Prevalence and Characterization of Non-O157 Shiga Toxin-Producing Escherichia coli on Carcasses in Commercial Beef Cattle Processing Plants

Beef carcass sponge samples collected from July to August 1999 at four large processing plants in the United States were surveyed for the presence of non-O157 Shiga toxin-producing Escherichia coli (STEC). Twenty-eight (93%) of 30 single-source lots surveyed included at least one sample containing non-O157 STEC. Of 334 carcasses sampled prior to evisceration, 180 (54%) were found to harbor non-O157 STEC. Non-O157 STEC isolates were also recovered from 27 (8%) of 326 carcasses sampled after the application of antimicrobial interventions. Altogether, 361 non-O157 STEC isolates, comprising 41 different O serogroups, were recovered. O serogroups that previously have been associated with human disease accounted for 178 (49%) of 361 isolates. Although 40 isolates (11%) carried a combination of virulence factor genes (enterohemorrhagic E. coli hlyA, eae, and at least one stx gene) frequently associated with STEC strains causing severe human disease, only 12 of these isolates also belonged to an O serogroup previously associated with human disease. Combining previously reported data on O157-positive samples (R. O. Elder, J. E. Keen, G. R. Siragusa, G. A. Barkocy-Gallagher, M. Koohmaraie, and W. W. Laegreid, Proc. Natl. Acad. Sci. USA 97:2999-3003, 2000) with these data regarding non-O157-positive samples indicated total STEC prevalences of 72 and 10% in preevisceration and postprocessing beef carcass samples, respectively, showing that the interventions used by the beef-processing industry effected a sevenfold reduction in carcass contamination by STEC.     

Detection and characterization of verocytotoxin-producing Escherichia coli (vtec) O157 and non-O157 in cattle at slaughter

Between September 2001 to June 2002, 145 samples of bovine caecal content were collected at slaughter for verocytotoxin-producing Escherichia coli (VTEC) serogroups O157 and non-O157 detection. For E. coli O157 the immunomagnetic-separation technique was performed. The enterohaemolytic phenotype was the target for non-O157 VTEC identification. The vero cell assay (VCA) was performed for toxic activity detection. The genomic sequence for VT1, VT2 and intimin (vt1, vt2, eae genes) were identified by PCR analysis. Eight VTEC O157 and eight non-O157 VTEC isolates were detected. VTEC O157, eae-positive strains were shed by 9.7% of feedlot cattle and by 2.5% of dairy cows. Non-O157 VTEC, eae-negative isolates were detected in the intestinal content of 12.5% dairy cows and of 2.1% feedlot cattle. VTEC-shedding cattle came from 18.1% of the farms included in the study. From cattle faeces, VTEC O91:H- (VT2-positive, eae-negative), responsible of human diarrhoeal disease in Europe, was recovered. Other VTEC serogroups identified in the present study were O74, O109, O110, O116, and O117.     

Genotypic Analyses of Shiga Toxin-Producing Escherichia coli O157 and Non-O157 Recovered from Feces of Domestic Animals on Rural Farms in Mexico

Shiga toxin-producing Escherichia coli (STEC) are zoonotic enteric pathogens associated with human gastroenteritis worldwide. Cattle and small ruminants are important animal reservoirs of STEC. The present study investigated animal reservoirs for STEC in small rural farms in the Culiacan Valley, an important agricultural region located in Northwest Mexico. A total of 240 fecal samples from domestic animals were collected from five sampling sites in the Culiacan Valley and were subjected to an enrichment protocol followed by either direct plating or immunomagnetic separation before plating on selective media. Serotype O157:H7 isolates with the virulence genes stx2, eae, and ehxA were identified in 40% (26/65) of the recovered isolates from cattle, sheep and chicken feces. Pulse-field gel electrophoresis (PFGE) analysis grouped most O157:H7 isolates into two clusters with 98.6% homology. The use of multiple-locus variable-number tandem repeat analysis (MLVA) differentiated isolates that were indistinguishable by PFGE. Analysis of the allelic diversity of MLVA loci suggested that the O157:H7 isolates from this region were highly related. In contrast to O157:H7 isolates, a greater genotypic diversity was observed in the non-O157 isolates, resulting in 23 PFGE types and 14 MLVA types. The relevant non-O157 serotypes O8:H19, O75:H8, O111:H8 and O146:H21 represented 35.4% (23/65) of the recovered isolates. In particular, 18.5% (12/65) of all the isolates were serotype O75:H8, which was the most variable serotype by both PFGE and MLVA. The non-O157 isolates were predominantly recovered from sheep and were identified to harbor either one or two stx genes. Most non-O157 isolates were ehxA-positive (86.5%, 32/37) but only 10.8% (4/37) harbored eae. These findings indicate that zoonotic STEC with genotypes associated with human illness are present in animals on small farms within rural communities in the Culiacan Valley and emphasize the need for the development of control measures to decrease risks associated with zoonotic STEC.     

Detection, isolation and characterization of Shiga toxin-producing Escherichia coli in retail-minced beef using PCR-based techniques, immunoassays and colony hybridization

AIMS: To provide information on detection of Shiga toxin-producing Escherichia coli (STEC) in retail-minced beef using an approach combining (i) PCR-based techniques and automated immunoassay for stx screening and detection of the five major serogroups associated with human infection, and (ii) immunomagnetic separation (IMS) and colony hybridization assays for bacterial strain isolation. METHODS AND RESULTS: Twenty-seven out of 164 minced beef samples were stx-positive by PCR-ELISA, nine of which were also positive by real-time PCR for at least one marker of the five main serogroups tested (O26, O103, O111, O145 and O157). Two E. coli O103 stx-negative strains were isolated from two out of 10 IMS and nine STEC strains that did not belong to the five main serogroups were isolated by colony hybridization. CONCLUSIONS: PCR techniques are applicable for rapid screening of samples containing both an stx gene and an O-group marker of the five main pathogenic STEC serogroups. Isolation of STEC strains belonging to the main non-O157 serogroups remains difficult. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents an evaluation of a multi-faceted approach for the detection of the most frequently reported human pathogenic STEC serogroups. The advantages and limits of this strategy are presented.     

Molecular risk assessment and epidemiological typing of Shiga toxin-producing Escherichia coli by using a novel PCR binary typing system

Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen that causes diarrheal disease in humans and is of public health concern because of its ability to cause outbreaks and severe disease such as hemorrhagic colitis or hemolytic-uremic syndrome. More than 400 serotypes of STEC have been implicated in outbreaks and sporadic human disease. The aim of this study was to develop a PCR binary typing (P-BIT) system that could be used to aid in risk assessment and epidemiological studies of STEC by using gene targets that would represent a broad range of STEC virulence genes. We investigated the distribution of 41 gene targets in 75 O157 and non-O157 STEC isolates and found that P-BIT provided 100% typeability for isolates, gave a diversity index of 97.33% (compared with 99.28% for XbaI pulsed-field gel electrophoresis [PFGE] typing), and produced 100% discrimination for non-O157 STEC isolates. We identified 24 gene targets that conferred the same level of discrimination and produced the same cluster dendrogram as the 41 gene targets initially examined. P-BIT clustering identified O157 from non-O157 isolates and identified seropathotypes associated with outbreaks and severe disease. Numerical analysis of the P-BIT data identified several genes associated with human or nonhuman sources as well as high-risk seropathotypes. We conclude that P-BIT is a useful approach for subtyping, offering the advantage of speed, low cost, and potential for strain risk assessment that can be used in tandem with current molecular typing schema for STEC.     

Genotypic analyses of Escherichia coli O157:H7 and O157 nonmotile isolates recovered from beef cattle and carcasses at processing plants in the Midwestern states of the United States

Escherichia coli O157:H7 and O157 nonmotile isolates (E. coli O157) previously were recovered from feces, hides, and carcasses at four large Midwestern beef processing plants (R. O. Elder, J. E. Keen, G. R. Siragusa, G. A. Barkocy-Gallagher, M. Koohmaraie, and W. W. Laegreid, Proc. Natl. Acad. Sci. USA 97:2999-3003, 2000). The study implied relationships between cattle infection and carcass contamination within single-source lots as well as between preevisceration and postprocessing carcass contamination, based on prevalence. These relationships now have been verified based on identification of isolates by genomic fingerprinting. E. coli O157 isolates from all positive samples were analyzed by pulsed-field gel electrophoresis of genomic DNA after digestion with XbaI. Seventy-seven individual subtypes (fingerprint patterns) grouping into 47 types were discerned among 343 isolates. Comparison of the fingerprint patterns revealed three clusters of isolates, two of which were closely related to each other. Remarkably, isolates carrying both Shiga toxin genes and nonmotile isolates largely fell into specific clusters. Within lots analyzed, 68.2% of the postharvest (carcass) isolates matched preharvest (animal) isolates. For individual carcasses, 65.3 and 66.7% of the isolates recovered postevisceration and in the cooler, respectively, matched those recovered preevisceration. Multiple isolates were analyzed from some carcass samples and were found to include strains with different genotypes. This study suggests that most E. coli O157 carcass contamination originates from animals within the same lot and not from cross-contamination between lots. In addition, the data demonstrate that most carcass contamination occurs very early during processing.     

Diagnosis of Shiga toxin producing Escherichia coli infection, contribution of genetic amplification technique

There has been no culture method of choice for detecting non-O157 Shiga toxin-producing Escherichia coli strains (STEC) because of their biochemical diversity The aim of this study was the assessment of verotoxin gene detection (VT1/VT2) within STEC PCR compared with the Vero cells cytotoxicity among O157 and non-O157 STEC serotypes. Stool cultures were performed on Tryptic Soy Broth and sorbitol MacConkey agar with cefixitime and tellurite supplements which were identified as Escherichia coli (E. coli) by BBL crystal. Further identifications were performed including verotoxin production assessment by Vero cells cytotoxicity assay, PCR for specific VT1/VT2 genotyping, and isolates were plated on blood agar and tested for enterohemolysis. Vero cells cytotoxicity assay revealed that 58 of E. coli isolates (71.6%) were STEC. In PCR, 33 (56.9%) of the 58 strains were positive for the VT2 gene, 24 (41.4%) were positive for the VT1 gene and one isolate was positive for both genes. In comparison to Vero cells cytotoxicity, the sensitivity, specificity of PCR were 100%. In comparative study between verotoxin assessment by Vero cells cytotoxicity and enterohemolytic activity, concordance positive results between both were 53 (91.4%). The most common serogroups of STEC were O157 (33%) and O26 (20%). From this study we can conclude that enterohemolysin production can be used as surrogate marker for STEC. The most rapid and promising approach for detection of STEC is by molecular method.     

Fecal carriage of Escherichia coli O157:H7 and carcass contamination in cattle at slaughter in northern Italy.

Feedlot cattle slaughtered at a large abattoir in northern Italy during 2002 were examined for intestinal carriage and carcass contamination with Escherichia coli O157:H7. Carcass samples were taken following the excision method described in the Decision 471/2001/EC, and fecal material was taken from the colon of the calves after evisceration. Bacteria were isolated and identified according to the MFLP-80 and MFLP-90 procedures (Food Directorate's Health Canada's). Eighty-eight non-sorbitol-fermenting E. coli O157:H7 isolates were obtained from 12 of the 45 calves examined. In particular, E. coli O157:H7 isolates were found in 11 (24%) fecal and five (11%) carcass samples. PCR analysis showed that all 11 fecal samples and five carcass samples carried eae-gamma1-positive E. coli O157:H7 isolates. In addition, genes encoding Shigatoxins were detected in O157:H7 isolates from nine and two of those 11 fecal and five carcasses, respectively. A representative group of 32 E. coli O157:H7 isolates was analyzed by phage typing and DNA macrorestriction fragment analysis (PFGE). Five phage types (PT8, PT32v, PT32, PT54, and PT not typable) and seven (I-VII) distinct restriction patterns of similarity >85% were detected. Up to three different O157:H7 strains in an individual fecal sample and up to four from the same animal could be isolated. These findings provide evidence of the epidemiological importance of subtyping more than one isolate from the same sample. Phage typing together with PFGE proved to be very useful tools to detect cross-contamination among carcasses and should therefore be included in HACCP programs at abattoirs. The results showed that the same PFGE-phage type E. coli O157:H7 profile was detected in the fecal and carcass samples from an animal, and also in two more carcasses corresponding to two animals slaughtered the same day.     

Non-O157:H7 Shiga toxin (verocytotoxin)-producing Escherichia coli strains: epidemiological significance and microbiological diagnosis

Shiga toxin (Stx)-producing Escherichia coli (STEC) are important causes of diarrhoea and the haemolytic uremic syndrome (HUS). The most common STEC serotype implicated worldwide is E. coli O157:H7 that is diagnosed using procedures based on its typical phenotypic feature, the lack of sorbitol fermentation. In addition to E. coli O157:H7, a variety of non-O157:H7 STEC strains that usually ferment sorbitol and are thus missed by using the diagnostic protocol for E.coli O157:H7 have been isolated from patients. Among these sorbitol-fermenting (SF) non-O157:H7 STEC, SF E. coli O157:H⁻ and non-O157 STEC strains of serogroups O26, O103, O111 and O145 have emerged as significant causes of HUS and diarrhoea in continental Europe and have been associated with human disease in other parts of the world. Microbiological diagnosis of non-O157:H7 STEC strains is difficult due to their serotype diversity and the absence of a simple biochemical property that distinguishes such strains from the physiological intestinal microflora. Screening for non-O157:H7 STEC and their isolation from stools is presently based on the detection of Stx production or stx genes that are common characteristics of such strains. Molecular subtyping of the most frequent non-O157 STEC demonstrated that strains of serogroups O26, O103 and O111 belong to their own clonal lineages and show unique virulence profiles. SF STEC O157:H⁻ strains that have been isolated mostly in Central Europe represent a new clone within E. coli O157 serogroup which has its own typical combination of virulence factors. This revised version was published online in November 2006 with corrections to the Cover Date.     

Occurrence of Escherichia coli O157:H7 in faeces, skin and carcasses from sheep and goats in Ethiopia

Aims:  To determine the occurrence and proportion of Escherichia coli O157:H7 in faeces, skin swabs and carcasses before and after washing, from sheep and goats in Ethiopia.
Method and Results:  Individual samples were enriched in modified tryptic soy broth with novobiocin, concentrated using immunomagnetic separation (IMS) and plated onto cefixime-tellurite containing sorbitol MacConkey agar. Presumptive colonies were confirmed by biochemical tests and subjected to latex agglutination tests. A PCR was performed on isolates for the detection of stx ₁, stx ₂ and eae genes. Escherichia coli O157:H7 was isolated from faeces (4·7%), skin swabs (8·7%) and carcasses before washing (8·1%) and after washing (8·7%) and on water samples (4·2%). The proportion of carcasses contaminated with E. coli O157:H7 was strongly associated with those recovered from faecal and skin samples. Both stx ₁ and stx ₂ genes were identified from one E. coli O157:H7 isolate from a goat carcass.
Conclusions:  Even though the numbers of samples examined in this study were limited to one abattoir, sheep and goats can be potential sources of E. coli O157:H7 for human infection in the country. Control measures to reduce the public health risks arising from E.   coli O157:H7 in reservoir animals need to be addressed at abattoir levels by reducing skin and faecal sources and carcass contaminations at different stages of slaughter operations.
Significance and Impact of the Study:  Escherichia coli O157:H7 was detected from carcasses before and after washing during slaughtering operations, and one O157 isolate was positive for verotoxins.     

Prevalence and characterization of non-O157 shiga toxin-producing Escherichia coli isolates from commercial ground beef in the United States

Escherichia coli O157:H7 is a Shiga toxin (stx)-producing E. coli (STEC) strain that has been classified as an adulterant in U.S. beef. However, numerous other non-O157 STEC strains are associated with diseases of various severities and have become an increasing concern to the beef industry, regulatory officials, and the public. This study reports on the prevalence and characterization of non-O157 STEC in commercial ground beef samples (n = 4,133) obtained from numerous manufacturers across the United States over a period of 24 months. All samples were screened by DNA amplification for the presence of Shiga toxin genes, which were present in 1,006 (24.3%) of the samples. Then, culture isolation of an STEC isolate from all samples that contained stx(1) and/or stx(2) was attempted. Of the 1,006 positive ground beef samples screened for stx, 300 (7.3% of the total of 4,133) were confirmed to have at least one strain of STEC present by culture isolation. In total, 338 unique STEC isolates were recovered from the 300 samples that yielded an STEC isolate. All unique STEC isolates were serotyped and were characterized for the presence of known virulence factors. These included Shiga toxin subtypes, intimin subtypes, and accessory virulence factors related to adherence (saa, iha, lifA), toxicity (cnf, subA, astA), iron acquisition (chuA), and the presence of the large 60-MDa virulence plasmid (espP, etpD, toxB, katP, toxB). The isolates were also characterized by use of a pathogenicity molecular risk assessment (MRA; based on the presence of various O-island nle genes). Results of this characterization identified 10 STEC isolates (0.24% of the 4,133 total) that may be considered a significant food safety threat, defined by the presence of eae, subA, and nle genes.     

The prevalence and spread of Escherichia coli O157:H7 at a commercial beef abattoir

AIMS: To investigate the prevalence and virulence characteristics of Escherichia coli O157:H7 after a number of beef process operations at a commercial Irish abattoir. METHODS AND RESULTS: Two 12-month studies were carried out. The first study (study 1) examined the prevalence of E. coli O157:H7 at up to six sites on carcasses at eight stages of the dressing, washing, chilling and boning process. The second study (study 2) examined the prevalence of E. coli O157:H7 in bovine faeces and rumen contents post-slaughter and on dressed, washed carcasses. Isolates from both studies were phage-typed and the presence of genes encoding verocytotoxin, enterohaemolysin and intimin production was determined. E. coli O157:H7 was isolated from four of 36 carcasses in study 1. E. coli O157:H7 was detected during hide removal and was detected at multiple carcass sites and multiple process stages, including boning. On two carcasses, contamination was first detected at the bung following its freeing and tying. All isolates from study 1 were phage type (PT) 2, eaeAO157 and ehlyA positive, but were verocytotoxin 1 (VT1) and verocytotoxin 2 (VT2) negative. In study 2, E. coli O157:H7 was isolated from 2.4% of faecal, 0.8% of rumen and 3.2% of carcass samples. In some cases, isolates recovered from the faeces of a particular animal, the resulting carcass and adjacent carcasses on the line had the same phage typing and virulence characteristic profile patterns. All isolates from study 2 were eaeAO157 and ehlyA positive and only one isolate was VT1 and VT2 negative. Most isolates were PT 32. A higher frequency of positive isolations was noted from samples taken during spring and late summer. CONCLUSION: These studies show that in a typical Irish beef abattoir, carcass contamination with E. coli O157:H7 can occur during hide removal and bung tying and this contamination can remain on the carcass during subsequent processing. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides data that is necessary for the understanding of how E. coli O157:H7 contamination of beef occurs.     

Saltelli Global Sensitivity Analysis and Simulation Modelling to Identify Intervention Strategies to Reduce the Prevalence of Escherichia coli O157 Contaminated Beef Carcasses

Introduction

Strains of Shiga-toxin producing Escherichia coli O157 (STEC O157) are important foodborne pathogens in humans, and outbreaks of illness have been associated with consumption of undercooked beef. Here, we determine the most effective intervention strategies to reduce the prevalence of STEC O157 contaminated beef carcasses using a modelling approach.

Method

A computational model simulated events and processes in the beef harvest chain. Information from empirical studies was used to parameterise the model. Variance-based global sensitivity analysis (GSA) using the Saltelli method identified variables with the greatest influence on the prevalence of STEC O157 contaminated carcasses. Following a baseline scenario (no interventions), a series of simulations systematically introduced and tested interventions based on influential variables identified by repeated Saltelli GSA, to determine the most effective intervention strategy.

Results

Transfer of STEC O157 from hide or gastro-intestinal tract to carcass (improved abattoir hygiene) had the greatest influence on the prevalence of contaminated carcases. Due to interactions between inputs (identified by Saltelli GSA), combinations of interventions based on improved abattoir hygiene achieved a greater reduction in maximum prevalence than would be expected from an additive effect of single interventions. The most effective combination was improved abattoir hygiene with vaccination, which achieved a greater than ten-fold decrease in maximum prevalence compared to the baseline scenario.

Conclusion

Study results suggest that effective interventions to reduce the prevalence of STEC O157 contaminated carcasses should initially be based on improved abattoir hygiene. However, the effect of improved abattoir hygiene on the distribution of STEC O157 concentration on carcasses is an important information gap—further empirical research is required to determine whether reduced prevalence of contaminated carcasses is likely to result in reduced incidence of STEC O157 associated illness in humans. This is the first use of variance-based GSA to assess the drivers of STEC O157 contamination of beef carcasses.     

Heterogeneity of Shiga toxin-producing Escherichia coli strains isolated from hemolytic-uremic syndrome patients, cattle, and food samples in central France

A detailed analysis of the molecular epidemiology of non-O157:H7 Shiga toxin-producing Escherichia coli (STEC) was performed by using isolates from sporadic cases of hemolytic-uremic syndrome (HUS), animal reservoirs, and food products. The isolates belonged to the O91 and OX3 serogroups and were collected in the same geographical area over a short period of time. Five typing methods were used; some of these were used to explore potentially mobile elements like the stx genes or the plasmids (stx(2)-restriction fragment length polymorphism [RFLP], stx(2) gene variant, and plasmid analyses), and others were used to study the whole genome (ribotyping and pulsed-field gel electrophoresis [PFGE]). The techniques revealed that there was great diversity among the O91 and OX3 STEC strains isolated in central France. A close relationship between strains of the same serotype having the same virulence factor pattern was first suggested by ribotyping. However, stx(2)-RFLP and stx(2) variant analyses differentiated all but 5 of 21 isolates, and plasmid analysis revealed further heterogeneity; a unique combination of characteristics was obtained for all strains except two O91:H21 isolates from beef. The latter strains were shown by PFGE to be the most closely related isolates, with >96% homology, and hence may be subtypes of the same strain. Overall, our results indicate that the combination of stx(2)-RFLP, stx(2) variant, and plasmid profile analyses is as powerful as PFGE for molecular investigation of STEC diversity. Finally, the non-O157:H7 STEC strains isolated from HUS patients were related to but not identical to those isolated from cattle and food samples in the same geographical area. The possibility that there are distinct lineages of non-O157:H7 STEC, some of which are more virulent for humans, should be investigated further.     

Subtypes of the plasmid-encoded serine protease EspP in Shiga toxin-producing Escherichia coli: distribution, secretion, and proteolytic activity

We investigated the prevalence, distribution, and structure of espP in Shiga toxin-producing Escherichia coli (STEC) and assessed the secretion and proteolytic activity of the encoded autotransporter protein EspP (extracellular serine protease, plasmid encoded). espP was identified in 56 of 107 different STEC serotypes. Sequencing of a 3,747-bp region of the 3,900-bp espP gene distinguished four alleles (espPalpha, espPbeta, espPgamma, and espPdelta), with 99.9%, 99.2%, 95.3%, and 95.1% homology, respectively, to espP of E. coli O157:H7 strain EDL933. The espPbeta, espPgamma, and espPdelta genes contained unique insertions and/or clustered point mutations that enabled allele-specific PCRs; these demonstrated the presence of espPalpha, espPbeta, espPgamma, and espPdelta in STEC isolates belonging to 17, 16, 15, and 8 serotypes, respectively. Among four subtypes of EspP encoded by these alleles, EspPalpha (produced by enterohemorrhagic E. coli [EHEC] O157:H7 and the major non-O157 EHEC serotypes) and EspPgamma cleaved pepsin A, human coagulation factor V, and an oligopeptide alanine-alanine-proline-leucine-para-nitroaniline, whereas EspPbeta and EspPdelta either were not secreted or were proteolytically inactive. The lack of proteolysis correlated with point mutations near the active serine protease site. We conclude that espP is widely distributed among STEC strains and displays genetic heterogeneity, which can be used for subtyping and which affects EspP activity. The presence of proteolytically active EspP in EHEC serogroups O157, O26, O111, and O145, which are bona fide human pathogens, suggests that EspP might play a role as an EHEC virulence factor.     

The potential use of chilling to control the growth of Enterobacteriaceae on porcine carcasses and the incidence of E. coli O157:H7 in pigs

Aims:  To (i) monitor the presence of Enterobacteriaceae as indicators of faecal contamination on pig carcasses, (ii) examine the potential use of chilling as a critical control point (CCP) and establish its influence on pig carcass categorization by Decision 471/EC and (iii) determine the incidence of E. coli O157:H7 in pigs.
Methods and Results:  Porcine faecal samples and carcass swabs were collected before and after chilling at four Irish pig abattoirs and examined for Enterobacteriaceae and E. coli O157:H7. Chilling generally reduced Enterobacteriaceae counts on carcasses, but increases were also observed, particularly in one abattoir. E. coli O157:H7 was absent from carcasses before chilling, present on 0·21% after chilling and was recovered from 0·63% of faecal samples. All of the isolates were found to contain virulence genes associated with clinical illness in humans.
Conclusions:  The data show that overall chilling had the capacity to reduce the numbers of carcasses positive for the presence of Enterobacteriaceae .
Significance and Impact of Study:  The influence of chilling on the categorization of pig carcasses suggests that it has the potential to improve the numbers of acceptable carcasses and the process could be used as a CCP within a HACCP plan.     

Prevalence and characteristics of shiga toxin-producing Escherichia coli from healthy cattle in Japan

The prevalence of Shiga toxin-producing Escherichia coli (STEC) in Japan was examined by using stool samples from 87 calves, 88 heifers, and 183 cows on 78 farms. As determined by screening with stx-PCR, the prevalence was 46% in calves, 66% in heifers, and 69% in cows; as determined by nested stx-PCR, the prevalence was 100% in all animal groups. Of the 962 isolates picked by colony stx hybridization, 92 isolates from 54 farms were characterized to determine their O serogroups, virulence factor genes, and antimicrobial resistance. Of these 92 isolates, 74 (80%) could be classified into O serogroups; 50% of these 74 isolates belonged to O serogroups O8, O26, O84, O113, and O116 and 1 isolate belonged to O serogroup O157. Locus of enterocyte effacement genes were detected in 24% of the isolates, and enterohemorrhagic E. coli (EHEC) hlyA genes were detected in 72% of the isolates. Neither the bundle-forming pilus gene nor the enteropathogenic E. coli adherence factor plasmid was found. STEC strains with characteristics typical of isolates from human EHEC infections, which were regarded as potential EHEC strains, were present on 11.5% of the farms.     

Shiga Toxin-Producing Escherichia coli O157 Is More Likely to Lead to Hospitalization and Death than Non-O157 Serogroups – Except O104

The clinical spectrum following infection with Shiga toxin-producing Escherichia coli (STEC) is wide ranging and includes hemorrhagic colitis and life-threatening hemolytic uremic syndrome (HUS). Severity of STEC illness depends on patients'' age and strongly on the infecting strains'' virulence. Serogroup O157 is often assumed to be more virulent than others. Age-adjusted population-based data supporting this view are lacking thus far.We conducted a large retrospective cohort study among patients of community-acquired gastroenteritis or HUS diagnosed with STEC infection, reported in Germany January 2004 through December 2011. Age-adjusted risks for reported hospitalization and death, as proxies for disease severity, were estimated for STEC serogroups separately, and compared with STEC O157 (reference group) using Poisson regression models with robust error estimation.A total of 8,400 case-patients were included in the analysis; for 2,454 (29%) and 30 (0.4%) hospitalization and death was reported, respectively. Highest risks for hospitalization, adjusted for age and region of residence, were estimated for STEC O104 (68%; risk ratio [RR], 1.33; 95% confidence interval [CI], 1.19–1.45), followed by STEC O157 (46%). Hospitalization risks for the most prevalent non-O157 serogroups (O26, O103, O91, O145, O128, O111) were consistently and markedly lower than for O157, with the highest RR for O145 (0.54; 95% CI, 0.41–0.70) and the lowest for O103 (0.27; 95% CI, 0.20–0.35). Mortality risk of O104 was similar to O157 (1.2% each), but the group of all other non-O157 STEC had only 1/10 the risk (RR, 0.09; 95% CI, 0.02–0.32) compared to O157.The study provides population-based and age-adjusted evidence for the exceptional high virulence of STEC O157 in relation to non-O157 STEC other than O104. Timely diagnosis and surveillance of STEC infections should prioritize HUS-associated E. coli, of which STEC O157 is the most important serogroup.     

Development of a Robust Method for Isolation of Shiga Toxin-Positive Escherichia coli (STEC) from Fecal,Plant, Soil and Water Samples from a Leafy Greens Production Region in California

During a 2.5-year survey of 33 farms and ranches in a major leafy greens production region in California, 13,650 produce, soil, livestock, wildlife, and water samples were tested for Shiga toxin (stx)-producing Escherichia coli (STEC). Overall, 357 and 1,912 samples were positive for E. coli O157:H7 (2.6%) or non-O157 STEC (14.0%), respectively. Isolates differentiated by O-typing ELISA and multilocus variable number tandem repeat analysis (MLVA) resulted in 697 O157:H7 and 3,256 non-O157 STEC isolates saved for further analysis. Cattle (7.1%), feral swine (4.7%), sediment (4.4%), and water (3.3%) samples were positive for E. coli O157:H7; 7/32 birds, 2/145 coyotes, 3/88 samples from elk also were positive. Non-O157 STEC were at approximately 5-fold higher incidence compared to O157 STEC: cattle (37.9%), feral swine (21.4%), birds (2.4%), small mammals (3.5%), deer or elk (8.3%), water (14.0%), sediment (12.3%), produce (0.3%) and soil adjacent to produce (0.6%). stx1, stx2 and stx1/stx2 genes were detected in 63%, 74% and 35% of STEC isolates, respectively. Subtilase, intimin and hemolysin genes were present in 28%, 25% and 79% of non-O157 STEC, respectively; 23% were of the “Top 6″ O-types. The initial method was modified twice during the study revealing evidence of culture bias based on differences in virulence and O-antigen profiles. MLVA typing revealed a diverse collection of O157 and non-O157 STEC strains isolated from multiple locations and sources and O157 STEC strains matching outbreak strains. These results emphasize the importance of multiple approaches for isolation of non-O157 STEC, that livestock and wildlife are common sources of potentially virulent STEC, and evidence of STEC persistence and movement in a leafy greens production environment.