Reproducibility of the Microdilution Checkerboard Method for ...

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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1993, P. 613-615 ... fourfold reduction in the MIC of both antibiotics in combi- nation, compared with ...
Vol. 37, No. 3

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1993, P. 613-615

0066-4804/93/030613-03$02.00/0 Copyright X) 1993, American Society for Microbiology

Reproducibility of the Microdilution Checkerboard Method for Antibiotic Synergy KENNETH H. RAND,1,2,3* HERBERT J. HOUCK,' PRINCE BROWN,1 AND DIANE BENNEWT4 Departments of Pathology and Laboratory Medicine' and Medicine,2 University of Florida, and Veterans Affairs Medical Center Gainesville,3 Gainesville, Florida 32610, and Alamar Biosciences, Sacramento, California 958344 Received 9 July 1992/Accepted 30 December 1992

We assessed the reproducibility of the microdilution checkerboard method for measuring antibiotic synergy. Five strains of Pseudomonas aeruginosa were tested with four antibiotic combinations by using 10 replicates each. Twenty-five percent of replicate sets gave discordant classification results (i.e., a 7:3 or worse split in categorization). Determination of the individual MICs of each antibiotic alone was excellent; all 10 replicates were within 1 twofold dilution for 95% of the 80 sets of 10 replicates. The microdilution checkerboard method either should not be used or should be used with at least five replicates per determination, with .80% agreement among the replicates required for classification.

The microdilution checkerboard has been one of the traditional methods for the measurement of antibiotic synergy. Synergy has generally been defined as requiring a fourfold reduction in the MIC of both antibiotics in combination, compared with each used alone, i.e., a fractional inhibitory concentration (FIC) index of c0.5 (5). Since there is an inherent 1-dilution variability in the determination of the MIC of each antibiotic used alone (8), this variability is additive and could become significant for the combination wells. We therefore decided to determine how much inherent variability occurs with microdilution checkerboard synergy testing under ideal conditions, namely, replicate microdilution plates inoculated on the same day with the same inoculum by as meticulous a technique as possible. We tested microdilution checkerboard plates prepared by two different methods, using five strains of Pseudomonas aeruginosa and four antibiotic combinations. (This work was presented at the 92nd General Meeting of the American Society for Microbiology, New Orleans, La., 26 to 30 May 1992.) ATCC 27853 and four laboratory strains of P. aeruginosa were studied. The following antibiotic powders were generously provided by their manufacturers: tobramycin (Lilly, Indianapolis, Ind.), aztreonam (Bristol-Myers Squibb, Bristol, Conn.), ceftazidime (Glaxo, Research Triangle Park, N.C.), piperacillin (Lederle, American Cyanamid Co., Pearl River, N.Y.), and ceftriaxone (Hoffmann-La Roche Inc., Nutley, N.J.). Recently, a new method for the microdilution determination of MICs in which disks containing appropriate concentrations of antibiotics and a proprietary colorimetric growth indicator are dispensed into microdilution wells has been reported. This method has shown .95% correlation with agar dilution MICs (4, 6). This method was used to prepare microdilution antibiotic synergy panels at Alamar Biosciences by adding the appropriate pairs of antibiotic disks to columns 1 to 11 and rows A to G in a microdilution plate. Column 12 and row H were reserved for the disks of each antibiotic alone to determine the MIC. *

Standard microdilution plates were prepared by one of us (D.B.), using Mueller-Hinton broth (Becton-Dickinson, Cockeysville, Md.) supplemented to 50 mg of Ca2" and 25 mg of Mg2+ per liter at Alamar Biosciences as described elsewhere (2, 7). These were shipped to the laboratory of one of us (K.H.R.) at the Veterans Affairs Medical Center Gainesville on dry ice and stored frozen at -70°C until used (within 2 weeks in all cases). The final volume was 0.1 ml per well containing 5 x 105 bacteria per ml for both methods. After 18 h of incubation at 36°C in a humidified non-CO2 incubator, plates were read by two observers. Colorimetric plates were read as recommended by the manufacturer; growth was considered present if there was any perceptible change of the blue color to red, however faint. Likewise, a well was considered positive or showing growth in the microdilution plates if even the faintest degree of turbidity was observed. Sometimes, instead of turbidity, tiny clumps that were clearly not present at higher antibiotic concentrations were observed, and these changes were considered to represent growth as well. Agreement between both observers was required, but there were no unresolved differences. The results are shown in Table 1, expressed as the number of replicates showing a majority/minority classification ratio. If 9 of 10 replicates showed synergy, it would be a 9:1 ratio. Alternatively, if 9 of 10 replicates showed no synergy, the agreement would also be 9:1. For the colorimetric method, agreement was 9:1 or better (i.e., only 1 or 0 of 10 replicates misclassified the strain) for 14 of 20 strain-antibiotic combinations. Six strain-antibiotic combinations showed a 7:3 or worse (6:4) discordance in classification. For the microdilution method, the corresponding figures were 12 of 20 showing 9:1 or better agreement among the 10 replicates, with 4 of 20 showing 7:3 or worse. There was no statistically significant difference between the colorimetric and microdilution results. Table 2 shows the data from Table 1 broken down for each method, strain, and specific antibiotic combination. The numbers in boldface type indicate that the colorimetric and microdilution methods for that strain and antibiotic combination were performed in the same test run with the same inoculum. We also compared the agreement in categorization be-

Corresponding author. 613

614

NOTES

ANTIMICROB. AGENTS CHEMOTHER. ratory (K.H.R.), overall results of agreement were, if anything, worse (data available upon request). Twofold variation in the determination of the MIC by either broth macro- or microdilution methods is considered acceptable variation (8). In a study by Ericsson and Sherris (3), MIC agreement was + 1 dilution of the mode for 94.8% of determinations. We achieved this degree of reproducibility for both the colorimetric and the microdilution methods. Exact MIC agreement was significantly better for the colorimetric method than for the microdilution method, and twofold variation in individual MIC determinations was achieved for 97.5 to 92.5% of sets of 10 replicates, respectively, for the colorimetric and microdilution checkerboard synergy plates. Despite this level of reproducibility, 20 to 30% of the sets yielded a 70:30 or worse split in categorical classification of antibiotic synergy. Even more disturbing was the observation that categorical agreement between these two methods was only 68.5% overall and only 71.4% even for those strains tested on the same day with the same inoculum. There are a number of potential methodological difficulties for the microdilution checkerboard method. Although microdilution equipment has an accuracy of measurement within 1 to 2%, there is no way to be certain that this degree of accuracy is achieved each time a particular pipet tip or dispensing step is used. The colorimetric method using standardized, rigidly quality controlled, commercially prepared antibiotic-impregnated disks probably minimizes variability in antibiotic concentration. Variability in the inoculum is certainly a major potential source of error. We controlled for this as well as possible by testing all replicates on the same day with the same inoculum. One measure of the success in controlling both antibiotic concentrations and inoculum is the reproducibility for the individual-antibiotic

TABLE 1. Distribution of discordant antibiotic synergy results No. of strain-antibiotic combinations with the

Method 10:0

Colorimetric Microdilution

10 10

indicated agreement:disagreement ratioa 9:1 8:2 7:3 6:4 or 5:5

4 2

0

5 2

4

1 2

a 10:0 means that all 10 replicates gave the same result, either synergy or no

synergy. A 9:1 ratio means that nine gave the same result and one was

discordant, and so on.

tween the colorimetric and microdilution methods. We used a simple majority to categorize a strain as showing synergy or not (at least 60% agreement among 10 replicates was required for classification). One strain was excluded because the microdilution result was 5:5 (strain U230, tobramycinaztreonam). Of 19 strain-antibiotic combinations, 13 (68.5%) were classified the same way by both the colorimetric and the microdilution methods. Even when the strain-antibiotic combination tests were performed in the same run with the same inoculum, there was agreement in classification between the colorimetric and microdilution methods for only 10 of 14 (71.4%). Thus, approximately 30% of strains were classified in opposite categories by the colorimetric and

microdilution methods. Determination of the MICs for each antibiotic used alone was highly reliable. For the colorimetric method, 39 of 40 sets (97.5%) of 10 individual MIC determinations were within a twofold range for all 10 replicates (for example, 8 or 16 ,g/ml as opposed to 8 ,g/ml + 1 dilution). Individual MICs determined by the microdilution method were within a twofold range for all 10 replicates in 37 of 40 determinations (92.5%). Overall, 368 of 398 individual antibiotic MIC determinations (92.5%) were in exact agreement for the colorimetric method, which was significantly higher than the 318 of 374 (85.0%) for the microdilution method (X2 = 10.0; P = 0.0015). There was no systematic pattern of error in the MIC determinations for any specific antibiotic, nor were there any systematic discrepancies or trends for the antibiotic combination wells. When the same strains and antibiotic combinations were tested with microdilution plates prepared in a second labo-

MIC determinations. Reader error is also another source of variability. All plates were read by two observers, and 100% agreement was achieved, albeit occasionally with some discussion. Although we did not undertake a systematic analysis of the between-reader discrepancies, it was clear that simply using either reader alone would not have changed the overall degree of variability observed. Finally, because of the small volume, microdilution methods are subject to error due to uneven evaporation, particularly from

TABLE 2. Replicate synergy testing of five strains of P. aeruginosa with four antibiotic combinations % of replicates of indicated strain showing synergya

combination

W17 U230 R70 W472 ATCC 27853 Colorimetric Microdilution Colorimetric Microdilution Colorimetric Microdilution Colorimetric Microdilution Colorimetric Microdilution

Tobramycin Ceftriaxone

100

100

0

20

10

60

10

30

100

0

Tobramycin Piperacillin

30

100"

0

0

0

14c

0

o0

0

0

Tobramycin Aztreonam

10

80

40

50

0

80

0

30

30

0

Amikacin Ceftazidime

70

20

30

0

30

0

0

10

10

0

a Each strain-antibiotic combination was tested with 10 replicate checkerboard plates unless indicated otherwise. Boldface numbers indicate that replicates were tested by both methods at the same time with the same inoculum. b Only five replicates were tested. c Only seven replicates were tested.

VOL. 37, 1993

the outermost wells. In the course of the experiments, we did not observe any problem from evaporation. The reproducibility of the individual-antibiotic MIC determinations suggests that evaporation was not a significant source of variability, since the individual antibiotics were always used in the outermost wells. Amsterdam (1) distinguishes between synergy and "marked" synergy in his discussion of checkerboard methodology. None of our strains of P. aeruginosa exhibited marked synergy, i.e., eightfold reductions in the MICs of antibiotics used in combination compared with the MICs of the antibiotics used alone (FIC index, .0.25). If one were to make the definition of antibiotic synergy more stringent, e.g., an FIC index of