Identification of high-risk Listeria monocytogenes serotypes in lineage I (serotype 1/2a, 1/2c, 3a and 3c) using multiplex PCR

Authors: NHO, SEONGWON - Mississippi State University; ABDELHAMES, HOSSAM - Mississippi State University; REDDY, SWETHA - Mississippi State University; KARSI, ATTILA - Mississippi State University; LAWRENCE, MARK - Mississippi State University

Submitted to: Journal of Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/4/2015
Publication Date: 9/1/2015
Citation: Nho, S., Abdelhames, H., Reddy, S., Karsi, A., Lawrence, M.L. 2015. Identification of high-risk Listeria monocytogenes serotypes in lineage I (serotype 1/2a, 1/2c, 3a and 3c) using multiplex PCR. Journal of Applied Microbiology. 119(3):845-852.

Interpretive Summary: Our results provide further confirmation that variation of genetic structure in L. monocytogenes can be useful for identification of serotype-specific genes. Not surprisingly, L. monocytogenes strains have considerable genetic variation; this intraspecies genetic diversity may result from strain-specific adaptations. Thus, these genes are useful not only for diagnostics and disease epidemiology, but they also represent physiological and virulence properties and reveal evolutionary relationships. The ability of multiplex PCR to identify the high-risk serotypes 1/2a and 1/2c is an important innovation, and this method can supplement the method of Doumith et al. (2004) that separates L. monocytogenes into five subgroups. Multiplex PCR is routinely performed in diagnostic and research laboratories to detect L. monocytogenes and classify strains into subgroups. Future research will focus on development of new PCR primers based on comparative genomics to distinguish the other high-risk L. monocytogenes serotypes 1/2b and 4b as well as refining the current assay into a more efficient real-time format.

Technical Abstract: Aims: Using molecular subtyping techniques, Listeria monocytogenes is divided into three major phylogenetic lineages, and a multiplex PCR method can differentiate five L. monocytogenes subgroups: 1/2a-3a, 1/2c-3c, 1/2b-3b-7, 4b-4d-4e, and 4a-4c. In the current study, we conducted genome comparisons and evaluated serotype-associated genes for their utility as a multiplex PCR-based method for distinguishing high-risk serotypes 1/2a and 1/2c in lineage I from low-risk serotypes 3a and 3c. Methods and Results: Primer sets were developed that are specific for serotype 1/2c (LMOSLCC2372_0308) and serotype 3a (LMLG_0742). These primers were then tested in a multiplex format with primers specific for serotype 1/2a (flaA) to separate serotypes 1/2a, 1/2c, 3a, and 3c using 25 strains of lineage I L. monocytogenes. Conclusions: Here for the first time we report primers specific for L. monocytogenes serotype 1/2c and serotype 3a, and we demonstrate a multiplex PCR method for separating the four serotypes of lineage I L. monocytogenes. Significance and Impact of the Study: The described multiplex PCR assay consistently showed successful separation of 1/2a and 1/2c strains from 3a and 3c strains. PCR is routinely performed in many diagnostic and epidemiologic investigations for L. monocytogenes, and these primers should increase the feasibility and accessibility of L. monocytogenes serotyping.