ACS 200 Pseudomonas aeruginosa Kill Time Study

Antimicrobial Activity of Experimental Disinfectants
Using Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella choleraesuis
Test Solutions: ACS 200 Extra Strength Advanced Cellular Silver Lot #: 1-1336401 & 2-1336401

Submitted February 24, 2014
March 8, 2014
PREPARED FOR:
Results RNA, LLC
BY:
Richard A. Robison, Ph.D.
Department of Microbiology
Brigham Young University

I. PURPOSE

The purpose of this study was to determine the antimicrobial activity of experimental disinfectant formulations on Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 15442, and S. choleraesuis ATCC 10708.This was accomplished by performing standard kill-time suspension tests using 3 and 5 min contact times.

II. MATERIALS AND METHODS

A. Test organism

The test suspensions were prepared by growing a 24 hr culture of Staphylococcus aureus ATCC 6538, in Nutrient broth at 37 °C.  The suspension was then centrifuged, washed with 5 ml of sterile distilled water (SDW) and re-suspended in 1ml SDW. The other two test suspensions, Pseudomonas aeruginosa ATCC 15442 and Salmonella choleraesuis ATCC 10708, were prepared in a similar manner to the S. aureus suspension.

B. Neutralizers

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

1.   9.9 ml of each disinfectant solution was added to separate sterile 50 ml polypropylene centrifuge tubes. The tubes containing the disinfectant were equilibrated in a 20 °C water bath. Then, 0.1 ml of the  bacterial suspension was added at time zero.
2.   After the specified contact times (3 min and 5 min), 1 ml of the organism/disinfectant suspension was added to 9 ml of neutralizer.  The tube was mixed thoroughly.
3.   After two min, the neutralized suspension was serially diluted using 9.0 ml blanks of sterile physiological saline solution (PSS).
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.
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 1:1×105 dilution of the titer. This produced about 426 CFU / ml in the S. aureus tube, 232 CFU / ml in the P. aeruginosa tube, and 68 CFU / ml in the S. choleraesuis tube. All tubes were 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: 1:1×107 1:1×108
Number of colonies: ~460 45
~520 41

 

ACS 200 Lot 1-1336401:
(Received 02/24/13)
Exposure Dilution of spore/Solution suspension:
Time 1:1×101 1:1×102 1:1×103
3 min 10 0 0
6 0 0
Time 1:1×101 1:1×102 1:1×103
5 min 0 0 0
0 0 0

 

Neutralization Control Expected Counts: Percent of Expected:
Undiluted 1:1×101 Undiluted 1:10 78.6%
~380 35 426 43
~460 32

 

ACS 200 Lot 2-1336401:
(Received 02/24/13)
Exposure Dilution of spore/Solution suspension:
Time 1:1×101 1:1×102 1:1×103
3 min 2 0 0
1 0 0
Time 1:1×101 1:1×102 1:1×103
5 min 0 0 0
0 0 0

 

Neutralization Control Expected Counts: Percent of Expected:
Undiluted 1:1×101 Undiluted 1:10 86.9%
~460 36 426 43
~460 38

 

P. aeruginosa suspension:
Titer.
Dilution: 1:1×107 1:1×108
Number of colonies: 212 31
203 21

 

ACS 200 Lot 1-1336401:
(Received 02/24/13)
Exposure Dilution of spore/Solution suspension:
Time 1:1×101 1:1×102 1:1×103
3 min 0 0 0
0 0 0
Time 1:1×101 1:1×102 1:1×103
5 min 0 0 0
0 0 0

 

Neutralization Control Expected Counts: Percent of Expected:
Undiluted 1:1×101 Undiluted 1:10 111
192 29 257 26
224 28

 

ACS 200 Lot 2-1336401:
(Received 02/24/13)
Exposure Dilution of spore/Solution suspension:
Time 1:1×101 1:1×102 1:1×103
3 min 1 0 0
0 0 0
Time 1:1×101 1:1×102 1:1×103
5 min 0 0 0
0 0 0

 

Neutralization Control Expected Counts: Percent of Expected:
Undiluted 1:1×101 Undiluted 1:10 96.1
231 23 257 26
238 17

 

S. choleraesuis suspension:
Titer.
Dilution: 1:1×107 1:1×108
Number of colonies: 61 9
53 7

 

ACS 200 Lot 1-1336401:
(Received 02/24/13)
Exposure Dilution of spore/Solution suspension:
Time 1:1×101 1:1×102 1:1×103
3 min 0 0 0
0 0 0
Time 1:1×101 1:1×102 1:1×103
5 min 0 0 0
0 0 0

 

Neutralization Control Expected Counts: Percent of Expected:
Undiluted 1:1×101 Undiluted 1:10 117
68 8 57 6
89 8

 

ACS 200 Lot 2-1336401:
(Received 02/24/13)
Exposure Dilution of spore/Solution suspension:
Time 1:1×101 1:1×102 1:1×103
3 min 1 0 0
0 0 0
Time 1:1×101 1:1×102 1:1×103
5 min 0 0 0
0 0 0

 

Neutralization Control Expected Counts: Percent of Expected:
Undiluted 1:1×101 Undiluted 1:10 101
70 6 57 6
75 7

 

Sterility Controls:
Material Counts
PSS-1 0, 0, 0
PSS-2 0, 0, 0
PSS-3 0, 0, 0
Neutralizer 0, 0, 0
Columbia Agar 0, 0, 0
ACS 200, lot: 1-1336401 0, 0, 0
ACS 200, lot: 2-1336401 0, 0, 0

IV. DISCUSSION

Results of the titer showed viable S. aureus, P. aeruginosa, and S. choleraesuis concentrations of 4.30 x 109 CFU/ml, 2.34 x 109 CFU/ml, and 6.85 x 108 CFU/ml, respectively, in the original suspensions. Inoculation of 9.9 ml of disinfectant with 0.1 ml of these suspensions produced an initial concentration of 4.30 x 107 S. aureus per ml, 2.34 x 107 P. aeruginosa per ml, and 6.85 x 106 S. choleraesuis per ml, in the assay tubes.

Results from these procedures allowed log reduction (LR) and percent kill (PK) values to be calculated using the formulas: 1) LR = -Log(S/S0); where S = concentration of viable organisms after the specified contact time; and S0 = the initial concentration of viable organisms at time zero. 2) PK = (1 – (S/S0)) × 100. These values are shown below.

 Test Solution Contact Time Log Reduction (LR) Percent Kill (PC)
S. aureus
1-1336401 3 min 5.73 >99.99981
1-1336401 5 min >6.93 >99.999988
2-1336401 3 min 6.46 99.999965
2-1336401 5 min 6.93 >99.999988
P. aeruginosa
1-1336401 3 min >6.67 >99.999979
1-1336401 5 min >6.67 >99.999979
2-1336401 3 min 6.67 99.999979
2-1336401 5 min >6.67 >99.999979
S. choleraesuis
1-1336401 3 min >6.14 >99.999927
1-1336401 5 min >6.14 >99.999927
2-1336401 3 min 6.14 99.999927
2-1336401 5 min >6.14 >99.999927

Neutralization control data revealed that the neutralizer was able to adequately neutralize the test disinfectants, with respect to each test organism. Observed counts were 78.6% to 117% of those expected. The ACS solutions tested showed relatively rapid kill against S. aureus, P. aeruginosa, and S. choleraesuis. Both solutions effected complete kill of these test organisms in 5 minutes. However, they were unable to effect complete kill in 3 minutes, with one lot for each organism showing a small number of counts.  That said, the mean log reduction for each test organism at 3 minutes was still greater than 6.