Reference Agar Dilution Method Research Articles

Cefotetan Susceptibility Testing Against AnaerobicBacteria From Obstetrical and Gynecologic Sources:Comparison of Five Different Methods

Five different antibiotic susceptibility methods were utilized to test the effectiveness of cefotetan against 200 anaerobic bacteria recovered from patients with obstetrical or gynecological infections. The object of this study was to determine if a more economical and rapid method for anaerobic susceptibility testing was as acceptable as the reference agar dilution method. The five methods were: 1) broth disk elution, 2) microbroth technique, 3) a commercially available microbroth technique, 4) a commercially available spiral gradient technique, and 5) reference agar dilution. The minimal inhibitory concentrations (MICs) calculated from the spiral gradient technique were equal to or within one doubling dilution of the reference system in 99.5% of cases, while the percentage for the commercially available microbroth system was 96.8%, very similar to the microbroth technique used in our laboratory that yielded a percentage of 96.3. The disk elution method correlated to the reference agar dilution method in 95.3% cases. While the overall agreement between these techniques is good, especially for the spiral gradient system, clustering of certain organisms near the breakpoint of the antibiotic tested results in variability in the labeling of these organisms as susceptible or resistant. This problem appears to be particularly significant for the disk elution method. Therefore, further refinements in these methods of suscleptibility testing are needed in order to provide a more clinically useful assessment of the susceptibility or resistance of certain bacterial isolates.

Review of methods for susceptibility testing of anaerobes.

In the USA, the National Committee for Clinical Laboratory Standards has studied and published a reference agar dilution method for the susceptibility testing of anaerobic bacteria. While numerous investigators both in Europe and the USA have evaluated a variety of methods with a variety of modifications, only the broth microdilution method appears to be appropriate for routine use. The problems of the choice of breakpoint, inoculum size, media, media additives, endpoint recognition and other parameters affecting test performance and interpretation, while troublesome for anaerobes, are not unique to this group of organisms. The increasing resistance of anaerobes and the ever existing need to provide therapeutic guidance, surveillance for resistance and susceptibility data on new drugs make the need for an accurate and reliable susceptibility test for anaerobes critical. The newer methods, while showing promise, need further evaluation with all agents that have a therapeutic indication for anaerobic infections.

Evaluation of the E test for susceptibility testing of anaerobic bacteria

The susceptibilities of 105 clinical isolates of anaerobic bacteria were determined by a new method, the E test (AB Biodisk, Solna, Sweden), and were compared with the MICs for these organisms obtained by the reference agar dilution method by using supplemented brucella and Wilkins-Chalgren agars. The E test is a plastic strip with a predefined antibiotic gradient immobilized on one side and a MIC interpretive scale printed on the other side. Strips with cefoxitin, cefotaxime, imipenem, penicillin, metronidazole, and clindamycin were used in this study. A suspension of the test strain equal to the visual turbidity of a no. 0.5 McFarland standard was prepared and swabbed onto a 150-mm-diameter plate. The strips were applied in a radial fashion, and the plates were incubated under anaerobic conditions. After growth had occurred, an ellipse of inhibition was seen around each strip. At the point of intersection of the ellipse with the strip, the MIC was read from the interpretive scale. For most antibiotic-organism combinations, the ellipse was clear and the endpoint was sharp. The E-test MICs were interpreted after overnight and 48-h incubation for 58 of the strains. After overnight incubation, 87% of the E-test MICs were within 1 dilution of the agar dilution MICs, and 98% were within 2 dilutions. After 48 h of incubation, agreement was 86 and 97% respectively. E-test MICs obtained for the Bacteroides fragilis group after overnight incubation were more comparable than those obtained after 48 h of incubation to agar dilution MICs determined at 48 h for all drugs except clindamycin. On brucella agar, there was a 2% categorical discrepancy rate between the E-test MICs and agar dilution MICs, which occurred mostly with cefoxitin. The E test is easy to perform and read, is suitable for all anaerobes, can be used to test single patient isolates as needed, and offers the laboratory a reliable method for susceptibility testing of anaerobic bacteria.

Susceptibilities of anaerobic Gram-negative bacilli to thirteen antimicrobials and β-lactamase inhibitor combinations

Thirteen antimicrobial agents and beta-lactamase inhibitor combinations were tested simultaneously for their in-vitro activity against a range of anaerobic Gram-negative bacilli with a standard reference agar dilution method. Overall, metronidazole, imipenem, ampicillin/sulbactam, ticarcillin/clavulanic acid and cefoperazone/sulbactam, followed by clindamycin, cefoxitin, and piperacillin, had the greatest activity. Cefotetan, ceftizoxime, and cefoperazone were moderately active, while ampicillin and penicillin were least active. Metronidazole was the only drug active against all strains, but only one strain was resistant to imipenem. Resistance was highest among certain members of the Bacteroides fragilis group, but was observed also among numerous other Bacteroides species. beta-Lactamase was produced by 94% of strains in the B. fragilis group, and by 64% of strains overall. The activities of clindamycin and cefoxitin were compared with those in previous surveys since 1982 at our institution. No clear evidence of increasing resistance was demonstrated, but the data emphasized the significant effects resulting from variations in susceptibility testing.

Antimicrobial Susceptibility Testing of Anaerobic Bacteria: Review and Update on the Role of the National Committee for Clinical Laboratory Standards

The National Committee for Clinical Laboratory Standards (NCCLS) has published a standard method (M11-A) and alternative methods (M17-P) for susceptibility testing of anaerobic bacteria. (These will be combined in the next edition of M11.) Even though M11-A describes the "standard reference" method, there is not agreement as to which method, if any, is the "best" method. In recent years NCCLS subcommittee members have realized that results obtained by microdilution or disk elution may vary markedly from those obtained with the reference agar dilution method. Furthermore, they have recognized the difficulty of relating in vitro susceptibility results to clinical success or failure. In response to these problems, the NCCLS convened a working group to consider recommendations about susceptibility testing. This group has concluded that for most individual patients a susceptibility test may not be required but that present methods can be useful for comparisons of the activity of various drugs and for surveillance of resistance patterns.

Effectiveness of cefotaxime alone and in combination with desacetylcefotaxime against Bacteroides fragilis

The synergistic activity of the combination cefotaxime-desacetylcefotaxime (CTX/dCTX) was compared to the effectiveness of seven other antimicrobial agents: cefoxitin (CFOX), cefotetan (CTAN), ceftizoxime (CTIZ), chloramphenicol (CLOR), clindamycin (CLIND), metronidazole (METR), and ampicillin-sulbactam (A/S) tested against 100 clinical isolates belonging to the Bacteroides fragilis group. All tests were performed using the NCCLS reference agar-dilution method. The overall susceptibility of these organisms to CTX/dCTX was 84% compared to CFOX at 78% or CTAN at 66%. The other antimicrobials inhibited greater than 90% of these isolates. There was no difference between the susceptibility rates of CTX/dCTX and CTX with the B fragilis (85%) or B. distasonis (75%) strains. Bacteroides thetaiotaomicron showed a 11% greater susceptibility to CTX/dCTX than to CTX. Of the 100 isolates tested, 40% showed either synergy or partial synergistic interactions between CTX and dCTX. Most of the isolates showed indifference (52%), while 8% demonstrated antagonism; a relatively unique finding to date.

Evaluation of in vitro methods for testing susceptibility of anaerobes to ampicillin-sulbactam and amoxicillin-clavulanic acid

A total of 97 anaerobic bacteria were tested for susceptibility to ampicillin, ampicillin-sulbactam, and amoxicillin-clavulanic acid by broth microdilution and disk elution methods, the results of which were compared with those of the reference agar dilution method. With the broth microdilution method, approximately 95% of MICs were within 1 dilution of those of the reference agar method, with a definite (0.6 to 0.7 dilution) trend toward lower MICs. The disk elution test performed satisfactorily, but additional anaerobic isolates resistant to ampicillin-sulbactam and/or amoxicillin-clavulanic acid (currently rare) are needed to assure the predictability of resistance by the disk elution test.

Evaluation of the AutoMicrobic system Gram-Negative General Susceptibility-Plus Card

The MICs of 10 antimicrobial agents were determined for 1,000 clinical aerobic and facultatively anaerobic gram-negative bacilli by the 6-h AutoMicrobic system Gram-Negative General Susceptibility-Plus Card (GSC+) (Vitek Systems, Inc., Hazelwood, Mo.) and by an 18-h reference agar dilution method. Results obtained by both systems were evaluated for twofold dilution discrepancies and for interpretive category discrepancies. MICs that differed by more than one twofold dilution between the two test systems were considered to be discrepant. The overall twofold dilution agreement of the GSC+ MICs to agar dilution MICs was 94.0%. The agreement for each drug tested ranged from 91.7% (ampicillin and chloramphenicol) to 96.5% (carbenicillin). Interpretive categories were considered in agreement when the susceptibility interpretations obtained by the two systems were both very susceptible, moderately susceptible, or resistant. The overall interpretive category agreement was 80.5%. This agreement increased to 95.8% when all minor errors (moderately susceptible, one system; very susceptible, the other system) that would not affect therapy were omitted. The AutoMicrobic system GSC+ provides rapid and accurate susceptibility test results for clinical aerobic and facultatively anaerobic gram-negative bacilli.

Evaluation of the Precept microdilution MIC system for single-drug testing in individual trays.

This study presents an evaluation of a commercial system for the MIC testing of single drugs dehydrated in disposable plastic trays (Precept, Austin Biological Laboratories, Inc., Austin, Tex.). The commercial system was compared with a reference agar dilution method, and 203 clinical bacterial isolates were tested by each method. For a total of 767 determinations, there was 94.2% agreement between the two methods, and of the discrepancies encountered, 0.8% were very major, 2.1% were major, and 2.9% were minor. The results suggest that the Precept system may provide a practical and reliable method for MIC determinations of individual antimicrobial agents.

Antimicrobial susceptibility testing of anaerobic bacteria.

Antimicrobial susceptibility testing of anaerobic bacteria has assumed greater importance in recent years because of (1) an increase in recognition of the clinical significance of anaerobes, (2) a decrease in predictability of susceptibility patterns, (3) the appearance of new antimicrobial agents with variable activity against anaerobes, (4) an increase in use of and demand for susceptibility data by clinicians, and (5) the availability of a standardized reference and practical methods for susceptibility testing of anaerobes. The broth microdilution and broth disk-elution techniques are suitable for clinical microbiology laboratories and have been shown to provide results comparable to those obtained with the reference agar-dilution method developed by the National Committee for Clinical Laboratory Standards. The ability to produce beta-lactamase has been demonstrated in most strains of the Bacteroides fragilis group and in over half of other Bacteroides. Increasing resistance of B. fragilis to clindamycin has been reported, and the newer beta-lactam antibiotics, including moxalactam, piperacillin, cefoperazone, and cefotaxime, have variable activity against this organism. Most medical centers should perform susceptibility tests on anaerobes isolated from patients with bacteremia and bone and joint and central nervous system infections and on those anaerobes isolated in pure culture from other specimens and also should test anaerobic isolates on special request of clinicians.

Susceptibility testing of filamentous fungi to amphotericin B by a rapid radiometric method.

A rapid, radiometric method was developed to determine the susceptibility of filamentous fungi to amphotericin B. The rapid, radiometric method depended on measurement of the inhibition of 14CO2 production in the presence of amphotericin B. Thirty isolates of filamentous fungi were tested by the rapid, radiometric method and a reference agar dilution method. There was 93% agreement between the two methods when an 80% or greater decrease in CO2 production was used to calculate the minimal inhibitory concentration with the rapid, radiometric method. Minimal inhibitory concentrations, based on 80% decrease of CO2 production, were achieved within 24 h of incubation with all of the fungi tested.

Variation in susceptibility patterns of species within the Bacteroides fragilis group.

Minimal inhibitory concentrations of eight antibiotics were determined for 159 clinical isolates of members of the Bacteroides fragilis group using the National Committee for Clinical Laboratory Standards proposed agar dilution reference method. Isolates were identified by standard techniques with deoxyribonucleic acid homolog confirmation. These closely related species demonstrated differences in susceptibility to commonly used drugs.

Rapid radiometric susceptibility testing of Mycobacterium tuberculosis.

A 48-hour radiometric test for determining the drug susceptibility of Mycobacterium tuberculosis has been developed. The test is based on the measurement of 14CO2 produced by the oxidation of formate labeled with carbon-14. The test system uses 5 X 10(7) organisms in 1 ml of Middlebrook 7H9 medium plus albumin-dextrose-catalase enrichment and 1 muCi of [14C]formate. The 14CO2 produced is measured in an ionization chamber at 24-, 48-, and 72-hour intervals, with and without the addition of antituberculous drugs. Isoniazid, streptomycin, rifampin, and ethambutol were each tested at 3 concentrations by the radiometric method and the reference (agar dilution) method. Six standard strains and 21 patient isolates were compared by both methods. Production of 14CO2 was quantitatively decreased in the presence of drugs that inhibit the organism. The radiometric method requires 2 days; the agar dilution, 14 to 21 days.

Antimicrobial susceptibility tests for anaerobic bacteria. Comparison of Wilkins-Chalgren agar reference method and a microdilution method, and determination of stability of antimicrobics frozen in broth

Susceptibility results for anaerobic bacteria obtained with a microdilution method with either Wilkins-Chalgren broth or Brain Heart Infusion broth were compared to results obtained with the reference agar dilution method proposed by the National Committee for Clinical Laboratory Standards. Results were generally comparable, but the minimal inhibitory concentrations obtained with the microdilution method were slightly lower than those obtained with the agar dilution method. Antibiotics stored in these broths in microdilution trays at-20°C were stable for up to 130 days.