detergents lethal to bacteria are insufficient to cause any general denaturation

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1 THE ACTION OF CATIONIC DETERGENTS ON BACTERIA AND BACTERIAL ENZYMES' W. E. KNOX, V. H. AUERBACH, K. ZARUDNAYA, AND M. SPIRTES Enzyme Laboratory, Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, N. Y. Received for publication July 5, 1949 The bactericidal action of the quaternary ammonium detergents has not yet been satisfactorily explained. Recent reviewers (Putnam, 1948; Rahn and Van Eseltine, 1947; cf. Glassman, 1948) have not agreed that the amounts of these detergents lethal to bacteria are insufficient to cause any general denaturation of the bacterial proteins (Valko, 1946). Action by enzyme inactivation, either primary or secondary to some other injury, has been repeatedly suggested. Respiratory and glycolytic activity is depressed by detergents, and the inhibiting amounts were later shown to parallel roughly the lethal amounts (Baker et al., 1941a,b). However, the frequent survival of cells at concentrations of detergent which produced marked respiratory inhibition has cast doubt on an action by direct inhibition of enzymes. Hotchkiss (1946) demonstrated that nitrogen and phosphate compounds diffuse out of the cells with bactericidal amounts of detergents. As an alternative to the action by enzyme inhibition he suggested that the loss of substrates and cofactors by diffusion from the cytolyzed cells might account for the death of the cells and the observed metabolic inhibitions. We have investigated the problem because objections can be made to much of the earlier work. Confirmation is apparently required that bactericidal amounts of detergents are well below those amounts causing general protein denaturation. The fact that some enzymes survive treatment with a bactericidal agent (Rahn and Schroeder, 1941; Greig and Hoogerheide, 1941; Bucca, 1943) has sometimes been interpreted as evidence against action by specific enzyme inhibition. More important is the lack of precise correlations between the lethal and the metabolic inhibitory amounts of detergent. Hotchkiss and others (Glassman, 1948) have pointed out that these irregularities may all be attributed to the comparison of quantitative metabolic measurements with the essentially qualitative determinations of viability by subculture. If cell death and metabolic inhibition are demonstrated to occur together with the same amount of detergent, an investigation limited to intact cells still leaves one unable to decide which of the results is cause and which effect (Roberts and Rahn, 1946). This decision would be simplified if the sensitivity of the enzymes in question, in cell-free form, to the detergents at the bactericidal levels was known. METHODS Escherichia coli was grown for 16 to 24 hours in 8-liter quantities of Difco casein digest broth, aerated by a stream of filtered air. Harvesting was accom- 1 This investigation was conducted under a contract of Dr. D. E. Green and Columbia University with the Committee on Medical Research of the Office of Scientific Research and Development and at the request of the Office of the Quartermaster General. 443

2 444 KNOX, AUERBACH, ZARUDNAYA, AND SPIRTES [VOL. 58 plished with a Sharples supercentrifuge. The bacteria were washed in the centrifuge with 3 to 5 liters of distilled water and then suspended as a thick paste in water and stored at 0 C. All suspensions of E. coli used for manometric experiments were again washed with 10 voluimes of water, centrifuged, and appropriately diluted according to the nitrogen content as determined by the micro- Kjeldahl method. Detergent concentrations were calculated from the percentage composition given by the manufacturers. In the manometric measurements of bacterial metabolism each cup contained 0.25 mg of bacterial protein N (1.5 X 10"1 bacteria per mg N), 0.5 ml of 0.2 M phosphate buffer ph 7.0, and detergent and water to make a total volume of 2.5 ml. The substrate, 0.5 ml of 0.1 M solution, was tipped in from the side arm after 10 minutes' equilibration at 38 C, and readings were made for 1 hour. The center well contained NaOH except in the glycolysis experiments; in these experiments bicarbonate buffer was used with 95 per cent N2 and 5 per cent C02 in the gas phase. Bacterial counts were made by diluting with sterile saline the contents of the manometric cups at the end of an experiment and inoculating aliquots into poured plates of Difco Endo agar. Counts were recorded after 24 hours' incubation. Each plate count was made in duplicate, and the average is expressed as a percentage of the average control count. Arginine decarboxylase was prepared and measured under the conditions used by Gale and Epps (1944). The lactic acid oxidase was prepared by grinding the stock suspension of E. coli cells in the Booth-Green mill for 3 hours and centrifuging the resulting slurry after diluting it twofold with water. The cell-free supernatant, referred to as crude enzyme, is stable for over 1 week at 0 C and was used in most experiments. The oxygen uptake with the lactic acid oxidase was assayed for 10 minutes after tipping 0.5 ml of 0.2 M lactate from the side arm into 1.0 ml of enzyme and 1.3 ml of 0.05 M phosphate (ph 7.0) with and without the addition of detergent. With 02 in the gas phase the activity under these conditions persists for 30 minutes and is proportional to the enzyme concentration between the limits of 10 and 140 mm8 02 per 10 minutes. Pyruvate formation was determined with the salicylaldehyde method. Measurement of the bactericidal amount of detergent. No one concentration of detergent wasfound to be generallybactericidal, contrary tothe general assumption. As was found to be the case with chlorine (Knox et al., 1948), a definite amount of detergent per amount of bacteria is reproducibly bactericidal. The lethal amount of detergent is therefore expressed as a bactericidal ratio, instead of a bactericidal concentration which would be meaningful only with given volumes of solution and numbers of bacteria. For convenience the bactericidal ratio is given as micrograms of detergent per mg of bacterial nitrogen, since in this form it is equally applicable to enzymes (per mg of protein nitrogen). That the inhibition of lactate oxidation by E. coli cells is proportional to the amount and not to the concentration of detergent is shown in table 1. The ratio of amounts of detergent and protein, and not the detergent concentration, as the determining factor may also be demonstrated from the experiments of previous workers in which sufficient data have been given. In the experiments of Sevag and Ross (1944), for example, a given amount of zephiran is lethal for only a certain

3 1949] CATIONIC DETERGENTS AND BACTERIA 445 amount of yeast, and with the addition of serum still more zephiran is necessary, proportional to the amount of extraneous protein added. The precipitation of proteins, which occurs with much higher amounts of detergent, is also dependent upon the detergent-protein mass ratio (Glassman, 1948). TABLE 1 Inhibition of the lactate oxidation by E. coli cell8: proportionality to the detergent-bacteria ratio and not to the detergent concentration BACTERIAL CONCENTRATION ZEPrIAN CONCENTRATION REPEZAN-BACTERIAL RATIO PER CENT INHIBITION mg N/lmi pg/m pg Z/mg N a5 ~~~5o~Q 00o- 50 K aV ptg ZEPHIRAN/mg N Figure 1. The amount of zephiran per mg of bacterial nitrogen to kill E. coli (A), to inhibit the glucose oxidation of E. coli (0), and to inhibit the lactic acid oxidase isolated from E. coli (@). Bacterial counts were made at the conclusion of the manometric determinations of glucose oxidation. Comparison of the bactericidal effect of a detergent with its metabolic inhibition, and comparison of the bactericidal effects of different detergents, have been made at levels giving 50 per cent mortality and 50 per cent inhibition. The determinations of viable cells and of metabolic activity are comparable at this point, whereas serious errors are inherent in such comparisons at the point of 100 per cent sterilization. Correlation of metabolic inhibition with bactericidal action. In figure 1 are given

4 446 KNOX, AUERBACH, ZARUMDNAYA, AND SPIRTES [VOL. 58 the percentages of bacteria killed by increasing amounts of zephiran per mg of bacterial nitrogen. A second curve shows the per cent of inhibition of glucose oxidation by these cells, measured in the same experiment. The agreement, within our experimental error, that half the cells are dead and half the glucose oxidation inhibited at the m e amount of detergent has been found in all instances with many different batches of bacteria. This same relationship was found to occur with a series of other cationic detergents. The amounts of these detergents which give 50 per cent killing and 50 per cent inhibition of glucose oxidation as determined from curves similar to that of figure 1 are given in table 2. The metabolism of other substrates is not inhibited by a given amount of detergent to the same degree as is glucose oxidation (table 3). The higher amount necessary to inhibit lactate oxidation in the intact cell may perhaps be associated with the considerable stimulation of lactate oxidation observed in intact cells TABLE 2 Comparison of the amounts of various detergents which kill E. coli, inhibit their oxidation of glucose, and inhibit the cell-free lactic acid oxidase 50%0 nzmitron S0%o nibnozi DETERGENT 50%o lled OF GLUCOSE 01 LACTIC OXIDATION ACD OIASZ pg/mg N pg8/mg N pg/mg N l-n-hexadecyl-pyridinium C1 (ceepryn) (C8-18) Alkyldimethyl-benzyl-ammonium C1 (zephiran) Cetyl-trimethyl-ammonium bromide N-(nonyl-naphthyl-methyl)-pyridinium chloride (emcol 888) N-(lauryl-colamino-formyl-methyl)-pyridinium C1 (emulsept) with less than bactericidal amounts of detergents. This variation between the sensitivities of different oxidative and glycolytic reactions is not large. The inhibition of reactions such as that of arginine decarboxylase and of catalase (Roberts and Rahn, 1946), on the other hand, occurs only with a large amount of detergent (table 4). The large amount necessary to inhibit arginine decarboxylase is of the order able to denature the protein. In experiments with the cell-free enzyme it can indeed be seen that inhibition occurs only with denaturation and precipitation of the protein. The mass ratio of detergent to protein necessary for denaturation of this bacterial enzyme is of the same order required to denature other proteins (Chinard, 1948) and is obviously much higher than the bactericidal ratio. The increase of arginine decarboxylase activity in the intact cell, which occurs with bactericidal and higher amounts of detergent, may be an expression of the increased cell permeability to the substrate, which occurs under these conditions. This effect has been recently used to measure the intracellular free amino acids

5 1949] CATIONIC DETERGENTS AND BACTERIA 447 upon their diffusion out of the cell (Gale and Taylor, 1947). The increased activity of this enzyme, which requires the coenzyme pyridoxal phosphate, does not provide any support to the idea that the diffusion of coenzymes and substrates from the cell might account for the observed inhibition of other enzyme reactions. TABLE 3 Amount of zephiran producing 50 per cent inhibition of various metabolic reactions of E. coli REACTION Glucose glycolysis... Glucose oxidation... Pyruvate oxidation... Formate oxidation... Alanine oxidation... Lactate oxidation... Succinate oxidation... Arginine decarboxylation... Hexose diphosphate oxidation... Hexose diphosphate glycolysis... Aldolase... INTACT CELLS pg/mg N * CELL-FREE ENZYXES pg/mg N , , 000 1,200 * Five thousand ;g per mg N produced a 4-fold increase in arginin deecarboxylase activity (table 4). TABLE 4 Effect of zephiran on arginine decarboxylase of E. coli ZEPHIRAN INTACT CELLS ISOLATED ENZYME pg/mg N QCo2 (mg N) Qc02 (mg N) , ,430 1,980 1,000 2,520 2,000 (+) 5,000 2,330 1,240 (++) 10,000 1, (+++) +-visible precipitation of the enzyme by detergent. Enzyme inactivation as a mechanism for bactericidal action of detergents. It remains to be demonstrated that the detergents in bactericidal amounts can inhibit essential enzymes apart from any action on cell structure. Inspection of table 3, which contains those reactions known to be inhibited in the intact cell, does not reveal any one reaction the inhibition of which would account for all the results. Several enzymes, the reactions of which appeared to be inhibited in the cell by detergents, were therefore isolated to test their sensitivity to the detergents in the absence of cellular organization. The enzyme aldolase, as well as the whole glycolytic system producing acid and CO2 from hexose diphosphate, were markedly resistant to detergents in the cell-free state, in

6 448 KNOX, AUERBACH, ZARUDNAYA, AND SPIRTES [VOL. 58 contrast to the glycolysis of glucose by the intact cell (table 3). The additional enzymes functioning in glycolysis in the intact cell, such as the phosphorylating enzymes, may be sensitive to detergents and account for this discrepancy, but such enzymes of E. coli have not yet been studied. However, two other systems, one oxidizing lactate and one oxidizing hexose diphosphate, could be prepared regularly in a soluble, cell-free form by the milling procedure used. Both of these isolated oxidizing systems were inhibited by bactericidal amounts of detergents (figure 1, tables 2 and 3). Both enzyme systems were unstable, the lactic acid oxidase perhaps less so, and it was selected for further study as a detergent-sensitive enzyme. The Detergent-sen8itive Lactic Acid Oxidase The enzyme catalyzes the one-step oxidation of lactate to pyruvate (table 5) by molecular oxygen. It is therefore a lactic acid oxidase. It appears to differ from other lactic acid enzymes (dehydrogenases) previously described by reacting directly with oxygen. The lactic acid dehydrogenase from E. coli described briefly by Still (1941) was not tested with oxygen. Neither Still's enzyme nor the lactic acid oxidase require diphosphopyridinonucleotide (coenzyme I), and TABLE 5 Oxidation of lactic acid to pyruvic acid by lactic acid oxidase REACON TM GN UPTAKE rleuvate FORMD j atoms p moles 40 min (1 ml enzyme) min (0.6 ml enzyme) min (1 ml enzyme) they are probably the same. The ph optimum of the enzyme is 7.0, with a marked decrease in activity below ph 6.0 or above ph 8.0. A relatively high concentration of lactate is required to saturate the enzyme, the Michaelis constant being approximately M under the assay conditions used (see "Methods"). Under these optimal conditions for assay, the crude enzyme maintains a linear uptake of oxygen amounting to about 30 mm8 per mg protein nitrogen per 10 minutes for 30 minutes, after which the activity falls off rapidly. The enzyme reacts most rapidly with oxygen, and less rapidly with air, and this reaction is prevented by cyanide. Methylene blue can function as a hydrogen acceptor in the place of oxygen, and will in this way reverse the inhibition produced by cyanide. The correspondence between the zephiran inhibition of the isolated lactic acid oxidase and the killing effect of zephiran on intact cells is shown in figure 1. Within the limits of error set by the techniques used, glucose oxidation in the intact cell, viability, and the cell-free lactic acid oxidase are all affected equally by this detergent. The effect of the detergent on the isolated enzyme, as on the intact cell, is dependent on the detergent-protein ratio and not simply on the concentration of detergent (table 6). The amounts of other detergents per mg of enzyme nitrogen which produce 50 per cent inhibition of the lactic acid oxidase

7 1949] CATIONIC DETERGENTS AND BACTERIA 449 are given in the third column of table 2. The inhibitions of lactic acid oxidase by these detergents parallel for the most part their effects on viability and glucose oxidation, as is the case with zephiran. This would tend to confirm the view that the cationic detergents are bactericidal because they affect a specific enzyme system such as the lactic acid oxidase and not because they primarily alter the cell structure, since this enzyme still possesses, in the absence of the cell structure, the intact cell's pattern of sensitivity to detergents. Such enzymes inhibited by detergents may in turn be necessary to maintain the dynamic integrity of the cell membranes, and their inhibition would then account for the observed permeability phenomena and the cell death. Mechanism of detergent inhibition of lactic acid oxidase. Further study of the lactic acid oxidase will be necessary to reveal the characteristic that produces the sensitivity of the enzyme, and therefore of the bacterial cell, to detergents. No added cofactors or coenzymes seem to be necessary for the reaction, since none of the known cofactors or coenzymes added increase the reaction, even after dialysis. Only threefold purification (by ammonium sulfate fractionation between 23 and 53 grams per cent) was possible due to the marked instability TABLE 6 Proportionality of lactic acid oxidase inhibition to zephiran-protein ratio and not to the concentration of zephiran ENZYME ZEPEIRAN ZEPEIAN OXYGEN UPTAE: INHI31TION ml pg/ml pg/ml N mmj/10 min % of the enzyme. Such purification as was achieved failed to reveal a need for cofactors or spectrophotometric evidence of a prosthetic group. In particular, the absorption curve of cytochrome b2 recently described as part of the yeast lactic dehydrogenase was not found (Bach et al., 1946). The reaction of lactic acid oxidase with oxygen and its inhibition by cyanide suggest the functions of a cytochrome component. Elimination of this cytochrome function by cyanide does not alter the detergent inhibition. Thus the anaerobic dehydrogenation reaction with the enzyme in the presence of methylene blue and cyanide shows the same sensitivity to detergents as does the reaction with oxygen. The cytochrome component, if it exists, is therefore not the site of detergent action. Enzyme which has been inhibited by detergent cannot be reactivated by removal of the detergent, by additional substrate, or by any known cofactors. There is no indication of prosthetic group separation or of protein denaturation in such an inhibited enzyme. DISCUSSION The most complete studies of inhibition of bacterial metabolism and bactericidal activity of surface-active agents (Baker et al., 1941a,b) have shown a

8 450 KNOX, AUERBACH, ZARUDNAYAk, AND SPIRTES [VOL. 58 general correlation of these two effects for a wide variety of detergents and for different bacterial species. However, the results, because of the methods used, do not support the hypothesis that a detergent-sensitive enzyme exists, the inhibition of which causes the metabolic depression and death of the cell. Although the two effects were determined in separate experiments and compared on the basis of detergent concentration instead of the bacteria-detergent mass ratio, identical conditions were maintained so that comparable bacteria-detergent ratios were achieved. In these experiments the occasional survival of cells with amounts of detergents which inhibited metabolic activity can then be attributed to the lack of precision of such comparisons at the point of 100 per cent sterilization. Metabolic measurements are essentially zero when 95 per cent of the cells in a suspension are inhibited, but subculture will still detect viable cells after more than per cent of the cells are dead. The unusually large amount of detergent required to kill the last few cells, which may be protected by clumping (DuBois and Dibblee, 1946), heightens the discrepancy between these two methods. From these considerations, and the results reported here, a parallelism between the inhibition of glycolysis and certain sensitive oxidations and the bactericidal activity of the cationic detergents may be accepted. Observations were restricted to E. coli in the present instance to minimize secondary effects which might arise from the greater tendency of cells of other species to clump or lyse with detergents. The experiments quoted and others (Ordal and Borg, 1942) suggest that a similar parallelism will hold for other species. However, no study of the sensitivity of the appropriate cell-free enzymes of other species has been made. The reported inability of evgn high amounts of detergent to inhibit bacterial metabolism more than 80 to 95 per cent (Baker et al., 1941a,b) is no doubt referable to the high metabolic blanks of the cell suspensions used, and does not represent a persistence of the particular metabolic reaction under study. The cell suspensions used in the present study were washed immediately before use to remove the traces of other substrates from the medium and from disintegrated cells. Suspensions so treated have no measurable metabolism without added substrate and are fully inhibited with appropriate amounts of detergent. It is clear from the information available that cationic detergents act in a specific manner on E. coli cells, and the cells of. some other species, to produce concomitant inhibition of certain essential metabolic reactions, cell death, and an increase in the cell permeability. The experiments reported show that cell death (by milling, for example) is not necessarily followed by loss of enzyme activity, nor are enzyme reactions necessarily inhibited by an increased permeability (arginine decarboxylase). Primary inhibition of an essential enzyme reaction, on the other hand, might be expected to produce the organizational derangements that result in cell death and increased permeability. The demonstration that E. coli possesses presumably essential enzymes, which in cell-free form can be inhibited by the amount of detergent bactericidal for the intact cell, strongly suggests that the cationic detergents act by the specific inhibition of such enzymes.

9 1949] CATlONIC DETERGENTS AND BACTERIA 451 The lactic acid oxidase is as yet insufficiently characterized to attempt any generalizations as to its relation with other detergent-sensitive enzymes of E. coli, or to the detergent-sensitive enzymes of other organisms. Sevag and Ross (1944) referred the action of zephiran on yeast to inhibition of the cytochrome c-cytochrome oxidase system. E. coli does not possess this familiar cytochrome system (Keilin and Harpley, 1941). Whatever cytochrome system is present in E. coli might well be present in the lactic acid oxidase complex (cf. the yeast cytochrome b2-lactic dehydrogenase described by Bach et al., 1946). Yet an action of the detergents on this moiety, as is assumed in the case of yeast, is apparently precluded in the E. coli lactic oxidase by the retention of detergent sensitivity even in the presence of cyanide. SlUMARY Several cationic detergents have been shown to kill Escherichia coli parallel with the inhibition of certain metabolic reactions of these cells. Other reactions persist in the presence of the lethal amounts of detergents. The detergents produce these effects of killing and inhibition proportional to the detergentbacterial ratio, and not to the detergent concentration. Escherichia coli possess certain enzymes which in cell-free form can be inhibited by the detergent-protein ratios which are bactericidal for the intact cells. One of these, the lactic acid oxidase, which does not require coenzyme I and which reacts directly with oxygen, is described. The specific inhibition of such detergentsensitive enzymes can account for the metabolic inhibition, cell death, and increased permeability observed in bacteria with bactericidal amounts of cationic detergents. REFERENCES BACH, S. J., DIxoN, M., AND ZERFAS, L. G Yeast lactic acid dehydrogenase and cytochrome b2. Biochem. J., 40, BAKER, Z., HARRISON, R. W., AND MILLER, B. F. 1941a Action of synthetic detergents on the metabolism of bacteria. J. Exptl. Med., 73, BAKER, Z., HARRIsoN, R. W., AND MILLER, B. F. 1941b The bactericidal action of synthetic detergents. J. Exptl. Med., 74, BUCCA, M. A The effect of germicides on the viability and on the respiratory enzyme activity of the gonococcus. J. Bact., 46, CHINARD, F. P Interaction of quaternary ammonium compounds and proteins. A simple method for the rapid estimation of urinary protein concefltration with alkyldimethylbenzyl ammonium compounds. J. Biol. Chem., 176, DuBois, A. S., AND DIBBLEE, D. D Death-rate study on a high molecular quaternary ammonium compound with Bacillus metiens. Science, 103, 734. GALE, E. F., AND EpPs, H. M. R Amino-acid decarboxylases. Biochem. J., 38, GALE, E. G., AND TAYLOR, E. S The action of tyrocidin and some detergent substances in releasing amino acids from the internal environment of Streptococcus faecalis. J. Gen. Microbiol., 1, GLASSMAN, H. N Surface active agents and their application in bacteriology. Bact. Revs., 12, GREIG, M. E., AND HOOGERHEIDE, J. C Evaluation of germicides by the manometric method. J. Bact., 41, HorCHKISS, R. D The nature of the bactericidal action of surface active agents. Ann. N. Y. Acad. Sci., 46,

10 452 KNOX, AUERBACH, ZARUDNAYA, AND SPIRTES [VOL. 58 KEILIN, D., AND HARPLEY, C. H Cytochrome system in B. coli. Biochem. J., 35, KNOX, W. E., STUMPF, P. K., GREEN, D. E., AND AUERBACH, V. H The inhibition of sulfhydryl enzymes as the basis of the bactericidal action of chlorine. J. Bact., 55, ORDAL, E. J., AND BORG, A. F. 1942,Effect of surface active agents on oxidation of lactate by bacteria. Proc. Soc. Exptl. Biol. Med., 50, PUTNAM, F. W The interaction of proteins and synthetic detergents. Advances in Protein Chem., IV, RAHN, O., AND SCHROEDER, W. R Inactivation of enzymes as the cause of death of bacteria. Biodynamica, 3, RAHN, O., AND VAN ESELTINE, W. P Quaternary ammonium compounds. Ann. Rev. Microbiol., 1, ROBERTS, M. H., AND RAHN, The amount of enzyme inactivation at bacteriostatic and bactericidal concentrations of disinfectants. J. Bact., 52, SEVAG, M. G., AND Ross, 0. A Studies on the mechanism of the inhibitory action of zephiran on yeast cells. J. Bact., 48, STILL, J. L Pyruvic acid dehydrogenase of B. coli. Biochem. J., 35, VALKO, E. I Surface active agents in biology and medicine. Ann. N. Y. Acad. Sci., 46, Downloaded from on July 27, 2018 by guest