Precision and accuracy of fluconazole susceptibility testing by broth microdilution, Etest, and disk diffusion methods.

Interpretive agreements among the results of fluconazole broth microdilution tests, Etests, and disk diffusion tests were documented by evaluating 495 Candida spp. Microdilution reference test results were in agreement with 96% of the Etest results; most discrepancies were minor differences. Fluconazole resistance of Candida krusei strains often required a full 48 h of incubation in order to be observed by the standard method. For the disk diffusion tests that were performed on Mueller-Hinton agar with glucose and methylene blue, 97% of results were in agreement with those of the reference test, especially when zones of inhibition were measured after the first 24 h of incubation. Some Candida glabrata isolates failed to grow satisfactorily until a full 48 h of incubation was completed. Precision was determined by testing 50 selected isolates in triplicate in each of three laboratories. The reproducibility of results of disk diffusion tests was comparable to that of the reference method. With all procedures, determination of test results was particularly challenging with some strains, and new methods are needed in order to improve endpoint definition.

Standardization of broth microdilution and disk diffusion susceptibility tests for Actinobacillus pleuropneumoniae and Haemophilus somnus: quality control standards for ceftiofur, enrofloxacin, florfenicol, gentamicin, penicillin, tetracycline, tilmicosin, and trimethoprim-sulfamethoxazole.

Quality control (QC) standards for the in vitro antimicrobial susceptibility testing of two fastidious veterinary pathogens, Actinobacillus pleuropneumoniae and Haemophilus somnus, were developed in a multilaboratory study according to procedures established by the National Committee for Clinical Laboratory Standards for broth microdilution and disk diffusion testing. The medium recommended for the broth microdilution testing is cation-adjusted Mueller-Hinton broth supplemented with 2% lysed horse blood, 2% yeast extract, and 2% supplement C. This medium has been designated veterinary fastidious medium. The medium recommended for the disk diffusion testing is chocolate Mueller-Hinton agar. The recommended QC organisms are A. pleuropneumoniae ATCC 27090 and H. somnus ATCC 700025. The QC MICs of ceftiofur, enrofloxacin, florfenicol, gentamicin, penicillin, tetracycline, tilmicosin, and trimethoprim-sulfamethoxazole were determined for each isolate, as were the zone size ranges. Of the results from the participating laboratories, 94.0% of the zone diameter results and 97.0% of the MIC results fell within the suggested QC ranges for all compounds. These QC guidelines should allow greater accuracy in interpreting results when testing these antimicrobial agents against fastidious pathogens.

Evaluation of daptomycin susceptibility testing by Etest and the effect of different batches of media.

One hundred and ninety-five Gram-positive bacteria representing 17 species were tested for susceptibility to daptomycin by broth microdilution and Etest methods. The geometric mean daptomycin MIC was 0.46 mg/L by broth microdilution tests and 0.73 mg/L by Etest. The concentration of calcium in 12 different batches of agar varied from 4 to 36 mg/L. Daptomycin Etest MICs varied inversely with the calcium concentration. Etest daptomycin MICs for quality control strains were within proposed quality control range on media with >20 mg/L of calcium. Monitoring the calcium levels of agar media by testing appropriate quality control strains is important for daptomycin Etests.

Relative potency of telithromycin, azithromycin and erythromycin against recent clinical isolates of gram-positive cocci.

A ketolide (telithromycin), an azalide (azithromycin) and a macrolide (erythromycin) were tested against 2,733 isolates of gram-positive cocci gathered from 11 different medical centers. Telithromycin was active against erythromycin-resistant staphylococci that were susceptible to clindamycin but was not active against those that were resistant to clindamycin. More than 99% of all Streptococcus pneumoniae and Streptococcus pyogenes isolates were susceptible to 1 microg/ml of telithromycin including erythromycin- and azithromycin-resistant strains. Telithromycin was not only more potent than azithromycin against macrolide-susceptible strains, it was also active against most macrolide-resistant strains. Although the prevalence of macrolide-resistant pneumococci increased from 19% to 27% between 1997 and 1999, macrolide resistance among other gram-positive cocci did not change substantially in that 2-year period.

In vitro activity of the ketolide ABT-773.

The in vitro activities of ABT-773, azithromycin, erythromycin, and clindamycin were compared by testing 1,223 clinical isolates selected to represent different species and phenotypes. ABT-773 was particularly potent against staphylococci (the MIC at which 90% of the strains tested were inhibited [MIC(90)] was < or =0.06 microg/ml), including all strains that were macrolide resistant but clindamycin susceptible. Streptococcus pneumoniae and other streptococci were inhibited by low concentrations of ABT-773, and that included most erythromycin-resistant strains. Against Haemophilus influenzae, ABT-773 and azithromycin were similar in their antibacterial potency (MIC(90), 4.0 and 2.0 microg/ml, respectively).

Interactions of quinupristin-dalfopristin with eight other antibiotics as measured by time-kill studies with 10 strains of Staphylococcus aureus for which quinupristin-dalfopristin alone was not bactericidal.

Quinupristin-dalfopristin (Q-D) and eight other antimicrobial agents were tested alone and in combination with Q-D in time-kill studies against 10 strains of macrolide-lincosamide-streptogramin B-resistant Staphylococcus aureus. Although Q-D is normally a bactericidal drug, it was only bacteriostatic for these isolates. Gentamicin alone was bactericidal against 7 of the 10 strains, and Q-D did not alter that killing effect. However, when vancomycin, cefepime, ceftazidime, imipenem, piperacillin-tazobactam, and ciprofloxacin were bactericidal when tested alone, the killing rates were reduced when combined with Q-D. The clinical significance of this in vitro antagonism is unknown at this time, and more studies are needed.

Comparative in vitro antimicrobial activity of a new carbapenem, E1010, and tentative disc diffusion test interpretative criteria.

The susceptibility of 705 bacterial isolates representing 46 different species to E1010 (ER-35786), imipenem, meropenem and cefepime was determined by the NCCLS broth microdilution test. The MIC(90)s for E1010 were < or =1.0 mg/L for Enterobacteriaceae, fastidious Gram-negative bacteria, streptococci and anaerobes. E1010 was two- to four-fold more active than imipenem and meropenem against Pseudomonas aeruginosa, and four-fold more active than the other carbapenems against methicillin-resistant Staphylococcus aureus. Vancomycin-resistant enterococci and most Enterococcus faecium were resistant to all four drugs tested. The NCCLS disc diffusion test was performed simultaneously on the non-fastidious organisms. Assuming the MIC breakpoints for E1010 will be the same as for the other carbapenems, the disc diffusion zone diameter breakpoints of imipenem and meropenem would also be applicable to E1010.

Interpretative criteria and quality control parameters for telithromycin disc diffusion susceptibility tests.

In vitro studies were carried out with 4175 Gram-positive clinical isolates, comparing telithromycin broth microdilution MICs with disc diffusion zone diameters. Provisional interpretive criteria for testing staphylococci were > or =23 mm for susceptible and < or =19 mm for resistant. Because pneumococci and other streptococci require increased CO(2), the corresponding zone sizes are 3 mm smaller (> or =20 mm and < or =16 mm). Two collaborative studies were performed in order to propose the following quality control limits: 24-30 mm for Staphylococcus aureus ATCC 25923 and 27-33 mm for Streptococcus pneumoniae ATCC 49619.

In vitro activities of daptomycin against 2,789 clinical isolates from 11 North American medical centers.

The in vitro activity of daptomycin is affected by the concentration of calcium cations in the test medium. Mueller-Hinton broth is currently adjusted to contain 10 to 12.5 mg of magnesium per liter and 20 to 25 mg of calcium per liter, but for testing of daptomycin, greater concentrations of calcium (50 mg/liter) are recommended to better resemble the normal concentration of ionized calcium in human serum. Two levels of calcium were used for broth microdilution tests of 2,789 recent clinical isolates of gram-positive bacterial pathogens. MICs of daptomycin were two- to fourfold lower when the broth contained additional calcium. For most species, however, the percentages of strains that were inhibited by 2.0 microg of daptomycin per ml were essentially identical with the two broth media. Enterococci were the important exception; i.e., 92% were inhibited when tested in calcium-supplemented broth but only 35% were inhibited by 2.0 microg/ml without the additional calcium. This type of information should be considered when selecting criteria for defining in vitro susceptibility to daptomycin.

In vitro activities of ertapenem (MK-0826) against clinical bacterial isolates from 11 North American medical centers.

This study compared the in vitro activities of the new long-half-life carbapenem ertapenem (also known as MK-0826 and L-749,345) with those of imipenem, amoxicillin-clavulanate, and ciprofloxacin against 5,558 recent clinical isolates from 11 North American medical centers. We confirmed the greater activity of ertapenem than of imipenem against the Enterobacteriaceae and the greater activity of imipenem against pseudomonads and gram-positive bacteria.

In vitro activities of three nonfluorinated quinolones against representative bacterial isolates.

In vitro susceptibility tests were performed to document the inhibitory activities of three nonfluorinated quinolone (NFQ) compounds (PGE 9262932, PGE 9509924, and PGE 4175997) compared to those of ciprofloxacin, levofloxacin, and trovafloxacin against 3,030 bacterial isolates. The spectra of the NFQ agents included most gram-positive species as well as quinolone-susceptible Enterobacteriaceae. Ciprofloxacin-resistant, methicillin-resistant Staphylococcus aureus strains were inhibited by the NFQ series at < or =1.0 microg/ml. The NFQ compounds were not very active against Pseudomonas aeruginosa and most other nonfermentative gram-negative bacilli. Against other species, the potency of the NFQ agents was similar to that of trovafloxacin. Continued investigation of the NFQ compounds seems to be warranted.

Identification of beta-lactamase-negative, ampicillin-resistant strains of Haemophilus influenzae with four methods and eight media.

A challenge set of 143 non-beta-lactamase-producing strains of Haemophilus influenzae was tested for ampicillin susceptibility on two broth media and six agar media, using broth microdilution, agar dilution, disk diffusion, and E-test procedures. When beta-lactamase-negative, ampicillin-resistant (BLNAR) strains were defined as those for which the ampicillin MIC was > or = 4.0 microg/ml, 5 to 44% of our selected strains were BLNAR depending on the medium and/or test method used. If nonsusceptible strains for which ampicillin MICs were intermediate were included in the BLNAR category, 32 to 50% of our isolates would be considered BLNAR. These data emphasize the need for a standardized testing procedure and a universal definition of BLNAR strains before the clinical relevance of such strains can be evaluated. NCCLS dilution tests with haemophilus test medium broth or agar are preferred for testing ampicillin against H. influenzae.

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.

Daptomycin susceptibility tests: interpretive criteria, quality control, and effect of calcium on in vitro tests.

Daptomycin MICs were determined for 844 Gram-positive bacteria in three concentrations of Ca(++) and compared with the MICs of vancomycin and teicoplanin. Daptomycin was twofold to fourfold more active against most species when tested in 50 microg/ml of Ca(++) than in 25 microg/ml. In 50 microg/ml of Ca(++) daptomycin was more active against methicillin-resistant staphylococci and vancomycin-resistant enterococci than teicoplanin or vancomycin; 100% of these isolates were susceptible to < or =2.0 microg/ml of daptomycin. Different lots of Mueller-Hinton agar were variable in Ca(++) content, and daptomycin disk diffusion zone diameters were affected, i.e., zones were 1 to 15 mm smaller on one lot of agar with only 6 microg/ml of Ca(++) compared to another lot with 28 microg/ml. The previously proposed daptomycin interpretive breakpoints performed satisfactorily when MICs were determined in Mueller-Hinton broth with 50 microg/ml of Ca(++) and when the agar gave appropriate zones with quality control strains. To define those control limits, replicate tests with four quality control strains were performed in ten laboratories using broth microdilution tests (with Ca(++) supplemented broth) and disk diffusion tests on Mueller-Hinton agar without cation adjustments.

Bactericidal activity of quinupristin-dalfopristin against Staphylococcus aureus: clindamycin susceptibility as a surrogate indicator.

Of 516 Staphylococcus aureus strains tested, 97.1% were susceptible to quinupristin-dalfopristin, which was bactericidal for 22 (56%) of the 39 strains tested, comparable to vancomycin. All 17 clindamycin and macrolide-resistant strains were inhibited but not killed by quinupristin-dalfopristin, whereas all 22 clindamycin-susceptible strains (5 were macrolide resistant) were killed.

Susceptibility of Streptococcus pneumoniae and Haemophilus influenzae to cefditoren, and provisional interpretive criteria.

In vitro cefditoren antimicrobial activity was tested by broth microdilution and disk diffusion methods against 300 Streptococcus pneumoniae and 299 Haemophilus influenzae isolates. MICs were also determined for three comparison drugs. The MICs of cefditoren were very comparable to those of cefotaxime against both species. If penicillin-resistant pneumococci are to be considered not susceptible to cefditoren, the tentative MIC breakpoints for cefditoren of < or = 0.25 microg/ml for susceptible and > or 1.0 microg/ml for resistant could be selected. With these breakpoints, all penicillin-susceptible pneumococci were cefditoren-susceptible, as were 85% of penicillin-intermediate strains. Provisional zone diameter breakpoints would be > or = 26 mm for susceptible and < or = 20 mm for resistant. If penicillin-resistant pneumococcal infections are shown to clinically respond to cefditoren therapy, then a susceptible MIC breakpoint of < or = 1.0 microg/ml would be appropriate, with a corresponding zone diameter breakpoint of > or = 21 mm. A susceptible MIC breakpoint of < or = 0.5 or < or = 1.0 microg/ml is appropriate for H. influenzae, but lack of correlation between cefditoren MICs and disk diffusion zone diameters when testing H. influenzae leads us to make no recommendations at this time regarding cefditoren disk tests for H. influenzae.

Quality control limits for broth microdilution susceptibility tests of ten antifungal agents.

Broth microdilution susceptibility tests of Candida species have now been standardized by the National Committee for Clinical Laboratory Standards (NCCLS). An eight-laboratory collaborative study was carried out in order to document reproducibility of tests of Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 by the NCCLS method. Replicate broth microdilution tests were used to define control limits for 24- and 48-h MICs of amphotericin B, flucytosine, fluconazole, voriconazole, ketoconazole, itraconazole, caspofungin (MK 0991), ravuconazole (BMS 207147), posaconazole (SCH 56592), and LY 303366.

Rapid automated antimicrobial susceptibility testing of Streptococcus pneumoniae by use of the bioMerieux VITEK 2.

The VITEK 2 is a new automated instrument for rapid organism identification and susceptibility testing. It has the capability of performing rapid susceptibility testing of Streptococcus pneumoniae with specially configured cards that contain enriched growth medium and antimicrobial agents relevant for this organism. The present study compared the results of testing of a group of 53 challenge strains of pneumococci with known resistance properties and a collection of clinical isolates examined in two study phases with a total of 402 and 416 isolates, respectively, with a prototype of the VITEK 2. Testing was conducted in three geographically separate laboratories; the challenge collection was tested by all three laboratories, and the unique clinical isolates were tested separately by the individual laboratories. The VITEK 2 results of tests with 10 antimicrobial agents were compared to the results generated by the National Committee for Clinical Laboratory Standards reference broth microdilution MIC test method. Excellent interlaboratory agreement was observed with the challenge strains. The overall agreement within a single twofold dilution of MICs defined by the VITEK 2 and reference method with the clinical isolates was 96.3%, although there were a number of off-scale MICs that could not be compared. The best agreement with the clinical isolates was achieved with ofloxacin and chloramphenicol (100%), and the lowest level of agreement among those drugs with sufficient on-scale MICs occurred with trimethoprim-sulfamethoxazole (89.7%). Overall there were 1.3% very major, 6.6% minor, and no major interpretive category errors encountered with the clinical isolates, although >80% of the minor interpretive errors involved only a single log(2) dilution difference. The mean time for generation of susceptibility results with the clinical isolates was 8.1 h. The VITEK 2 provided rapid, reliable susceptibility category determinations with both the challenge and clinical isolates examined in this study.

In vitro activity of gemifloxacin against contemporary clinical bacterial isolates from eleven North American medical centers, and assessment of disk diffusion test interpretive criteria.

A total of 5499 contemporary clinical bacterial isolates were tested for susceptibility to gemifloxacin and four comparison agents by the broth microdilution method. Gemifloxacin activity against Enterobacteriaceae was generally comparable to that of ciprofloxacin and trovafloxacin, but because the gemifloxacin susceptible MIC breakpoint is lower, the percent susceptible to gemifloxacin was less than that to the other quinolones for some species. All agents were less active against Pseudomonas spp. Gemifloxacin was the most active agent tested against Gram-positive species, though Corynebacterium jeikeium and vancomycin-resistant enterococci were uniformly resistant to all agents tested. With staphylococci, a bimodal distribution of gemifloxacin MICs corresponded with susceptibility or resistance to ciprofloxacin. The significance of ciprofloxacin-resistant staphylococci that have susceptible gemifloxacin MICs is not known at this time. Disk diffusion tests were performed simultaneously with gemifloxacin and trovafloxacin as a control drug. Gemifloxacin MIC-zone diameter scattergrams indicated that interpretive discrepancy rates based on previously proposed criteria when using < or = 0.5 microg/ml as the susceptible MIC breakpoint was within acceptable limits. However, with the currently proposed MIC breakpoint of < or = 0.25 microg/ml, tentative zone diameter breakpoints of > or = 22 mm for susceptible, 19-21 mm for intermediate and < or = 18 mm for resistant are proposed.