Insights into mycobacteriome composition in Mycobacterium bovis-infected African buffalo (Syncerus caffer) tissue samples.

Animal tuberculosis significantly challenges global health, agriculture, and wildlife conservation efforts. Mycobacterial cultures are resource-intensive, time-consuming, and challenged by heterogeneous populations. In this study, we employed a culture-independent approach, using targeted long-read-based next-generation sequencing (tNGS), to investigate the mycobacterial composition in 60 DNA samples extracted from Mycobacterium bovis infected culture-confirmed African buffalo tissue. We detected mycobacterial DNA in 93.3% of the samples and the sensitivity for detecting Mycobacterium tuberculosis complex (MTBC) was 91.7%, demonstrating a high concordance of our culture-independent tNGS approach with mycobacterial culture results. In five samples, we identified heterogenous mycobacterial populations with various non-tuberculous mycobacteria, including members of the Mycobacterium avium complex (MAC), M. smegmatis, and M. komaniense. The latter Mycobacterium species was described in South Africa from bovine nasal swabs and environmental samples from the Hluhluwe-iMfolozi Park, which was the origin of the buffalo samples in the present study. This finding suggests that exposure to environmental mycobacteria may confound detection of MTBC in wildlife. In conclusion, our approach represents a promising alternative to conventional methods for detecting mycobacterial DNA. This high-throughput technique enables rapid differentiation of heterogeneous mycobacterial populations, which will contribute valuable insights into the epidemiology, pathogenesis, and microbial synergy during mycobacterial infections.