Reservoir Of Shiga Toxin-producing Escherichia Coli Research Articles

Detection of Shiga toxin- and extended-spectrum -lactamase-producing Escherichia coli O145:NM and Ont:NM from calves with diarrhoea

Sir, Shiga toxin-producing Escherichia coli (STEC) are recognized as important human pathogens. Ruminants, in particular bovines, are the primary reservoir of STEC and transmission principally occurs via the food chain. So far, extended-spectrum b-lactamases (ESBLs) have been rarely observed among STEC strains. In humans, such findings are currently limited to five isolates belonging to serogroups O26:NM, O26:H11, O64 and O157:H7, expressing b-lactamases TEM-52, CTX-M-2, CTX-M-3, CTX-M-18 or CTX-M-1, and to the O104:H4-CTX-M-15 strain associated with an outbreak of haemolytic uraemic syndrome (HUS) in Germany. – 7 Even rarer is the finding of ESBL-producing STEC strains from animal sources, where only one O157-CTX-M-2 chicken isolate and one O111:H8-CTX-M-15 bovine isolate have been described so far. In the present study, two E. coli strain collections either known for a positive ESBL phenotype or for possession of Shiga toxin genes were screened for a combination of these two determinants. In detail, 62 ESBL-positive E. coli isolates recovered through routine diagnostics (Vet Med Labor GmbH, Germany) from the faecal contents of cattle/calves with diarrhoea between 2010 and 2012 in Germany, determined as ESBL producers by MIC testing (Vitek 2; AST-GN38 or AST-N062) and the combination disc test (CLSI document M31-A3), were investigated for the possession of Shiga toxin genes in this study. A DNA array (Identibac EC, Alere, Germany) identified the stx2 gene in one strain (VB932491), which was further determined as stx2a by sequence analysis (Table 1). Another 87 E. coli, predominantly isolated from calves with diarrhoea in the past 30 years in the diagnostic laboratory of the Institute of Hygiene and Infectious Diseases of Animals, Germany, and known to harbour either an stx1 or an stx2 gene, as determined by PCR analyses, were screened by overnight incubation on Mueller–Hinton agar containing 4 mg/L cefotaxime. Here, one stx1-positive isolate (IHIT23778), further typed as stx1a by sequence analysis, showed growth, was further examined by MIC testing and could be finally confirmed as an ESBL producer by the combination disc test. The ESBL-producing STEC strains were determined as Ont:NM and O145:NM, respectively. O145 represents a serotype that is counted among the ‘big six’ of non-O157 STEC O antigens, namely O26, O45, O103, O111, O121 and O145. Multilocus sequence typing (MLST) assigned this strain to sequence type (ST) 32, which is currently represented by another seven E. coli isolates from Germany and Norway, six of which were defined as human STEC or enterohaemorrhagic E. coli (EHEC) strains associated with HUS in the years 1996 to 2009, all expressing O145 (http://mlst.ucc.ie/ mlst/dbs/Ecoli). The Ont:NM strain was assigned to ST301. Another two bovine ST301 STEC strains (O80:NM and O4:NM), isolated in 1992 and 1994, respectively, are available in the MLST database. However, none of these strains showed an ESBL phenotype, as confirmed in the present study. Both ESBL-producing STEC strains possessed genes formerly detected in plasmid pO157, i.e. the putative serine protease precursor-encoding gene espP and EHEC haemolysin-encoding gene hlyA. Genes located on the genetic element O island 122 (efa1 and nleABC) initially identified in EHEC strain EDL933 and associated with STEC capable of causing HUS and foodborne outbreaks were further detected. In addition, both strains harboured adherence-conferring protein gene iha, which is located on a chromosomal island recently acquired by O157:H7 STEC strains. Genes aggR, irp2 and pAA, which are related to the 2011 German O104:H4-CTX-M-15 Shiga toxin-producing enteroaggregative haemorrhagic E. coli outbreak strain, were absent. The ESBL-producing STEC strains possessed different types of eae genes (variants j andg). They also differed partly in the genes linked with the type III secretion system and with effector proteins located on the locus of enterocyte effacement (LEE) pathogenicity island (Table 1). Here, O145:NM strain IHIT23778 harboured tccp, which encodes a Tir cytoskeleton coupling protein, and cif, which encodes type III secretion effector cycle inhibiting factor, in addition to esp genes, nleABC and tir. Finally, it differs from the Ont:NM strain in the possession of the catalase-peroxidaseencoding gene katP. These three genes are tightly associated with strains causing complicated EHEC infections underlining the serious threat of the acquisition of an ESBL plasmid by such a strain. PCR and sequence analysis revealed blaCTX-M-1 in our STEC strains. The transferability of ESBL plasmids was explored by filter mating using E. coli K-12 strain J53 as the recipient strain and tryptic soy agar (TSA) plates containing 100 mg/L sodium azide and 4 mg/L cefotaxime. According to PCR-based replicon typing, the donor strains contained four and three plasmids of different sizes and replicon types, respectively. Self-transferable plasmids carrying the blaCTX-M-1 genes were either of replicon type IncFII (VB932491) or Inc1I (IHIT23778).

Evidence of Non-O157 Shiga Toxin—Producing Escherichia coli in the Feces of Meat Goats at a U.S. Slaughter Plant

Shiga toxin-producing Escherichia coli (STEC) are important human pathogens, and attention to non-O157 serogroups has increased in recent years. Although cattle are normally considered the primary reservoir for STEC, recent illnesses associated with goat contact have indicated that these animals are important potential reservoirs for the organisms. The prevalence of STEC, particularly non-O157 serogroups, in U.S. goats has not been well described. Our objective was to determine the prevalence of six major non-O157 STEC serogroups in the feces of meat goats. Rectal contents from 296 goats were collected postevisceration at a slaughter plant in the southeastern United States over 9 days during a 12-week period from August through October 2012. Samples were enriched in E. coli broth, and DNA was extracted and used as template in an 11-gene multiplex PCR that detected six non-O157 serogroups (O26, O45, O103, O121, O111, and O145) and virulence genes. Samples were considered positive when at least one non-O157 STEC serotype was present with either stx₁ or stx₂. All six non-O157 serogroups were detected by PCR in our samples, and 14.5% of samples were positive for at least one serogroup. Prevalence of O26 was highest, with 6.4% of goat fecal samples positive. The prevalence of O45 was 3.4%, O103 was 4.4%, O111 was 4.1%, O121 was 1.4%, and O145 was 3.0%. Twenty-two (7.4%) of 296 fecal samples had more than one non-O157 serogroup detected in the feces. Two samples had evidence of three non-O157 STEC serogroups. Goats appear to be an important reservoir for non-O157 STEC, and further work to understand the characteristics, epidemiology, and ecology of STEC in these animals is warranted.

Shiga toxin-producing Escherichia coli in Central Greece: prevalence and virulence genes of O157:H7 and non-O157 in animal feces, vegetables, and humans

In Greece, Shiga toxin-producing Escherichia coli (STEC) have only been sporadically reported. The objective of this study was to estimate the prevalence of STEC and Escherichia coli O157:H7 in farm animals, vegetables, and humans in Greece. A total number of 1,010 fecal samples were collected from farm animals (sheep, goats, cattle, chickens, pigs), 667 diarrheal samples from humans, and 60 from vegetables, which were cultured in specific media for STEC isolates. Enzyme-linked immunosorbent assay (ELISA) was used to detect toxin-producing colonies, which, subsequently, were subjected to a multiplex polymerase chain reaction (PCR) for stx1, stx2, eae, rfbE O157, and fliC h7 genes. Eighty isolates (7.9 %) from animal samples were found to produce Shiga toxin by ELISA, while by PCR, O157 STEC isolates were detected from 8 (0.8 %) samples and non-O157 STEC isolates from 43 (4.2 %) samples. STEC isolates were recovered mainly from sheep and goats, rarely from cattle, and not from pigs and chickens, suggesting that small ruminants constitute a potential risk for human infections. However, only three human specimens (0.4 %) were positive for the detection of Shiga toxins and all were PCR-negative. Similarly, all 60 vegetable samples were negative for toxin production and for toxin genes, but three samples (two roman rockets and one spinach) were positive by PCR for rfbE O157 and fliC h7 genes. These findings indicate that sheep, goats, cattle, and leafy vegetables can be a reservoir of STEC and Escherichia coli O157:H7 isolates in Greece, which are still rarely detected among humans.

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.

USE OF A MIXTURE OF PROBIOTIC STRAINS AGAINST SHIGA TOXIN‐PRODUCING ESCHERICHIA COLI (STEC) COLONIZATION IN SHEEP

Shiga toxin‐producing Escherichia coli (STEC) strains are food‐borne pathogens that cause human diseases, and ruminants are usually important reservoirs of STEC. The first step of enteric infection is colonization of the host's gut mucosal surface by pathogenic strains of bacteria. Probiotic bacteria can decrease the severity of infection by competing for receptors and nutrients and by synthesizing an acid that creates an unfavorable environment for the growth of several bacterial species. The aim of this study was to determine whether the inoculation of sheep with a mixture containing 3 × 108 (CFU) of Bacillus cereus, Lactobacillus acidophilus and Enterococcus faecium strains decrease the colonization of STEC. Sheep that received oral inoculums containing 2 × 109 viable bacteria of STEC carriers of stx1, stx2 and eae genes were compared with others groups that did not receive inoculums. When mixture was inoculated together with the STEC, the numbers of STEC in a population were lower than the group did not receive during the 3a, 5a, 6a and 7a weeks post‐inoculation. Thus, we conclude that this mixture likely presented a potential protective effect in reducing colonization by STEC and can be used as an alternative method to decrease STEC infection in sheep, thereby reducing transmission to humans.Financial Support: Fapesp Process: 2009/14923‐8

Shedding of foodborne pathogens and microbial carcass contamination of hunted wild ruminants

To assess the shedding of selected bacterial foodborne pathogens, fecal samples from 239 hunted wild red deer, roe deer, chamois, and ibex were examined. All samples tested negative for Salmonella spp. and L. monocytogenes, but other Listeria species were occasionally found. Of the 239 fecal samples, 32.6% tested positive for stx (Shiga toxins), 6.7% for eae (intimin) and 13.8% for both stx and eae genes. Among the 56 isolated Shiga toxin-producing Escherichia coli (STEC) strains, 44.6% harbored genes for the Stx2 group, 30.4% for the Stx1 group, and 21.4% for both Stx1 and Stx2. Only two of these strains harbored eae. Hence, wild ruminants constitute a reservoir for STEC, but further characterization data of the isolated strains are required to assess their actual human pathogenicity. In addition, 328 carcasses from hunted wild red deer, roe deer, and chamois were examined for total viable counts (TVC) and Enterobacteriaceae by swabbing. For the examined animal species, average TVC (4.0–4.2logCFUcm−2) and average Enterobacteriaceae counts/detection rates (2.3–2.6logCFUcm−2; 87.5–90%) were at comparable levels. On the other hand, the microbial status of carcasses differed between certain abattoirs by several orders of magnitude. Strict compliance with good hunting and hygiene practices during any step from shooting, through evisceration in the field, to dehiding, cooling, and processing is therefore of central importance to avoid contaminations and to prevent foodborne pathogens carried by the animals from entering the food chain.

Prevalence, Molecular Characterization and Serology of Shiga toxin-Producing Escherichia coli Isolated from Buffaloes in West Azerbaijan, Iran

This present study is the first to report the presence of Shiga toxin-producing Escherichia coli (STEC) in buffaloes in Iran. A total of 360 fecal samples were collected from buffaloes from different regions in the west Azerbaijan province of Iran and cultured for the isolation of E. coli using routine biochemical tests. From the fecal samples, 340 E. coli were isolated and, of these, 26 STEC isolates were identified. The STEC isolates were further analyzed for the presence of specific virulence genes. Among the STEC isolates, 11 (42.3%) isolates were positive for the stx1 gene, nine (34.6%) were positive for the stx2 gene and six (23%) were positive for both of these genes. Six (23%) STEC isolates harbored the hly gene and two (7.6%) isolates were positive for the eae gene. Based on serotyping, only one (3.8%) isolate was of the O157 serotype, while the other 25 (96.1%) belonged to non-O157 serotypes. The results of the present study provide the first evidence that buffaloes could be a reservoir for STEC in Iran, especially those belonging to non-0157 serotypes.

Fates of Acid-Resistant and Non-Acid-Resistant Shiga Toxin-Producing Escherichia coli Strains in Ruminant Digestive Contents in the Absence and Presence of Probiotics

Healthy ruminants are the main reservoir of Shiga toxin-producing Escherichia coli (STEC). During their transit through the ruminant gastrointestinal tract, STEC encounters a number of acidic environments. As all STEC strains are not equally resistant to acidic conditions, the purpose of this study was to investigate whether acid resistance confers an ecological advantage to STEC strains in ruminant digestive contents and whether acid resistance mechanisms are induced in the rumen compartment. We found that acid-resistant STEC survived at higher rates during prolonged incubation in rumen fluid than acid-sensitive STEC and that they resisted the highly acidic conditions of the abomasum fluid, whereas acid-sensitive strains were killed. However, transit through the rumen contents allowed acid-sensitive strains to survive in the abomasum fluid at levels similar to those of acid-resistant STEC. The acid resistance status of the strains had little influence on STEC growth in jejunal and cecal contents. Supplementation with the probiotic Saccharomyces cerevisiae CNCM I-1077 or Lactobacillus acidophilus BT-1386 led to killing of all of the strains tested during prolonged incubation in the rumen contents, but it did not have any influence in the other digestive compartments. In addition, S. cerevisiae did not limit the induction of acid resistance in the rumen fluid. Our results indicate that the rumen compartment could be a relevant target for intervention strategies that could both limit STEC survival and eliminate induction of acid resistance mechanisms in order to decrease the number of viable STEC cells reaching the hindgut and thus STEC shedding and food contamination.

Shiga Toxins, and the Genes Encoding Them, in Fecal Samples from Native Idaho Ungulates

Cattle are a known reservoir of Shiga toxin-producing Escherichia coli. The prevalence and stability of Shiga toxin and/or Shiga toxin genes among native wild ungulates in Idaho were investigated. The frequency of both Shiga genes and toxin was similar to that reported for Idaho cattle ( approximately 19%).

Detection and characterisation of Shiga toxin-producing Escherichia coli other than Escherichia coli O157:H7 in wild ruminants

Shiga toxin-producing Escherichia coli (STEC) are an important group of emerging pathogens, with ruminants recognised as their main natural reservoir. The aim of this work was to establish the prevalence of non-O157 STEC in free-ranging wild ruminants in the Extremadura region of Spain and to characterise them phenogenotypically. Faecal samples were collected from 243 wild ruminants, including Cervus elaphus, Capreolus capreolus, Dama dama and Ovis musimon and were examined for STEC using both phenotypic (Vero cells) and genotypic (PCR and PFGE) methods. Shiga toxin-producing Escherichia coli were isolated from 58 (23.9%) of the samples and a total of 65 isolates were characterised. A PCR method indicated that 11 (16.9%) strains carried the stx 1 gene, 44 (67.7%) carried the stx 2 gene and 10 (15.4%) carried both these genes. The ehxA gene was detected in 37 (57%) of the isolates but none contained either the eae or saa genes. The isolates were from a total of 12 ‘O’ serogroups, although 80% were restricted to the O2, O8, O128, O146, O166 and O174 serogroups. The most commonly isolated STEC bacteria, which were from the O146 serogroup, exhibited a high degree of polymorphism as indicated by PFGE. Shiga toxin-producing Escherichia coli isolates of serogroups O20, O25, O166, O171, O174 and O176 had not previously been found in wild ruminants. This is the first study to confirm that wild ruminants in Spain are a reservoir of STEC and are thus a potential source of human infection.

Prevalence and genetic profiles of Shiga toxin-producing Escherichia coli strains isolated from buffaloes, cattle, and goats in central Vietnam

We investigated the prevalence of Shiga toxin-producing Escherichia coli (STEC) in 568 healthy domestic animals (buffaloes, cattle, and goats) from 98 farms in the central region of Vietnam. The aims of this study were to determine if the prevalence of STEC in South East Asia is similar to that in other parts of the world, to characterize the virulence gene profiles from the recovered STEC and to determine if the recovered STEC belong to serotypes commonly associated with human disease. STEC and intimin-positive strains were recovered from 27% of buffaloes, 23% of cattle, and 38.5% of goats. Seventy percent of buffalo farms, 60% of cattle farms and 100% goat farms were positive for STEC. Of 170 STEC strains, 99 carried both stx1 and stx2 genes, 36 carried the stx2 gene, and 35 carried the stx1 gene. The eae gene was found in six caprine isolates, but not in buffalo or bovine isolates. Among 173 E. coli strains (170 STEC and 3 intimin-positive), 110 carried the ehxA gene, 106 possessed the s aa gene. Further characterization of stx subtypes demonstrated that among 134 stx1-containing isolates, 107 belonged to the stx1c subtype and 27 were the stx1 subtype. Of the 132 stx2-containing isolates, 36 were stx2, 34 were stx2c, 43 were stx2d subtype, 3 belonged to stx2g, and 16 strains were stx2d act . The stx2c variant was dominant in strains isolated from buffalo while the stx2d variant occurred more frequently in caprine isolates. Only 9 (5%) STEC strains contained genes encoding for serotypes O26, O91, O121, O145, and O157 LPS, which are more frequently associated with human infections. The results of this study provide data for understanding of epidemiology of STEC among domestic animals in Vietnam and indicate that buffaloes are also an important reservoir of STEC.

Growth of Shiga-Toxin producing Escherichia coli (STEC) and bovine feces background microflora in various enrichment protocols

Cattle are an important reservoir for STEC and eating food contaminated with fecal material is a frequent source of human STEC infection. It is thus essential to reliably determine the prevalence of STEC contamination in cattle. Currently, different enrichment protocols are used before the detection of Shiga-Toxin producing Escherichia coli (STEC) in fecal samples. However, there have not been any studies performed that have compared the effectiveness of these various enrichment protocols for the growth of non-O157 STEC in fecal samples. The objective of this present study was to characterize the effects of different enrichment factors on the simultaneous growth of the feces background microflora (BM) and two non-O157 STEC strains. The different factors studied were the basal medium (TSB and EC), the effect of novobiocin in the broth (N+ or N−) and the incubation temperature (37 or 40 °C). The BM and STEC growth data were simultaneously fitted by using a competitive growth model. The STEC final levels obtained after 24 h were higher for the protocols with novobiocin and/or EC compared to the others. However, novobiocin inhibited the growth of one STEC strain. We observed that the addition of novobiocin into broths is not advisable for optimal growth conditions. Moreover, given high BM and low STEC levels often observed in feces, predictions made with the growth model highlighted that false negative results could more likely appear with protocols using TSB without novobiocin than with protocols using EC. In conclusion, the use of EC broth in enrichment protocols seems to be more appropriate for detecting non-O157 STEC from bovine fecal samples. This can help avoid false negative results that cause an underestimation of the STEC prevalence in cattle.

Serotypes, virulence genes, and intimin types of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) isolated from calves in São Paulo, Brazil

Shiga toxin-producing Escherichia coli (STEC), is the most important recently emerged group of foodborne pathogens. Ruminants, especially cattle, have been implicated as a principal reservoir of STEC, undercooked ground beef and raw milk being the major vehicles of foodborne outbreaks. Enteropathogenic E. coli (EPEC) strains are defined as eae-harboring diarrheagenic E. coli that possess the ability to form A/E lesions on intestinal cells and that do not possess Shiga toxin genes. In order to determine the occurrence, serotypes and virulence markers of STEC and EPEC strains, 546 fecal samples from 264 diarrheic calves and 282 healthy calves in beef farms in São Paulo, Brazil, were screened by PCR. STEC and EPEC were isolated in 10% and 2.7% of the 546 animals, respectively. Although IMS test was used, the STEC serotype O157:H7 was not detected. The most frequent serotypes among STEC strains were O7:H10, O22:H16, O111:H −, O119:H − and O174:H21, whereas O26:H11, O123:H11 and O177:H11 were the most prevalent among EPEC strains. In this study, serotypes not previously reported were found among STEC strains: O7:H7, O7:H10, O48:H7, O111:H19, O123:H2, O132:H51, O173:H −, and O175:H49. The eae gene was detected in 25% of the STEC and 100% of EPEC strains. The intimin type θ/γ2 was the most frequent among STEC, whereas the intimin β1 was the most frequent intimin type among EPEC strains. To our knowledge, this is the first report of the occurrence of the new intimin μB in one strain of animal origin. This new intimin was detected in one atypical EPEC strain of serotype O123:H? isolated from diarrheic cattle. The enterohemolysin ( ehxA) was detected in 51% of the STEC and 80% of the EPEC strains, whereas STEC autoagglutinating adhesin ( saa) virulence gene was detected only in those STEC strains negative for eae gene. All 15 bovine EPEC strains isolated in this study were negative for both eaf and bfp genes. Our data shows that in Brazil cattle are not only a reservoir of STEC and atypical EPEC, but also a potential source of infection in humans, since the important STEC serotypes previously described and associated with severe diseases in humans, such as O111:H −, O113:H21, O118:H16, and O174:H21 were isolated.

Shiga toxin-producing Escherichia coli and enteropathogenic Escherichia coli in healthy goats in India: occurrence and virulence properties

To describe the occurrence and virulence gene pattern of shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) in healthy goats of Jammu and Kashmir, India. A total of 220 E. coli strains belonging to 60 different 'O' serogroups was isolated from 206 local (nonmigratory) and 69 migratory goats. All the 220 strains were screened for the presence of stx(1), stx(2), eaeA and hlyA genes. Twenty-eight E. coli (75.6%) strains from local and nine (24.3%) strains from migratory goats belonging to 18 different serogroups showed at least presence of one virulence gene studied. Twenty-eight strains (16.47%) (belonging to 13 different serogroups) from local goats carried stx(1) gene alone or in combination with stx(2) gene, while as only one strain (2%) from migratory goats possessed stx(2) gene alone. Interestingly in the present study none of the STEC strains carried eaeA gene. Similarly, none of the strains from local goats possessed eaeA and none of the migratory goats possessed stx(1) gene. Eight strains (16%) (belonging to four different serogroups) from migratory goats carried eaeA gene. Twenty-five (14.7%) and seven (14%) strains from local and migratory goats harboured hlyA gene respectively. Healthy goats of Jammu and Kashmir state serve as a reservoir of STEC and EPEC. Further studies in this direction are needed to work out whether or not they are transmitted to humans in this part of world. This study is the first report of isolation of STEC and EPEC strains from healthy goats in Jammu and Kashmir State of India, which could be a source of infection to humans.

Virulence genes and intimin types of Shiga-toxin-producing Escherichia coli isolated from cattle and beef products in Argentina.

A total of 153 Shiga-toxin-producing Escherichia coli (STEC) isolates from feces of cattle and beef products (hamburgers and ground beef) in Argentina were characterized in this study. PCR showed that 22 (14%) isolates carried stx1 genes, 113 (74%) possessed stx2 genes and 18 (12%) both stx1 and stx2. Intimin (eae), enterohemolysin (ehxA), and STEC autoagglutinating adhesin (saa) virulence genes were detected in 36 (24%), 70 (46%) and in 34 (22%) of the isolates, respectively. None of 34 saa-positive isolates carried the gene eae, and 31 were ehxA-positive. Fourteen (7 of serotype O26:H11 and 4 of serotype O5:H-) isolates had intimin b1, 16 isolates possessed intimin g1 (11 of serotype O145:H- and 5 of serotype O157:H7), 5 isolates had intimin type e1 (4 of serotypes O103:H- and O103:H2), and one isolate O111:H- showed intimin type q/g2. Although the 153 STEC isolates belonged to 63 different seropathotypes, only 12 accounted for 58% of isolates. Seropathotype ONT:H- stx2 (18 isolates) was the most common, followed by O171:H2 stx2 (12 isolates), etc. The majority (84%) of STEC isolates belonged to serotypes previously found in human STEC and 56% to serotypes associated with STEC isolated from patients with hemolytic uremic syndrome (HUS). Thus, this study confirms that cattle are a major reservoir of STEC pathogenic for humans. To our knowledge, this is the first study that described the presence of saa gene in STEC of serotypes O20:H19, O39:H49, O74:H28, O79:H19, O116:H21, O120:H19, O141:H7, O141:H8, O174:H21, and ONT:H21. The serotypes O120:H19 and O185:H7 were not previously reported in bovine STEC.

Prevalence of Shiga Toxin–Producing Escherichia coli in Ground Beef and Cattle Feces from King County, Washington

Shiga toxin–producing Escherichia coli (STEC) is increasingly recognized as a common cause of diarrhea. STEC infection is a major public health threat because of its ability to cause serious and potentially life-threatening illnesses. The main reservoirs of STEC are believed to be the intestinal tracts of animals. Several studies have investigated the prevalence of STEC in various food items. The objective of this study was to determine the prevalence of STEC in the Seattle ground beef supply. In addition, the relative amount of STEC contamination between stores was compared, and possible differences between types of ground beef based on fat content (9, 16, and 23%) were investigated. A survey of Stx-I and/or Stx-II genes in fecal samples from cattle at a local slaughter house was also conducted. Of 296 ground beef samples tested from area retail grocery stores, 16.8% were positive for the presence of the toxin genes. Our data showed that there was no statistically significant difference (P > 0.05) in the prevalence of STEC between the ground beef samples of different fat contents and between grocery store chains. Of the 103 cattle fecal samples tested, 19 (18.4%) were found positive for the presence of Stx-I and/or Stx-II genes. The presence of a rather high percentage of STEC in the food supply in the absence of large number of cases suggests that not all STEC lineages are pathogenic for humans.

Methods for the detection and isolation of Shiga toxin-producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) are an important cause of haemorrhagic colitis and the diarrhoea-associated form of the haemolytic uraemic syndrome. Of the numerous serotypes of E. coli that have been shown to produce Shiga toxin (Stx), E. coli O157:H7 and E. coli O157:NM (non-motile) are most frequently implicated in human disease. Early recognition of STEC infections is critical for effective treatment of patients. Furthermore, rapid microbiological diagnosis of individual patients enables the prompt notification of outbreaks and implementation of control measures to prevent more cases. Most human infections caused by STEC have been acquired by the consumption of contaminated foods, especially those of bovine origin such as undercooked ground beef and unpasteurized cows' milk, and by person-to-person contacts. To identify the reservoirs of STEC and the routes of transmission to man, sensitive methods are needed as these pathogens may only be present in food, environmental and faecal samples in small numbers. In addition, sensitive and rapid detection methods are necessary for the food industry to ensure a safe supply of foods. Sensitive methods are also needed for surveillance programmes in risk assessment studies, and for studies on survival and growth of STEC strains. Cultural methods for the enrichment, isolation and confirmation of O157 STEC are still evolving. Several selective enrichment media have been described, of which modified tryptone soy broth with novobiocin and modified E. coli broth with novobiocin, seem to be the most appropriate. These media are minimally-selective broths that give a somewhat limited differential specificity favouring isolation of O157 STEC, as opposed to other Gram-negative bacteria, in the sample. An incubation temperature of 41-42 degrees C further enhances selectivity. The occurrence of heat-, freeze-, acid- or salt-stressed STEC in foods means that it is important to be able to detect cells that are in a stressed state, as STEC generally have a very low infectious dose, and injured cells mostly retain their pathogenic properties. For the isolation of stressed O157 STEC, pre-enrichment in a non-selective broth is necessary. The most widely used plating medium for the isolation of typical sorbitol-non-fermenting strains of STEC of serogroup O157 is sorbitol MacConkey agar with cefixime and tellurite (CT-SMAC). As some STEC strains are sensitive for tellurite and/or are sorbitol-fermenting, the use of a second isolation medium, such as one of the newer chromogenic media, is recommended. Immunomagnetic separation (IMS) following selective enrichment, and subsequent spread-plating of the concentrated target cells onto CT-SMAC agar, appears to be the most sensitive and cost-effective method for the isolation of E. coli O157 from raw foods. IMS increases sensitivity by concentrating E. coli O157 relative to background microflora, which may overgrow or mimic O157 STEC cells on selective agars. While cultural isolation of O157 STEC from foods and faeces is time-consuming, labour-intensive and hence, costly, rapid immunological detection systems have been developed which significantly reduce the analysis time. These methods include enzyme-linked immunosorbent assays (ELISAs), colony immunoblot assays, direct immunofluorescent filter techniques, and several immunocapture techniques. Both polyclonal and monoclonal antibodies specific for the O and H antigens are used for these methods. Many of these test systems are able to detect less than one O157 STEC cell g(-1) of raw meat after overnight enrichment. Presumptive results are available after just one day, but need to be completed with the isolation of the organisms. The primary use of these procedures is therefore to identify food and faecal samples that possibly contain O157 STEC.

Long-term survival of shiga toxin-producing Escherichia coli O26, O111, and O157 in bovine feces.

Cattle are an important reservoir of Shiga toxin-producing Escherichia coli (STEC) O26, O111, and O157. The fate of these pathogens in bovine feces at 5, 15, and 25 degrees C was examined. The feces of a cow naturally infected with STEC O26:H11 and two STEC-free cows were studied. STEC O26, O111, and O157 were inoculated into bovine feces at 10(1), 10(3), and 10(5) CFU/g. All three pathogens survived at 5 and 25 degrees C for 1 to 4 weeks and at 15 degrees C for 1 to 8 weeks when inoculated at the low concentration. On samples inoculated with the middle and high concentrations, O26, O111, and O157 survived at 25 degrees C for 3 to 12 weeks, at 15 degrees C for 1 to 18 weeks, and at 5 degrees C for 2 to 14 weeks, respectively. Therefore, these pathogens can survive in feces for a long time, especially at 15 degrees C. The surprising long-term survival of STEC O26, O111, and O157 in bovine feces shows that such feces are a potential vehicle for transmitting not only O157 but also O26 and O111 to cattle, food, and the environment. Appropriate handling of bovine feces is emphasized.