A Novel Amino Acid Deletion and Substitution in amrB Gene Associated with Gentamicin Susceptibility in Burkholderia pseudomallei from Malaysian Borneo

Abstract

Burkholderia pseudomallei is a highly pathogenic saprophyte that is intrinsically resistant to a wide variety of antibiotics. Resistance to gentamicin is considered as an earmark of B. pseudomallei. However, rare susceptible strains have been isolated in certain regions due to gene mutations. Currently, data on the susceptible strains prevalence and the actual causal mutations are still scarce, particularly in Malaysian Borneo. A pool of B. pseudomallei isolates (n = 46) were screened for gentamicin susceptibility and phenotypically confirmed using the gradient minimum inhibitory concentration method. Three isolates were gentamicin-susceptible strains and were identified as having originated from Bintulu, Sarawak, Malaysian Borneo. The amrB gene mutation in these mutant strains was analysed, and the effect of amino acid substitution on the stability of the amrB protein was determined by using in silico analysis. The mutagenesis analysis identified a polymorphism-associated mutation, g.1056T>G, and two susceptible-associated mutations identified as novel in-frame amino acid deletion p.Val412del and amino acid substitution p.Thr368Arg that compromised gentamicin resistance. In silico analysis using amrB homology-modelled and AlphaFold-solved structures proposed the role of p.Thr368Arg amino acid substitution in conferring GEN susceptibility by other mechanisms than destabilising the structure of amrB protein, which is most probably due to the mutations location in the highly conserved region. The findings have shed light on the phenotypic characteristics and mutations involved in the amrB gene of the gentamicin-susceptible B. pseudomallei.