Sulfamethoxazole and Trimethoprim: Correlation with a Disk

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 1981, p Vol. 20, No /81/ $02.00/0 Agar Dilution Susceptibility of Bacteroides spp. to Sulfamethoxazole and Trimethoprim: Correlation with a Disk Diffusion Technique THOMAS V. RILEY Department of Clinical Microbiology, Queen Elizabeth II Medical Centre, University of Western Australia, Nedlands, Australia 6009 Received 9 July 1981/Accepted 16 September 1981 The minimum inhibitory concentrations of sulfamethoxazole and trimethoprim against 105 strains of Bacteroides spp. were determined by an agar dilution method, using Diagnostic Sensitivity Test agar supplemented with lysed blood. A total of 92% of the strains were susceptible to sulfamethoxazole alone (minimum inhibitory concentration, s-50,ug/ml), whereas only 28% were susceptible to trimethoprim alone (minimum inhibitory concentration, <1,Ig/ml). Susceptibility was also determined by a disk diffusion technique. For sulfamethoxazole, there was good correlation between inhibition zone diameters and miniimum inhibitory concentrations. Hence, the disk diffusion test for susceptibility of Bacteroides spp. to sulfamethoxazole is suitable for routine laboratory testing. However, for trimethoprim there was a lack of correlation between the agar diffusion method and the minimum inhibitory concentrations. Three different methods for detecting synergy between sulfamethoxazole and trimethoprim against Bacteroides spp. were evaluated. With a checkerboard method, synergy was demonstrated in 96% of the strains which could be evaluated. However, this technique is unsuitable for routine testing. Two other techniques, which have been successfully used for detecting synergy with aerobic bacteria, were found to be unsatisfactory for Bacteroides spp. Cotrimoxazole, a combination of sulfamethoxazole (SMZ) and trimethoprim (TMP), is widely used for the treatment of not only urinary tract infections, but also infections of the respiratory tract, septicemia, and gonorrhea (5, 7). It has never been strongly advocated for the treatment of anaerobic infections, and this may reflect the paucity of information in this area. Previous reports with regard to its efficacy against anaerobes have tended to be contradictory (10-12, 17). This disparity in results almost certainly reflects the difficulties in in vitro testing of these two antimicrobial agents because of variations in the choice of media, inoculum size, and time of incubation. The two purposes of this investigation were to provide additional information on the susceptibility of Bacteroides spp. to SMZ and TMP, alone and in combination, and to establish whether or not a disk diffusion technique would be suitable for the in vitro testing of these antimicrobial agents. Although it is recognized that the disk diffusion technique is not the best method for determining the susceptibility of anaerobes to some antibiotics, it appears to be commonly used in Australia. MATERLALS AND METHODS Bacterial strains. A total of 105 strains of Bacteroides spp. were obtained from the University Department of Microbiology Anaerobe Laboratory culture collection. They were identified by the criteria of Holdeman et al. (6) and represented the most commonly isolated Bacteroides spp. from human infections at the Sir Charles Gairdner Hospital. The 105 strains were comprised of the following: 39 B. fragilis, 16 B. capillosus, 16 B. ruminicola, 11 B. fragilis group (4 B. distasonis, 2 B. ovatus, 2 B. thetaiotaomicron, 3 B. vulgatus) (3), 8 B. disiens, 6 B. oralis, 5 B. multiacidus, 3 B. bivius, and 1 B. uniformis. The cultures were maintained in prereduced supplemented brain heart infusion broth and prereduced cookedmeat broth (6) at room temperature. Antimicrobial agents. SMZ and TMP-lactate were supplied as powders of known purity by the Wellcome Foundation, Ltd. SMZ was dissolved in 0.5 N NaOH to give a final concentration of 10,000,g/ml, and TMP-lactate was dissolved in 0.02 N lactic acid to give a final concentration of 4,000,tg/ml. These stock solutions were kept at -20 C. Susceptibility testing. Minimum inhibitory concentrations (MICs) of each agent were determined by an agar dilution method. Diagnostic Sensitivity Test agar (Oxoid Ltd.) supplemented by the addition of 5% lysed horse blood was used as this medium was least 731

2 732 RILEY inhibitory to the action of sulfonamides (11). Plates containing 20 ml of agar were poured aerobically on the day of the experiment, with SMZ concentrations ranging from 200 to 1,ug/ml and TMP concentrations ranging from 20 to 1,ug/ml. Before use, the plates were dried at 370C for 20 min. The inoculum was prepared by diluting an overnight culture in supplemented brain heart infusion broth to the turbidity of a McFarland no. 1 turbidity standard and by making a 1:500 dilution in supplemented brain heart infusion broth. The agar plates were inoculated with a Steers replicator, resulting in a final inoculum of approximately 103 bacteria per spot. This low inoculum size was used to avoid the inoculum effect when sulfonamides were tested as described by Waterworth (15). A susceptible strain of Escherichia coli (NCTC 10418) was used as a control together with agar plates containing no antimicrobial agent incubated aerobically and anaerobically. The plates were incubated in GasPak (BBL Microbiology Systems, Cockeysville, Md.) jars at 370C for 24 h. After incubation, the MIC was determined as the lowest concentration of antimicrobial agent that caused complete inhibition of growth or a barely visible haze (4). Disk diffusion susceptibility was determined by the method of Sutter et al. (13), except that the diagnostic sensitivity test agar containing 5% lysed blood was used instead of brucella agar, and the inoculum was prepared in supplemented brain heart infusion broth instead of brucella broth. Disks containing jig of SMZ, 1.25,tg of TMP, and 25,ug of the combination (Oxoid Ltd.) were added to the inoculated plates. E. coli was used again as a susceptible control, and B. fragilis (NCTC 8560) was tested over several days to check the reproducibility of the method. The tests were carried out in duplicate. After overnight incubation at 370C in GasPak jars, the diameters of the zones of inhibition around each disk were measured, and the average of two measurements was recorded. The average diameters were correlated with MIC values, and a regression line was calculated by least-squares analysis. Tests for synergy. Tests for synergistic activity between SMZ and TMP were carried out on Diagnostic Sensitivity Test agar plus 5% lysed blood. Three approaches were made. First, the size of the zone of inhibition around the ILg SMZ disk was compared with the zone around the 25-,ug combined disk. An alternative approach involved placing the SMZ and TMP disks at a suitable distance apart on the agar plate (about 30 mm), and any deviation of circu- ANTIMICROB. AGENTS CHEMOTHER. larity around the SMZ disk was suggestive of synergism. Finally, SMZ and TMP were incorporated in agar plates in suitable doubling dilutions with SMZ/ TMP ratios of 20:1, 3:1, 1:1, 1:3, and 1:20. These plates were inoculated at the same time as the MIC determinations were carried out. For evaluation of synergistic activity of the components the fractional inhibitory concentration was calculated as follows: fractional inhibitory concentration index = (MIC of SMZ in combination/mic of SMZ alone) + (MIC of TMP in combination/mic of TMP alone). Any value of the fractional inhibitory concentration index that was less than 0.7 was considered to indicate synergy (8). The fractional inhibitory concentration index was not calculated when the MIC of either drug was at the lowest or next-to-lowest concentration tested for that drug. RESULTS Susceptibility testing. The susceptibility of 105 strains of Bacteroides spp. to SMZ and TMP, expressed as the range of MIC and concentration of antimicrobial agents yielding inhibition of 50 and 90% of the strains, is shown in Table 1. With the low inoculum used, no difficulty was experienced in recording the MIC determinations. When the inoculum recommended by Sutter et al. (13) was used (500 times denser), MICs increased by as much as four times and endpoints became hard to establish. With many strains there was quite a heavy haze, of growth at antibiotic levels much higher than the MIC. When the zones of inhibition obtained by disk diffusion were correlated with the MICs for SMZ, the regression line shown in Fig. 1 was obtained. Zone diameters ranged from 20 to 60 mm. When the control strain of B. fragilis was tested against SMZ on 10 separate occasions, the zones of inhibition ranged from 40 to 45 mm with a standard deviation of 1.4 mm. There was no significant deviation of the regression line from linearity, and the correlation coefficient was calculated to be An arbitrary MIC breakpoint of 100,tg/ml was chosen for SMZ based on achievable blood levels after oral administration of cotrimoxazole (2). Interpretive zone standards for the 105 organisms tested are TABLE 1. In vitro susceptibilities of Bacteroides spp. to SMZ and TMP SMZ (,Ug/ml) TMP (,ug/nml) Species na MIC range MICfob MICsof MIC range MIC50 MIC90 B. fragilis 39 <1-25 <1 5 <1-> B. fragilis group 11 <1-200 <1 2.5 <1-> Bacteroides spp. 55 <1-> <1-> a n, Number of strains b tested. Concentration inhibiting 50% of the strains. 'Concentration inhibiting 90% of the strains.

3 VOL. 20, MR I j.:5 2.5 I.I....\. I.. lifm..1.. W,o.. _ ZONE DIAMETER I I Zot Zs (mm ) FIG. 1. Comparison of SMZ MICs with zone sizes for a p.g SMZ disk, using 105 strains of Bacteroides spp. c33 mm for resistant isolates and :35 mm for susceptible strains by the error rate-bound method (9). None of the 105 strains of Bacteroides spp. produced a zone of inhibition around a 1.25-,ug TMP disk. All were considered resistant, and no regression line was calculated. Tests for synergy. A comparison of the zones of inhibition around the three disks showed that in many cases the zones around the combined disk were significantly smaller than those around the SMZ disk, suggesting that synergism could not be detected using this technique. Table 2 shows the diameters of zones of inhibition for 12 representative strains of Bacteroides spp. around the three disks. Of the 105 strains of Bacteroides spp. tested by the second approach, 47 (44%) displayed a deviation from circularity around the SMZ disk, suggesting a synergistic effect. The number of Bacteroides spp. which were synergistically affected by the combination of SMZ and TMP at a ratio of 20:1 is shown in Table 3. Of the organisms evaluated, only one strain of B. fragilis, with a fractional inhibitory concentration value of 0.9, was not influenced synergistically. Results for the other ratios of SMZ and TMP tested were identical to the 20:1 ratio, and synergism was evident for all ratios. DISCUSSION The two objectives of this study were (i) to provide more information on the susceptibility of Bacteroides spp. to SMZ and TMP and to the combination and (ii) to establish whether a disk diffusion technique was suitable for deter- BACTEROIDES SPP. AND COTRIMOXAZOLE 733 mining susceptibility of Bacteroides spp. to these antimicrobial agents. A total of 39 strains (100%) ofb. fragilis tested were sensitive to 25,ug or less of SMZ per ml. In general, most other Bacteroides spp. were susceptible to 25,ug/ml, although eight strains were resistant to 200,ug or more of SMZ per ml. These fmdings are in agreement with previous reports (11, 17): Bacteroides spp. were generally resistant to TMP (11, 17), and this resistance was due to the decreased susceptibility of the dihydrofolate reductase in this organism (14). In the present study, however, 29 (28%) of 105 strains examined had MICs of G1,ug/ml. This apparent discrepancy in results may be due to the wider range of Bacteroides spp. tested. Susceptibility testing of anaerobes is not carried out in many institutions in Australia because of the lack of a simple standardized method. Routine MIC determinations are not TABLE 2. Diameters of zones of inhibition of 12 Bacteroides spp. around disks containing SMZ and TMP alone and in combination Diameter (mm) of zone of inhibition' Organism SMZ SMZ (23.75 yg) (23.75 tg) + TMP (1.25 jig) B. fragilis B capillosus B. ruminicola B. disiens B. multiacidus B. ovatus B. uniformis B. thetaiotaomicron B. oralis B. vulgatus B. distasonis B. bivius a For TMP (1.25 ug), the diameter of the zone of inhibition for all organisms was zero. TABLE 3. Synergistic activity of SMZ and TMP No. of Frac- No. of Noof tional instrains.. Organism strains showing hibitory evalu- concn inated syner- dex gism (mean) B. fragilis B. capillosus B. ruminicola B. disiens B. multiacidus B. ovatus B. uniformis B. thetaiotaomicron

4 734 RILEY practicable for most clinical laboratories. The broth-disk dilution technique of Wilkins and Thiel (16) has a disadvantage in its need for prereduced media and inoculating the tubes under a stream of oxygen-free C02 and hence is only used in large laboratories. The disk diffusion technique was shown to be unsatisfactory for sensitivity testing of anaerobes with several antibiotics (1). SMZ and TMP, however, were not tested in this series. Wust and Wilkins (17) found poor correlation between zone of inhibition and MIC for SMZ. However, many bacteria in their series were slow-growing organisms, such as fusobacteria, eubacteria, and actinomyces, which failed to produce confluent growth and distinct inhibition zones. All of the Bacteroides spp. in the present series produced confluent growth and distinct inhibition zones, and hence an investigation of disk diffusion sensitivity testing for this group of organisms, with SMZ and TMP, was thought warranted. The results suggest good correlation between the zone of inhibition apd MIC for SMZ, and therefore this technique can be recommended for the susceptibility testing of SMZ against Bacteroides spp., providing that confluent growth is achievable after overnight incubation. The results for TMP testing by disk diffusion were unsatisfactory with no organism producing a zone of inhibition, and hence the technique cannot be recommended. The results for the three different methods of detecting synergy are interesting. From the results of the third experiment in this series, it was apparent that there was a synergistic effect when SMZ and TMP were combined in the ratio of 20:1, which is the ratio achieved in blood. This was observed in 28 (96%) of 29 strains which could be evaluated. Because this method of synergy testing is not feasible for clinical laboratories, two other methods were also tried. With a comparison ofzones of inhibition around a ,ug SMZ disk and a combined SMZ and TMP disk, none of the 105 strains of Bacteroides spp. tested showed evidence of synergism. With this method, Phillips and Warren (11) found that 3 of 13 strains of B. fragilis produced larger zones ANTIMICROB. AGENTS CHEMOTHER. around the combined disk, indicating synergy. However, in two of these strains, the difference in the zone diameter was only 2 mm, and this small difference may not be significant. Although this technique is suitable for synergy testing of many aerobic bacteria, it is not suitable for Bacteroides spp. The technique of looking at the shape of the zone of inhibition around SMZ and TMP disks has been successfully used to detect synergism in aerobic bacteria (15). Of the 105 Bacteroides spp. tested in this way, synergism was detected in 47 (45%) strains. This technique has a disadvantage in that the optimal distance between the SMZ and TMP disk can vary from strain to strain. Hence, only positive results can be considered valid. Both disk diffusion techniques for detecting synergy suffer, because some strains of Bacteroides spp. produced very large zones of inhibition (>50 mm), making small variations in shape and size difficult to detect. At present, there appears to be no suitable method for routinely testing SMZ and TMP for synergistic activity against Bacteroides spp. The in vitro results of activity of SMZ, either alone or in combination with TMP, against Bacteroides spp., suggest that the antimicrobial agents might be useful altematives in the therapy of Bacteroides infections. Because most facultative bacteria are susceptible to the combination of SMZ and TMP and the results of Wiist and Wilkins (17) suggest that other anaerobes are also susceptible, the treatment of mixed infections could be simplified. Clinical trials are now needed to resolve this problem. ACKNOWLEDGMENT I thank Brian Mee for helpful discussions during the preparation of this manuscript. LITERATURE CITED 1. Bodner, S. J., M. G. Koenig, L L Treanor, and J. 8. Goodman Antibiotic susceptibility teting of Bacteroides. Antimicrob. Agents Chemother. 2: Bruun, J. N., N. 0stby, J. E. Bredesen, P. Kierulf, and P. K. M. Lunde Sulfonamide and trimethopnm concentrations in human serum and skin blister fluid. Antimicrob. Agents Chemother. 19: Cato, E. P., and J. L Johnson Reinstatement of species rank for Bacteroides fragilis, B. ovatus, B. distasoniw, B. thetaiotaomicron, and B. vulgatus: designation of neotype strains for Bacteroides fragilis (Veilon and Zuber) Castellani and Chalmers and Bacteroides thetaiotaomicron (Distaso) Castellani and Chalmers. Int. J. Syst. Bacteriol. 26: Ericsson, H. M., and J. C. Sherris Antibiotic sensitivity testing: report of an international collaborative study. Acta Pathol. Microbiol. Scand. 217(Suppl.): Finland, M., and E. H. Kass (ed.) Trimethoprimsulfamethoxazole. Universty of Chicago Press, Chicago, mi. 6. Holdeman, L. V., E. P. Cato, and W. E. C. Moore (ed.) Anaerobe laboratory manual, 4th ed. Virginia Polytechnic Institute and State University, Blacksburg, Va. 7. Kallings, L, O., and J. E. Johanson (ed.) Sulphamethoxazole-trimethoprim. Scand. J. Infect. Dis. 8(Suppl.): Kerry, D. W., J. M. T. Hamilton-Miller, and W. Brumfftt Trimethoprim and rifampicin: in vitro activities separately and in combination. J. Antimicrob. Chemother. 1: Metzler, C. M., and R. M. DeHaan Susceptibility tests of anaerobic bacteria: statistical and clinical considerations. J. Infect. Dis. 130: Okubadejo, 0. A Susceptibility of Bacteroides fragilis to cotrimoxazole. Lancet i: Phillips, I., and C. Warren Susceptibility of Bacteroides fragilis to trimethoprim and sulphamethoxa-

5 VOL. 20, 1981 BACTEROIDES SPP. AND COTRIMOXAZOLE 735 zole. Lancet i: Rosenblatt, J. E., and P. R. Stewart Lack of activity of sulfamethoxazole and trimethoprim against anaerobic bacteria. Antimicrob. Agents Chemother. 6: Sutter, V. L., V. L. Vargo, and S. M. Finegold Wadsworth anaerobic bacteriology manual, 2nd ed. Anaerobic bacteriology laboratory. Wadsworth Hospital Center and Department of Medicine. University of California at Los Angeles, Los Angeles, Calif. 14. Then, R. L., and P. Angehrn Low trimethoprim susceptibility of anaerobic bacteria due to insensitive dihydrofolate reductases. Antimicrob. Agents Chemother. 15: Waterworth, P. M Laboratory control, p In L. P. Garrod, H. P. Lambert, and F. O'Grady (ed.), Antibiotic and chemotherapy. Churchill Livingstone, Ltd., Edinburgh. 16. Wilkins, T. D., and T. Thiel Modified broth-disk method for testing the antibiotic susceptibility of anaerobic bacteria. Antimiicrob. Agents Chemother. 3: Wust, J., and T. D. Wilkins Susceptibility of anaerobic bacteria to sulfamethoxazole/trimethoprim and routine susceptibility testing. Antimicrob. Agents Chemother. 14: Downloaded from on October 30, 2018 by guest