Development of a capture/enrichment sandwich ELISA for the rapid detection of enteropathogenic and enterohaemorrhagic Escherichia coli O26 strains

AIMS: To improve the sensitivity of a monoclonal antibody (MAb 2F3) based enteropathogenic Escherichia coli (EPEC)/enterohaemorrhagic E. coli (EHEC) serogroup O26-specific sandwich ELISA (sELISA), using a capture/enrichment format of the assay. METHODS AND RESULTS: The sELISA utilized an EPEC/EHEC O26-specific MAb 2F3 as the capture reagent and an E. coli serogroup O26 lipopolysaccharide-specific polyclonal antibody in the development stage. Wells containing faeces test samples from bovine enteritis cases and agar colony sweep cultures from human diarrhoea cases, after a 2-h capture stage, were washed and enrichment of the captured cells was encouraged by addition of tryptone soya broth. After overnight incubation, the contents of each well were transferred to sterile wells and the sELISA completed. Any sELISA positive samples were then subcultured onto blood agar to recover and further characterize the positive cultures. The assay had a sensitivity of 10(3) CFU ml(-1). ELISA positive samples consisted of 21 (4.8%) of the 442 bovine and 19 (3.7%) of the 519 human samples tested, and ELISA positive EPEC/EHEC O26 strains were isolated from 11 and three of these samples respectively. CONCLUSION: The capture/enrichment method improved the sensitivity of a MAb-based sELISA for the detection of EPEC/EHEC O26 strains, and also contributed to an improved isolation rate of the organism from field samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of a specific MAb in a capture/enrichment format of the sELISA, provides a prospectively suitable screening method for the detection of pathogenic bacteria from mixed culture samples.     

Shiga-toxigenic Escherichia coli O157 and non-Shiga-toxigenic E. coli O157 respond differently to culture and isolation from naturally contaminated bovine faeces

AIM: To quantify the effect of enrichment, immunomagnetic separation (IMS), and selective plating procedures on isolation of Shiga-toxigenic Escherichia coli O157 (STEC O157) and non-Shiga-toxigenic Escherichia coli O157 (non-STEC O157) from naturally contaminated bovine faeces. METHODS AND RESULTS: Two broth enrichment times, two IMS strategies, and two selective plating media were evaluated. STEC O157 and non-STEC O157 strains were often isolated from the same faecal specimen and responded differently to the isolation protocols. A large-volume IMS system was more sensitive than a conventional small-volume IMS method, but was also more expensive. STEC O157 was more frequently isolated from 6 h enriched broth and ChromAgar plates containing 0.63 mg l(-1) potassium tellurite (TCA). Non-STEC O157 was more frequently isolated from un-enriched broth and ChromAgar plates without tellurite (CA). CONCLUSIONS: The combination of 6-h enrichment in Gram-negative broth containing vancomycin, cefixime and cefsuludin, large volume IMS and selective plating on TCA maximized STEC O157 recovery from naturally contaminated cattle faecal specimens. SIGNIFICANCE AND IMPACT OF THE STUDY: The pairing of proper enrichment with a specific plating procedure is key for STEC O157 recovery from naturally contaminated bovine faeces. Incorporating tellurite into an E. coli O157 detection strategy may select for the subset of E. coli O157 that contains the Shiga-toxin genes.     

The optimization of isolation media used in immunomagnetic separation methods for the detection of Escherichia coli O157 in foods

AIMS: To compare media used in immunomagnetic separation (IMS) techniques for the isolation of Escherichia coli O157 from food. METHODS AND RESULTS: Foods, both naturally contaminated and spiked, with low numbers (< 1 g(-1)) of stressed E. coli O157 were enriched in media based on buffered peptone water (BPW), tryptone soya and EC broths incubated at 30, 37, 40 and 42 degrees C. Following immunomagnetic separation, beads were plated on a range of selective agars. CONCLUSION: BPW supplemented with vancomycin (8 mg l(-1)) incubated at 42 degrees C, followed by IMS and subsequent plating of immunobeads onto cefixime tellurite sorbitol MacConkey agar plus either Rainbow or CHROMagar agars, proved optimum for the recovery of spiked, stressed E. coli O157 in minced beef, cheese, apple juice and pepperoni. The same protocol was optimum for recovery from naturally-contaminated minced beef and cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimum protocol would increase isolation rates of E. coli O157 from foods.     

Cefixime–tellurite rhamnose MacConkey agar for isolation of Vero cytotoxin-producing Escherichia coli serogroup O26 from Scottish cattle and sheep faeces

Aims:  To compare rhamnose MacConkey agar supplemented with cefixime and tellurite (CT-RMac) and tryptone bile X-glucuronide (TBX) agars as isolation media for Vero cytotoxin-producing Escherichia coli (VTEC) serogroup O26 from animal faeces.
Methods and Results:  Nine VTEC O26 were isolated from sheep faeces; out of which six were isolated only on CT-RMac and one was isolated only on TBX. One hundred and twelve VTEC O26 were isolated from calf faeces; out of which 97% were from CT-RMac and 52% were from TBX. In a study of E. coli O26 strains, 84% of VT-positive O26 did not ferment rhamnose when compared with 16% of VT-negative O26. VT-positive (19%) and VT-negative (39%) E. coli O26 strains did not grow on CT-RMac agar.
Conclusions:  It is important to consider that VTEC O26 strains either may ferment rhamnose or may be sensitive to the CT supplement of CT-RMac agar.
Significance and Impact of the Study:  This work compares CT-RMac and TBX agars as isolation medium for VTEC O26 from Scottish animal faeces and highlights that VTEC O26 may be missed if only CT-RMac agar is used.     

Optimization of enrichment and plating procedures for the recovery of Escherichia coli O111 and O26 from minced beef

AIM: Optimization of enrichment media and selective agars for the detection of Escherichia coli O26 and O111 from minced beef. METHODS AND RESULTS: This study compared a number of different enrichment conditions and plating media for the recovery of E. coli O26 and E. coli O111 from minced beef. The optimum enrichment conditions for E. coli O26 was observed in beef samples enriched at 41.5 degrees C in tryptone soya broth supplemented with cefixime (50 microg l(-1)), vancomycin (40 mg l(-1)) and potassium tellurite (2.5 mg l(-1)). Similar enrichment conditions were optimal for E. coli O111 with the omission of potassium tellurite. The optimum agar for recovery of E. coli O26 and giving the most effective suppression of contaminants was MacConkey agar [lactose replaced by rhamnose (20 g l(-1))] and supplemented with cefixime (50 microg ml(-1)) and potassium tellurite (2.5 mg l(-1)). Optimum recovery of E. coli O111 was on chromocult agar, supplemented with cefixime (50 microg ml(-1)), cefsulodin (5 mg l(-1)) and vancomycin (8 mg l(-1)). Minced beef samples were inoculated with a number of strains of E. coli O26 (n=9) and O111 (n=8), and the developed enrichment and plating methods, used in combination with immunomagnetic separation, were shown to be an effective method for the recovery of all strains. CONCLUSIONS: Routine cultural methods for the recovery of E. coli O26 and O111 from minced beef are described. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimized enrichment and plating procedure described for the recovery of E. coli O111 and O26 from meat can be used to extend research on these emerging pathogens in beef.     

Detection of genetic diversity by pulsed-field gel electrophoresis among Escherichia coli O157 isolated from bovine faecal samples by immunomagnetic separation technique

AIMS: Escherichia coli O157 is considered to be one of most important human pathogens of animal origin which causes serious clinical complications. One of the most common methods to isolate E. coli O157 is the immunomagnetic separation (IMS) technique which employs specific antibodies coupled to magnetic beads to bind and extract cells from enrichment broths followed by plating onto sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (CT-SMAC) plates. The aim of this study was to determine strain variation by pulsed-field gel electrophoresis (PFGE) among E. coli O157 on IMS/CT-SMAC plates. METHODS AND RESULTS: Every suspect colony of E. coli O157 was tested following isolation by the IMS/CT-SMAC technique. From 124 colonies detected; six XbaI-PFGE profiles were identified. CONCLUSIONS: Our results demonstrate that mixed populations of E. coli O157 with distinguishable PFGE profiles that are simultaneously present in bovine faeces can be isolated with IMS/CT-SMAC technique. SIGNIFICANCE AND IMPACT OF THE STUDY: If the aim of the study was to analyse diversity of PFGE profiles of E. coli O157 in a faecal sample following isolation by the IMS/CT-SMAC technique, at least five colonies per sample should be analysed to detect different PFGE subtypes if present.     

Comparison of the sensitivity of manual and automated immunomagnetic separation methods for detection of Shiga toxin-producing Escherichia coli O157:H7 in milk

AIM: To determine the sensitivity of methods for detection of injured and uninjured Escherichia coli O157:H7 (E. coli O157) in raw and pasteurized milk. METHODS AND RESULTS: Raw milk, pasteurized milk with 1.5% fat content and pasteurized milk with 3.5% fat content were spiked with E. coli O157 at low levels. The samples were enriched in modified tryptone soya broth with novobiocin (mTSBn) at 37 degrees C. Aliquots of the enriched culture were analysed either by manual immunomagnetic separation (MIMS) and culturing on sorbitol MacConkey agar with or without cefixime and potassium tellurite (SMACct or SMAC), or by automated immunomagnetic separation and integrated ELISA (EiaFosstrade mark). Uninjured E. coli O157 organisms were detected in milk by both methods at 1 cfu 10 ml-1 sample). Injured organisms were detected at levels of about 4 cfu 10 ml-1 sample. Direct enrichment in mTSBn (22 h incubation) showed better sensitivity for injured cells than enrichment in buffered peptone water (BPW, 22 h incubation), or in a two-step enrichment consisting of BPW (6 h, 37 degrees C) and mTSBn (16 h, 37 degrees C), successively. CONCLUSIONS: The methods showed equal sensitivity in that they were both able to detect 1 cfu 10 ml-1 milk sample. Injured organisms can be detected and isolated at a level almost as low as this. A resuscitation step is not recommended for the detection and isolation of injured and non-injured E. coli O157 from milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the dilution of contamination in the bulk tank, analysis of milk for the presence of E. coli O157 requires a very sensitive method. Both methods described here are useful for such analysis.     

Polymyxin-based enzyme-linked immunosorbent assay for the detection of Escherichia coli O111 and O26

Polymyxin-based enzyme-linked immunosorbent assay (polymyxin-ELISA) systems were developed for the detection of Escherichia coli O111 and O26 in ground beef after enrichment. Polymyxin immobilized in the wells of a microtiter plate served as a high affinity adsorbent for lipopolysaccharide (LPS) antigens, which were detected immunoenzymatically using commercially available anti-E. coli O111 or anti-E. coli O26 antisera. The polymyxin-ELISA sensitively detected E. coli strains bearing the O111 and O26 LPS antigens, discriminating between these target strains and a panel of various non-target Gram negative and Gram positive bacteria. The detection of E. coli O111 and O26 strains inoculated into ground beef was achieved after enrichment in either modified trypticase soy broth (TSB) with novobiocin, or the serotype-specific medium TSB supplemented with cefixime and vancomycin (E. coli O111), and the same medium containing potassium tellurite (E. coli O26). The polymyxin-ELISA shows promise as a rapid, simple and inexpensive screening tool for E. coli O111 and O26 in enrichment cultures of ground beef.     

A comparison of two pre-enrichment media prior to immunomagnetic separation for the isolation of E. coli O157 from bovine faeces

AIMS: To compare the sensitivity of two pre-enrichment broth media prior to immunomagnetic separation for the isolation of Escherichia coli O157 from cattle faeces. METHODS AND RESULTS: One-gram portions of 721 cattle faeces collected from 43 farms were pre-enriched in buffered peptone water containing vancomycin, cefixime and cefsulodin (BPW-VCC) and buffered peptone water without additives (BPW-WOA), respectively. A total of 137 samples were positive for E. coli O157: 127 pre-enriched with BPW-WOA and 89 pre-enriched in BPW-VCC. Representative isolates were tested for phage type, verotoxin and eae (E. coli attaching and effacing) gene sequences, resulting in the recognition of eight different types. All the E. coli O157 types recognized were isolated by both methods except for three different strains, each of which were isolated only on a single occasion: two by BPW-WOA and another by BPW-VCC. CONCLUSIONS: The results clearly demonstrate, under the conditions of this study, that BPW without antibiotics was the superior pre-enrichment medium for the isolation of E. coli O157 from cattle faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of BPW-WOA in preference to BPW-VCC for the isolation of E. coli O157 from cattle faeces in future research and outbreak studies should lead to a higher number of positive isolates.     

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.     

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 Escherichia coli O157 on pasture following an outbreak associated with sheep at a scout camp

AIMS: To monitor the decay of E. coli O157 in soil (loamy sand) on a scout campsite following an outbreak in humans. METHODS AND RESULTS: Samples of soil and sheep faeces were collected from the campsite and tested for the presence of E. coli O157 by immunomagnetic separation (IMS) after enrichment in buffered peptone water + vancomycin at 42 degrees C for 6 h. Enumeration of target was carried out by direct plating onto sorbitol MacConkey agar plates supplemented with cefixime and tellurite (CTSMAC) incubated at 37 degrees C for 24 h. Low numbers (< 100 g(-1)) were estimated by the most probable number (3-tube MPN) technique. CONCLUSIONS: Survival was observed for 15 weeks. SIGNIFICANCE AND IMPACT OF THE STUDY: A number of laboratory studies have followed the decay of E. coli O157 in soil, animal faeces and water. This study follows (for the first time) the decay of the organism in soil after an outbreak associated with sheep. It demonstrates the long-term persistence of the organism in the environment and the results will be potentially important in performing risk assessments for both human and animal infection.     

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.     

The detection of non-O157 E. coli in food by immunomagnetic separation

AIMS: To compare immunomagnetic separation (IMS) protocols (enrichment media and temperature) for the isolation of Escherichia coli serotypes O26 and O111 from four different foods. METHODS AND RESULTS: Foods (minced beef, cheese, apple juice and pepperoni) spiked with low numbers (<100 g(-1)) of stressed nalidixic mutant E. coli serotypes O26 and O111 were enriched in media based on buffered peptone water (BPW), tryptone soya and EC broths incubated at temperatures of 37 and 42 degrees C to optimize the IMS technique. BPW enrichments gave increased recoveries of both serotypes compared with tryptone soya and EC broths. Elevated temperatures of incubation at 42 degrees C were superior to 37 degrees C. CONCLUSIONS: Positive detection of low numbers of stressed target pathogens in all replicate tests was only possible using BPW enrichments. The majority of tests from alternative enrichments resulted in zero or single colonies recovered post-IMS. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimum IMS protocol would improve isolation rates of E. coli O26 and O111 from foods and lead to increased safety for the consumer. Sub-optimal IMS protocols could lead to foods being incorrectly labelled free from these pathogens.     

Isolation of Salmonella strains from reptile faeces and comparison of different culture media

AIMS: To provide information on epidemiology and isolation of Salmonella strains from reptiles. METHODS AND RESULTS: Ninety-one samples collected from reptiles of the zoo of Rome or belonging to private owners were analysed using a standard protocol for isolation of Salmonella from food. Salmonella strains were tested for susceptibility to 15 antimicrobics by a disc-agar diffusion method. Forty-six samples (50.5%) were positive for Salmonella. Of the 22 strains serotyped, 17 belonged to Salmonella enterica subsp. I, four to the subsp. IIIa and one strain resulted untypeable. Rappaport-Vassiliadis broth (RVB) allowed to recover more Salmonella strains when bacterial growth in buffered peptone water (BPW) was scarce, while selenite cystine broth (SCB) was more efficient, whereas growth in BPW was abundant. The maximum isolation score was obtained by plating onto xylose lysine desoxycholate agar (XLD). The strains exhibited resistance at high percentages to colistin sulphate (58.7%), sulphamethoxazole (55.5%), streptomycin (32.6%), tetracycline (19.6%), ampicillin (17.4%) and nalidixic acid (13.1%). CONCLUSIONS: A high prevalence of Salmonella in reptiles was observed. For isolation, the choice of the enrichment broth depending on the degree of growth in BPW followed by plating onto XLD may be suggested. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provides epidemiological data on the prevalence of Salmonella and laboratory protocols useful for isolation of Salmonella from faeces of reptiles.     

Evaluation of methods for the isolation and detection of Escherichia coli O157 in minced beef

This study has evaluated enrichment and detection procedures for the isolation and detection of Escherichia coli O157 inoculated into minced beef. The use of a 24 h enrichment in modified EC broth containing novobiocin allowed low numbers of contaminating cells to multiply to levels detectable on culture media and by ELISA test kits. Total analysis time was reduced by the use of the Dynabead^TM immunomagnetic separation system. The use of the Petrifilm^TM Test Kit-HEC for E. coli O157: H7 and Organon Teknika EHEC-TEK system detected low numbers of contaminating cells following enrichment and reduced analysis time by 1 d. The incorporation of cefixime and tellurite into Sorbitol MacConkey Agar increased the rate and ease of isolation of E. coli O157 and its use is therefore recommended.     

RAPID detection and quantification of E. coli O157/O26/O111 in minced beef by real-time PCR

AIM: To develop a real-time PCR detection procedure for Escherichia coli O111, O26 and O157 from minced meat. METHODS AND RESULTS: Strains (n = 8) of each of E. coli O26, E. coli O111 and E. coli O157 were inoculated at ca 10-20 CFU g(-1) into minced retail meat and enriched for 6 h at 41.5 degrees C as follows: E. coli O26 in tryptone soya broth (TSB) supplemented with cefixime (50 microg l(-1)), vancomycin (40 mg l(-1)) and potassium tellurite (2.5 mg l(-1)); E. coli O111 in TSB supplemented with cefixime (50 microg l(-1)) and vancomycin (40 mg l(-1)); E. coli O157 in E. coli broth supplemented with novobiocin (20 mg l(-1)). DNA was extracted from the enriched cultures, and detected and quantified by real-time PCR using verotoxin (vt1 and vt2) and serogroup (O157 per gene; O26 fliC-fliA genes and O111 wzy gene) specific primers. CONCLUSIONS: The methods outlined were found to be sensitive and specific for the routine detection of E. coli O111, O26 and O157 in minced beef. SIGNIFICANCE AND IMPACT OF THE STUDY: The enrichment, isolation and detection procedures used in this study provide a rapid routine-based molecular method for the detection and differentiation of E. coli O26, O111 and O157 from minced meat.     

Occurrence of Escherichia coli O157 in feces of healthy persons, from selected groups, in the country

The purpose of the study was determination of the occurrence of E. coli O157 in faeces samples of healthy subjects and characterization of the isolated strains with respect to their potential pathogenicity. The study was carried out in two stages. In the first one in 5 sanitary-epidemiological stations samples were tested from healthy subjects after inoculation onto McConkey (MC) or/and McConkey with sorbitol (SMC) media and isolating from each culture 10 lactose-positive (on MC medium) or sorbitol-negative (on SMC) colonies. Then latex test was done with each isolate for E. coli O157 presence. In all, 1005 samples were studied, including 260 taken from children aged 0-2 years, 180 samples from children aged 3-10 years, and 565 samples from older children and adults. E. coli O157 rods were cultured from 6 adults (0.6%). In the second stage carried out at the Laboratory of Enterobacteriaceae, Bacteriology Department, National Institute of Hygiene strains obtained from territorial laboratories were studied determining their phenotypic and genotypic traits regarded as virulence markers of verotoxic E. coli O157 strains, such as inability to ferment sorbitol and MUG breakdown, and production of verotoxins and enterohaemolysin. By the PCR method fragments were sought of genes coding for production of verotoxins, intimin and enterohaemolysin. The results showed that no E. coli O157 strain obtained from healthy individuals produced verotoxins, but three studied strains contained the eae gene determining intimin production and they were regarded as enteropathogenic.     

High numbers of Shiga toxin-producing Escherichia coli found in bovine faeces collected at slaughter in Japan

The prevalence and concentration of Shiga toxin-producing Escherichia coli (STEC) in cattle faeces (n=605) at the time of slaughter was studied in Shimane Prefecture, Japan on a monthly basis between April 2000 and March 2001. Screening with stx-PCR determined a prevalence of 37.5%. After analysis of spread faeces and enriched samples on cefixime, tellurite and sorbitol-MacConkey agar using HCl treatment, 114 STEC strains were singly or concomitantly isolated from 97 cattle (15.9%). Of the 605 cattle, 31 (5.1%) harbored O26:H11, O111:H-, O121:H19 or O157:H7, which had the stx1 and/or stx2 and eae and hlyA genes, and 7 (23%) of these 31 cattle were high level carriers that contained these typical STEC at concentrations of 10(5)-10(8) CFU/g of faeces. The predominant serotype was O26:H11 (20 strains) and the second most frequent was O157:H7 (9 strains). Of the 605 cattle, 68 (11.2%) harboured 36 other serotypes and 6 (5.9%) of the 67 cattle were high level carriers. As a comparison between the prevalence of STEC and the faecal pH, it was demonstrated that STEC can be isolated from cattle with a wide range of faecal pH values. The presence of a high-carriage animal at the abattoir increases the potential risk of meat contamination during the slaughtering process, regardless of faecal pH.     

Efficacy of supplemented buffered peptone water for the isolation of Campylobacter jejuni and C. coli from broiler retail products

Broiler retail samples (n=113) were analyzed to determine (i) the effectiveness of buffered peptone water (BPW) supplemented with blood and antibiotics for the isolation of Campylobacter jejuni and C. coli, (ii) if a 1:4 enrichment ratio performs similarly as a 1:9 ratio, and (iii) if BPW is similar to Bolton broth for enumeration of Campylobacter spp. in retail broiler meat using the most probably number (MPN) procedure. Chi-square comparison showed that BPW performed similarly as Bolton broth (P< or =0.05) for Campylobacter isolation in breast tenders, boneless breasts, split breasts and skin samples. However, BPW showed a lower detection rate (P> or =0.05) for thighs and boneless thighs. When the results were combined, BPW performed similarly as Bolton broth for the isolation of Campylobacter spp. (P< or =0.05). BPW at an enrichment ratio of 1:4 was statistically similar to Bolton broth or BPW at a ratio of 1:9. No differences were observed between the MPN data from Bolton broth and the MPN data from BPW (P< or =0.50). A multiplex PCR assay revealed that ca. 48% of the isolates obtained from Bolton broth and 59% of the isolates obtained with BPW were C. coli. Both Bolton broth and BPW allowed for the growth of C. jejuni and C. coli from the same sample. Remarkably, a large genomic variability was observed by PFGE analysis of the isolates collected from the same sample with Bolton broth or BPW, which confirms that more than one genotype can successfully multiply during enrichment and be recoverable on agar plates. These findings suggest that BPW could be used as an enrichment medium for isolation of Campylobacter from retail broiler samples. The implications of the high number of C. coli isolates found in this study is discussed.