose agar (PDA) for 7 days and transferred to a coconut agar medium, incubated at a temperature of 25 deg C for 14 days to produce the laboratorial wastes (coconut agar) containing aflatoxins. The samples were conditioned in petri dish for radiation treatment of contaminated material and processed in the Electron Accelerator with 0 kGy and 5.0 kGy. Aflatoxin B{sub 1} was extracted with chloroform and separated on a thin layer chromatography plate (TLC) with chloroform: acetone (9:1). All the control and irradiated samples were analyzed in a Shimadzu Densitometer. The detection limit of this methodology is 0.1{mu}g kg{sup -1}. The results indicate that the irradiated samples had a reduction of 75.49 % in the analyzed dose. (author)

  20. Enzymatic desulfation of the red seaweeds agar by Marinomonas arylsulfatase.

    Science.gov (United States)

    Wang, Xueyan; Duan, Delin; Fu, Xiaoting

    2016-12-01

    Agar and sulfated galactans were isolated from the red seaweeds Gracilariopsis lemaneiformis and Gelidium amansii. A previously purified arylsulfatase from Marinomonas sp. FW-1 was used to remove sulfate groups in agar and sulfated galactans. After enzymatic desulfation, the sulfate content decreased to about 0.16% and gel strength increased about two folds. Moreover, there was no difference between the DNA electrophoresis spectrum on the gel of the arylsulfatase-treated agar and that of the commercial agarose. In order to reveal the desulfation ratio and site, chemical and structural identification of sulfated galactan were carried out. G. amansii sulfated galactan with 7.4% sulfated content was composed of galactose and 3,6-anhydro-l-galactose. Meanwhile, G. lemaneiformis sulfated galactan with 8.5% sulfated content was composed of galactose, 3,6-anhydro-l-galactose, 2-O-methyl-3,6-anhydro-l-galactose and xylose. Data from (13)C NMR, FT-IR, GC-MS provided evidence of sulfate groups at C-4 and C-6 of d-galactose and C-6 of l-galactose both in GRAP and GEAP. Data from GC-MS revealed that desulfation was carried out by the arylsulfatase at the sulfate bonds at C-4 and C-6 of d-galactose and C-6 of l-galactose, with a desulfation ratio of 83.4% and 86.0% against GEAP and GRAP, respectively. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Agar Plates Made from Common Supermarket Substances and Bacillus subtilis Natto as an Inexpensive Approach to Microbiology Education

    Directory of Open Access Journals (Sweden)

    Franz-Josef Scharfenberg

    2015-08-01

    Full Text Available To address the possible limitations that financial restrictions may have on students’ independent experimentation at school, we developed and implemented an inexpensive approach for basic microbiology education. We describe four nutrient agars consisting only of everyday substances available from the supermarket or online that we developed to replace standard agars and specific agars. Additionally, we selected Bacillus subtilis natto as an example of a pure-culture species. Our tip first reports the four supermarket-substance agar variants; second, it suggests utilizing them to introduce basic microbiological techniques; and third, it introduces B. subtilis natto in the context of the antibacterial effects of antibiotics as well as of supermarket products which students can bring to class from home. We implemented our approach in microbiology education at school as well as in pre-service teacher education and in in-service teacher professional development courses at our university. Finally, our paper provides worksheets for all the experiments. Editor's Note:The ASM advocates that students must successfully demonstrate the ability to explain and practice safe laboratory techniques. For more information, read the laboratory safety section of the ASM Curriculum Recommendations: Introductory Course in Microbiology and the Guidelines for Biosafety in Teaching Laboratories, available at www.asm.org. The Editors of JMBE recommend that adopters of the protocols included in this article follow a minimum of Biosafety Level 1 practices. If the soil plates described in the activity are opened, a minimum of Biosafety Level 2 is required.

  2. Development of agar diffusion method for dosage of gramicidin

    Directory of Open Access Journals (Sweden)

    Ana Gabriela Reis Solano

    2011-09-01

    Full Text Available Gramicidin, an antimicrobial peptide active against Gram positive bacteria, is commonly used in pharmaceutical preparations for topical use. Considering that only the turbidimetric method has been described in the literature, the present study sought to develop and validate an agar diffusion method for the dosage of gramicidin. The method was developed and validated using the Kocuria rhizophila ATCC 9341 as a test microorganism. Two designs were used: a 3x3 parallel-line model, and a 5x1 standard curve. The validation demonstrated that the method follows the linear model (r²= 0.994, presenting a significant regression between the zone diameter of growth inhibition and the logarithm of the concentration within the range of 5 to 25.3 µg/mL. The results obtained for both designs were precise, having a relative standard deviation (R.S.D. for intra-day precision of 0.81 for the 3x3 assay and 1.90 for the 5x1 assay. For the inter-day precision, the R.S.D. was 1.35 for the 3x3 and 2.64 for the 5x1. The accuracy was verified and results confirmed to be accurate, having a tolerance interval of 95%, which lay within permitted limits and appropriate trueness. In addition, the method was considered selective, with limit of detection and upper and lower limits of quantification of 2.00, 5.00 and 25.3 µg/mL, respectively. No difference in precision between the designs used in the agar diffusion method was evident (p>0.05. The method proved to be appropriate for the microbiological dosage of the raw material gramicidin.A gramicidina, um peptídeo antimicrobiano ativo contra bactérias Gram positivo, é utilizada em preparações farmacêuticas de uso tópico. Neste trabalho procurou-se desenvolver e validar outro método para o doseamento de gramicidina tendo em vista que somente o método turbidimétrico é descrito. O método de difusão em ágar foi desenvolvido e validado utilizando como microrganismo teste Kocuria rhizophila ATCC 9341. Foram utilizados

  3. Comparing in vitro and in vivo virulence phenotypes of Burkholderia pseudomallei type G strains.

    Science.gov (United States)

    Lewis, Eric R G; Kilgore, Paul B; Mott, Tiffany M; Pradenas, Gonzalo A; Torres, Alfredo G

    2017-01-01

    Burkholderia pseudomallei (Bpm) is a saprophytic rod-shaped gram-negative bacterium and the causative agent of melioidosis. This disease has previously been described as endemic in areas such as northern Australia and Southeast Asia, but, more recently, a better understanding of the epidemiology of melioidosis indicated that the disease is distributed worldwide, including regions of the Americas and Africa. A 16S-23S rDNA internal transcribed spacer (ITS) typing system has been developed for Bpm and has revealed that ITS types C, E, and hybrid CE are mainly associated with Australia and Southeast Asia while type G strains are more associated with cases of melioidosis in the Western Hemisphere. The purpose of the current study was to determine the in vitro and in vivo virulence profiles of the understudied Bpm type G strains Ca2009, Ca2013a, Mx2013, and 724644 and compared such phenotypes to the commonly studied Bpm type C strain K96243. We evaluated virulence by measuring invasion/uptake and survival of these Bpm strains in murine respiratory epithelial LA-4 cells and alveolar macrophage MH-S cells using different multiplicity of infections (MOIs of 1 and 10). We also calculated the lethal dose 50 values (LD50) in BALB/c mice that were inoculated intranasally with either Ca2009, Ca2013a, or Mx2013. Overall, the virulence and lethality phenotypes of Bpm type G strains were similar to the Bpm type C strain K96243. Additional comparative analyses between the Bpm ITS types may lead to a better understanding of the contribution of the ITS type to the epidemiology and ecology of Bpm strains.

  4. Agar-agar entrapment increases the stability of endo-β-1,4-xylanase for repeated biodegradation of xylan.

    Science.gov (United States)

    Bibi, Zainab; Shahid, Faiza; Ul Qader, Shah Ali; Aman, Afsheen

    2015-04-01

    Microbial xylanases, specially endo-β-1,4-xylanase catalyzes the hydrolysis of xylan, is considered one of the most significant hydrolases. It has numerous applications but most extensively is utilized in paper and pulp industry as a bio-bleaching agent. Immobilization technique is comprehensively studied with the expectation of modifying and improving enzyme stability and characteristics for commercial purposes. Currently, matrix entrapment technique is applied to immobilize endo-β-1,4-xylanase within agar-agar gel beads produced by Geobacillus stearothermophilus KIBGE-IB29. Maximal enzyme immobilization yield was achieved at 2.5% of agar-agar concentration. Optimized conditions demonstrated an increase in the optimal reaction time from 05 min to 30 min and incubation temperature from 50 °C to 60 °C with reference to free enzyme whereas; no effect was observed for optimum pH. Entrapment technique uniquely changed the kinetic parameters of immobilized endo-β-1,4-xylanase (Km: 0.5074 mg min(-1) to 0.5230 mg min(-1) and Vmax: 4773 U min(-1) to 968 U min(-1)) as compared to free enzyme. However, immobilized enzyme displayed broad thermal stability and retained 79.0% of its initial activity at 80 °C up to 30 min whereas; free enzyme completely lost its activity at this temperature. With respect to economic feasibility, the immobilized enzyme showed impressive recycling efficiency up to six reaction cycles. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Alternative to the soft-agar assay that permits high-throughput drug and genetic screens for cellular transformation

    Science.gov (United States)

    Rotem, Asaf; Janzer, Andreas; Izar, Benjamin; Ji, Zhe; Doench, John G.; Garraway, Levi A.; Struhl, Kevin

    2015-01-01

    Colony formation in soft agar is the gold-standard assay for cellular transformation in vitro, but it is unsuited for high-throughput screening. Here, we describe an assay for cellular transformation that involves growth in low attachment (GILA) conditions and is strongly correlated with the soft-agar assay. Using GILA, we describe high-throughput screens for drugs and genes that selectively inhibit or increase transformation, but not proliferation. Such molecules are unlikely to be found through conventional drug screening, and they include kinase inhibitors and drugs for noncancer diseases. In addition to known oncogenes, the genetic screen identifies genes that contribute to cellular transformation. Lastly, we demonstrate the ability of Food and Drug Administration-approved noncancer drugs to selectively kill ovarian cancer cells derived from patients with chemotherapy-resistant disease, suggesting this approach may provide useful information for personalized cancer treatment. PMID:25902495

  6. Use of a Real-Time PCR TaqMan Assay for Rapid Identification and Differentiation of Burkholderia pseudomallei and Burkholderia mallei

    OpenAIRE

    U?Ren, Jana M.; Van Ert, Matthew N.; Schupp, James M.; Easterday, W Ryan; Simonson, Tatum S.; Okinaka, Richard T.; Pearson, Talima; Keim, Paul

    2005-01-01

    A TaqMan allelic-discrimination assay designed around a synonymous single-nucleotide polymorphism was used to genotype Burkholderia pseudomallei and Burkholderia mallei isolates. The assay rapidly identifies and discriminates between these two highly pathogenic bacteria and does not cross-react with genetic near neighbors, such as Burkholderia thailandensis and Burkholderia cepacia.

  7. Development of a multiplex PCR assay for the detection and differentiation of Burkholderia pseudomallei, Burkholderia mallei, Burkholderia thailandensis, and Burkholderia cepacia complex.

    Science.gov (United States)

    Zakharova, Irina; Teteryatnikova, Natalya; Toporkov, Andrey; Viktorov, Dmitry

    2017-10-01

    Two species of Burkholderia pseudomallei complex (Bpc), B. pseudomallei and B. mallei, can cause severe life-threatening infections. Rapidly discerning individual species within the group and separating them from other opportunistic pathogens of the Burkholderia cepacia complex (Bcc) is essential to establish a correct diagnosis and for epidemiological surveillance. In this study, a multiplex PCR assay based on the detection of an individual set of chromosomal beta-lactamase genes for single-step identification and differentiation of B. pseudomallei, B. mallei, B. thailandensis, and Bcc was developed. Two pairs of primers specific to a distinct class of B metallo-beta-lactamase genes and a pair of primers specific to the oxacillin-hydrolyzing class D beta-lactamase gene were demonstrated to successfully discriminate species within Bpc and from Bcc. The assay sensitivity was 9561 genomic equivalents (GE) for B. pseudomallei, 7827 GE for B. mallei, 8749 GE for B. thailandensis and 6023 GE for B. cepacia. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Improved toluidine blue-DNA agar for detection of DNA hydrolysis by campylobacters.

    OpenAIRE

    Lior, H.; PATEL, A.

    1987-01-01

    Our improved toluidine blue-DNA agar was compared with methyl green DNase test agar for the detection of DNA hydrolysis by campylobacters. The toluidine blue-DNA agar gave clear-cut positive and negative reactions with campylobacter strains belonging to several species.

  9. Macrophage and Galleria mellonella infection models reflect the virulence of naturally occurring isolates of B. pseudomallei, B. thailandensis and B. oklahomensis

    Directory of Open Access Journals (Sweden)

    Michell Stephen L

    2011-01-01

    Full Text Available Abstract Background Burkholderia pseudomallei is the causative agent of melioidosis, a tropical disease of humans with a variable and often fatal outcome. In murine models of infection, different strains exhibit varying degrees of virulence. In contrast, two related species, B. thailandensis and B. oklahomensis, are highly attenuated in mice. Our aim was to determine whether virulence in mice is reflected in macrophage or wax moth larvae (Galleria mellonella infection models. Results B. pseudomallei strains 576 and K96243, which have low median lethal dose (MLD values in mice, were able to replicate and induce cellular damage in macrophages and caused rapid death of G. mellonella. In contrast, B. pseudomallei strain 708a, which is attenuated in mice, showed reduced replication in macrophages, negligible cellular damage and was avirulent in G. mellonella larvae. B. thailandensis isolates were less virulent than B. pseudomallei in all of the models tested. However, we did record strain dependent differences. B. oklahomensis isolates were the least virulent isolates. They showed minimal ability to replicate in macrophages, were unable to evoke actin-based motility or to form multinucleated giant cells and were markedly attenuated in G. mellonella compared to B. thailandensis. Conclusions We have shown that the alternative infection models tested here, namely macrophages and Galleria mellonella, are able to distinguish between strains of B. pseudomallei, B. thailandensis and B. oklahomensis and that these differences reflect the observed virulence in murine infection models. Our results indicate that B. oklahomensis is the least pathogenic of the species investigated. They also show a correlation between isolates of B. thailandensis associated with human infection and virulence in macrophage and Galleria infection models.

  10. Hydrological connectivity and Burkholderia pseudomallei prevalence in wetland environments: investigating rice-farming community's risk of exposure to melioidosis in North-East Thailand.

    Science.gov (United States)

    Chuah, C Joon; Tan, Esther K H; Sermswan, Rasana W; Ziegler, Alan D

    2017-06-01

    In our analysis of 136 water samples from wetland environments (rice paddies, natural wetland sites, man-made water bodies) in rural areas of North-East Thailand, Burkholderia pseudomallei was most prevalent in rice paddies (15 of the 30 positive sites). The high prevalence in the water of rice fields is indicative of the inherent vulnerability of farmers in rural agricultural areas in this area of Thailand and likely other locations in the tropics. Nearly all B. pseudomallei-positive sites were found within the vicinity of a large wetland associated with the Chi River, in the month of July 2014. Positive samples were found in water ranging in pH from 5.9 to 8.7, salinity ranging from 0.04 to 1.58 ppt, nitrate ranging from 0 to 10.8 ppm, and iron ranging from 0.003 to 1.519 ppm. Of these variables, only iron content was statistically higher in B. pseudomallei-positive versus B. pseudomallei-negative sites, suggesting that increasing concentrations of iron may encourage the growth of this bacterium, which is responsible for melioidosis. Our results, when combined with data from other published studies, support the notion that B. pseudomallei can exist in a wide range of environmental conditions. Thus, we argue that health safety education is a more appropriate means of addressing farmer vulnerability than chemical or physical alterations to fields at large scales. Further, it may be important to investigate melioidosis through transdisciplinary approaches that consider the complex social and ecological contexts in which the disease occurs.

  11. Agar films containing silver nanoparticles as new supports for electromembrane extraction.

    Science.gov (United States)

    Hidalgo, Cristina Román; Ramos-Payán, María; Ocaña-González, Juan Antonio; Martín-Valero, María Jesús; Bello-López, Miguel Ángel

    2015-02-01

    A new support containing silver nanoparticles to assist electromembrane extraction (EME) procedures is proposed. For the first time, synthesized agar films containing silver nanoparticles (AgNPs) have been used as a support for liquid membranes in EME. Agarose films of 20 μm thickness containing 107.9 mg Ag/g agar were synthesized and characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM), showing isolated spherical silver nanoparticles of 20-30 nm diameter with homogeneous distribution. Nanometallic films were cut and glued to narrow bore glass tubes and used as supports for a dihexyl ether liquid membrane for use in an EME procedure. EME conditions were optimized and applied to the extraction of selected non-steroidal anti-inflammatory drugs (NSAIDs). The results were compared to those using polypropylene membranes (450 μm and 100 μm thickness), achieving 10- to 70-fold higher extraction efficiency. This article opens a new line of research into electrically assisted microextraction systems by combining other possible kinds of nanometallic films, including different metals, film functionalization through metallic NPs, and the use of low polarity solvents. Also, very low currents are obtained during the extraction process, which lead to high electromigration of the analytes.

  12. Evaluation of the Bruker Biotyper matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system for identification of clinical and environmental isolates of Burkholderia pseudomallei

    Directory of Open Access Journals (Sweden)

    He eWang

    2016-04-01

    Full Text Available Burkholderia pseudomallei is not represented in the current version of Bruker Biotyper matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS system. A total of 66 isolates of B. pseudomallei, including 30 clinical isolates collected from National Taiwan University Hospital (NTUH, n=27 and Peking Union Medical College Hospital (PUMCH, n=3, and 36 isolates of genetically confirmed strains, including 13 from clinical samples and 23 from environmental samples, collected from southern Taiwan were included in this study. All these isolates were identified by partial 16S rDNA gene sequencing analysis and the Bruker Biotyper MALDI-TOF MS system. Among the 30 isolates initially identified as B. pseudomallei by conventional identification methods, one was identified as B. cepacia complex (NTUH and three were identified as B. putida (PUMCH by partial 16S rDNA gene sequencing analysis and Bruker Biotyper MALDI-TOF MS system. The Bruker Biotyper MALDI-TOF MS system misidentified 62 genetically confirmed B. pseudomallei isolates as B. thailandensis or Burkholderia species (score values, 1.803-2.063 when the currently available database (DB 5627 was used. However, using a newly created MALDI-TOF MS database (including B. pseudomallei NTUH-3 strain, all isolates were correctly identified as B. pseudomallei (score values >2.000, 100%. An additional 60 isolates of genetically confirmed B. cepacia complex and B. putida were also evaluated by the Bruker Biotyper MALDI-TOF MS system using the newly created database and none of these isolates were identified as B. pseudomallei. MALDI-TOF MS is a versatile and robust tool for the rapid identification of B. pseudomallei using the enhanced database.

  13. Application of agar liquid-gel transition in cultivation and harvesting of microalgae for biodiesel production.

    Science.gov (United States)

    Kumar, Vinod; Nanda, Manisha; Verma, Monu

    2017-11-01

    In order to increase microalgal biomass productivity efficient cultivation and harvesting methods are needed against the available traditional methods. The present study focuses on the same by harvesting microalgae using agar gel. Agar medium containing bold's basal medium (BBM) undergoes a thermoreversible gel transition. As compared to the traditional protocols, this gel is used to cultivate microalgae without even affecting the total productivity. To develop the gel for microalgae cultivation, agar was boiled in BBM. Then the agar was cooled to 35°C and microalgae culture was added to it. After seeding the microalgae the temperature of the agar was further decreased by 10°C to induce gelation. Instead of isolated cells microalgae were grown in clusters within the agar gel. Microalgal clusters gravimetrically settle at the bottom within 2h. In this method agar can be reused. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Karakterisasi Enzim Selulase PMP 0126Y dari Limbah Pengolahan Agar

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    Ekowati Chasanah

    2013-12-01

    Full Text Available Hasil penapisan bakteri penghasil enzim selulase terdahulu mendapatkan isolat PMP 0126 sebagai isolat yang berpotensi yang diisolasi dari limbah pengolahan agar skala UKM di Pamengpeuk, Garut. Isolat tersebut ternyata belum merupakan koloni tunggal, terdiri dari 2 isolat bakteri yaitu PMP 0126Y dan PMP 0126W. Isolat PMP 0126Y memiliki kemampuan mendegradasi selulosa yang lebih besar dibanding PMP 0126W. Tujuan dari penelitian ini adalah untuk memproduksi dan mengkarakterisasi enzim selulase dari isolat PMP 0126Y, serta mengidentifikasi isolat tersebut. Hasil penelitian menunjukkan bahwa enzim selulase diproduksi optimum pada hari ke-3 kultivasi menggunakan medium cair berisi  CMC 1%. Enzim kasar yang diperoleh dapat bekerja optimal pada suhu 30 °C dan pH 5, dapat ditingkatkan aktivitasnya dengan ion logam dalam bentuk garam CaCl2 dan ZnCl2 5 mM.. Pemurnian dengan sistem penukar anion dapat meningkatkan aktivitas enzim 15x dengan perolehan 20%. Dari hasil SDS-PAGE terlihat bahwa ada 3 selulase dengan perkiraan berat molekul 39, 30, dan 14 kDa. Enzim kasar ini memiliki kemampuan menghidrolisis limbah pengolahan agar sebaik ketika memecah substrat CMC,  yang mengindikasikan bahwa enzim dari isolat ini berpotensi sebagai kandidat agen sakarifikasi pada produksi bioetanol. Identifikasi bakteri dengan 16S-rDNA menunjukkan bahwa isolat ini memiliki kemiripan 96% dengan bakteri Chryseobacterium indologenes McR-1.

  15. Comparison of Mannitol Salt Agar and Blood Agar Plates for Identification and Susceptibility Testing of Staphylococcus aureus in Specimens from Cystic Fibrosis Patients▿

    OpenAIRE

    Sharp, Susan E.; Searcy, Cindy

    2006-01-01

    Antimicrobial susceptibilities of Staphylococcus aureus strains can be determined accurately by using isolates from mannitol salt agar, and yellow isolates on mannitol salt agar at quantities of >1+ can be reported as S. aureus. These methods decrease the time to identification/antimicrobial susceptibility testing of S. aureus and decrease costs through eliminating additional testing.

  16. Thermal inactivation and sublethal injury kinetics of Salmonella enterica and Listeria monocytogenes in broth versus agar surface.

    Science.gov (United States)

    Wang, Xiang; Devlieghere, Frank; Geeraerd, Annemie; Uyttendaele, Mieke

    2017-02-21

    The objective of the present study was to compare the thermal inactivation and sublethal injury kinetics of Salmonella enterica and Listeria monocytogenes in broth (suspended cells) and on solid surface (agar-seeded cells). A 3-strain cocktail of S. enterica or L. monocytogenes inoculated in broth or on agar was subjected to heating in a water bath at various set temperatures (55.0, 57.5 and 60.0°C for S. enterica and 60.0, 62.5 and 65°C for L. monocytogenes). The occurrence of sublethally injured cells was determined by comparing enumerations on nonselective (TSAYE) and selective (XLD or ALOA) media. Results showed that the inactivation curves obtained from selective media were log-linear, and significant shoulders (pagar surface exhibited higher heat resistance than those in broth. For S. enterica, cell injury increased with the exposure time, no difference was observed when treated at temperatures from 55.0 to 60.0°C, while for L. monocytogenes, cell injury increased significantly with heating time and treatment temperature (from 60.0 to 65°C). Moreover, the degree of sublethal injury affected by thermal treatment in broth or on agar surface depended upon the target microorganism. Higher proportions of injured S. enterica cells were observed for treatment in broth than on agar surface, while the opposite was found for L. monocytogenes. The provided information may be used to assess the efficacy of thermal treatment processes on surfaces for inactivation of S. enterica and L. monocytogenes, and it provides insight into the sublethally injured survival state of S. enterica and L. monocytogenes treated in liquid or on solid food. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Characterization of the mrgRS locus of the opportunistic pathogen Burkholderia pseudomallei: temperature regulates the expression of a two-component signal transduction system

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    Dance David AB

    2006-08-01

    Full Text Available Abstract Background Burkholderia pseudomallei is a saprophyte in tropical environments and an opportunistic human pathogen. This versatility requires a sensing mechanism that allows the bacterium to respond rapidly to altered environmental conditions. We characterized a two-component signal transduction locus from B. pseudomallei 204, mrgR and mrgS, encoding products with extensive homology with response regulators and histidine protein kinases of Escherichia coli, Bordetella pertussis, and Vibrio cholerae. Results The locus was present and expressed in a variety of B. pseudomallei human and environmental isolates but was absent from other Burkholderia species, B. cepacia, B. cocovenenans, B. plantarii, B. thailandensis, B. vandii, and B. vietnamiensis. A 2128 bp sequence, including the full response regulator mrgR, but not the sensor kinase mrgS, was present in the B. mallei genome. Restriction fragment length polymorphism downstream from mrgRS showed two distinct groups were present among B. pseudomallei isolates. Our analysis of the open reading frames in this region of the genome revealed that transposase and bacteriophage activity may help explain this variation. MrgR and MrgS proteins were expressed in B. pseudomallei 204 cultured at different pH, salinity and temperatures and the expression was substantially reduced at 25°C compared with 37°C or 42°C but was mostly unaffected by pH or salinity, although at 25°C and 0.15% NaCl a small increase in MrgR expression was observed at pH 5. MrgR was recognized by antibodies in convalescent sera pooled from melioidosis patients. Conclusion The results suggest that mrgRS regulates an adaptive response to temperature that may be essential for pathogenesis, particularly during the initial phases of infection. B. pseudomallei and B. mallei are very closely related species that differ in their capacity to adapt to changing environmental conditions. Modifications in this region of the genome may

  18. Characterization of in vitro phenotypes of Burkholderia pseudomallei and Burkholderia mallei strains potentially associated with persistent infection in mice.

    Science.gov (United States)

    Bernhards, R C; Cote, C K; Amemiya, K; Waag, D M; Klimko, C P; Worsham, P L; Welkos, S L

    2017-03-01

    Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm), the agents of melioidosis and glanders, respectively, are Tier 1 biothreats. They infect humans and animals, causing disease ranging from acute and fatal to protracted and chronic. Chronic infections are especially challenging to treat, and the identification of in vitro phenotypic markers which signal progression from acute to persistent infection would be extremely valuable. First, a phenotyping strategy was developed employing colony morphotyping, chemical sensitivity testing, macrophage infection, and lipopolysaccharide fingerprint analyses to distinguish Burkholderia strains. Then mouse spleen isolates collected 3-180 days after infection were characterized phenotypically. Isolates from long-term infections often exhibited increased colony morphology differences and altered patterns of antimicrobial sensitivity and macrophage infection. Some of the Bp and Bm persistent infection isolates clearly displayed enhanced virulence in mice. Future studies will evaluate the potential role and significance of these phenotypic markers in signaling the establishment of a chronic infection.

  19. Burkholderia mallei and Burkholderia pseudomallei cluster 1 type VI secretion system gene expression is negatively regulated by iron and zinc.

    Science.gov (United States)

    Burtnick, Mary N; Brett, Paul J

    2013-01-01

    Burkholderia mallei is a facultative intracellular pathogen that causes glanders in humans and animals. Previous studies have demonstrated that the cluster 1 type VI secretion system (T6SS-1) expressed by this organism is essential for virulence in hamsters and is positively regulated by the VirAG two-component system. Recently, we have shown that T6SS-1 gene expression is up-regulated following internalization of this pathogen into phagocytic cells and that this system promotes multinucleated giant cell formation in infected tissue culture monolayers. In the present study, we further investigated the complex regulation of this important virulence factor. To assess T6SS-1 expression, B. mallei strains were cultured in various media conditions and Hcp1 production was analyzed by Western immunoblotting. Transcript levels of several VirAG-regulated genes (bimA, tssA, hcp1 and tssM) were also determined using quantitative real time PCR. Consistent with previous observations, T6SS-1 was not expressed during growth of B. mallei in rich media. Curiously, growth of the organism in minimal media (M9G) or minimal media plus casamino acids (M9CG) facilitated robust expression of T6SS-1 genes whereas growth in minimal media plus tryptone (M9TG) did not. Investigation of this phenomenon confirmed a regulatory role for VirAG in this process. Additionally, T6SS-1 gene expression was significantly down-regulated by the addition of iron and zinc to M9CG. Other genes under the control of VirAG did not appear to be as tightly regulated by these divalent metals. Similar results were observed for B. pseudomallei, but not for B. thailandensis. Collectively, our findings indicate that in addition to being positively regulated by VirAG, B. mallei and B. pseudomallei T6SS-1 gene expression is negatively regulated by iron and zinc.

  20. Burkholderia mallei and Burkholderia pseudomallei cluster 1 type VI secretion system gene expression is negatively regulated by iron and zinc.

    Directory of Open Access Journals (Sweden)

    Mary N Burtnick

    Full Text Available Burkholderia mallei is a facultative intracellular pathogen that causes glanders in humans and animals. Previous studies have demonstrated that the cluster 1 type VI secretion system (T6SS-1 expressed by this organism is essential for virulence in hamsters and is positively regulated by the VirAG two-component system. Recently, we have shown that T6SS-1 gene expression is up-regulated following internalization of this pathogen into phagocytic cells and that this system promotes multinucleated giant cell formation in infected tissue culture monolayers. In the present study, we further investigated the complex regulation of this important virulence factor. To assess T6SS-1 expression, B. mallei strains were cultured in various media conditions and Hcp1 production was analyzed by Western immunoblotting. Transcript levels of several VirAG-regulated genes (bimA, tssA, hcp1 and tssM were also determined using quantitative real time PCR. Consistent with previous observations, T6SS-1 was not expressed during growth of B. mallei in rich media. Curiously, growth of the organism in minimal media (M9G or minimal media plus casamino acids (M9CG facilitated robust expression of T6SS-1 genes whereas growth in minimal media plus tryptone (M9TG did not. Investigation of this phenomenon confirmed a regulatory role for VirAG in this process. Additionally, T6SS-1 gene expression was significantly down-regulated by the addition of iron and zinc to M9CG. Other genes under the control of VirAG did not appear to be as tightly regulated by these divalent metals. Similar results were observed for B. pseudomallei, but not for B. thailandensis. Collectively, our findings indicate that in addition to being positively regulated by VirAG, B. mallei and B. pseudomallei T6SS-1 gene expression is negatively regulated by iron and zinc.

  1. Burkholderia mallei and Burkholderia pseudomallei Cluster 1 Type VI Secretion System Gene Expression Is Negatively Regulated by Iron and Zinc

    Science.gov (United States)

    Burtnick, Mary N.; Brett, Paul J.

    2013-01-01

    Burkholderia mallei is a facultative intracellular pathogen that causes glanders in humans and animals. Previous studies have demonstrated that the cluster 1 type VI secretion system (T6SS-1) expressed by this organism is essential for virulence in hamsters and is positively regulated by the VirAG two-component system. Recently, we have shown that T6SS-1 gene expression is up-regulated following internalization of this pathogen into phagocytic cells and that this system promotes multinucleated giant cell formation in infected tissue culture monolayers. In the present study, we further investigated the complex regulation of this important virulence factor. To assess T6SS-1 expression, B. mallei strains were cultured in various media conditions and Hcp1 production was analyzed by Western immunoblotting. Transcript levels of several VirAG-regulated genes (bimA, tssA, hcp1 and tssM) were also determined using quantitative real time PCR. Consistent with previous observations, T6SS-1 was not expressed during growth of B. mallei in rich media. Curiously, growth of the organism in minimal media (M9G) or minimal media plus casamino acids (M9CG) facilitated robust expression of T6SS-1 genes whereas growth in minimal media plus tryptone (M9TG) did not. Investigation of this phenomenon confirmed a regulatory role for VirAG in this process. Additionally, T6SS-1 gene expression was significantly down-regulated by the addition of iron and zinc to M9CG. Other genes under the control of VirAG did not appear to be as tightly regulated by these divalent metals. Similar results were observed for B. pseudomallei, but not for B. thailandensis. Collectively, our findings indicate that in addition to being positively regulated by VirAG, B. mallei and B. pseudomallei T6SS-1 gene expression is negatively regulated by iron and zinc. PMID:24146925

  2. Preparation and Characterization of Chitosan/Agar Blended Films: Part 2. Thermal, Mechanical, and Surface Properties

    Directory of Open Access Journals (Sweden)

    Esam A. Elhefian

    2012-01-01

    Full Text Available Chitosan/agar (CS/AG films were prepared by blending different proportions of chitosan and agar (considering chitosan as the major component in solution forms. The thermal stability of the blended films was studied using thermal gravimetric analysis (TGA. It was revealed that chitosan and agar form a compatible blend. Studying the mechanical properties of the films showed a decrease in the tensile strength and elongation at break with increasing agar content. Blending of agar with chitosan at all proportions was found to form hydrogel films with enhanced swelling compared to the pure chitosan one. Static water contact angle measurements confirmed the increasing affinity of the blended films towards water suggesting that blending of agar with chitosan improves the wettability of the obtained films.

  3. Colony growth of Philippine isolates of Blastocystis hominis in simplified, soft agar medium.

    Science.gov (United States)

    Valido, Ezra M; Rivera, Windell L

    2007-06-01

    The agar-cloning technique of Blastocystis hominis has been observed in both solid and semisolid agar using Iscove's modified Dulbecco's medium. In this study, Philippine isolates of B. hominis were grown by pour-plate method in semisolid agar using Locke's solution. Inoculated plates contained 0.7% agar, 10% horse serum, and 0.1% sodium thioglycollate. Plates were incubated at 37 degrees C in a microaerophilic jar for 7-10 days. Biconvex disk-shaped colonies were seen abound at the bottom half of the medium. Colonies growing at the agar-glass interface were flat and consisted of thin layers of cells. From these colonies, large amoeboid cells were frequently seen on the periphery, whereas smaller cells were concentrated at the core. Analysis of the SSU rDNA genetically established the identity of the clones to be B. hominis. This is the first report on agar cloning of Blastocystis in a compound medium.

  4. Beyond Agar: Gel Substrates with Improved Optical Clarity and Drug Efficiency and Reduced Autofluorescence for Microbial Growth Experiments

    Science.gov (United States)

    McElfresh, Cameron; Wong, Lily R.

    2015-01-01

    Agar, a seaweed extract, has been the standard support matrix for microbial experiments for over a century. Recent developments in high-throughput genetic screens have created a need to reevaluate the suitability of agar for use as colony support, as modern robotic printing systems now routinely spot thousands of colonies within the area of a single microtiter plate. Identifying optimal biophysical, biochemical, and biological properties of the gel support matrix in these extreme experimental conditions is instrumental to achieving the best possible reproducibility and sensitivity. Here we systematically evaluate a range of gelling agents by using the yeast Saccharomyces cerevisiae as a model microbe. We find that carrageenan and Phytagel have superior optical clarity and reduced autofluorescence, crucial for high-resolution imaging and fluorescent reporter screens. Nutrient choice and use of refined Noble agar or pure agarose reduce the effective dose of numerous selective drugs by >50%, potentially enabling large cost savings in genetic screens. Using thousands of mutant yeast strains to compare colony growth between substrates, we found no evidence of significant growth or nutrient biases between gel substrates, indicating that researchers could freely pick and choose the optimal gel for their respective application and experimental condition. PMID:26070672

  5. Characterization of the species Malassezia pachydermatis and re-evaluation of its lipid dependence using a synthetic agar medium.

    Science.gov (United States)

    Puig, Laura; Bragulat, M Rosa; Castellá, Gemma; Cabañes, F Javier

    2017-01-01

    The genus Malassezia includes lipophilic yeasts, which are part of the skin microbiota of various mammals and birds. Unlike the rest of Malassezia species, M. pachydermatis is described as non-lipid-dependent, as it is able to grow on Sabouraud glucose agar (SGA) without lipid supplementation. In this study we have examined the phenotypic variability within M. pachydermatis and confirmed its lipid-dependent nature using a synthetic agar medium. We used a selection of representative non-lipid-dependent strains from different animal species and three atypical lipid-dependent strains of this species, which were not able to grow after multiple passages on SGA. More than 400 lipid-dependent Malassezia isolates from animals were studied in order to detect the three lipid-dependent strains of M. pachydermatis. The identity of the atypical strains was confirmed by DNA sequencing. On the other hand, we have modified the Tween diffusion test, which is widely used in the characterization of these yeasts, by using a synthetic agar-based medium instead of SGA. This modification has proved to be useful for differentiation of M. pachydermatis strains, providing reproducible results and a straightforward interpretation. The finding of these peculiar lipid-dependent strains exemplifies the large variability within the species M. pachydermatis, which involves rare atypical strains with particular growth requirements.

  6. Comparison of different fungal agar for the environmental monitoring of pharmaceutical-grade cleanrooms.

    Science.gov (United States)

    Gebala, Barbara; Sandle, Tim

    2013-01-01

    In relation to a growth in reported incidents of fungal contamination of pharmaceutical products, there has been a developing interest by U.S. and U.K. regulators concerning the risk of fungi. This paper describes a study undertaken to examine the suitability of different commercially available mycological agars for the environmental monitoring of pharmaceutical-grade cleanrooms. Five agars were evaluated in relation to the detection of both numbers and different species of fungi (yeasts and moulds). The objective was to determine if one mycological medium is more suitable than another. Data was collected using different sampling techniques (settle plates, active air samples, and contact plates) from different locations within representative cleanrooms. Samples were taken over a 3 month time period. The study results indicated that fungi are not distributed evenly across cleanrooms and that that the prevalence of fungi partly relates to the room design and operation. In relation to the different agar types, the study indicated that Sabouraud dextrose agar was the most effective at detecting the widest number of different types of isolates, and that Sabouraud dextrose agar and malt extract agar were the most efficient in terms of the numbers of recovered isolates. Other media, notably potato dextrose agar, was relatively less effective. There has been an increased regulatory concern about the presence of fungi in cleanrooms. Some environmental monitoring regimes are not especially orientated towards the examination of fungi, and it may be that special agars are required. Given the choice of different agars available, this paper outlines a case study where different fungal agars were evaluated. The study showed that Sabouraud dextrose agar was the optimal agar.

  7. Use of a safe, reproducible, and rapid aerosol delivery method to study infection by Burkholderia pseudomallei and Burkholderia mallei in mice.

    Directory of Open Access Journals (Sweden)

    Eric R Lafontaine

    Full Text Available Burkholderia pseudomallei, the etiologic agent of melioidosis, is a saprophytic bacterium readily isolated from wet soils of countries bordering the equator. Burkholderia mallei is a host-adapted clone of B. pseudomallei that does not persist outside of its equine reservoir and causes the zoonosis glanders, which is endemic in Asia, Africa, the Middle East and South America. Infection by these organisms typically occurs via percutaneous inoculation or inhalation of aerosols, and the most common manifestation is severe pneumonia leading to fatal bacteremia. Glanders and melioidosis are difficult to diagnose and require prolonged antibiotic therapy with low success rates. There are no vaccines available to protect against either Burkholderia species, and there is concern regarding their use as biological warfare agents given that B. mallei has previously been utilized in this manner. Hence, experiments were performed to establish a mouse model of aerosol infection to study the organisms and develop countermeasures. Using a hand-held aerosolizer, BALB/c mice were inoculated intratracheally with strains B. pseudomallei 1026b and B. mallei ATCC23344 and growth of the agents in the lungs, as well as dissemination to the spleen, were examined. Mice infected with 10(2, 10(3 and 10(4 organisms were unable to control growth of B. mallei in the lungs and bacteria rapidly disseminated to the spleen. Though similar results were observed in mice inoculated with 10(3 and 10(4 B. pseudomallei cells, animals infected with 10(2 organisms controlled bacterial replication in the lungs, dissemination to the spleen, and the extent of bacteremia. Analysis of sera from mice surviving acute infection revealed that animals produced antibodies against antigens known to be targets of the immune response in humans. Taken together, these data show that small volume aerosol inoculation of mice results in acute disease, dose-dependent chronic infection, and immune responses

  8. Use of a safe, reproducible, and rapid aerosol delivery method to study infection by Burkholderia pseudomallei and Burkholderia mallei in mice.

    Science.gov (United States)

    Lafontaine, Eric R; Zimmerman, Shawn M; Shaffer, Teresa L; Michel, Frank; Gao, Xiudan; Hogan, Robert J

    2013-01-01

    Burkholderia pseudomallei, the etiologic agent of melioidosis, is a saprophytic bacterium readily isolated from wet soils of countries bordering the equator. Burkholderia mallei is a host-adapted clone of B. pseudomallei that does not persist outside of its equine reservoir and causes the zoonosis glanders, which is endemic in Asia, Africa, the Middle East and South America. Infection by these organisms typically occurs via percutaneous inoculation or inhalation of aerosols, and the most common manifestation is severe pneumonia leading to fatal bacteremia. Glanders and melioidosis are difficult to diagnose and require prolonged antibiotic therapy with low success rates. There are no vaccines available to protect against either Burkholderia species, and there is concern regarding their use as biological warfare agents given that B. mallei has previously been utilized in this manner. Hence, experiments were performed to establish a mouse model of aerosol infection to study the organisms and develop countermeasures. Using a hand-held aerosolizer, BALB/c mice were inoculated intratracheally with strains B. pseudomallei 1026b and B. mallei ATCC23344 and growth of the agents in the lungs, as well as dissemination to the spleen, were examined. Mice infected with 10(2), 10(3) and 10(4) organisms were unable to control growth of B. mallei in the lungs and bacteria rapidly disseminated to the spleen. Though similar results were observed in mice inoculated with 10(3) and 10(4) B. pseudomallei cells, animals infected with 10(2) organisms controlled bacterial replication in the lungs, dissemination to the spleen, and the extent of bacteremia. Analysis of sera from mice surviving acute infection revealed that animals produced antibodies against antigens known to be targets of the immune response in humans. Taken together, these data show that small volume aerosol inoculation of mice results in acute disease, dose-dependent chronic infection, and immune responses that correlate

  9. In vitro activities of amoxicillin-clavulanate, doxycycline, ceftazidime, imipenem, and trimethoprim-sulfamethoxazole against biofilm of Brazilian strains of Burkholderia pseudomallei.

    Science.gov (United States)

    Bandeira, Tereza de Jesus Pinheiro Gomes; Moreira, Camila Alencar; Brilhante, Raimunda Sâmia Nogueira; Castelo-Branco, Débora de Souza Collares Maia; Neto, Manoel Paiva de Araújo; Cordeiro, Rossana de Aguiar; Rodrigues, Terezinha de Jesus Santos; Rocha, Marcos Fábio Gadelha; Sidrim, José Júlio Costa

    2013-11-01

    This study aimed at investigating the in vitro activities of amoxicillin-clavulanate, doxycycline, ceftazidime, imipenem, and trimethoprim-sulfamethoxazole against Burkholderia pseudomallei in planktonic and biofilm forms, through broth microdilution and resazurin-based viability staining, respectively. In planktonic growth, the strains were susceptible to the drugs, while in biofilm growth, significantly higher antimicrobial concentrations were required, especially for ceftazidime and imipenem, surpassing the resistance breakpoints. These results highlight the importance of the routine evaluation of biofilm antimicrobial susceptibility.

  10. Evaluation of mannitol salt agar with oxacillin as a screening medium for methicillin-resistant Staphylococcus aureus.

    OpenAIRE

    Lally, R T; Ederer, M N; Woolfrey, B F

    1985-01-01

    We evaluated the use of mannitol salt agar with oxacillin for use as a primary screening medium for the simultaneous detection and identification of methicillin-resistant Staphylococcus aureus in clinical surveillance specimens. Oxacillin agar dilution susceptibility tests with mannitol salt agar and Mueller-Hinton agar were performed in parallel with disk-agar diffusion testing on 95 oxacillin-susceptible and 105 oxacillin-resistant S. aureus stock isolates. MICs were found to be comparable,...

  11. Heavy metal accumulation by carrageenan and agar producing algae

    Energy Technology Data Exchange (ETDEWEB)

    Burdin, K.S. [Moscow State Univ. (Russian Federation). Faculty of Biology; Bird, K.T. [North Carolina Univ., Wilmington, NC (United States). Center for Marine Science Research

    1994-09-01

    The accumulation of six heavy metals Cu, Cd, Ni, Zn, Mn and Pb was measured in living and lzophilized algal thalli. The agar producing algae were Gracilaria tikvahiae and Gelidium pusillum. The carrageenan producing macroalgae were Agardhiella subulata and the gametophyte and tetrasporophyte phases of Chondrus crispus. These produce primarily iota, kappa and lambda carrageenans, respectively. At heavy metal concentrations of 0.5 mg L{sup -1}, living thalli of Gracilaria tikvahiae generally showed the greatest amount of accumulation of the 6 heavy metals tested. The accumulation of Pb was greater in the living thalli of all four species than in the lyophilized thalli. Except for Agardhiella subulata, lyophilized thalli showed greater accumulation of Ni, Cu and Zn. There was no difference in heavy metal accumulation between living and lyophilized thalli in the accumulation of Cd. Manganese showed no accumulation at the tested concentration. There did not appear to be a relationship between algal hydrocolloid characteristics and the amounts of heavy metals accumulated. (orig.)

  12. Agar dilution method for susceptibility testing of Neisseria gonorrhoeae

    Directory of Open Access Journals (Sweden)

    Marta C de Castillo

    1996-12-01

    Full Text Available The antibiotic susceptibilities of Neisseria gonorrhoeae isolates obtained from patients attending a clinic for sexually transmitted diseases in Tucumán, Argentina, were determined by the agar dilution method (MIC. 3.5% of the isolates produced ²-lactamase. A total of 96.5% of ²-lactamase negative isolates tested were susceptible to penicillin (MIC < 2 µgml-1; 14.03% of the tested isolates were resistant to tetracycline (MIC < 2 µgml-1, and 98% of the tested isolates were susceptible to spectinomycin (MIC < 64 µgml-1. The MICs for 95% of the isolates, tested for other drugs were: < 2 µgml-1 for cefoxitin, < 0.06 µgml-1 for cefotaxime, < 0.25 µgml-1 for norfloxacin, < 10 µgml-1 for cephaloridine, < 10 µgml-1 for cephalexin, and < 50 µgml-1 for kanamycin. Antibiotic resistance among N. gonorrhoeae isolates from Tucumán, Argentina, appeared to be primarily limited to penicillin and tetracycline, which has been a general use against gonorrhoeae in Tucumán since 1960. Periodic monitoring of the underlying susceptibility profiles of the N. gonorrhoeae strains prevalent in areas of frequent transmission may provide clues regarding treatment options and emerging of drug resistance.

  13. A modified agar pad method for mycobacterial live-cell imaging

    Directory of Open Access Journals (Sweden)

    Robertson Brian D

    2011-03-01

    Full Text Available Abstract Background Two general approaches to prokaryotic live-cell imaging have been employed to date, growing bacteria on thin agar pads or growing bacteria in micro-channels. The methods using agar pads 'sandwich' the cells between the agar pad on the bottom and a glass cover slip on top, before sealing the cover slip. The advantages of this technique are that it is simple and relatively inexpensive to set up. However, once the cover slip is sealed, the environmental conditions cannot be manipulated. Furthermore, desiccation of the agar pad, and the growth of cells in a sealed environment where the oxygen concentration will be in gradual decline, may not permit longer term studies such as those required for the slower growing mycobacteria. Findings We report here a modified agar pad method where the cells are sandwiched between a cover slip on the bottom and an agar pad on top of the cover slip (rather than the reverse and the cells viewed from below using an inverted microscope. This critical modification overcomes some of the current limitations with agar pad methods and was used to produce time-lapse images and movies of cell growth for Mycobacterium smegmatis and Mycobacterium bovis BCG. Conclusions This method offers improvement on the current agar pad methods in that long term live cell imaging studies can be performed and modification of the media during the experiment is permitted.

  14. Growth and study of barium oxalate single crystals in agar gel

    Indian Academy of Sciences (India)

    Keywords. Barium oxalate; agar gel; XRD; IR; TGA/DTA. Abstract. Barium oxalate was grown in agar gel at ambient temperature. The effect of various parameters like gel concentration, gel setting time and concentration of the reactants on the growth of these crystals was studied. Prismatic platy shaped spherulites and ...

  15. Tapioca-a new and cheaper alternative to agar for direct in vitro ...

    African Journals Online (AJOL)

    Solanum tuberosum L.) from nodal explants was studied using agar and other new and cheaper gelling agents- tapioca and sago in Murashige & Skoog (MS) salt medium. For shoot regeneration, agar was maintained at 8 mg l -1, tapioca and ...

  16. Pembuatan Bakto Agar dari Rumput Laut Gelidium rigidum untuk Media Tumbuh bagi Mikroorganisme

    Directory of Open Access Journals (Sweden)

    Murdinah murdinah

    2008-06-01

    Full Text Available ABSTRAK Telah dilakukan penelitian tentang pembuatan bakto agar dari rumput laut Gelidium rigidum untuk media tumbuh bagi mikroorganisme. Pembuatan bakto agar dilakukan dengan variasi waktu ekstraksi yaitu 1, 2, dan 3 jam pada suhu 121°C dan tekanan 1,1 atm. Bakto agar dianalisis rendemen dan mutunya yang meliputi kadar air, kadar abu, kadar abu tak larut asam, kadar sulfat, kekuatan gel, pH, titik leleh, dan titik jendal. Uji mikrobiologi yang diamati meliputi angka lempeng total bakteri (ALT dan diameter koloni. Dari hasil pengamatan diketahui bahwa bakto agar hasil ekstraksi dari rumput laut jenis Gelidium rigidum selama 2 jam mutunya menyamai bakto agar komersial, khususnya dari nilai kadar air, pH, kadar abu, kadar abu tak larut asam, kekuatan gel, serta kemampuannya menumbuhkan bakteri yang terdapat pada ikan segar dan kultur murni yaitu E. coli dan L. lactis. Tetapi dalam hal kadar sulfat, titik leleh, dan titik jendal masih di bawah mutu bakto agar komersial. Hasil penelitian juga menunjukkan bahwa waktu ekstraksi selama 2 jam menghasilkan bakto agar yang memenuhi standar bakto agar komersial dengan karakteristik kadar air 10,41%, kadar abu 2,1%, kadar abu tak larut asam 0,18%, kekuatan gel 670,72 g/cm2, dan pH 7,1.

  17. A modified agar pad method for mycobacterial live-cell imaging.

    Science.gov (United States)

    Joyce, Graham; Robertson, Brian D; Williams, Kerstin J

    2011-03-21

    Two general approaches to prokaryotic live-cell imaging have been employed to date, growing bacteria on thin agar pads or growing bacteria in micro-channels. The methods using agar pads 'sandwich' the cells between the agar pad on the bottom and a glass cover slip on top, before sealing the cover slip. The advantages of this technique are that it is simple and relatively inexpensive to set up. However, once the cover slip is sealed, the environmental conditions cannot be manipulated. Furthermore, desiccation of the agar pad, and the growth of cells in a sealed environment where the oxygen concentration will be in gradual decline, may not permit longer term studies such as those required for the slower growing mycobacteria. We report here a modified agar pad method where the cells are sandwiched between a cover slip on the bottom and an agar pad on top of the cover slip (rather than the reverse) and the cells viewed from below using an inverted microscope. This critical modification overcomes some of the current limitations with agar pad methods and was used to produce time-lapse images and movies of cell growth for Mycobacterium smegmatis and Mycobacterium bovis BCG. This method offers improvement on the current agar pad methods in that long term live cell imaging studies can be performed and modification of the media during the experiment is permitted.

  18. Agar composition affects in vitro screening of biocontrol activity of antagonistic microorganisms

    NARCIS (Netherlands)

    Bosmans, Lien; De Bruijn, I.; de Mot, Rene; Readers, Hans; Lievens, Bart

    2016-01-01

    Agar-based screening assays are the method of choice when evaluating antagonistic potential of bacterial biocontrol-candidates against pathogens.Weshowed thatwhen using the samemedium, but different agar compositions, the activity of a bacterial antagonist against Agrobacteriumwas strongly affected.

  19. Physicochemical and morphological properties of plasticized poly(vinyl alcohol)-agar biodegradable films.

    Science.gov (United States)

    Madera-Santana, T J; Freile-Pelegrín, Y; Azamar-Barrios, J A

    2014-08-01

    The effects of the addition of glycerol (GLY) on the physicochemical and morphological properties of poly(vinyl alcohol) (PVA)-agar films were reported. PVA-agar films were prepared by solution cast method, and the addition of GLY in PVA-agar films altered the optical properties, resulting in a decrease in opacity values and in the color difference (ΔE) of the films. Structural characterization using Fourier transformation infrared (FTIR) spectroscopy and X-ray diffraction (XRD) indicated that the presence of GLY altered the intensity of the bands (from 1200 to 800cm(-1)) and crystallinity. The characterization of the thermal properties indicated that an increase in the agar content produces a decrease in the melting temperature and augments the heat of fusion. Similar tendencies were observed in plasticized films, but at different magnification. The formulation that demonstrated the lowest mechanical properties contained 25wt.% agar, whereas the formulation that contained 75wt.% agar demonstrated a significant improvement. The water vapor transmission rate (WVTR) and surface morphology analysis demonstrated that the structure of PVA-agar films is reorganized upon GLY addition. The physicochemical properties of PVA-agar films using GLY as a plasticizer provide information for the application of this formulation as packaging material for specific food applications. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Electrospinning of agar/PVA aqueous solutions and its relation with rheological properties.

    Science.gov (United States)

    Sousa, Ana M M; Souza, Hiléia K S; Uknalis, Joseph; Liu, Shih-Chuan; Gonçalves, Maria P; Liu, LinShu

    2015-01-22

    In this work, we report the successful fabrication of agar-based nanofibers by electrospinning technique, using water as solvent media. A tubeless spinneret was attached inside the electrospinning chamber, operating at 50°C, to avoid agar gelation. Agar pure solution (1 wt%) showed inadequate spinnability regardless of the used electrospinning conditions. The addition of a co-blending polymer such as PVA (10 wt% starting solution) improved the solutions viscoelasticity and hence, the solutions spinnability. Agar/PVA solutions were prepared with different mass ratios (100/0, 50/50, 40/60, 30/70, 20/80 and 0/100) and electrospun at various sets of electrospinning conditions. Best nanofibers were obtained with 30/70 and 20/80 agar/PVA blends while samples with higher agar contents (50/50 and 40/60 agar/PVA) were harder to process and led to discontinuous fibrous mats. This first set of encouraging results can open a new window of opportunities for agar-based biomaterials in the form of nanofibers. Published by Elsevier Ltd.

  1. Characteristics of thermoplastic sugar palm Starch/Agar blend: Thermal, tensile, and physical properties.

    Science.gov (United States)

    Jumaidin, R; Sapuan, S M; Jawaid, M; Ishak, M R; Sahari, J

    2016-08-01

    The aim of this work is to study the behavior of biodegradable sugar palm starch (SPS) based thermoplastic containing agar in the range of 10-40wt%. The thermoplastics were melt-mixed and then hot pressed at 140°C for 10min. SEM investigation showed good miscibility between SPS and agar. FT-IR analysis confirmed that SPS and agar were compatible and inter-molecular hydrogen bonds existed between them. Incorporation of agar increased the thermoplastic starch tensile properties (Young's modulus and tensile strength). The thermal stability and moisture uptake increased with increasing agar content. The present work shows that starch-based thermoplastics with 30wt% agar content have the highest tensile strength. Higher content of agar (40wt%) resulted to more rough cleavage fracture and slight decrease in the tensile strength. In conclusion, the addition of agar improved the thermal and tensile properties of thermoplastic SPS which widened the potential application of this eco-friendly material. The most promising applications for this eco-friendly material are short-life products such as packaging, container, tray, etc. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Agar composition affects in vitro screening of biocontrol activity of antagonistic microorganisms.

    Science.gov (United States)

    Bosmans, L; De Bruijn, I; De Mot, R; Rediers, H; Lievens, B

    2016-08-01

    Agar-based screening assays are the method of choice when evaluating antagonistic potential of bacterial biocontrol-candidates against pathogens. We showed that when using the same medium, but different agar compositions, the activity of a bacterial antagonist against Agrobacterium was strongly affected. Consequently, results from in vitro screenings should be interpreted cautiously. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Adsorptive removal of methylene blue by agar: effects of NaCl and ethanol

    Science.gov (United States)

    2012-01-01

    Adsorption of methylene blue (MB) on agar was investigated as a function of temperature (308-328 K), different concentrations of NaCl and HCl and various weight percentages of binary mixtures of ethanol with water. It was observed that the maximum experimental adsorption capacity, qm, exp, in water is up to 50 mg g-1 and decreases with increase in weight percentage of ethanol and NaCl and HCl concentration compared to that of water. Analysis of data using ARIAN model showed that MB adsorbs as monomer and dimer on the surface of agar. Binding constants of MB to agar were calculated using the Temkin isotherm. The process is exothermic in water and other solutions. The mean adsorption energy (E) value indicated binding of MB to agar is chemical adsorption. Kinetics of this interaction obeys from the pseudo-second-order model and diffusion of the MB molecules into the agar is the main rate-controlling step. PMID:22339759

  4. E. coli swimming over agar in a thin aqueous film

    Science.gov (United States)

    Berg, Howard

    2010-11-01

    When cells of Escherichia coli are grown in a rich medium over somewhat soft agar (0.45%) they elongate, produce more flagella, and swarm (or flock). Their behavior is dominated by collisions: an individual cell's velocity is randomized in about 0.2 s [1]. However, cells do not swim in spirals, as they do when in a thick layer of fluid near a solid boundary [2]. This suggests that the surface of the swarm is stationary, i.e., that the cells swim in a thin film of fluid between two fixed surfaces. We showed that this is the case by following the motion of MgO smoke particles deposited at the fluid-air interface [3]. By visualizing flagella of cells in swarms, we found that cells can escape from a confined environment by swimming back through the flagellar bundle, without changing the orientation of the cell body. This maneuver involves normal-to-curly and curly-to-normal polymorphic transformations [4]. These phenomena will be illustrated.[4pt] [1] Darnton NC, Turner L, Rojevsky S, & Berg HC (2010) Dynamics of bacterial swarming. Biophys. J. 98:2082-2090.[0pt] [2] Lauga E, DiLuzio WR, Whitesides GM, & Stone HA (2006) Swimming in circles: motion of bacteria near solid boundaries. Biophys. J. 90:400-412.[0pt] [3] Zhang R, Turner L, & Berg HC (2010) The upper surface of an Escherichia coli swarm is stationary. Proc. Natl. Acad. Sci. USA 107:288-290.[0pt] [4] Turner L, Zhang R, Darnton NC, & Berg HC (2010) Visualization of flagella during bacterial swarming. J. Bacteriol. 192:3259-3267.

  5. Comparison of 3 selective media for enumeration of Bacillus cereus in several food matrixes.

    Science.gov (United States)

    Chon, Jung-Whan; Song, Kwang-Young; Kim, Hyunsook; Seo, Kun-Ho

    2014-12-01

    In this study, we compared the inclusivity, exclusivity, recoverability, and selectivity of the 3 selective agars (mannitol yolk polymyxin B agar [MYPA], polymyxin pyruvate egg yolk mannitol bromothymol blue agar [PEMBA], and Brillance Bacillus cereus agar [BBC agar]) for Bacillus cereus (B. cereus) from pure culture and several food samples. BBC agar showed greater exclusivity and selectivity in pure culture and in foods with high background flora, respectively; however, all the tested media showed similar recoverability (P > 0.05) of B. cereus in pure culture and in most foods. Our results suggest that BBC agar could be useful to enumerate B. cereus from, in particular, food matrixes with high background competing micro flora. © 2014 Institute of Food Technologists®

  6. Continuous degradation of maltose: improvement in stability and catalytic properties of maltase (α-glucosidase) through immobilization using agar-agar gel as a support.

    Science.gov (United States)

    Nawaz, Muhammad Asif; Karim, Asad; Aman, Afsheen; Marchetti, Roberta; Qader, Shah Ali Ul; Molinaro, Antonio

    2015-04-01

    Maltose degrading enzyme was immobilized within agar-agar support via entrapment method due to its industrial utilization. The maximum immobilization efficiency (82.77%) was achieved using 4.0% agar-agar keeping the diameter of bead up to 3.0 mm. The matrix entrapment showed maximum catalytic activity at pH 7.0 and temperature 65 °C. Substrate saturation kinetics showed that the K m of immobilized enzyme increased from 1.717 to 2.117 mM ml(-1) where as Vmax decreased from 8,411 to 7,450 U ml(-1 )min(-1) as compared to free enzyme. The immobilization significantly increased the stability of maltase against various temperatures and immobilized maltase retain 100% of its original activity after 2 h at 50 °C, whereas the free maltase only showed 60% residual activity under same condition. The reusability of entrapped maltase showed activity up to 12 cycles and retained 50% of activity even after 5th cycle. Storage stability of agar entrapped maltase retain 73% of its initial activity even after 2 months when stored at 30 °C while free enzyme showed only 37% activity at same storage conditions.

  7. Complexation of WPI and microwave-assisted extracted agars with different physicochemical properties.

    Science.gov (United States)

    Souza, Hiléia K S; Sousa, Ana M M; Gómez, Javier; Gonçalves, Maria Pilar

    2012-08-01

    The complex formation between whey protein isolate (WPI) and agar has been investigated and their interactions were monitored as a function of the physicochemical properties of agar, the pH and the ionic strength of the medium. Agars from Gracilaria vermiculophylla were extracted under different MAE conditions and characterized according to their physicochemical properties. By using microwave irradiation a wide variety of agars was obtained, as different MAE conditions results in polyelectrolytes with distinct properties. UV-vis (in optical dispersion (O.D.) model) spectroscopy and isothermal titration calorimetry (ITC) have been used to study the formation of insoluble (coacervate) complexes. MAE agars revealed excellent properties for complex formation with WPI. The binding of WPI to MAE agar samples has been shown to be the result of different contributions. O.D. and ITC results showed that the molecular mass and the sulfate content of different agars had a determinant effect on coacervate formation. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. Blood Agar Validation for Susceptibility Testing of Isoniazid, Rifampicin, Ethambutol, and Streptomycin to Mycobacterium tuberculosis Isolates

    Science.gov (United States)

    Coban, Ahmet Yilmaz

    2013-01-01

    In recent studies, it was shown that blood agar can be used at least as effectively as Löwenstein-Jensen medium for growing Mycobacterium tuberculosis. It was also shown that susceptibility testing can be performed on blood agar. Additional validation of blood agar was performed on regional M. tuberculosis isolates from Turkey to determine critical concentrations of isoniazid (INH), rifampicin (RIF), ethambutol (ETM), and streptomycin (STR). In the current study, 40 M. tuberculosis clinical isolates were tested. H37Rv, which is susceptible to all antituberculosis agents, ATCC 35822 (INH-resistant), ATCC 35838 (RIF-resistant), ATCC 35837 (ETM-resistant), and ATCC 35820 (STR-resistant) quality control strains were used as control strains. Proportion method on 7H11 agar was considered as gold standard in the study. MIC values of the control strains and clinical isolates were detected on blood and 7H11 agar. Categorical agreements were 100% for each antibiotic, and essential agreements were 100%, 97.5%, 82.5%, and 95% for INH, RIF, ETM, and STR, respectively. According to the data, 0.2 µg/mL for INH, 1 µg/mL for RIF, 4 µg/mL for ETM, and 2 µg/mL for STR were appropriate breakpoint values for susceptibility testing on blood agar. Blood agar may be recommended for use in both developed and developing countries for the susceptibility testing of M. tuberculosis isolates to primary antituberculosis drugs. PMID:23405140

  9. Redefining the PF06864 Pfam family based on Burkholderia pseudomallei PilO2(Bp S-SAD crystal structure.

    Directory of Open Access Journals (Sweden)

    Patricia Lassaux

    Full Text Available Type IV pili are surface-exposed filaments and bacterial virulence factors, represented by the Tfpa and Tfpb types, which assemble via specific machineries. The Tfpb group is further divided into seven variants, linked to heterogeneity in the assembly machineries. Here we focus on PilO2(Bp, a protein component of the Tfpb R64 thin pilus variant assembly machinery from the pathogen Burkholderia pseudomallei. PilO2(Bp belongs to the PF06864 Pfam family, for which an improved definition is presented based on newly derived Hidden Markov Model (HMM profiles. The 3D structure of the N-terminal domain of PilO2(Bp (N-PilO2(Bp, here reported, is the first structural representative of the PF06864 family. N-PilO2(Bp presents an actin-like ATPase fold that is shown to be present in BfpC, a different variant assembly protein; the new HMM profiles classify BfpC as a PF06864 member. Our results provide structural insight into the PF06864 family and on the Type IV pili assembly machinery.

  10. Nasal-associated lymphoid tissue and olfactory epithelium as portals of entry for Burkholderia pseudomallei in murine melioidosis.

    Science.gov (United States)

    Owen, Suzzanne J; Batzloff, Michael; Chehrehasa, Fatemeh; Meedeniya, Adrian; Casart, Yveth; Logue, Carie-Anne; Hirst, Robert G; Peak, Ian R; Mackay-Sim, Alan; Beacham, Ifor R

    2009-06-15

    Burkholderia pseudomallei, the causative agent of melioidosis, is generally considered to be acquired via inhalation of dust or water droplets from the environment. In this study, we show that infection of the nasal mucosa is potentially an important portal of entry in melioidosis. After intranasal inoculation of mice, infection was monitored by bioluminescence imaging and by immunohistological analysis of coronal sections. The bacterial loads in organ and tissue specimens were also monitored. Bioluminescence imaging showed colonization and replication in the nasal cavity, including the nasal-associated lymphoid tissue (NALT). Analysis of coronal sections and immunofluorescence microscopy further demonstrated the presence of infection in the respiratory epithelium and the olfactory epithelium (including associated nerve bundles), as well as in the NALT. Of significance, the olfactory epithelium and the brain were rapidly infected before bacteria were detected in blood, and a capsule-deficient mutant infected the brain without significantly infecting blood. These data suggest that the olfactory nerve is the route of entry into the brain and that this route of entry may be paralleled in cases of human neurologic melioidosis. This study focuses attention on the upper respiratory tract as a portal of entry, specifically focusing on NALT as a route for the development of systemic infection via the bloodstream and on the olfactory epithelium as a direct route to the brain.

  11. Structural characterization of the Ser324Thr variant of the catalase-peroxidase (KatG) from Burkholderia pseudomallei.

    Science.gov (United States)

    Deemagarn, Taweewat; Carpena, Xavier; Singh, Rahul; Wiseman, Ben; Fita, Ignacio; Loewen, Peter C

    2005-01-07

    The Ser315Thr variant of the catalase-peroxidase KatG from Mycobacterium tuberculosis imparts resistance to the pro-drug isonicotinic acid hydrazide (isoniazid) through a failure to convert it to the active drug, isonicotinoyl-NAD. The equivalent variant in KatG from Burkholderia pseudomallei, Ser324Thr, has been constructed, revealing catalase and peroxidase activities that are similar to those of the native enzyme. The other activities of the variant protein, including the NADH oxidase, the isoniazid hydrazinolysis and isonicotinoyl-NAD synthase activities are reduced by 60-70%. The crystal structure of the variant differs from that of the native enzyme in having the methyl group of Thr324 situated in the entrance channel to the heme cavity, in a modified water matrix in the entrance channel and heme cavity, in lacking the putative perhydroxy modification on the heme, in the multiple locations of a few side-chains, and in the presence of an apparent perhydroxy modification on the indole nitrogen atom of the active-site Trp111. The position of the methyl group of Thr324 creates a constriction or narrowing of the channel leading to the heme cavity, providing an explanation for the lower reactivity towards isoniazid and the slower rate of isonicotinoyl-NAD synthesis.

  12. [Comparison of the results of determining antibiotic sensitivity on AGV medium and on Mueller-Hinton and isosensitest agars].

    Science.gov (United States)

    Kozlov, R S; Strachunskiĭ, L S; Livermor, D M; Stetsiuk, O U; Shavrikova, E P

    1996-01-01

    To evaluate the adequacy of AGV agar for antimicrobial susceptibility testing, the susceptibility of a range of bacteria to 10 antimicrobials on AGV, Mueller-Hinton and isoSensitest agars, all supplemented with 5 per cent lyzed horse blood was determined. Disc tests were used. In general, AGV agar gave identical susceptibility results to Mueller-Hinton and isoSensitest agars for common gram positive and gram negative bacteria with most of the tested microbials excluding sulphonamides and trimethoprim. With those latter antimicrobials inhibition zones for susceptible organisms were not formed on AGV agar whereas large zones were present on Mueller-Hinton and isoSensitest agars. This discrepancy probably can be explained by the presence of high levels of thymidine in AGV agar; too high to be corrected even by the addition of 5 per cent lysed horse blood. AGV agar is possible to use for susceptibility testing with most of the microbials excluding trimethoprim and sulphonamides.

  13. Shelf life of prepared Bordet-Gengou and Regan-Lowe agar plates for isolation of Bordetella pertussis.

    Science.gov (United States)

    Ruijs, G J; Groenendijk, T W; Biever, M

    1991-11-01

    The shelf life of prepared agar plates used for the isolation of Bordetella pertussis was studied. They contained Bordet-Gengou agar, Bordet-Gengou agar with cephalexin, Regan-Lowe agar, Regan-Lowe agar with cephalexin, or Regan-Lowe agar with both cephalexin and amphotericin B. Plates stored were compared to freshly prepared control plates for up to a maximum of 18 weeks. They were inoculated with clinical isolates of Bordetella pertussis, either in pure culture, or mixed with a defined oropharyngeal flora. Bordet-Gengou agar plates may be used, with proper storage at 4 degrees C in airtight-sealed plastic bags, for up to 10 weeks, Regan-Lowe agar plates for up to 14 weeks. Field studies are needed to substantiate our findings.

  14. Preparation and Properties of Non-Crosslinked and Ionically Crosslinked Chitosan/Agar Blended Hydrogel Films

    Directory of Open Access Journals (Sweden)

    Mohamed Mahmoud Nasef

    2011-01-01

    Full Text Available Hydrogel films of chitosan (Cs and agar blends of various proportions were prepared using physical solution blending. Some of the obtained films were ionically cross-linked by treatment with calcium chloride solution. The obtained films were characterized by Fourier transform infrared (FTIR spectroscopy, thermogravimetry analysis (TGA, differential scanning calorimetery (DSC and universal mechanical tester. The non-crosslinked Cs/agar blended films showed lower water swelling, melting temperature, stiffer and less elastic properties than that of pure Cs films and higher than pure agar films. Crosslinked blended films displayed further reduction in the swelling and melting temperature while maintained more hydrogel stability.

  15. Modeling of the Bacillus subtilis Bacterial Biofilm Growing on an Agar Substrate

    Science.gov (United States)

    Wang, Xiaoling; Wang, Guoqing; Hao, Mudong

    2015-01-01

    Bacterial biofilms are organized communities composed of millions of microorganisms that accumulate on almost any kinds of surfaces. In this paper, a biofilm growth model on an agar substrate is developed based on mass conservation principles, Fick's first law, and Monod's kinetic reaction, by considering nutrient diffusion between biofilm and agar substrate. Our results show biofilm growth evolution characteristics such as biofilm thickness, active biomass, and nutrient concentration in the agar substrate. We quantitatively obtain biofilm growth dependence on different parameters. We provide an alternative mathematical method to describe other kinds of biofilm growth such as multiple bacterial species biofilm and also biofilm growth on various complex substrates. PMID:26355542

  16. DEVELOPMENT AND EVALUATION OF A SELECTIVE AND INDICATIVE MEDIUM FOR ISOLATION OF ACTINOBACILLUS-PLEUROPNEUMONIAE FROM TONSILS

    DEFF Research Database (Denmark)

    Jakobsen, Marianne; Nielsen, Jens

    1995-01-01

    In order to isolate ActinobacillIus pleuropneumoniae from mixed bacterial flora a selective and indicative medium was developed. The optimal concentrations of antibiotics were determined for selective chocolate agar (S-TSA) and selective blood agar (S-MBA) using a set of 25 strains of A. pleuropn......In order to isolate ActinobacillIus pleuropneumoniae from mixed bacterial flora a selective and indicative medium was developed. The optimal concentrations of antibiotics were determined for selective chocolate agar (S-TSA) and selective blood agar (S-MBA) using a set of 25 strains of A...

  17. Rendimiento diagnóstico del agar sangre con filtro versus agar karmali para el diagnóstico de Campylobacter en coprocultivo

    OpenAIRE

    Moya-Salazar, Jeel; 1. MSc(c), M.T., Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Perú.; Pio-Dávila, Liz; 2. MLT., Hospital Nacional Dos de Mayo, Lima, Perú.; Terán-Vásquez, Alfonso; 3. M.Ed., MLT., Área de Microbiología, Hospital Nacional Docente Madre-Niño San Bartolomé, Lima, Perú.; Olivo-López, José; 4. M.T., Servicio de Microbiología. Instituto Nacional de Salud del Niño, Lima, Perú.

    2016-01-01

    Objetivo: Evaluar el rendimiento diagnóstico del agar sangre con filtro (ASF) en comparación con el agar Karmali (AK) para el diagnóstico de Campylobacter spp en coprocultivo.Material y métodos: Se realizó una investigación de tipo experimental prospectiva de corte transversal en el Hospital Nacional Docente Madre-Niño “San Bartolomé” se evaluaron muestra de heces con examen coprolóico funcional reactivo para inflamación. Las muestras fueron sembradas en ASF y en AK e incubadas hasta 72 horas...

  18. Enhanced catalytic efficiency of endo-β-agarase I by fusion of carbohydrate-binding modules for agar prehydrolysis.

    Science.gov (United States)

    Alkotaini, Bassam; Han, Nam Soo; Kim, Beom Soo

    2016-11-01

    Recently, Microbulbifer thermotolerans JAMB-A94 endo-β-agarase I was expressed as catalytic domain (GH16) without a carbohydrate-binding module (CBM). In this study, we successfully constructed different fusions of GH16 with its original CBM6 and CBM13 derived from Catenovulum agarivorans. The optimum temperature and pH for fusions GH16-CBM6, GH16-CBM13, GH16-CBM6-CBM13 and GH16-CBM13-CBM6 were similar to GH16, at 55°C and pH 7. All the constructed fusions significantly enhanced the GH16 affinity (Km) and the catalytic efficiency (Kcat/Km) toward agar. Among them, GH16-CBM6-CBM13 exhibited the highest agarolytic activity, for which Km decreased from 3.67 to 2.11mg/mL and Kcat/Km increased from 98.6 (mg/mL)(-1)sec(-1) to 400.6 (mg/mL)(-1)sec(-1). Moreover, all fusions selectively increased GH16 binding ability to agar, in which the highest binding ability of 95% was obtained with fusion GH16-CBM6-CBM13. Melted agar was prehydrolyzed with GH16-CBM6-CBM13, resulting in a degree of liquefaction of 45.3% and reducing sugar yield of 14.2%. Further addition of Saccharophagus degradans agarolytic enzymes resulted in mono-sugar yields of 35.4% for galactose and 31.5% for 3,6-anhydro-l-galactose. There was no pH neutralization step required and no 5-hydroxymethylfurfural detected, suggesting the potential of a new enzymatic prehydrolysis process for efficient production of bio-products such as biofuels. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Improved detection of Burkholderia pseudomallei from non-blood clinical specimens using enrichment culture and PCR: narrowing diagnostic gap in resource-constrained settings.

    Science.gov (United States)

    Tellapragada, Chaitanya; Shaw, Tushar; D'Souza, Annet; Eshwara, Vandana Kalwaje; Mukhopadhyay, Chiranjay

    2017-07-01

    To evaluate the diagnostic utility of enrichment culture and PCR for improved case detection rates of non-bacteraemic form of melioidosis in limited resource settings. Clinical specimens (n = 525) obtained from patients presenting at a tertiary care hospital of South India with clinical symptoms suggestive of community-acquired pneumonia, lower respiratory tract infections, superficial or internal abscesses, chronic skin ulcers and bone or joint infections were tested for the presence of Burkholderia pseudomallei using conventional culture (CC), enrichment culture (EC) and PCR. Sensitivity, specificity, positive and negative predictive values of CC and PCR were initially deduced using EC as the gold standard method. Further, diagnostic accuracies of all the three methods were analysed using Bayesian latent class modelling (BLCM). Detection rates of B. pseudomallei using CC, EC and PCR were 3.8%, 5.3% and 6%, respectively. Diagnostic sensitivities and specificities of CC and PCR were 71.4, 98.4% and 100 and 99.4%, respectively in comparison with EC as the gold standard test. With Bayesian latent class modelling, EC and PCR demonstrated sensitivities of 98.7 and 99.3%, respectively, while CC showed a sensitivity of 70.3% for detection of B. pseudomallei. An increase of 1.6% (95% CI: 1.08-4.32%) in the case detection rate of melioidosis was observed in the study population when EC and/or PCR were used in adjunct to the conventional culture technique. Our study findings underscore the diagnostic superiority of enrichment culture and/or PCR over conventional microbiological culture for improved case detection of melioidosis from non-blood clinical specimens. © 2017 John Wiley & Sons Ltd.

  20. Potato carrot agar with manganese as an isolation medium for Alternaria, Epicoccum and Phoma.

    Science.gov (United States)

    Sørensen, Jens Laurids; Mogensen, Jesper Mølgaard; Thrane, Ulf; Andersen, Birgitte

    2009-03-15

    A semi-selective medium for isolation of Alternaria spp., Epicoccum sp. and Phoma spp. from soil and plant samples was developed. The basal medium was a modified potato carrot agar (PCA), containing 10 g/L of potato and carrot. It is known that the target genera sporulate well on standard PCA when grown at 25 degrees C with an alternating light/dark cycle consisting of 8 h of cool-white daylight followed by 16 h darkness. Addition of 1.5% MnCl(2) 4 H(2)O (w/v) inhibited most other fungi than Alternaria, Epicoccum and Phoma species when tested on pure cultures. The mycobiota of two soil samples and eight grain samples were examined using PCA-Mn and three commonly used isolation media, DRYES, DG18 and V8. On the three conventional media growth of several genera was observed with the predominant being Aspergillus, Eurotium, Fusarium, Mucor, Penicillium and Rhizopus. Of these only F. oxysporum and F. verticillioides were able to grow on PCA-Mn. Alternaria infectoria and Epicoccum nigrum were present in three cereal grain samples, but emerged to a far lower degree on the three conventional media compared to PCA-Mn. Three black spored fungi, identified as Phoma eupyrena, Paraconiothyrium minitan and one unknown species, were isolated from the two soil samples when incubated on PCA-Mn but were absent on the three conventional media.

  1. Proton beam writing of microstructures in Agar gel for patterned cell growth

    Energy Technology Data Exchange (ETDEWEB)

    Larisch, Wolfgang, E-mail: wolfgang.larisch@studserv.uni-leipzig.de [Nukleare Festkoerperphysik, Universitaet Leipzig, Linnestr. 5, 04103 Leipzig (Germany); Koal, Torsten; Werner, Ronald; Hohlweg, Marcus; Reinert, Tilo; Butz, Tilman [Nukleare Festkoerperphysik, Universitaet Leipzig, Linnestr. 5, 04103 Leipzig (Germany)

    2011-10-15

    A rather useful prerequisite for many biological and biophysical studies, e.g., for cell-cell communication or neuronal networks, is confined cell growth on micro-structured surfaces. Solidified Agar layers have smooth surfaces which are electrically neutral and thus inhibit receptor binding and cell adhesion. For the first time, Agar microstructures have been manufactured using proton beam writing (PBW). In the irradiated Agar material the polysaccharides are split into oligosaccharides which can easily be washed off leaving Agar-free areas for cell adhesion. The beam diameter of 1 {mu}m allows the fabrication of compartments accommodating single cells which are connected by micrometer-sized channels. Using the external beam the production process is very fast. Up to 50 Petri dishes can be produced per day which makes this technique very suitable for biological investigations which require large throughputs.

  2. Proton beam writing of microstructures in Agar gel for patterned cell growth

    Science.gov (United States)

    Larisch, Wolfgang; Koal, Torsten; Werner, Ronald; Hohlweg, Marcus; Reinert, Tilo; Butz, Tilman

    2011-10-01

    A rather useful prerequisite for many biological and biophysical studies, e.g., for cell-cell communication or neuronal networks, is confined cell growth on micro-structured surfaces. Solidified Agar layers have smooth surfaces which are electrically neutral and thus inhibit receptor binding and cell adhesion. For the first time, Agar microstructures have been manufactured using proton beam writing (PBW). In the irradiated Agar material the polysaccharides are split into oligosaccharides which can easily be washed off leaving Agar-free areas for cell adhesion. The beam diameter of 1 μm allows the fabrication of compartments accommodating single cells which are connected by micrometer-sized channels. Using the external beam the production process is very fast. Up to 50 Petri dishes can be produced per day which makes this technique very suitable for biological investigations which require large throughputs.

  3. Use of agar-based diet to fulfil the food and water requirements of mice.

    Science.gov (United States)

    Lang, J A; Lang, C M; White, W J

    1984-01-01

    Mice were fed an agar-based diet without an additional source of water for 5 weeks. In comparison with a similar group of mice fed a commercial diet and water ad libitum, there were no significant changes in bodyweight.

  4. A fresh liver agar substrate for rearing small numbers of forensically important blow flies (Diptera: Calliphoridae)

    Science.gov (United States)

    Gruner, Susan V.; Slone, Daniel H.

    2014-01-01

    Forensically important calliphorids can be reared on a mixture of beef liver and agar. Small pieces of meat, especially fresh or frozen beef liver, will desiccate in 2–6 h, but this simple-to-make feeding substrate remains moist for at least 12 h at 25 and 30°C without desiccation, even in small (5 g) amounts. We determined the survivorship of small numbers of Chrysomya megacephala (F.) (first-instar larvae to adult eclosion) raised on 5 g of liver agar and fresh beef liver. We found that all larvae raised on 5 g of liver died due to desiccation, but survivorship on 5 g of liver agar was equivalent to that on larger (50 g) pieces of either liver agar or beef liver.

  5. Agar Dish Isopiestic Equilibration Method for Controlling the Water Potential of Solid Substrates 1

    Science.gov (United States)

    Harris, R. F.; Gardner, W. R.; Adebayo, A. A.; Sommers, L. E.

    1970-01-01

    Maintenance of substrate water potential in petri dishes is achieved by using vapor-pressure controlling, solute-amended agar gel discs attached to the inside of the top halves of the dishes. PMID:4909356

  6. Effect of Gravity on the Colonial Morphology of Staphylococci in Soft Agar

    Science.gov (United States)

    Wilkins, Judd R.; Hatten, Mary B.; Salmirs, Seymour

    1969-01-01

    After horizontal rotation on a clinostat at 1 rev/min, subsurface colonies of staphylococci in soft agar were compact and spherical; nonrotated colonies were diffuse and elongated. Images PMID:5369302

  7. Green fabrication of agar-conjugated Fe{sub 3}O{sub 4} magnetic nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Hsieh, S; Huang, B Y; Lin, P Y; Chang, C W [Department of Chemistry and Center for Nanoscience and Nanotechnology, National Sun Yat-sen University, Kaohsiung 80424, Taiwan (China); Hsieh, S L [Department of Seafood Science, National Kaohsiung Marine University, Kaohsiung 81157, Taiwan (China); Wu, C C [Department of Nutrition and Health Sciences, Chang Jung Christian University, Tainan 71101, Taiwan (China); Wu, C H [Department of Computer Science and Information Engineering, National University of Kaohsiung, Kaohsiung 80811, Taiwan (China); Huang, Y S, E-mail: shsieh@facmail.NSYSU.edu.tw [Department of Food Science and Technology, Tajen University, Pingtung 90741, Taiwan (China)

    2010-11-05

    Magnetic nanoparticles are of great interest both for fundamental research and emerging applications. In the biomedical field, magnetite (Fe{sub 3}O{sub 4}) has shown promise as a hyperthermia-based tumor therapeutic. However, preparing suitable solubilized magnetite nanoparticles is challenging, primarily due to aggregation and poor biocompatibility. Thus methods for coating Fe{sub 3}O{sub 4} NPs with biocompatible stabilizers are required. We report a new method for preparing Fe{sub 3}O{sub 4} nanoparticles by co-precipitation within the pores of agar gel samples. Permeated agar gels were then dried and ground into a powder, yielding agar-conjugated Fe{sub 3}O{sub 4} nanoparticles. Samples were characterized using XRD, FTIR, TGA, TEM and SQUID. This method for preparing agar-coated Fe{sub 3}O{sub 4} nanoparticles is environmentally friendly, inexpensive and scalable.

  8. Effect of directly compressible excipient and treated agar on drug release of clopidogrel oral disintegrating tablets.

    Science.gov (United States)

    Venkateswarlu, Kambham; Nirosha, Manyam; Kumar Reddy, Budideti Kishore; Sai Kiran, Badithala Siva

    2017-07-01

    In the present investigation, oral dissolving/dispersible/disintegrating tablets (ODTs) of clopidogrel were designed with a view to enhance the bioavailability and patient compliance by two different methods, namely, direct compression and effervescent methods using directly compressible excipient and treated agar (TAG). In the direct compression method, TAG was used as a disintegrant and another method used a mixture of sodium bicarbonate and tartaric acid along with TAG as disintegrants. Among the directly compressed tablets, treated agar formulation 3 and among the effervescent tablets, treated agar and effervescent formulation 4 was found to be promising. Treated agar formulation 3 prepared by direct compression method emerged as an overall best formulation based on the in vitro drug release characteristics.

  9. AGAR FROM MALAYSIAN RED SEAWEED AS POTENTIAL MATERIAL FOR SYNTHESIS OF BIOPLASTIC FILM

    National Research Council Canada - National Science Library

    SIEW-LING HII; JIA-YEE LIM; WAN-TECK ONG; CHING-LEE WONG

    2016-01-01

    The main aim of this study was to identify the potential use of agar extracted from red seaweed, Gracilaria salicornia, collected from the coastal area of Malaysia as the raw material for synthesis of bioplastic film...

  10. PacBio But Not Illumina Technology Can Achieve Fast, Accurate and Complete Closure of the High GC, Complex Burkholderia pseudomallei Two-Chromosome Genome

    Directory of Open Access Journals (Sweden)

    Jade L. L. Teng

    2017-08-01

    Full Text Available Although PacBio third-generation sequencers have improved the read lengths of genome sequencing which facilitates the assembly of complete genomes, no study has reported success in using PacBio data alone to completely sequence a two-chromosome bacterial genome from a single library in a single run. Previous studies using earlier versions of sequencing chemistries have at most been able to finish bacterial genomes containing only one chromosome with de novo assembly. In this study, we compared the robustness of PacBio RS II, using one SMRT cell and the latest P6-C4 chemistry, with Illumina HiSeq 1500 in sequencing the genome of Burkholderia pseudomallei, a bacterium which contains two large circular chromosomes, very high G+C content of 68–69%, highly repetitive regions and substantial genomic diversity, and represents one of the largest and most complex bacterial genomes sequenced, using a reference genome generated by hybrid assembly using PacBio and Illumina datasets with subsequent manual validation. Results showed that PacBio data with de novo assembly, but not Illumina, was able to completely sequence the B. pseudomallei genome without any gaps or mis-assemblies. The two large contigs of the PacBio assembly aligned unambiguously to the reference genome, sharing >99.9% nucleotide identities. Conversely, Illumina data assembled using three different assemblers resulted in fragmented assemblies (201–366 contigs, sharing only 92.2–100% and 92.0–100% nucleotide identities to chromosomes I and II reference sequences, respectively, with no indication that the B. pseudomallei genome consisted of two chromosomes with four copies of ribosomal operons. Among all assemblies, the PacBio assembly recovered the highest number of core and virulence proteins, and housekeeping genes based on whole-genome multilocus sequence typing (wgMLST. Most notably, assembly solely based on PacBio outperformed even hybrid assembly using both PacBio and Illumina

  11. Low density, microcellular, dopable, agar/gelatin foams for pulsed power experiments

    Energy Technology Data Exchange (ETDEWEB)

    McNamara, W.F. [Orion International Technologies, Inc., Albuquerque, NM (United States); Aubert, J.H. [Sandia National Lab., Albuquerque, NM (United States)

    1997-04-01

    Low-density, microcellular foams prepared from the natural polymers agar and gelatin have been developed for pulsed-power physics experiments. Numerous experiments were supported with foams having densities at or below 10 mg/cm{sup 3}. For some of the experiments, the agar/gelatin foam was uniformly doped with metallic elements using soluble salts. Depending on the method of preparation, cell sizes were typically below 10 microns and for one process were below 1.0 micron.

  12. [GROWTH OF MICROMYCETES FROM DIFFERENT ECOLOGICAL NICHES ON AGAR NUTRIENT MEDIA].

    Science.gov (United States)

    Kurchenko, I M; Yurieva, E M; Voychuk, S I

    2015-01-01

    Radial growth rate of (K(r)) 153 strains 6 species of micromycetes from different ecological niches was studied on 7 agar media: three standard (malt extract agar, potato-dextrose agar, Czapek's agar), and on agar media with plant polymers (carboxymethylcellulose, xylan, soluble starch and apple pectin). Endophytic and plant pathogenic strains (biotrophs) of all studied species did not differ significantly in their ability to grow on nutrient media of different composition--average values of K(r) for these two groups were the same (0,200 and 0,199 mm/h, respectively). Soil micromycetes (saprophytes) characterized by the lowest average growth rate (0,169 mm/h) and significantly differed from the endophytic and plant pathogenic ones. Average of the radial growth rates of studied microscopic fungi were higher on standard nutrient media than with plant polymers ones. Growth parameters of endophytes and plant pathogens of all studied species on various agar media differed from the soil strains. High growth rate of endophytic and plant pathogenic strains of Fusarium poae, Alternaria alternata and Ceratocystis sp. provides them the rapid colonization of plants. Penicillium funiculosum strains equally can exist as saprophytes in soil and as endophytic plant symbionts. A wide range of K(r) variation of endophytic dark pigmented Mycelia sterilia indicates the presence in this group of different species of micromycetes, which have no sporulation.

  13. Broth versus solid agar culture of swab samples of cadaveric allograft musculoskeletal tissue.

    Science.gov (United States)

    Varettas, Kerry

    2013-12-01

    As part of the donor assessment protocol, bioburden assessment must be performed on allograft musculoskeletal tissue samples collected at the time of tissue retrieval. Swab samples of musculoskeletal tissue allografts from cadaveric donors are received at the microbiology department of the South Eastern Area Laboratory Services (Australia) to determine the presence of bacteria and fungi. This study will review the isolation rate of organisms from solid agar and broth culture of swab samples of cadaveric allograft musculoskeletal tissue over a 6-year period, 2006-2011. Swabs were inoculated onto horse blood agar (anaerobic, 35 °C) and chocolate agar (CO2, 35 °C) and then placed into a cooked meat broth (aerobic, 35 °C). A total of 1,912 swabs from 389 donors were received during the study period. 557 (29.1 %) swabs were culture positive with the isolation of 713 organisms, 249 (34.9 %) from solid agar culture and an additional 464 (65.1 %) from broth culture only. This study has shown that the broth culture of cadaveric allograft musculoskeletal swab samples recovered a greater amount of organisms than solid agar culture. Isolates such as Clostridium species and Staphylococcus aureus would not have been isolated from solid agar culture alone. Broth culture is an essential part of the bioburden assessment protocol of swab samples of cadaveric allograft musculoskeletal tissue in this laboratory.

  14. Fluconazole-containing agar Sabouraud dextrose plates are not useful when screening for susceptibility in Candida albicans.

    Science.gov (United States)

    Bordallo-Cardona, M A; Marcos-Zambrano, L J; Gómez G de la Pedrosa, E; Escribano, P; Bouza, E; Guinea, J; Cantón, R

    2017-04-01

    Fluconazole is an alternative for candidemic patients who are not critically ill. Fluconazole is mainly fungistatic and does not completely inhibit visual Candida albicans growth. We studied the usefulness of fluconazole-containing Sabouraud dextrose agar plates for detecting susceptibility to fluconazole in C. albicans. Adjusted inocula of 19 isolates were transferred directly onto fluconazole-containing Sabouraud dextrose plates (concentrations ranging from 0.125 mg/L to 128 mg/L). The fluconazole MIC in fresh isolates and after growth on the fluconazole-containing plate at 128 mg/L was recorded following the EUCAST EDef 7.2 guidelines. Then isolates were classified according to their degree of trailing production, based on microdilution procedure. All isolates were able to grow on all fluconazole-containing plates, even those isolates susceptible to fluconazole. In fact, we selected isolates with different degrees of trailing based on microdilution procedures. 50% of isolates classified as heavy trailers, 35.71% as moderate trailers, and 14.28% as slight trailers. The use of fluconazole-containing Sabouraud dextrose agar plates was not a reliable method to detect fluconazole susceptibility in C. albicans isolates; growth of the isolates was a trailing effect rather than true resistance.

  15. Comparison of the Sulfonamide Inhibition Profiles of the β- and γ-Carbonic Anhydrases from the Pathogenic Bacterium Burkholderia pseudomallei

    Directory of Open Access Journals (Sweden)

    Daniela Vullo

    2017-03-01

    Full Text Available We have cloned, purified, and characterized a β-carbonic anhydrase (CA, EC 4.2.1.1, BpsCAβ, from the pathogenic bacterium Burkholderia pseudomallei, responsible for the tropical disease melioidosis. The enzyme showed high catalytic activity for the physiologic CO2 hydration reaction to bicarbonate and protons, with the following kinetic parameters: kcat of 1.6 × 105 s−1 and kcat/KM of 3.4 × 107 M−1 s−1. An inhibition study with a panel of 38 sulfonamides and one sulfamate—including 15 compounds that are used clinically—revealed an interesting structure–activity relationship for the interaction of this enzyme with these inhibitors. Many simple sulfonamides and clinically used agents such as topiramate, sulpiride, celecoxib, valdecoxib, and sulthiame were ineffective BpsCAβ inhibitors (KI > 50 µM. Other drugs, such as ethoxzolamide, dorzolamide, brinzolamide, zonisamide, indisulam, and hydrochlorothiazide were moderately potent micromolar inhibitors. The best inhibition was observed with benzene-1,3-disulfonamides—benzolamide and its analogs acetazolamide and methazolamide—which showed KI in the range of 185–745 nM. The inhibition profile of BpsCAβ is very different from that of the γ-class enzyme from the same pathogen, BpsCAγ. Thus, identifying compounds that would effectively interact with both enzymes is relatively challenging. However, benzolamide was one of the best inhibitors of both of these CAs with KI of 653 and 185 nM, respectively, making it an interesting lead compound for the design of more effective agents, which may be useful tools for understanding the pathogenicity of this bacterium.

  16. Burkholderia mallei and Burkholderia pseudomallei stimulate differential inflammatory responses from human alveolar type II cells (ATII) and macrophages.

    Science.gov (United States)

    Lu, Richard; Popov, Vsevolod; Patel, Jignesh; Eaves-Pyles, Tonyia

    2012-01-01

    Alveolar type II pneumocytes (ATII) and alveolar macrophages (AM) play a crucial role in the lung's innate immune response. Burkholderia pseudomallei (BP) and Burkholderia mallei (BM) are facultative Gram-negative bacilli that cause melioidosis and glanders, respectively. The inhalation of these pathogens can cause lethal disease and death in humans. We sought to compare the pathogenesis of and host responses to BP and BM through contact with human primary ATII cells and monocytes-derived macrophages (MDM). We hypothesized that because BP and BM induce different disease outcomes, each pathogen would induce distinct, unique host immune responses from resident pulmonary cells. Our findings showed that BP adhered readily to ATII cells compared to BM. BP, but not BM, was rapidly internalized by macrophages where it replicated to high numbers. Further, BP-induced significantly higher levels of pro-inflammatory cytokine secretion from ATII cells (IL-6, IL-8) and macrophages (IL-6, TNFα) at 6 h post-infection compared to BM (p < 0.05). Interestingly, BM-induced the anti-inflammatory cytokine, IL-10, in ATII cells and macrophages at 6 h post-infection, with delayed induction of inflammatory cytokines at 24 h post-infection. Because BP is flagellated and produces LPS, we confirmed that it stimulated both Toll-like receptor (TLR) 4 and TLR5 via NF-κb activation while the non-flagellated BM stimulated only TLR4. These data show the differences in BP and BM pathogenicity in the lung when infecting human ATII cells and macrophages and demonstrate the ability of these pathogens to elicit distinct immune responses from resident lung cells which may open new targets for therapeutic intervention to fight against these pathogens.

  17. Burkholderia mallei and Burkholderia pseudomallei stimulate differential inflammatory responses from human alveolar type II cells (ATII and macrophages.

    Directory of Open Access Journals (Sweden)

    Richard eLu

    2012-12-01

    Full Text Available Alveolar type II pneumocytes (ATII and alveolar macrophages (AM play a crucial role in the lung’s innate immune response. Burkholderia pseudomallei (BP and Burkholderia mallei (BM are facultative Gram-negative bacilli that cause melioidosis and glanders, respectively. The inhalation of these pathogens can cause lethal disease and death in humans. We sought to compare the pathogenesis of and host responses to BP and BM through contact with human primary ATII cells and monocytes-derived macrophages (MDM. We hypothesized that because BP and BM induce different disease outcomes, each pathogen would induce distinct, unique host immune responses from resident pulmonary cells. Our findings showed that BP adhered readily to ATII cells compared to BM. BP, but not BM, was rapidly internalized by macrophages where it replicated to high numbers. Further, BP induced significantly higher levels of pro-inflammatory cytokine secretion from ATII cells (IL-6, IL-8 and macrophages (IL-6, TNFα at 6h post-infection compared to BM (p<0.05. Interestingly, BM induced the anti-inflammatory cytokine, IL-10, in ATII cells and macrophages at 6h post-infection, with delayed induction of inflammatory cytokines at 24h post-infection. Because BP is flagellated and produces LPS, we confirmed that it stimulated both Toll-like receptor (TLR 4 and TLR5 via NF-κb activation while the non-flagellated BM stimulated only TLR4. These data show the differences in BP and BM pathogenicity in the lung when infecting human ATII cells and macrophages and demonstrate the ability of these pathogens to elicit distinct immune responses from resident lung cells which may open new targets for therapeutic intervention to fight against these pathogens.

  18. Development of hydrolysis probe-based real-time PCR for identification of virulent gene targets of Burkholderia pseudomallei and B. mallei--a retrospective study on archival cases of service members with melioidosis and glanders.

    Science.gov (United States)

    Zhang, Binxue; Wear, Douglas J; Kim, H S; Weina, Peter; Stojadinovic, Alexander; Izadjoo, Mina

    2012-02-01

    Burkholderia pseudomallei and B. mallei are two highly pathogenic bacteria responsible for melioidosis and glanders, respectively. Our laboratory developed hydrolysis probe-based real-time polymerase chain reaction assays targeting type three secretion system (TTS) and transposase family protein (TFP) of B. pseudomallei and B. malli, respectively. The assays were validated for target specificity, amplification sensitivity, and reproducibility. A bacterial DNA panel, composed of B. pseudomallei (13 strains), B. mallei (11 strains), Burkholderia species close neighbors (5 strains), and other bacterial species (17 strains), was prepared for specificity testing. Reference DNAs from B. pseudomallei and B. mallei bacterial cultures were used as controls for amplification, limit of detection, and reproducibility testing. The two TaqMan assays, Bp-TTS 1 and Bm-TFP, were optimized and applied in a retrospective study of archived cases from the Armed Forces Institute of Pathology. We tested 10 formalin-fixed paraffin-embedded blocks originally from autopsy specimens of patients who died of melioidosis or glanders during or after overseas tours in 1960s. Polymerase chain reaction results confirmed that DNA samples from formalin-fixed paraffin-embedded blocks of eight patients with melioidosis were positive for Bp-TTS 1 target and two patients with glanders were positive for Bm-TFP target.

  19. Gas chromatography-mass spectrometry method for rapid identification and differentiation of Burkholderia pseudomallei and Burkholderia mallei from each other, Burkholderia thailandensis and several members of the Burkholderia cepacia complex.

    Science.gov (United States)

    Li, D; March, J K; Bills, T M; Holt, B C; Wilson, C E; Lowe, W; Tolley, H D; Lee, M L; Robison, R A

    2013-11-01

    To develop a simple gas chromatography-mass spectrometry (GC-MS) method for the detection and differentiation of Burkholderia pseudomallei and Burkholderia mallei from each other, Burkholderia thailandensis and several members of the Burkholderia cepacia complex. Biomarkers were generated by one-step thermochemolysis (TCM) and analysed using a GC-MS system. Fragments of poly-3-hydroxybutyrate-co-hydroxyvalerate [poly(3HBA-co-3HVA)] produced by TCM were useful biomarkers. Several cellular fatty acid methyl esters were important in differentiating the various Burkholderia species. A statistical discrimination algorithm was constructed using a combination of biomarkers. The identities of four B. pseudomallei strains, four B. mallei strains and one strain of each near neighbour were confirmed in a statistically designed test using the algorithm. The detection limit for this method was found to be approximately 4000 cells. The method is fast, accurate and easy to use. The algorithm is robust against different growth conditions (medium and temperature). This assay may prove beneficial in a clinical diagnostic setting, where the rapid identification of B. pseudomallei is essential to effective treatment. This method could also be easily employed after a biological attack to confirm the presence of either B. pseudomallei or B. mallei. © 2013 The Society for Applied Microbiology.

  20. Intramolecular aglycon delivery enables the synthesis of 6-deoxy-β-D-manno-heptosides as fragments of Burkholderia pseudomallei and Burkholderia mallei capsular polysaccharide.

    Science.gov (United States)

    Tamigney Kenfack, Marielle; Blériot, Yves; Gauthier, Charles

    2014-05-16

    Burkholderia pseudomallei and Burkholderia mallei are potential bioterrorism agents. They express the same capsular polysaccharide (CPS), a homopolymer featuring an unusual [→3)-2-O-acetyl-6-deoxy-β-D-manno-heptopyranosyl-(1→] as the repeating unit. This CPS is known to be one of the main targets of the adaptive immune response in humans and therefore represents a crucial subunit candidate for vaccine development. Herein, the stereoselective synthesis of mono- and disaccharidic fragments of the B. pseudomallei and B. mallei CPS repeating unit is reported. The synthesis of 6-deoxy-β-D-manno-heptosides was investigated using both inter- and intramolecular glycosylation strategies from thio-manno-heptose that was modified with 2-naphthylmethyl (NAP) at C2. We show here that NAP-mediated intramolecular aglycon delivery (IAD) represents a suitable approach for the stereocontrolled synthesis of 6-deoxy-β-D-manno-heptosides without the need for rigid 4,6-O-cyclic protection of the sugar skeleton. The IAD strategy is highly modular, as it can be applied to structurally diverse acceptors with complete control of stereoselectivity. Problematic hydrogenation of the acetylated disaccharides was overcome by using a microfluidic continuous flow reactor.

  1. A hidden pitfall in the preparation of agar media undermines microorganism cultivability.

    Science.gov (United States)

    Tanaka, Tomohiro; Kawasaki, Kosei; Daimon, Serina; Kitagawa, Wataru; Yamamoto, Kyosuke; Tamaki, Hideyuki; Tanaka, Michiko; Nakatsu, Cindy H; Kamagata, Yoichi

    2014-12-01

    Microbiologists have been using agar growth medium for over 120 years. It revolutionized microbiology in the 1890s when microbiologists were seeking effective methods to isolate microorganisms, which led to the successful cultivation of microorganisms as single clones. But there has been a disparity between total cell counts and cultivable cell counts on plates, often referred to as the "great plate count anomaly," that has long been a phenomenon that still remains unsolved. Here, we report that a common practice microbiologists have employed to prepare agar medium has a hidden pitfall: when phosphate was autoclaved together with agar to prepare solid growth media (PT medium), total colony counts were remarkably lower than those grown on agar plates in which phosphate and agar were separately autoclaved and mixed right before solidification (PS medium). We used a pure culture of Gemmatimonas aurantiaca T-27(T) and three representative sources of environmental samples, soil, sediment, and water, as inocula and compared colony counts between PT and PS agar plates. There were higher numbers of CFU on PS medium than on PT medium using G. aurantiaca or any of the environmental samples. Chemical analysis of PT agar plates suggested that hydrogen peroxide was contributing to growth inhibition. Comparison of 454 pyrosequences of the environmental samples to the isolates revealed that taxa grown on PS medium were more reflective of the original community structure than those grown on PT medium. Moreover, more hitherto-uncultivated microbes grew on PS than on PT medium. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  2. Mass production of spores of lactic acid-producing Rhizopus oryzae NBRC 5384 on agar plate.

    Science.gov (United States)

    Yamane, Tsuneo; Tanaka, Ryosuke

    2013-01-01

    Mass production of sporangiospores (spores) of Rhizopus oryzae NBRC 5384 (identical to NRRL 395 and ATCC 9363) on potato-dextrose-agar medium was studied aiming at starting its L(+)-lactic acid fermentation directly from spore inoculation. Various parameters including harvest time, sowed spore density, size of agar plate, height of air space, and incubation mode of plate (agar-on-bottom or agar-on-top) were studied. Ordinarily used shallow Petri dishes were found out to be unsuitable for the full growth of R. oryzae sporangiophores. In a very wide range of the sowed spore density, the smaller it was, the greater the number of the harvested spores was. It was also interesting to find out that R. oryzae grown downward vertically with a deep air space in an agar-on-top mode gave larger amount of spores than in an agar-on-bottom mode at 30°C for 7-day cultivation. Scale-up of the agar plate culture from 26.4 to 292 cm(2) was studied, resulting in the proportional relationship between the number of the harvested spores/plate and the plate area in the deep Petri dishes. The number of plates of 50 cm in diameter needed for 100 m(3) industrial submerged fermentation started directly from 2 × 10(5) spores/mL inoculum size was estimated as about 6, from which it was inferred that such a fermentation would be feasible. Designing a 50 cm plate and a method of spreading and collecting the spores were suggested. Bioprocess technological significance of the "full-scale industrial submerged fermentation started directly from spore inoculation omitting pre-culture" has been discussed. © 2013 American Institute of Chemical Engineers.

  3. Performance of CHROMAGAR candida and BIGGY agar for identification of yeast species

    Directory of Open Access Journals (Sweden)

    Marol Serhat

    2003-10-01

    Full Text Available Abstract Background The importance of identifying the pathogenic fungi rapidly has encouraged the development of differential media for the presumptive identification of yeasts. In this study two differential media, CHROMagar Candida and bismuth sulphite glucose glycine yeast agar, were evaluated for the presumptive identification of yeast species. Methods A total number of 270 yeast strains including 169 Candida albicans, 33 C. tropicalis, 24 C. glabrata, 18 C. parapsilosis, 12 C. krusei, 5 Trichosporon spp., 4 C. kefyr, 2 C. lusitaniae, 1 Saccharomyces cerevisiae and 1 Geotrichum candidum were included. The strains were first identified by germ tube test, morphological characteristics on cornmeal tween 80 agar and Vitek 32 and API 20 C AUX systems. In parallel, they were also streaked onto CHROMagar Candida and bismuth sulphite glucose glycine yeast agar plates. The results were read according to the color, morphology of the colonies and the existance of halo around them after 48 hours of incubation at 37°C. Results The sensitivity and specificity values for C. albicans strains were found to be 99.4, 100% for CHROMagar Candida and 87.0, 75.2% for BiGGY agar, respectively. The sensitivity of CHROMagar Candida to identify C. tropicalis, C. glabrata and C. krusei ranged between 90.9 and 100% while the specificity was 100%. The sensitivity rates for BiGGY agar were 66.6 and 100% while the specificity values were found to be 95.4 and 100% for C. tropicalis and C. krusei, respectively. Conclusions It can be concluded that the use of CHROMagar Candida is an easy and reliable method for the presumptive identification of most commonly isolated Candida species especially C. albicans, C. tropicalis and C. krusei. The lower sensitivity and specificity of BiGGY agar to identify commonly isolated Candida species potentially limits the clinical usefulness of this agar.

  4. Preparation and characterization of agar/clay nanocomposite films: the effect of clay type.

    Science.gov (United States)

    Rhim, Jong-Whan; Lee, Soo-Bin; Hong, Seok-In

    2011-04-01

    Agar-based nanocomposite films with different types of nanoclays, such as Cloisite Na+, Cloisite 30B, and Cloisite 20A, were prepared using a solvent casting method, and their tensile, water vapor barrier, and antimicrobial properties were tested. Tensile strength (TS), elongation at break (E), and water vapor permeability (WVP) of control agar film were 29.7±1.7 MPa, 45.3±9.6%, and (2.22±0.19)×10(-9) g·m/m2·s·Pa, respectively. All the film properties tested, including transmittance, tensile properties, WVP, and X-ray diffraction patterns, indicated that Cloisite Na+ was the most compatible with agar matrix. TS of the nanocomposite films prepared with 5% Cloisite Na+ increased by 18%, while WVP of the nanocomposite films decreased by 24% through nanoclay compounding. Among the agar/clay nanocomposite films tested, only agar/Cloisite 30B nanocomposite film showed a bacteriostatic function against Listeria monocytogenes.

  5. Antimicrobial and physical-mechanical properties of agar-based films incorporated with grapefruit seed extract.

    Science.gov (United States)

    Kanmani, Paulraj; Rhim, Jong-Whan

    2014-02-15

    The use of synthetic petroleum based packaging films caused serious environmental problems due to their difficulty in recycling and poor biodegradability. Therefore, present study was aimed to develop natural biopolymer-based antimicrobial packaging films as an alternative for the synthetic packaging films. As a natural antimicrobial agent, grapefruit seed extract (GSE) has been incorporated into agar to prepare antimicrobial packaging film. The films with different concentrations of GSE were prepared by a solvent casting method and the resulting composite films were examined physically and mechanically. In addition, the films were characterized by FE-SEM, XRD, FT-IR and TGA. The incorporation of GSE caused increase in color, UV barrier, moisture content, water solubility and water vapor permeability, while decrease in surface hydrophobicity, tensile strength and elastic modulus of the films. As the concentration of GSE increased from 0.6 to 13.3 μg/mL, the physical and mechanical properties of the films were affected significantly. The addition of GSE changed film microstructure of the film, but did not influence the crystallinity of agar and thermal stability of the agar-based films. The agar/GSE films exhibited distinctive antimicrobial activity against three test food pathogens, such as Listeria monocytogenes, Bacillus cereus and Escherichia coli. These results suggest that agar/GSE films have potential to be used in an active food packaging systems for maintaining food safety and extending the shelf-life of the packaged food. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Effects of shape and size of agar gels on heating uniformity during pulsed microwave treatment.

    Science.gov (United States)

    Soto-Reyes, Nohemí; Temis-Pérez, Ana L; López-Malo, Aurelio; Rojas-Laguna, Roberto; Sosa-Morales, María Elena

    2015-05-01

    Model gel systems with different shape (sphere, cylinder, and slab) and size (180 and 290 g) were prepared with agar (5%) and sucrose (5%). Dielectric constant (ε'), loss factor (ε"), thermophysical properties, and temperature distribution of the model system were measured. Each agar model system was immersed and suspended in water, and then, heated in a microwave oven with intermittent heating until the core temperature reached 50 °C. The ε' and ε" of agar gels decreased when frequency increased. The density and thermal conductivity values of the agar gels were 1033 kg/m(3) and 0.55 W/m °C, respectively. The temperature distribution of sphere, cylinder, and slab was different when similar power doses were applied. The slab reached 50 °C in less time (10 min) and showed a more uniform heating than spheres and cylinders in both sizes. Agar model systems of 180 g heated faster than those of 290 g. The coldest point was the center of the model systems in all studied cases. Shape and size are critical food factors that affect the heating uniformity during microwave heating processes. © 2015 Institute of Food Technologists®

  7. [Screening and identification of a bacterium capable of converting agar to neoagaro oligosaccharides].

    Science.gov (United States)

    Han, Junping; Huang, Yayan; Ye, Jing; Xiao, Meitian

    2015-09-04

    To screen and identify a bacterium capable of converting agar to neoagaro oligosaccharides. We took samples of porphyra haitanensis and nearby seawater, and then used the medium containing 1 per thousand agar to enrich the target bacteria. The target isolates were obtained by dilution-plate method, of which crude enzymes were further obtained by liquid culture. We adopted DNS method to determine the target bacteria which can convert agar to neoagaro oligosaccharides. The phylogenetics was identified by analyzing 16S rDNA sequence and combining the strain's morphological and bacterial colonial physiological biochemical characteristics. We isolated a gram-negative bacterial strain HJPHYXJ-1 capable of transforming agar to neoagaro oligosaccharides. Basic Local Alignment Search Tool (BLAST) search of HJPHYXJ-1's 16S rDNA sequence on GenBank suggested that the similarity between this strain and Vibrio natriegens reached 99% . In addition, the morphological and physiological biochemical characteristics of HJPHYXJ-1 also showed highly similarity to Vibrio natriegens. So we identified HJPHYXJ-1 as Vibrio natriegens. The results of HPLC suggested that the metabolite of enzymatic degradation was neoagaro oligosaccharides. HJPHYXJ-1 or the new isolate of Vibrio natriegens was capable of converting agar to neoagaro oligosaccharides.

  8. Migration of Chemotactic Bacteria in Soft Agar: Role of Gel Concentration

    Science.gov (United States)

    Croze, Ottavio A.; Ferguson, Gail P.; Cates, Michael E.; Poon, Wilson C.K.

    2011-01-01

    We study the migration of chemotactic wild-type Escherichia coli populations in semisolid (soft) agar in the concentration range C = 0.15–0.5% (w/v). For C≲0.35%, expanding bacterial colonies display characteristic chemotactic rings. At C = 0.35%, however, bacteria migrate as broad circular bands rather than sharp rings. These are growth/diffusion waves arising because of suppression of chemotaxis by the agar and have not been previously reported experimentally to our knowledge. For C = 0.4–0.5%, expanding colonies do not span the depth of the agar and develop pronounced front instabilities. The migration front speed is weakly dependent on agar concentration at C agar. The model makes it possible to fit the observed front speed decay in the range C = 0.15–0.35%, and its solutions qualitatively reproduce the observed transition from chemotactic to growth/diffusion bands. We discuss the implications of our results for the study of bacteria in porous media and for the design of improved bacteriological chemotaxis assays. PMID:21806920

  9. Rheological and structural characterization of agar/whey proteins insoluble complexes.

    Science.gov (United States)

    Rocha, Cristina M R; Souza, Hiléia K S; Magalhães, Natália F; Andrade, Cristina T; Gonçalves, Maria Pilar

    2014-09-22

    Complex coacervation between whey proteins and carboxylated or highly sulphated polysaccharides has been widely studied. The aim of this work was to characterise a slightly sulphated polysaccharide (agar) and whey protein insoluble complexes in terms of yield, composition and physicochemical properties as well as to study their rheological behaviour for better understanding their structure. Unlike other sulphated polysaccharides, complexation of agar and whey protein at pH 3 in the absence of a buffering agent resulted in a coacervate that was a gel at 20°C with rheological properties and structure similar to those of simple agar gels, reinforced by proteins electrostatically aggregated to the agar network. The behaviour towards heat treatment was similar to that of agar alone, with a high thermal hysteresis and almost full reversibility. In the presence of citrate buffer, the result was a "flocculated solid", with low water content (75-81%), whose properties were governed by protein behaviour. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. The effect of agar jelly on energy expenditure, appetite, gastric emptying and glycaemic response.

    Science.gov (United States)

    Clegg, Miriam E; Shafat, Amir

    2014-01-01

    Agar contains a high amount of soluble fibre and has been shown to delay gastric emptying (GE) without impacting on glycaemic response (GR). The current study aimed to further the limited data on the effect of agar on metabolism by assessing the effects on GE and GR as well as appetite- and diet-induced thermogenesis (DIT). In this randomized control trial, eleven healthy volunteers were tested on two occasions following an overnight fast. Following baseline and resting measurements, volunteers were either fed a fruit-flavoured drink (liquid) or consumed a fruit-flavoured jelly (jelly). The two were exactly the same in composition except the jelly contained 4 g of agar crystals. Both contained 50 g of available carbohydrate. DIT was measured using indirect calorimetry, GE using the (13)C sodium acetate breath test, appetite using visual analogue scale and GR using finger prick blood samples. The jelly significantly delayed GE across all time points-latency phase (p = 0.07), lag phase (p = 0.04), half-time (p Agar delays GE and increases appetite but does not change GR or DIT most probably due to the increase in viscosity caused by the agar jelly.

  11. Novel grafted agar disks for the covalent immobilization of β-D-galactosidase.

    Science.gov (United States)

    Wahba, Marwa I; Hassan, Mohamed E

    2015-12-01

    Novel grafted agar disks were prepared for the covalent immobilization of β-D-galactosidase (β-gal). The agar disks were activated through reacting with ethylenediamine or different molecular weights of Polyethyleneimine (PEI), followed by glutaraldehyde (GA). The modification of the agar gel and the binding of the enzyme were verified by Fourier Transform Infrared (FTIR) and elemental analysis. Moreover, the agar's activation process was optimized, and the amount of immobilized enzyme increased 3.44 folds, from 38.1 to 131.2 U/g gel, during the course of the optimization process. The immobilization of β-gal onto the activated agar disks caused its optimum temperature to increase from 45°C to 45-55°C. The optimum pH of the enzyme was also shifted towards the acidic side (3.6-4.6) after its immobilization. Additionally, the Michaelis-Menten constant (Km ) increased for the immobilized β-gal as compared to its free counterpart whereas the maximum reaction rate (Vmax ) decreased. The immobilized enzyme was also shown to retain 92.99% of its initial activity after being used for 15 consecutive times. © 2015 Wiley Periodicals, Inc.

  12. [Effect of molar root canal angles on the integrity of impression made by agar].

    Science.gov (United States)

    Cao, Yong; Wei, Chun-hua; Liao, Hong-bing; Li, Xiao-jie

    2014-08-01

    To evaluate the effect of impressions made by agar/alginate compared with silicon rubber material. Five moulds of bi-root canal molar, with different root canal angles of 0°, 10°, 20°, 30° and 40° were made. Twenty impressions were made for each mould with either silicon rubber or agar/alginate. The effects of two methods were evaluated and the influence of angles between root canals on the impression methods was analyzed. The data was analyzed with SPSS16.0 software package. Significant difference of integrality rate (IR) of impression was not found in silicon rubber group among different angle groups (P>0.05). IR of 40° group (60%) was significantly lower than that of 0°(95%) and general IR (83%) when using agar (Pagar (60%) was statistically lower than silicon rubber (90%) in 40° group (Pagar method was significantly lower than silicon rubber method in 40° group. The general IR of agar (83%) was significantly lower than that of silicon rubber (95%) (Pagar/alginate impression method is qualified for smaller angles. The general IR of silicon rubber material is better than that of agar when taking the impression of post and core in molar.

  13. Strategies to improve the mechanical strength and water resistance of agar films for food packaging applications.

    Science.gov (United States)

    Sousa, Ana M M; Gonçalves, Maria P

    2015-11-05

    Agar films possess several properties adequate for food packaging applications. However, their high cost-production and quality variations caused by physiological and environmental factors affecting wild seaweeds make them less attractive for industries. In this work, native (NA) and alkali-modified (AA) agars obtained from sustainably grown seaweeds (integrated multi-trophic aquaculture) were mixed with locust bean gum (LBG) to make 'knife-coated' films with fixed final concentration (1 wt%) and variable agar/LBG ratios. Agar films were easier to process upon LBG addition (viscosity increase and gelling character decrease of the film-forming solutions observed by dynamic oscillatory and steady shear measurements). The mechanical properties and water resistance were optimal for films with 50 and/or 75% LBG contents and best in the case of NA (cheaper to extract). These findings can help reduce the cost-production of agar packaging films. Moreover, the controlled cultivation of seaweeds can provide continuous and reliable feedstock for transformation industries. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Time domain NMR study of Agar-gelatin blends; Estudo por RMN no dominio do tempo de blendas de Agar- gelatina

    Energy Technology Data Exchange (ETDEWEB)

    Mattos, Ritamara; Pericini, H.A.; Tambelli, Caio E., E-mail: tambelli@usp.br [Universidade de Sao Paulo (USP), Pirassununga, SP (Brazil). Faculdade de Zootecnia e Engenharia de Alimentos; Raphael, Ellen [Universidade Federal de Sao Joao del Rei (UFSJ), MG (Brazil). Departamento de Ciencias Naturais; Magon, Claudio J.; Donoso, P. [Universidade de Sao Paulo (USP), Sao Carlos, SP (Brazil). Instituto de Fisica

    2015-07-01

    This communication presents results of {sup 1}H time domain nuclear magnetic resonance of Agar-Gelatin blends plasticised with glycerol, cross-linked with formaldehyde and containing acetic acid. The spin-spin relaxation decay curves of samples obtained from CPMG experiments were inverted into corresponding distributions of relaxation times using NNLS (Non Negative Least Square) algorithm. The continuous distributions reveals up to four components of spin-spin relaxation times (T{sub 2}). The two components at short T{sub 2} were associated with protons in different environments of Agar and gelatin polymer chain. The two components at longer T{sub 2} can be associated with the glycerol that is the responsible to promote the proton conduction in the blend. (author)

  15. [Evaluation of oxacillin resistance screening agar and chromogenic MRSA agar media for the detection of methicillin resistance in Staphylococcus aureus clinical isolates].

    Science.gov (United States)

    Cesur, Salih; Yildiz, Eda; Irmak, Hasan; Aygün, Züleyha; Karakoç, Esra; Kinikli, Sami; Demiröz, Ali Pekcan

    2010-04-01

    Methicillin-resistant Staphylococcus aureus (MRSA) strains which are the most frequent causes of hospital acquired infections, are also currently encountered with increasing frequency in community acquired infections. Therefore rapid and accurate identification of MRSA strains is essential in both implementation of infection control measures and prevention of the nosocomial spread of this microorganism. The aim of this study was to determine the specifisity, sensitivity, positive and negative predictive values of two commercial media, one was Oxacillin Resistance Screening Agar Base (ORSAB; Oxoid, England) and the other was chromogenic MRSA agar (BBL CHROMagar MRSA; BD, Paris, France), for the identification of MRSA strains. A total of 175 clinical S. aureus isolates, of which 45 were MRSA, and 130 were methicillin-susceptible S. aureus (MSSA), whose susceptibility to methicillin were determined by disk diffusion method using oxacillin and cefoxitin disks in Mueller-Hinton agar medium, were included in the study. When oxacillin disk diffusion test was accepted as the reference method, the specificity, sensitivity, positive and negative predictive values of ORSAB were found as 97.7%, 40%, 36.5% and 98.1%, respectively; while these values were detected as 95.5%, 37.6%, 35.7% and 96.1% for CHROMagar MRSA, respectively. These results indicated that both media may be used in laboratories where work load is high and the number of personnel is inadequate especially in screening studies together or in addition to another medium (mannitol-salt agar). However, since these methods exhibit low specifity (high false positive results), positive results should be confirmed using other methods such as disk diffusion, E-test or microdilution susceptibility testing.

  16. Evaluation of semisolid agar method for antifungal susceptibility test of T. rubrum

    Directory of Open Access Journals (Sweden)

    Sultana Razia

    2016-08-01

    Full Text Available Background: With increasing fungal disease many newer antifungal drugs are available with different spectrum of activ­ity. Antifungal susceptibility test will help clinicians for selection of effective drug and thereby treatment of patient. Objective: The study was undertaken to perform a simple screening drug susceptibility test of T. rnbrum by Semi Solid Agar Antifungal Susceptibility (SAAS Method: Perfonnance of susceptibility method was assessed by comparing the MICs of three commonly prescribed antifungal agents namely- tluconazole (FCZ, itraconazole (ITZ and terbinafine (TER to the CLSI (Clinical and Laboratory Standard Institute recommended M-38, a broth microdilution method. Results: In SAAS method, among twenty nine T. rubrum, twenty five (86.2% were susceptible (MIC range 0.5-64 µg/ml to Fluconazole (FCZ and four (13.7% were resistant (MIC value >64 µg/ml. In broth microdilution method, among twenty nine T. rubrum, twenty six (89.6% were susceptible (MIC range 0.3-64 µg/ml to FCZ and three (10.3% were resistant (MIC value >64 µg/ml. In case of both ITZ and TER, all were susceptible (MIC range 0.3-64 µg/ml to both methods. The SAAS method demonstrated the susceptibility pattern of T. rubrum against FCZ, ITZ and TER usually within 72 to 96 hours after organism isolation and results were concordance with the results of CLSI broth microdilution method. Conclusion: Though it is a newer method with proper standardization of the test method, SAAS method is simple and easily applicable screening method for susceptibility testing of antifungal agents against dermatophytes in any microbiology laboratories.

  17. Homogeneous Matrix Deposition on Dried Agar for MALDI Imaging Mass Spectrometry of Microbial Cultures

    Science.gov (United States)

    Hoffmann, Thomas; Dorrestein, Pieter C.

    2015-11-01

    Matrix deposition on agar-based microbial colonies for MALDI imaging mass spectrometry is often complicated by the complex media on which microbes are grown. This Application Note demonstrates how consecutive short spray pulses of a matrix solution can form an evenly closed matrix layer on dried agar. Compared with sieving dry matrix onto wet agar, this method supports analyte cocrystallization, which results in significantly more signals, higher signal-to-noise ratios, and improved ionization efficiency. The even matrix layer improves spot-to-spot precision of measured m/z values when using TOF mass spectrometers. With this technique, we established reproducible imaging mass spectrometry of myxobacterial cultures on nutrient-rich cultivation media, which was not possible with the sieving technique.

  18. Mineralized agar-based nanocomposite films: Potential food packaging materials with antimicrobial properties.

    Science.gov (United States)

    Malagurski, Ivana; Levic, Steva; Nesic, Aleksandra; Mitric, Miodrag; Pavlovic, Vladimir; Dimitrijevic-Brankovic, Suzana

    2017-11-01

    New mineralized, agar-based nanocomposite films (Zn-carbonate and Zn-phosphate/agar) were produced by a combination of in situ precipitation and a casting method. The presence of minerals significantly influenced the morphology, properties and functionality of the obtained nanocomposites. Reinforcement with the Zn-mineral phase improved the mechanical properties of the carbonate-mineralized films, but had a negligible effect on the phosphate-mineralized samples. Both nanocomposites showed improved optical and thermal properties, better Zn(II) release potential in a slightly acidic environment and exhibited antimicrobial activity against S. aureus. These results suggest that Zn-mineralized agar nanocomposite films could be potentially used as affordable, eco-friendly and active food packaging materials. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Homogeneous matrix deposition on dried agar for MALDI imaging mass spectrometry of microbial cultures.

    Science.gov (United States)

    Hoffmann, Thomas; Dorrestein, Pieter C

    2015-11-01

    Matrix deposition on agar-based microbial colonies for MALDI imaging mass spectrometry is often complicated by the complex media on which microbes are grown. This Application Note demonstrates how consecutive short spray pulses of a matrix solution can form an evenly closed matrix layer on dried agar. Compared with sieving dry matrix onto wet agar, this method supports analyte cocrystallization, which results in significantly more signals, higher signal-to-noise ratios, and improved ionization efficiency. The even matrix layer improves spot-to-spot precision of measured m/z values when using TOF mass spectrometers. With this technique, we established reproducible imaging mass spectrometry of myxobacterial cultures on nutrient-rich cultivation media, which was not possible with the sieving technique. Graphical Abstract ᅟ.

  20. Glass bead cultivation of fungi: combining the best of liquid and agar media.

    Science.gov (United States)

    Droce, Aida; Sørensen, Jens Laurids; Giese, Henriette; Sondergaard, Teis Esben

    2013-09-01

    Production of bioactive compounds and enzymes from filamentous fungi is highly dependent on cultivation conditions. Here we present an easy way to cultivate filamentous fungi on glass beads that allow complete control of nutrient supply. Secondary metabolite production in Fusarium graminearum and Fusarium solani cultivated on agar plates, in shaking liquid culture or on glass beads was compared. Agar plate culture and glass bead cultivation yielded comparable results while liquid culture had lower production of secondary metabolites. RNA extraction from glass beads and liquid cultures was easier than from agar plates and the quality was superior. The system allows simple control of nutrient availability throughout fungal cultivation. This combined with the ease of extraction of nucleic acids and metabolites makes the system highly suitable for the study of gene regulation in response to specific nutrient factors. © 2013.

  1. Synthesis and characterization of agar-based silver nanoparticles and nanocomposite film with antibacterial applications.

    Science.gov (United States)

    Shukla, Mahendra K; Singh, Ravindra Pal; Reddy, C R K; Jha, Bhavanath

    2012-03-01

    This study describes the synthesis and characterization of silver nanoparticles and nanocomposite material using agar extracted from the red alga Gracilaria dura. Characterization of silver nanoparticles was carried out based on UV-Vis spectroscopy (421 nm), transmission electron microscopy, EDX, SAED and XRD analysis. The thermal stability of agar/silver nanocomposite film determined by TGA and DSC analysis showed distinct patterns when compared with their raw material (agar and AgNO(3)). The TEM findings revealed that the silver nanoparticles synthesized were spherical in shape, 6 nm in size with uniform dispersal. The synthesized nanoparticles had the great bactericidal activity with reduction of 99.9% of bacteria over the control value. The time required for synthesis of silver nanoparticles was found to be temperature dependent and higher the temperature less the time for nanoparticles formation. DSC and XRD showed approximately the same crystalline index (CI(DSC) 0.73). Copyright © 2011 Elsevier Ltd. All rights reserved.

  2. [Presumptive identification of Candida spp. and other clinically important yeasts: usefulness of Brilliance Candida Agar].

    Science.gov (United States)

    Alfonso, Claudia; López, Mónica; Arechavala, Alicia; Perrone, María Del Carmen; Guelfand, Liliana; Bianchi, Mario

    2010-06-30

    Fungal infections caused by yeasts have increased during the last decades and invasive forms represent a serious problem for human health. Candida albicans is the species most frequently isolated from clinical samples. However, other emerging yeast pathogens are increasingly responsible for mycotic infections, and some of them are resistant to some antifungal drugs. Consequently, it is necessary to have methods that can provide a rapid presumptive identification at species level. Numerous chromogenic agar media have been shown to be of value as diagnostic tools. We have compared a chromogenic medium, Brilliance Candida Agar, with CHROMagar Candida, the chromogenic medium most used in our country. A multicentre study was conducted in 16 Hospitals belonging to the Mycology Net of Buenos Aires City Government. A total of 240 yeast isolates were included in this research. The new chromogenic agar showed results very similar to those obtained with CHROMagar Candida. Copyright 2009 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

  3. Assessment of Etest as an Alternative to Agar Dilution for Antimicrobial Susceptibility Testing of Neisseria gonorrhoeae

    Science.gov (United States)

    Taylor, Thomas H.; Pettus, Kevin; Trees, David

    2014-01-01

    We studied whether the Etest can be used as an alternative to agar dilution to determine antimicrobial susceptibilities of ceftriaxone, cefixime, and cefpodoxime in Neisseria gonorrhoeae surveillance. One hundred fifteen clinical and laboratory isolates of N. gonorrhoeae were tested following the Clinical Laboratory Improvement Amendments (CLIA)-approved CLSI standard agar dilution method and, separately, by the Etest according to the manufacturer's recommendations. The MICs were determined and compared. Ten laboratory-generated mutants were used to simulate substantially nonsusceptible specimens. The Etest and agar dilution methods were well correlated. Statistical tests produced regression R2 values of 88%, 82%, and 85% and Pearson correlation coefficients of 92%, 91%, and 92% for ceftriaxone, cefixime, and cefpodoxime, respectively. When paired comparisons were made, the two tests were 88.7%, 80%, and 87% within 1 log2 dilution from each other for ceftriaxone, cefixime, and cefpodoxime, respectively. The within-2-log2 agreements were 99.1%, 98.3%, and 94.8% for ceftriaxone, cefixime, and cefpodoxime, respectively. Notwithstanding the good correlations and the within-2-log2 general agreement, the Etest results produced slightly lower MICs than the agar dilution results. In conclusion, we found that the Etest can be effectively used as an alternative to agar dilution testing to determine the susceptibility of N. gonorrhoeae to ceftriaxone, cefixime, and cefpodoxime, although we recommend further research into extremely resistant isolates. For isolates within the typical range of clinical MICs, reexamination of the Etest interpretation of susceptible and nonsusceptible categories would likely allow for successful transition from agar dilution to the Etest. PMID:24554750

  4. Influence of agar concentration on in vitro multiplication of Cymbopogon citratus (D. C. Stapf

    Directory of Open Access Journals (Sweden)

    Ricardo J. Licea Moreno

    2001-04-01

    Full Text Available Here are presented the results on in vitro multiplication of lemon grass (Cymbopogon citratus (D. C. Stapf.; it is a very important medicinal plant because its analgesic, antinflamatory and hipotensor properties, among others, useful to elaborate several medicaments with a high popular acceptation. The main aim of this research was to set up the influence of agar concentration in culture medium during in vitro establishment on multiplication of lemon grass. Were used three treatments: (1 liquid medium with filter paper bridges, (2 3 g.l-1 of agar (BIOCEN and (3 6 g.l-1 of agar (BIOCEN. The explants were inoculated on a culture media containing Murashige and Skoog salts (1962, Heinz and Mee vitamins (1969, myoinositol 100 mg.l-1, 6-BAP 0.2 mg.l-1 and sucrose 20 g.l-1. Meristematic tips were inoculated on the treatments described above under sun light conditions, once desinfected. The explants Influence of agar concentration on in vitro multiplication of Cymbopogon citratus (D. C. Stapf. were maintained 21 days in this culture media and later it is were subcultured 5 times each 21 days, on the same multiplication culture media containing Murashige and Skoog salts (1962, tiamine 1 mg.l-1, myoinositol 100 mg.l-1, 6-BAP 0.3 mg.l-1 and sucrose 30 g.l-1. The pH was 5.7 for all culture media. The results showed the relevance of agar concentration during in vitro establishment on multiplication of lemon grass. Differences among treatments until the 2nd subculture was observed. 3.43 new axillary shoots from each explant cultured on a culture media supplemented with 3 g.l-1 of agar was reached. Key words: lemon grass, medicinal plants, micropropagation, tissue culture

  5. Agar-block microcosms for controlled plant tissue decomposition by aerobic fungi.

    Science.gov (United States)

    Schilling, Jonathan S; Jacobson, K Brook

    2011-02-03

    The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; agar-block). It is well-accepted that soil-block microcosms yield higher decay rates, fewer moisture issues, lower variability among studies, and higher thresholds of preservative toxicity. Soil-block testing is thus the more utilized technique and has been standardized by American Society for Testing and Materials (ASTM) (method D 1413-07). The soil-block design has drawbacks, however, using locally-variable soil sources and in limiting the control of nutrients external (exogenous) to the decaying tissues. These drawbacks have emerged as a problem in applying this method to other, increasingly popular research aims. These modern aims include degrading lignocellulosics for bioenergy research, testing bioremediation of co-metabolized toxics, evaluating oxidative mechanisms, and tracking translocated elements along hyphal networks. Soil-blocks do not lend enough control in these applications. A refined agar-block approach is necessary. Here, we use the brown rot wood-degrading fungus Serpula lacrymans to degrade wood in agar-block microcosms, using deep Petri dishes with low-calcium agar. We test the role of exogenous gypsum on decay in a time-series, to demonstrate the utility and expected variability. Blocks from a single board rip (longitudinal cut) are conditioned, weighed, autoclaved, and introduced aseptically atop plastic mesh. Fungal inoculations are at each block face, with exogenous gypsum added at interfaces. Harvests are aseptic until the final destructive harvest. These microcosms are designed to avoid block contact with agar or Petri dish walls. Condensation is minimized during plate pours and during incubation. Finally, inoculum/gypsum/wood spacing is minimized but without allowing contact. These less technical aspects of agar-block design are also the most common causes of failure and the key source of

  6. Spectroscopic and kinetic investigation of the reactions of peroxyacetic acid with Burkholderia pseudomallei catalase-peroxidase, KatG.

    Science.gov (United States)

    Ivancich, Anabella; Donald, Lynda J; Villanueva, Jacylyn; Wiseman, Ben; Fita, Ignacio; Loewen, Peter C

    2013-10-15

    Catalase-peroxidases or KatGs can utilize organic peroxyacids and peroxides instead of hydrogen peroxide to generate the high-valent ferryl-oxo intermediates involved in the catalase and peroxidase reactions. In the absence of peroxidatic one-electron donors, the ferryl intermediates generated with a low excess (10-fold) of peroxyacetic acid (PAA) slowly decay to the ferric resting state after several minutes, a reaction that is demonstrated in this work by both stopped-flow UV-vis absorption measurements and EPR spectroscopic characterization of Burkholderia pseudomallei KatG (BpKatG). EPR spectroscopy showed that the [Fe(IV)═O Trp330(•+)], [Fe(IV)═O Trp139(•)], and [Fe(IV)═O Trp153(•)] intermediates of the peroxidase-like cycle of BpKatG ( Colin, J. Wiseman, B. Switala, J. Loewen, P. C. Ivancich, A. ( 2009 ) J. Am. Chem. Soc. 131 , 8557 - 8563 ), formed with a low excess of PAA at low temperature, are also generated with a high excess (1000-fold) of PAA at room temperature. However, under high excess conditions, there is a rapid conversion to a persistent [Fe(IV)═O] intermediate. Analysis of tryptic peptides of BpKatG by mass spectrometry before and after treatment with PAA showed that specific tryptophan (including W330, W139, and W153), methionine (including Met264 of the M-Y-W adduct), and cysteine residues are either modified with one, two, or three oxygen atoms or could not be identified in the spectrum because of other undetermined modifications. It was concluded that these oxidized residues were the source of electrons used to reduce the excess of PAA to acetic acid and return the enzyme to the ferric state. Treatment of BpKatG with PAA also caused a loss of catalase activity towards certain substrates, consistent with oxidative disruption of the M-Y-W adduct, and a loss of peroxidase activity, consistent with accumulation of the [Fe(IV)═O] intermediate and the oxidative modification of the W330, W139, and W153. PAA, but not H2O2 or tert

  7. Formation of Ramified Colony of Fungus Aspergillus Oryzae on Agar Media

    Science.gov (United States)

    Matsuura, Shu; Miyazima, Sasuke

    Ramified colonies of fungus Aspergillus oryzae have been found to grow at a low growth rate on "liquid-like" agar media with low concentrations of agar and glucose. Box-counting fractal dimensions of the individual colony branches have been found to decrease with the time of incubation. Addition of glucose solution in the interior of branched colonies has brought about the production of the hyphal filaments almost only at the apical region of the colony branches. Active growth of the ramified colonies is localized in the peripheral zone, and this growth manner implies that the fungus is exhibiting a positive exploitation.

  8. Influence of Adjacent teeth on the Three-dimensional Accuracy of Die Obtained from Agar-alginete Combined Impressions

    OpenAIRE

    鈴木, 郁男; スズキ, イクオ; Ikuo, SUZUKI

    2003-01-01

    The purpose of this study was to elucidate the influence of adjacent teeth with natural morphology on the three-dimensional accuracy of the bridge die obtained from agar-alginate combined impressions. In this study, three kinds of cartilage type agar impression materials, 3 kinds of alginate impression materials combined with these agar impression materials and one kind of dental stone were used. The metal cast was composed of the original abutment tooth cast, a mounting plate and its fixture...

  9. Systematic mutagenesis of genes encoding predicted autotransported proteins of Burkholderia pseudomallei identifies factors mediating virulence in mice, net intracellular replication and a novel protein conferring serum resistance.

    Directory of Open Access Journals (Sweden)

    Natalie R Lazar Adler

    Full Text Available Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA. Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE. A single mutant (bpaC was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA, those attenuated for virulence and net intracellular replication (BpaE, the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA. Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors

  10. Comparison of Agar Media for Detection and Quantification of Shiga Toxin-Producing Escherichia coli in Cattle Feces.

    Science.gov (United States)

    Stromberg, Zachary R; Lewis, Gentry L; Moxley, Rodney A

    2016-06-01

    The isolation and quantification of non-O157 Shiga toxin-producing Escherichia coli (STEC) from cattle feces are challenging. The primary objective of this study was to evaluate the performance of selected agar media in an attempt to identify an optimal medium for the detection and quantification of non-O157 STEC in cattle feces. Comparison studies were performed using CHROMagar STEC, Possé differential agar (Possé), Possé modified by the reduction or addition of antimicrobials, STEC heart infusion washed blood agar with mitomycin C (SHIBAM), and SHIBAM modified by the addition of antimicrobials. Fourteen STEC strains, two each belonging to serogroups O26, O45, O103, O111, O121, O145, and O157, were used to test detection in inoculated fecal suspensions at concentrations of 10(2) or 10(3) CFU/g. One STEC strain from each of these seven serogroups was used to estimate the concentration of recovered STEC in feces inoculated at 10(3), 10(4), or 10(5) CFU/g. Significantly more suspensions (P < 0.05) were positive for STEC when plated on Possé containing reduced concentrations of novobiocin and potassium tellurite compared with SHIBAM, but not SHIBAM modified by containing these same antimicrobials at the same concentrations. Numerically, more suspensions were positive for STEC by using this same form of modified Possé compared with Possé, but this difference was not statistically significant. More suspensions were positive for STEC cultured on CHROMagar STEC compared with those on Possé (P < 0.05) and on modified Possé (P = 0.05). Most inoculated fecal suspensions below 10(4) CFU/g of feces were underestimated or not quantifiable for the concentration of STEC by using CHROMagar STEC or modified Possé. These results suggest that CHROMagar STEC performs better than Possé or SHIBAM for detection of STEC in bovine feces, but adjustments in the concentrations of novobiocin and potassium tellurite in the latter two media result in significant improvements in their

  11. Observing Chemotaxis in Vibrio fischeri Using Soft Agar Assays in an Undergraduate Microbiology Laboratory

    Directory of Open Access Journals (Sweden)

    Cindy R. DeLoney-Marino

    2013-08-01

    Full Text Available Chemotaxis, the directed movement of cells towards or away from a chemical, is both an exciting and complicated behavior observed in many bacterial species. Attempting to adequately visualize or demonstrate the chemotaxic response of bacteria in the classroom is difficult at best, with good models to illustrate the concept lacking. The BSL-1 marine bacterium Vibrio fischeri (a.k.a. Aliivibrio fischeri is easy to culture, making it an ideal candidate for experiments in an undergraduate microbiology course. A number of chemoattractants for V. fischeri have been identified, including a variety of sugars, nucleosides, and amino acids (1, 2. Below presents how the soft agar-based chemotaxis assay can be implemented in the undergraduate laboratory. As bacterial cells migrate towards one or more attractants in soft agar, students can directly observe the chemotaxic behavior of V. fischeri without the need to learn complicated techniques or use specialized equipment. Once the bands of bacterial cells are observed, the migration can then be disrupted by the addition of excess attractant to the soft agar, thereby visualizing what happens once cells are no longer in a gradient of attractant. In addition, soft agar plates lacking attractants can be used to visualize the random movements of bacterial cells that are non-chemotaxing. These exercises can be used in the microbiology laboratory to help students understand the complex behavior of bacterial chemotaxis.

  12. Reconstructing the evolution of agarics from nuclear gene sequences and basidiospore ultrastructure.

    Science.gov (United States)

    Garnica, Sigisfredo; Weiss, Michael; Walther, Grit; Oberwinkler, Franz

    2007-09-01

    Traditional classifications of agaric fungi involve gross morphology of their fruit bodies and meiospore print-colour. However, the phylogeny of these fungi and the evolution of their morphological and ecological traits are poorly understood. Phylogenetic analyses have already demonstrated that characters used in traditional classifications of basidiomycetes may be heavily affected by homoplasy, and that non-gilled taxa evolved within the agarics several times. By integrating molecular phylogenetic analyses including domains D1-D3 and D7-D8 of nucLSU rDNA and domains A-C of the RPB1 gene with morphological and chemical data from representative species of 88 genera, we were able to resolve higher groups of agarics. We found that the species with thick-walled and pigmented basidiospores constitute a derived group, and hypothesize that this specific combination of characters represents an evolutionary advantage by increasing the tolerance of the basidiospores to dehydration and solar radiation and so opened up new ecological niches, e.g. the colonization of dung substrates by enabling basidiospores to survive gut passages through herbivores. Our results confirm the validity of basidiospore morphology as a phylogenetic marker in the agarics.

  13. Evolutionary consequences of putative intra- and interspecific hybridiation in agaric fungi

    Science.gov (United States)

    Karen W. Hughes; Ronald H. Petersen; D. Jean Lodge; Sarah E. Bergemann; Kendra Baumgartner; Rodham E. Tulloss; Edgar Lickey; Joaquin. Cifuentes

    2013-01-01

    Agaric fungi of the southern Appalachian Mountains including Great Smoky Mountains National Park are often heterozygous for the rDNA internal transcribed spacer region (ITS) with .42% of collections showing some heterozygosity for indels and/or base-pair substitutions. For these collections, intra-individual haplotype divergence is typically less than 2%, but for 3% of...

  14. Can serums be replaced by Mueller‑Hinton agar in germ tube test?

    African Journals Online (AJOL)

    2016-03-03

    Mar 3, 2016 ... various serums (i.e., human, rabbit, horse, and fetal bovine serum) used in the GTT and Mueller‑Hinton agar (MHA). Materials and Methods: Fifty ... parent yeast cell and with no constriction at the point of origin. Results: When the use of ... marrow and organ transplants, use of broad‑spectrum antibiotics ...

  15. Medium dependant production of corymbiferone a novel product from Penicillium hordei cultured on plant tissue agar

    DEFF Research Database (Denmark)

    Overy, David Patrick; Zidorn, C.; Petersen, B.O.

    2005-01-01

    Medium dependant production and the structure elucidation of corymbiferone (1) from the fungus Penicillitan hordei grown on oatmeal and macerated tulip, yellow onion and red onion agars are reported. Compound 1 possesses an unusual oxygenated aromatic structure with a lactone bridge preventing full...

  16. Immediate differentiation of salmonella-resembling colonies on brilliant green agar

    DEFF Research Database (Denmark)

    Jensen, Annette Nygaard; Hoorfar, Jeffrey

    2000-01-01

    A rapid biochemical system (OBIS) based on immediate enzymatic differentiation of Citrobacter, Proteus, Providencia, Hafnia and Morganella spp. from Salmonella on brilliant green agar was evaluated A total of 96 field isolates from various Salmonella serotypes, 18 Citrobacter freundii and 25...

  17. Mercury in fruiting bodies of Fly Agaric Amanita muscaria (L.: Fr.) Pers. collected from Poland

    Science.gov (United States)

    Falandysz, J.; Lipka, K.

    2003-05-01

    Total mercury concentrations were determined in the fruiting bodies of Fly Agaric Amanita muscaria (L.: FL) Pers. and underlying soil substrate collected from several sites in Poland in 1993-2000 to evaluate mercury status as contaminant and bioindicating features of this species. The samples were collected from the spatially distant sites such as: Zaborski Landscape Park, Mierzeja Wiślana Landscape Park, Wdzydzki Landscape Park, Borecka Forest, Tucholskie Forest, Wieluńska Upland, the communities of Gubin, Manowo, Lubiana and Morag. Total mercury content of caps and stalks of Fly agaric varied widely depending on the sites examined. The range of the mean mercury concentrations for all 17 sites was between 96±10 and 1900±1400 ng/g dry wt for the caps and between 6l±32 and 920±760 ng/g dry wt for the stalks, while between 4.4±3.1 and 150±20 ng/g were noted for soil substrate samples from 9 sites examined. Fly agaric independently of the site examined showed relatively good capacity to accumulate total mercury and BCF values varied between 16±10 and 74±15 for the caps and between 11±8 and 42±10 for the stalks. Nevertheless, relatively high bioconcentration potential of mercury by Fly agaric seems to be specific for that species and under soil mercury concentrations noted no bioindication properties of this mushroom could be observed.

  18. Mechanical, Thermal and Surface Investigations of Chitosan/Agar/PVA Ternary Blended Films

    Directory of Open Access Journals (Sweden)

    Esam A. El-Hefian

    2011-01-01

    Full Text Available The mechanical and thermal properties of chitosan/agar/poly vinyl alcohol (CS/AG/PVA ternary blended films having various proportions considering chitosan as the main component were investigated. The various variables static water contact angle such as contact angle, drop base area, drop volume and drop height was also studied in correlation with the variation of time. Results obtained from mechanical measurements showed a noticeable increase in the tensile strength (TS coincided with a sharp decrease in elongation percent at break (E% of blended films with increasing agar and PVA contents. The DSC results prevailed the development of an interaction between chitosan individual components: agar and PVA. Moreover, an enhancement of the wettability of the blends was obtained with increasing agar and PVA contents. It was also found that the pure CS film and the blended films with 90/05/05 and 80/10/10 compositions were more affected by time than blended films with other compositions when the contact angle, the drop height and the drop length were studied as a function of time. In addition, when the drop is initially placed on the substrate, the drop area and the drop volume of all films remained almost constant up to a certain time after which they showed a slight difference with the elapse of time.

  19. Comparison of the antibacterial activity of chelating agents using the agar diffusion method

    Science.gov (United States)

    The agar diffusion assay was used to examine antibacterial activity of 2 metal chelators. Concentrations of 0 to 40 mM of ethylenediaminetetraacetic acid (EDTA) and ethylenediamine-N,N’-disuccinic acid (EDDS) were prepared in 1.0 M potassium hydroxide (KOH). The pH of the solutions was adjusted to 1...

  20. Ringonderzoek : bepaling van avoparcine gehalte in diervoeders met behulp van agar-diffusie

    NARCIS (Netherlands)

    Broex, N.J.G.; Huf, F.

    1988-01-01

    De microbiologische agar-diffusie methode voor de bepaling van het gehalte in diervoeders is geringtest door 12 laboratoria. Ieder laboratorium heeft van 3 monsters het gehalte bepaald. Een voormengsel, een gepelleteerd en een ongepelleteerd diervoer. De monsters zijn door de deelnemers in drievoud

  1. Structural, physical, and chemical modifications induced by microwave heating on native agar-like galactans.

    Science.gov (United States)

    Sousa, Ana M M; Morais, Simone; Abreu, Maria H; Pereira, Rui; Sousa-Pinto, Isabel; Cabrita, Eurico J; Delerue-Matos, Cristina; Gonçalves, Maria Pilar

    2012-05-16

    Native agars from Gracilaria vermiculophylla produced in sustainable aquaculture systems (IMTA) were extracted under conventional (TWE) and microwave (MAE) heating. The optimal extracts from both processes were compared in terms of their properties. The agars' structure was further investigated through Fourier transform infrared and NMR spectroscopy. Both samples showed a regular structure with an identical backbone, β-d-galactose (G) and 3,6-anhydro-α-l-galactose (LA) units; a considerable degree of methylation was found at C6 of the G units and, to a lesser extent, at C2 of the LA residues. The methylation degree in the G units was lower for MAE(opt) agar; the sulfate content was also reduced. MAE led to higher agar recoveries with drastic extraction time and solvent volume reductions. Two times lower values of [η] and M(v) obtained for the MAE(opt) sample indicate substantial depolymerization of the polysaccharide backbone; this was reflected in its gelling properties; yet it was clearly appropriate for commercial application in soft-texture food products.

  2. Long-term maintenance of fungal cultures on perlite in cryovials - an alternative for agar slants.

    Science.gov (United States)

    Homolka, L; Lisá, L

    2008-01-01

    Cultures of 33 basidiomycete strains out of 35 tested were viable with unchanged characteristics after four years of maintenance on perlite in cryovials. These cultures can be a good substitute for agar cultures in long-term maintenance of fungi. For comparison, the storage under oil was evaluated but it turned out to be unsuitable for the majority of our cultures.

  3. A modified micro chamber agar spot slide culture technique for microscopic examination of filamentous fungi.

    Science.gov (United States)

    Prakash, Peralam Yegneswaran; Bhargava, Kanika

    2016-04-01

    The slide culture technique aids in the study of undisturbed microscopic morphological details of filamentous fungi. The existing methods for setting up of slide culture are quite cumbersome, time-consuming and require elaborate preparation. We describe a modified and easy to perform micro chamber agar spot slide culture technique. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Preparation and characterization of bio-nanocomposite films of agar and silver nanoparticles: laser ablation method.

    Science.gov (United States)

    Rhim, Jong-Whan; Wang, Long-Feng; Lee, Yonghoon; Hong, Seok-In

    2014-03-15

    Silver nanoparticles (AgNPs) were prepared by a laser ablation method and composite films with the AgNPs and agar were prepared by solvent casting method. UV-vis absorbance test and transmission electron microscopy (TEM) analysis results revealed that non-agglomerated spherical AgNPs were formed by the laser ablation method. The surface color of the resulting agar/AgNPs films exhibited the characteristic plasmonic effect of the AgNPs with the maximum absorption peaks of 400-407 nm. X-ray diffraction (XRD) test results also exhibited characteristic AgNPs crystals with diffraction peaks observed at 2θ values of 38.39°, 44.49°, and 64.45°, which were corresponding to (111), (200), and (220) crystallographic planes of face-centered cubic (fcc) silver crystals, respectively. Thermogravimetric analysis (TGA) results showed that thermal stability of the agar/AgNPs composite films was increased by the inclusion of metallic silver. Water vapor barrier properties and surface hydrophobicity of the agar/AgNPs films increased slightly with the increase in AgNPs content but they were not statistically significant (p>0.05), while mechanical strength and stiffness of the composite films decreased slightly (pagar/AgNPs films exhibited distinctive antimicrobial activity against both Gram-positive (Listeria monocytogenes) and Gram-negative (Escherichia coli O157:H7) bacterial pathogens. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Use of Mueller-Hinton broth and agar in the germ tube test.

    Science.gov (United States)

    Mattei, Antonella Souza; Alves, Sydney Hartz; Severo, Cecília Bittencourt; Guazzelli, Luciana da Silva; Oliveira, Flávio de Mattos; Severo, Luiz Carlos

    2014-01-01

    Candida albicans is often isolated from clinical samples, thus its presumptive differentiation from other species of the same genus can be based on its ability to form the germ tube in human serum. Nevertheless, there are two other species that share this characteristic: C. dubliniensis and C. africana. The aim of this study was to compare four different substrates to perform the germ tube (GT) test. The Candida spp. isolates were identified using a manual system (135 C. albicans, 24 C. tropicalis and one C. dubliniensis). The germ tube test was performed with fresh, previously frozen serum and Mueller-Hinton (MH) broth and agar. GT was observed in 96% (130/136) of the isolates through the fresh serum technique, 94% (128/136) through previously frozen serum, 92% (125/136) in MH agar, and 90% (122/136) in MH broth. The sensitivity of each test was higher than 90%, with 100% specificity. Both the MH agar and broth were able to identify the true positives, and false positives were not found. However, some C. albicans isolates were not identified. MH agar and broth may be used in laboratory for the rapid presumptive identification of C. albicans, as an alternative method for germ tube test.

  6. Colony formation in agar: in vitro assay for haemopoietic stem cells

    NARCIS (Netherlands)

    Dicke, K.A.; Platenburg, M.G.C.; Bekkum, D.W. van

    1971-01-01

    Using a method in which embryo fibroblasts were used as feeder layers, the colony forming capacity in agar of a variety of mouse haemopoietic suspensions was compared with their CFUs content. A striking parallelism between the results of the two assays was found. In addition, under certain

  7. Characterization and immobilization of arylsulfatase on modified magnetic nanoparticles for desulfation of agar.

    Science.gov (United States)

    Xiao, Qiong; Yin, Qin; Ni, Hui; Cai, Huinong; Wu, Changzheng; Xiao, Anfeng

    2017-01-01

    Carboxyl functioned magnetic nanoparticles (CMNPs) were prepared by a simple co-precipitation method and characterized by Fourier transform infrared spedtroscopy and scanning electron microscope. The prepared CMNPs were used for covalent immobilization of the arylsulfatase which could be applied in desulfation of agar. The optimal immobilizaion conditions were obtained as follows: glutaraldehyde concentration 1.0% (v/v), cross-linking time 3h, immobilization time 3h, immobilization temperature 5°C and enzyme dose 0.62U. Increase in properties of the arylsulfatase such as optimum temperature and pH was observed after immobilization. Immobilization led to increased tolerance of enzyme to some metal ions, inhibitors and detergents. The Km and kcat of the immobilized enzyme for hydrolysis of p-NPS at pH 7.5 and at 50°C were determined to be 0.89mmol/L and 256.91s(-1), respectively. The relative desulfuration rates of immobilized arylsulfatase maintained 61.7% of its initial desulfuration rates after seven cycles. After the reaction of agar with immobilized arylsulfatase for 90min at 50°C, 46% of the sulfate in the agar was removed. These results showed that the immobilization of arylsulfatase onto CMNPs is an efficient and simple way for preparation of stable arylsulfatase and have a great potential for application in enzymatic desulfation of agar. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Fusion of agarase and neoagarobiose hydrolase for mono-sugar production from agar.

    Science.gov (United States)

    Alkotaini, Bassam; Han, Nam Soo; Kim, Beom Soo

    2017-02-01

    In enzymatic saccharification of agar, endo- and exo-agarases together with neoagarobiose hydrolase (NABH) are important key enzymes for the sequential hydrolysis reactions. In this study, a bifunctional endo/exo-agarase was fused with NABH for production of mono-sugars (D-galactose and 3,6-anhydro-L-galactose) from agar using only one fusion enzyme. Two fusion enzymes with either bifunctional agarase (Sco3476) or NABH (Zg4663) at the N-terminus, Sco3476-Zg4663 (SZ) and Zg4663-Sco3476 (ZS), were constructed. Both fusion enzymes exhibited their optimal agarase and NABH activities at 40 and 35 °C, respectively. Fusions SZ and ZS enhanced the thermostability of the NABH activity, while only fusion SZ showed a slight enhancement in the NABH catalytic efficiency (K cat/K M) from 14.8 (mg/mL)(-1) s(-1) to 15.8 (mg/mL)(-1) s(-1). Saccharification of agar using fusion SZ resulted in 2-fold higher mono-sugar production and 3-fold lower neoagarobiose accumulation when compared to the physical mixture of Sco3476 and Zg4663. Therefore, this fusion has the potential to reduce enzyme production cost, decrease intermediate accumulation, and increase mono-sugar yield in agar saccharification.

  9. Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds.

    Science.gov (United States)

    Hockett, Kevin L; Baltrus, David A

    2017-01-14

    The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

  10. THE MICROGARDENING COOKBOOK, DIRECTIONS FOR PREPARING DISHES AND TUBES OF STERILE NUTRIENT AGAR.

    Science.gov (United States)

    CHANDLER, MARION N.

    THIS BOOKLET WAS PREPARED FOR TEACHER USE IN ASSOCIATION WITH THE ELEMENTARY SCIENCE STUDY UNIT "MICROGARDENING." IT CONTAINS DIRECTIONS FOR PREPARING CULTURE DISHES AND TUBES OF NUTRIENT STERILE AGAR FOR FUNGAL AND/OR BACTERIAL GROWTH. IT INCLUDES (1) LISTS OF NEEDED SUPPLIES AND EQUIPMENT, (2) DIRECTIONS FOR THE PREPARATION AND…

  11. Characterization of bionanocomposite films prepared with agar and paper-mulberry pulp nanocellulose.

    Science.gov (United States)

    Reddy, Jeevan Prasad; Rhim, Jong-Whan

    2014-09-22

    Crystallized nanocellulose (CNC) was separated from paper-mulberry (Broussonetia kazinoki Siebold) bast pulp by sulfuric acid hydrolysis method and they were blended with agar to prepare bionanocomposite films. The effect of CNC content (1, 3, 5 and 10 wt% based on agar) on the mechanical, water vapor permeability (WVP), and thermal properties of the nanocomposites were studied. Changes of the cellulose fibers in structure, morphology, crystallinity, and thermal properties of the films were evaluated using FT-IR, TEM, SEM, XRD, and TGA analysis methods. The CNC was composed of fibrous and spherical or elliptic granules of nano-cellulose with sizes of 50-60 nm. Properties of agar film such as mechanical and water vapor barrier properties were improved significantly (pagar film increased by 40% and 25%, respectively, in the composite film with 5 wt% of CNC, and the WVP of agar film decreased by 25% after formation of nanocomposite with 3 wt% of CNC. The CNC obtained from the paper-mulberry bast pulp can be used as a reinforcing agent for the preparation of bio-nanocomposites, and they have a high potential for the development of completely biodegradable food packaging materials. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Applying Agar's Concept of "Languaculture" to Explain Asian Students' Experiences in the Australian Tertiary Context

    Science.gov (United States)

    Norris, Lindy; Tsedendamba, Nara

    2015-01-01

    This paper reports part of a broader qualitative case study of Asian students "translation" (Agar, 2006) to study in an Australian university. The paper is concerned with the experiences of eight participants and their involvement in a training programme in the use of language learning strategies (LLS) to support their engagement with…

  13. Investigation of dental alginate and agar impression materials as a brain simulant for ballistic testing.

    Science.gov (United States)

    Falland-Cheung, Lisa; Piccione, Neil; Zhao, Tianqi; Lazarjan, Milad Soltanipour; Hanlin, Suzanne; Jermy, Mark; Waddell, J Neil

    2016-06-01

    Routine forensic research into in vitro skin/skull/brain ballistic blood backspatter behavior has traditionally used gelatin at a 1:10 Water:Powder (W:P) ratio by volume as a brain simulant. A limitation of gelatin is its high elasticity compared to brain tissue. Therefore this study investigated the use of dental alginate and agar impression materials as a brain simulant for ballistic testing. Fresh deer brain, alginate (W:P ratio 91.5:8.5) and agar (W:P ratio 81:19) specimens (n=10) (11×22×33mm) were placed in transparent Perspex boxes of the same internal dimensions prior to shooting with a 0.22inch caliber high velocity air gun. Quantitative analysis to establish kinetic energy loss, vertical displacement elastic behavior and qualitative analysis to establish elasticity behavior was done via high-speed camera footage (SA5, Photron, Japan) using Photron Fastcam Viewer software (Version 3.5.1, Photron, Japan) and visual observation. Damage mechanisms and behavior were qualitatively established by observation of the materials during and after shooting. The qualitative analysis found that of the two simulant materials tested, agar behaved more like brain in terms of damage and showed similar mechanical response to brain during the passage of the projectile, in terms of energy absorption and vertical velocity displacement. In conclusion agar showed a mechanical and subsequent damage response that was similar to brain compared to alginate. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  14. USE OF MUELLER-HINTON BROTH AND AGAR IN THE GERM TUBE TEST

    Directory of Open Access Journals (Sweden)

    Antonella Souza Mattei

    2014-12-01

    Full Text Available Candida albicans is often isolated from clinical samples, thus its presumptive differentiation from other species of the same genus can be based on its ability to form the germ tube in human serum. Nevertheless, there are two other species that share this characteristic: C. dubliniensis and C. africana. The aim of this study was to compare four different substrates to perform the germ tube (GT test. The Candida spp. isolates were identified using a manual system (135 C. albicans, 24 C. tropicalis and one C. dubliniensis. The germ tube test was performed with fresh, previously frozen serum and Mueller-Hinton (MH broth and agar. GT was observed in 96% (130/136 of the isolates through the fresh serum technique, 94% (128/136 through previously frozen serum, 92% (125/136 in MH agar, and 90% (122/136 in MH broth. The sensitivity of each test was higher than 90%, with 100% specificity. Both the MH agar and broth were able to identify the true positives, and false positives were not found. However, some C. albicans isolates were not identified. MH agar and broth may be used in laboratory for the rapid presumptive identification of C. albicans, as an alternative method for germ tube test.

  15. In vitro susceptibility of filamentous fungi to copper nanoparticles assessed by rapid XTT colorimetry and agar dilution method.

    Science.gov (United States)

    Ghasemian, E; Naghoni, A; Tabaraie, B; Tabaraie, T

    2012-12-01

    Metal nanoparticles and their uses in various aspects have recently drawn a great deal of attention. One of the major applications is that it can be used as an antimicrobial agent. They can be considered in approaches targeted to decrease the harms caused by microorganisms, specifically fungi, threatening the medical and industrial areas. The aim of this study was to investigate the antifungal activity of synthesized copper nanoparticles (CuNPs) against four filamentous fungi including Alternaria alternata, Aspergillus flavus, Fusarium solani, and Penicillium chrysogenum. Zerovalent copper nanoparticles of mean size 8nm were synthesized by inert gas condensation (IGC) method. The antifungal activity of these synthesized copper nanoparticles was measured against selected fungi by using two different techniques including agar dilution method and XTT reduction assay. The minimal inhibitory concentrations (MICs) for copper nanoparticles by agar dilution method were less or equal to 40mg/L for P. chrysogenum, less or equal to 60mg/L for A. alternata, less or equal to 60mg/L for F. solani, and less or equal to 80mg/L for A. flavus. And also MICs obtained by XTT reduction assay ranged from 40 to 80mg/L. Our data demonstrated that the copper nanoparticles inhibited fungal growth, but the fungal sensitivity to copper nanoparticles varies depending on the fungal species. Therefore, it is advisable that the minimal inhibitory concentrations (MICs) be examined before using these compounds. It is hoped that, in future, copper nanoparticles could replace some antifungal agents, making them applicable to many different medical devices and antimicrobial control system. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  16. Structural characterization of Burkholderia pseudomallei adenylate kinase (Adk): Profound asymmetry in the crystal structure of the 'open' state

    Energy Technology Data Exchange (ETDEWEB)

    Buchko, G.W.; Robinson, H.; Abendroth, J.; Staker, B. L.; Myler, P. J.

    2010-04-16

    In all organisms adenylate kinases (Adks) play a vital role in cellular energy metabolism and nucleic acid synthesis. Due to differences in catalytic properties between the Adks found in prokaryotes and in the cytoplasm of eukaryotes, there is interest in targeting this enzyme for new drug therapies against infectious bacterial agents. Here we report the 2.1 {angstrom} resolution crystal structure for the 220-residue Adk from Burkholderia pseudomallei (BpAdk), the etiological agent responsible for the infectious disease melioidosis. The general structure of apo BpAdk is similar to other Adk structures, composed of a CORE subdomain with peripheral ATP-binding (ATP{sub bd}) and LID subdomains. The two molecules in the asymmetric unit have significantly different conformations, with a backbone RMSD of 1.46 {angstrom}. These two BpAdk conformations may represent 'open' Adk sub-states along the preferential pathway to the 'closed' substrate-bound state.

  17. Production of microbial medium from defatted brebra (Milletia ferruginea) seed flour to substitute commercial peptone agar.

    Science.gov (United States)

    Andualem, Berhanu; Gessesse, Amare

    2013-10-01

    To investigate and optimize microbial media that substitute peptone agar using brebra seed defatted flour. Defatted process, inoculums preparation, evaluation of bacterial growth, preparation of cooked and hydrolyzed media and growth turbidity of tested bacteria were determined. Two percent defatted flour was found to be suitable concentration for the growth of pathogenic bacteria: Escherichia coli (ATCC 25922) (E. coli), Pseudomonas aeruginosa (ATCC 27853), Salmonella (NCTC 8385) and Shigella flexneri (ATCC 12022) (S. flexneri), while 3% defatted flour was suitable for Staphylococcus aureus (ATCC 25923) (S. aureus). E. coli (93±1) and S. flexneri (524±1) colony count were significantly (P≤0.05) greater in defatted flour without supplement than in supplemented medium. E. coli [(3.72×10(9)±2) CFU/mL], S. aureus [(7.4×10(9)±2) CFU/mL], S. flexneri [(4.03×10(9)±2) CFU/mL] and Salmonella [(2.37×10(9)±1) CFU/mL] in non-hydrolyzed sample were statistically (P≤0.05) greater than hydrolyzed one and commercial peptone agar. Colony count of Salmonella [(4.55×10(9)±3) CFU/mL], S. flexneri [(5.40×10(9)±3) CFU/mL] and Lyesria moncytogenes (ATCC 19116) [(5.4×10(9)±3) CFU/mL] on raw defatted flour agar was significantly (P≤0.05) greater than cooked defatted flour and commercial peptone agar. Biomass of E. coli, S. aureus, Salmonella and Enterococcus faecalis in non-hydrolyzed defatted flour is highly increased over hydrolyzed defatted flour and commercial peptone broth. The defatted flour agar was found to be better microbial media or comparable with peptone agar. The substances in it can serve as sources of carbon, nitrogen, vitamins and minerals that are essential to support the growth of microorganisms without any supplements. Currently, all supplements of peptone agar are very expensive in the market. Copyright © 2013 Asian Pacific Tropical Biomedical Magazine. Published by Elsevier B.V. All rights reserved.

  18. Antibodies against In Vivo-Expressed Antigens Are Sufficient To Protect against Lethal Aerosol Infection with Burkholderia mallei and Burkholderia pseudomallei.

    Science.gov (United States)

    Zimmerman, Shawn M; Dyke, Jeremy S; Jelesijevic, Tomislav P; Michel, Frank; Lafontaine, Eric R; Hogan, Robert J

    2017-08-01

    Burkholderia mallei, a facultative intracellular bacterium and tier 1 biothreat, causes the fatal zoonotic disease glanders. The organism possesses multiple genes encoding autotransporter proteins, which represent important virulence factors and targets for developing countermeasures in pathogenic Gram-negative bacteria. In the present study, we investigated one of these autotransporters, BatA, and demonstrate that it displays lipolytic activity, aids in intracellular survival, is expressed in vivo, elicits production of antibodies during infection, and contributes to pathogenicity in a mouse aerosol challenge model. A mutation in the batA gene of wild-type strain ATCC 23344 was found to be particularly attenuating, as BALB/c mice infected with the equivalent of 80 median lethal doses cleared the organism. This finding prompted us to test the hypothesis that vaccination with the batA mutant strain elicits protective immunity against subsequent infection with wild-type bacteria. We discovered that not only does vaccination provide high levels of protection against lethal aerosol challenge with B. mallei ATCC 23344, it also protects against infection with multiple isolates of the closely related organism and causative agent of melioidosis, Burkholderia pseudomallei Passive-transfer experiments also revealed that the protective immunity afforded by vaccination with the batA mutant strain is predominantly mediated by IgG antibodies binding to antigens expressed exclusively in vivo Collectively, our data demonstrate that BatA is a target for developing medical countermeasures and that vaccination with a mutant lacking expression of the protein provides a platform to gain insights regarding mechanisms of protective immunity against B. mallei and B. pseudomallei, including antigen discovery. Copyright © 2017 American Society for Microbiology.

  19. Correlation between Agar Plate Screening and Solid-State Fermentation for the Prediction of Cellulase Production by Trichoderma Strains

    Science.gov (United States)

    Florencio, Camila; Couri, Sonia; Farinas, Cristiane Sanchez

    2012-01-01

    The viability of converting biomass into biofuels and chemicals still requires further development towards the reduction of the enzyme production costs. Thus, there is a growing demand for the development of efficient procedures for selection of cellulase-producing microorganisms. This work correlates qualitative screening using agar plate assays with quantitative measurements of cellulase production during cultivation under solid-state fermentation (SSF). The initial screening step consisted of observation of the growth of 78 preselected strains of the genus Trichoderma on plates, using microcrystalline cellulose as carbon source. The 49 strains that were able to grow on this substrate were then subjected to a second screening step using the Congo red test. From this test it was possible to select 10 strains that presented the highest enzymatic indices (EI), with values ranging from 1.51 to 1.90. SSF cultivations using sugarcane bagasse and wheat bran as substrates were performed using selected strains. The CG 104NH strain presented the highest EGase activity (25.93 UI·g−1). The EI results obtained in the screening procedure using plates were compared with cellulase production under SSF. A correlation coefficient (R 2) of 0.977 was obtained between the Congo red test and SSF, demonstrating that the two methodologies were in good agreement. PMID:23227312

  20. Evaluation of MRSA chrome agar for the detection of methicillin resistant staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Durdana Chowdhury

    2013-01-01

    Full Text Available The aim of this study was to evaluate the efficacy of MRSA Chrome agar to detect methicillin resistant Staphylococcus aureus (MRSA and compare it with 1µg oxacillin disc diffusion tests and detection of mecA gene by PCR. A total 116 Staphylococcus aureus (S. aureus, isolated from various clinical samples, were obtained from three tertiary care hospitals of Dhaka city. S. aureus was identified by colony characters, Gram stain and standard biochemical procedures. MRSA was detected by susceptibility to 1µg oxacillin disc, growth of denim blue color colonies of S. aureus on the Brilliance MRSA Chrome agar at 24 and 48 hours of incubation. PCR was performed for amplification of mecA gene as a gold standard method. Out of 116 isolated S. aureus, 33 (28.44% were MRSA by oxacillin disc diffusion test where mecA gene was detected in 28 strains. On MRSA Chrome agar, 29 (25.0% S. aureus produced denim blue colonies at 24 hours, of which 28 isolates possessed mecA gene. At 48 hours incubation, an additional 4 isolates yielded denim blue colonies from which mecA gene could not be identified. All the strains of S. aureus that produced denim blue colonies at 24 and 48 hours were resistant to oxacillin. The sensitivity, specificity and accuracy of oxacillin disc diffusion test were 100%, 94.31% and 95.68% and Chrome agar at 24 hours were 100%, 98.86% and 99.13% respectively. Thus MRSA Chrome agar could be good choice in clinical microbiology laboratory for rapid and accurate identification of MRSA. Ibrahim Med. Coll. J. 2013; 7(1: 1-4

  1. Disk diffusion, agar dilution and the E-test for susceptibility testing of Corynebacterium jeikeium.

    Science.gov (United States)

    Pennekamp, Andreas; Pünter, Verena; Zbinden, Reinhard

    1996-02-01

    OBJECTIVE: The susceptibilities to penicillin, tetracycline, erythromycin, gentamicin, vancomycin and teicoplanin of 58 strains of Corynebacterium jeikeium were assessed by disk diffusion and agar dilution reference methods. METHODS: Zone sizes and minimal inhibitory concentrations (MIC) by agar dilution were interpreted using the ranges in the NCCLS tables for organisms other than Haemophilus, Neisseria gonorrhoeae, and Streptococcus pneumoniae. RESULTS: By agar dilution, 14%, 88%, 17% and 26% of the 58 isolates were susceptible to penicillin, tetracycline, erythromycin, and gentamicin, respectively. Using the breakpoints for Listeria monocytogenes, all strains showed concordant results for penicillin by disk diffusion. Discrepancies in the interpretative categories by disk diffusion were found in four cases (two very major and two minor) for tetracycline, in nine (two very major, two major, and five minor) for erythromycin, and in 1 case (very major) for gentamicin. All 58 strains were susceptible to vancomycin and teicoplanin by agar dilution and disk diffusion. The overall agreement of interpretative disk diffusion for all six antibiotics was 95.9%. In addition, all strains were susceptible to both glycopeptides by E-test. However, for vancomycin the MIC results in 58.6% were two log2 dilutions and in 1.7% more than two log2 dilutions higher by E-test than by agar dilution, whereas for teicoplanin agreement within one log2 dilution was 100%. CONCLUSIONS: Further evaluation of methodologies of disk diffusion is required to obtain a better agreement for erythromycin and tetracycline. The criteria of the NCCLS for interpretation of disk diffusion are adequate for susceptibility testing of C. jeikeium to penicillin, gentamicin, vancomycin and teicoplanin.

  2. Use of Dehydrated Agar to Estimate Microbial Water Quality for Horticulture Irrigation.

    Science.gov (United States)

    Meador, Dustin P; Fisher, Paul R; Guy, Charles L; Harmon, Philip F; Peres, Natalia A; Teplitski, Max

    2016-07-01

    Petrifilms are dehydrated agar culture plates that have been used to quantify colony forming units (CFU) mL of either aerobic bacteria (Petrifilm-AC) or fungus (Petrifilm-YM), depending on substrate composition. Microbes in irrigation systems can indicate biofilm risk and potential clogging of irrigation emitters. The research objective was to compare counts on Petrifilms versus traditional, hydrated-agar plates using samples collected from recirculated irrigation waters and cultures of isolated known species. The estimated count (in CFU mL) from a recirculated irrigation sample after 7 d of incubation on Petrifilm-YM was only 5.5% of the count quantified using sabouraud dextrose agar (SDA) with chloramphenicol after 14 d. In a separate experiment with a known species, Petrifilm-YM did not successfully culture zoospores of . Isolates of viable zoospores were cultured successfully on potato-dextrose agar (PDA), with comparable counts with a vegetable juice medium supplemented with the antibiotics pimaricin, ampicillin, rifamycin, pentochloronitrobenzene and hymexazol (PARP-H). The quantification of pv. Begoniaceae on Petrifilm-AC was not significantly different ( < 0.05) than on PDA, but was lower than on Reasoner and Goldrich agar (R2A) or with a hemocytometer. The current formulation of Petrifilm-YM is unlikely to be a useful monitoring method for plant pathogens in irrigation water because of the inability to successfully culture oomycetes. However, Petrifilm-AC was an effective method to quantify bacteria and can provide an easy-to-use on-farm tool to monitor biofilm risk and microbial density. Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.

  3. A preliminary study on the application of natural sweet proteins in agar-based gels.

    Science.gov (United States)

    Miele, Nicoletta A; Di Monaco, Rossella; Dell'Amura, Francesca; Rega, Michele F; Picone, Delia; Cavella, Silvana

    2017-04-01

    Natural sweet proteins may be used as sugar replacer in simple liquid food systems but their applicability in more complex matrices has not been investigated yet. Gelling agent nature and texture characteristics as well as type and distribution of a stimulus in a gel could affect taste perception through inhibition or enhancement of tastants migration to the receptors. The mechanical, nonoral texture and time-intensity sweetness characteristics of sweet proteins MNEI and super sweet Y65R mutant, aspartame and saccharin added at a concentration iso-sweet to 40 g/L of sucrose in three agar gel concentrations (1%, 1.5%, and 2%) were evaluated. The results have shown that agar concentration and agar sweetener interaction particularly affect mechanical fracture stress and non oral hardness of the sweetened gels. Time intensity results illustrated that unlike in solution, the intensity of sweet taste in a gelled system over time decreases. Indeed, the behavior of the sweet proteins differed greatly in the gelled system compared to when they are in solution. MNEI has been proved to be a high-potency sweetener for beverages, but the possibility to use it in semisolid foodstuff was not investigated yet. This study represented a preliminary characterization of two variants of natural sweetener monellin, MNEI and Y65R in semisolid model foodstuff. The data were an important scientific contribution to the knowledge of sweet proteins in agar-based gels and could be useful in order to extend the possible application of these sweet proteins as low calorie sweeteners in semisolid foodstuffs. Some problems concerning their delivered sweetness in agar gels were underlined and their application should be optimized in order to improve sweetness conveyed. © 2016 Wiley Periodicals, Inc.

  4. Differentiating non-0157:H7 STEC serogroups from ground beef plated on agar media by hyperspetral imaging

    Science.gov (United States)

    Introduction: The development of an assay to detect and confirm a positive non-O157:H7 isolate is challenging when mixed morphologically results are obtained from the serogroups growing on Rainbow agar. Rainbow agar is only claimed by the manufacturer to be very specific for E.coli O157:H7 strain...

  5. Evaluation of CLSI agar dilution method and Trek Sensititre broth microdilution panel for determining antimicrobial susceptibility of Streptococcus pneumoniae.

    Science.gov (United States)

    Zhang, Sean X; Rawte, Prasad; Brown, Shirley; Lo, Steven; Siebert, Heather; Pong-Porter, Sylvia; Low, Donald E; Jamieson, Frances B

    2011-02-01

    Both the CLSI agar dilution method and Trek Sensititre broth microdilution panel for Streptococcus pneumoniae antimicrobial susceptibility testing were evaluated against the reference CLSI broth microdilution method using the most recently published CLSI breakpoints. While agar dilution was not an optimal method, the commercial panel appeared to be an acceptable method, with minor errors encountered for ceftriaxone, penicillin, and meropenem.

  6. Evaluation of CLSI Agar Dilution Method and Trek Sensititre Broth Microdilution Panel for Determining Antimicrobial Susceptibility of Streptococcus pneumoniae▿

    OpenAIRE

    Zhang, Sean X.; Rawte, Prasad; Brown, Shirley; Lo, Steven; Siebert, Heather; Pong-Porter, Sylvia; Low, Donald E; Frances B Jamieson

    2011-01-01

    Both the CLSI agar dilution method and Trek Sensititre broth microdilution panel for Streptococcus pneumoniae antimicrobial susceptibility testing were evaluated against the reference CLSI broth microdilution method using the most recently published CLSI breakpoints. While agar dilution was not an optimal method, the commercial panel appeared to be an acceptable method, with minor errors encountered for ceftriaxone, penicillin, and meropenem.

  7. On the quantitative Amido Black B staining of protein spots in agar gel at low local protein concentrations

    NARCIS (Netherlands)

    Jansen, M.T.

    1962-01-01

    Protein spots in agar gel of identical protein content but different in surface area are found to bind different amounts of dye upon staining with Amido Black B. The lower the protein concentration within the agar gel, the more the Amido Black B content of the spot falls short of the value expected

  8. Effect of lignin on water vapor barrier, mechanical, and structural properties of agar/lignin composite films.

    Science.gov (United States)

    Shankar, Shiv; Reddy, Jeevan Prasad; Rhim, Jong-Whan

    2015-11-01

    Biodegradable composite films were prepared using two renewable resources based biopolymers, agar and lignin alkali. The lignin was used as a reinforcing material and agar as a biopolymer matrix. The effect of lignin concentration (1, 3, 5, and 10wt%) on the performance of the composite films was studied. In addition, the mechanical, water vapor barrier, UV light barrier properties, FE-SEM, and TGA of the films were analyzed. The agar/lignin films exhibited higher mechanical and UV barrier properties along with lower water vapor permeability compared to the neat agar film. The FTIR and SEM results showed the compatibility of lignin with agar polymer. The swelling ratio and moisture content of agar/lignin composite films were decreased with increase in lignin content. The thermostability and char content of agar/lignin composite films increased with increased lignin content. The results suggested that agar/lignin films have a potential to be used as a UV barrier food packaging material for maintaining food safety and extending the shelf-life of the packaged food. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Agar Sediment Test for Assessing the Suitability of Organic Waste Streams for Recovering Nutrients by the Aquatic Worm Lumbriculus variegatus

    NARCIS (Netherlands)

    Laarhoven, Bob; Elissen, H.J.H.; Temmink, H.; Buisman, C.J.N.

    2016-01-01

    An agar sediment test was developed to evaluate the suitability of organic waste streams from the food industry for recovering nutrients by the aquatic worm Lumbriculus variegatus (Lv). The effects of agar gel, sand, and food quantities in the sediment test on worm growth, reproduction, and water

  10. Detection of methicillin-resistant Staphylococcus pseudintermedius with commercially available selective media.

    Science.gov (United States)

    Horstmann, C; Mueller, R S; Straubinger, R K; Werckenthin, C

    2012-01-01

    Commercially available selective media for methicillin-resistant Staphylococcus aureus (MRSA) were tested for the detection and isolation of methicillin-resistant Staphylococcus pseudintermedius (MRSP). Five different screening agars [mannitol salt agar with oxacillin and BD BBL™ Chromagar™ MRSA (BD Diagnostics); chromID™ MRSA agar (bioMérieux); Oxacillin resistance screening agar base (ORSAB); and Brilliance MRSA agar (Oxoid)] were analysed for the detection of MRSP. Bacteria that may be isolated together with MRSP and may grow on the screening agars were included in the study to determine possible interference with the growth of MRSP. MRSP grew well on all selective media except on BD BBL™ Chromagar™ MRSA (BD Diagnostics) and chromID™ MRSA agar (bioMérieux), on which a low to moderate growth rate was noted. ORSAB (Oxoid) and Brilliance MRSA agar (Oxoid) are most suitable for the detection and isolation of MRSP from clinical material. The importance of MRSP in veterinary medicine is increasing. Diagnostic systems are needed to detect MRSP carrier as soon as possible. This study provides information about selected MRSA screening agars for the detection of MRSP to the clinical microbiologists. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

  11. Gamma radiation effect on agar viscosity for use in food industry; Efeito da radiacao gama na viscosidade de agar para uso na industria alimenticia

    Energy Technology Data Exchange (ETDEWEB)

    Aliste, Antonio J.; Del Mastro, Nelida L. [Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, SP (Brazil)

    1999-11-01

    The application of food radiation processing is increasing worldwide mainly because of its efficiency in the industrial decontamination of packaged food products. Indeed, the process neither introduces any undesirable elements nor increases the temperature, thus allowing the preparation of ready-to-use products which remain stable for long periods at room temperature. The aim of this work was to study the effect of Co-60 gamma radiation on the viscosity of agar. This hydrocolloid derived from seaweed is a galactose polymer with a high hysteresis capacity (great difference among melting and gelification temperature) which is extremely important when used as additive for the food industry. Commercial agar was irradiated with doses of 0, 1, 5 and 10 kGy. Proper dilutions were prepared and the viscosity was measured in a Brookfield model LVDVIII viscosimeters. The relationships viscosity/dose for the temperatures of 45 deg C and 60 deg C were established. The decrease of the viscosity was 71.4% and 49.6% respectively when the applied dose was 10 kGy. The implications of the use of this additive in food irradiation are discussed. (author) 9 refs., 2 figs.

  12. Comparative evaluation of Borelli's lactritmel agar and Lowenstein-Jensen agar for conidiation in the Trichophyton mentagrophytes and Trichophyton rubrum complexes.

    Science.gov (United States)

    Döğen, Aylin; Ilkit, Macit

    2013-02-01

    The characteristics of macroconidia are the key to the identification of dermatophytic fungi. In this study, we compared Lowenstein-Jensen agar (LJA) with Borelli's lactritmel agar (BLA) for the ability to induce macroconidia production in a collection of Trichophyton mentagrophytes (12) and Trichophyton rubrum (12) complexes and in Arthroderma isolates (4). We evaluated 28 strains from across the world, which were mainly obtained from the culture collection of the Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, The Netherlands. All cultures were incubated at 26 °C on the bench, and conidia formation was investigated every 5 days, over a period of 30 days after inoculation. At 15 days, strains grown on BLA demonstrated better macroconidia production than those grown on LJA, 23 (82.1%) versus 16 (57.1%) isolates, respectively. Thus, the use of BLA, an inexpensive medium that can be prepared in the laboratory, should facilitate conidiation and the presumptive identification of dermatophytic fungi in mycology and clinical microbiology laboratories.

  13. Agar Sediment Test for Assessing the Suitability of Organic Waste Streams for Recovering Nutrients by the Aquatic Worm Lumbriculus variegatus.

    Directory of Open Access Journals (Sweden)

    Bob Laarhoven

    Full Text Available An agar sediment test was developed to evaluate the suitability of organic waste streams from the food industry for recovering nutrients by the aquatic worm Lumbriculus variegatus (Lv. The effects of agar gel, sand, and food quantities in the sediment test on worm growth, reproduction, and water quality were studied. Agar gel addition ameliorated growth conditions by reducing food hydrolysis and altering sediment structure. Best results for combined reproduction and growth were obtained with 0.6% agar-gel (20 ml, 10 g. fine sand, 40 g. coarse sand, and 105 mg fish food (Tetramin. With agar gel, ingestion and growth is more the result of addition of food in its original quality. Final tests with secondary potato starch sludge and wheat bran demonstrated that this test is appropriate for the comparison of solid feedstuffs and suspended organic waste streams. This test method is expected to be suitable for organic waste studies using other sediment dwelling invertebrates.

  14. Agar Sediment Test for Assessing the Suitability of Organic Waste Streams for Recovering Nutrients by the Aquatic Worm Lumbriculus variegatus.

    Science.gov (United States)

    Laarhoven, Bob; Elissen, H J H; Temmink, H; Buisman, C J N

    2016-01-01

    An agar sediment test was developed to evaluate the suitability of organic waste streams from the food industry for recovering nutrients by the aquatic worm Lumbriculus variegatus (Lv). The effects of agar gel, sand, and food quantities in the sediment test on worm growth, reproduction, and water quality were studied. Agar gel addition ameliorated growth conditions by reducing food hydrolysis and altering sediment structure. Best results for combined reproduction and growth were obtained with 0.6% agar-gel (20 ml), 10 g. fine sand, 40 g. coarse sand, and 105 mg fish food (Tetramin). With agar gel, ingestion and growth is more the result of addition of food in its original quality. Final tests with secondary potato starch sludge and wheat bran demonstrated that this test is appropriate for the comparison of solid feedstuffs and suspended organic waste streams. This test method is expected to be suitable for organic waste studies using other sediment dwelling invertebrates.

  15. The bactericidal effect of carbon nanotube/agar composites irradiated with near-infrared light on Streptococcus mutans

    Energy Technology Data Exchange (ETDEWEB)

    Akasaka, Tsukasa, E-mail: akasaka@den.hokudai.ac.jp [Graduate School of Dental Medicine, Hokkaido University, Kita13 Nishi7, Kita-ku, Sapporo 060-8586 (Japan); Matsuoka, Makoto [Graduate School of Dental Medicine, Hokkaido University, Kita13 Nishi7, Kita-ku, Sapporo 060-8586 (Japan); Hashimoto, Takeshi [Meijo Nano Carbon Co. Ltd., Otsubashi bldg. 4F, 3-4-10 Marunouchi, Naka-ku, Nagoya 460-0002 (Japan); Abe, Shigeaki; Uo, Motohiro; Watari, Fumio [Graduate School of Dental Medicine, Hokkaido University, Kita13 Nishi7, Kita-ku, Sapporo 060-8586 (Japan)

    2010-10-15

    Dental caries are mainly associated with oral pathogens, and Streptococcus mutans is a primary cariogenic organism. Many methods have been established to eliminate S. mutans from the oral cavity. This study aimed to evaluate the effect of carbon nanotube (CNT)/agar composites irradiated with near-infrared (NIR) light on S. mutans, as a potential photothermal antimicrobial nanotherapy. A colony-forming unit assay clearly showed that CNT/agar composites attain bactericidal activity after NIR light irradiation; this bactericidal activity is higher than that of graphite (GP)/agar and activated carbon (AC)/agar composites. Furthermore, it was observed that longer irradiation times immobilized S. mutans in the CNT/agar composite.

  16. Optimum use of selective plated media in primary processing of respiratory tract specimens from patients with cystic fibrosis.

    Science.gov (United States)

    Doern, G V; Brogden-Torres, B

    1992-10-01

    A total of 258 respiratory tract specimens from patients with cystic fibrosis were inoculated onto nine different plated media, and the rates of recovery of potential pathogens were compared. Media included sheep blood agar, enriched chocolate agar, MacConkey agar for gram-negative bacilli, chocolate agar containing bacitracin for Haemophilus spp., bromcresol green agar for yeasts, cetrimide agar for Pseudomonas spp., sheep blood agar containing colistin and nalidixic acid for gram-positive cocci, mannitol salt agar for Staphylococcus aureus, and oxidation-fermentation agar containing 300 U of polymyxin B per ml and 2 U of bac