Feasibility of a Defined Microflora Challenge Method for Evaluating the Efficacy of Foodborne Listeria monocytogenes Selective Enrichments

Abstract

Comparison of isolation methods for microbial pathogens is complicated by the variable interference caused by the competitive microflora present in test samples such as foods. In principle, using measured amounts of a standard competitor in a defined surrogate food matrix might control the effect of variable interference. This possibility was investigated using Listeria monocytogenes and enrichment broths belonging to the acriflavine-nalidixate selective agent class. Triplicate test sample sets were prepared. Each set consisted of suspensions of variable levels of the standard competitor, Enterococcus faecium strain 111 (≈10 to 109 CFU/25 g), mixed with a low constant level (10 to 100 CFU/25 g) of L. monocytogenes. These test samples were enriched at 30°C for 48 h in different selective media and streaked onto selective isolation agars. The input CFU ratio (E. faecium/L. monocytogenes) that permitted a 50% end point L. monocytogenes recovery was 2.2 × 106 or higher for the Food and Drug Administration one-step enrichments and 0.8 × 106 for the International Standards Organization (ISO) two-step enrichment. These and other results show that this evaluation method is feasible with this class of enrichments. Interestingly, L. monocytogenes could be detected in enrichment cultures at high-input E. faecium/L. monocytogenes ratios even when the enriched samples were plated onto nonselective media. The pinpoint colonies of L. monocytogenes embedded in a confluent lawn of E. faecium 111 were detectable by their contrasting coloration in Henry obliquely transmitted illumination.