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Persister cells are a small subpopulation of non-growing bacteria within a population that can survive long exposures to antibiotic treatment. Following antibiotic removal, persister cells can regrow and populate, playing a key role in the chronic reoccurrence of bacterial infections. The development of new molecules and methods to kill bacterial persisters is critical. Essential oils and other natural products have long been studied for their antimicrobial effects. Here, we studied the effectiveness of tea tree essential oil (TTO), a common component in many commercial care products, against Escherichia coli and Staphylococcus epidermidis persister cells. Using biphasic kill curve assays, we found that concentrations of 0.5% and 1.0% TTO for E. coli and S. epidermidis , respectively, completely eradicated persister cells over a period of 24 h, with the component terpinen-4-ol responsible for most of the killing. Using a colorimetric assay, it was determined that the TTO exhibited its anti-persister effects through a membrane disruption mechanism.

Nitric oxide (NO) is a chemical weapon within the arsenal of immune cells, but is also generated endogenously by different bacteria. Pseudomonas aeruginosa are pathogens that contain an NO-generating nitrite (NO 2 - ) reductase (NirS), and NO has been shown to influence their virulence. Interestingly, P. aeruginosa also contain NO dioxygenase (Fhp) and nitrate (NO 3 - ) reductases, which together with NirS provide the potential for NO to be metabolically cycled (NO→NO 3 - →NO 2 - →NO). Deeper understanding of NO metabolism in P. aeruginosa will increase knowledge of its pathogenesis, and computational models have proven to be useful tools for the quantitative dissection of NO biochemical networks. Here we developed such a model for P. aeruginosa and confirmed its predictive accuracy with measurements of NO, O 2 , NO 2 - , and NO 3 - in mutant cultures devoid of Fhp or NorCB (NO reductase) activity. Using the model, we assessed whether NO was metabolically cycled in aerobic P. aeruginosa cultures. Calculated fluxes indicated a bottleneck at NO 3 - , which was relieved upon O 2 depletion. As cell growth depleted dissolved O 2 levels, NO 3 - was converted to NO 2 - at near-stoichiometric levels, whereas NO 2 - consumption did not coincide with NO or NO 3 - accumulation. Assimilatory NO 2 - reductase (NirBD) or NorCB activity could have prevented NO cycling, and experiments with ΔnirB, ΔnirS, and ΔnorC showed that NorCB was responsible for loss of flux from the cycle. Collectively, this work provides a computational tool to analyze NO metabolism in P. aeruginosa, and establishes that P. aeruginosa use NorCB to prevent metabolic cycling of NO., (Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Staphylococcus aureus is a notorious pathogen with a propensity to cause chronic, non-healing wounds. Bacterial persisters have been implicated in the recalcitrance of S. aureus infections, and this motivated us to examine the persistence of S. aureus to ciprofloxacin, a quinolone antibiotic. Upon treatment of exponential phase S. aureus with ciprofloxacin, we observed that survival was a non-monotonic function of ciprofloxacin concentration. Maximal killing occurred at 1 µg/mL ciprofloxacin, which corresponded to survival that was up to ~40-fold lower than that obtained with concentrations ≥ 5 µg/mL. Investigation of this phenomenon revealed that the non-monotonic response was associated with prophage induction, which facilitated killing of S. aureus persisters. Elimination of prophage induction with tetracycline was found to prevent cell lysis and persister killing. We anticipate that these findings may be useful for the design of quinolone treatments.