Direct electric current treatment under physiologic saline conditions kills Staphylococcus epidermidis biofilms via electrolytic generation of hypochlorous acid.

The purpose of this study was to investigate the mechanism by which a direct electrical current reduced the viability of Staphylococcus epidermidis biofilms in conjunction with ciprofloxacin at physiologic saline conditions meant to approximate those in an infected artificial joint. Biofilms grown in CDC biofilm reactors were exposed to current for 24 hours in 1/10(th) strength tryptic soy broth containing 9 g/L total NaCl. Dose-dependent log reductions up to 6.7 log(10) CFU/cm(2) were observed with the application of direct current at all four levels (0.7 to 1.8 mA/cm(2)) both in the presence and absence of ciprofloxacin. There were no significant differences in log reductions for wells with ciprofloxacin compared to those without at the same current levels. When current exposures were repeated without biofilm or organics in the medium, significant generation of free chlorine was measured. Free chlorine doses equivalent to the 24 hour endpoint concentration for each current level were shown to mimic killing achieved by current application. Current exposure (1.8 mA/cm(2)) in medium lacking chloride and amended with sulfate, nitrate, or phosphate as alternative electrolytes produced diminished kills of 3, 2, and 0 log reduction, respectively. Direct current also killed Pseudomonas aeruginosa biofilms when NaCl was present. Together these results indicate that electrolysis reactions generating hypochlorous acid from chloride are likely a main contributor to the efficacy of direct current application. A physiologically relevant NaCl concentration is thus a critical parameter in experimental design if direct current is to be investigated for in vivo medical applications

Non-Monotonic Survival of Staphylococcus aureus with Respect to Ciprofloxacin Concentration Arises from Prophage-Dependent Killing of Persisters

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

Treatment of <i>P. aeruginosa</i> biofilm with direct current.

<p>Direct current was applied to biofilm in wells containing (A) no antibiotic and (B) 10 µg/mL tobramycin sulfate for 20 hours. All wells contained 0.1 g/L TSB with varying amounts of total NaCl. References to theoretical salt limitation or excess salt apply only to wells with current. Each symbol indicates a mean LD on coupons across one well and lines show connected means for each current level.</p

Treatment of <i>S. epidermidis</i> biofilm with electric current using various electrolytes.

<p>Direct current was applied to biofilms for 24 hours. The treatment solutions had all of the components of TSB except NaCl. Chloride was replaced with nitrate, phosphate, or sulfate at the same molarity (0.154 M) used for chloride experiments. The mean chloride results for 2 and 5 mA are shown for comparison (full data set in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055118#pone-0055118-g002" target="_blank">Figure 2A</a>). Each symbol indicates the LD for an individual coupon and lines show the connected means for each current level.</p

Treatment of <i>S. epidermidis</i> biofilm with electric current or a mimicked current exposure using chlorine.

<p>Direct current was applied in parallel to wells (A) with no antibiotic and (B) with 2.5 µg/mL ciprofloxacin at one or two current levels for <i>N</i> = 11 experiments. (C) An initial dose of free chlorine corresponding to each current level was used to mimic the direct current exposure in the absence of current for <i>N</i> = 1 experiment (no antibiotic). All wells contained 3 g/L TSB and 9 g/L total NaCl and treatments lasted 24 hours. Each data point denotes the LD for an individual coupon and lines show connected mean LDs for each current level.</p

Increased killing of <i>S. epidermidis</i> biofilm adjacent to the cathode.

<p>Within each well, a trend of larger reductions at coupon 3 (adjacent to the cathode) compared to coupon 1 (near the center of the well) was observed following electric current treatment, as indicated by positive values. Each symbol denotes the measurement of the difference in LR between the extreme coupon positions (coupon 3 minus coupon 1) in a current-exposed well.</p

Images of <i>S. epidermidis</i> biofilm after 24 hours of no current and 3 mA direct current exposure.

<p>Biofilms are stained with LIVE/DEAD BacLight stains. Green color indicates cells with intact membranes (“live” cells) while red color indicates cells with damaged membranes (“dead” cells). (A ) Confocal and (D) cryosection images of the control biofilm. Confocal and cryosection images of biofilm exposed to 3 mA of current are shown for position 1 (B and E) and position 3 (C and F) from the same treatment well. The scale bar for confocal images is 100 µm. The scale bar for cryosection images is 50 µm.</p

Chlorine generation and pH changes with electric current in the absence of biofilm.

<p>(A) Free chlorine was measured at each electrode after 24 hours of current when the direct current exposures were repeated without sources of chlorine demand (biofilm and organics in the medium). These “endpoint free chlorine concentrations” approximate the cumulative chlorine generated over the course of treatment without reaction. (B) The free chlorine differences between electrodes (cathode minus anode) in each well indicated a trend of higher free chlorine concentrations at the cathode as indicated by positive values. (C) pH measurements were taken at each electrode after direct current was applied for 24 hours to the standard treatment solution (with organics). Symbols indicate individual measurements and lines (A) show linear mixed effects models.</p

Polycarbonate treatment well.

<p>A piece of rubber sheeting on the bottom of the well held the biofilm-coated coupons at the same position (labeled 1–3) for each experiment. Platinum electrodes were inserted through the lid at opposite ends of the well and current was applied lengthwise. The anode was connected to the positive terminal of the power supply and the cathode to the negative.</p

The role of metabolism in bacterial persistence

Bacterial persisters are phenotypic variants with extraordinary tolerances toward antibiotics. Persister survival has been attributed to inhibition of essential cell functions during antibiotic stress, followed by reversal of the process and resumption of growth upon removal of the antibiotic. Metabolism plays a critical role in this process, since it participates in the entry, maintenance, and exit from the persister phenotype. Here, we review the experimental evidence that demonstrates the importance of metabolism to persistence, highlight the successes and potential for targeting metabolism in the search for anti-persister therapies, and discuss the current methods and challenges to understand persister physiology