UV Light Inactivation of MRSA (Methicillin Resistant Staphylococcus aureus)

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1 UV Light Inactivation of MRSA (Methicillin Resistant Staphylococcus aureus) Item Type text; Electronic Thesis Authors Lemons, Katherine Faye Publisher The University of Arizona. Rights Copyright is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 25/07/ :04:20 Link to Item

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4 Abstract: UV light is commonly used as a method of disinfection, as it greatly reduces the amount of bacteria present on a surface or in a solution. The objective of this study was to determine the effect of low pressure ultra violet (UV) light on the inactivation of Methicillin resistant staphylococcus aureus (MRSA) on inanimate objects (fomites). There has been increased use of UV light systems for disinfecting health care environments, but no data is available on the effective doses to kill microorganisms on fomites. Stainless steel, Formica, and flame retardant polyester surfaces were chosen as the substrates as they are most commonly found in the healthcare setting. Methicillinresistant staphylococcus aureus (MRSA) was chosen as the test organism because it is of concern in hospital settings and results in ~10,000 deaths a year. The substrates were inoculated with MRSA and exposed to approximately 1,597μWs/cm 2, 6,042μWs/cm 2, 12,107μWs/cm 2, 17,594μWs/cm 2 or 35,014μWs/cm 2 of UV light to determine the rate of inactivation. The results show that the Stainless Steel had an average log reduction of 4.20 when exposed to a UV intensity of around 35,014μWs/cm 2, the Formica showed around an average log reduction of 3.70 when exposed to a UV intensity of around 35,014μWs/cm 2, and the flame retardant polyester fabric showed an average log reduction of 1.11 when exposed to a UV intensity of 260,825μWs/cm 2.It was found that the killing of the bacterium followed a non linear curve on fomites. Furthermore, there is a dependence on the type of surface as to what UV light intensities are necessary for a reduction in bacteria to be observed. These findings indicate that UV disinfection could be used as an alternative method used to prevent nosocomial infections taking into consideration the type of fomite and the non linear nature of the killing curve. Introduction: Bacteria such as MRSA (methicillin resistant staphylococcus aureus), VRE (vancomycin resistant enterococci) and Clostridium difficile are commonly found on surfaces and are of particular importance in hospital settings as transfer to ill patients is highly likely (Boyce et al. 2011). This can be by direct transfer or transfer through touch (Boyce et al. 2011). MRSA (methicillin resistant staphylococcus aureus) is a bacterium that is a natural inhabitant of human skin and nasal passages as well as it can be commonly found in certain foods such as raw meats (Ferreira J et al. 2014). MRSA has the potential to cause necrotic lesions, abscesses, endocarditis and bacteremia (Ferreira J et al. 2014). Furthermore, this bacterium produces a protein encoded by the gene meca that is able to counteract the effects of certain antibiotics (Ferreira J et al. 2014). This effect is due to the inability of the antibiotics to bind to the cell wall proteins (Ferreira J et al. 2014). Therefore, MRSA can be of particular significance to individuals residing in hospitals or nursing homes as these individuals tend to have lowered defense systems or are immune compromised and therefore more susceptible to infection by these common pathogens (Ferreira J et al. 2014). Information concerning UV light inactivation of bacteria and viruses in liquids has been investigated extensively for the treatment of drinking water and wastewater; however information concerning UV light inactivation on hard surfaces has received little study. This is important because of the increased interest in using UV light for disinfecting health care environments. Data is needed on what doses are needed to kill/inactivate antibiotic resistant bacteria like MRSA. A study conducted by

5 Rönnqvist M. et al. found that norovirus that was allowed to dry on a hard surface could be inactivated by exposure to UV light; however the techniques that were used in the experiment were reported to give an underestimate of the inactivation (Rönnqvist M et al., 2014). The goal of this study was to determine the doses of UV light needed to reduce the level of MRSA on the types of surfaces common in health care environments. Bacterial infections can be induced through many pathways with touch transfer being a very common route. Touch transfer could be due to improper hygiene in which hands are not washed regularly promoting contamination of anything a person touches (Umezawa 2012). However, some studies have shown that UV light decontamination has a significant effect on reducing the levels of Clostridium difficile in hospital patient rooms (Levin et al. 2013). There does seem to be variation in the level of disinfection depending on the type of surface, how long the surface is exposed to UV light, and how close the surface is to the UV light (Boyce, 2011). In a study conducted by Boyce et al. different areas of patient rooms were exposed to 22,000 μws/cm 2 of UV light for varying amounts of time, between 34.2 and minutes (Boyce, 2011). A mean log reduction of 1.7 and 2.9 was determined for C. difficile spores (Boyce, 2011). When a 2 stage UV light treatment was used in which the UV light was placed directly in the area that was contaminated and allowed to run and then the light was placed in a general area in the room and allowed to run (Boyce, 2011). The results showed a log reduction of 1.4 to 3.2 in C. difficile spores, in which the light was run for 72.1 to minutes at the same intensity as the one stage treatment (Boyce, 2011). It is important to determine the effect that UV light has on disinfecting these surfaces as an alternative to traditional cleaning practices as these may not be as effective at disinfecting these hard surfaces. Furthermore, cleaning may not be performed regularly enough to maintain proper disinfection of the surface (Umezawa 2012). UV light decontamination may be a more practical method of disinfecting areas such as hospital rooms or devices as it would not require personnel to spend the time individually cleaning the rooms or devices after each use. One study has shown that UV light disinfection can reduce the working hours of personnel by half when compared to disinfection using ethanol wipes (Umezawa 2012). Additionally, a study conducted by k. Umezawa et al. showed that current measures to disinfect surfaces, such as the use of ethanol wipes, are not as effective because certain strains of bacteria have acquired antimicrobial resistance (Umezawa 2012). This same study has shown that pulsed UV irradiation however is effective at decontaminating patient rooms (Umezawa 2012). Pulsed UV irradiation provides an advantage to continuous UV exposure as it may be more effective at disinfecting the area and does not need as long of an exposure time, however further studies need to be conducted to further elucidate its safety and efficacy (Umezawa 2012). One complication with UV disinfection is that the room may need to be exposed to the light for up to an hour which could be problematic when available rooms are a necessity in hospitals (Umezawa 2012).

6 Methods: MRSA was grown overnight at 37 0 C for 24 hours in tryptic soy broth (TSB). Then the solution was centrifuged and the TSB was poured off. The bacteria pellet was then re suspended in a PBS solution and made to a concentration of A turbidity meter and a McFarland Standard were used to obtain an estimate of the concentration of bacteria so that the same amount could be placed on each test surface every time the experiment was conducted. Once this was completed 100 μl of the solution was used to inoculate each coupon. The coupons were previously wiped down with 70% ethanol and placed under UV light for at least fifteen minutes. The solution was then spread over the coupon using a hockey stick and allowed to dry for 30 minutes. Once dry the coupon was placed under the UV lamp being tested, for a picture of the experimental setup refer to Figure 1. The coupon was exposed to the UV light for approximately 1,597 μws/cm 2, 6,042 μws/cm 2, 12,107 μws/cm 2, 17,594 μws/cm 2 or 35,014 μws/cm 2.Three coupons (triplicate) were used for each exposure. After exposure the coupon was swabbed using sterile swabs and then placed in dilution tubes containing 10 ml of phosphate buffered saline (PBS) solution. Dilutions of this solution were then made, from 10 1 to 10 5, and then 100 μl of each solution was plated on trypticase soy agar (TSA). All samples were plated in duplicate. A control of 100 μl of the solution with the bacterium was placed on the coupon and allowed to dry (30 minutes) but the coupon was never exposed to the UV light. The plates were incubated at 37 0 C for 24 hours and then counted. This method was also used to inoculate the Formica coupons with the only difference being the drying time was shorter, 15 minutes. For the fabric experiments the method was the same for inoculating the surface however instead of using a hockey stick the 100 μl of bacterium solution was pipetted relatively evenly onto the surface in small droplets and then allowed to soak into the fabric. To determine the amount of bacteria that was killed by the UV light 10 ml of PBS was poured into the Petri dish containing the fabric coupon and then the liquid and the fabric were poured into a stomacher plastic bag. The liquid was then squeezed from the fabric and around 2 3 mls of liquid were removed using a pipet. The amount of liquid that was removed was recorded for each sample. The method from this point on was the same as for the hard surfaces. For the fabric the coupon was exposed to higher doses of UV light: 124,716μWs/cm 2 and 236,256μWs/cm 2. The equation used to calculate the intensity of the UV light was: I ave = I o (1 e a(e)(l) )/(a(e)(l)), in which I o is the average measured intensity in μw/cm 2, a is a constant of 0.008, e is a constant of 2.303, and L is the distance from the UV light device to the surface that the UV light is intended to disinfect

7 Figure 1: Depiction of the experimental setup Results: Both the Stainless steel and the Formica surfaces showed a significant decline in bacteria present on the coupon when the coupon was exposed to UV intensities of 1,597 μws/cm 2, 6,042 μws/cm 2, 12,107 μws/cm 2, 17,594 μws/cm 2 or 35,014 μws/cm 2. The stainless steel had an average log reduction of 4.20 and the Formica had an average log reduction of The flame retardant polyester fabric showed significantly less reduction in bacteria with an average of 1.11 log reduction when the coupon was exposed to an intensity of 260,825μWs/cm 2.

8 Table 1: Effect of UV light dose on killing of MRSA on Stainless Steel (experiment 1) UV Exposure Stainless Steel Experiment 1 Control Replicate CFU/mL CFU/cm 2 CFU/mL CFU/cm2 Standard Deviation Average REDUCTION A 1.1E E B 1.2E E C 1.2E E A 1.7E E B 2.0E E C 5.6E E A 2.4E E B 9.7E E C 2.3E E A 1.8E E B 1.3E E C 7.8E E Figure 2: Log reduction vs. UV light dose. STAINLESS STEEL EXPERIMENT REDUCTION UV EXPOSURE (MW/SEC/CM2)

9 Table 2: Effect of UV light dose on killing of MRSA on Stainless Steel (experiment 2) UV Exposure Stainless Steel Experiment 2 Control Replicate CFU/mL CFU/cm 2 CFU/mL CFU/cm2 Standard Deviation Average A 2.6E E B 6.6E E C 3.4E E Log Reduction CFU/mL A 1.4E E B 3.1E E C 6.3E E A 7.0E E B 1.2E E C 3.5E E A 6.0E E B 1.5E E C 2.0E E Figure 3: Effect of UV light dose on killing of MRSA on Stainless Steel (experiment 2) STAINLESS STEEL EXPERIMENT REDUCTION Series1 3.5 UV EXPOSURE (MW/SEC/CM2)

10 Table 3: Effect of UV light dose on killing of MRSA on Stainless Steel (experiment 3) UV Exposure Stainless Steel Experiment 3 Control Replicate CFU/mL CFU/cm 2 CFU/mL CFU/cm2 Standard Deviation Average A 9.8E E B 7.2E E Log Reduction C 5.6E E A 1.9E E B 1.4E E C 1.0E E A 1.5E E B 1.0E E C 1.5E E A 4.0E E B 3.0E E C 1.0E E A 9.0E E B 1.5E E C 1.0E E Figure 4: Effect of UV light dose on killing of MRSA on Stainless Steel (experiment 4) STAINLESS STEEL EXPERIMENT 3 REDUCTION UV EXPOSURE (MW/SEC/CM2)

11 Table 4: Effect of UV light dose on killing of MRSA on Stainless Steel (experiment 4) UV Exposure Stainless Steel Experiment 4 Replicate CFU/mL CFU/cm 2 CFU/mL CFU/cm2 Standard Deviation Average A 6.6E E Log Reduction Control B 4.2E E C 1.2E E A 1.7E E B 1.9E E C 1.5E E A 9.1E E B 2.7E E C 1.8E E A 5.0E E B 2.5E E C 1.4E E A 1.5E E B 1.7E E C 6.0E E A 1.2E E B 7.0E E C 1.8E E

12 Figure 5: Effect of UV light dose on killing of MRSA on Stainless Steel (experiment 4) REDUCTION STAINLESS STEEL EXPERIMENT UV EXPOSURE (MW/SEC/CM2) Table 5: Effect of UV light dose on killing of MRSA on Stainless Steel (experiment 5) UV Exposure Stainless Steel Experiment 5 Control Replicate CFU/mL CFU/cm 2 CFU/mL CFU/cm2 Standard Deviation Average Log Reduction A 1.8E E B 1.5E E C 2.5E E A 1.2E E B 1.1E E C 2.0E E A 2.3E E B 1.5E E C 1.1E E A 5.1E E B 5.6E E C 3.9E E A 4.5E E B 6.5E E C 1.5E E A 9.0E E B 8.5E E C 1.3E E

13 Figure 6: Effect of UV light dose on killing of MRSA on Stainless Steel (experiment 5) STAINLESS STEEL EXPERIMENT 5 REDUCTION UV EXPOSURE (MW/SEC/CM2) Series1

14 UV Exposure Control Table 6: Effect of UV light dose on killing of MRSA on Stainless Steel (experiment 6) Formica Experiment 1 Replicate CFU/mL CFU/cm 2 CFU/mL CFU/cm2 Standard Deviation Average Log Reduction A 2.5E E B 2.7E E C 7.9E E A 5.7E E B 4.1E E C 1.5E E A 2.6E E B 7.4E E C 3.3E E A 3.0E E B 2.7E E C 3.0E E A 1.5E E B 2.3E E C 3.5E E A 1.0E E B 1.5E E C 1.0E E Figure 7: Effect of UV light dose on killing of MRSA on Stainless Steel (experiment 6) REDCUTION FORMICA EXPERIMENT UV EXPOSURE (ΜW/SEC/CM2)

15 UV Exposure Control Table 7: Effect of UV light dose on killing of MRSA on Formica (experiment 2) Formica Experiment 2 Replicate CFU/mL CFU/cm 2 CFU/mL CFU/cm2 Standard Deviation Average Log Reduction A 7.2E E B 1.1E E C 7.0E E A 1.8E E B 7.0E E C 1.4E E A 2.2E E B 2.5E E C 8.0E E A 7.3E E B 2.2E E C 6.0E E A 2.5E E B 4.2E E C 3.0E E A 8.0E E B 1.3E E C 2.5E E Figure 8: Effect of UV light dose on killing of MRSA on Formica (experiment 2) REDCUTION FORMICA EXPERIMENT UV EXPOSURE (ΜW/SEC/CM2)

16 UV Exposure Formica Experiment 3 UV Exposure Control Table 8: Effect of UV light dose on killing of MRSA on Formica (experiment 3) Formica Experiment 3 Replicate CFU/mL CFU/cm 2 CFU/mL CFU/cm2 Standard Deviation Average Log Reduction A 3.0E E B 2.7E E C 6.9E E A 5.7E E B 3.3E E C 1.5E E A 2.6E E B 7.4E E C 3.3E E A 3.0E E B 2.7E E C 3.0E E A 1.6E E B 2.3E E C 3.5E E A 1.5E E B 1.5E E C 1.0E E Figure 9: Effect of UV light dose on killing of MRSA on Formica (experiment 3) REDCUTION FORMICA EXPERIMENT UV EXPOSURE (ΜW/SEC/CM2)

17 UV Exposure Control Table 9: Effect of UV light dose on killing of MRSA on flame retardant polyester fabric (experiment 2) Experiment 2 Replicate CFU/mL CFU/cm 2 CFU/mL CFU/cm2 Standard Deviation Average A 1.6E E B 1.6E E Log Reduction C 1.0E E A 8.1E E B 1.2E E C 1.1E E A 1.2E E B 2.3E E C 2.2E E Figure 10: Effect of UV light dose on killing of MRSA on flame retardant polyester fabric (experiment 2) REDCUTION FABRIC EXPERIMENT UV EXPOSURE (ΜW/SEC/CM2)

18 Table 10: Effect of UV light dose on killing of MRSA on flame retardant polyester fabric (experiment 3) Control Replicate CFU/mL CFU/cm 2 CFU/mL CFU/cm2 Standard Deviation Average A 1.3E E B 1.2E E Log Reduction C 1.2E E A 1.2E E B 7.9E E C 9.4E E A 2.2E E B 6.1E E C 1.2E E Figure 11: Effect of UV light dose on killing of MRSA on flame retardant polyester fabric (experiment 3) REDCUTION FABRIC EXPERIMENT UV EXPOSURE (ΜW/SEC/CM2)

19 Table 11: Log reductions observed for the Stainless Steel surface. UV Exposure Average Log Reduction Standard Deviation 1,558 1, ,788 6, ,948 12, ,594 17, ,014 35, Table 12: Log reductions observed for the Formica surface. UV Exposure Average Log Reduction Standard Deviation 1,193 1, ,543 6, ,951 12, ,762 18, ,433 37, Figure 12: Log reductions observed for the Stainless Steel and Formica surfaces. 6 UV Inactivation of MRSA Average Log 10 Reduction UV Exposure 1,193 1,632 5,543 6,540 10,951 12,637 15,762 18,872 30,433 37,032 0 STAINLESS STEEL FORMICA 1

20 Table 13: Log reductions of MRSA on flame retardant polyester fabric. UV Exposure Average Log Reducion Standard Deviation 121, , , , Figure 13: Log reductions of flame retardant polyester fabric Average Log 10 Reduction , , , ,395 UV Exposure

21 Discussion: The results of this study indicate that inactivation of bacteria on hard surfaces differs from inactivation of bacteria in liquids. Previous studies demonstrate that there is a linear decline in various pathogens when in solutions such as water (Hijnen, 2006). However, on hard surfaces such as Stainless Steel and Formica there was a steep decline in the bacteria present, followed by a leveling off as the exposure time to UV light was increased (Figures 2 thru 9). As the dose of UV light was increased from 1,597 μws/cm 2 to 6,042 μws/cm 2 and then to 12,107 μws/cm 2, it was found that the inactivation or kill rate declined significantly. This could be due to the possibility that the bacteria aggregated together or may have grouped on top of one another in layers. The top layer may be killed by the UV light however the UV light may not reach the lower layers. This could be particularly significant in the instances in which the solution tended to bead up more on the coupon and did not dry evenly on the surface. The first experiment performed with the flame retardant polyester fabric did not yield any significant reduction in the amount of bacteria on the surface of the coupon; therefore the dosage of UV light was significantly increased, as can be seen in Figures 10 and 11. Further considerations to take into account are the various drying periods. The nature of the surface caused the bacteria to bead up more on some of the coupons; while on others it spread out evenly, indicating that the hydrophobic nature of the Stainless Steel coupon had an effect on drying. Future experimentation concerning UV light inactivation should be performed in order to determine how UV light affects different strains of bacteria as well as to determine if this relationship is seen on all types of hard surfaces or if there is considerable variation, as the fabric experiment might indicate. The fabric also seemed to need significantly increased exposure to the UV light indicating that UV inactivation on hard surfaces depends on the surface that is being disinfected. This can be seen in the tables in which the fabric was only starting to show reductions in the bacteria by the time that the Stainless Steel and Formica surfaces had started to level off (Graphs 11 and 12). Another possible consideration might be that the bacterium has photo repair capabilities that might be more significant depending on what surface it is on. Conclusions: A non linear killing curve was observed for MRSA on hard surfaces. The Stainless Steel and Formica showed a steep curve that leveled off while the flame retardant polyester fabric showed more of a linear relationship. Therefore, there is a dependence on the type of surface as to what UV light intensities are necessary for a reduction in bacteria. These findings indicate that UV disinfection could be used as an alternative method used to prevent nosocomial infections in particular on surfaces such as Stainless Steel and Formica.

22 References: Boyce M. John, Nancy L. Havill, and Brent A. Moore. Terminal Decontamination of Patient Rooms Using an Automated Mobile UV Light Unit. Chicago Journals (2011): Web. 27 Dec Levin, Joanne, Linda S. Riley, Christine Parrish, Daniel English, and Sehoon Ahn, The effect of portable pulsed xenon ultraviolet light after terminal cleaning on hospital associated Clostridium difficile infection in a community hospital. American journal of Infection Control (2013): Web. 27 Dec Ferreira J, Costa W, Cerqueira E, Carvalho J, Oliveira L, Almeida R., Food handler associated methicillinresistant Staphylococcus aureus in public hospitals in Salvador, Brazil. Food Control. March 2014;37: Web. 18 Feb Hijnen, W. M., Beerendonk, E. F., & Medema, G. J. (2006). Inactivation credit of UV radiation for viruses, bacteria and protozoan (oo)cysts in water: A review. Water Research, 40(1), doi: /j.watres Rönnqvist M., Mikkelä A., Tuominen P., Salo S., Maunula L., Ultraviolet Light Inactivation of Murine Nororvirus and Human Norovirus GII: PCR May Overestimate the Persistence of Noroviruses Even When Combined with Pre PCR Treatments. Food Environment Virology. 6 (2014): Web. 16 Feb Nerandzic M., Michelle, Jennifer Cadnum L., Kevin E. Eckart, and Curtis J. Dnskey, "Evaluation of a handheld far ultraviolet radiation device for decontamination of Clostridium difficile and other healthcare associated pathogens." BMC Infectious Diseases (2012): Web. 27 Dec Umezawa Kazuo, Satomi Asai, Sadaki Inokuchi, and Hayato Miyachi. A Comparative Study of the Bactericidal Activity and Daily Disinfection Housekeeping Surfaces by a New Portable Pulsed UV Radiation Device. Curr Microbiol. 64 (2012): Web. 27 Dec