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Non-O157 Shiga toxin (stx)–producing Escherichia coli (STEC) is recognized as an important human diarrheal pathogen. Cattle are the principal reservoirs of STEC, although other animals can be carri...

Background:This research work focused on ascertaining the presence of virulence and antibiotic-resistant genes inEscherichia coli(E. coli) O157 and non-O157 recovered from drinking water sources.Methods:Identification ofE. coliO157 and non-O157 was carried out using standard serological and PCR techniques. Virulence genes (rfbO157,fliCH7,stx1,stx2,eaeandhlygenes) and antibiotic-resistant gene (BlaTEM) were detected using PCR method on selected isolates (n= 15) from different water sources which demonstrated multiple antibiotic-resistance in a previous study.Results:The serological identification result revealed that a total of 68 out of 382E. coliisolates, recovered in a previous work, were identified as a presumptiveE. coliO157. These included 19.1 %, 21.7 %, 33.3 %, 14.3 % and 9.1 % ofE. coliisolates from wells, boreholes, sachets, streams and pipe-borne respectively. Statistical analysis revealed that there was no significant difference in the frequency ofE. coliO157 from the different water sources (p> 0.05). Also, there was a statistically significant positive correlation between theE. coliisolates andE. coliO157 (Pearson’sr= 0.996). Detection of virulence and antibiotic-resistant genes showed that only 46.7 %, 33.3 %, 33.3 %, 93.3 %, and 66.7 % carriedrfbO157,fliCH7,stx1,stx2 andrpoSgene respectively. In contrast, all the isolates possessedhlyand BlaTEMgenes but none hadeaegene.Conclusion:The presence of one or combination of these genes in these isolates depicts their virulence and resistance nature.

This study aimed to detect presence of RpoS gene in Escherichia coli O157:H7 and non-O157 and investigate their survival pattern in different water treatment methods. A total of fifteen serologically and molecularly identified E. coli was selected from a previous work, out of which eight were Escherichia coli O157 and seven were E. coli non-O157. From among these, S30 and S89 identified isolates served as presentative E. coli O157:H7 and non-O157 respectively for survival studies. The water treatment methods used employed included: use of silver, lime, storage, acidification (low pH), high temperature and Moringa oleifera. Survival pattern of the test organisms under the influence of these methods were carried out using standard techniques. Molecular detection of stress response gene, RpoS, in the fifteen (15) test organisms was performed following manufacturer’s instruction. Results showed that for both test organisms, silver was bactericidal at high concentration while storage allows their survival up till 21 days though with a reduction in cfu. Both organisms showed low survival at pH 9 while E. coli O157:H7 and non-O157 could survive at pH 4 and 6 respectively. E. coli O157:H7 survived better than non-O157 at high concentration of lime. While both survived at low temperature, E. coli O157:H7 survive better at 60oC. Sunlight and chlorine showed mild and complete bactericidal action respectively with increased exposure time for both test organisms. Moringa oleifera was only effective at a reduced concentration on the two organisms. Detection of RpoS genes showed that only 66.7% carried the gene in them while 33.3% did not. Findings from this study show that the possession of stressed genes in bacteria causing waterborne disease could allow these organisms to survive water treatment methods adopted in many under developed countries or rural communities. This suggests a threat to health of these communities.

The objective of this study was to quantify the frequency, distribution, and variability of fecal shedding and super-shedding of Shiga toxin–producing Escherichia coli (STEC) serogroups O26, O45, O...

This study examined the biofilm-forming ability of six non-O157 Shiga-toxin-producing Escherichia coli (STEC) strains: O116:H21, wzx-Onovel5:H19, O129:H21, O129:H23, O26:H11, and O154:H10 on stainless steel coupons after 24, 48, and 72 h of incubation at 22 °C and after 168 h at 10 °C. The results of crystal violet staining revealed that strains O129:H23 and O154:H10 were able to form biofilms on both the submerged surface and the air–liquid interface of coupons, whereas strains O116:H21, wzx-Onovel5:H19, O129:H21, and O26:H11 formed biofilm only at the air–liquid interface. Viable cell counts and scanning electron microscopy showed that biofilm formation increased (p < 0.05) over time. The biofilm-forming ability of non-O157 STEC was strongest (p < 0.05) at 22 °C after 48 h of incubation. The strongest biofilm former regardless of temperature was O129:H23. Generally, at 10 °C, weak to no biofilm was observed for isolates O154:H10, O116:H21, wzx-Onovel5:H19, O26:H11, and O129:H21 after 168 h. This study found that temperature affected the biofilm-forming ability of non-O157 STEC strains. Overall, our data indicate a high potential for biofilm formation by the isolates at 22 °C, suggesting that non-O157 STEC strains could colonize stainless steel within food-processing facilities. This could serve as a potential source of adulteration and promote the dissemination of these potential pathogens in food.

Shiga toxin producing Escherichia coli is a contaminant of food and water that causes a diarrheal syndrome followed by more severe disease of the kidneys and symptoms of the central nervous system in humans. The isolation of Shiga toxin producing Escherichia coli (STEC) from diarrheic and apparently health calves is difficult due to lack of differential phenotypic characteristics from nonpathogenic Escherichia coli. The improvement of molecular technology allows identification of both toxin and serogroup specific genetic determinants. In this study, 300 fecal samples from diarrheic and apparently healthy calves were screened for STEC using PCR targeting Shiga toxin determinants. In addition routine culture methods for isolating O157 and non O157 STEC were also performed. The screening assays of serotyping isolates revealed 7 (4.1%) of O157H7, 156 (92.8%) of non O157 and 5 (3.1%) for untypable strains. These included STEC serotypes of O157H7 and O26 from diarrheic samples, and O78, O55 and O126 from apparently healthy calves. The high rate of STEC isolation and the diversity of STEC serogroup from calves focus the light on the importance of calves as reservoir of E. coli as well as motivate us to improve biosecurity measures in dairy farms.

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

The objectives of the current study were detection and characterization of the isolated Escherichia coli non O157 using PCR assay. A total of 90 cheese samples high salt soft cheese, Kareish cheese and Tallaga cheese were examined for E. coli non O157 using modified vancomycin- trypticase soy broth and Sorbitol MacConkey agar plates. Serodiagnosis of E. coli non-O157 has been done using slide agglutination test. Toxigenic E. coli non-O157 isolates were detected using PCR assay. Results postulated that the detection rate of non O157 using biochemical technique was 63.33% in Kariesh cheese, 20% in high salt soft cheese cheese, and 33.33% in Tallaga cheese. Ten different serotypes of E. coli non O157 have been distributed as following O1 (4.44%), O18 (1.11%), O20 (1.11%), O25 (1.11%), O26 (3.33%), O125 (1.11%), O126 (1.11%), O127 (1.11%) and untyped E. coli (18.89%). Regarding PCR results, serotypes group (O1) was positive for Stx2 gene, (O1 and O20) were positive for both Stx2 and hly genes, one serotype group O25 was positive for eaeA gene, one serotype group O26 was positive for hly gene while no detection for Stx1and STa genes. From the aforementioned data, attention must be paid to the problems of E. coli non O157 in foods. Consequently, more restriction and preventive measures should be taken in milk herds, milk production and dairy factories in respect to quality control sanitation and health care.

Background The implementation by diagnostic laboratories in England of polymerase chain reaction (PCR) to screen faecal specimens for Shiga toxin-producing Escherichia coli (STEC) has resulted in a significant increase in notifications mainly due to non-O157 strains. The purpose of this study was to develop an approach to public health risk assessment that prioritizes follow-up to cases caused by haemolytic uraemic syndrome (HUS) associated E. coli (HUSEC) strains and minimizes unnecessary actions. Methods Epidemiological and microbiological data were prospectively collected from 1 November 2013 to 31 March 2017 and used to compare three risk assessment approaches. Results A history of HUS/bloody diarrhoea/age under 6 years and faecal specimens positive for stx-predicted HUSEC with a diagnostic accuracy of 84% (95% CI; 81–88%). STEC isolated by Gastrointestinal Bacteria Reference Unit (GBRU) and stx2 and eae positive predicted HUSEC with a diagnostic accuracy of 99% (95% CI; 98–100%). Risk assessment combining these two tests predicts the most efficient use of resources, predicting that 18% (97/552) of cases would be eligible for follow-up at some stage, 16% (86/552) following local stx PCR results, 1% (7/552) following GBRU results of stx2 and eae status and 0.7% (4/552) following whole-genome sequencing. Follow-up could be stopped in 78% (76/97) of these cases, 97% (74/76) following second stage risk assessment. Conclusions This three-stage risk assessment approach prioritizes follow-up to HUSEC and minimizes unnecessary public health actions. We developed it into the algorithm for public health actions included in the updated PHE Guidance for management of STEC published in August 2018.

Resistance of Shiga toxin-producing Escherichia coli (STEC) O157:H7 and serogroups O103, O26 and O145 to synthetic gastric fluid (SGF, pH 1.5) were investigated during frankfurter storage. Pathogens were inoculated (5 ± 1 log(10 )cfu g(−1)) on frankfurters and frankfurters were stored at 4 °C for 75 days in vacuum packages. Population changes of the competitive flora and STEC, changes in the pH of the frankfurters and resistance of STEC to SGF were monitored on days 0, 15, 30, 45, 60 and 75 of frankfurter storage. Direct synthetic gastric fluid (DSGF) challenges were also conducted to assess pathogen resistance without being effected by frankfurters, by inoculating pathogen cultures directly into SGF. Results showed that acid resistance of O145 and O26 was stronger than that of O103 and O157 during frankfurter storage. Resistance of O103 to SGF was better than that of O157 during frankfurter storage but, was similar to that of O157 during DSGF challenges. Results indicate that acid resistance of some strains of STEC pathogens might differentiate during storage of frankfurters. Different resistance capabilities to SGF were observed in the STEC strains when inoculated and stored on frankfurters than directly inoculated in the SGF.

Forty-eight Escherichia coli strains were chosen due to variable detection of stx or serogroup by PCR. Although all strains were initially determined to be Shiga toxin-producing Escherichia coli (STEC), their genomes revealed 11 isolates carrying stx1a, stx1b, stx2a, and/or stx2b. Assembled genome sizes varied between 4,667,418 and 5,556,121 bp, with N50 values between 79,648 and 294,166 bp and G+C contents between 50., Forty-eight Escherichia coli strains were chosen due to variable detection of stx or serogroup by PCR. Although all strains were initially determined to be Shiga toxin-producing Escherichia coli (STEC), their genomes revealed 11 isolates carrying stx1a, stx1b, stx2a, and/or stx2b. Assembled genome sizes varied between 4,667,418 and 5,556,121 bp, with N50 values between 79,648 and 294,166 bp and G+C contents between 50.3% and 51.4%.

Shiga toxin-producing Escherichia coli, especially E. coli O157 is an important foodborne pathogen capable ofcoexisting in multispecies biofilms found in almost all the natural environments. Biofilm cells are usually more resistant than planktonic cells against environmental stresses. Thus, E. coli O157 in biofilms is a serious food safety concern. This study describes the characterization of a bacteriophage FP43 isolated from bovine intestine and the ability of FP43 to inhibit and remove multi species biofilms of E. coli O157 strain 196 and non-O157 strain 104. Phage FP43 has a short latent period of 15 min and a large burst size of 98 PFU/cell, with great stability at temperatures ranging from 4 to 60°C and pH from 4 to 9. To evaluate the effects of FP43 on E. coli,in microplate, biofilm formation was determined by crystal violet stainingas well as viable counts of biofilm and planktonic cells by conventional plating method. Phage FP43 decreased biofilm adhesionof E. coli cells with equal proportions of E. coli O157 and non-O157 by 82.4%. Viable counts were also reduced by 2.76 and 2.85 log in E. coli O157 and total biofilm cells after 6-h infection, respectively, compared with control. In planktonic cells, E. coli O157 and total counts decreased by 3.44 and 3.62 log after a 4-h phage treatment, respectively. Moreover, after a 6-h exposure to phage FP43, more than 60% of established biofilms were removed, and E. coli O157 and total viable counts in biofilm were decreased by 2.07 and 1.93 log, respectively. These findings suggest that phage FP43 seems to be a potential agent against E. coli O157 in multi species biofilms.

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.

Shiga toxin-producing Escherichia coli (STEC) cause illnesses ranging from mild diarrhea to ischemic colitis and hemolytic uremic syndrome (HUS), serogroup O157 is the most common cause. We describe the epidemiology and transmission routes for U.S. STEC outbreaks during 2010–2017. Health departments reported 466 STEC outbreaks affecting 4769 persons, 459 outbreaks had a serogroup identified (330 O157, 124 non-O157, 5 both). Among these, 361 (77%) had a known transmission route: 200 foodborne (44% of O157 outbreaks, 41% of non-O157 outbreaks), 87 person-to-person (16%, 24%), 49 animal contact (11%, 9%), 20 water (4%, 5%), and 5 environmental contamination (2%, 0%). The most common food category implicated was vegetable row crops. The distribution of O157 and non-O157 outbreaks varied by age, sex, and severity. A significantly higher percentage of STEC O157 than non-O157 outbreaks were transmitted by beef (p = 0.02). STEC O157 outbreaks also had significantly higher rates of hospitalization and HUS (p &lt, 0.001).

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.

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

Summary Non-O157 Shiga toxin-producing Escherichia coli (STEC) are a frequent cause of STEC-related infections such as diarrhoea. Fermentation by presumptive probiotic Lactobacillus plantarum strain B411 isolated from cereal fermentation was investigated to prevent the growth of acid-adapted (AA) and non-acid-adapted (NAA) non-O157 STEC in traditionally fermented maize gruel, a widely used complementary food in Africa. L. plantarum strain B411 possessed probiotic characteristics and antimicrobial activity against selected pathogenic bacteria. Growth of AA and NAA non-O157 STEC strains was substantially inhibited by 3.6 and 4.8 log reductions, respectively, in the maize gruel fermented with the L. plantarum B411, while their growth was only inhibited by 1.0 and 1.2 log reductions, respectively, by traditional fermentation alone. Inclusion of fermentative strains of L. plantarum exhibiting probiotic activity is a feasible method to ensure safety of traditionally fermented African cereal porridges through inhibition of non-O157 STEC.

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.

peer-reviewed Phenotypic, chemotaxonomic and genotypic data from 12 strains of Escherichia coli were collected, including carbon source utilisation profiles, ribotypes, sequencing data of the 16S–23S rRNA internal transcribed region (ITS) and Fourier transform-infrared (FT-IR) spectroscopic profiles. The objectives were to compare several identification systems for E. coli and to develop and test a polyphasic taxonomic approach using the four methodologies combined for the sub-typing of O157 and non-O157 E. coli. The nucleotide sequences of the 16S–23S rRNA ITS regions were amplified by polymerase chain reaction (PCR), sequenced and compared with reference data available at the GenBank database using the Basic Local Alignment Search Tool (BLAST) . Additional information comprising the utilisation of carbon sources, riboprint profiles and FT-IR spectra was also collected. The capacity of the methods for the identification and typing of E. coli to species and subspecies levels was evaluated. Data were transformed and integrated to present polyphasic hierarchical clusters and relationships. The study reports the use of an integrated scheme comprising phenotypic, chemotaxonomic and genotypic information (carbon source profile, sequencing of the 16S–23S rRNA ITS, ribotyping and FT-IR spectroscopy) for a more precise characterisation and identification of E. coli. The results showed that identification of E. coli strains by each individual method was limited mainly by the extension and quality of reference databases. On the contrary, the polyphasic approach, whereby heterogeneous taxonomic data were combined and weighted, improved the identification results, gave more consistency to the final clustering and provided additional information on the taxonomic structure and phenotypic behaviour of strains, as shown by the close clustering of strains with similar stress resistance patterns. The authors acknowledge the financial contribution of the Spanish INIA, the Research Council of Norway (project 178230/I10), Foundation for Levy on Foods, the Norwegian Research Fees Fund for Agricultural Goods, the Norwegian Independent Meat and Poultry Association, Nortura SA and NHO Matog Landbruk.

Background: The Polyskope 1.0 Multiplex Assay is a novel test to simultaneously detect Escherichia coli O157, non-O157 Shiga Toxin-Producing E. coli (STEC), Listeria monocytogenes, and Salmonella species in a single enrichment using real-time PCR. Objective: A Performance Tested MethodSM study was conducted to validate Polyskope 1.0 for inclusivity and exclusivity as well as a matrix comparison study. Method: This assay was evaluated in an unpaired independent validation study compared with reference methods according to AOAC INTERNATIONAL validation guidelines. Polyskope 1.0 evaluated raw ground beef (25 g), deli turkey (25 g), baby spinach (25 g), and stainless-steel environmental surface sponges (4 × 4 in. test area) after inoculation with a suspension of the three target microorganisms. All matrices were compared with appropriate reference methods from the U.S. Food and Drug Administration Bacteriological Analytical Manual, U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook, or International Organization for Standardization standards. Results: Polyskope 1.0 demonstrated no statistically significant differences between candidate and reference method results or between presumptive and confirmed results for three food matrices and one environmental surface. Results from inclusivity and exclusivity evaluations indicated the test method can accurately detect the target analytes and excluded all nontarget organisms. No differences were observed with the stability or lot-to-lot evaluations. Polyskope 1.0 demonstrated robustness by remaining unaffected by small variations in method parameters, which had no statistically significant effect on the results for all eight variations. Conclusions and Highlights: Polyskope 1.0 was shown to be a specific, highly accurate, and robust method for the detection of Listeria monocytogenes, Salmonella species, non-O157 STECs, and E. coli O157 across four matrices.

Shiga toxin-producing Escherichia coli (STEC) serogroups O26, O45, O103, O111, O121, and O145, referred to as the top six non-O157 serogroups, are responsible for more than 70% of human non-O157 STEC infections in North America. Cattle harbor non-O157 strains in the hindgut and shed them in the feces. The objective of this study was to use the U.S. Food and Drug Administration (FDA) E. coli identification (ECID) DNA microarray to identify the serotype, assess the virulence potential of each, and determine the phylogenetic relationships among five of the six non-O157 E. coli serogroups isolated from feedlot cattle feces. Forty-four strains of STEC, enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), or putative nonpathotype E. coli (NPEC) of cattle origin and five human clinical strains of EHEC were assayed with the FDA-ECID DNA microarray. The cattle strains harbored diverse flagellar genes. The bovine and human strains belonging to serogroups O26, O45, and O103 carried stx

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.

In this study, specific and rapid enrichment and growth conditions for the most important, classic non-O157 enterohemorrhagic Escherichia coli (EHEC) serogroups were assessed. Three enrichment broth types, namely, EC medium with MUG broth, BRILA broth, and mTSB broth with novobiocin, were compared to identify the optimum enrichment broth for EHEC isolation. Four kinds of selective media, namely, ENDO agar, Chromocult agar, TBX agar, and CHROMagar TM STEC medium, were compared to identify the optimum one for non-O157 EHEC isolation. The results suggested that incubation in EC medium with MUG broth at 42℃ for 20 h is optimum for the enrichment of non-O157 EHEC. TBX agar was identified to have the highest specificity among the tested media. Consequently, a combination of complementary selective media according to serotype must be considered for comprehensive isolation of specific EHEC.

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.

We compared 47 enterohemorrhagic Escherichia coli (EHEC) O157 isolates with 184 EHEC non-O157 isolates from Korean patients with diarrhea. In the O157 group, the strains harboring both Shiga toxin genes (stx1 and stx2) were detected with highest frequency, whereas the strains harboring only stx1 gene were most frequently detected in the non-O157 group. Eight virulence genes (eaeA, hlyA, ehx, iha, efa1, tir, toxB, and espA) were found to show a higher frequency of occurrence in the O157 group than in the non-O157 group. In addition, the symptom of bloody diarrhea was exhibited at a higher rate in the O157 group (51.1%) than in the non-O157 group (16.8%). Our findings demonstrate that EHEC O157 strains are more frequently implicated in cases of bloody diarrhea in the Korean population than EHEC non-O157 strains.

In August 2016, an outbreak of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) with 237 cases occurred in the Helsinki metropolitan area, Finland. Gastroenteritis cases were reported at 11 events served by one catering company. Microbiological and epidemiological investigations suggested rocket salad as the cause of the outbreak. STEC ONT:H11 and EPEC O111:H8 strains isolated from food samples containing rocket were identical to the patient isolates. In this outbreak, the reported symptoms were milder than considered before for STEC infection, and the guidelines for STEC control measures need to be updated based on the severity of the illness. Based on our experience in this outbreak, national surveillance criteria for STEC have been updated to meet the practice in reporting laboratories covering both PCR-positive and culture-confirmed findings. We suggest that EPEC could be added to the national surveillance since diagnostics for EPEC are routinely done in clinical laboratories.

Non-O157 strains of Shiga toxin-producing Escherichia coli (STEC) are more common causes of acute diarrhea than the better-known O157 strains and have the potential for large outbreaks. This systematic review of the literature identified 129 serogroups as well as 262 different O and H antigen combinations of STEC in cases of epidemic and sporadic disease worldwide. Excluding the results from a single large outbreak of STEC O104:H4 in Germany and France in 2011, the reported frequency of dysenteric illness in patients was 26% (119 of 464) for epidemic disease and 25% (646 of 2588) for sporadic cases. Hemolytic uremic syndrome was identified in 14% of epidemic disease cases and 9% of sporadic illness cases. With the increasing use of PCR-based diagnostics, STEC strain identification may not be possible. Rapid diagnostics are needed for STEC infections to aid the clinician while allowing epidemiologists the opportunity to identify outbreaks and to trace the source of infection. Keywords: Shigatoxin producing E. coli, hemolytic uremic syndrome, dysentery

Microbiological safety of beef products can be protected by application of antimicrobial interventions throughout the beef chain. This study evaluated a commercial prototype antimicrobial intervention comprised of lytic bacteriophages formulated to reduce O157 and non-O157 Shiga-toxigenic Escherichia coli (STEC) on beef cattle hide pieces, simulating commercial pre-harvest hide decontamination. STEC reduction in vitro by individual and cocktailed phages was determined by efficiency of plating (EOP). Following STEC inoculation onto hide pieces, the phage intervention was applied and hide pieces were analyzed to quantify reductions in STEC counts. Phage intervention treatment resulted in 0.4 to 0.7 log10 CFU/cm2 (p < 0.01) E. coli O157, O121, and O103 reduction. Conversely, E. coli O111 and O45 did not show any significant reduction after application of bacteriophage intervention (p > 0.05). Multiplicity of infection (MOI) evaluation indicated E. coli O157 and O121 isolates required the fewest numbers of phages per host cell to produce host lysis. STEC-attacking phages may be applied to assist in preventing STEC transmission to beef products.

The aim of this study was to assess the performance of the Diatheva STEC FLUO and BAX System real-time PCR assays for detection of Shiga toxin-producing Escherichia coli (STEC) (stx1/stx2 and eae target genes) and O-group identification in ground beef and bean sprout samples. Ground beef (325 g or 25 g) and mung bean sprout (25 g) samples were inoculated with ~ 10 CFU of the “top five” STEC (O157:H7, O26, O103, O111, and O145 as specified in EU regulation ISO13136:2012), enriched using different broths and incubation temperatures, and tested using the Diatheva and BAX real-time PCR assays. In ground beef, both molecular methods were able to detect the “top five” STEC, and lower Ct values were observed for the Diatheva kits compared to BAX System. The O111-contaminated samples gave negative results with both methods using mTSB + novobiocin for enrichment. In bean sprouts, both methods provided positive results, although detection was not possible using mTSB + acriflavin/cefsulodin/vancomycin for enrichment. In conclusion, the Diatheva and BAX methods detected the “top five” STEC in ground beef and bean sprouts when inoculated at low levels. Both assays provided equivalent results in terms of performance and reliability. Thus, the Diatheva kits are comparable to reference STEC-detection methods and could be used by the food industry to reliably detect the “top five” STEC.

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.

Virulence markers in Shiga toxin-producing Escherichia coli (STEC) and their association with diseases remain largely unknown. This study determines the importance of 44 genetic markers for STEC (O157 and non-O157) from human clinical cases and their correlation to disease outcome. STEC isolated from a cattle surveillance program were also included. The virulence genes tested were present in almost all O157:H7 isolates but highly variable in non-O157 STEC isolates. Patient age was a significant determinant of clinical outcome.

RAPID-B® is a high performance, integrated microbiology/infectious disease diagnostic system. The system uses hardware and software that are specifically designed for optimal detection using custom, immuno-based reagents designed to react to cell surface antigens of the target bacteria. The Vivione Bioscience RAPID-B Escherichia coli O157 and non-O157 Shiga toxin-producing E. coli (STEC) kits were validated alongside the U.S. Department of Agriculture, Food Safety and Inspection Service (FSIS), Microbiology Laboratory Guidebook (MLG) 5.07 (for E. coli O157) and FSIS MLG 5B.04 (fornon-O157 STEC) reference methods for the detection of E. coli O157 and STEC. The matrixes, ground beef and beef trim, were inoculated with appropriate CFU/test portion of E. coli O157 and STEC so as to generate fractional positives results, 5 to 15 positives out of 20 inoculated samples. Samples were enriched in prewarmed Brain Heart Infusion broth at 42 ± 1°C for 6.5–7.5 h or 8.5–9.5 h depending on thesample size. All samples were confirmed using the MLG reference method, regardless of initial screen result. The RAPID-B test methods were statistically equivalent to the reference method for the detection ofE. coli O157 and STEC in all testedsamples. Inclusivity and exclusivity testing of the RAPID-B methods showed 100% specificity for both kits. Finally, the RAPID-B test methods were shown to be robust when variations were applied to enrichment time, broth temperature, and vortexing time.

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.

Fermented sausages have been identified as source of several outbreaks of Shiga toxin-producing Escherichia coli (STEC). Illnesses linked to non-O157 STEC serotypes appear to be on the rise worldwide, and serogroup O26 is the second most reported in Europe after O157. However, data on the behavior of serogroup O26 in food are rare, so that the aim of this study was to investigate the survival of STEC O26:H11 in different types of fermented sausages (“Teewurst”, fast-ripened and long-fermented salami). Challenge studies were performed with an inoculation cocktail which consisted of three STEC O26:H11 strains isolated from human, cattle and food sources. In the short-ripened spreadable sausage type “Teewurst” STEC counts decreased by only 0.5 log10 within 28 days. In contrast, STEC reductions from 2.2 to 2.6 log10 units were observed in the different salami products, while the most pronounced decrease of 1.0 log10 unit within one day was detected in fast-ripened sausages with glucono delta-lactone (GdL). Moreover, numbers of the food-associated E. coli O26:H11 strain were significantly higher (p

Cattle are a major reservoir of the six major Shiga toxin-producing non-O157 Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145) responsible for foodborne illnesses in humans. Besides prevalence in feces, the concentrations of STEC in cattle feces play a major role in their transmission dynamics. A subset of cattle, referred to as super shedders, shed E. coli O157 at high concentrations (≥4 log CFU/g of feces). It is not known whether a similar pattern of fecal shedding exists for non-O157. Our objectives were to initially validate the spiral plating method to quantify the six non-O157 E. coli serogroups with pure cultures and culture-spiked fecal samples and then determine the applicability of the method and compare it with multiplex quantitative PCR (mqPCR) assays for the quantification of the six non-O157 E. coli serogroups in cattle fecal samples collected from commercial feedlots. Quantification limits of the spiral plating method were 3 log, 3 to 4 log, and 3 to 5 log CFU/mL or CFU/g for individual cultures, pooled pure cultures, and cattle fecal samples spiked with pooled pure cultures, respectively. Of the 1,152 cattle fecal samples tested from eight commercial feedlots, 122 (10.6%) and 320 (27.8%) harbored concentrations ≥4 log CFU/g of one or more of the six serogroups of non-O157 by spiral plating and mqPCR methods, respectively. A majority of quantifiable samples, detected by either spiral plating (135 of 137, 98.5%) or mqPCR (239 of 320, 74.7%), were shedding only one serogroup. Only one of the quantifiable samples was positive for a serogroup carrying Shiga toxin (stx

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.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups such as O26, O45, O103, O111, O121, and O145 are recognized as serious risks to human health due to their toxicity. The conventional microbiological detection method of cell counting on agar plates is laborious and time-consuming. Because optical methods are promising for real-time, in situ foodborne pathogen detection, a hyperspectral microscope imaging (HMI) method based on acousto-optic tunable filters (AOTF) was developed for detecting pathogenic bacteria with a capability to differentiate the spectral characteristics of each bacterial cell from microcolony samples. Using the AOTF-based HMI method, a total of 89 contiguous spectral images were acquired within approximately 45 s with 250 ms exposure time. In this study, we developed a protocol for successfully immobilizing live cells on glass slides to acquire quality spectral images of STEC bacterial cells using a modified drying method. Among the contiguous spectral imagery between 450 and 800 nm, the intensities at 458, 498, 522, 546, 570, 586, 670, and 690 nm were distinct for STEC bacteria under dark-field illumination. With two different classification algorithms, i.e., support vector machine (SVM) and sparse kernel-based ensemble learning (SKEL), STEC serogroup O45 could be classified with 92% detection accuracy. However, the mean accuracies in identifying the six STEC serogroups with SVM and SKEL were not high enough for use in classification models.

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.