We have tested the ability of hyperoxia (98% 02-2% CO2 at 2.8 atmospheres absolute [ca. 284.6 kPa]) to enhance killing of Escherichia coli (serotype 018 or ATCC 25922) by nitrofurantoin, sulfamethoxazole, trimethoprim, gentamicin, and tobramycin. We have also looked for interactions between hyperoxia and the aminoglycosides against Pseudomonas aeruginosa ATCC 27853. Hyperoxia significantly enhanced bacteriostatic activity of nitrofurantoin and trimethoprim as measured by MIC testing. The possibility exists that these effects might be due to the method required to test MICs under hyperoxic conditions rather than to the effect of hyperoxia itself. In addition, hyperoxia enhanced killing of bacteria by trimethoprim as measured by MBC testing. Hyperoxia decreased numbers of E. coli by 1.3 loglo and P. aeruginosa by 2.7 log1o in cationsupplemented Mueller-Hinton broth medium. The bacteriostatic effects of hyperoxia did not affect MICs of gentamicin or tobramycin. The lack of interaction between hyperoxia and gentamicin or tobramycin was confirmed by determining the number of viable bacteria remaining after 24 h of exposure to hyperoxia by using a pour plate method. We conclude that hyperoxia potentiates the antimicrobial activity of the reductionoxidation-cycling antibiotic tested (nitrofurantoin) and of one of the antimetabolites tested (trimethoprim). Hyperoxia does not enhance the bactericidal effects of gentamicin and tobramycin, which require oxidative metabolism for transport into bacterial cells.

Hyperoxia prolongs the postantibiotic effect (PAE) of the aminoglycoside tobramycin in Pseudomonas aeruginosa. We tested the hypothesis that the PAE is prolonged because hyperoxia increases free radical flux while tobramycin inhibits the induction of antioxidant defenses. Exposure ofP. aeruginosa to hyperoxia (100%O 02) for 1 h increased superoxide dismutase, catalase, and glutathione levels. In the presence of tobramycin (1 x the MIC), the induction of antioxidant defenses by hyperoxia was nearly abrogated. Neither preexposure ofP. aeruginosa to hyperoxia nor supplementation with the antioxidants copper(II) (diisopropylsalicylate)2 (superoxide dismutase-like), catalase, or dimethyl sulfoxide abolished prolongation of the PAE of tobramycin induced by hyperoxia.We recently showed that hyperoxia acts in synergy with an aminoglycoside antibiotic to prolong the postantibiotic effect (PAE) of tobramycin against Pseudomonas aeruginosa (18). The PAE describes the period of bacterial growth suppression that results from a brief exposure to an antimicrobial agent (14). For aminoglycosides, the PAE represents the time required for synthesis of new ribosomes (4).Hyperoxia increases the intracellular flux of 02 and other reactive oxygen species (8). In the study described here, we tested the hypothesis that hyperoxia enhances the PAE of tobramycin against P. aeruginosa by increasing the intracel- MIC of tobramycin for P. aeruginosa ATCC 27853 was 0.5 p,g/ml.The antioxidant enzymes and free radical scavengers were used at the highest concentration which did not inhibit bacterial growth, namely, CuDIPS at 1 x 10-4 M, CAT at 4 x 10' M, and DMSO at 0.4 M. The antioxidants were added before exposure to hyperoxia. The PAE was measured as described previously (18).For enymatic and thiol assays, bacteria (final concentration, -10' CFU/ml) were grown for 2 h in a shaking water bath under ambient conditions (21% 02, 37°C, 100 rpm) before a 1-h exposure to hyperoxia and/or tobramycin (lx the MIC). Bacteria were then filtered onto 0.2-,m-pore-size filters, washed, and resuspended in 50 mM phosphate buffer (pH 7.0); filters were removed after vortexing. Bacteria were centrifuged at 10,000 x g for 15 min at 4°C. After decanting and resuspending with 50 mM phosphate buffer (pH 7.0) with 0.1 mM EDTA, bacterial cells were lysed by sonication (model W185 sonifier; Branson, Plainview, N.Y.) at 60 W (45 s, eight times). Membranes were sedimented by centrifugation at 27,500 x g for 30 min.