Horizontal gene transfer and loss of serotype-specific genes in Listeria monocytogenes can lead to incorrect serotype designations using a commonly-employed molecular serotyping scheme

Authors: BROWN, PHILLIP - North Carolina State University; KUCEROVA, ZUZANA - Centers For Disease Control And Prevention (CDC) - United States; Gorski, Lisa; CHEN, YI - Food And Drug Administration(FDA); IVANOVA, MIRENA - North Carolina State University; LEEKITCHAROENPHON, PIMLAPAS - North Carolina State University; PARSONS, CAMERON - North Carolina State University; NIEDERMEYER, JEFFERY - North Carolina State University; JACKSON, JAMES - North Carolina State University; KATHARIOU, SOPHIA - North Carolina State University

Submitted to: Microbiology Spectrum
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/9/2022
Publication Date: 2/14/2023
Citation: Brown, P., Kucerova, Z., Gorski, L.A., Chen, Y., Ivanova, M., Leekitcharoenphon, P., Parsons, C., Niedermeyer, J., Jackson, J., Kathariou, S. 2023. Horizontal gene transfer and loss of serotype-specific genes in Listeria monocytogenes can lead to incorrect serotype designations using a commonly-employed molecular serotyping scheme. Microbiology Spectrum. 11(1). Article e02745-22. https://doi.org/10.1128/spectrum.02745-22.
DOI: https://doi.org/10.1128/spectrum.02745-22

Interpretive Summary: Listeria monocytogenes is a foodborne pathogen capable of causing severe, invasive illness. Three serotypes (1/2a, 1/2b, and 4b) are the leading contributors to human listeriosis. Classical methods for serotype assessment have utilized specific antisera that are mixed with cells and measure agglutination reactions visually or enzymatically. The antisera used in these reactions is expensive, and the interpretation of results may vary between labs. In the last two decades, a multiplex polymerase chain reaction (PCR) scheme targeting five sets of genes has been widely adopted for determining serotype in L. monocytogenes. In the last decade whole genome sequencing (WGS) has been more widely adopted for serotype determination. The multiplex PCR method is widely used because of cost effectiveness, ease, and reproducibility of results between laboratories. However, for certain strains of L. monocytogenes, the serotype designations differ depending on the method used to assess it. Specifically, all tested strains of Sequence Type 782 (ST782) appear as serotype 1/2b by the PCR scheme, while they are serotype 4b when tested directly by antisera or assessed by WGS. WGS analysis indicated an absence of the gene LMOf2365_1900 in all tested ST782 strains, while the genomes retained ORF2819 (LMOf2365_2059) that, together with prs, yields the serotype 1/2b profile with the PCR scheme. Furthermore, additional strains of serotype 1/2 were discovered that appear as 1/2b in the PCR scheme. These findings suggest that for certain strains of L. monocytogenes, PCR serotype designations should be examined with caution and complemented by alternative tools such as those based on serotype-specific antibodies or WGS.

Technical Abstract: Listeria monocytogenes is a Gram-positive, facultative intracellular foodborne pathogen. L. monocytogenes capable of causing severe, invasive illness (listeriosis). Three serotypes, 1/2a, 1/2b and 4b, are leading contributors to human listeriosis, with 4b also including several hypervirulent clones. The multiplex polymerase chain reaction scheme developed by Doumith and collaborators (Doumith scheme) employs five sets of primers and has become extensively employed to determine serotype designations for L. monocytogenes, due to its high accuracy, ease and affordability. However, we have become aware that for certain strains the serotype designations determined by the Doumith scheme are in conflict with those based on antibody-based schemes or analysis of whole genome sequence (WGS) data. Specifically, all tested strains of sequence type (ST) 782, member of the hypervirulent clonal complex (CC) 2 serotyped as 1/2b using the Doumith scheme, WGS analysis revealed absence of LMOf2365_1900 in all tested ST782 strains, while the genomes retained ORF2819 (LMOf2365_2059) that, together with prs, yields the serotype 1/2b profile with the Doumith scheme. Furthermore, strains of several different serotype 1/2a STs serotyped as 1/2b using the Doumith scheme. WGS analysis revealed that these strains lacked the lmo0737 cassette but harbored a genomic island with the ORF2819 (LMOf2365_2059), thus yielding the serotype 1/2b profile with the Doumith scheme. Lastly, we investigated a novel, dual 1/2a-1/2b profile that was obtained with strains of several STs of serotype 1/2a using the Doumith scheme, and found that the genomes harbored both the lmo0737 cassette and genomic islands with the ORF2819 (LMOf2365_2059). These findings suggest that for certain strains and clones of L. monocytogenes, molecular serotype designations should be examined with caution and complemented by alternative tools such as those based on serotype-specific antibodies or WGS.