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...

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Photosystem I-hydrogenase chimera intercepts electron flow directly from the photosynthetic electron transport chain and directs it to hydrogen production.