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From January to December 2016, Alberta Agriculture and Forestry (AAF) conducted a provincial survey of selected pathogens and indicator organisms on beef carcasses processed at Provincially Licensed Abattoirs (PLAs) in Alberta. The survey was conducted in seven small and medium scale slaughterhouses located in southern and northern Alberta that process beef cattle and cows. Paired samples were collected from the same carcass immediately after hide removal (pre-evisceration n = 401) and at pre-chill (n = 402) after application of a carcass wash and/or anti-microbial interventions. Swab samples were screened for the presence of Shiga toxin-producing Escherichia coli (STEC) O157:H7, six non-O157 STECs (O26, O45, O103, O111, O121 and O145), and Salmonella. In addition, samples were enumerated for indicator organisms (aerobic colony count (ACC), Enterobacteriaceae, coliforms, and generic E. coli). At pre-evisceration, 30 samples (7.4%) were confirmed positive for E. coli O157:H7; 13 samples (3.2%) were confirmed positive for non-O157 STEC; and 7 samples (1.7%) were confirmed positive for Salmonella. Pre-chill swabs had 21 samples (5.2%) positive for E. coli O157:H7, 16 samples (3.9%) positive for non-O157 STEC, and 1 sample (0.2%) positive for Salmonella. At pre-chill the prevalence of Salmonella was significantly lower (P

Shiga toxin-producing Escherichia coli (STEC) is one of the main cause of foodborne disease worldwide, but isolation rates or characteristics of this bacteria from ground beef in Chile are unknown. The present study aimed to isolate and characterize non-O157 STEC from ground beef sold at retail in the city of Santiago, Chile. We analyzed 430 ground beef samples for the presence of STEC, and isolated the microorganism in 10% of samples (43/430). We obtained 56 isolates from the 43 positive samples; 55 of these (98.2%) fermented sorbitol. Most isolates (98.2%; 55/56) showed β-glucoronidase activity, and only six (10.7%; 6/56) were resistant to tellurite. Among the virulence factors studied (stx1, stx2, eae, and hlyA), stx2 was the only virulence factor in 41% of the isolates (23/56), whereas 10.7% (6/56) of isolates carried a combination of three virulence factors (stx1 + stx2 + hlyA). None of the isolates carried the gene eae. Finally, isolates were neither serogroups O157 nor “big six”. In conclusion, ground beef sold in Santiago, Chile is contaminated with STEC; however, further studies are required for understanding their virulence potential.

Shiga toxin-producing Escherichia coli (STEC) are globally important food-borne pathogens. The isolation of non-O157 STEC is a significant public health challenge due to the dramatic diversity of their phenotypes and genotypes. In the present study, 476 non-O157 STEC strains representing 95 different O-serogroups were used to evaluate tellurite resistance and the performance of 12 different chromogenic agars. Of 476 strains, only 108 (22.7%) strains showed the minimal inhibitory concentration (MIC) values for potassium tellurite being higher than 4μg/ml, and 96 (20.2%) strains harbored intact ter genes cluster. The presence of ter genes was significantly correlated with tellurite resistance. Six commercial chromogenic agars (TBX, MAC, SMAC, Rainbow® Agar O157, CHROMagar™ ECC, and Fluorocult O157) supported the growth of all strains. However, CT-SMAC, CHROMagar™ O157, and CHROMagar™ STEC agars exhibited 12.2%, 31.1%, and 38.0% of growth inhibition, respectively. Furthermore, 4.6%, 33.2%, and 45.0% of strains were inhibited on RBA-USDA, RBA-NT, and BCM O157 agar media. Variations in tellurite resistance and colony appearance might result in discrepant performance of non-O157 STEC recovery from different chromogenic agars. Using inclusive agars or less selective agar in combination with highly selective agar should be suggested to recover most non-O157 STEC strains, which would increase the probability of recovering STECs from complex background microflora.

Dairy compost with 20, 30, or 40% moisture content (MC) was inoculated with a mixture of six non-O157 Shiga toxin-producing Escherichia coli (STEC) serovars at a final concentration of 5.1 log CFU/g and then stored at 22 and 4°C for 125 days. Six storage conditions-4°C and 20% MC, 4°C and 30% MC, 4°C and 40% MC, 22°C and 20% MC, 22°C and 30% MC, and 22°C and 40% MC-were investigated for the persistence of non-O157 STEC in the dairy compost. During the entire storage, fluctuations in indigenous mesophilic bacterial levels were observed within the first 28 days of storage. After inoculation, the non-O157 STEC population increased 0.69 and 0.79 log CFU/g in the dairy compost with 30 and 40% MC at 22°C within the first day, respectively; for all other storage conditions, the pathogen population decreased rapidly. After the 125-day storage, the reductions of non-O157 STEC for 4°C and 20% MC, 4°C and 30% MC, 4°C and 40% MC, 22°C and 20% MC, 22°C and 30% MC, and 22°C and 40% MC storage conditions were >4.52, >4.55, 3.89, >4.61, 3.60, and 3.17 log CFU/g, respectively. All the survival curves showed an extensive tail, indicating non-O157 STEC can survive at least for 125 days in the dairy compost. The survival data were analyzed with log-linear with tailing and Weibull models. Compared with the log-linear with tailing model, the Weibull model was found to be a better choice for predicting the survival of non-O157 STEC in dairy compost owing to a high overall R2 value (0.8738 to 0.9909). The decay rate of non-O157 STEC was higher in dairy compost stored at 4°C compared with at 22°C, and the same trend was found for the compost with 40% MC versus 20% MC. In addition, two non-O157 STEC serotypes (STEC O145 and O45) were detected on the last day of the longitudinal study and may deserve special attention in the Big 6 STEC group. Our results have provided scientific data for risk assessment of the microbiological safety of dairy compost to control non-O157 STEC during subsequent storage of dairy compost.

The study examined the relative fate of the top six non-O157 shiga-toxin producing Escherichia coli (STEC) and E. coli O157:H7 during the manufacture of dry fermented sausages (DFS). Three separate batches of sausages containing a five-strain cocktail for each serogroup and uninoculated control were manufactured and subjected to identical fermentation, maturation and dry curing conditions. Changes in physicochemical properties and inoculated STEC numbers were enumerated during the DFS production stages and log reduction and log reduction rates were calculated. Inoculation of very high concentrations (8logCFUg-1) of STEC in the sausage batter did not significantly (P>0.05) affect the changes in the pH, aw, moisture, protein, fat content compared to the uninoculated DFS. There was a significant (P 0.05) from each other. These results indicate that the lethality of DFS production processes observed against E. coli O157:H7 would result in a similar inactivation of the top six non-O157 STEC.

Six major Shiga toxin producing Escherichia coli (STEC) serogroups: O26, O103, O145, O111, O121, and O45 have been declared as adulterants in federally inspected raw beef in the USA effective June 4th, 2012 in addition to the routinely tested STEC O157: H7. This study tests a real-time multiplex PCR assay and pooling of the samples to optimize the detection and quantification (prevalence and contamination) of six major non-O157 STEC, regardless of possessing Shiga toxins. To demonstrate the practicality, one large-scale slaughter plant (Plant LS) and one small-scale slaughter plant (Plant SS) located in the Mid-Western USA were sampled, in 2011, before the establishment of 2013 USDA laboratory protocols. Carcasses were sampled at consecutive intervention stations and beef trimmings were collected at the end of the fabrication process. Plant SS had marginally more contaminated samples than Plant LS (p-value 0.08). The post-hide removal wash, steam pasteurization, and lactic acid (≤5%) spray used in Plant LS seemed to reduce the six serogroups effectively, compared to the hot-water wash and 7-day chilling at Plant SS. Compared to the culture isolation methods, quantification of the non-O157 STEC using real-time PCR may be an efficient way to monitor the efficacy of slaughter line interventions.

We investigated for virulence genes ( stx1 , stx2 and hlyA ), serotypes and extended-spectrum β-lactamases (ESBLs) producing capacity in O157 and non-O157 Escherichia coli isolated from producer-distributor bulk milk (PDBM). Fifteen different E. coli O-serogroups were observed from the isolates (n = 121). The prevalence of stx1 and stx2 genes among the E. coli isolates was 8.3% and 11.6%, respectively, while 5.8% harboured both stx1 and stx2 . Four E. coli isolates (3.3%) had ESBLs producing capacity, resisted multiple cephalosporins and aztreonam, and carried stx genes. Cluster analysis using GTG 5 finger printing revealed a diversity of E. coli seropathotypes in PDBM that are known to be associated with human diarrhoeal diseases. These results highlight a potential risk posed on human health by the consumption of PDBM contaminated with pathogenic E. coli . A further quantitative risk assessment of the impact of pathogenic E. coli contamination in PDBM on human health is therefore recommended.

Strawberries are vulnerable to harboring microbial pathogens because they are generally not washed due to their perishable nature. The focus of this study was to quantify the reduction in infection risks associated with non-O157 Shiga toxin producing Escherichia coli serotypes contaminated strawberries if the strawberries are exposed to low doses ∼1 kGy (kiloGray) of electron beam (eBeam) irradiation. A cocktail of six serotypes of non O157 E. coli namely, O26:H11, O45:H2, O103:H2, O111:NM, O121:H19, and O145 was employed. Strawberry puree rather than whole strawberries were used in this study to ensure dose uniformity that is critical for accurate interpretation of microbial reduction. The results show that when these serotypes are exposed to ≤1 kGy eBeam dose, there is approximately 4-log reduction in their numbers when they are present within a strawberry matrix (puree). Quantitative microbial risk assessments suggest that if a typical strawberry serving (150 g) was heavily contaminated (∼105 CFU/serving size), 2 out of 10 susceptible individuals (20%) would get sick (without eBeam treatment). However, if these contaminated strawberries had been treated with 1 kGy of eBeam dose, the infection risks would have be significantly reduced to approximately 4 out of every 100,000 individuals (0.004%). Similarly, even at low levels of contamination (∼102 CFU/serving), the infection risks would be reduced from 6 out of 10,000 susceptible individuals to approximately 4 out of 100 million susceptible individuals.

According to the United States Food and Drug Administration (FDA) agency, bacteriophage solutions targeting the serotype O157:H7 are Generally Recognized as Safe (GRAS) to control STEC during beef processing. However, outbreaks involving the "Big Six" STEC increased the industry concern about those serotypes. The objective of this study was to test the efficacy of MS bacteriophages to reduce the "Big Six" non-O157 STEC in beef. The lysing efficacy of phages isolated for each specific serotype varied from 96.2% to 99.9% in vitro. When applied to contaminated trim, reductions ranging from 0.7 to 1.3 Log of all STEC were observed in ground beef. Bacteriophages may provide an additional barrier against the "Big Six" STEC in ground beef. Results of this research provide support documentation to the FDA to extend GRAS status for bacteriophages as processing aids against all adulterant STEC.

Escherichia coli O157:H7 has frequently been associated with foodborne infections and is considered an adulterant in raw non-intact beef in the U.S. Shiga toxin-producing E. coli (STEC) belonging to serogroups O26, O45, O103, O111, O121, and O145 (known as the “big six” non-O157) were estimated to cause > 70% of foodborne infections attributed to non-O157 serogroups in the U.S., as a result, these six serogroups have also been targeted by regulation in the U.S. The purpose of this study was to develop a rapid and high-throughput molecular method to group STEC isolates into seven clinically important serogroups (i.e., O157 and the “big six” non-O157 serogroups) targeted by regulation in the U.S. by interrogating single nucleotide polymorphisms (SNPs) in gnd. A collection of 195 STEC isolates, including isolates belonging to O157:H7 (n = 18), O26 (n = 21), O45 (n = 19), O103 (n = 24), O111 (n = 24), O121 (n = 23), O145 (n = 21), and ten other STEC serogroups (n = 45), was assembled and characterized by full gnd sequencing to identify informative SNPs for molecular serogrouping. A multiplex SNP typing assay was developed to interrogate twelve informative gnd SNPs by single base pair extension chemistry and used to characterize the STEC isolate collection assembled here. SNP types were assigned to each isolate by the assay and polymorphisms were confirmed with gnd sequence data. O-serogroup-specific SNP types were identified for each of the seven clinically important STEC serogroups, which allowed the differentiation of these seven STEC serogroups from other non-O157 STEC serogroups. Although serogroups of the “big six” non-O157 STEC and O157:H7 contained multiple SNP types per O-serogroup, there were no overlapping SNP types between serogroups. Our results demonstrate that molecular serogrouping of STEC isolates by interrogation of informative SNPs in gnd represents an alternative to traditional serogrouping by agglutination for rapid and high-throughput identification of clinically important STEC serogroups targeted by regulation for surveillance and epidemiological investigations.

Detection of Shiga toxin-producing Escherichia coli (STEC) has evolved significantly since the introduction of sorbitol-MacConkey agar. This study compares four chromogenic media (CHROMagar™ STEC, Rainbow® O157 agar, CHROMagar™ O157, and Colorex® O157) in their identification of non-O157 STEC. When 161 non-O157 STEC were directly inoculated onto each medium, detection rates on CHROMagar™ STEC, Rainbow® O157 agar, CHROMagar™ O157 and Colorex® O157 were 90%, 70%, 3.7% and 6.8%, respectively. Tellurite minimal inhibitory concentrations (MICs) correlated with growth on CHROMagar™ STEC as 20 of 22 isolates with poor or no growth had MICs ≤1μg/mL. Stool spiking experiments revealed that CHROMagar™ STEC had the highest recovery of the six most common non-O157 STEC, ranging from 30% (in mucoid stool) to 98% (in watery stool). When using clinical stool samples, CHROMagar™ STEC had a sensitivity, specificity, positive predictive value, and negative predictive value of 84.6%, 87%, 13.9%, and 99.6%, respectively.

The goal of this study was to evaluate the efficacy of a novel antimicrobial solution made with chitosan, lauric arginate ester, and organic acids on Escherichia coli O157:H7, Salmonella spp., Listeria monocytogenes, and non-O157 shiga toxin-producing E. coli cocktails and to test its potential to be used as a marinade for raw beef. Fresh beef top round steaks were surface-inoculated with the pathogen cocktails at approximately 2.5 or 4.5 Log CFU/cm2, marinated with the antimicrobial solution (AMS), and then stored at 4 °C for 6, 24, and 48 h. Three commercially available marinades were used for comparison. Results revealed that AMS had the most antimicrobial effect regardless of the type or inoculation level of pathogens (P

Shiga toxin-producing Escherichia coli (STEC) is one of the most significant causes of food-borne infections capable of causing serious health complications in humans. Even though ruminants are known to be the major reservoirs of STEC, other non-ruminant food producing animals may also harbour pathogenic E. coli strains. In this study, we investigated the prevalence of E. coli serogroups O26, O111, O121, O145, and O157 and their associated virulence genes (stx1, stx2, eae, and ehxA) in swine faecal samples obtained from the two major commercial farms located in the Nkonkobe Municipality, Eastern Cape, South Africa. The proportions of serogroups detected were O26; 35 (7%), O145; 14 (2.8%), and O157:H7; 43 (8.6%) of the total animals sampled. Out of the 500 animals sampled, 22 isolates of E. coli (1.4%) tested positive for the stx2 gene, and 7 of these isolates belonged to E. coli O26 serogroup, while the remaining 15 most likely belonged to serogroups untargeted in this study. Other virulence genes (stx1, eae, and ehxA) that we screened for were not detected. These findings reveal that pigs within the Eastern Cape Province of South Africa can harbour Shiga toxin-producing E. coli.

The prevalence of potentially positive Shiga toxin-producing Escherichia coli (STEC) bovine hides and carcasses in three abattoirs in Costa Rica was estimated. Two export facilities (A and B) and one non-export establishment (C) were visited during the dry and rainy seasons of 2013. Swabs of hides pre-eviscerated and treated (180–220 peroxyacetic acid spray) carcasses were tested for the potential presence of STEC serogroups O26, O45, O103, O111, O121, and O145. The prevalence on hides during the rainy season was 86.7, 96.7 and 96.7% for facilities A, B, and C, respectively. During the dry season, the prevalence on hides was significantly lower in plants A and B (40% and 26.7%, respectively), but was marginally associated with the season in plant C (76.7%, P = 0.0523). The prevalence of non-O157 STEC markers on treated carcasses was low (0 to 3.3%), suggesting that all plants were effective in minimizing the target non-O157 STEC in beef destined for export and for domestic consumption.

Latex agglutination tests (LAT) were developed and evaluated for the rapid detection of LPS antibodies against E. coli serogroup O157, O26, O104, O111 and O145. The latex tests have been proved to be sensitive, fast, easy-to-perform and cost-efficient tools for the screening serodiagnosis of VTEC infections causing haemolytic-uraemic syndrome.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups such as O26, O45, O103, O111, O121 and O145 often cause illness to people in the United States and the conventional identification of these “Big-Six” are complex. The label-free hyperspectral microscope imaging (HMI) method, which provides spectral “fingerprints” information of bacterial cells, was employed to classify serogroups at the cellular level. In spectral analysis, principal component analysis (PCA) method and stacked auto-encoder (SAE) method were conducted to extract principal spectral features for classification task. Based on these features, multiple classifiers including linear discriminant analysis (LDA), support vector machine (SVM) and soft-max regression (SR) methods were evaluated. Different sizes of datasets were also tested in search for the suitable classification models. Among the results, SAE-based classification models performed better than PCA-based models, achieving classification accuracy of SAE-LDA (93.5%), SAE-SVM (94.9%) and SAE-SR (94.6%), respectively. In contrast, classification results of PCA-based methods such as PCA-LDA, PCA-SVM and PCA-SR were only 75.5%, 85.7% and 77.1%, respectively. The results also suggested the increasing number of training samples have positive effects on classification models. Taking advantage of increasing dataset, the SAE-SR classification model finally performed better than others with average accuracy of 94.9% in classifying STEC serogroups. Specifically, O103 serogroup was classified with the highest accuracy of 97.4%, followed by O111 (96.5%), O26 (95.3%), O121 (95%), O145 (92.9%) and O45 (92.4%), respectively. Thus, the HMI technology coupled with SAE-SR classification model has the potential for “Big-Six” identification.

Shiga toxin-producing E. coli (STEC) is an important group of foodborne pathogens in the United States and worldwide. Nearly half of STEC-induced diarrheal disease in the United States is caused by serotype O157:H7, while non-O157 STEC account for the remaining illnesses. Thus, the U.S. Department of Agriculture (USDA) Food Safety and Inspection Service has instituted regulatory testing of beef products and has a zero-tolerance policy for regulatory samples that test positive for STEC O157:H7 and six other non-O157 STEC (serogroups O26, O45, O103, O111, O121, and O145). In this study, positive control (PC) strains for the detection of STEC O157:H7 and the six USDA-regulated non-O157 STEC were constructed. To ensure that the food testing samples are not cross-contaminated by the PC sample, it is important that the STEC-PC strains are distinguishable from STEC isolated from test samples. The PC strains were constructed by integrating a unique DNA target sequence and a gene for spectinomycin (Sp) resistance into the chromosomes of the seven STEC strains. End-point and real-time PCR assays were developed for the specific detection of the PC strains and were tested using 93 strains of E. coli (38 STEC O157:H7, at least 6 strains of each of the USDA-regulated non-O157 STEC, and 2 commensal E. coli) and 51 strains of other bacteria (30 species from 20 genera). The PCR assays demonstrated high specificity for the unique target sequence. The target sequence was detectable by PCR after 10 culture passages (∼100 generations), demonstrating the stability of the integrated target sequence. In addition, the strains were tested for their potential use in modeling the growth of STEC. Plating the PC strains mixed with ground beef flora on modified rainbow agar containing Sp eliminated the growth of the background flora that grew on modified rainbow agar without Sp. Thus, these strains could be used to enumerate and model the growth of STEC in the presence of foodborne background flora.

The beef industry continues to be interested in reliable rapid detection technologies for shiga toxin-producing Escherichia coli (STEC). Current rapid technologies require several hours of pre-enrichment and additional time on the rapid technology instrument. A flow cytometer-based system (RAPID-B®) has been shown to improve the turn-around for results with a more rapid pre-enrichment requiring only 6.5 h pre-enrichment for a 25 g and 8.5 h for a 375 g sample, followed by an additional 30 min time to achieve final results using the screening technology. The purpose of this study was to validate the RAPID-B® technology for non-O157 STEC detection as compared to the USDA-FSIS reference method which utilizes the BAX® system. A total of 180 STEC isolates from various sources and 20 non-STEC strains were used to evaluate specificity and sensitivity using the RAPID-B® flow cytometer. Also, three different weights (25, 325 and 375 g) of beef trim and ground beef samples were spiked with each STEC to verify detection sensitivity of BAX® system and RAPID-B® flow cytometer. For both methods, samples were confirmed by culturing using the USDA-FSIS reference method regardless of the screening result. The RAPID-B® flow cytometer showed that 180 isolates were all positive and the 20 non-STEC strains were all negative. For spiked beef samples, overall detection sensitivity was the same for both the BAX® system and RAPID-B® flow cytometer. When detection sensitivity was based on sample weight, there was no differences in 25 and 375 g samples between RAPID-B® flow cytometer and USDA-FSIS reference method. The RAPID-B® system yielded the same sensitivity as the reference method with a decrease of over 10 h of pre-enrichment time and 3 h of rapid screening detection time. In conclusion, the RAPID-B® flow cytometer based on whole cell detection generated similar results as BAX® system therefore the RAPID-B® flow cytometer system could be a valuable rapid method for the detection of non-O157 STEC in beef products.

Seven non-pathogenic Escherichia coli strains: ATCC 25253, ATCC 25922, ATCC 11775, ATCC 8739, ATCC 11229, NAR and O157 Dm3Na were tested as potential candidates for the surrogate of pathogenic O157:H7 and non-O157 STEC strains (O111:NM, O26:H11, O145:NM and O103:H2) in apple juice treated by UV-C light. The survival studies in apple juice (pH 3.5) revealed that all non-O157 pathogens exhibit acid resistance similar to O157:H7. Subsequently the acid tolerant bacteria, i.e.: all STEC strains, ATCC 11229, ATCC 11775, ATCC 8739 and O157 Dm3Na, were exposed to three UV-C sources emitting light at the wavelengths of 222 nm, 254 nm and 282 nm. The inactivation studies of the nine most UV resistant strains indicated that ATCC 11229 strain can be recommended as a potential surrogate organism for UV treatments of non-O157 STEC strains at 222 nm. In the case of UV-C at 254 nm and 282 nm E. coli ATCC 8739 strain met the criteria for the surrogate organism for O157:H7 and non-O157 STEC strains. The manuscript presents the systematic procedure for the selection and verification of the potential surrogate organisms that can be recommended for validation of UV light processing of high acid juices and beverages.

Five Escherichia coli O157:H7 strains and one nonpathogenic E. coli strain were used. All strains were cultured in brain heart infusion broth and were inoculated in 16-well disposable module cassettes of a Bactometer impedance system. Two initial concentrations were obtained in the wells: 1.37 × 10(3) and 1.36 × 10(5) CFU/ml. The impedance measurements were monitored for 72 h at 5, 10, or 15°C, 48 h at 20°C, and 24 h at 25, 30, 35, 40, 45, 50 or 55°C. The lag time and the generation time of each culture were calculated from the detection time data. The coefficients of variation between the strains' growth parameters were low (0.009 to 0.105 for generation time and 0.074 to 0.475 for lag time). An F test showed no significant differences between strains at 5 or 1% confidence levels.

Understanding the survival and growth of non-O157 Shiga toxin-producing Escherichia coli (STEC) strains under cold temperatures may be important for protecting public health. The aim of this study was to compare the growth of three strains of each of the major non-O157 STEC serogroups (O26, O45, O103, O111, O121, and O145) with the growth of six O157:H7 STEC strains in broth at 10°C. Brain heart infusion broth (BHIB; pH 7.4) was inoculated with a single strain of stationary-phase STEC culture to produce a starting inoculum of ∼10(6) CFU/ml and stored at 10°C for up to 96 h (three trials per strain). Populations over time were fitted to the Baranyi and Roberts model, and lag-phase duration (LPD) and growth rate were calculated for each strain per trial. Average LPD ranged from 9.2 to 32.8 h for non-O157 STEC and from 10.5 to 17.2 h for O157 STEC. One strain of O26 STEC had a significantly longer LPD (P0.05) than did the other strains (32.8 h); otherwise, no significant differences were noted (P0.05). Growth rate ranged from 0.031 to 0.060 log CFU/ml/h for non-O157 STEC strains and from 0.034 to 0.046 log CFU/ml/h for O157 STEC strains. No significant difference in growth rate was noted among strains in BHIB at pH 7.4 and 10°C. In subsequent trials, growth of a single strain of each of the non-O157 STEC serogroups was compared with growth of four acid-tolerant O157 STEC strains in BHIB acidified to pH 5.6 with lactic acid. Acidification generally increased LPD and decreased the growth rate for strains, although the effect was variable and not significant. These findings suggest that growth patterns for strains of non-O157 STEC are similar to those for strains of O157 STEC in neutral and pH 5.6 BHIB at 10°C. Further research is needed to determine whether strains behave similarly in meat systems.

STEC isolates and also stx-negative Escherichia coli isolates from sheep meat from the Chaharmahal-va-Bakhtiari province, Iran were analyzed for nine clinically important non-O157 serotypes by PCR. A total of 90 E. coli isolates were tested. Stx-positive and eae-positive E. coli isolates did not belong to the nine most clinically relevant non-O157 STECs. Of the 80 non-STEC isolates, two belonged to the O103 and two belonged to the O128 groups. Stx-negative E. coli O103 and O128 strains isolated have potential in acquiring stx genes and continuing into the digestive system of consumers. Further studies are needed to analyze virulence characteristics of these E. coli strains to determine their potential role in causing disease in humans. For the sake of public health, it is important to monitor and investigate non-O157 diarrheagenic E. coli strains in meat in order to control and prevent them.

Shiga toxin producing Escherichia coli O157 and non-O157 serogroups are known to cause serious diseases in human. However, research on the persistence of E. coli non-O157 serogroups in preharvest environment is limited. In the current study, we compared the survival behavior of E. coli O157 to that of non-O157 E. coli strains in agricultural soils collected from three major fresh produce growing areas of California (CA) and Arizona (AZ). Results showed that the nonpathogenic E. coli O157:H7 4554 survived longer than the pathogenic E. coli O157:H7 EDL933 in Imperial Valley CA and Yuma AZ, but not in soils from the Salinas area. However, E. coli O157:NM was found to persist significantly longer than E. coli O157:H7 EDL933 in all soil tested from the three regions. Furthermore, two non-O157 (E. coli O26:H21 and E. coli O103:H2) survived significantly longer than E. coli O157:H7 EDL933 in all soils tested. Pearson correlation analysis showed that survival of the E. coli strains was affected by different environmental factors. Our data suggest that survival of E. coli O157 and non-O157 may be strain and soil specific, and therefore, care must be taken in data interpretation with respect to survival of this pathogen in different soils.

Shiga toxin-producing Escherichia coli (STEC) serotype O157:H7 and serogroups O26, O45, O103, O111, O121, and O145 are the leading cause of STEC-associated infections in humans in the United States. In the United States, these organisms are considered adulterants in raw nonintact beef products and in intact beef destined to be made into or used in nonintact raw beef products. The objective of this study was to provide an estimate of the burden of the six serogroups of non-O157 STEC in ground beef obtained from retail stores across the United States. A convenience sample of commercial ground beef products (n = 1,129) were purchased from retail stores in 24 states from October 2011 to May 2012. The samples had various lean/fat proportions, muscle group of origin (chuck, round, sirloin, or not specified), and packaging types. For each ground beef sample, 25 g was inoculated in 225 ml of modified tryptic soy broth, stomached for 1 min, and then incubated at 41°C for 18 ± 2 h. These enrichment cultures were then screened for stx, eae, and O group genes using a commercially available, closed-platform PCR-based method. The potential positive samples were subjected to immunomagnetic separation and plated on modified Rainbow agar. Morphologically typical colonies were subjected to latex agglutination and PCR determination of stx and eae genes. Nine (0.8%) of the ground beef samples were potentially positive for at least one STEC serogroup after PCR screening. The serogroups detected by PCR assay were O26 (four samples), O103 (four samples), O145 (three samples), O45 (two samples), and O121 (one sample). No STEC isolates belonging to these serogroups were recovered from the sample cultures. The current research provides updated surveillance data for non-O157 STEC isolates among commercial ground beef products and information regarding the potential sources of contamination from different parts of beef trims destined for ground beef production.

Preflattened veal cutlets (ca. 71.5 g, ca. 0.32 cm thick) were surface inoculated with ca. 6.8 log CFU/g of a multistrain cocktail of Escherichia coli O157:H7 (ECOH) or a cocktail made of single strains of serogroups O26, O45, O103, O104, O111, O121, and O145 of Shiga toxin-producing E. coli (STEC) cells and then were mechanically tenderized by passing once through a "Sir Steak" tenderizer. For each cooking time, in each of at least three trials, three inoculated and tenderized cutlets, with and without breading, were individually cooked in 15 or 30 ml of canola oil for 0.0, 0.75, 1.0, 1.25, 1.5, 1.75, or 2.25 min per side on an electric skillet set at 191.5°C. The temperatures of the meat and of the skillet were monitored and recorded using a type J thermocouple. Regardless of the breading or volume of oil used to cook the meat, the longer the cooking times, the higher was the internal temperature of the meat, along with a greater reduction of both ECOH and STEC. The average final internal temperature of the meat at the approximate geometric center ranged from 56.8 to 93.1°C. Microbial reductions of ca. 2.0 to 6.7 log CFU/g and ca. 2.6 to 6.2 log CFU/g were achieved for ECOH and STEC, respectively. Our data also revealed no differences in thermal inactivation of ECOH relative to the volume of oil used to cook nonbreaded cutlets. However, when cooking breaded cutlets, the use of more (30 ml) compared with less (15 ml) cooking oil resulted in greater reductions in pathogen numbers. To deliver about a 5.0-log reduction of ECOH and STEC, and to achieve the recommended internal temperature of 71.1°C, it was necessary to cook mechanically tenderized veal cutlets for at least 1.5 min per side on a preheated electric skillet set at 191.5°C and containing 15 ml of cooking oil. These data also established that cooking times and temperatures effective for inactivating serotype O157:H7 strains of E. coli in tenderized veal are equally effective against the additional six non-O157 Shiga toxin-producing strains investigated herein.

This study determined the prevalence, serotypes and virulence genes distribution of non-O157 Shiga toxin-producing Escherichia coli in meat products collected from butchers shops and supermarkets in Mansoura city, Egypt. We have characterized 18 non-O157 STEC strains among the identified 100 E. coli isolates recovered from the examined 87 meat product samples. The prevalence of non-O157 STEC strains in fresh beef, ground beef and beef burger samples were 11.1% (3/27), 16.7% (5/30), and 33.3% (10/30), respectively. The eighteen non-O157 STEC isolated strains were serotyped into seven (38.9%) O111:H8, six (33.3%) O26:H11, two (11.1%) O111:H–, and one (5.56%) for each of O55:H7, O126:H5 and O128:H2. PCR assays for different virulence genes showed that nine (50%), eleven (61.1%), and nine (50%) strains carry stx1, stx2, and eae genes, respectively. The distribution of shiga toxin genes among the isolated strains indicated that seven (38.9%) strains harbored stx1 only, nine (50%) strains harbored stx2 only, and two (11.1%) strains harbored both stx1 and stx2. The eae gene was present in association with five (27.8%), three (16.7%), and one (5.6%) strains that harbored stx1 only, stx2 only, and both stx1 and stx2, respectively. This study concluded that the examined meat products, particularly beef burger, consumed in Egypt are considerably contaminated with a variety of non-O157 STEC serotypes, and hence consumption of such products may constitute a potential health risk for consumers.

This study determined the extent that irradiation of fresh beef surfaces with an absorbed dose of 1 kGy electron (e-) beam irradiation might reduce the viability of mixtures of O157 and non-O157 verotoxigenic Escherichia coli (VTEC) and Salmonella. These were grouped together based on similar resistances to irradiation and inoculated on beef surfaces (outside flat and inside round, top and bottom muscle cuts), and then e-beam irradiated. Salmonella serovars were most resistant to 1 kGy treatment, showing a reduction of ≤1.9 log CFU/g. This treatment reduced the viability of two groups of non-O157 E. coli mixtures by ≤4.5 and ≤3.9 log CFU/g. Log reductions of ≤4.0 log CFU/g were observed for E. coli O157:H7 cocktails. Since under normal processing conditions the levels of these pathogens on beef carcasses would be lower than the lethality caused by the treatment used, irradiation at 1 kGy would be expected to eliminate the hazard represented by VTEC E. coli.

The behavior of enteroaggregative Escherichia coli (EAEC), non-O157 shiga toxin-producing E. coli (non-O157-STEC), enteroinvasive E. coli (EIEC), enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC) on mung bean seeds at 25±2 °C and during germination and sprouting of mung bean seeds at 20±2 ° and 30±2 °C and on mung bean sprouts at 3±2 °C was determined. When mung bean seeds were inoculated with EAEC, non-O157 STEC, EIEC, EPEC or ETEC strains, all these diarrheagenic E. coli pathotypes (DEPs) survived at least 90 days on mung bean seeds at 25±2 °C. All DEPs grew during germination and sprouting of seeds, reaching counts of approximately 5 Log and 7 Log CFU/g after 2 days at 20±2 ° and 30±2 °C, respectively. However, when the sprouts were inoculated after 1 day of seeds germination and stored at 20±2 ° or 30±2 °C, no growth was observed for any DEPs during sprouting at 20±2 °C per 9 d; however, a significant increase in the concentration of DEPs of approximately 0.7 log CFU/g was observed during sprouting at 30±2 °C after 1 day of sprout contamination. Refrigeration reduced the number of viable DEPs strains on sprouts after 10 days in storage; nevertheless, these decreases have no practical significance in the safety of the sprouts.

Shiga toxin-producing Escherichia coli (STEC) are important human pathogens, and attention to non-O157 serogroups has increased in recent years. Although cattle are normally considered the primary reservoir for STEC, recent illnesses associated with goat contact have indicated that these animals are important potential reservoirs for the organisms. The prevalence of STEC, particularly non-O157 serogroups, in U.S. goats has not been well described. Our objective was to determine the prevalence of six major non-O157 STEC serogroups in the feces of meat goats. Rectal contents from 296 goats were collected postevisceration at a slaughter plant in the southeastern United States over 9 days during a 12-week period from August through October 2012. Samples were enriched in E. coli broth, and DNA was extracted and used as template in an 11-gene multiplex PCR that detected six non-O157 serogroups (O26, O45, O103, O121, O111, and O145) and virulence genes. Samples were considered positive when at least one non-O157 STEC serotype was present with either stx 1 or stx 2. All six non-O157 serogroups were detected by PCR in our samples, and 14.5% of samples were positive for at least one serogroup. Prevalence of O26 was highest, with 6.4% of goat fecal samples positive. The prevalence of O45 was 3.4%, O103 was 4.4%, O111 was 4.1%, O121 was 1.4%, and O145 was 3.0%. Twenty-two (7.4%) of 296 fecal samples had more than one non-O157 serogroup detected in the feces. Two samples had evidence of three non-O157 STEC serogroups. Goats appear to be an important reservoir for non-O157 STEC, and further work to understand the characteristics, epidemiology, and ecology of STEC in these animals is warranted.

An increasing number of non-O157 Shiga toxin-producing Escherichia coli (STEC) infections and outbreaks have been reported. In this study, we evaluated the performance of ImmunoCard STAT!(®) (Meridian Bioscience, Inc., Cincinnati, OH, USA) as a method to screen stool specimens for STEC (O157 and non-O157). An in-house real-time PCR method was used as the "gold standard". We also evaluated the prevalence and clinical characteristics of STEC infections in the Alberta South West Zone. From July to November 2011, 819 stool specimens submitted for routine stool culture were tested. With our in-house real-time PCR, 7 O157:H7 and 10 non-O157 STEC isolates were identified for a total of 17 STECs. In comparison, ImmunoCard STAT!(®) identified a total of 6, resulting in a sensitivity and specificity of 35% and 99%, respectively (P0.05). Because of the low sensitivity, ImmunoCard STAT!(®) cannot be recommended as a routine screening test for STEC from enriched stool specimens. The rate of STEC positivity as detected by PCR was 2.08%, of which 0.86% was O157:H7 and 1.22% non-O157 STEC. Five of the 7 cases of STEC O157 infection experienced bloody diarrhea, and 1 developed hemolytic uremic syndrome.

Both high-fat and low-fat ground beef (percent lean:fat = ca. 70:30 and 93:7, respectively) were inoculated with a 6-strain cocktail of non-O157:H7 Shiga toxin-producing Escherichia coli (STEC) or a five-strain cocktail of E. coli O157:H7 (ca. 7.0 log CFU/g). Patties were pressed (ca. 2.54 cm thick, ca. 300 g each) and then refrigerated (4°C, 18 to 24 h), or frozen (-18°C, 3 weeks), or frozen (-18°C, 3 weeks) and then thawed (4°C for 18 h or 21°C for 10 h) before being cooked on commercial gas or electric grills to internal temperatures of 60 to 76.6°C. For E. coli O157:H7, regardless of grill type or fat level, cooking refrigerated patties to 71.1 or 76.6°C decreased E. coli O157:H7 numbers from an initial level of ca. 7.0 log CFU/g to a final level of ≤1.0 log CFU/g, whereas decreases to ca. 1.1 to 3.1 log CFU/g were observed when refrigerated patties were cooked to 60.0 or 65.5°C. For patties that were frozen or freeze-thawed and cooked to 71.1 or 76.6°C, E. coli O157:H7 numbers decreased to ca. 1.7 or ≤0.7 log CFU/g. Likewise, pathogen numbers decreased to ca. 0.7 to 3.7 log CFU/g in patties that were frozen or freeze-thawed and cooked to 60.0 or 65.5°C. For STEC, regardless of grill type or fat level, cooking refrigerated patties to 71.1 or 76.6°C decreased pathogen numbers from ca. 7.0 to ≤0.7 log CFU/g, whereas decreases to ca. 0.7 to 3.6 log CFU/g were observed when refrigerated patties were cooked to 60.0 or 65.5°C. For patties that were frozen or freeze-thawed and cooked to 71.1 or 76.6°C, STEC numbers decreased to a final level of ca. 1.5 to ≤0.7 log CFU/g. Likewise, pathogen numbers decreased from ca. 7.0 to ca. 0.8 to 4.3 log CFU/g in patties that were frozen or freeze-thawed and cooked to 60.0 or 65.5°C. Thus, cooking ground beef patties that were refrigerated, frozen, or freeze-thawed to internal temperatures of 71.1 and 76.6°C was effective for eliminating ca. 5.1 to 7.0 log CFU of E. coli O157:H7 and STEC per g.

Data about the behavior of non-O157 Shiga toxin-producing Escherichia coli (non-O157 STEC), enteroinvasive E. coli (EIEC), enterotoxigenic E. coli (ETEC), and enteropathogenic E. coli (EPEC) on seeds and alfalfa sprouts are not available. The behavior of STEC, EIEC, ETEC, and EPEC was determined during germination and sprouting of alfalfa seeds at 20 ± 2°C and 30 ± 2°C and on alfalfa sprouts at 3 ± 2°C. When alfalfa seeds were inoculated with STEC, EIEC, ETEC, or EPEC strains, all these diarrheagenic E. coli pathotypes (DEPs) grew during germination and sprouting of seeds, reaching counts of approximately 5 and 6 log CFU/g after 1 day at 20 ± 2°C and 30 ± 2°C, respectively. However, when the sprouts were inoculated after 1 day of seed germination and stored at 20 ± 2°C or 30 ± 2°C, no growth was observed for any DEP during sprouting at 20 ± 2°C or 30 ± 2°C for 9 days. Refrigeration reduced significantly (P < 0.0.5) the number of viable DEPs on sprouts after 20 days in storage; nevertheless, these decreases have no practical significance for the safety of the sprouts.

The non-O157 Shiga toxigenic Escherichia coli (STEC) serogroups most commonly associated with illness are O26, O45, O103, O111, O121, and O145. In the United States, these serogroups are considered adulterants in raw nonintact beef. To begin to understand the behavior of these pathogens in meat systems, we compared the thermal tolerance of acid-adapted cells of non-O157 STEC and O157:H7 STEC in a beef-derived broth. D58°C-values were determined for at least three strains per serogroup, and D54.6°C-values and D63.6°C-values were determined for one strain per serogroup. Each strain was grown to stationary phase in brain heart infusion broth (BHIB; pH 7.0) and inoculated into prewarmed BHIB in a shaking water bath for thermotolerance experiments at 54.6, 58.0, or 63.6°C (three trials per strain). Samples were heated for up to 160 min at 54.6°C, 3 min at 58.0°C, or 45 s at 63.6°C, with periodic sampling followed by rapid cooling and plating on modified Levine's eosin methylene blue agar. For each strain and temperature, the log CFU per milliliter was plotted versus time, and D-values were determined. Across all strains, the least and most heat tolerant STEC serogroups at 58°C were O145 and O157, respectively. D58°C-values in BHIB ranged from 0.44 min for an O145 strain to 1.42 min for an O157:H7 strain. D58°C-values for O157 STEC strains were significantly higher than those for at least one strain in each of the non-O157 STEC serogroups (P0.05) except for serogroup O103. At 54.6°C, the most heat-resistant STEC strain belonged to serogroup O103 and was significantly more heat tolerant than the O157:H7 strains (P0.05). Grouping the strains, there were no significant differences in heat tolerance between O157 and non-O157 STEC at 63.6°C (P ≥ 0.05). The z-values for non-O157 STEC strains were comparable to those for O157:H7 STEC strains (P ≥ 0.05), ranging from 4.10 to 5.21°C. These results suggest that thermal processing interventions that target destruction of E. coli O157:H7 may have adequate lethality against non-O157 STEC.

Four hundred and fifty beef animal hides and a similar number of carcasses were screened for STEC in 3 beef abattoirs over a 12 month period using PCR and culture based methods. 67% (301/450) of hides and 27% (122/450) of carcasses were STEC PCR positive. Forty isolates representing 12 STEC serotypes (O5:H-, O13:H2, O26:H11, O33:H11, O55:H11, O113:H4, O128:H8, O136:H12, O138:H48, O150:H2, O168:H8 and ONT:H11) and 15 serotype–virulotype combinations were identified. This study provides much needed non-O157 STEC surveillance data and also provides further evidence of bovines as a source of clinically significant STEC as well as identifying 3 emerging serotypes O5:H- ( eae -β1), O13:H2 ( eae -ζ), and O150:H2 ( eae -ζ) that should be considered when developing beef testing procedures for non-O157 STEC.

The prevalence and characteristics of non-O157 Shiga toxin-producing Escherichia coli (STEC) in retail ground meat from the Washington D.C. area were investigated in this study. STEC from 480 ground beef and pork samples were identified using PCR screening followed by colony hybridization. The STEC isolates were serogrouped and examined for the presence of virulence genes (stx1, stx2, eae and hlyA), and antimicrobial susceptibility. PFGE was used to identify the clonal relationships of STEC isolates, and PCR-RFLP was employed to determine stx subtypes. In addition, the cytotoxicity of STEC isolates was determined using a Vero cell assay. STEC were identified in 12 (5.2%) of 231 ground pork and 13 (5.2%) of 249 ground beef samples. Among 32 STEC isolates recovered from the 25 samples, 12 (37.5%) carried stx2dact and 7 (21.9%) carried hlyA, but none carried eae. Nine isolates were identified as O91, and 17 (53.1%) isolates were resistant to two or more antimicrobials. Verotoxicity was detected in 26 (81.3%) of the STEC isolates. Thus, the retail ground meat was contaminated with a heterogeneous population of non-O157 STEC, some of which were potential human pathogens.

Non-O157 Shiga toxin-producing Escherichia coli (non-O157 STEC) strains have emerged as important foodborne pathogens worldwide. Non-O157 STEC serogroups O26, O45, O103, O111, O121, and O145 have been declared as adulterants in beef by the U. S. Department of Agriculture Food Safety and Inspection Service. While documentation is limited, treatments including heat and acid that have been shown to inactivate E. coli O157:H7 will likely also destroy non-O157 STEC; however, non-O157 STEC strains show variability in their responses to stress. It has been shown that non-O157 STEC may survive in fermented sausages and cheeses, and treatments such as high pressure may be necessary to eliminate non-O157 STEC from these products. The mechanisms used by non-O157 STEC to resist acid environments are similar to those used by O157:H7 strains and include the acid tolerance response, the oxidative system, and the glutamate and arginine decarboxylase systems. However, one study demonstrated that some non-O157 STEC strains utilize a chaperone-based acid stress response (HdeA and HdeB) to combat acidic conditions, which is lacking in E. coli O157:H7. Genomic studies suggest that while non-O157 STEC can cause diseases similar to those caused by E. coli O157:H7, O157 and non-O157 STECs have different evolutionary histories. Non-O157 STECs are a heterogeneous group of organisms, and there is currently a limited amount of information on their virulence, fitness, and stress responses, rendering it difficult to draw firm conclusions on their behavior when exposed to stress in the environment, in food, and during processing.

The objective of this research was to compare the effectiveness of two application methods (dip versus spray) of 4.4% lactic acid for reducing pathogens on inoculated beef trim and in ground beef. Beef trim inoculated with cocktail mixtures of E. coli O157:H7, non-O157 Shiga toxigenic E. coli (STEC), or Salmonella (10(5) to 10(6) CFU/g) at separate times was subjected to five treatments: lactic acid spray (LS), lactic acid dip (LD), water spray (WS), water dip (WD), and untreated control (CTL). Intervention effectiveness for pathogen reduction was measured at 1 and 20 h after treatment on beef trim. Trim was then ground and intervention effectiveness was measured 1 h, 24 h, 72 h, and 7 days after grinding. The LD treatment reduced all pathogens significantly (P0.05); E. coli O157:H7 was reduced by 0.91 to 1.41 log CFU/g on beef trim and ground beef, non-O157 STEC by 0.48 to 0.82 log CFU/g, and Salmonella by 0.51 to 0.81 log CFU/g. No other treatment significantly reduced any pathogen, although the WD treatment noticeably reduced (P0.05) both E. coli O157:H7 and non-O157 STEC populations compared with the CTL. The LS treatment reduced E. coli O157:H7 and Salmonella by up to 0.5 log CFU/g on beef trim, but these reduced counts did not significantly differ (P0.05) from the CTL counts. Overall, the LD treatment was most effective for reducing all pathogens and is the best of these options for improving the safety of beef trim and subsequently produced ground beef.

This study was conducted to investigate the growth of non-O157 Shiga toxin-producing Escherichia coli (STEC) in spinach leaves and to develop kinetic models to describe the bacterial growth. Six serogroups of non-O157 STEC, including O26, O45, O103, O111, O121, and O145, were used in the growth studies conducted isothermally at 4, 8, 15, 20, 25, 30, and 35 °C. Both STEC and background microflora were enumerated to develop kinetic models. Growth of STEC in spinach leaves was observed at elevated temperatures (15–35 °C), but not at 4 and 8 °C. This study considered the dynamic interactions between the STEC cells and the background microflora. A modified Lotka–Volterra and logistic equation was used to simulate the bacterial growth. In combination with an unconstrained optimization procedure, the differential growth equations were solved numerically to evaluate the dynamic interactions between the STEC cells and the background microflora, and to determine the kinetic parameters by fitting each growth curve to the growth equations. A close agreement between the experimental growth curves and the numerical analysis results was obtained. The analytical results showed that the growth of STEC in spinach leaves was unhindered when the population was low, but the growth was suppressed by the background microflora as the STEC population approached the maximum population density. The effect of temperature on the growth of both STEC and background microflora was also evaluated. Secondary models, evaluating the effect of temperature on growth rates, were also developed. The estimated apparent minimum growth temperature for STEC was 11 °C in commercial spinach leaves. The methodology and results of this study can be used to examine the dynamic interactions and growth between different bacteria in foods, and to conduct risk assessments of STEC in spinach leaves.

5 páginas, 1 figura, 2 tablas.-- et al., Shiga toxin-producing Escherichia coli (STEC) are an important group of emerging pathogens, with ruminants recognised as their main natural reservoir. The aim of this longitudinal study was to provide information on the prevalence and existence of seasonal variation in the occurrence of non-O157 STEC in four sheep flocks over a 12-month period. A total of 504 faecal samples from 48 adult sheep in four flocks were collected and examined for STEC using both phenotypic and genotypic methods. STEC were isolated from 407 (80.8%) faecal samples representing all the animals sampled. The overall monthly prevalence of STEC varied between 71.1 and 94.4% and no seasonal variation in the occurrence of STEC could be observed over the study period. A total of 521 STEC isolates were characterised. The PCR procedure indicated that 275 (52.8%) isolates carried the stx1 gene, 44 (8.4%) carried the stx2 gene and 202 (38.8%) contained both of these genes. The eae and ehxA genes were detected in 4 (0.8%) and 368 (70.6%) isolates, respectively. The isolates belonged to 28 O serogroups, although 72.4% were restricted to only 10 serogroups (O5, O6, O76, O87, O91, O123, O128, O146, O166 and O176). None of the isolates belonged to the O157 STEC serogroup. STEC isolates of serogroups O33, O53, O105 and O162 have not previously been reported in sheep. This is the first study to report the maintenance of high frequencies of non-O157 STEC infection in sheep flocks over long time periods., This study was supported by grants from the Fondo de Investigación Sanitaria (G03-025-COLIRED-O157 and REIPI-RD06/0008-1018), from the Ministerio de Educación y Ciencia (AGL-2008-02129), from the Xunta de Galicia (PGIDIT05BTF26101P, PGIDIT065TAL26101P and 07MRU036261PR) and from the Junta de Extremadura and FEDER (3PR05A009-III Plan Regional de Investigación). S. Sánchez acknowledges the Consejería de Educación y Ciencia de la Junta de Comunidades de Castilla-La Mancha and Fondo Social Europeo for his research fellowship (09/02- C). R. Martínez acknowledges the Junta de Extremadura for his research fellowship (PRE06053). A. Mora acknowledges the Ramón y Cajal programme from the Ministerio de Educación y Ciencia.

The rapid and economical detection of human pathogens in animal and food production systems would enhance food safety efforts. An instrument based on gas sensors coupled with an artificial neural network (ANN) was developed for the detection of and differentiation between laboratory isolates of Escherichia coli O157:H7 and non-O157:H7 E. coli. The purpose of this study was to use field isolates of E. coli to further evaluate the sensor system. This gas sensor-based, computer-controlled detection system was used to monitor gas emissions from 12 isolates of E. coli O157:H7 and 8 non-O157:H7 E. coli isolates. A standard concentration of each isolate was grown in 10 ml of nutrient broth at 37 degrees C for 16 h, and gas sampling was carried out every 5 min. Readings were continuously plotted to generate gas signatures. A back-propagation ANN algorithm was used to interpret the gas patterns. By analysis of the response of the ANN, the sensitivity and specificity of the instrument were calculated. Detectable differences between the gas signatures of the E. coli O157:H7 isolates and the non-O157:H7 isolates were observed. The instruments degree of sensitivity was high for E. coli O157:H7 isolates, but a lower degree of accuracy was observed for non-O157:H7 isolates because of increased strain variation. The sensitivity of the detection system was improved by the normalization of the data generated from the gas sensors. Because of its ability to detect differences in gas patterns, this instrument has a broad range of potential food safety applications.

The increasing frequency of Escherichia coli O157:H7 outbreaks, especially in acidic foods, raises the concern of an acid tolerance response (ATR). Organic acids can be present in processed and preserved foods: shifts in the acid levels of foods due to these acids may allow E. coli to adapt and later tolerate pH levels that would normally inactivate the organism. The effect of temperature and agitation on the ATRs of three E. coli O157:H7 and two non-O157:H7 isolates were determined. Triggered at pH 5.0, the adaptive system of the ATR allowed for up to nearly 1,000-fold enhanced survival of E. coli O157:H7 cells in some cases compared to survival of nonadapted cells at pH 4.0. E. coli O157:H7 isolates revealed greater acid tolerance responses when incubated statically at 32 degrees C, whereas the non-O157:H7 E. coli isolates exhibited a greater acid tolerance response with orbital agitation at 25 degrees C. The magnitude of response changed over the incubation period.

12 www.thelancet.com/infection Vol 12 January 2012 4 Bettelheim KA, Faiers M, Shooter RA. Serotypes of Escherichia coli in normal stools. Lancet 1972; 300: 1224–26. 5 Pennington H. Escherichia coli O104, Germany 2011. Lancet Infect Dis 2011; 11: 652–53. The full investigation of outbreaks is essential to understand their causes. In the Comment about the German O104 outbreak, Hugh Pennington notes that four of nine outbreaks of intestinal infections linked to bean sprouts were caused by E coli O157. These bean sprouts could have been contaminated with animal faecal matter, and other STEC serotypes might have been involved. The development of a medium that will select for all STEC rather than specifi cally for one or a few could help our understanding of human infection and is needed urgently. An organisation such as the European Centre for Disease Prevention and Control should initiate such an endeavour.