title: 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 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.
authors.affiliation: Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, Arizona, United States of America.
authors.forename: Derek S
authors.initials: DS
authors.lastname: Sarovich
authors.forename: Erin P
authors.initials: EP
authors.lastname: Price
authors.forename: Alex T
authors.initials: AT
authors.lastname: Von Schulze
authors.forename: James M
authors.initials: JM
authors.lastname: Cook
authors.forename: Mark
authors.initials: M
authors.lastname: Mayo
authors.forename: Lindsey M
authors.initials: LM
authors.lastname: Watson
authors.forename: Leisha
authors.initials: L
authors.lastname: Richardson
authors.forename: Meagan L
authors.initials: ML
authors.lastname: Seymour
authors.forename: Apichai
authors.initials: A
authors.lastname: Tuanyok
authors.forename: David M
authors.initials: DM
authors.lastname: Engelthaler
authors.forename: Talima
authors.initials: T
authors.lastname: Pearson
authors.forename: Sharon J
authors.initials: SJ
authors.lastname: Peacock
authors.forename: Bart J
authors.initials: BJ
authors.lastname: Currie
authors.forename: Paul
authors.initials: P
authors.lastname: Keim
authors.forename: David M
authors.initials: DM
authors.lastname: Wagner
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology / Q000627:therapeutic use; D001315:Australia; D016957:Burkholderia pseudomallei / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification*; D002442:Ceftazidime / Q000494:pharmacology* / Q000627:therapeutic use; D024881:Drug Resistance, Bacterial / Q000187:drug effects*; D055786:Gene Knockout Techniques; D005798:Genes, Bacterial / Q000235:genetics; D005816:Genetic Complementation Test; D006801:Humans; D008554:Melioidosis / Q000188:drug therapy / Q000382:microbiology; D008826:Microbial Sensitivity Tests; D009154:Mutation / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D000900:Anti-Bacterial Agents; D002442:Ceftazidime