I. PURPOSE. II. MATERIALS AND METHODS.

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1 KILL-TIME STUDIES Antimicrobial Activity of Experimental Disinfectants Using Methicillin-Resistant Staphylococcus aureus (MRSA) Test Solutions: Silver Immune Booster and ASAP-10 Lot # December 9, 2013 I. PURPOSE. The purpose of this study was to determine the antimicrobial activity of two disinfectant formulations on methicillin-resistant Staphylococcus aureus (MRSA) ATCC This was accomplished by performing standard kill-time suspension tests using 10, 20, 40, and 80 min contact times. II. MATERIALS AND METHODS. A. Test organism suspension. The test suspension was prepared by growing a 24 hr culture of Staphylococcus aureus, ATCC 43300, in Nutrient broth at 37 C. The suspension was then centrifuged, washed with 5 ml of sterile distilled water and re-suspended in 1ml sterile distilled water. B. Neutralizer. The Neutralizer solution consisted of 9ml tubes of 12.7% Tween 80, 6.0% Tamol, 1.7% lecithin, 1% Peptone, 1.0% Cysteine and 500 mm Tris (ph 7.0). C. Kill-Time Procedure ml of each disinfectant solution was added to separate sterile 50 ml polypropylene centrifuge tubes. The tubes were equilibrated in a 20 C water bath. Then, 0.1 ml of the S. aureus test suspension was added at time zero. 2. After the specified contact times (10 min, 20 min, 40 min, and 80 min), 1 ml of the organism/disinfectant suspension was added to 9 ml of neutralizer. The tubes were mixed thoroughly. 3. After two min, the neutralized suspensions were serially diluted using 9.0 ml blanks of sterile saline solution. 4. The number of viable organisms in selected dilution tubes was assayed by membrane filtration. One ml aliquots were plated in duplicate. The membranes were washed with about 100 ml of sterile PSS and removed to Columbia Agar plates. The plates were incubated at 37 C for 24 and 48 hours. 5. The number of colonies on each filter was counted and log reduction and percent kill values were computed. D. Controls. 1. A titer of the test suspension was computed by performing membrane filtration assays on selected 1:10 dilutions in PSS of the test suspension. Page 1 of 5

2 2. A neutralizer control for each disinfectant was performed by inoculating a mixture of 9.0 ml of neutralizer and 1 ml of disinfectant with 0.1 ml of the 1:1x10 6 dilution of the titer. This produced about 35 CFU / ml in the tube, which was allowed to stand for 20 minutes prior to dilution and assay by membrane filtration using duplicate 1 ml samples III. RESULTS. S. aureus suspension: Titer. Dilution: 1:1x10 8 1:1x10 9 Number of colonies: Silver Immune: (Received 12/9/13) Exposure Dilution of spore/disinfectant suspension: Time 1:1x10 3 1:1x10 4 1:1x min ~ ~ Time 1:1x10 2 1:1x10 3 1:1x min min min Neutralization Control Expected Counts: Percent of Expected: Undiluted 1:10 Undiluted 1:10 102% Page 2 of 5

3 ASAP-10 (Lot #13254): (Received 12/9/13) Exposure Dilution of spore/disinfectant suspension: Time 1:1x10 3 1:1x10 4 1:1x min TNC ~ TNC ~ Time 1:1x10 2 1:1x10 3 1:1x min ~ ~ min min Neutralization Control Expected Counts: Percent of Expected: Undiluted 1:10 Undiluted 1: % Sterility Controls: Material Counts PSS-1 0, 0 PSS-2 0, 0 PSS-3 0, 0 Silver Immune 0, 0 ASAP-10 0, 0 Neutralizer 0, 0 Columbia Agar 0, 0 Page 3 of 5

4 IV. DISCUSSION. Results of the titer showed a viable S. aureus concentration of 3.5 x 10 9 CFU per ml in the original suspension. Inoculation of 9.9 ml of disinfectant with 0.1 ml of this suspension produced an initial concentration of 3.5 x 10 7 CFU per ml in the assay tube. Results from these procedures allowed log reduction (LR) and percent kill (PK) values to be calculated using the formulas: 1) LR = -Log(S/S 0 ); where S = concentration of viable organisms after the specified contact time; and S 0 = the initial concentration of viable organisms at time zero. 2) PK = (1 - (S/S 0 )) 100. These values are shown below. Test Solution Contact Time Log Reduction (LR) Percent Kill (PK) Silver Immune 10 min Silver Immune 20 min Silver Immune 40 min Silver Immune 80 min >6.85 > ASAP min ASAP min ASAP min ASAP min Neutralization control data revealed that the neutralizer was able to adequately neutralize the test disinfectants. Observed counts were 102% and 76.4% of those expected, for Silver Immune and ASAP-10 solutions, respectively. The Silver Immune and ASAP-10 disinfectants exhibited significant antimicrobial kill of methicillin-resistant S. aureus. The Silver Immune solution produced a faster kill on this test organism than the ASAP-10 solution. After 80 minutes of contact, the Silver Immune solution was able to effect greater than a 6.85 log reduction of S. aureus. The ASAP solution produced a 5.89 log reduction after this same period. Linear regressions of the kill kinetics of these two solutions are shown in Figures 1 and 2 below. Since an exact log reduction after 80 minutes of contact, could not be obtained for the Silver Immune solution (because it effected complete kill sometime before 80 minutes), only the first 3 contact times were used to generate these figures. Using the regression equations, 6 log reduction times (time required to kill 1 million MRSA organisms) for the two solutions were estimated as: Silver Immune = 38.7 min; ASAP = 44.4 min. Faster kill times would likely be observed if the concentration of active ingredient was increased. Page 4 of 5

5 Figure 1: Kill kinetics of Silver Immune solution for 10, 20, and 40 minute contact times. Figure 2: Kill kinetics of ASAP-10 solution for 10, 20, and 40 minute contact times. Page 5 of 5

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