Whole Genome Sequencing of Brucella melitensis Isolates from Kuwait for the Identification of Biovars, Variants and Relationship within a Biovar.

Abstract

Objective: The identification of Brucella genotypes is essential for epidemiological studies. The whole genome sequencing is emerging as a novel tool for genetic characterization of infectious microbes. The aim of this study was to genotype B. melitensis isolates from Kuwait using whole genome sequencing and variant analysis of the sequence data.

Methods: DNA were purified from 15 heat-inactivated B. melitensis isolates and used to prepare sequencing libraries employing Nextera XT DNA Sample Preparation Kit (Illumina San Diego, CA, USA) and sequenced on a MiSeq (Illumina). The sequence files were aligned to three biovars of B. melitensis, i.e., biovar 1 str. 16M, biovar 2 str. 63/9, and biovar 3 str. Ether. The alignment and variant calling were performed using 'bwa mem' and SAMtools/VCFtools, respectively.

Results: The genome size of all the isolates was around 3.3 mega base pairs and resembled B. melitensis biovar 2. Single nucleotide polymorphisms (SNPs), insertions and deletions, (indels) were spread all over the genome; but 138 SNPs were common among the 14 isolates, supporting the same ancestral origin. A neighbor-joining tree analysis identified isolate 2 as an outlier. In addition, SNPs (2 - 478) specific to each isolate were also identified, which divided the B. melitensis biovar 2 into two major groups/genotypes. A further analysis showed that the Kuwaiti isolates of the present study shared phylogeny mainly with strains from the Middle Eastern countries.

Conclusions: Among the 15 isolates studied in Kuwait, biovar 2 is the most prevalent biovar of B. melitensis. Furthermore, isolate-specific genetic variations were identified, which may be useful in epidemiological investigations.