Insights into Mycobacteriome Composition in Mycobacterium bovis-Infected African Buffalo (Syncerus caffer) Tissue Samples: A Culture-Independent Approach

Abstract

<title>Abstract</title> Animal tuberculosis significantly challenges global health, agriculture, and wildlife conservation efforts. Mycobacterial cultures necessitate stringent biosafety measures due to the risk of laboratory-acquired infections. In this study, we employed a culture-independent approach, using targeted long-read-based next-generation sequencing (tNGS), to investigate the mycobacterial composition in DNA extracted from <italic>Mycobacterium bovis</italic> infected culture-confirmed African buffalo tissue. We detected mycobacterial DNA in 93.3% of the samples and the sensitivity for detecting <italic>Mycobacterium tuberculosis</italic> complex (MTBC) was 91.7%, demonstrating a high concordance of our culture-independent tNGS approach with mycobacterial culture results. We identified heterogenous mycobacterial populations with various non-tuberculous mycobacteria, including members of the <italic>Mycobacterium avium</italic> complex, <italic>M. smegmatis</italic>, and <italic>M. komaniense</italic>. The latter <italic>Mycobacterium</italic> 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 mycobacterial DNA found in the environment may confound detection of MTBC in wildlife. In conclusion, our approach represents an alternative to conventional methods for detecting mycobacterial DNA. This high-throughput technique enables the differentiation of heterogeneous mycobacterial populations and facilitates relative quantification, which will contribute valuable insights into the epidemiology, pathogenesis, and microbial synergy during mycobacterial infections.