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.

Staphylococcus fleurettii sp. nov., isolated from goat's milk cheeses.

A new coagulase-negative and novobiocin-resistant species of the genus Staphylococcus, Staphylococcus fleurettii, isolated from raw-milk cheeses, is described. This species is differentiated from the other novobiocin-resistant staphylococci on the basis of ribotype and intergenic transcribed spacer patterns, DNA-DNA reassociation reactions, cell wall composition and phenotypic characteristics. S. fleurettii could be distinguished by its oxidase activity, by its ability to produce acid aerobically from D-trehalose, D-mannose, D-turanose and maltose and by its inability to produce acid from D-cellobiose. The type strain of S. fleurettii is CIP 106114T (= DSM 13212T).

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.

Growth and enterotoxin production of Staphylococcus aureus during the manufacture and ripening of Camembert-type cheeses from raw goats' milk.

Tests were carried out to determine the effect of manufacturing procedures for a Camembert-type cheese from raw goats' milk on the growth and survival of Staphylococcus aureus organisms added to milk at the start of the process, and to study the possible presence of staphylococcal enterotoxin A in these cheeses. The initial staphylococcal counts were, respectively, 2, 3, 4, 5 and 6 log cfu ml-1. Cheese was prepared following the industrial specifications and ripened for 41 d. Detection of enterotoxins was done by the Vidas SET test and by an indirect double-sandwich ELISA technique using antienterotoxin monoclonal antibodies. Generally, numbers of microbes increased at a similar rate during manufacture in all cheeses until salting. During the ripening period, the aerobic plate count population and Staph. aureus levels remained stable and high. There was an approximately 1 log reduction of Staph. aureus in cheeses made with an initial inoculum of Staph. aureus greater than 10(3) cfu ml-1 at the end of the ripening period (41 d) compared with the count at 22 h. The level of staphylococcal enterotoxin A recovered varied from 1 to 3.2 ng g-1 of cheese made with an initial population of 10(3)-10(6) cfu ml-1. No trace of enterotoxin A was detected in cheeses made with the lowest Staph. aureus inoculum used in this study.

Evaluation of the VIDAS methodology for detection of Escherichia coli O157 in food samples.

An automated enzyme-linked fluorescence immunoassay (ELFA), the VIDAS E. coli O157 method, was compared with immunomagnetic separation (IMS) followed by culture on cefixime tellurite sorbitol MacConkey agar (CT-SMAC) for detecting Escherichia coli O157 in artificially and naturally contaminated food samples including raw milk cheeses, poultry, raw sausages, and ground beef retail 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. A total of 496 retail food samples were examined. Seventeen food samples gave positive values with the ELFA method, and among them 9 food samples were confirmed by the ICE method. Eight were shown to contain sorbitol-positive, O157-positive, H7-negative, motile, non-verotoxin-producing E. coli. The ninth positive sample contained an O157-positive, H7-negative, sorbitol-negative, non-verotoxin-producing E. coli. The IMS technique only allowed confirmation of this sorbitol-negative, non-verotoxin-producing E. coli O157.

Behaviour and enterotoxin production by Staphylococcus aureus during the manufacture and ripening of raw goats' milk lactic cheeses.

To study the possible presence of staphylococcal enterotoxin A in raw goats' milk lactic cheese, milk was inoculated with an enterotoxigenic Staphylococcus aureus strain to a final concentration of 4, 5 and 6 log(cfu/ml). Cheese was prepared following industrial specifications and ripened for 42 d. Detection of the enterotoxins was by the Vidas Staph enterotoxin test (BioMérieux) and by an indirect double-sandwich ELISA technique using anti-enterotoxin monoclonal antibodies. Staphylococcal counts declined markedly after draining, and by the end of ripening they had disappeared from some cheeses. In contrast, aerobic mesophilic organisms grew well. The level of staphylococcal enterotoxin A recovered varied from 1 to 2.5 ng/g cheese made with an initial population of 10(5) or 10(6) cfu/ml. Only traces of enterotoxin A (0.5 ng/g) were detected in cheeses made with the lowest Staph. aureus inoculum used in this study. Enterotoxin A was also detected in cheeses from which Staph. aureus had disappeared.

Detection of Escherichia cole O157 in French food samples using an immunomagnetic separation method and the VIDAS E. coli O157.

Two commercially available screening methods, an automated enzyme-linked fluorescent immunoassay (VIDAS E. coli O157) and an immunomagnetic separation followed by culture onto cefixime tellurite sorbitol MacConkey agar (CT-SMAC), were compared for detection of Escherichia coli O157 in naturally and artificially contaminated food samples. A total of 250 naturally contaminated food samples, including raw milk cheeses, poultry, raw sausages and ground beef retail samples, were examined. Four poultry, one raw sausage and one ground beef sample were found to be positive for E. coli O157 by both methods. Of the six positive samples, five were shown to contain sorbitol-positive, O157-positive, H7-negative, motile and non-verotoxin-producing E. coli.

Identification and ribotyping of Staphylococcus xylosus and Staphylococcus equorum strains isolated from goat milk and cheese.

Twenty-five strains of staphylococci isolated from goat milk and cheese were identified as belonging to the Staphylococcus xylosus/equorum group using the ID 32 Staph system (bioMérieux, Marcy-L'Etoile, France). This system, however, was not able to discriminate between these two species for 19 of the strains tested. Ribotyping was performed on these 25 strains, as well as on three reference strains of each of these two species. Hybridization membranes were scanned and analyzed using the Taxotron software package (Taxolab, Institut Pasteur, Paris, France). A dendrogram representation showed that ribotypes were distributed in two clear-cut clusters corresponding to S. equorum (21 strains) and S. xylosus (four strains).

Enterotoxin production by coagulase-negative staphylococci isolated from goats' milk and cheese.

An antigen related to the Enterotoxin E from Staphylococcus aureus was produced by ten of 187 coagulase-negative staphylococci (CNS) isolated from goats' milk, whey and cheese in quantities ranging from 10 to 90 ng/ml supernatant. The enterotoxin-producing strains were identified at the species level as S. simulans, S. xylosus, S. equorum, S. lentus and S. capitis. Detection of the enterotoxins was done by the VIDAS SET test (bioMérieux) and by an indirect double-sandwich ELISA technique using anti-enterotoxin monoclonal antibodies. The results obtained were further confirmed by Southern blotting, using two radioactive oligonucleotide probes that hybridized specifically with the gene of S. aureus coding for the enterotoxin E.

Identification of micrococcaceae isolated from goat's milk and cheese in the Poitou-Charentes region.

One hundred and ninety strains of coagulase-negative staphylococci were isolated from goat's milk, whey and cheese at various stages of manufacture. Sixteen different coagulase negative Staphylococci (CNS) species were recovered, 3 of which were predominant: Staphylococcus simulans, Staphylococcus epidermidis and Staphylococcus xylosus. The prevalent species were recovered at least at two different stages of cheese manufacturing, suggesting a better adaptation to the environment. After 15 days of ripening, the cheeses showed lower counts of Micrococcaceae.