Typing and whole-genome sequencing of Brucella isolated from clinical samples in Lebanon. (c2016)

Abstract

Brucella is a Gram-negative intracellular bacterium that causes brucellosis characterized by several symptoms including fever, nausea and myalgia. Routes of infection include direct contact through skin or mucous membranes, inhalation, handling manure of infected animals and ingestion of infected dairy products. Brucella melitensis is the main causative agent of infection in Lebanon, with increasing incidence as a result of occupational contact and ingestion of infected meat and dairy products. Whole-genome sequencing (WGS) is essential for pathogenomics and evolutionary analysis of different species of Brucella. In this study, and as a follow-up to previous studies done in Lebanon, resistance profiles to common antibiotics used against Brucella by antibiotic disc diffusion method were obtained. The isolates were shown to be resistant to fluoroquinolones and co-trimoxazole. Typing of Brucella was performed using 16S rRNA sequencing, Bruce-ladder multiplex PCR, and PCR-RFLP of omp2a and omp31 genes. 16S rRNA sequencing confirmed the genus Brucella, while PCR-RFLP of omp31 and omp2a proved more accurate and practical in detecting the species than Bruce-ladder PCR. Sixteen isolates were chosen for WGS and their subsequent genomes were analyzed for presence of virulence and resistance determinants, genomic islands, CRISPRs, prophages, insertion sequences and phylogenetic analysis. Several virulence genes vital for Brucella pathogenicity were detected in our isolates including type IV secretion system, flagellar and LPS genes, ureases and BvrR/S two component system. Additionally, antibiotic resistance genes against quinolones and efflux pumps mediating multidrug efflux were also found, indicating the emerging resistance patterns in Lebanon. Prophages, insertion sequences and genomic islands confirmed that lateral gene transfer occurred in our B. melitensis strains and further contributed to the genome evolution, diversity, and virulence. To our best knowledge, this is the first kind of study done on Brucella in Lebanon. It will provide an insight into brucellosis and its prevalence in addition to highlighting the importance of taking control measures to limit future spread of the disease.