Development and assessment of a rapid method to detect Escherichia coli O26, O111 and O157 in retail minced beef

A molecular-based detection method was developed to detect Escherichia coli O26, O111 and O157 in minced (ground) beef samples. This method consists of an initial overnight enrichment in modified tryptone soya broth (mTSB) and novobiocin prior to DNA extraction and subsequent serogrouping using a triplex PCR. This method has a low limit of detection and results are available within 24 hours of receipt of samples. Once optimized, this rapid method was utilized to determine the prevalence of these E. coli serogroups in six hundred minced beef samples all of which were previously examined by immunomagnetic separation (IMS) and selective plating for E. coli O26 and O111. Using IMS, two E. coli O26 isolates were detected. No E. coli O111 were recovered. The multiplex PCR technique described here did not detect E. coli O111 nor O157 in any of the samples, however six minced beef samples were positive for E. coli O26 using our method, only two of these were previously detected by IMS and culture. Application of molecular methods are useful to support culture-based approaches thereby further contributing to risk reduction along the food chain.     

Polymerase chain reaction screening for Salmonella and enterohemorrhagic Escherichia coli on beef products in processing establishments

Pathogenic Escherichia coli strains on raw or insufficiently cooked foods are of public health concern as serious disease may result from their ingestion. Therefore, many commercial producers of beef products screen for E. coli O157:H7 before shipment. While Salmonella is not considered an adulterant on raw beef products, it is used as an indication of process control. To detect these microorganisms, rapid screening methods are often used to provide results within 8-24 hours after sampling. During 2005-2008, about 971,389 samples from several commercial beef production plants were tested using a rapid screening method based on the polymerase chain reaction to determine if they were presumptively positive for bacterial cells carrying Salmonella or Shiga toxin-producing E. coli-specific genes. Of the product lots sampled (trim, ground beef, and variety meats), 15% were positive for the stx(1) and/or stx(2) (Shiga toxin genes), 9.1% for the eae gene (the attaching and effacing gene [eae] encoding intimin), 3.0% for an rfb gene region (encoding the O157-specific O side chain polysaccharide), and 1.67% for Salmonella by the polymerase chain reaction assay. In general, lots of ground beef showed the lowest frequency of contamination, and variety meats (by-products of carcass evisceration), the highest. Overall, 4.6%, 4.6%, and 0.8% samples were screen-positive for enteropathogenic E. coli, enterohemorrhagic E. coli, and E. coli O157, respectively. Of the E. coli O157-positive samples, 14% were also Salmonella positive. The frequency of screen-positive samples increases during the summer months, probably because of the prevalence of climatic conditions more conducive to microbial growth. The presence of fecal organisms in beef products suggests a failure of sanitary controls during processing and the more prevalent relatives of E. coli O157, Shiga toxin-producing Escherichia coli, enteropathogenic E. coli, and enterohemorrhagic E. coli, serve as more sensitive indicators of contamination than O157 strains alone.     

Isolation and characterization of Shiga toxin-producing Escherichia coli (STEC) in retail edible beef by-products

The prevalence of Shiga toxin-producing Escherichia coli (STEC) was investigated in 350 edible beef intestinal samples, including omasum (n=110), abomasum (n=120), and large intestines (n=120), collected from traditional beef markets in Seoul, Korea. A total of 23 STEC strains were isolated from 15 samples (four strains from three omasa, 10 from five abomasa, and nine from seven large intestines). The O serotypes and toxin gene types of all STEC isolates were identified, and antimicrobial resistance was assessed using the disk diffusion method. The isolation rates of STEC from edible beef intestines were 2.8% in omasum, 4.2% in abomasums, and 5.9% in large intestines. All STEC isolates harbored either stx1, or both stx1 and stx2 genes simultaneously. Among the 23 isolates, 13 strains were identified as 11 different O serogroups, and 10 strains were untypable. However, enterohemorrhagic Esherichia coli O157, O26, and O111 strains were not isolated. The highest resistance rate observed was against tetracycline (39%), followed by streptomycin (35%) and ampicillin (22%). Of the 23 isolates, 12 isolates (52%) were resistant to at least one antibiotic, nine (39%) isolates were resistant to two or more antibiotics, and one isolate from an abmasum carried resistance against nine antibiotics, including beta-lactam/beta-lactamase inhibitor in combination and cephalosporins. This study shows that edible beef by-products, which are often consumed as raw food in many countries, including Korea, can be potential vehicles for transmission of antimicrobial-resistant pathogenic E. coli to humans.     

Isolation of Escherichia coli O157:H7 from intact colon fecal samples of swine

Escherichia coli O157:H7 was recovered from colon fecal samples of pigs. Polymerase chain reaction confirmed two genotypes: isolates harboring the eaeA, stx(1), and stx(2) genes and isolates harboring the eaeA, stx(1), and hly(933) genes. We demonstrate that swine in the United States can harbor potentially pathogenic E. coli O157:H7.     

Detection by multiplex real-time polymerase chain reaction assays and isolation of Shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121, and O145 in ground beef

Six Shiga toxin-producing Escherichia coli (STEC) serogroups, which include O26, O45, O103, O111, O121, and O145, are responsible for the majority of non-O157 STEC infections in the United States, representing a growing public health concern. Cattle and other ruminants are reservoirs for these pathogens; thus, food of bovine origin may be a vehicle for infection with non-O157 STEC. Methods for detection of these pathogens in animal reservoirs and in food are needed to determine their prevalence and to develop intervention strategies. This study describes a method for detection of non-O157 STEC in ground beef, consisting of enrichment in modified tryptic soy broth at 42°C, followed by real-time multiplex polymerase chain reaction (PCR) assays targeting stx(1), stx(2), and genes in the O-antigen gene clusters of the six serogroups, [corrected] and then immunomagnetic separation (IMS) followed by plating onto Rainbow? Agar O157 and PCR assays for confirmation of isolates. All ground beef samples artificially inoculated with 1-2 and 10-20 CFU/25?g of ground beef consistently gave positive results for all of the target genes, including the internal amplification control using the multiplex real-time PCR assays after enrichment in modified tryptic soy broth for a total of 24?h (6?h at 37°C and 18?h at 42°C). The detection limit of the real-time multiplex PCR assays was ～50 CFU per PCR. IMS for O26, O103, O111, and O145 was performed with commercially available magnetic beads, and the IMS beads for O45 and O121 were prepared using polyclonal antiserum against these serogroups. A large percentage of the presumptive colonies of each serogroup picked from Rainbow Agar O157 were confirmed as the respective serogroups; however, the percent recovery of STEC O111 was somewhat lower than that of the other serogroups. This work provides a method for detection and isolation in ground beef and potentially other foods of non-O157 STEC of major public health concern.     

Phenotypic and genetic markers for serotype-specific detection of Shiga toxin-producing Escherichia coli O26 strains from North America

Phenotypic and genetic markers of Shiga toxin-producing Escherichia coli (STEC) O26 from North America were used to develop serotype-specific protocols for detection of this pathogen. Carbohydrate fermentation profiles and prevalence of gene sequences associated with STEC O26 (n = 20) were examined. Non-STEC O26 (n = 17), E. coli O157 (n = 20), E. coli O111 (n = 22), and generic E. coli (n = 21) were used as comparison strains. Effects of supplements: cefixime-tellurite, 4-methylumbelliferyl-beta-D-glucuronide (MUG) and chromogenic additives (5-bromo4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal), 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-GlcA) and o-nitrophenyl-beta-D-galactopyranoside (ONPG), added to isolation agar media were examined. Tests for presence of gene sequences encoding beta intimin (eae beta), Shiga toxin 1 and 2 (stx1 and stx2), H7 flagella (flicCh7), enterohemolysin (ehlyA), O26 somatic antigen (wzx), and high pathogenicity island genes (irp2 and fyuA) were conducted using multiplex polymerase chain reaction. Pulsed-field gel electrophoresis (PFGE) of XbaI restriction endonuclease genomic DNA digests was used to establish clonality among E. coli O26 strains. Of the 26 carbohydrates tested, only rhamnose had diagnostic value. Rhamnose non-fermenters included STEC O26 (100%), non-STEC O26 (40%), generic E. coli (29%), E. coli O111 (23%), and E. coli O157 (0%). Rhamnose non-fermenting colonies growing on Rhamnose-McConkey agar supplemented with X-GlcA, X-Gal, or ONPG, respectively, were blue, white, or faint yellow, whereas rhamnose-fermenters were red. Blue colonies from X-GlcA-containing media were the most well-defined and easiest to pick for further tests. All STEC O26 were MUG-fluorescent, while STEC O157 (n = 18) were non-fluorescent. E. coli O111 and generic E. coli strains were either MUG-positive or-negative. Serotype-specific detection of STEC O26 was achieved by selecting cefixime-tellurite-resistant, MUG-fluorescent, rhamnose-nonfermenting colonies, which carried stx1, eae beta, irp2, and wzx gene sequences. STEC O26 prevalence in dairy farm environmental samples determined using the developed isolation and genetic detection protocols was 4%. PFGE indicated the presence of one major cluster of E. coli O26 with 72-100% DNA fragment-length digest similarity among test strains. The serotype-specific detection methods described herein have potential for routine application in STEC O26 diagnosis.     

Applicability of a multiplex PCR to detect the seven major Shiga toxin-producing Escherichia coli based on genes that code for serogroup-specific O-antigens and major virulence factors in cattle feces

An 11-gene multiplex polymerase chain reaction (mPCR) was developed based on genes that code for serogroup-specific O-antigens and four major virulence factors (intimin, enterohemorrhagic hemolysin, and Shiga toxins [Stx] 1 and 2), to detect O157 and the "top six" non-O157 (O26, O45, O103, O111, O121, and O145) Shiga toxin-producing Escherichia coli (STEC). The assay specificity was validated with pure cultures of seven major STEC (185 strains), 26 other STEC (65 strains), non-STEC (five strains), and 33 strains of other genera and species. Sensitivity of the assay with cattle fecal sample spiked with pooled cultures of seven major STEC was 10(5) colony-forming units (CFU)/g before enrichment and 10(2) CFU/g after enrichment. The applicability of the assay to detect STEC in fecal samples (n=50), before and after enrichment, was evaluated by comparing with culture-based methods for O26, O111, and O157. The mPCR assay of 50 fecal samples showed seven (14%) positive before enrichment and 23 (46%) positive after enrichment for one or more of the seven O-groups. Overall, 17 isolates from 17 fecal samples and 27 isolates (four for O26, three for O45, and 20 for O103) from 19 fecal samples were obtained, by culture-based methods, for O157 and non-O157 serogroups, respectively. None of the 27 non-O157 isolates possessed the stx genes, suggesting that cattle harbor Shiga toxin-negative E. coli belonging to the "top six" non-O157 serogroups. Our data, although based on a limited number of samples, suggest that the sensitivities of the mPCR and culture-based methods in detecting the seven serogroups of STEC in feces differed between O-groups. An obvious limitation of our mPCR is that the concurrent detection of virulence genes and the serogroups in a sample does not necessarily associate the virulence genes with the prevalent serogroups in the same sample. The major application of our 11-gene mPCR assay may be in identifying putative colonies of STEC obtained by culture-based methods.     

Comparison between human and animal isolates of Shiga toxin-producing Escherichia coli O157 from Australia

There is very little human disease associated with enterohaemorrhagic Escherichia coli O157 in Australia even though these organisms are present in the animal population. A group of Australian isolates of E. coli O157:H7 and O157:H- from human and animal sources were tested for the presence of virulence markers and compared by XbaI DNA macrorestriction analysis using pulsed-field gel electrophoresis (PFGE). Each of 102 isolates tested contained the gene eae which encodes the E. coli attaching and effacing factor and all but one carried the enterohaemolysin gene, ehxA, found on the EHEC plasmid. The most common Shiga toxin gene carried was stx2c, either alone (16%) or in combination with stx1 (74%) or stx2 (3%). PFGE grouped the isolates based on H serotype and some clusters were source specific. Australian E. coli O157:H7 and H- isolates from human, animal and meat sources carry all the virulence markers associated with EHEC disease in humans therefore other factors must be responsible for the low rates of human infection in Australia.     

Comparison of Escherichia coli Isolates from humans, food, and farm and companion animals for presence of Shiga toxin-producing E. coli virulence markers

The objective of this study was to characterize Escherichia coli isolates from dairy cows/feedlots, calves, mastitis, pigs, dogs, parrot, iguana, human disease, and food products for prevalence of Shiga toxin-producing E. coli (STEC) virulence markers. The rationale of the study was that, isolates of the same serotypes that were obtained from different sources and possessed the same marker profiles, could be cross-species transmissible. Multiplex polymerase chain reaction (PCR) was used to detect presence of genes encoding Shiga toxin 1 and 2 (stx1 and stx2), H7 flagella (flicC), enterohemolysin (hly) and intimin (eaeA) in E. coli isolates (n = 400). Shiga toxin-producing isolates were tested for production of Shiga toxins (Stx1 and Stx2 and enterohemolysin. Of the E. coli O157:H7/H- strains, 150 of 164 (mostly human, cattle, and food) isolates were stx+. Sixty-five percent of O157 STEC produced both Stx1 and Stx2; 32% and 0.7% produced Stx2 or Stx1, respectively. Ninety-eight percent of O157 STEC had sequences for genes encoding intimin and enterohemolysin. Five of 20 E. coli O111, 4 of 14 O128 and 4 of 10 O26 were stx+ . Five of 6 stx+ O26 and O111 produced Stx1, however, stx+ O128 were Stx-negative. Acid resistance (93.3%) and tellurite resistance (87.3%) were common attributes of O157 STEC, whereas, non-O157 stx+ strains exhibited 38.5% and 30.8% of the respective resistances. stx-positive isolates were mostly associated with humans and cattle, whereas, all isolates from mastitis (n = 105), and pigs, dogs, parrot and iguanas (n = 48) were stx-negative. Multiplex PCR was an effective tool for characterizing STEC pathogenic profiles and distinguished STEC O157:H7 from other STEC. Isolates from cattle and human disease shared similar toxigenic profiles, whereas isolates from other disease sources had few characteristics in common with the former isolates. These data suggest interspecies transmissibility of certain serotypes, in particular, STEC O157:H7, between humans and cattle.     

Cattle as a possible source of verocytotoxin-producing Escherichia coli O157 infections in man.

In May-June 1992 cases of infection with verocytotoxin-producing (VT+) Escherichia coli O157 in South Yorkshire could have been associated with prior consumption of beef from a local abattoir. During investigation of the abattoir, bovine rectal swabs and samples of meat and surface swabs from beef carcasses were examined for E. coli O157, isolates of which were tested for toxigenicity, plasmid content and phage type. E. coli O157 was isolated from 84 (4%) of 2103 bovine rectal swabs; of these 84, 78 (93%) were VT+, the most common phage types being 2 and 8, the types implicated in the cluster of human cases. Positive cattle were from diverse sources within England. E. coli O157 was isolated from 7 (30%) of 23 carcasses of rectal swab-positive cattle and from 2 (8%) of 25 carcasses of rectal swab-negative cattle. The study has shown that cattle may be a reservoir of VT+ E. coli O157, and that contamination of carcasses during slaughter and processing may be how beef and beef products become contaminated and thereby transmit the organism to man.     

Validation of a method for simultaneous isolation of Shiga toxin-producing Escherichia coli O26, O103, O111, and O145 from minced beef by an international ring-trial

An isolation method described by Possé et al. (FEMS Microbiol Lett 2008;282:124-131) was satisfactorily validated in an international ring-trial using artificially contaminated minced beef samples. Until now, no validated method existed for the simultaneous isolation of Shiga toxin-producing Escherichia coli serogroups O26, O103, O111, and O145 in food. Twelve laboratories from five European countries participated and received 16 inoculated beef samples contaminated with cold-stressed cells of the four serogroups O26, O103, O111, and O145 in two levels (approximately 30 and 300 CFU 25?g?1) in duplicate. In addition, they received four non-inoculated samples. The isolation protocol comprised a selective enrichment step, a selective isolation step on a non-O157 agar plate differentiating the serogroups by color, followed by confirmation by plating on confirmation agar media and agglutination. All laboratories were able to isolate the inoculated serogroups from the samples, both for the high and the low inoculation level. Results did not differ whether in-house-prepared or ready-to-use non-O157 agar plates were used, demonstrating that by following the instructions laboratories managed to perform the complete protocol with success.     

Pathogenic potential to humans of bovine Escherichia coli O26, Scotland

Escherichia coli O26 and O157 have similar overall prevalences in cattle in Scotland, but in humans, Shiga toxin-producing E. coli O26 infections are fewer and clinically less severe than E. coli O157 infections. To investigate this discrepancy, we genotyped E. coli O26 isolates from cattle and humans in Scotland and continental Europe. The genetic background of some strains from Scotland was closely related to that of strains causing severe infections in Europe. Nonmetric multidimensional scaling found an association between hemolytic uremic syndrome (HUS) and multilocus sequence type 21 strains and confirmed the role of stx(2) in severe human disease. Although the prevalences of E. coli O26 and O157 on cattle farms in Scotland are equivalent, prevalence of more virulent strains is low, reducing human infection risk. However, new data on E. coli O26-associated HUS in humans highlight the need for surveillance of non-O157 enterohemorrhagic E. coli and for understanding stx(2) phage acquisition.     

Occurrence of shigatoxinogenic Escherichia coli O157 in Norwegian cattle herds.

To investigate if there is a reservoir of Escherichia coli O157 in Norwegian cattle, faecal samples from 197 cattle herds were screened for E. coli O157 by the use of immunomagnetic separation (IMS) and PCR during the 1995 grazing season. Six E. coli O157:H-isolates were detected in two herds, one isolate in one and five in the other. The isolates carried the stx1, stx2, and eae genes, and a 90 MDa virulence plasmid. They were toxinogenic in a Vero cell assay. From 57 other herds, 137 faecal samples were positive for stx1 and/or stx2 genes detected by PCR run directly on IMS-isolated material. Among these samples, stx2 were the most widely distributed toxin encoding genes. No difference was found among milking cows and heifers in the rate of stx1 and/or stx2 in positive samples.     

The prevalence and characterization of verotoxin-producing Escherichia coli isolated from cattle and pigs in an abattoir in Hong Kong

The aim of the study was to define the prevalence of verotoxin-producing Escherichia coli (VTEC) in cattle and pigs in a Hong Kong abattoir. Faecal and carcass samples collected from 986 cattle and 487 pigs from an abattoir were tested for verotoxin (VT) by PCR and cytotoxicity assays. VTEC was isolated from 415 and 1-8% of cattle faecal and carcass samples and from 2.1 and 0.2% of porcine faecal and carcass samples, respectively. Amongst 409 VTEC isolates from cattle, 9 were serotype O157:H7 and eaeA+. The most prevalent vt genotype among bovine VTEC was vtl+vt2 (73.8%) and in porcine VTEC was vt2e+ (30%). None of the porcine VTEC isolates and 9.3% of the bovine VTEC isolates was eaeA+. The non-O157 serogroup VTEC isolates carrying eaeA and EHEC-hlyA belonged to serogroups O172, O15, O84, O91, O110 and O121. The local dietary preference for pork or chicken (rather than beef), the low VTEC carriage in pigs, the rarity of additional virulence factors (caeA) in VTEC isolated from cattle may explain the apparently low incidence of human diarrhoeal disease associated with VTEC in Hong Kong hitherto. However, the presence of non-O157 VTEC strains carrying the eacA virulence marker in cattle highlights the fact that sole reliance on sorbitol-MacConkey agar for screening human VTEC isolates may underestimate the human disease burden. The changing dietary habits of the population in Hong Kong reinforce the need for continued vigilance.     

Contamination of potable water distribution systems by multiantimicrobial-resistant enterohemorrhagic Escherichia coli

BACKGROUND: The contamination of processed or unprocessed drinking water by fecal coliform bacteria has been reported worldwide. Despite a high incidence of waterborne diseases, entero-hemorrhagic Escherichia coli (EHEC) is an underacknowledged pathogen of concern to public health in India. Although the presence of EHEC is recorded in surface water resources of India, drinking water sources are yet to be investigated. OBJECTIVES: The goal of this study was to analyze potable water samples for the presence of virulence determinants of EHEC and to determine the sensitivity of the virulence determinants to antimicrobials. METHODS: We enumerated coliform bacteria in potable water samples collected from six locations in Lucknow, a major city in northern India, using the most probable number method. E. coli (n = 81), randomly isolated by membrane-filtration technique from four sites, were identified by biochemical characterization. E. coli were not detected in samples from two other sites. We screened 15 randomly selected isolates from each site for virulence determinants of EHEC using polymerase chain reaction (PCR). The isolates positive for virulence determinants (n = 18) were screened for sensitivity to 15 antimicrobials by the disk diffusion method. RESULTS: Both stx1 and stx2 genes were present in 33.3% of isolates, whereas others possessed either stx1 (11.1%) or stx2 (55.6%). eaeA, hlyA, and chuA genes were present in 100, 23.3, and 16.7% of isolates, respectively. Resistance to multiple antimicrobials was observed in potential EHEC. CONCLUSIONS: The occurrence of multiantimicrobial-resistant EHEC in potable water is an important health concern because of the risk of waterborne outbreaks.     

Antimicrobial activity of a combination of Mume fructus, Schizandrae fructus, and Coptidis rhizoma on enterohemorrhagic Escherichia coli O26, O111, and O157 and its effect on Shiga toxin releases

The antibacterial activity of an herbal combination composed of Mume Fructus, Coptidis Rhizoma, and Schizandrae Fructus extracts on enterohemorrhagic Escherichia coli (EHEC) was evaluated in the present study. The combination demonstrated antibacterial activity against all EHEC strains tested in this study, including those resistant to multiple antibiotics; minimum inhibitory concentration values ranged from 0.49 to 31.25?mg/mL. In in vivo antibacterial activity assay, the herbal combination was administered to mice after initial E. coli O157 infection and had significant effects on mouse mortality. The effects of the herbal combination on Shiga toxin release from EHEC O26, EHEC O111, and EHEC O157 strains containing the stx1 and stx2 genes were assessed by the reversed passive latex agglutination method, and there was no increased Shiga toxin release in the strain cultures containing the herbal combination. These results suggested that the herbal combination may be a safe and effective remedy for EHEC inhibition.     

Shiga Toxin Subtypes Associated with Shiga Toxin-Producing Escherichia coli Strains Isolated from Red Deer, Roe Deer, Chamois, and Ibex

Abstract A total of 52 Shiga toxin-producing Escherichia coli (STEC) strains, isolated from fecal samples of six ibex, 12 chamois, 15 roe deer, and 19 red deer were further characterized by subtyping the stx genes, examining strains for the top nine serogroups and testing for the presence of eae and ehxA. Eleven of the 52 strains belonged to one of the top nine STEC O groups (O26, O45, O91, O103, O111, O113, O121, O145, and O157). Eight STEC strains were of serogroup O145, two strains of serogroup O113, and one strain of serogroup O157. None of the strains harbored stx2a, stx2e, or stx2f. Stx2b (24 strains) and stx1c (21 strains) were the most frequently detected stx subtypes, occurring alone or in combination with another stx subtype. Eight strains harbored stx2g, five strains stx2d, three strains stx1a, two strains stx2c, and one strain stx1d. Stx2g and stx1d were detected in strains not harboring any other stx subtype. The eae and ehxA genes were detected in two and 24 STEC strains, respectively. Considering both, the serogroups and the virulence factors, the majority of the STEC strains isolated from red deer, roe deer, chamois, and ibex do not show the typical patterns of highly pathogenic STEC strains. To assess the potential pathogenicity of STEC for humans, strain isolation and characterization is therefore of central importance.     

Antimicrobial resistance of Escherichia coli O26, O103, O111, O128, and O145 from animals and humans

Susceptibilities to fourteen antimicrobial agents important in clinical medicine and agriculture were determined for 752 Escherichia coli isolates of serotypes O26, O103, O111, O128, and O145. Strains of these serotypes may cause urinary tract and enteric infections in humans and have been implicated in infections with Shiga toxin-producing E. coli (STEC). Approximately 50% of the 137 isolates from humans were resistant to ampicillin, sulfamethoxazole, cephalothin, tetracycline, or streptomycin, and approximately 25% were resistant to chloramphenicol, trimethoprim-sulfamethoxazole, or amoxicillin-clavulanic acid. Approximately 50% of the 534 isolates from food animals were resistant to sulfamethoxazole, tetracycline, or streptomycin. Of 195 isolates with STEC-related virulence genes, approximately 40% were resistant to sulfamethoxazole, tetracycline, or streptomycin. Findings from this study suggest antimicrobial resistance is widespread among E. coli O26, O103, O111, O128, and O145 inhabiting humans and food animals.     

Prevalence and growth kinetics of Shiga toxin-producing Escherichia coli (STEC) in bovine offal products in Japan

Recent epidemiological data suggest a link between the consumption of bovine offal products and Shiga toxin-producing Escherichia coli (STEC) infection in Japan. This study thus examined the prevalence of STEC in various types of these foods. PCR screened 229 bovine offal products for the presence of Shiga toxin (stx) gene. Thirty-eight (16·6%) samples were stx positive, of which eight were positive for rfbE(O157) and three were positive for wzy(O26). Four O157 and one O26 STEC isolates were finally obtained from small-intestine and omasum products. Notably, homogenates of bovine intestinal products significantly reduced the extent of growth of O157 in the enrichment process compared to homogenates of beef carcass. As co-incubation of O157 with background microbiota complex from bovine intestinal products in buffered peptone water, in the absence of meat samples, tended to reduce the extent of growth of O157, we reasoned that certain microbiota present in offal products played a role. In support of this, inoculation of generic E. coli from bovine intestinal products into the homogenates significantly reduced the extent of growth of O157 in the homogenates of bovine intestinal and loin-beef products, and this effect was markedly increased when these homogenates were heat-treated prior to inoculation. Together, this report provides first evidence of the prevalence of STEC in a variety of bovine offal products in Japan. The prevalence data herein may be useful for risk assessment of those products as a potential source of human STEC infection beyond the epidemiological background. The growth characteristic of STEC O157 in offal products also indicates the importance of being aware when to test these food products.     

Evaluation of a multiplex real-time polymerase chain reaction for the quantification of Escherichia coli O157 in cattle feces

Cattle are asymptomatic reservoirs for Escherichia coli O157, a major foodborne pathogen. The organism generally colonizes the hindgut of cattle and is shed in the feces at low concentrations. The objective of this research was to evaluate a multiplex, real-time polymerase chain reaction (mqPCR) assay for quantification of E. coli O157 in cattle feces using stx1, stx2, and rfbE gene targets. Primer efficiency and analytical sensitivity of the assay were evaluated with a single or pooled (five strain) culture of E. coli O157. In pure culture, the minimum detection limit of the assay was 1.4×10(3) CFU/mL and 3.6×10(3) CFU/mL for the single and five-strain mixture of E. coli O157, respectively. Diagnostic sensitivity was analyzed using DNA extracted from cattle feces spiked with E. coli O157. In feces spiked with the pooled mixture of five E. coli O157 strains, the minimum detection limit was 3.6×10(4) CFU/g. We also evaluated the assay with feces from cattle experimentally inoculated with E. coli O157 by comparing the results to a culture-based method. For the majority of samples tested, the concentration of E. coli O157 detected by the real-time and culture methods was within one log difference. However, the assay could only be evaluated for cattle shedding high concentrations of E. coli O157. In conclusion, the mqPCR quantifying E. coli O157 in cattle feces using stx1, stx2, and rfbE gene targets may have use in detecting and quantifying super shedders, but is not applicable for quantification in animals shedding low concentrations (10(2) to 10(3) CFU/g feces).