Burkholderia pseudomallei Isolates from Sarawak, Malaysian Borneo, Are Predominantly Susceptible to Aminoglycosides and Macrolides

Abstract

h Melioidosis is a potentially fatal disease caused by the saprophytic bacterium Burkholderia pseudomallei. Resistance to gentamicin is generally a hallmark of B. pseudomallei, and gentamicin is a selective agent in media used for diagnosis of melioidosis. In this study, we determined the prevalence and mechanism of gentamicin susceptibility found in B. pseudomallei isolates from Sarawak, Malaysian Borneo. We performed multilocus sequence typing and antibiotic susceptibility testing on 44 B. pseudomallei clinical isolates from melioidosis patients in Sarawak district hospitals. Whole-genome sequencing was used to identify the mechanism of gentamicin susceptibility. A novel allelic-specific PCR was designed to differentiate gentamicin-sensitive isolates from wild-type B. pseudomallei. A reversion assay was performed to confirm the involvement of this mechanism in gentamicin susceptibility. A substantial proportion (86%) of B. pseudomallei clinical isolates in Sarawak, Malaysian Borneo, were found to be susceptible to the aminoglycoside gentamicin, a rare occurrence in other regions where B. pseudomallei is endemic. Gentamicin sensitivity was restricted to genetically related strains belonging to sequence type 881 or its single-locus variant, sequence type 997. Whole-genome sequencing identified a novel nonsynonymous mutation within amrB, encoding an essential component of the AmrAB-OprA multidrug efflux pump. We confirmed the role of this mutation in conferring aminoglycoside and macrolide sensitivity by reversion of this mutation to the wild-type sequence. Our study demonstrates that alternative B. pseudomallei selective media without gentamicin are needed for accurate melioidosis laboratory diagnosis in Sarawak. This finding may also have implications for environmental sampling of other locations to test for B. pseudomallei endemicity.

Loss of Methyltransferase Function and Increased Efflux Activity Leads to Doxycycline Resistance in Burkholderia pseudomallei

“…This antibiotic is a known substrate of AmrAB-OprA (15,25,35). Curiously, in the closely related species Burkholderia thailandensis, overexpression of AmrAB-OprA can lead to DOX MICs up to 32 g/ml (36), which was not observed in this study.…”

“…Unlike the BPSL3085 knockouts, removal of amrR did not detectably alter the DOX MIC in either strain (Table 2). To determine the effect of the Ser174Pro protein change on AmrR activity and verify functional upregulation AmrAB-OprA, we measured the MIC of gentamicin, a known substrate for this efflux pump (15,25). For MSHR0293 ΔamrR, the gentamicin MIC increased 8-fold (from 96 to 768 g/ml).…”

Antibiotic Resistance Markers in Burkholderia pseudomallei Strain Bp1651 Identified by Genome Sequence Analysis

“…This failure is likely due to the deletions of nucleotides G760 and C761 in amrB, resulting in the nonfunctional efflux pump AmrAB-OprA and susceptibility of Bp1651 to GEN. A majority of clinical isolates of B. pseudomallei found in Sarawak, Malaysian Borneo, are also GEN susceptible (67). Some of this susceptibility is attributed to a nonsynonymous SNP, C1102G, within amrB (67). We previously showed that the GEN-susceptible phenotypes of GEN-susceptible Thai isolates were due to either deletion of the amrAB-oprA operon or regulatory mutations affecting its expression (42).…”

“…This failure is likely due to the deletions of nucleotides G760 and C761 in amrB, resulting in the nonfunctional efflux pump AmrAB-OprA and susceptibility of Bp1651 to GEN. A majority of clinical isolates of B. pseudomallei found in Sarawak, Malaysian Borneo, are also GEN susceptible (67). Some of this susceptibility is attributed to a nonsynonymous SNP, C1102G, within amrB (67).…”

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

“…0.5 to 2 g/ml) were isolated from both clinical and environmental samples and contained a point mutation in amrB that inactivated AmrAB-OprA. The reversion of this mutation via in vitro exposure of the susceptible strains to gentamicin and kanamycin (40 and 30 g/ml, respectively) was able to restore aminoglycoside and macrolide resistance (625), highlighting the clinical concerns on current gentamicin use (e.g., 4 g/ml) in medium to isolate B. pseudomallei (626). In B. thailandensis, multidrug-resistant mutants selected by doxycycline overproduced AmrAB-OprA and BpeEF-OprC.…”