Sulfamethoxazoleと Trimethoprimの感受性測定法

抄録

An ad hoc committee has been organized by members of the Japan Society for Chemotherapy with an aim to establish acceptable methods for determination of minimal inhibitory concentrations (MICs) of sulfamethoxazole (SMX) -trimethoprim (TMP) combination product. The following summary outlines the conclusion derived from investigations by this committee.
1. Sensitivities shall be assayed by the agar plate dilution method and activities shall be expressed in terms of MIC (the minimal concentration at which bacterial growth is completely inhibited).
2. Culture medium for inoculation
MUELLER-HINTON broth (Difco) shall be used without modifying its pH (7. 4 ± 0. 2). For those test organisms which require specific nutrients, appropriate media shall be selected.
3. Dilution of culture broth
Sterilized physiological saline shall be used in general as a diluting medium but other appropriate material may be employed for specific organisms.
4. Culture medium for sensitivity testing
MUELLER-HINTON agar (Eiken) or Medium for Sensitivity Disc Testing (Nissan) shall be employed enriched with 7.5% hemolyzed horse blood (pH 7.4).
5. Method of inoculating test organisms
Culture medium for inoculation purposes shall be incubated at 37°C for 18-20 hours. The culture broth thus obtained shall be diluted 100-fold for gram positive cocci, or 1000-fold for gram negative rods to make inoculating materials for sensitivity testings. A loopful of this culture broth will be streaked over 2 cm on a sensitivity plate.
6. Judgement
After incubating the plate at 37°C for 18-20 hours, the minimal concentration at which the bacterial growth is completely inhibited shall be examined macroscopically to determine MIC. The growth of a single colony, or of a very thin bacterial film shall be judged as bacterial growth (in case the bacterial growth is insufficient after the aforementioned conditions, the judgement shall be made after 48 hours' incubation).
7. Concentrations of drugs
Two-fold dilutions shall be made in the following order : 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.2, 0.1, 0.05 and 0.025 mcg/ml. When employing concentrations in excess of 100 mcg/m I, concentrations shall be 200, 400 and 800 mcg/ml.
8. Preparation of standard solution of TMP and SMX
Ten mg of TMP are dissolved in a small volume of 4/100 N HCI, to which water is added to make a total volume of 10 ml. Ten mg of SMX is dissolved in a small volume of 1/8 N NaOH, to which water is added to make a total volume of 10 ml. The following alternative may be acceptable for TMP : Ten mg of TMP is dissolved in 0.5 ml of dimethylformamide or 0.5 ml propyleneglycol, to which water is added to make a total of 10 ml.
9. Preparation of plate for sensitivity testing
Hemolyzed horse blood shall be added when the temperature of culture medium is cooled down to approximately 50-60°C. Drugs in prescribed quantities are poured into the plate and the culture medium in 9-fold volume is added while the plate is gently shaken.
10. Test organisms (clinical isolates and standard bacterial strains)
Test organisms must be fresh within 2-3 culture generations after the clinical isolation. As reference standard of organisms, the following 2 strains as recommended by the Japan Society for Chemotherapy shall be employed ;Staphylococcus aureus FDA 209-P JC-1 and Escherichia coli NIHJ JC-2. The MICs of these standard strains as assayed by the foregoing procedures are as follows :
SMX (mcg/ml) TMP (mcg/ml)
Staphylococcus aureus FDA 209-P JC-1 25-50 0.39-0.78
Escherichia coli NIHJ JC-2 3.12-6.25 O.1-0.2