(5) It Appears That Methylated Di- and Triphenylmethane Derivatives Act Against Gram-Positive Germs Chiefly, If Not Exclusively, in Their Quinoid Dye Form

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COMPARATIVE BACTERIOSTATIC ASSAYS WITH ROSANILINE AND ITS PHENOLIC ANALOG (ROSOLIC ACID) E. FISCHER AND R. MUROZ Experimental Laboratory S. A. Organa and the Institute of Physiology, Universidad de Chile, Santiago, Chile Received for publication November 19, 1946 Certain relations between the chemical structure and the bacteriostatic action of di- and triphenylmethane dyes have been studied by Browning and Gilmour (1913) and by Kligler (1918). The fi.ndings of these workers are in agreement, as are likewise those of Thornberry (1931). Partially contrasting findings have been published by Fairbrother and Renshaw (1922), who used, however, a different technique, based on bactericidal effects. Their results will therefore not be considered here. The experimental data of the above-mentioned authors, confirmed and somewhat extended by one of the writers and coworkers (Fischer, Garc6s, and L6pez, 1946, and unpublished experiments) permit the following conclusions to be deduced: (1) The presence of three phenylic rings attached to the central methyl radical (i.e., the triphenylmethane structure) results in a considerably stronger action than does the presence of only two such rings (diphenylmethanes) (Kligler, 1918; Fischer et al., 1946). (2) The presence of an amino group attached to the third phenylic ring is of little or no significance, since diaino derivatives are nearly or completely as active as the corresponding triamino derivatives malachite green vs. methyl violet (Browning and Gilmour, 1913; Kligler, 1918; Thornberry, 1931). (3) The methylating, and especially the ethylating, of the amino groups increase the action substantially, whereas the phenylating of the same groups has a contrary effect (rosaniline vs. methyl violet, ethyl violet, and aniline blue) (Browning and Gilmour, 1913; Kligler, 1918; Thornberry, 1931). (4) Transforming one amine into a quaternary ammonium group greatly diminishes the action (methyl violet vs. methyl green) (Browning and Gilmour, 1913; Kligler, 1918). In our opinion this circumstance cannot be related to the reduction of the three resonating structures of methyl violet to two in methyl green, because malachite green with only two resonating structures is as active as methyl violet with three (see no. 2 above). (5) It appears that methylated di- and triphenylmethane derivatives act against gram-positive germs chiefly, if not exclusively, in their quinoid dye form. The nonmethylated dye rosaniline, however, also is effective in the form of leucobase, although only in a lower degree (Fischer et al., 1944, 1946). Recently Kahn and Petrow (1945) published experiments on some pyridyl analogs of triphenylmethane, according to which the substitution of one or two phenyl radicals for pyridyl ones diminishes the bacteriostatic action. From our 381 382 E. FISCHER AND R. MUR[OZ[VOL. 53 point of viewone statement of these authors is of particular interest,namely, that leuco compounds of these derivatives seem to show approximately the same (low) degree of activity against gram-negative as against gram-positive organisms, whereas their oxidation products are many times more active against the latter than the former. Looking at the results of these authors one finds, furthermore, that the difference between the action of leuco compounds and that of oxidized products against gram-positivegerms is less in the case of a monomethyl- amino derivative (1: 2,000 vs. 1:256,000) than it is with dimethylamino derivative (1:2,000 vs. 1:1,024,000). This fact corresponds to our findings (1944), that there is a relatively small difference between the action of the leuco base and that of the oxidized form in the case of the nonmethylated dye rosaniline, whereas the actions of the corresponding forms of methylated dyes (gentian violet, malachite green) differ widely. In order to explain the bacteriostatic activity of the triphenylmethane dyes, several hypotheses have been proposed. We shall consider three of them. (1) The hypothesis of Stearn and Steam (1923, 1924, 1926, 1930) assumes the formation of un-ionized complexes from amphoteric cell constituents and dye radicals, the basic dyes neutralizing the acidic groups of these constituents, and acid dyes the basic groups. (2) Ingraham's hypothesis (1933) is based on a supposed poising effect of the dyes upon the oxidation-reduction potential (cf. Hoffmann and Rahn, 1944). (3) Churchman's hypothesis (1912, 1923) is that the dyes in question saturate certain specific protoplasmic groups, which have an affinity for the former. Steam and Stearn point out, among other arguments, that the stronger action of basic dyes in more alkaline media, and correspondingly the stronger action of acid dyes in more acid media, are in accordance with their hypothesis. These authors explain furthermore the more potentiated bacteriostatic effects of alkylated derivatives by their increased basic character. Hence, phenylating, which lowers the basic property, diminishes the action. It is also possible to explain the importance of the quinoid structure on the same basis, such a structure being linked to the presence of an imino group of strong alkaline character. The theory of Stearn and Steam provides no explanation for the significance of the third phenylic ring, nor for the unimportance of the third group or the weak action of derivatives with one quaternary group, unless it can be shown that the corresponding groupings bear relationship with the ability to form un-ionized complexes. Ingraham (1933) formulates certain objections against the exclusive validity of the hypothesis of Steam and Steamn, some of which we shall mention below. (1) Ingraham contradicts the statement that acid dyes act more strongly in acid media, affirming that the action of acid dyes depends only in a small degree on the pH of the media, but except for this acid dyes as well as basic dyes are invariably more effective in alkaline media. (2) Gram-positive anaerobes are relatively dye-tolerant, as are in general those organisms which possess powerful reducing mechanisms. 1947] BACTERIOSTATIC ASSAYS WITH ROSANILINE 383 Ingraham's own hypothesis was inspired by the studies of Dubos (1929), who concludes that methylene blue inhibits microbial growth by poising the oxidation- reduction potential at a point unfavorable for germ multiplication. It seems questionable, however, whether such a conclusion can be extended to include triphenylmethane dyes, since methylene blue is a typical oxidation- reduction indicator and buffer,whereas,as Ingraham herself states, gentian violet does not significantly change the oxidation-reduction potential of bacteriological media. We can only confirm this for malachite green (Fischer et al., 1944). Otherwise, some of the arguments and experimental results of Ingraham (1933) and Hoffmann and Rahn (1944) support the assumption of an interference by gentian violet with microbial oxidation processes. Such an interference, however, does not necessarily bear any relation to a poi8ing of the potential, but may be explained by some other mechanism, such as blocking or inactivating ferments or other biological substances (cf. Quastel, 1932; Quastel and Wheatley, 1931). In fact, some of Ingraham's arguments and experiments intended to prove a poising effect of gentian violet are not fully convincing. Such heterogeneous processes as the simple formation of a carbinol base from the dye by NaOH and the necessarily destructive oxidation by peroxide are indistinctly designated by this author as more or less reversible "oxidation," without taking into con- sideration the chemical structure of the derivatives produced. The ineffectiveness of gentian violet after decolorization by iron dust and by peroxide is used, furthermore, as an argument in favor of the causal importance of changes of potential, and yet this ineffectiveness may be explained equally well as a conse- quence of structural alterations of very different character, such as the simple loss of quinoid structure by reduction (iron dust) and the destruction of the molecule by oxidation (peroxide). In our opinion a modernized form of Churchman's hypothesis may be accepted, namely, that triphenylmethane dyes act by blocking some important biological mnechanisms, possibly connected with oxidation processes (cf. Davies, Hinshel- wood, and Pryce, 1944). EXPERIMENTAL In the experiments to be reported here the bacteriostatic effects of rosaniline and its phenolic analog (rosolic acid) have been studied comparatively. The commercial preparations labeled "rosaniline" or "fuchsine" consist generally of a mixture of this dye with a near homolog inaccurately called "pararosaniline" or "parafuchsine" (nor-rosaniline would be a more appropriate name),which differsfrom rosaniline onlybythe absence ofa methyl groupattached to one of the phenylic rings in an ortho position with respect to the amino group. The same relation exists between the corresponding phenolic analogs, rosolic acid and aurine. In order to be sure that the preparations used in our experiments corresponded to one another in every respect, we prepared our "rosolic acid" from the "rosaniline" used in these experiments according to the method of Caro and Graebe (diazotization and hydrolysis). Figure 1 shows the structures of these dyes, both of them in the form of a monovalent ion. 384 E. FISCHER AND R. MUSOZ [VOL. 53 Rosaniline is a basic dye, which in moderately acid solutions forms a monovalent cation. The ionized group has an immonium structure (methenyl- quinimine ionized). Rosolic acid is a phenolic dye, which in moderately basic

+ H,N-7-C7NH

R R

NH2 OH R-H pararosaniline R-H auriue RCH roaniline R-CHs rosolic acid FiGeuz 1

TABLE 1 Growth of yea8t in the presence of different concentrations of malachite green at varying pH values pH 42 428 4.50 5.30 6.22 6. 1:100,000 - - - - - 1:200,000 _ _ _ - _ _ 1:400,000 + + + _ _ _ 1:800,000 + + + _ _ _ 1:1,600,000 ++ ++ ++ + + _ 0 ++ ++ ++ ++ ++ +

TABLE 2 Growth of yeast in the presence of different concentrations of rosaniline and rosolic acid at varying pH values

ZOBANIINE tOSOLIC ACID DYE CO TIONS pH 48 5.30 5.80 5.30 6.60 1:1,000 + - - - - 1:2,000 + _ - + + 1:4,000 ++ + _ ++ + 1:8,000 ++ ++ + ++ + 1:16,000 ++ ++ ++ ++ + 0 ++ ++ ++ ++ + solutions forms a monovalent anion. The ionized group in this case is a phenol, whereas the quinoid part of the molecule is a quinone (methenylquinone). Besides these dyes we employed malachite green, a methylated diamino- triphenylmethane dye, with a strong bacteriostatic action. The basic dyes malachite green and rosniline have been used in the form of chlorides and rosolic 1947] BACTERIOSTATIC ASSAYS WITH ROSANILINE 385 acid in the form of sodium salt. The first series of assays has been dedicated to the study of the influence of the pH of the medium upon the bacteriostatic action of malachite green against bakers' yeast. This germ is particularly interesting in this respect as it grows optimally in acid media. The yeast was cultivated in yeast water (1:8) containing 0.5 per cent glucose, buffered by acetate mixtures (M/15) and inoculated with 0.1 ml of a 1 per cent fresh yeast suspension. It was incubated for 24 hours at 20 C (table 1). The results clearly show the dependence of the bacteriostatic action of malachite green on the pH of the medium. Similar experiments with rosaniline and rosolic acid gave the results shown in table 2. It can be observed that the order of magnitude of the action of malachite green (table 1) is quite different from that of both rosaniline and rosolic acid (table 2), the methylated dye malachite green being effective at pH 5.3 in a dilution of

TABLE 3 Growth of gervs in preence of varying concentrations of maachite green, rosaniline, and rosolic acid

DYES STAPYLOCOCCUS YEAT SIILA IITELA ICERCI Malachitegreen 1:8,000,000- 1:800,000- 1:400,000- 1:80,000- 1:80,000- 1:16,000,000+ 1:1,600,000+ 1:800,000+ 1:160,000+ 1:160,000+ Rosaniline 1:80,000- 1:2,000- 1:2,000- 1:2,000- 1:600- 1:160,000+ 1:4,000+ 1:4,000+ 1:4,000+ 1:1,000+ Rosolic acid 1:40,000- 1:1,000- 1:1,000- 1:2,000- 1:500- 1:80,000+ 1:2,000+ 1:2,000+ 1:4,000+ 1:1,000+ pH 7.6 5.3 7.1 7.3 7.3

1:800,000, whereas both rosaniline and rosolic acid acted at the same pH only until they were diluted to 1:2,000 and 1:1,000, respectively. Furthermore there is clear evidence that the actioa of the basic dyes malachite green (table 1) and rosaniline (table 2) depends on the pH range. On the other hand, no such dependence appears in the case of the phenolic dye rosolic acid between the pH values of 5.3 and 6.6. More acid solutions could not be tested for this dye precipitated in such conditions. In more basic solutions there was no regular growth of yeast. In the following assays malachite green, rosaniline, and rosolic acid were tested against Staphylococcus aureus, Shigella, Escherihia coli, and EbertheUa typhosa, cultivated in peptone water and in broth. Inoculation was with 0.05 ml of a 24-hour culture; readings occurred after 24 hours at 37 C (see table 3). There appears a very close parallelism between the bacteriostatic strength of rosaniline and that of rosolic acid against all the germs studied here, which belong to very different classes of microorganisms and have very different dye sensi- tivities. The action of the methylated dye malachite green is in every instance 386 E. FISCHER AND R. MUROZ [[VOI.. 53 of a much higher order of magnitude than that of both rosaniline and rosolic acid. It further seems that all three dyes have a stronger effect against staphylococcus than against the members of the coli-typhoid group. In the case of gram-positive germs and Shigella, rosaniline acts somewhat more strongly than does rosolic acid, whereas both of them act with equal strength against E8- cherichia and EbertheUa. DISCUSSION It is a well-established fact that basic dyes act more strongly in more alkaline solutions, thus supporting the hypothesis of Steam and Stearn. But apparently this hypothesis cannot be applied so easily to the action of acid dyes, as they do not act more strongly in more acid solutions, as would be required by this hypothesis. On the contrary, they behave either like the basic dyes (showing only a weaker dependence on the pH value), as Ingraham states, or they are not influenced by pH ranges at all, as observed in our assays. To explain this discrepancy between the bacteriostatic behavior of basic and acid dyes, one may assume that the mode of action of both classes of triphenylmethane dyes is different and that the hypothesis proposed by Steamn and Steam is only valid for basic dyes. We think, however, that the close parallelism observed between the action of rosaniline and that of rosolic acid against very different kinds of microbes makes the existence of a similar mechanism of action in both cases more probable. If this is so, the basic character would not have any fundamental importance but would represent only one of the factors able to re- inforce the effectiveness. The importance of the quinoid structure and its presence in both rosaniline and rosolic acid may be used as an argument in favor of Ingraham's thesis of poising action on the oxidation-reduction potential. However, as set forth earlier, this hypothesis is not well supported asyet. It would be necessary to determine, in any case, the potentials of the different triphenylmethane dyes and to compare the values obtained with the strength of the bacteriostatic action of the corresponding dyes, as has already been done in the case of acridine dyes by Breyer, Buchanan, and Duewell (1944; cf. Albert et al., 1945). One is impressed, furthermore, by the great difference existing between the action of alkylated dyes, on one hand, and that of a nonalkylated basic dye and an acid dye, on the other hand. Unless it can be shown that the change in the strength of the basic character of the oxidation-reduction potential caused by alkylation of the amino groups can be responsible for that difference, this circumstance may rather indicate a partially different mode of action for alkylated and nonalkylated dyes (cf. Thomberry, 1931). There seems to exist some further evidence in favor of such an assumption, for alkylation not only strengthens in a considerable degree the bacteriostatic action, but it also seems to increase the importance of the quinoid structure for this action (see earlier discussion of the varying relations between the activity of leuco bases and of quinoid dye salts in nonmethylated and methylated dyes). In pyridyl analogs nf triDhenvlmethane. alkylating and quinoid structure are of importance solely 194711RA9CTERIOSTATIC ASSAYS WITH ROSANILINE 387 for the action against gram-positive organisms but not for gram-negative organisms. Apparently alkylation of the amino groups alters the bacteriostatic activity not only quantitatively but also qualitatively. It seems, therefore, not only that the bactericidal and bacteriostatic effects of triphenylmethane dyes may have different mechanisms (Churchman, 1912; Hoffman and Rahn, 1944), but the mechanisms of the bacteriostatic effects of different derivatives against different microorganisms also may not be entirely identical. Finally the conclusion reached by Breyer et al. (1944) in the case of acridine derivatives may be valid also for triphenylmethane dyes, namely, that the activity cannot be connected to any single chemical or physical property, but represents the sum total of such properties. ACKNOWLEDGMENT We are very much indebted to Prof. C. Garc6s, Chief of the Department of Bacteriological Research, Instituto Bacteriol6gico de Chile, for his generous help in carrying out our experiments. SUMMARY Whereas the basic dye rosaniline acts more strongly in a more alkaline medium, the action of the phenolic (acidic) dye rosolic acid shows no dependence on the pH value. The bacteriostatic effects of both rosaniline and rosolic acid are of the same order of magnitude, which is considerably lower than that of the methylated basic dye malachite green. REFERENCES ALBERT, A., RUBBO, S. D., GOLDACRE, R. J., DAVEY, M. E., AND STONE, J. D. 1945 The influence of chemical constitution on antibacterial activity. II. A general survey of the acridine series. Brit. J. Exptl. Path., 26, 160-192. BREYER, B., BUCHANAN, G. S., AND DUEWELL, H. 1944 Reduction potential of acridines with reference to their antiseptic activity. J. Chem. Soc., 1944, 360-363. BROWNING, C. H., AND GILMOUR, W. 1913 Bactericidal action and chemical constitution with special reference to basic benzol derivatives. J. Path. Bact., 18, 144-146. CHURCHMAN, J. W. 1912 The selective bactericide action of gentian violet. J. 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