Standardized disk diffusion susceptibility test for Haemophilus 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.