Molecular identification and resistance-associated gene profiling of non-tuberculous mycobacteria isolated from bovine clinical samples.

BACKGROUND: Non-tuberculous mycobacteria (NTMs) are environmental opportunistic pathogens capable of producing tuberculosis-like lesions in bovines, potentially complicating the diagnosis and surveillance of bovine tuberculosis. Molecular data on resistance-associated genes in bovine NTMs from India remain limited. This study aimed to isolate and characterize NTMs from bovine clinical samples and to investigate selected resistance-associated genes at molecular level. METHODS AND RESULTS: A total of 193 samples comprising faeces (n = 101), nasal swabs (n = 21), trans-tracheal washes (n = 13), tissues (n = 30), and blood (n = 28) were analysed using direct PCR and culture-based isolation. Genus-level identification was performed using hsp65 PCR followed by species-specific PCR assays. Eighteen confirmed NTM isolates were screened for resistance-associated genes (rpoB, katG, rpsL, and gyrA) and selected amplicons were sequenced. Statistical analysis was conducted using chi-square or Fisher's exact test. Mycobacterium vaccae predominated among isolates (66.7%). The rpoB gene was detected in 88.9% of isolates and was mainly associated with M. vaccae. The katG (16.7%) and gyrA (5.6%) genes were detected exclusively in M. vaccae, while rpsL gene was not amplified in any isolate. Sequence analysis showed no mutations in rpoB or katG hotspots, but non-synonymous substitutions were detected in quinolone resistance-determining region of gyrA. CONCLUSION: Resistance-associated genes were predominantly detected in M. vaccae, with rpoB most frequent, followed by katG and gyrA. These findings indicate genotypic variation only and cannot confirm phenotypic resistance because susceptibility testing was not performed. Further, phenotypic and species-specific analyses are required to establish functional significance.