C. N. Wendakoon, R. Zapata, W. M. Fedio, Food Safety Laboratory, New Mexico Mexico State University
Background: The FDA-BAM cultural method for detecting Shigella spp. requires anaerobic incubation followed by presumptive and confirmatory tests. This study was conducted to evaluate aerobic enrichment for the isolation and detection of low numbers of Shigella spp. by selective plating and by PCR.
Methods: Artificially contaminated Shigella broth, potato salad and cilantro rinse samples were enriched in Shigella Broth (SB), SB with 0.5% novobiocin and SB with 3% novobiocin at 40°C. Evaluations were performed with S. sonnei, S. flexneri, S. boydii and S. dysenteriae at four inoculum levels (0-1, 1-10, 10-100 and 100-1000 colony forming units). After enrichment for 20 h, samples were streaked onto selective agar and the presumptive shigellae were confirmed by biochemical tests. Enriched samples were also used to detect Shigella spp. by nested PCR.
Results: In inoculated SB, both PCR and selective plating detected shigellae at the lowest inoculum. For potato salad enrichments, the PCR procedure was found to be approximately 10 times more sensitive than the selective plating procedure for detecting Shigella spp. While for cilantro, a 100-1000 fold increase of sensitivity was seen for the PCR procedure. Different novobiocin levels in the enrichments did not improve isolation on the plates.
Conclusion: Followingaerobic enrichment, the PCR procedure detected Shigella spp. at very low levels of contamination. Competing microorganisms in the cilantro rinse enrichments made the isolation of shigellae difficult on the selective agar plates. Use of more than one selective agar improved the isolation of Shigella in highly contaminated food matrices.