Whole Genome Sequencing of A Pooled Sample of Bedaquiline and Clarithromycin Resistant Mycolicibacterium smegmatis Exhibits Mutations in Genes Associated with Efflux Family Protein and ATP Synthase
Abstract
Objective: This study aimed to evaluate the pooled sequencing of mixed samples with different minimum inhibitory concentrations (MIC) of bedaquiline (BDQ) and clarithromycin to identify genes associated with drug resistance and explore those that could predict cross-resistance to both drugs. Additionally, it aimed to preliminarily investigate the association between single-nucleotide polymorphisms (SNPs) from pooled samples and the progression of drug resistance.
Material and Methods: Mycolicibacterium smegmatis wild type (WT) and hup B (MDP1) knock-out strains were obtained from the Bacteriology Laboratory, Graduate School of Medical and Dental Science, Niigata University. Drug-resistant strains were generated through stepwise passaging on drug-containing agar plates, followed by MIC determination. Whole-genome sequencing was performed using the MinION Nanopore platform, with pooled samples categorized based on MIC levels. Bioinformatics analysis, including SNP identification and annotation, was conducted using burrows wheeler aligner, Samtools, genome analysis toolkit, and SnpEff pipelines. Molecular docking and molecular dynamics simulations were employed for in silico validation of SNP-drug interactions. SNP frequency was calculated, and statistical analysis was performed using a paired t-test in Prism® software.
Results: High-impact SNPs that changed the amino acid were found in both pooled samples, one in pooled sample A (atpA p.Glu429Gln) and 5 in pooled sample B (MSMEG_0639, MSMEG_1867, MSMEG_4765, and MSMEG_6302).
Conclusion: Pooled samples of BDQ and clarithromycin-resistant M. smegmatis strains exhibited mutations in the genes associated with the efflux family and the adenosine triphosphate synthase protein. A pooled sample sequencing method can be used to identify variant sites and predict genes associated with cross-resistance.
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