Persister cells are able to survive in the presence of high concentrations of antibiotic, and re-grow once the antibiotic has been removed. Unlike conventional antibiotic resistance, the antibiotic tolerance of persister cells is due to phenotypic switching, and is non-inherited. There is growing evidence for a role of persisters in various persistent bacterial diseases. Burkholderia pseudomallei is a pathogen which causes melioidosis, which often persists in the host despite antibiotic treatment. As persister cells may contribute to persistent melioidosis, this study investigated persisters in B. thailandensis, as a model for B. pseudomallei. Treatment of B. thailandensis with ceftazidime, ciprofloxacin, imipenem or trimethoprim demonstrated persister cells which survived antibiotic treatment. Persister frequencies were increased in the absence of oxygen, and higher in stationary phase cultures compared with growing cultures. Drug concentration did not affect persister frequencies, and inherited antibiotic resistance was not detected. Different persister fractions were detected using treatment with multiple antibiotics, indicating heterogeneous susceptibility to antibiotics. In order to increase understanding of the molecular basis of B. thailandensis persister cells, a transposon mutagenesis-based sequencing approach was used on persister cultures. This indicated some issues with genome coverage and mutant diversity. Genes were identified from mutants present before and/or after ciprofloxacin treatment. In order to try to eradicate persister cells from a culture, two anti-persister strategies were tested. Itaconate appeared to stimulate growth of B. thailandensis, increasing susceptibility to the antibiotic ceftazidime. However, the overall effect of the combination was no greater than ceftazidime alone in the conditions tested. Metronidazole was effective against a persister culture under anaerobic conditions, suggesting it may be useful in treating anaerobic persisters. Treatment of B. pseudomallei infected mice with metronidazole and ceftazidime did not improve survival over ceftazidime treatment alone.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic in Northern Australia and Southeast Asia. Melioidosis can present with acute, chronic and latent infections and can relapse several months or years after initial presentation. Currently not much is known about the ways in which B. pseudomallei can persist within the host, although it has been speculated that the ability to survive within an anaerobic environment will play some role. B. pseudomallei is able to survive anaerobically for extended periods of time but little is known about the molecular basis of anaerobic respiration in this pathogenic species. Bioinformatic analysis was used to determine the respiratory flexibility of both B. pseudomallei and B. thailandensis, identifying multiple genes required for aerobic and anaerobic respiration, and molybdopterin biosynthesis. Using B. thailandensis as a model organism a transposon mutant library was created in order to identify genes required for anaerobic respiration. From this library one transposon mutant was identified to have disrupted moeA, a gene required for the molybdopterin biosynthetic pathway. This B. thailandensis transposon mutant (CA01) was unable to respire anaerobically on nitrate, exhibiting a significant reduction in nitrate reductase activity, altered motility and biofilm formation, but did not affect virulence in Galleria mellonella. It was hypothesised that the reduction in nitrate reductase activity was contributing to the phenotypes exhibited by the B. thailandensis moeA transposon mutant. To determine whether this was true an in-frame narG deletion mutant was created in B. pseudomallei. Deletion of B. pseudomallei narG (ΔnarG) resulted in a significant reduction in nitrate reductase activity, anaerobic growth, motility and altered persister cell formation, and but did not affect virulence in G. mellonella or intracellular survival within J774A.1 murine macrophages. This study has highlighted the importance of anaerobic respiration in the survival of B. thailandensis and B. pseudomallei.

Burkholderia pseudomallei is an opportunistic soil pathogen that causes melioidosis, a life-threatening human disease prevalent in Southeast Asia, northern Australia, the Middle East, Africa, and South America. The organism also causes disease in plants and animals. Persons with severe melioidosis usually die of septicemia. Relatively little is known regarding the virulence mechanisms of B. pseudomallei; however, several putative virulence determinants have been identified. The organism is able to invade and replicate within phagocytic cells and is particularly pathogenic in males with diabetes mellitus. B. thailandensis is closely related to B. pseudomallei, but is not pathogenic. This study examines various in vitro monocyte / macrophage infection models used to study innate immune responses to B. pseudomallei. Several monocyte and macrophage models showed little or no significant differences between proinflammatory cytokines secreted in response to infection with B. pseudomallei and B. thailandensis. Peripheral blood monocytes from diabetic males produced lower normalized levels of proinflammatory cytokines IL-1α, IL-1β, IL-6, and IL-8 than monocytes from healthy males in response to infection with B. pseudomallei, B. thailandensis, and E. coli. Surprisingly, normalized levels of secreted IL-1β from B. pseudomallei-infected monocytes from diabetic females were higher than levels from healthy females. The results revealed a significant interactive effect of gender and diabetic state on peripheral blood monocyte secretion of IL-1β (p = 0.0370) and IL-8 (p = 0.0390), as well as a significant interactive effect of diabetic state and type of infectious agent on peripheral blood monocyte secretion of IL-1α (p=0.0210) and IL-6 (p=0.0204). These results may help explain why diabetic males are unusually susceptible to infection with B. pseudomallei.