Comparative evaluation of the E test, agar dilution, and broth microdilution for testing susceptibilities of Helicobacter pylori strains to 20 antimicrobial agents.

The Epsilometer test (E test; AB Biodisk, Solna, Sweden), a new quantitative technique for the determination of antimicrobial susceptibility, was compared to reference methods (agar dilution and broth microdilution) for the antimicrobial susceptibility testing of Helicobacter pylori. Seventy-one H. pylori strains isolated from patients with duodenal ulcers were tested against 20 antimicrobial agents. The E test and the agar dilution method were carried out on Mueller-Hinton agar; the broth microdilution method was performed with Mueller-Hinton broth. The E-test results showed excellent correlation with the agar dilution results, with 91.3 and 98.8% agreement within 1 and 2 log2 dilution steps, respectively, in a total of 1,350 tests. The correlation between the E-test results and the broth microdilution results was slightly higher, with 91.6 and 99.1% agreement within 1 and 2 log2 dilution steps, respectively, in a total of 1,317 tests. There were six major errors and two very major errors by the metronidazole E test compared to the results obtained by reference methods. Excellent agreement between E-test, agar dilution, and broth microdilution results was found for resistance to erythromycin (8%), clarithromycin (6%), and tetracycline (6%). Our results confirm that the E test is comparable to standardized methods for susceptibility testing. Therefore, the E test is a reliable and alternative method for testing H. pylori susceptibility to a wide range of antimicrobial agents in clinical practice.     

Fluconazole and amphotericin B antifungal susceptibility testing by National Committee for Clinical Laboratory Standards broth macrodilution method compared with E-test and semiautomated broth microdilution test.

A comparative study of fluconazole and amphotericin B susceptibility testing was performed with 68 clinical Candida species isolates and three test methods. The methods used were an agar diffusion method (E-test) and two broth dilution methods, the National Committee for Clinical Laboratory Standards (NCCLS) reference broth macrodilution method and an in-house-prepared semiautomated broth microdilution method based on the Bioscreen turbidometer. In the microdilution method, growth of the yeasts was measured continuously by the automatic turbidometer (Bioscreen), which permitted precise and objective determination of endpoints. MIC endpoints were read after 24 h for the microdilution method and the E-test. Amphotericin B susceptibility testing with the NCCLS method and the E-test yielded comparable results in 89% of the tests, meaning that the endpoints obtained were identical or differed by no more than 2 twofold dilutions. The NCCLS and broth microdilution tests scored 97% comparable results, and the E-test and the broth microdilution test yielded 90% comparable results. Fluconazole susceptibility testing produced 96% comparable results with the NCCLS test and the E-test, 100% comparable results with the NCCLS and the microdilution methods, and 98.5% comparable results with the microdilution method and the E-test. We conclude that the E-test and the Bioscreen microdilution method are valuable alternatives to the NCCLS reference method for routine susceptibility testing of Candida species with fluconazole and amphotericin B.     

Comparison of Agar Dilution, Microdilution, E-Test, and Disk Diffusion Methods for Testing Activity of Cefditoren against Streptococcus pneumoniae

This study evaluated the susceptibility of pneumococci to cefditoren by agar dilution and microdilution methods (both in air) and by E-test (AB Biodisk, Solna, Sweden) and disk diffusion methods (both in CO(2)). By the three MIC tests, the MICs at which 50 and 90% of isolates were inhibited (MIC(50)s and MIC(90)s) were, respectively, as follows (in micrograms per milliliter): for the 65 penicillin-susceptible strains tested, 0.016 and 0.03 (by agar dilution), 0.016 and 0.03 (by microdilution), and 0.016 and 0.03 (by E test); for the 68 penicillin-intermediate strains tested, 0.125 and 0.5 (by agar dilution), 0.125 and 0.5 (by microdilution), and 0. 25 and 0.5 (by E test); and for the 67 penicillin-resistant strains tested, 1.0 and 1.0 (by agar dilution), 0.5 and 1.0 (by microdilution), and 1.0 and 1.0 (by E test). With tentative cefditoren breakpoints (in micrograms per milliliter) of /=8.0 (resistant), all strains were susceptible to cefditoren by agar, microdilution, and E-test results; with breakpoints of /=4.0 microg/ml, 97% of strains were cefditoren susceptible by agar dilution results, 98% were susceptible by microdilution results, and 99% were susceptible by E-test results. When microdilution and E-test results were compared to those from the reference agar dilution method, 191 (95.5%) and 183 (91.5%) of strains gave essential agreement (+/-1 log(2) dilution); 8 (2.7%) minor discrepancies were found for both methods with a breakpoint of /=20 (susceptible), 17 to 19 (intermediate), and /=16 mm (susceptible). All three methods for testing the MIC of cefditoren showed excellent correlation.     

Discrepancies in susceptibility test results for imipenem employing different in vitro test methods and DIN 58 940 breakpoints

During the first half of 1993, bacteria that were isolated from clinical materials and found to have intermediate susceptibility by an agar dilution breakpoint method were collected in a large service laboratory in Germany. All of these isolates were gramnegative bacteria. They were re-tested employing full-scale agar dilution, broth microdilution, E-test and agar diffusion procedures. The results obtained indicated that 76.9% of the isolates were actually susceptible upon re-testing with a reference agar dilution technique. The reason for the discrepant results remained largely unclear. There was a high correlation between agar dilution and E-test results while the agreement with broth microdilution and agar diffusion was less satisfactory. It is suggested that the breakpoint between susceptible and intermediate categories currently recommended by DIN 58 940 (standard set by Deutsches Institut für Normung e.V.) be raised to reduce erroneous interpretations of minimum inhibitory concentrations.     

Comparison of agar dilution, broth dilution, disk diffusion, and the E-test for susceptibility testing of penicillin-susceptible and penicillin-resistantStreptococcus pneumoniae

An evaluation to determine the optimal methods for the in vitro susceptibility testing of 41 clinical isolates and the ATCC 49619 strain ofStreptococcus pneumoniae to penicillin was undertaken. No very major or major interpretive errors were observed with the following test methods and media: agar dilution using either Mueller-Hinton medium with lysed horse blood or Haemophilus test medium; broth dilution using cation-adjusted Mueller-Hinton medium with lysed horse blood, Haemophilus test medium, or Todd-Hewitt medium; and the epsilometer test (E-test) using agar containing Mueller-Hinton medium and 5% sheep blood. The disk diffusion method using agar containing Mueller-Hinton medium and 5% sheep blood agar was an effective screening method, requiring confirmation by a dilution susceptibility test method.     

Comparison of agar dilution, tube dilution, and broth microdilution susceptibility tests for determination of ramoplanin MICs.

Standard broth microdilution (with and without bovine serum albumin [BSA] supplementation), tube dilution, and agar dilution susceptibility tests were compared for determining ramoplanin MICs. With a data base of 246 clinical isolates of gram-positive bacteria from 33 U.S. sites, it was shown that (i) agar and tube dilution susceptibility tests gave essentially the same results (93.9% of the test results were within 1 doubling dilution of equivalence), (ii) broth microdilution susceptibility tests gave results up to 5 doubling dilutions higher than agar or tube assays, and (iii) this data skewing could be reversed by BSA supplementation (final concentration, 0.02%) of the broth microdilution test medium.     

Evaluation of CLSI agar dilution method and Trek Sensititre broth microdilution panel for determining antimicrobial susceptibility of Streptococcus pneumoniae

Both the CLSI agar dilution method and Trek Sensititre broth microdilution panel for Streptococcus pneumoniae antimicrobial susceptibility testing were evaluated against the reference CLSI broth microdilution method using the most recently published CLSI breakpoints. While agar dilution was not an optimal method, the commercial panel appeared to be an acceptable method, with minor errors encountered for ceftriaxone, penicillin, and meropenem.     

Quantitative antimicrobial susceptibility testing of Haemophilus influenzae and Streptococcus pneumoniae by using the E-test.

The E-test (PDM Epsilometer; AB Biodisk, Solna, Sweden) is an antimicrobial agent gradient-coated plastic test strip which allows MIC determinations on agar media. The test is performed in a manner similar to the agar disk diffusion procedure. A collection of Haemophilus influenzae and Streptococcus pneumoniae strains possessing various resistance mechanisms was used to evaluate the E-test method. H. influenzae strains were tested with both Haemophilus test medium (HTM) and PDM ASM II chocolate agar, while the S. pneumoniae strains were tested on Mueller-Hinton sheep blood agar. E-test MICs for a total of 10 antimicrobial agents were compared with broth microdilution MICs determined according to National Committee for Clinical Laboratory Standards methods. In general, E-test MICs for both species were quickly and easily interpreted and agreed within one log2 MIC increment in 89.8% of tests with H. influenzae and in 80.4% of pneumococcal tests. The majority of disagreements between the E-test and conventional MICs occurred with trimethoprim-sulfamethoxazole because of trailing and diffuse E-test MIC endpoints with both species. Ampicillin MICs for beta-lactamase-producing H. influenzae determined by the E-test differed at times from those determined by conventional testing because of the vagaries of interpreting colonies growing within the E-test inhibition ellipses. E-test penicillin MICs for pneumococci tended to be 1 to 2 log2 dilutions lower than those determined by using Mueller-Hinton broth supplemented with lysed horse blood. Nevertheless, strains of both species with documented resistance to the study drugs were detected by E-tests, i.e., 0.7% of the tests had very major errors with H. influenzae and 0.8% had very major errors with S. pneumoniae. Thus, the E-test represents a potential alternative method for antimicrobial susceptibility testing of these two fastidious bacterial species.     

Comparison of broth microdilution,E Test,and agar dilution methods for antibiotic susceptibility testing of Campylobacter jejuni and Campylobacter coli

A standardized broth microdilution method was compared to the E test and an agar dilution method for the antimicrobial susceptibility testing of Campylobacter jejuni and C. coli isolates. A group of 47 human clinical isolates, 37 isolates from retail poultry, and 29 isolates from living turkeys (total, 113 isolates) was included in the study. These encompassed 92 C. jejuni and 21 C. coli strains. The MICs of six antimicrobial agents were determined by the broth microdilution and E test methods, and the strains of human origin were additionally tested by the agar dilution method. In general, broth microdilution MICs agreed within 1 log(2) MIC increment with 90.0% of E test results and 78.7% of agar dilution test results. The agar dilution method gave much lower gentamicin MICs than the broth microdilution method, but the data were significantly (P < 0.01) correlated and there was 100% agreement in the sensitivities and specificities in the comparison of the tests. The broth microdilution method had the highest sensitivity for analysis of the susceptibilities of Campylobacter to nalidixic acid and trimethoprim-sulfamethoxazole. The MICs of ciprofloxacin and erythromycin complied numerically by all three methods. The classification of the results and the correlation of the data demonstrated a high degree of agreement. All methods were equally suitable for the testing of the sensitivity of Campylobacter to tetracycline. Thus, the broth microdilution method appears to be an easy and reliable method for determination of the MICs of antibiotics for C. jejuni and C. coli, and it may offer an interesting alternative to MIC determination by the agar dilution technique or the E test.     

Comparison of the E Test to agar dilution, broth microdilution, and agar diffusion susceptibility testing techniques by using a special challenge set of bacteria.

The E Test (AB Biodisk, Solna, Sweden) is a new method for performing antimicrobial susceptibility tests. It consists of an impervious carrier (5- by 50-mm strip) with a predefined antimicrobic gradient which is placed on an inoculated agar plate and processed like a disk diffusion test. Results are generated directly as MICs from a continuous concentration gradient covering 15 twofold dilutions, and MICs are read where the edge of the inhibition zone intersects the strip. We compared the E Test with disk diffusion, broth microdilution, and agar dilution tests by using a challenge set of 195 gram-positive and gram-negative bacteria for 14 antimicrobial agents. Also, disk diffusion, broth microdilution, and agar dilution tests were compared with each other. All test method comparisons gave greater than 94% agreement for the category of susceptibility. The E Test category agreement with disk diffusion and broth microdilution was 95.1%, and with agar dilution it was 95.2%. The E Test results were as reliable as the results obtained by the standard antimicrobial susceptibility testing methods.     

Comparison of E-test with agar dilution for determining susceptibility ofStreptococcus pneumoniae to penicillin

Minimum inhibitory concentrations (MICs) of penicillin forStreptococcus pneumoniae were determined by the E-test and the agar dilution method. NinetyStreptococcus pneumoniae strains were tested, of which 16 were resistant, 33 intermediate, and 41 susceptible by agar dilution. By the E-test, 80 (88.9%) strains agreed with these determinations within one log₂ dilution step, and no strains disagreed by more than two dilution steps. Sixty-eight of the 70 strains with discrepant MICs read lower in the E-test, resulting in 15 strains being placed in different susceptibility categories when classified by this test. Exact MICs rather than classification groups should be used to determine appropriate antibiotic therapy, since small differences in MICs determined by different methods can lead to a significant degree of misclassification.     

Activity of Gatifloxacin against Haemophilus influenzae and Moraxella catarrhalis, Including Susceptibility Test Development, E-Test Comparisons, and Quality Control Guidelines for H. influenzae

In vitro antimicrobial activity and susceptibility testing interpretation criteria and quality control were studied for gatifloxacin, a new 8-methoxy fluoroquinolone, tested against Haemophilus influenzae. Moraxella catarrhalis (600 strains) and H. influenzae (1,400 strains) from the SENTRY Antimicrobial Surveillance Program in North America (Canada and the United States) were also tested against gatifloxacin and 12 other antimicrobial agents. Gatifloxacin (MIC at which 90% of the isolates are inhibited [MIC90], /=18 mm) was also suggested for H. influenzae testing. No interpretive errors were observed. Quality control guidelines for H. influenzae ATCC 49247 were determined by using the NCCLS M23-T3 (1998) study design. The results from the nine-laboratory protocol suggested the following control ranges: for broth microdilution tests, 0.004 to 0.03 microg/ml; for disk diffusion testing, 33 to 41 mm. Gatifloxacin appears to be a potent anti-Haemophilus fluoroquinolone compound with in vitro testing interpretive criteria that will produce accurate results (disk diffusion, broth microdilution, and E-test).     

Reliability of the E-test method for detection of colistin resistance in clinical isolates of Acinetobacter baumannii

We compared the E-test to the broth microdilution method for testing the susceptibility of 115 clinical isolates of Acinetobacter baumannii to colistin. Twenty-two (19.1%) strains were resistant to colistin and 93 (80.8%) strains were susceptible according to the reference broth microdilution method. A categorical agreement of 98.2% was found, with only two (1.7%) very major errors. Agreement within 1 twofold dilution between the E-test and the broth microdilution was 16.5%. Complete agreement was found for the strains for which MICs fell within the range of 0.25 to 1 microg of colistin/ml. However, there was poor concordance, particularly in extreme dilutions with higher MICs by the E-test method.     

Comparison of five methods, including the PDM Epsilometer test (E test), for antimicrobial susceptibility testing of Pseudomonas aeruginosa.

The antimicrobial susceptibilities of 100 clinical isolates of Pseudomonas aeruginosa to six antipseudomonal antibiotics were tested by five methods: the National Committee for Clinical Laboratory Standards (NCCLS) methods for broth microdilution, agar dilution, and agar disk diffusion; the Vitek Automicrobic System method (Vitek Systems, Hazelwood, Mo.); and the PDM Epsilometer test (E test) (AB Biodisk, Solna, Sweden). The E test results showed excellent correlation with agar dilution results, with over 90% agreement within 1 doubling dilution between the E test and reference agar dilution MICs for all antimicrobial agents tested. The E test results also showed good correlation with the results from the reference agar disk diffusion method, with 90 to 99% complete agreement and 100% essential agreement on categories for all antibiotics tested (essential agreement is the agreement obtained when minor discrepancies are ignored). Comparison of categories with the E test and broth microdilution methods, using the broth microdilution method as the reference method, gave only 59% complete agreement for gentamicin, with 28 minor discrepancies and 13 very major discrepancies. Some discrepancies were observed between results from the E test and broth methods for gentamicin, with the broth microdilution and Vitek methods giving higher MICs than the E test and other methods using agar. The most recent NCCLS guidelines for broth dilution testing have reduced the recommended levels of cation supplementation, which may enhance future agreement between results for the aminoglycosides and P. aeruginosa on broth and on agar. We found that the E test offers a simple, labor-efficient, and accurate method for MIC determination on an agar medium.     

Comparison of susceptibility test methods to detect penicillin susceptibility in Streptococcus pneumoniae isolates

The increasing prevalence of penicillin-resistant Streptococuus pneumoniae urges for fast and accurate susceptibility testing methods. This study evaluated the comparability of three commonly used techniques; disk diffusion, E-test and agar dilution, to detect penicillin susceptibility in clinical isolates of S. pneumoniae. Fifty pneumococcal isolates, obtained from patients at the University of Malaya Medical Centre, were selected to include both penicillin-susceptible strains and those that had decreased susceptibility (resistant and intermediate) to penicillin. The minimum inhibitory concentration (MIC) values of penicillin to serve as the reference was determined by the agar dilution method in which, based on the MIC breakpoints recommended by the National Committee for Clinical Laboratory Standards (NCCLS), 27 strains had decreased susceptibility to penicillin with 17 strains resistant and 10 intermediate. Comparing to the agar dilution method, oxacillin disk diffusion test detected all strains with decreased penicillin susceptibility as such while E-test showed a close agreement of susceptibility (92%) of the isolates to penicillin. This confirmed that oxacillin is a good screening test for S. pneumoniae isolates with decreased susceptibility to penicillin while E-test is very reliable for rapid and accurate detection of penicillin susceptibility.     

Influence of variations in test methods on susceptibility of Haemophilus influenzae to ampicillin, azithromycin, clarithromycin, and telithromycin

The National Committee for Clinical Laboratory Standards standard broth microdilution method for testing the susceptibility of Haemophilus influenzae to ampicillin, azithromycin, clarithromycin, and telithromycin was evaluated by altering one variable at a time. Variables that were tested included age of colony for inoculum preparation, inoculum density, test medium, incubation atmosphere, and incubation time. For the macrolide, azalide, and ketolide agents, incubation in 5 to 7% CO(2) most significantly affected the MICs, producing nearly twofold increases for clarithromycin and telithromycin and a greater than threefold increase for azithromycin. For ampicillin, a 10-fold increase in inoculum density increased the geometric mean MICs for beta-lactamase-negative strains from 1. 50 to 2.45 microg/ml. In addition, 206 H. influenzae strains were tested for their susceptibilities to the same drugs by the broth microdilution tests in two media, as well as by agar dilution tests, disk diffusion tests, and Etests, on six different agar media. The three standard methods with Haemophilus test medium (HTM) compared favorably with each other except for a high minor discrepancy rate (27%) by the disk diffusion test with ampicillin and clarithromycin. Agar dilution test MICs on the five comparative media were generally higher than those on HTM agar but were only rarely more than one twofold concentration higher. Etest MICs of azithromycin and telithromycin were more than twofold higher than agar dilution and broth microdilution MICs on HTM; ampicillin Etest MICs were nearly twofold lower. The use of media other than HTM agar appears to have a minimal effect on susceptibility test results for the ketolide, azalide, or macrolide drugs that we tested against H. influenzae.     

Comparison of the agar dilution, tube dilution, and broth microdilution susceptibility tests for determination of teicoplanin MICs.

The purpose of this study was to evaluate the National Committee for Clinical Laboratory Standards agar dilution, tube dilution, and broth microdilution susceptibility tests for the measurement of teicoplanin MICs. The three standardized tests gave equivalent (within a twofold dilution) results with 98.8 to 99.0% of the 508 gram-positive clinical isolates tested, indicating that either method may be used for teicoplanin MIC determination.     

Evaluation of the Merlin,Micronaut System for Automated Antimicrobial Susceptibility Testing of<Emphasis Type="Italic"> Pseudomonas aeruginosa</Emphasis> and<Emphasis Type="Italic"> Burkholderia</Emphasis> Species Isolated from Cystic Fibrosis Patients

Since accurate antimicrobial susceptibility testing of bacterial cystic fibrosis isolates is known to be problematic and an optimal in vitro testing method has not yet been evaluated, the study presented here was conducted to compare the performance of the reference agar dilution method and broth microdilution with a commercially available automated susceptibility test system (Merlin; Micronaut, Germany). In this pilot study, the susceptibility of 70 clinical strains of Pseudomonas aeruginosa and Burkholderia cepacia-like organisms to nine antimicrobial agents was tested using these methods. Susceptibility results generated by broth microdilution (both automated and according to the National Committee for Clinical Laboratory Standards recommendations) were demonstrated to be of good reproducibility, and they compared favourably to the time- and material-consuming standard agar dilution reference method, especially after a prolonged incubation time (48 h).     

Antipneumococcal Activities of Levofloxacin and Clarithromycin as Determined by Agar Dilution, Microdilution, E-Test, and Disk Diffusion Methodologies

The activities of levofloxacin and clarithromycin against 199 penicillin- and macrolide-susceptible and -resistant pneumococci were tested by agar and microdilution methods in air and by disk diffusion and E-test methods in air and CO₂. For levofloxacin, ≥99.0% of strains were susceptible at ≤2.0 μg/ml with zone diameters of ≥17 mm, regardless of incubation in air or CO₂. Although zone sizes were smaller and E-test MICs were higher for clarithromycin in CO₂ than those in air, category differences were minor, and susceptibility rates for clarithromycin were similar to those obtained by agar and microdilution in air (range, 76.9 to 80.9% by all methods). For clarithromycin, adjustment of breakpoints based upon distribution of results resulted in susceptibility rates which were similar by all methods (75.8 to 76.9% susceptible, 0 to 1.5% intermediate, 22.6 to 23.1% resistant). Minor discrepancies were obtained with levofloxacin for one strain (0.5%) by microdilution and two strains (1.0%) by disk diffusion in CO₂. For clarithromycin, minor discrepancies were found in three strains (1.5%) by microdilution, seven strains (3.5%) by agar dilution, four strains (2.0%) by E-test in air, six strains (3.0%) by disk diffusion in air, and five strains (2.5%) by disk diffusion in CO₂. Major discrepancies occurred with levofloxacin in one strain (0.5%) by microdilution but were not found with clarithromycin. Very major discrepancies were not seen with levofloxacin, but occurred with clarithromycin in five strains (2.5%) by microdilution, three strains (1.5%) by agar dilution, two strains (1.0%) by E-test in air, eight strains (4.0%) by disk diffusion in air, and one strain (0.5%) by disk diffusion in CO₂.     

Evaluation of Reference Dilution Test Methods for Antimicrobial Susceptibility Testing of Pseudomonas aeruginosa Strains Isolated from Patients with Cystic Fibrosis

The development of multidrug-resistant Pseudomonas aeruginosa in patients with cystic fibrosis (CF) is most likely a consequence of increasing life expectancy and more prolonged exposure to antibiotics. The optimal method for antibiotic susceptibility testing of CF strains, particularly mucoid P. aeruginosa strains, is unknown. Antimicrobial susceptibilities of 48 CF strains (25 mucoid) and 50 non-CF strains to 12 anti-Pseudomonas agents were tested by both agar dilution and commercially custom-prepared broth microdilution plates (PML Microbiologicals, Portland, Oreg.) in three laboratories simultaneously to determine if broth microdilution could substitute for agar dilution as the reference method in subsequent studies. Comparison of MICs generated by agar dilution and broth microdilution demonstrated correlation coefficients (r) exceeding 0.85 for all agents tested; correlation was excellent for aminoglycosides (r >/= 0.92) and very good for beta-lactam agents including agents paired with a beta-lactamase inhibitor (r >/= 0.87) and for ciprofloxacin (r = 0.86). Correlation was not improved by 48-h readings, but correlation between 24- and 48-h readings ranged between 0.91 and 0.98 for both methods. Interlaboratory variations were minimal, as the percentage of acceptable variations was 94% for both methods, and serious discords were infrequent (<2% of comparisons). However, CF strains were more likely to have serious discords than were non-CF strains (P < 0. 0001), although mucoid strains were not more likely to have serious discords than were nonmucoid strains. In this study, MICs determined by custom-prepared broth microdilution compared favorably with MICs determined by agar dilution. Thus, this broth microdilution assay can serve as a reference method and facilitate future studies to determine the optimal method for antibiotic susceptibility testing of CF strains.