ANTIMICROBIL AGENTS AND CHEMOTHERAPY, Apr. 1977, p. 631-637 Copyright © 1977 American Society for Microbiology
Vol. 11, No. 4 Printed in U.S.A.
Susceptibility of Anaerobic Bacteria to Metronidazole, Ornidazole, and Tinidazole and Routine Susceptibility Testing by Standardized Methods JURG WtJST Institute for Medical Microbiology, University ofZurich, CH-8028 Zurich, Switzerland
Received for publication 1 December 1976
A total of 114 strains of anaerobic bacteria were examined for their susceptibility to metronidazole, ornidazole, and tinidazole by measuring the minimum inhibitory concentration (MIC) and minimum bactericidal concentration in different media. All strains, with the exception of the isolates ofPropionibacterium acnes, were inhibited by 3.1 ug each and killed by 6.3 ,ug each of all three nitroimidazole compounds per ml. No significant differences in MIC values were found among metronidazole, ornidazole, and tinidazole. Only minor differences were detected by comparing MIC values obtained in brain heart infusion agar with and without sheep blood, brucella agar, and Mueller-Hinton agar (both containing blood). When the strains were tested by the modified broth-disk method proposed by the Anaerobe Laboratory of the Virginia Polytechnic Institute (VPI), there was good correlation with the MIC values (97.4% agreement for metronidazole and 94.7% for ornidazole and tinidazole). For routine testing, use of a 30-,ug-class disk of either nitroimidazole derivative is proposed for the brothdisk method, resulting in a final concentration of 6 ug/ml in the test tubes, a concentration easily attainable in body fluids. In contrast to the broth-disk method, there was very poor correlation between inhibition zone diameters by the standardized VPI agar diffusion test and MIC values.
Metronidazole and related nitroimidazole derivatives, including ornidazole and tinidazole, are receiving increasing attention in the treatment of anaerobic infections. This interest rests on the results ofintensive in vitro susceptibility evaluations (2-4, 9, 10, 20, 24, 26, 28, 32) and limited but promising trials of these agents in patients infected with various anaerobic bacteria (14, 19, 31, 39). All in vitro data have been obtained by determination of minimal inhibitory concentrations (MIC), a procedure that is not suitable for routine testing in the clinical laboratory, unless a sufficient number of strains have to be tested every day. Currently, there are several methods proposed for routine susceptibility testing of anaerobic bacteria (5, 8, 21, 25, 29, 36, 37). The purpose of this study was to investigate the reliability of susceptibility testing of anaerobes against metronidazole, ornidazole, and tinidazole by two routine procedures proposed by the Anaerobe Laboratory of the Virginia Polytechnic Institute (VPI), Blacksburg, Va., namely, the modified broth-disk method proposed by Wilkins and Thiel (37) and the agar diffusion method of Wilkins et al. (36). In addition, the
influence of media most used in anaerobic bacteriological work on the results of MIC determinations was evaluated, since Chow et al. (3) have found higher MIC values on Mueller-Hinton agar than authors using other media (2, 4, 9, 24, 26, 28, 32). MATERIALS AND METHODS Bacterial strains.Of the 114 strains examined in this study, 9 were provided from VPI and 105 were isolated from clinical specimens at the Institute for Medical Microbiology of the University of Zurich. They were identified according to the criteria outlined by Holdeman and Moore (13). Identification of subspecies of Bacteroides fragilis was performed by considering acid production from mannitol, rhamnose, and trehalose and indole production; carbohydrate fermentation was considered positive only if a pH of less than 5.75 was achieved (L. V. Holdeman, personal communication). The isolated strains comprised the following species: 39B. fragilis (2 B. fragilis subsp. distasonis, 28 B. fragilis subsp. fragilis, 6 B. fragilis subsp. thetaiotaomicron, 1 B. fragilis subsp. vulgatus, and 2 B. fragilis strains that could not be subspecified), 2 Bacteroides melaninogenicus subsp. asaccharolyticus, 2 Fusobacterium mortiferum, 3 Fusobacterium necrophorum, 7 Fusobacterium nucleatum, 1 Fuso631
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bacterium varium, 7 Peptococcus asaccharolyticus, 8 Peptococcus magnus, 2 Peptococcus prevotii, 7 Peptostreptococcus anaerobius, 4 Veillonella sp., 1 Eubacterium cylindroides, 1 Eubacterium lentum, 7 Propionibacterium acnes, 1 Clostridium butyricum, 1 Clostridium chauvoei, 1 Clostridium novyi A, 9 Clostridium perfringens, 3 Clostridium ramosum, 6 Clostridium sporogenes, 1 Clostridium tertium, 1 Clostridium sp. The strains were maintained at room temperature in chopped-meat broth (13). Media. Throughout the experiments, prereduced, anaerobically sterilized media were used. These were prepared according to methods described by Holdeman and Moore (13). The basic media were supplemented brain heart infusion (BHI) broth and chopped-meat carbohydrate (CMC) broth. The basal peptone-yeast extract medium used for testing biochemical reactions contained 0.5% peptone (Difco) and 0.5% Trypticase (BBL) instead of 1% peptone (W. E. C. Moore, personal communication). The media used for MIC determinations were: BHI agar (Difco), supplemented with 0.5% yeast extract (Difco), with and without 5% citrated sheep blood (BHIA); brucella agar (kindly supplied by Pfizer Overseas, Inc., New York) with 5% sheep blood; Mueller-Hinton agar (BBL), supplemented with 0.5% yeast extract (Difco) and 5% sheep blood. All media for MIC determinations were supplemented with menadione (0.5 ,g/ml) and hemin (5
,tg/ml).
Antimicrobial agents. Metronidazole and ornidazole were kindly supplied by P. Angehrn, Hoffmann-La Roche & Co. AG, Basel, Switzerland, and tinidazole was supplied by E. Milek, Pfizer AG, Zurich, Switzerland. Stock solutions containing 1,000 ,ug of each antimicrobial agent per ml were prepared in distilled water, filter-sterilized (Millipore, 0.22 um), and kept frozen at -20°C. MIC determinations. MIC determinations were performed according to Blazevic (1). For the agar dilution technique, plates were prepared containing serial twofold dilutions of each antimicrobial agent from 100 to 0.1 ,ug/ml. The plates were held overnight at room temperature in GasPak jars (BBL) or prepared on the day of the experiment. Prior to use, they were dried with the lids slightly opened, at 370C. All strains were checked for purity by subculturing to an anaerobic blood agar plate. One colony was inoculated into CMC broth. After overnight incubation at 37°C, a 1:100 dilution was made in BHI broth. The agar plates were inoculated with a device similar to the Steers replicator (27), resulting in an inoculum of approximately 2 x 103 to 2 x 104 bacteria per spot. Only 19 organisms were replicated at a time to avoid prolonged exposure to air. Anaerobic control plates without antimicrobial agents were inoculated last to ensure that viable organisms were present throughout the experiment and that the strains were able to grow on the media (35). The plates were incubated in GasPak jars at 370C for 48 h. Plates incubated aerobically and in an atmosphere with 10% C02 were included as additional controls. After incubation, the MIC was determined as
ANTIMICROB. AGENTS CHEMOTHER. the lowest concentration of the antimicrobial agent showing no growth, a barely visible haze, or one discrete colony (7). One strain of B. fragilis subsp. fragilis was included in each experiment to determine the reproducibility of the method. MIC determinations of nine swarming clostridia were performed separately for each strain and each concentration of metronidazole, ornidazole, and tinidazole, using the cups of disposable plastic trays as miniaturized petri dishes. These experiments were performed only with BHIA, and therefore these strains were excluded from the comparative evaluation of the different media. Determination of the MBC. The minimal bactericidal concentration (MBC) in BHIA without blood was determined by replica plating (6). The velvet surface of a sterilized pad was pressed against the primary BHI master plate and then to a BHIA plate without antimicrobial agent. The MBC was defined as the lowest concentration showing no growth or only one single colony in the subculture after 48 h of incubation at 370C in GasPak jars. Disks for susceptibility testing. Disks containing 10 and 30 ug of antimicrobial agent were prepared by adding 10 ul of a 1,000-,ul/ml solution and 20 ul of a 1,500-,ul/ml solution, respectively, to 6-mm filter paper disks. The disks were dried for about 16 h at 370C and stored at 4°C together with a desiccant. Broth-disk method. The broth-disk method described by Wilkins and Thiel (37) was strictly followed. One drop of an overnight culture in CMC broth was added to 5 ml of BHI broth. A disk containing 30 ,g of metronidazole, ornidazole, or tinidazole was added, resulting in a final concentration of 6 ug/ml of the respective antimicrobial agent. A control culture without antimicrobial agent was included for each strain. Tncubation was at 370C for 18 to 24 h. Susceptibility was defined as either absence of growth or less than 50% of the turbidity of the control culture. When there was growth in the tubes, indicating resistance, the strain was subcultured anaerobically and aerobically to exclude the possibility of contamination. Agar diffusion test. The method for the agar diffusion test described by Wilkins et al. (36) was used. A 1.5-ml portion of an 18- to 24-h culture at maximal turbidity in CMC broth was added to 10 ml of melted and cooled (500C) supplemented BHIA (13). The contents were mixed by inversion and poured into standard 90-mm petri dishes. After the agar had solidified, filter-paper disks containing 10 and 30 ug of metronidazole, ornidazole, and tinidazole were applied. After incubation at 370C in GasPak jars for 18 to 24 h, the zone diameters were measured with a ruler. Some strains of B. fragilis exhibited an inner area of light growth around the disk, with a clear area of inhibition outside this hazy growth. In these instances, the outer zone of inhibition was measured.
RESULTS The cumulative percentages of anaerobic bacterial strains inhibited and killed at various concentrations of metronidazole, ornidazole,
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NITROIMIDAZOLE SUSCEPTIBILITY OF ANAEROBES
and tinidazole are summarized in Table 1. All strains except the isolates of P. acnes were inhibited by 3.1 Mg each and killed by 6.3 ,g each of all three nitroimidazoles per ml. For most strains the MBC was equal to the MIC; in some-instances the MBC was one or two twofold dilutions higher than the MIC. In a few rare cases the MBC was one dilution step lower than the MIC, indicating that the antimicrobial agent became effective only after several generations (22) or that the microorganisms were killed by exposure to oxygen during the transfer. Table 2 contains the geometric means of the MICs of metronidazole, ornidazole, and tinidazole in the different media tested. A two-way analysis of variance showed no significant difference betwen the three antimicrobial agents in all four media (P > 5%). Whereas no difference was detected between BHIA with and without blood (P > 5%), the geometric means of the MICs in brucella agar with 5% sheep blood and Mueller-Hinton agar with 5% sheep blood were slightly higher (P < 1%) than in BHIA.
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Table 3 compares the results obtained by the broth-disk test and the agar dilution method. Three organisms considered as susceptible to metronidazole according to the MICs were classified as resistant by the broth-disk method. For ornidazole and tinidazole there were six strains each showing similar behavior. These false resistance results in the broth-disk test were primarily found in testing clostridia; in addition, there was one strain of F. necrophorum resistant to all three nitroimidazoles in the broth-disk test. Upon repetition, part of the strains again showed false growth in the broth disk test tubes. When the tests were repeated with a 1:100 dilution of the CMC broth culture as inoculum, no false resistant classifications were encountered any more. In general, the results of the broth-disk method were easy to read. The tubes showed either heavy growth or were clear. With some B. fragilis strains there was a slight turbidity in the drug-containing tubes, but it was easy to ascertain a marked difference from the heavily grown control tube without drug. Difficulties
TABLE 1. Inhibitory and bactericidal effect of metronidazole, ornidazole, and tinidazole on anaerobic bacteria Organisms
No. of strains
Antimicrobial
Cumulative percent of isolates inhibited/killed at various concn
mI) a
agentI a0.1 0.2 0.4 0.8 1.6
tete tee
Bacteroides fragilis
Bacteroides melaninogenicus
Fusobacterium sp. Clostridium perfringens
Clostridium sp. (other) Peptococcus sp. and
Peptostreptococcus sp. Veillonella sp.
39 2 13 9
Metronidazole Ornidazole 3/5 Tinidazole 3/3 Metronidazole 100/100 Ornidazole 100/100 Tinidazole 100/100 Metronidazole 62/62 Ornidazole 62/54 Trinidazole 62/54 Metronidazole
Ornidazole
14/5b 24
Tinidazole Metronidazole Ornidazole Tinidazole Metronidazole
Ornidazole
15/15 18/15 28/23
85/85 85/85 85/77 33/33 11/22 11/11
14/0
64/0
14/0 14/0 4/8 4/4 4/8
57/0 57/0 58/50 50/46 42/25
38/31 44/44 56/56
62/67 72/79 74/82
92/100
97/90 97/95 92/95
6.3 3.1 100/100 100/97 100/100 100/97 100/100
(Qig/
>100
100/100
92/92 92/92
100/100
44/44 56/44 56/33
89/89 100/89 89/78
71/0 64/0 71/67 92/79 75/58
93/80
86/60 92/88 100/96
100/100 100/100 100/100 100/100 100/80 100/100 100/100 93/80 100/100 100/96 100/100 100/100 100/100
Tinidazole 96/92 4 Metronidazole 100/100 Ornidazole 50/50 100/100 Tinidazole 50/50 50/100 100/100 Eubacterium sp. 2 Metronidazole 50/50 100/50 100/100 Ornidazole 50/50 100/100 Tinidazole 50/50 100/100 7 Propionibacterium Metronidazole 100/100 acnes Ornidazole 100/100 Tinidazole 100/100 Total 114/105b Metronidazole 11/11 39/32 53/46 75/74 93/88 94/93 100/100 Ornidazole 12/11 36/30 62/54 83/83 93/91 94/92 94/93 100/100 Tinidazole 12/11 38/27 62/53 75/74 90/90 94/92 94/93 100/100 a In BHIA without blood. b MIC determinations of nine swarming clostridia were performed separately in small petri dishes. Therefore, the MBC assay could not be performed by the method used.
634
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ANTIMICROB. AGENTS CHEMOTHER.
were encountered with five Peptococcus and Peptostreptococcus species (i.e., 4.3% of the strains tested), which showed only very fair growth in the control tube. Prolonged incubation for another 24 h produced growth sufficient to read, although this procedure does not conform to the test described originally and has to be interpreted with caution, because of possible inactivation of the antimicrobial agent. Figure 1 shows the relationship of zone diameters around a 10-,ug metronidazole disk and MIC values in BHIA without blood. Twenty-six organisms (22.8%) that grew slowly in CMC broth and did not produce confluent growth in the pour plates, or produced indistinct zones, were excluded as proposed by Wilkins et al. (36). These strains comprised mainly Peptococcus sp. and some clostridia other than C. per-
TABLE 2. Comparison of MIC values of metronidazole, ornidazole, and tinidazole against anaerobic bacteria in different mediaa MIC (,ug/ml) in: b Antimicrobial agent
BHIA
BA
MHA
Without With blood blood
0.87 0.72 0.55 0.56 Metronidazole 0.84 0.59 0.49 0.50 Ornidazole 0.60 0.79 0.54 0.53 Tinidazole a Geometric means of the MIC values of 105 strains. b BA, Brucella agar with 5% sheep blood; MHA, Mueller-Hinton agar with 5% sheep blood.
fringens. Due to the extensive spread of diameters at the various MIC values and the relative narrow range of MICs, no regression line was calculated and drawn (7, 16, 18). The problems met in reading the diffusion tests were mainly cloudy zones due to the dense inoculum or organisms that grew well close to the disk but were inhibited further away from the disk (33). The complications of spreading were also encountered when 30-,ug disks were used and with ornidazole and tinidazole as well as with metronidazole. The reproducibility of the agar diffusion and the agar dilution method (see Table 4) was tested by performing the assays on different days with the same strain of B. fragilis subsp. fragilis. Direct numerical comparisons of the extent of variability of the two test procedures cannot be made, because the results were obtained in two systems not directly transferable (7). DISCUSSION There is controversy over the questions of whether the susceptibility testing of anaerobic bacteria should be performed routinely or whether the management of anaerobic infections should rely upon published susceptibility patterns for the different bacterial speeies (11, 16, 26, 34). I believe that routine testing should be done for clinically significant anaerobes from sources not contaminated with normal flora, since the susceptibility pattern of various anaerobes against several antimicrobial agents is known to change (12, 15-17, 21, 28, 30). For
TABLE 3. Comparison of results with the broth-disk and the agar dilution methods |Resistant strainsa
Sustrainsbl
Overall
Strains with false reaction in broth-disk test
agreement AntimicroNo. of No. of (%) between bial agent No. of false sus- No. of false rethe two strains ceptible strains sistant methods Species readingsb readingsb 0 3 7 107 97.4 Clostridium perfringens Metronidazole Clostridium sp. Fusobacterium mortiferum 7 Ornidazole 0 107 6 94.7 Clostridium perfringens Clostridium perfringens Clostridium sp. Fusobacterium nortiferum 7 Tinidazole 0 107 6 94.7 Bacteroides fragilis Clostridium perfringens Clostridium perfringens Clostridium perfringens Clostridium sp. Fusobacterium mortiferum a According to MIC in BHIA without blood. b In broth-disk test. c In BHIA without blood.
MIC (,g/ml)c
0.8 0.8
0.4
0.4 (2 strains) 0.8 (2 strains) 0.8 0.8 0.4 0.4 0.8 1.6 1.6 1.6
635
NITROIMIDAZOLE SUSCEPTIBILITY OF ANAEROBES
VOL. 11, 1977 2100
Metronidazole (10-wg disk) o Bacteroides fraglis o Fusobacterium sp. * Anaerobic cocci *Clostridium sp. *Eubacterium and Propionibacterium sp.
12.5L 6.31-
0
3.11E cn
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i800
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80 *
0.2-
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12
18
24
* t
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54
Zone Diameters (mm) FIG. 1. Lack of correlation of MIC values in BHIA without blood and inhibition zone diameters of anaerobic bacteria around 10-pg metronidazole disks. Similar patterns were found when ornidazole and tinidazole were tested and when 30-pg disks were used instead of10-pg disks for all three nitroimidazoles.
TABLE 4. Reproducibility of antimicrobial susceptibility testsa Agar diffusion test (zone diam, mm)
Agar dilution test (MIC, ,ug/ml)
Antimicrobial agent Mean
Svtionde
22.2 Metronidazole 19.3 Ornidazole 18.7 Tinidazole a A strain of B. fragilis subsp. fragilis
Range
Mean
viation 0.73 17-25 2.8 0;47 0.21 0.53 15-22 2.7 0.44 0.80 15-22 2.5 was tested seven times on different days.
instance, several strains with an unusual susceptibility pattern have been isolated in our
laboratory over a period of only a few months, e.g., C. perfringens resistant to penicillin (MIC, 25 ug/ml in BiH broth, determined with an inoculum of approximately 105 bacteria/ml) or chloramphenicol (MIC, 50 pg/ml) andB. fragilis resistant to chloramphenicol (MIC, 100 pg/ml). The broth-disk method of Wilkins and Thiel (37) is easy to perform and has been found to give reliable results (1, 37). When this method was used for testing susceptibility to metronidazole, ornidazole, and tinidazole and the results were compared with MIC values, growth
Range 0.4-1.6 0.4-0.8 0.4-1.6
in the tubes gave contradictory results in 2.6% of the tests with metronidazole and in 5.3% of the tests with the latter two drugs. This problem was encountered mainly with a few clostridial strains and could be overcome by using .a 1:100 dilution of the original inoculum in CMC broth. A possible explanation of this phenomenon may be found in the observation by Plant and Edwards (22), who have shown that growth of Clostridium bifermentans was inhibited by metronidazole and tinidazole only after 3 to 4 h of incubation, a period long enough to result in dense growth when 1 drop of an overnight CMC broth culture is added to 5 ml of BHI broth.
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This initial growth phase in the presence of the nitroimidazole drug has, however, not been unifornly observed by other workers using C. perfringens and B. fragilis (23; R. Luthy, F. Weber, and W. Siegenthaler, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother., 16th, Chicago, Ill., Abstr. 454, 1976). For clostridia, therefore, a 1:100 dilution of a CMC broth culture is recommended as the inoculum for the broth-disk test. A level of 6 ,ug/ml was chosen to differentiate between susceptible and resistant organisms, because such a concentration and a higher level of all three nitroimidazoles in serum are easily obtained (4, 23, 24, 28, 31, 32; additional data were provided by Hoffmann-La Roche and Co. AG and Pfizer AG). The level of 6 ug/ml also takes into account the deviation in disk content allowed by the Food and Drug Administration, i.e., 67 to 150% of the labeled value (37). Because no significant differences in MIC values of metronidazole, ornidazole, and tinidazole could be detected, use of only one representative disk in the modified broth-disk test is recommended (class disk). In contrast to the modified broth-disk test, very poor correlation was found betwen zone size in the agar diffusion test (36) and MIC values. For a given MIC value, the range of zone diameters obtained was very large. A similar observation has been made by Reynolds et al. (24) using a different diffusion test procedure. Due to the lack of strains with an intermediate susceptibility, no standard zone diameters representing susceptibility or resistance can be given at present. The reproducibility of the agar diffusion test was low, and when the same strain of B. fragilis was tested several times, the standard deviation was about double the standard deviation allowed by Sutter et al. (29) with their method. Therefore, the agar diffusion test cannot be recommended, at least for the time being, for detection of strains resistant to the nitroimidazole compounds, with the exception of extremely resistant bacteria such as P. acnes which show no inhibition zone at all. The results of MIC determinations were essentially identical in the various test media, BIHI agar with and without blood, brucella agar, and Mueller-Hinton agar (both with blood). The current study has shown that the modified broth-disk test provides a reliable and comparatively uncomplicated approach to measuring drug susceptibilities of various anaerobic bacteria. Clinical laboratories should be encouraged to apply this method to isolates of
ANTIMICROB. AGENTS CHEMOTHER.
medically important anaerobic organisms. Metronidazole, or another nitroimidazole, should be included among the agents evaluated in these tests. The results of such determinations could play an important role in the treatment of infections with a group of organisms that is exhibiting changing patterns of drug susceptibility. ACKNOWLEDGMENTS I thank F. H. Kayser and P. Santanam for critically reviewing the manuscript, J. Iten for skillful technical assistance, P. Landis for preparing the prereduced media, and L. Ruegg for help in preparing the manuscript. LITERATURE CITED 1. Blazevic, D. J. 1975. Evaluation of the modified brothdisk method for determining antibiotic susceptibilities of anaerobic bacteria. Antimicrob. Agents Chemother. 7:721-723. 2. Busch, D. F., V. L. Sutter, and S. M. Finegold. 1976. Activity of combinations of antimicrobial agents against Bacteroides fragilis. J. Infect. Dis. 133:321328. 3. Chow, A. W., V. Patten, and L. B. Guze. 1975. Susceptibility of anaerobic bacteria to metronidazole: relative resistance of non-spore-forming gram-positive bacilli. J. Infect. Dis. 131:182-185. 4. Dornbusch, K., and C.-E. Nord. 1974. In vitro effect of metronidazole and tinidazole on anaerobic bacteria. Med. Microbiol. Immunol. 160:265-267. 5. Dornbusch, K., C.-E. Nord, and T. Wadstrom. 1974. Biochemical characterization and in vitro determination of antibiotic susceptibility of clinical isolates of Bacteroides fragilis. Scand. J. Infect. Dis. 6:253-258. 6. Elek, S. D., and G. R. F. Hilson. 1954. Combined agar diffusion and replica plating techniques in the study of antibacterial substances. J. Clin. Pathol. 7:37-44. 7. Ericsson, H. M., and J. C. Sherris. 1971. Antibiotic sensitivity testing. Report of an international collaborative study. Acta Pathol. Microbiol. Scand. Suppl. 217:1-90. 8. Fass, R. J., R. B. Prior, and C. A. Rotilie. 1975. Simplified method for antimicrobial susceptibility testing of anaerobic bacteria. Antimicrob. Agents Chemother. 8:444-452. 9. Ferguson, I. R., and L. L. Smith. 1976. Bacteroides fragilis and nitroimidazoles. J. Antimicrob. Chemother. 2:220-221. 10. Finegold, S. M., J. G. Bartlett, A. W. Chow, D. J. Flora, S. L. Gorbach, E. J. Harder, and F. P. Tally. 1975. Management of anaerobic infections. Ann. Intern. Med. 83:375-389. 11. Gorbach, S. L., and J. G. Bartlett. 1974. Anaerobic infections. N. Engl. J. Med. 290:1177-1184, 12371245, 1289-1294. 12. Hamilton-Miller, J. M. T. 1975. Antimicrobial agents acting against anaerobes. J. Antimicrob. Chemother. 1:273-289. 13. Holdeman, L. V., and W. E. C. Moore (ed.). 1975. Anaerobe laboratory manual, 3rd ed. Virginia Polytechnic Institute and State University, Blacksburg. 14. Ingham, H. R., G. E. Rich, J. B. Selkon, J. H. Hale, C. M. Roxby, M. J. Betty, R. W. G. Johnson, and P. R. Uldall. 1975. Treatment with metronidazole of three patients with serious infections due to Bacteroides fragilis. J. Antimicrob. Chemother. 1:235-242. 15. Kislak, J. W. 1972. The susceptibility of Bacteroides fragilis to 24 antibiotics. J. Infect. Dis. 125:295-299.
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16. Laslie, W. W., and D. W. Lambe, Jr. 1976. Susceptibility testing of anaerobic bacteria with 100-.g carbenicillin disks. Antimicrob. Agents Chemother. 10:112118. 17. Martin, J. W., M. Gardner, and J. A. Washington II. 1972. In vitro antimicrobial susceptibility of anaerobic bacteria isolated from clinical specimens. Antimicrob. Agents Chemother. 1:148-158. 18. Metzler, C. M., and R. M. DeHaan. 1974. Susceptibility tests of anaerobic bacteria: statistical and clinical considerations. J. Infect. Dis. 130:588-594. 19. Mitre, R. J., and E. B. Rotheram, Jr. 1974. Anaerobic septicemia from thrombophlebitis of the internal jugular vein. J. Am. Med. Assoc. 230:1168-1169. 20. Nastro, J. L., and S. M. Finegold. 1972. Bactericidal activity of five antimicrobial agents against Bacteroides fragilis. J. Infect. Dis. 126:104-107. 21. Overman, S. B., D. W. Lambe, and J. V. Bennett. 1974. Proposed standardized method for testing and interpreting susceptibility of Bacteroides fragilis to tetracycline. Antimicrob. Agents Chemother. 5:357-361. 22. Plant, C. W., and D. I. Edwards. 1976. The effect of tinidazole, metronidazole and nitrofurazone on nucleic acid synthesis in Clostridium bifermentans. J. Antimicrob. Chemother. 2:203-209. 23. Ralph, E. D., and W. M. M. Kirby. 1975. Unique bactericidal action of metronidazole against Bacteroides fragilis and Clostridium perfringens. Antimicrob. Agents Chemother. 8:409-414. 24. Reynolds, A. V., J. M. T. Hamilton-Miller, and W. Brumfitt. 1975. A comparison of the in vitro activity of metronidazole,, tinidazole and nimorazole against gramnegative anaerobic bacilli. J. Clin. Pathol. 28:775-778. 25. Stalons, D. R., and C. Thornsberry. 1975. Broth-dilution method for determining the antibiotic susceptibility of anaerobic bacteria. Antimicrob. Agents Chemother. 7:15-21. 26. Staneck, J. L., and J. A. Washington II. 1974. Antimicrobial susceptibilities of anaerobic bacteria: recent clinical isolates. Antimicrob. Agents Chemother. 6:311-315. 27. Steers, E., E. L. Foltz, B. S. Graves, and J. Riders. 1959. An inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9:307-311. 28. Sutter, V. L., and S. M. Finegold. 1975. Susceptibility
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of anaerobic bacteria to carbenicillin, cefoxitin, and related drugs. J. Infect. Dis. 131:417-422. 29. Sutter, V. L., V. L. Vargo, and S. M. Finegold. 1975. Wadsworth anaerobic bacteriology manual, 2nd ed. Anaerobic Bacteriology Laboratory, Wadsworth Hospital Center and the Department of Medicine, Los
Angeles. 30. Tally, F. P., A. Y. Armfield, V. R. Dowell, Jr., Y. Y. Kwok, V. L. Sutter, and S. M. Finegold. 1974. Susceptibility of Clostridium ramosum to antimicrobial agents. Antimicrob. Agents. Chemother. 5:589-593. 31. Tally, F. P., V. L. Sutter, and S. M. Finegold. 1975. Treatment of anaerobic infections with metronidazole. Antimicrob. Agents Chemother. 7:672-675. 32. Whelan, J. P. F., and J. H. Hale. 1973. Bactericidal activity of metronidazole against Bacteroides fragilis. J. Clin. Pathol. 26:393-395. 33. Wilkins, T. D. 1974. Antibiotic susceptibility testing of anaerobic bacteria, p. 451-455. In A. Balows, R. M. DeHaan, V. R. Dowell, Jr., and L. B. Guze (ed.), Anaerobic bacteria: role in disease. Charles C Thomas, Springfield, Ill. 34. Wilkins, T. D., and M. D. Appleman. 1976. Review of methods for antibiotic susceptibility testing of anaerobic bacteria. Lab. Med. 7:12-15. 35. Wilkins, T. D., S. L. Chalgren, F. Jimenez-Ulate, C. R. Drake, Jr., and J. L. Johnson. 1976. Inhibition of Bacteroides fragilis on blood agar plates and reversal of inhibition by added hemin. J. Clin. Microbiol. 3:359-363. 36. Wilkins, T. D., L. V. Holdeman, I. J. Abramson, and W. E. C. Moore. 1972. Standardized single-disc method for antibiotic susceptibility testing of anaerobic bacteria. Antimicrob. Agents Chemother. 1:451459. 37. Wilkins, T. D., and T. Thiel. 1973. Modified broth-disk method for testing the antibiotic susceptibility of anaerobic bacteria. Antimicrob. Agents Chemother. 3:350-356. 38. Willis, A. T., C. L. Bullen, I. R. Ferguson, et al. 1974. Metronidazole in the prevention and treatment of bacteriodes infections in gynaecological patients. Lancet ii:1540-1543. 39. Willis, A. T., I. R. Ferguson, P. M. Jones, et al. 1976. Metronidazole in the prevention and treatment of bacteroides infections following appendicectomy. Br. Med. J. 1:318-321.
Vol. 11, No. 4 Printed in U.S.A.
Susceptibility of Anaerobic Bacteria to Metronidazole, Ornidazole, and Tinidazole and Routine Susceptibility Testing by Standardized Methods JURG WtJST Institute for Medical Microbiology, University ofZurich, CH-8028 Zurich, Switzerland
Received for publication 1 December 1976
A total of 114 strains of anaerobic bacteria were examined for their susceptibility to metronidazole, ornidazole, and tinidazole by measuring the minimum inhibitory concentration (MIC) and minimum bactericidal concentration in different media. All strains, with the exception of the isolates ofPropionibacterium acnes, were inhibited by 3.1 ug each and killed by 6.3 ,ug each of all three nitroimidazole compounds per ml. No significant differences in MIC values were found among metronidazole, ornidazole, and tinidazole. Only minor differences were detected by comparing MIC values obtained in brain heart infusion agar with and without sheep blood, brucella agar, and Mueller-Hinton agar (both containing blood). When the strains were tested by the modified broth-disk method proposed by the Anaerobe Laboratory of the Virginia Polytechnic Institute (VPI), there was good correlation with the MIC values (97.4% agreement for metronidazole and 94.7% for ornidazole and tinidazole). For routine testing, use of a 30-,ug-class disk of either nitroimidazole derivative is proposed for the brothdisk method, resulting in a final concentration of 6 ug/ml in the test tubes, a concentration easily attainable in body fluids. In contrast to the broth-disk method, there was very poor correlation between inhibition zone diameters by the standardized VPI agar diffusion test and MIC values.
Metronidazole and related nitroimidazole derivatives, including ornidazole and tinidazole, are receiving increasing attention in the treatment of anaerobic infections. This interest rests on the results ofintensive in vitro susceptibility evaluations (2-4, 9, 10, 20, 24, 26, 28, 32) and limited but promising trials of these agents in patients infected with various anaerobic bacteria (14, 19, 31, 39). All in vitro data have been obtained by determination of minimal inhibitory concentrations (MIC), a procedure that is not suitable for routine testing in the clinical laboratory, unless a sufficient number of strains have to be tested every day. Currently, there are several methods proposed for routine susceptibility testing of anaerobic bacteria (5, 8, 21, 25, 29, 36, 37). The purpose of this study was to investigate the reliability of susceptibility testing of anaerobes against metronidazole, ornidazole, and tinidazole by two routine procedures proposed by the Anaerobe Laboratory of the Virginia Polytechnic Institute (VPI), Blacksburg, Va., namely, the modified broth-disk method proposed by Wilkins and Thiel (37) and the agar diffusion method of Wilkins et al. (36). In addition, the
influence of media most used in anaerobic bacteriological work on the results of MIC determinations was evaluated, since Chow et al. (3) have found higher MIC values on Mueller-Hinton agar than authors using other media (2, 4, 9, 24, 26, 28, 32). MATERIALS AND METHODS Bacterial strains.Of the 114 strains examined in this study, 9 were provided from VPI and 105 were isolated from clinical specimens at the Institute for Medical Microbiology of the University of Zurich. They were identified according to the criteria outlined by Holdeman and Moore (13). Identification of subspecies of Bacteroides fragilis was performed by considering acid production from mannitol, rhamnose, and trehalose and indole production; carbohydrate fermentation was considered positive only if a pH of less than 5.75 was achieved (L. V. Holdeman, personal communication). The isolated strains comprised the following species: 39B. fragilis (2 B. fragilis subsp. distasonis, 28 B. fragilis subsp. fragilis, 6 B. fragilis subsp. thetaiotaomicron, 1 B. fragilis subsp. vulgatus, and 2 B. fragilis strains that could not be subspecified), 2 Bacteroides melaninogenicus subsp. asaccharolyticus, 2 Fusobacterium mortiferum, 3 Fusobacterium necrophorum, 7 Fusobacterium nucleatum, 1 Fuso631
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WUST
bacterium varium, 7 Peptococcus asaccharolyticus, 8 Peptococcus magnus, 2 Peptococcus prevotii, 7 Peptostreptococcus anaerobius, 4 Veillonella sp., 1 Eubacterium cylindroides, 1 Eubacterium lentum, 7 Propionibacterium acnes, 1 Clostridium butyricum, 1 Clostridium chauvoei, 1 Clostridium novyi A, 9 Clostridium perfringens, 3 Clostridium ramosum, 6 Clostridium sporogenes, 1 Clostridium tertium, 1 Clostridium sp. The strains were maintained at room temperature in chopped-meat broth (13). Media. Throughout the experiments, prereduced, anaerobically sterilized media were used. These were prepared according to methods described by Holdeman and Moore (13). The basic media were supplemented brain heart infusion (BHI) broth and chopped-meat carbohydrate (CMC) broth. The basal peptone-yeast extract medium used for testing biochemical reactions contained 0.5% peptone (Difco) and 0.5% Trypticase (BBL) instead of 1% peptone (W. E. C. Moore, personal communication). The media used for MIC determinations were: BHI agar (Difco), supplemented with 0.5% yeast extract (Difco), with and without 5% citrated sheep blood (BHIA); brucella agar (kindly supplied by Pfizer Overseas, Inc., New York) with 5% sheep blood; Mueller-Hinton agar (BBL), supplemented with 0.5% yeast extract (Difco) and 5% sheep blood. All media for MIC determinations were supplemented with menadione (0.5 ,g/ml) and hemin (5
,tg/ml).
Antimicrobial agents. Metronidazole and ornidazole were kindly supplied by P. Angehrn, Hoffmann-La Roche & Co. AG, Basel, Switzerland, and tinidazole was supplied by E. Milek, Pfizer AG, Zurich, Switzerland. Stock solutions containing 1,000 ,ug of each antimicrobial agent per ml were prepared in distilled water, filter-sterilized (Millipore, 0.22 um), and kept frozen at -20°C. MIC determinations. MIC determinations were performed according to Blazevic (1). For the agar dilution technique, plates were prepared containing serial twofold dilutions of each antimicrobial agent from 100 to 0.1 ,ug/ml. The plates were held overnight at room temperature in GasPak jars (BBL) or prepared on the day of the experiment. Prior to use, they were dried with the lids slightly opened, at 370C. All strains were checked for purity by subculturing to an anaerobic blood agar plate. One colony was inoculated into CMC broth. After overnight incubation at 37°C, a 1:100 dilution was made in BHI broth. The agar plates were inoculated with a device similar to the Steers replicator (27), resulting in an inoculum of approximately 2 x 103 to 2 x 104 bacteria per spot. Only 19 organisms were replicated at a time to avoid prolonged exposure to air. Anaerobic control plates without antimicrobial agents were inoculated last to ensure that viable organisms were present throughout the experiment and that the strains were able to grow on the media (35). The plates were incubated in GasPak jars at 370C for 48 h. Plates incubated aerobically and in an atmosphere with 10% C02 were included as additional controls. After incubation, the MIC was determined as
ANTIMICROB. AGENTS CHEMOTHER. the lowest concentration of the antimicrobial agent showing no growth, a barely visible haze, or one discrete colony (7). One strain of B. fragilis subsp. fragilis was included in each experiment to determine the reproducibility of the method. MIC determinations of nine swarming clostridia were performed separately for each strain and each concentration of metronidazole, ornidazole, and tinidazole, using the cups of disposable plastic trays as miniaturized petri dishes. These experiments were performed only with BHIA, and therefore these strains were excluded from the comparative evaluation of the different media. Determination of the MBC. The minimal bactericidal concentration (MBC) in BHIA without blood was determined by replica plating (6). The velvet surface of a sterilized pad was pressed against the primary BHI master plate and then to a BHIA plate without antimicrobial agent. The MBC was defined as the lowest concentration showing no growth or only one single colony in the subculture after 48 h of incubation at 370C in GasPak jars. Disks for susceptibility testing. Disks containing 10 and 30 ug of antimicrobial agent were prepared by adding 10 ul of a 1,000-,ul/ml solution and 20 ul of a 1,500-,ul/ml solution, respectively, to 6-mm filter paper disks. The disks were dried for about 16 h at 370C and stored at 4°C together with a desiccant. Broth-disk method. The broth-disk method described by Wilkins and Thiel (37) was strictly followed. One drop of an overnight culture in CMC broth was added to 5 ml of BHI broth. A disk containing 30 ,g of metronidazole, ornidazole, or tinidazole was added, resulting in a final concentration of 6 ug/ml of the respective antimicrobial agent. A control culture without antimicrobial agent was included for each strain. Tncubation was at 370C for 18 to 24 h. Susceptibility was defined as either absence of growth or less than 50% of the turbidity of the control culture. When there was growth in the tubes, indicating resistance, the strain was subcultured anaerobically and aerobically to exclude the possibility of contamination. Agar diffusion test. The method for the agar diffusion test described by Wilkins et al. (36) was used. A 1.5-ml portion of an 18- to 24-h culture at maximal turbidity in CMC broth was added to 10 ml of melted and cooled (500C) supplemented BHIA (13). The contents were mixed by inversion and poured into standard 90-mm petri dishes. After the agar had solidified, filter-paper disks containing 10 and 30 ug of metronidazole, ornidazole, and tinidazole were applied. After incubation at 370C in GasPak jars for 18 to 24 h, the zone diameters were measured with a ruler. Some strains of B. fragilis exhibited an inner area of light growth around the disk, with a clear area of inhibition outside this hazy growth. In these instances, the outer zone of inhibition was measured.
RESULTS The cumulative percentages of anaerobic bacterial strains inhibited and killed at various concentrations of metronidazole, ornidazole,
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NITROIMIDAZOLE SUSCEPTIBILITY OF ANAEROBES
and tinidazole are summarized in Table 1. All strains except the isolates of P. acnes were inhibited by 3.1 Mg each and killed by 6.3 ,g each of all three nitroimidazoles per ml. For most strains the MBC was equal to the MIC; in some-instances the MBC was one or two twofold dilutions higher than the MIC. In a few rare cases the MBC was one dilution step lower than the MIC, indicating that the antimicrobial agent became effective only after several generations (22) or that the microorganisms were killed by exposure to oxygen during the transfer. Table 2 contains the geometric means of the MICs of metronidazole, ornidazole, and tinidazole in the different media tested. A two-way analysis of variance showed no significant difference betwen the three antimicrobial agents in all four media (P > 5%). Whereas no difference was detected between BHIA with and without blood (P > 5%), the geometric means of the MICs in brucella agar with 5% sheep blood and Mueller-Hinton agar with 5% sheep blood were slightly higher (P < 1%) than in BHIA.
633
Table 3 compares the results obtained by the broth-disk test and the agar dilution method. Three organisms considered as susceptible to metronidazole according to the MICs were classified as resistant by the broth-disk method. For ornidazole and tinidazole there were six strains each showing similar behavior. These false resistance results in the broth-disk test were primarily found in testing clostridia; in addition, there was one strain of F. necrophorum resistant to all three nitroimidazoles in the broth-disk test. Upon repetition, part of the strains again showed false growth in the broth disk test tubes. When the tests were repeated with a 1:100 dilution of the CMC broth culture as inoculum, no false resistant classifications were encountered any more. In general, the results of the broth-disk method were easy to read. The tubes showed either heavy growth or were clear. With some B. fragilis strains there was a slight turbidity in the drug-containing tubes, but it was easy to ascertain a marked difference from the heavily grown control tube without drug. Difficulties
TABLE 1. Inhibitory and bactericidal effect of metronidazole, ornidazole, and tinidazole on anaerobic bacteria Organisms
No. of strains
Antimicrobial
Cumulative percent of isolates inhibited/killed at various concn
mI) a
agentI a0.1 0.2 0.4 0.8 1.6
tete tee
Bacteroides fragilis
Bacteroides melaninogenicus
Fusobacterium sp. Clostridium perfringens
Clostridium sp. (other) Peptococcus sp. and
Peptostreptococcus sp. Veillonella sp.
39 2 13 9
Metronidazole Ornidazole 3/5 Tinidazole 3/3 Metronidazole 100/100 Ornidazole 100/100 Tinidazole 100/100 Metronidazole 62/62 Ornidazole 62/54 Trinidazole 62/54 Metronidazole
Ornidazole
14/5b 24
Tinidazole Metronidazole Ornidazole Tinidazole Metronidazole
Ornidazole
15/15 18/15 28/23
85/85 85/85 85/77 33/33 11/22 11/11
14/0
64/0
14/0 14/0 4/8 4/4 4/8
57/0 57/0 58/50 50/46 42/25
38/31 44/44 56/56
62/67 72/79 74/82
92/100
97/90 97/95 92/95
6.3 3.1 100/100 100/97 100/100 100/97 100/100
(Qig/
>100
100/100
92/92 92/92
100/100
44/44 56/44 56/33
89/89 100/89 89/78
71/0 64/0 71/67 92/79 75/58
93/80
86/60 92/88 100/96
100/100 100/100 100/100 100/100 100/80 100/100 100/100 93/80 100/100 100/96 100/100 100/100 100/100
Tinidazole 96/92 4 Metronidazole 100/100 Ornidazole 50/50 100/100 Tinidazole 50/50 50/100 100/100 Eubacterium sp. 2 Metronidazole 50/50 100/50 100/100 Ornidazole 50/50 100/100 Tinidazole 50/50 100/100 7 Propionibacterium Metronidazole 100/100 acnes Ornidazole 100/100 Tinidazole 100/100 Total 114/105b Metronidazole 11/11 39/32 53/46 75/74 93/88 94/93 100/100 Ornidazole 12/11 36/30 62/54 83/83 93/91 94/92 94/93 100/100 Tinidazole 12/11 38/27 62/53 75/74 90/90 94/92 94/93 100/100 a In BHIA without blood. b MIC determinations of nine swarming clostridia were performed separately in small petri dishes. Therefore, the MBC assay could not be performed by the method used.
634
"8ST
ANTIMICROB. AGENTS CHEMOTHER.
were encountered with five Peptococcus and Peptostreptococcus species (i.e., 4.3% of the strains tested), which showed only very fair growth in the control tube. Prolonged incubation for another 24 h produced growth sufficient to read, although this procedure does not conform to the test described originally and has to be interpreted with caution, because of possible inactivation of the antimicrobial agent. Figure 1 shows the relationship of zone diameters around a 10-,ug metronidazole disk and MIC values in BHIA without blood. Twenty-six organisms (22.8%) that grew slowly in CMC broth and did not produce confluent growth in the pour plates, or produced indistinct zones, were excluded as proposed by Wilkins et al. (36). These strains comprised mainly Peptococcus sp. and some clostridia other than C. per-
TABLE 2. Comparison of MIC values of metronidazole, ornidazole, and tinidazole against anaerobic bacteria in different mediaa MIC (,ug/ml) in: b Antimicrobial agent
BHIA
BA
MHA
Without With blood blood
0.87 0.72 0.55 0.56 Metronidazole 0.84 0.59 0.49 0.50 Ornidazole 0.60 0.79 0.54 0.53 Tinidazole a Geometric means of the MIC values of 105 strains. b BA, Brucella agar with 5% sheep blood; MHA, Mueller-Hinton agar with 5% sheep blood.
fringens. Due to the extensive spread of diameters at the various MIC values and the relative narrow range of MICs, no regression line was calculated and drawn (7, 16, 18). The problems met in reading the diffusion tests were mainly cloudy zones due to the dense inoculum or organisms that grew well close to the disk but were inhibited further away from the disk (33). The complications of spreading were also encountered when 30-,ug disks were used and with ornidazole and tinidazole as well as with metronidazole. The reproducibility of the agar diffusion and the agar dilution method (see Table 4) was tested by performing the assays on different days with the same strain of B. fragilis subsp. fragilis. Direct numerical comparisons of the extent of variability of the two test procedures cannot be made, because the results were obtained in two systems not directly transferable (7). DISCUSSION There is controversy over the questions of whether the susceptibility testing of anaerobic bacteria should be performed routinely or whether the management of anaerobic infections should rely upon published susceptibility patterns for the different bacterial speeies (11, 16, 26, 34). I believe that routine testing should be done for clinically significant anaerobes from sources not contaminated with normal flora, since the susceptibility pattern of various anaerobes against several antimicrobial agents is known to change (12, 15-17, 21, 28, 30). For
TABLE 3. Comparison of results with the broth-disk and the agar dilution methods |Resistant strainsa
Sustrainsbl
Overall
Strains with false reaction in broth-disk test
agreement AntimicroNo. of No. of (%) between bial agent No. of false sus- No. of false rethe two strains ceptible strains sistant methods Species readingsb readingsb 0 3 7 107 97.4 Clostridium perfringens Metronidazole Clostridium sp. Fusobacterium mortiferum 7 Ornidazole 0 107 6 94.7 Clostridium perfringens Clostridium perfringens Clostridium sp. Fusobacterium nortiferum 7 Tinidazole 0 107 6 94.7 Bacteroides fragilis Clostridium perfringens Clostridium perfringens Clostridium perfringens Clostridium sp. Fusobacterium mortiferum a According to MIC in BHIA without blood. b In broth-disk test. c In BHIA without blood.
MIC (,g/ml)c
0.8 0.8
0.4
0.4 (2 strains) 0.8 (2 strains) 0.8 0.8 0.4 0.4 0.8 1.6 1.6 1.6
635
NITROIMIDAZOLE SUSCEPTIBILITY OF ANAEROBES
VOL. 11, 1977 2100
Metronidazole (10-wg disk) o Bacteroides fraglis o Fusobacterium sp. * Anaerobic cocci *Clostridium sp. *Eubacterium and Propionibacterium sp.
12.5L 6.31-
0
3.11E cn
00 o0o8
1.6[
8 0o
00
0
::
0o8[
* 8 8 0
o
i800
0
0
0
*00 o t
0
80 *
0.2-
s. *
e0.1
0
6
12
18
24
* t
S
00
0
30
*
N
0
0
36
U
U
i
42
48
54
Zone Diameters (mm) FIG. 1. Lack of correlation of MIC values in BHIA without blood and inhibition zone diameters of anaerobic bacteria around 10-pg metronidazole disks. Similar patterns were found when ornidazole and tinidazole were tested and when 30-pg disks were used instead of10-pg disks for all three nitroimidazoles.
TABLE 4. Reproducibility of antimicrobial susceptibility testsa Agar diffusion test (zone diam, mm)
Agar dilution test (MIC, ,ug/ml)
Antimicrobial agent Mean
Svtionde
22.2 Metronidazole 19.3 Ornidazole 18.7 Tinidazole a A strain of B. fragilis subsp. fragilis
Range
Mean
viation 0.73 17-25 2.8 0;47 0.21 0.53 15-22 2.7 0.44 0.80 15-22 2.5 was tested seven times on different days.
instance, several strains with an unusual susceptibility pattern have been isolated in our
laboratory over a period of only a few months, e.g., C. perfringens resistant to penicillin (MIC, 25 ug/ml in BiH broth, determined with an inoculum of approximately 105 bacteria/ml) or chloramphenicol (MIC, 50 pg/ml) andB. fragilis resistant to chloramphenicol (MIC, 100 pg/ml). The broth-disk method of Wilkins and Thiel (37) is easy to perform and has been found to give reliable results (1, 37). When this method was used for testing susceptibility to metronidazole, ornidazole, and tinidazole and the results were compared with MIC values, growth
Range 0.4-1.6 0.4-0.8 0.4-1.6
in the tubes gave contradictory results in 2.6% of the tests with metronidazole and in 5.3% of the tests with the latter two drugs. This problem was encountered mainly with a few clostridial strains and could be overcome by using .a 1:100 dilution of the original inoculum in CMC broth. A possible explanation of this phenomenon may be found in the observation by Plant and Edwards (22), who have shown that growth of Clostridium bifermentans was inhibited by metronidazole and tinidazole only after 3 to 4 h of incubation, a period long enough to result in dense growth when 1 drop of an overnight CMC broth culture is added to 5 ml of BHI broth.
636
WUOST
This initial growth phase in the presence of the nitroimidazole drug has, however, not been unifornly observed by other workers using C. perfringens and B. fragilis (23; R. Luthy, F. Weber, and W. Siegenthaler, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother., 16th, Chicago, Ill., Abstr. 454, 1976). For clostridia, therefore, a 1:100 dilution of a CMC broth culture is recommended as the inoculum for the broth-disk test. A level of 6 ,ug/ml was chosen to differentiate between susceptible and resistant organisms, because such a concentration and a higher level of all three nitroimidazoles in serum are easily obtained (4, 23, 24, 28, 31, 32; additional data were provided by Hoffmann-La Roche and Co. AG and Pfizer AG). The level of 6 ug/ml also takes into account the deviation in disk content allowed by the Food and Drug Administration, i.e., 67 to 150% of the labeled value (37). Because no significant differences in MIC values of metronidazole, ornidazole, and tinidazole could be detected, use of only one representative disk in the modified broth-disk test is recommended (class disk). In contrast to the modified broth-disk test, very poor correlation was found betwen zone size in the agar diffusion test (36) and MIC values. For a given MIC value, the range of zone diameters obtained was very large. A similar observation has been made by Reynolds et al. (24) using a different diffusion test procedure. Due to the lack of strains with an intermediate susceptibility, no standard zone diameters representing susceptibility or resistance can be given at present. The reproducibility of the agar diffusion test was low, and when the same strain of B. fragilis was tested several times, the standard deviation was about double the standard deviation allowed by Sutter et al. (29) with their method. Therefore, the agar diffusion test cannot be recommended, at least for the time being, for detection of strains resistant to the nitroimidazole compounds, with the exception of extremely resistant bacteria such as P. acnes which show no inhibition zone at all. The results of MIC determinations were essentially identical in the various test media, BIHI agar with and without blood, brucella agar, and Mueller-Hinton agar (both with blood). The current study has shown that the modified broth-disk test provides a reliable and comparatively uncomplicated approach to measuring drug susceptibilities of various anaerobic bacteria. Clinical laboratories should be encouraged to apply this method to isolates of
ANTIMICROB. AGENTS CHEMOTHER.
medically important anaerobic organisms. Metronidazole, or another nitroimidazole, should be included among the agents evaluated in these tests. The results of such determinations could play an important role in the treatment of infections with a group of organisms that is exhibiting changing patterns of drug susceptibility. ACKNOWLEDGMENTS I thank F. H. Kayser and P. Santanam for critically reviewing the manuscript, J. Iten for skillful technical assistance, P. Landis for preparing the prereduced media, and L. Ruegg for help in preparing the manuscript. LITERATURE CITED 1. Blazevic, D. J. 1975. Evaluation of the modified brothdisk method for determining antibiotic susceptibilities of anaerobic bacteria. Antimicrob. Agents Chemother. 7:721-723. 2. Busch, D. F., V. L. Sutter, and S. M. Finegold. 1976. Activity of combinations of antimicrobial agents against Bacteroides fragilis. J. Infect. Dis. 133:321328. 3. Chow, A. W., V. Patten, and L. B. Guze. 1975. Susceptibility of anaerobic bacteria to metronidazole: relative resistance of non-spore-forming gram-positive bacilli. J. Infect. Dis. 131:182-185. 4. Dornbusch, K., and C.-E. Nord. 1974. In vitro effect of metronidazole and tinidazole on anaerobic bacteria. Med. Microbiol. Immunol. 160:265-267. 5. Dornbusch, K., C.-E. Nord, and T. Wadstrom. 1974. Biochemical characterization and in vitro determination of antibiotic susceptibility of clinical isolates of Bacteroides fragilis. Scand. J. Infect. Dis. 6:253-258. 6. Elek, S. D., and G. R. F. Hilson. 1954. Combined agar diffusion and replica plating techniques in the study of antibacterial substances. J. Clin. Pathol. 7:37-44. 7. Ericsson, H. M., and J. C. Sherris. 1971. Antibiotic sensitivity testing. Report of an international collaborative study. Acta Pathol. Microbiol. Scand. Suppl. 217:1-90. 8. Fass, R. J., R. B. Prior, and C. A. Rotilie. 1975. Simplified method for antimicrobial susceptibility testing of anaerobic bacteria. Antimicrob. Agents Chemother. 8:444-452. 9. Ferguson, I. R., and L. L. Smith. 1976. Bacteroides fragilis and nitroimidazoles. J. Antimicrob. Chemother. 2:220-221. 10. Finegold, S. M., J. G. Bartlett, A. W. Chow, D. J. Flora, S. L. Gorbach, E. J. Harder, and F. P. Tally. 1975. Management of anaerobic infections. Ann. Intern. Med. 83:375-389. 11. Gorbach, S. L., and J. G. Bartlett. 1974. Anaerobic infections. N. Engl. J. Med. 290:1177-1184, 12371245, 1289-1294. 12. Hamilton-Miller, J. M. T. 1975. Antimicrobial agents acting against anaerobes. J. Antimicrob. Chemother. 1:273-289. 13. Holdeman, L. V., and W. E. C. Moore (ed.). 1975. Anaerobe laboratory manual, 3rd ed. Virginia Polytechnic Institute and State University, Blacksburg. 14. Ingham, H. R., G. E. Rich, J. B. Selkon, J. H. Hale, C. M. Roxby, M. J. Betty, R. W. G. Johnson, and P. R. Uldall. 1975. Treatment with metronidazole of three patients with serious infections due to Bacteroides fragilis. J. Antimicrob. Chemother. 1:235-242. 15. Kislak, J. W. 1972. The susceptibility of Bacteroides fragilis to 24 antibiotics. J. Infect. Dis. 125:295-299.
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16. Laslie, W. W., and D. W. Lambe, Jr. 1976. Susceptibility testing of anaerobic bacteria with 100-.g carbenicillin disks. Antimicrob. Agents Chemother. 10:112118. 17. Martin, J. W., M. Gardner, and J. A. Washington II. 1972. In vitro antimicrobial susceptibility of anaerobic bacteria isolated from clinical specimens. Antimicrob. Agents Chemother. 1:148-158. 18. Metzler, C. M., and R. M. DeHaan. 1974. Susceptibility tests of anaerobic bacteria: statistical and clinical considerations. J. Infect. Dis. 130:588-594. 19. Mitre, R. J., and E. B. Rotheram, Jr. 1974. Anaerobic septicemia from thrombophlebitis of the internal jugular vein. J. Am. Med. Assoc. 230:1168-1169. 20. Nastro, J. L., and S. M. Finegold. 1972. Bactericidal activity of five antimicrobial agents against Bacteroides fragilis. J. Infect. Dis. 126:104-107. 21. Overman, S. B., D. W. Lambe, and J. V. Bennett. 1974. Proposed standardized method for testing and interpreting susceptibility of Bacteroides fragilis to tetracycline. Antimicrob. Agents Chemother. 5:357-361. 22. Plant, C. W., and D. I. Edwards. 1976. The effect of tinidazole, metronidazole and nitrofurazone on nucleic acid synthesis in Clostridium bifermentans. J. Antimicrob. Chemother. 2:203-209. 23. Ralph, E. D., and W. M. M. Kirby. 1975. Unique bactericidal action of metronidazole against Bacteroides fragilis and Clostridium perfringens. Antimicrob. Agents Chemother. 8:409-414. 24. Reynolds, A. V., J. M. T. Hamilton-Miller, and W. Brumfitt. 1975. A comparison of the in vitro activity of metronidazole,, tinidazole and nimorazole against gramnegative anaerobic bacilli. J. Clin. Pathol. 28:775-778. 25. Stalons, D. R., and C. Thornsberry. 1975. Broth-dilution method for determining the antibiotic susceptibility of anaerobic bacteria. Antimicrob. Agents Chemother. 7:15-21. 26. Staneck, J. L., and J. A. Washington II. 1974. Antimicrobial susceptibilities of anaerobic bacteria: recent clinical isolates. Antimicrob. Agents Chemother. 6:311-315. 27. Steers, E., E. L. Foltz, B. S. Graves, and J. Riders. 1959. An inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9:307-311. 28. Sutter, V. L., and S. M. Finegold. 1975. Susceptibility
637
of anaerobic bacteria to carbenicillin, cefoxitin, and related drugs. J. Infect. Dis. 131:417-422. 29. Sutter, V. L., V. L. Vargo, and S. M. Finegold. 1975. Wadsworth anaerobic bacteriology manual, 2nd ed. Anaerobic Bacteriology Laboratory, Wadsworth Hospital Center and the Department of Medicine, Los
Angeles. 30. Tally, F. P., A. Y. Armfield, V. R. Dowell, Jr., Y. Y. Kwok, V. L. Sutter, and S. M. Finegold. 1974. Susceptibility of Clostridium ramosum to antimicrobial agents. Antimicrob. Agents. Chemother. 5:589-593. 31. Tally, F. P., V. L. Sutter, and S. M. Finegold. 1975. Treatment of anaerobic infections with metronidazole. Antimicrob. Agents Chemother. 7:672-675. 32. Whelan, J. P. F., and J. H. Hale. 1973. Bactericidal activity of metronidazole against Bacteroides fragilis. J. Clin. Pathol. 26:393-395. 33. Wilkins, T. D. 1974. Antibiotic susceptibility testing of anaerobic bacteria, p. 451-455. In A. Balows, R. M. DeHaan, V. R. Dowell, Jr., and L. B. Guze (ed.), Anaerobic bacteria: role in disease. Charles C Thomas, Springfield, Ill. 34. Wilkins, T. D., and M. D. Appleman. 1976. Review of methods for antibiotic susceptibility testing of anaerobic bacteria. Lab. Med. 7:12-15. 35. Wilkins, T. D., S. L. Chalgren, F. Jimenez-Ulate, C. R. Drake, Jr., and J. L. Johnson. 1976. Inhibition of Bacteroides fragilis on blood agar plates and reversal of inhibition by added hemin. J. Clin. Microbiol. 3:359-363. 36. Wilkins, T. D., L. V. Holdeman, I. J. Abramson, and W. E. C. Moore. 1972. Standardized single-disc method for antibiotic susceptibility testing of anaerobic bacteria. Antimicrob. Agents Chemother. 1:451459. 37. Wilkins, T. D., and T. Thiel. 1973. Modified broth-disk method for testing the antibiotic susceptibility of anaerobic bacteria. Antimicrob. Agents Chemother. 3:350-356. 38. Willis, A. T., C. L. Bullen, I. R. Ferguson, et al. 1974. Metronidazole in the prevention and treatment of bacteriodes infections in gynaecological patients. Lancet ii:1540-1543. 39. Willis, A. T., I. R. Ferguson, P. M. Jones, et al. 1976. Metronidazole in the prevention and treatment of bacteroides infections following appendicectomy. Br. Med. J. 1:318-321.
