Simplified Method for Antimicrobial Susceptibility Testing of Anaerobic Bacteria

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, OCt. 1975, p Copyright American Society for Microbiology Vol. 8, No. 4 Printed in U.S.A. Simplified Method for Antimicrobial Susceptibility Testing of Anaerobic Bacteria ROBERT J. FASS,* RICHARD B. PRIOR, AND CAROL A. ROTILIE Division of Infectious Diseases, Department of Medicine, The Ohio State University College of Medicine, Columbus, Ohio Received for publication 30 May 1975 A simple, abbreviated broth dilution test (tube test) utilizing a commercially available medium and inexpensive disposable materials, and which could be performed entirely in room air, was developed and used to test the susceptibility of 100 strains of anaerobic bacteria to clindamycin, chloramphenicol, ampicillin, and tetracycline. Results are reported in categories of susceptibility: susceptible to concentrations surpassed in vivo with usual dosage, susceptible to concentrations surpassed in vivo with high dosage, and resistant to concentrations achievable in vivo. Results are compared to minimal inhibitory concentrations which were determined simultaneously by using a microdilution method in an anaerobic glove box. Twenty strains of Bacteroides fragilis, 10 strains of Fusobacterium, 20 strains of Clostridium, 10 strains of gram-positive nonsporeforming bacilli, and 30 strains of cocci grew to visible turbidity within 1 day of incubation. Of the 360 antibiotic-organism combinations tested, 98% were in a susceptibility category that corresponded (within one concentration) to the actual minimal inhibitory concentration as determined by the microdilution method. After 2 days of incubation, growth was more abundant, but results often indicated inappropriate degrees of resistance. Variation in inoculum size had little effect on results. Ten strains of B. melaninogenicus did not grow enough for susceptibility to be categorized accurately. The tube test could be used in any clinical microbiology laboratory for a limited number of susceptibility tests on anaerobic bacteria other than B. melaninogenicus without preparation of special media or purchase of special equipment. Antimicrobial susceptibility testing of anaerobic bacteria is usually done by agar or broth dilution methods (16). Disk diffusion methods (2, 3, 6, 8, 11, 14, 15, 17, 19), analogous to that described by Bauer et al. (1) for rapidly growing aerobic and facultative bacteria, have also been developed in attempts to simplify and standardize the approach to antimicrobial susceptibility testing of anaerobes. These methods have been limited in their usefulness, however, since a significant percentage of clinical isolates did not grow or grew insufficiently for zones of inhibition to be determined. For those that did grow, extreme variations in growth rates required that separate linear regression plots of minimal inhibitory concentrations (MICs) versus zone diameters be used for different organisms. In addition, there was much greater variation about the regression lines than occurs with aerobic tests, and accurate separation into the categories susceptible, intermediate, and resistant, based on zones of inhibition, was not consistently achieved with statistical validity (7). 444 A modified broth dilution method, the "broth-disk" method, has been proposed by Wilkins and Thiel (20), which provides a simple method for testing all anaerobes including slowgrowing and very oxygen-sensitive strains that could not be tested by disk diffusion techniques. A concentration of antibiotic approximately equal to that achievable in blood is put into a tube of broth medium, with commercial filter paper disks as the carrier of the antibiotic. Growth at that concentration is considered to constitute resistance. Ninety-seven percent of the strains tested by Wilkins and Thiel grew sufficiently for readable results, but the method requires the use of a completely anaerobic environment, and there is considerable variation (73 to 148% of the labeled values in their tests) in the actual antibiotic concentrations delivered by the filter paper disks. A modified broth dilution method has also been proposed by Stalons and Thornsberry (12), which simplifies the susceptibility testing of anaerobic bacteria. Selected concentrations of antibiotics are used so that results are divided

2 VOL. 8, 1975 SUSCEPTIBILITY TESTING OF ANAEROBIC BACTERIA 445 into three categories of susceptibility. Although the method utilizes a commercially available medium, Schaedler broth (BBL, Cockeysville, Md.), and a reduced number of tubes for each susceptibility test performed, it requires the use of an anaerobic glove box or incubation in a GasPak (BBL) system. The present report deals with'another modified broth dilution method which will be referred to as the "tube test." As in the methods of Wilkins and Thiel (20) and Stalons and Thornsberry (12), results are reported in categories of susceptibility and are compared with MICs. The tube test is unique for several reasons. Antibiotic dispensing and inoculum preparation are simplified. All materials are inexpensive, disposable and, like the medium, commercially available. All manipulations including incubation are performed in room air so that an anaerobic glove box, GasPak jar, or other special equipment necessary to provide an anaerobic environment is not required. MATERIALS AND METHODS Bacteria. One hundred strains of anaerobic bacteria were tested. Seventy-eight were clinical isolates from patients in University Hospital, Columbus, Ohio. Twenty-two were stock cultures identified and provided by the Center for Disease Control, Atlanta, Ga. The anaerobes were grouped into the following categories: Bacteroides fragilis (20 strains), B. melaninogenicus (10 strains), Fusobacterium (10 strains), Clostridium (10 strains of C. perfringens and 10 strains of other Clostridium species), gram-positive nonsporeforming bacilli (10 strains including Propionibacterium, Eubacterium, and Bifidobacterium), and cocci (30 strains including Peptococcus, Peptostreptococcus, and Veillonella). Cultures in Schaedler broth (BBL) with 10% glycerol were maintained frozen in liquid nitrogen and were subcultured onto anaerobically stored 5% sheep blood agar plates prior to use. Antibiotics. Commercially available laboratory standards of clindamycin, chloramphenicol, ampicillin, and tetracycline were dissolved in sterile, distilled water. For the microdilution susceptibility tests, working solutions were prepared by dilution to concentrations of 100,g/ml. For the tube tests, working solutions were prepared by dilution to concentrations of 306 ug/ml for clindamycin and chloramphenicol, 306 and 1,224 ;g/ml for ampicillin, and 153 ug/ml for tetracycline. The working solutions were stored for a maximum of 1 month at -20 C in sterile, plastic Microdel bottles (Cooke Laboratory Products, Alexandria, Va.); a fresh set of bottles was thawed for each run of tests. Media. Schaedler broth was used for all tests. For the microdilution tests, it was autoclaved and then stored for up to 5 days in an anaerobic glove box (Coy Manufacturing, Ann Arbor, Mich.) that contained 80% nitrogen, 10% hydrogen, and 10% carbon dioxide at 75 to 85% relative humidity. For the tube test, flint glass tubes (13 by 100 mm) with rubber-lined screw caps (Scientific Products, McGaw Park, Ill.), each containing one 6-mm solid glass bead, were filled with Schaedler broth containing % resazurin and then autoclaved. The tubes were stored at room temperature for up to 48 h and were not used if the medium turned pink due to oxidation of the resazurin. The final ph of the media was 7.2. For both tests, heat-inactivated (56 C for 0.5 h) horse serum was added to a final concentration of 1% immediately before the testing of B. melaninogenicus, Fusobacterium, and cocci. MICs. MICs were determined in an anerobic glove box by a microdilution method (10). Each of the four antibiotics studied was added to the first two wells of two horizontal rows in the microdilution plate, and serial twofold dilutions were made from the second set of wells. For inoculum preparation, colonies of each test strain were subcultured from 48-h 5% sheep blood agar plates to 5 ml of Schaedler broth and incubated at 35 C in a glove box overnight. For faintly turbid suspensions (optical density approximately 0.1 to 0.3 at 650 nm), a 1:10 dilution was used; for suspensions that were more turbid, a 1:100 dilution was used. The final inocula ranged from 105 to 107 colony-forming units (CFU) per ml. The final antibiotic concentrations ranged from 50 to 0.05 Ag ml. Plates were sealed with cellophane tape (Cooke Laboratory Products, Alexandria, Va.) to prevent evaporation and were incubated at 35 C in the glove box. The humidity in the incubator was kept at 65 to 75% to further reduce evaporation from the plates. MICs were read at 20 to 22 h (1 day) and 44 to 46 h (2 days) as the lowest concentrations of antibiotics that inhibited visible growth as judged visually with the aid of a test-reading mirror (Cooke Laboratory Products, Alexandria, Va.). When the duplicate tests varied by one concentration, the higher concentration was considered to be the MIC. When they varied by more than one concentration the test was repeated. A strain of B. fragilis with known MICs was tested each day and served as a system control. Tube test. The tube test was performed simultaneously with the microdilution test for each organism. In room air, the glass tubes were uncapped. One to eight drops (0.05 ml/drop) of antibiotic were added to tubes with the Microdel bottles so that for each organism being tested there was a tube with a high and a low concentration of each of the four antibiotics. The concentrations in the Microdel bottles were calculated so that: 1 and 4 drops of clindamycin (306 jg/ml) resulted in final tube concentrations of 1.6 and 6.2 jg/ml, respectively; 1 and 8 drops of chloramphenicol (306 jg/ml) resulted in final tube concentrations of 1.6 and 12.5 Mg/ml, respectively; 1 drop of ampicillin (306 Mg/ml) and 4 drops of ampicillin (1,224 Ag/ml) resulted in final tube concentrations of 1.6 and 25 Ag/ml, respectively; and 1 and 8 drops of tetracycline (153 Mg/ml) resulted in final tube concentrations of 0.8 and 6.2 ug/ml, respectively. (These calculations were based on a tube volume of 9.2 [+0.24 standard deviation I ml and took into consideration the % [+2.7 standard deviation] error of the Microdel bottles.) For tests requiring the use of horse serum, two drops (0.05 ml/drop, with a -3.86% [±4.5 standard

3 446 FASS, PRIOR, AND ROTILIE deviation J error) were added with a disposable pipette (Cooke Laboratory Products, Alexandria, Va.) at the same time as the antibiotics. The tube caps were replaced and the tube contents were mixed by inversion. The caps were again removed, and 1 drop (0.05 ml) of undiluted inoculum was added with a disposable pipette. This resulted in a final inoculum that was 110% of that used in the microdilution method when a 1:100 dilution was employed and 11% of that used in the microdilution test when a 1:10 dilution was employed. If any space remained in the tubes after the addition of antibiotic, horse serum, and inoculum, Schaedler broth was added to fill the tubes completely and the caps were replaced tightly. During the two brief exposures to room air, the media in the tubes became slightly pink as the resazurin was oxidized. Within 15 min after the caps were retightened, the indicator became colorless as it was reduced in the Schaedler broth. With each set of eight antibioticcontaining tubes, a ninth tube without antibiotic was similarly inoculated with the organism being tested and served as a growth control. The nine inoculated tubes for each test organism and an uninoculated sterility control tube were then incubated in a standard incubator at 35 C. After 20 to 22 h (1 day) and 44 to 46 h (2 days) of incubation, tubes were observed for turbidity and were read as "growth" or "no growth." Inhibition of growth by both concentrations of a given antibiotic was interpreted as indicating susceptibility to concentrations easily surpassed in vivo with "usual" dosage. Inhibition by only "high" concentration of a given antibiotic was interpreted as indicating susceptibility to concentrations surpassed in vivo with the high dosage. Lack of inhibition by both antibiotic concentrations was interpreted as indicating resistance to concentrations achievable in vivo. The breakpoints for the three categories of susceptibility for each of the four antibiotics used are summarized in Table 1. Inoculum effect. The microdilution and tube tests were performed simultaneously with 10-fold variations in inoculum size. One strain each of B. fragilis, C. perfringens, and Veillonella and two strains of B. melaninogenicus were used. For comparison of results obtained with the two tests, a 1:100 dilution of the inoculum for the microdilution test was considered to be equivalent to an undiluted inoculum for the tube test. RESULTS Microdilution method. The 2-day MICs of the four antibiotics tested against the 100 anaerobic strains are shown in Fig. 1. Clindamycin inhibited 97% of the strains at concentrations of 1.6 Mg or less per ml and 99% of the strains at concentrations of 6.2,g or less per ml. Chloramphenicol inhibited 33% of the strains at concentrations of 1.6 Mg or less per ml and 99% of the strains at concentrations of 12.5 Mg or less per ml. Ampicillin inhibited 99% of the strains other than Bacteroides at concentrations of 1.6,Mg or less per ml; concentrations of 25 Ag or less TABLE 1. Categories of susceptibility as defined by MICsa Antibiotic ANTIMICROB. AGENTS CHEMOTHER. MIC (yg/ml) SS S R Clindamycin < 1.6 > >6.2 Chloramphenicol < 1.6 > > 12.5 Ampicillin < 1.6 > > 25 Tetracycline <0.8 > >6.2 a SS, Susceptible to concentrations surpassed' in vivo with usual dosage; S, susceptible to concentrations surpassed in vivo with high dosage; R, resistant to concentrations achievable in vivo. per ml inhibited 98% of all strains. Tetracycline inhibited 50% of the strains at concentrations of 0.8,ug or less per ml and 69% of the strains at concentrations of 6.2 jig or less per ml. By day 1, 4% of the test strains had not grown sufficiently for MICs to be read. For the 96 strains that grew, 91% of the MICs (total: 384) were the same or only one concentration lower than the 2-day MICs; 9% had MICs two to five concentrations lower. No growth or low MICs at day 1 occurred sporadically with all anaerobes and with all four antibiotics. When the test was repeated with those strains, 1-day MICs were often readable and closer to the 2-day MICs. Conversely, when tests with organisms that had 1-day MICs that were the same or only one concentration lower than 2-day MICs were repeated, the 1-day MICs were occasionally unreadable or low. The reproducibility of MICs is shown in Fig. 2 for the control strain of B. fragilis. In 23 tests run on separate days, all 1-day and 2-day MICs were within one concentration of those respective mean MICs. Tube test. The number of strains in each group of anaerobes that fell into each category of susceptibility for each of the four antibiotics is shown in Table 2. The results obtained with the microdilution test, by category, are included for comparison. Except for B. melaninogenicus, the results of the 2-day microdilution tests and 1-day tube tests correlated best (Fig. 3). Oneday tube test results for only 8 (2%) of 360 tests were in categories of susceptibility that indicated MICs differing from the 2-day microdilu,- tion MICs by more than one concentration (Table 3). For seven of the eight tests, the 1-day tube test categories indicated greater susceptibility than did the 2-day MICs. Of the ten strains of B. melaninogenicus, only one grew sufficiently in the tubes to be read by 1 day and only two strains grew sufficiently to be read by 2 days. When the tube tests were

4 VOL. 8, 1975 SUSCEPTIBILITY TESTING OF ANAEROBIC BACTERIA z a-20f A-XEB z06t. X 0~~~~~~~~~~~~~~~~~~~~~'0 LIL 49 -j MBELANIN0IM I T C f~~~~~oc f U.S ww,acteri,um'w ww MINIMAL INHIBITORY CONCENTRATION (jig/mi) FIG. 1. Cumulative percentage of strains inhibited by (A) clindamycin, (B) chloramphenicol, (C) ampicillin, and (D) tetracycline. GPNSB, Gram-positive nonsporeforming bacilli. Q) I-i Cl) M Ui. 0 a: m z 25 IS 5- D ,b 25J CLINDAMYCIN CHLORAMPHENICOL AMPICILLIN ; MINIMAL INHIBITORY CONCENTRA procedure was repeated in room air with a 10- TETRACYCLINE fold concentration of the inocula, six strains grew by 1 day and eight grew by 2 days. Both 1-day and 2-day results with either modification were frequently unreliable. Inoculum effect. The effects of varying the inoculum size in both the microdilution and tube tests with four of the five strains tested are shown in Tables 4 through 7. The results with the second strain of B. melaninogenicus were similar to those shown in Table 7. With the microdilution test there was little inoculum g effect observed, although the use of a 1:10 dilution occasionally resulted in high MICs. No growth at day 1 occurred with inocula of B. melaninogenicus diluted 1:1,000 or more. The tube test was affected to a similar degree by variations in inoculum size. The use of concentrated inocula (corresponding to a 1:10 dilution in the microdilution test) occasionally resulted TION (jaq/ml) in an apparent increased resistance, although FIG. 2. Frequency of 1-day (top) and 2 this was not so with one strain of B..day (bottom) MICs for the control strain of B. fragilis melaninogenicus (Table 7). With both strains of B. melaninogenicus, 1:10 dilution of inocula repeated in the anaerobic glove boxc, six strains (corresponding to 1:1,000 in the microdilution grew by 1 day and ten grew by 2 dayrs. When the test) or more resulted in no visible growth.

5 448 FASS, PRIOR, AND ROTILIE ANTIMICROB. AGENTS CHEMOTHER. TABLE 2. Number of strains in each category of susceptibility as determined by the microdilution and tube tests after 1 and 2 days of incubationa Microdilution test (no.) Tube test (no.) Determination 1 Day 2 Days 1 Day 2 Days NG 88 S R NG 88 R S NG S R NG 88 S R Clindamycin B. fragilis Fusobacterium Clostridium GPNSB Cocci B. melaninogenicus Chloramphenicol B. fragilis Fusobacterium Clostridium GPNSB Cocci B. melaninogenicus Ampicillin B. fragilis Fusobacterium Clostridium GPNSB Cocci B. melaninogenicus Tetracycline B. fragilis Fusobacterium Clostridium GPNSB Cocci B. melaninogenicus a NG, No growth; GPNSB, gram-positive nonsporeforming bacilli. For other abbreviations, see footnote a of Table MICRODILUTION TEST TUBE TEST I-DAY 2-DAY -DAY NG SS S R NG SS S R NG SS S R CATEGORY OF SUSCEPTIBILITY FIG. 3. Susceptibility of anaerobes after I day and 2 days of incubation by the microdilution and tube test methods. Results with B. melaninogenicus are excluded. NG, No growth. For other abbreviations see footnote a of Table 1. A 2-DAY - DISCUSSION Our previous (5, 10) and current experiences with the microdilution method have indicated that it is a reliable method for determining the antimicrobial susceptibility of anaerobic bacteria. Certain materials and procedures make it relatively simple and convenient and, therefore, have been incorporated into the tube test. Thus, Schaedler broth, a commercially available medium which has been reported to be excellent for the growth of most anaerobes (13) is used. The addition of 1% horse serum improves the growth of the more fastidious genera (5, 10). Microdel bottles calibrated to deliver 0.05 ml of predetermined concentrations of antibiotics are prepared in advance, and disposable pipettes

6 VOL. 8, 1975 SUSCEPTIBILITY TESTING OF ANAEROBIC BACTERIA 449 TABLE 3. Results of the simultaneously performed microdilution and tube tests for which the 1-day tube test categories of susceptibility indicated MICs more than one concentration different from the 2-day microdilution MICsa Organism Antibiotic Microdilution MIC (,g/ml) Tube test category 1 Day 2 Days 1 Day 2 Days B. fragilis Ampic;llin SS S B. fragilis Tetracycline S R B. fragilis Tetracycline S R B. fragilis Tetracycline SS R C. innocuum Chloramphenicol S S F. necrophorum Chloramphenicol SS S Peptostreptococcus Chloramphenicol SS S C. sporogenes Clindamycin SS S a See footnote a of Table 1 for abbreviations. Results for B. melaninogenicus are not included. TABLE 4. Effect of inoculum size on susceptibility testing of B. fragilis Microdilution test Tube test Antibiotic Inoculum MIC (lsg/ml) Inoculum Categorya dilution 1 Day 2 Days dilution 1 Day 2 Days Clindamycin 1:10 <0.05 < :1 SS SS 1:100 < 0.05 < 0.05 None SS SS 1:1,000 <0.05 <0.05 1:10 SS SS 1:10,000 <0.05 <0.05 1:100 SS SS Chloramphenicol 1: :1 S R 1: None S S 1:1, :10 S S 1:10, :100 SS S Ampicillin 1: :1 S R 1: None S R 1:1, :10, S R 1:10, :100 S S Tetracycline 1: :1 R R 1: None R R 1:1, :10 S R 1:10, :100 S R a See footnote a of Table 1 for abbreviations. are used to deliver 0.05-ml aliquots of medium, inoculum, and horse serum. Both the bottles and the pipettes are inexpensive, commercially available, and disposable. Although neither conform to the advertised 2% error limits, as determined by our tests, both are sufficiently accurate for serial dilution tests where the anticipated accuracy of the system is a one concentration variation in MICs. Standardization of inocula to a specific turbidity standard is eliminated. Our experience and that of others (13) have indicated that suspensions of organisms that vary as much in size and conformation as the various genera of anaerobes cannot be adjusted to a given turbidity standard and be expected to contain the same number of CFUs per milliliter. By dividing inocula into categories of low and high turbidity and diluting them 1:10 and 1:100, respectively, we have found that inocula are generally 105 to 107 CFU/ml. Since inoculum variation did not have a marked effect on MIC results, this seemed acceptable. The lack of a marked inoculum effect with anaerobes has also been observed by others with both agar dilution (9) and broth dilution (12) methods of susceptibility testing. Although the microdilution test is a convenient and accurate method for performing large numbers of susceptibility tests, it would not be feasible in a clinical laboratory unable to pur-

7 450 FASS, PRIOR, AND ROTILIE ANTIMICROB. AGENTS CHEMOTHER. TABLE 5. Effect of inoculum size on susceptibility testing of C. perfringens Microdilution test Tube test Antibiotic Inoculum MIC (Mg/ml) Inoculum Categorya dilution 1 Day 2 Days dilution 1 Day 2 Days Clindamycin 1: :1 SS S 1: None SS SS 1:1, :10 Ss SS 1:10, :100 Ss SS Chloramphenicol 1: :1 R R 1: None S R 1:1, :10 S S 1:10, :100 S S Ampicillin 1:10 <0.05 < :1 SS SS 1:1,000 <0.05 <0.05 1:10 Ss SS 1:10,000 <0.05 <0.05 1:100 Ss SS Tetracycline 1:10 <0.05 < :1 SS SS 1:1,000 <0.05 <0.05 1:10 Ss SS 1:10,000 <0.05 <0.05 1:100 Ss SS See footnote a of Table 1 for abbreviations. TABLE 6. Effect of inoculum size on susceptibility testing of Veillonella Microdilution test Tube test Antibiotic Inoculum MIC (,Rg/ml) Inoculum Categorya dilution 1 Day 2 Days dilution 1 Day 2 Days Clindamycin 1:10 <0.05 < :1 SS SS 1:1,000 <0.05 <0.05 1:10 SS SS 1:10,000 <0.05 <0.05 1:100 SS SS Chloramphenicol 1: :1 S S 1: None S S 1:1, :10 SS S 1:10, :100 SS SS Ampicillin 1: :1 SS SS 1:1,000 <0.05 <0.05 1:10 SS SS 1:10,000 <0.05 <0.05 1:100 SS SS Tetracycline 1: :1 S S 1:100 < None SS S 1:1,000 <0.05 <0.05 1:10 SS SS 1:10,000 <0.05 <0.05 1:100 SS SS a See footnote a of Table 1 for abbreviations. chase microdilution equipment and an anaerobic glove box or which performed only small numbers of tests at a given time. With the tube test, no special equipment is required, and all inoculum preparation and incubation can be done in room air. Oxygen is excluded from the tubes by filling them to the top with broth to displace all air and by only briefly exposing the media in the tubes to air when introducing antibiotic, inoculum and, in some cases, horse serum. Oxygen that does enter the system is adequately eliminated by the reducing agent

8 VOL. 8, 1975 SUSCEPTIBILITY TESTING OF ANAEROBIC BACTERIA 451 TABLE 7. Effect of inoculum size on susceptibility testing of B. melaninogenicus Microdilution test Tube test Antibiotic Inoculum MIC (gg/ml) Inoculum Categorya dilution 1 Day 2 Days dilution 1 Day 2 Days Clindamycin 1:10 <0.05 < :1 SS SS 1:1,000 NG <0.05 1:10 NG NG 1:10,000 NG <0.05 1:100 NG NG Chloramphenicol 1: :1 SS SS 1: None SS SS 1:1,000 NG 0.8 1:10 NG NG 1:10,000 NG 0.8 1:100 NG NG Ampicillin 1: :1 SS SS 1: None SS SS 1:1,000 NG 0.4 1:10 NG NG 1:10,000 NG 0.4 1:100 NG NG Tetracycline 1: :1 SS SS 1:1,000 NG <0.05 1:10 NG NG 1:10,000 NG <0.05 1:100 NG NG a NG, No growth. For additional abbreviations see footnote a of Table 1. contained in the Schaedler broth. Tally et al. (18) have shown that clinically significant anaerobes can tolerate exposure to room air for far longer periods than necessary for preparing and inoculating the tubes in the tube test. The only organism that did not grow reliably in the tube test was B. melaninogenicus. We believe the problem was due to both an inadequate inoculum size and suboptimal anaerobiasis. Correction of either of these factors alone improved results, but neither was adequate since performance of the tube test in the anaerobic glove box or in the air with concentrated inocula did not yield satisfactory results. When both factors were corrected (as in the microdilution test that employed a more concentrated inoculum than was routinely used in the tube test with more fastidious organisms and which was performed entirely within the glove box), growth of B. melaninogenicus was consistently satisfactory. Wilkins and Thiel (20) have also found that dilutions of B. melaninogenicus inocula greater than 1:10 resulted in erratic growth. Rahimi et al. (9) reported a similar problem with B. melaninogenicus in an agar dilution method with three different media. Growth was sufficiently unreliable with inocula of 104 or 105 that the test could not be performed. The tube test further simplifies the basic broth dilution test by adopting a categorization approach to susceptibility testing in which two clinically significant concentrations of each antibiotic are used in individual glass tubes. This approach has been used by others for testing anaerobes (12) and has been advocated by Ericsson and Sherris and the World Health Organization-International Collaborative Study Group (4) as a practical approach to all in vitro susceptibility testing. In the tube test, we selected four antibiotics commonly used for treating anaerobic infections and chose the breakpoints we felt to be most useful. Concentrations of the antibiotics can be varied by changing either the number of drops delivered to each tube or the concentrations in the working solutions, and the test can presumably be performed with other antimicrobial agents. The ideal duration of incubation for reading susceptibility tests for anaerobic bacteria has never been determined. Because lag phases of growth may be as long as 18 to 24 h (13), a 2-day incubation period has been used to insure adequate time for cultures to become turbid. On the other hand, incubations of 18 to 24 h have been considered preferable because longer incubation has resulted in gradual increases in MICs (20). Although the microdilution and tube tests use the same medium and inocula, we believe that the optimal duration of incubation for reading results is 2 days for the former and 1 day for the latter. In the microdilution test, 1-day results were erratic with some strains; 4% of strains did not grow to turbidity, 9% of the MICs for those that did grow were two or more concentrations lower

9 452 FASS, PRIOR, AND ROTILIE than the 2-day MICs. In a previous study (10), 8% of strains did not grow by day 1 and 7% of the MICs for those that did grow were two or more concentrations lower than the 2-day MICs. Two-day MICs were reproducible, and unexpectedly high MICs after 2 days of incubation due to breakthrough growth did not seem to occur. Some evaporation from the wells in the microdilution plates occurred despite covering the plates and maintaining high humidity in the incubator, and this presumably resulted in progressive increases in antibiotic concentrations or other unfavorable growth conditions that prevented breakthrough growth with prolonged incubation. In the tube test, growth after 1 day of incubation was always detected except with B. melaninogenicus strains. Slight turbidity, which might not have been detectable in the microdilution plates where small volumes were used and reading was done through the plastic glove box, could be observed by slowly inverting the tubes, rolling the glass bead, and observing a swirl of organsims. Growth after 2 days of incubation was often easier to read, but 1-day results correlated better with 2-day microdilution MICs. The 2-day tube test results often indicated inappropriate resistance when correlated with 2-day microdilution MICs. The choice of 1-day readings for the tube test was not ideal, however, since seven of the eight results that varied significantly from 2-day microdilution MICs indicated erroneous susceptibility which became apparent after 2 days of incubation and subsequent comparison of results obtained by both tests. In three instances, these errors were considered to be serious since tetracycline-resistant strains of B. fragilis were categorized as susceptible by day 1. The tube test is a simple, rapid, and inexpensive method that clinical laboratories can use without special media or equipment for antimicrobial susceptibility testing of anaerobes. It is not perfect since there is no infallibly reliable incubation period after which results can be read, and the test needs to be modified for testing B. melaninogenicus strains. ACKNOWLEDGMENTS We- thank Beth Ann Bailey for her excellent technical assistance. LITERATURE CITED 1. Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turck Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45: Blazevic, D. J., and J. M. Matsen Susceptibility of anaerobic bacteria to carbenicillin. Antimicrob. Agents Chemother. 5: ANTIMICROB. AGENTS CHEMOTHER. 3. Bodner, S. J., M. G. Koenig, L. L. Treanor, and J. S. Goodman Antibiotic susceptibility testing of Bacteroides. Antimicrob. Agents Chemother. 2: Ericsson, H. M., and J. C. Sherris Antibiotic sensitivity testing: report of an international collaborative study. Acta Pathol. Microbiol. Scand. Sect. B. Suppl Fass, R. J., C. A. Rotilie, and R. B. Prior Interaction of clindamycin and gentamicin in vitro. Antimicrob. Agents Chemother. 6: Kwok, Y.-Y., F. P. Tally, V. L. Sutter, and S. M. Finegold Disk susceptibility testing of slowgrowing anaerobic bacteria. Antimicrob. Agents Chemother. 7: Metzler, C. M., and R. M. DeHaan Susceptibility tests of anaerobic bacteria: statistical and clinical considerations. J. Infect. Dis. 130: Overman, S. B., D. W. Lambe, Jr., and J. V. Bennett Proposed standardized method for testing and interpreting susceptibility of Bacteroides fragilis to tetracycline. Antimicrob. Agents Chemother. 5: Rahimi, A., W. J. Martin, and J. A. Washington II Effects of medium and inoculum on antimicrobial susceptibility of anaerobic bacteria. Am. J. Clin. Pathol. 62: Rotilie, C. A., R. J. Fass, R. B. Prior, and R. L. Perkins Microdilution technique for antimicrobial susceptibility testing of anaerobic bacteria. Antimicrob. Agents Chemother. 7: Sapico, F. L., Y.-Y. Kwok, V. L. Sutter,, and S. M. Finegold Standardized antimicrobial disc susceptibility testing of anaerobic bacteria: in vitro susceptibility of Clostridium perfringens to nine antibiotics. Antimicrob. Agents Chemother. 2: Stalons, D. R., and C. Thornsberry Broth-dilution method for determining the antibiotic susceptibility of anaerobic bacteria. Antimicrob. Agents Chemother. 7: Stalons, D. R., C. Thornsberry, and V. R. Dowell, Jr Effect of culture medium and carbon dioxide concentration on growth of anaerobic bacteria commonly encountered in clinical specimens. Appl. Microbiol. 27: Sutter, V. L., Y.-Y. Kwok, and S. M. Finegold Standardized antimicrobial disc susceptibility testing of anaerobic bacteria. I. Susceptibility of Bacteroides fragilis to tetracycline. Appl. Microbiol. 23: Sutter, V. L., Y.-Y. Kwok, and S. M. Finegold Susceptibility of Bactgroides fragilis to six antibiotics determined by standardized antimicrobial disc susceptibility testing. Antimicrob. Agents Chemother. 3: Sutter, V. L., and J. A. Washington II Susceptibility testing of anaerobes, p In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C. 17. Tally, F. P., A. Y. Armfield, V. R. Dowell, Jr., Y.-Y. Kwok, V. L. Sutter, and S. M. Finegold Susceptibility of Clostridium ramosum to antimicrobial agents. Antimicrob. Agents Chemother. 5: Tally, F. P., P. R. Stewart, V. L. Sutter, and J. E. Rosenblatt Oxygen tolerance of fresh clinical anaerobic bacteria. J. Clin. Microbiol. 1: Wilkins, T. D., L. V. Holdeman, I. J. Abramson, and W. E. C. Moore Standardized single-disc method for antibiotic susceptibility testing of anaerobic bacteria. Antimicrob. Agents Chemother. 1: Wilkins, T. D., and T. Thiel Modified broth-disk method for testing the antibiotic susceptibility of anaerobic bacteria. Antimicrob. Agents Chemother. 3: