J. Van Hoof

Isolation and molecular characterization of Escherichia coli O157 isolated from cattle, pigs and chickens at slaughter

From 1999 until 2001, 3625 food samples were examined for the presence of Escherichia coli O157. Samples were from bovine origin (ground beef, n=549; carcasses, n=2452), calves (carcasses, n=147), chicken (breast, n=203; carcasses, n=71) and pigs (carcasses, n=85; trimmings, n=118). Vidas ECO detected 451 (12%) samples positive, but from only 27 (0.74%) samples was E. coli O157 isolated. One strain was isolated from bovine ground beef (0.18%), one from a pig carcass (1.17%) and all others were isolated from bovine carcasses (1.02%). All strains possessed the attaching-and-effacing gene, the enterohemorrhagic plasmid and verotoxin (VT) genes, except the strain isolated from the pig carcass that was therefore eliminated. Six of the strains were urease-positive. Strains were typed by two DNA fingerprinting methods: random amplification of polymorphic DNA (RAPD) and pulsed field gel electrophoresis (PFGE). PFGE revealed a similarity of 71.05%, while RAPD was 77.36% similar. None of the typing methods were able to classify all urease-positive strains to one pattern. Strains in the same PFGE cluster did not belong to one RAPD cluster. This paper highlights that Belgian fresh meat at retail level can be contaminated with E. coli O157 and that two different typing methods divide strains into different types.

Sensitivity of methods for the isolation of Escherichia coli O157 from naturally infected bovine faeces

At present, no standard protocol has been described to detect the presence of Escherichia coli O157 in cattle faeces. Therefore, the sensitivity of 26 different isolation methods was determined in order to recommend a method of choice. Faeces samples from 17 different beef cattle at a farm previously found positive for E. coli O157 were subdivided into a total of 40 samples. It was not known whether the 17 cattle shed E. coli O157 at the time of sampling. At another farm where cattle have been found negative for E. coli O157 on different occasions, five faeces samples were collected. Two methods yielded the highest sensitivity (74%): 6h enrichment in modified tryptone soya broth supplemented with novobiocin (mTSBn) followed by an immunomagnetic separation (IMS) with (i) Dynal beads or (ii) Captivate beads and selective plating on Rainbow agar (RA) plates. Enrichment for 6h was significantly better than 24h enrichment. Only after 24h, buffered peptone water (BPw) was significantly better than mTSBn. A sensitivity of 82% was obtained only when the two most sensitive tests were done simultaneously. Because none of the tests gave 100% sensitivity, it can be concluded that isolation rates of E. coli O157 from bovine faeces using only one of the tested procedures results in an underestimation of the incidence of E. coli O157 in cattle. Performing more than one test on the samples must be considered.

A one-plate microbiological screening test for antibiotic residue testing in kidney tissue and meat: an alternative to the EEC four-plate method?

A one-plate screening method is described for the microbiological detection of antibiotic residues by growth inhibition of Bacillus subtilis in agar medium, at pH 7, which contains trimethoprim for better detection of sulphonamides. beta-Glucuronidase is added to the samples to enable the detection of chloramphenicol residues. The sensitivity was determined for 16 antimicrobial substances frequently used in animal husbandry. A comparison was made with the sensitivity of the EEC four-plate test and existing maximum residue levels. The method has been used in Belgium for 5 years for the monitoring of antimicrobial residues in kidney tissue and meat of slaughter animals.

Evaluation and establishing the performance of different screening tests for tetracycline residues in animal tissues

Four methods intended for screening muscle tissue for residues belonging to the tetracycline group were compared using artificially contaminated as well as incurred samples. Two agar diffusion methods were studied: one with Bacillus subtilis as a test strain, the second with Bacillus cereus. Two variants of each method were compared: thin plates for analysis of intact or minced meat, and thick plates for analysis of meat fluid. The thin plate variants could not be evaluated with artificially contaminated samples because it was impossible to prepare homogeneously spiked, undiluted meat. The thick plates were suited for doxycycline and chlortetracycline, but they did not detect oxytetracycline or tetracycline in spiked meat fluid. The results of these tests done on incurred meat were very good for doxycycline and satisfying or just failing for oxytetracycline, while the best detection capability was obtained when intact frozen meat was examined on thin plates seeded with B. cereus. Two commercially available screening tests were also evaluated. The Premi® test, an inhibitor test with Bacillus stearothermophilus as a test strain and an indicator for growth, was not suited for detection of tetracyclines up to the maximum residue limit. Tetrasensor®, a receptor test specific for tetracyclines, proved a quick and simple test able to detect meat samples artificially contaminated with tetracycline, oxytetracycline, doxycycline or chlortetracycline, as well as meat incurred with oxytetracycline or doxycycline.

Isolation and characterization of enterohaemorrhagic Escherichia coli O157:H7 from cattle in Belgium and Poland

EHEC O157 were isolated from faeces of Belgian and Polish beef slaughter cattle. In Belgium, 1281 faecal samples were analysed by immunomagnetic separation [IMS] after enrichment in buffered peptone water from June 1998 till July 1999. Eighty-one samples (6.3%) were positive for E. coli O157. Phage type 8 was most frequently found. Bulls between 1 and 2 years old, slaughtered in September and October were most frequently found positive. Atypical biochemical features were observed in some isolates: 22 (27%) isolates were urease positive and 1 (1.2%) isolate was unable to ferment lactose. In Poland, 551 faecal samples, taken from January 1999 till December 1999, were examined using exactly the same techniques. Four faecal samples (0.7%) were positive for O157 EHEC, yielding seven phage type 8 isolates. All positive samples were from cattle younger than 2 years. Positive samples occurred in August, September and October.