Integrative genomic and molecular dynamics characterisation of acrB_R717L/Q mutations in azithromycin-resistant...

Azithromycin remains a highly effective oral therapy for uncomplicated typhoid fever caused by extensively drug-resistant (XDR) Salmonella Typhi strains. However, azithromycin resistance has been reported in low and middle-income countries (LMICs). In this study, we identified three azithromycin-resistant S. Typhi isolates and performed phylogenetic analysis along with molecular dynamics simulation to assess the structural stability and conformational dynamics of the acrB efflux pump harbouring R717L/Q mutations.

Antimicrobial susceptibility of three azithromycin-resistant S. Typhi isolates was determined using the minimum inhibitory concentration (MIC) assay following CLSI guidelines. Whole genome sequencing was performed to elucidate the molecular determinants of resistance and to assess phylogenetic relatedness within a global genomic framework. In addition, computational analysis was conducted to evaluate the impact of mutations on drug resistance.

All three azithromycin-resistant S. Typhi exhibited an MIC value of 32 mg/L. Phylogenetic analysis demonstrated that two isolates clustered within a distinct sublineage (genotype 4.3.1.2.1) of lineage II (4.3.1.2), whereas the third isolate belonged to lineage 4.3.1.3, consistent with the patient's geographic link to Bangladesh. Antimicrobial resistance gene analysis identified the acrB_R717Q mutation in the two isolates and acrB_R717L in the single isolate, both associated with azithromycin resistance. Molecular dynamics simulations indicated greater structural deviation in the R717L variant compared to R717Q, with possible increased conformational flexibility that could influence efflux pump dynamics.

Our findings underscore the need for genomic surveillance frameworks to incorporate both plasmid-mediated resistance genes (e.g., mphA) and chromosomal mutations, such as acrB_R717L/Q variants, to effectively track emerging azithromycin resistance and guide typhoid conjugate vaccine deployment alongside antibiotic stewardship.