Distribution of Escherichia coli O157 in bovine fecal pats and its impact on estimates of the prevalence of fecal shedding

The distribution of Escherichia coli O157 in bovine feces was examined by testing multiple samples from fecal pats and determining the density of E. coli O157 in immunomagnetic separation (IMS)-positive fecal samples. The density of E. coli O157 in bovine feces was highly variable, differing by as much as 76,800 CFU g(-1) between samples from the same fecal pat. The density in most positive samples was <100 CFU g(-1), the limit of reliable detection by IMS. Testing only one 1-g sample of feces per pat with IMS may result in a sensitivity of detection as low as 20 to 50%. It is therefore probable that most surveys have greatly underestimated the prevalence of E. coli O157 shedding in cattle and the proportion of farms with shedding cattle. The sensitivity of the detection of E. coli O157 in bovine feces can be as much as doubled by testing two 1-g samples per pat rather than one 1-g sample.     

A comparison of immunomagnetic separation and culture, Reveal and VIP for the detection of E. coli O157 in enrichment cultures of naturally-contaminated raw beef, lamb and mixed meat products

AIMS: The aims of this study were (i) to evaluate the specificity and sensitivity of three previously described PCR assays for the detection of E. coli O157 and, (ii) to compare PCR, culture, and two visual immunoassays (VIAs), BioSign and Path-Stik, for detecting E. coli O157 after enrichment culture and immunomagnetic separation (IMS) performed on various naturally contaminated raw beef, lamb and mixed meat products. METHODS AND RESULTS: Twelve sorbitol non fermenting (SNF) verocytotoxin-producing (VT+) E. coli O157, 6 SNF VT- E. coli O157, 4 sorbitol fermenting (SF) VT+ E. coli O157, 3 SF VT- E. coli O157, 23 E. coli belonging to 17 other serogroups and 12 organisms of other species were used to check the specificity of PCR reactions. Only one primer pair generated amplimers only with E. coli O157 and was used for all subsequent work. After enrichment culture and on inoculated minced beef samples, PCR was as sensitive as culture for detecting 9 of the 12 strains of E. coli O157, but up to 4 log10 more sensitive than culture for detecting three strains. Of the 120 samples of naturally contaminated meat products examined, 80 (67%) were positive by PCR, 70 (58%) were positive by BioSign, 69 (58%) were positive by culture and 67 (56%) were positive by Path-Stik. Eleven samples were positive by PCR and both VIAs, but negative by culture because culture plates were heavily overgrown with SF organisms making detection of any E. coli O157 present impossible. CONCLUSIONS: PCR and both VIAs compared well with culture of beads to CT-SMAC for detecting E. coli O157 after enrichment culture and IMS. PCR appeared to be the most sensitive method, but needed specialised equipment and was also the most expensive, laborious and technically demanding technique. Although lacking the sensitivity of PCR, the VIAs were of comparable sensitivity to culture and were extremely quick and easy to perform giving a result in less than 15 minutes. SIGNIFICANCE AND IMPACT OF THE STUDY: Culture techniques may fail to detect E. coli O157 retrieved by IMS due to overgrowth with other organisms.     

A comparison of a monoclonal antibody-based sandwich ELISA and immunomagnetic bead selective enrichment for the detection of Escherichia coli O157 from bovine faeces

AIMS: To compare a recently developed monoclonal antibody (MAb) based sandwich ELISA (sELISA) with an immunomagnetic separation (IMS) method for the detection of Escherichia coli O157 in bovine faeces. METHODS AND RESULTS: Faecal samples from 345 cattle were obtained from eight farms in Northern Ireland, in which human disease due to E. coli O157 had occurred. Both assays detected E. coli O157 on five of the farms and the phage-type of the majority of the bovine strains were the same as the corresponding human isolates. Similar numbers of the organism were detected by the two methods, 59 by the sELISA and 53 by the IMS procedure, 39 of the positive samples being common to both. Twenty samples were sELISA positive/IMS negative. CONCLUSIONS: If the IMS is regarded as the gold standard, then the sELISA is less sensitive and less specific, but under the conditions used sELISA positive results were obtained from all positive farms, and the sELISA gave a presumptive positive a day earlier than the IMS method. SIGNIFICANCE AND IMPACT OF THE STUDY: The sELISA has the potential to be used as a rapid method for screening large numbers of samples for E. coli O157, but further work is required to determine its specificity.     

An adapted ImmunoMagnetic cell separation method for use in quantification of Escherichia coli O157:H7 from bovine faeces

Detection of Escherichia coli O157:H7 organisms in food, clinical or environmental samples is necessary for diagnosis of infection and epidemiological investigations. However, this pathogen may be present in low numbers and difficult to identify among high numbers of other background bacteria. In order to increase the sensitivity of culture- and PCR detection, pre-enrichment of E. coli O157:H7 in broth culture combined with ImmunoMagnetic cell Separation (IMS) is routinely employed. These methods, although able to detect levels as low as 2 cfu/g (from 10 to 25 g samples), are qualitative detection strategies only. If the actual numbers of E. coli O157:H7 are to be quantified, growth enrichment must be excluded and the organisms isolated directly from the sample of interest. Such quantification is necessary, for example, to determinate contamination levels on beef carcasses and for determination of bacterial numbers in in vivo gene expression studies. In the present study, it was not possible to recover organisms from bovine faecal suspensions using the customary IMS system and so a range of alternative buffers and other paramagnetic beads was tested. Combination of a 6.2-microm diameter bead with a detergent-based buffer gave optimal recovery of E. coli O157:H7 organisms from faecal suspensions. This system was validated for recovery of E. coli O157:H7 by comparing it with that obtained with the standard Dynabeads IMS protocol, using both the traditional broth enrichment method and a quantitative detection approach. We conclude that a 6.2-microm diameter Aureon bead can be used for quantitative isolation of E. coli O157:H7 directly from bovine faeces and, for this purpose, is preferred to the 2.8-microm diameter Dynal bead.     

RAPID TEST FOR DIAGNOSIS OF ENTEROHEMORRHAGIC E. COLI O157:H7 IN HUMAN STOOL SAMPLES

A new rapid test platform for the direct detection of E. coli O157:H7 in stool samples of infected patients was compared with the current standard methods. The new test, DIAPRO FAST-Q®, used biochip ICEflo ®technology that provided results within 20 min. Twenty-one stool samples from patients infected with E. coli O157:H7 or of unknown status were studied. Using the DIAPRO FAST-Q® method, within 20 min, we confirmed positive results for the seven known E. coli O157:H7 samples. While the standard culture method gave rise to only four positives in the remaining 14 unknown samples, the DIAPRO FAST-Q® detected E. coli O157:H7 in eight of these samples, which was confirmed subsequently by broth enrichment culture method.     

EVALUATION OF BEADRETRIEVERTM, AN AUTOMATED SYSTEM FOR CONCENTRATION OF ESCHERICHIA COLI O157 FROM ENRICHMENT CULTURES BY IMMUNOMAGNETIC SEPARATION

The aim of this study was to compare automated immunomagnetic separation (AIMS) with manual IMS (MIMS) for detection of E. coli O157 in natural and artificially inoculated samples. E. coli O157 was isolated from 27 (14.7%) of 184 natural bovine fecal samples by MIMS and from 26 (14.1%) by AIMS. Of 94 natural carcass samples, E. coli O157 was isolated from 6 by both MIMS and AIMS and from a further 6 by AIMS only. Three of the six positive only by AIMS were from tubes processed adjacently to one containing an inoculated sample and strains isolated from these adjacent tubes were indistinguishable by various typing techniques. Of the 36 carcass samples inoculated with E. coli O157, the organism was isolated from 25 by both MIMS and AIMS and from a further 9 by AIMS only. To process 100 samples by MIMS took 5h whereas by AIMS this took only 3h 20 min which represented a considerable saving of staff time.     

DynabeadsTM plus 3 M Petrifilm HECTM versus Vitek Immunodiagnostic Assay SystemTM for detection of E. coli O157 in minced meat

The potentially low infective dose of Escherichia coli O157 makes it necessary to be able to detect low numbers in food, and the lack of sensitivitiy of direct plating has led to the development of various enrichment and detection methods. Until now, the most selective procedure for detection of E. coli O157 isolates was the immunomagnetic separation (IMS) method. The number of sorbitol non-fermenting micro-organisms other than E. coli O157 that adhere non-specifically to the magnetic beads hampers the application of IMS. The use of IMS in conjunction with 3 M Petrifilm-HEC^TM yielded EHEC O157 in 21 of 165 samples of minced meat (12·7%). Without advance application of IMS, Petrifilm plates often yield confluent growth and colonies too numerous to count. The Vitek Immunodiagnostic Assay System^TM (VIDAS-ECO) showed good sensitivity when testing artificially contaminated beef samples, but only four of 21 naturally contaminated samples were recognized. The addition of 3 M Petrifilm to IMS resulted in less growth of contaminants and eliminated much of the need to test presumed colonies for confirmation. The combination of IMS with 3 M Petrifilm-HEC^TM is a fast and efficient screening procedure for E. coli O157 in minced meat.     

COMPARISON OF RAPID TECHNIQUES FOR THE DETECTION OF ESCHERICHIA COLI O157:H7

Four different commercial kits (EHEC-TEK of Organon Teknika, Durham, NC; HEC O157 of 3M, St. Paul, MN; and Coline dipstick and Coline one-step of AMP-COR Inc., Camden, NJ) were evaluated for the detection and recovery of E. coli O157:H7from 75 fresh meat samples and 23 artificially inoculated beef and pork samples. Of the total 75 samples tested, 21 (28%) were presumptive positive by HEC O157 and Coline dipstick, 18 (24%) by Coline one-step, and 12 (16%) by EHEC-TEK. None of the presumptive positive samples by any of the methods was confirmed as E. coli O157:H7 (false positive). Of the 23 positive spiked samples tested, 23 were recovered by Coline dipstick and one-step (100%), 22 (95.6%) by HEC O157, and 20 (86.9) were recovered by EHEC-TEK. In the confirmation step 17 of the 23 spiked samples produced characteristic colonies on MacConkey sorbitol agar (Difco) with 5-bromo-4-chloro-3-indoxy-β-D-glucuronide (Biosynth International) (MSA-BCIG) and were confirmed as E. coli O157:H7. No characteristic colonies on MSA-BCIG were detected for 6 of the spiked samples with initial inoculum levels of between 2 to 70 cells/g and, therefore, were not confirmed as E. coli O157:H7. A better enrichment medium, as well as improved selective plating and confirmation techniques, are needed to enhance the selective growth of E. coli O157:H7 and provide lower detection levels.     

Distribution of Escherichia coli O157 in Bovine Fecal Pats and Its Impact on Estimates of the Prevalence of Fecal Shedding

The distribution of Escherichia coli O157 in bovine feces was examined by testing multiple samples from fecal pats and determining the density of E. coli O157 in immunomagnetic separation (IMS)-positive fecal samples. The density of E. coli O157 in bovine feces was highly variable, differing by as much as 76,800 CFU g⁻¹ between samples from the same fecal pat. The density in most positive samples was <100 CFU g⁻¹, the limit of reliable detection by IMS. Testing only one 1-g sample of feces per pat with IMS may result in a sensitivity of detection as low as 20 to 50%. It is therefore probable that most surveys have greatly underestimated the prevalence of E. coli O157 shedding in cattle and the proportion of farms with shedding cattle. The sensitivity of the detection of E. coli O157 in bovine feces can be as much as doubled by testing two 1-g samples per pat rather than one 1-g sample.     

The isolation and detection of Escherichia coli O157 by use of immunomagnetic separation and immunoassay procedures

A.R. BENNETT, S. MACPHEE AND R.P. BETTS. 1996. The use of immunomagnetic separation (IMS) techniques has been reported to reduce the total test time, and improve the sensitivity, of microbiological tests done on foods. This approach is being adopted in epidemiological investigations into suspected foodborne outbreaks of Escherichia coli O157 infection and has gained acceptance by public health laboratories and the food industry. This study demonstrated the ability of a commercially available IMS procedure, Dynabeads anti- E. coli O157, to enable detection of a few cells of E. coli O157 in 25 g of inoculated minced beef, giving results 1 d earlier than a cultural analysis of similar sensitivity. With correct choice of enrichment broths, IMS may increase isolation rate of E. coli O157 compared to that obtained using conventional cultural methods. It is suggested that this may be due to an increase in relative concentration of E. coli O157 compared with the background microflora present in minced beef, which may reduce reliability of non-IMS detection procedures by masking or mimicking target cells on selective/differential solid media. The use of an immunoassay incorporating an IMS step, EHEC-Tek (Organon-Teknika), enabled detection of a few cells of E. coli O157 in 25 g of minced beef. Comparison of the IMS-ELISA with a standard ELISA procedure (Tecra) indicated the sensitivity of the latter system to be greater, perhaps resulting in the higher isolation rate. The use of a method to reliability isolate and detect extremely low levels of E. coli O157 in a food is necessary to aid reduction in the incidence of this most serious of foodborne pathogens.     

Methods for the isolation of water-borne Escherichia coli O157

AIMS: To develop improved methods for the detection of Escherichia coli O157 from water and sediments. METHODS AND RESULTS: The effects of different broth enrichment media (unsupplemented tryptic soya broth, tryptic soya broth with antibiotics, and gram-negative broth), incubation durations (5 and 24 hrs), incubation temperatures (37 and 44.5 degrees C) and the use of immunomagnetic separation (IMS) on the sensitivity of E. coli O157 detection were evaluated on artificially and naturally-contaminated water and sediment samples. The sensitivity of recovery of E. coli O157 from samples was dependent upon the media composition, temperature duration of incubation and the use of IMS. CONCLUSION: Use of high temperature (44.5 degrees C) incubation for 24 hrs in unsupplemented tryptic soya broth and the use of IMS improved the sensitivity of E. coli O157 culture from water and sediment samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods described can be used to increase the sensitivity of E. coli O157 detection from water and sediments.     

Comparison of a membrane surface adhesion recovery method with an IMS method for use in a polymerase chain reaction method to detect Escherichia coli O157:H7 in minced beef

In this study, enrichment procedures and two recovery methods, a membrane surface adhesion technique and an immunomagnetic separation (IMS), were compared for use in conjunction with a multiplex polymerase chain reaction (PCR) method with a view to describing a fast (24 h) and economical test for detection of Escherichia coli O157:H7 in meat samples. The study showed no significant difference between three different enrichment media (BHI, E. coli (E.C.) broth+novobiocin, modified tryptone soya broth (mTSB)+novobiocin) or two incubation temperatures (37 or 41.5 degrees C) for growth of E. coli O157:H7 in minced beef. Minced beef samples inoculated with E. coli O157:H7 at 40 cfu g(-1) were incubated at 37 degrees C for 16 h in E.C. broth+novobiocin reaching numbers of (log(10)7.82-8.70). E. coli O157:H7 were recovered by attachment to polycarbonate membranes immersed in the enriched cultures for 15 min or by immunomagnetic separation. Subsequent treatment of recovered membranes or IMS beads with lysis buffer and phenol/chloroform/isoamyl alcohol was used to extract the DNA from the extracted E. coli O157:H7 cells. The results show when E. coli O157:H7 was present at high levels in the enriched meat sample (log(10)9.6-7.5 cfu ml(-1); >16-h enrichment), the membrane and IMS techniques recovered similar levels of the pathogen and the microorganism was detectable by PCR using both methods. At lower levels of E. coli O157:H7 (log(10)6.4), only the IMS method could recover the pathogen but at levels below this neither method could recover sufficient numbers of the pathogens to allow detection. The conclusion of the study is that with sufficient enrichment time (16 h) the membrane surface adhesion membrane extraction method used in combination with multiplex PCR has the potential for a rapid and economical detection method.     

Biocontrol of Escherichia coli O157 with O157-specific bacteriophages.

Escherichia coli O157 antigen-specific bacteriophages were isolated and tested to determine their ability to lyse laboratory cultures of Escherichia coli O157:H7. A total of 53 bovine or ovine fecal samples were enriched for phage, and 5 of these samples were found to contain lytic phages that grow on E. coli O157:H7. Three bacteriophages, designated KH1, KH4, and KH5, were evaluated. At 37 or 4 degrees C, a mixture of these three O157-specific phages lysed all of the E. coli O157 cultures tested and none of the non-O157 E. coli or non-E. coli cultures tested. These results required culture aeration and a high multiplicity of infection. Without aeration, complete lysis of the bacterial cells occurred only after 5 days of incubation and only at 4 degrees C. Phage infection and plaque formation were influenced by the nature of the host cell O157 lipopolysaccharide (LPS). Strains that did not express the O157 antigen or expressed a truncated LPS were not susceptible to plaque formation or lysis by phage. In addition, strains that expressed abundant mid-range-molecular-weight LPS did not support plaque formation but were lysed in liquid culture. Virulent O157 antigen-specific phages could play a role in biocontrol of E. coli O157:H7 in animals and fresh foods without compromising the viability of other normal flora or food quality.     

Longitudinal study of Escherichia coli O157 in a cattle finishing unit

In a longitudinal study in a Finnish cattle finishing unit we investigated excretion and sources of Escherichia coli O157 in bulls from postweaning until slaughter. Three groups of 31 to 42 calves were sampled in a calf transporter before they entered the farm and four to seven times at approximately monthly intervals at the farm. All calves sampled in the livestock transporter were negative for E. coli O157 on arrival, whereas positive animals were detected 1 day later. During the fattening period the E. coli O157 infection rate varied between 0 and 38.5%. The animals were also found to be shedding during the cold months. E. coli O157 was isolated from samples taken from water cups, floors, and feed passages. E. coli O157 was detected in 9.7 to 38.9% of the fecal samples taken at slaughter, while only two rumen samples and one carcass surface sample were found to be positive. E. coli O157 was isolated from barn surface samples more often when the enrichment time was 6 h than when the enrichment time was 24 h (P < 0.0001). Fecal samples taken at the abattoir had lower counts (< or = 0.4 MPN/g) than fecal samples at the farm (P < 0.05). E. coli O157 was isolated more often from 10-g fecal samples than from 1-g fecal samples (P < 0.0001). Most farm isolates belonged to one pulsed-field gel electrophoresis (PFGE) genotype (79.6%), and the rest belonged to closely related PFGE genotypes. In conclusion, this study indicated that the finishing unit rather than introduction of new cattle was the source of E. coli O157 at the farm and that E. coli O157 seemed to persist well on barn surfaces.     

Comparison of cultures from rectoanal-junction mucosal swabs and feces for detection of Escherichia coli O157 in dairy heifers

Fecal culture for Escherichia coli O157:H7 was compared to rectoanal mucosal swab (RAMS) culture in dairy heifers over a 1-year period. RAMS enrichment culture was as sensitive as fecal culture using immunomagnetic separation (IMS) (P = 0.98, as determined by a chi-square test). RAMS culture is less costly than fecal IMS culture and can yield quantitative data.     

Evaluation of a PCR detection method for Escherichia coli O157:H7/H- bovine faecal samples

AIMS: Combinations of PCR primer sets were evaluated to establish a multiplex PCR method to specifically detect Escherichia coli O157:H7 genes in bovine faecal samples. METHODS AND RESULTS: A multiplex PCR method combining three primer sets for the E. coli O157:H7 genes rfbE, uidA and E. coli H7 fliC was developed and tested for sensitivity and specificity with pure cultures of 27 E. coli serotype O157 strains, 88 non-O157 E. coli strains, predominantly bovine in origin and five bacterial strains other than E. coli. The PCR method was very specific in the detection of E. coli O157:H7 and O157:H- strains, and the detection limit in seeded bovine faecal samples was <10 CFU g(-1) faeces, following an 18-h enrichment at 37 degrees C, and could be performed using crude DNA extracts as template. CONCLUSIONS: A new multiplex PCR method was developed to detect E. coli O157:H7 and O157:H-, and was shown to be highly specific and sensitive for these strains both in pure culture and in crude DNA extracts prepared from inoculated bovine faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This new multiplex PCR method is suitable for the rapid detection of E. coli O157:H7 and O157:H- genes in ruminant faecal samples.     

Evaluation of DNA probes for detection of Shiga-like-toxin-producing Escherichia coli in food and calf fecal samples

The use of DNA probes for Shiga-like toxin I (SLT-I) and SLT-II for detection of SLT-producing Escherichia coli (SLTEC) in foods and calf fecal samples was evaluated. Enrichment cultures were prepared from food or fecal samples. Colonies formed by plating of enrichment cultures were probed for SLTEC by colony hybridization. Alternatively, enrichment cultures were analyzed for SLTEC presence by dot blot. The lowest detected concentration of SLTEC in sample homogenates inoculated with E. coli O157:H7 corresponded to 1.3 CFU/g of sample. Of the 44 food samples and 28 fecal samples from dairy calves tested by the colony hybridization method, 4 food samples, including ground beef, raw goat milk, blueberries, and surimi-based delicatessen salad, and 9 calf fecal samples were positive with the SLT probes. The dot blot technique yielded results within 48 h and can be used as a fast and sensitive method of detection for SLTEC in foods and calf fecal samples. The colony hybridization technique took 3 to 4 days but permits recovery of the positive colonies when desired.     

Detection of Salmonella strains and Escherichia coli O157:H7 in feces of small ruminants and their isolation with various media

Salmonella strains and Escherichia coli O157:H7 were detected in 17 and 5 small ruminants in Virginia, respectively, of 287 tested. Background microflora interfered with the fecal analysis. The combination of Salmonella enzyme immunoassay (EIA) detection and xylose-lysine-deoxycholate agar isolation was satisfactory. Modifying enrichment to a 1:100 dilution enabled effective E. coli O157:H7 detection by EIA and isolation by sorbitol-MacConkey agar with cefixime-tellurite.     

Optimizing enrichment conditions for the isolation of Escherichia coli O157 in soils by immunomagnetic separation

AIMS: To compare a range of enrichment broths and enrichment temperatures for the isolation of Escherichia coli O157 by immunomagnetic separation (IMS) from sandy, loam and clay soils. METHODS AND RESULTS: Soils were spiked with cocktails of four atoxigenic strains of E. coli O157 and four strains of commensal E. coli. The organisms were stressed by subjecting soils to cycles of freeze/thawing, followed by drying at 20 degrees C for up to 4 days. Nine enrichment broths were trialled based on buffered peptone water, tryptone soya broths and EC broths supplemented with a range of selective additions. Enrichments were incubated for 6 h and assessed by target recovery after IMS on cefixime tellurite sorbitol MacConkey agar (CTSMAC) incubated at 37 degrees C for 24 h. A comparison of enrichment temperatures (37 and 42 degrees C) was also performed. Buffered peptone water (with or without vancomycin) and tryptone soya broth (with or without novobiocin) gave significant increases in recovery of E. coli O157 compared to others tested. In addition, broths incubated at 42 degrees C were superior to those at 37 degrees C for the recovery of E. coli O157. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that sub-lethally damaged E. coli O157 surviving in soil can be sensitive to antimicrobial additions. The choice and concentration of these additions is vitally important to optimize target recovery. Some IMS protocols, established for the isolation of E. coli O157, may be unsuitable for the examination of soil samples.     

Evaluation of DNA probes for detection of Shiga-like-toxin-producing Escherichia coli in food and calf fecal samples.

The use of DNA probes for Shiga-like toxin I (SLT-I) and SLT-II for detection of SLT-producing Escherichia coli (SLTEC) in foods and calf fecal samples was evaluated. Enrichment cultures were prepared from food or fecal samples. Colonies formed by plating of enrichment cultures were probed for SLTEC by colony hybridization. Alternatively, enrichment cultures were analyzed for SLTEC presence by dot blot. The lowest detected concentration of SLTEC in sample homogenates inoculated with E. coli O157:H7 corresponded to 1.3 CFU/g of sample. Of the 44 food samples and 28 fecal samples from dairy calves tested by the colony hybridization method, 4 food samples, including ground beef, raw goat milk, blueberries, and surimi-based delicatessen salad, and 9 calf fecal samples were positive with the SLT probes. The dot blot technique yielded results within 48 h and can be used as a fast and sensitive method of detection for SLTEC in foods and calf fecal samples. The colony hybridization technique took 3 to 4 days but permits recovery of the positive colonies when desired.