Spiral Gradient Endpoint Method Compared to Standard Agar Dilution for Susceptibility Testing of Anaerobic Gram-Negative Bacilli

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, May 1991, p /91/ $02.00/0 Copyright 1991, American Society for Microbiology Vol. 29, No. 5 Spiral Gradient Endpoint Method Compared to Standard Agar Dilution for Susceptibility Testing of Anaerobic Gram-Negative Bacilli GALE B. HILL Departments of Obstetrics & Gynecology and Microbiology & Immunology and Anaerobe Section, Clinical Microbiology Laboratory, Duke University Medical Center, Durham, North Carolina Received 3 December 1990/Accepted 30 January 1991 More efficient and reproducible alternative methods of performing agar dilution susceptibility testing are desirable, particularly for anaerobic bacteria. Anaerobes generally grow more reliably on solid media than they do in broth microdilution wells. A new method, the revised spiral gradient endpoint (SGE) method, was evaluated against the standard agar dilution (SAD) method by using a wide variety of anaerobic gram-negative bacilli (161 strains) and eight antimicrobial agents. For the SGE method, a spiral plater was used to set up a concentration gradient of an antimicrobial agent within an agar plate across which bacterial strains were inoculated as radial streaks. After incubation, the MIC of the antimicrobial agent was calculated from the radial endpoint location where bacterial growth ceased along the streak. The MICs for 90% of strains tested (in micrograms per milliliter) and the cumulative percentages of susceptible strains at the breakpoints for the SGE and SAD methods, respectively, and for afl 161 strains were as follows: for metronidazole, 2 and 100 versus 2 and 100; for imipenem, 1 and 99 versus 0.5 and 98; for ampicillin-sulbactam, 8 and 97 versus 8 and 98; for clindamycin, 4 and 90 versus 4 and 91; for cefoxitin, 32 and 95 versus 32 and 95; for mezlocillin, 256 and 88 versus >128 and 86; for ampicillin,.256 and 51 versus >64 and 51; and for penicillin (in units per milliliter),.512 and 71 versus >64 and 65. The excellent agreement of these data and the greater sensitivity, reproducibility, and efficiency of the revised SGE method warrant further evaluations. Assuming that these advantages are confirmed, the revised SGE method should be a useful alternative test method when detailed susceptibility data are desired. Antimicrobial susceptibility testing of anaerobes was discussed in a recent review (4). Differences in testing methodologies affect results, particularly for certain bacterial species and drug combinations. There are certain advantages to testing anaerobic bacteria on solid media by agar dilution rather than microbroth dilution. Yet, the standard procedure for agar dilution testing requires considerable expenditure of technical time and materials. Additional information and methods are needed to improve the efficiency, reproducibility, and reliability of susceptibility testing for anaerobes. This report describes the results of an evaluation of a new agar dilution method, the revised spiral gradient endpoint (SGE) method (6, 11). In the SGE method it is possible to circumvent the need for preparation of serial dilutions and incremental plates (11, 12). A spiral plater (11) is used to deposit a known concentration of an antimicrobial agent onto the surface of an agar plate in a precise spiral pattern of increasing dilution of the compound from near the center of the plate to the periphery, thus setting up a concentration gradient. Bacteria then are inoculated as radial streaks onto the surface of the plate across (perpendicular to) the concentration gradient of the antimicrobial agent in the medium. Following incubation, growth of the bacteria ceases along the radial streak if the bacteriostatic or bactericidal concentration of the antimicrobial agent is reached. This drug concentration can be calculated from the radial location of the growth endpoint based on mathematical formulas that consider the volume and concentration of drug deposited at that position on the plate plus the diffusion characteristics of the drug. A single 15-cm agar plate accommodates testing of up to 17 bacterial strains over a concentration range of an antimicrobial agent covering up to eight twofold dilutions (6, 11). The application of the spiral plater for performing antimicrobial susceptibility testing for anaerobes required additional development and standardization beyond that proposed in a preliminary users' guide (6, 11). As reported here, MIC results determined by the revised SGE method correlated very well with those obtained by a standard agar dilution (SAD) method, a reference test of the National Committee for Clinical Laboratory Standards for anaerobes (9). A wide variety of species of anaerobic gram-negative bacilli and different classes of antimicrobial agents were tested. The SGE method was more efficient, reproducible, and sensitive than the SAD method. MATERIALS AND METHODS Microorganisms. One hundred sixty-one anaerobic gramnegative bacilli were tested; the species groups and species consisted of the Bacteroides fragilis group (70 strains, including 36 Bacteroides fragilis, 12 Bacteroides ovatus, 11 Bacteroides thetaiotaomicron, 7 Bacteroides vulgatus, and 4 Bacteroides distasonis), the black pigmenting group (15 strain including -6 species of Bacteroides and Porphyromonas), Bacteroides bivius (16 strains), all other species of Bacteroides (54 strains encompassing -12 species and including strains among Bacteroides uniformis, Bacteroides eggerthii, and Bacteroides caccae), and Fusobacterium strains (6 strains including 4 species). These 161 strains were isolated from clinical specimens collected from appropriate sites and submitted to the Anaerobe Section, Clinical Microbiology Laboratory, Duke University Medical Center. Two 975

2 976 HILL quality control strains, B. fragilis ATCC and B. thetaiotaomicron ATCC 29741, were included for susceptibility testing. The no. 0.5 McFarland standard inocula for susceptibility testing were prepared from growth on supplemented brucella blood agar. Other details of the methods for bacterial cultivation, identification, and preparation for susceptibility testing have been described previously (6, 9). Antimicrobial agents. Eight antimicrobial agents were selected for this evaluation of the SGE method on the basis of the following considerations, among others: applicability to treatment of anaerobic infections, representation of a class of compounds (macrolide, nitroimidazole, beta-lactam, combinations with a P-lactamase inhibitor), and drugs for which MICs for quality control strains were published. The following antimicrobial agents were obtained as laboratory standard powders from the indicated sources: ampicillin and sulbactam, Pfizer Pharmaceuticals, Groton, Conn.; cefoxitin and imipenem, Merck Sharp & Dohme, Rahway, N.J.; mezlocillin, Miles Pharmaceuticals, West Haven, Conn.; metronidazole, G. D. Searle & Co., Skokie, Ill.; penicillin G, Lilly Research Laboratories, Indianapolis, Ind.; and clindamycin, The Upjohn Co., Kalamazoo, Mich. The dry powders were stored, diluted, and used within the time period recommended by the manufacturer. plus sulbactam was tested in a constant 2:1 concentration ratio. Other details of the preparation and handling of these antimicrobial agents for susceptibility testing have been described previously (6, 9). Susceptibility tests. The revised SGE method for susceptibility testing was simultaneously performed along with a reference agar dilution method (9) for sets of bacterial strains and all antimicrobial agents. Five percent lysed sheep blood was added to Wilkins-Chalgren agar (Difco Laboratories, Detroit, Mich.), to permit susceptibility testing of more fastidious strains. A summary of the evaluation protocol and the revised SGE method follows. Approximately 7 h prior to the inoculation of bacteria, antimicrobial stock solutions were deposited onto 15-cm-diameter plates by using a model DU spiral plater (Spiral System Instruments, Bethesda, Md.). These plates were returned to an anaerobic glove box (Coy Laboratory Products, Inc., Ann Arbor, Mich.), where the plates remained at room temperature until their use. Soon after deposition, a concentration gradient of the antimicrobial agent was established within the media. Inocula for susceptibility testing were prepared (turbidity to match a no. 0.5 McFarland standard) just prior to testing and then were divided so that the same inoculum suspensions of all strains were set up simultaneously for testing by the SAD and SGE methods. A prototype SGE inoculum replicator device (courtesy of Spiral System Instruments, Inc.) was used to replicate the inocula onto sets of the antimicrobial gradient plates (6). Seventeen troughs in a metal plate held the inocula, which were transferred by means of 17 corresponding metal fins on a replicator head which could be repeatedly lowered into the troughs and stamped onto fresh plates, analogous to the well arrangement of a Steers replicator. Fifteen test strains and two quality control strains were inoculated onto each agar plate as radial streaks across the concentration gradient of the antimicrobial compound. Quality control plates not containing antibiotic likewise were stamped with inocula and were incubated anaerobically and aerobically to detect contamination and to ensure that adequate confluent growth occurred along the streak of each test and control strain. After the inocula were absorbed into the surface of the agar plates, the plates were inverted and incubated anaerobically within an anaerobic glove box. The J. CLIN. MICROBIOL. plates for the SAD method, which were inoculated by using a Steers replicator within another glove box, were handled similarly. The sets of SGE and SAD plates were read independently by two different readers for MIC determinations after 48 h of incubation at 37 C. The endpoint of growth for the SGE test was chosen to correspond with the SAD endpoint definition, i.e., no growth, one discrete colony, or only a fine, barely visible haze remaining (9). One colony that was well separated from the zone of continuous growth along the streak was considered an outlier. The radial location of the growth endpoint from the center of the plate (spiral) was measured in millimeters by using Vernier calipers, and values between 20 and 60 mm were accepted for computation of the antimicrobial agent concentration at that location by revised formulas (6). The MIC results obtained by the revised SGE method were directly compared with those generated from the reference SAD tests. Additional details of SAD and SGE testing have been described previously (6, 9, 11). The results of testing by the SAD and revised SGE methods are given as the MIC for 50% of strains tested (MIC50), the MIC for 90% of strains tested (MIC0), and the cumulative percentage of susceptible strains at moderate and high-breakpoint concentrations. Two values for each of these parameters are presented for the SGE results for the following reason. The MICs obtained by the SGE method are continuous and discrete rather than incremental twofold dilution steps. The continuous or actual concentration at which growth inhibition occurred, termed the tail-ending concentration (TEC), was used as one of the SGE test endpoints. However, for a more direct comparison with SAD method values, each TEC growth endpoint concentration of the SGE test was rounded up to the next higher twofold dilution value corresponding to that of the SAD method series. Thus, if TEC was 1.8 jig/ml, the SGE or gradient MIC (GMIC) would be 2,ug/ml; this presents the data in another form which are equivalent to the incremental MIC by the SAD method. The GMIC previously was termed the SGE MIC (6). RESULTS SGE method versus SAD method susceptibility.comparison. Susceptibility data are compared in Table 1 for the SAD and the revised SGE methods. The overall agreement was excellent, which is most apparent from the comparative figures on the cumulative percentage of susceptible strains at breakpoint concentrations (Table 1). For the SGE method, the MIC50s and MIC90s are presented both for the TEC endpoint (the concentration at which growth had ceased [6, 11]) and the GMIC endpoint (the TEC value rounded up to the next discrete twofold dilution). The GMIC MIC50s and MIC90s could be compared most easily with the SAD MIC50s and MIC90s that were obtained. In some instances, however, exact comparisons were not possible because the MIC50s or MIC90s were below the lowest or above the highest concentration range of the drug tested by one or both of the methods. Also, the range of concentrations tested for each drug were not the same for the two methods, because it was intended that the concentration range for the SGE method extend beyond that of the concentration range for the SAD method (6). Nevertheless, these boundary MIC50s and MIC90s were consistent in direction. As an example, for the B. fragilis group, all species, and mezlocillin, the SAD MIC90 of >128,ug/ml was consistent with the GMIC MIC%0 of 512,ug/ml. Values also were consistent for the very

3 TABLE 1. Comparison of the SAID and SGE methods for determination of the in vitro susceptibilities of anaerobic gram-negative bacilli (161 strains) to eight antimicrobial agentsa MIC50, MIC9b Cumulative % Organism susceptible at (no. of strains) Antimicrobial agent SGE breakpoint concn TEC GMIC SADc SGEd Bacteroides fragilis group, all species (70) Bacteroides fragilis (36) Bacteroides fragilis group, other members (34) Black pigmenting group, all species (15) Bacteroides bivius (16) Bacteroides, all other species (54) All anaerobic gram-negative bacillie (161) -sulbactam -sulbactam -sulbactam -sulbactam -sulbactam -sulbactam -sulbactam 0.50, , , >8 8.00, > , > , > , , , > , > , , , > , > , > , > , 1.00 s0.015, 0.03 <0.125, <0.015, , 4.00 <0.125, 4.00 <0.125, , , , , , , , > , , , , , , > , > , > , , , , , > , > , , , , > , , , > , > , , , , , , , > , > , , , , > , , , > , > , , , , , , , , , , , , , , , > , , , , , , , , > , > , , , , , , , > , > , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , 99 99, 99 90, 96 90, 96 84, 84 84, 84 64, 87 72, 89 70, 76 69, 79 9, 23 13, 23 19, 44 20, 54 97, 97 97, 97 92, 97 97, 97 97, 97 97, 97 89, 89 93, 93 64, 76 64, 82 6, 21 12, 24 18, 55 15, 58 89, 94 71, 71 44, 85 77, 77 11, 26 19, 33 94, , 69 96,98 96, , 96 89, , 89 54, 62 62, 79 98, 98 94, 98 91, 91 81, 95 82, 86 41, 51 48, 65 83, 94 71, 71 56, 85 74, 77 14, 23 25, 50 94, , , 96 94, 94 90, 98 85, 91 56, 62 62, 81 99, , 99 94, 97 90, 90 85, 95 81, 88 43, 51 48, 71 a SAD, Reference agar dilution procedure for antimicrobial susceptibility testing of anaerobic bacteria (9); SGE, revised spiral gradient endpoint method (6, 11, 13). b MICs are in micrograms per milliliter except for penicillin, which are in units per milliliter. Percentage of strains susceptible to moderate (first number) and high (second number) breakpoint concentrations. The moderate- and high-breakpoint concentrations, respectively, used for analysis of the activities of these drugs were as follows: penicillin G, 16 and 32 U/ml; ampicillin, 8 and 16,ug/ml; ampicillin plus sulbactam, 8 and 16,ug/ml (based on ampiciltin); mezlocillin, 64 and 128.Lg/ml; cefoxitin, 16 and 32 p.g/ml; clindamycin, 4 and 8 ±g/ml; metronidazole, 8 and 16 p.g/ml; imipenem, 4 and 8 Rg/ml. d Percentage of strains susceptible to moderate (first number) and high (second number) breakpoint concentrations. For the SGE method, the cumulative percentage of susceptible strains was the same whether it was derived from the TEC or the corresponding GMIC. ' Includes all strains already listed in the table plus six strains of Fusobacterium. 977

4 978 HILL susceptible species, such as the black pigmenting group, all species, at the low end of the concentration range (Table 1). Drug susceptibilities. The susceptibility data for the strains tested by the SAD and SGE methods (Table 1) demonstrated high activity, in general, with metronidazole, imipenem, and ampicillin-sulbactam. was significantly less active alone than it was when it was combined with sulbactam, a,b-lactamase inhibitor. For the other drugs, their activities sometimes markedly depended on the particular strains or species tested. For example, strains of B. fragilis were much more susceptible than were certain species and strains among the B. fragilis group, other members (Table 1). The black pigmenting group, all species, generally were very susceptible to almost all of the drugs tested (Table 1), and cumulative susceptibilities usually were 100% at the high (and moderate) breakpoint. B. bivius was susceptible to the majority of drugs at the high breakpoint. Strains among Bacteroides, all other species, in general were very susceptible to metronidazole, imipenem, ampicillin-sulbactam, clindamycin, and cefoxitin, but somewhat less so to the other drugs tested (Table 1), depending on the species and strain. Virtually all strains of Fusobacterium were very susceptible to all of these drugs. DISCUSSION Excellent agreement was demonstrated between the MIC data obtained by the revised SGE method compared with those obtained by the SAD method. Overall, there was a 94% correlation at ±+1 twofold dilution for GMICs matched against SAD test MICs for these antimicrobial agents within the range of concentrations which generally would be used for clinical susceptibility testing. Also, the intralaboratory repeatability of the revised SGE method (TEC endpoint) was approximately ±24%, compared with the accepted reproducibility of +100 and -50% (±1 twofold dilution) for the SAD test (6, 9). This advantage probably is due primarily to the more precise mechanical dilution of the antimicrobial agent by the spiral plater than is possible by manual dilution. Prior to the revision of the SGE method (6), limited data on the application of the spiral plater to susceptibility testing were encouraging and supported further studies for anaerobic (3) and aerobic (7, 14) bacteria. These studies were based on a preliminary users' guide (11). Recently, a new edition of the users' guide has become available that encompasses the revised procedures and formulas (13). Further evaluations of the revised method are needed, since the data set reported here also was used to derive the formulas and changes that make up much of the revised SGE method (6). However, results of a subsequent separate study (8) indicate that results of the revised SGE method correlate well with MIC data obtained by serial twofold agar dilution susceptibility testing. Other investigators, in general, have reported similar susceptibilities (1, 2, 5, 10, 15, 16) for the species of anaerobic gram-negative bacilli tested in the present study by the SAD method. This consistency of the present SAD data with data given in these previously published reports also supports the validity of the SGE test data, which correlated well with the SAD results. The added precision and reproducibility of the SGE method and the TEC endpoint could be expected to decrease intra- and interlaboratory variabilities. Yet, some irreducible variability regarding the percentage of susceptible strains is likely, since MICs tend to cluster around the breakpoint for certain drug and organism combinations. The more precise J. CLIN. MICROBIOL. and continuous TEC endpoint often indicated that the MICgo really was closer to the susceptible range than might be apparent by the SAD MICg. Examples within the Bacteroides, all other species, group were as follows: for mezlocillin, the SAD MIC%0 was >128,ug/ml (resistant), but the TEC MIC90 was 127.7,ug/ml, just below the susceptible breakpoint; and for cefoxitin, the SAD MIC90 was 32,ug/ml, but the TEC MIC9J was 15.5,ug/ml. Thus, very small differences in endpoint concentrations near a breakpoint can markedly affect the perceived activity of a drug, compounding differences caused by technical factors, when twofold concentration intervals are reported, as illustrated here by the composite MIC90s. The revised SGE test also was more efficient than the SAD method in the present study, insofar as labor and materials were concerned. Additional technical advances worked out in collaboration with Spiral System Instruments, Inc., during and after this study have further markedly improved the efficiency of the test and have enhanced the test's convenience. A reusable template with a precise millimeter scale is available so that endpoints can be read on an agar plate. A computer program calculates the required stock concentrations for the various antimicrobial agents according to the range of test concentrations desired for plates and calculates the TEC and GMIC of an antimicrobial agent for each strain. For comparative purposes, data on the MICs from parallel SAD tests can be entered in the computer program, and these can be directly compared with the corresponding GMICs. This comparative function is intended to aid investigators who wish to further validate the SGE method, to serve as a quality control for laboratories beginning to use the method, and to confirm whether the SGE method, formulas, and so forth would be applicable to new or other antimicrobial agents not previously tested. The continuous TEC endpoints for the strains tested with an antimicrobial agent can be rank-ordered along with the corresponding cumulative percentage of susceptible strains; this conveniently provides the range, the M'C50, the MIC%, and the full spectrum of susceptibilities in terms of the more precise TEC. Other convenient analyses also are available. The SGE inoculum replicator has been improved for easier loading of the inocula and repetitive inoculation of plates. Previous work (6) demonstrated that comparable results were obtained for intervals of 0.75 to 8 h in between deposition of the antimicrobial agent and inoculation of the bacteria onto plates, providing for greater flexibility in scheduling. Thus, this revised SGE method has many observed and potential advantages (6) as a modified procedure for agar dilution susceptibility testing for anaerobic (and aerobic) bacteria. In summary, the present evaluation and certain advantages (6) of the revised SGE method support its application when detailed susceptibility data are needed or desirable. If further testing confirms the added precision, reproducibility, and efficiency of the revised SGE method for determining MICs, this should warrant its consideration and recognition as an alternative agar dilution method for testing anaerobic (and aerobic) bacteria. Acquisition of the necessary spiral gradient equipment would be most practical for laboratories that perform many MIC susceptibility tests, for laboratories that are active in developing standardized procedures, or for both types of laboratories. ACKNOWLEDGMENTS This work was supported in part by Merck Sharp & Dohme and by the Roerig Division of Pfizer Pharmaceuticals.

5 VOL. 29, 1991 SPIRAL GRADIENT ENDPOINT SUSCEPTIBILITY TEST 979 I thank Ouida Ayers and Barbara Everett for technical assistance, Donna Dzubay for special strain identifications, Melanie Blakely for data entry, and Samuel Schalkowsky of Spiral System Instruments, Inc., for aid in data analysis. REFERENCES 1. Brown, W. J National Committee for Clinical Laboratory Standards agar dilution susceptibility testing of anaerobic gramnegative bacteria. Antimicrob. Agents Chemother. 32: Cornick, N. A., G. J. Cuchural, Jr., D. R. Snydman, N. V. Jacobus, P. Iannini, G. Hill, T. Cleary, J. P. O'Keefe, C. Pierson, and S. M. Finegold The antimicrobial susceptibility patterns of the Bacteroides fragilis group in the United States, J. Antimicrob. Chemother. 25: Cuchural, G., D. Stearns, and M. Barza Program Abstr. 27th Intersci. Conf. Antimicrob. Agents Chemother., abstr Finegold, S. M., and the National Committee for Clinical Laboratory Standards Working Group on Anaerobic Susceptibility Testing Susceptibility testing of anaerobic bacteria. J. Clin. Microbiol. 26: Goldstein, E. J. C., and D. M. Citron Annual incidence, epidemiology, and comparative in vitro susceptibilities to cefoxitin, cefotetan, cefmetazole, and ceftizoxime of recent community-acquired isolates of the Bacteroides fragilis group. J. Clin. Microbiol. 26: Hill, G. B., and S. Schalkowsky Development and evaluation of the spiral gradient endpoint method for susceptibility testing of anaerobic gram-negative bacilli. Rev. Infect. Dis. 12(Suppl. 2):S200-S Hindle, M. and A. J. Westwell Preliminary evaluation of the spiral plater method for antimicrobial endpoint determination, p In Proceedings of the symposium on spiral system in antimicrobial susceptibility testing. Don Whitley Scientific Ltd., West Yorkshire, United Kingdom. 8. Molitoris, E., F. Jashnian, H. M. Wexler, and S. M. Finegold Abstr. Annu. Meet. Am. Soc. Microbiol. 1990, C 260, p National Committee for Clinical Laboratory Standards Reference agar dilution procedure for antimicrobic susceptibility testing of anaerobic bacteria. Approved standard Mll-A. National Committee for Clinical Laboratory Standards, Villanova, Pa. 10. O'Keefe, J. P., F. R. Venezio, C. A. Divincenzo, and K. L. Shatzer Activity of newer P-lactam agents against clinical isolates of Bacteroides fragilis and other Bacteroides species. Antimicrob. Agents Chemother. 31: Schalkowsky, S Preliminary user guide: determination of antimicrobial susceptibility by the spiral gradient endpoint test. Spiral System Instruments, Inc., Bethesda, Md. 12. Schalkowsky, S Plating systems, p In M. D. Pierson and N. J. Stern (ed.), Foodborne microorganisms and their toxins: developmental methodology. Marcel Dekker, Inc., New York. 13. Spiral System Instruments, Inc User guide spiral gradient endpoint SGET antimicrobial susceptibility test. Spiral System Instruments, Inc., Bethesda, Md. 14. Weckbach, L. S., and J. L. Staneck Abstr. Annu. Meet. Am. Soc. Microbiol. 1987, C 65, p Wexler, H. M., and S. M. Finegold In vitro activity of cefoperazone plus sulbactam compared with that of other antimicrobial agents against anaerobic bacteria. Antimicrob. Agents Chemother. 32: Wexler, H. M., B. Harris, W. T. Carter, and S. M. Finegold In vitro efficacy of sulbactam combined with ampicillin against anaerobic bacteria. Antimicrob. Agents Chemother. 27: Downloaded from on January 22, 2019 by guest