Melioidosis is caused by Burkholderia pseudomallei, a Gram-negative bacterium found in soil and water in tropical regions predominantly in Southeast Asia and northern Australia. Treating melioidosis is complicated because the pathogen is resistant to most antibiotics used in the initial empirical management of sepsis, including ceftazidime. Ceftazidime (CAZ) is the primary intravenous (IV) drug of choice recommended for treating melioidosis. CAZ is a third-generation cephalosporin antibiotic with bactericidal activity. The primary goal of this project was to identify genetic and molecular basis of CAZ resistance mechanisms in a large collection of B. pseudomallei collected from a longitudinal cohort study in Thailand. This study has demonstrated that mutations of penA, a class A β-lactamase gene, can potentially cause CAZ resistance. Based on the findings from patients with treatment failures in Thailand during 1987-2007, our study has suggested strong associations between the increased minimal inhibitory concentrations of CAZ and multiple amino acid substitution (AAS) mutations in PenA and the multiplication of penA, which is one of the most common mechanisms that B. pseudomallei uses to survive the antibiotic treatment. We are currently developing real-time PCR assays to detect these mutations in clinical settings. This project will not only empower clinicians with innovative tools to monitor drug susceptibility for B. pseudomallei in real-time, but also has a significant impact on alteration of treatment(s) for melioidosis.