Bactericidal Effect of Ultraviolet C (UVC), Direct and Filtered Through Transparent Plastic, on Gram-positive Cocci: An In Vitro Study

Abstract

  The prevalence of wound infections caused by multidrug-resistant (MDR) bacteria is increasing along with concern about widespread use of antibiotics. In vitro studies have shown that ultraviolet radiation, especially UVC, is both an effective bactericidal and antifungal.

However, evidence about its bactericidal effect on wounds covered with transparent dressings remains inconclusive. Transparent dressings are used to retain moisture over the wound as part of an intermittent negative pressure dressing—the Limited Access Dressing (LAD) technique. Because this dressing is designed to remain in place for a number of days, an in vitro study was conducted to explore the bactericidal effect of direct and indirect UVR through a transparent 0.15-mm thick transparent polythene sheet on Gram-positive cocci. Six bacterial strains were inoculated to sheep blood agar (SBA) plates and exposed to direct and filtered UVC (254 nm) for 5, 10, 15, 20, 25, and 30 seconds with one media serving as a control (no UVC exposure). Plates were subsequently incubated for 24 hours and bacterial growth observed. Each set of experiment was repeated three times. Direct UVC was shown to have good bactericidal effect (100% eradication of organisms inoculated) at durations ranging from a minimum of 5 seconds (methicillin-resistant, coagulase-negative Staphylococcus and Streptococcus pyogenes) to a maximum of 15 seconds (methicillin-susceptible Staphylococcus aureus and Enterococci species). No bactericidal effect was observed when UVC was filtered through a 0.15-mm thick transparent polythene sheet. The results confirm the bactericidal effect of UVC in vitro and suggest that this effect can be achieved after a very short period of time. At the same time, film dressings appear to filter UVC. This may help protect skin from exposure to UVC but also limits its utility for use with the LAD technique. In vivo studies to evaluate the shortest effective UVC treatment duration and follow-up clinical studies to ascertain treatment efficacy and effectiveness are needed.

Potential Conflicts of Interest: none disclosed

  Ultraviolet rays (UVR) in general (UVA: 315 nm to 400 nm; UVB: 280 nm to 315 nm; and UVC: 100 nm to 280 nm)¹ have the ability to trigger cellular actions—namely, cell proliferation,² increased epidermal thickness,³ and enhanced blood flow in the cutaneous capillaries,⁴ which help facilitate wound debridement.⁵ UVC specifically has been shown to have bactericidal effects in vitro.^6-9 In randomized controlled trials,⁵ a case study,¹⁰ and a prospective case series,¹¹ the beneficial effects of UVR have been used in treating chronic infected wounds and for preventing exit site infection.¹² Different frequently studied organisms treated using UVC are Staphylococci,⁸ Streptococcus pyogenes,⁶ Enterococcus faecalis,⁸ Escherichia coli,⁷ Pseudomonas aeruginosa,⁷ and fungal strains.   UVC causes cellular damage by inducing changes in the chemical structure of deoxyribonucleic acid (DNA) chains,⁷which yields a photoproduct —intrastrand cyclobutane-type pyrimidine dimers. UVC can cause adjacent pyrimidine bases (thymine and cytosine) to bond to one another instead of to the complementary DNA strand.13 These and other photoproducts can be lethal to bacterial cells, possibly impairing their viability and ability to replicate. The ultimate fate of irradiated cells depends on their ability to repair the UV-induced damage.14

  Many researchers have identified the prevalence of S. aureus,15 S. pyogenes, Klebsiella species, P. aeruginosa, and E. coli in different types of wound infections,16-19 but widespread use of antimicrobials in the management of wound infection has led to major concern regarding multidrug-resistant (MDR) micro-organisms.20 In addition, long-term use of topical antiseptics and antimicrobials such as iodine, hydrogen peroxide, and silver preparations may reduce the bacterial burden but comes with side effects.21,22

  The widespread use of negative pressure wound therapy (NPWT) is a relatively new trend in wound care. However, in extensive open wounds, NPWT may be difficult to provide. In these instances, a modification using sterilized plastic covers and two drainage tubes—a technique called limited access dressing (LAD)—has been used successfully in more than 1,000 patients by 2009 at Kasturba Hospital (Manipal, India).23,24 LAD uses intermittent negative pressure (-30 mm Hg for 30 minutes) through the tube(s) and has been shown in a case report25 and case series26 to maintain moisture at the wound site (for 3 hours, 30 minutes without negative pressure) with the use of transparent polythene material (a total of 21 hours of moist dressing and 3 hours of negative pressure dressing in a 24-hour period).

  Previous research (in vitro8 and human studies10,11) has shown that UVC has a bactericidal effect against MDR bacteria. In recent years, MDR bacteria20-23 as well as clinician understanding of the benefits of both LAD (ie, its cost effectiveness, transparency, reduced chances of anaerobic infection, and reduced smell) and UVR on bacterial growth have increased. In an empirical study, MacKinnon and Cleek27 reported inconclusive results regarding the bactericidal effects of UV light on a wound covered with the transparent dressing. Although UVC has proven a definitive adjunct in wound healing, not many related studies of late have been conducted. As such, the authors designed this prospective in vitro study to ascertain the bactericidal effect of UVC, direct and filtered, through a transparent plastic sheet, on Gram-positive cocci.

Materials and Methods

  Gram-positive cocci were collected from the pus section of the Department of Microbiology at KMC Manipal and researchers identified organisms using standard microbiological methods, tested for antibiotic sensitivity by disk diffusion method, and recorded sensitivity patterns.28 The following organisms were collected: methicillin-resistant S. aureus (MRSA), methicillin-susceptible S. aureus (MSSA), methicillin-resistant, coagulase-negative Staphylococcus (MRCONS), methicillin-susceptible, coagulase-negative Staphylococcus (MSCONS), S. pyogenes (sensitive, moderately sensitive, and antibiotic-resistant sensitivity pattern, designated 1, 2, 3, respectively), and Enterococcus species (sensitive, moderately sensitive, and antibiotic-resistant sensitivity pattern, designated 1, 2, 3, respectively).

 Preparation of bacterial cultures. A standard inoculum for the study was prepared from the isolated bacterial colony as follows: A single bacterial colony was inoculated into Peptone water and incubated for 6 hours at 37˚ C. After 6 hours, the turbidity of the Peptone water was standardized to McFarlands standard tube to obtain a final concentration of 105 organisms/mL. One standard loop (4 mm holding 0.01 mL of inoculum) of the broth culture was inoculated to sheep blood agar (SBA) plate by semiquantitative method by continuous streaking without intermittent heating on four quadrants.29-31

 UVC dosimetry. An Endolamp 474 (Enraf Nonius, Holland) was used to provide the UVR rays. According to the manufacturer’s manual, the lamp emits mostly (74%) UVC at 254 nm (see Figure 1, spectrum analysis), UVB 5%, UVA 2.5%, and visible light 18.5%. The calibrated energy output per unit area (culture medium of 9-cm diameter) at a distance of 10 cm is 0.318J/s/m2. The total energy per unit area is 1.59 J/m2 for 5 seconds of use, 3.18 J/m2 for 10 seconds, 4.77 J/m2 (15 seconds), 6.36 J/m2 (20 seconds), 7.95 J/m2 (25 seconds), and 9.54 J/m2 following 30 seconds of use. Unit output was regularly monitored using a broadband power energy meter (13PEM001 Melles Griot, Albuquerque, NM).

UVC exposure protocol. Each culture medium was exposed to direct UVC (see Figure 2) and to UVC filtered through a plastic sheet typically used for the LAD technique (0.15-mm thick, see Figure 3) for 5, 10, 15, 20, 25, or 30 seconds. Following the UVC manufacturer instructions, the distance between the UVC source and culture plate was maintained at 10 cm by using an investigator-designed rigid box made of thermocol (see Figure 2); the applicator was kept on the box before irradiation. One untreated culture plate was kept as a control and 18 plates were exposed to UVC (three plates containing the same bacteria for each duration). All experiments were repeated three times with the different organisms and various antibiotic susceptibility patterns.

  Incubation and bacterial growth. All plates (control and UVR-exposed) were incubated at 37˚ C for 24 hours and observed for bacterial growth. The exposure duration required to achieve no growth of the organism was noted. Media growth patterns were assessed and recorded as percentages of growth; no growth was considered zero. Growth was recorded as follows: scanty growth was first quadrant only. Colonies were counted and then multiplied by 100.32,33 Scanty growth and <10 colonies or <1,000 CFU/mL was <1%. Scanty growth and approximately 50 colonies or 5,000 CFU/mL was 5%. Scanty growth and 50 to 100 colonies or 10,000 CFU/mL was 10%; 25,000 CFU/mL growth or difficult to count in first quadrant was 25%. Growth in first and second quadrant and 50,000 CFU/mL was 50%; first, second and third quadrant growth and 75,000 CFU/mL was 75%; and >105 CFU/mL and growth in all four quadrants as 100%.

Data collection and analysis. Specimen collection, preparation of bacterial culture, and results interpretation were performed by the authors.

Results

  Using direct UVC, the exposure time required to achieve 0% growth (eradication) ranged from 5 seconds for S. pyogenes and MRCONS to 15 seconds for MSSA and Enterococcus species (see Table 1). All Staphylococci and Streptococci organisms were eradicated after a maximum direct exposure of 15 seconds: 90% of MRSA was eradicated by 5 seconds and 100% by 10 seconds (see Table 2). MSSA was 90% eradicated within 5 seconds, more than 99% eradicated within 10 seconds, and 100% eradicated by 15 seconds (see Table 3). MSCONS was 99.9% eradicated by 5 seconds and 100% eradicated by 10 seconds (see Table 4). MRCONS and S. pyogenes (irrespective of their susceptibility patterns) had 100% eradication by 5 seconds (see Table 5). Enterococcus species required 15 seconds of exposure (see Table 6).

  Filtering UVC through the 0.15-mm plastic sheet had no effect on the growth of any of the bacterial strains tested at any time point. Results regarding duration and various antibiotic susceptibility patterns also are presented in Figures 4 through 7. A line diagram shows the trend among the different Gram-positive cocci responses to direct UVC (see Figure 8).

Discussion

  This study demonstrated the excellent in vitro bactericidal effect (100% eradication of organisms) of direct UVC exposure on wound-infecting Gram-positive cocci. The effective irradiation time for 100% eradication varied from 5 to 15 seconds. The short time required to produce the bactericidal effect in vitro should encourage clinicians to use this modality in the treatment of chronic infected wounds; however, the duration of exposure required to produce the bactericidal effect in the clinical setting needs further investigation because it is not a controlled environment (such as an agar plate).

  The depth of UVR penetration in skin depends on wavelength and distance. UVC reaches only into the upper layers of the human epidermis; thus, it demonstrates the least epidermal absorption. Consequently, erythemal reaction would require long exposure duration, which may produce significant cellular necrosis. Hence, UVC exposure time should be selected on the basis of in vitro and in vivo tests of bactericidal efficacy rather than erythemal dose, as is the case when using UVA and UVB.34

   The intensity of illumination varies with the square of the distance between the lamp and treatment surface.35 This means that shortening the distance by 50% between the treating surface and the lamp will increase the dosage by 400%. In the current study, even though the distance from the source to the culture plate was 10 cm as prescribed by the manufacturer, the results were comparable to those of other researchers who may not have used that distance.6-8 The in vitro study conducted by Sullivan and Conner-Kerr6 used a distance of 2.54 cm and a calibrated output of 15.54 mW/cm2 to assess effect on group A S. pyogenes. The authors concluded that short exposure times to UVC (4 seconds, 99.9% kill rate) are detrimental to group A S. pyogenes. In the current study, 100% eradication was obtained in 5 to 15 seconds for Gram-positive cocci, even at a distance of 10 cm and at 5 mJ/s/cm2 (= 5 mW/cm2).   

Staphylococci. UVC (254 nm wavelength) has been reported in previous in vitro study8 to have the ability to kill bacteria, including antibiotic-resistant bacteria such as MRSA. The current in vitro study showed better bacterial efficacy with shorter exposure duration compared to the Conner et al6 study. This could be because of the small proportion (5%) of UVB delivered by the current study equipment, which causes both direct and indirect damage to the DNA molecule as an effect of the strong absorption at wavelengths below 320 nm.36 The current study demonstrated sensitivity to UVR (254 nm) was maximum for MSCONS and minimum for MSSA. The order of UVR sensitivity was MRCONS >MSCONS and MRSA >MSSA. In contrast to the current study, Conner et al27 demonstrated equal sensitivity of MRSA and MSSA to UVR exposure.   

Streptococci. The current study demonstrated 100% eradication of S. pyogenes by 5 seconds, irrespective of their susceptibility patterns. Further study is needed to observe the efficacy of irradiation with <5 seconds, which may minimize the side effects on host cells. In their in vitro study, Sullivan and Conner-Kerr6 showed a 99% kill rate of S. pyogenes (group A Streptococcus—GAS) after 4 seconds of UVC exposure. Due to the technical limitations of the equipment used in the current study, the effect of <5 seconds of UVR exposure could not be assessed. The current study indicates that resistant strains (to fewer than six antibiotics) of Enterococcus species were eradicated with shorter duration (10 seconds) of exposure as compared to that of sensitive strains (15 seconds). In a study by Conner et al8 on E. faecalis (vancomycin-resistant—VRE—and vancomycin-sensitive—VSE) were eradicated by 8 seconds, while VRE were eradicated by 45 seconds.

  In vitro filtered effect of UVC through plastic on common wound infecting organisms. No bactericidal effect was achieved when UVC was filtered through plastic. Subsequently, an experiment was conducted to assess the amount of radiation that passed through the plastic sheet used in this study. Using a UV photometer (UV 1700 series) available at the Department of Biotechnology, Manipal University, the highest optical density (0.079) was 292 nm. Optical densities showed a reduction from 297 nm to 400 nm (100% transmission), indicating complete absorption of the rays below 297 nm (0% transmission). This may be the reason why filtered UVC failed to produce any bactericidal effect through the transparent plastic sheet. The study by MacKinnon and Cleek27 reported inconclusive results regarding the bacteriocidal effects of UV light on a wound covered with a transparent dressing; however, the current study is consistent with findings from Conner et al,34 who reported UVR did not have any effect when filtered. This implies UVC should not be used in clinical situations where frequent removal of dressing material is not feasible. Current reports were consistent with those from Conner et al. However, at this juncture, the current authors believe they can safely conclude that a 0.15-mm thick plastic sheet may be used to protect the surrounding skin from exposure (below 297 nm) during therapy. When using UVR with LAD, a specialized UVC delivery system should be developed similar to the protype device designed by Dai et al,37 constructed to prevent catheter-related infections.

  Physicians, physiotherapists, and nursing professionals frequently use ultraviolet lamps to treat difficult wounds. Many UVR sources available in hospitals do not indicate energy delivered per unit area and percentage of UVA, UVB, and UVC emission. Before initiating treatment and also periodically throughout therapy, energy per unit area should be checked and documented. Duration of each treatment and total number of treatment days with UVR should be determined on the basis of well-designed, randomized controlled trails and also consider permissible exposure limits (1 to 3 minutes) for germicidal UVR (UVC).38

Conclusion

  In an in vitro study, direct UVC has shown bactericidal effect after short exposure (5 seconds) on Gram-positive cocci, but when filtered through a transparent plastic sheet, UVC is ineffective. This beneficial effect of direct UVC in short duration may reduce the bacterial load and simultaneously may minimize the side effects related to longer exposure. From the findings, it also can be concluded that using a 0.15-mm thick plastic sheet may protect the surrounding skin from exposure during therapy. These results can be extrapolated to the clinical situation only with great prudence; the longer exposure times that may be required in clinical situations were not investigated. In vivo studies (animal experiments) should be conducted to ascertain the shortest treatment duration and minimum number of days of treatment to produce bactericidal efficacy using UVC to achieve appropriate microbiological outcome measures. In addition, further studies are required to assess the change in the sensitivity pattern for S. pyogenes and MRCONS with exposure duration <5 seconds. Professionals using UVR equipment in clinical situations should first conduct well-designed in vitro and in vivo experiments to obtain minimum duration of exposure for therapeutic bactericidal efficacy.

Acknowledgment

  The authors gratefully acknowledge the contribution of K. Jagadishchandra, MD during the initial phases of planning of the study and Ms. Asha Kamath for organizing the overall manuscript.

 Ms. B. Rao is an Associate Professor, Department of Physiotherapy, MCOAHS, Manipal University, Manipal, India. Dr. Kumar is Professor and Head, Department of Plastic Surgery; Dr. S. Rao is a Professor, Department of Microbiology; and Dr. Gurung is a postgraduate student, Department of Microbiology, Kasturba Medical College, Manipal, Manipal University. Please address correspondence to: Bhamini K. Rao, Associate Professor, Department of Physiotherapy, MCOAHS, Manipal University, Manipal-575104, Karnataka, India: email: bhaminikr@yahoo.co.in.

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