Reduced susceptibility of Clinical Strains of Mycobacterium tuberculosis to Reactive Nitrogen Species. TB_NO

Background: Drugs such as isoniazid (INH) and pretomanid (PRT), used against Mycobacterium tuberculosis are active partly through generation of reactive nitrogen species (RNS). The aim of this study was to explore variability in intracellular susceptibility to nitric oxide (NO) in clinical strains of M. tuberculosis. Method: Clinical M. tuberculosis strains with or without INH resistance were transformed with a plasmid allowing for constitutive luciferase expression and the effects of RNS donors (DETA/NO and SIN-1) were analysed by luminometry. NO-dependent intracellular killing in clinical isolates was studied in RAW 264.7 murine macrophages activated with interferon gamma and lipopolysaccharide in combination with the NO inhibitor L-NMMA. Results: There was a significant difference in NO susceptibility for clinical isolates compared to M tuberculosis H37Rv. All strains showed a dose-dependent susceptibility to RNS donors. The reduced mycobacterial susceptibility to NO observed in broth cultures correlated to a reduced capacity of activated macrophages to limit mycobacterial growth. Furthermore, the most NO-tolerant clinical isolate showed impaired intracellular susceptibility to PRT compared to H37Rv in the absence of mutational resistance in genes associated to reduced susceptibility against the drug or NO. Conclusion: NO inhibited growth of M. tuberculosis with a significant difference between strains. Impaired intracellular susceptibility to nitric oxide was associated with reduced susceptibility to PRT. Our results indicate that susceptibility to cellular host defence mechanisms such as NO need to be taken into consideration when designing new therapeutic strategies.