Nitrate Reduction: New Method for Testing the Antibiotic Susceptibility ofHaemophilus influenzae

Abstract

We have developed a new micro-broth-dilution assay for determining the antimicrobial susceptibility ofHaemophilus influenzae. This assay is based on the ability of viableH. influenzaeto reduce nitrates to nitrites. Bacterial viability is detected by a positive nitrite reaction rather than visible turbidity. The nitrate reduction assay was compared with a standard microassay using 51 isolates ofH. influenzaeand six beta-lactam antibiotics. Although there was good agreement between the two methods, the nitrate reduction assay was more sensitive in detecting viable bacteria, and so established a more accurate estimate of the minimal inhibitory concentration. The nitrate reduction assay offered the additional advantage that it could be used to determine the minimal bactericidal concentration without having to subculture the broth. Ampicillin, penicillin, and cefamandole were equally effective in vitro against susceptible strains (minimal inhibitory concentrations, 0.125 to 0.5 μg/ml), whereas all three antibiotics were ineffective against two beta-lactamase-producing strains. Using the nitrate reduction assay, resistance to cefamandole was detectable with inoculum sizes ranging from 104to 106colony-forming units per ml, while the turbidity assay detected resistance only with the largest inoculum.