Haemophilus Test Medium Agar Research Articles

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.

Activity of quinupristin/dalfopristin and its components against Haemophilus influenzae

Quinupristin/dalfopristin is an injectable streptogramin antibiotic that is constituted in a 30:70 (w/w) ratio of the two components. Quinupristin and dalfopristin are thought to act synergically by binding to two separate sites on the bacterial 50S ribosomal subunit. The in-vitro activities of the two components separately and together in different ratios were determined for a collection of 100 Haemophilus influenzae strains representing various antimicrobial resistance phenotypes. The NCCLS microdilution susceptibility testing procedure incorporating Haemophilus test medium (HTM) broth was used to determine MICs of quinupristin, dalfopristin and seven other antimicrobial agents. The MIC50 and MIC90 values were 4 and 8, 4 and 16, and 64 and 128 mg/L for quinupristin/dalfopristin (30:70), dalfopristin and quinupristin, respectively. MICs of quinupristin and dalfopristin were also determined in Mueller-Hinton lysed horse blood broth and by HTM agar dilution testing. Compared with HTM broth-derived results, the MICs of quinupristin/dalfopristin and its components were the same or one dilution higher in lysed horse blood and HTM agar incubated in air, and were equivalent or one dilution lower in HTM agar incubated in a CO2 atmosphere. The MICs of quinupristin and dalfopristin separately or together were directly proportional to erythromycin MICs, but were otherwise unaffected by any of the resistance mechanisms represented in these strains. MICs of quinupristin and dalfopristin combined in ratios of 10:90, 70:30 and 90:10 did not differ significantly from those of the 30:70 ratio. Thus, unlike the synergic activity noted against Gram-positive bacteria, the activity of quinupristin/dalfopristin against H. influenzae appears to be due almost entirely to the dalfopristin component of the combination.

Interpretive criteria for susceptibilities of Haemophilus influenzae to ampicillin, amoxicillin, and amoxicillin-clavulanic acid.

One hundred fifty isolates of Haemophilus influenzae (including 30 beta-lactamase-positive strains and 23 beta-lactamase-negative, ampicillin-resistant strains) were tested for susceptibilities to ampicillin, amoxicillin, and amoxicillin-clavulanic acid (A/C) by the broth microdilution method in Haemophilus test medium (HTM) and in Mueller-Hinton medium with lysed horse blood and by the disk diffusion method on HTM agar. Our results support the use of HTM for susceptibility testing of H. influenzae but raise a number of questions regarding the interpretive criteria currently in use, particularly with respect to the fourfold difference in MIC susceptibility breakpoints for ampicillin and A/C and a resulting high proportion of A/C-susceptible beta-lactamase-negative, ampicillin-resistant strains.

Interpretive criteria and quality control guidelines for lomefloxacin and meropenem in susceptibility tests of Haemophilus influenzae using Haemophilus test medium.

Lomefloxacin and meropenem were tested in a multilaboratory study to establish susceptibility testing interpretive criteria and quality control (QC) guidelines for Haemophilus influenzae using Haemophilus test medium (HTM). Interpretive criteria were established by using triplicate testing of 102 representative H. influenzae strains. Only a susceptible category was proposed for lomefloxacin (greater than or equal to 22 mm and less than or equal to 2 micrograms/ml) and meropenem (greater than or equal to 13 mm and less than or equal to 4 micrograms/ml) due to the lack of resistant isolates. QC range for H. Influenzae ATCC 49247 were established using multiple HTM agar and broth base lots, three disk lots for each drug, and a number of test replicates consistent with the National Committee for Clinical Laboratory Standards M23-T guideline.

Antimicrobial susceptibility testing of less commonly isolated Haemophilus species using Haemophilus test medium

Haemophilus test medium (HTM) was developed recently for dilution and disk diffusion antimicrobial agent susceptibility testing of Haemophilus influenzae. The application of HTM to the testing of other, less frequently encountered Haemophilus species recovered from humans was evaluated in this study by using commercially prepared HTM (BBL Microbiology Systems, Cockeysville, Md.) in broth microdilution and agar disk diffusion susceptibility tests with 18 antimicrobial agents. A total of 93.3% of 90 isolates belonging to six Haemophilus species provided acceptable growth in HTM agar disk diffusion tests, while only 63.3% (57 of 90) provided acceptable growth in the broth microdilution tests. However, HTM agar dilution testing provided an alternative method for those strains (primarily H. haemolyticus) which failed to grow adequately in broth. Based on the latest National Committee for Clinical Laboratory Standards guidelines (standard M2-T4) for interpretation of HTM disk tests of H. influenzae, the overall very major, major, and minor errors for all 18 drugs and six species tested were 0.2, 0.7, and 3.4%, respectively. Thus, the use of HTM in agar or broth susceptibility tests can be recommended for testing the less commonly encountered Haemophilus species by using the same test conditions and interpretive guidelines developed for H. influenzae.