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.

Antibiotic Resistance Markers in Burkholderia pseudomallei Strain Bp1651 Identified by Genome Sequence Analysis

“…3). A common feature of CAZ-resistant B. pseudomallei clinical isolates is the combination of the G-to-A mutation in the penA upstream region and mutations affecting PenA structure (16), as was also found in Bp1651.…”

“…This mutation changed a putative weak Ϫ10 sequence (5=-TACGCT-3=) that is present in B. pseudomallei strains such as K96243 and 1026b to 5=-TACACT-3=, a sequence that is closer to consensus (TATAAT). The G-78A SNP has been identified in several CAZ-resistant clinical isolates (16,22). In a laboratory-selected CAZ-resistant Burkholderia thailandensis mutant, this mutation leads to increased penA transcription (18).…”

Exposing a β-Lactamase “Twist”: the Mechanistic Basis for the High Level of Ceftazidime Resistance in the C69F Variant of the Burkholderia pseudomallei PenI β-Lactamase

“…In some parts of the world, melioidosis is characterized by a 20 to 50% mortality rate with treatment, and the death rate increases to Ͼ80% for patients with sepsis (2,3). Resistance to ceftazidime is common in clinical isolates of B. pseudomallei and is linked to increased expression of serine ␤-lactamases (classes A and D) and expression of different isoforms of the class A ␤-lactamase, as well as to deletion of penicillin binding protein 3 (⌬PBP3) (4)(5)(6)(7)(8)(9)(10)(11)(12).…”

Membrane-Bound PenA β-Lactamase of Burkholderia pseudomallei

“…Although still rare, ceftazidime resistance as a result of therapy has been observed (7). Ceftazidime-resistant mutants are the consequence of mutations affecting the expression or amino acid sequence of chromosomally encoded class A PenA ␤-lactamase (8)(9)(10)(11)(12) and deletion of the ceftazidime target penicillin binding protein 3 (PBP3) BPSS1219 (13). However, PenA is the dominant player in ceftazidime resistance.…”

“…While ceftazidime is a poor substrate for wild-type PenA, point mutations causing amino acid substitutions in several of the Ambler domains result in an enzyme bestowing significant ceftazidime resistance. Previously documented amino acid changes implicated in ceftazidime resistance in clinical isolates include C69Y and P167S (8,9,11,12), but other mutations continue to be identified. We previously demonstrated that PenA is secreted by the twin arginine translocation (TAT) system and that the enzyme was located in the spheroplastic fraction rather than the periplasmic fraction (10).…”