Article pp.93-96 du Vol.23 n°1 (2003)

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SCIENCES DES ALIMENTS, 23(2003) 93-96

FOCUS : JSMTV

Characterization of shiga toxin producing E. coli and O157 serotype E. coli isolated

from food and environment in France

C. Vernozy-Rozand¹

Verotoxin-producing Escherichia coli (VTEC) is one of the most important, recently emerged, group of food-borne pathogens. In patients, VTEC strains are associated with watery or bloody diarrhea, haemorrhagic colitis, and haemolytic uraemic syndrome Stx. Apart from the ability to produce at least one Shiga-like toxin or verotoxin (encoded by stx₁ or stx₂), VTEC may possess many other virulence factors such as those associated with the attaching and effacing lesions: intimin encoded by eae or enterohemolysin encoded by ehx. Although E. coli O157:H7 and O157:H- are currently the most common VTEC reported in many regions of the world, other serogroups including O26, O111 and O103 are also recognized as a serious threat to public health and have been recovered from infected patients.

Many outbreaks of E. coli O157:H7 – related food-borne illness have been linked with the consumption of contaminated meat, since the intestinal tract of cattle is the primary reservoir for this pathogen. Other foodstuffs, however, have been implicated in many outbreaks, including water, lettuce, alfalfa sprouts and apple juice.

The lack of baseline data on the prevalence of VTEC in food and environment in France prompted us to survey raw milk cheeses, minced beef, pork and slurry, farmyard manure and sewage sludge. Except for minced beef for whom only E. coli O157: H7 was searched, VTEC strains were isolated from the other matrixes. More precisely, PCR detection of stx genes was performed with dege- nerate primers which amplified a conserved sequence of the stx₁and stx₂ genes. 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 and geneti- cally characterized.

All the studies described below have been made by the Food Research Unit of the Veterinary School of Lyon and they have been published in 2002 or are still in press.

1. École Nationale Vétérinaire de Lyon.

Unité de Microbiologie Alimentaire et Prévisionnelle.

1, avenue Bourgelat, BP 83, 69280 Marcy-l’Étoile.

4-FOCUS (093-96) Page 93 Jeudi, 15. mai 2003 8:21 20

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94 Sci. Aliments 23(1), 2003 C. Vernozy-Rozand

SURVEY OF RETAIL RAW MILK CHEESES FOR VEROTOXIN-PRODUCING ESCHERICHIA COLI (VTEC) AND E. COLI O157

The consumption of unpasteurized milk and dairy products manufactured from unpasteurized milk has also been associated with transmission of E. coli O157:H7. In many parts of Europe, indigenous cheeses manufactured from unpasteurized milk are consumed, giving rise to concern that these products may be a threat to consumer safety as a result of the presence of pathogens such as E. coli O157:H7 and verotoxin-producing Escherichia coli (VTEC).

The lack of baseline data on the prevalence of VTEC in retail raw milk cheeses in France prompted us to survey both industrial and artisanal cheeses production. One thousand and thirty nine raw milk cheeses including soft, hard, boiled and blue mould cheeses were sampled. The different types of the raw milk cheeses collected were representative of the French production.

One hundred and thirty six cheeses (13.1%) were positive for stx genes.

These cheeses were mainly soft cheeses (washed rinded or white mould rinded soft cheeses) and uncooked hard cheeses.

Thirty two VTEC strains were recovered from positive samples by colony hybridization. These strains were tested for serogroups O26, O55, O111, O103 and O157:H7 which are the VTEC serotypes isolated most frequently from French human patients. No isolate belonged to these serogroups. All the isolates were positive for stx2 gene, 90% for stx1 gene and 6% for eae gene.

Data on the presence of the eae gene, which is carried by the majority of non-O157:H7 STEC strains isolated from human disease, showed that 30 strains out of 32 did not have this additional virulence factor, and no isolate belonged to the major serogroups linked to disease. These two elements sug- gest that the human pathogenic potential of the STEC strains isolated during the course of this study was probably limited.

SURVEY OF PORK FOR VEROTOXIN-PRODUCING ESCHERICHIA COLI (VTEC) AND E. COLI O157

Since healthy domestic animals, particularly ruminants can harbour VTEC in their faeces, they are regarded as natural reservoirs for these organisms. How- ever, other animals, including pigs, poultry, cats and dogs, can also harbour VTEC . But only a few data are available about the occurrence of VTEC in other species than domestic ruminants especially for pigs and pork meat in France.

Five hundred and forty six pig carcasses from three French slaughterhouses were sampled by swabbing a surface of 300 cm². One thousand and six hundred pork items from three French cutting plants were sampled by excising a surface of 25 cm². Different sites in each slaughterhouse and cutting plant were sampled before and after the commencement of the work day. PCR detection of stx genes was performed on a total of 3022 samples.

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Characterization of shiga toxin producing E. coli and O157 serotype E. coli 95

No E. coli O157:H7 was isolated. Among the 2146 carcass and pork sam- ples, and among the 876 environmental samples (swabs of surfaces or mate- rials), 15% and 19% were PCR-positive for stx genes respectively. Ninety six VTEC strains were recovered from positive samples by colony hybridization:

98% were stx_2-positive, 2% stx₁-positive and 86% stx_2e-positive. These strains possessed neither eae gene nor ehx gene or uidA gene. One strain belonged to serogroup O103.

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 seems to be low. General hygiene measures seem to be sufficient and specific hygiene measures for VTEC may not be necessary at this time.

SURVEY OF SLURRY, FARMYARD MANURE AND SEWAGE SLUDGE FOR VEROTOXIN-PRODUCING ESCHERICHIA COLI (VTEC) AND E. COLI O157

Ruminants are particularly regarded as natural reservoirs for these organisms. Consequently these and certain other farm animals can excrete a large numbers of this organism in their faeces. Humans are also a reservoir for VTEC.

The surprising long term survival of VTEC in slurry, farmyard manure and sludge, on pasture-land and in associated water systems has important implica- tions for their spread to crops by direct application of manure, irrigation with infected water or directly to man by contact with animals or contaminated soil.

Therefore, an attempt must be made to evaluate the potential risks from sewage sludge and the animal excreta handled on farms, a significant propor- tion of which will, inevitably, be contaminated with VTEC. Data on the presence of VTEC in sewage or polluted water and slurries are limited and difficult to compare and data on the prevalence of VTEC in the environment in France are lacking.

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 of them were PCR positive for stx gene. Twenty one VTEC strains were recovered from positive samples by colony hybridization:

76% of them were positive for stx₂gene, 33% for stx₁gene, and 19% for eae gene. One strain belonged to serotype O157:H7 and two others to serogroups O26 and O55 respectively.

Some of the VTEC strains isolated from environment in France, should be considered as potentially pathogenic for humans. 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.

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96 Sci. Aliments 23(1), 2003 C. Vernozy-Rozand

SURVEY OF MINCED BEEF FOR VEROTOXIN-PRODUCING ESCHERICHIA COLI (VTEC) AND E. COLI O157

An automated enzyme-linked fluorescence immunoassay (ELFA), the VIDAS E. coli O157 method, was used for detecting Escherichia coli O157 in industrial French minced beef samples.

Confirmation of the samples positive according to the ELFA, was performed by use of an automated immunoconcentration system, VIDAS ICE, which allows selective capture and release of target organisms. The ICE was followed by culture on cefixime tellurite sorbitol Mac Conkey agar (CT-SMAC) and a chromogenic medium, O157:H7 ID.

Among the 3450 minced beef samples tested, 175 samples positive with the ELFA method, and of them, 4 were confirmed by the ICE method. The four strains were sorbitol negative, β glucuronidase (-), uidA (+), and flic (+).

They were identified as sorbitol-negative, O157-positive, H7-positive, mobile, verotoxin-producing E. coli. As they had the ability to produce both Stx1 and Stx2 and possessed eaeA, ehx genes., they can be considered as pathogenic strains. The four strains were isolated from four different minced beef samples and these samples came from three different mixes.

Prevalence of E. coli O157:H7 in industrial French minced beef was: 0,12%

(4 of 3450). This low percentage is consistent with other reports in which E. coli O157:H7 occur at low frequency in meat.

No outbreaks linked to food contaminated by VTEC have been described in France. However the incidence of haemolytic–uremic syndrome in French peo- ple is close to those described in the other European countries. The previous surveys will be completed by epidemiological studies including the faecal car- riage in cattle and the vehicles of transmission of VTEC inside dairy herds.