No description available.

No description available.

No description available.

Numerous outbreaks of foodborne illness have been attributed to non-intact beef (e.g., tenderized, marinated, and enhanced) products contaminated with Escherichia coli O157:H7. Organic acids are commonly utilized in the beef industry as antimicrobial interventions, which must be validated to eliminate or reduce E. coli O157:H7 to an undetectable level. Rifampicin-resistant Biotype I E. coli O157:H7 surrogate microorganisms (ATCC BAA-1427, BAA-1428, and BAA-1430) were applied as a cocktail (7.8 log10 CFU/ml) to three beef products (boneless strip loins, top sirloin butts, and bottom sirloin flaps) prior to treatment with an antimicrobial intervention (2.5% Beefxide or 2.9% lactic acid). Products were then subjected to a single or multiple pass tenderization and/or marination process. Beefxide and lactic acid treatments resulted in statistically significant log reductions of the microorganisms (P < 0.05) on the surfaces for all three products. Surrogate microorganisms were recovered from interior samples of all three products after mechanical tenderization. Additionally, surrogate concentrations recovered from flap surface and internal samples taken post-tumbling and marination were statistically similar (P < 0.05). These data indicate that tenderization and marination processes can transfer microorganisms into the interior of whole-muscle cuts, and suggest Beefxide and lactic acid may be similar in their efficacy as an antimicrobial applied as an intervention in the production of non-intact beef products.

Bacterial biofilms are microbial communities that adhere to abiotic or biotic surfaces. Biofilm formation (BF) studies in E. coli have primarily concentrated on uropathogenic E. coli, commensal K-12 and enterohemorrhagic E. coli O157:H7. This does not include the vast diversity of environmental strains. Quorum sensing (QS) is a means by which bacteria can communication with one another through the production of signalling molecules. The autoinducer 2 (AI-2) QS system is utilized by E. coli and several other bacterial species for controlling gene expression. The role of AI-2 in E. coli BF varies among different strains. For example in the K-12 strain, AI-2 regulates motility, and thus can affect BF; whereas in O157:H7, AI-2 has a more metabolic role. Interestingly, in strain O157:H7, motility is controlled by a newly discovered QS system regulated by the autoinducer 3 (AI-3) molecule plus the mammalian hormones epinephrine (Epi) and norepinephrine (Ne). The purpose of this study was to investigate the ability of a panel of environmental E. coli strains to form biofilms and to determine whether QS is involved in the process. A new pathotype of E. coli, adherent invasive E. coli (AIEC) which is associated with Crohn’s disease was included in the investigation. Study 1 sought to determine whether BF under different media conditions correlated with the presence of genes involved in the AI-2 QS system or adhesin factors. Media conditions were the principal variable affecting the BF. Study 2 examined the role of the AI-2 and AI-3/Epi/Ne QS systems in motility and BF by the AIEC strain. It was discovered that the AI-3 system is involved in motility; whereas the AI-2 system had no effect on BF or motility. In Study 3, microarray gene expression analysis and invasion assays were performed using qseB or qseC mutants. These genes encode the two-component regulatory system recognizing AI-3 or its cognate, epinephrine. Our findings indicate that alternative pathways likely account for the BF observed for the qseB and qseC mutants. It was concluded that the AI-3/Epi/Ne QS system partially controls AIEC motility and the invasion of epithelial cells.

No description available.

SurA is a molecular chaperone in the periplasm of E.coli and has been implicated in the maturation of outer membrane proteins (OMPs). SurA consists of four domains, but only two of them, namely the N and C-­terminal domains, are necessary for chaperone function. Very little is known about which residues drive the interaction between the N and C-­termini that facilitates normal activity. We mutated several conserved residues on the C-terminus and generated additive truncations to observe the effects of each on the fitness of the cell. We found one mutation E(408):A was sufficient to reduce SurA activity by 3-­fold, but structural characterization of the mutated protein revealed little variation from wild-­‐type SurA. Most notable, we found that when at least 10 residues are removed from the C-­‐terminus, the protein is completely non-­‐functional. We introduced a random peptide library to substitute these 10 residues and found that ~1.5% of all possible sequences in the library can restore SurA function to at least 50% activity. Moreover, we observed no pattern in the sequences of 26 different variants that were chosen and characterized. Here we show for the first time that SurA can tolerate many mutations at the C-­terminus and still be active.

The viable but non-culturable (VBNC) state is outstanding among bacterial stress responses as being completely uncharacterised at the molecular level. The aim of this investigation was to gain an insight into the molecular basis of the condition by identifying genes whose expression was up-regulated in response to VBNC-inducing stimuli. First, a model experimental system was established where bacteria were induced to enter the state in a routine and predictable manner. <I>Escherichia coli </I>HB101 exhibited a partial viable but non-culturable phenotype when inoculated into microcosms of artificial seawater at 37°C, <I>Pseudomonas fluorescens </I>10586 became viable but non-culturable in microcosms of drinking water incubated at 37°C, and <I>Vibrio vulnificus </I>MO6-24/T entered a viable, non-culturable state in artificial seawater at 5°C. A transposon mutagenesis strategy utilising a promoter-less bioluminescent reporter cassette, <I>lux</I>AB, was employed in the search for VBNC-associated genes. The mini-Tn<I>5 lux</I>AB transposon was induced to transform into arbitrary positions of the <I>P. fluorescens </I>10586 chromosome, thus creating a library of <I>P. fluorescens lux</I>AB mutants. This library (consisting of over 1200 transformants) was screened for those which were dark under normal circumstances, but luminesced in response to VBNC stimuli, indicating that the transposon had integrated downstream of a gene up-regulated during the VBNC response. Unfortunately, no mutant examined exhibited such a bioluminescence profile. Differential display of RNA technology was employed subsequently and resulted in the cloning and sequencing of several <I>V. vulnificus </I>transcripts thought to be associated with the VBNC state. Although absolute verification of the involvement of these transcripts was not achieved, hints as to what mechanisms lay at the basis of the VBNC state were gained. Some findings indicated that VBNC cells experience considerable levels of oxidative stress, and it was proposed that this physiological state may lie at the crux of the VBNC phenotype.

To enhance outbreak investigations of Shiga toxin-producing Escherichia coli O157:H7, PulseNet Canada has recently applied Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) as a supplemental subtyping tool in combination with the gold standard subtyping method Pulsed-field Gel Electrophoresis (PFGE) for enhanced resolution of isolates exhibiting indistinguishable/highly similar PFGE patterns. The objective was to assess the discriminatory power and level of specificity MLVA offers for outbreak detection and response. Results demonstrate that MLVA provides a statistically significant increase in discriminatory power for outbreak investigations (0.998) compared to PFGE alone (0.993). MLVA was able to provide additional resolution over PFGE analysis and generally agreed with PFGE when isolates were identical and epidemiologically linked. MLVA shows great promise as a molecular epidemiological tool to complement PFGE, as it improves case categorization during outbreak investigations, and the greatest benefits of MLVA may be realized during routine surveillance, when epidemiological information is not available.

Bacterial biofilms are microbial communities that adhere to abiotic or biotic surfaces. Biofilm formation (BF) studies in E. coli have primarily concentrated on uropathogenic E. coli, commensal K-12 and enterohemorrhagic E. coli O157:H7. This does not include the vast diversity of environmental strains. Quorum sensing (QS) is a means by which bacteria can communication with one another through the production of signalling molecules. The autoinducer 2 (AI-2) QS system is utilized by E. coli and several other bacterial species for controlling gene expression. The role of AI-2 in E. coli BF varies among different strains. For example in the K-12 strain, AI-2 regulates motility, and thus can affect BF; whereas in O157:H7, AI-2 has a more metabolic role. Interestingly, in strain O157:H7, motility is controlled by a newly discovered QS system regulated by the autoinducer 3 (AI-3) molecule plus the mammalian hormones epinephrine (Epi) and norepinephrine (Ne). The purpose of this study was to investigate the ability of a panel of environmental E. coli strains to form biofilms and to determine whether QS is involved in the process. A new pathotype of E. coli, adherent invasive E. coli (AIEC) which is associated with Crohn’s disease was included in the investigation. Study 1 sought to determine whether BF under different media conditions correlated with the presence of genes involved in the AI-2 QS system or adhesin factors. Media conditions were the principal variable affecting the BF. Study 2 examined the role of the AI-2 and AI-3/Epi/Ne QS systems in motility and BF by the AIEC strain. It was discovered that the AI-3 system is involved in motility; whereas the AI-2 system had no effect on BF or motility. In Study 3, microarray gene expression analysis and invasion assays were performed using qseB or qseC mutants. These genes encode the two-component regulatory system recognizing AI-3 or its cognate, epinephrine. Our findings indicate that alternative pathways likely account for the BF observed for the qseB and qseC mutants. It was concluded that the AI-3/Epi/Ne QS system partially controls AIEC motility and the invasion of epithelial cells.