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Drug-specific differential culturability in diverse strains of Mycobacterium tuberculosis

S Borrell(1,2) V F.A March(1,2) S Gagneux(1,2)

1:Swiss Tropical and Public Health Institute-University of Basel; 2:University of Basel

Recent years have highlighted the importance of drug-pathogen interactions on clinical outcomes in microbial infections. This includes assessing phenomena such as tolerance, persistence and more recently, resilience on the clearance of bacteria by antibiotic chemotherapy. In Mycobacterium tuberculosis, it has previously been shown that differentially culturable bacteria often grow poorly on nutrient rich solid media but are detectable using other culture methods due to metabolic changes in oxidative stress. We hypothesized that antibiotic treatment itself can induce oxidative stress in Mtb, resulting in differential culturability, which has implications for assay design when investigating drug-pathogen interactions.

To investigate the effect of antibiotic treatment on Mtb culturability, we subjected a set of clinical strains from different phylogenetic lineages to high-dose short-interval exposure to Rifampicin (RIF), Isoniazid (INH) or RIF and INH in combination at clinically relevant doses for 72 hours. After treatment, drugs were washed out by two cycles of centrifugation and resuspension in 7H9.  Survival was assessed by plating on 7H11 and counting colony-forming units (CFUs), and recovery was assessed by allowing recovered bacteria to regrow in falcons tubes shaking at 37°C shaking at 140rpm.

Interestingly, our investigation revealed a disparity between INH-treated cultures and RIF-treated cultures in terms of recovery in broth compared to solid media. RIF-treated cultures indicated higher survival compared to INH when considering CFUs, however, INH-treated cultures recovered faster in broth.

Our initial investigation has indicated that drug-specific action on Mtb can affect subsequent culturability. This requires further evaluation to inform relevant and reliable experimental design when investigating drug-pathogen interactions.

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