Neutralization of antimicrobial chemicals by recovery media

Journal of Microbiological Methods, 19 (1994) 51-58 © 1994 Elsevier Science Publishers B.V. All rights reserved 0167 7012/94/$07.00

51

MIMET 00613

Neutralization of antimicrobial chemicals by recovery media B.P. Dey* a n d F . B . E n g l e y , Jr. Department of Microbiology, School of Medicine, University of Missouri, Columbia, MO, USA (Received 12 August 1992; accepted 20 June 1993)

Summary The ability to neutralize a variety of antimicrobial agents by commerciallyprepared Dey and Engley (D/E) Neutralizirig broth and the solid medium with a 2% agar was tested against the broth and the agar formulations of Letheen (LT) and Standard Methods (SM) medium. Paper disc assay of aqueous solutions of 1:50 phenol, 1:750 benzalkonium chloride (Zephiran®), 1:100 alkalinized glutaraldehyde (Cidex;~, 1:128 phenol (Mikro-Bac®), 1:128 quaternary ammonium compound (Mikro-Quat®) and tinctures of 1:1000 mercuric chloride (Merthiolate®) and 1:50 iodine did not inhibit Staphylococcus aureus ATCC 6538, Salmonella typhimurium ATCC 14028, Escherichia coli ATCC 25922, Bacillus subtilis ATCC 6633 and Pseudomonas aeruginosa ATCC 27853 growth on D/E agar. Except for 1:100 phenol and 1:128 Mikro-Bac on LT, all agents inhibited organism growth on LT and SM agar. In the presence of all test agents all test organisms grew in D/E broth. Except for 1:100phenol and 1:128 Mikro-Bac in LT broth, all agents inhibited growth of test organisms in LT and SM broth. The recovery of viable organisms unable to grow in LT broth in the presence of test agents was better with D/E Agar than with LT and SM Agar. Both formulations of D/E medium show promise for studying and evaluating known and unknown compounds, mixtures and formulations of antimicrobial chemicals.

Key words: Antimicrobials; Recovery media

Introduction Partial loss of antibacterial activity of a n a n t i m i c r o b i a l was first observed with bichloride of m e r c u r y in the presence o f organic m a t t e r [1]. Again, partial loss of activity for other mercurials was reported in the presence o f s o d i u m thioglycollate [2] a n d ethyl glycol [3]. Similarly, reduced activity o f p h e n o l in the presence o f organic a n d i n o r g a n i c m a t t e r [4], q u a t e r n a r y a m m o n i u m c o m p o u n d s (Quat) in the presence o f b o t h organic a n d i n o r g a n i c m a t t e r a n d high acidic c o n d i t i o n s [5-10], loss of chlorine potency in the presence of c a r b o h y d r a t e , alcohols, s o d i u m oleate, s o d i u m *Corresponding author. Present address: USDA, FSIS, S & T, Building 322, BARC-East, Beltsville,MD 20705, USA.

52 and Fe 2÷ salts [11-13] and that of iodine in the presence of organic matter, alcohols, urine, mercury salts, sodium thioglycollate, and high alkaline conditions were reported [14-16]. Realizing the fact that antimicrobials are used under various conditions whereby their potency is reduced, organic matter was included in an antimicrobial testing method [4]. Later, inclusion of a specific neutralizer in the recovery medium was recommended for testing antimicrobial agents [17,18]. Presently, the Letheen medium (Difco laboratories) is recommended as the recovery medium for evaluating phenols and Quats [19]. The only other routine neutralizing medium used over the years consisted of various modifications of thioglycollate broth for neutralizing mercurials. This medium was originally intended to create an anaerobic condition [20]. Presently, there is no single recovery medium which has the ability to inactivate the range of antimicrobial agents that are in current use. Depending upon an antimicrobial under test, a specific neutralizer is added in the recovery medium [21]. Recognizing the need of a universal neutralizing medium which can be used in the evaluation of a broad range ofantimicrobial agents, the Dey and Engley (D/E) Neutralizing Medium was developed to neutralize phenols, Quats, mercurials, halogens and aldehydes [22,23]. This medium was found useful for sterility testing of antimicrobial cream [24] and recovering chemically treated organisms [25]. The present paper reports the neutralizing and recovery capacity of commercially prepared D/E agar and broth [26] which are essential for a recovery medium used in antimicrobial testing. Material and Methods

Organisms Staphylococcus aureus ATCC 6538, Salmonella typhimurium ATCC 14028, Escherichia eoli ATCC 25922, Bacillus subtilis ATCC 6633 and Pseudomonas aeruginosa ATCC 27853 were used in the study. Media The liquid and the solid forms of Dey and Engley (D/E) Neutralizing Medium (Difco) which additionally contains 2% agar were tested against Letheen (LT) Medium for neutralizing antimicrobial agents and recovering chemically treated organisms with Standard Methods (SM) Medium (Difco) as control. Antimicrobial agents The dilutions of test agents in double distilled water used in the study were: 1:750 benzalkonium chloride (Zephiran®), Sterling Drug, Inc., New York; 1:100 alkalinized glutaraldehyde (Cidex®), Arbrook & Co., New York; 1:128 phenol (MikroBac ®) and 1:128 quaternary ammonium compound (Mikro-Quat®), Economics Laboratories, St. Paul, MN., and 1:100 phenol (A-91I), Fisher Scientific Co., Pittsburgh, PA. The dilutions of Zephiran, Mikro-Bac and Mikro-Quat were recommended by the manufacturers. Other preparatory agents used were: 1:1000 tinctures of mercuric chloride (Merthiolate®), Eli Lilly & Co., New York and 1:50 iodine (Medical College, University of Missouri, Columbia, MO).

53

Neutralization of test chemicals by test agars An aliquot of 0.1 ml of a 10 -5 dilution of an 18 h culture of each test organism was pipetted onto triplicate plates of SM, LT and D/E agar and spread with glass angle rods. After 30 min, 12.7 mm paper discs soaked with 0.1 ml of an antimicrobial were placed on the plates. After incubation at 37°C for 24 h, the zones of inhibition by agents on plates were measured [27]. Neutral&ation of test chemicals by test broths One ml of each antimicrobial was pipetted into 8.9 ml of each broth and mixed thoroughly (all broths upon addition of mercurial, glutaraldehyde and iodine became turbid). After 30 min at room temperature, each broth mixture was inoculated with 0.1 ml of a 10-5 dilution of an 18 h culture of a test organism and incubated at 37°C for 48 h. In addition to turbidity, ropy growth in some tubes at the bottom and pellicle growth in others at the top, prevented determining microbial growth in these tubes by light transmission. After thorough shaking, a 0.1 ml aliquot from each tube was plated on to a fresh SM agar plate. After incubation at 37°C for 48 h, organism growth on plates was noted. Recovering of chemically treated organbms The procedure was the same as described above, except that a 0.1 ml aliquot from those tubes which did not show organism growth on SM agar were plated on to LT and D/E agar. After incubation at 37°C for 48 h, organism growth on all plates was noted. Results

Neutralization The zones of inhibition by antimicrobial agents against all test organisms reported (Table 1) are mean values of 9 individual measurements, 3 replicate measurements done 3 times. The data indicate that zone sizes on a medium varied with test agents and organisms. All agents produced zones of inhibition against all test organisms on SM agar as expected because the medium contains no neutralizer. Zones of inhibition by all antimicrobials on LT except for 1:100 phenol and Mikro-Bac indicate the neutralizing effect of polysorbate 80 present in this medium. Absence of zone on D/E agar by any chemicals indicates that all agents were neutralized by specific neutralizers present in this medium. Statistical analysis of the data presented in Table 1 indicates that the changes in zone sizes among media was due to antimicrobial agents. Standard deviation between any two zone sizes indicates that the neutralizing capacity of D/E for all test agents was significantly higher than SM. Except for phenols (Phenol 1:100 and 1:128 MikroBac), the neutralizing capacity of LT for other test agents was significantly less than D/E and equal to SM agar. The ability of test broth to neutralize test agents and support organism growth confirmed by SM agar presented in Table 2 agrees with the Table 1 observations. Due to the absence of any neutralizer, the SM broth was unable to support organism growth in the presence of any test agents. Organism growth in LT broth only in the

54 TABL E 1 Inhibition of organisms by antimicrobials on agar media Antimicrobials

Medium

Organisms and inhibitory zones (mm)

S. aureus

E. coli

S. typh#nurium

P. aeruginosa

B. subtilis

Phenol Mikro-Bac Zephiran Mikro-Quat Merthiolate Iodine Cidex

SM SM SM SM SM SM SM

21.7 24.1 17.9 19.4 34.2 50.1 18.9

21.1 21.7 16.9 19.9 41.0 43.0 21.9

21.8 l 7.9 17.9 18.7 40.8 43.0 18.0

16.9 17.0 16.9 17.2 30.7 35.6 18.7

20.0 20.9 20.4 21.3 44.9 36.0 19.0

Phenol Mikro-Bac Zephiran Mikro-Quat Merthiolate Iodine Cidex

LT LT LT LT LT LT LT

0.0 0.0 16.1 17.6 31.9 48.0 17.9

0.0 0.0 16.3 16.9 39.0 41.0 20.8

0.0 0.0 17.0 17.6 39.4 41.9 17.3

0.0 0.0 15.0 16.1 30.0 34.4 18.3

0.0 0.0 17.0 21.3 43.9 35.0 18.0

All 7

D/E

0.0

0.0

0.0

0.0

0.0

TABL E 2 Organism growth in broths in the presence of antimicrobials as confirmed on SM agar Antimicrobials

Medium

Organisms and inhibitory zones (mm)

S. aureus Phenol Mikro-Bac Zephiran Mikro-Quat Merthiolate Iodine Cidex

SM SM SM SM SM SM SM

. . . . . . .

Phenol Mikro-Bac Zephiran Mikro-Quat Merthiolate Iodine Cidex

LT LT LT LT LT LT LT

+ + . . . . .

All 7

D/E

+

E. coli . . . . . . .

S. typhimurium . . . . . . .

+ + . . . . .

. . . . . . .

+

. . . . . +

B. subtilis

+ +

+ +

+

+

. . . . . . .

+ + . . . . .

P. aeruginosa

. . . . .

55 TABLE 3 Antimicrobial treated organisms from SM and LT broths recovered by LT and D/E agar Test organisms

Treated with antimicrobials

From broth

Recovery on agar LT

D/E

All 5

All 7

SM

_a

_

S. aureus

Zephiran Mikro-Quat MerthiolateLT

LT LT +

q_b + ++

_~+ c ++

E. coli and S. typhimurium

Zephiran Merthiolate

LT LT

-

+ +

P. aeruginosa

Zephiran Mikro-Quat Merthiolate

LT LT LT

-

+ + +

B. subtilis

All 7

LT

a(__) NO growth; b(+) Growth; c(+ +) More growth.

presence of phenol 1:100 and Mikro-Bac 1:128 indicate neutralization achieved by polysorbate 80, whereas growth of all organisms in D/E in the presence of all test agents indicates that the specific neutralizers in this medium inactivated all test agents. Organism recovery

The ability of a medium to recover antimicrobial treated organisms (Table 3) indicates that the organism survival depended upon an agent, organism resistance to chemicals and the specific neutralizer present in the final recovery medium. N o recovery of any test organism from SM broth by LT and D/E indicates that in the absence of neutralizers, the organisms were lethally damaged initially. Recovery of only Zephiran, Mikro-Quat and Merthiolate treated S. a u r e u s and non-recovery of other test organisms from LT broth indicate that the recovery by LT was affected by the agent action, organism resistance and sensitivity to agents and the neutralizers in the medium. Compared to LT, better recovery results by D/E agar indicate that in addition to S. a u r e u s the dormant organisms were also revived due to inactivation of antimicrobials by specific and adequate amounts of neutralizers present in the medium. Non-recovery of all iodine and glutaraldehyde organisms from LT broth by LT and D/E agar indicate that the agents were lethal to organisms whereas nonrecovery of B. s u b t i l i s indicates greater sensitivity to test chemicals.

56 Discussion

In the evaluation of antimicrobial agents, the presence of specific neutralizer(s) in the recovery medium is essential to counteract the inhibitory action of carried-over test chemical(s) on remaining viable organisms at a given time [28,29]. In the absence of a neutralizer, a strongly bacteriostatic agent can produce erroneous results and may appear as a bactericidal agent. In order to avoid such discrepancy, many compounds have been found as suitable neutralizers for various antimicrobials [30]. Both LT agar and LT broth are commonly used for evaluating phenols and Quats. Their usage is based on the Quat-neutralizing capacity of LT broth [31]. This study indicates that both LT agar and broth have the ability to neutralize only phenol but not Quats as both contain a sufficient amount ofpolysorbate 80, a specific neutralizer for phenols. Their inability to neutralize quat completely indicates an error in reporting the neutralizing capacity of LT broth [31]. The study did not consider reduction in the Quat concentration resulting from transferring the aliquot of the antimicrobial into a 10 ml volume of the LT broth [32]. As a result, the Quat neutralizing capacity of the medium was reported 10-times higher [31]. Our earlier study has shown that a concentration of 0.7 g/1 lecithin present in LT medium is not sufficient, and that a recovery medium must contain 7.0 g/1 of lecithin in order to achieve complete neutralization of Quats [33]. This concentration of lecithin in a medium was later found essential for supporting organism growth in the presence of Quats and also for recovering Quat-treated organisms [23,25]. The study indicates that both SM media have no neutralizing capacity for antimicrobial chemicals whereas LT media have limited ability. Except for phenol, LT is not suitable as a recovery medium for evaluating many other antimicrobial chemicals used today. The importance and the effect of neutralizers in a recovery medium is also evident in this study. As D/E contains polysorbate 80, lecithin, sodium thioglycollate, sodium thiosulfate and sodium bisulfite specific neutralizers for phenol, Quat, mercurial, iodine and aldehyde respectively, the medium has the ability to support organism growth in the presence of these agents. Due to these neutralizers, the D/E medium has the ability to inactivate a range of carried over antimicrobials and therefore can recover chemically treated viable organisms. With these advantages, the D/E medium can safely be used in evaluating a variety of antimicrobial agents. It has been well documented that a successful evaluation of an antimicrobial agent besides organism resistance and recovery method largely depends upon the capability of the recovery medium [29,30,34]. Besides neutralization of antimicrobials, easy detection of organisms on D/E medium is an added advantage. All test organisms produce yellow colonies against the purple background of D/E agar except Pseudomonas, which produces grayishpurple colonies. The D/E broth with organism growth changes from purple to yellow except in the case of Pseudomonas, which forms a pellicle.

57

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