P-016. Comparative Analysis of PFGE and DNA Sequence-Based Subtyping of Listeria monocytogenes

T. J. Ward1, P. Evans2, G. Tillman3, K. Pupedis3, K. Volpe Sperry4, T. F. Ducey5, L. Graves6, R. Broeker6;
1USDA-ARS, Peoria, IL, 2USDA-FSIS, Washington, DC, 3USDA-FSIS, Athens, GA, 4North Carolina State Lab. of Pub. Hlth., Raleigh, NC, 5USDA-ARS, Florence, SC, 6CDC, Atlanta, GA.

Listeria monocytogenes is the etiologic agent of an invasive, food-borne disease that represents a serious threat to public health and the agricultural economy. In order to enhance current subtyping capabilities for this important pathogen, multilocus genotyping (MLGT) assays were developed for high-throughput subtype determination based on interrogation of single nucleotide polymorphisms via multiplexed primer extension reactions. The MLGT assays differentiate >140 subtypes, major serotype groups, epidemic clones, and individual outbreaks. In addition, MLGT identified isolates with premature truncation mutations in internalin (inlA) associated with virulence attenuated phenotypes, and produced data amenable to analysis of strain relatedness. MLGT subtyping of virtually all product isolates obtained by the Food Safety and Inspection Service (USDA-FSIS) in the last five years was used to determine the frequency and distribution of epidemic clones and virulence-attenuated subtypes in ready-to-eat (RTE) food products. These survey data were also used to evaluate the virulence gene attributes, and the phylogenetic, serotype clade, and epidemic clone associations for common pulsed-field gel electrophoresis (PFGE) patterns. Analyses based on panels of food and clinical isolates revealed that the discriminatory power of MLGT (SI = 0.91-0.93) was equivalent or greater than that provided by multilocus sequence typing (MLST; SI = 0.8-0.91) or typing based on multilocus variable number tandem repeat analysis (MLVA; SI = 0.81-0.91). However, PFGE had greater discriminatory power (SI = 0.97-0.98) than MLGT, MLST and MLVA combined. While this highlights the utility of PFGE for strain discrimination, it also indicates that PFGE patterns may be unstable over very short evolutionary time scales, Combined with the additional information provided by DNA sequence-based typing methods, these results suggest that epidemiological investigations and routine product sampling programs could be enhanced by complementing PFGE analysis with DNA sequence-based subtyping.