Abstract
Burkholderia pseudomallei is a Gram-negative bacterium that causes the serious human disease, melioidosis. There is no vaccine against melioidosis and it can be fatal if not treated with a specific antibiotic regimen, which typically includes the third-generation cephalosporin, ceftazidime (CAZ). There have been several resistance mechanisms described for B. pseudomallei, of which the best described are amino acid changes that alter substrate specificity in the highly conserved class A β-lactamase, PenA. In the current study, we sequenced penA from isolates sequentially derived from two melioidosis patients with wild-type (1.5 µg/mL) and, subsequently, resistant (16 or ≥256 µg/mL) CAZ phenotypes. We identified two single-nucleotide polymorphisms (SNPs) that directly increased CAZ hydrolysis. One SNP caused an amino acid substitution (C69Y) near the active site of PenA, whereas a second novel SNP was found within the penA promoter region. In both instances, the CAZ resistance phenotype corresponded directly with the SNP genotype. Interestingly, these SNPs appeared after infection and under selection from CAZ chemotherapy. Through heterologous cloning and expression, and subsequent allelic exchange in the native bacterium, we confirmed the role of penA in generating both low-level and high-level CAZ resistance in these clinical isolates. Similar to previous studies, the amino acid substitution altered substrate specificity to other β-lactams, suggesting a potential fitness cost associated with this mutation, a finding that could be exploited to improve therapeutic outcomes in patients harboring CAZ resistant B. pseudomallei. Our study is the first to functionally characterize CAZ resistance in clinical isolates of B. pseudomallei and to provide proven and clinically relevant signatures for monitoring the development of antibiotic resistance in this important pathogen.
Highlights
Burkholderia pseudomallei, the etiologic agent of melioidosis, is a saprophytic bacterium that is commonly found in surface waters and soil of Australia and Thailand
Sequencing of penA from Patient 21 (P21) and P337 isolates We sequenced penA of B. pseudomallei isolates obtained from two melioidosis patients (P21 and P337; Table 1; Genbank accession numbers JQ364927 through JQ364935) where CAZ resistance (CAZR) appeared to have developed directly in response to CAZ chemotherapy
The G to A transition corresponded to a 10-fold increase in CAZR (16 mg/mL), suggesting this single-nucleotide polymorphisms (SNPs) is involved in up-regulation of penA expression
Summary
Burkholderia pseudomallei, the etiologic agent of melioidosis, is a saprophytic bacterium that is commonly found in surface waters and soil of Australia and Thailand. There are several different mechanisms of antibiotic resistance in B. pseudomallei, including multi-drug efflux pumps [5,6], enzymatic inactivation [7,8], impermeability of the bacterial cell membrane [9] and mutations in the antibiotic target site [10]. This impressive array of intrinsic resistance and broad-spectrum mechanisms limits the number of treatment options for melioidosis. CAZ is the single most important antibiotic for the treatment of melioidosis
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