Genome-guided pooled infection and whole-genome sequencing identify clinical Mycobacterium avium complex isolates that persist in a murine infection model.

Clinical isolates of Mycobacterium avium complex (MAC) differ markedly in their capacity to persist in mouse lungs, complicating strain selection for experimental infection and treatment-response studies. Here, we applied a screening strategy that combined genome-guided pooled infection with whole-genome sequencing to identify mouse-persistent MAC clinical isolates. Thirty-nine MAC clinical isolates, including 13 clarithromycin-resistant isolates, were classified into eight genome-based groups and used for pooled intranasal infection in BALB/c mice. At 8 weeks postinfection, pooled groups that maintained or increased lung bacterial burdens underwent bacterial whole-genome sequencing and deconvolution of isolate proportions based on isolate-specific single-nucleotide variants. Candidate isolates exhibiting increased relative representation after infection were selected for single-isolate validation. MAV22, MI01, and MI05 increased lung bacterial burdens, whereas MAV17 showed a modest decline but retained measurable burdens suitable for downstream assessment. These isolates were subsequently evaluated in a multidrug treatment assay comprising clarithromycin, rifampicin, and ethambutol. Treatment reduced lung bacterial burdens in mice infected with clarithromycin-susceptible isolates, whereas the clarithromycin-resistant isolate MAV22 exhibited a smaller reduction. This screening strategy provides a practical first-pass approach for identifying MAC clinical isolates that maintain measurable lung bacterial burdens and are suitable for CFU-based treatment-response assessment.