P020

1:Statens Serum Institut

Disease caused by nontuberculosis mycobacteria (NTM) is rising worldwide, and in several low-tuberculosis-incidence countries, clinical NTM is approaching the incidence of tuberculosis (TB) disease. Routine species identification still relies on line-probe assays (LPAs) such as GenoType Mycobacterium CM/AS, which recognise only the 25–30 most common taxa. Divergent or novel lineages therefore hybridise solely to the genus control band and are reported as “Mycobacterium sp.”, obscuring clinically relevant diversity. Targeted sequencing of marker regions (hsp65, 16S, ITS, 23S) offers higher resolution but falls short for very closely related taxa, making whole-genome sequencing (WGS) the current gold standard. We analysed every Danish NTM high-quality genome generated in the past decade (n=131) together with representative genomes for all described NTM species in RefSeq. Pair-wise average nucleotide identity (ANI) was calculated and clustered with UPGMA. Eighty-six percent of isolates shared >95% with recognised type strains while the remainder (n=18) did not. Twelve of these grouped within the M. avium complex (MAC), forming tight clusters, consistent with at least six novel MAC taxa. As MAC is responsible for >60% of NTM cases in Denmark, we chose to focus on these unknown members of this clinically relevant group. One representative from each cluster underwent Oxford Nanopore sequencing, and hybrid Illumina/Nanopore assembly produced complete, high-quality circular genomes (5.5–6.3 Mbp) for all six candidates, each harbouring one to three plasmids. These findings broaden the known membership of the MAC and highlight the limitations of probe-based diagnostics for emerging NTM pathogens.