ACS 200 Legionella pneumophila Kill Time Study

Antimicrobial Activity of Experimental Solutions Using Legionella pneumophila
Test Solution: ACS 200
Submitted June 6, 2012
October 11, 2012

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 ACS 200 on Legionella pneumophila. This was accomplished by performing standard kill-time suspension tests using 2, 5, 10, and 20 min contact times.

II. MATERIALS AND METHODS

A. Test organism

The test suspension was prepared by growing a 24 hr culture of Legionella pneumophila, ATCC 33156, in Legionella Broth at 37 °C with 5% CO2, then 10 ml of culture was centrifuged at 3200 xg for 5 minutes, the supernatant fluid was removed, and the pellet was re-suspended in 1 ml of sterile deionized water.

B. Media

Legionella Agar was made as follows: 1.0 % Yeast Extract, 0.2% Charcoal, 1.0% ACES buffer, 1.7% agar, 4.0% L-Cysteine-HCL, and 2.5% Fe-pyrophosphate.  The pH of the agar was adjusted to 6.9 using KOH prior to autoclaving.
Legionella Broth was made as follows: 1.0 % Yeast Extract, 1.0% ACES buffer, 4.0% L-Cysteine-HCL, 2.5% Fe-pyrophosphate, and 0.05% bovine serum albumin (BSA).  The pH of the solution was adjusted to 6.9 using KOH.

C. Neutralizers

(9-ml tubes with the following formulations for each trial were used)
Trial 1: 12.7% Tween 80, 6.0% Tamol, 1.7% lecithin, 1.0% Peptone, 1.0% Cysteine and 500 mM Tris (pH 7.0).
Trial 2: 0.1% Tween 80, 1% peptone, 1.0% Cysteine and 500 mM Tris (pH 7.0).
Trial 3: heat-inactivated fetal bovine serum with 500 mM Tris (pH 7.0).
Trial 4: heat-inactivated fetal bovine serum with 500 mM Tris (pH 7.0), and 1.0% Cysteine.

D. Kill-Time Procedure

1.   9.9 ml of the disinfectant was added to a 50 ml polypropylene sterile centrifuge tube. The tube was equilibrated in a 20 °C water bath. Next, 0.1 ml of the L. pneumophila test suspension was added at time zero.
2.   After the specified contact times (2, 5, 10 and 20 minutes), 1 ml of this mixture was added to 9 ml of neutralizer.  The tube was mixed thoroughly.
3.   After two min, the neutralized suspension was serially diluted in sterile 9-ml physiological saline solution (PSS) blanks.
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 Legionella Agar plates. The plates were incubated at 37 °C with 5% CO2 for four days.
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 organism 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 somewhere between13.6 and 72.3 CFU / ml in the tube (depending on the Trial), which was allowed to stand for 20 minutes prior to dilution and assay by membrane filtration using duplicate 1 ml samples

III. RESULTS

 

Trial 1 L. pneumophila suspension: Titer.
Dilution:	1:1×106	1:1×107
Number of colonies:	140	54
	135	14

 

ACS 200: (Received 06/06/12)
Exposure	Dilution of spore/Solution suspension:
Time	1:1×101	1:1×102	1:1×103	1:1×104
2 min	0	0	0	0
	0	0	0	0
5 min	0	0	0
	0	0	0
10 min	0	0	0
	0	0	0
20 min	0	0
	0	0

 

Neutralization Control		Expected Counts:		Percent of Expected:
Undiluted	1:1×101	Undiluted	1:10	< 10%
0	0	13.6	1
0	0

 

Trial 2 L. pneumophila suspension: Titer.
Dilution:	1:1×106	1:1×107
Number of colonies:	58	3
	52	5

 

Titer with Neutralizer in tube 1.
Dilution:	1:1×107	1:1×108
Number of colonies:	52	4
	48	7

 

ACS 200: (Received 06/06/12)
Exposure	Dilution of spore/Solution suspension:
Time	1:1×101	1:1×102	1:1×103	1:1×104
2 min	0	0	0	0
	0	0	0	0
5 min	0	0	0
	0	0	0
10 min	0	0	0
	0	0	0
20 min	0	0
	0	0

 

Neutralization Control		Expected Counts:		Percent of Expected:
Undiluted	1:1×101	Undiluted	1:10	12.5
8	0	52	5
5	0

 

Trial 3 L. pneumophila suspension: Titer.
Dilution:	1:1×107	1:1×108
Number of colonies:	74	12
	66	12

 

Titer with Neutralizer in tube 1.
Dilution:	1:1×107	1:1×108
Number of colonies:	64	10
	82	10

 

ACS 200: (Received 06/06/12)
Exposure	Dilution of spore/Solution suspension:
Time	1:1×101	1:1×102	1:1×103	1:1×104
2 min	0	0	0	0
	0	0	0	0
5 min	0	0	0
	0	0	0
10 min	0	0	0
	0	0	0
20 min	0	0
	0	0

 

Neutralization Control		Expected Counts:		Percent of Expected:
Undiluted	1:1×101	Undiluted	1:10	44
32	5	71	7
30	0

 

Trial 4 L. pneumophila suspension: Titer.
Dilution:	1:1×107	1:1×108
Number of colonies:	70	8
	65	6

 

Titer with Neutralizer in tube 1.
Dilution:	1:1×107	1:1×108
Number of colonies:	0	0
	0	0

 

ACS 200: (Received 06/06/12)
Exposure	Dilution of spore/Solution suspension:
Time	1:1×101	1:1×102	1:1×103	1:1×104
2 min	0	0	0	0
	0	0	0	0
5 min	0	0	0
	0	0	0
10 min	0	0	0
	0	0	0
20 min	0	0
	0	0

 

Neutralization Control		Expected Counts:		Percent of Expected:
Undiluted	1:1×101	Undiluted	1:10	22
24	0	66	7
5	0

 

Sterility Controls:
Material	Counts
PSS	0, 0, 0
Neutralizer	0, 0, 0
Legionella Agar	0, 0, 0
ACS 200	0, 0, 0

IV. DISCUSSION

Results of the four different trials showed a viable Legionella pneumophila concentration ranging from 1.4 x 108 to 7.3 x 108 colony forming units (CFU) per ml in the test suspensions. Inoculation of 9.9 ml of disinfectant with 0.1 ml of this suspension produced an initial concentration of 1.4 x 106 to 7.3 x 106 CFUper 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/So); where S = concentration of viable organisms after the specified contact time; and So = the initial concentration of viable organisms at time zero. 2) PK = (1 – (S/So)) × 100. These values are shown below.

Test Solution	Contact Time	Log Reduction (LR)	Percent Kill (PC)
1	2 min	>5.44	>99.9996
	5 min	>5.44	>99.9996
	10 min	>5.44	>99.9996
	20 min	>5.44	>99.9996
2	2 min	>6.02	>99.99990
	5 min	>6.02	>99.99990
	10 min	>6.02	>99.99990
	20 min	>6.02	>99.99990
3	2 min	>6.16	>99.99993
	5 min	>6.16	>99.99993
	10 min	>6.16	>99.99993
	20 min	>6.16	>99.99993
4	2 min	>6.13	>99.99993
	5 min	>6.13	>99.99993
	10 min	>6.13	>99.99993
	20 min	>6.13	>99.99993

Neutralization control data for each trial showed general improvement in the level of neutralization with each trial.  However, observed counts were consistently lower than those expected, indicating that there may still be small amounts of residual toxicity in the neutralizer tubes.  In trials 2 and 3, duplicate titers were performed using a neutralizer tube as the first dilution blank, demonstrating that the neutralizer formulations were not themselves toxic to the test organism.  Since neutralizer controls did not produce counts similar to those expected, the exact log reductions corresponding to each exposure time may be slightly inflated, since theoretically, some small un-neutralized residual could still be effecting kill past the exposure time specified.  However, considering all the data, these effects are probably low.  The neutralizer formulation used in trial 3 produced 44% of the expected counts, which is close to the 50% minimum usually required.
Therefore, it can be generally concluded from these results that ACS 200 has rapid bactericidal activity against Legionella pneumophila, effecting complete kill of the test organism within a few minutes.