Abstract

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.

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