Genomics-optimized Sequence Typing of Listeria monocytogenes Isolates

Listeria monocytogenes is an important foodborne pathogen and persistent contaminant in many food processing facilities. Strain typing is critical to detecting and investigating outbreaks, and can be used to track down sources of contamination. Available typing systems have limitations ranging from low resolution and data portability to high cost and technical complexity. The aim of this study was to develop an outsourcing option for L. monocytogenes strain typing that addresses these limitations. The NCBI Genomes database representing 109 strains was screened for informative, tandem repeat-containing loci. The most promising, LmMT1, encoded an internalin-like protein with Pro-Val-Asp repeats. It exhibited a complex pattern of polymorphism (insertions/deletions and SNPs), and was present in all L. monocytogenes (and Listeria innocua) database strains. The repeat region within this locus was previously validated as typing target in studies examining length polymorphism alone (diversity index 0.87-0.93); its information content was significantly enhanced by sequence analysis (diversity index 0.99). Phylogenetic analysis of LmMT1 sequences generated distinct clusters corresponding to serotype (4b, 1/2a, and 4a complexes) and evolutionary lineage (I-IV). Comparisons to PFGE, the current gold standard, suggests that LmMT1 typing is comparably discriminatory. Importantly, strains from four outbreaks formed corresponding clusters, although those from a 2002 outbreak were resolved into related environmental and food/human isolates that challenges their epidemiological connection. In the laboratory, LmMT1 typing proved to be robust, generating high quality sequence from colonies submitted as non-hazardous heat-inactivated suspensions. Phylogenetic analysis of these sequences from 62 diverse strains demonstrated overall agreement between LmMT1 and SNP-based typing.