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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.

Extended-spectrum β-lactamases (ESBLs) are active against oxyimino cephalosporins and monobactams. Twenty-oneKlebsiella pneumoniaeisolates obtained between 1991 and 1995 at the Princess Alexandra Hospital in Brisbane, Australia, were subject to amplification and sequencing of the SHV β-lactamase-encoding genes. Thirteen strains were phenotypically ESBL positive. Of these, six strains carried theblaSHV-2agene and seven strains carried theblaSHV-12gene. Eight strains were phenotypically ESBL negative. Of these, seven strains carried the non-ESBLblaSHV-11gene and one strain carried the non-ESBLblaSHV-1gene. There was complete correspondence between the ESBL phenotype and the presence or absence of an ESBL-encoding gene(s). In addition, it was determined that of the 13 ESBL-positive strains, at least 4 carried copies of a non-ESBL-encoding gene in addition to theblaSHV-2aorblaSHV12gene. A minisequencing-based assay was developed to discriminate the different SHV classes. This technique, termed “first-nucleotide change,” involves the identification of the base added to a primer in a single-nucleotide extension reaction. The assay targeted polymorphisms at the first bases of codons 238 and 240 and reliably discriminated ESBL-positive strains from ESBL-negative strains and also distinguished strains carryingblaSHV-2afrom strains carryingblaSHV-12. In addition, this method was used to demonstrate an association between the relative copy numbers ofblaSHVgenes in individual strains and the levels of antibiotic resistance.

Klebsiella pneumoniae isolates frequently contain complex mixtures of bla SHV alleles. A high-resolution melting-based method for interrogating the extended-spectrum activity conferring codon 238 and 240 polymorphisms was developed. This detects minority extended-spectrum β-lactamase-encoding alleles, allows estimation of allele ratios, and discriminates between single and double mutants.

In Klebsiella pneumoniae , it is common for plasmid-located and chromosome-located bla SHV copies to coexist within single cells. The plasmid-borne genes are mainly derived from two separate IS 26 -mediated mobilizations of bla SHV . The objective of this study was to test the hypothesis that the presence of a non-extended-spectrum β-lactamase (non-ESBL) encoding plasmid-borne form of bla SHV facilitates the cefotaxime (CTX)-mediated selection of ESBL-expressing mutants, even when there is a chromosomal copy of the same gene. Twenty-one diverse ESBL-negative, bla TEM -negative K. pneumoniae clinical isolates were tested for the IS 26 insertions characteristic of the two mobilization events. The isolates were then tested for their ability to be selected for ESBL-mediated CTX resistance by serial subculturing with a doubling of the CTX concentration at every subculture. Fourteen isolates possessed neither of the IS 26 insertions. None of these became ESBL positive, and all died during the course of the experiment, despite possessing chromosomal bla SHV copies. The other isolates all became ESBL positive and grew abundantly up to a CTX concentration of 128 μg/ml. Similar results were obtained with ceftazidime. ESBL expression was associated with the appearance of the expected G→A mutation at position 1 of codon 238 and also with bla SHV copy number amplification. It was concluded that plasmid-borne bla SHV greatly facilitates the selection of ESBL expression, even when the same gene is on the chromosome, and that gene dosage effects are likely to contribute to this phenomenon.