Unravelling the genetic contributors to linezolid resistance in Mycobacterium smegmatis: insights from a transposon library.

Objectives: This study aimed to identify and characterize genes associated with linezolid resistance in Mycobacterium smegmatis using a transposon mutagenesis approach.

Methods: This research conducted three replicative experiments where 16 isolates displaying pronounced resistance to linezolid were examined, revealing two distinct morphologies. WGS was employed to investigate these isolates, uncovering mutations in specific genes. The binding affinity of linezolid to relevant proteins was assessed through molecular docking studies and validated by drug affinity responsive target stability (DARTS) assays.

Results: Complementation of the mspA gene in mutant strains restored linezolid susceptibility, but the Ala127Gln substitution in MSMEG_0965 did not, suggesting a critical role of this residue in resistance. Further investigations revealed that the resistance mechanism in the △MSMEG_0965 mutant involves impaired linezolid uptake.

Conclusions: The research successfully identified two genes, MSMEG_4189 and MSMEG_0965, associated with linezolid resistance in M. smegmatis. It also elucidated the role of MSMEG_0965 in the resistance mechanism, providing significant targets and reference points for future studies on clinical strains.