Development of ceftazidime resistance in an acute Burkholderia pseudomallei infection

Abstract

Burkholderia pseudomallei, a bacterium that causes the disease melioidosis, is intrinsically resistant to many antibiotics. First-line antibiotic therapy for treating melioidosis is usually the synthetic β-lactam, ceftazidime (CAZ), as almost all B. pseudomallei strains are susceptible to this drug. However, acquired CAZ resistance can develop in vivo during treatment with CAZ, which can lead to mortality if therapy is not switched to a different drug in a timely manner. Serial B. pseudomallei isolates obtained from an acute Thai melioidosis patient infected by a CAZ susceptible strain, who ultimately succumbed to infection despite being on CAZ therapy for the duration of their infection, were analyzed. Isolates that developed CAZ resistance due to a proline to serine change at position 167 in the β-lactamase PenA were identified. Importantly, these CAZ resistant isolates remained sensitive to the alternative melioidosis treatments; namely, amoxicillin-clavulanate, imipenem, and meropenem. Lastly, real-time polymerase chain reaction-based assays capable of rapidly identifying CAZ resistance in B. pseudomallei isolates at the position 167 mutation site were developed. The ability to rapidly identify the emergence of CAZ resistant B. pseudomallei populations in melioidosis patients will allow timely alterations in treatment strategies, thereby improving patient outcomes for this serious disease.

Keywords: Burkholderia pseudomallei; antibiotic resistance; ceftazidime; melioidosis; penA; β-lactamase.

Figure 1

DNA alignments of penA from patient 45 wild-type and mutant alleles against three other wild-type Burkholderia pseudomallei strains – K96243, 1710b, and 668 – showing a cytosine/thymine nucleotide transition at position 517 (according to penA annotation in B. pseudomallei K96243). Note: This transition results in an amino acid substitution from proline to serine at position 167 in PenA. Abbreviation: P45, patient 45.

Figure 2

SYBR Green mismatch amplification mutation assay interrogation of the proline to serine at position 167 (P167S) PenA mutation, conferred by a cytosine/thymine nucleotide transition in penA at position 517 (according to penA annotation in Burkholderia pseudomallei K96243; see Figure 1) in patient 45 B. pseudomallei isolates. (A) Example amplification of the wild-type P167S penA allele. (B) Example amplification of the mutated P167S penA allele. All mutant P167S B. pseudomallei strains from patient 45 contained an elevated ceftazidime minimum inhibitory concentration of 64 μg/mL compared with the wild-type strains (2 μg/mL). Abbreviation: Wt, wild-type.