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Characterization of ceftazidime resistance mechanisms in clinical isolates of Burkholderia pseudomallei from Australia.

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 ?-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 ?-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.

SUBMITTER: Sarovich DS

PROVIDER: S-EPMC3283585 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Characterization of ceftazidime resistance mechanisms in clinical isolates of Burkholderia pseudomallei from Australia.

Sarovich Derek S DS Price Erin P EP Von Schulze Alex T AT Cook James M JM Mayo Mark M Watson Lindsey M LM Richardson Leisha L Seymour Meagan L ML Tuanyok Apichai A Engelthaler David M DM Pearson Talima T Peacock Sharon J SJ Currie Bart J BJ Keim Paul P Wagner David M DM

PloS one 20120221 2

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-lactama ...[more]

PMID: 22363490

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Project description:Much is known about the mode of action of drugs and resistance mechanisms under laboratory growth conditions, but research on the bacterial transcriptional response to drug pressure in vivo or efficacious mode of action and transient resistance mechanisms of clinically employed drugs is limited. Accordingly, to assess active alternative metabolism and transient resistance mechanisms, and identify molecular markers of treatment response, the in vivo transcriptional response of Burkholderia pseudomallei 1026b to treatment with ceftazidime in infected lungs was compared to the in vitro bacterial response in the presence of drug. There were 1,688 transcriptionally active bacterial genes identified that were unique to in vivo treated conditions. Of the in vivo transcriptionally active bacterial genes, 591 (9.4% coding capacity) genes were differentially expressed by ceftazidime treatment. In contrast, only 186 genes (2.7% coding capacity) were differentially responsive to ceftazidime treatment under in vitro culturing conditions. Within the genes identified were alternative PBP proteins that may compensate for target inactivation and transient resistance mechanisms, such as ?-lactamses that may influence the potency of ceftazidime. This disparate observation is consistent with the thought that the host environment significantly alters the bacterial metabolic response to drug exposure compared to the response observed under in vitro growth. Notably, this study revealed 184 bacterial genes and ORFs that were unique to in vivo ceftazidime treatment and thus provide candidate molecular markers for treatment response. This is the first report of the unique transcriptional response of B. pseudomallei from host tissues in an animal model of infection and elucidates the in vivo metabolic vulnerabilities, which is important in terms of defining the efficacious mode of action and transient resistance mechanisms of a frontline meliodosis chemotherapeutic, and biomarkers for monitoring treatment outcome.

Dataset Information

Characterization of ceftazidime resistance mechanisms in clinical isolates of Burkholderia pseudomallei from Australia.

Publications

Characterization of ceftazidime resistance mechanisms in clinical isolates of Burkholderia pseudomallei from Australia.

Similar Datasets

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