A four gene genotyping panel for Listeria monocytogenes driven by amino-acid repeat hotspots

Listeria monocytogenes remains a major foodborne threat despite decades of research, with persistent outbreaks linked to ready-to-eat foods underscore the limitations in current food safety monitoring. Therefore, rapid, interpretable markers that complement gold standard surveillance are valuable for monitoring of Listeria monocytogenes in food settings. We analyzed 67 publicly available L. monocytogenes genomes and quantified amino-acid repeat (AAR) variations in four proteins viz., Internalin (PVD-rich), LPXTG cell-wall anchor (DA-rich), ActA (GPN-rich) and FtsH (EPKE). We summarized each genome by a four-element AAR profile, and clustering these profiles by similarity yielded 14 groups among 33 strains. These AAR groups showed strong agreement with MLST numbers (Adjusted Rand Index 0.972), indicating that AAR loci carry same genetic background signal comparable to that of to seven-locus MLST. In contrast, AAR patterns did not correlate with core-genome SNP. Further, the gene mapping of genomes yielded four structures (A–D) with structural differences primarily driven by LPXTG and Internalin repeat content and, were concordant with AAR and MLST patterns. Together, these results demonstrate a four-protein AAR genotyping panel as a complementary tool in discriminating L. monocytogenes in line with MLST.