Vol. 35, No. 4
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1991, p. 691-695
0066-4804/91/040691-05$02.00/0 Copyright ©D 1991, American Society for Microbiology
Hyperoxia Prolongs the Aminoglycoside-Induced Postantibiotic Effect in Pseudomonas aeruginosa MATTHEW K. PARK,"2 KENNETH H. MUHVICH,1 2t ROY A. M. MYERS,' AND LOUIS MARZELLAl2* Division of Hyperbaric Medicine, Maryland Institute for Emergency Medical Services Systems,' and Department of Pathology, University of Maryland School of Medicine, 10 South Pine Street,2 Baltimore, Maryland 21201 Received 2 October 1990/Accepted 16 January 1991
The objective of this study was to determine whether hyperoxia enhances aminoglycoside activity against Pseudomonas aeruginosa. The existence of tobramycin-oxygen synergy was determined by using the in vitro postantibiotic effect (PAE). P. aeruginosa strains were incubated for 1 h in medium containing tobramycin at four times the MIC in the following gas mixtures: normoxia (21% 02), hyperoxia (100% 02 101.3 kPa), or hyperbaric oxygen (100% 02, 274.5 kPa). Tobramycin was removed after 1 h and bacteria were incubated under normoxic conditions; growth rates were measured for 5 h. Exposure of three P. aeruginosa strains to hyperoxia prolonged the PAE of tobramycin approximately twofold compared with the PAE after exposure to normoxia (P < 0.05). Exposure of P. aeruginosa ATCC 27853 to tobramycin and hyperbaric oxygen prolonged the time required for bacteria to increase 1 loglo CFU/ml compared with the time after exposure for this increase to occur in tobramycin-treated, normoxic or hyperoxic groups (P < 0.02). Pulse-chase labeling of bacteria with L-[35S]methionine, immediately after removal of tobramycin, showed that protein synthesis rates were decreased compared with those in controls (P = 0.0001). Moreover, in tobramycin-treated groups, hyperoxia and hyperbaric oxygen induced 2- and 16-fold decreases, respectively, in protein synthesis rates compared with normoxia; these results did not achieve statistical significance. In the absence of tobramycin, hyperoxia increased bacterial growth (134%; P < 0.01) and protein synthesis (24%; not significant) compared with normoxia. Hyperbaric oxygen, however, delayed the growth recovery of bacteria (P < 0.05). We conclude that hyperoxia enhances the bacteriostatic effects of tobramycin in a synergistic manner. Hyperbaric oxygen also enhances the bacteriostatic effects of tobramycin against P. aeruginosa. Our findings may have relevance to the in vivo growth rates of P. aeruginosa in tissues of patients treated with high inspired oxygen tensions.
The activities of many antimicrobial agents are altered by changes in oxygen tension. Anaerobic (anoxic) conditions markedly diminish aminoglycoside activity (32, 36, 37), because energy derived from quinone-associated electron transport is required to facilitate uptake of the antimicrobial agent into gram-negative bacteria (9). The loss of aminoglycoside bactericidal activity induced by an anoxic environ-
kPa) and a sulfonamide-trimethoprim combination against Vibrio anguillarum in vitro. Hyperoxia enhanced trimethoprim activity, presumably by oxidizing enzymes or metabolic intermediates involved in folate metabolism in bacteria (14). Hyperbaric oxygen and sodium sulfisoxazole increased the survival of V. anguillarum-infected goldfish more than threefold (20). Hyperoxia enhanced the bacteriostatic activity of nitrofurantoin and both the bacteriostatic and bactericidal activities of trimethoprim against E. coli (25). The purpose of the current study was to test the hypothesis that elevated oxygen tensions enhance the activity of antimicrobial agents that inhibit bacterial protein synthesis. Experiments were designed so that hyperoxia could act synergistically with tobramycin to suppress the growth of P. aeruginosa, independent of bacterial killing. To this end, we used the PAE, defined as the period of bacterial growth suppression that results from the brief exposure of bacteria to an antimicrobial agent (12, 23).
ment can be restored by hyperbaric oxygen (22). The activity of vancomycin against Staphylococcus aureus is also markedly diminished in an anaerobic environment, and this may account for the failure of the antimicrobial agent to sterilize infected tissues, such as bone, where low oxygen tensions are found (30). In addition, the bactericidal activity of ciprofloxacin is abolished under anaerobic conditions (35). Oxygen tensions within the physiologic range (20 to 150 mm Hg [2.7 to 20 kPa]) found in body tissues (18, 39) can also influence the bactericidal activities of antimicrobial agents. For example, amikacin showed less killing and a shorter postantibiotic effect (PAE) against Pseudomonas aeruginosa at a partial pressure (PO2) of 80 mm Hg (10.6 kPa) than it did at a P02 of 40 mm Hg (5.3 kPa) (2). Concentrations of oxygen in the normoxic range also appear to be required for optimal killing of Escherichia coli by streptonigrin (16). Elevated oxygen tensions have been shown to potentiate the activities of some antimicrobial agents. Keck et al. (20) found synergy between hyperbaric oxygen (100% 02, 324.2
MATERIALS AND METHODS
Reagents and media. Tobramycin was obtained from Sigma Chemical Co. (St. Louis, Mo.). Stock solutions (1,000 p.g/ml) were stored frozen at -135°C in 0.1 M phosphate buffer (pH 7.0) (28). L-[35S]methionine, in vivo cell labeling grade (specific activity 1,371 Ci/mmol), was purchased from Amersham (Arlington Heights, Ill.), subdivided, stored at -70°C, and used within 3 weeks. L-Methionine was purchased from Sigma. Mueller-Hinton broth (Difco, Detroit, Mich.) was supplemented with Mg2+ (25 mg/ml) and Ca2+ (50 mg/ml) (28). Bacterial strains. Two American Type Culture Collection
* Corresponding author. t Present address: Division of Altitude and Hyperbaric Physiology, Armed Forces Institute of Pathology, Washington, DC 20306-
6000.
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(ATCC; Rockville, Md.) strains and six clinical strains (Sinai Hospital, Baltimore, Md.) of P. aeruginosa were initially screened to determine whether hyperoxia could prolong the PAE of tobramycin. Five of the eight Pseudomonas strains showed enhanced PAEs following hyperoxic exposure. Of these five strains, three strains (see Table 1) were selected for further examination. Bacteria were stored in 15% glycerol at -70°C. Bacteria were grown in tryptic soy broth overnight at 37°C. A 1:30 dilution of overnight culture was made into tryptic soy broth, and bacteria were grown in a shaking water bath (100 rpm, 4 h, 37°C). Bacteria were optically matched with a 0.5 McFarland standard and diluted to 107 CFU/ml in prewarmed, cation-supplemented MuellerHinton broth. In vitro PAE. Six assay conditions were tested: 21% 02 21% 02 and tobramycin, hyperoxia (100% 02 101.3 kPa), hyperoxia and tobramycin, hyperbaric oxygen (100% 02, 274.5 kPa), and hyperbaric oxygen and tobramycin. MICs for P. aeruginosa ATCC 27853, SH 6468, and SH 7132 were 0.5, 0.5, and 2 .g/ml, respectively, as determined by a microdilution method (28). Suspensions of P. aeruginosa were exposed to various oxygen tensions and/or tobramycin (4x the MIC) for 1 h, and then a 0.1-ml aliquot was sampled from the bacterial culture. Phosphate buffer was added in place of tobramycin in control cultures. Tobramycin was then removed from cultures by filtering bacteria onto 0.2,um-pore-size filters (MSI, Westboro, Mass.) prerinsed with prewarmed cation-supplemented Mueller-Hinton broth and washing three times. The filter was transferred into 20 ml of prewarmed cation-supplemented Mueller-Hinton broth. After 30 s of vortexing, the filters were removed aseptically, and another 0.1-ml aliquot was sampled; this time point was denoted as time zero. No further bacterial killing was evident after the removal of tobramycin. Bacterial suspensions were incubated in a shaking water bath (37°C) and grown under normoxic conditions (21% 02) for the remainder of the experiment. Removal of tobramycin resulted in a 3-log1o-CFU/ml reduction in bacterial numbers when the standard 1:1,000 drug dilution method was used (12). Other investigators (1, 11) have used filtration for drug removal to determine the PAE. Because of the 3.5-log1o-CFU/ml killing of P. aeruginosa by tobramycin at the standard 4 x MIC (see Table 4), a filtration modification (13a) was used to remove tobramycin, and less than 1 log1o CFU of bacteria per ml was lost by this procedure. This particular filtration method was previously compared with the standard 1: 1,000 dilution method and showed an excellent correlation (32a). Bacterial growth and viability were measured by enumeration, because it has recently been shown that this method is suitable for quantification of bacteria exposed to reactive oxygen intermediates (24). Aliquots of 0.1 ml were removed every 0.5 h for the first 3 h of incubation and at two additional 1-h time points. Less than 10% of the starting volume was removed for serial dilutions over the course of the experiment. Serial 10- or 100-fold dilutions of the 0.1-ml aliquots were performed in duplicate by using cold, sterile 0.9% saline. Pour plates were made of selected dilutions by using tryptic soy agar. Colonies were counted between 24 and 48 h after incubation at 37°C. Duplicate determinations were made for each time point. The in vitro PAE was calculated by using the following formula (12): PAE = T C, where T is the time required for the CFU per milliliter in the antimicrobial agent-treated bacterial culture to increase 1 log1o unit above the CFU per milliliter observed immediately after resuspension of bacteria into drug-free medium, and C represents the time re-
an untreated control culture to increase 1 log1o unit above the CFU per milliliter observed immediately after resuspension of bacteria into drug-free medium. Exposure of bacterial cultures to hyperbaric oxygen. Bacterial cultures were exposed to 100% 02 at a pressure of 274.5 kPa in a steel hyperbaric chamber (Bethlehem Corp., Bethlehem, Pa.). The hyperbaric chamber was fitted with brass penetrators which housed oxygen lines and polyethylene tubing for bacterial inoculation. Battery-operated magnetic stirrers (Bel-Art Products, Pennaquinnock, N.J.) were used to ensure adequate oxygen saturation of the media. Cation-supplemented Mueller-Hinton broth containing tobramycin or phosphate buffer was gassed with 100% 02 at 6 liters/min for 30 min before the addition of bacteria. After a 1-h incubation, the chamber was decompressed within 10 s. Determination of protein synthesis rates. Bacteria were filtered onto 0.2-[Lm-pore-size filters, washed, and resuspended in 10 ml of methionine-free, minimal salts acetate medium (8), because cation-supplemented Mueller-Hinton broth was found to contain 1 mM methionine (unpublished data). After vortexing for 30 s, filters were removed aseptically. Bacteria were collected on 25-mm-diameter filters (pore size, 0.2 [Lm; Millipore, Bedford, Mass.) which were prerinsed with 1 mM unlabeled methionine. Protein synthesis rates in P. aeruginosa were measured by pulsing bacteria with 0.5 pXCi of L-[35S]methionine for 5 min at 37°C and by chasing with 1 mM unlabeled methionine for 5 min at room temperature. Each measurement was performed in triplicate, and background radioactivity was subtracted. Background radioactivity was due to nonspecific binding of L-[35S]methionine to filters after washing in the absence of bacteria. Filters were dried for 30 min at 60°C before 5 ml of Biosafe-II scintillation fluid (Research Products International Corp., Mount Prospect, Ill.) was added. After an overnight incubation in the dark, the filters were counted in a Beckman LS 7800 scintillation counter. Data analysis. The Mann-Whitney test, Kruskal-Wallis one-way analysis of variance by rank sums, and a repeated measures design analysis of variance (SAS procedure GLM) were used for statistical analysis. When necessary, the Tukey honestly significant difference procedure was used as a posttest. Data were considered significant at P < 0.05.
quired for the CFU per milliliter in
RESULTS Hyperoxia and the in vitro PAE of tobramycin. To determine the PAE under hyperoxic conditions, 18 ml of cationsupplemented Mueller-Hinton broth, containing 4x the MIC of tobramycin, was pregassed at a rate of 3 liters/min for 10 min before adding 2 ml of bacteria (108 CFU/ml). Ten minutes was sufficient for >95% oxygen saturation of the medium. The data in Table 1 show that under normoxic conditions, tobramycin had a PAE that ranged from 0.7 to 1.9 h for ATCC 27853 and two clinical isolates of P. aeruginosa. Hyperoxia (100% 02 101.3 kPa) extended the PAE of tobramycin approximately twofold compared with normoxia in all three strains of P. aeruginosa (P < 0.05). The effect of oxygen tensions normally found in tissues (20 to 40 mm Hg [2.7 to 5.3 kPa]) on the PAE was also tested by exposing P. aeruginosa ATCC 27853 to 3.7% 02 (26 mm Hg [3.5 kPa]). The PAE (0.82 h) at 26 mm Hg (3.5 kPa) of 02 was not statistically different from the PAE (0.70 h) at 150 mm Hg
of 02 (20 kPa) (data not shown). Hyperbaric oxygen and the growth recovery of P. aeruginosa ATCC 27853. A reference ATCC strain of P. aerugi-
HYPEROXIA PROLONGS THE PAE OF TOBRAMYCIN
VOL. 35, 1991 TABLE 1. Effect of hyperoxia on the PAE of tobramycin in P. aeruginosa
ATCC 27853 (9) SH 6468 (6) SH 7132 (4)
L-[35Slmethionine incorporation (dpm/logio CFU [n])a
21% 02c
100% 02d
0.70 ± 0.13 1.00 ± 0.23 1.91 + 0.05
1.42 ± 0.l9e 2.50 ± 0.58f 3.11 ± 0.17e
a n, Number of experiments. b PAE = T C (where T is the time required for bacterial growth in tobramycin-treated group to increase 1.0 loglo CFU/ml after removal of the antibiotic by filtration, and C is the time required for bacteria not treated with tobramycin to increase 1.0 loglo CFU/ml). Numbers are means + standard errors of the mean and represent the duration of PAE. c Bacteria were exposed to 21%02 and tobramycin (4x the MIC) for 1 h and were maintained at 21%02 after the removal of tobramycin. d Bacteria were exposed to 100% 02 (101.3 kPa) and tobramycin (4x the MIC) for 1 h and subsequently returned to normoxic conditions (21% 02) after the removal of tobramycin. e Significantly different (P < 0.02) from the value found under normoxic conditions. f Significantly different (P < 0.05) from the value found under normoxic conditions.
nosa was used to determine whether the PAE of tobramycin could be further enhanced by using the maximal oxygen dose that is used clinically. As seen in Table 2, hyperbaric oxygen (100% 02, 274.5 kPa) alone significantly prolonged the time necessary for a 1-log1o-CFU/ml increase in bacterial growth compared with normoxia (P < 0.05) and hyperoxia (P < 0.01). In the presence of tobramycin, hyperbaric oxygen significantly prolonged the time necessary for a 1-log1oCFU/ml increase in bacterial growth compared with normoxia (P < 0.001) or hyperoxia (P = 0.01). The delayed recovery of bacterial growth seen in the hyperbaric oxygen controls was responsible for the apparent lack of difference in PAE between hyperoxic and hyperbaric oxygen-exposed cultures. Effects of hyperoxia and hyperbaric oxygen on protein synthesis. Bacterial suspensions were incubated under hyperoxic or hyperbaric oxygen conditions in the presence of TABLE 2. Effect of hyperoxia or hyperbaric oxygen on growth recovery of P. aeruginosa ATCC 27853 Time interval for growth recovery (h)b TobramycinControl treated
Oxygen exposure (na)
Normoxia (9) Hyperoxia (9) Hyperbaric oxygen (4)
2.20 ± 0.13 2.03 ± 0.12 2.67 ± O.O9fg
2.89 ± 0.13c 3.45 + 0. 9d.e 4.42 + 0.18c'h,i
n, Number of experiments. Bacteria were exposed to tobramycin and one of three oxygen tensions for 1 h. Tobramycin was removed by filtration. Numbers are means ± standard errors of the mean and represent the time needed for bacteria to increase 1.0 CFU/ml under normoxic conditions after the removal of tobramycin. log1o I Significantly different (P < 0.02) from respective controls without tobraa
b
mycin. d Significantly different (P < 0.002) from respective controls without tobramycin. eSignificantly different (P < 0.02) from the normoxia and tobramycintreated group. f Significantly different (P < 0.05) from normoxia. g Significantly different (P < 0.01) from hyperoxia. h Significantly different (P < 0.001) from the normoxia and
treated group. i Significantly different (P treated group.
TABLE 3. Effect of hyperoxia or hyperbaric oxygen on protein synthesis in P. aeruginosa ATCC 27853
PAE (h)b
Strain (na)
=
693
tobramycin-
0.01) from the hyperoxia and tobramycin-
Oxygen
0h
exposure
Normoxia Hyperoxia Hyperbaric oxygen
1 h,
Control
Tobramycin
415 + 185 (9) 514 ± 237 (9) 202 ± 52 (9)
16.7 6.5b (9) 7.4 ± 4.3b (9) -7.2 + 11.6b.c (9)
Tobramycin
18.5 7.3b (6) 13.8 + 6.3b (6) 6.4 ± 12.1b (3)
a Bacteria were exposed to tobramycin and one of three oxygen tensions for 1 h. Tobramycin was removed by filtration and bacteria were pulsed for 5 min (35°C) under normoxic conditions with 0.5 ,Ci of L-[35S]methionine. Bacteria were then chased for 5 min with 1 mM unlabeled methionine. Data are means + standard errors of the mean. n, Number of experiments. The value 0 h refers to protein synthesis measured immediately after removal of tobramycin; the value 1 h refers to protein synthesis measured 1 h after removal of
tobramycin. b Significantly different (P = 0.0001) from controls at 0 h. c Value approximating background.
tobramycin or phosphate buffer. After 1 h, the bacteria were filtered and resuspended in minimal salts acetate medium (8) under normoxic conditions. Protein synthesis was measured at 0 and 1 h after the removal of tobramycin. Table 3 shows that in the control groups, hyperoxia increased protein synthesis by 24%, while hyperbaric oxygen decreased protein synthesis by 50%; these differences did not achieve statistical significance. In the tobramycin-treated groups, protein synthesis was markedly diminished (P = 0.0001). Table 3 also shows that 1 h after the removal of tobramycin, protein synthesis rates remained essentially unchanged from time zero. At approximately 3 h after tobramycin and normoxia exposure, protein synthesis in bacteria approached normal rates (287 dpm/log1o CFU). Rates of protein synthesis were also measured at 3 h after exposure to tobramycin and hyperoxia and were found to remain low (69 dpm/log1o CFU) (data not shown). Effects of hyperoxia and hyperbaric oxygen on the growth of P. aeruginosa ATCC 27853. During the course of the PAE experiments, bacteria were enumerated before and immediately after a 1-h exposure to hyperoxia or hyperbaric oxygen. Table 4 shows that hyperoxia alone caused a significant increase in bacterial growth (P < 0.05). Tobramycin significantly decreased growth under normoxic, hyperoxic, and hyperbaric oxygen conditions compared with the growth of controls (P = 0.0001). However, killing of P. aeruginosa by tobramycin was maximal under normoxic conditions. DISCUSSION
Hyperoxia significantly enhanced the PAE of tobramycin against P. aeruginosa compared with normoxia. P. aeruginosa was incubated for 1 h with 4x the MIC of tobramycin under normoxic, hyperoxic, or hyperbaric oxygen conditions. After the 1-h exposure, tobramycin was removed and the bacteria were incubated under normoxic conditions. The maximal killing of P. aeruginosa which occurred under normoxic conditions is in agreement with our previous work (25). In the case of amikacin, antimicrobial activity was found to be significantly diminished at 80 mm Hg (10.7 kPa) of 02 compared with that at 40 mm Hg (5.3 kPa) of 02, as evidenced by decreased killing and shortened PAEs in P. aeruginosa (2). However, at lowered oxygen tensions, such as those present physiologically in tissues (26 mm Hg [3.5
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TABLE 4. Effect of hyperoxia or hyperbaric oxygen on the growth of P. aeruginosa ATCC 27853 Oxygen exposure (na)
Viable bacteria (mean logl0 CFU/ml ± SEM) TobramycinOriginal Controlb treatedb inoculum
Normoxia (9) Hyperoxia (9) Hyperbaric oxygen (3)
6.96 ± 0.04 7.02 + 0.07 3.33 ± 0.20' 6.96 ± 0.04 7.33 ± 0.04d 3.41 ± 0.13c 6.86 ± 0.01 6.74 ± 0.18e 3.61 ± 0.26C
n, Number of experiments. Viable bacteria remaining after a 1-h exposure to oxygen and/or tobramycin prior to filtration. c Significantly different (P = 0.0001) from similar oxygen-exposed controls. d Significantly different (P < 0.05) from original inoculum. e Significantly different (P = 0.0023) from hyperoxia. a
b
kPa]), we found no significant difference compared with normoxia in the PAE caused by tobramycin. Hyperoxia increased protein synthesis and the growth rates of P. aeruginosa. These results are in agreement with those of Ollodart (31), who found that hyperoxia enhances the growth of E. coli. Aminoglycoside uptake by bacteria has been shown to be dependent upon growth rates or quinonedependent bacterial respiration (26, 27, 29). Based on these findings, the prolongation by hyperoxia of the PAE in P. aeruginosa may be the result of increased tobramycin uptake because of enhanced growth and/or respiration rates. To determine the effect of growth rates on the PAE, P. aeruginosa was preexposed to hyperoxic conditions for 1 h and then incubated with tobramycin under normoxic conditions. Despite the increased growth rates induced by preexposure to hyperoxia, there was no significant difference in the PAE (data not shown). These results indicate that the observed synergy between hyperoxia and tobramycin is not related to changes in bacterial growth. However, it is possible that hyperoxia may enhance the PAE of tobramycin by inducing oxidative stress in P. aeruginosa. Hyperoxia generates reactive oxygen intermediates, which inhibit respiration (6) and protein synthesis (3) and decrease NAD levels (8) in bacteria. These reactive oxygen intermediates can induce the synthesis of superoxide dismutase (15, 38) and other antioxidant or repair enzymes (38). In the presence of tobramycin, which induces misreading of proteins at low concentrations and inhibits protein synthesis at high concentrations (13), synthesis of functional antioxidant or repair enzymes does not occur. The reactive oxygen intermediates generated under hyperoxic conditions, in the absence of newly synthesized antioxidant enzymes, may then inactivate the proteins (5, 7, 17) essential for bacterial growth. Aminoglycosides are thought to bind to ribosomes irreversibly, and it is postulated that the PAE of tobramycin may represent the additional time needed to synthesize functional ribosomes (12). The enhancement of the PAE by hyperoxia may be the result of the effects of oxidative stress on the biosynthesis of ribosomal proteins, either directly by inactivation or indirectly through reduced energy levels. The significantly delayed growth recovery of P. aeruginosa by hyperbaric oxygen (100% 02, 274.5 kPa) was independent of the presence of tobramycin (Table 2). Exposure to hyperbaric oxygen for 18 to 24 h inhibited the growth of P. aeruginosa (4, 19, 25). Brunker and Brown (8) found that a 2-h exposure of E. coli to 100% 02 (607.8 kPa) was bacteriostatic. Interestingly, in this study, we found that only a 1-h exposure of P. aeruginosa to hyperbaric oxygen
(100% 02, 274.5 kPa) was needed to delay the subsequent recovery of bacterial growth under normoxic conditions (Table 2). This delay in recovery of bacterial growth war; associated with a twofold decrease in protein synthesis (Table 3). It is known that hyperbaric oxygen can inhibit the biosynthesis of amino acids (3, 7) and can induce a stringent response (33). The resultant increase in tetra- and pentaphosphorylated guanosine levels can inhibit the synthesis of carbohydrates, lipids, and nucleotides, in addition to increasing proteolysis (10). These metabolic effects could explain the delay in recovery of bacterial growth by shortterm exposure (1 h) to hyperbaric oxygen. Because differences in P02 exist in various body tissues, it is likely that the variation in aminoglycoside activity seen in vitro will also be found in vivo. Under physiologic conditions, different compartments of the body are exposed to a wide range of pO2s. For example, under normoxic conditions inspired P02S of 150 mm Hg (20 kPa) and alveolar P02S of 100 mm Hg (13.3 kPa) are found (39); P02s are approximately 95 mm Hg (12.7 kPa) in arterioles and are reduced to 40 mm Hg (5.3 kPa) in the venous circulation (39). pO2s range from 20 to 40 mm Hg (2.7 to 5.3 kPa) in many visceral tissues (18). In the presence of hyperoxia, alveolar and arterial P02s are ideally 673 and 660 mm Hg, respectively (89.7 and 88.0 kPa, respectively) (34). Under hyperbaric oxygen conditions (308.0 kPa), arterial P02 levels can exceed 1,700 mm Hg (226.6 kPa) (40). At a lower concentration of hyperbaric oxygen (253.2 kPa), pO2s of 800 mm Hg (106.6 kPa) can be found in subcutaneous tissue (21). It can be concluded that different body compartments can have elevated P02S that approach or exceed the levels attained in vitro under hyperoxic conditions. Elevated oxygen tensions may thus influence the effectiveness of aminoglycosides for infections at different body sites. In summary, hyperoxia enhanced the PAE of tobramycin against P. aeruginosa in vitro. The PAE of tobramycin has been demonstrated to be up to three times longer in vivo than in vitro (12). Hyperoxia may extend the in vivo PAE and therefore prolong the therapeutic activity of aminoglycosides in patients exposed to hyperoxia or hyperbaric oxygen. ACKNOWLEDGMENTS This study was supported by the Maryland Institute for Emergency Medical Services Systems Research Fund. We thank George L. Drusano for helpful advice and Harry L. T. Mobley for critical review of the manuscript. We thank Patricia L. Schleiff and William F. McCarthy for statistical analysis of the data. We gratefully acknowledge the excellent help of Gargi Raval in determining the PAEs. The technical assistance of Paul P. Rodier and Bret Holliday in the maintenance and operation of the hyperbaric chamber is gratefully appreciated. We thank Clifton Bennett, Jr., for providing the clinical strains of P. aeruginosa. REFERENCES 1. Baquero, F., E. Culebras, C. Patron, J. C. Prez-Diaz, J. C. Medrano, and M. F. Vicente. 1986. Postantibiotic effect of imipenem on gram-positive and gram-negative micro-organisms. J. Antimicrob. Chemother. 18(Suppl. E):47-59. 2. Bayer, A. S., T. O'Brien, D. C. Norman, and C. C. Nast. 1989. Oxygen-dependent differences in exopolysaccharide production and aminoglycoside inhibitory-bactericidal interactions with Pseudomonas aeruginosa-implications for endocarditis. J. Antimicrob. Chemother. 23:21-35. 3. Boehme, D. E., K. Vincent, and 0. R. Brown. 1976. Oxygen and toxicity inhibition of amino acid biosynthesis. Nature (London) 262:418-420. 4. Bornside, G. H., L. M. Pakman, and A. A. Ordofiez, Jr. 1975.
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Inhibition of pathogenic enteric bacteria by hyperbaric oxygen: enhanced antibacterial activity in the absence of carbon dioxide. Antimicrob. Agents Chemother. 7:682-687. 5. Brot, N., L. Weissbach, J. Werth, and H. Weissbach. 1981. Enzymatic reduction of protein-bound methionine sulfoxide. Proc. Natl. Acad. Sci. USA 78:2155-2158. 6. Brown, 0. R. 1972. Reversible inhibition of respiration of Escherichia coli by hyperoxia. Microbios 5:7-16. 7. Brown, 0. R., and F. Yein. 1978. Dihydroxyacid dehydratase: the site of hyperbaric oxygen poisoning in branch-chain amino acid biosynthesis. Biochem. Biophys. Res. Commun. 85:12191224. 8. Brunker, R. L., and 0. R. Brown. 1971. Effects of hyperoxia on oxidized and reduced NAD and NADP concentrations in Escherichia coli. Microbios 4:193-203. 9. Bryan, L. E., and S. Kwan. 1983. Roles of ribosomal binding, membrane potential, and electron transport in bacterial uptake of streptomycin and gentamicin. Antimicrob. Agents Chemother. 23:835-845. 10. Cashel, M. 1975. Regulation of bacterial ppGpp and pppGpp. Annu. Rev. Microbiol. 29:301-318. 11. Chin, N.-X., A. Novelli, and H. C. Neu. 1988. In vitro activity of lomefloxacin (SC-47111; NY-198), a difluoroquinolone 3-carboxylic acid, compared with those of other quinolones. Antimicrob. Agents Chemother. 32:656-662. 12. Craig, W. A., and S. Gudmundsson. 1986. The postantibiotic effect, p. 515-536. In V. Lorian (ed.), Antibiotics in laboratory medicine, 2nd ed. The Williams & Wilkins Co., Baltimore. 13. Davis, B. D. 1987. Mechanism of bactericidal action of aminoglycosides. Microbiol. Rev. 51:341-350. 13a.Drusano, G. Personal communication. 14. Gottlieb, S. F. 1971. Effect of hyperbaric oxygen on microorganisms. Annu. Rev. Microbiol. 25:111-152. 15. Gregory, E. M., and I. Fridovich. 1973. Induction of superoxide dismutase by molecular oxygen. J. Bacteriol. 114:543-548. 16. Harley, J. B., C. J. Fetterolf, C. A. Bello, and J. G. Flaks. 1982. Streptonigrin toxicity in Escherichia coli: oxygen dependence and the role of the intracellular oxidation-reduction state. Can. J. Microbiol. 28:545-552. 17. Jamieson, D., B. Chance, E. Cadenas, and A. Boveris. 1986. The relation of free radical production to hyperoxia. Annu. Rev. Physiol. 48:703-719. 18. Jamieson, D., and H. A. S. van den Brenk. 1965. Electrode size and tissue P02 measurement in rats exposed to air or high pressure oxygen. J. Appl. Physiol. 20:514-518. 19. Kaye, D. 1967. Effect of hyperbaric oxygen on aerobic bacteria in vitro and in vivo. Proc. Soc. Exp. Biol. Med. 124:1090-1093. 20. Keck, P. E., S. F. Gottlieb, and J. Conley. 1980. Interaction of increased pressures of oxygen and sulfonamides on the in vitro and in vivo growth of pathogenic bacteria. Undersea Biomed. Res. 7:95-106. 21. Kivisaari, J., and J. Niinikoski. 1975. Effects of hyperbaric oxygenation and prolonged hypoxia on the healing of open wounds. Acta Chir. Scand. 141:14-19. 22. Mader, J. T., C. A. Hicks, and J. Calhoun. 1989. Bacterial osteomyelitis. Adjunctive hyperbaric oxygen therapy. Orthop. Rev. 18:581-585. 23. McDonald, P. J., W. A. Craig, and C. M. Kunin. 1977. Persistent effect of antibiotics on Staphylococcus aureus after exposure for limited periods of time. J. Infect. Dis. 135:217-223. 24. Minakami, H., and I. Fridovich. 1990. Effects of paraquat on
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cultures of Escherichia coli: turbidity versus enumeration. Free Radical Biol. Med. 8:387-391. 25. Muhvich, K. H., M. K. Park, R. A. M. Myers, and L. Marzella. 1989. Hyperoxia and the antimicrobial susceptibility of Escherichia coli and Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 33:1526-1530. 26. Muir, M. E., M. Ballesteros, and B. J. Wallace. 1985. Respiration rate, growth rate and the accumulation of streptomycin in Escherichia coli. J. Gen. Microbiol. 131:2573-2579. 27. Muir, M. E., R. S. van Heeswyck, and B. J. Wallace. 1984. Effect of growth rate on streptomycin accumulation by Escherichia coli and Bacillus megaterium. J. Gen. Microbiol. 130:20152022. 28. National Committee for Clinical Laboratory Standards. 1985. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. National Committee for Clinical Laboratory Standards, Villanova, Pa. 29. Nichols, W. W., and S. N. Young. 1985. Respiration-dependent uptake of dihydrostreptomycin by Escherichia coli. Its irreversible nature and lack of evidence for a uniport process. Biochem. J. 228:505-512. 30. Norden, C. W., and M. Shaffer. 1983. Treatment of experimental chronic osteomyelitis due to Staphylococcus aureus with vancomycin and rifampin. J. Infect. Dis. 147:352-357. 31. Ollodart, R. M. 1966. Effects of hyperbaric oxygenation and antibiotics on aerobic microorganisms, p. 565-571. In I. W. Brown, Jr., and B. G. Cox (ed.), Proceedings of the Third International Conference on Hyperbaric Medicine. National Academy of Sciences, Washington, D.C. 32. Reynolds, A. V., J. M. T. Hamilton-Miller, and W. Brumfitt. 1976. Diminished effect of gentamicin under anaerobic or hypercapnic conditions. Lancet i:447-449. 32a.Rider, D. A., G. L. Drusano, and H. C. Standiford. 1989. Abstr. Annu. Meet. Am. Soc. Microbiol. 1989, A 115, p. 20. 33. Seither, R. L., and 0. R. Brown. 1982. Induction of stringency by hyperoxia in Escherichia coli. Cell. Mol. Biol. 28:285-291. 34. Sheffield, P. J. 1988. Tissue oxygen measurements, p. 17-51. In J. C. Davis and T. K. Hunt (ed.), Problem wounds. The role of oxygen. Elsevier Science Publishing Co., New York. 35. Smith, J. T., and C. S. Lewin. 1988. Chemistry and mechanisms of action of the quinolone antibacterials, p. 23-82. In V. T. Andriole (ed.), The quinolones. Academic Press, Inc., New York. 36. Tack, K. J., and L. D. Sabath. 1985. Increased minimum inhibitory concentrations with anaerobiosis for tobramycin, gentamicin, and amikacin compared to latamoxef, piperacillin, chloramphenicol, and clindamycin. Chemotherapy 31:204-210. 37. Verklin, R. M., Jr., and G. L. Mandell. 1977. Alteration of effectiveness of antibiotics by anaerobiosis. J. Lab. Clin. Med. 89:65-71. 38. Walkup, L. K. B., and T. Kogoma. 1989. Escherichia coli proteins inducible by oxidative stress mediated by the superoxide radical. J. Bacteriol. 171:1476-1484. 39. Weibel, E. R. 1984. The pathway for oxygen. Structure and function in the mammalian respiratory system, p. 178-179, 343-345. Harvard University Press, Cambridge, Mass. 40. Whalen, R. E., H. A. Saltzman, D. H. Holloway, Jr., H. D. McIntosh, H. 0. Sieker, and I. W. Brown, Jr. 1965. Cardiovascular and blood gas responses to hyperbaric oxygenation. Am. J. Cardiol. 15:638-646.
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0066-4804/91/040691-05$02.00/0 Copyright ©D 1991, American Society for Microbiology
Hyperoxia Prolongs the Aminoglycoside-Induced Postantibiotic Effect in Pseudomonas aeruginosa MATTHEW K. PARK,"2 KENNETH H. MUHVICH,1 2t ROY A. M. MYERS,' AND LOUIS MARZELLAl2* Division of Hyperbaric Medicine, Maryland Institute for Emergency Medical Services Systems,' and Department of Pathology, University of Maryland School of Medicine, 10 South Pine Street,2 Baltimore, Maryland 21201 Received 2 October 1990/Accepted 16 January 1991
The objective of this study was to determine whether hyperoxia enhances aminoglycoside activity against Pseudomonas aeruginosa. The existence of tobramycin-oxygen synergy was determined by using the in vitro postantibiotic effect (PAE). P. aeruginosa strains were incubated for 1 h in medium containing tobramycin at four times the MIC in the following gas mixtures: normoxia (21% 02), hyperoxia (100% 02 101.3 kPa), or hyperbaric oxygen (100% 02, 274.5 kPa). Tobramycin was removed after 1 h and bacteria were incubated under normoxic conditions; growth rates were measured for 5 h. Exposure of three P. aeruginosa strains to hyperoxia prolonged the PAE of tobramycin approximately twofold compared with the PAE after exposure to normoxia (P < 0.05). Exposure of P. aeruginosa ATCC 27853 to tobramycin and hyperbaric oxygen prolonged the time required for bacteria to increase 1 loglo CFU/ml compared with the time after exposure for this increase to occur in tobramycin-treated, normoxic or hyperoxic groups (P < 0.02). Pulse-chase labeling of bacteria with L-[35S]methionine, immediately after removal of tobramycin, showed that protein synthesis rates were decreased compared with those in controls (P = 0.0001). Moreover, in tobramycin-treated groups, hyperoxia and hyperbaric oxygen induced 2- and 16-fold decreases, respectively, in protein synthesis rates compared with normoxia; these results did not achieve statistical significance. In the absence of tobramycin, hyperoxia increased bacterial growth (134%; P < 0.01) and protein synthesis (24%; not significant) compared with normoxia. Hyperbaric oxygen, however, delayed the growth recovery of bacteria (P < 0.05). We conclude that hyperoxia enhances the bacteriostatic effects of tobramycin in a synergistic manner. Hyperbaric oxygen also enhances the bacteriostatic effects of tobramycin against P. aeruginosa. Our findings may have relevance to the in vivo growth rates of P. aeruginosa in tissues of patients treated with high inspired oxygen tensions.
The activities of many antimicrobial agents are altered by changes in oxygen tension. Anaerobic (anoxic) conditions markedly diminish aminoglycoside activity (32, 36, 37), because energy derived from quinone-associated electron transport is required to facilitate uptake of the antimicrobial agent into gram-negative bacteria (9). The loss of aminoglycoside bactericidal activity induced by an anoxic environ-
kPa) and a sulfonamide-trimethoprim combination against Vibrio anguillarum in vitro. Hyperoxia enhanced trimethoprim activity, presumably by oxidizing enzymes or metabolic intermediates involved in folate metabolism in bacteria (14). Hyperbaric oxygen and sodium sulfisoxazole increased the survival of V. anguillarum-infected goldfish more than threefold (20). Hyperoxia enhanced the bacteriostatic activity of nitrofurantoin and both the bacteriostatic and bactericidal activities of trimethoprim against E. coli (25). The purpose of the current study was to test the hypothesis that elevated oxygen tensions enhance the activity of antimicrobial agents that inhibit bacterial protein synthesis. Experiments were designed so that hyperoxia could act synergistically with tobramycin to suppress the growth of P. aeruginosa, independent of bacterial killing. To this end, we used the PAE, defined as the period of bacterial growth suppression that results from the brief exposure of bacteria to an antimicrobial agent (12, 23).
ment can be restored by hyperbaric oxygen (22). The activity of vancomycin against Staphylococcus aureus is also markedly diminished in an anaerobic environment, and this may account for the failure of the antimicrobial agent to sterilize infected tissues, such as bone, where low oxygen tensions are found (30). In addition, the bactericidal activity of ciprofloxacin is abolished under anaerobic conditions (35). Oxygen tensions within the physiologic range (20 to 150 mm Hg [2.7 to 20 kPa]) found in body tissues (18, 39) can also influence the bactericidal activities of antimicrobial agents. For example, amikacin showed less killing and a shorter postantibiotic effect (PAE) against Pseudomonas aeruginosa at a partial pressure (PO2) of 80 mm Hg (10.6 kPa) than it did at a P02 of 40 mm Hg (5.3 kPa) (2). Concentrations of oxygen in the normoxic range also appear to be required for optimal killing of Escherichia coli by streptonigrin (16). Elevated oxygen tensions have been shown to potentiate the activities of some antimicrobial agents. Keck et al. (20) found synergy between hyperbaric oxygen (100% 02, 324.2
MATERIALS AND METHODS
Reagents and media. Tobramycin was obtained from Sigma Chemical Co. (St. Louis, Mo.). Stock solutions (1,000 p.g/ml) were stored frozen at -135°C in 0.1 M phosphate buffer (pH 7.0) (28). L-[35S]methionine, in vivo cell labeling grade (specific activity 1,371 Ci/mmol), was purchased from Amersham (Arlington Heights, Ill.), subdivided, stored at -70°C, and used within 3 weeks. L-Methionine was purchased from Sigma. Mueller-Hinton broth (Difco, Detroit, Mich.) was supplemented with Mg2+ (25 mg/ml) and Ca2+ (50 mg/ml) (28). Bacterial strains. Two American Type Culture Collection
* Corresponding author. t Present address: Division of Altitude and Hyperbaric Physiology, Armed Forces Institute of Pathology, Washington, DC 20306-
6000.
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(ATCC; Rockville, Md.) strains and six clinical strains (Sinai Hospital, Baltimore, Md.) of P. aeruginosa were initially screened to determine whether hyperoxia could prolong the PAE of tobramycin. Five of the eight Pseudomonas strains showed enhanced PAEs following hyperoxic exposure. Of these five strains, three strains (see Table 1) were selected for further examination. Bacteria were stored in 15% glycerol at -70°C. Bacteria were grown in tryptic soy broth overnight at 37°C. A 1:30 dilution of overnight culture was made into tryptic soy broth, and bacteria were grown in a shaking water bath (100 rpm, 4 h, 37°C). Bacteria were optically matched with a 0.5 McFarland standard and diluted to 107 CFU/ml in prewarmed, cation-supplemented MuellerHinton broth. In vitro PAE. Six assay conditions were tested: 21% 02 21% 02 and tobramycin, hyperoxia (100% 02 101.3 kPa), hyperoxia and tobramycin, hyperbaric oxygen (100% 02, 274.5 kPa), and hyperbaric oxygen and tobramycin. MICs for P. aeruginosa ATCC 27853, SH 6468, and SH 7132 were 0.5, 0.5, and 2 .g/ml, respectively, as determined by a microdilution method (28). Suspensions of P. aeruginosa were exposed to various oxygen tensions and/or tobramycin (4x the MIC) for 1 h, and then a 0.1-ml aliquot was sampled from the bacterial culture. Phosphate buffer was added in place of tobramycin in control cultures. Tobramycin was then removed from cultures by filtering bacteria onto 0.2,um-pore-size filters (MSI, Westboro, Mass.) prerinsed with prewarmed cation-supplemented Mueller-Hinton broth and washing three times. The filter was transferred into 20 ml of prewarmed cation-supplemented Mueller-Hinton broth. After 30 s of vortexing, the filters were removed aseptically, and another 0.1-ml aliquot was sampled; this time point was denoted as time zero. No further bacterial killing was evident after the removal of tobramycin. Bacterial suspensions were incubated in a shaking water bath (37°C) and grown under normoxic conditions (21% 02) for the remainder of the experiment. Removal of tobramycin resulted in a 3-log1o-CFU/ml reduction in bacterial numbers when the standard 1:1,000 drug dilution method was used (12). Other investigators (1, 11) have used filtration for drug removal to determine the PAE. Because of the 3.5-log1o-CFU/ml killing of P. aeruginosa by tobramycin at the standard 4 x MIC (see Table 4), a filtration modification (13a) was used to remove tobramycin, and less than 1 log1o CFU of bacteria per ml was lost by this procedure. This particular filtration method was previously compared with the standard 1: 1,000 dilution method and showed an excellent correlation (32a). Bacterial growth and viability were measured by enumeration, because it has recently been shown that this method is suitable for quantification of bacteria exposed to reactive oxygen intermediates (24). Aliquots of 0.1 ml were removed every 0.5 h for the first 3 h of incubation and at two additional 1-h time points. Less than 10% of the starting volume was removed for serial dilutions over the course of the experiment. Serial 10- or 100-fold dilutions of the 0.1-ml aliquots were performed in duplicate by using cold, sterile 0.9% saline. Pour plates were made of selected dilutions by using tryptic soy agar. Colonies were counted between 24 and 48 h after incubation at 37°C. Duplicate determinations were made for each time point. The in vitro PAE was calculated by using the following formula (12): PAE = T C, where T is the time required for the CFU per milliliter in the antimicrobial agent-treated bacterial culture to increase 1 log1o unit above the CFU per milliliter observed immediately after resuspension of bacteria into drug-free medium, and C represents the time re-
an untreated control culture to increase 1 log1o unit above the CFU per milliliter observed immediately after resuspension of bacteria into drug-free medium. Exposure of bacterial cultures to hyperbaric oxygen. Bacterial cultures were exposed to 100% 02 at a pressure of 274.5 kPa in a steel hyperbaric chamber (Bethlehem Corp., Bethlehem, Pa.). The hyperbaric chamber was fitted with brass penetrators which housed oxygen lines and polyethylene tubing for bacterial inoculation. Battery-operated magnetic stirrers (Bel-Art Products, Pennaquinnock, N.J.) were used to ensure adequate oxygen saturation of the media. Cation-supplemented Mueller-Hinton broth containing tobramycin or phosphate buffer was gassed with 100% 02 at 6 liters/min for 30 min before the addition of bacteria. After a 1-h incubation, the chamber was decompressed within 10 s. Determination of protein synthesis rates. Bacteria were filtered onto 0.2-[Lm-pore-size filters, washed, and resuspended in 10 ml of methionine-free, minimal salts acetate medium (8), because cation-supplemented Mueller-Hinton broth was found to contain 1 mM methionine (unpublished data). After vortexing for 30 s, filters were removed aseptically. Bacteria were collected on 25-mm-diameter filters (pore size, 0.2 [Lm; Millipore, Bedford, Mass.) which were prerinsed with 1 mM unlabeled methionine. Protein synthesis rates in P. aeruginosa were measured by pulsing bacteria with 0.5 pXCi of L-[35S]methionine for 5 min at 37°C and by chasing with 1 mM unlabeled methionine for 5 min at room temperature. Each measurement was performed in triplicate, and background radioactivity was subtracted. Background radioactivity was due to nonspecific binding of L-[35S]methionine to filters after washing in the absence of bacteria. Filters were dried for 30 min at 60°C before 5 ml of Biosafe-II scintillation fluid (Research Products International Corp., Mount Prospect, Ill.) was added. After an overnight incubation in the dark, the filters were counted in a Beckman LS 7800 scintillation counter. Data analysis. The Mann-Whitney test, Kruskal-Wallis one-way analysis of variance by rank sums, and a repeated measures design analysis of variance (SAS procedure GLM) were used for statistical analysis. When necessary, the Tukey honestly significant difference procedure was used as a posttest. Data were considered significant at P < 0.05.
quired for the CFU per milliliter in
RESULTS Hyperoxia and the in vitro PAE of tobramycin. To determine the PAE under hyperoxic conditions, 18 ml of cationsupplemented Mueller-Hinton broth, containing 4x the MIC of tobramycin, was pregassed at a rate of 3 liters/min for 10 min before adding 2 ml of bacteria (108 CFU/ml). Ten minutes was sufficient for >95% oxygen saturation of the medium. The data in Table 1 show that under normoxic conditions, tobramycin had a PAE that ranged from 0.7 to 1.9 h for ATCC 27853 and two clinical isolates of P. aeruginosa. Hyperoxia (100% 02 101.3 kPa) extended the PAE of tobramycin approximately twofold compared with normoxia in all three strains of P. aeruginosa (P < 0.05). The effect of oxygen tensions normally found in tissues (20 to 40 mm Hg [2.7 to 5.3 kPa]) on the PAE was also tested by exposing P. aeruginosa ATCC 27853 to 3.7% 02 (26 mm Hg [3.5 kPa]). The PAE (0.82 h) at 26 mm Hg (3.5 kPa) of 02 was not statistically different from the PAE (0.70 h) at 150 mm Hg
of 02 (20 kPa) (data not shown). Hyperbaric oxygen and the growth recovery of P. aeruginosa ATCC 27853. A reference ATCC strain of P. aerugi-
HYPEROXIA PROLONGS THE PAE OF TOBRAMYCIN
VOL. 35, 1991 TABLE 1. Effect of hyperoxia on the PAE of tobramycin in P. aeruginosa
ATCC 27853 (9) SH 6468 (6) SH 7132 (4)
L-[35Slmethionine incorporation (dpm/logio CFU [n])a
21% 02c
100% 02d
0.70 ± 0.13 1.00 ± 0.23 1.91 + 0.05
1.42 ± 0.l9e 2.50 ± 0.58f 3.11 ± 0.17e
a n, Number of experiments. b PAE = T C (where T is the time required for bacterial growth in tobramycin-treated group to increase 1.0 loglo CFU/ml after removal of the antibiotic by filtration, and C is the time required for bacteria not treated with tobramycin to increase 1.0 loglo CFU/ml). Numbers are means + standard errors of the mean and represent the duration of PAE. c Bacteria were exposed to 21%02 and tobramycin (4x the MIC) for 1 h and were maintained at 21%02 after the removal of tobramycin. d Bacteria were exposed to 100% 02 (101.3 kPa) and tobramycin (4x the MIC) for 1 h and subsequently returned to normoxic conditions (21% 02) after the removal of tobramycin. e Significantly different (P < 0.02) from the value found under normoxic conditions. f Significantly different (P < 0.05) from the value found under normoxic conditions.
nosa was used to determine whether the PAE of tobramycin could be further enhanced by using the maximal oxygen dose that is used clinically. As seen in Table 2, hyperbaric oxygen (100% 02, 274.5 kPa) alone significantly prolonged the time necessary for a 1-log1o-CFU/ml increase in bacterial growth compared with normoxia (P < 0.05) and hyperoxia (P < 0.01). In the presence of tobramycin, hyperbaric oxygen significantly prolonged the time necessary for a 1-log1oCFU/ml increase in bacterial growth compared with normoxia (P < 0.001) or hyperoxia (P = 0.01). The delayed recovery of bacterial growth seen in the hyperbaric oxygen controls was responsible for the apparent lack of difference in PAE between hyperoxic and hyperbaric oxygen-exposed cultures. Effects of hyperoxia and hyperbaric oxygen on protein synthesis. Bacterial suspensions were incubated under hyperoxic or hyperbaric oxygen conditions in the presence of TABLE 2. Effect of hyperoxia or hyperbaric oxygen on growth recovery of P. aeruginosa ATCC 27853 Time interval for growth recovery (h)b TobramycinControl treated
Oxygen exposure (na)
Normoxia (9) Hyperoxia (9) Hyperbaric oxygen (4)
2.20 ± 0.13 2.03 ± 0.12 2.67 ± O.O9fg
2.89 ± 0.13c 3.45 + 0. 9d.e 4.42 + 0.18c'h,i
n, Number of experiments. Bacteria were exposed to tobramycin and one of three oxygen tensions for 1 h. Tobramycin was removed by filtration. Numbers are means ± standard errors of the mean and represent the time needed for bacteria to increase 1.0 CFU/ml under normoxic conditions after the removal of tobramycin. log1o I Significantly different (P < 0.02) from respective controls without tobraa
b
mycin. d Significantly different (P < 0.002) from respective controls without tobramycin. eSignificantly different (P < 0.02) from the normoxia and tobramycintreated group. f Significantly different (P < 0.05) from normoxia. g Significantly different (P < 0.01) from hyperoxia. h Significantly different (P < 0.001) from the normoxia and
treated group. i Significantly different (P treated group.
TABLE 3. Effect of hyperoxia or hyperbaric oxygen on protein synthesis in P. aeruginosa ATCC 27853
PAE (h)b
Strain (na)
=
693
tobramycin-
0.01) from the hyperoxia and tobramycin-
Oxygen
0h
exposure
Normoxia Hyperoxia Hyperbaric oxygen
1 h,
Control
Tobramycin
415 + 185 (9) 514 ± 237 (9) 202 ± 52 (9)
16.7 6.5b (9) 7.4 ± 4.3b (9) -7.2 + 11.6b.c (9)
Tobramycin
18.5 7.3b (6) 13.8 + 6.3b (6) 6.4 ± 12.1b (3)
a Bacteria were exposed to tobramycin and one of three oxygen tensions for 1 h. Tobramycin was removed by filtration and bacteria were pulsed for 5 min (35°C) under normoxic conditions with 0.5 ,Ci of L-[35S]methionine. Bacteria were then chased for 5 min with 1 mM unlabeled methionine. Data are means + standard errors of the mean. n, Number of experiments. The value 0 h refers to protein synthesis measured immediately after removal of tobramycin; the value 1 h refers to protein synthesis measured 1 h after removal of
tobramycin. b Significantly different (P = 0.0001) from controls at 0 h. c Value approximating background.
tobramycin or phosphate buffer. After 1 h, the bacteria were filtered and resuspended in minimal salts acetate medium (8) under normoxic conditions. Protein synthesis was measured at 0 and 1 h after the removal of tobramycin. Table 3 shows that in the control groups, hyperoxia increased protein synthesis by 24%, while hyperbaric oxygen decreased protein synthesis by 50%; these differences did not achieve statistical significance. In the tobramycin-treated groups, protein synthesis was markedly diminished (P = 0.0001). Table 3 also shows that 1 h after the removal of tobramycin, protein synthesis rates remained essentially unchanged from time zero. At approximately 3 h after tobramycin and normoxia exposure, protein synthesis in bacteria approached normal rates (287 dpm/log1o CFU). Rates of protein synthesis were also measured at 3 h after exposure to tobramycin and hyperoxia and were found to remain low (69 dpm/log1o CFU) (data not shown). Effects of hyperoxia and hyperbaric oxygen on the growth of P. aeruginosa ATCC 27853. During the course of the PAE experiments, bacteria were enumerated before and immediately after a 1-h exposure to hyperoxia or hyperbaric oxygen. Table 4 shows that hyperoxia alone caused a significant increase in bacterial growth (P < 0.05). Tobramycin significantly decreased growth under normoxic, hyperoxic, and hyperbaric oxygen conditions compared with the growth of controls (P = 0.0001). However, killing of P. aeruginosa by tobramycin was maximal under normoxic conditions. DISCUSSION
Hyperoxia significantly enhanced the PAE of tobramycin against P. aeruginosa compared with normoxia. P. aeruginosa was incubated for 1 h with 4x the MIC of tobramycin under normoxic, hyperoxic, or hyperbaric oxygen conditions. After the 1-h exposure, tobramycin was removed and the bacteria were incubated under normoxic conditions. The maximal killing of P. aeruginosa which occurred under normoxic conditions is in agreement with our previous work (25). In the case of amikacin, antimicrobial activity was found to be significantly diminished at 80 mm Hg (10.7 kPa) of 02 compared with that at 40 mm Hg (5.3 kPa) of 02, as evidenced by decreased killing and shortened PAEs in P. aeruginosa (2). However, at lowered oxygen tensions, such as those present physiologically in tissues (26 mm Hg [3.5
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TABLE 4. Effect of hyperoxia or hyperbaric oxygen on the growth of P. aeruginosa ATCC 27853 Oxygen exposure (na)
Viable bacteria (mean logl0 CFU/ml ± SEM) TobramycinOriginal Controlb treatedb inoculum
Normoxia (9) Hyperoxia (9) Hyperbaric oxygen (3)
6.96 ± 0.04 7.02 + 0.07 3.33 ± 0.20' 6.96 ± 0.04 7.33 ± 0.04d 3.41 ± 0.13c 6.86 ± 0.01 6.74 ± 0.18e 3.61 ± 0.26C
n, Number of experiments. Viable bacteria remaining after a 1-h exposure to oxygen and/or tobramycin prior to filtration. c Significantly different (P = 0.0001) from similar oxygen-exposed controls. d Significantly different (P < 0.05) from original inoculum. e Significantly different (P = 0.0023) from hyperoxia. a
b
kPa]), we found no significant difference compared with normoxia in the PAE caused by tobramycin. Hyperoxia increased protein synthesis and the growth rates of P. aeruginosa. These results are in agreement with those of Ollodart (31), who found that hyperoxia enhances the growth of E. coli. Aminoglycoside uptake by bacteria has been shown to be dependent upon growth rates or quinonedependent bacterial respiration (26, 27, 29). Based on these findings, the prolongation by hyperoxia of the PAE in P. aeruginosa may be the result of increased tobramycin uptake because of enhanced growth and/or respiration rates. To determine the effect of growth rates on the PAE, P. aeruginosa was preexposed to hyperoxic conditions for 1 h and then incubated with tobramycin under normoxic conditions. Despite the increased growth rates induced by preexposure to hyperoxia, there was no significant difference in the PAE (data not shown). These results indicate that the observed synergy between hyperoxia and tobramycin is not related to changes in bacterial growth. However, it is possible that hyperoxia may enhance the PAE of tobramycin by inducing oxidative stress in P. aeruginosa. Hyperoxia generates reactive oxygen intermediates, which inhibit respiration (6) and protein synthesis (3) and decrease NAD levels (8) in bacteria. These reactive oxygen intermediates can induce the synthesis of superoxide dismutase (15, 38) and other antioxidant or repair enzymes (38). In the presence of tobramycin, which induces misreading of proteins at low concentrations and inhibits protein synthesis at high concentrations (13), synthesis of functional antioxidant or repair enzymes does not occur. The reactive oxygen intermediates generated under hyperoxic conditions, in the absence of newly synthesized antioxidant enzymes, may then inactivate the proteins (5, 7, 17) essential for bacterial growth. Aminoglycosides are thought to bind to ribosomes irreversibly, and it is postulated that the PAE of tobramycin may represent the additional time needed to synthesize functional ribosomes (12). The enhancement of the PAE by hyperoxia may be the result of the effects of oxidative stress on the biosynthesis of ribosomal proteins, either directly by inactivation or indirectly through reduced energy levels. The significantly delayed growth recovery of P. aeruginosa by hyperbaric oxygen (100% 02, 274.5 kPa) was independent of the presence of tobramycin (Table 2). Exposure to hyperbaric oxygen for 18 to 24 h inhibited the growth of P. aeruginosa (4, 19, 25). Brunker and Brown (8) found that a 2-h exposure of E. coli to 100% 02 (607.8 kPa) was bacteriostatic. Interestingly, in this study, we found that only a 1-h exposure of P. aeruginosa to hyperbaric oxygen
(100% 02, 274.5 kPa) was needed to delay the subsequent recovery of bacterial growth under normoxic conditions (Table 2). This delay in recovery of bacterial growth war; associated with a twofold decrease in protein synthesis (Table 3). It is known that hyperbaric oxygen can inhibit the biosynthesis of amino acids (3, 7) and can induce a stringent response (33). The resultant increase in tetra- and pentaphosphorylated guanosine levels can inhibit the synthesis of carbohydrates, lipids, and nucleotides, in addition to increasing proteolysis (10). These metabolic effects could explain the delay in recovery of bacterial growth by shortterm exposure (1 h) to hyperbaric oxygen. Because differences in P02 exist in various body tissues, it is likely that the variation in aminoglycoside activity seen in vitro will also be found in vivo. Under physiologic conditions, different compartments of the body are exposed to a wide range of pO2s. For example, under normoxic conditions inspired P02S of 150 mm Hg (20 kPa) and alveolar P02S of 100 mm Hg (13.3 kPa) are found (39); P02s are approximately 95 mm Hg (12.7 kPa) in arterioles and are reduced to 40 mm Hg (5.3 kPa) in the venous circulation (39). pO2s range from 20 to 40 mm Hg (2.7 to 5.3 kPa) in many visceral tissues (18). In the presence of hyperoxia, alveolar and arterial P02s are ideally 673 and 660 mm Hg, respectively (89.7 and 88.0 kPa, respectively) (34). Under hyperbaric oxygen conditions (308.0 kPa), arterial P02 levels can exceed 1,700 mm Hg (226.6 kPa) (40). At a lower concentration of hyperbaric oxygen (253.2 kPa), pO2s of 800 mm Hg (106.6 kPa) can be found in subcutaneous tissue (21). It can be concluded that different body compartments can have elevated P02S that approach or exceed the levels attained in vitro under hyperoxic conditions. Elevated oxygen tensions may thus influence the effectiveness of aminoglycosides for infections at different body sites. In summary, hyperoxia enhanced the PAE of tobramycin against P. aeruginosa in vitro. The PAE of tobramycin has been demonstrated to be up to three times longer in vivo than in vitro (12). Hyperoxia may extend the in vivo PAE and therefore prolong the therapeutic activity of aminoglycosides in patients exposed to hyperoxia or hyperbaric oxygen. ACKNOWLEDGMENTS This study was supported by the Maryland Institute for Emergency Medical Services Systems Research Fund. We thank George L. Drusano for helpful advice and Harry L. T. Mobley for critical review of the manuscript. We thank Patricia L. Schleiff and William F. McCarthy for statistical analysis of the data. We gratefully acknowledge the excellent help of Gargi Raval in determining the PAEs. The technical assistance of Paul P. Rodier and Bret Holliday in the maintenance and operation of the hyperbaric chamber is gratefully appreciated. We thank Clifton Bennett, Jr., for providing the clinical strains of P. aeruginosa. REFERENCES 1. Baquero, F., E. Culebras, C. Patron, J. C. Prez-Diaz, J. C. Medrano, and M. F. Vicente. 1986. Postantibiotic effect of imipenem on gram-positive and gram-negative micro-organisms. J. Antimicrob. Chemother. 18(Suppl. E):47-59. 2. Bayer, A. S., T. O'Brien, D. C. Norman, and C. C. Nast. 1989. Oxygen-dependent differences in exopolysaccharide production and aminoglycoside inhibitory-bactericidal interactions with Pseudomonas aeruginosa-implications for endocarditis. J. Antimicrob. Chemother. 23:21-35. 3. Boehme, D. E., K. Vincent, and 0. R. Brown. 1976. Oxygen and toxicity inhibition of amino acid biosynthesis. Nature (London) 262:418-420. 4. Bornside, G. H., L. M. Pakman, and A. A. Ordofiez, Jr. 1975.
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Inhibition of pathogenic enteric bacteria by hyperbaric oxygen: enhanced antibacterial activity in the absence of carbon dioxide. Antimicrob. Agents Chemother. 7:682-687. 5. Brot, N., L. Weissbach, J. Werth, and H. Weissbach. 1981. Enzymatic reduction of protein-bound methionine sulfoxide. Proc. Natl. Acad. Sci. USA 78:2155-2158. 6. Brown, 0. R. 1972. Reversible inhibition of respiration of Escherichia coli by hyperoxia. Microbios 5:7-16. 7. Brown, 0. R., and F. Yein. 1978. Dihydroxyacid dehydratase: the site of hyperbaric oxygen poisoning in branch-chain amino acid biosynthesis. Biochem. Biophys. Res. Commun. 85:12191224. 8. Brunker, R. L., and 0. R. Brown. 1971. Effects of hyperoxia on oxidized and reduced NAD and NADP concentrations in Escherichia coli. Microbios 4:193-203. 9. Bryan, L. E., and S. Kwan. 1983. Roles of ribosomal binding, membrane potential, and electron transport in bacterial uptake of streptomycin and gentamicin. Antimicrob. Agents Chemother. 23:835-845. 10. Cashel, M. 1975. Regulation of bacterial ppGpp and pppGpp. Annu. Rev. Microbiol. 29:301-318. 11. Chin, N.-X., A. Novelli, and H. C. Neu. 1988. In vitro activity of lomefloxacin (SC-47111; NY-198), a difluoroquinolone 3-carboxylic acid, compared with those of other quinolones. Antimicrob. Agents Chemother. 32:656-662. 12. Craig, W. A., and S. Gudmundsson. 1986. The postantibiotic effect, p. 515-536. In V. Lorian (ed.), Antibiotics in laboratory medicine, 2nd ed. The Williams & Wilkins Co., Baltimore. 13. Davis, B. D. 1987. Mechanism of bactericidal action of aminoglycosides. Microbiol. Rev. 51:341-350. 13a.Drusano, G. Personal communication. 14. Gottlieb, S. F. 1971. Effect of hyperbaric oxygen on microorganisms. Annu. Rev. Microbiol. 25:111-152. 15. Gregory, E. M., and I. Fridovich. 1973. Induction of superoxide dismutase by molecular oxygen. J. Bacteriol. 114:543-548. 16. Harley, J. B., C. J. Fetterolf, C. A. Bello, and J. G. Flaks. 1982. Streptonigrin toxicity in Escherichia coli: oxygen dependence and the role of the intracellular oxidation-reduction state. Can. J. Microbiol. 28:545-552. 17. Jamieson, D., B. Chance, E. Cadenas, and A. Boveris. 1986. The relation of free radical production to hyperoxia. Annu. Rev. Physiol. 48:703-719. 18. Jamieson, D., and H. A. S. van den Brenk. 1965. Electrode size and tissue P02 measurement in rats exposed to air or high pressure oxygen. J. Appl. Physiol. 20:514-518. 19. Kaye, D. 1967. Effect of hyperbaric oxygen on aerobic bacteria in vitro and in vivo. Proc. Soc. Exp. Biol. Med. 124:1090-1093. 20. Keck, P. E., S. F. Gottlieb, and J. Conley. 1980. Interaction of increased pressures of oxygen and sulfonamides on the in vitro and in vivo growth of pathogenic bacteria. Undersea Biomed. Res. 7:95-106. 21. Kivisaari, J., and J. Niinikoski. 1975. Effects of hyperbaric oxygenation and prolonged hypoxia on the healing of open wounds. Acta Chir. Scand. 141:14-19. 22. Mader, J. T., C. A. Hicks, and J. Calhoun. 1989. Bacterial osteomyelitis. Adjunctive hyperbaric oxygen therapy. Orthop. Rev. 18:581-585. 23. McDonald, P. J., W. A. Craig, and C. M. Kunin. 1977. Persistent effect of antibiotics on Staphylococcus aureus after exposure for limited periods of time. J. Infect. Dis. 135:217-223. 24. Minakami, H., and I. Fridovich. 1990. Effects of paraquat on
HYPEROXIA PROLONGS THE PAE OF TOBRAMYCIN
695
cultures of Escherichia coli: turbidity versus enumeration. Free Radical Biol. Med. 8:387-391. 25. Muhvich, K. H., M. K. Park, R. A. M. Myers, and L. Marzella. 1989. Hyperoxia and the antimicrobial susceptibility of Escherichia coli and Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 33:1526-1530. 26. Muir, M. E., M. Ballesteros, and B. J. Wallace. 1985. Respiration rate, growth rate and the accumulation of streptomycin in Escherichia coli. J. Gen. Microbiol. 131:2573-2579. 27. Muir, M. E., R. S. van Heeswyck, and B. J. Wallace. 1984. Effect of growth rate on streptomycin accumulation by Escherichia coli and Bacillus megaterium. J. Gen. Microbiol. 130:20152022. 28. National Committee for Clinical Laboratory Standards. 1985. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. National Committee for Clinical Laboratory Standards, Villanova, Pa. 29. Nichols, W. W., and S. N. Young. 1985. Respiration-dependent uptake of dihydrostreptomycin by Escherichia coli. Its irreversible nature and lack of evidence for a uniport process. Biochem. J. 228:505-512. 30. Norden, C. W., and M. Shaffer. 1983. Treatment of experimental chronic osteomyelitis due to Staphylococcus aureus with vancomycin and rifampin. J. Infect. Dis. 147:352-357. 31. Ollodart, R. M. 1966. Effects of hyperbaric oxygenation and antibiotics on aerobic microorganisms, p. 565-571. In I. W. Brown, Jr., and B. G. Cox (ed.), Proceedings of the Third International Conference on Hyperbaric Medicine. National Academy of Sciences, Washington, D.C. 32. Reynolds, A. V., J. M. T. Hamilton-Miller, and W. Brumfitt. 1976. Diminished effect of gentamicin under anaerobic or hypercapnic conditions. Lancet i:447-449. 32a.Rider, D. A., G. L. Drusano, and H. C. Standiford. 1989. Abstr. Annu. Meet. Am. Soc. Microbiol. 1989, A 115, p. 20. 33. Seither, R. L., and 0. R. Brown. 1982. Induction of stringency by hyperoxia in Escherichia coli. Cell. Mol. Biol. 28:285-291. 34. Sheffield, P. J. 1988. Tissue oxygen measurements, p. 17-51. In J. C. Davis and T. K. Hunt (ed.), Problem wounds. The role of oxygen. Elsevier Science Publishing Co., New York. 35. Smith, J. T., and C. S. Lewin. 1988. Chemistry and mechanisms of action of the quinolone antibacterials, p. 23-82. In V. T. Andriole (ed.), The quinolones. Academic Press, Inc., New York. 36. Tack, K. J., and L. D. Sabath. 1985. Increased minimum inhibitory concentrations with anaerobiosis for tobramycin, gentamicin, and amikacin compared to latamoxef, piperacillin, chloramphenicol, and clindamycin. Chemotherapy 31:204-210. 37. Verklin, R. M., Jr., and G. L. Mandell. 1977. Alteration of effectiveness of antibiotics by anaerobiosis. J. Lab. Clin. Med. 89:65-71. 38. Walkup, L. K. B., and T. Kogoma. 1989. Escherichia coli proteins inducible by oxidative stress mediated by the superoxide radical. J. Bacteriol. 171:1476-1484. 39. Weibel, E. R. 1984. The pathway for oxygen. Structure and function in the mammalian respiratory system, p. 178-179, 343-345. Harvard University Press, Cambridge, Mass. 40. Whalen, R. E., H. A. Saltzman, D. H. Holloway, Jr., H. D. McIntosh, H. 0. Sieker, and I. W. Brown, Jr. 1965. Cardiovascular and blood gas responses to hyperbaric oxygenation. Am. J. Cardiol. 15:638-646.
