No abstract

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.

The effect of volume of liquid medium (Hunt broth) supplemented with Oxyrase® on growth of Campylobacter jejuni was evaluated by using four sizes of containers and two concentrations of Oxyrase® (0.15 units/mL and 0.6 units/mL). We used 13×100 mm screw‐capped tubes containing 1, 3, and 6 mL of medium; 18x150 mm screw‐capped tubes containing 1, 5, 10, and 20 mL of medium; 45x45x120 mm bottles containing 25, 50, and 100 mL of medium; and Stomacher® 400 bags containing 250 mL of medium. Counts of four strains of C. jejuni were determined on blood agar plates at 0, 6, 12, 20, and 28 h of incubation. For 13×100 mm tubes, 3 mL of broth resulted the highest bacterial counts with increases of 4–5 log CFU/mL at 12 h and 7–8 log CFU/mL at 20 h. For 18x150 mm tubes, 10 mL and 20 ml of broth both provided increases of 3–4 log CFU/mL and of 6–7 log CFU/mL at 12 h and 20 h, respectively. Increases of 3.5 log CFU/mL at 12 h and of 7–8 log CFU/mL at 20 h were found in bottles containing 100 mL of broth. Overall, 0.15 units/mL Oxyrase® enhanced Campylobacter growth as effectively as 0.6 units/mL in all containers with no significant differences (p>0.05). However, supplementation with 0.6 units/mL of Oxyrase® gave slightly higher bacterial counts than addition of 0.15 units/mL in containers with high ratios of head space volume to medium volume (H:M). Ratios between 1.0 and 3.0 generally gave better results with all strains. The dissolved oxygen levels in each container were lower with high medium volume than with low medium volume. This study positively supported the efficiency of using Oxyrase® for recovery of C. jejuni under normal atmospheric conditions. In addition, various sizes of containers could be used for culturing campylobacters by maintaining the H:M ratio between 1.0 and 3.0.

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.