Burkholderia pseudomallei Class A β-Lactamase Mutations That Confer Selective Resistance against Ceftazidime or Clavulanic Acid Inhibition

Abstract

Burkholderia pseudomallei, the causative agent of melioidosis, is inherently resistant to a variety of antibiotics including aminoglycosides, macrolides, polymyxins, and ␤-lactam antibiotics. Despite resistance to many ␤-lactams, ceftazidime and ␤-lactamase inhibitor-␤-lactam combinations are commonly used for treatment of melioidosis. Here, we examine the enzyme kinetics of ␤-lactamase isolated from mutants resistant to ceftazidime and clavulanic acid inhibition and describe specific mutations within conserved motifs of the ␤-lactamase enzyme which account for these resistance patterns. Sequence analysis of regions flanking the B. pseudomallei penA gene revealed a putative regulator gene located downstream of penA. We have cloned and sequenced the penA gene from B. mallei and found it to be identical to penA from B. pseudomallei.Burkholderia pseudomallei is the causative agent of melioidosis, an endemic disease of Southeast Asia and Northern Australia (6). The severity of the disease can vary from asymptomatic infection to a severe form leading to acute sepsis and death. B. pseudomallei is a facultative intracellular pathogen which is able to survive inside phagocytic cells and thereby escape the host's humoral response. The disease can be reactivated after a very long remission (3,5,12). Currently, prolonged antibiotic treatment is advised to ensure complete eradication of the organism. Unfortunately, this practice creates a strong positive selection for antibiotic resistant strains resulting in many cases of treatment failure. Many reports have described successful treatment using a combination of ␤-lactam antibiotics and a ␤-lactamase inhibitor, such as amoxicillin plus clavulanic acid (19). Livermore, et al. described a clavulanic acid-inhibitable ␤-lactam resistance phenotype of B. pseudomallei (13), and recently, the cloning of B. pseudomallei class A and D ␤-lactamases has been reported (4, 14). Godfrey et al. described three different phenotypes of clinical isolates from three patients which had undergone antibiotic treatment, and demonstrated that the resistance was due to derepressed ␤-lactamase production and structural mutations in the enzyme (10). Here, we examine the B. pseudomallei penA gene encoding a class A ␤-lactamase in the clinical isolates of B. pseudomallei described by Godfrey et al. and from B. mallei ATCC 23344. We have identified point mutations in two of the isolates which likely account for their altered phenotypes. Finally, the enzyme kinetics of these mutants were compared to the wild type enzyme. MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains and plasmid used in this study are shown in Table 1. B. pseudomallei strains used in this study were collected from blood and urine samples from melioidosis patients both before and during antibiotic treatment at Sappasitprasong Hospital, Ubon Ratchatani, Thailand, between 1986 and 1989 and have been described previously (7, 10). All bacterial strains were grown at 37°C on Luria-Bertani (LB) agar or in LB broth. M...

Melioidosis: Epidemiology, Pathophysiology, and Management

“…Acquired resistance to beta-lactam antibiotics while on treatment with a beta-lactam-beta-lactamase inhibitor combination or ceftazidime resulted from three distinct phenotypic changes: derepression of the chromosomal enzyme, an insensitivity to inhibition by beta-lactamase inhibitors, and a betalactamase specific for ceftazidime (169). These were associated with mutations in the blaA gene (439). Overexpression of the class D beta-lactamases, OXA-42 and OXA-43, may also be responsible for ceftazidime resistance in some isolates (316).…”

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

“…3). The S72F change is the likely cause of AMC resistance of Bp1651 because it is present in other AMC-resistant clinical isolates, and genetic and biochemical evidence with purified protein showed its involvement in AMC resistance (14,45). T147A amino acid substitution that increased AMC and IPM resistance in Bp1651 is located between the conserved 130 SDN 132 loop and the Omega loop (amino acids 164 to 179) (Fig.…”

“…The protein encoded by this gene is actually classified as an extended-spectrum ␤-lactamase (ESBL), PenI (45). Several point mutations within penA have been implicated in conferring resistance to ␤-lactam antibiotics in B. pseudomallei (13,14). Mutations that result in the amino acid changes C69Y and P167S, known to confer CAZ resistance, were not present in the Bp1651 penA (TR70_6344) sequence (Fig.…”

A Structure-Based Classification of Class A β-Lactamases, a Broadly Diverse Family of Enzymes

“…Substitutions involving other residues can lead to cephalosporin resistance (161)(162)(163)(164). For example, Pro167 is a classically variable residue identified in ceftazidimeresistant isolates of B. pseudomallei and in in vitro mutants.…”

“…Twelve positions displaying single-amino-acid substitutions are located in the active-site pocket, such as Cys69Phe and Cys69Tyr, or in the ⍀-loop (positions 162,164,166,169,170,171,172,174,176, and 179 but not position 167). Interestingly, a single-amino-acid deletion (Glu168del) may expand the in vitro spectrum of inactivation to ceftazidime in B. thailandensis (165).…”