Variation in drug penetration does not account for the natural resistance of Mycobacterium abscessus biofilms to antibiotic

Mycobacterium abscessus, an inherently drug-resistant, opportunistic, nontuberculous mycobacterium (NTM) predominantly causes pulmonary infections in immunocompromised patients, notably those with cystic fibrosis. M. abscessus subspecies display distinct colony morphologies (rough and smooth), with the prevalent view that M. abscessus (smooth) is a persistent, biofilm-forming phenotype, whilst M. abscessus (rough) is unable to form biofilms. Biofilm formation contributes to persistent infections and exhibits increased antibiotic resistance. We used the chemical mapping technique, nanoscale secondary ion spectrometry (NanoSIMS), to investigate if variations in the biofilm morphology and antibiotic penetration account for the antibiotic susceptibility amongst M. abscessus subspecies, contributing to increased antimicrobial resistance (AMR) and potentially explaining the protracted treatment duration. The susceptibility to bedaquiline (BDQ) of M. abscessus grown as planktonic bacilli and biofilms was measured. The minimum biofilm eradication concentration (MBEC) of BDQ was 8-16 times higher (2-4{micro}g/ml) compared with the minimum inhibitory concentration (MIC) (0.25{micro}g/ml), indicating reduced efficacy against biofilms. Correlative imaging with electron microscopy revealed that M. abscessus (irrespective of the colony morphotype) formed biofilms and that BDQ treatment influenced biofilm morphology. We determined that M. abscessus morphotypes exhibit differential uptake of the antibiotic BDQ in biofilms. M. abscessus subsp. abscessus (smooth) biofilms exhibited the least uptake of BDQ, whereas M. abscessus subsp. bolletii biofilms showed the greatest antibiotic penetration. NanoSIMS analysis revealed no correlation between antibiotic penetration and drug efficacy within the biofilm. This challenges the previous assumption linking biofilm architecture to drug efficacy. Investigating other biofilm characteristics like antibiotic persistence could lead to enhanced treatment approaches. Significance StatementMycobacterium abscessus is an increasingly prevalent pathogen, most often causing lung infections in immunocompromised individuals. Their distinct morphotypes and biofilm-forming capabilities contribute to persistent infections, rendering them challenging to treat with increased antibiotic resistance. This research demonstrates that the antibiotic, bedaquiline exhibits significantly reduced efficacy against M. abscessus growing as a biofilm compared to planktonic growth, but that the efficiency of antibiotic penetration was not the main explanation for the different susceptibilities of MABC biofilms to treatment.