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°C for 18-20 hours.
4. The inoculum size is one loopful (inner diameter I mm : weight of wet bacteria ca. 2 mg) of a 10-2dilution of a gram-positive broth culture or 10-3dilution 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°C 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.
