Core Genome Multi Locus Sequence Typing and Single Nucleotide Polymorphism Analysis in the Epidemiology of Brucella melitensis Infections
AbstractThe use of whole genome sequencing (WGS) using next generation sequencing (NGS) technology has become a widely accepted method for microbiology laboratories in the application of molecular typing for outbreak tracing and genomic epidemiology. Several studies demonstrated the usefulness of WGS data analysis through Single Nucleotide Polymorphism (SNP) calling from a reference sequence analysis for Brucella melitensis, whereas gene-by-gene comparison through core-genome Multilocus Sequence Typing (cgMLST) has not been explored so far. The current study developed an allele-based method cgMLST and compared its performance to the genome-wide SNP approach and the traditional MLVA on a defined sample collection. The dataset comprised of 37 epidemiologically linked animal cases of brucellosis as well as 71 epidemiologically unrelated human and animal isolates collected in Italy. The cgMLST scheme generated in this study contained 2,687 targets of the B. melitensis 16M reference genome (75.4% of the complete genome). We established the potential criteria necessary for inclusion of an isolate into a brucellosis outbreak cluster to be ≤4 loci in the cgMLST and ≤10 in WGS SNP analysis. CgMLST and SNP analysis provided much higher phylogenetic distance resolution than MLVA, particularly for strains belonging to the same lineage thus allowing diverse and unrelated genotypes to be identified with greater confidence. The application of this cgMLST scheme to the characterization of B. melitensis strains provided insights into the epidemiology of this pathogen and it is a candidate to be a benchmark tool for outbreak investigations in human and animal brucellosis.