(210i) Eradication of Bacterial Persister Cells By Targeting Membrane Potential

Bacteria are well known to enter dormancy and form persister cells which is a subpopulation exhibiting high-level tolerance to antibiotics. This feature allows persister cells to survive the treatment of potent antibiotic and cause reoccurring infections. Despite extensive research, controlling persister cells still remains a challenge. Because persister cells have a lower membrane potential than normal cells, we hypothesize that persister cells have reduced efflux activities, which can lead to higher accumulation of antibiotics intracellularly. To test this hypothesis, we compared the concentration of minocycline, a semi-synthetic tetracycline antibiotic, in normal and persister cells of Escherichia coli (E. coli) HM22. We show that with a concentration of 100μg/mL of minocycline we can kill E.coli persister cells by 70.8 ± 5.9% while it only killed normal cells by 10.3 ± 3.7%. In addition, the results show that persister cells accumulated ~4 times more minocycline per cell compared to normal cell. Consistently, the results were corroborated with tests using efflux pump mutants and efflux pump inhibitors. Specifically, the acrAB mutant (of the Resistance Nodulation Division efflux pumps) exhibited an increased susceptibility to minocycline; e.g. minocycline killed the acrA mutant by 99.0 ± 0.1% at a concentration of 100μg/mL. Consistently, treatment with carbonyl cyanide m-chlorophenylhydrazone (CCCP), an efflux pump inhibitor, at 10μM led to 94.7 ± 2.5% killing of E.coli normal cells by minocycline. This is a ~4 times increase compared to treatment without membrane depotentiation which only led to 22.0 ± 3.3% killing. Collectively, these findings reveal that persister formation may cause enhanced penetration for certain antibiotics which can lead to more killing of tolerant population under normal conditions.