Inhibition of pathogenic enteric bacteria by hyperbaric oxygen: enhanced antibacterial activity in the absence of carbon dioxide.

The antibacterial effects of 24-h exposures to high-pressure oxygen in relation to environmental CO(2) were studied at 3 atm absolute (ata) and at 1 ata. Eight gram-negative, aerobic and facultatively aerobic, pathogenic enteric bacteria (Salmonella typhosa, Salmonella paratyphi, Salmonella schottmuelleri, Shigella dysenteriae, Shigella flexneri, Proteus vulgaris, Pseudomonas aeruginosa, and Escherichia coli) were exposed as shallow-broth cultures and agar surface cultures. Although broths supplemented with 0.2% glucose permitted some growth of Salmonella typhosa, Salmonella schottmuelleri, Shigella dysenteriae, and Shigella flexneri during exposure to high-pressure oxygen in the presence of CO(2), the other species grew only after the exposure, indicating a bacteriostatic effect. Both bacteriostatic and bactericidal effects were demonstrated on the surface of Trypticase soy agar, where killing of Salmonellea typhosa, Proteus vulgaris, and Pseudomonas aeruginosa was significantly greater after exposure to pure O(2) at 3 ata than at 1 ata. At 3 ata, significantly more killing occurred upon exposure of all species (except Shigella dysenteriae and S. flexneri) on an agar surface to 100% O(2) as compared with exposure to a mixture of 95% O(2) + 5% CO(2). Thus, deprivation of CO(2) during exposure to pure O(2) enhanced the bactericidal effect of high-pressure oxygen.

Inhibition of Pseudomonas aeruginosa by hyperbaric oxygen: interaction with mouse peritoneal exudate cells.

High-pressure oxygen (HPO) therapy for Pseudomonas aeruginosa infections of burn wounds has not been as effective as in vitro studies predicted. Mitigation of HPO toxicity for P. aeruginosa by nutrients present at the burn site could explain the lack of in vivo success. Alternatively, HPO-induced depression of host defense mechanisms could negate beneficial effects arising from HPOs known toxicity for P. aeruginosa. Accordingly, mouse peritoneal exudate cells (PEC), preincubated for 24 h in 1 atm of air-CO(2), were used to study the in vitro effects of HPO or air-CO(2) on phagocytosis of P. aeruginosa or sheep erythrocytes (SRBC). Subsequent 2-h exposures of PEC to increasing numbers of bacteria, in an air-CO(2) atmosphere, decreased the percentage of bacteria cleared as well as PEC viability. Similar exposures of PEC to bacteria in an HPO atmosphere prevented the loss of PEC viability and increased bacterial clearance. In control experiments, increasing the number of SRBC relative to PEC decreased the percentage of SRBC cleared without decreasing PEC viability, as determined under air-CO(2); short (2 h) exposure to HPO did not affect SRBC clearance. Microscopic examination of PEC indicated that a 24-h preincubation in HPO decreased the percentage of PEC which could ingest SRBC during subsequent experimental exposures (2 h) to air-CO(2) or HPO. These data suggest that short periods of exposure to HPO promote the ability of PEC to clear pseudomonads by adversely affecting the bacteria. This in turn prevents a pseudomonad-induced depression of PEC viability and function. In contrast, prolonged HPO exposure may be detrimental to phagocytic activity.

Inhibition of Pseudomonas aeruginosa by hyperbaric oxygen. II. Ultrastructural changes.

The ultrastructure of Pseudomonas aeruginosa cells treated with hyperbaric oxygen for 20 to 24 hr was examined by electron microscopy. A marked difference in the morphology of the nuclear area and cytoplasm of experimental cells was noted when compared to control cells grown under normobaric conditions. This difference was characterized by the absence of a definitive nuclear area and a reduced granularity of the cytoplasm.

Inhibition of Pseudomonas aeruginosa by hyperbaric oxygen. I. Sulfonamide activity enhancement and reversal.

To elucidate an explanation for in vitro sulfonamide enhancement by high-pressure oxygen (HPO) and the reported absence of enhancement with in vivo therapy, Pseudomonas aeruginosa cultures were exposed to selected antifolate antimicrobials in the presence of 1.87 atm absolute of O(2) and compared with non-HPO treated controls. Under these conditions, HPO alone retarded growth. Trimethoprim, a non-sulfonamide which inhibits dihydrofolate reductase, was not bactericidal, nor did HPO enhance existent bacteriostatic activity. The sulfonamide, sulfisozazole, was not bactericidal, but HPO enhanced bacteriostatic activity twofold; bacteriostasis was mitigated in HPO-treated and control cultures by p-aminobenzoate but not by a mixture of compounds involved in folate-mediated "1-C" biosynthesis. Mafenide, a unique sulfonamide, at high concentrations with HPO, was synergistically bactericidal; non-HPO-treated cultures were bacteriostatically inhibited. Bacteriostatic activity of lower mafenide concentrations was also enhanced at least twofold by HPO. These inhibitory effects of mafenide, acting with or without HPO, were mitigated by the above mixture, but not by p-aminobenzoate. This may explain the lack of in vivo HPO-mafenide enhancement in burn-wound sepsis where exudates would contain such a mixture. Lastly, HPO itself was largely bactericidal at 2.87 atm absolute of O(2). This was reversed to various degrees by the above mixture, or its components, or by folic, folinic, or p-aminobenzoic acids. These in vitro interactions suggest HPO per se may act at the same site as some sulfonamides to inhibit folate synthesis (not primarily at the dihydrofolate reductase level), or coenzyme functions of folate, or both.

Effect of high oxygen tensions on the growth of selected, aerobic, gram-negative, athogenic bacteria.

The in vitro effects of high O(2) tensions (P(O2)) on aerobic, enteric pathogens were examined at pressures of up to 3 atm absolute. Organisms from the genera Salmonella, Shigella, and Vibrio were usually subjected to 24-hr exposures. Tensions of 0.87, 1.87, and 2.87 atm absolute of O(2) (plus traces of CO(2) and N(2)) became progressively inhibitory for Salmonella and Shigella growth, but were bactericidal only for V. comma strains at tensions greater than 0.87 atm absolute of O(2). Growth inhibition of enteric organisms resulted from increased P(O2), rather than pressure per se, and could be mitigated nutritionally; an appropriate carbohydrate source is at least partially involved. Further studies with vibrios indicated that such mitigation was independent of medium pH. In addition, a synergistic relationship existed between O(2) and sulfisoxazole when tensions from 0.87 to 2.87 atm absolute of O(2) were maintained for 3 to 24 hr. Synergism occurred even under nutritional conditions which negated growth inhibition by O(2) alone. Bactericidal concentrations of sulfisoxazole, in the presence of increased P(O2), were reducible up to 4,000-fold. The combined procedure employed in this investigation, by use of an antimicrobial drug of known action, which also synergizes with O(2), plus nutritional studies, suggests a means for establishing a site of O(2) toxicity. These data support the concept that O(2) inhibition of growth represents a metabolic disturbance and that metabolic pathways involving p-aminobenzoic acid may be O(2)-labile. Such an approach could also guide development of antimicrobial agents as O(2) substitutes for promoting synergism.