Kevin J. Allen

Examination of Stress and Virulence Gene Expression in Escherichia coli O157:H7 Using Targeted Microarray Analysis

Escherichia coli O157:H7 poses a threat to humans through food- and water-borne transmission. To investigate how environmental stresses affect the Escherichia coli O157:H7 transcriptome, we designed a targeted microarray consisting of stress response and virulence genes (n = 125) to analyze the impact of acidified (pH 3.5), cold (7.5 degrees C), and fresh tryptic soy broth (TSB) (37 degrees C) on E. coli O157:H7 stress response and virulence gene expression. Nutrient replenishment with fresh TSB resulted in 72 differentially expressed genes (> or = 1.5-fold change; p < 0.05), with 65 induced. All queried global and specific stress regulators were affected, as were 12 virulence genes. Cold-shocked cells displayed 17 differentially expressed genes, with 10 being induced. Induction of rpoS, members of the sigma(H) regulon (clpB, dnaK, ftsH), and acid resistance (AR) genes (gadA, gadX) was observed. Porin transcript (ompC, ompF) and gapA and tufA ancillary genes were repressed. Acid shock resulted in 24 differentially expressed genes, with 21 induced. No induction of any stationary phase AR system was observed, though acid-coping mechanisms were recruited, including mar and phoB induction, and repression of ompC and ompF. Stress regulators were induced, including relA, soxS, rpoE, and rpoH. The microarray data were validated by quantitative real-time polymerase chain reaction. Exposure to sublethal stress events led to the induction of diverse stress response networks. In the food chain, sublethal events may render cells increasingly resistant to future stresses, potentially leading to increased survival.

Occurrence and characterization of Listeria spp. in ready-to-eat retail foods from Vancouver, British Columbia

The occurrence of Listeria spp. and Listeria monocytogenes in retail RTE meat and fish products in Vancouver, British Columbia (B.C.) was investigated. To assess potential consumer health risk, recovered L. monocytogenes isolates were subjected to genotypic and phenotypic characterization. Conventional methods were used to recover Listeria spp. from deli meat (n = 40) and fish (n = 40) samples collected from 17 stores. Listeria spp. were recovered only from fish samples (20%); 5% harboured Listeria innocua, 5% had L. monocytogenes and 10% contained Listeria welshimeri. L. monocytogenes isolates serotyped as 1/2a and 1/2b, possessed dissimilar PFGE patterns, and had full-length InlA. Three 1/2a clonal isolates encoded the 50 kb genomic island, LGI1. Antimicrobial resistance (AMR) profiling showed all Listeria spp. possessed resistance to cefoxitin and nalidixic acid. L. monocytogenes were resistant to clindamycin, two were resistant to streptomycin, and one to amikacin. Reduced susceptibility to ciprofloxacin was seen in all L. monocytogenes, L. innocua and three L. welshimeri isolates. Reduced susceptibility to amikacin and chloramphenicol was also observed in one L. monocytogenes and three L. welshimeri isolates, respectively. Recovery of L. monocytogenes in fish samples possessing AMR, full-length InlA, LGI1, and serotypes frequently associated with listeriosis suggest B.C. consumers are exposed to high-risk strains.

Use of luminescent Campylobacter jejuni ATCC 33291 to assess eggshell colonization and penetration in fresh and retail eggs.

A luminescent phenotype in Campylobacter jejuni ATCC 33291, generated by a transcriptional fusion between the C. jejuni flaA sigma28 promoter and the luxCDABE genes of Xenorhabdus luminescens on plasmid pRYluxCDABE, was used to examine colonization and penetration of fresh and retail eggs. C. jejuni colonized both fresh and retail eggs at 37, 40, and 42 degrees under microaerophilic conditions. Fresh eggs were more heavily colonized than retail eggs. Under aerobic conditions, fresh eggs were colonized at similar levels for all three temperatures. C. jejuni was found to penetrate the eggshell in 2 of 48 (4.2%) fresh eggs assessed. Although the lux+ phenotype did not provide an effective means of predicting penetration sites, it was effective at visualizing eggshell colonization. Also, it effectively demonstrated the organisms opportunistic nature, as eggshell surfaces with flaws and slight cracks were extensively colonized and easily detected by a photon counting charge-coupled device camera. Using scanning electron microscopy, C. jejuni ATCC 33291 was visualized on both the exterior and interior surfaces of the egg membranes indicating penetration of these barriers.

Microbiological survey of imported produce available at retail across Canada.

Increasing consumption and year-round consumer demand for fresh, minimally processed green vegetables have been observed in Canada and other developed countries. However, in the past two decades, produce has been increasingly implicated in outbreaks and correspondingly recognized as a vector for the transmission of pathogenic microorganisms. To this end, we examined the microbiological quality of imported produce available at retail across Canada during a period of limited domestic availability. In total, 106 samples obtained from five Canadian cities were purchased from retail outlets and subjected to microbiological analyses, including aerobic plate (APC) and coliform counts, and enrichments for enterococci, indicator Escherichia coli, E. coli O157:H7 and Salmonella spp. Also, recovered Enterococcus faecalis and Enterococcus faecium were screened for antimicrobial resistance (AMR). Overall, samples included herbs (n=61), leafy greens (n=25), and spinach (n=20) deriving from five countries (Columbia, Dominican Republic, Guatemala, Mexico, and the United States [US]). APCs were consistent across commodities regardless of country, ranging from mean log10 CFU/g of 6.1 to 7.4, with no significant differences observed. Excluding a single leafy green sample from Guatemala, the lowest prevalence of coliforms was for Mexican herbs (22.2%), with a high of 66.7% on US leafy greens. With the exception of spinach, concentrations of coliforms varied widely, ranging from undetectable to too numerous to count (>8.5 log10 CFU/g). Of the commodities assessed, Mexican and US spinach had the lowest coliform concentrations (undetectable to 4.0 log10 CFU/g). Organic herbs and conventional leafy greens possessed significantly lower (p<0.05) prevalence of coliforms compared to conventional herbs and organic leafy greens, respectively. The most frequent recovery of indicator E. coli was observed for herbs, with 11.1, 8.3, and 3.7% prevalence observed in samples from Columbia, US, and Mexico, respectively. For spinach, 0 and 6.7% of Mexican and US samples tested positive, while no leafy green samples from either country were positive. No E. coli O157:H7 or Salmonella spp. were detected. E. faecium and E. faecalis were recovered from 15.1 and 5.7% of samples, respectively. Although no glycopeptide resistance was observed, resistance to other clinically relevant antibiotics was noteworthy in both species. Overall, though microbiological quality indicators were frequently high, E. coli O157:H7 and Salmonella were not detected. However, the presence of resistance and reduced susceptibility to clinically relevant antimicrobials in recovered enterococci demonstrate imported fresh produce may serve as a vehicle for the transmission of antimicrobial resistance across national borders.

Examination of Food Chain-Derived Listeria monocytogenes Strains of Different Serotypes Reveals Considerable Diversity in inlA Genotypes, Mutability, and Adaptation to Cold Temperatures

ABSTRACT Listeria monocytogenes strains belonging to serotypes 1/2a and 4b are frequently linked to listeriosis. While inlA mutations leading to premature stop codons (PMSCs) and attenuated virulence are common in 1/2a, they are rare in serotype 4b. We observed PMSCs in 35% of L. monocytogenes isolates (n = 54) recovered from the British Columbia food supply, including serotypes 1/2a (30%), 1/2c (100%), and 3a (100%), and a 3-codon deletion (amino acid positions 738 to 740) seen in 57% of 4b isolates from fish-processing facilities. Caco-2 invasion assays showed that two isolates with the deletion were significantly more invasive than EGD-SmR (P < 0.0001) and were either as (FF19-1) or more (FE13-1) invasive than a clinical control strain (08-5578) (P = 0.006). To examine whether serotype 1/2a was more likely to acquire mutations than other serotypes, strains were plated on agar with rifampin, revealing 4b isolates to be significantly more mutable than 1/2a, 1/2c, and 3a serotypes (P = 0.0002). We also examined the ability of 33 strains to adapt to cold temperature following a downshift from 37�C to 4�C. Overall, three distinct cold-adapting groups (CAG) were observed: 46% were fast (<70 h), 39% were intermediate (70 to 200 h), and 15% were slow (>200 h) adaptors. Intermediate CAG strains (70%) more frequently possessed inlA PMSCs than did fast (20%) and slow (10%) CAGs; in contrast, 87% of fast adaptors lacked inlA PMSCs. In conclusion, we report food chain-derived 1/2a and 4b serotypes with a 3-codon deletion possessing invasive behavior and the novel association of inlA genotypes encoding a full-length InlA with fast cold-adaptation phenotypes.

Phenotypic and genotypic characterization of food animal isolates of Salmonella with reduced sensitivity to ciprofloxacin.

Reports of nontyphoidal Salmonella enterica subsp. enterica showing reduced sensitivity to ciprofloxacin (RSC) have increased rapidly during the past decade. Infection in humans with Salmonella possessing RSC may compromise the effectiveness of ciprofloxacin therapy. Nineteen among 4,357 Salmonella strains isolated from food animals in Canada from 1998 to 1999 showed RSC; 17 were from turkeys and 2 from chickens. All were resistant to nalidixic acid and sulfisoxazole and possessed RSC at a level of 0.125-0.5 microg/ml. PCR-RFLP of the gyrA quinolone resistance-determining region (QRDR) with Hinfl revealed that S. Bredeney and S. Heidelberg isolates possessed a mutation in this region. Single-strand conformational polymorphism (SSCP) analysis showed that S. Schwarzengrund and S. Senftenberg isolates also possessed a point mutation in the QRDR. DNA sequencing confirmed the findings and showed that all isolates possessed a base substitution in the gyrA QRDR. Sequencing revealed no mutations in the gyrB and silent wobble mutations in the parC QRDR. Reserpine, a known efflux pump inhibitor, did not effect the MICs for ciprofloxacin, nalidixic acid, and tetracycline. The mar operon could be induced in all isolates at 37 degrees C and in 18 of 19 at 30 degrees C; induction resulted in a two- to four-fold increase in the MIC of ciprofloxacin. In 14 of the 19 isolates, the mutation rate was two-fold or higher than in a ciprofloxacin sensitive S. Bredeney and S. Typhimurium LT2 control strain. Examination of clonal relatedness using pulsed-field gel electrophoresis (PFGE) and plasmid profiles indicated that some degree of clonal dispersion may have occurred, but the majority of isolates may have arisen from de novo mutations.

Modulation of Mucin mRNA (MUC5AC and MUC5B) Expression and Protein Production and Secretion in Caco-2/HT29-MTX Co-cultures Following Exposure to Individual and Combined Fusarium Mycotoxins

Intestinal epithelial cells (IECs) are a critical component of the innate local immune response. In order to reduce the risk of pathogen infection or xenobiotic intoxication, different host defense mechanisms have been evolved. Evidence has shown that upon ingestion of food or feed contaminated with toxins (e.g., mycotoxins), IECs respond by regulating mucin secretions, which act as a physical barrier inhibiting bacterial attachment and subsequent infection-related processes. However, the effect of Fusarium mycotoxins on mucin production remains unclear. Consequently, the aim of this study was to evaluate individual and interactive effects of four common Fusarium mycotoxins, deoxynivalenol, nivalenol, zearalenone, and fumonisins B1 on mRNA expression and secretion of mucins, MUC5AC, and MUC5B, as well as total mucin-like glycoprotein secretion, using Caco-2 (absorptive-type) and HT29-MTX (secretive-type) cells and their co-cultures (initial seeding ratios Caco-2/HT29-MTX: 90/10 and 70/30). Our results showed that individual and mixtures of mycotoxins significantly modulated MUC5AC and MUC5B mRNA and protein, and total mucin-like glycoprotein secretion as measured by quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and enzyme-linked lectin assay, respectively. Additive effects were not always observed for mixtures. Also, the present study showed that in co-cultures, lower MUC5AC and MUC5B mRNA, protein and total mucin production occurred following exposure, which might suggest higher intestinal permeability and susceptibility to toxin exposure. This study demonstrates the importance of selecting an appropriate cell model for the in vitro investigation of Fusarium mycotoxin effects either alone or in combinations on the immunological defense mechanisms of IECs, and will contribute to improved toxin risk assessments.

Modulation of Porcine β-Defensins 1 and 2 upon Individual and Combined Fusarium Toxin Exposure in a Swine Jejunal Epithelial Cell Line

ABSTRACT Defensins are small antimicrobial peptides (AMPs) that play an important role in the innate immune system of mammals. Since the effect of mycotoxin contamination of food and feed on the secretion of intestinal AMPs is poorly understood, the aim of this study was to elucidate the individual and combined effects of four common Fusarium toxins, deoxynivalenol (DON), nivalenol (NIV), zearalenone (ZEA), and fumonisin B1 (FB1), on the mRNA expression, protein secretion, and corresponding antimicrobial effects of porcine β-defensins 1 and 2 (pBD-1 and pBD-2) using a porcine jejunal epithelial cell line, IPEC-J2. In general, upregulation of pBD-1 and pBD-2 mRNA expression occurred following exposure to Fusarium toxins, individually and in mixtures (P < 0.05). However, no significant increase in secreted pBD-1 and pBD-2 protein levels was observed, as measured by enzyme-linked immunosorbent assay (ELISA). Supernatants from IPEC-J2 cells exposed to toxins, singly or in combination, however, possessed significantly less antimicrobial activity against Escherichia coli than untreated supernatants. When single toxins and two-toxin combinations were assessed, toxicity effects were shown to be nonadditive (including synergism, potentiation, and antagonism), suggesting interactive toxin effects when cells are exposed to mycotoxin combinations. The results show that Fusarium toxins, individually and in mixtures, activate distinct antimicrobial defense mechanisms possessing the potential to alter the intestinal microbiota through diminished antimicrobial effects. Moreover, by evaluating toxin mixtures, this improved understanding of toxin effects will enable more effective risk assessments for common mycotoxin combinations observed in contaminated food and feed.

Genotypes Associated with Listeria monocytogenes Isolates Displaying Impaired or Enhanced Tolerances to Cold, Salt, Acid, or Desiccation Stress

The human pathogen Listeria monocytogenes is a large concern in the food industry where its continuous detection in food products has caused a string of recalls in North America and Europe. Most recognized for its ability to grow in foods during refrigerated storage, L. monocytogenes can also tolerate several other food-related stresses with some strains possessing higher levels of tolerances than others. The objective of this study was to use a combination of phenotypic analyses and whole genome sequencing to elucidate potential relationships between L. monocytogenes genotypes and food-related stress tolerance phenotypes. To accomplish this, 166 L. monocytogenes isolates were sequenced and evaluated for their ability to grow in cold (4°C), salt (6% NaCl, 25°C), and acid (pH 5, 25°C) stress conditions as well as survive desiccation (33% RH, 20°C). The results revealed that the stress tolerance of L. monocytogenes is associated with serotype, clonal complex (CC), full length inlA profiles, and the presence of a plasmid which was identified in 55% of isolates. Isolates with full length inlA exhibited significantly (p < 0.001) enhanced cold tolerance relative to those harboring a premature stop codon (PMSC) in this gene. Similarly, isolates possessing a plasmid demonstrated significantly (p = 0.013) enhanced acid tolerance. We also identified nine new L. monocytogenes sequence types, a new inlA PMSC, and several connections between CCs and the presence/absence or variations of specific genetic elements. A whole genome single-nucleotide-variants phylogeny revealed sporadic distribution of tolerant isolates and closely related sensitive and tolerant isolates, highlighting that minor genetic differences can influence the stress tolerance of L. monocytogenes. Specifically, a number of cold and desiccation sensitive isolates contained PMSCs in σB regulator genes (rsbS, rsbU, rsbV). Collectively, the results suggest that knowing the sequence type of an isolate in addition to screening for the presence of full-length inlA and a plasmid, could help food processors and food agency investigators determine why certain isolates might be persisting in a food processing environment. Additionally, increased sequencing of L. monocytogenes isolates in combination with stress tolerance profiling, will enhance the ability to identify genetic elements associated with higher risk strains.

Targeted microarray analysis of stationary phase Escherichia coli O157:H7 subjected to disparate nutrient conditions

Aims:  To determine how stress response and virulence gene expression of stationary phase (SP) Escherichia coli O157:H7 are affected by nutrient levels.