sulfapyridine, sulfaguanidine, sulfathiazole, sulfadiazine, and sulfapyrazine upon

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1 THE ACTION OF SULFONAMIDES AND OF PARA-AMINOBENZOIC ACID ON BACTERIUM TULARENSE JOSEPH T. TAMURA Department of Bacteriology, College of Medicine, University of Cincinnati and the Cincinnati General Hospital Received for publication January 1, 1944 An inhibitory effect of sulfadiazine on growth of Bacterium tularense on bloodglucose-cystine agar was reported by Ransmeier (1943). Inhibition occurred with concentrations of 50, 100 and 200 mg per cent, but not with concentrations of 10 and 20 mg per cent. Experience here and elsewhere indicates that results of drug inhibition studies in which solid media were used are difficult to duplicate. Strauss and Finland (1941) demonstrated the importance of the medium for comparison of efficacy of various sulfonamides. This study deals with the bacteriostatic effect of sulfanilamide, neoprontosil, sulfapyridine, sulfaguanidine, sulfathiazole, sulfadiazine, and sulfapyrazine upon Bacterium tularense cultivated in an inhibitor-free gelatin hydrolyzate medium. Each drug was tested repeatedly by the same method but, since inhibition with sulfanilamide, neprontosil, sulfapyridine, and sulfaguanidine was observed only in concentrations in excess of 20 mg per cent, the results with these four drugs are omitted. MATERIALS AND METHODS The composition of the medium was described previously (Tamura and Gibby, 1943). Fresh sulfonamide stock solutions were made in distilled water for each day's tests. Drug solutions were added to the medium in desired concentrations, dispensed in 4.5 ml quantities in 25 mm diameter tubes, and autoclaved at 10 pounds for 10 minutes. The virulent Memp strain of B. tularense was used throughout. Graded inocula were obtained as follows. Growth from 24-hr cultures on blood-gluposecystine agar was suspended in a tube of normal liquid medium. The turbidity was adjusted to that of 500 p.p.m. of the U.S.P.H.S. fullers' earth standard by means of a photoelectric comparometer. From this T-500 suspension, designated as 100, decimal dilutions were prepared in the usual way. One half ml of the desired suspension formed the inoculum for each tube. No satisfactory method exists to determine the number of organisms per unit volume, but many titrations of virulence in white mice indicate that a T-500 suspension of highly virulent strains contains approximately 2 billions of organisms per ml. Incubation at 37.5 C was continued for 8 days. The criterion of growth was the appearance of turbidity in the media. Para-aminobenzoic acid (PABA) was prepared as a 10-1 molar solution in normal medium and was added to complete media in desired concentrations before tubing. 529

2 530 JOSEPH T. TAMURA OBSERVED RESULTS Representative bacteriostatic effects of sulfapyrazine, sulfadiazine, and sulfathiazole are shown in table 1. Sulfapyrazine was always the most inhibitory; cor~pletely bacteriostatic in 2 X 104 molar concentration (equivalent to 5 mg per cent) throughout the inocula range from 20 to 20,000 orgaisms per ml of medium. Sulfadiazine and sulfathiazole, in the order stated, were decreasingly inhibitory in equivalent concentrations. Sulfadiazine was completely bacteriostatic in 3.2 X 10- concentration (8 mg per cent) only when inocula were 200 TABLE 1 Effect of sulfonamides upon Bacterium tularene INOCTuLm, 0.5 3tL MOLAR CONCE1N ' DRIUGS TRATION OF DRUG M x 10-' _ o O oo SD o o o O O ST O Control medium Inoculum: 0.5 ml of 10o- represents approximately 20,000 organisms per ml of medium. 0 - no visible growth; + visible growth. organisms, or less, per ml of medium. With these same inocula it required 6 X 10-4 molar sulfathiazole (approximately 10 mg per cent) to achieve complete bacteriostasis. EFFECT OF PABA ON SULFONAMIDE INEIBIMON It was demonstrated by Woods (1940) and confirmed by others (Strauss, Lowell and Finland, 1941; Strauss, Dingle and Finland, 1941; Spink and Jermsta, 1941; Rose and Fox, 1942; Wyss, Grubaugh and Schmelkes, 1942) that PABA inhibited the bacteriostatic action of sulfonamides on various bacteria, and that the molar ratios of antagonistic pairs were constant throughout the range of sulfonamide inhibition.

3 SULFONAMIDES AND BACTERIUM TULARENSE 531 Contrary to anticipation PABA produced no inhibition of effects of sulfapyrazine, sulfadiazine, or sulfathiazole upon B. tularense. Furthermore, PABA itself inhibited growth of B. tularense in concentrations greater than M X Table 2 illustrates the experience with sulfapyrazine, the most effective of the sulfonamides studied; also the effect of PABA on pure cultures in gelatin hydrolyzate medium. This unusual effect of PABA on B. tularense is not due to any substance contained in the medium. Sulfonamide bacteriostasis and PABA inhibition were TABLE 2 Effect of p-aminobenzoic acid on sulfapyrazine inhibition and upon Bacterium tularense alone in gelatin hydrolyzate cultures MOLAR CON- CENTRATION OF DRUG 2 x 10-4 MOIAR CONCENTRA- TION 01 PABA 1 X X X 10 1 X 10 5 X 1O-' 2.5 X X 10-1 X X X X X X 10-' 2.5 X 10-' 1 X 10-1 X 10-' inocululm, ' 10-j 10-' Control medium... + Inoculum: 0.5 ml. of no visible growth; + visible growth., sultapyrazine; PABA, p-aminobenzoic acid :6.7 8 O O O 0 0, ' Y O represents approximately 20,000 organisms per ml of medium. studied in relation to Escherichia coli in the same hydrolyzate medium. The lowest molar concentrations of sulfapyrazine, sulfadiazine, and sulfathiazole that prevented growth of E. coli (incubation for 4 days with inocula of 12 to 20 organisms per ml of medium) were identical. The amounts of PABA required to inhibit bacteriostasis were found to be the same for the least effective molar concentration of each sulfonamide. The results of a typical experiment are shown in table 3. Similarly, the minimal molar concentrations of these three sulfonamides that induced complete bacteriostasis of PasteureUa pestis in the same medium was 3.2 X 10-4 for each. And, as shown in table 4, PABA in

4 532 JOSEPH T. TAMURA concentration of 7.3 X 10-c completely inhibited the bacteriostatic action of each drug. The m bacteriostatic concentrations of these drugs against E. coli in gelatin hydrolyzate medium, and the inhibitory concentrations of PABA, are TABLE 3 Sudfonamide baderiostasis and inhibition by PABA (7.8 X 107 M) with Eacherichia coli in gelatin hydrolyzate medium MIOLAR CONCENTRATION OF DRUG DRtUGS 4 X1" 8 X1" 2 X 19'f 4 X 1" 8 X 10' 2 X 1O t PABA SD + + t SD-PABA ST + + t ST-PABA Inoculum: 12 to 20 organisms per ml of medium. 0 = no visible growth; t - trace of growth; + = visible growth. TABLE 4 Sudfonamide bacteriostasis and inhibition by PABA (7.8 X 10-6) with Pasteurella pestiu in gelatin hydrolyzate medium MOLAR CONCENTRATION OF DRUG 1.6 X 1" 3.2 X X 104 6X1" ttt PABA SD ttt SD-PABA ST ST-PABA Inoculum: 3,000 organisms per ml of medium. 0 = no visible growth; t = trace of growth; + = visible growth. comparable to those determined for this organism by other investigators who used other liquid media. Thus the failure of PABA to inhibit sulfonamide bacteriostasis of B. tularense is in marked contrast to its effect on many bacteria, and

5 SULFONAMIDES AND BACTERIUM TUIJARENSE 533 it adds another item to a large and growing list of unusual or so-called unique features recorded for this organism. It is interesting to note that among all reports of clinical chemotherapeutic trials in tularemia no mention is made of sulfapyrazine, the only sulfonamide hitherto available for which in vitro studies justify an anticipation of clinical usefulness. SUMMARY Sulfapyrazine in molar concentration of 2 X 10-4 (5 mg per cent) induced complete bacteriostasis of Bacterium tularense with moderate and large inocula in a very favorable liquid medium. Sulfadiazine and sulfathiazole, in this order, were decreasingly effective inhibitory agents, requiring higher concentrations and smaller inocula to produce complete bacteriostasis. P-aminobenzoic acid did not inhibit sulfonamide bacteriostasis of B. tularense. In concentrations in excess ofm X 10-' PABA alone inhibited growth of this organism. Insofar as in vitro inhibition of virulent organisms is an index of clinical expectation the outlook for therapeutic usefulness of sulfanilamide, neoprontosil, sulfapyridine, sulfaguanidine, sulfathiazole, and sulfadiazine in tularemia is not good. Of the drugs studied only sulfapyrazine seems worthy of clinical trial. REFERENCES RANSMEIER, J. C. Studies on the activity of sodium sulfadiazine against Bacterium tularenee. J. Infectious Diseases, 72, ROSE, H. M., AND Fox, C. L., JR A quantitative analysis of sulfonamide bacteriostasis. Science, 95, SPINK, W. W., AND JERMSTA, J Effect of sulfonamide compounds upon growth of staphylococci in presence and absence of p-aminobenzoic acid. Proc. Soc. Exptl. Biol. Med., 47, STRAUSS, E., DINGLE, J. H., AND FINLAND, M Studies on mechanism of sulfonamide bacteriostasis, inhibition and resistance; experiments with E. coli in synthetic medium. J. Immunol., 42, STRAUSS, E., AND FINLAND, M Selective inhibition of sulfonamide drugs by various media. Proc. Soc. Exptl. Biol. Med., 47, ; Bacteriostatic and bactericidal action of sulfadiazine in vitro on gram-negative bacteria. Ibid., 47, STRAuss, E., LOWELL, F. C., AND FINLAND, M Observations on inhibition of sulfonamide action by para-aminobenzoic acid. J. Clin. Investigation, 20, TAIuRA, J. T., AND GIBBY, I. W Cultivation of Bacterium tularense in simplified liquid media. J. Bact., 45, WOODS, D. D Relation of p-aminobenzoic acid to mechanism of action of sulphanilamide. Brit. J. Exptl. Path., 21, Wyss, O., GRuBAuGH, K. K., AND SCHMEKEs, F. C Non-specificity of sulfonamides. Proc. Soc. Exptl. Biol. Med., 49,