Effect of colistin-based antibiotic combinations on the eradication of persister cells in Pseudomonas aeruginosa

Objectives: Persister cells are responsible for antibiotic treatment failure and the emergence of antibiotic resistance. The synergistic lethal effects of antibiotic combinations on persister cells were investigated using Pseudomonas aeruginosa isolates. Methods: Persister assays were performed on P. aeruginosa clinical isolates using colistin, amikacin, ciprofloxacin and cefepime, individually and in combination. ATP concentrations were measured and morphological changes in persister cells were observed using transmission electron microscopy (TEM). The expression of relA, spoT and obg genes was evaluated and persister-cell formation was investigated in a relA and spoT double mutant (Delta relA Delta spoT). Results: The P. aeruginosa persister cells were eradicated upon exposure to the colistin-based antibiotic combination colistin + ciprofloxacin. Simultaneous treatment with both antibiotics, rather than sequential treatment, caused more effective eradication. The intracellular ATP concentration was most reduced in colistin persisters. While the spoT gene was only overexpressed in colistin-persister cells, the relA gene was overexpressed in all persister cells compared with untreated parent cells. TEM analysis suggested the possibility that persister cells might be formed by different mechanisms depending on the antibiotic. Cell elongation and cell wall or membrane damage in colistin persisters, DNA condensation in amikacin persisters and outer membrane vesicles in ciprofloxacin persisters were identified. Conclusions: In P. aeruginosa, the colistin-based antibiotic combination (colistin + ciprofloxacin) was effective for the eradication of persister cells, probably due to the different persister cell-formation mechanisms between the two antibiotics. Simultaneous, rather than sequential, treatment with two antibiotics could be more effective for eradicating persister P. aeruginosa cells.