Comparison of spiral gradient endpoint and agar dilution methods for ...

JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1996, p. 170–174 0095-1137/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 34, No. 1

Comparison of Spiral Gradient Endpoint and Agar Dilution Methods for Susceptibility Testing of Anaerobic Bacteria: a Multilaboratory Collaborative Evaluation HANNAH M. WEXLER,1,2* ERIC MOLITORIS,2 PATRICK R. MURRAY,3 JOHN WASHINGTON,4 RONALD J. ZABRANSKY,5 PAUL H. EDELSTEIN,6 AND SYDNEY M. FINEGOLD1,2,7,8 Medical7 and Research1 Services, Veterans Affairs Medical Center West Los Angeles, and Departments of Medicine2 and Microbiology and Immunology,8 School of Medicine, University of California, Los Angeles, Los Angeles, California; Department of Pathology, Washington University School of Medicine, St. Louis, Missouri3; Department of Clinical Pathology, The Cleveland Clinic Foundation, Cleveland, Ohio4; Department of Clinical Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas5; and Department of Pathology and Laboratory Medicine and Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania6 Received 12 December 1994/Returned for modification 14 December 1994/Accepted 2 October 1995

A multilaboratory collaborative study was carried out to assess the utility of the spiral gradient endpoint (SGE) method for the determination of the antimicrobial susceptibilities of anaerobes and to evaluate the equivalence of the MICs obtained by the SGE method with those obtained by the reference agar dilution method of the National Committee for Clinical Laboratory Standards. The standard deviation of the MIC obtained by the SGE method for the five participating laboratories was 60.26 of a twofold dilution, whereas it was 61 twofold dilution by the reference method. The interlaboratory reproducibility of the results for two control strains tested with imipenem, chloramphenicol, and metronidazole indicated that 96% of the measurements fell within 61 twofold dilution of the mode. The equivalence of the SGE method with the agar dilution method was assessed with a wide variety of anaerobic organisms. The MICs by both methods were within 1 doubling dilution in 93% of the measurements (n 5 1,074). Discrepancies generally occurred with those organism-drug combinations that resulted in tailing endpoints (Fusobacterium nucleatum, 86% agreement) or in cases of light growth (Peptostreptococcus spp., 86% agreement). obes, evaluate the equivalence of the MICs obtained by the SGE method (SGE MICs) with those obtained by the reference agar dilution method, and assess the causes for the differences between them. Phase 1 of the analysis measured the intralaboratory repeatability and the interlaboratory reproducibility of the method, and phase 2 assessed the equivalence of the SGE and agar dilution methods.

The necessity of performing susceptibility tests on anaerobic isolates has been reviewed (3, 13), and revised recommendations have recently been published by the National Committee for Clinical Laboratory Standards (NCCLS) (6). The recommendations outline the circumstances (i.e., patient status, type of infection, and method of treatment) which warrant susceptibility testing. Because commonly isolated pathogenic anaerobes may have unpredictable susceptibility patterns, the resistance patterns of sufficient numbers of anaerobes should be periodically monitored to detect trends in the development of resistance. While laboratories may use the NCCLS reference agar dilution method, this method is time- and labor-intensive, and the inherent 61 dilution error results in ambiguous results for a large proportion of the isolates normally tested. The spiral plater (Spiral Biotech, Bethesda, Md.) has been used for bacterial enumeration for almost two decades (2), and the use of the spiral gradient endpoint (SGE) method for determining MICs was introduced in 1990 (4, 7). The antibiotic concentration gradient on one plate typically spans 8 twofold dilutions, thus considerably minimizing the time and materials needed to perform the test (compared with those required by the NCCLS reference agar dilution method). The purpose of the multilaboratory collaborative study described here was to assess the utility of the SGE method for the determination of the antimicrobial susceptibilities of anaer-

MATERIALS AND METHODS Organisms used. (i) Phase 1. Control strains (Bacteroides fragilis ATCC 25285 and Bacteroides thetaiotaomicron ATCC 29741) were purchased and sent to each laboratory to eliminate any possibility that the laboratories’ own quality control strains had developed variations. Five laboratories (laboratories A to E) participated in the study. (ii) Phase 2. Laboratory C was not able to participate in the study; four laboratories (laboratories A, B, D, and E) participated, and laboratory A tested the isolates originally designated for laboratory C. Each laboratory tested 40 clinical isolates. In some cases, strains were sent from the Wadsworth Anaerobe Laboratories (Veterans Affairs Medical Center, West Los Angeles) to the testing laboratory (if that laboratory did not have sufficient strains of the particular species). Bacteria were identified by established procedures (9). Preparation of inocula. (i) Phase 1. The inoculum was prepared in accordance with NCCLS approved standard M11-A2 (6) by using overnight incubation of colonies transferred from blood agar plates (the direct suspension method was not used to avoid differences that may be due to the selection of colonies of different sizes by the different laboratories). (ii) Phase 2. The inoculum for each isolate was prepared by either of the alternatives of NCCLS approved standard M11-A2 (6); the density was adjusted to match that of a 0.5 McFarland barium sulfate standard and was used for both SGE and agar dilution plates. Media. (i) Phase 1: repeatability (within the same laboratory), reproducibility (between laboratories), and presence of systematic errors (bias) in SGE endpoint measurements. Plates (diameter, 150 mm) prepared from the same lot of medium (brucella laked-blood agar [9]) by Anaerobe Systems (San Jose, Calif.) were provided to all participating laboratories.

* Corresponding author. Mailing address: Microbial Diseases Research Laboratory, Bldg. 304, Room E3-224, VA Wadsworth Medical Center 691/151J, Los Angeles, CA 90073. Phone: (310) 268-3404. Fax: (310) 268-4646. 170

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(ii) Phase 2: equivalence of the SGE and agar dilution methods. Plates were prepared by the participating laboratories as specified in NCCLS publication M11-A2 (6). Either the reference (Wilkins Chalgren medium to which blood was added) or the Wadsworth (brucella base laked blood) agars could be used. Each laboratory used the same lot of medium for the two methods. Antimicrobial agents and antimicrobial stock preparation. Antimicrobial agents were obtained as powders from their respective manufacturers, as follows: cefoxitin and imipenem, Merck Sharp & Dohme (Rahway, N.J.); metronidazole, Searle Laboratories (Chicago, Ill.); clindamycin, Upjohn (Kalamazoo, Mich.); ampicillin-sulbactam, Pfizer Laboratories (New York, N.Y.); ceftizoxime, Fujisawa (Philadelphia, Pa.); and piperacillin and ticarcillin-clavulanic acid, American Cyanamid (Pearl River, N.Y.). Powders were divided into aliquots at the Wadsworth Anaerobe Laboratories and were sent to the participating laboratories. Identical lots were used by all participating laboratories. (i) Phase 1. Two different stock concentrations for the same antibiotic-control strain combination were used for the SGE plates to locate the minimum activity concentration (MAC) at radii of approximately 30 and 50 mm. Imipenem, metronidazole, and clindamycin were tested. Each laboratory was told the stock concentrations that were necessary and the expected endpoint locations. Each control strain was inoculated into locations 1 through 7 and 9 through 15; these ‘‘paired streaks’’ (1 and 9, 2 and 10, etc.) provided seven pairs of diametrically opposite measurements on each plate. Duplicate plates were made, resulting in 840 determinations from paired streaks (5 laboratories 3 7 radii 3 24 plates [2 control strains 3 2 plates each 3 2 concentrations 3 3 antimicrobial agents]). If single radii, as opposed to paired streaks, were measured, 1,680 measurements were made. Level plates, i.e., those with uniform agar depths, were defined as those plates on which duplicate streaks (on opposite sides of the plate) had endpoint radii within 3 mm of each other. (ii) Phase 2. Ticarcillin-clavulanate, imipenem, metronidazole, cefoxitin, clindamycin, ceftizoxime, piperacillin, and ampicillin-sulbactam (2:1) were tested. Separate stock solutions were prepared for the agar dilution and SGE procedures. Two SGE plates were required to test the same range of concentrations tested by the agar dilution procedure. The lower-concentration stock solution was made by diluting the higher-concentration stock solution. To minimize errors, dilutions did not exceed 100:1, and the minimum volume transferred was at least 10% of the total pipette volume. Determination of MICs. The conventional incremental agar dilution technique was performed as described previously (6). The spiral plater (model DU), radial replicator, templates for measurement, and computer software were provided by Spiral Biotech (Bethesda, Md.) and were used according to the manufacturer’s instructions. The plates were incubated in GasPak jars (BBL Microbiology Systems, Cockeysville, Md.) or in anaerobic chambers (Anaerobe Systems or Coy Manufacturing, Ann Arbor, Mich.) for 48 h at 378C. Anaerobic and aerobic antibiotic-free control plates were inoculated at the start and end of the inoculation sequence. In phase 2, parallel SGE and standard agar dilution tests were conducted. Determination of endpoints. The instructions sent to participating laboratories included a copy of the NCCLS criterion for endpoint reading that recommends reading the endpoint at that concentration at which there is a ‘‘marked change’’ in the appearance of growth compared with the growth on the control plate. The marked change might be a change to no growth or lighter growth, a haze, multiple tiny colonies, or one to several normal-size colonies. For certain organism-antimicrobial agent combinations at least, this point is correlated with the viability of the organisms (5, 10). SGEs were measured as the radius from the center of the plate to the endpoint of growth. The radius at which growth ends is termed a ‘‘tail ending radius’’ (Fig. 1). The point at which a heavy, confluent line of growth became much less dense (i.e., a ‘‘marked’’ change from the control growth) was called a ‘‘tail beginning radius’’. The radial measurements were entered into a software program provided by the manufacturer; the program uses the molecular weight and diffusion characteristics of the antimicrobial agent to calculate the corresponding concentration. The concentration corresponding to the tail beginning radius is referred to as MAC. The MIC was the next highest twofold dilution greater than or equal to the MAC. If both the tail ending radius and the tail beginning radius were entered into the database, the tail beginning radius was used to calculate the MIC.

FIG. 1. Representation of SGE endpoint measurements. TBR, tail beginning radius; TER, tail ending radius; OR, outlier colony radius.

82% for single streaks and from 71 to 88% if the average of the paired streaks was measured (which should compensate to some degree for nonlevel plates). Level plates, i.e., those with uniform agar depths, were defined as those plates on which duplicate streaks (on opposite sides of the plate) had endpoint radii within 3 mm of each other. The standard deviations of MICs for the control strain in laboratories B and E (0.36 [58%] and 0.25 [63%] level plates, respectively) were higher than those in laboratory A (0.19, 75% level plates), laboratory C (0.20, 75% level plates), and laboratory D (0.20, 77% level plates). The average standard deviation of MAC was 60.26 (of a twofold dilution); the inherent error in the agar dilution technique is 61 twofold dilution. (ii) Interlaboratory reproducibility. For the two control strains, MICs were equal to the modal value for 58% of the values measured, and 96% of the values fell within 61 twofold dilution of the mode (Table 2). Phase 2: equivalence of the SGE and the agar dilution techniques. Tables 3 and 4 list the concordance values of the four participating laboratories for each antimicrobial agent. Offscale data (i.e., when the agar dilution or SGE method yielded MICs outside the range of concentrations tested) were elimi-

TABLE 1. Intralaboratory repeatability of data for control strains

Laboratory

% Level platesa

RESULTS Phase 1. (i) Intralaboratory repeatability. The antimicrobial agents were chosen to span the range of molecular weights of most commonly tested agents: metronidazole (molecular weight, 171), imipenem (molecular weight, 317), and clindamycin (molecular weight, 461). Intralaboratory repeatability was measured by determining the percentage of MICs equal to the modal MIC (Table 1). We found that two factors could increase the repeatability of the SGE MIC: use of the average of duplicate paired streaks and use of level plates. The proportion of MICs equal to the modal MICs ranged from 67 to

171

% MICs equal to modal MICs Single streak

Duplicate streak (avg)c

% MICs (SD of MACb) within 61 twofold dilution of mode MIC

A B C D E

75 58 75 77 63

79 63 82 68 67

88 65 86 77 71

100 (0.19) 100 (0.36) 100 (0.20) 100 (0.20) 95 (0.25)

All

69

72

77

99 (0.26)

a Percentage of plates on which the duplicate streaks had a tail beginning radius difference of ,3 mm. b Standard deviation of MAC in ln2 (twofold dilution) units. c On the basis of duplicate streak analyses.

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WEXLER ET AL. TABLE 2. Interlaboratory reproducibility of MICs for control strains Antimicrobial agent

Organism

No. of determinations

Mode MIC (mg/ml)

No. (%) of values within 2 dilutions of mode 22

21

Mode

11

Metronidazole

B. fragilis B. thetaiotaomicron

140 140

0.5 1.0

23 (16.4) 101 (72.1) 38 (27.1) 66 (47.1)

16 (11.4) 36 (25.7)

Imipenem

B. fragilis B. thetaiotaomicron

140 126

0.0312 0.125

8 (5.7) 11 (7.9)

70 (50) 106 (75.7)

62 (44.3) 9 (6.4)

Clindamycin

B. fragilis B. thetaiotaomicron

140 140

0.5 1

27 (19.3) 3 (2.1)

66 (47.1) 74 (52.9)

Total

826

Percent Percent within 6 1 twofold dilution of mode

nated from the analysis, since a comparison of the two methods was not possible. Data are organized by antimicrobial agent and participating laboratory in Table 3 and by organism group in Table 4. The MICs were within 1 doubling dilution in 93% of the comparisons (n 5 1,074). For laboratory B, concordance values for clindamycin and metronidazole were extremely low; repeat testing of certain strains confirmed that technical errors were the cause (a dilution error in the case of metronidazole and a reading error [referred to below] in the case of clindamycin). This laboratory’s data (marked as NI in Table 3) for these two agents was deleted from the analysis since technical errors (i.e., not the method itself) were responsible for the low concordance. Concordance was poorer than expected for laboratory E (see footnote a of Table 3). Upon review of the data, it became clear that the criteria for reading the SGE and agar dilution plates were not equivalent. Fortunately, the agar dilution data were recorded in a manner that allowed one of us (H.M.W.) to essentially reread the plates by reading the data sheets (without any knowledge of the SGE data for those strains) and using the endpoint criterion of ‘‘a marked change from the control growth.’’ The data in Table 3 reflect those for both the original and the reread plates. The data in Table 4 support our earlier comments (5, 11, 12) about the difficulty of determining endpoints with certain antimicrobial agent-organism combinations and the observation that discrepancies generally occurred with those organismdrug combinations that resulted in tailing endpoints (Fusobacterium nucleatum, 86% agreement) or in cases of light growth (Peptostreptococcus spp., 86% agreement). As in earlier studies, the b-lactam agents (particularly ceftizoxime) were the most likely to result in tailing endpoints (12). DISCUSSION The approved techniques of NCCLS for susceptibility testing of anaerobes include tests by the agar dilution, broth microdilution, and broth macrodilution methods (6). The broth macrodilution method is quite cumbersome and is used infrequently. While practical, the broth microdilution technique will not support the growth of some fastidious anaerobes. The agar dilution test will support the growth of most clinically relevant anaerobes, but it is labor-intensive and the inherent error of the test results in a high degree of ambiguous results. For many anaerobes, particularly the B. fragilis group, MICs of many

10 10 1.2

110 13.3

483 58.5 96.1

12

33 (23.6) 4 (2.9) 45 (32.1) 18 (12.9) 201 24.3

22 2.7

antibiotics cluster near the breakpoint. Thus, the inherent twofold dilution error may result in the same organism being called variably susceptible or resistant when it is tested on different occasions. This clustering is a characteristic of the organism-drug interaction and is present to some degree in every technique. However, the greater precision of the SGE test in determining MICs (in our study, the standard deviation was 60.26 of a twofold dilution) reduces the percentage of strains within this population. Recent studies found the overall agreement between the two techniques (within 1 doubling dilution) to be .90% (4, 15), which is also confirmed by the present collaborative study. Data from those earlier studies were used to develop a diffusion correction protocol to be used in determining the concentrations of antimicrobial agents along the gradient. In the present study, we confirmed the validity of this correction by testing two different stock concentrations of the antimicrobial agents used in phase 1 (which span the range of molecular weights of most antimicrobial agents) intended to result in radii of 30 and 50 mm, respectively. The two concentrations gave equivalent results, confirming the validity of the correction in the SGE analysis (unpublished data). Some sources of error that occur in traditional susceptibility testing were highlighted by the present study. In phase 1, the MICs for control strains tended to be on the low end of the approved quality control range and, in one case, were below the acceptable range. The inoculum for phase 1 was prepared from an overnight broth culture; the culture was at an earlier stage of growth than the inoculum normally used in agar dilution testing. These same strains were well within the NCCLS quality control range during phase 2 (which used the normal inoculum). We confirmed that MICs for log-phase inocula were typically lower (unpublished data). Other errors included a 10-fold dilution error, ‘‘skipped’’ inoculum spots on the agar dilution plates (i.e., growth at 0.25 mg/ml, no growth from 0.5 to 4 mg/ml, growth at 8 mg/ml, and the MIC was recorded as 0.5 mg/ml), and inconsistent application of the criterion for reading endpoints. As mentioned above, when the reread plates from laboratory E were compared with the SGE MICs, the concordance improved dramatically (from 73.8 to 92.9%), giving credence to the general sense among many microbiologists that interpretative criteria are highly subjective. Laboratory D had the highest level of concordance between the two methods. All plates in laboratory D were read against

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TABLE 3. Gradient to incremental (agar dilution) MIC comparison Percent within 61 twofold dilution (no. of strains tested)

Antimicrobial agent Laboratory A

Ampicillin-sulbactam Cefoxitin Ceftizoxime Clindamycin Imipenem Metronidazole Piperacillin Ticarcillin-clavulanic acid

97.6 (41) 95.5 (66) 91.2 (34) 97.2 (36) 87.0 (46) 100 (52) 92.5 (53) 87.9 (33)

Percent within 1 twofold dilution

93.9 (339/361)

Laboratory B

Laboratory D

91.3 (23) 80.0 (40) 85.2 (27) NIb 80.8 (52) NI 96.8 (31) 89.5 (19)

95.0 (40) 97.6 (42) 97.3 (37) 100 (38) 90.0 (40) 100.0 (30) 97.0 (33) 94.4 (36)

85.9 (165/192)

96.3 (285/296)

Laboratory E

100 (23) 57.9 (38) 75.8 (29) 59.4 (32) 78.4 (37) 74.1 (31) 80.7 (31) 72.2 (18) 73.8 (177/239)

Laboratory Ea

100 (25) 94.0 (33) 89.3 (28) 92.9 (28) 91.0 (33) 82.1 (28) 100 (32) 94.4 (18)

All laboratories

96 (124/129) 92 (167/181) 91 (115/126) 97 (99/102) 87 (148/171) 96 (105/110) 96 (143/149) 91.5 (97/106)

92.9 (209/225)

93 (998/1074)

a

Values are the ‘‘corrected’’ values when the plates were reevaluated by rereading the actual agar dilution data sheets (without any knowledge of the SGE data for those strains) by using the current NCCLS endpoint criterion (see Discussion). Totals are calculated by using the corrected values. b NI, not included in the data analysis because of technical errors in the laboratory.

a dark background, which minimizes the appearance of tailing, and few tail entries were recorded in the data set. Concordance levels were high even for the difficult organisms (e.g., 95% for Fusobacterium mortiferum-Fusobacterium varium versus 79, 80, and 91% for the other laboratories). Partly on the basis of these results, the NCCLS protocol now recommends that agar dilution plates be read against a dark background (6). The problems that occurred with the SGE method included a mechanical error (the stylus on the SGE plater in one laboratory was bent and deposited the antimicrobial solution in an off-center spiral; this will be avoided with the newer model of the plater). Our data also underscored the importance of using level plates for the SGE method. In other words, it is important that the agar depth be even throughout the plate (agar depth is one of the variables entered into the SGE software and is used to calculate the concentration at any point in the gradient). For the present study, level plates were defined as those in which the duplicate streaks (at opposite ends of the plate) have a tail beginning radius difference of ,3 mm. In an unpublished study from the Wadsworth Anaerobe Laboratories, the MICs of five antimicrobial agents were obtained by the SGE method for the NCCLS controls strains by using (i) plates poured in the laboratory (97% level) and (ii) commercially available plates (45% level). The percentage of MICs equal to the modal MIC (a measure of repeatability) was significantly greater (85%) for the laboratory-poured plates than for the commercially obtained plates (59%). New methods are generally evaluated in terms of error rates

vis-a`-vis the standard method. This may be inappropriate in the case of the reference agar dilution method for anaerobes, which is not sufficiently accurate or reproducible to be used in such comparisons. We have written extensively about the problems involved in interpreting the results obtained by the standard method (11, 12). A significant percentage of strain-antimicrobial agent combinations yield endpoints which are difficult to pinpoint. Most troubling is that the inherent error in the reference method (61 twofold dilution [14]) combined with the clustering of many MICs about the breakpoint results in a very significant percentage of isolates whose categorical placement (i.e., susceptible or resistant) is subject to day-today variability. The lower inherent error in the SGE method (60.26 of a twofold dilution) necessarily means that the percentage of isolates with questionable categorical placements is reduced. It would be ideal if the MICs obtained by all methods could be compared with an MIC defined only by the organismantimicrobial agent interaction (i.e., not method dependent). If an absolute MIC standard could be described (perhaps by using kill curves [8] with a low initial inoculum concentration to obtain a more homogeneous cell population), the MICs obtained by all other methods could then be evaluated relative to this MIC standard. Such a comparison would be markedly superior to one based on a standard (i.e., the agar dilution reference method) which has such a high degree of inherent error. In any case, the MICs obtained by the SGE and agar dilution methods were within 1 doubling dilution (i.e., essential agreement) in 93% of comparisons (the U.S. Food and Drug

TABLE 4. Comparison of gradient with incremental agar dilution MICs for different groups of anaerobic organisms % within 61 twofold dilution (no. of strains) Group

Ampicillinsulbactam

Cefoxitin

Ceftizoxime

Clindamycin

Imipenem

Metronidazole

Piperacillin

Ticarcillinclavulanate

All

B. fragilis B. thetaiotaomicron-B. ovatus F. nucleatum F. mortiferum-F. varium Porphyromonas spp. Peptostreptococcus spp. Clostridium spp. GPRa Total

97 (34) 91 (33) 97 (33) 92 (39) 88 (8) 73 (11) 94 (18) 92 (25) 100 (5) 100 (5) 100 (4) 83 (6) 100 (19) 100 (22) 88 (8) 97 (30) 96 (129) 92 (181)

89 (35) 93 (30) 80 (5) 79 (19) 100 (5) 50 (2) 100 (15) 100 (16) 90 (127)

100 (25) 100 (21) 100 (10) 89.5 (19) 100 (1) 100 (1) 100 (16) 89 (9) 97 (102)

83 (47) 89 (44) 80 (10) 87 (31) 86 (7) 100 (2) 83 (18) 100 (12) 86 (171)

94 (32) 100 (30) 100 (1) 92 (13) 71 (7) 100 (3) 100 (17) 100 (7) 96 (110)

100 (38) 92 (37) 100 (4) 92 (24) 100 (8) 100 (3) 94 (16) 100 (19) 96 (149)

93 (28) 91 (35) 100 (1) 100 (17) 50 (2) 0 (1) 90 (21) 100 (1) 92 (106)

93 (282) 94 (269) 86 (49) 90 (166) 90 (40) 86 (22) 96 (144) 97 (102) 93 (1,074)

Control strains

100 (69)

87 (90)

94 (70)

80 (98)

97 (68)

88 (95)

81 (64)

a

84 (93)

GPR, gram-positive rods; includes Propionibacterium, Eubacterium, and Actinomyces species.

88 (647)

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WEXLER ET AL.

Administration generally requires .90% essential agreement with a standard method). The initial expense for the equipment needed for the SGE method is high, but this method is not designed for testing small numbers of isolates in a clinical laboratory. The Etest, another gradient method, also has the advantage of using agar and would be a more appropriate alternative in this setting (1). The SGE method is a more economical and more precise alternative (in terms of reduced inherent error) to the reference agar dilution technique for both research purposes and batch testing (in a reference laboratory). Also, more information may be gained about the transition from growth to no growth. Another setting in which the SGE method may prove to be quite useful is in calibrating disk diffusion measurements since both techniques are based on continuous diffusion (8). ACKNOWLEDGMENTS The technical assistance of Martha A. C. Edelstein, Mei Yu, Cindy Knapp, Ann Niles, and Lourdes Bayola-Mueller is gratefully acknowledged. This work was supported in part by Veterans Affairs Medical Research Funds and in part by Pfizer Pharmaceuticals (New York, N.Y.), Spiral Biotech (Bethesda, Md.), and SmithKline Beecham (Philadelphia, Pa.). REFERENCES 1. Citron, D. M., M. I. Ostavari, A. Karlsson, and E. J. C. Goldstein. 1991. Evaluation of the epsilometer (E-test) for susceptibility testing of anaerobic bacteria. J. Clin. Microbiol. 29:2197–2203. 2. Donnelly, C. B., J. E. Gilchrist, J. T. Peeler, and J. E. Campbell. 1976. Spiral plate count method for the examination of raw and pasteurized milk. Appl. Environ. Microbiol. 32:21–27.

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