THE "REVERSAL," NEUTRALIZATION, AND SELECTIVITY

THE "REVERSAL," NEUTRALIZATION, AND SELECTIVITY OF GERMICIDAL CATIONIC DETERGENTS' MORTON KLEIN AND ZELMA G. KARDON Department of Bacteriology, School of Medicine, University of Pennsylvania, Philadelphia 4, Pennsylvania Received for publication May 22, 1947 The reversal of the inhibitory action of antiseptics by substances showing a specific affinity for the inhibiting agent has been reported for many compounds, the most striking effect probably being the reversal of the action of mercurials by H2S, glutathione, sodium thioglycolate, etc. Valko and DuBois (1944) have reported that the germicidal action of cationic detergents against both gram-positive and gram-negative bacteria can be reversed by the anionic detergent duponol PC (sodium lauryl sulfate). In the present work we have studied the ability of anionic detergents to reverse or neutralize the action of cationic detergents. We have found that anionic detergents did not reverse the germicidal action of cationic detergents against either gram-positive or gram-negative bacteria. If the cationic detergent inactivated the bacteria, the addition of an anionic detergent did not result in any reactivation. However, when the anionic detergent was added before all the bacteria in a given inoculum were inactivated by the cation, the anionic detergent could neutralize the action of the cation against gram-negative bacteria and prevent any further germicidal action on the surviving bacteria. Against gram-positive bacteria the bacteriostatic action of the cationic detergent was not neutralized by an anionic detergent even though the anion was added before the cation. The failure to neutralize the action of a cationic detergent against gram-positive bacteria was found to be related to the high degree of selectivity shown by the compound for gram-positive bacteria. EXPERIMENTAL Choice of neutralizing agent. The anionic detergent duponol PC (sodium lauryl sulfate) was used as a neutralizing agent by Valko and DuBois in their studies on the reversibility of the bactericidal action of the cationic detergents. However, in extract broth, ph 7.2, sodium lauryl sulfate was itself inhibitory against Staphylococcus albus in dilutions as high as 1:2,. We therefore examined a group of anionic detergents in an effort to find an agent that was not bacteriostatic against the relatively susceptible gram-positive bacteria but was effective as a neutralizing agent against the cationic detergents. We determined the bacteriostatic action of 12 anionic detergents2 by seeding various 1 This investigation has been aided by a grant from the Josiah Macy, Jr., Foundation. 2 The anionic detergents tested were aerosol OT, tergitol 7, triton W-3, triton 72, igepon AP, igepon TD, duponol C, sodium octyl, decyl, lauryl, myristyl, and cetyl sulfates. References to the formulae of these compounds and the cationic detergents have previously been given (Klein and Stevens, 1945). 245 Downloaded from http://jb.asm.org/ on January 6, 219 by guest

246 MORTON KLEIN AND ZELMA G. KARDON [VOL. 54 dilutions of the anionic detergents in extract broth, ph 7.2, with.1 ml of a 2- to 24-hour broth culture of S. albus and determining the degree of inhibition after 24 hours' incubation at 37 C. The neutralizing action of the compounds against the cationic detergent zephiran was determined by adding various dilutions of the anions to equal volumes of 1:5, zephiran broth. The solutions were seeded with.1 ml of a 2- to 24-hour broth culture of Escherichia coli, and the lowest concentration of anionic detergent inhibiting the action of zephiran was determined. E. coli was used as the test strain in the neutralization tests since anionic detergents have little activity against gram-negative bacteria at ph 7.2. The anionic detergents were found to vary markedly in their ability to neutralize the action of cationic detergents, and there was in general a correlation between the degree of inhibition of S. albus and the extent of the neutralization of zephiran against E. coli. No compound was found which was completely noninhibitory against S. albus8 and still effective in neutralizing the action of zephiran against E. coli. Sodium decyl sulfate was found to be the best neutralizing agent and the only compound which combined a low bacteriostatic activity with a high degree of neutralizing action against E. coli. It was only slightly inhibitory against S. albus in a 1:5, broth dilution, was not inhibitory at a 1:1, dilution, and was completely inactive against the gram-negative bacilli in a 1:5, broth dilution, ph 7.2. The addition of an equivalent volume of a 1:5, dilution of the compound permitted the growth of E. coli in a 1:5, broth dilution of zephiran. Sodium decyl sulfate was also found to neutralize effectively the action of the cationic detergents ceepryn, phemerol, and emulsol 67 against E. coli. Failure to obtain reversal of the germicidal action of the cationic detergents. In our studies on the ability of the anions to reverse the action of cationic detergents, we first repeated the procedure used by Valko and DuBois. Three anionic detergents were used as neutralizing agents: duponol C, a commercial grade of alkyl sulfates, primarily sodium lauryl sulfate; sodium lauryl sulfate; and sodium decyl sulfate. Our test procedure followed that of Valko and DuBois though, in addition to broth subcultures, plate counts were used to determine quantitatively the degree of reversal. To varying dilutions of the cationic detergents in distilled water,.5 ml of a 2- to 24-hour broth culture of the test bacteria were added. After 5 minutes at room temperature (several tests were run at 37 C with similar results), 4 loops were subcultured into extract broth and a 1-ml sample was removed and diluted in saline; counts were determined in extract agar. Immediately after the removal of the sample for plating.5 ml of the test concentration of the anionic detergent were added to the solution of catiotic detergents. Five or 1 minutes (several tests were also done at 3 minutes) after the addition of the anionic detergents, broth subcultures were again made and a l-ml sample was removed, diluted in saline, and plated out to determine the degree of reversal effected by the anionic detergent. Four test strains were used, Staphylococcus aureus, Esherichia coli, ShigeUa paradysenteriae (Flexner), and Salmoiiella schottmuelleri. A total of 3 asays were done with Downloaded from http://jb.asm.org/ on January 6, 219 by guest

1947] GERMICIDAL CATIONIC DETERGENTS 247 the cationic detergents zephiran and phemerol. Though the dilutions of the cationic detergents and the anionic detergents varied from 1:3, to 1:2, and the ratio of cation to anion varied from 1: 1 to 1:6, in no case was any evidence of reversal obtained in the germicidal range of the cationic detergents. As determined by plate counts, there was never any increase in the number of viable bacteria after the addition of the neutralizing agent. Broth subcultures from the germicidal concentrations of zephiran and phemerol were always negative, and all subcultures remained negative after the addition of the neutralizing agent. Neutralization of the action of cationic detergents against gram-negativebacteria. Though no reversal was obtained with the germicidal concentrations of zephiran or phemerol, it was thought that reversal might be effected with the more dilute concentrations of zephiran in the bacteriostatic range. With sodium decyl sulfate as the neutralizing agent, the following test procedure was used. For each test a duplicate series of broth tubes containing dilutions of zephiran ranging from 1:1, to 1:3,, were seeded with.1 ml of a 2- to 24-hour broth culture of the test bacteria. After the bacteria were in contact with zephiran for 15 minutes at room temperature, equal volumes of sodium decyl sulfate in a broth dilution of 1:5, (1:15, and 1:25, sodium decyl sulfate were also used against the gram-positive bacteria) were added to one series of tubes, and equal volumes of broth were added to the control zephiran tubes. The cultures were incubated at 37 C for 24 hours, and the titers obtained with the zephiran alone were compared with the titers obtained with the sephiran and sodium decyl sulfate. A total of 17 gram-negative and gram-positive bacteria were studied. As shown in table 1, sodium decyl sulfate differed markedly in its zephiran-neutralizing action when tested against the grampositive bacteria (and meningococcus) and the gram-negative bacteria. There was only a slight degree of neutralization of zephiran action against 3 of the 1 gram-positive bacteria, but effective neutralization was obtained with all of the gram-negative bacteria. In order to determine whether growth in broth tubes to which sodium decyl sulfate was added was an actual reversal of zephiran action on the gram-negative bacteria or merely an interruption of the continued action of zephiran (neutralization) the foregoing test procedure was modified as shown in table 2. The data reveal that the growth obtained after the addition of sodium decyl sulfate was not a reversal phenomenon, as indicated by the absence of any significant increase in the bacterial count. Sodium decyl sulfate merely interrupted the continued action of the zephiran; upon further incubation the viable gramnegative bacteria present at 15 minutes in zephiran alone were inhibited, whereas the addition of the sodium decyl sulfate neutralized the zephiran and permitted the surviving bacteria to grow out. Failure to obtain neutralization of the action of zephiran against the gram-positve bacteria. Though viable gram-positive bacteria were present in the high zephiran dilutions at the time of the addition of the sodium decyl sulfate, no significant degree of neutralization was obtained (table 1). Inhibition of growth by the Downloaded from http://jb.asm.org/ on January 6, 219 by guest

248 MORTON KLEIN AND ZELMA, G. KARDON [VOL. 54 sodium decyl sulfate was not a factor in the failure to obtain neutralization since n 1:3, broth dilution (equivalent to the 1:15, sodium decyl sulfate added to an equal volume of zephiran) permitted the growth of an inoculum of 1 to 1 bacteria from a S. aureus broth culture. As shown in table 3, neither sodium decyl sulfate nor sodium lauryl sulfate was able to neutralize the bacterio- TABLE 1 Neutralization of thp bacteriostatic action of zephiran broth by sodium decyl sulfate ZlzItA BIOTE* ZEDrIA DUZT US 1:5, QR"I"TIvEr,.ucrmI SODIU MM DXCI ULATEt Inhibited by Growth n Inhibitd by G in Staphylococcu aureus AA... 1:1,OOO 1:2, 1:8 1:1, Streptococcus pyogenes... 1:8 1:1, 1:6 1:8 Bacillus subtilis... 1:1, 1:2, 1:8 1:1, Staphylococcus aurew 4A... 1:1,ooo 1:2, 1:1, 1:2, Staphylococcu albs... 1:8 1:1, 1:8 1:1, BaciUus mycoides... 1:8 1:1, 1:8 1:1, Sarcina lutea... 1:2, 1:3, 1:2, 1:3, -Gafflya tetragena... 1:2, 1:3, 1:2, 1:3, Diplococcus pneumoniae... 1:2 1:4 1:2 1:4 Neisseria intracellularis... 1:4 1:5 1:4 1:5 GAMNEATIVE BACTEI Eberthella typhosa... 1:4 1:8 1:5 1:1 Shigella paradysenteriae (Flexner)... 1:2 1:4 1:2 1:4 E8cherichia coli... 1:1 1:2 1:5 1:1 Proteus vulgaris... 1:2 1:4 1:1 1:5 Salmonella schottmuelleri... 1:1 1:2 1:1 1:2 Salmonella paratyphi... 1:1 1:2 1:1 1:5 Pseudomona aeruginosa... 1:1 1:3 1:1 * Neisseria intracellularis, Streptococcus pyogenes, and Diplococcus pneumoniae were grown in Difco phenol red glucose broth, ph 7.2. The activity of zephiran-in this medium was less than in the extract broth, ph 7.2, used in all other assays. t A 1:15, sodium decyl sulfate broth was also used as a neutralizing agent against all of the gram-positive bacteria and Neisseria intracellularis. A 1:25, sodium decyl sulfate broth was also used against Streptococcus pyogenes, Diplococcus pneumoniae, and Neisseria intracellularis. Titers were similar at all concentrations of the neutralizing agents. All readings were taken after 24 hours' incubation at 37 C. t Indicated dilutions X 1'. static action of zephiran against gram-positive bacteria when added before the bacteria. The addition of sodium decyl sulfate to the zephiran before the addition of the gram-negative bacteria did result in effective neutralization. Though the anionic detergent did neutralize the immediate germicidal action of zephiran against gram-positive bacteria, the bacteria were not capable of growing. For example, when 1:1, or 1:2, sodium lauryl sulfate broth was added to equal volumes of 1:1, or 1:2, zephiran broth and then seeded with S. aureus, the rapid germicidal action of zephiran was neutralized, but after 24 hours' incubation the viable bacteria failed to grow and plate counts Downloaded from http://jb.asm.org/ on January 6, 219 by guest

TABLE 2 Neuralization of action of sephiran by sodium decyl sulfae (Escherichia coli) ZURAN DJE DmfiUnONS 1:1, 1:2, 1:4, Broth Contru A. 15 min in sephiran broth. Bac- 7 X 1 11 X 14 3.2 X 16 1.5 X 18 tenra per ml Growth in 24 hr ++++ B. 15 min in zephiran broth. 1:4, 17 X 1 7.8 X 14 5.4 X 1' sodium decyl sulfate broth added. After 15 min bacteria per ml Growth in 24 hr +++ ++++ ++++ ++++ The titer of sephiran in this assay was lower than it was in previous assays. Similar results were obtained in several asays with Shigella paradysenteriac (Flezner) and Salmonella paratyphi. TABLE 3 Neutralization of action of zephiran by sodium decyl sulfate (Staphylococcus aurueu ptrain 4A) Zephiranbroth. in 24 hr Growth Zephiran broth plus 1:l1, sodium decyl sulfate. Growth in 24 hr Zephiran broth plus 1:5, sodium decyl sulfate. Growth in 24 hr 1:25 1:5 I 1:1 USNIMAN 31T 1:2 1 1:4 (Eberthella typhosa) DILVUOS 1:6 1:8 g 1:1, Control 1:S 1:1 1:2 1:4 1:8 Control Zephiran broth. Growth in 24 +++ ++++ hr Zephiran broth plus 1:1, +++ +++ +++ +++ ++++ ++++ sodium decyl sulfate. Growth in 24 hr Equal volumes of zephiran broth and sodium decyl sulfate broth combined before seeding with 1 loop of a 2-hour culture of S. aureus or E. typhosa. Similar results were obtained with S. aureus when- 1:25, and 1:5, sodium lauryl sulfate were used as the neutralizing agent. * Indicated dilution X 1'. 249 Downloaded from http://jb.asm.org/ on January 6, 219 by guest

25 MORTON KLEIN AND ZELMA G. KARDON [vol. 54 made after 6 and 24 hours' incubation showed a gradual reduction in the number of viable bacteria. Selectivity of the action ofcationic detergents. On the basis of germicial activity and the inhibition of bacterial respiration (Baker, Harrison, and Miller, 1941) the cationic detergents have been found to be relatively nonselective in their activity against gram-positive and gram-negative bacteria, showing only a slightly greater activity against gram-positive bacteria than against gramnegative bacteria. In the present studies on the bacteriostatic activity of zephiran, however, the compound, as shown in table 1, has, after neutralization, several hundredfold greater activity against the gram-positive bacteria (and Neisseria intraceuularis) than against the gram-negative bacteria, a selectivity quite similar to inhibitors such as penicillin or gentian violet. DISCUSSION Our results have shown that anionic detergents are not capable of reversing the action of cationic detergents in a manner analogous to the reversal obtained with H2S acting on HgCl,. Hotchkiss (1946) has reported that relatively high concentrations of cationic detergents damage the bacterial cell with a subsequent release of the intracellular constituents into the surrounding medium. HgCl2 did not effect this destruction of the cell. Under such conditions one would not expect any reversal of the action of high concentrations of zephiran, and no reversal was obtained in the present work. Our results on the selectivity of action of zephiran on gram-positive bacteria indicate that it has essentially the same bacterial spectrum as an agent such as penicillin. The recognition of the selectivity of action of penicillin and the classification of zephiran as a relatively nonselective inhibitor is probably due to the fact that we routinely test our chemotherapeutic agents by means of bacteriostatic tests, whereas germicides are ordinarily assayed by means of the phenol coefficient test. The high concentrations required for the bactericidal action of the compound obscured the selective action in the bacteriostatic range. The high bacteriostatic activity of zephiran against the gram-positive bacteria and the failure of anionic detergents to neutralize this action indicates a specificity of action not revealed in the studies on the inhibition of bacterial respiration (Baker, Harrison, and Miller, 1941). Gale and Taylor (1946) have found that the assimilation and concentration of glutamic acid is restricted to the grampositive bacteria and this mechanism is specifically inhibited by penicillin. In view of the similarities in the susceptibility of gram-positive and gram-negative bacteria to penicillin and zephiran, it is possible that the relatively selective gram-positive bacteriostatic activity of zephiran may be related to a similar mechanism. Downloaded from http://jb.asm.org/ on January 6, 219 by guest The activity of the cationic detergents zephiran and phemerol against grampositive and gram-negative bacteria could not be reversed by the anionic detergents sodium decyl sulfate or sodium lauryl sulfate.

1947] GERMICIDAL CATIONIC DETERGENTS 251 Anionic detergents neutralize the bacteriostatic action of zephiran against gram-negative bacteria but do not neutralize the bacteriostatic action of zephiran against gram-positive bacteria. The cationic detergent zephiran in its bacteriostatic range has a high degree selectivity and possesses several hundredfold greater activity against grampositive bacteria than against gram-negative bacteria. REFERENCES BAKER, Z., HARRISON, R. W., AND MILLER, B. F; 1941a Action of synthetic detergents on the metabolism of bacteria. J. Exptl. Med., 73, 249-271. BAKER, Z., HARRISON, R. W., AND MILLER, B. F. 1941b The bactericidal action of synthetic detergents. J. Exptl. Med., 74, 611-62. GALE, E. F., AND TAYLOR, E. S. 1946 Action of penicillin in preventing the assimilation of glutamic acid by Staphylococcus aureus. Nature, 158, 676. HOTCHKISS, R. D. 1946 The nature of the bactericidal action of surface active agents. Ann. N. Y. Acad. Sci., 46, 479-493. KLEIN, M., AND STEVENS, D. A. 1945 In vitro and in vivo activity of synthetic detergents against influenza A virus. J. Immunol., 5, 265-273. VALKO, E. I., AND DuBois, A. S. 1944 The antibacterial action of surface active cations. J. Bact., 47, 15-25. Downloaded from http://jb.asm.org/ on January 6, 219 by guest