Whole-Genome Sequence Typing shows extensive diversity of Listeria monocytogenes in the outdoor environment and poultry processing plants.

A reliable and standardized classification of Listeria monocytogenes (Lm) is important for accurate strain identification during outbreak investigations. Current whole-genome sequencing (WGS) based approaches for strain characterization either lack standardization, rendering them less suitable for data exchange, or are not freely available. Thus, we developed a portable and open-source tool Haplo-ST to improve standardization and provide maximum discriminatory potential to WGS data tied to an MLST (multi locus sequence typing) framework. Haplo-ST performs whole-genome MLST (wgMLST) for Lm while allowing for data exchangeability worldwide. This tool takes in (i) raw WGS reads as input, (ii) cleans the raw data according to user specified parameters, (iii) assembles genes across loci by mapping to genes from reference strains, (iv) assigns allelic profiles to assembled genes and provides a wgMLST subtyping for each isolate. Data exchangeability relies on the tool assigning allelic profiles based on a centralized nomenclature defined by the widely-used BIGSdb-Lm database. Tests on Haplo-STs performance with simulated reads from Lm reference strains yielded a high sensitivity of 97.5%, and coverage depths of [≥] 20x was found to be sufficient for wgMLST profiling. We used Haplo-ST to characterize and differentiate between two groups of Lm isolates, derived from the natural environment and poultry processing plants. Phylogenetic reconstruction showed sharp delineation of lineages within each group and no lineage-specificity was observed with isolate phenotypes (transient vs. persistent) or origins. Genetic differentiation analyses between isolate groups identified 21 significantly differentiated loci, potentially enriched for adaptation and persistence of Lm within poultry processing plants. IMPORTANCEWe have developed an open-source tool that provides allele-based subtyping of Lm isolates at the whole genome level. Along with allelic profiles, this tool also generates allele sequences, and identifies paralogs, which is useful for phylogenetic tree reconstruction and deciphering relationships between closely related isolates. More broadly, Haplo-ST is flexible and can be adapted to characterize the genome of any haploid organism simply by installing an organism-specific gene database. Haplo-ST also allows for scalable subtyping of isolates; fewer reference genes can be used for low resolution typing, whereas higher resolution can be achieved by increasing the number of genes used in the analysis. Our tool enabled clustering of Lm isolates into lineages and detection of potential loci for adaptation and persistence in food processing environments. Findings from these analyses highlights the effectiveness of Haplo-ST in subtyping and evaluating relationships among isolates for routine surveillance, outbreak investigations and source tracking.