Core Genome Multi Locus Sequence Typing and Single Nucleotide Polymorphism Analysis in the Epidemiology of Brucella melitensis Infections

Abstract

28The use of whole genome sequencing (WGS) using next generation sequencing (NGS) 29 technology has become a widely accepted method for microbiology laboratories in the 30 application of molecular typing for outbreak tracing and genomic epidemiology. Several 31 studies demonstrated the usefulness of WGS data analysis through Single Nucleotide 32 Polymorphism (SNP) calling from a reference sequence analysis for Brucella melitensis, 33whereas gene-by-gene comparison through core-genome Multilocus Sequence Typing 34 (cgMLST) has not been explored so far. The current study developed an allele-based 35 method cgMLST and compared its performance to the genome-wide SNP approach and 36 the traditional MLVA on a defined sample collection. The dataset comprised of 37 37 epidemiologically linked animal cases of brucellosis as well as 71 epidemiologically 38 unrelated human and animal isolates collected in Italy. The cgMLST scheme generated in 39 this study contained 2,687 targets of the B. melitensis 16M reference genome (75.4% of 40 the complete genome). We established the potential criteria necessary for inclusion of an 41 isolate into a brucellosis outbreak cluster to be ≤4 loci in the cgMLST and ≤10 in WGS 42 SNP analysis. CgMLST and SNP analysis provided much higher phylogenetic distance 43 resolution than MLVA, particularly for strains belonging to the same lineage thus allowing 44 diverse and unrelated genotypes to be identified with greater confidence. The application 45 of this cgMLST scheme to the characterization of B. melitensis strains provided insights 46 into the epidemiology of this pathogen and it is a candidate to be a benchmark tool for 47 outbreak investigations in human and animal brucellosis. 48 49 50 51 52 53 3 Introduction 54

Native circulating Brucella melitensis lineages causing a brucellosis epidemic in Qinghai, China

“…The multilocus variable-number tandem repeat analysis (MLVA) has been used as the gold standard for genotyping Brucella strains, with the results combined with epidemiological data to investigate the relationships between the Brucella strains (Kiliç et al, 2011;Liu et al, 2017). Furthermore, single-nucleotide polymorphisms (SNPs) based on whole-genome sequencing have excellent power to discriminate strains and allow for the characterization of the phylogenetic relationships of strains from different scales (Janowicz et al, 2018;Abdel-Glil et al, 2022). Importantly, molecular typing tools can not only limit control costs and test time but also, improve the surveillance and evaluation of control measure effects.…”