Complete genome sequences and multidrug resistance genotypes of nontuberculous mycobacteria isolates from the Central Tuberculosis Reference Laboratory Muhimbili Tanzania

Abstract

Background: Nontuberculous mycobacteria (NTM) usually comprise a group of environmental bacteria, with emerging but elusive coinfection with tuberculous mycobacteria, causing pulmonary tuberculosis. Whole genome sequencing may give insight into potential antimicrobial resistance genotypes, giving clinicians and policymakers proper directions in clinical applications and management regimens. Methods: WGS was performed on twenty-four gDNA isolates from archival samples at the Central Tuberculosis Reference Laboratory using the MinION Oxford Nanopore Sequencing approach. Out of twenty-four, two were confirmed to belong to the NTM group. Further analysis was done to resolve the complete genomes of two nontuberculous mycobacteria strains isolated from tuberculosis patients. We then combined phylogenomics, reference-based scaffolding and average nucleotide identity (ANI) analysis to delineate each strain's taxonomic position and corresponding features. Results: Our findings reveal that the two strains fit into the genus Mycolicibacterium, and the closest relative is Mycolicibacterium novocastrense. Coupling BacAnt and CARD-based antibiotic resistance analyses revealed multidrug-resistant genotypes of diverse spectra and mechanisms. While the BC02 strain is genetically resistant to beta-lactams, macrolides and rifamycins, the BC05 strain portrays an extended drug resistance genotype encompassing beta-lactams, macrolides, polyamines, and aminoglycosides. Both strains possess a single nucleotide polymorphism (SNP) of the RNA polymerase beta-subunit (rpoB), representing resistance to the first-line rifampicin. Additionally, the BC05 strain genetically portrays resistance to ethambutol, isoniazid and fosfomycin through mechanisms involving target alteration through SNPs, drug inactivation and efflux. Conclusion: Our findings strongly suggest the potential implication of multidrug-resistant NTM clinical isolates in the pathogenesis of pulmonary tuberculosis.