Genomic and RT-qPCR analysis of trimethoprim-sulfamethoxazole and meropenem resistance in Burkholderia pseudomallei clinical isolates

Abstract

Melioidosis is an endemic disease in southeast Asia and northern Australia caused by the saprophytic bacteria Burkholderia pseudomallei, with a high mortality rate. The clinical presentation is multifaceted, with symptoms ranging from acute septicemia to multiple chronic abscesses. Here, we report a chronic case of melioidosis in a patient who lived in Malaysia in the 70s and was suspected of contracting tuberculosis. Approximately 40 years later, in 2014, he was diagnosed with pauci-symptomatic melioidosis during a routine examination. Four strains were isolated from a single sample. They showed divergent morphotypes and divergent antibiotic susceptibility, with some strains showing resistance to trimethoprim-sulfamethoxazole and fluoroquinolones. In 2016, clinical samples were still positive for B. pseudomallei, and only one type of strain, showing atypical resistance to meropenem, was isolated. We performed whole genome sequencing and RT-qPCR analysis on the strains isolated during this study to gain further insights into their differences. We thus identified two types of resistance mechanisms in these clinical strains. The first one was an adaptive and transient mechanism that disappeared during the course of laboratory sub-cultures; the second was a mutation in the efflux pump regulator amrR, associated with the overexpression of the related transporter. The development of such mechanisms may have a clinical impact on antibiotic treatment. Indeed, their transient nature could lead to an undiagnosed resistance. Efflux overexpression due to mutation leads to an important multiple resistance, reducing the effectiveness of antibiotics during treatment. B. pseudomallei is a Gram-negative bacterium that causes melioidosis, a tropical disease. The mortality rate is high, the treatment long and harsh, and the therapeutic arsenal is limited due to the natural resistance of the bacteria to antibiotics. Eleven percent of melioidosis cases are chronic. Here, we studied a chronic melioidosis case in a French male patient who lived in Malaysia in the 70s. B. pseudomallei was identified in 2014 and in a relapse in 2016. Analysis revealed several strains from the same clinical sample with different morphotypes and divergent antibiotic-resistance profiles. Two atypical multidrug resistance profiles were observed for two strains: one possessed multiple resistance to trimethoprim-sulfamethoxazole, fluoroquinolones, and chloramphenicol and the other multiple resistance to fluoroquinolones and meropenem. Trimethoprim-sulfamethoxazole or meropenem resistance have rarely been described in clinical cases and are probably underdiagnosed. Here, we show that trimethoprim-sulfamethoxazole resistance can be transient in clinical strains and easily lost in the laboratory after sub-culture during identification, resulting in an underestimation of trimethoprim-sulfamethoxazole resistance and therapeutic failure. We also identified a mutation in the AmrAB-OprA efflux pump regulator, leading to high level meropenem resistance, but this resistance is also transient.