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Foodborne illnesses, caused by harmful microorganisms in food, are a significant global health issue. Current methods for identifying these pathogens are both labor-intensive and time-consuming. In this research, we devised a swift and precise detection technique using recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) for three foodborne pathogens found in meat. By employing a dedicated detection device, RPA-LFD allows for the rapid analysis of DNA from Escherichia coli O157 (E. coli O157), Salmonella, and Shigella-pathogens that are prohibited in food. The detection thresholds for E. coli O157, Salmonella, and Shigella are 0.168 fg/μl (1.04 CFU/ml), 0.72 fg/μl (27.49 CFU/ml), and 1.25 fg/μl (48.84 CFU/ml), respectively. This method provides a short detection window, operates at low temperatures, follows simple procedures, and exhibits high sensitivity. Our study establishes the RPA-LFD method for simultaneously identifying the nucleic acid of three foodborne pathogens, offering an efficient solution for quickly identifying multiple contaminants., Competing Interests: Declaration of competing interest No conflict of interest exits in the submission of this manu, and manu is approved by all authors for publication. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. All the authors listed have approved the manu that is enclosed., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Romaine lettuce in the U.S. is primarily grown in California or Arizona and either processed near the growing regions (source processing) or transported long distance for processing in facilities serving distant markets (forward processing). Recurring outbreaks of Escherichia coli O157:H7 implicating romaine lettuce in recent years, which sometimes exhibited patterns of case clustering in Northeast and Midwest, have raised industry concerns over the potential impact of forward processing on romaine lettuce food safety and quality. In this study, freshly harvested romaine lettuce from a commercial field destined for both forward and source processing channels was tracked from farm to processing facility in two separate trials. Whole-head romaine lettuce and packaged fresh-cut products were collected from both forward and source facilities for microbiological and product quality analyses. High-throughput amplicon sequencing targeting16S rRNA gene was performed to describe shifts in lettuce microbiota. Total aerobic bacteria and coliform counts on whole-head lettuce and on fresh-cut lettuce at different storage times were significantly (p < 0.05) higher for those from the forward processing facility than those from the source processing facility. Microbiota on whole-head lettuce and on fresh-cut lettuce showed differential shifting after lettuce being subjected to source or forward processing, and after product storage. Consistent with the length of pre-processing delays between harvest and processing, the lettuce quality scores of source-processed romaine lettuce, especially at late stages of 2-week storage, was significantly higher than of forward-processed product (p < 0.05)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

Escherichia coli O157:H7 is a notorious foodborne pathogen known to cause severe illnesses such as hemolytic colitis and hemolytic uremic syndrome, with fresh produce consumption being implicated in recent outbreaks. The inappropriate use of antimicrobials to combat pathogens has led to the emergence and rapid dissemination of antimicrobial-resistant microorganisms including pathogenic E. coli, presenting a significant risk to humans. Here, we isolated two E. coli O157:H7 infecting bacteriophages, PECP14 and PECP20, from irrigation water and city sewage, respectively, as alternatives to antimicrobials. Both phages were stable for at least 16 h in a broad range of pH (pH 3-11) and temperature (4-40 °C) conditions and have a double-stranded DNA chromosome. PECP14 and PECP20, classified under the Epseptimavirus and Mosigvirus genera, respectively, exhibit specificity in targeting different host receptors, BtuB protein and lipopolysaccharide. Interestingly, these phages demonstrate the ability to infect not only E. coli O157:H7 but also other foodborne enteric pathogens like Shigella sonnei and S. flexneri. Upon mixing phages with their respective host bacteria, rapid adsorption (at least 68 % adsorption within 10 min) and substantial bacterial lysis were observed. The efficacy of phage treatment was further validated through the reduction of E. coli O157:H7 on radish sprouts. Moreover, purified endolysins, LysPECP14 and LysPECP20, derived from each phage exhibited remarkable bacteriolytic activity against E. coli O157:H7 cells pretreated with EDTA. In particular, the activity of LysPECP20 was also noticeable against Listeria monocytogenes and Bacillus cereus, suggesting its potential for broader antimicrobial applications in food industry. The combined results showed that the phages PECP14, PECP20, and their endolysins could be used for biological control of E. coli O157:H7 in various circumstances, from production, harvesting, and storage stages to processing and distribution steps of agricultural products., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

This study aimed to investigate the contamination of carcasses and slaughterhouse environment with Escherichia coli O157:H7 and non-O157 serogroups (O45:H2, O103:H2, O121:H19, O145:H28, O26:H11, O111:H8). For this purpose, a total of 150 samples (30 carcasses, 30 shredding units, 30 knives, 30 slaughterhouse waste water and 30 wall surfaces) were collected from 5 different slaughterhouses in Kayseri, Turkey. The conventional and molecular methods were performed in order to detect Escherichia coli and its serogroups. Of the 150 samples, 55 (36%) were found to be contaminated with E. coli. Among isolates, E. coli serogroup (O157:H7) were detected in 2 (11%) carcass and 2 (11%) wastewater samples. None of the E. coli isolates harbored tested genes (stx1, stx2, eaeA, and hylA). Effective infection control measures and antibiotic stewardship programs should be adopted to limit the spread of multidrug-resistant bacteria. It was also deduced that these isolates resistance to different antibiotics could be hazardous for public health., (Copyright © 2022. Published by Elsevier B.V.)

Fresh beef and meat products have been implicated in outbreaks of Shiga toxin-producing Escherichia coli (STEC) worldwide. This study investigated the prevalence of E. coli O157: H7 and non-O157 STEC serogroups in fresh beef in the open market and street vended meat products (n = 180) in Lagos metropolis, Nigeria. A combination of culture media and immunomagnetic separation followed by typing for associated virulence factors and serotypes was performed. Antimicrobial susceptibility testing was performed on the isolated STEC serotypes using the disk diffusion method. A total of 72 STEC serogroup isolates were detected from 61 out of 180 samples. The O157 STEC serotypes were detected in fresh beef, suya, minced meat and tsire with prevalence of 20.8% while non-O157 STEC serogroups were detected in all the samples. Molecular typing revealed 25% (n = 18) of the STEC serogroups showed presence of all the stx1, stx2, eaeA, fliCH7 and rfbEO157 virulence factors while 54.2% (n = 39) possessed a combination of two virulence genes. Multidrug resistance was discovered in 23.6% (n = 17) of the total STEC serogroups. Locally processed ready-to-eat meat products in Lagos metropolis, Nigeria harbour potentially pathogenic multi-drug resistant STEC serogroups that can constitute public health hazard., (Copyright © 2021. Published by Elsevier B.V.)

Shiga toxin-producing Escherichia coli (STEC) can cause severe human illness, which are frequently linked to the consumption of contaminated beef or dairy products. However, recent outbreaks associated with contaminated flour and undercooked dough in the United States and Canada, highlight the potential of plant based food as transmission routes for STEC. In Germany STEC has been isolated from flour, but no cases of illness have been linked to flour. In this study, we characterized 123 STEC strains isolated from flour and flour products collected between 2015 and 2019 across Germany. In addition to determination of serotype and Shiga toxin subtype, whole genome sequencing (WGS) was used for isolates collected in 2018 to determine phylogenetic relationships, sequence type (ST), and virulence-associated genes (VAGs). We found a high diversity of serotypes including those frequently associated with human illness and outbreaks, such as O157:H7 (stx2c/d, eae), O145:H28 (stx2a, eae), O146:H28 (stx2b), and O103:H2 (stx1a, eae). Serotypes O187:H28 (ST200, stx2g) and O154:H31 (ST1892, stx1d) were most prevalent, but are rarely linked to human cases. However, WGS analysis revealed that these strains, as well as, O156:H25 (ST300, stx1a) harbour high numbers of VAGs, including eae, nleB and est1a/sta1. Although STEC-contaminated flour products have yet not been epidemiologically linked to human clinical cases in Germany, this study revealed that flour can serve as a vector for STEC strains with a high pathogenic potential. Further investigation is needed to determine the sources of STEC contamination in flour and flour products particularly in regards to these rare serotypes., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Escherichia coli O157:H7 is an important food-borne pathogenic microorganism that has been used as a model organism for studying microbial inactivation effects and inactivation mechanism in various sterilization technologies. The objective of this study was to investigate the effects of high voltage short time ohmic- (HVST), low voltage long time ohmic- (LVLT), and water bath- (WB) heating on inactivation and proteome changes of E. coli O157:H7 cells at the same endpoint temperature of 72 °C, and to analyze whether a non-thermal death effect existed in ohmic heating. The inactivation effect of E. coli cells after HVST was comparable to WB, and the largest inactivation was observed after LVLT. There was lower intracellular protein content detected in LVLT and HVST samples than those of WB (P < 0.05). Quantitative proteomic profiles using capillary-HPLC-MS/MS technology identified 2626 proteins, among them, a total of 142 (62 up-regulated and 80 down-regulated), 129 (37 up-regulated and 92 down-regulated), and 61 (20 up-regulated and 41 down-regulated) differential proteins were obtained by comparisons of HVST vs. CT (control), LVLT vs. CT, and WB vs. CT samples, respectively, and revealing a strongest cell response to HVST followed by LVLT and WB. Compared with WB samples, more protein changes in HVST and LVLT samples were mainly attributed to the leakage of intracellular proteins due to the damage of cell membrane by current of ohmic heating. Bioinformatics analysis indicated that the differential proteins were mainly involved in transcription, translation, cell wall and membrane biogenesis, amino acid, carbohydrate, and lipid metabolism. KEGG enrichment analysis indicated that the ribosome, terpenoid backbone biosynthesis, glycerophospholipid metabolism, ABC transporters, and folate biosynthesis were significantly enriched. Overall, the application of both HVST and LVLT treatments had the potential to inactivate E. coli cells, especially HVST with a shorter heating time, and the results in this study presented an important step toward understanding the response of E. coli cells to ohmic heating on proteome level., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This study aimed to evaluate the presence of pathogens in, and the hygienic-sanitary quality of, commercialized foods of animal origin at the international border region of Rio Grande do Sul, Brazil. In total, 270 samples of raw and processed foods of animal origin were collected in Paso de los Libres, Argentina (n = 65 raw meat, n = 47 dairy products, n = 28 processed meat) and Rivera, Uruguay (n = 60 raw meat, n = 31 dairy products, n = 29 processed meat), or were seized by the Brazilian International Agricultural Surveillance System (Brazil-Argentina border) (n = 9 raw meat, n = 1 bush meat). The samples were subjected to the enumeration of aerobic mesophilic bacteria, enterobacteria, and coagulase-positive staphylococci, and were tested for Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7. The virulence genes for Salmonella spp. (hilA, invA, spvC, pefA, and sefA), L. monocytogenes (prs, inlA, inlC, and inlJ) and E. coli O157:H7 (uspA, eae, rfb O157 , fliC H7 , stx1, stx2, and hlyA) were investigated using PCR assays. Raw products showed higher counts of aerobic mesophiles and enterobacteria compared to processed products (P < 0.05). There were no significant differences in aerobic mesophile or in enterobacterial counts between identical products according to origin (Argentina vs. Uruguay, P > 0.05). Escherichia coli O157:H7 was not detected in any of the samples tested. Salmonella spp. was detected in six (8%) raw products from Argentina. Listeria monocytogenes was isolated from five (6.66%) raw products originating in Argentina and 20 (16.66%) raw products from Uruguay. All 52 E. coli isolates carried the uspA gene, but only one carried the eae gene. The rfb O157 , fliC H7 , stx1, stx2, and hlyA genes were not detected. All Salmonella spp. isolates carried hilA and invA genes, but spvC, pefA, and sefA were not found. All L. monocytogenes isolates carried the prs gene; however, inlA, inlC, and inlJ genes were found in 20% of the isolates from Argentina and 95% of those from Uruguay. To our knowledge, this is the first microbiological study into the hygienic-sanitary quality of animal products in Brazil's land border region. Salmonella spp. and L. monocytogenes were detected in products of animal origin, constituting a public health concern and emphasizing the need for an active surveillance system to reduce the risk of foodborne pathogen introduction into Brazil., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Seventeen clusters of Shiga toxin-producing Escherichia coli O157:H7/- (O157) strains, determined by cluster analysis of pulsed-field gel electrophoresis patterns, were analyzed using whole genome sequence (WGS) data to investigate this pathogen's molecular epidemiology. The 17 clusters included 136 strains containing strains from nine outbreaks, with each outbreak caused by a single source contaminated with the organism, as shown by epidemiological contact surveys. WGS data of these strains were used to identify single nucleotide polymorphisms (SNPs) by two methods: short read data were directly mapped to a reference genome (mapping derived SNPs) and common SNPs between the mapping derived SNPs and SNPs in assembled data of short read data (common SNPs). Among both SNPs, those that were detected in genes with a gap were excluded to remove ambiguous SNPs from further analysis. The effectiveness of both SNPs was investigated among all the concatenated SNPs that were detected (whole SNP set); SNPs were divided into three categories based on the genes in which they were located (i.e., backbone SNP set, O-island SNP set, and mobile element SNP set); and SNPs in non-coding regions (intergenic region SNP set). When SNPs from strains isolated from the nine single source derived outbreaks were analyzed using an unweighted pair group method with arithmetic mean tree (UPGMA) and a minimum spanning tree (MST), the maximum pair-wise distances of the backbone SNP set of the mapping derived SNPs were significantly smaller than those of the whole and intergenic region SNP set on both UPGMAs and MSTs. This significant difference was also observed when the backbone SNP set of the common SNPs were examined (Steel-Dwass test, P≤0.01). When the maximum pair-wise distances were compared between the mapping derived and common SNPs, significant differences were observed in those of the whole, mobile element, and intergenic region SNP set (Wilcoxon signed rank test, P≤0.01). When all the strains included in one complex on an MST or one cluster on a UPGMA were designated as the same genotype, the values of the Hunter-Gaston Discriminatory Power Index for the backbone SNP set of the mapping derived and common SNPs were higher than those of other SNP sets. In contrast, the mobile element SNP set could not robustly subdivide lineage I strains of tested O157 strains using both the mapping derived and common SNPs. These results suggested that the backbone SNP set were the most effective for analysis of WGS data for O157 in enabling an appropriation of its molecular epidemiology., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Despite continuing efforts to reduce foodborne pathogen contamination of fresh produce, significant outbreaks continue to occur. Identification of appropriate surrogates for foodborne pathogens facilitates relevant research to identify reservoirs and amplifiers of these contaminants in production and processing environments. Therefore, the objective of this study was to identify environmental Escherichia coli isolates from manures (poultry, swine and dairy) and surface water sources with properties similar to those of the produce associated foodborne pathogens E. coli O157:H7 and Salmonella enterica serotype Typhimurium. The most similar environmental E. coli isolates were from poultry (n=3) and surface water (n=1) sources. The best environmental E. coli surrogates had cell surface characteristics (zeta potential, hydrophobicity and exopolysaccharide composition) that were similar (i.e., within 15%) to those of S. Typhimurium and/or formed biofilms more often when grown in low nutrient media prepared from lettuce lysates (24%) than when grown on high nutrient broth (7%). The rate of attachment of environmental isolates to lettuce leaves was also similar to that of S. Typhimurium. In contrast, E. coli O157:H7, a commonly used E. coli quality control strain and swine isolates behaved similarly; all were in the lowest 10% of isolates for biofilm formation and leaf attachment. These data suggest that the environment may provide a valuable resource for selection of surrogates for foodborne pathogens., (Published by Elsevier B.V.)

Escherichia coli O157:H7, a major foodborne pathogen, is a major public health concern associated with life-threatening diseases such as hemolytic uremic syndrome. To alleviate this burden, a sensitive and rapid system is required to detect this pathogen in various kinds of foods. Herein, we propose a phage-based pathogen detection method to replace laborious and time-consuming conventional methods. We engineered an E. coli O157:H7-specific phage phiV10 to rapidly and sensitively detect this notorious pathogen. The luxCDABE operon was introduced into the phiV10 genome and allowed the engineered phage phiV10lux to generate bioluminescence proportional to the number of viable E. coli O157:H7 cells without any substrate addition. The phage phiV10lux was able to detect at least 1CFU/ml of E. coli O157:H7 in a pure culture within 40min after 5h of pre-incubation. In artificially contaminated romaine lettuce, apple juice (pH3.51), and ground beef, the reporter phage could detect approximately 10CFU/cm 2 , 13CFU/ml, and 17CFU/g of E. coli O157:H7, respectively. Taken together, the constructed reporter phage phiV10lux could be applied as a powerful tool for rapid and sensitive detection of live E. coli O157:H7 in foods., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Bacterial foodborne diseases remain major threats to food safety and public health, especially in developing countries. In this study a novel assay, combining gold nanoparticle (GNP)-based multiplex oligonucleotide ligation-PCR and universal oligonucleotide microarray technology, was developed for inexpensive, specific, sensitive, and multiplex detection of eight common foodborne pathogens, including Shigella spp., Campylobacter jejuni, Bacillus cereus, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica, Staphylococcus aureus, and Vibrio parahaemolyticus. The target fragments of the eight pathogens were enriched by multiplex PCR and subjected to multiplex ligase detection reaction. Ligation products were enriched and labeled with GNPs by universal asymmetric PCR, using excess GNP-conjugated primers. The labeled single-stranded amplicons containing complementary tag sequences were captured by the corresponding tag sequences immobilized on microarrays, followed by silver staining for signal enhancement. Black images of microarray spots were visualized by naked eyes or scanned on a simple flatbed scanner, and quantified. The results indicated that this assay could unambiguously discriminate all eight pathogens in single and multiple infections, with detection sensitivity of 3.3-85CFU/mL for pure cultures. Microarray results of ninety-five artificially contaminated and retail food samples were consistent with traditional culture, biochemical and real-time PCR findings. Therefore, the novel assay has the potential to be used for routine detection due to rapidity, low cost, and high specificity and sensitivity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Pathogenic Shiga toxin-producing E. coli (STEC) are recognized worldwide as environment and foodborne pathogens which can be transmitted by ingestion of ready-to-eat food such as raw milk-derived products. STEC show a prevalence rate in dairy products of 0.9%, yet comparably few outbreaks have been related to dairy products consumption. In this study, we used rt-qPCR to identify the virulence potential of O157, O26 and O103 STEC strains isolated from raw-milk dairy products by analyzing virulence-related gene frequencies and associations with O-island (OI) 44, OI-48, OI-50, OI-57, OI-71 and OI-122. Results showed that 100% of STEC strains investigated harbored genes associated with EHEC-related virulence profile patterns (eae and stx, with either espK, espV, ureD and/or Z2098). We also found similarities in virulence-related gene content between O157:H7 and O103:H2 dairy and non-dairy STEC strains, especially isolates from human cases. The O26:H11-serotype STEC strains investigated harbor the arcA-allele 2 gene associated with specific genetic markers. These profiles are associated with high-virulence seropathotype-A STEC. However, the low frequency of stx2 gene associated with absence of other virulence genes in dairy isolates of O26:H11 remains a promising avenue of investigation to estimate their real pathogenicity. All O26:H11 attaching-effacing E. coli (AEEC) strains carried CRISPR O26:H11 SP&#95;O26&#95;E but not genetic markers espK, espV, ureD and/or Z2098 associated with the emerging potentially high-virulence "new French clone". These strains are potentially as "EHEC-like" strains because they may acquire (or have lost) stx gene. In this study, O157:H7, O103:H2 and O26:H11 STEC strains isolated from dairy products were assigned as potential pathogens. However, research now needs to investigate the impact of dairy product environment and dairy processing on the expression of their pathogenicity., (Copyright © 2017. Published by Elsevier B.V.)

The objective of this study was to examine the effect of dry chilling on the genetic diversity of naturally occurring Escherichia coli on beef carcasses, and to examine whether two populations of E. coli recovered from carcasses during chilling and E. coli O157 differed in their response to desiccation. Isolates of E. coli were obtained from beef carcasses during a 67h dry chilling process and were genotyped using multiple-locus variable-number tandem-repeat analysis (MLVA). Ten E. coli genotypes found only at 0h (group A) and found more than once (group B), as well as five strains of E. coli O157 (group C) were inoculated on stainless steel coupons and their survival was examined after exposure to 75 and 100% relative humidity (RH) at 0 or 35°C for 67h. A total of 450 E. coli isolates were obtained, with 254, 49, 49, 51, 23, 20, and 4 from 0, 1, 2, 4, 6, 8 and 24h of chilling, respectively. No E. coli were recovered at 67h. MLVA of the isolates revealed 173 distinct genotypes. Genetic diversity of E. coli isolates, defined as ratio of the number of isolates to the number of genotypes, remained between 2.3 and 1.3 during the 24h of chilling. All strains inoculated on stainless steel coupons and exposed to 75% RH at 35°C were completely inactivated, irrespective of their groups. Inactivation of E. coli of the three groups was not significantly (P>0.05) different by exposure to 75% RH at 0°C. The findings indicate that the genetic diversity of E. coli on beef carcasses was not affected by dry chilling. In addition, inactivation of E. coli genotypes and E. coli O157 by desiccation on stainless steel simulating dry chilling conditions did not differ significantly (P>0.05). Thus, dry chilling may be used as an effective antimicrobial intervention for beef carcasses., (Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.)

Shiga toxin-producing Escherichia coli (STEC) are widely recognized as pathogens causing food borne disease. Here we evaluate the genetic diversity of 197 strains, mainly STEC, from serotypes O157:H7, O26:H11, O103:H2, O111:H8 and O145:28 and compared strains recovered in dairy products against strains from human, meat and environment cases. For this purpose, we characterized a set of reference-collection STEC isolates from dairy products by PFGE DNA fingerprinting and a subset of these by virulence-gene profiling. PFGE profiles of restricted STEC total DNA showed high genomic variability (0.9976 on Simpson's discriminatory index), enabling all dairy isolates to be differentiated. High-throughput real-time PCR screening of STEC virulence genes were applied on the O157:H7 and O26:H11 STEC isolates from dairy products and human cases. The virulence gene profiles of dairy and human STEC strains were similar. Nevertheless, frequency-wise, stx1 was more prevalent among dairy O26:H11 isolates than in human cases ones (87% vs. 44%) while stx2 was more prevalent among O26:H11 human isolates (23% vs. 81%). For O157:H7 isolates, stx1 (0% vs. 39%), nleF (40% vs 94%) and Z6065 (40% vs 100%) were more prevalent among human than dairy strains. Our data point to differences between human and dairy strains but these differences were not sufficient to associate PFGE and virulence gene profiles to a putative lower pathogenicity of dairy strains based on their lower incidence in disease. Further comparison of whole-genome expression and virulence gene profiles should be investigated in cheese and intestinal tract samples., (Copyright © 2016 Elsevier B.V. All rights reserved.)

To establish an efficient detection method for Shiga toxin (Stx)-producing Escherichia coli (STEC) O26, O103, O111, O121, O145, and O157 in food, an interlaboratory study using all the serogroups of detection targets was firstly conducted. We employed a series of tests including enrichment, real-time PCR assays, and concentration by immunomagnetic separation, followed by plating onto selective agar media (IMS-plating methods). This study was particularly focused on the efficiencies of real-time PCR assays in detecting stx and O-antigen genes of the six serogroups and of IMS-plating methods onto selective agar media including chromogenic agar. Ground beef and radish sprouts samples were inoculated with the six STEC serogroups either at 4-6CFU/25g (low levels) or at 22-29CFU/25g (high levels). The sensitivity of stx detection in ground beef at both levels of inoculation with all six STEC serogroups was 100%. The sensitivity of stx detection was also 100% in radish sprouts at high levels of inoculation with all six STEC serogroups, and 66.7%-91.7% at low levels of inoculation. The sensitivity of detection of O-antigen genes was 100% in both ground beef and radish sprouts at high inoculation levels, while at low inoculation levels, it was 95.8%-100% in ground beef and 66.7%-91.7% in radish sprouts. The sensitivity of detection with IMS-plating was either the same or lower than those of the real-time PCR assays targeting stx and O-antigen genes. The relationship between the results of IMS-plating methods and Ct values of real-time PCR assays were firstly analyzed in detail. Ct values in most samples that tested negative in the IMS-plating method were higher than the maximum Ct values in samples that tested positive in the IMS-plating method. This study indicates that all six STEC serogroups in food contaminated with more than 29CFU/25g were detected by real-time PCR assays targeting stx and O-antigen genes and IMS-plating onto selective agar media. Therefore, screening of stx and O-antigen genes followed by isolation of STECs by IMS-plating methods may be an efficient method to detect the six STEC serogroups., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This study rigorously evaluated a previously developed immunobead array method to simultaneously detect three important foodborne pathogens, Campylobacter jejuni, Listeria monocytogenes, and Salmonella spp., for its actual application in routine food testing. Due to the limitation of the detection limit of the developed method, an enrichment step was included in this study by using Campylobacter Enrichment Broth for C. jejuni and Universal Pre-enrichment Broth for L. monocytogenes and Salmonella spp.. The findings showed that the immunobead array method was capable of detecting as low as 1CFU of the pathogens spiked in the culture media after being cultured for 24h for all three pathogens. The immunobead array method was further evaluated for its pathogen detection capabilities in ready-to-eat (RTE) and ready-to-cook (RTC) chicken samples and proven to be able to detect as low as 1CFU of the pathogens spiked in the food samples after being cultured for 24h in the case of Salmonella spp., and L. monocytogenes and 48 h in the case of C. jejuni. The method was subsequently validated with three types of chicken products (RTE, n=30; RTC, n=20; raw chicken, n=20) and was found to give the same results as the conventional plating method. Our findings demonstrated that the previously developed immunobead array method could be used for actual food testing with minimal enrichment period of only 52 h, whereas the conventional ISO protocols for the same pathogens take 90-144 h. The immunobead array was therefore an inexpensive, rapid and simple method for the food testing., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The potential for using whole genome sequencing (WGS) data in microbiological risk assessment (MRA) has been discussed on several occasions since the beginning of this century. Still, the proposed heuristic approaches have never been applied in a practical framework. This is due to the non-trivial problem of mapping microbial information consisting of thousands of loci onto a probabilistic scale for risks. The paradigm change for MRA involves translation of multidimensional microbial genotypic information to much reduced (integrated) phenotypic information and onwards to a single measure of human risk (i.e. probability of illness). In this paper a first approach in methodology development is described for the application of WGS data in MRA; this is supported by a practical example. That is, combining genetic data (single nucleotide polymorphisms; SNPs) for Shiga toxin-producing Escherichia coli (STEC) O157 with phenotypic data (in vitro adherence to epithelial cells as a proxy for virulence) leads to hazard identification in a Genome Wide Association Study (GWAS). This application revealed practical implications when using SNP data for MRA. These can be summarized by considering the following main issues: optimum sample size for valid inference on population level, correction for population structure, quantification and calibration of results, reproducibility of the analysis, links with epidemiological data, anchoring and integration of results into a systems biology approach for the translation of molecular studies to human health risk. Future developments in genetic data analysis for MRA should aim at resolving the mapping problem of processing genetic sequences to come to a quantitative description of risk. The development of a clustering scheme focusing on biologically relevant information of the microbe involved would be a useful approach in molecular data reduction for risk assessment., (Copyright © 2015. Published by Elsevier B.V.)

This work examined Escherichia coli O157:H7 strain 02-0304 for putative genes responsible for sinigrin hydrolysis. Sinigrin is a glucosinolate present in Oriental mustard (Brassica juncea), and its hydrolysis is mediated in plants by the enzyme myrosinase. Sinigrin hydrolysis by plant or bacterial myrosinase yields allyl isothiocyanate (AITC) which is bactericidal. In silico analysis using public databases found sequence similarity between plant myrosinase and enzymes encoded by genes from β-glucosidase families in E. coli O157:H7. Specifically, 6-phospho-β-glucosidase encoded by the genes bglA and ascB (family 1), and chbF (family 4) present in E. coli O157:H7 showed the highest similarity. Polymerase chain reaction (PCR) confirmed the presence of bglA, ascB, and chbF in the clinical E. coli strain tested. Disruption of these genes in wild-type E. coli O157:H7 strain 02-0304 using lambda-red replacement created single and double mutants. The relative importance of each gene in the hydrolysis of sinigrin by E. coli O157:H7 was also assessed by comparing gene expression and sinigrin degradation rates among the E. coli O157:H7 wild-type strain and its mutants. The results suggested that the genes bglA and ascB play a substantial role in the degradation of sinigrin by E. coli O157:H7 strain 02-0304., (Copyright © 2015. Published by Elsevier B.V.)

Shiga toxin-producing Escherichia coli (STEC) O157:H7 is an important food-borne pathogen that has been implicated in numerous disease outbreaks worldwide. Little is known about the extent and molecular basis of antimicrobial resistance in STEC O157:H7 of food origin. Therefore, the current study aimed to characterize the genetic basis of multidrug resistance in 54 STEC O157:H7 strains isolated from 1600 food samples (800 meat products and 800 dairy products) collected from different street venders, butchers, retail markets, and slaughterhouses in Egypt. Thirty-one of 54 (57.4%) isolates showed multidrug resistance phenotypes to at least three classes of antimicrobials. The highest incidence of antimicrobial resistance was to kanamycin (96.8%), followed by spectinomycin (93.6%), ampicillin (90.3%), streptomycin (87.1%), and tetracycline (80.6%). PCR and DNA sequencing were used to screen and characterize integrons and antibiotic resistance genes, and 29.6% and 5.6% of isolates were positive for class 1 and class 2 integrons, respectively. β-Lactamase-encoding genes were identified in 63.0% of isolates as follows: blaTEM₋₁ and blaTEM₋₅₂ in 35.2% and 1.9% isolates respectively; blaCMY₋₂ in 13.0% isolates; blaCTX-M in 5.6% isolates; blaSHV₋₁₂ in 5.6% isolates; and blaOXA₋₁ in 1.9% isolate. The plasmid-mediated quinolone resistance genes were identified in 13.0% of isolates as follows: qnrB, qnrS, and aac(6')-Ib-cr in 5.6%, 3.7%, and 3.7% isolates, respectively. Finally, the florfenicol resistance gene floR was identified in 7.4% of isolates. This study demonstrated that meat and dairy products are potential sources of multidrug resistant STEC O157:H7. To our knowledge, this is the first report of the occurrence of class 2 integrons, qnrB, qnrS, and aac(6')-Ib-cr in STEC O157:H7., (Copyright © 2014. Published by Elsevier B.V.)

Enterohemorrhagic Escherichia coli (EHEC) are anthropozoonotic agents that range third among food-borne pathogens respective to their incidence and dangerousness in the European Union. EHEC are Shiga-toxin producing E. coli (STEC) responsible for foodborne poisoning mainly incriminated to the consumption of contaminated beef meat. Among the hundreds of STEC serotypes identified, EHEC mainly belong to O157:H7 but non-O157 can represent 20 to 70% of EHEC infections per year. Seven of those serogroups are especially of high-risk for human health, i.e. O26, O45, O103, O111, O121, O145 and O104. While meat can be contaminated all along the food processing chain, EHEC contamination essentially occurs at the dehiding stage of slaughtering. Investigating bacterial colonization to the skeletal-muscle extracellular matrix (ECM) proteins, it appeared that environmental factors influenced specific and non-specific bacterial adhesion of O157 and non-O157 EHEC as well as biofilm formation. Importantly, mechanical treatment (i.e. shaking, centrifugation, pipetting and vortexing) inhibited and biased the results of bacterial adhesion assay. Besides stressing the importance of the protocol to investigate bacterial adhesion to ECM proteins, this study demonstrated that the colonization abilities to ECM proteins vary among EHEC serogroups and should ultimately be taken into consideration to evaluate the risk of contamination for different types of food matrices., (Copyright © 2014. Published by Elsevier B.V.)

Quantitative real-time polymerase chain reaction (qPCR) can be a convenient alternative to the Most Probable Number (MPN) methods to count VTEC in milk. The number of VTEC is normally very low in milk; therefore with the aim of increasing the method sensitivity a qPCR protocol that relies on preliminary enrichment was developed. The growth pattern of six VTEC strains (serogroups O157 and O26) was studied using enrichment in Buffered Peptone Water (BPW) with or without acriflavine for 4-24h. Milk samples were inoculated with these strains over a five Log concentration range between 0.24-0.50 and 4.24-4.50 Log CFU/ml. DNA was extracted from the enriched samples in duplicate and each extract was analysed in duplicate by qPCR using pairs of primers specific for the serogroups O157 and O26. When samples were pre-enriched in BPW at 37°C for 8h, the relationship between threshold cycles (CT values) and VTEC Log numbers was linear over a five Log concentration range. The regression of PCR threshold cycle numbers on VTEC Log CFU/ml had a slope coefficient equal to -3.10 (R(2)=0.96) which is indicative of a 10-fold difference of the gene copy numbers between samples (with a 100 ± 10% PCR efficiency). The same 10-fold proportion used for inoculating the milk samples with VTEC was observed, therefore, also in the enriched samples at 8h. A comparison of the CT values of milk samples and controls revealed that the strains inoculated in milk grew with 3 Log increments in the 8h enrichment period. Regression lines that fitted the qPCR and MPN data revealed that the error of the qPCR estimates is lower than the error of the estimated MPN (r=0.982, R(2)=0.965 vs. r=0.967, R(2)=0.935). The growth rates of VTEC strains isolated from milk should be comparatively assessed before qPCR estimates based on the regression model are considered valid. Comparative assessment of the growth rates can be done using spectrophotometric measurements of standardized cultures of isolates and reference strains cultured in BPW at 37°C for 8h. The method developed for the serogroups O157 and O26 can be easily adapted to the other VTEC serogroups that are relevant for human health. The qPCR method is less laborious and faster than the standard MPN method and has been shown to be a good technique for quantifying VTEC in milk., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The prevalence and seasonal distribution of E. coli O157:H7(+)/H7(-) in an array of aged cattle at slaughter and its dissemination with slaughterhouse wastewater over a two year period in Turkey were investigated. For this purpose, a total of 720 samples (240 rectoanal mucosal swap [RAMS], 240 carcass sponge and 240 bile samples) of 240 cattle categorized according to age, gender, breed and sampling site were collected along with additional 24 wastewater samples and were subjected to immunomagnetic separation based cultivation technique to efficiently isolate E. coli O157 from the background flora. Identification (rfbEO157, fliCh7), detection of major virulence factors (stx1, stx2, eaeA, hly, lpfA1-3 and espA), intimin variants (eae-α1, eae-α2, eae-β, eae-β1, eae-β2, eae-γ1 and eae-γ2/θ) and shiga toxin variants (stx1c, stx1d, stx2c, stx2d, stx2e, stx2f and stx2g) of all the isolates were assessed by PCR. From 10 (4.2%) of RAMS and 11 (4.6%) of carcass sponge samples and 5 (20.8%) of slaughterhouse wastewater samples, a total of 102 colonies (99 sorbitol negative and 3 sorbitol positive) were isolated. Overall, 17 (7.1%) and 15 (6.3%) of 240 sampled cattle were shown to harbor E. coli O157 and E. coli O157:H7, respectively either in their RAMS or carcass sponge samples analyzed. Statistically significant differences between categories; season, age, gender and breed of cattle were not observed (p>0.05). None of the isolated E. coli O157:H7(+)/H7(-) strains harbored any of the investigated intimin types other than eaeγ1 or shiga toxin variants stx1d, stx2e, stx2f or stx2g while all were lpfA1-3(+) except 5 E. coli O157:H7(-) strains. Intimin variant eaeγ1 and shiga toxin 1 variant stx1c were detected from all of the eaeA(+) (97/102, 95.1%) and stx1(+) (32/102, 31.3%) strains, respectively while from stx2(+) (80/102, 78.4%) isolates, both stx2c (68/80, 85.0%) and stx2d (12/80, 15.0%) variants were determined. In the last decade, prevalence of E. coli O157:H7 has an increasing trend in cattle. Slaughterhouses are the significant sources of environmental contamination with E. coli O157:H7. Isolation and molecular characterization of sorbitol fermenting E. coli O157:H7 are a novel finding and may lead to a revision of reference isolation procedure of E. coli O157:H7 in future., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Infection by enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a worldwide problem, and there is no effective therapy. Biofilm formation is closely related to EHEC infection and is also a mechanism of antimicrobial resistance. Antibiofilm screening of 560 purified phytochemicals against EHEC showed that ginkgolic acids C15:1 and C17:1 at 5μg/ml and Ginkgo biloba extract at 100μg/ml significantly inhibited EHEC biofilm formation on the surfaces of polystyrene and glass, and on nylon membranes. Importantly, at their working concentrations, ginkgolic acids and G. biloba extract did not affect bacterial growth. Transcriptional analyses showed that ginkgolic acid C15:1 repressed curli genes and prophage genes in EHEC, and these findings were in-line with reduced fimbriae production and biofilm reductions. Interestingly, ginkgolic acids and G. biloba extract did not inhibit the biofilm formation of a commensal E. coli K-12 strain. In addition, ginkgolic acids and G. biloba extract inhibited the biofilm formation of three Staphylococcus aureus strains. The findings of this study suggest that plant secondary metabolites represent an important resource for biofilm inhibitors., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Contamination of fresh produce could represent a public health concern because no terminal kill step is applied during harvest or at the processing facility to kill pathogens. In addition, once contaminated, pathogens may internalize into produce and be protected from disinfectants during the postharvest processing step. The objective of the current study was to determine the potential internalization of Escherichia coli O157:H7 into spinach roots and subsequent transfer to the edible parts. Because curli are involved in biofilm formation, we investigated whether their presence influence the internalization of E. coli O157:H7 into spinach. Further, the effect of the spinach cultivar on E. coli O157:H7 internalization was evaluated. Spinach plants were grown in contaminated soil as well as hydroponically to prevent mechanical wounding of the roots and inadvertent transfer of pathogens from the contamination source to the non-exposed plant surfaces. Results showed that E. coli O157:H7 could internalize into hydroponically grown intact spinach plants through the root system and move to the stem and leaf level. The incidence of internalization was significantly higher in hydroponically grown plants when roots were exposed to 7 log CFU/mL compared to those exposed to 5 log CFU/mL. The effect of cultivar on E. coli O157:H7 internalization was not significant (P>0.05) for the analyzed spinach varieties, internalization incidences showing almost equal distribution between Space and Waitiki, 49.06% and 50.94% respectively. Wounding of the root system in hydroponically grown spinach increased the incidence of E. coli O157:H7 internalization and translocation to the edible portions of the plant. Experimental contamination of the plants grown in soil resulted in a greater number of internalization events then in those grown hydroponically, suggesting that E. coli O157:H7 internalization is dependent on root damage, which is more likely to occur when plants are grown in soil. Curli expression by E. coli O157:H7 had no significant effect on its root uptake by spinach plants., (Published by Elsevier B.V.)

Escherichia coli O157:H7 associated with food has caused many serious public health problems in recent years. However, only viable cells of this pathogen can cause infections, and false-positive detection caused by dead cells can lead to unnecessary product recalls. The objective of this study was to develop and optimize a method that combines propidium monoazide (PMA) staining with real-time PCR to detect only viable cells of E. coli O157:H7 in ground beef. PMA is a DNA intercalating dye that can penetrate compromised membranes of dead cells and bind to cellular DNA, preventing its amplification via a subsequent PCR. Three strains of E. coli O157:H7 (505B, G5310 and C7927) at concentrations of 10(0) to 10(8)CFU/mL were used as live cells. Dead cells were obtained by heating cell suspensions at 85°C for 15 min. Suspensions were treated with PMA and the optimized assay was applied to artificially contaminated ground beef with two different fat contents (10% and 27%). DNA was extracted and amplified by TaqMan® real-time PCR assay targeting the uidA gene for detection of E. coli O157:H7. Plasmid pUC19 was added as an internal amplification control (IAC). A treatment of 25 μM PMA with a 10-min light exposure on ice was sufficient to eliminate DNA from 10(8) dead E. coli O157:H7 cells/mL. The optimized assay could detect as low as 10(2) CFU/mL viable E. coli O157:H7 in pure culture and 10(5) CFU/g in ground beef, in the presence of 10(6)/mL or g of dead cells. With an 8-h enrichment, 1 CFU/g viable E. coli O157:H7 in ground beef was detectable without interference from 10(6) dead cells/g. In conclusion, the PMA real-time PCR could effectively detect viable E. coli O157:H7 without being compromised by dead cells., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Foodborne pathogens are a major threat to food safety, especially in developing countries where hygiene and sanitation facilities are often poor. Salmonella enterica, Escherichia coli O157:H7 and Shigella spp. are among the major causes of outbreaks of foodborne diseases. This large-scale study investigated the prevalence of these foodborne pathogens in meat (beef and chicken) and dairy products collected from street vendors, butchers, retail markets and slaughterhouses in Egypt. A total of 1600 food samples (800 meat products and 800 dairy products) were analyzed using culture and PCR based methods. S. enterica, E. coli O157:H7 and Shigella spp. were detected in 69 (4.3%), 54 (3.4%) and 27 (1.7%) samples respectively. S. enterica serovar Typhimurium, S. enterica serovar Enteritidis, S. enterica serovar Infantis and non-typable serovars were detected in 28 (1.8%), 22 (1.4%), 16 (1.0%) and 3 (0.1%) samples respectively. All E. coli O157:H7 isolates were positive for stx1 and/or stx2 virulence toxin genes. Shigella flexneri, Shigella sonnei and Shigella dysenteriae were detected in 18 (1.2%), 7 (0.4%) and 2 (0.1%) samples respectively. The incidences of S. enterica and Shigella spp. were higher in meat products (53; 6.6% and 16; 2.0%, respectively) than in dairy products (16; 2.0% and 11; 1.4%, respectively), while, E. coli O157:H7 was higher in dairy products (29; 3.6%) than in meat products (25; 3.1%). The incidence of foodborne pathogens in meat and dairy products was determined in a large-scale survey in Africa., (© 2013.)

Application of high hydrostatic pressure (HHP) constitutes a valuable non-thermal pasteurization process in modern food conservation. Triggered by our interest in the rapid adaptive evolution towards HHP resistance in the food-borne pathogen E. coli O157:H7 (strain ATCC 43888) that was demonstrated earlier, we used genetic screening to identify specific loci in which a loss-of-function mutation would be sufficient to markedly increase HHP survival. As such, individual loss of RssB (anti RpoS-factor), CRP (catabolite response protein) and CyaA (adenylate cyclase) were each found to confer significant HHP resistance in the 300MPa range (i.e. >1,000-fold), and this phenotype invariably coincided with increased resistance against heat as well. In contrast to loss of RssB, however, loss of CRP or CyaA also conferred significantly increased resistance to 600MPa (i.e. >10,000-fold), suggesting cAMP/CRP homeostasis to affect extreme HHP resistance independently of increased RpoS activity. Surprisingly, none of the rapidly emerging HHP-resistant mutants of ATCC 43888 that were isolated previously did incur any mutations in rssB, crp or cyaA, indicating that a number of other loci can guide the rapid emergence of HHP resistance in E. coli O157:H7 as well. The inability of spontaneous rssB, crp or cyaA mutants to emerge during selective enrichment under HHP selection likely stems from their decreased competitive fitness during growth. Overall, this study is the first to shed light on the possible genetic strategies supporting the acquisition of HHP resistance in E. coli O157:H7., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Sorbitol-fermenting (SF) Escherichia coli O157:H- strains have emerged as important pathogens and have been associated with a higher incidence of progression to hemolytic-uremic syndrome (HUS) than non-sorbitol fermenting (NSF) E. coli O157:H7. The present study was carried out to determine the prevalence of SF E. coli O157:H- and NSF E. coli O157:H7 strains in retail beef products in Mansoura, Egypt. The contamination rates with rfbEO157-positive E. coli O157 strains were 26.7% (8/30), 10% (3/30) and 3.7% (1/27) in ground beef, beef burger, and fresh beef samples, respectively with an overall mean of 13.8% (12/87) among all meat products tested. SF E. coli O157:H- were the most dominant among the isolated O157 strains. Of the fifteen O157 strains isolated, 11 (73.3%) were SF E. coli O157:H-, while the remaining 4 (26.7%) were NSF E. coli O157:H7. The 11 SF O157H- strains were genetically positive for sfpA gene. Restriction fragment length polymorphism (RFLP) analysis for fliC gene demonstrated a similar pattern for both SF and NSF O157 isolates. PCR assays verified the existence of stx1 gene in 7 (46.7%) and stx2 gene in 13 (86.7%) of the 15 O157 strains isolated. Unexpectedly, two of the 15 O157 strains isolated were negative for Shiga toxin genes. The eae gene was identified in all of the 15 O157 strains except in one NSF O157:H7 strain. EHEC-hlyA gene was detected in 14 (93.3%) of the 15 O157 isolates, nonetheless only 11 strains showed enterohemolytic phenotype on blood agar. A combination of the four virulence genes, stx1, stx2, eae and EHEC-hlyA were detected in 7 (46.7%) strains, while six (40%) strains were positive for stx2, eae and hlyA genes. This is the first record for isolation of E. coli O157: H- in Egypt as well as in the African continent., (Copyright © 2013. Published by Elsevier B.V.)

A wide variety of qPCR methods currently exist for Salmonella spp., Escherichia coli O157 and Listeria monocytogenes detection. These methods target several genes and use different detection chemistries, either in simplex or in multiplex formats. However, the majority of these methods have not been carefully validated, and the number of validated methods that use multiplex qPCR is even lower. The aim of the present study was to develop and validate a multiplex qPCR method from previously validated simplex qPCR primers and probes. A modified broth medium was selected and primary and secondary enrichment times were further optimized. Efficiency of the newly combined qPCR system was comprised between 91% and 108%, for simplex and multiplex analyses. A total of 152 food and environmental, natural and spiked samples, were analyzed for the evaluation of the method obtaining values above 91% that were reached for all the quality parameters analyzed. A very low limit of detection (5 cfu/25 g after enrichment) for simultaneous identification of these 3 pathogens was obtained., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Exposure to high hydrostatic pressure (HHP) is increasingly being used in food preservation as a non-thermal pasteurization process, and its further implementation necessitates a more thorough understanding of bacterial resistance development and intraspecies variability with regard to inactivation by HHP. In this report, we discovered that exposure to high hydrostatic pressure stress can rapidly select for strongly increased RpoS activity in a hypersensitive Escherichia coli O157:H7 strain (ATCC 43888), leading to a simultaneous increase in HHP and heat resistance. Moreover, the level of RpoS activity correlated well with the original hypersensitivity and the extent of acquired HHP resistance, and extremely HHP-resistant mutants of ATCC 43888 clearly incurred a number of additional RpoS-dependent phenotypes. These findings suggest that implementation of novel processing techniques in the food production chain can readily affect the physiology of food-borne pathogens., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This study assessed the resistance of ten verocytotoxigenic Escherichia coli (VTEC) isolates of commonly encountered serogroups/-types and two non-pathogenic E. coli strains to various food-related stresses (acid, alkaline, heat and high hydrostatic pressure treatments) and their biofilm formation ability. In addition, the global changes in the cellular composition in response to the exposure to these adverse environments were monitored by Fourier Transform Infrared (FT-IR) spectroscopy for two of the strains. Large inter-strain variations in stress resistance were observed. The most tolerant strains belonged to serogroup O157 which included both the O157:H7 type strain EDL933 and a representative isolate of the sorbitol fermenting O157:H- VTEC clone (strain MF3582). Strain C-600, a non-pathogenic laboratory strain, was sensitive to multiple stresses. Although wide variation in biofilm-forming ability was observed among VTEC isolates, no consistent relationships between biofilm-forming ability and capacity to withstand stress exposures were found. Analysis of the allelic status of the rpoS gene, involved in the general stress response of stationary-phase cells, allowed detection of loss-of-function mutations for two strains, E218/02 and MF2411, both of them showing as common features a high sensitivity to alkaline and heat treatments and a poor ability to form mature biofilms. Evidences found in this study confirm rpoS as a highly mutable gene in nature, and suggest its relevance not only for the mount of an active stress response but also for the establishment of mature biofilm communities. Our findings contribute to increase the knowledge on the resistance of VTEC to environmental stresses commonly encountered in the food chain, which can lead to improved strategies for preventing VTEC infections., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Escherichia coli (E. coli) O157:H7 remains a major food safety concern associated with meat, especially beef products. Shiga toxins (Stx) are key virulence factors produced by E. coli O157:H7 that are responsible for hemorrhagic colitis and Hemolytic Uremic Syndrome. Stx are heat stable and can be absorbed after oral ingestion. Despite the extensive study of E. coli O157:H7 survival during meat processing, little attention is paid to the production of Stx during meat processing. The objective of this study was to elucidate the effect of salt, an essential additive to processed meat, at concentrations relevant to meat processing (0%, 1%, 2%, 3%, W/V) on Stx2 production and Stx2 prophage induction by E. coli O157:H7 strains. For both E. coli O157:H7 86-24 and EDL933 strains, including 2% salt in LB broth decreased (P<0.05) E. coli O157:H7 population, but increased (P<0.05) Stx2 production (as measured relative to Log(10)CFU) compared to that of the control (1% salt). Supplementing 3% salt decreased (P<0.05) both E. coli O157:H7 number and Stx2 production. Quantitative RT-PCR indicated that stx2 mRNA expression in culture media containing 2% salt was greatly increased (P<0.05) compared to other salt concentrations. Consistent with enhanced Stx2 production and stx2 expression, the 2% salt group had highest lambdoid phage titer and stx2 prophage induction among all salt treatments. RecA is a key mediator of bacterial response to stress, which mediates prophage activation. Quantitative RT-PCR further indicated that recA mRNA expression was higher in both 2% and 3% salt than that of 0% and 1% salt treatments, indicating that stress was involved in enhanced Stx2 production. In conclusion, salt at the concentration used for meat processing enhances Stx production, a process linked to bacterial stress response and lambdoid prophage induction., (Published by Elsevier B.V.)

The use of propidium monoazide (PMA) is enjoying increased popularity among researchers in different fields of microbiology. Its use in combination with real-time PCR (qPCR) represents one of the most successful approaches to detect viable cells. PMA-qPCR has successfully been used to evaluate the efficacy of various disinfection technologies in different microorganisms. Initially, in this study the effect of four essential oils (EOs), cumin, clove, oregano and cinnamon, was evaluated on suspensions of the enterohemorrhagic Escherichia coli O157:H7 by PMA-qPCR, LIVE/DEAD BacLight flow cytometry analysis (LIVE/DEAD-FCM), and plate count. E. coli O157:H7 cells treated with EOs at killing concentrations were permeable to PMA which was confirmed by LIVE/DEAD-FCM. However, the PMA-qPCR assay allows specific quantification among the autochthonous microbiota of food products. Therefore, the PMA-qPCR assay was used to evaluate its applicability in artificially contaminated iceberg lettuce and soya sprouts. Amplification signal was negative for the spiking tests performed with any of the EO-killed E. coli cells. It demonstrates that the PMA-qPCR assay is a suitable technique for monitoring E. coli O157:H7 inactivation by essential oils in fresh-cut vegetables., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A mid-log phase broth culture of Escherichia (E.) coli O157:H7 381 (final concentration 10(4) cfu/mL) was monitored by conventional liquid- and solid-based enumeration techniques combined with PCR while it was subjected to thermal stress in gradually more complex systems (i.e., Tryptone Soya Broth, pasteurized milk and during lab-scale productions of a pasta filata fior di latte cheese obtained from raw or pasteurized milk). Our results highlighted: i) the incapability of the selective medium, ii) the effectiveness of the thin agar layer-PCR method, and iii) the effectiveness of the most probable number (MPN)-PCR method (in comparison with both plating-based methods) in recovering and selectively counting viable and stressed or injured E. coli O157:H7. Moreover, MPN-PCR was superior to both plating-based methods in terms of speed and easiness to get results. The thermal stresses herein applied (heating at 55 °C for 5 and 8 min) were less effective on the pasteurized milk than on the Tryptone Soya Broth and the pathogen was more protected in the raw milk-based matrices than in the pasteurized ones. Moreover, given the contamination level (10(4) cfu/mL of milk) of the strain, the temperature/time of stretching and the hardening and brining conditions herein used, the complete inactivation of the pathogen is not achievable., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The results of this study demonstrate the occurrence of the non-spore-forming pathogens, Staphylococcus aureus, Escherichia coli (total count and O157:H7), Listeria, Campylobacter and Salmonella, in New Zealand's raw milk supply. Samples of raw milk were collected monthly within five major dairying regions over one year. Each month, samples from five randomly selected farm vats in each region were collected for analysis (297 samples in total). Methods based on plate count techniques were used to enumerate S. aureus and E. coli. Enrichment methods in combination with a modified most probable number detection method were used to monitor samples for the presence of E. coli O157:H7, Listeria, Campylobacter and Salmonella. Salmonella was not detected in this study, and Campylobacter was isolated once (0.34%). E. coli was present at <100 cfu/ml in 99% of samples and exceeded 10(3)cfu/ml in 0.7% of samples. E. coli O157:H7 was not detected whereas non-pathogenic E. coli O157 strains (i.e. lacking genes for stx1, stx2, eae and Hly A) were detected in 1% of samples. S. aureus was not detected (<1 cfu/ml) in 21% of samples; levels were >1 but <100 cfu/ml in 60% of samples and on one occasion (0.34%) S. aureus exceeded 10(4)cfu/ml. L. monocytogenes was isolated from 0.68% of samples and L. innocua was present in 4% of samples. The results demonstrate that raw milk sampled from farm vats in New Zealand, as in other countries, inevitably contains recognised pathogens and, hence, control by pasteurisation or an equivalent treatment of raw milk remains paramount. Even so, the prevalence of most of these pathogens was lower than those reported in many of the studies performed in other countries., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The purpose of this study was to investigate carriage and transfer of verocytotoxigenic Escherichia coli (VTEC) O157, O26, O111, O103 and O145 from faeces and hide to dressed carcasses of Irish cattle as well as establishing the virulence potential of VTEC carried by these cattle. Individual cattle was tracked and faecal samples, hide and carcass (pre-evisceration and post-wash) swabs were analysed for verotoxin (vt1 and vt2) genes using a duplex real-time PCR assay. Positive samples were screened for the five serogroups of interest by real-time PCR. Isolates were recovered from PCR positive samples using immunomagnetic separation and confirmed by latex agglutination and PCR. Isolates were subject to a virulence screen (vt1, vt2, eaeA and hlyA) by PCR. Isolates carrying vt genes were examined by Pulsed-Field Gel Electrophoresis (PFGE). Of the VTEC isolated, E. coli O157 was the most frequently recovered from hide (17.6%), faeces (2.3%) and pre-evisceration/post-wash carcass (0.7%) samples. VTEC O26 was isolated from 0.2% of hide swabs and 1.5% of faeces samples. VTEC O145 was isolated from 0.7% of faeces samples. VTEC O26 and VTEC O145 were not recovered from carcass swabs. Non-VTEC O103 was recovered from all sample types (27.1% hide, 8.5% faeces, 5.5% pre-evisceration carcass, 2.2% post-wash carcass), with 0.2% of hide swabs and 1.0% of faeces samples found to be positive for VTEC O103 isolates. E. coli O111 was not detected in any samples. For the four serogroups recovered, the direct transfer from hide to carcass was not observed. This study shows that while VTEC O157 are being carried by cattle presented for slaughter in Ireland, a number of other verotoxin producing strains are beginning to emerge., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In the environment, bacteriophages are regarded as natural vector for the transmission of Shiga-toxin genes among Shiga-toxin Escherichia coli strains. The possibility of transduction has been noticed in intestinal tract of various animals but experimental observations on this phenomenon in food processes are lacking. To investigate the transduction in milk at different temperature profiles and cell concentrations, an experimental plan including two different Stx(2)-phages (ϕGV2412 and ϕL34), induced respectively from E. coli O157:H7 181181/2 and E. coli O157:H7 EC34, and two recipient E. coli strains (CNCTC 6896, WG5) was performed. The donor strains were generated by lysogenization of CNCTC 6896 with ϕGV2412 and ϕL34 respectively. Spectinomycin resistance gene (aadA) was inserted into stx(2) operon in order to select transduced cells. Transductants were never observed at 4°C up to 24 h, whereas after a treatment at 37°C for 2 h and at 25°C for 22 h they were detected in 67% of the trials with a ratio of transduction varying from 1.13 10(-6) to 7.87 10(-8). A treatment at 48°C for 2 h followed by a second step at 25°C for 22 h showed an occurrence of transduction events in only 19% of cases with a ratio of transduction varying from 2.22 10(-7) to 2.67 10(-8). The generation of transductants and the spontaneous induction of phages in milk were not affected by initial or final concentration of the donor or recipient strains. The results show that transduction phenomenon occurs when the cells are metabolically active and it does not take place at low temperatures. Therefore, the maintenance of the chilling chain proved to be a main factor to prevent the spread of Stx-genes in dairy processes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Escherichia coli O157:H7 has been associated in multiple outbreaks linked to the consumption of whole produce and fresh-cut leafy vegetables. However, plant-based foods had not been traditionally recognized as a host for enteric pathogens until the elevated incidence of produce-related outbreaks became apparent. The survival dynamics of two cocktails of generic E. coli (environmental water, plant and soil isolates) and E. coli O157:H7 within the phyllosphere of Mizuna, Red Chard and Tatsoi during their production, harvest, minimal processing, packaging and storage over two greenhouse production cycles were studied. Genotyping of applied generic E. coli strains to evaluate their comparative survival and relative abundance in the phyllosphere by REP-PCR is also reported. The Mizuna, Red Chard and Tatsoi shoots were grown under standard greenhouse conditions and fertility management. Both E. coli cocktails were spray-inoculated separately and determined to result in an initial mean population density of log 4.2 CFU/cm². Leaves were harvested as mini-greens approximating commercial maturity, minimally processed in a model washing system treated with 3 mg/L of ClO₂ and stored for 7 days at 5 °C. Rapid decline of generic E. coli and E. coli O157:H7 populations was observed for all plant types regardless of the leaf age at the time of inoculation and the irrigation type across both seasonal growth cycle trials. The decline rate of the surviving populations for the fall season was slower than for the summer season. The minimal processing with 3 mg/L of ClO₂ was not sufficient to fully disinfect the inoculated leaves prior to packaging and refrigerated storage. Viable populations of E. coli and E. coli O157:H7 were confirmed throughout storage, including the final time point at the end of acceptable visual leaf quality. In this study, the ability of low populations of E. coli to survive during production and postharvest operations in selected mini-greens has been demonstrated. However, further field-based trials are needed to expand understanding of the post-contamination fate of enteric bacterial pathogens on leafy vegetables. In summary, this research work provides baseline data upon which to develop food safety preventive control guidance during the production and minimal processing of these crops., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Zn(2+) uptake systems are required for many enteric pathogens to survive and form biofilm in zinc-deficient conditions. ykgM and zinT (formerly yodA), regulated by Zur (zinc uptake regulator), have been reported as being highly induced during zinc shortage. This work reports that ykgM and zinT in enterohemorrhagic Escherichia coli (EHEC) O157:H7 biofilms under fluidic conditions were highly expressed compared to those in stationary-phase planktonic cells and a mutation of either ykgM or zinT genes led to the inhibition of curli biosynthesis. Inductively coupled plasma mass spectroscopy showed that the ykgM and zinT mutants contained lower concentrations of Zn(2+) than the wild type. Both mutants were less attached to both the glass surface of a microchannel and epithelial cells than the wild type. Quantitative reverse-transcription PCR data indicated that the expression of csgA, which encodes the major curli subunit, was inhibited in both mutants with a zinc deficiency. Scanning electron microscopy showed that the mutants grown under zinc-deficient condition were covered with a lower amount of curli compared to the wild type and often became filamentous. Zn(2+) supplementation restored curli production and prevented filamentation in the mutants. Overall, under zinc-deficient conditions, YkgM and ZinT proteins are required for maintaining optimal zinc concentration in EHEC and intracellular zinc deficiency inhibits curli production., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Phenotypic analyses were performed using an enterohemorrhagic Escherichia coli O157 (EHEC) strain (B-1) and a commensal E. coli K-12 strain, exposed to prolonged cold stress. The EHEC E. coli O157 showed significantly (P<0.05) higher resistance to cold stress compared to non-pathogenic E. coli K-12 DH5α. Further, it was found that RpoS sigma factor plays a significant (P<0.05) role in the cold stress physiology of the enterohemorrhagic E. coli strain. Using comparative proteomic analysis of hypo-thermally adapted E. coli O157 wild-type and rpoS mutant strains, we identified 21 proteins that were differentially expressed upon cold temperature shifts or rpoS mutation. All identified proteins of cold post-acclimation stimulons fell into two large sub-groups: (i) stress proteins, and (ii) housekeeping proteins. This prolonged cold stress response included proteins involved in mRNA turnover, cell replication efficiency, conditional and post-synthetic modification of membrane lipid bilayers, biosynthetic processes, and the uptake of different sugars. The RpoS sigma factor had no control over the key stress proteins, polynucleotide phosphorylase and elongation factor G, in prolonged stress stimulon. However, RpoS was shown to regulate the expression of proteins involved in homeoviscous adaptation during cold shock, as well as various proteins involved in central metabolic pathways of this food-borne pathogen., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The occurrence of Escherichia coli O157, O111 and O26 in 159 raw ewe's milk samples was examined. Sample-aliquots were incubated simultaneously in TSB added with yeast extract (YETSB) and mTSB with novobiocin (N-mTSB). Serogroup-specific immunomagnetic separation (IMS) was then used and IMS beads were plated in a cefixime tellurite (CT)-containing media (CT-SMAC, CT-SBMAC and CT-RMAC for E. coli O157, O111 and O26, respectively) and E. coli O157:H7 chromogenic ID agar. A sweep of confluent growth from each medium was examined for the presence of E. coli O157 and O111 using PCR, and for E. coli O26 using a latex agglutination test. Enumeration of E. coli O157 and O111 was performed in the samples tested positive for the correspondent serogroup using the most probable number (MPN) method combined with PCR. Percentage occurrences of E. coli O157, O111 and O26 were 18.2, 8.2 and 5.7, respectively. Mean E. coli O157 and O111 levels were 0.22 and <0.04 MPN/mL, respectively. Enrichment in YETSB resulted in higher detection rates of E. coli O157 and O26 than in N-mTSB. When YETSB was used as enrichment broth and for these last two serogroups, the analysis of the confluent growth from the CT-media gave more positive results than that from E. coli O157:H7-ID medium., (Copyright © 2010 Elsevier B.V. All rights reserved.)

This study aimed to investigate verocytotoxigenic Escherichia coli O157 in the largest beef and sheep slaughter plants in Ireland over a one-year period. Samples consisted of pooled rectal swabs (n=407) and pooled carcass swabs (n=407) from 5 animals belonging to the same herd or flock and minced meat (n=91) from the same sampling date. E. coli O157 isolates were characterised using PCR for a range of genes, i.e. 16S, rfbE, fliC, vtx1, vtx2, eaeA and confirmed VTEC O157 isolates were tested for antimicrobial susceptibility and typed using Pulsed-Field Gel Electrophoresis (PFGE) and Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA). VTEC O157 was isolated from 7.6% and 3.9% of bovine rectal and carcass swab samples and from 5.8% and 2.9% of ovine rectal and carcass swab samples respectively. None of the bovine minced meat samples (n=77) and only one of the 14 ovine minced meat samples was positive for VTEC O157. Following PFGE and MLVA, cross contamination from faeces to carcasses was identified. While PFGE and MLVA identified the same clusters for highly related strains, MLVA discriminated better than PFGE in addition to being more rapid and less labour intensive. Results showed that cattle and sheep presented for slaughter in Ireland harbour VTEC O157, and although the levels entering the food chain are low, this should not be overlooked as possible sources of zoonotic infection; molecular typing was able to demonstrate relationships among strains and could be used to elucidate the sources of human infection., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A micro-array has been developed, based on the GeneDisc(R) array, for the genetic identification of 12 O-types and 7 H-types of Shiga toxin-producing Escherichia coli (STEC) including the most clinically relevant enterohemorrhagic E. coli (EHEC) serotypes. The genes selected for determination of the O antigens (rfbE(O157), wzx(O26), wzx(O103), wbd1(O111), ihp1(O145), wzx(O121), wzy(O113), wzy(O91), wzx(O104), wzy(O118), wzx(O45), and wbgN(O55)) and H-types (fliC(H2), fliC(H7), fliC(H8), fliC(H11), fliC(H19), fliC(H21), and fliC(H28)) showed a high specificity and concordance with serology. The micro-array also had a high specificity for EHEC-associated virulence factors, including Shiga toxins 1 and 2 (stx1 and stx2), intimin (eae), enterohemolysin (ehxA), serine protease (espP), catalase peroxidase (katP), the type II secretion system (etpD), subtilase cytotoxin (subA), autoagglutinating adhesin (Saa) and type III secreted effectors encoded in the genomic islands OI-122 (ent/espL2, nleB, and nleE) and OI-71 (nleF, nleH1-2, and nleA). The eae gene was detected in all typical EHEC strains, and the pattern of nle genes encoded in OI-71 and OI-122 was found to be closely associated with certain serotypes of typical EHEC and emerging EHEC strains. Virulence plasmid associated genes such as katP, espP, and etpD were more common in EHEC than in STEC strains; this supports their association with virulence. This array constitutes a valuable approach for the identification of STEC strains with a high potential for human virulence., (Copyright 2010 Elsevier B.V. All rights reserved.)

Relative gene transcription and virulence potential, as measured by a Caco-2 adhesion assay, were investigated for three enterohemorrhagic Escherichia coli (EHEC) strains after long-term adaptation for 24h to acid (BHI pH 5.5) and salt (BHI 4.5% (w/v) NaCl) stress. Five virulence genes (eae, lpfA, stx1A, stx2A and tir) and three stress response genes (asr, gadA and rpoS) were selected for gene transcription studies and compared to the relative transcription after growth at standard condition (BHI pH 7.0). In general, long-term adaptation to acid and salt stress significantly repressed the relative transcription of the virulence genes and induced relative transcription of the stress response genes though some strain variations were observed: the stress dependent gene transcription profiles obtained for STEC2 (O157:H7, minced beef) and c196-01 (O157:H-, clinical origin) were more similar compared to EDL933 (O157:H7, raw hamburger). Long-term adaptation to salt stress significantly increased the adhesion of all three EHEC strains to Caco-2 compared to the non-stressed controls. The present study shows that long-term adaptation to food related stress factors such as acid and salt is capable of changing the relative transcription of important virulence and stress response genes and increasing the virulence potential as measured by adhesion to the human colonic epithelial cell line, Caco-2., (2010 Elsevier B.V. All rights reserved.)

The prevalence of verocytotoxin producing Escherichia coli (VTEC), E. coli O157, and VTEC O157 in 474 swab samples from Danish beef carcasses was determined. The presence of E. coli O157 was determined by a culture method that included immunomagnetic separation (IMS) followed by real time PCR testing of isolates for verocytotoxin (vtx) genes. E. coli O157 was recovered from 4.2% of the carcass samples and VTEC O157 from 3.4% of the samples. All VTEC O157 contaminated carcasses were from bull calves and the VTEC O157 prevalence on bull calf carcasses was 7.3%. The VTEC O157 contaminated beef carcasses were sampled again after one week of cold storage, and 15 of the 16 carcasses were then VTEC O157 negative. The presence of VTEC was determined by a duplex real time PCR assay for vtx1 and vtx2 in DNA from enrichment cultures of swabs. In total 45.4% of the samples were VTEC positive. VTEC were isolated from 21% of 77 vtx-positive samples that were identified by replication of colonies on hydrophobic grid membrane filters followed by hybridisation with vtx specific DNA probes. Fourteen of the 16 VTEC isolates were non-O157 and these strains were negative for the virulence gene eae. A real time PCR assay for the E. coli O157 specific rfbE gene was developed. In total 22.4% of the enriched samples were positive for the O157 rfbE gene. The combined results of the vtx and rfbE real time PCR screening showed that 17.5% of the carcasses potentially were contaminated with VTEC O157. Screening of carcass swabs was expanded by real time PCR testing for eae in a subset of the samples. Of 244 samples, 25.4% were positive for both vtx and eae. The eae gene was detected in 81% of the vtx-positive samples and in 46% of 67 vtx-negative samples, indicating that bacteria harbouring eae are widespread on bovine carcasses. The present study shows that real time PCR screening of carcass samples for genes encoding virulence or other genetic markers is a reliable method for rapid identification of carcasses that potentially are contaminated with VTEC., (2010 Elsevier B.V. All rights reserved.)

Limonoids are important constituents of the grapefruit and other citrus fruits. Research on health benefits suggests that citrus limonoids may act as anti-cancer, cholesterol lowering, anti-HIV and anti-feedant compounds. However, antimicrobial activities of citrus limonoids are not reported. In the present investigation, limonoids were purified from grapefruit seed and evaluated for their potential to antagonize cell-to-cell communication, biofilm formation and expression of Enterohemorrhagic Escherichia coli (EHEC) type three secretion system (TTSS). The results of the present study suggest that, certain limonoids are inhibitory to the cell-to-cell communication, biofilm formation and EHEC TTSS. Specifically, obacunone demonstrated strong inhibition of EHEC biofilm formation and TTSS. Furthermore, obacunone and other limonoids seem to inhibit the biofilm formation and TTSS in quorum sensing dependent fashion. The results indicate that certain grapefruit limonoids may possibly help in antagonizing the EHEC infection process, and may serve as lead compound in development of new antipathogenic molecules., (Published by Elsevier B.V.)

Here, the gene expression profiles of EHEC O157:H7 and HT-29 during the attachment stage were investigated by using duplex whole transcriptome analysis. After the initial attachment (3 h), the gene regulation systems of both the EHEC O157:H7 and HT-29 host cells were immediately remodeled. A total of 326 genes of the HT-29 cells, which involved proteins associated with the detoxification process, stress response proteins, anti-apoptosis/inflammation proteins, immune response protein, and oxidative stress proteins, were differentially regulated by more than 2.0-fold during EHEC attachment. In contrast, when HT-29 was attached to EHEC the expression of 611 genes was induced and the expression of 384 genes was reduced by more than twofold when compared to RPMI 1640-grown EHEC (16.14% of the total hybridized genes). Among the genes that were classified according to biological function, the mRNA levels of the genes involved in stress response, oxidative stress, cell signaling and cell surface proteins were significantly altered after the attachment of EHEC O157:H7. Therefore, the results of this study provide crucial insight into the genetic networks that provide host cell protection and the strategy of EHEC O157:H7 pathogenesis in gastro-intestinal (GI) tracts.

Escherichia coli O157 strains cause diseases in humans that result from the consumption of food and water contaminated with faeces of infected animals and/or individuals. The objectives of this study were to isolate and characterise E. coli O157 strains from humans, cattle and pigs and to determine their antibiotic resistant profiles as well as detection of virulence genes by PCR. Eight hundred faecal samples were analysed for typical E. coli O157 and 76 isolates were positively identified as E. coli O157 strains. 16S rRNA sequence data were used to confirm the identity of the isolates. Susceptibility profiles to 9 antibiotics were determined and the multiple antibiotic resistant (MAR) patterns were compiled. A large proportion (52.6%-92.1%) of the isolates from pigs, cattle and humans were resistant to tetracycline, sulphamethoxazole and erythromycin. Thus the phenotype Smx-T-E (sulphamethozaxole-tetracycline-erythromycin) was present in most of the predominant MAR phenotypes obtained. Cluster analysis of antibiotic resistances revealed a closer relationship between isolates from pig and human faeces than cattle and humans. PCR were performed to amplify STEC virulence and tetracycline resistance gene fragments. A tetB gene fragment was amplified among the isolates. Eighteen (60%) of the isolates possessed the hlyA gene and 7(23.3%) the eae gene while only 5(16.7%) possessed both genes. Although shiga toxin genes were detected in the E. coli O157:H7 positive control strain none of the isolates that were screened possessed these genes. In a related study we reported that the prevalence of E. coli O157 was higher in pigs than cattle and humans. A high market demand for pork and beef in South Africa amplifies the risk that diseased animals pose to human health. This highlighted the need for proper hygiene management to reduce the prevalence of E. coli O157 in farm animals and prevent the spread from animals to humans.

The present study was conducted in Lima Metropolitana to evaluate the prevalence of Shiga toxin-producing Escherichia coli (STEC) O157:H7 in raw beef, raw ground beef, soft cheese and fresh vegetables, sampled at different markets in the city. Between October 2000 and February 2001, 407 food samples were collected from different markets in the 42 districts of Lima Metropolitana. Samples were assayed for E. coli O157 by selective enrichment in modified Tryptic Soy Broth containing novobiocin, followed by immunomagnetic separation (IMS) and plating onto sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Fifty (12.3%) of 407 food samples resulted positive for E. coli O157 isolation (23 of 102 ground beef; 15 of 102 beef meat; eight of 102 soft cheese and four of 101 fresh vegetables). Thirty-five E. coli O157 isolates were further analysed for the presence of virulence genes. All 35 were positive by PCR for O157 rfbE, fliCh7, eae-gamma1 and ehxA genes. In addition, genes encoding Shiga toxins were detected in 33 of 35 isolates, five isolates (14%) encoded stx(1), stx(2), and 28 (80%) stx2 only. The isolates were of seven different phage types (PT4, PT8, PT14, PT21, PT34, PT54, and PT87) with three phage types accounting for 80% of isolates: PT4 (15 isolates), PT14 (8 isolates), and PT21 (5 isolates). Interestingly, the majority (31 of 35; 89%) of E. coli O157:H7 isolates characterized in this study belonged mainly to the phage types previously found in STEC O157:H7 strains associated with severe human disease in Europe and Canada. Pulsed-field gel electrophoresis (PFGE) of 32 isolates revealed 14 XbaI-PFGE groups (I to XIV) of similarity >85%, with 23 (72%) isolates grouped in five clusters. Some isolates from different districts presented a high clonal relatedness. Thus, PFGE group VIII clustered eleven strains from nine different districts. The broad range of PFGE subtypes found in this study demonstrates the natural occurrence of many genetic variants among STEC O157:H7 spread in Lima.