Subspecies Identification and Characterization of Drug Resistance and Virulence Factors in Clinical Strains of Mycobacterium abscessus Complex Isolated from South India.

BACKGROUND

Mycobacterium abscessus complex (MABC), comprising Mycobacterium abscessus subsp. abscessus (MABa), Mycobacterium abscessus subsp. bolletii (MABb), and Mycobacterium abscessus subsp. massiliense (MABm), is an emerging group of non-tuberculous mycobacteria with clinically significant infections and challenging treatment outcomes due to extensive antimicrobial resistance. Accurate subspecies identification and characterization of resistance- and virulence-associated determinants are essential for effective disease management. This study aimed to determine the prevalence and subspecies distribution of MABC and to characterize resistance-associated mutations and virulence factors, including biofilm formation.

METHODS

A total of 1110 NTM-suspected clinical samples were screened during the study period, between January 2024 and October 2025. Samples negative by GeneXpert MTB/RIF were subjected to Mycobacteria Growth Indicator Tube (MGIT) culture, followed by Ziehl-Neelsen staining and MPT64 antigen testing. Acid-fast bacilli-positive, MPT64-negative isolates were identified as NTM and analyzed using GenoType CM and NTM-DR line probe assays (LPA) for species identification and detection of resistance-associated mutations. A polymerase chain reaction (PCR) assay was optimized to differentiate MABa and MABm. All MABC clinical strains were further characterized for colony morphology (smooth and rough) and biofilm formation. Three biofilm-producing MABa strains (2 rough and 1 smooth) that were detected as macrolide-resistant by NTM-DR were subjected to whole-genome sequencing (WGS).

RESULTS

Among 1110 clinical samples, MABC was identified in 2.25% (n = 25) of cases, while other NTM species accounted for 4.41% (n = 49). Among 25 MABC clinical strains, 14 (56%) were MABm, and 11 (44%) were MABa, as confirmed by both LPA and PCR. LPA-NTM DR detected erm(41) T28 sequevar (n = 9) and C28 mutation (n = 2) among MABa strains, with one strain exhibiting aminoglycoside resistance-associated rrs mutation. Nineteen isolates displayed a smooth morphotype (MABa = 8 and MABm = 11), and six were rough (MABa = 3 and MABm = 3). Biofilm formation was observed in both smooth (n = 5) and rough (n = 4) morphotypes. WGS analysis confirmed erm(41) T28 sequevar, identified a missense mutation (A238G), and revealed genes associated with glycopeptidolipid biosynthesis.

CONCLUSIONS

Our findings provide important insights into subspecies identification and genetic determinants associated with drug resistance and virulence in MABC. The biofilm-forming ability observed in both smooth and rough morphotypes emphasizes its potential role in persistence and treatment challenges, emphasizing the need for comprehensive diagnostic strategies.