Persister cells in Burkholderia thailandensis

Abstract

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.