Assessment of Antibacterial Efficacy of against MRSA and VRE Sheri Maxwell, B.S. Charles P. Gerba, Ph.D. Department of Soil, Water and Environmental Science University of Arizona Tucson, Arizona. 85721 April 2, 2008
Objective The purpose of this project was to demonstrate the efficacy and residual effects of in inactivating Methicillin resistant Staphylococcus aureus (MRSA) and Vancomycin resistant Enterococcus (VRE) on stainless steel, plastic, vinyl and ceramic tiles. Methods Stainless steel, acrylic plastic (Altuglas International, Philadelphia, PA) and glazed almond colored ceramic tiles (4x4 inch) (Dal-Tile Corporation, Dallas Texas) were purchased at a local building supply store in Tucson, Arizona. Vinyl, product # PLCX4 (Spralding, Pelham, AL) (1x1 inch squares were used) was purchased at a local sewing supply store in Tucson, Arizona. Test Procedure Efficacy The test surfaces were inoculated with 100 µl of the bacterial suspension applied in 10 micro liter droplets using a micropipetter. After allowing the bacterial suspension to air dry for 20 minutes, was applied to the surface using a pump spray bottle held approximately 12 inches from the surface. The product was applied in a sweeping motion and allowed to dry before testing. After various periods of time the, surfaces were swabbed using cotton-tipped applicator swabs premoistened in DE neutralizing broth (Remel, Lenexa, KS). The swabs were then placed into 1 ml of DE broth and vortexed for 30 seconds to recover the organisms. Bacteria were assayed by the spread plate method onto tryptic soy agar (Difco, Sparks, MD) amended with 5% sheep blood (Hardy Diagnostics, Mesa, AZ) and incubated at 37 C for 24 hours. Agar plates were incubated at 37 C for 24-48 hours. After incubation, bacteria colonies were counted and log reduction was calculated ([Initial concentration/final Concentration] x log = log reduction). Residual Surfaces were sprayed with and allowed to stand at room temperature for 3, 5, 10 and 14 days. To determine the residual effects of, surfaces that had been treated with 3 days before were then inoculated with 100 µl of the bacterial suspension applied in 10 micro liter droplets using a micropipetter. The residual procedure was repeated with surfaces that had been treated with for 5, 10, and 14 days prior to testing. At the designated time points, surfaces were swabbed using cotton-tipped applicator swabs pre-moistened in DE neutralizing broth (Remel, Lenexa, KS). The swabs were then placed into 1 ml of DE broth and vortexed for 30 seconds to recover the organisms. Bacteria were assayed by the spread plate method onto tryptic soy agar (Difco) amended with 5% sheep blood (Hardy Diagnostics, Mesa, AZ) and incubated at 37 C for 24 hours. Agar plates were incubated at 37 C for 24-48 hours. After 2
incubation, bacterial colonies were counted and log reduction was calculated ([Initial concentration/final Concentration] x log = log reduction). Test Organisms MRSA - An overnight culture of MRSA obtained from the American Type Culture Collection (ATCC 700698) was grown in Trypticase Soy Broth (TSB) (Difco, Sparks, MD). Bacteria were prepared by centrifugation and resuspension in sterile physiological saline. This bacterial suspension was used as an inoculum on the test surfaces. VRE - An overnight culture of VRE (ATCC 51559) was grown in Trypticase Soy Broth (TSB) (Difco, Sparks, MD). Bacteria were prepared by centrifugation and resuspension in sterile physiological saline. This bacterial suspension was used as an inoculum on the test surfaces. Results MRSA Stainless Steel The efficacy of against MRSA on stainless steel is shown in Table 1. An initial sample was collected as soon as the surfaces were inoculated. A reduction of 99.9% of MRSA occurred immediately after exposure to. After one minute of exposure to, MRSA was reduced by 99.9%. No MRSA could be detected after three minutes of contact (>99.99% reduction). Table 1. Efficacy of against MRSA on Stainless Steel 7.70E+05 2.00E+05 2.70E+05 2.90E+05 1.30E+05 1.10E+03 193 <10 <10 <10 Log Reduction 2.85 3 >4.43 >4.46 >4.11 Percent Reduction 99.9 99.9 >99.99 >99.99 >99.99 3
Figure 1. Colonies of MRSA on Blood Agar before and after Exposure to Table 2 shows the residual action of on stainless steel with MRSA over a 14 day period. MRSA was reduced by greater than 99% in one minute after three, five, ten and fourteen days post application of. Table 2. Residual Action of against MRSA on Stainless Steel 3 day Residual 1.23E+06 4.90E+06 8.20E+06 3.60E+06 2.20E+06 1.80E+04 3.90E+03 3.50E+03 427 143 Log Reduction 1.83 3.1 3.37 3.92 4.2 Percent Reduction 98.5 99.92 99.96 99.99 99.99 5 day Residual 2.10E+06 3.10E+06 4.30E+06 3.60E+06 1.60E+06 7.40E+04 1.80E+04 1.90E+04 1.20E+03 643 Log Reduction 1.45 2.24 2.35 3.48 3.4 Percent Reduction 96.5 99.4 99.6 99.97 99.96 10 day Residual 3.60E+06 6.60E+06 7.40E+06 2.50E+06 8.50E+06 2.80E+04 4.00E+04 3.30E+04 1.20E+04 4.10E+04 Log Reduction 2.1 2.21 2.35 2.22 2.32 Percent Reduction 99.22 99.4 99.55 99.52 99.52 14 day Residual 1.50E+06 4.50E+06 3.40E+06 3.00E+06 2.00E+06 3.10E+05 3.10E+04 2.00E+04 2.10E+04 1.00E+03 Log Reduction 0.68 2.16 2.23 2.15 3.3 Percent Reduction 79.33 99.31 99.41 99.3 99.95 4
MRSA Plastic In this test, the efficacy of was tested against MRSA on acrylic plastic. A reduction of 99.9% of MRSA occurred immediately after exposure to (Table 3). After one minute of exposure to, MRSA was reduced by 99.99% and by 99.99% after three minutes. Table 3. Efficacy of against MRSA on Plastic 1.50E+05 2.50E+05 2.30E+05 3.60E+05 5.80E+05 760 17 54 40 60 Log Reduction 2.3 4.17 3.63 3.95 3.99 Percent Reduction 99.5 99.99 99.99 99.99 99.99 Figure 3. Colonies of MRSA on Blood Agar Before and After Exposure to on a Plastic Surface Table 4 shows the residual action of on plastic MRSA over a 14 day period. MRSA was reduced by greater than 99% after one minute but did not increase after for after ten minutes of exposure. After 14 days post application the MRSA was reduced by 99% in 10 minutes. 5
Table 4. Residual Action of against MRSA on Plastic 3 day Residual 1.70E+07 1.20E+07 1.20E+07 1.30E+07 8.90E+06 2.10E+04 3.00E+04 4.10E+04 2.60E+04 1.40E+04 Log Reduction 2.91 2.6 2.47 2.7 2.8 Percent Reduction 99.88 99.75 99.66 99.8 99.84 5 day Residual 1.00E+07 9.90E+07 1.00E+07 9.80E+06 8.00E+06 9.50E+04 4.20E+04 6.70E+04 1.30E+04 8.80E+03 Log Reduction 2.02 3.37 2.17 3.12 2.96 Percent Reduction 99.05 99.96 99.33 98.87 99.89 10 day Residual 7.00E+06 6.30E+06 9.40E+06 1.10E+06 7.00E+06 1.10E+05 1.00E+04 6.80E+04 1.30E+04 9.50E+04 Log Reduction 1.8 3.2 2.14 1.93 1.87 Percent Reduction 98.43 99.84 99.28 98.82 98.64 14 day Residual 6.40E+06 5.40E+06 4.10E+06 1.20E+06 8.90E+06 5.50E+06 4.10E+05 3.80E+05 4.30E+04 4.10E+04 Log Reduction 0.06 1.12 1.03 1.45 2.34 Percent Reduction 14.06 92.4 90.73 96.4 99.54 MRSA - Vinyl reduced MRSA by greater than 99.9% immediately after exposure. Table 5. Efficacy of against MRSA on Vinyl 1.40E+04 1.30E+04 1.40E+04 1.40E+04 1.50E+04 <10 <10 <10 <10 <10 Log Reduction >2.59 >2.62 >2.59 >2.59 >2.56 Percent Reduction >99.92 >99.92 >99.92 >99.92 >99.93 6
slowly loss efficacy 10 days after application on the Vinyl (Table 6 and Figure 5). After 5 days application, bacterial numbers were reduced by 99% at 3, 5 and 10 minutes of exposure. A 97% reduction occurred after 10 days of application with all exposure times. After 14 days of application this was reduced to 88% even after ten minutes of exposure. Table 6. Residual Action of against MRSA on Vinyl 3 day Residual 4.90E+06 1.00E+06 9.50E+06 9.60E+06 8.00E+06 5.20E+04 4.30E+04 1.50E+04 1.00E+04 280 Log Reduction 1.97 1.37 2.8 2.98 4.5 Percent Reduction 98.9 95.7 99.8 99.9 99.99 5 day Residual 8.10E+06 1.10E+06 8.60E+06 1.10E+06 1.50E+06 9.00E+04 1.20E+04 1.10E+04 9.00E+03 1.10E+03 Log Reduction 1.95 1.96 2.89 2.1 3.13 Percent Reduction 98.9 98.9 99.9 99.2 99.9 10 day Residual 6.70E+06 6.90E+06 7.30E+06 7.90E+06 7.70E+06 1.70E+05 1.80E+05 1.90E+05 1.90E+05 2.00E+05 Log Reduction 1.6 1.58 1.58 1.62 1.59 Percent Reduction 97.5 97.4 97.4 97.6 97.4 14 day Residual 9.90E+06 8.50E+06 9.90E+06 9.50E+06 7.50E+06 1.20E+06 1.00E+06 1.10E+06 1.20E+06 1.00E+06 Log Reduction 0.92 0.93 0.95 0.9 0.88 Percent Reduction 87.9 88.2 88.9 87.4 86.7 7
MRSA Tile In this test, the efficacy of was tested against MRSA on ceramic tiles (Table 7). A 71.2% reduction was observed initially increasing to 98.6% after 10 minutes. Table 7. Efficacy of against MRSA on Tiles 1.70E+06 1.60E+06 1.50E+06 1.20E+06 1.00E+06 4.90E+05 8.80E+04 5.20E+04 2.30E+04 1.40E+04 Log Reduction 0.54 1.26 1.46 1.72 1.85 Percent Reduction 71.2 94.5 96.5 98.1 98.6 Figure 6. Colonies of MRSA on Blood Agar Before and After Exposure to on a Plastic Surface 8
Table 8 shows the residual action of on tiles against MRSA over a 14day period. MRSA was reduced by greater than 99% in three minutes after three and five days post application of. After ten and fourteen days post application MRSA was reduced by greater than 99% at 10 minutes. Table 8. Residual Action of against MRSA on Tiles 3 day Residual 4.90E+05 8.70E+05 7.50E+05 7.10E+05 6.90E+05 6.40E+04 5.80E+04 1.60E+03 357 387 Log Reduction 0.88 1.18 2.67 3.3 3.3 Percent Reduction 86.9 93.3 99.8 99.95 99.9 5 day Residual 3.20E+06 4.50E+06 5.10E+06 2.40E+06 1.50E+06 1.70E+04 8.60E+04 3.90E+04 6.60E+04 1.60E+04 Log Reduction 2.27 1.71 2.12 1.56 1.97 Percent Reduction 99.5 98.1 99.2 97.3 98.9 10 day Residual 4.50E+06 5.90E+06 5.90E+06 6.60E+06 2.50E+06 3.80E+04 4.00E+04 6.30E+04 7.70E+04 2.30E+04 Log Reduction 2.07 2.17 1.97 1.93 2.03 Percent Reduction 99.2 99.32 98.9 98.8 99.1 14 day Residual 3.20E+06 2.70E+06 5.80E+06 6.60E+06 7.10E+06 2.50E+06 3.10E+05 4.50E+05 5.10E+05 1.00E+04 Log Reduction 0.1 0.9 1.11 1.11 2.85 Percent Reduction 21.9 88.5 92.2 92.3 99.9 9
VRE Stainless Steel VRE was reduced by greater than 99.99% after one minute. Table 9. Efficacy of against VRE on Stainless Steel 4.60E+05 3.10E+05 2.40E+05 1.50E+05 4.40E+05 970 <10 <10 <10 <10 Log Reduction 2.68 >4.49 >4.38 >4.2 >4.64 Percent Reduction 99.8 >99.99 >99.99 >99.99 >99.99 Figure 8. Colonies of VRE on Blood Agar Before and After Exposure to on Stainless Steel 10
Residual action of against VRE on stainless steel is shown in Table 10. VRE was reduced by greater than 99.99% after 3 days post application after five minutes. After 5 days post application the bacteria were reduced by 99% in 1 and 3 minutes and greater than 99.9% reduction was seen at the 5 and 10 minute points. VRE continued to be reduced after 10 and 14 days of application by greater than 99% in 3 minutes. Table 10. Residual Action of against VRE on Stainless Steel 3 day Residual 4.10E+06 1.30E+06 1.20E+06 1.00E+06 3.30E+06 2.60E+03 687 567 110 220 Log Reduction 3.2 3.28 2.67 3.96 4.18 Percent Reduction 99.94 99.95 99.95 99.99 99.99 5 day Residual 4.70E+05 8.00E+05 1.30E+05 1.20E+05 1.10E+05 7.60E+03 2.10E+03 1.30E+03 97 43 Log Reduction 1.79 2.58 2.0 3.09 3.41 Percent Reduction 98.38 99.73 99.0 99.92 99.96 10 day Residual 2.20E+06 1.70E+06 3.50E+06 6.60E+06 6.70E+06 2.90E+04 2.40E+04 3.10E+04 1.30E+04 3.10E+04 Log Reduction 1.88 1.85 2.05 2.7 2.33 Percent Reduction 98.69 98.59 99.11 99.8 99.54 14 day Residual 7.40E+06 4.40E+06 3.00E+06 3.10E+06 1.20E+06 2.30E+05 1.50E+05 1.30E+04 1.10E+04 8.80E+03 Log Reduction 1.51 1.47 2.36 2.43 2.13 Percent Reduction 96.89 96.6 99.57 99.65 99.27 VRE Plastic was very effective against VRE on plastic. A reduction of greater than 99.99% occurred after just one minute (Table 11). Table 11. Efficacy of against VRE on Plastic 2.70E+05 2.80E+05 2.00E+05 4.00E+05 4.90E+05 1.10E+03 <10 <10 <10 <10 Log Reduction 2.39 >4.45 >4.3 >4.6 >4.69 Percent Reduction 99.6 >99.99 >99.99 >99.998 >99.998 11
The residual action of against VRE on plastic is shown in Table 12. After 3, 5 and 10 days post application, VRE was reduced by greater than 99% after 1 minute. After 14 days application, VRE was reduced 99% after 3 minutes of exposure. Table 12. Residual Action of against VRE on Plastic 3 day Residual 3.80E+06 2.90E+06 4.40E+06 5.30E+06 2.90E+06 4.50E+03 7.00E+03 1.10E+03 1.80E+03 387 Log Reduction 2.93 2.62 3.6 3.47 3.88 Percent Reduction 99.88 99.76 99.98 99.66 99.99 5 day Residual 3.30E+06 4.90E+06 3.90E+06 2.70E+06 3.10E+06 7.20E+03 5.40E+03 1.70E+03 567 357 Log Reduction 2.66 2.96 3.36 3.67 3.94 Percent Reduction 99.78 99.99 99.96 99.98 99.99 10 day Residual 3.40E+06 3.60E+06 3.50E+06 4.80E+06 4.00E+06 3.40E+04 1.70E+04 2.40E+04 4.80E+04 2.70E+04 Log Reduction 2 2.33 2.16 2 2.17 Percent Reduction 98.87 99.53 99.31 99.0 99.33 14 day Residual 7.40E+06 4.60E+06 6.40E+06 8.10E+06 3.30E+06 1.00E+05 2.10E+05 4.00E+04 6.00E+04 4.00E+04 Log Reduction 1.87 1.28 2.2 2.13 1.92 Percent Reduction 98.65 95.43 99.38 99.26 98.79 VRE Vinyl reduced VRE on vinyl by greater than 99.9% immediately after the initial application (Table 13). Table 13. Efficacy of against VRE on Vinyl 3.30E+04 2.50E+04 2.50E+04 2.40E+04 2.30E+04 <10 <10 <10 <10 <10 Log Reduction >2.21 >2.34 >3.40 >2.36 >2.38 Percent Reduction >99.97 >99.96 >99.96 >99.96 >99.96 12
VRE was reduced by 99.99% after 3 days post application after one minute of exposure (Table 14). After 5 days post application, VRE was reduced by greater than 99% after just one minute, greater than 99.9% and after 10 minutes by greater than 99.999. At 10 days post application, VRE was reduced by 99.7% after ten minutes of exposure. After 14 days post application, the percent reduction was less, reaching 94% after ten minutes. Table 14. Residual Action of against VRE on Vinyl 3 day Residual 2.00E+06 3.70E+06 2.60E+06 4.80E+06 3.30E+06 4.00E+03 190 110 90 90 Log Reduction 2.7 4.3 4.34 4.73 4.56 Percent Reduction 99.8 99.99 99.99 99.99 99.99 5 day Residual 2.50E+06 3.20E+06 3.60E+06 2.60E+06 2.90E+06 1.80E+04 5.00E+03 1.20E+03 2.00E+03 <10 Log Reduction 2.14 2.81 3.48 3.11 >5.46 Percent Reduction 99.3 99.8 99.97 99.9 >99.999 10 day Residual 4.20E+06 2.40E+06 2.50E+06 3.10E+06 2.50E+06 1.80E+04 1.50E+04 7.70E+03 9.20E+03 7.30E+03 Log Reduction 2.37 2.2 2.51 2.53 2.53 Percent Reduction 99.6 99.4 99.7 99.7 99.7 14 day Residual 1.40E+06 2.50E+06 2.30E+06 2.90E+06 2.00E+06 1.50E+05 1.30E+05 1.60E+05 1.50E+05 1.50E+05 Log Reduction 0.97 1.28 1.16 1.29 1.1 Percent Reduction 89.3 94.8 93.0 94.8 92.5 13
VRE Ceramic Tiles VRE was reduced by 99% after 1 and 3 minutes, and greater than 99.98% after 10 minutes exposure on ceramic tiles (Table 15). Table 15. Efficacy of against VRE on Tiles 6.50E+04 5.90E+04 5.60E+04 6.20E+04 4.00E+04 2.40E+03 370 180 37 <10 Log Reduction 1.43 2.2 2.8 3.22 >3.6 Percent Reduction 96.3 99.4 99.7 99.94 >99.98 Figure 11. Colonies of VRE on Blood Agar Before and After Exposure to on Tiles 14
After 3 days post application of, VRE was reduced by 99.9% after one minute and by 99.99% after 10 minutes (Table 16). Five days after application reduction of VRE was still reduced by 99.97% after ten minutes, after 10 and 14 days this was reduced to approximately 99%. Table 16. Residual Action of against VRE on Tiles 3 day Residual 8.70E+06 7.40E+06 8.80E+06 1.40E+06 5.70E+06 1.30E+04 2.80E+03 77 20 310 Log Reduction 2.83 3.42 5.1 4.84 4.26 Percent Reduction 99.9 99.9 99.99 99.99 99.99 5 day Residual 5.50E+06 4.00E+06 6.20E+06 2.30E+06 1.70E+06 2.70E+04 1.00E+03 1.20E+03 150 430 Log Reduction 2.31 3.6 3.7 4.18 3.6 Percent Reduction 99.5 99.98 99.98 99.99 99.97 10 day Residual 4.50E+05 1.90E+05 9.50E+05 9.30E+05 5.00E+05 4.67E+04 3.10E+03 5.60E+03 1.40E+03 1.30E+03 Log Reduction 0.98 1.78 2.22 2.82 2.58 Percent Reduction 89.6 98.4 99.4 99.8 99.7 14 day Residual 5.10E+06 7.20E+06 4.40E+06 5.10E+06 3.00E+06 4.80E+05 6.60E+05 2.20E+05 1.30E+05 3.20E+04 Log Reduction 1.02 1.03 1.3 1.59 1.97 Percent Reduction 90.6 90.8 95.0 97.5 98.9 15
Summary of Test Results MRSA exhibited different rates of inactivation on the different test surfaces, however within ten minutes contact the bacterium was inactivated by 99.99% or greater on all the surfaces except the ceramic tiles (Table 17). The degree of inactivation of MRSA did not increase significantly after one minute of exposure on the ceramic tiles, although there was a 98.6% reduction in the organism after ten minutes. Table 17. Summary of the efficacy of against MRSA Surface Steel 99.9 99.9 >99.99 >99.99 >99.99 Plastic 99.5 99.99 99.99 99.99 99.99 Vinyl >99.92 >99.92 >99.92 >99.92 >99.93 Tiles 71.2 94.5 96.5 98.1 98.6 was more effective against VRE with more than 99.99% of the initial titer being inactivated within less than a minute on all the test surfaces except the ceramic tile (Table 18). In the case of the tiles, 10 minutes of contact was require to achieve greater than a 99.99% reduction. Table 18. Summary of the efficacy of against VRE Surface Steel 99.8 >99.99 >99.99 >99.99 >99.99 Plastic 99.6 >99.99 >99.99 >99.998 >99.998 Vinyl >99.97 >99.96 >99.96 >99.96 >99.96 Tiles 96.3 99.4 99.7 99.94 >99.98 16
MRSA The residual effects of against MRSA are shown in Table 19. Some loss of efficacy of the was observed on all the surfaces studied over the 14 days of the study. However, this did not appear to be significant for the ceramic tiles. The residual effect decreased the most on the vinyl surface. However, on the other surfaces a 99% or more of the MRSA occurred after 10 minutes exposure even 14 days after treatment. Table 19. Summary of Residual Action of against MRSA Number of Days Post Application of 3 Days 5 Days 10 Days 14 Days Minutes of Exposure Steel Plastic Tiles Vinyl Steel Plastic Tiles Vinyl Steel Plastic Tiles Vinyl Steel Plastic Tiles Vinyl 1 99.92 99.75 93.3 95.7 99.4 99.96 98.1 98.9 99.4 99.84 99.32 97.4 99.31 92.4 88.5 88.2 3 99.96 99.66 99.8 99.8 99.6 99.33 99.2 99.9 99.55 99.28 98.9 97.4 99.41 90.73 92.2 88.9 5 99.99 99.8 99.95 99.9 99.97 98.87 97.3 99.2 99.52 98.82 98.8 97.6 99.3 96.4 92.3 87.4 10 99.99 99.84 99.9 99.99 99.96 99.89 98.9 99.9 99.52 98.64 99.1 97.4 99.95 99.54 99.9 86.7
VRE was more effective against VRE than MRSA on all of the surfaces tested. After five days it was still capable of inactivating at least 99.9% of VRE and at 10 days at least 99%. As with MRSA the action against VRE decreased the most over time on vinyl surfaces. Still 92 to 99% of the VRE could be inactivated after a period of ten minutes contact. Table 20. Summary of Residual Action of against VRE Number of Days Post Application of 3 Days 5 Days 10 Days 14 Days Minutes of Exposure Steel Plastic Tiles Vinyl Steel Plastic Tiles Vinyl Steel Plastic Tiles Vinyl Steel Plastic Tiles Vinyl 1 99.95 99.76 99.9 99.99 99.73 99.99 99.98 99.8 98.59 99.53 98.4 99.4 96.6 95.43 90.8 94.8 3 99.95 99.98 99.99 99.99 99.0 99.96 99.98 99.97 99.11 99.31 99.4 99.7 99.57 99.38 95.0 93.0 5 99.99 99.66 99.99 99.99 99.92 99.98 99.99 99.9 99.8 99.0 99.8 99.7 99.65 99.26 97.5 94.8 10 99.9999 99.99 99.99 99.99 99.96 99.99 99.97 >99.999 99.54 99.33 99.7 99.7 99.27 98.79 98.9 92.5 Overall exhibit anti-bacterial activity for 14 days, although it slowly decreased over that period of time. The residual activity appears to decrease more on the vinyl than the other surfaces tested. Even after three days 99.99% of the test organisms were inactivated after ten minutes on all the test surfaces after 10 minutes of contact. After five days post application this was reduced to approximately 99.9% on all the test surfaces. After 10 days VRE could still be reduced by 99% on all the test surfaces in ten minutes. 18