Whole genome sequencing based analysis of Brucella species isolated from ruminants in various regions of Türkiye

This study was conducted to perform whole genome sequencing (WGS) of Brucella isolates originated from ruminants. Brucellosis is an important animal and public health problem in Türkiye and many countries around the world. In this context, single nucleotide polymorphisms (SNPs) and WGS-SNP-based phylogenetic analysis and double SNP distance calculation, WGS-based virulence gene and resistance gene analyses, identification and biotyping by conventional microbiological analyses, phenotypic antimicrobial resistance and phage susceptibility analyses were performed and the obtained data were compared with each other within the scope of compatibility. In the study, 21 Brucella spp. isolates from ruminants and two Brucella spp. isolates from humans in various regions of Türkiye were used. As a result of cultural methods, polymerase chain reaction (PCR) and WGS analyses, 11 Brucella melitensis and 12 B. abortus were identified. It was determined that all B. abortus isolates were of bovine origin, while B. melitensis isolates were isolated from sheep, goat, ram and human. Eleven (91.66%) B. abortus isolates were biotyped as biovar (bv) 3, one (8.33%) as B. abortus bv 9. Nine (81.8%). B. melitensis isolates were biotyped as B. melitensis bv 3, while one isolate was found as bv 1 and the other was identified as bv 2. The average GC content of 23 whole genome sequenced isolates was 57% and the genome length was 3.2 megabases (Mb). In the whole genome SNP-based phylogenetic tree, it was determined that all of the B. melitensis isolates were located in the IIb subgenotype of genotype II, which is associated with the Eastern Mediterranean lineage. Among the local isolates that clustered into the IIb subgenotype, the highest SNP distance was observed between the isolates from Siirt and Istanbul (22RB24671). According to the WGS-SNP-based phylogenetic tree constructed with B. abortus isolates, all local isolates were found to be in clade C1. Among the local isolates showing two main clusters in clade C1, all isolates from Iğdır province and isolates from Şanlıurfa/Hilvan and Istanbul (22RB24674) formed one cluster with Iranian strains, while the other B. abortus isolates formed the second cluster including the Chinese strain. In the pairwise SNP distance comparison analysis, it was determined that the SNP differences between local B. abortus isolates ranged between 0-316. In some B. melitensis and B. abortus isolates isolated from different animals in the same period and in the same herd, no SNP difference was detected and this was considered as a common transmission-induced infection. In the virulence gene analysis performed with WGS data of 23 B. abortus and B. melitensis isolates included in the study, 43 virulence gene-related regions were detected in all isolates regardless of species, host and isolation year. It was determined that most of the detected genes were located on chromosome I (Chr I) and the majority of the genes associated with lipopolysaccharide (LPS) and type 4 secretion system (T4SS). In the WGS-based antimicrobial resistance gene analysis, no classical resistance-related genes were detected, while 58.33% of B. abortus isolates were resistant to at least two of the antibiotics tested in phenotypic resistance analysis. This rate was 63.63% in B. melitensis isolates. In conclusion, WGS-based epidemiological analysis of B. melitensis and B. abortus isolates, the two most common causative agents of brucellosis, was performed and the circulating genotypes in Türkiye were determined. It was concluded that these data are important for monitoring the disease and controlling outbreaks in the country. It was also thought that antimicrobial resistance reached an alarming level and it should be monitored.