Express Yourself: Quantitative Real-Time PCR Assays for Rapid Chromosomal Antimicrobial Resistance Detection in Pseudomonas aeruginosa.

The rise of antimicrobial-resistant (AMR) bacteria is a global health emergency. One critical facet of tackling this epidemic is more rapid AMR diagnosis in serious multidrug-resistant pathogens like Pseudomonas aeruginosa. Here, we designed and then validated two multiplex quantitative real-time PCR (qPCR) assays to simultaneously detect differential expression of the resistance-nodulation-division efflux pumps MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM, the AmpC ß-lactamase, and the porin OprD, which are commonly associated with chromosomally encoded AMR. Next, qPCRs were tested on 15 sputa from 11 participants with P. aeruginosa respiratory infections to determine AMR profiles in vivo. We confirmed multiplex qPCR testing feasibility directly on sputa, representing a key advancement in in vivo AMR diagnosis. Notably, comparison of sputa with their derived isolates grown in Luria-Bertani broth (±2.5% NaCl) or a 5-antibiotic cocktail showed marked expression differences, illustrating the difficulty in replicating in vivo expression profiles in vitro. Cystic fibrosis sputa showed significantly reduced mexE and mexY expression compared with chronic obstructive pulmonary disease sputa, despite harboring fluoroquinolone- and aminoglycoside-resistant strains, indicating that these loci do not contribute to AMR in vivo. oprD was also significantly downregulated in cystic fibrosis sputa, even in the absence of contemporaneous carbapenem use, suggesting a common adaptive trait in chronic infections that may affect carbapenem efficacy. Sputum ampC expression was highest in participants receiving carbapenems (6.7 to 15×), some of whom were simultaneously receiving cephalosporins, the latter of which would be rendered ineffective by the upregulated ampC. Our qPCR assays provide valuable insights into the P. aeruginosa resistome, and their use on clinical specimens will permit timely treatment alterations that will improve patient outcomes and antimicrobial stewardship measures.

Pathogen to commensal? Longitudinal within-host population dynamics, evolution, and adaptation during a chronic >16-year Burkholderia pseudomallei infection.

Although acute melioidosis is the most common outcome of Burkholderia pseudomallei infection, we have documented a case, P314, where disease severity lessened with time, and the pathogen evolved towards a commensal relationship with the host. In the current study, we used whole-genome sequencing to monitor this long-term symbiotic relationship to better understand B. pseudomallei persistence in P314's sputum despite intensive initial therapeutic regimens. We collected and sequenced 118 B. pseudomallei isolates from P314's airways over a >16-year period, and also sampled the patient's home environment, recovering six closely related B. pseudomallei isolates from the household water system. Using comparative genomics, we identified 126 SNPs in the core genome of the 124 isolates or 162 SNPs/indels when the accessory genome was included. The core SNPs were used to construct a phylogenetic tree, which demonstrated a close relationship between environmental and clinical isolates and detailed within-host evolutionary patterns. The phylogeny had little homoplasy, consistent with a strictly clonal mode of genetic inheritance. Repeated sampling revealed evidence of genetic diversification, but frequent extinctions left only one successful lineage through the first four years and two lineages after that. Overall, the evolution of this population is nonadaptive and best explained by genetic drift. However, some genetic and phenotypic changes are consistent with in situ adaptation. Using a mouse model, P314 isolates caused greatly reduced morbidity and mortality compared to the environmental isolates. Additionally, potentially adaptive phenotypes emerged and included differences in the O-antigen, capsular polysaccharide, motility, and colony morphology. The >13-year co-existence of two long-lived lineages presents interesting hypotheses that can be tested in future studies to provide additional insights into selective pressures, niche differentiation, and microbial adaptation. This unusual melioidosis case presents a rare example of the evolutionary progression towards commensalism by a highly virulent pathogen within a single human host.

Pharmacodynamic Evaluation of Plasma and Epithelial Lining Fluid Exposures of Amikacin against Pseudomonas aeruginosa in a Dynamic In Vitro Hollow-Fiber Infection Model.

Given that aminoglycosides, such as amikacin, may be used for multidrug-resistant Pseudomonas aeruginosa infections, optimization of therapy is paramount for improved treatment outcomes. This study aims to investigate the pharmacodynamics of different simulated intravenous amikacin doses on susceptible P. aeruginosa to inform ventilator-associated pneumonia (VAP) and sepsis treatment choices. A hollow-fiber infection model with two P. aeruginosa isolates (MICs of 2 and 8 mg/liter) with an initial inoculum of ∼108 CFU/ml was used to test different amikacin dosing regimens. Three regimens (15, 25, and 50 mg/kg) were tested to simulate a blood exposure, while a 30 mg/kg regimen simulated the epithelial lining fluid (ELF) for potential respiratory tract infection. Data were described using a semimechanistic pharmacokinetic/pharmacodynamic (PK/PD) model. Whole-genome sequencing was used to identify mutations associated with resistance emergence. While bacterial density was reduced by >6 logs within the first 12 h in simulated blood exposures following this initial bacterial kill, there was amplification of a resistant subpopulation with ribosomal mutations that were likely mediating amikacin resistance. No appreciable bacterial killing occurred with subsequent doses. There was less (<5 log) bacterial killing in the simulated ELF exposure for either isolate tested. Simulation studies suggested that a dose of 30 and 50 mg/kg may provide maximal bacterial killing for bloodstream and VAP infections, respectively. Our results suggest that amikacin efficacy may be improved with the use of high-dose therapy to rapidly eliminate susceptible bacteria. Subsequent doses may have reduced efficacy given the rapid amplification of less-susceptible bacterial subpopulations with amikacin monotherapy.

Comparative Genomics and Antimicrobial Resistance Profiling of Elizabethkingia Isolates Reveal Nosocomial Transmission and In Vitro Susceptibility to Fluoroquinolones, Tetracyclines, and Trimethoprim-Sulfamethoxazole.

The Elizabethkingia genus has gained global attention in recent years as containing sporadic, worldwide, nosocomial pathogens. Elizabethkingia spp. are intrinsically multidrug resistant, primarily infect immunocompromised individuals, and are associated with high mortality (∼20 to 40%). As yet, gaps remain in our understanding of transmission, global strain relatedness, antimicrobial resistance, and effective therapy. Over a 16-year period, 22 clinical and 6 hospital environmental isolates were collected from Queensland, Australia. Identification using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Vitek MS) and whole-genome sequencing was compared with a global strain data set. Phylogenomic reconstruction robustly identified 22 Elizabethkingia anophelis, 3 Elizabethkingia miricola, 2 Elizabethkingia meningoseptica, and 1 Elizabethkingia bruuniana isolates, most of which branched as unique lineages. Global analysis revealed that some Australian E. anophelis isolates are genetically closely related to strains from the United States, England, and Asia. Comparative genomics of clinical and environmental strains identified evidence of nosocomial transmission in patients, indicating probable infection from a hospital reservoir. Furthermore, broth microdilution against 39 antimicrobials revealed almost ubiquitous resistance to aminoglycosides, carbapenems, cephalosporins, and penicillins. Like other international strains, our isolates expressed susceptibility to minocycline and levofloxacin and the less common trimethoprim-sulfamethoxazole. Our study demonstrates important new insights into the genetic diversity, environmental persistence, and transmission of and potential effective therapy for Australian Elizabethkingia species.

Peptidyl-Prolyl Isomerase ppiB Is Essential for Proteome Homeostasis and Virulence in Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic to Southeast Asia and northern Australia. Mortality rates in these areas are high even with antimicrobial treatment, and there are few options for effective therapy. Therefore, there is a need to identify antibacterial targets for the development of novel treatments. Cyclophilins are a family of highly conserved enzymes important in multiple cellular processes. Cyclophilins catalyze the cis-trans isomerization of xaa-proline bonds, a rate-limiting step in protein folding which has been shown to be important for bacterial virulence. B. pseudomallei carries a putative cyclophilin B gene, ppiB, the role of which was investigated. A B. pseudomalleiΔppiB (BpsΔppiB) mutant strain demonstrates impaired biofilm formation and reduced motility. Macrophage invasion and survival assays showed that although the BpsΔppiB strain retained the ability to infect macrophages, it had reduced survival and lacked the ability to spread cell to cell, indicating ppiB is essential for B. pseudomallei virulence. This is reflected in the BALB/c mouse infection model, demonstrating the requirement of ppiB for in vivo disease dissemination and progression. Proteomic analysis demonstrates that the loss of PpiB leads to pleiotropic effects, supporting the role of PpiB in maintaining proteome homeostasis. The loss of PpiB leads to decreased abundance of multiple virulence determinants, including flagellar machinery and alterations in type VI secretion system proteins. In addition, the loss of ppiB leads to increased sensitivity toward multiple antibiotics, including meropenem and doxycycline, highlighting ppiB inhibition as a promising antivirulence target to both treat B. pseudomallei infections and increase antibiotic efficacy.

Virulence of the Melioidosis Pathogen Burkholderia pseudomallei Requires the Oxidoreductase Membrane Protein DsbB.

The naturally antibiotic-resistant bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a disease with stubbornly high mortality and a complex, protracted treatment regimen. The worldwide incidence of melioidosis is likely grossly underreported, though it is known to be highly endemic in northern Australia and Southeast Asia. Bacterial disulfide bond (DSB) proteins catalyze the oxidative folding and isomerization of disulfide bonds in substrate proteins. In the present study, we demonstrate that B. pseudomallei membrane protein disulfide bond protein B (BpsDsbB) forms a functional redox relay with the previously characterized virulence mediator B. pseudomallei disulfide bond protein A (BpsDsbA). Genomic analysis of diverse B. pseudomallei clinical isolates demonstrated that dsbB is a highly conserved core gene. Critically, we show that DsbB is required for virulence in B. pseudomallei A panel of B. pseudomalleidsbB deletion strains (K96243, 576, MSHR2511, MSHR0305b, and MSHR5858) were phenotypically diverse according to the results of in vitro assays that assess hallmarks of virulence. Irrespective of their in vitro virulence phenotypes, two deletion strains were attenuated in a BALB/c mouse model of infection. A crystal structure of a DsbB-derived peptide complexed with BpsDsbA provides the first molecular characterization of their interaction. This work contributes to our broader understanding of DSB redox biology and will support the design of antimicrobial drugs active against this important family of bacterial virulence targets.

Raising the Stakes: Loss of Efflux Pump Regulation Decreases Meropenem Susceptibility in Burkholderia pseudomallei.

Background: Burkholderia pseudomallei, the causative agent of the high-mortality disease melioidosis, is a gram-negative bacterium that is naturally resistant to many antibiotics. There is no vaccine for melioidosis, and effective eradication is reliant on biphasic and prolonged antibiotic administration. The carbapenem drug meropenem is the current gold standard option for treating severe melioidosis. Intrinsic B. pseudomallei resistance toward meropenem has not yet been documented; however, resistance could conceivably develop over the course of infection, leading to prolonged sepsis and treatment failure. Methods: We examined our 30-year clinical collection of melioidosis cases to identify B. pseudomallei isolates with reduced meropenem susceptibility. Isolates were subjected to minimum inhibitory concentration (MIC) testing toward meropenem. Paired isolates from patients who had evolved decreased susceptibility were subjected to whole-genome sequencing. Select agent-compliant genetic manipulation was carried out to confirm the molecular mechanisms conferring resistance. Results: We identified 11 melioidosis cases where B. pseudomallei isolates developed decreased susceptibility toward meropenem during treatment, including 2 cases not treated with this antibiotic. Meropenem MICs increased from 0.5-0.75 µg/mL to 3-8 µg/mL. Comparative genomics identified multiple mutations affecting multidrug resistance-nodulation-division (RND) efflux pump regulators, with concomitant overexpression of their corresponding pumps. All cases were refractory to treatment despite aggressive, targeted therapy, and 2 were associated with a fatal outcome. Conclusions: This study confirms the role of RND efflux pumps in decreased meropenem susceptibility in B. pseudomallei. These findings have important ramifications for the diagnosis, treatment, and management of life-threatening melioidosis cases.

Staphylococcus aureus from patients with chronic rhinosinusitis show minimal genetic association between polyp and non-polyp phenotypes.

BACKGROUND: Staphylococcus aureus has a high prevalence in chronic rhinosinusitis (CRS) patients and is suggested to play a more etiopathogenic role in CRS patients with nasal polyps (CRSwNP), a severe form of the CRS spectrum with poorer surgical outcomes. We performed a microbial genome-wide association study (mGWAS) to investigate whether S. aureus isolates from CRS patients have particular genetic markers associated with CRS with nasal polyps (CRSwNP) or CRS without nasal polyps (CRSsNP). METHODS: Whole genome sequencing was performed on S. aureus isolates collected from 28 CRSsNP and 30 CRSwNP patients. A mGWAS approach was employed using large-scale comparative genomics to identify genetic variation within our dataset. RESULTS: Considerable genetic variation was observed, with > 90,000 single nucleotide polymorphisms (SNPs) sites identified. There was little correlation with CRS subtype based on SNPs and Insertion/Delection (Indels). One indel was found to significantly correlate with CRSwNP and occurred in the promoter region of a bacitracin transport system ATP-binding protein. Additionally, two variants of the highly variable superantigen-like (SSL) proteins were found to significantly correlate with each CRS phenotype. No significant association with other virulence or antibiotic resistance genes were observed, consistent with previous studies. CONCLUSION: To our knowledge this study is the first to use mGWAS to investigate the contribution of microbial genetic variation to CRS presentations. Utilising the most comprehensive genome-wide analysis methods available, our results suggest that CRS phenotype may be influenced by genetic factors other than specific virulence mechanisms within the S. aureus genome.

Increased Neurotropic Threat from Burkholderia pseudomallei Strains with a B. mallei-like Variation in the bimA Motility Gene, Australia.

Neurologic melioidosis is a serious, potentially fatal form of Burkholderia pseudomallei infection. Recently, we reported that a subset of clinical isolates of B. pseudomallei from Australia have heightened virulence and potential for dissemination to the central nervous system. In this study, we demonstrate that this subset has a B. mallei-like sequence variation of the actin-based motility gene, bimA. Compared with B. pseudomallei isolates having typical bimA alleles, isolates that contain the B. mallei-like variation demonstrate increased persistence in phagocytic cells and increased virulence with rapid systemic dissemination and replication within multiple tissues, including the brain and spinal cord, in an experimental model. These findings highlight the implications of bimA variation on disease progression of B. pseudomallei infection and have considerable clinical and public health implications with respect to the degree of neurotropic threat posed to human health.

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

The soil-dwelling bacterium Burkholderia pseudomallei is the causative agent of the potentially fatal disease melioidosis. The lack of a vaccine toward B. pseudomallei means that melioidosis treatment relies on prolonged antibiotic therapy, which can last up to 6 months in duration or longer. Due to intrinsic resistance, few antibiotics are effective against B. pseudomallei The lengthy treatment regimen required increases the likelihood of resistance development, with subsequent potentially fatal relapse. Doxycycline (DOX) has historically played an important role in the eradication phase of melioidosis treatment. Both primary and acquired DOX resistances have been documented in B. pseudomallei; however, the molecular mechanisms underpinning DOX resistance have remained elusive. Here, we identify and functionally characterize the molecular mechanisms conferring acquired DOX resistance in an isogenic B. pseudomallei pair. Two synergistic mechanisms were identified. The first mutation occurred in a putative S-adenosyl-l-methionine-dependent methyltransferase (encoded by BPSL3085), which we propose leads to altered ribosomal methylation, thereby decreasing DOX binding efficiency. The second mutation altered the function of the efflux pump repressor gene, amrR, resulting in increased expression of the resistance-nodulation-division efflux pump, AmrAB-OprA. Our findings highlight the diverse mechanisms by which B. pseudomallei can become resistant to antibiotics used in melioidosis therapy and the need for resistance monitoring during treatment regimens, especially in patients with prolonged or recrudesced positive cultures for B. pseudomallei.

Unprecedented Melioidosis Cases in Northern Australia Caused by an Asian Burkholderia pseudomallei Strain Identified by Using Large-Scale Comparative Genomics.

Melioidosis is a disease of humans and animals that is caused by the saprophytic bacterium Burkholderia pseudomallei. Once thought to be confined to certain locations, the known presence of B. pseudomallei is expanding as more regions of endemicity are uncovered. There is no vaccine for melioidosis, and even with antibiotic administration, the mortality rate is as high as 40% in some regions that are endemic for the infection. Despite high levels of recombination, phylogenetic reconstruction of B. pseudomallei populations using whole-genome sequencing (WGS) has revealed surprisingly robust biogeographic separation between isolates from Australia and Asia. To date, there have been no confirmed autochthonous melioidosis cases in Australia caused by an Asian isolate; likewise, no autochthonous cases in Asia have been identified as Australian in origin. Here, we used comparative genomic analysis of 455 B. pseudomallei genomes to confirm the unprecedented presence of an Asian clone, sequence type 562 (ST-562), in Darwin, northern Australia. First observed in Darwin in 2005, the incidence of melioidosis cases attributable to ST-562 infection has steadily risen, and it is now a common strain in Darwin. Intriguingly, the Australian ST-562 appears to be geographically restricted to a single locale and is genetically less diverse than other common STs from this region, indicating a recent introduction of this clone into northern Australia. Detailed genomic and epidemiological investigations of new clinical and environmental B. pseudomallei isolates in the Darwin region and ST-562 isolates from Asia will be critical for understanding the origin, distribution, and dissemination of this emerging clone in northern Australia.

Haemophilus influenzae: using comparative genomics to accurately identify a highly recombinogenic human pathogen.

BACKGROUND: Haemophilus influenzae is an opportunistic bacterial pathogen that exclusively colonises humans and is associated with both acute and chronic disease. Despite its clinical significance, accurate identification of H. influenzae is a non-trivial endeavour. H. haemolyticus can be misidentified as H. influenzae from clinical specimens using selective culturing methods, reflecting both the shared environmental niche and phenotypic similarities of these species. On the molecular level, frequent genetic exchange amongst Haemophilus spp. has confounded accurate identification of H. influenzae, leading to both false-positive and false-negative results with existing speciation assays. RESULTS: Whole-genome single-nucleotide polymorphism data from 246 closely related global Haemophilus isolates, including 107 Australian isolate genomes generated in this study, were used to construct a whole-genome phylogeny. Based on this phylogeny, H. influenzae could be differentiated from closely related species. Next, a H. influenzae-specific locus, fucP, was identified, and a novel TaqMan real-time PCR assay targeting fucP was designed. PCR specificity screening across a panel of clinically relevant species, coupled with in silico analysis of all species within the order Pasteurellales, demonstrated that the fucP assay was 100 % specific for H. influenzae; all other examined species failed to amplify. CONCLUSIONS: This study is the first of its kind to use large-scale comparative genomic analysis of Haemophilus spp. to accurately delineate H. influenzae and to identify a species-specific molecular signature for this species. The fucP assay outperforms existing H. influenzae targets, most of which were identified prior to the next-generation genomics era and thus lack validation across a large number of Haemophilus spp. We recommend use of the fucP assay in clinical and research laboratories for the most accurate detection and diagnosis of H. influenzae infection and colonisation.

Single-molecule sequencing reveals the molecular basis of multidrug-resistance in ST772 methicillin-resistant Staphylococcus aureus.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-associated infection, but there is growing awareness of the emergence of multidrug-resistant lineages in community settings around the world. One such lineage is ST772-MRSA-V, which has disseminated globally and is increasingly prevalent in India. Here, we present the complete genome sequence of DAR4145, a strain of the ST772-MRSA-V lineage from India, and investigate its genomic characteristics in regards to antibiotic resistance and virulence factors. RESULTS: Sequencing using single-molecule real-time technology resulted in the assembly of a single continuous chromosomal sequence, which was error-corrected, annotated and compared to nine draft genome assemblies of ST772-MRSA-V from Australia, Malaysia and India. We discovered numerous and redundant resistance genes associated with mobile genetic elements (MGEs) and known core genome mutations that explain the highly antibiotic resistant phenotype of DAR4145. Staphylococcal toxins and superantigens, including the leukotoxin Panton-Valentinin Leukocidin, were predominantly associated with genomic islands and the phage φ-IND772PVL. Some of these mobile resistance and virulence factors were variably present in other strains of the ST772-MRSA-V lineage. CONCLUSIONS: The genomic characteristics presented here emphasize the contribution of MGEs to the emergence of multidrug-resistant and highly virulent strains of community-associated MRSA. Antibiotic resistance was further augmented by chromosomal mutations and redundancy of resistance genes. The complete genome of DAR4145 provides a valuable resource for future investigations into the global dissemination and phylogeography of ST772-MRSA-V.

Use of Whole-Genome Sequencing to Link Burkholderia pseudomallei from Air Sampling to Mediastinal Melioidosis, Australia.

The frequency with which melioidosis results from inhalation rather than percutaneous inoculation or ingestion is unknown. We recovered Burkholderia pseudomallei from air samples at the residence of a patient with presumptive inhalational melioidosis and used whole-genome sequencing to link the environmental bacteria to B. pseudomallei recovered from the patient.

Endemic melioidosis in residents of desert region after atypically intense rainfall in central Australia, 2011.

After heavy rains and flooding during early 2011 in the normally arid interior of Australia, melioidosis was diagnosed in 6 persons over a 4-month period. Although the precise global distribution of the causal bacterium Burkholderia pseudomallei remains to be determined, this organism can clearly survive in harsh and even desert environments outside the wet tropics.

Tracing melioidosis back to the source: using whole-genome sequencing to investigate an outbreak originating from a contaminated domestic water supply.

Melioidosis, a disease of public health importance in Southeast Asia and northern Australia, is caused by the Gram-negative soil bacillus Burkholderia pseudomallei. Melioidosis is typically acquired through environmental exposure, and case clusters are rare, even in regions where the disease is endemic. B. pseudomallei is classed as a tier 1 select agent by the Centers for Disease Control and Prevention; from a biodefense perspective, source attribution is vital in an outbreak scenario to rule out a deliberate release. Two cases of melioidosis within a 3-month period at a residence in rural northern Australia prompted an investigation to determine the source of exposure. B. pseudomallei isolates from the property's groundwater supply matched the multilocus sequence type of the clinical isolates. Whole-genome sequencing confirmed the water supply as the probable source of infection in both cases, with the clinical isolates differing from the likely infecting environmental strain by just one single nucleotide polymorphism (SNP) each. For the first time, we report a phylogenetic analysis of genomewide insertion/deletion (indel) data, an approach conventionally viewed as problematic due to high mutation rates and homoplasy. Our whole-genome indel analysis was concordant with the SNP phylogeny, and these two combined data sets provided greater resolution and a better fit with our epidemiological chronology of events. Collectively, this investigation represents a highly accurate account of source attribution in a melioidosis outbreak and gives further insight into a frequently overlooked reservoir of B. pseudomallei. Our methods and findings have important implications for outbreak source tracing of this bacterium and other highly recombinogenic pathogens.

Whole-genome sequencing of Burkholderia pseudomallei isolates from an unusual melioidosis case identifies a polyclonal infection with the same multilocus sequence type.

Twelve Burkholderia pseudomallei isolates collected over a 32-month period from a patient with chronic melioidosis demonstrated identical multilocus sequence types (STs). However, whole-genome sequencing suggests a polyclonal infection. This study is the first to report a mixed infection with the same ST.

Whole-genome sequencing confirms that Burkholderia pseudomallei multilocus sequence types common to both Cambodia and Australia are due to homoplasy.

Burkholderia pseudomallei isolates with shared multilocus sequence types (STs) have not been isolated from different continents. We identified two STs shared between Australia and Cambodia. Whole-genome analysis revealed substantial diversity within STs, correctly identified the Asian or Australian origin, and confirmed that these shared STs were due to homoplasy.

Autochthonous melioidosis in humans, Madagascar, 2012 and 2013.

Melioidosis is an often fatal infectious disease affecting humans and animals in the tropics. Only sporadic cases have been reported from Africa and the Indian Ocean region. We describe 2 confirmed autochthonous cases of human melioidosis in Madagascar, both from novel genotypes of Burkholderia pseudomallei.

Burkholderia pseudomallei isolates from Sarawak, Malaysian Borneo, are predominantly susceptible to aminoglycosides and macrolides.

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.