Inhibition of bacterial growth in vitro following stimulation with high voltage, monophasic pulsed current.

Inhibition of Bacterial Growth In Vitro Following Stimulation with High Voltage, Monophasic, Pulsed Current

Low-intensity direct current has been reported to be effective in promoting healing of infected wounds, and these results have been assumed to apply to stimulation of wound tissue with monophasic high voltage pulsed current (HVPC). The purpose of this study was to determine whether HVPC has an inhibitory effect on growth in vitro of three bacterial species--Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa--commonly isolated from open wounds. Following exposure to HVPC, the measured zone of inhibition of bacterial growth was not significantly different between bacterial species. Inhibition at the anode (positive pole) occurred secondary to build-up of toxic end products, and inhibition at the cathode (negative pole) resulted from exposure to HVPC. Duration of exposure and voltage showed a highly significant linear relationship. Exposure to more than 250 V of HVPC for at least two hours resulted in some degree of inhibition of growth in all three bacterial species. [Kincaid CB, Lavoie KH: Inhibition of bacterial growth in vitro following stimulation with high voltage, monophasic, pulsed current. Phys Ther 69:651-655, 1989] Key Words: Bacterial infections, Electric stimulation, Electrotherapy, Wound healing. The use of electrotherapy to promote healing of superficial and deep dermal wounds has been reported sporadically in the literature. A historical review of literature on the use of low-intensity direct current (LIDC) revealed that most studies suggest that LIDC enhances wound healing.(1-13) Two reasons cited for this beneficial effect are the bactericidal effects of electrical current(14) and the stimulation of granulation tissue growth by the use of electrical current.(15) Clinicians have been applying high voltage pulsed current (HVPC) for its assumed antibacterial and wound-healing effects in recent years.(16) These assumptions are based almost entirely on results obtained in LIDC studies. Thurman and Christian reported a single-case study involving the successful use of HVPC for the treatment of a persistent toe abscess.(16) High voltage pulsed current instruments produce a waveform markedly different from the waveform generated by LIDC instruments. High voltage pulsed current characteristics consist of twin-peaked, paired pulses of high peak and low total current having a fixed duration of 100 to 200 [Mu] sec. Low-intensity direct current is characterized by a low-intensity, continuous, unidirectional flow of current.(15) The actual current used in this study was delivered in modified form from a Rich-Mar HV-20* HVPC device. To date, there are no published reports regarding the effects of HVPC on bacterial growth. The purpose of this study was to establish whether HVPC has an inhibitory effect in vitro on the growth of bacterial pathogens commonly found as infectious agents in wounds.

Method

Organisms Tested

Three bacterial species commonly isolated from wounds were used as test organisms.(17) Isolates of Staphylococcus aureus (gram-positive cocci) and Escherichia coli and Pseudomonas aeruginosa (both gram-negative rods) were obtained from American Type Culture Collection stocks.

Procedure and Instrumentation

Our procedure was a slight modification of the procedure used by Barranco et al to test the in vitro effect of weak direct current on S aureus.(14) Sterile disposable plastic petri dishes were used throughout the experiment. Stainless-steel wires (0.035 gauge) used as electrodes were positioned parallel 50 mm apart and covered with growth medium containing the test organisms. With a heated wire, four holes were melted 3 mm from the bottom edge of the dish. The holes allowed parallel placement of two 15-cm pieces of sterile stainless-steel wire extending across the entire width of the dish with the ends bent to prevent the wires from rolling and breaking contact with the medium. Test organisms were grown in trypticase soy broth overnight at 37 [degrees] C in a shaking water bath, and enough culture was added to the test medium to reach a final concentration of 1 x [10.sup.7] colony-forming units per milliliter, as determined by standard use of a hemocytometer. The test medium selected was Mueller-Hinton agar,([subsection]) which is used in the semiquantitative Kirby-Bauer technique for determining effectiveness of antibiotics, because of the consistency of the widths of zones indicating inhibition of bacterial growth.[18] A sufficient quantity of medium inoculated with the test organism was poured into the prepared petri dishes to completely cover the wire electrodes, covered, and allowed to solidify. Plates were refrigerated for use within 24 hours of preparation. For test runs, wires in the petri dishes were connected by alligator-clip leads to a HVPC stimulator. The experimental setup is shown in Figure 3. The pulse rate was set at 120 pulses per second (pps) and the interpulse interval (measured at 50% of peak pulse amplitude) at 55 [Mu] sec. Voltages of 150, 200, 250, and 300 V were applied to the test organisms for 1, 2, 3, and 4 hours' duration. According to Roberta A Newton (RA Newton, PhD; unpublished data), these voltages are within the normal therapeutic range for motor effect. Exposure durations were chosen based on the results of a pilot study that showed no bactericidal effect of HVPC at these voltages with shorter exposure duration. Each petri dish was incubated at 37 [degrees] C for 24 hours following exposure to HVPC. After incubation, the width of the zone paralleling the wire electrodes where no bacterial growth occurred was measured to the nearest 0.1 mm using a millimeter ruler. Test results represent the average of four measurements per zone of inhibition per plate. Subcultures from the zone of inhibition were checked for sterility by using an inoculating needle to transfer medium from the zone into tubes of sterile nutrient broth and incubating them at 37 [degrees] C for 24 hours. Visible turbidity was used as an indication of growth. Changes in pH of the medium were determined by using pH paper touched to the surface of the medium. Determination of the actual current waveform was made using an oscilloscope. The pattern was determined for the output at 250 V from the HVPC leads and for the electrodes through a petri dish setup with the test medium (Fig. 2).

Controls

Seeded plates with the wire electrodes in place were incubated without exposure to HVPC to determine whether the wires themselves, the medium, or a combination of both would be inhibitory to bacterial growth. Plates were also prepared with sterile medium (ie, without organisms added) to determine whether wire-insertion preparation introduced contamination. The presence of potential toxic electrochemical end products from the interaction of current, wire, and medium was determined by sending current through sterile medium without organisms. Extreme conditions (ie, beyond a normal therapeutic range) of 500 V for either 30 minutes or 2 hours were used to allow maximum potential toxin production. Overlays of medium containing either S aureus or E coli were poured into the plates, allowed to solidify, and then incubated and evaluated as previously described.

Data Analysis

Significance of zone width, as a function of the organism, was analyzed by a two-way analysis of variance (ANOVA).[20] A regression analysis was performed using the MIDAS (Michigan Interactive Data Analysis System) program on the Michigan Terminal System.

Results

Inhibition of growth of S aureus, E coli and P aeruginosa at the cathode (negative electrode) following exposure to HVPC is shown in Figure 4. Each line represents pooled data from three organisms, three replicates each, at 300, 250, 200, and 150 V for 1 to 4 hours of exposure. The ANOVA of the data at 4 hours of exposure revealed that differences using different microorganisms were not significant (F = 2.18; df = 2,18; p [is less than] .10), whereas varying the voltage resulted in highly significant differences (F = 152.98; df = 2,18; p [is less than] .001). The linear relationship between the effects of time and voltage on zone width was highly significant (F = 254.34; df = 2,105; p [is less than] .0001), as expressed by the following equation: Zone width (mm) = 0.04295(Time [min])

                  (0.00236)
                + 0.05156(EMF [V])
                  (0.00388)
                - 15.98
                  (1.05)

where EMF refers to electromotive force and values in parentheses are standard errors of the means. This relationship has a coefficient of determination ([r.sup.2]) of .828. Exposure of all test organisms to HVPC for 2 hours at 250 V was enough to cause inhibition of growth of each test organism. Inhibition was noted at both the anode (positive electrode) and the cathode. Sterility checks of the zone of inhibition established that exposure to the current had a bactericidal effect on the test organisms as opposed to a static effect; that is, the organisms were killed as opposed to having growth inhibited. Motile test organisms were able to recolonize the zone of inhibition around the cathode, causing false-positive sterility checks. Sterile control plates with no organisms added to them remained sterile after incubation, indicating that the experimental procedure did not introduce any contaminants. Several control plates established that exposure to the current was the cause of the bactericidal effect on the test organisms at the cathode. Plates containing seeded medium that were not exposed to HVPC showed no inhibition of growth, indicating that the wires themselves, the medium, or both, were not inhibitory to bacterial growth. The presence of toxic electrochemical end products arising from HVPC was determined by overlaying seeded medium on plates that had been subjected to 500 V for either 30 minutes or 2 hours. No inhibition of growth at the cathode was observed, but the test organisms would not grow around the anode, and motile organisms could not recolonize the zone. All plates showed some degree of discoloration around the anode. The pH of the medium at the cathode was observed to increase to between 8.5 and 9 with increasing time or voltage to a maximum with 4 hours of exposure to HVPC at 500 V. The increased pH was transient, reverting to neutrality within 18 hours after exposure to HVPC, presumably because of the highly buffered nature of the test medium.[13] The pH at the anode was not increased at any time during or after the experiment. The current waveform from the instrument changed greatly as it flowed through the test medium. The resistance to flow varied, making it impossible to measure actual current flow. Figure 2 shows a reduction in total current and resistance to flow, as indicated by the baseline not returning to zero, as the waveform leaves the stimulator and passes through the medium.

Discussion

High voltage pulsed current can be effective in killing common wound-infecting bacteria in vitro. All organisms tested were equally affected by 2 hours of exposure to HVPC above 250 V. The data analysis revealed a strong positive linear relationship between the voltage and the duration of exposure to HVPC. Exposure to HVPC at the cathode accounted for most or all killing of bacterial cells. The increasing pH observed at the cathode was transient and probably did not reach levels extreme enough to directly kill bacterial cells. No effect on skin pH following a 30-minute application of HVPC at 100 V was reported by Newton and Karselis.[20] Such a rise in pH could have a static effect on growth that, combined with the lethal effect of HVPC, would help to keep bacterial population levels down and enable body defenses to fight off the infection. Effects of HVPC at the anode were complicated by production of some toxic electrochemical end products created by passing current through the wire. Zones of inhibition around the cathode were recolonized by motile bacteria, suggesting that no permanent change had occurred there. In contrast, organisms were unable to recolonize the zone of discoloration around the anode, suggesting that lethal end products had accumulated and persisted. These results are comparable to those reported by Barranco et al.[14] Caution must be exercised when attempting to extrapolate the findings of in vitro studies to predict results when applying the same intervention to infected wounds in human subjects. Present treatment protocols for use of HVPC on infected wounds, however, generally indicate a treatment duration of 20 to 45 minutes once or twice a day, with voltage amplitude adjusted to a subthreshold level for muscle contraction. Our study used a much higher voltage setting for a much longer exposure duration than those used in current clinical practice. Human subjects may not be able to tolerate such high voltage applications. Alternatively, the actual current flow through the petri plates was very low because of resistance from the test medium. If there is less resistance to current flow in human skin, then lower settings might be bactericidally effective in a clinical setting. Future avenues of investigation include in vivo application of HVPC to infected wounds as well as the application of other types of electrical current to microorganisms in vitro. If future studies indicate that the exposure of infected wounds to electrical current results in decreased infection, then it may be possible to effectively treat infected wounds on a home-care basis with portable electrical stimulators, thereby making wound management more cost effective.

Conclusion

Some clinicians use HVPC to inhibit bacterial growth in infected wounds. The results of this study indicate that, although there is inhibition or killing of bacteria in vitro at the cathode with the application of HVPC, either the voltage applied or duration of treatment, or both, may need to be substantially increased to achieve healing of infected wounds. Studies conducted in vivo are necessary to determine the effectiveness of HVPC in wound healing in clinical practice.

(*)Rich-Mar Corp, Rt 2, PO Box 879, Inola, OK 74036-0879. ([dagger])American Type Culture Collection, 12301 Parklawn Dr, Rockville, MD 20852. ([double dagger])Phoenix Wire Cloth, Inc, 585 Stevenson Hwy, Troy, MI 48083. ([subsection])Difco Laboratories, Inc, 920 Henry St, Detroit, MI 48232.

PHOTO : Fig. 3. Experimental setup. Stainless-steel wires were placed 50 mm apart through sides of

PHOTO : plastic petri dish. Medium seeded with test organisms was poured over wires and allowed

PHOTO : to solidify. Current from high voltage pulsed current stimulator was applied, and effects

PHOTO : of varying time and voltage on bacterial growth were recorded. References [1]Assimacopoulos D: Low intensity negative electric current in the treatment of ulcers of the leg due to chronic venous insufficiency. Am J Surg 115:683-687, 1968 [2]Assimacopoulos D: Wound healing promotion by the use of negative electrical current. Am Surg 34:423-431, 1968 [3]Dueland R, Hoffer RE, Scleen WA, et al: The effects of low voltage current on healing of thermal third degree burns. Cornell Vet 68:51-59, 1978 [4]Edel H, Freund R: Direct current treatment of chronic skin ulcer and wound healing by second intention. Physiotherapy 27:457-464, 1975 [5]Gault WR, Gatens PF Jr: Use of low intensity direct current in management of ischemic skin ulcers. Phys Ther 56:265-269, 1976 [6]Moore AD: Electrostatic discharge for treating skin lesions. Med Instrum 9:274-275, 1975 [7]Tyurlikova LD, Lassi NI: The effect of the anode of a constant intermittent current on regenerative process in skeletal muscle. Byulleten' Eksperimental' noi Biologii i Meditsky 63:71-74, 1967 [8]Wolcott LE, Wheeler PC, Hardwicke HM, et al: Accelerated healing of skin ulcers by electrotherapy: Preliminary clinical results. South Med J 62:795-801, 1969 [9]Kloth LC, Feedar JA: Accelaration of wound healing with high voltage, monophasic, pulsed current. Phys Ther 68:503-508, 1988 [10]Akers TK, Gabrielson AL: The effect of high voltage galvanic stimulation on the rate of healing of decubitus ulcers. Biomed Sci Instrum 20:99-100, 1984 [11]Rowley BA, McKenna JM, Chase GR, et al: The influence of electrical current on an infecting microorganism in wounds. Ann NY Acad Sci 238:543-551, 1974 [12]Rowley BA: Electrical current effects on E coli growth rates. Proc Soc Exp Biol Med 139: 929-934, 1972 [13]Carley PJ, Wainapel SF: Electrotherapy for acceleration of wound healing: Low intensity direct current. Arch Phys Med Rehabil 66:443-446, 1985 [14]Barranco SD, Spadaro JA, Berger TJ, et al: In vitro effect of weak indirect current on Staphylococcus aureus. Clin Orthop 100:250-255, 1974 [15]Nelson RM, Currier DP (eds): Clinical Electrotherapy. East Norwalk, CT, Appleton & Lange, 1987, chap 8 [16]Thurman BF, Christian EL: Response of a serious circulatory lesion to electrical stimulation: A case report. Phys Ther 51:1107-1110, 1971 [17]Finegold SM, Martin WJ, Scott EG: Bailey and Scott's Diagnostic Microbiology. St Louis, MO, C V Mosby Co, 1978, chap 20 [18]Difco Manual: Dehydrated Culture Media and Reagents for Microbiology, ed 10. Detroit, MI, Difco Laboratories, 1984, pp 582-585 [19]Brownlee KA: Statistical Theory and Methodology in Science and Engineering, ed 2. New York, NY, John Wiley & Sons Inc, 1965, chap 14 [20]Newton RA, Karselis TC: Skin pH following high voltage pulsed galvanic stimulation. Phys Ther 63:1593-1596, 1983 C Kincaid, MS, PT, is Assistant Professor and Associate Director for Clinical Education, Physical Therapy Program, The University of Michigan-Flint, 1108 Lapeer St, Flint, MI 48502-2186 (USA). K Lavoie, PhD, is Associate Professor, Department of Biology, The University of Michigan-Flint.