Improved medium for antimicrobial susceptibility testing of Haemophilus influenzae

Abstract

The need for complex growth media has complicated routine susceptibility testing of Haemophilus influenzae because of antagonism of certain antimicrobial agents by the medium or because of difficulties in interpretation of growth endpoints. Haemophilus test medium (HTM) is a simple, transparent medium for brothor agar-based tests with H. influenzae. HTM incorporates Mueller-Hinton medium with additions of 15 ,ug of hematin per ml, 15 ,ug of NAD per ml, and 5 mg of yeast extract per ml as growth-promoting additives. Agar or broth microdilution MICs of 10 antimicrobial agents for a collection of 179 H. influenzae isolates determined by using HTM compared favorably with MICs determined by the conventional agar or broth dilution methods recommended by the National Committee for Clinical Laboratory Standards. Disk diffusion tests performed with HTM allowed accurate categorization of susceptible and resistant strains and were easier to interpret than tests performed with Mueller-Hinton chocolate agar. A particular advantage of HTM was the reliability of brothor agar-based test results with trimethoprim-sulfamethoxazole. The results of the study suggest modification of current National Committee for Clinical Laboratory Standards MIC-interpretive criteria for H. influenzae with amoxicillin-clavulanate, chloramphenicol, and trimethoprim-sulfamethoxazole. Error rate-bounded analysis of MICs and disk diffusion zone sizes also suggest modified zone-interpretive criteria for ampicillin, amoxicillin-clavulanate, chloramphenicol, and tetracycline with HTM or conventional media. Interpretive zone sizes are newly proposed for cefaclor and rifampin disk diffusion tests.

Investigation of ampicillin-intermediate strains of Haemophilus influenzae by using the disk diffusion procedure and current National Committee for Clinical Laboratory Standards guidelines

“…Until recently, CMH was recommended for H. influenzae. However, complex constituents of this medium have been shown to antagonize certain antimicrobial agents, and its opaque nature makes interpretation of zone sizes difficult (11). Also, no strain of H. influenzae has been recommended by NCCLS to ensure quality control of this medium.…”

“…Jorgensen et al (11) described a new, simplified medium, HTM, which avoids many of these problems. The medium is transparent, which allows measurement of zone sizes from the back of the plate as is done with nonfastidious organisms.…”

“…It is stable, reproducible from lot to lot, and commercially available. This medium is recommended by the current NCCLS standards (15) (11). On the basis of MIC studies involving a large number of H. influenzae isolates, it was proposed that strains having zone sizes of <21 mm be considered resistant, while those with zone diameters of .22 mm be considered sensitive.…”

“…The opacity of media such as chocolate-Mueller-Hinton agar (CMH) often makes interpretation difficult (7). A new medium called Haemophilus test medium (HTM), consisting of Mueller-Hinton agar supplemented with yeast extract, hematin (X factor), and NAD (V factor) (11), supports the growth of H. influenzae and has the advantage of clarity, permitting easier measurement of zone diameters. This medium is recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (standards M2-A4) for disk susceptibility testing of H. influenzae.…”

Haemophilus influenzae ATCC 49766, an alternative quality control strain for monitoring broth microdilution susceptibility tests with selected beta-lactams

“…For testing the susceptibility ofHaemophilus influenzae to antimicrobial agents, supplemented Mueller-Hinton medium has been recommended (2)(3)(4)6). However, Mendelman et al (5) have described unsatisfactory results with the agar version of Haemophilus test medium (HTM).…”