Genomic characterization of novel Listeria monocytogenes serotype 4b variant strains

Abstract

Over 90% of the human listeriosis cases are caused by Listeria monocytogenes serotypes 1/2a, 1/2b and 4b strains. As an alternative to antigen-antibody based serotyping, a PCR-based method for serogrouping has been developed and validated. In this communication, we report an in-depth analysis of five 4b variant strains, four clinical isolates from Australia and one environmental isolate from USA. Although these five strains were serotype 4b by classical serotyping method, the serogrouping PCR profiles of these strains show the presence of a 1/2a-3a specific amplicon in addition to the standard 4b-4d-4e specific amplicons. These strains were further analyzed by pulsed field gel electrophoresis, binary gene typing, multi-locus variable-number-tandem-repeat analysis and a high density pan-genomic Listeria microarray. Using these sub-typing results, the clinical isolates were grouped into two distinct genomic groups- one of which could be part of an unidentified outbreak. The microarray results when compared with our database of other 4b outbreak isolates indicated that the serotype 4b variant strains represent very different genotypic profiles than the known reported 4b outbreak strains representing major epidemic clones. The acquisition of serotype 1/2a gene clusters by the 4b variant strains appears to be independent in origin, spanning large areas of geographical and temporal space and may indicate predisposition of some 4b strains towards accepting DNA from related organisms.

Figure 1. Multiplex PCR profiles of L. monocytogenes strains.

Lanes: MW, Molecular weight markers. Lane 1: LS1 serotype 1/2a, Lane 2: LS402, serotype 4b; Lane3-7: serotype IVb-v1 strains LS542; LS642; LS643; LS644; LS645, respectively.

Figure 2. ApaI/AscI PFGE profiles of L. monocytogenes strains 4b and IVb-v1.

The dendrogram was calculated and drawn using Bionumerics software. NSW; New South Wales, VIC; Victoria, CA; California, CT; Connecticut, and NK;Not known.

Figure 3. A neighbor-net constructed from the gene contents of 28 strains belonging to serotype 4b.

The parallel edges represent incompatible signals indicative of independent gene loss or gain due to the multiple transduction or recombination. Serotypes and epidemic clones are grouped in different color as indicated. Node labels refer to strain names (Table 1 and Laksanalamai et al 2012 (7). Scale bar represents number of gene differences (present or absent) per gene site.

Figure 4. Hierarchical clustering based on the Robust Multi Array (RMA) analysis of the L. monocytogenes strains serotypes 4b (LS406, LS412) and IVb-v1 (LS542, LS642, LS643, LS644, LS645).

Figure 5. Scatter plots of the summarized Robust Multi-Array Averaging (RMA) intensities.

A–C, between serotypes 4b (LS412) and IVb-v1 (LS642, A; LS644, B; LS645, C). D–E, between the same serotype, IVb-v1, isolated from Australia; from the different states (LS644 and LS645, D); from the same state (LS642 and LS644, E). F, between the same serotype, IVb-v1, isolated from the different countries (LS642; Australia and LS542; USA).

Figure 6. The organization of the genomic region containing the lmo0737 to lmo0739 cassette that is present only in the L. monocytogenes serotypes IVb-v1 and 1/2a strains, confirmed by microarray analysis.