Utility of RGM medium for recovery of Mycobacterium abscessus from presumptive pulmonary tuberculosis patients and its molecular phylogenetic characterization

Abstract

Background and Objectives: Non-tuberculous mycobacteria (NTM), especially Mycobacterium abscessus, are emerging causes of pulmonary disease and are often misdiagnosed as tuberculosis in high TB burden countries. Conventional culture methods are time-consuming and may reduce viable organism recovery. Rapidly growing mycobacteria (RGM) medium en- ables direct inoculation of respiratory specimens and improved recovery of rapidly growing NTM. This study evaluated the utility of RGM medium for isolation of M. abscessus from presumptive pulmonary tuberculosis patients and characterized the isolates using molecular and phylogenetic analyses.

Materials and Methods: A total of 223 sputum samples collected from presumptive pulmonary tuberculosis patients. Un- treated sputum samples were inoculated onto RGM medium with selective antibiotics and incubated at 30°C for 10 days. Suspected colonies were confirmed by Ziehl–Neelsen staining. Identification was performed by PCR and sequencing of hsp65 and rpoB genes. Phylogenetic analysis was done using the Neighbour-Joining method in MEGA12. Mycobacterium abscessus isolates were further analysed using the GenoType NTM-DR assay.

Results: Among the 223 sputum samples processed, 7.6% (17/223) demonstrated positive growth on RGM medium. Of the culture-positive isolates, 64.7% (11/17) were identified as Mycobacterium abscessus, while 35.3% (6/17) were identified as other non-tuberculous mycobacterial (NTM) species. All isolates were positive for the rpoB and hsp65 genes. Eleven M. abscessus isolates were further analysed using the GenoType NTM-DR assay, and no resistance-associated mutations were detected in the erm(41), rrl, or rrs genes. Additionally, all isolates were found to be susceptible to macrolides and amino-glycosides.

Conclusion: RGM medium demonstrated good utility for recovery of clinically significant rapidly growing mycobacteria, particularly M. abscessus, directly from sputum samples. Molecular and phylogenetic analyses enabled accurate species identification and characterization. The absence of resistance-associated mutations suggests retained susceptibility to key therapeutic agents. These findings support the use of RGM medium for improved detection of pulmonary NTM infections in TB-endemic settings.