Standardized Disk-diffusion Susceptibility Test forHaemophilus influenzae

The emergence of ampicillin-resistant strains of Haemophilus influenzae has emphasized the need for an improved practical method for routine susceptibility testing of clinical isolates. We have previously described a simplified medium for quantitative dilution susceptibility testing that is composed of Mueller-Hinton medium plus Supplement C (Difco). In the present study, paired broth-dilution and disk-diffusion susceptibility tests with ampicillin and chloramphenicol were performed on 100 strains of Haemophilus (95 H. influenzae and five H. parainfluenzae), including 30 strains with previously documented ampicillin resistance. Disk-diffusion tests were performed in exactly the same manner as the standardized Kirby-Bauer procedure used for less fastidious organisms, except that supplemented Mueller-Hinton agar plates were incubated in an increased-CO2 atmosphere. Using this method, ampicillin-susceptible strains of Haemophilus produced zone diameters of 22 mm or more, while ampicillin-resistant strains produced zones of 18 mm or less. All strains were chloramphenicol-susceptible and produced zone diameters of 30 mm or more. This method would allow routine disk-diffusion testing of isolates of H. influenzae by hospital diagnostic laboratories, using a clear medium that closely resembles unsupplemented Mueller-Hinton agar.