Whole-Genome Sequencing and Search for Determinants of Resistance to Benzalkonium Chloride in Burkholderia pseudomallei

Abstract

The aim of the study was to carry out whole-genome sequencing and comparative analysis of the original and benzalkonium chloride-resistant strains of Burkholderia pseudomallei.Materials and methods. We used the strain B. pseudomallei 134 and resistant to benzalkonium chloride B. pseudomallei 134K. Whole-genome sequencing was conducted on the MiSeq Reagent Kit v3 platform (600-cucle).Genome assembly for both strains was performed with the help of SPAdes v3.11.1. In order to compare genome sequences of the studied strains, Snippy v4.6.0 software was applied. MEGA X program was used to align the nucleotide and amino acid sequences.Results and discussion. The search and analysis of determinants responsible for the emergence of resistance to biocides in B. pseudomallei 134K have revealed two genes: the TetR transcriptional repressor gene and the AmrAB-OprA efflux pump gene. A single nucleotide polymorphism has been found in the TetR regulator, which led to the replacement of serine by proline in the mutant protein, and, as a result, a change in its secondary structure. It is believed that this mutation causes the loss of regulatory protein functionality, resulting in an increased expression of the efflux pump genes (AcrB/AcrD/AcrF) regulated by it. This follows by both, decrease in the level of sensitivity to benzalkonium chloride and the emergence of resistance to ceftazidime. In the AmrAB-OprA efflux pump gene, an insertion of 16 nucleotides has been detected at the position 544 of the amrA operon, which led to an increase in the length of the cistron, a shift in the reading frame, a change in the amino acid composition, and, as a result, a change in the secondary structure of the encoded protein. It is most likely that this mutation contributes to the loss of AmrAB-OprA operon function and the failure of normal outflow of xenobiotics from the cytoplasm of the microorganism. This assumption is evidenced by the loss of resistance to gentamicin in the mutant strain.