VOL. 21 NO. 2 CHEMOTHERAPY 137

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VOL. 21 NO. 2 CHEMOTHERAPY 137

Minimal inhibitory concentration (mcg/m1) of TM? and SMX in MUELLER-HINTON agar with 7.5% lysed horse blood, Diagnostic sensitivity test medium with 7. 596 lysed horse blood and SR-medium Minimal inhibitory concentration (mcg/ml)

Effect of inoculum size on in vitro antibacterial activity of SMX and TMP Effect of medium ph on in vitro antibacterial activities of combinations of SMX and TMP in MUELLER-HINTON agar with 7.5% lysed horse blood

VOL. 21 NO. 2 CHEMOTHERAPY 141 a) ph 6.0 Effect of medium ph on in vitro antibacterial activities of combinations of TMP and SMX against Staph. aureus CN 491 b) ph 7.0 c) ph 7.4

142 CHEMOTHERAPY MAR. 1973 Enhancement of activity against Staphylococcus aureus FDA 209-P JC-1 by various combinations of SMX and TM? \8 Enhancement of activity against Escherichia coli NIHJ JC-2 by various combinations of SMX and TMP

VOL. 21 NO. 2 CHEMOTHERAPY 143 Calculation of FIC index Calculation of FIC index Graphical representation of the enhancement of activity against Staphylococcus aureus FDA 209-P JC-1 by various combinations of SMX and TM? Graphical representation of the enhancement of activity against E. coli NIHJ JC-2 by various combinations of SMX and TMP

144 CHEMOTHERAPY MAR. 1973

146 CHEMOTHERAPY MAR. 1973

VOL. 21 NO. 2 CHEMOTHERAPY 147 1) BUSHBY, S.R.M. & G.H. HITCHINGS: Trimethoprim, a sulphonamide potentiator. Brit. J. Pharmacol. 33: 72 `90, 1968 2) BOHNI, E.: Vergleichende bakteriologische Untersuchungen mit der Kombination Trimethoprim/Sulfamethoxazole in Vitro and in Vivo. Chemotherapy. Suppl. ad. 14: 1 `21, 1969 3) WATERWORTH, P.M.: Practical aspects of testing sensitivity to trimethoprim and sulphonamide. Postgraduate Med. J. Suppl. 45: 21 `27, 1969 4) PECHERE, J. C.: Combination of trimethoprim with sulfamethoxazole: Study of antibacterial activity in vivo and comparison with gentamycin in the treatment of urinary tract infections. Prog. in antimicrobial and anticancer chemotherapy-proc. 6th International Cong. of Chemotherapy. 959 `965. Univ. of Tokyo Press. 1970 5) MOSSER, G.: Klinishe Ergebniss mit dem Kombinationspraparat Sulfamethoxazole + Trimethoprim. ibid. 966 `970. 6) BRUMFIT, W.: Trimethoprim-Sulfamethoxazole combinations in treatment of urinary infections in hospital and domiciliary patients. ibid. 981 `985. 7) DARREL, J. H.; L.P. GARROD & P. M. WATER- WORTH : Trimethoprim: laboratory and clinical studies. J. Clin. Path. 21: 202 ` 209, 1968 8) ELION, G. B. ; S. SINGER & G. H. HITCHINGS: Antagonists of nucleic acid derivatives. VIII. Synergism in combinations of biochemically related antimetabolites. J. Biol. Chem. 208: 477 `488, 1954 9) HARPER. G.J. & W.C. CAWSTON: The in vitro determination of the sulphonamide sensitivity of bacteria. J. Path. Bact. 57:59 1945 10) WALKER, N.; R. P. PHILIP, M. W. SNYTH & J. W. MACLEOD: Observations on the prevention of bacterial growth by sulphonamides with special reference to the Harper & Cawston effect. J. Path. Bact. 59: 631 `645, 1947 11) BUSHBY, S. R. M.: Combined antibacterial action in vitro of trimethoprim and sulphonamide. The in vitro nature of synergy. Postgraduate Med. J. Suppl. 45: 10 `18, 1969 12) HITCHINGS, G. H.: Species differences among dihydrofolate reductase as a basis for chemotherapy. ibid. 45: 7 `40, 1969 (1)

148 CHEMOTHERAPY MAR. 1973 HEWLETT, P. S.: Measurement of potencies of drug mixtures. Biometrics 477487, 1969

VOL. 21 NO. 2 CHEMOTHERAPY 149 STUDIES ON SULFAMETHOXAZOLE AND TRIMETHOPRIM Fundamental Research I Antibacterial Activities of Sulfamethoxazole and Trimethoprim Alone and in Combination In Vitro SHIGEMI AWATAGUCHI and CHIKAKO SHIRANE Biological Research Laboratory, Tanabe Seiyaku Co., Ltd. MIKAO MAYAMA, HIROSHI NAGATA, KIYOJI KANAZAWA, TADASHI YOSHIDA and KEN KATAGIRI Shionogi Research Laboratory, Shionogi & Co., Ltd. This study was undertaken to investigate the antibacterial activities of sulfamethoxazole (SMX) and trimethoprim (TMP), alone and in combination in vitro. Activities of the 2 drugs, alone and in combination, were found to be dependent on the composition of culture medium, medium ph and inoculum size of the organisms to be tested. SMX and TMP were active against a wide range of gram-positive and gram-negative organisms. The potentiation of the antibacterial activity of SMX by TMP was confirmed. The maximum potentiation by the combination was achieved when the 2 components were combined at the ratio of their respective minimal inhibitory concentrations acting alone. A guiding manual for sensitivity testing was established as follows: 1. In vitro antibacterial activity is determined according to the agar dilution method. The sensitivity is expressed as minimal inhibitory concentration (MIC). 2. MUELLER-HINTON broth (Difco) is recommended for culturing test organisms and MUELLER- HINTON agar (Eiken) with hemolyzed (freeze-thaw) horse blood (7. 5% v/v) is recommended as a medium for sensitivity testing. 3. Test organisms are inoculated in MUELLER HINTON broth and incubated at 37 Ž for 18 `20 hours. 4. The inoculum size is one loopful (inner diameter I mm: weight of wet bacteria ca. 2 mg) of a 10-2 dilution of a gram-positive broth culture or 10 dilution of a gram-negative broth culture; the inoculum is streaked over 2 cm in length on the plate. 5. The interpretation is made macroscopically after 18 `20 hours of incubation at 37 Ž and the lowest concentration at which bacterial growth is completely inhibited is designated as MIC. 6. Bacterial strains isolated from clinical materials should be tested while they remain fresh within 2-3 culture generations. For reference strains, Staphylococcus aureus FDA 209-P JC-1 and Escherichia coli NIHJ JC-2 are used. The following guidelines have been proposed for estimation of potentiation of activities by SMX and TMP. a) When organisms are sensitive to both SMX (MIC 800 mcg/ml) and TMP (MIC 100 mcg/ml): Potentiation is estimated by calculating FIC (fractional inhibitory concentration) index. FIC values can be calculated by dividing the MICs of SMX and TMP in the combination by the MICs of SMX and TMP when used alone. FIC index is the sum of FIC values. When this index is below 1. 0, potentiation is indicated. b) When organisms are resistant to either SMX (>800 mcg/ml) or TMP (>100 mcg/ml): If the MIC of SMX or TMP in the combination against a test organism is less than one fourth that of SMX or TMP when used alone, potentiation is indicated. c) When organisms are resistant to both SMX (>800 mcg/ml)and TMP (>100 mcg/ml): If the growth of a test organism is completely inhibited by the combination of SMX-TMP (the range of their combination concentrations: SMX 800, 400, 200 mcg/ml and TMP 100, 50, 25 mcg/ml, respectively), potentiation is indicated.

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