DNA sequence-based subtyping of Listeria monocytogenes

Listeria monocytogenes is a Gram-positive, facultative and asporogenous bacterium that can cause a potentially fatal foodborne disease, listeriosis, among high risk populations. L. monocytogenes is ubiquitous in nature and persistent in colonizing food processing environments, which makes food contamination by this organism very difficult to control. Molecular subtyping has been widely used for studying the epidemiology of L. monocytogenes. Commonly used subtyping methods include ribotyping (RT), randomly-amplified polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE), and most recently, multilocus sequence typing (MLST). Comparison of PFGE and MLST suggested that PFGE targets rapidly indexed genomic variations in L. monocytogenes and is probably more suitable for studying short-term epidemiology, whereas MLST is based on the selectively neutral variability in housekeeping genes of L. monocytogenes, and therefore is less discriminatory than PFGE thus more suitable for studying long-term epidemiology.; To improve the discriminatory power of housekeeping-loci-based MLST, a multi-virulence-locus sequence typing (MVLST) scheme was developed using L. monocytogenes as a model microorganism. Intragenic regions of three virulence genes (prfA, inlB, inlC) and three virulence-associated genes (dal, lisR, and clpP) were targeted and analyzed. Results showed that MVLST provided higher discriminatory power than RT, PFGE and MLST. In addition, cluster analysis based on the intragenic sequences of the selected virulence genes indicated a strain phylogeny closely related to serotypes and genetic lineages. To simplify MVLST for practical use in the food industry, a multiplex PCR assay was developed to provide simultaneous serotype differentiation and sequence typing of L. monocytogenes. Intragenic regions of two putative serotype-specific genes, ORF2372 and lmo0171, and two virulence genes, inlB and inlC, were analyzed. Multiplex PCR-based sequence typing reduced the time and cost for MVLST, but maintained high discriminatory power similar to that of PFGE.