ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1989, p. 1526-1530 Vol. 33, No. 9 0066-4804/89/091526-05$02.00/0 Copyright © 1989, American Society for Microbiology Hyperoxia and the Antimicrobial Susceptibility of Escherichia coli and Pseudomonas aeruginosa K. H. MUHVICH, M. K. PARK, R. A. M. MYERS, AND L. MARZELLA* Department of Pathology, University of Maryland School of Medicine, and Division of Hyperbaric Medicine, Maryland Institute for Emergency Medical Services Systems, 10 South Pine Street, Baltimore, Maryland 21201 Received 22 December 1988/Accepted 26 June 1989 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 bacterio- static 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 cation- supplemented 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 reduction- oxidation-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. Hypoxic tissues are particularly susceptible to the estab- (14). Adriamycin, for example, generates fourfold more lishment of mixed aerobic-anaerobic bacterial infections. oxygen-derived free radicals at 100% 02 compared with at These types of infections cause localized abscesses or may 21% 02 (19). spread along fascial planes to cause necrosis of muscle and Finally, hyperoxia may be lethal to bacteria (17) and skin (7). Standard treatment consists ofincision and drainage parasites (5) that lack adequate antioxidant defenses by of infected tissues and intravenous administration of antimi- increasing the generation of 02- and other toxic oxygen crobial agents (6). The rationale for using hyperoxia in these species by cellular metabolic reactions. infections is to restore normal 02 tensions in ischemic tissue, In this work, we have tested representative compounds improve oxidative cellular metabolism (13), and enhance from three classes of antimicrobial agents for interactions phagocytic killing of bacteria (12, 18). with hyperoxia in bacterial killing. The following interac- It has also been suggested that hyperoxia may be benefi- tions may exist between hyperoxia and antimicrobial agents: cial by increasing the activity of specific antimicrobial hyperoxia alone has no effect but potentiates the mode of agents. The best evidence in favor of this proposal was action of the antimicrobial agent, and hyperoxia does not presented by Gottlieb and co-workers (8, 15). In a series of potentiate the mode of action of the antimicrobial agent but in vitro and in vivo experiments, they reported synergy has an effect which is either additive or synergistic with the between sulfonamides and hyperoxia against Vibrio anguil- antimicrobial agent. Synergy exists when oxygen and an larum (15) and Moraxella (Branhamella) catarrhalis (8). antimicrobial agent combine to produce an effect which is Evidence of decreased bacteriostatic and bactericidal activ- greater than the sum of their activities taken separately. ity of aminoglycosides in anaerobic environments has been Additive effects result when the actions of oxygen and an presented by several laboratories (24, 27-29); hyperoxia can antimicrobial agent are equal to the sum of their activities restore the bactericidal activity of tobramycin (1). However, taken separately. The use of a bacterial strain which is it is not known whether hyperoxia potentiates the bacteri- sensitive to hyperoxia maximized the chances of seeing an cidal activity of aminoglycosides in comparison with normal effect, whether it was additive or synergistic. Endpoint oxygen conditions. Hyperoxia may also increase bacteri- colony counting of bacteria by a pour plate method was used cidal activity of antimicrobial agents, such as nitrofurantoin in addition to MIC and MBC testing for the detection of and nifurtimox, by increasing the generation of superoxide interactions between oxygen and antimicrobial agents. anion (027). These antimicrobial agents undergo one-elec- (Portions of this study were presented at the Annual tron reduction to free radicals (nitrofurantoin + e-- Scientific Meeting of the Undersea and Hyperbaric Medical nitrofurantoin-). In the presence of oxygen, the antimicro- Society, New Orleans, La., 1988.) bial agent free radical undergoes oxidation to generate 02 (5) (nitrofurantoin- + 02 -- nitrofurantoin + 027). The sum MATERIALS AND METHODS of these reactions is termed reduction-oxidation cycling. Chemicals. Nitrofurantoin, trimethoprim, sulfamethox- The generation of superoxide anion from the drug free azole, gentamicin, tobramycin, nitrofurazone, menadione, radical follows first-order kinetics with respect to oxygen rifamycin SV, and NADPH were purchased from Sigma Chemical Co., St. Louis, Mo. Nitrofurantoin was solubilized * Corresponding author. in dimethylsulfoxide. Sulfamethoxazole and trimethoprim 1526 VOL. 33, 1989 02 INTERACTIONS WITH ANTIMICROBIAL AGENTS AND BACTERIA 1527 were diluted in 0.1 N NaOH and 0.05 N HCI, respectively. pour plates of serial 10-fold dilutions at 24 h of incubation. Stock solutions of gentamicin and tobramycin were made in The growth control well and the wells containing concentra- 0.1 M phosphate buffer (pH 7.8) and supplemented with 25 tions of antibiotics two and four times the MIC for each ,ug of Mg2+ and 50 ,ug of Ca2+ per ml (22). The stock organism were chosen for quantification of bacterial num- solutions of antibiotics were adjusted to pH 7.4, filter steril- bers. ized (pore size, 0.20 ,um), and diluted to desired concentra- Exposure of bacterial cultures to hyperoxia. Bacterial cul- tions. tures were treated in a small (27 by 50 cm) Plexiglas Bacterial strains. A clinical isolate of Escherichia coli hyperbaric chamber (model 1224; Bethlehem Corporation, (serotype 018) was used for MIC and MBC determinations, Bethlehem, Pa.). Cultures were exposed to 98% 02-2% CO2 endpoint quantitation of bacterial numbers, and polaro- at a pressure of 2.8 ATA for 24 h. The pressure at which the graphic measurement of 02 consumption. Two reference 02 was administered (2.8 ATA) is used clinically and does strains, E. coli ATCC 25922 and Pseudomonas aeruginosa not induce toxicity in human patients. Before compression, ATCC 27853, were used for MIC and MBC determinations the chamber was thoroughly flushed with 98% 02-2% CO2. and endpoint colony counting of bacteria. The temperature in the chamber was maintained at 35 to MIC and MBC determinations. MICs were determined by 37°C by circulating heated water through coils of copper broth macrodilution testing (22). MIC testing was performed tubing located in the bottom of the chamber. in 12-well (22-mm-diameter) culture plates (Costar, Cam- Polarographic measurement of antimicrobial agent reduc- bridge, Mass.). Serial twofold antimicrobial agent dilutions tion-oxidation cycling. The ability of nitrofurantoin, ni- were made in either minimal salts-acetate broth medium trofurazone, menadione, and rifamycin SV to generate su- (MSA) as described by Brunker and Brown (3) or Mueller- peroxide anion was measured by oxygen consumption Hinton broth supplemented with 50 ,ug of Ca2' and 25 ,ug of induced in rat liver microsomes and in E. coli serotype 018. Mg2+ per ml. Each well contained 0.5 ml of antimicrobial Oxygen consumption was measured with a YSI model 53 agent diluted in specific medium, 0.4 ml of bacterial biological oxygen monitor equipped with a Clark-type elec- inoculum in the same medium, and 0.1 ml of 1 M HEPES (N- trode, standard bath stirrer assembly (Yellow Springs Instru- 2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer ment Co., Yellow Springs, Ohio), and strip chart recorder (pH 7.4). A growth control well (bacterial inoculum and (Beckman Instruments Co., Irvine, Calif.) by using a modi- HEPES buffer) and a sterility control well (growth medium fication of the polarographic assay described by Hassan (11). and HEPES buffer) were included with each MIC test. The A microsomal fraction was prepared from rat liver (2) to total volume per well was 1.0 ml, with a depth of 3 mm. This test for reduction of nitrofurantoin, nitrofurazone, menadi- depth was selected to maximize 02 penetration into the one, and rifamycin SV by cytochrome P-450 reductase. For medium as shown by Gudewicz et al. (10). The final bacterial microsomal assays, each antimicrobial agent (0.7 mM) and inoculum was approximately 5 x 105 CFU/ml and was NADPH (0.4 mM) were added to 50 mM potassium