Antimicrobial resistance to ceftazidime involving loss of penicillin-binding protein 3 in Burkholderia pseudomallei

Abstract

Known mechanisms of resistance to β-lactam antibiotics include β-lactamase expression, altered drug target, decreased bacterial permeability, and increased drug efflux. Here, we describe a unique mechanism of β-lactam resistance in the biothreat organism Burkholderia pseudomallei (the cause of melioidosis), associated with treatment failure during prolonged ceftazidime therapy of natural infection. Detailed comparisons of the initial ceftazidime-susceptible infecting isolate and subsequent ceftazidime-resistant variants from six patients led us to identify a common, large-scale genomic loss involving a minimum of 49 genes in all six resistant strains. Mutational analysis of wild-type B. pseudomallei demonstrated that ceftazidime resistance was due to deletion of a gene encoding a penicillin-binding protein 3 (BPSS1219) present within the region of genomic loss. The clinical ceftazidime-resistant variants failed to grow using commonly used laboratory culture media, including commercial blood cultures, rendering the variants almost undetectable in the diagnostic laboratory. Melioidosis is notoriously difficult to cure and clinical treatment failure is common in patients treated with ceftazidime, the drug of first choice across most of Southeast Asia where the majority of cases are reported. The mechanism described here represents an explanation for ceftazidime treatment failure, and may be a frequent but undetected resistance event.

Fig. 1.

Comparison of the appearance of an initial ceftazidime-susceptible B. pseudomallei strain 415a and the ceftazidime-resistant variant strain 415e isolated from the same patient after prolonged ceftazidime therapy. Colony morphology (A and D), Gram stain and light microscopy (B and E), and unstained appearance by real-time microscopy (C and F) of initial (A–C) and variant strain (D–F). Colony morphology was observed after spread-plating on Ashdown agar and incubation for 4 d at 37 °C in air. Gram stain was observed through a 40× objective. Real-time microscopy was performed using an RTM-3 at 1,000× magnification.

Fig. 2.

Variant ceftazidime-resistant B. pseudomallei strains exhibit a recurrent genomic deletion in chromosome 2. (A) PFGE banding patterns of paired B. pseudomallei strains from six patients in whom the infecting isolate developed secondary ceftazidime resistance during therapy. M represents lambda ladder. (B) Variant B. pseudomallei strains exhibited a common region of genomic loss in chromosome 2. (Upper) Chromosome 2 aCGH (variant/initial) signals for all six B. pseudomallei pairs. A region of deletion was identified as a drop in the aCGH signal. Each variant strain exhibited a regional deletion of chromosome 2 compared with the initial strain. The 71-kb minimal common region (MCR) of deletion is demarcated by red lines. (Lower) Genomic organization of the 49 genes in the 71-kb MCR region. The two PBP 3 homologs (BPSS1219 and BPSS1240) are red, and BPSS1239 (carboxypeptidase) is green. (C) PCR validation of aCGH results. PCR primers designed to amplify a 1-Kb region in the MCR were used to amplify the cognate region in initial and variant strains. M, molecular marker; the 1-kb size range is indicated. Strains are ordered as follows: K9 (BpK96243 reference strain) and the six pairs (initial followed by variant).

Fig. 3.

Deletion of BPSS1219 from B. pseudomallei strain 1026b. (A) Deletion of the BPSS1219 gene from chromosome 2 (Chr 2) was achieved in several steps. In step 1, a mini-Tn7 element containing the BPSS1219 gene flanked by loxP sites and expressed from a B. thailandensis s12 promoter (Ps12) was inserted at the glmS2 attTn7 site on chromosome 1 (Chr 1), resulting in Bp483 (1026b::mini-Tn7-Ps12-BPSS1219⁺ cBPSS1219⁺). In step 2, the resident chromosomal BPSS1219 gene (cBPSS1219) was deleted from Chr 2 using a gene replacement method yielding Bp561(1026b::mini-Tn7-Ps12-BPSS1219⁺ ΔcBPSS1219). In step 3, the BPSS1219 rescue copy was deleted using Cre recombinase-mediated excision, resulting in Bp560 (1026b::mini-Tn7-ΔBPSS1219 ΔcBPSS1219). (B) Growth curves of 1026b (red), Bp561 (magenta), Bp483 (green), and Bp560 (blue) of bacteria in LB medium.