Molecular Basis of Rare Aminoglycoside Susceptibility and Pathogenesis of Burkholderia pseudomallei Clinical Isolates from Thailand

Abstract

Burkholderia pseudomallei is intrinsically resistant to aminoglycosides and macrolides, mostly due to AmrAB-OprA efflux pump expression. We investigated the molecular mechanisms of aminoglycoside susceptibility exhibited by Thai strains 708a, 2188a, and 3799a. qRT-PCR revealed absence of amrB transcripts in 708a and greatly reduced levels in 2188a and 3799a. Serial passage on increasing gentamicin concentrations yielded 2188a and 3799a mutants that became simultaneously resistant to other aminoglycosides and macrolides, whereas such mutants could not be obtained with 708a. Transcript analysis showed that the resistance of the 2188a and 3799a mutants was due to upregulation of amrAB-oprA expression by unknown mechanism(s). Use of a PCR walking strategy revealed that the amrAB-oprA operon was missing in 708a and that this loss was associated with deletion of more than 70 kb of genetic material. Rescue of the amrAB-oprB region from a 708a fosmid library and sequencing showed the presence of a large chromosome 1 deletion (131 kb and 141 kb compared to strains K96243 and 1710b, respectively). This deletion not only removed the amrAB-oprA operon, but also the entire gene clusters for malleobactin and cobalamin synthesis. Other genes deleted included the anaerobic arginine deiminase pathway, putative type 1 fimbriae and secreted chitinase. Whole genome sequencing and PCR analysis confirmed absence of these genes from 708a. Despite missing several putative virulence genes, 708a was fully virulent in a murine melioidosis model. Strain 708a may be a natural candidate for genetic manipulation experiments that use Select Agent compliant antibiotics for selection and validates the use of laboratory-constructed Δ(amrAB-oprA) mutants in such experiments. Burkholderia pseudomallei is the etiologic agent of melioidosis, an emerging tropical disease. Because of low infectious dose, broad-host-range infectivity, intrinsic antibiotic resistance and historic precedent as a bioweapon, B. pseudomallei was listed in the United States as a Select Agent and Priority Pathogen of biodefense concern by the US Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases. The mechanisms governing antibiotic resistance and/or susceptibility and virulence in this bacterium are not well understood. Most clinical and environmental B. pseudomallei isolates are highly resistant to aminoglycosides, but susceptible variants do exist. The results of our studies with three such variants from Thailand reveal that lack of expression or deletion of an efflux pump is responsible for this susceptibility. The large deletion present in one strain not only removes an efflux pump but also several putative virulence genes, including an entire siderophore gene cluster. Despite this deletion, the strain is fully virulent in an acute mouse melioidosis model. In summary, our findings shed light on mechanisms of antibiotic resistance and pathogenesis. They also validate the previously advocated use of laboratory-constructed, aminoglycoside susceptible efflux pump mutants in genetic manipulation experiments.