Growth and Survival of Acid-Resistant and Non-Acid-Resistant Shiga-Toxin-Producing Escherichia coli Strains during the Manufacture and Ripening of Camembert Cheese.

Growth and survival of acid-resistant (AR) and non-acid-resistant (NAR) Shiga-toxin-producing Escherichia coli (STEC) strains were investigated during the manufacture and ripening of microfiltered milk Camembert cheeses. The induction of acid resistance of the STEC strains in cheeses was also studied. Six different mixtures of AR and/or NAR STEC strains were inoculated separately into microfiltered milk at a level of 10(3) CFU mL(-1). The STEC counts (AR and NAR) initially increased by 1 to 2 log(10) CFU g(-1) during cheese-making. Thereafter, the populations stabilized during salting/drying and then decreased during the early stages of ripening. Exposing the STEC strains in artificially inoculated cheeses to simulated gastric fluid (SGF - pH: 2.0) reduced the number of NAR strains to undetectable levels within 40 minutes, versus 120 minutes for the AR STEC strains. AR and NAR STEC were able to survive during the manufacture and ripening of Camembert cheese prepared from microfiltered milk with no evidence of induced acid tolerance in NAR STEC strains.

Fate of acid-resistant and non-acid resistant Shiga toxin-producing Escherichia coli strains in experimentally contaminated French fermented raw meat sausages.

Both pathogenic and nonpathogenic E. coli exhibit a stress response to sublethal environmental stresses. Several studies have reported acid tolerance and survival characteristics of E. coli O157:H7 in foodstuffs, but there are few reports about the tolerance of non-O157 serogroups (STEC) to organic acids in foods. The purpose of this study was to examine the effect of the manufacturing process of French fermented raw meat sausages on the growth and survival of acid-resistant (AR) and non-acid resistant (NAR) STEC strains. The six strains, 3 AR and 3 NAR, were inoculated separately into raw sausage mixture at a level of 10(4)-10(5) CFU/g. A total of 19 batches of sausages were manufactured. A rapid and similar decrease in the number of both AR and NAR STEC strains, from less than 1 to 1.5 log(10) CFU/g, was observed during the first 5 days of fermentation at 20-24 degrees C. This rapid decrease was followed by a more gradual but continuous decrease in STEC counts after drying at 13-14 degrees C, up to day 35. The STEC counts were <10 CFU/g after 35 days for the NAR strains and the same concentration for the AR strains on the best before date (day 60). It was not possible to detect any NAR STEC after 60 days. The present study shows that the process used in the manufacture of French sausages results in a complete destruction of NAR STEC strains after 60 days, but it does not have the same effect on the AR STEC strains.

Community-wide outbreak of Escherichia coli O157:H7 associated with consumption of frozen beef burgers.

On 24-25 October 2005 a cluster of five haemolytic uraemic syndrome (HUS) cases was reported in southwest France. An investigation was undertaken to identify the outbreak source and implement control measures. Cases were defined as individuals with HUS or diarrhoea with isolation of Escherichia coli O157:H7 in stools or a positive antibody response to E. coli O157 lipopolysaccharide, resident in southwest France with symptom onset after 19 September 2005. Sixty-nine identified patients had symptom onset between 5 October and 3 November 2005, including 17 cases of HUS. One brand of frozen beef burgers produced on 22 August 2005 was consumed by all patients in the week before symptom onset. E. coli O157:H7 strains from patients, patients' burgers and the manufacturing plant were genetically related. This is the largest community-wide outbreak of E. coli O157:H7 in France to date and the first associated with consumption of contaminated frozen beef burgers.

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 degrees C). The BM and STEC growth data were simultaneously fitted by using a competitive growth model. The STEC final levels obtained after 24h 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.

O148 Shiga toxin-producing Escherichia coli outbreak: microbiological investigation as a useful complement to epidemiological investigation.

An outbreak of Shiga toxin-producing Escherichia coli (STEC) O148 infection occurred among wedding attendees in France in June 2002. A retrospective cohort study was performed and ten cases were identified, including two adults with haemolytic uraemic syndrome (HUS). The analytical study revealed that > 80% of affected individuals had eaten lightly roasted mutton and poultry pâté, but only the consumption of pâté tended to be associated with illness (relative risk 3.4; 95% CI 0.8-14.4). Left-overs (cooked mutton and raw offal) and processed foods (pâté) from the same batches as served at the party were sampled. Human, food and environmental samples were examined for the Shiga toxin (stx) gene and virulence traits by PCR. Stx-positive samples were cultured for STEC. HUS cases were tested for serum antibodies against 26 major STEC serogroups. An STEC O26 strain (stx1, eae, ehxA) was isolated from one case with diarrhoea, and an STEC O148 strain (stx2c) from one case of HUS. Serum antibodies against O26 were not detected in either of these patients; antibodies against O148 were not tested. Three STEC strains were isolated from the mutton and the offal (stx2c, O148), and two from the pâté (stx2c, O-X and O-Y). The isolates from the mutton were indistinguishable from the human stx2c isolate, whereas the pâté isolates differed. Although four different STEC strains were identified in patients and foods, the results of molecular subtyping, in conjunction with analysis of food consumption patterns, strongly suggested that this outbreak was caused by mutton contaminated with STEC O148.

Modeling and predicting the simultaneous growth of Escherichia coli O157:H7 and ground beef background microflora for various enrichment protocols.

The simultaneous growth of Escherichia coli O157:H7 (O157) and the ground beef background microflora (BM) was described in order to characterize the effects of enrichment factors on the growth of these organisms. The different enrichment factors studied were basal medium (Trypticase soy broth and E. coli broth), the presence of novobiocin in the broth, and the incubation temperature (37 degrees C or 40 degrees C). BM and O157 kinetics were simultaneously fitted by using a competitive growth model. The simple competition between the two microfloras implied that O157 growth stopped as soon as the maximal bacterial density in the BM was reached. The present study shows that the enrichment protocol factors had little impact on the simultaneous growth of BM and O157. The selective factors (i.e., bile salts and novobiocin) and the higher incubation temperature (40 degrees C) did not inhibit BM growth, and incubation at 40 degrees C only slightly improved O157 growth. The results also emphasize that when the level of O157 contamination in ground beef is low, the 6-h enrichment step recommended in the immunomagnetic separation protocol (ISO EN 16654) is not sufficient to detect O157 by screening methods. In this case, prior enrichment for approximately 10 h appears to be the optimal duration for enrichment. However, more experiments must be carried out with ground beef packaged in different ways in order to confirm the results obtained in the present study for non-vacuum- and non-modified-atmosphere-packed ground beef.

First isolation of Shiga toxin 1d producing Escherichia coli variant strains in shellfish from coastal areas in France.

AIMS: This study was carried out to evaluate the presence of Shiga toxin-producing Escherichia coli (STEC) and E. coli O157:H7 in shellfish from French coastal environments. METHODS AND RESULTS: Shellfish were collected in six growing areas or natural beds (B category) and nonfarming areas (D category) from July 2002 to August 2004. PCR detection of stx genes was performed on homogenized whole shellfish and digestive gland tissues enrichments. STEC strains were detected by colony DNA hybridization using a stx-specific gene probe and E. coli O157 strains were additionally searched by immunomagnetic separation with O157-specific magnetic beads. Stx genes were detected in 40 of 144 (27.8%) sample enrichments from mussels, oysters or cockles, 32 of 130 enrichments (24.6%) were from B-category areas and eight of 14 (57.1%) from the D-category area. Five strains carrying stx(1) or stx(1d) genes and one stx negative, eae and ehxA positive E. coli O157:H7 were isolated from six of 40 stx-positive enrichments. No relation was found between the total E. coli counts in shellfish and the presence of STEC strains in the samples. CONCLUSIONS: The STEC strains of different serotypes and stx types are present in shellfish from French coastal environments. It is the first isolation of STEC stx1d strains in France. SIGNIFICANCE AND IMPACT OF THE STUDY: Shellfish collected in coastal environments can serve as a vehicle for STEC transmission.

Isolation and characterization of Shiga toxin-producing Escherichia coli strains from raw milk cheeses in France.

AIMS: To evaluate Shiga toxin-producing Eschericha coli (STEC) prevalence in 1039 French raw milk cheeses including soft, hard, unripened and blue mould cheeses, and to characterize the STEC strains isolated (virulence genes and serotypes). METHODS AND RESULTS: STEC strains were recovered from cheese samples by colony hybridization. These strains were then serotyped and genetically characterized. These strains (32 STEC) were then recovered from 18 of 136 stx-positive samples: 19 strains had stx2 variant genes stx(2vh-a) (n = 2), stx(2NV206) (n = 2), stx(2EDL933) (n = 4) and stx2d (n = 11). Thirty strains had the stx1 gene and one strain, the eae gene. Combinations of stx2 and stx1 genes were present in 17 (81%) of the STEC strains. Nineteen strains belonged to the O6 serogroup and the other strains belonged to the O174, O175, O176, O109, O76, O162 and O22 serogroups in decreasing frequency. CONCLUSIONS: No conclusion can be drawn at the moment concerning the potential risk to consumers because the O6:H1 serotype has already been found associated with the haemolytic uremic syndrome and almost no isolate had the eae gene. SIGNIFICANCE AND IMPACT OF THE STUDY: The large number of STEC strains recovered from the cheese samples evaluated in this study emphasizes the health risks associated with raw milk cheeses. This further emphasizes the immediate need to identify and implement effective pre- and postharvest control methods that decrease STEC carriage by dairy cattle and to eliminate contamination of their cheeses during processing.

Growth and survival of Escherichia coli O157:H7 during the manufacture and ripening of raw goat milk lactic cheeses.

The behaviour of Escherichia coli O157:H7 was studied during the manufacture and ripening of raw goat milk lactic cheeses. Cheese was manufactured from raw milk in the laboratory and inoculated with E. coli O157:H7 to a final concentration of 10, 100 and 1000 cfu ml(-1). E. coli O157:H7 was counted by CT-SMAC (Mac Conkey Sorbitol Agar with cefixim and tellurite) and O157:H7 ID throughout the manufacturing and ripening processes. When the milk was inoculated with 10, 100 or 1000 cfu ml(-1), counts decreased to less than 1 log(10) g(-1) in curds just prior to moulding. However, viable E. coli O157:H7 were found in cheeses throughout processing, and even after 42 days of ripening. Results indicate that E. coli O157:H7 survives the lactic cheese manufacturing process. Thus, the presence of low numbers of E. coli O157:H7 in milk destined for the production of raw milk lactic cheeses can constitute a threat to the consumer.

Serotyping, stx2 subtyping, and characterization of the locus of enterocyte effacement island of shiga toxin-producing Escherichia coli and E. coli O157:H7 strains isolated from the environment in France.

Twenty-seven Shiga toxin-producing Escherichia coli (STEC) strains were isolated from 207 stx-positive French environmental samples. Ten of these strains were positive for stx(1), and 24 were positive for stx(2) (10 were positive for stx(2vh-a) or stx(2vh-b), 19 were positive for stx(2d), and 15 were positive for stx(2e)). One strain belonged to serotype O157:H7, and the others belonged to serogroups O2, O8, O11, O26, O76, O103, O113, O121, O141, O166, and O174. The environment is a reservoir in which new clones of STEC that are pathogenic for humans can emerge.

Prevalence of verotoxin-producing Escherichia coli (VTEC) in slurry, farmyard manure and sewage sludge in France.

AIMS: The aims of the present study were to determine VTEC prevalence in manure, slurry and sewage sludge in France and to characterize the VTEC strains isolated (virulence genes and serotype). METHODS AND RESULTS: Seven hundred and fifty-two samples from 55 farmyard manures, 136 bovine and porcine faeces, 114 slurries, 10 composts, and 437 samples from outflows of sewage wastewater treatment plants were analysed. Twenty-four percent contained isolates which were PCR positive for stx gene. Twenty-one VTEC strains were recovered from positive samples by colony hybridization: 76% of them were positive for stx(2) gene, 33% for stx(1) gene,and 19% for eae gene. One strain belonged to serotype O157:H7 and two others to serogroups O26 and O55, respectively. CONCLUSIONS: Some of the VTEC strains isolated from environments in France should be considered as potentially pathogenic for humans. SIGNIFICANCE AND IMPACT OF THE STUDY: Appropriate handling or use of manure, slurry and sewage sludge is necessary so that contamination of the environment and food by VTEC can be prevented.

Effects of cutting process on pork meat contamination by verotoxin-producing Escherichia coli (VTEC) and E. coli O157:H7.

The aims of the present study were: (i) to evaluate verotoxin-producing Escherichia coli (VTEC) prevalence in pork cutting meat; (ii) to determine the effects of cutting process on pork meat contamination by VTEC; (iii) to characterise the VTEC strains isolated from pork and pork cutting plants (virulence genes and serotype); and (iv) to compare the strains isolated the same day in the same cutting plant in order to identify the routes of contamination inside the cutting plant. Pork carcasses from three French cutting plants were sampled before carcass cutting (carcass samples), after carcasses were divided into big portions (untrimmed cuts) and after preparation of primal cuts (rindless boneless cuts), and different environmental sites in each cutting plant were sampled at three different times in the work day. Potable water was also collected. PCR detection of stx genes was performed on a total of 2042 samples. In addition, a second PCR specific for E. coli O157:H7 detection was carried out on the stx-positive samples. VTEC strains were recovered from positive samples by colony hybridisation or immunoconcentration, then serotyped, genetically characterised (eae, ehx, stx1, stx2, stx2e, uidA and genes which are associated with virulence) and pulsotyped. No E. coli O157:H7 was detected. Meat contamination decreased from carcass (12%) and primary cuts (19%) to secondary cuts (5%), whereas environmental contamination increased after 2 h of activity (from 3% before the commencement of the work day to 25% and 20%, 2 and 6 h after commencement of cutting). No VTEC isolates harboured eae, ehx and uidA genes. VTEC contamination routes were not clearly identified.

Effects of slaughter processes on pig carcass contamination by verotoxin-producing Escherichia coli and E. coli O157:H7.

The aims of the present study were: (i) to evaluate verotoxin-producing Escherichia coli (VTEC) faecal carriage of slaughtered pigs; (ii) to determine the effects of three different pig slaughtering processes on pig carcass contamination by VTEC; (iii) to characterise the VTEC strains isolated from pig and pig slaughterhouses (virulence genes and serotype); and (iv) to compare the strains isolated in the same slaughterhouse in order to identify the routes of contamination inside the slaughterhouse. Pork carcasses from three French slaughterhouses were sampled at three steps of the slaughter process and different sites in each slaughterhouse were sampled at three different times in the work day. Faecal material from each sampled carcass, potable water and scalding water were also collected. Detection of stx genes was performed by polymerase chain reaction (PCR) on a total of 1227 samples. In addition, a second PCR specific for E. coli O157:H7 detection was carried out on the stx-positive samples. VTEC strains were recovered from positive samples by colony hybridisation or immunoconcentration, then serotyped, genetically characterised (eae, ehx, stx1, stx2, stx2c, uidA genes associated with virulence) and pulsotyped. No E. coli O157:H7 was isolated from the three uidA-positive samples. VTEC faecal carriage was 31%. Global carcass contamination decreased with slaughter process (from 46% to 15%), whereas environmental contamination increased (from 7% to 29%). No VTEC isolates harboured eae, ehx, and uidA genes. VTEC contamination routes were not clearly identified.

Prevalence of verotoxin-producing Escherichia coli (VTEC) and E. coli O157:H7 in French pork.

AIMS: To determination the prevalence of VTEC in pork products and the surrounding environment of the pork plant (slaughterhouse and cutting plant), and characterization of the VTEC strains isolated (virulence genes and serotype). METHODS AND RESULTS: Among the 2146 carcass and pork samples and 876 environmental samples (swabs of surfaces or materials), 328 (15%) and 170 (19%) were PCR-positive for stx genes respectively. VTEC strains were recovered from positive samples by colony hybridization or immunoconcentration, serotyped and genetically characterized. Strains of E. coli O157:H7 were not isolated from 3 uidA-positive samples detected by PCR. The VTEC isolates did not harbour eae, ehx and uidA genes. CONCLUSIONS: Pigs and pork meat may contain VTEC strains but characterization of the strains based on virulence factors showed that the potential danger of pork meat appears to be low since although all strains harboured a stx gene, they did not have other virulence genes. SIGNIFICANCE OF THE STUDY: General hygiene measures appear to be sufficient and specific hygiene measures for VTEC are not necessary at this time. The porcine VTEC strains isolated in our study probably do not present a hazard.

Prevalence of Escherichia coli O157:H7 in industrial minced beef.

AIMS: The lack of baseline data on the prevalence of Escherichia coli O157:H7 in retail minced beef in France prompted this survey of industrial minced beef production. METHODS AND RESULTS: An automated enzyme-linked fluorescence immunoassay (ELFA), the VIDAS E. coli O157 method, was used to detect E. coli O157 in industrial minced beef samples. Confirmation of samples positive according to the ELFA was performed using an automated immunoconcentration (ICE) system, VIDAS ICE, which allows the selective capture and release of target organisms. The ICE was followed by culture on cefixime tellurite sorbitol MacConkey agar and a chromogenic medium, O157:H7 ID. Of the 3450 minced beef samples tested, 175 samples were positive with the ELFA method and, of these, four were confirmed by the ICE method. They were identified as sorbitol-negative, O157-positive, H7-positive, mobile, verotoxin-producing E. coli. CONCLUSIONS: The prevalence of E. coli O157:H7 in industrial French minced beef was 0.12%, consistent with many other reports. SIGNIFICANCE AND IMPACT OF THE STUDY: The low infective dose of E. coli O157:H7 presents a major threat. The main means of combating this organism are thermal destruction and good food hygiene covering activities on-farm, in the abattoir and in minced beef industries.

Prevalence of verotoxin-producing Escherichia coli and E. coli O157:H7 in pig carcasses from three French slaughterhouses.

Verotoxin-producing Escherichia coli (VTEC) are important food-borne pathogens in humans. Several studies have demonstrated that cattle are a major reservoir of VTEC but few data are available about the occurrence of VTEC in other species. In France, there is no data about pigs and pork meat. The main purpose of this study was to evaluate the prevalence of E. coli O157:H7 and other VTEC in pork carcasses. The second aim of the study was to get a picture of pork carcass contamination by VTEC. Pork carcasses from three French slaughterhouses (50 carcasses per slaughterhouse) were tested for the presence of VTEC and E. coli O157:H7. For each carcass, both internal and external sites were investigated (five on pig skin and three on muscles) and samples were collected by cutting out a surface of 25 cm2. A total of 1200 samples were analyzed by the polymerase chain reaction (PCR) after an enrichment step. Primers used were degenerate-sequences which allowed amplification of various types of verotoxin genes (stx). In addition, a second PCR which specifically detected E. coli O157:H7 was carried out on the stx-positive samples. The percentage of stx-positive PCR samples and carcasses was 12.7% (152/1200) and 50% (75/150), respectively. No E. coli O157:H7 was detected. The prevalence for each slaughterhouse was not significantly different. Skin samples of belly, leg and shoulder allowed detection of more than 80% of the VTEC positive carcasses.

Detection of Escherichia coli O157:H7 in heifers' faecal samples using an automated immunoconcentration system.

Pre-treatment of a 5-h enrichment culture with an automated immunoconcentration (ICE) system greatly improved the isolation of Escherichia coli O157:H7 from spiked heifer faecal samples. Enrichment samples plated directly onto sorbitol MacConkey agar (SMAC) and SMAC agar supplemented with cefixime and potassium tellurite (CT-SMAC) showed recovery rates of 8% and 56%, respectively. However, after ICE treatment, E. coli O157:H7 was recovered from 92% of the samples on SMAC and 100% on CT-SMAC. Immunoconcentration analysis of heifers' faecal samples collected from a slaughter-house in France, during March to June 1998, showed that 1% (three of 300) was positive for E. coli O157:H7. Phenotypic and genotypic analysis showed that all three isolates carried both the O157 and H7 antigens, did not ferment sorbitol or had beta-glucuronidase activity and carried trait virulence factors for E. coli O157:H7 (uidA allele, eaeA and pO157 plasmid). However, only one strain was toxigenic and this strain produced a single toxin, namely verotoxin 2.

An automated method for the detection of staphylococcal heat stable deoxyribonuclease in dairy products.

An automated sandwich immunoassay with specific polyclonal antibodies for the detection of Staphylococcus aureus thermostable nuclease (DNase) is described. To evaluate this assay, different quantities of purified S. aureus nuclease were added to dairy products. Additionally, staphylococcal counts and nuclease activity of milk samples inoculated with S. aureus were determined. Different extraction procedures were performed and compared. The results indicated that the automated test was a reliable method for detecting DNase activity in milk products. The procedure was completed in 2 h and detected 1 ng of DNase ml-1. Detection of the DNase was especially useful in cheeses and could be used to confirm positive enterotoxin results.

Evaluation of an alternative extraction procedure for enterotoxin determination in dairy products.

A concentration protocol based on trichloroacetic acid precipitation was evaluated and compared with the reference method using dialysis concentration. Different quantities of purified staphylococcal enterotoxins were added to pasteurized Camembert-type cheeses. Detection of enterotoxins in these cheeses was performed using an automated detection system. Raw goat milk Camembert-type cheeses involved in a staphylococcal food poisoning were also tested. Both enterotoxin extraction methods allowed detection of the lowest enterotoxin concentration level used in this study (0.5 ng g-1). Compared with the dialysis concentration method, TCA precipitation of staphylococcal enterotoxins was 'user-friendly' and less time-consuming. These results suggest that TCA precipitation is a rapid (1 h), simple and reliable method of extracting enterotoxin from food which gives excellent recovery from dairy products.