Genomic profiling and drug resistance patterns of Mycobacterium tuberculosis in Indonesia: whole-genome sequencing of isolates from the 2017–2018 national Tuberculosis Drug Resistance Survey

Objective Drug-resistant tuberculosis remains a major public health challenge in Indonesia. This study aimed to characterize the genetic diversity, lineage distribution, and genotypic resistance profiles of Mycobacterium tuberculosis isolates across the Indonesian archipelago using whole-genome sequencing. Methods A cross-sectional study was conducted using 1,281 analyzed MTB isolates derived from the 2017–2018 National Tuberculosis Drug Resistance Survey (DRS TB). Samples were screened via Xpert MTB/RIF and phenotypic drug susceptibility testing using the MGIT 960 system. Genomic DNA was extracted using the CTAB protocol. Whole-genome sequencing (WGS) libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA). Sequencing was performed on an Illumina MiSeq platform using the MiSeq Reagent Kit v3 (600 cycles), generating paired-end reads of 2 × 300 bp. To identify drug resistance-associated mutations and determine strain lineage/sub-lineages, raw sequencing reads were initially processed with Trimmomatic. The filtered reads were subsequently aligned to the Mycobacterium tuberculosis H37Rv reference genome (ATCC 27294;NC_000962.3) using the MTBseq pipeline, which the BWA-MEM algorithm (v0.7.17) for sequence mapping. Variant calling was carried out with Samtools mpileup (v1.6). Results Four major MTB lineages were identified: Lineage 4 (43.1%) was dominant, followed by Lineage 2 (30.4%) and Lineage 1 (26.3%), while Lineage 3 was rare, with only 0.16% detected. At the sub-lineage level, L2.2.1 (18.5%), L1.2.1 (11.3%), and L4.1.2.1 (9.7%) were the most prevalent. Resistance to first-line drugs was the most frequent, primarily driven by mutations in the katG p.Ser315Thr (7.5%) and rpoB p.Ser450Leu (5%), conferring isoniazid and rifampicin resistance, respectively. Second-line resistance was highest for Fluoroquinolones (2.03%). Notably, four pre-XDR strains (0.31%) were identified and validated across Java-Bali and Sumatera, all harboring critical gyrA mutations and rpoB mutations (p.Asp435Tyr, p.Asn437Asp, p.Ser450Leu, and p.Ser450Trp). Conclusion The study confirms a high prevalence of L2 and L4 in Indonesia. The identification of pre-XDR-TB isolates among the national survey samples highlights an urgent need for expanded molecular surveillance. While newer drugs like Bedaquiline remain effective, the presence of specific gyrA mutations underscores the necessity of integrating WGS into national TB control programs to proactively manage resistance and optimize treatment regimens.