Physiological concentrations of calcium in serum antagonize the activities of colistin, polymyxin B, and gentamicin against Pseudomonas aeruginosa. Studies were carried out to determine whether tobramycin, a new aminoglycoside antibiotic, is also antagonized by calcium. The activity of tobramycin in vitro was shown to be antagonized by human serum and by physiological concentrations of calcium. The addition of human serum in broth-dilution tests produced a fourfold rise in the minimal inhibitory concentrations of tobramycin for five strains of Pseudomonas. In disc diffusion tests, the addition of calcium to the agar significantly decreased the size of inhibition zones, and the addition of a chelating agent to the agar increased the zone sizes. In a limited comparative study, tobramycin and gentamicin were tested against both light and heavy bacterial inocula of two strains of Pseudomonas. Tobramycin appeared to be antagonized less by serum than was gentamicin at equal antibiotic concentrations.Tobramycin, formerly designated nebramycin factor 6 (6), is a new aminoglycoside antibiotic that has a broad spectrum and high activity against Pseudomonas aeruginosa (1, 8). Physiological concentrations of calcium are known to antagonize the activities of gentamicin, colistin, and polymyxin B against P. aeruginosa (2-4). The present study was designed to determine whether the activity of tobramycin against Pseudomonas is also antagonized by calcium.
MATERIALS AND METHODSA group of 21 strains of P. aeruginosa, representing seven immunotypes, were obtained from Myron Fisher of Detroit, Mich. (5). Five strains of Escherichia coli were obtained from the Clinical Microbiology Laboratory of the University Hospital. Tobramycin was supplied by Eli Lilly & Co. as a 1 mg/ml solution and was stored at 4 C. Gentamicin powder, a gift of Schering Corp., was reconstituted in sterile water at 100 mg/ml and was stored at -65 C until used. Serum from healthy adult donors was also stored at -65 C until used.Studies on the interaction of antibiotic, serum, and other reagents in broth were carried out as described previously (2-4). Tubes contained 0.4 ml of antibiotic in Mueller-Hinton broth (BBL), 0.4 ml of bacterial inoculum in broth, and 0.8 ml of other reagents in broth. Bacteria from an overnight culture were grown at 37 C in broth for 2 to 3 hr and then were diluted in broth to give a final concentration of about 3 X 103 organisms per ml. Antibiotic, bacteria, and other reagents were mixed at time zero by agitation, and incubation was begun at 37 C in a shaker-water bath.At time zero and at 3 hr, samples of 0.1 ml were taken and streaked onto the surface of agar plates. The plates were incubated overnight and the colonies were counted.Tests for antibiotic susceptibility were carried out by a twofold dilution technique in Mueller-Hinton broth with and without various concentrations of human serum. Tubes contained 0.4 ml of antibiotic in broth, 0.4 ml of bacterial inoculum in broth, and 0.8 ml of serum diluted in broth. The bacterial inoc...
