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  • 1
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    Using whole-genome sequencing to determine appropriate streptomycin epidemiological cutoffs for Salmonella and Escherichia coli (2016)
    Oxford University Press
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    Publication Date: 2016-02-03
    Description: For Enterobacteriaceae such as Salmonella spp. and Escherichia coli , no unified interpretive resistance criteria exist for streptomycin, an epidemiologically important antibiotic. As part of the National Antimicrobial Resistance Monitoring System, we had previously used a minimum inhibitory concentration of ≥64 μg mL –1 as an epidemiological cutoff value (ECV) to define non-wild-type isolates. To identify whether this ECV correlated with genetic determinants of resistance, we performed whole-genome sequencing of 463 Salmonella and E. coli isolates to identify streptomycin resistance genotypes. From this analysis, we found that using a streptomycin resistance breakpoint of ≥64 μg mL –1 classified over 20% of strains possessing aadA or strA/strB resistance genes as wild-type. Therefore, to improve the concordance between genotypic and phenotypic data, we propose reducing the phenotypic cutoff values to ≥32 μg mL –1 for both Salmonella and E. coli , to be used widely as ECVs to categorize non-wild-type isolates.
    Keywords: Food Microbiology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
    Published by Oxford University Press
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  • 2
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    Protective roles of katG-homologous genes against extrinsic peroxides in Vibrio parahaemolyticus (2016)
    Oxford University Press
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    Publication Date: 2016-03-04
    Description: The marine foodborne enteropathogen, Vibrio parahaemolyticus , has four putative catalase genes. Function of the katG -homologous genes, katG1 (VPA0768) and katG2 (VPA0453), was examined using gene deletion mutants, and compared with those of the katE -homologous genes, katE1 (VPA1418) and katE2 (VPA0305). Bacterial growth of katG1 was significantly delayed in the presence of 200–300 μM H 2 O 2 , and such inhibition was enhanced when incubation temperature was lowered from 37°C to 22°C. In the stationary phase, the katG1 strain was more susceptible to the lethal dosage of H 2 O 2 than the katE1 strain. The minimum inhibitory concentrations and minimum bactericidal concentrations revealed that katE1/katE2 strains were more susceptible to H 2 O 2 than the katG1/katG2 strains in exponential phase, while katG1 was more susceptible than the katE1/katE2 strains in the starved culture. This study demonstrated the chief antioxidative role of katG1 in the stationary phase and starved culture of V. parahaemolyticus , while katG1 and katG2 were also responsive to H 2 O 2 and cumene hydroperoxide in the exponential phase.
    Keywords: Food Microbiology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
    Published by Oxford University Press
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  • 3
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    Acquisition of the yeast Kluyveromyces marxianus from unpasteurised milk by a kefir grain enhances kefir quality (2016)
    Oxford University Press
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    Publication Date: 2016-07-24
    Description: Kefir is a fermented milk beverage consumed for nutritional and health tonic benefits in many parts of the world. It is produced by the fermentation of milk with a consortium of bacteria and yeast embedded within a polysaccharide matrix. This consortium is not well defined and can vary substantially between kefir grains. There are little data on the microbial stability of kefir grains, nor on interactions between microbes in the grain and in the milk. To study this, a grain was split, with one half of each stored at –20°C and the other half passaged repeatedly in whole unpasteurised milk. Grains passaged in the unpasteurised milk recovered vigour and acquired the yeast Kluyveromyces marxainus from the milk which was confirmed to be the same strain by molecular typing. Furthermore, these passaged grains produced kefir that was distinguished chemically and organoleptically from the stored grains. Some changes in ultrastructure were also observed by scanning electron microscopy. The study showed that kefir grains can acquire yeast from their environment and the final product can be influenced by these newly acquired yeasts. Kluyveromyces marxianus is considered to be responsible for some of the most important characteristics of kefir so the finding that this yeast is part of the less stable microbiota is significant.
    Keywords: Food Microbiology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
    Published by Oxford University Press
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  • 4
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    Expression of the genes encoding the CasK/R two-component system and the DesA desaturase during Bacillus cereus cold adaptation (2016)
    Oxford University Press
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    Publication Date: 2016-07-31
    Description: Two-component systems (TCS) allow a cell to elaborate a variety of adaptive responses to environment changes. The recently discovered CasK/R TCS plays a role in the optimal unsaturation of fatty acids necessary for cold adaptation of the foodborne-pathogen Bacillus cereus . Here, we showed that the promoter activity of the operon encoding this TCS was repressed during growth at low temperature in the stationary phase in the parental strain when compared to the casK/R mutant, suggesting that CasR negatively regulates the activity of its own promoter in these conditions. The promoter activity of the desA gene encoding the 5 fatty acid desaturase, providing unsaturated fatty acids (UFAs) required for low temperature adaptation, was repressed in the casK/R mutant grown at 12°C versus 37°C. This result suggests that CasK/R activates desA expression during B. cereus growth at low temperature, allowing an optimal unsaturation of the fatty acids. In contrast, desA expression was repressed during the lag phase at low temperature in presence of UFAs, in a CasK/R-independent manner. Our findings confirm that the involvement of this major TCS in B. cereus cold adaptation is linked to the upregulation of a fatty acid desaturase.
    Keywords: Food Microbiology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
    Published by Oxford University Press
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  • 5
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    Characterization of a highly potent antimicrobial peptide microcin N from uropathogenic Escherichia coli (2016)
    Oxford University Press
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    Publication Date: 2016-05-12
    Description: Microcin N is a low-molecular weight, highly active antimicrobial peptide produced by uropathogenic Escherichia coli . In this study, the native peptide was expressed and purified from pGOB18 plasmid carrying E. coli in low yield. The pure peptide was characterized using mass spectrometry, N-terminal sequencing by Edman degradation as well as trypsin digestion. We found that the peptide is 74-residue long, cationic (+2 total charge), highly hydrophobic and consists of glycine as the first N-terminal residue. The minimum inhibitory concentration of the peptide against Salmonella enteritidis was found to be 150 nM. Evaluation of the solution conformation of the peptide using circular dichroism spectroscopy showed that the peptide is well folded in 40% trifluoroethanol with helical structure whereas the folded structure is lost in aqueous solution. To increase the yield of this potent peptide, we overexpressed GST-tagged microcin N using E. coli BL21. Recombinant GST-tagged microcin N was successfully expressed in E. coli BL21; however, the cleaved mature microcin N did not show activity against the indicator strain ( S. enterica ) most likely due to the extreme hydrophobic nature of the peptide. Efforts to produce active microcin N in large scale are discussed as this peptide has huge potential to be the next generation antimicrobial agent.
    Keywords: Food Microbiology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
    Published by Oxford University Press
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  • 6
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    Stx1 prophage excision in Escherichia coli strain PA20 confers strong curli and biofilm formation by restoring native mlrA (2016)
    Oxford University Press
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    Publication Date: 2016-06-04
    Description: Prophage insertions in Escherichia coli O157:H7 mlrA contribute to the low expression of curli fimbriae and biofilm observed in many clinical isolates. Varying levels of CsgD-dependent curli/biofilm expression are restored to strains bearing prophage insertions in mlrA by mutation of regulatory genes affecting csgD . Our previous study identified strong biofilm- and curli-producing variants in O157:H7 cultures that had lost the mlrA -imbedded prophage characteristic of the parent population, suggesting prophage excision as a mechanism for restoring biofilm properties. In this study, we compared genomic, transcriptomic and phenotypic properties of parent strain PA20 ( stx 1 , stx 2 ) and its prophage-cured variant, 20R2R ( stx 2 ), and confirmed the mechanism underlying the differences in biofilm formation.
    Keywords: Food Microbiology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
    Published by Oxford University Press
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  • 7
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    Characterization of LysPBC4, a novel Bacillus cereus-specific endolysin of bacteriophage PBC4 (2016)
    Oxford University Press
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    Publication Date: 2016-05-20
    Description: Bacillus cereus is a spore-forming, Gram-positive bacterium and is a major food-borne pathogen. A B. cereus -specific bacteriophage PBC4 was isolated from the soil of a stock farm, and its genome was analyzed. PBC4 belongs to the Siphoviridae family and has a genome consisting of 80 647-bp-long double-stranded DNA, including 123 genes and two tRNAs. LysPBC4, the endolysin of PBC4, has an enzymatically active domain (EAD) on its N-terminal region and a putative cell wall-binding domain (CBD) on its C-terminal region, respectively. Although the phage PBC4 showed a very limited host range, LysPBC4 could lyse all of the B. cereus strains tested. However, LysPBC4 did not kill other bacteria such as B. subtilis or Listeria , indicating that the endolysin has specific lytic activity against the B. cereus group species. Furthermore, LysPBC4_CBD fused with enhanced green fluorescent protein (EGFP) could decorate limited strains of B. cereus group, suggesting that the LysPBC4_CBD may be a promising material for specific detection of B. cereus .
    Keywords: Food Microbiology
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    Electronic ISSN: 1574-6968
    Topics: Biology
    Published by Oxford University Press
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  • 8
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    Characterization of Bacillus cereus isolates from local dairy farms in China (2016)
    Oxford University Press
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    Publication Date: 2016-05-20
    Description: Bacillus cereus is an important opportunistic foodborne pathogen. In the present work, a total of 306 milk and environmental samples were collected from 10 local dairy farms in Beijing, China. Of the 92 B. cereus -like isolates, 88 and 4 belonged to B. cereus and B. thuringiensis , respectively. The prevalence of B. cereus isolates in bedding, feces, feed, liquid manure and raw milk was 93.3%, 78.9%, 41.2%, 100.0% and 9.8%, respectively. Three main toxin genes nhe, hbl and ces were detected with rates of 100.0%, 78.3% and 1.1%, but no strain harbored cytK1 . The production of Nhe, Hbl and cereulide could be confirmed by specific monoclonal antibodies-based enzyme immunoassays in 94.6%, 70.7% and 1.1% of all isolates, respectively. Cytotoxicity tests were used to further corroborate the results of genetic and protein-based assays; 91.3% of the isolates showed cytotoxicity to Vero cells. All isolates were tested for antimicrobial resistance against 17 antibiotics. All isolates were resistant to lincomycin, retapamulin, tiamulin and valnemulin, while two strains were susceptible to ampicillin and ceftiofur. A total of 16 isolated strains were resistant to tetracycline. Since spores of B. cereus are not inactivated during manufacturing of most milk products, contamination of milk with B. cereus on the farm level may represent a potential hazard, particularly with respect to emetic toxin-producing strains.
    Keywords: Food Microbiology
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    Topics: Biology
    Published by Oxford University Press
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  • 9
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    Lactobacillus acidophilus binds to MUC3 component of cultured intestinal epithelial cells with highest affinity (2016)
    Oxford University Press
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    Publication Date: 2016-04-10
    Description: Lactobacillus strains have been shown to adhere to the mucosal components of intestinal epithelial cells. However, established in vitro adhesion assays have several drawbacks in assessing the adhesion of new Lactobacillus strains. The present study aimed to compare the adhesion of four different Lactobacillus strains and select the most adherent microbe, based on in silico approach supported by in vitro results. The mucus-binding proteins in Lactobacillus acidophilus , L. plantarum, L. brevis and L. fermentum were identified and their capacities to interact with intestinal mucin were compared by molecular docking analysis. Lactobacillus acidophilus had the maximal affinity of binding to mucin with predicted free energy of –6.066 kcal mol –1 . Further, in vitro experimental assay of adhesion was performed to validate the in silico results. The adhesion of L. acidophilus to mucous secreting colon epithelial HT-29 MTX cells was highest at 12%, and it formed biofilm with maximum depth (Z = 84 μm). Lactobacillus acidophilus was determined to be the most adherent strain in the study. All the Lactobacillus strains tested in this study, displayed maximum affinity of binding to MUC3 component of mucus as compared to other gastrointestinal mucins. These findings may have importance in the design of probiotics and health care management.
    Keywords: Food Microbiology
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    Topics: Biology
    Published by Oxford University Press
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  • 10
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    Susceptibility of Klebsiella pneumoniae on coriander leaves to liquid- and vapor-phase ethanol (2016)
    Oxford University Press
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    Publication Date: 2016-04-20
    Description: The bio-control of ethanol on Klebsiella pneumoniae on fresh coriander leaves for significantly reducing consumer health risk was investigated. Washed and sterilized leaves of coriander were inoculated with K. pneumoniae cultured in Trypticase Soy broth. Susceptibility of the K. pneumoniae to liquid- and evaporated vapor-phase ethanol (EVE) was then examined in vitro . Complete inhibition of K. pneumoniae was found with 18% (v/v) liquid ethanol. Exposure for 15 min to EVE (9.00 ± 0.8 mmol L –1 ) completely destroyed K. pneumoniae (4.04 ± 0.02 log CFU/ml) spread on Mueller Hilton agar at 30 ± 2°C. The effect of EVE with and without evaporated water vapor (EWV) on the susceptibility of K. pneumoniae on fresh coriander leaves was examined. While exposure to EVE affected the survival of K. pneumoniae , the degree of reduction depended on both the inoculation level and the EWV. Complete reduction of K. pneumoniae was achieved for the low inoculation level by EVE alone (37 ± 2% relative humidity; RH) but susceptibility was reduced with EWV (high RH; 80 ± 2%). Scanning electron microscope (SEM) images of inoculated coriander leaves confirm the effects of EVE in reducing levels of K. pneumoniae . Exposure to EVE alone proved an effective bio-control for K. pneumoniae on fresh coriander leaves.
    Keywords: Food Microbiology
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    Electronic ISSN: 1574-6968
    Topics: Biology
    Published by Oxford University Press
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