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AIMS: Exposure of Listeria monocytogenes to osmotic stress can induce increased resistance to subsequent lethal exposure to cell envelope stressors, such as nisin and bile salts. We wanted to determine if similar cross-protection phenotypes could occur when L. monocytogenes strains were treated with osmotic stress and exposed to sublethal levels of the cell envelope stressor, bile. METHOD AND RESULTS: Growth phenotypes were measured for six L. monocytogenes strains exposed to 6% NaCl, 0·3 and 1% bile in BHI. To evaluate cross-protection, cells were pre-exposed to 6% NaCl, followed by exposure to BHI+1% bile for 26 h and vice versa. Significant increases in λ (lag phase) and doubling time were observed under salt and bile stresses compared with BHI alone. Average λ and Nmax (maximum cell density) in 0·3 and 1% bile for all strains were significantly lower than that in 6% NaCl. Pre-exposure to 6% NaCl followed by exposure to 1% bile significantly increased λ (P < 0·05), whereas pre-exposure to 1% bile followed by exposure to 6% NaCl led to formation of filamentous cells, with no changes in cell density over 26 h. CONCLUSIONS: Variation in growth characteristics was observed among strains exposed to bile. Exposure to osmotic stress did not lead to increased resistance to bile. Exposure to bile significantly impacted the ability of L. monocytogenes to adapt to grow under osmotic stress, where cells did not multiply but formed filamentous cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Pre-exposure to a cell envelope stress and subsequent exposure to an osmotic stress appears to pose a significant stress to L. monocytogenes cells.

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Immunomagnetic separation used with culture based methods has been a useful technique in the detection of pathogens. However, previous studies have not answered many of the necessary questions for real world applications. The objective of this study was to assess the efficacy of different immunomagnetic separation (IMS) bead types in recovery of the correct serogroup from a mixture of big six non-O157 Shiga toxin-producing Escherichia coli strains. To determine the impact of different matrices on recovery, samples of sterile phosphate buffered saline (PBS), sterile and non-sterile cattle faeces, ground beef and lettuce were inoculated with 10 CFU per ml mixture of isolates representing the six serogroups. After a 6 h incubation at 37°C, samples were mixed with IMS beads from three different commercial sources and plated on eosin methylene blue agar (EMB). Three suspect E. coli colonies were selected from each EMB plate and multiplex polymerase chain reaction was used to determine the serogroup. The rate of correct identification varied with the serogroup, IMS bead manufacturer and matrix. Overall, recovery of the correct serogroup became less likely with increase in matrix complexity, with enrichments containing lettuce having the greatest number of bead types with significantly lower likelihood of correct recovery compared to recovery in PBS. SIGNIFICANCE AND IMPACT OF THE STUDY: The need to accurately and efficiently detect Shiga toxin-producing Escherichia coli (STEC) O26, O45, O103, O111, O121 and O145, which have caused outbreaks on numerous occasions, is a major public health and food safety concern in the United States. Detecting these STEC serogroups can be challenging because methods to detect non-O157 serogroups have not been refined as compared to those for O157. Immunomagnetic separation (IMS) has the potential to isolate STEC from a mixture in complex matrices. Our results highlight the need for optimization of IMS-based detection of STEC to effectively recover the targeted serogroup from a variety of sample matrices.

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AIMS: Listeria monocytogenes nisin resistance increases when first exposed to NaCl and other stresses, such as low pH. In addition to environmental stressors, specific genomic elements can confer nisin resistance, such as the stress survival islet (SSI-1). As SSI-1 is variably present among L. monocytogenes strains, we wanted to determine if SSI-1 was associated with salt-induced nisin resistance. METHODS AND RESULTS: The presence of SSI-1 was determined using PCR for 48 strains of L. monocytogenes. When combined with multilocus sequence typing data, we found that the distribution of SSI-1 is clonal, where strains from clonal complexes (CC) 2, 6 and 11 do not have SSI-1, while strains from CCs 3, 5, 7 and 9 contain SSI-1. The impact of SSI-1 on salt-induced nisin resistance was dependent on CC. The average log decrease after 24 h of exposure to nisin at 7°C under salt-inducing conditions was 2·6 ± 1·1 for CC 9 strains and 2·3 ± 0·7 for CC 11 strains, which had significantly lower survival compared to the other CCs, such as 1·3 ± 0·3 for CC 6. Deletion of SSI-1 from a CC 7 strain demonstrated the role SSI-1 plays in salt-induced nisin resistance, as the deletion mutant had lower resistance compared to the parent strain. CONCLUSIONS: These data suggest that inducible nisin resistance in L. monocytogenes can be influenced by environmental conditions as well as the genetic composition of the strain, which should be considered when selecting control measures for ready-to-eat foods. SIGNIFICANCE AND IMPACT OF THE STUDY: The foodborne pathogen L. monocytogenes can grow in suboptimal conditions, including low temperature and high osmolarity, which makes it a safety concern for ready-to-eat foods. When using antimicrobial peptide inhibitors such as nisin, it is important to understand how food components can impact antimicrobial resistance across the genetic diversity of L. monocytogenes.

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The objective of this study was to assess the genetic diversity of non-O157 Shiga toxin-producing Escherichia coli (STEC) isolates from cattle. Multi-locus sequence typing (MLST) was used to identify and compare the sequence types (STs) of 43 non-O157 STEC cattle isolates using the EcMLST database curated by the STEC Center at Michigan State University. For the 43 isolates, 19 STs were identified and 10 of those STs were novel compared to those in EcMLST. For the 43 isolates, 19 different serotypes were identified. STEC O22:H8, O174:H28 and O8:H19 were most common, and STEC O8 isolates were the most diverse, with seven different STs for isolates with that O group. STEC strains with O types identified in this study have been isolated from cattle by other researchers, as well as from cases of human gastroenteritis. Of the 10 novel STs identified, six were found to be closely related to previously identified STs, indicating that populations of non-O157 STEC in cattle are similar to those from other sources, including human clinical cases. Significance and impact of the study: The foodborne pathogen Shiga toxin-producing Escherichia coli (STEC) is a significant public health concern. One of the main reservoirs for STEC are cattle, which can directly or indirectly contribute to STEC in the food supply. The genetic subtype data presented here highlight the diversity of STEC that can be isolated from cattle. These results further our understanding of the ecology of STEC in the primary production environment, which is important for developing effective control measures to reduce this pathogen in the food supply.

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Listeria monocytogenes is well known to survive and grow under several stress conditions, including salt stress, which is important for growth in certain foods as well as for host infection. To characterize the contributions, to salt stress response, of transcriptional regulators important for stress response and virulence (i.e., σ(B) and PrfA), we analyzed three L. monocytogenes parent strains and isogenic mutants (ΔsigB, ΔprfA, and ΔsigBΔprfA), representing different serotypes and lineages, for their ability to grow, at 25°C, in BHI with 1.9 M NaCl. With regard to growth rate, only the lineage IV strain presented a significant difference between the parent strain and both of its respective mutants lacking prfA (ΔprfA and ΔsigBΔprfA). Conversely, the lineage I and II parent strains showed significantly shorter lag phase in comparison to their respective ΔsigB mutant strains. Intestinal epithelial cell invasion assay and hemolytic activity assays showed a significant role for σ(B) in the former and for PrfA in the latter. To explore the mechanism that may contribute to the extended lag phase in the ΔsigB mutant strain and survival and growth of the parent strain upon salt shock, whole genome transcription profiling was performed to compare transcript levels between the lineage I, serotype 1/2b, parent strain and its isogenic ΔsigB mutant after 30 min of lag phase growth at 25°C in the presence of 1.9M NaCl (salt shock) without aeration. Microarray data showed significantly higher transcript levels for 173 genes in the parent strain as compared to the ΔsigB strain. Overall, 102 of the 173 σ(B) up-regulated genes had been identified in previous studies, indicating that 71 genes were newly identified as being up-regulated by σ(B) in this study. We hypothesize that, among these genes newly identified as σ(B) up-regulated, four genes (lmo2174, lmo0530, lmo0527 and lmo0529) may play a major role in response to salt stress. Lmo2174 contains domains that facilitate sensing and producing a transduction signal in the form of cyclic di-GMP, which may activate the enzymes Lmo0527, Lmo0529 and Lmo0530, which encode proteins similar to those responsible for synthesis of exopolysaccharides that may protect the cell by changing the cell wall structure during salt stress. Overall, our data showed that σ(B), but not PrfA, contributes to growth under salt stress. Moreover, we show that the σ(B) regulon of a L. monocytogenes lineage I strain challenged with salt shock includes salt stress-specific as well as previously unidentified σ(B) up-regulated genes.

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AIMS: To compare survival of enterohaemorrhagic Escherichia coli (EHEC) strains of two clonal groups in a simulated gastric environment and to quantify the effect of storage in an acidic food, apple juice, on subsequent survival of EHEC in the simulated gastric environment. METHODS AND RESULTS: To characterize acid resistance of EHEC under conditions simulating the gastric environment, survival was measured in a model stomach system (MSS) for two clonal groups of EHEC: 14 EHEC 1 strains of serotype O157:H7 and 12 EHEC 2 strains of serotypes O26:H11 and O111:H8. There were significant differences between the two EHEC groups, with the average survival rate of O157 strains in the MSS twice as great as the O26/O111 strains. Strains of the two groups also differed in the quantity of injured cells in MSS and in the transcript levels of the glutamate decarboxylase genes (measured by quantitative PCR) in stationary phase before cultures entered the MSS. CONCLUSIONS: The results indicate that E. coli O157:H7 strains have superior ability to survive simulated gastric acidity compared with the non-O157 EHEC. SIGNIFICANCE AND IMPACT OF THE STUDY: E. coli O157:H7 becomes acid resistant rapidly upon entry into stationary phase, which may underlie the low infectious dose of this pathogen.

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