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.