THE JOURNAL OF INFECTIOUS DISEASES • VOL. 132. NO.5· © 1975 by the University of Chicago. All rights reserved.
NOVEMBER 1975
Bioassay of Metronidazole with Either Anaerobic or Aerobic Incubation From the Department of Medicine, University of Washington School of Medicine, Seattle. Washington
Edward D. Ralph and William M. M. Kirby
Metronidazole [1-(,8-hydroxyethyl)-2-methyl-5 nitroimidazole] is active in vitro against many anaerobes and appears to be promising in the treatment of anaerobic bacterial infections [1-5]. In most assays reported previously, physical or chemical properties of the drug have been used, and the parent compound and its metabolites have been measured to various degrees without attention to their antibacterial activity [6-9]. However, two bioassays have been described in which the agar disk diffusion technique is used [10, 11], clostridial species are used as the test organisms, and anaerobic incubation for 24-48 hr is required. The present report describes a bioassay for metronidazole adapted from the agar well diffusion technique of Bennett et al. [12]. A wide range of concentrations (0.25-128 ~/ml) was measurable in serum or urine without dilution by use of two clostridial species with different sensitivities to metronidazole. Moreover, with minor modifications of the method, the option of either anaerobic or aerobic incubation was available. Materials and Methods
Organisms. Two rapidly growing, partially aerotolerant clostridial species were chosen for measurement of a wide range of concentra-
Received for publication February 11, 1975, and in revised form July 24, 1975. Please address requests for reprints to Dr. William M. M. Kirby, Department of Medicine, University of Washington School of Medicine, Seattle, Washington 98195. 587
tions. A strain of Clostridium sporogenes (Wadsworth no. 2253; MIC, 0.125 ~glml), kindly supplied by Dr. Vera Sutter (Wadsworth Veterans Administration Hospital, Los Angeles, Calif.), was used as the test organism for levels of 0.25-2.0 ~ of metronidazole/ml. A clinical isolate of Clostridium perfringens (Seattle no. 1; MIC, 1.6-3.1 J,tg/ml) , obtained from the Microbiology Laboratory, University Hospital, Seattle, was used for levels of 2-128 ~/ml. Because it has a higher MIC, this strain did not yield excessively large zones with high drug concentrations, and it was also less malodorous. Cultures. Both organisms were maintained anaerobically in GasPak jars (Baltimore Biological Laboratories [BBL], Baltimore, Md.) on brain-heart infusion agar (Difco, Detroit, Mich.) with 5 ~ of hemin/ml. For assay, the organisms were subcultured in approximately 10 ml of thioglycolate broth (BBL) containing 5 ug of hemin/ml in capped tubes. After incubation overnight, the C. perfringens culture was diluted 1:200 and the C. sporogenes 1:50 in 0.9% N aCI, for a final colony count of about 107 viable cfu/ml. Standards. Metronidazole standard powder was supplied by Searle and Co., San Juan, Puerto Rico. Stock solutions of 2,000 ~/ml were made up in sterile water and frozen; these solutions lost no potency for at least four weeks when stored at - 20 C. Dilutions containing 0.25-128 ~/ml were made in 100% normal human serum for determinations of serum concentrations or in 0.15 M phosphate buffer (pH 7.35) for urine assays. No deterioration in metronidazole activity was noted in standard solu-
Downloaded from http://jid.oxfordjournals.org/ at University of New South Wales on July 22, 2015
In a bioassay for metronidazole, a modified agar well diffusion technique was used. Two clostridial species were used as the test organisms, and, with minor variations, the method was as effective with aerobic as with anaerobic incubation. Serum and urine levels of 0.25-128 ~ml were measurable by this method without dilution of the specimens being assayed. The principal acid and alcohol metabolites of metronidazole were found to possess only approximately 5% and 30%, respectively, of the activity of the parent compound.
Ralph and Kirby
588
procedure was identical to that described above for the anaerobic method except that (l) 30 ml instead of 40 ml of inoculated anaerobic agar was poured into the petri dish; (2) after it had hardened, a second layer of 40 ml of uninoculated agar was poured on top of the inoculated agar; and (3) after the agar wells were punched and filled with standard and unknown specimens, the plates were incubated overnight at 37 C in a regular aerobic incubator before the zone diameters were recorded. Large plates. Large glass assay plates (12 x 12 inches) have been used routinely in this laboratory for the measurement of antibiotic levels, since they permit many more unknown samples to be assayed in relation to the number of standards needed for each plate [12]. Thus considerable time and human serum are saved. One milliliter of the diluted culture of the test organism (see above) was added to 200 ml of agar for the seed layer, and this preparation was overlaid with 250 ml of uninoculated agar. The procedure was in all other respects the same as that with the small plates. Accuracy of the bioassay method. Accuracy of the method was tested by assay of samples of pooled human plasma to which known amounts of metronidazole had been added at the Searle Laboratories (Chicago), and the code was not broken until Searle received our results. In addition, known concentrations in plasma of the two principal metabolites of metronidazole, I-acetic acid-2-methyl-5-nitroimidazole and 1-(f3hydroxyethyl) - 2 - hydroxymethyl- 5 - nitroimidazole, known as the acid and alcohol metabolites, respectively, were sent by Searle to determine biological activity in relation to the actual amounts known to be present. In both of the above studies, standard curves were constructed by the addition of known amounts of metronidazole to pooled human plasma (supplied by Searle) so that they would be comparable to the unknown specimens. Results
Assay of unknowns. The 35 specimens of plasma with unknown concentrations (Searle) actually contained only seven different concentrations, each of which was present in five tubes.
Downloaded from http://jid.oxfordjournals.org/ at University of New South Wales on July 22, 2015
tions after they had been frozen and thawed several times. Assay procedure. (l)Anaerobic method. For each assay plate, 40 ml of melted anaerobic agar (Difco) containing 5 1J,g of hemin/ml and maintained at 50 C in a 125-ml Erlenmeyer flask was inoculated with 0.5 ml of the diluted culture of C. sporogenes or with 0.2 ml of C. perfringens, depending on the probable concentrations of metronidazole to be assayed. A larger volume of C. sporogenes was used for this method because it was found to produce more distinct zones of inhibition. The contents were mixed well, poured into a disposable plastic 150- x 15-mm petri dish (no. 1058, Falcon Plastics, Los Angeles, Calif.), and spread uniformly. The agar was allowed to harden for 20 min, and one central and eight equally spaced peripheral wells were punched in each plate. Adequate zone sizes were produced by wells 5.5 mm in diameter for concentrations of metronidazole of ~ 2 ~ml (C. perfringensinoculated plate) but for concentrations of < 2 1J,g/ml, 6.5-mm wells were required (C. sporogenes-inoculated plate). Four wells on each plate were filled with serum containing known concentrations of metronidazole and the remaining five wells with unknown samples. Enough assay plates were prepared to determine zone sizes for each standard concentration and unknown sample in quadruplicate. After a 15-30-min interval at room temperature (about 24 C) for diffusion of the metronidazole, the plates were incubated at 37 C for 12-15 hr in a GasPak 150 anaerobic jar (BBL) with a CO 2-H2 generator (BBL). Zones of inhibition of growth were measured by calipers to the nearest 1110 mm, and values were averaged. Unknown concentrations of metronidazole in the samples were determined by comparisons with the standard curve [12], which included a fairly wide range of concentrations, so that dilutions of the unknowns were not necessary. The "best-fitting curve" was calculated [12], and the concentrations of the unknowns were then determined by extrapolation from the "graph." The calculations were made quickly and easily with a scientific calculator (HP-45, Hewlett-Packard, Cupertino, Calif.). (2) Aerobic method. Small plates. The
Bioassay of Metronidazole
589
Table 1. Accuracy of the method as tested by bioassay of serum specimens containing unknown concentrations of metronidazole. Known concentration
o
47.00 14.60 7.57 2.77 1.40 0.68 0.28
NOVEMBER 1975
Bioassay of Metronidazole with Either Anaerobic or Aerobic Incubation From the Department of Medicine, University of Washington School of Medicine, Seattle. Washington
Edward D. Ralph and William M. M. Kirby
Metronidazole [1-(,8-hydroxyethyl)-2-methyl-5 nitroimidazole] is active in vitro against many anaerobes and appears to be promising in the treatment of anaerobic bacterial infections [1-5]. In most assays reported previously, physical or chemical properties of the drug have been used, and the parent compound and its metabolites have been measured to various degrees without attention to their antibacterial activity [6-9]. However, two bioassays have been described in which the agar disk diffusion technique is used [10, 11], clostridial species are used as the test organisms, and anaerobic incubation for 24-48 hr is required. The present report describes a bioassay for metronidazole adapted from the agar well diffusion technique of Bennett et al. [12]. A wide range of concentrations (0.25-128 ~/ml) was measurable in serum or urine without dilution by use of two clostridial species with different sensitivities to metronidazole. Moreover, with minor modifications of the method, the option of either anaerobic or aerobic incubation was available. Materials and Methods
Organisms. Two rapidly growing, partially aerotolerant clostridial species were chosen for measurement of a wide range of concentra-
Received for publication February 11, 1975, and in revised form July 24, 1975. Please address requests for reprints to Dr. William M. M. Kirby, Department of Medicine, University of Washington School of Medicine, Seattle, Washington 98195. 587
tions. A strain of Clostridium sporogenes (Wadsworth no. 2253; MIC, 0.125 ~glml), kindly supplied by Dr. Vera Sutter (Wadsworth Veterans Administration Hospital, Los Angeles, Calif.), was used as the test organism for levels of 0.25-2.0 ~ of metronidazole/ml. A clinical isolate of Clostridium perfringens (Seattle no. 1; MIC, 1.6-3.1 J,tg/ml) , obtained from the Microbiology Laboratory, University Hospital, Seattle, was used for levels of 2-128 ~/ml. Because it has a higher MIC, this strain did not yield excessively large zones with high drug concentrations, and it was also less malodorous. Cultures. Both organisms were maintained anaerobically in GasPak jars (Baltimore Biological Laboratories [BBL], Baltimore, Md.) on brain-heart infusion agar (Difco, Detroit, Mich.) with 5 ~ of hemin/ml. For assay, the organisms were subcultured in approximately 10 ml of thioglycolate broth (BBL) containing 5 ug of hemin/ml in capped tubes. After incubation overnight, the C. perfringens culture was diluted 1:200 and the C. sporogenes 1:50 in 0.9% N aCI, for a final colony count of about 107 viable cfu/ml. Standards. Metronidazole standard powder was supplied by Searle and Co., San Juan, Puerto Rico. Stock solutions of 2,000 ~/ml were made up in sterile water and frozen; these solutions lost no potency for at least four weeks when stored at - 20 C. Dilutions containing 0.25-128 ~/ml were made in 100% normal human serum for determinations of serum concentrations or in 0.15 M phosphate buffer (pH 7.35) for urine assays. No deterioration in metronidazole activity was noted in standard solu-
Downloaded from http://jid.oxfordjournals.org/ at University of New South Wales on July 22, 2015
In a bioassay for metronidazole, a modified agar well diffusion technique was used. Two clostridial species were used as the test organisms, and, with minor variations, the method was as effective with aerobic as with anaerobic incubation. Serum and urine levels of 0.25-128 ~ml were measurable by this method without dilution of the specimens being assayed. The principal acid and alcohol metabolites of metronidazole were found to possess only approximately 5% and 30%, respectively, of the activity of the parent compound.
Ralph and Kirby
588
procedure was identical to that described above for the anaerobic method except that (l) 30 ml instead of 40 ml of inoculated anaerobic agar was poured into the petri dish; (2) after it had hardened, a second layer of 40 ml of uninoculated agar was poured on top of the inoculated agar; and (3) after the agar wells were punched and filled with standard and unknown specimens, the plates were incubated overnight at 37 C in a regular aerobic incubator before the zone diameters were recorded. Large plates. Large glass assay plates (12 x 12 inches) have been used routinely in this laboratory for the measurement of antibiotic levels, since they permit many more unknown samples to be assayed in relation to the number of standards needed for each plate [12]. Thus considerable time and human serum are saved. One milliliter of the diluted culture of the test organism (see above) was added to 200 ml of agar for the seed layer, and this preparation was overlaid with 250 ml of uninoculated agar. The procedure was in all other respects the same as that with the small plates. Accuracy of the bioassay method. Accuracy of the method was tested by assay of samples of pooled human plasma to which known amounts of metronidazole had been added at the Searle Laboratories (Chicago), and the code was not broken until Searle received our results. In addition, known concentrations in plasma of the two principal metabolites of metronidazole, I-acetic acid-2-methyl-5-nitroimidazole and 1-(f3hydroxyethyl) - 2 - hydroxymethyl- 5 - nitroimidazole, known as the acid and alcohol metabolites, respectively, were sent by Searle to determine biological activity in relation to the actual amounts known to be present. In both of the above studies, standard curves were constructed by the addition of known amounts of metronidazole to pooled human plasma (supplied by Searle) so that they would be comparable to the unknown specimens. Results
Assay of unknowns. The 35 specimens of plasma with unknown concentrations (Searle) actually contained only seven different concentrations, each of which was present in five tubes.
Downloaded from http://jid.oxfordjournals.org/ at University of New South Wales on July 22, 2015
tions after they had been frozen and thawed several times. Assay procedure. (l)Anaerobic method. For each assay plate, 40 ml of melted anaerobic agar (Difco) containing 5 1J,g of hemin/ml and maintained at 50 C in a 125-ml Erlenmeyer flask was inoculated with 0.5 ml of the diluted culture of C. sporogenes or with 0.2 ml of C. perfringens, depending on the probable concentrations of metronidazole to be assayed. A larger volume of C. sporogenes was used for this method because it was found to produce more distinct zones of inhibition. The contents were mixed well, poured into a disposable plastic 150- x 15-mm petri dish (no. 1058, Falcon Plastics, Los Angeles, Calif.), and spread uniformly. The agar was allowed to harden for 20 min, and one central and eight equally spaced peripheral wells were punched in each plate. Adequate zone sizes were produced by wells 5.5 mm in diameter for concentrations of metronidazole of ~ 2 ~ml (C. perfringensinoculated plate) but for concentrations of < 2 1J,g/ml, 6.5-mm wells were required (C. sporogenes-inoculated plate). Four wells on each plate were filled with serum containing known concentrations of metronidazole and the remaining five wells with unknown samples. Enough assay plates were prepared to determine zone sizes for each standard concentration and unknown sample in quadruplicate. After a 15-30-min interval at room temperature (about 24 C) for diffusion of the metronidazole, the plates were incubated at 37 C for 12-15 hr in a GasPak 150 anaerobic jar (BBL) with a CO 2-H2 generator (BBL). Zones of inhibition of growth were measured by calipers to the nearest 1110 mm, and values were averaged. Unknown concentrations of metronidazole in the samples were determined by comparisons with the standard curve [12], which included a fairly wide range of concentrations, so that dilutions of the unknowns were not necessary. The "best-fitting curve" was calculated [12], and the concentrations of the unknowns were then determined by extrapolation from the "graph." The calculations were made quickly and easily with a scientific calculator (HP-45, Hewlett-Packard, Cupertino, Calif.). (2) Aerobic method. Small plates. The
Bioassay of Metronidazole
589
Table 1. Accuracy of the method as tested by bioassay of serum specimens containing unknown concentrations of metronidazole. Known concentration
o
47.00 14.60 7.57 2.77 1.40 0.68 0.28
