Characterization of ceftazidime resistance mechanisms in clinical isolates of burkholderia pseudomallei from Australia

Derek S. Sarovich, Erin P. Price, Alex T. von Schulze, James M. Cook, Mark Mayo, Lindsey M. Watson, Leisha Richardson, Meagan L. Seymour, Apichai Tuanyok, David M. Engelthaler, Talima R Pearson, Sharon J. Peacock, Bart J. Currie, Paul S Keim, David M Wagner

Research output: Contribution to journalArticle

37 Citations (Scopus)

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.

Original languageEnglish (US)
Article numbere30789
JournalPLoS One
Volume7
Issue number2
DOIs
StatePublished - Feb 21 2012

Fingerprint

Burkholderia pseudomallei
Ceftazidime
resistance mechanisms
Polymorphism
single nucleotide polymorphism
Single Nucleotide Polymorphism
Melioidosis
Nucleotides
amino acid substitution
Amino Acid Substitution
substrate specificity
Substrate Specificity
Amino Acids
Bacteria
Substitution reactions
lactams
Anti-Bacterial Agents
Phenotype
phenotype
Lactams

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Characterization of ceftazidime resistance mechanisms in clinical isolates of burkholderia pseudomallei from Australia. / Sarovich, Derek S.; Price, Erin P.; von Schulze, Alex T.; Cook, James M.; Mayo, Mark; Watson, Lindsey M.; Richardson, Leisha; Seymour, Meagan L.; Tuanyok, Apichai; Engelthaler, David M.; Pearson, Talima R; Peacock, Sharon J.; Currie, Bart J.; Keim, Paul S; Wagner, David M.

In: PLoS One, Vol. 7, No. 2, e30789, 21.02.2012.

Research output: Contribution to journalArticle

Sarovich, DS, Price, EP, von Schulze, AT, Cook, JM, Mayo, M, Watson, LM, Richardson, L, Seymour, ML, Tuanyok, A, Engelthaler, DM, Pearson, TR, Peacock, SJ, Currie, BJ, Keim, PS & Wagner, DM 2012, 'Characterization of ceftazidime resistance mechanisms in clinical isolates of burkholderia pseudomallei from Australia', PLoS One, vol. 7, no. 2, e30789. https://doi.org/10.1371/journal.pone.0030789
Sarovich, Derek S. ; Price, Erin P. ; von Schulze, Alex T. ; Cook, James M. ; Mayo, Mark ; Watson, Lindsey M. ; Richardson, Leisha ; Seymour, Meagan L. ; Tuanyok, Apichai ; Engelthaler, David M. ; Pearson, Talima R ; Peacock, Sharon J. ; Currie, Bart J. ; Keim, Paul S ; Wagner, David M. / Characterization of ceftazidime resistance mechanisms in clinical isolates of burkholderia pseudomallei from Australia. In: PLoS One. 2012 ; Vol. 7, No. 2.
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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.",
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