Nitrate Reduction: New Method for Testing the Antibiotic Susceptibility of Haemophilus influenzae

Abstract

We have developed a new micro-broth-dilution assay for determining the antimicrobial susceptibility of Haemophilus influenzae. This assay is based on the ability of viable H. influenzae to 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 of H. influenzae and 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 ,g/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 104 to 106 colony-forming units per ml, while the turbidity assay detected resistance only with the largest inoculum.The appearance of ampicillin-and chloramphenicol-resistant strains of Haemophilus influenzae (9, 14) has stimulated interest in developing better techniques for assaying the antimicrobial susceptibility of these fastidious bacteria (5, 6). Broth dilution assays, though commonly employed to measure both the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of antimicrobial agents, can be used to test the susceptibility of H. influenzae only if the medium is supplemented with hemin and nicotinamide adenine dinucleotide. Some broths will support the growth of H. influenzae, but even under good growth conditions turbidity may be difficult to detect (4, 7).We have developed a new assay procedure for testing the antimicrobial susceptibility of H. influenzae. The assay is based on the nitrate reductase activity of viable H. influenzae and the resultant production of nitrite (4, 16). Our method has the distinct advantage of increased reproducibility and accuracy, since the MIC is indicated by a color reaction, which is more easily detected than turbidity. This report includes a description of the assay and the results obtained with several beta-lactam antibiotics. MATERIALS AND METHODSBacteria. Fifty-one isolates of H. influenzae were tested. Thirty-nine isolates were smooth, nontypable H. influenzae recovered from sputum specimens submitted to the bacteriology laboratory, San Diego Veterans Administration Hospital. Ten isolates from various clinical specimens were classified serologically as type B and were ampicillin susceptible. Two strains were ampicillin-resistant, beta-lactamase-producing type B strains; one was obtained from Clyde Thornsberry, Center for Disease Control, Atlanta, Ga., ...

Interrogation of Essentiality in the Reconstructed Haemophilus influenzae Metabolic Network Identifies Lipid Metabolism Antimicrobial Targets: Preclinical Evaluation of a FabH β-Ketoacyl-ACP Synthase Inhibitor

“…Despite the presence of amino acids in the CDM composition, i NL638 grew in amino acid absence using nitrate as the nitrogen source, suggesting the lack of amino acid auxotrophies. Interestingly, utilization of nitrate as nitrogen source has been reported in H. influenzae ( 30 – 32 ). Similarly, i NL638 predicted as essential only a few additional CDM nutrients, including uracil, NAD, protoheme, choline, pantothenate, and thiamine.…”

Susceptibility of Haemophilus influenzae to chloramphenicol and eight beta-lactam antibiotics

“…Others have noted that the MBCs of cefamandole for f8-lac' isolates are 4 to 8 times higher than the MICs (9,23 40 ,ug/ml when the inoculum was increased from 105 to 107 CFU/ ml. Fleming and Fierer, using a sensitive nitratereduction method, found that both the MIC and the MBC of cefamandole were higher for ,B-lac+ strains than for fi-lac-strains, even with a 104-CFU/ml inoculum (9).…”

“…Fleming and Fierer, using a sensitive nitratereduction method, found that both the MIC and the MBC of cefamandole were higher for ,B-lac+ strains than for fi-lac-strains, even with a 104-CFU/ml inoculum (9). Yourassowsky et al (23) stated that the cefamandole MIC for such strains did not change when the inoculum was increased to 107 CFU/ml, although the MBC increased from 0.8 to 6.4 utg/ml.…”

Limited in vitro activity of cefamandole against 100 beta-lactamase- and non-beta-lactamase-producing Haemophilus influenzae strains: comparison of moxalactam, chloramphenicol, and ampicillin

“…Although we have no explanation for this observation, which was reproduced in three successive experiments, it may represent an anomaly inherent to serotype differences, 9 of the 11 non-beta-lactamase-producing strains being non-type B H. influenzae. There does not seem to be a good correlation between the biotype We do not believe that the inoculum (7,9,25), the medium, a misreading of the plates (7) …”