Effect of narrow, pulsed high voltages on bacterial viability.

High-voltage pulsed current (HVPC) has been used to promote healing of decubitus ulcers and surgical wounds.[1] The mechanism by which HVPC is thought to promote healing is not known, but among the hypothesized mechanisms is antimicrobial action. This effect, if observed, may be produced by the direct action of the current on the organism; the electrochemical generation of antimicrobial factors, including changes in pH; localized heat generation; or the electrophoretic recruitment of antimicrobial factors already present in the body (eg, those of the immune system). Whereas low-voltage (< 10 V) direct current (DC) potentials have been shown to be bactericidal in vitro[2] and in vivo[,3,4] the antimicrobial effects of pulsed high voltages have not been systematically explored.

Guffey and Asmussen's[5] failure to observe inhibition of bacterial growth may have been due to their experimental setup, in which they controlled current flow rather than voltage during treatment. As a result, with the highly conductive trypticase soy agar media they were using, their applied potentials were fairly low, possibly even below levels that should be considered high voltage. Kincaid and Lavoie[6] observed direct and indirect inhibition of bacterial growth at both electrodes with prolonged exposure or higher voltages. The authors state, however, that they grew their organisms overnight in trypticase soy broth (TSB) and adjusted the inoculum to give 1 x [10.sup.7] colony-forming units per milliliter (CFU/mL) using a hemocytometer. Because, we believe, it is almost impossible to count bacteria in a hemocytometer and because by using this method both viable and nonviable organisms would be counted, a significant number of organisms in the culture may not have been viable after overnight incubation. Kincaid and Lavoie's assertion, therefore, that they were using 1 x [10.sup.7] CFU/mL is questionable. They also. state that they used pH paper (type and manufacturer not stated) to measure the agar pH following treatment. Because most pH papers are intended for saturation with the liquid to be measured, the reported pH results are questionable. Kincaid and Lavoie also report that motile organisms recolonized the zones of inhibition, an unlikely occurrence as the concentration of agar in Mueller-Hinton media, at 1.7%, is too high for motility to occur. As the agar concentration increases, the gel matrix becomes too dense for the organisms to move through it. Culture media with agar concentrations greater than 0.4% produce gels through which motile organisms cannot spread.[7]

We have developed an in vitro model with which it is possible to determine the manner in which high-voltage pulsed electrical stimulation may exert an antimicrobial action. In our system, the constituents present during the HVPC application can be controlled while concomitantly providing an environment suitable for the subsequent growth of the organisms. The organisms studied include representative gram-negative and gram-positive organisms that are typically isolated from decubitus ulcers.

Materials and Methods

Organisms

Exponential growth phase clinical isolates of Escherichia coli, Klebsiella, Pseudomonas aeruginosa, and Staphylococcus aureus (final dilution [10.sup.3] CFU/mL in pour plates; [10.sup.5] CFU/mL when plates were inoculated after the HVPC treatment) were utilized.

Pulsed High-Voltage Potential

Pulsed high voltage was applied to solidified agarose in 60-mm petri plates with a Chattanooga Intelect Model 500 high-voltage galvanic stimulator(*) using two 5-mm-diameter (3/16-in) stainless steel electrodes placed 3 cm apart in the center of the plate on top of the solidified agarose. A 500-V potential (peak-voltage, twin 7-microsecond pulses spaced 70 microseconds apart and delivered at 120 pulses per second) was applied for 30 minutes at room temperature (22[degrees]C) (Fig. 1).

Antimicrobial Effects

Both direct and indirect effects of HVPC were examined in 60-mm petri plates containing 9 mL of 1% agarose([dagger]) (weight per volume) in one of the following: (1) distilled water, (2) 0.85% sodium chloride (weight per volume), (3) 0.1-M phosphate-buffered saline (PBS), or (4) TSB([double dagger]) in PBS (TSB-PBS).

The direct antimicrobial action of HVPC was examined with the organisms throughout the agarose (pour plate) during the application of the electrical potential. To determine whether the HVPC produced antimicrobial factors (ie, an indirect effect), the potential was applied to agarose without the organisms present in the media. Immediately following application of the potential, [10.sup.5] CFU/mL was inoculated onto the surface using a saturated cotton swab with excess suspension removed by rolling the swab against the side of the tube. Control plates for each test were identical except for the HVPC application.

In those instances in which the agarose did not contain nutrients, subsequent to the application of the HVPC, 3 mL of TSB (three times normal concentration) in 1% agarose was layered over the treated plates. After overnight incubation at 35[degrees]C, growth inhibition was determined. Two individuals, using calipers, measured the diameters of the zones of inhibition and recorded the readings to the nearest millimeter. These readings were converted into a grading scale developed in our laboratory in which inhibition was graded as 1+ when the zones were 5 through 8 mm in diameter (electrodes were 5 mm in diameter), 2+ when the zones were 9 through 12 mm in diameter, and 3 when the zones were [greater than or equal to] 13 mm in diameter. The largest zones of inhibition observed were 16 mm in diameter. Photographs were taken for archival storage and reference in case of differences in zone sizes recorded by the two readers. All tests for antimicrobial effects of HVPC were performed in triplicate (n=3).

Determination of pH and Temperature After the Application of HVPC

The HVPC was applied to the agarose plates at room temperature without organisms present, and determinations of pH and temperature were made immediately afterward. Changes in pH were determined both with a flat-surface pH electrode[sections] and with chemical indicators. The potentiometric pH measurements were taken, using an Orion 701A/Meter,[parallel] at 13 predefined grid coordinates spaced 1.27 cm (1/2-in) apart. Because it took approximately 5 to 10 minutes to take the 13 measurements, five or six sets of replicate experiments were run for each system and the pH was measured in each experiment. The measurements were taken from right to left in half of the experiments and from left to right in the other half of the experiments. The chemical pH indicator was a mixture of 0.01% methyl red(#) (weight per volume) and 0.01% thymol blue(#) (weight per volume), giving a range of colors such that a dark red color indicates a pH of [less than or equal to] 4.4, a lighter red indicates a pH of 4.4 to 6.2, yellow indicates a pH of 6.2 to 8.0, and purple indicates a pH of [greater than or equal to] 8.0. Approximately 1 mL of the pH indicator mixture was layered over the agarose following HVPC treatment. This experiment was performed once for each system. Photographs were taken for archival storage and reference.

Temperature measurements were made using the temperature sensor of a Fisher Accumet 750 ion analyzer.(#) These measurements were taken in a separate experimental run at the same coordinates as the pH measurements. This experiment was performed once for each system,

Determination of pH and Temperature Changes During Application of HVPC

Temperature and pH changes near the electrodes were measured during the application of the HVPC to agarose. The temperature probe or the pH electrode** was placed within 2 mm of each electrode 10 minutes prior to the start of the HVPC treatment, and baseline values were obtained. The HVPC treatment was then initiated, and the temperature or pH potential was monitored during the 30-minute treatment. The pH was monitored for an additional 10 to 20 minutes posttreatment. These experiments were performed once for each system.

Effects of pH on Viability of Organisms

To determine the effects of pH on the Viability of the organisms, 1:5 dilutions of [10.sup.5] CFU/mL exponential growth-phase organisms were made in buffers at pH 2.0, 3.0, 4.0, 6.4, 7.0, 8.0, 9.0, and 10.0,([double dagger]) using Nutrient Broth([double dagger]) as the control. The 1:5 dilution ensured that the pH was determined by that of the buffer and not the nutrient broth in which the organisms had been grown. Furthermore, the effects of pH on the viability of the organisms were determined over the same time (30 minutes) and temperature (22[degrees]C) chosen for the treatment of the petri plates. Using a calibrated loop (0.001 mL), the suspensions were sampled at 5, 10, 15, and 30 minutes. The samples were streaked onto Nutrient Agar and incubated at 35[degrees]C overnight, and CFU/mL was determined.

Results

Antimicrobial Effects

Small amounts of gas, visible as small bubbles at the edge of the electrode, were generated at the positive electrode and were occasionally observed at the negative electrodes during the HVPC treatment. Following treatment, there was generally a depression at both electrode positions, possibly resulting from compression or liquefaction of the agarose. Therefore, lack of growth directly under the electrode was not considered inhibition.

As would be expected, current flow depended greatly on the makeup of the supporting electrolyte in which the agarose was made. As shown in Table 1, the lowest peak current flows (25-50 mA) were observed for agarose in distilled water, whereas the greatest peak current flows were observed with agarose in saline. Table 2 summarizes the results of the inhibition experiments. In each experiment, there was agreement between the readers for zone sizes. The results of the triplicate experiments were consistently within the same graded zone size for each set of experimental conditions. In most instances, the results are the same for both the indirect and the direct inhibition experiments (ie, when inhibition was observed, the inhibition was seen in both the direct and indirect experiments for the same media). In the four instances in which inhibition occurred in the indirect experiments but not in the direct experiments (E coli, Klebsiella, and S aureus in TSB-PBS combination and Klebsiella in PBS), this difference was observed at the cathode. In the five instances in which the opposite was observed (ie, direct inhibition but no indirect inhibition), four (E coli, Klebsiella, and P aeruginosa in agarose and S aureus in PBS) were at the anode and one (S aureus in agarose) was at the cathode.

Table 1. Observed Peak Currents
                              Observed Peak
Medium                         Current (mA)
Agarose in distilled water        25-50
Agarose in saline
  (0.85% NaCl)                 2,000-2,400
Agarose in phosphate buffer    1,100-1,400
Agarose in TSB-PBS(a)          1,500-1,800
(a) TSB-PBS = trypticase soy broth in phosphate-buffered
saline.

[TABULAR DATA 2 OMITTED]

In the plates with agarose in distilled water, there was no indirect inhibition, but slight direct inhibition was observed at the anode for the gram-negative rods and at the cathode for a gram-positive coccus. In the agarose in saline plates, there was marked direct inhibition of all organisms (3+) at the cathode, with slightly less inhibition (ie, 2+) at the anode. In the indirect experiments, there was also marked indirect inhibition (ie, 3+ for all except Klebsiella, which was 2+) at the cathode, with only slight inhibition (ie, 1+) at the anode with all organisms.

In the agarose in PBS experiments, there was no direct inhibition at the cathode for Klebsiella and S aureas, but we found 2+ inhibition of E coli and P aeruginosa. In the indirect tests, there is 2+ inhibition at each electrode for the gram-negative organisms, but no inhibition was observed in the S aureus experiments.

In the PBS-TSB experiments, the results were the same in both direct and indirect experiments at the anode (ie, 2+ inhibition). At the cathode, the results were the same only for P aeruginosa; with the other organisms, there was indirect inhibition but no direct inhibition at the cathode.

Determination of pH After Treatment

Figures 2 through 4 show the results of the potentiometric and indicator pH measurements, The numerical values shown are the mean of five or six separate experimental runs with pH measurements at the predetermined locations on the surface of the agarose. This replication was done to minimize the effect of changes in pH with time (5-10 minutes), as the measurements were made at the 13 locations on the plates. The standard deviation of the replicate measurements at any one point was generally less than 0.5 pH units. The pH changes are toward acid at the positive electrode (anode) and, conversely, toward basic at the negative electrode (cathode). As would be expected, the extent of the pH change was less in those plates in which a buffer was incorporated.

The results obtained when the two pH indicator dyes methyl red and thymol blue were layered over the treated agarose confirmed the potentiometric pH determinations (Fig. 2). The pH change is quickly ameliorated when there is any buffering capacity within the agarose. For agarose in PBS, the pH reverted to its original value within 2 hours (Fig. 3). When the agarose was unbuffered, as was the case with agarose in saline, the zone of pH change continued to spread so that the zones met in the center of the plate approximately 3 hours after the cessation of HVPC treatment and continued to spread for the 8 hours it was monitored (Fig. 4)

Effect on pH During Treatment

These measurements were complicated by an instantaneous jump in the observed pH readings on the pH meter upon application of the HVPC and an immediate drop in the pH reading upon cessation of the treatment. These changes were most likely due to an interaction of the applied potential and the solid-state pH-sensing element of the Lazar PHR-142 electrode.

Discounting this artifactual change, when the pH was monitored near the negative electrode (Fig. 5), the greatest pH change was with agarose in saline. The pH rose to approximately 11.5 within 15 minutes following initiation of treatment. The pH remained elevated for the 20 minutes it was monitored after the treatment. For agarose in TSB, a slow positive pH change was observed throughout the treatment and the posttreatment monitoring period, in which a final value of 9.0 was observed. No change in pH near the negative electrode was observed for agarose in PBS except for the artifactual jump.

At the positive electrode (Fig. 6), the artifactual change makes interpretation slightly more difficult because it was of opposite sign to the actual pH changes. The greatest pH change was again observed for agarose in saline, in which the pH declined to approximately 3.5 by 15 minutes into the treatment. During the 15-minute posttreatment monitoring period, the pH showed a slow rise to 4.5. Interpretation of the results from the agarose in TSB is especially complicated by the artifactual change, but it is believed to indicate a small, slow decline in pH during the treatment and the posttreatment monitoring period. For agarose in PBS, the pH near the positive electrode, like that near the negative electrode, showed little change.

Effect of pH on Viability

There was no effect on the viability of organisms; that is, the number of viable organisms did not change at pH [greater than or equal to] 6.4 when incubated at room temperature for 30 minutes, Lower pH values, as shown in Table 3, did have an effect on the viability of the organisms. When exposed to pH 4.0 for 15 minutes, the E coli and S aureus bacteria were unaffected and were only slightly inhibited at 30 minutes (Fig, 4). Half of the P aeruginosa bacteria were nonviable by 5 minutes in PH 4.0, with a decreasing loss of viability until there were 3 x [10.sup.3] CFU/mL at 30 minutes. The Klebsiella bacteria were markedly inhibited in pH 4.0 after 5 minutes, with no further decrease in viability observed until the 30-minute sample when there were 3 x [10.sup.3] CFU/ml. After 30 minutes of exposure to pH 3.0, 5 x [10.sup.3] CFU/mL of the P aeruginosa bacteria and [10.sup.3] CFU/mL of the S aureus bacteria survived, but there was no survival of E coli and Klebsiella. Klebsiella did not survive exposure to pH 3.0 even for 5 minutes; all other organisms were markedly reduced after 5 minutes' exposure. There was no growth on any of the plates from the 10-minute exposure to pH 2.0, and few organisms were viable after 5 minutes at this pH.

[TABULAR DATA 3 OMITTED]

Temperature

Temperature measurements made during the application of the HVPC did not reveal any striking changes. The maximum change between any two points on the plate was less than 1[degree]C. Similarly, temperature measurements determined subsequent to the application of the HVPC did not show more than a 1[degree]C change, with no difference in temperature between the untreated and treated plates.

Discussion and Conclusion

The results show that, at least in this simple in vitro system, HVPC exerts an antimicrobial action. This antimicrobial action appears to be due to a direct antimicrobial effect that occurs during application of the current and to the generation of indirect antimicrobial factors (ie, effects that persist when the current is discontinued). When a stepped current is applied to a conductive solution, the total current that flows is the sum of the current flow due to capacitive charging currents and ionic flow as well as the faradaic current that occurs due to any redox electrochemical reactions that take place. Charging and ionic currents are most dependant on the makeup of the supporting electrolyte and would induce only direct antimicrobial effects because these currents would not produce residual changes (other than pH) within the media. The faradaic current, which will depend on the potential applied, could generate reactive species that would give rise to indirect antimicrobial effects. Because the antimicrobial effects are not observed in all agarose combinations, the inhibition is not likely to be due to generation of metal ions from the electrodes but rather to some other factor or factors produced by the flow of the HVPC through the media.

Two factors, pH and temperature, were examined as possible mechanisms for the indirect effect. The temperature changes observed during the application of HVPC at room temperature (20[degrees]-22[degrees]C) were minimal (< 1[degrees]C). Because all the organisms used in this study are mesophilic, that is, they have optimum growth at temperatures of 30[degrees] to 40[degrees]C, the temperature changes observed would not have induced an antimicrobial effect. Some evidence suggests that extreme pH changes are not the major cause of the antimicrobial effects. For the indirect effect experiments, the zones of inhibition observed were generally larger than the zones of extremely acid or basic pHs. This was especially true in those cases in which the agarose was supplemented with a buffer. Furthermore, the pH change was quickly eliminated in the buffered systems. At the anode, the greatest pH changes were observed in the unbuffered (saline or water) systems, yet the zones of inhibition were smaller or equal to those of the buffered (PBS or TSB) systems in which pH changes were minimal. At the cathode, the largest zones of inhibition were found in the saline system. When the effects of pH on bacterial viability were examined, however, no antimicrobial effects were observed in the range of alkaline pHs that were generated at the cathode. At the anode, the lowest pH observed in the pH determination experiments was with the unbuffered systems. In the agarose-in-water system, however, only slight inhibition was observed in the direct experiments, and no inhibition was observed in the indirect experiments. Greater inhibition was observed in the buffered agarose systems, in which it is unlikely that the pH fell to levels (pH <4.0) needed to cause complete loss of viability in the experiment examining the effect of pH on the bacteria. All of this evidence supports the contention that pH changes occurring with the application of HVPC are not the primary cause of the inhibition observed.

Because the mechanisms by which HVPC would exert an antimicrobial effect are unknown, changes in inhibition zone diameter may not represent differences in antimicrobial activity. Rigorous comparison should only be made once the mechanisms have been elucidated. With this caution in mind, it appears that although the antimicrobial effects (both direct and indirect) in our system were the same for several media-organism combinations at both the anode and cathode, differences were observed in each combination. When we compared the results using any magnitude of difference (not just inhibition versus no inhibition), no consistent differences were found for the gram-negative organisms versus the gram-positive organisms (ie, S aureus). Additionally, as many differences were noted at the anode as at the cathode. There were, however, 10 instances in which the direct inhibition was greater than the indirect inhibition and only 4 instances in which the indirect experiments had more inhibition than the direct experiments. Thus, overall, it appears that the direct effects are more important than the indirect effects in the inhibition of microorganisms, although both contribute to the antimicrobial effects. Further work must be done to elucidate the antimicrobial mechanisms of HVPC and to determine whether these effects take place in vivo as used in the management of decubitus ulcers. It appears that the practice of alternating the electrodes in patient treatment regimens is supported by both direct and indirect inhibition occurring at each electrode in the different in vitro systems examined. Until evidence of in vivo antimicrobial effects are found and data on clinical effectiveness are available, however, findings such as those of this study must be interpreted with caution. (*) Chattanooga Corp, 101 Memorial Dr, PO Box 4287, Chattanooga, TN 37405. ([dagger]) SeaKem agarose, Marine Colloids Inc, PO Box 308, Rockland, ME 04841, ([double dagger]) Difco Laboratories, PO Box 1058, Detroit, MI 48232. ([sections]) Corning Scientific Instruments, Medfield, MA 02052. ([parallel]) Orion Research Inc, 529 Main St, Cambridge, MA 02139. (#) Fisher Scientific, 711 Forbes Ave, Pittsburgh, PA 15219. (**) PHR-142 microcombination electrode, Lazar Research Labs, Los Angeles, CA 90046. ([double dagger)] Metrepak, Micro Essential Laboratories, Brooklyn, NY 11210.

References

[1] Kloth LC, Feedar JA. Acceleration of wound healing with high voltage, monophasic, pulsed current. Phys Ther 1988;68:503-508. [2] Barranco SD, Spadaro JA, Berger TJ, Becker RO. In vitro effect of weak direct current on Staphylococcus aureus. Clin Orthop. 1974; 100: 250-255, [3] Carley PJ, Wainapel SF. Electrotherapy for acceleration of wound healing: low intensity direct current. Arch Phys Med Rehabil. 1985;66: 443-446. [4] Wolcott LE, Wheeler OC, Hardwicke HM, Rowley BA. Accelerated healing of skin ulcers by electrotherapy: preliminary clinical results. South Med J. 1969;62:795-801. [5] Guffey JS, Asmussen MD. In vitro bactericidal effects of high voltage pulsed current versus direct current against Staphylococcus aureus. J Clin Electrophysiol. 1989; 1:5-9. [6] Kincade CB, Lavoie KH. Inhibition of bacterial growth in vitro following stimulation with high voltage, monophasic, pulsed current, Phys Ther. 1989;69:651-655. [7] Hendrickson DA, Krenz MM. Reagents and stains. In: Belows A, Huasler WJ Jr, Herrman KL, et al, eds. Manual of Clinical Microbiology. 5th ed. Washington, DC: American Society for Microbiology; 1991: chap 122.

NJ Szuminsky, MT(ASCP), is Assistant Professor, Department of Medical Technology, School of Health and Rehabilitation Sciences, University of Pittsburgh, 209 Pennsylvania Hall, Pittsburgh, PA 15261 (USA). Address all correspondence to Mr Szuminsky.

AC Albers, DRPH, MY(ASCP), is Chair and Associate Professor, Department of Medical Technology, School of Health and Rehabilitation Sciences, University of Pittsburgh.

PG Unger, PT, is an independent consultant for utilization review and wound management, Her practice is based in Kutztown, PA.

JG Eddy, PT, ECS, is a physical therapist in the Pittsburgh area and is currently working on his graduate degree at the School of Health and Rehabilitation Sciences, University of Pittsburgh.