Growth medium and nitric oxide alter Mycobacterium abscessus morphotype and virulence

Mycobacterium abscessus complex (MABC) infections cause significant morbidity and mortality among patients with chronic lung disease, like cystic fibrosis. MABC exists in smooth (S) and rough (R) morphotypes, but triggers of morphotype switching and associated pathogenicity or antimicrobial susceptibility are poorly understood. We demonstrate that M. abscessus subspecies abscessus (Mab), massiliense (Mms), and bolletii (Mbl) cultured in Middlebrook (MB) broth exhibit S morphotype, whereas the bacteria grown in Luria Bertani (LB) broth adopt the R morphotype, characterized by low glycopeptidolipid (GPL) expression. The components of broth that mediate this selection are complex, with albumin supplementation promoting growth of S morphotype, but not sufficient for complete selection. Consistent with the findings of other groups, R forms of Mab, Mms and Mbl selected by LB broth were internalized in RAW 264.7 macrophages with higher efficiency than S. Intracellular survival of broth-selected organisms was variable and was higher for S Mab, but lower for S Mms and Mbl. It is proposed that growth in R morphotype is induced during stress conditions, such as nutrient poor environments or during inflammation. One key component of inflammation is release of nitric oxide. We demonstrated that a nitric oxide donor (DETA-NONOate) appears to induce growth in an R morphotype, as indicated by reduced GPL expression of Mab. Mab treated with DETA-NONOate also enhanced susceptibility to azithromycin at sub-MIC concentrations. In conclusion, morphotype and macrophage intracellular Bacterial load of MABC subspecies can be manipulated by growing the bacteria in different culture conditions. Nitric oxide may also drive morphotype selection and enhanced azithromycin activity against Mab and macrophage killing.

Antibiotic resistance; Glycopeptidolipids; Morphotypes; Mycobacterium abscessus; Nitric oxide; Phenotypic switching.