Enrichment media for isolation of Campylobacter jejuni from inoculated ground beef and chicken skin under normal atmosphere.

The efficiency of Hunt broth containing Oxyrase was compared with the gas replacement method for detection of Campylobacter jejuni in inoculated ground beef and chicken skin. Five strains of C. jejuni were inoculated individually into samples and cultured with various media under conditions generated by either flushing with a mixture of gases or supplementing with Oxyrase. Oxyrase media added with 7% lysed blood, 2.5% charcoal, or 6% ground cooked meat were compared with examinations from chicken skin samples. Campylobacter counts from enrichments were performed at 6, 12, 20, and 28 h of incubation. From inoculated ground beef, counts at 20 h increased by 4 to 7 log CFU/ml depending on strains and initial concentration of inocula. The efficiencies of Hunt medium using gassing and those with Oxyrase added were similar (P > 0.05). Broth containing 0.15 U/ml of Oxyrase without blood effectively supported the growth of all strains (P > 0.05). From inoculated chicken skin, 20-h incubation counts increased by 3.0 to 7.5 log CFU/ml for the gassing method and by 2.7 to 7.3 log CFU/ml for supplementation with 0.6 U/ml of Oxyrase and blood. The addition of 7% lysed sheep blood provided better Campylobacter growth than supplementing with 2.5% charcoal or 6% ground cooked meat. Enrichment media incorporating with Oxyrase is a simple, convenient, and time-saving method to replace flushing with mixed gas for isolation of Campylobacter jejuni.

Evaluation of a 5'-nuclease (TaqMan) assay for the detection of virulent strains of Yersinia enterocolitica in raw meat and tofu samples.

Culture methods for detecting virulent Yersinia enterocolitica require selective enrichment and a series of confirmatory tests that are time-consuming, costly, and laborious. The objective of this study was to evaluate a fluorogenic 5'-nuclease assay for detecting the enterotoxin yst gene of virulent Y. enterocolitica in pure cultures, inoculated ground pork samples, and naturally contaminated food samples. These results were then compared with "gold standard" methods recommended by the U.S. Food and Drug Administration in the Bacteriological Analytical Manual for detecting pathogenic Y. enterocolitica. The 5'-nuclease assay was able to identify the organism in 100% of the repetitions when 10(2) CFU/ml or more organisms were present in pure cultures and 10(3) CFU/g or more organisms were present in ground pork. Similar recovery efficiency on cefsulodin-irgasan-novobiocin (CIN) agar plates was only evident when 10(5) CFU/ml or more organisms were present in pure culture and 10(6) CFU/g or more organisms were present in inoculated ground pork. The 5'-nuclease assay indicated a contamination rate of 35.5% (94/265) in various meats and tofu, whereas the CIN plating method indicated a contamination rate of 28.3% (75/265). This resulted in 100% sensitivity and 64.5% specificity for the 5'-nuclease assay when compared with the standard culture recovery method. Only 75% (60/80) of the Yersinia spp. isolated on CIN was identified as containing a virulence plasmid by autoagglutination and crystal violet binding tests. These results indicate that the true rate of contamination of virulent Y. enterocolitica in pork and other processed meats and foods is being underestimated using current detection methods. This study demonstrates the potential of the 5'-nuclease assay for rapidly and specifically detecting virulent Y. enterocolitica in processed foods with the added advantage of being an automated detection system with high-throughput capability.

Rapid 5' nuclease (TaqMan) assay for detection of virulent strains of Yersinia enterocolitica.

We have developed a rapid procedure for the detection of virulent Yersinia enterocolitica in ground pork by combining a previously described PCR with fluorescent dye technologies. The detection method, known as the fluorogenic 5' nuclease assay (TaqMan), produces results by measuring the fluorescence produced during PCR amplification, requiring no post-PCR processing. The specificity of the chromosomal yst gene-based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 nonpathogenic serotypes of Y. enterocolitica, other species of Yersinia (Y. aldovae, Y. pseudotuberculosis, Y. mollaretti, Y. intermedia, Y. bercovieri, Y. ruckeri, Y. frederiksenii, and Y. kristensenii), and other enteric bacteria (Escherichia, Salmonella, Citrobacter, and Flavobacterium). The assay was 100% specific in identifying the pathogenic strains of Y. enterocolitica. The sensitivity of the assay was found to be >/=10(2) CFU/ml in pure cultures and >/=10(3) CFU/g in spiked ground pork samples. Results of the assay with food enrichments prespiked with Y. enterocolitica serotypes O:3 and O:9 were comparable to standard culture results. Of the 100 field samples (ground pork) tested, 35 were positive for virulent Y. enterocolitica with both 5' nuclease assay and conventional virulence tests. After overnight enrichment the entire assay, including DNA extraction, amplification, and detection, could be completed within 5 h.