INFECTION AND IMMUNITY, OCt. 1971, p. 479-487 Vol. 4, No. 4Copyright © 1971 American Society for Microbiology Printed hi U.S.A.

Inhibition of Pseudomonas aeruginosa byHyperbaric Oxygen

I. Sulfonamide Activity Enhancement and ReversalLEONARD M. PAKMAN

Department of Microbiology, Temple University School ofDentistry, Philadelphia, Pennsylvantia 19140

Received for publication 5 April 1971

To elucidate an explanation for in vitro sulfonamide enhancement by high-pres-sure oxygen (HPO) and the reported absence of enhancement with in vivo therapy,Pseudomonas aeruginosa cultures were exposed to selected antifolate antimicrobialsin the presence of 1.87 atm absolute of 02 and compared with non-HPO treated con-trols. Under these conditions, HPO alone retarded growth. Trimethoprim, a non-sulfonamide which inhibits dihydrofolate reductase, was not bactericidal, nor didHPO enhance existent bacteriostatic activity. The sulfonamide, sulfisozazole, was notbactericidal, but HPO enhanced bacteriostatic activity twofold; bacteriostasis wasmitigated in HPO-treated and control cultures by p-aminobenzoate but not by amixture of compounds involved in folate-mediated "1-C" biosynthesis. Mafenide, aunique sulfonamide, at high concentrations with HPO, was synergistically bacteri-cidal; non-HPO-treated cultures were bacteriostatically inhibited. Bacteriostatic ac-tivity of lower mafenide concentrations was also enhanced at least twofold by HPO.These inhibitory effects of mafenide, acting with or without HPO, were mitigated bythe above mixture, but not by p-aminobenzoate. This may explain the lack of in vivoHPO-mafenide enhancement in burn-wound sepsis where exudates would containsuch a mixture. Lastly, HPO itself was largely bactericidal at 2.87 atm absolute of 02.This was reversed to various degrees by the above mixture, or its components, or byfolic, folinic, or p-aminobenzoic acids. These in vitro interactions suggest HPO per semay act at the same site as some sulfonamides to inhibit folate synthesis (not pri-marily at the dihydrofolate reductase level), or coenzyme functions of folate, or both.

The in vitro growth of several gram-negative,aerobic, pathogenic bacteria of enteric originhas been reported to be inhibited by increasedoxygen tensions (Po2). At 3 atm absolute, 02 wasbacteriostatic for species of Salmonella andShigella and bactericidal for Vibrio commastrains (10). Brown et al. (5) reported Pseudomo-nas aeruginosa to be killed by direct exposure to02-enriched air, and Bornside (2) noted 02 at 3atm absolute inhibited growth of several strainsin broth cultures. In addition, in vitro studieswith P. aeruginosa indicated that the inhibitoryeffects of streptomycin and kanamycin (5), aswell as polymyxin B (2), could be enhanced byPo2. Such enhancement was generally mani-fested by maintained high levels of antimicrobialdrug activity at greatly reduced drug concentra-tions. Combinations of high-pressure oxygen(HPO) and antibiotics were accordingly suggestedfor increased therapeutic effectiveness in pseu-domonad infections. Because of the prevalenceand seriousness of burn wounds infected with

pseudomonads, as well as an ideal opportunityto expose such surface lesions to HPO, Bornsideand Nance (3) employed P. aeruginosa strain 283to produce a burn-wound sepsis in rats. This in-fection was treated with HPO plus antimicrobialdrugs demonstrated to have HPO-enhanced,anti-pseudomonad activity in vitro, i.e., the mini-mal inhibitory concentrations of polymyxin Band mafenide acetate (a topically applied sulfona-mide) could be reduced, respectively, 30 and 45%,in vitro, in the presence of HPO. Unfortunately,the activity of neither drug was enhanced in vivo,and no explanation was given for this difference.Argamaso and Wiseman, using mice infectedwith P. aeruginosa, reported a similar lack ofincreased survival after treatment with polymyxinplus HPO at 3 atm absolute (1). To explain thesedifferences, it is appropriate to consider reasonsfor HPO toxicity and for in vitro HPO-anti-microbial drug interactions.

Several diverse mechanisms have been pro-posed to account for 02 toxicity in microorga-

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nisms (15, 21). One has incorporated an explana-tion of in vitro HPO-antimicrobial drug enhance-ment; Gottlieb and Pakman (10) suggested thatHPO, like sulfonamides, may interfere withmetabolic pathways involving p-aminobenzoate(PAB). This was based on observations that forV. comma, bactericidal concentrations of sul-fisoxazole, in the presence of increased P02,were reducible up to 4,000-fold. Since such en-hancement occurred even under nutritionalconditions which negated growth inhibitioncaused by HPO alone, a synergistic relationshipwas said to exist between HPO and the drug.Synergism was taken to denote cooperative actionof discrete agents producing an effect greaterthan the sum of the two effects taken independ-ently. Because of the magnitude of this effect andthe nature of the relationship, the authors hy-pothesized that HPO and the sulfonamide mightact at similar metabolic sites.The current investigation explores the above

hypothesis and attempts to explain the reportedlack of in vivo enhancement of mafenide andpolymyxin by HPO (1, 3). The inhibitory effectsof three antifolate antimicrobials, used in con-junction with HPO, are examined. Usingtrimethoprim, sulfisoxazole, and mafenide, at-tempts were made to overcome any HPO en-hancement of their activity, as well as the in-hibitory effect of HPO itself. To accomplish this,compounds involved in PAB and folic acid me-tabolism were used to enrich the growth medium.The nature of the compounds responsible forsuch reversal of inhibition could implicate theexistence of a possible 02-labile metabolic se-quence.

MATERIALS AND METHODSBacterial strain and maintenance. The organism

used throughout this study was P. aeruginosa 283,kindly furnished by G. H. Bornside (Department ofSurgery, Louisiana State University School of Medi-cine, New Orleans, La.). Originally isolated from abum patient, it was sensitive to HPO in vitro and wasused for the antimicrobial-HPO enhancement studiesdescribed by Bornside and Nance (3). Stock culturesof this motile organism were stored at room tempera-ture, under mineral oil, on slants of Trypticase SoyBroth (TSB; BBL) solidified with 2.0% agar (TSA).Growth medium and drug additives. Growth medium

consisted of TSB, distributed in 2.0-ml portions tocotton-plugged, optically matched test tubes. Thesewere sterilized by autoclaving, inoculated, and placedin the high-pressure chamber at a 1300 angle from thehorizontal to decrease the diffusion limitation (10).Experiments were performed in duplicate or triplicateand repeated on three separate occasions; data fromtypical experiments are presented unless otherwiseindicated. Although use of a completely syntheticmedium was attempted, difficulties encounted in

growing the organism in such media, under HPO,necessitated use of a complex broth (TSB).

Trimethoprim, obtained from Hoffmann-LaRocheInc., Nutley, N.J., was solubilized in TSB adjusted topH 1.4 with 5 N H2SO4 and with 5 N KOH, brought topH 6.7. Crystallization occurred at higher pH values.Sulfisoxazole (sodium Gantrisin), also obtained fromHoffmann-LaRoche, was dissolved directly in TSB.Mafenide acetate (the antimicrobial of SulfamylonCream) was supplied by Sterling-Winthrop ResearchInstitute, Rensselaer, N.Y.; after solubilization inTSB, the pH was adjusted back to neutrality with 2 NHCl. These three antimicrobial stock solutions weresterilized by filtration, and samples were ascepticallyadded to tubes containing sterilized growth medium;the final pH of any such medium was 6.9 + 0.2. Serialtwofold dilutions of each drug were made in thegrowth medium, maintaining a total final volume of2.0 ml per tube. The first tube in any such series con-tained 10,000 ,g/ml, the maximum concentration ofthe drug examined, except for trimethoprim, wheresolubility limitations restricted the concentration to5,000 ,sg/ml.

Inoculation and growth measurements. Inocula wereprepared by inoculating 10 ml of TSB, followed byincubation at 37 C for 24 hr. This culture was thendiluted 1:1,000 in TSB, and 0.05 ml was used toinoculate each tube of experimental liquid media.Such tubes contained 2.68 + 0.14 X 104 colony-forming units (CFU) per ml. This was determined bythe use of plate counts; the plating medium employedwas TSB solidified with 2.0% agar and containing0.1% KNO3. The KNO, promoted rapid developmentand visualization of subsurface colonies and was sug-gested by 0. S. Weislow of this laboratory (personalcommunication). This plating medium was also utilizedfor all other viable cell counts.

Inoculated liquid media were incubated at 37 Cfor 24 hr in a candle jar (CO2 environment) or in ahigh-pressure chamber. After such incubation, growthin liquid media was measured turbidimetrically at 650nm with a Lumetron colorimeter, no. 401A (Photo-volt, New York, N.Y.), modified to read small vol-umes. To facilitate these measurements, the volume ofthe original 2.0-ml culture was adjusted to 4.0 ml bythe addition of uninoculated, sterile TSB. The finaloptical density (OD) values as given in this paper thusrepresent a 1:2 dilution of the actual growth. Bac-tericidal or bacteriostatic conditions were determinedas previously detailed (10) and consisted of sampletransfer from tubes devoid of visible growth to en-riched broth which was examined for turbidity afterprolonged incubation.

For studies involving inhibition of surface growthby 02, TSA plates replaced TSB, although nutrientagar (Difco) and Brain Heart Infusion agar (Difco)were also used as noted. Inocula were prepared asdescribed above, except that the 24-hr-old TSB seedcultures were used undiluted; 0.2 ml was ascepticallyspread on the agar surface with a glass rod. This vol-ume of inoculum contained approximately 108 CFU.The predried agar permitted rapid absorption ofinoculum fluid. After 24 hr of gaseous exposurewithin the chamber (37 C), the plates were examined

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for surface growth and compared to control platesincubated in a candle jar or in air (37 C, 24 hr). Sincethe latter two control cultures always demonstratedequivalent amounts of profuse and confluent surfacegrowth, all normobaric controls described in the text,for broth and agar cultures, were incubated in a candlejar where the CO2 tension was more in keeping withthe experimental conditions employed. It was as-sumed that plates which, upon removal from thechamber, showed only a slight haze over their surface(minimal growth) had been subjected to conditionsthat retarded the growth rate. This assumption wasmade if profuse and confluent growth subsequentlydeveloped on these plates within 24 hr of candle jarincubation at 37 C. Experimental conditions wereconsidered bactericidal if plates showed no growthupon removal from the chamber and failed to developprofuse and confluent growth upon subsequent in-cubation in a candle jar (24 hr, 37 C).

Chamber pressurization. The chamber into whichthe freshly inoculated cultures were placed was amodified, cylindrical, ethylene oxide sterilizer, withinternal dimensions of 31 cm diameter by 61 cm height,enclosing a volume of approximately 44 liters andsimilar in other details to a commercial version (modelno. 614 Table-Top Hyperbaric Chamber, The Bethle-hem Corp., Bethlehem, Pa.). The air in this chamberwas evacuated to a pressure of 76 mm of Hg, leavingtherein a calculated, residual N2 content of 60 mm ofHg (0.08 atm). Thehamber c was then filled to 1 atmabsolute (760 mm of Hg) with a gas mixture consist-ing of95% 02 plus 5.0% C02; for oxygen atmospheresgreater than 1 atm absolute, 100% oxygen was super-imposed on this original mixture already in the cham-ber. The final, subsequent partial pressures of N2, 02,and CO2 were readily calculated and are presented inthe text for each experiment. For pressure controlexperiments, the desired pressure was attained byadding 100% N2 to the air (at 1 atm) already in thechamber, thus maintaining the partial pressure of 02equivalent to air at 1 atm absolute. A similar proce-dure has been employed previously (10). After pres-surization, the chamber and its contents were placedin an incubator and the internal chamber temperaturewas maintained at 36 ±fi 1 C throughout each experi-ment.

Agents used to reverse growth inhibition. Compoundsinvolved in folic acid-mediated transfer and reductionof 1-carbon fragments were added singly or in groupsto TSB cultures in attempts to reverse the inhibitoryeffects of antimicrobials, or HPO, or a combinationthereof. These compounds, whose biosynthesis de-pends on folic acid, spare part of the folate require-ment of an organism and are described as classical orprinciple reversing agents of sulfonamide inhibitionin bacteria (8, 14). Three separate stock solutions ofthese agents (amino acids, heterocyclic bases, andvitamins) were prepared in TSB and sterilized byfiltration, and samples were added to growth mediaprior to inoculation. These stock solutions were pre-pared as follows. (i) Amino acids: L-serine andL-methionine were dissolved in TSB at pH 7.1 suchthat, when a 0.1-ml sample was added to 1.9 ml ofgrowth broth, the final concentration of each amino

acid was 200,Ig/ml. (ii) Bases: adenine, guanine, andthymine were solubilized in TSB adjusted to less thanpH 1 with 12 N HCI, and thepH was then raised to 2.6with KOH pellets (increased pH promoted precipita-tion) such that, when 0.2 ml of this solution was addedto 1.8 ml of growth broth, the final concentration ofeach base was 70,ug/ml. (iii) Vitamins: thiamine HCIand the Ca salt of DL-pantothenic acid were dissolvedin TSB at pH 6.9 and the solution was diluted suchthat, when a 0.1-ml sample was added to 1.9 ml ofgrowth broth, the final concentration of each vitaminwas 0.8 ,ug/ml. The final concentrations of these addi-tives approximated 2 to 4 times the concentration ofsimilar compounds as used in synthetic growth mediafor streptococci (20).

In addition to the above compounds, folic acid, fo-linic acid (a synthetic tetrahydroformyl folic acid), andPAB were also used as supplements for growth broth.Separate stock solutions of each were prepared asfollows. (i) Folic acid (Sigma Chemical Co., St. Louis,Mo.) was dissolved in TSB, and the pH was adjustedto 7.1 with 1 N KOH; (ii) folinic acid (General Bio-chemicals, Chagrin Falls, Ohio), as Ca leucovorin,and (iii) PAB (Sigma Chernical Co.) were treatedsimilarly to folic acid. All three stock solutions weresterilized by filtration. When 0.1- or 1.0-ml samplesfrom any stock solution were incorporated in a totalfinal volume of 2.0 ml of growth broth, the concentra-tion of any additive was 10 or 100 ,ug/ml, respectively.The final pH of any supplemented growth mediumused in this work was 6.9 :+1 0.2.

RESULTS

Inhibition of growth by HPO per se. Initial ex-periments established the sensitivity of P. aeru-ginosa 283 to the growth-inhibitory effects of 02alone. These conditions would subsequently beemployed with antimicrobial drugs in the pres-ence or absence of (±) compounds potentiallycapable of inhibition reversal. Table 1 showsthat on various types of agar, as well as in TSB,this aerobic organism was affected by Po2 in asimilar fashion at any given pressure. During a24-hr exposure period at 37 C to 0.87 atm ab-solute of 02 plus 0.05 atm absolute of CO2 plus0.08 atm absolute of N2, growth on agar was notrepressed and, in broth, apparently was stimu-lated somewhat when compared with controlcultures. At 2 atm absolute, 02(1.87 atm absolute;CO2 and N2 as above) retarded growth. In broth,turbidity was depressed approximately 15-fold.Agar cultures which, after HPO incubation,showed only a slight surface haze were capable ofprofuse outgrowth after reincubation underconditions employed for control cultures. At 3atm absolute, 02 (2.87 atm absolute; CO2 and N2as above) was largely bactericidal; of the 108 CFUinoculated per plate, approximately 103 survived.It seemed unlikely that these could be HPO-resistant mutants, since this would represent an

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TABLE 1. Growth on agar surfaces and in brotha upon exposure to isereased oxygen tensionsb

Oxygen tension (atm absolute)Medium Controls

0.87 1.87 2.87

Nutrient agar; Brain Heart Infu- Confluent Minimal None Confluentsion agar; Trypticase soy agar (confluent)c (103 CFU/plate)c

Trypticase soy broth 0.89 0.05 0 0.73(0.52)c

a Growth in broth expressed as optical density units at 650 nm.bAll 02 exposures were at 37 C for 24 hr with 0.05 atm absolute of CO2 and 0.08 atm absolute of N2

present; controls were incubated in a candle jar at 1 atm absolute.c Growth which appears after incubation of 02-exposed (inhibited) cultures to control culture con-

ditions. CFU, colony-forming units.

unusually high frequency (10-5). The change wassubsequently taken to represent a populationreduction of over 99%. In broth, similar condi-tions apparently prevailed. No turbidity was evi-dent after HPO exposure, although two-thirds ofthe turbidity seen in control cultures appearedwhen the inhibited cultures were reincubated atcontrol conditions. This supported results ob-tained with agar cultures indicating that mostcells in the original inoculum were killed at threeatmospheres; the bacteriostatically inhibited re-mainder were capable of growth when returnedto normobaric conditions. A similar interpreta-tion was made by other workers (2, 5). In addi-tion, the inhibitory effects of HPO were notovertly influenced by media pH, which variedfrom 6.8 (nutrient agar) to 7.2 (TSA) to 7.4(Brain Heart Infusion agar).The inhibition of growth noted was caused by

the Po, and not by elevated pressures per se.Similar levels of growth thus appeared on agaror in broth when cultures were incubated for 24hr at 37 C either at 1 atm absolute in a candlejar or at 3 atm absolute in a gaseous environmentconsisting of 0.2 atm absolute of 02 plus 0.05 atmabsolute of CO2 plus 2.75 atm absolute of N2(Po2 as in 1 atm absolute of air). The lattercondition thus served as a pressure control.Other control experiments indicated that thevacuum drawn on the chamber (to 76 mm of Hg)prior to charging with the indicated gaseous mix-ture had no measurable effect on growth. Thishas also been reported for other aerobes (10).

Inhibition of growth by antifolate agents. Thepartial inhibition of growth in broth culturesproduced by incubation under 1.87 atm absolute02 permitted quantitation of the combined effectsof HPO and antimicrobial drugs. Three antifolatedrugs (trimethoprim, sulfisoxazole, and mafenide)were examined. These were individually employedin twofold serial dilutions with TSB. After inocu-lation, these cultures were subjected to HPO,

examined after 24 hr for growth inhibition, andcompared with normobaric controls. The bac-teriostatic (static) and bactericidal (cidal) con-centrations found for each drug are shown inTable 2; HPO was considered to have enhanceddrug activity if the inhibitory concentration ofthe drug could be reduced in the presence of 02.

(i) Trimethoprim, a non-sulfonamide, is asubstituted pyrimidine which inhibits dihydro-folate reductase activity and thus the conversionof dihydrofolate to tetrahydrofolate (14). Atconcentrations up to 5,000 ,ug/ml, the drug wasnot found to be bactericidal with or withoutHPO. Although it did exhibit bacteriostatic ac-tivity, trimethoprim was not directly enhanced inthis activity by HPO.

(ii) Sulfisoxazole, a sulfonamide possessingthe classical sulfanilamido-radical, presumablyinterferes with the PAB metabolism of the micro-organism (12) and, predictably, was not foundto be bactericidal with or without HPO in con-centrations up to 10,000 ,ug/ml. This drug, liketrimethoprim, exhibited bacteriostatic activity.Unlike trimethoprim, this activity was enhancedtwofold by HPO since the concentration range inHPO was one-half that required in control cul-tures.

(iii) Mafenide, as described by Winthrop Labo-ratories, New York, N.Y., is a-amino-p-toluene-sulfonamide (personal communication). At posi-tion 1 of the benzene ring, both mafenide andsulfisoxazole contain the most important func-tional group, the sulfamyl moiety. The aminogroup nitrogen of mafenide, however, is notdirectly linked to position 4 of the ring, as is thecase with sulfisoxazole. The mechanism of actionof the uniquely structured mafenide is as yet un-known; however, the structure differs from PABto a degree which may render the simple PAB-sulfonamide competition hypothesis improbable(12). Unlike both trimethoprim and sulfisoxazole,

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TABLE 2. Inhibitory effects of increased oxygen tensiona anzd antimicrobials on growth of Trypticase SoyBroth cultures

Test atmosphereAntimicrobial Potentiation

1.87 atm 02 Control

Trimethoprim Noncidal up to 5,000 Noncidal up to 5,000 None(pg/mi) Static: >313 _ 625 Static: >313 < 625 None

Sulfisoxazole Noncidal up to 10,000 Noncidal up to 10,000 None(Ag/mI) Static: >1,250 < 2,500 Static: >2,500 < 5,000 2X

Mafenide Cidal: >5,000 < 10,000 Noncidal up to 10,000 2X(pg/ml) Static: >156 < 313 Static: >313 < 625 2X

(>625 < 1,250) (4X)

a All 02 exposures were at 37 C for 24 hr with 0.05 atm absolute of CO and 0.08 atm absolute of N2present; controls were incubated in a candle jar at 1 atm absolute.

mafenide (>5,000 ±10,000 ,g/ml) acts bac-tericidally (Table 2), but only in the presence ofHPO and at approximately one-half the concen-

tration which was bacteriostatic in normobariccontrol cultures. Since 1.87 atm absolute of 02per se was earlier found only to retard growthand mafenide in non-HPO-treated cultures be-haved bacteriostatically, the interaction of ma-fenide with HPO to produce bactericidal condi-tions may be considered synergistic. The bac-teriostatic activity of lesser mafenide concentra-tions was also found to be enhanced twofold intwo experiments; a third experiment (data shownin parenthesis in Table 2) indicated the concen-

tration range of mafenide required for bacterio-stasis in normobaric controls was fourfold greaterthan in HPO-treated cultures.

Reversal of sulfonamide activity and HPO-drug enhancement by nutritional enrichment. Theminimum inhibitory concentrations of sulfisoza-zole and mafenide were adopted for use in experi-ments where reversal of HPO-drug enhancementand sulfonamide activity alone would be at-tempted. Trimethoprim was not examined be-cause HPO failed to enhance its activity. Sincesulfisoxazole acted bacteriostatically with HPO atconcentrations less than 2,500 ,ug/ml and innormobaric controls at less than 5,000 ,ug/ml, thelatter concentration was adopted for use withHPO and in controls. This concentration pro-vided a margin of safety for reliably achievingbacteriostasis. From the data in Table 2, it isseen that minimum bacteriostatic concentrationsof mafenide could be of different orders of magni-tude depending on the presence or absence ofHPO. Thus, 400 Ag/mnl was selected for use withHPO-treated cultures; 1,400 Ag/ml was adoptedfor reliable use with controls. The bactericidalconcentration of 10,000 ,ug/ml, applicable onlywith HPO exposures, was selected.

TABLE 3. Effect of additives on bacteriostaticconditions produced by sulfisoxazole plusincreased oxygen tensiona in Trypticase Soy

Broth cultures

Growthb at test atmosphereAdditive Sulfi-Agll Isoxazole-

(gm)1.87 atm 02 Control

None .......... 0 0.05 0.73None.......... 5,000 0 (static) 0 (static)Folic or folinic

acid....... 5,000 0 (static) 0 (static)(10-100)

p-Amino-benzoate... 5,000 0.03 0.60

(10-100)Mixturec....... 5,000 0 (static) 0 (static)

a All 02 exposures were at 37 C for 24 hr with0.05 atm absolute of CO2 and 0.08 atm absolute ofN2 present; controls were incubated in a candlejar at 1 atm absolute.bGrowth expressed as optical density units at

650 nm.C L-Serine and L-methionine, 200 Aglml, respec-

tively; guanine, thymine, and adenine, 70 ,g/ml,respectively; thiamine*HCI and calcium panto-thenate, 0.8 ,ug/ml, respectively.

(i) The effect of compounds involved in folatemetabolism on the bacteriostatic conditionsproduced by sulfisoxazole with or without HPOis shown in Table 3. These compounds were in-corporated in TSB at the concentrations indi-cated. As expected, HPO alone retarded growth15-fold. Sulfisoxazole itself acted bacteriostati-cally with or without HPO. Neither folic norfolinic acids, at concentrations of 10 and 100Asg/ml, reversed this. These PAB derivatives arenot utilized by most bacteria that respond tosulfonamide treatment, presumably because ofimpermeability (7, 8); this strain responds to

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TABLE 4. Effect of additives on bactericidalconditions produced by mafenide plus increasedoxygen tensiona in Trypticase Soy Broth cultures

Additive (/Mafe- Growthb at test atmosphere(;Agml) nide(gm) (pg/mI) 1.87 atm O2 Control

None .......... 0 0.05 0.73None.......... 10,000 0 (cidal) 0 (static)Folic or folinic

acid....... 10,000 0 (cidal) 0 (static)(10-100)

p-Amino-benzoate... 10,000 0 (cidal) 0 (static)

(10-100)Mixture- ....... 10,000 0 (static) 0 (static)

All 02 exposures were at 37 C for 24 hr with0.05 atm absolute of CO2 and 0.08 atm absolute ofN2 present; controls were incubated in a candlejar at 1 atm absolute.bGrowth expressed as optical density units at

650 nm.cL-Serine and L-methionine, 200 ,Ag/ml, re-

spectively; guanine, thymine, and adenine, 70,ug/ml, respectively; thiamine-HCl and calciumpantothenate, 0.8 ,Ag/ml, respectively.

such treatment in vivo (3). At concentrations of10 and 100,ug/ml, PAB predictably reversed thebacteriostatic activity of sulfisoxazole with orwithout HPO, since growth occurred. A mixtureof compounds considered to be the principal,noncompetitive reversing agents of sulfonamideinhibition failed to reverse the bacteriostaticconditions produced by sulfisoxazole with orwithout HPO. This mixture was composed ofamino acids, heterocyclic bases, and vitamins.

(ii) The effects of the same additives, as usedabove, on the bactericidal conditions produced bymafenide (10,000 ,ug/ml) plus HPO are shown inTable 4. In this case, folic, folinic, and p-amino-benzoic acids, respectively, failed to alter theseconditions, but the reversing mixture caused thebactericidal conditions to become bacteriostatic.Similar results are shown in Table 5, where ma-fenide was used at minimal concentrations whichwere bacteriostatic with HPO (400 Ag/ml) andwith normobaric controls (1,400 ,ug/ml). Again,only the mixture was active; growth occurred incontrols and HPO-exposed cultures, reversingthe bacteriostatic conditions produced by ma-fenide with or without HPO.

Reversal of HPO inhibition per se by specificnutritional enrichment. Having noted earlier that2.87 atm absolute of 02 (plus traces of CO2 andN2) was largely bactericidal, this condition wasemployed to establish quantitatively that theeffects of HPO alone could be reversed by the

TABLE 5. Effect of additives on bacteriostaticconditions produced by mafenide plus increasedoxygen tensiona in Trypticase Soy Broth cultures

Additive(pg/ml)

None ..........None ..........

Folic or folinicacid

(10-100).....(10-100) ....

p-Amino-benzoate

(10-100) .....(10-100).....

Mixturec.......

Mafe-nide

(pg/ml)

01 ,400400

1,400400

1,400400

1,400400

Growthb at test atmosphere

1.87 atm 02

0.050 (static)0 (static)

0 (static)0 (static)

0 (static)0 (static)0 (static)0.01

Control

0.730 (static)0.01

0 (static)0.01

0 (static)0.010.010.12

a All 02 exposures were at 37 C for 24 hr with0.05 atm absolute of CO2 and 0.08 atm absolute ofN2 present; controls were incubated in a candlejar at 1 atm absolute.

b Growth expressed as optical density units at650 nm.

C L-Serine and L-methionine, 200 ,Ag/ml, respec-tively; guanine, thymine, and adenine, 70 ,ug/ml,respectively; thiamine * HCI and calcium pantothe-nate, 0.8 ,ug/ml, respectively.

same additives used previously. Figure 1 showsthe results obtained. Viable cell counts were made,and the data indicated that when broth cultureswere exposed to 2.87 atm absolute of 02 approxi-mately 85% of the cells in the original inocu-lum were killed within a 24-hr period. Folic,folinic, and p-aminobenzoic acids, respectively,decreased killing by 20%. Small amounts of folicand folinic acids presumably permeated thesecells, permitting increased survival but notgrowth. The vitamin and amino acid portions ofthe reversing mixture respectively decreasedHPO-killing by approximately 15%, whereas thedecrease approximated 35% when the entiremixture of vitamins, amino acids, and bases wasadded. The most effective portions of this mix-ture were the purine and pyrimidine bases, ofwhich adenine was the most active, decreasingthe lethal effects of HPO by approximately 60%.

DISCUSSIONThis investigation was directed at an explana-

tion for the reported (3) lack of in vivo HPO-sulfonamide enhancement in the treatment ofburn-wound sepsis, as caused by P. aeruginosa.In vitro experiments with a sulfonamide (ma-fenide) indicated enhancement should have oc-

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NO ADDITIVES

p-AMINOBENZOATE _

FOLINIC ACID

FOLIC ACID

VITAMINS (THIAMINE; PANTOTHENATE)

AMINO ACIDS (SERINE; METHIONINE)

AMINO ACIDS, HETEROCYCLIC BASES, VITAMINS

HETEROCYCLIC BASES (GUANINE ,THYMINE ,ADENINE)-

GUANINE

THYMINE

ADENINE

__*1~~~~~~~~~~~~1

_,,

0 20 400 60 80 100ADDITIVES TO TSB PERCENTAGE CFU KILLED

FIG. 1. Effect of additives on bactericidal conditions produced by 2.87 atm absolute of 02 plus 0.05 atmabsolute Of CO2 plus 0.08 atm absolute ofN2. Cultures of 2 ml of TSB were each inoculated with 2.68 4 0.14X 104 CFU/ml and incubated at 37 C for 24 hr. The extent of each bar represents a numerical average ofviable counts from at least two separate experiments; each condition was performed in triplicate in each ex-periment. Brackets indicate the maximum deviation ofany count from the average figure. The concentration ofeach additive follows: p-aminobenzoic, folic, and folinic acids, 10 ,ug/ml, respectively; L-serine and L-me-thionine, 200 ;&g/ml, respectively; guanine, thymine, and adenine, 70 ,ug/ml, respectively; thiamine * HC1 andcalcium pantothenate, 0.8,ug/ml, respectively.

curred (2, 3). The approach used in this paperwas based on the premise that observed in vitroenhancement of sulfonamide and PAS antibac-terial activity by HPO (3, 10, 11) may have re-sulted from an inhibition of folic acid metabolismby these drugs and HPO, respectively. Such in-hibition should then be reversible nutritionally,and possibly in vivo by body fluids (burn exu-date).A comparison of the data presented in Tables

2, 3 and 4 plus Fig. 1 lends support to this hy-pothesis. In vitro inhibition produced by sulfona-mides, by HPO per se, and by the enhancedinteraction of the two, was reversed by com-pounds which are products of the coenzyme ac-tivity of folate or which are involved in folatebiosynthesis. Although it was necessary to con-duct these experiments in a complex nutrientmedium (TSB), the reversal effects noted oc-curred only upon supplementation with thesefolate-related compounds. The concentrations ofthese nutritional supplements were arbitrarily se-lected to produce a qualitative response ratherthan to define a mechanism (competitive versusnoncompetitive) for the observed inhibitions andtheir reversals (18). These concentrations are notnecessarily critical since inhibition reversals were

often possible at considerably reduced levels(unpublished data). The high sulfonamide con-centrations employed were as recommended byRoche Laboratories, Nutley, N.J., (personalcommunication) for total growth inhibition incomplex media. They do not necessarily reflect in

vivo requirements, the amounts necessary forenzymatic level inhibition, nor the low inhibitoryconcentrations active in reducing growth by50% in semisynthetic growth media (18).The above results taken collectively suggest

that HPO-labile reactions may exist in pathwaysinvolved with folic acid coenzymes or folatebiosynthesis. This suggestion is not intended toexclude other possible explanations for 02 tox-icity although it correlates well with observa-tions that HPO can enhance antifolate drug ac-tivity against a variety of aerobes. Thus, in vitro,HPO has been reported to enhance PAS activityagainst mycobacteria (11), and sulfonamide ac-tivity against vibrios (10) and pseudomonads (3).

It is not known whether similar inhibitorymechanisms are functional, at the enzymaticlevel, for HPO and sulfisoxazole, respectively. Inview of the mechanism of sulfonamide inhibitionof PAB metabolism, whereby the drug acts as acompetitive substrate in the synthesis of dihy-dropteroic acid (4), it is difficult to ascribe a simi-lar role to HPO. It is, nonetheless, possible that asimilar metabolic sequence may be fortuitouslysensitive to both HPO and sulfonamides althoughthe inhibitory mechanisms may differ at the enzy-matic level.

Reversal of HPO-sulfisoxazole inhibition. Theenhanced, bacteriostatic effects of 1.87 atm ab-solute of 02 plus sulfisoxazole, as well as thebacteriostatic activity of sulfisoxazole alone, wereovercome by PAB. The inability of the non-competitive reversing mixture (composed of

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compounds whose biosynthesis is mediated byfolate derivatives) to overcome the bacteriostaticactivity of sulfisoxazole with or without HPOmay be due to an intracellular accumulation ofPAB or to toxic products of its aberrant metabo-lism. Such mechanisms have been reviewed byDavis (7). This phenomenon could be caused bysulfisoxazole replacing PAB in the condensationreaction with a pteridine such that PAB could ac-cumulate. The synthesis of dihydropteroic acidand subsequently dihydrofolic and tetrahydro-folic acids (4) would also be inhibited.

Reversal of HPO-mafenide inhibition. A dif-ferent situation was observed with inhibitionproduced by mafenide. The bacteriostatic andbactericidal effects of this drug, as enhanced byHPO, as well as the bacteriostasis produced bymafenide alone, were not reversed by PAB addi-tions. These phenomena were, however, miti-gated by the mixture of amino acids, purine andpyrimidine bases, and vitamins. This mixture alsopartially overcame the bactericidal effects of 2.87atm absolute of 02. The mechanism of mafenideaction is as yet unreported; the inability of PABto reverse mafenide activity indicates that it is anatypical sulfonamide. However, the mitigation ofmafenide activity achieved by the mixture offolate-related compounds suggests this antimicro-bial is nonetheless inhibitory to folate metabolismat a non-PAB site. If this is the case, the aboveobservations could be explained by speculatingthat mafenide and HPO respectively may inhibitat one of two metabolic stages, or both: (i) in theformation of dihydrofolic acid from its precursor,dihydropteroic acid, or (ii) in the conversion oftetrahydrofolate into its active, coenzyme forms.In the latter case, the concomitant synthesis ofthose products contained in the noncompetitivereversing mixture would also be inhibited.Trimethoprim was not enhanced in its inhibi-

tory activity by HPO. It is thus not necessary topostulate that dihydrofolate reductase is HPO-labile. Subsequently, the conversion of dihy-drofolate to tetrahydrofolate (14) would not beinhibited by HPO in this organism. This observedlack of HPO-trimethoprim enhancement mayalso indicate that the above enzymatic step is notmafenide-sensitive since the activity of mafenidewas enhanced by HPO. In this discussion thepathway of folate synthesis was assumed to be thegenerally accepted scheme as indicated by Brown(4) for Escherichia coli and later reviewed else-where (14). It is of interest that Gottlieb, usingNeisseria catarrhalis, also reported trimethoprimactivity was not enhanced by HPO. However,sulfisoxazole plus trimethoprim apparentlyshowed increased effectiveness in the presence ofHPO such that folic acid reductase was considered

to be HPO-labile in this organism (S. F. Gottlieb,Bacteriol. Proc., p. 80, 1969).

Reversal of HPO-inhibition per se. The dataindicated that 02 per se, at 3 atm absolute, had abactericidal effect, killing 85% of the inoculumwithin 24 hr. The magnitude of this effect, as wellas the bacteriostatic effects encountered in thisstudy, are dependent on time, pressure, and evenstrain of organism (2, 3, 5, 10). The bactericidaleffect reported, however, could be mitigated byfolic, folinic, and p-aminobenzoic acids, and bycompounds whose synthesis is mediated by folatederivatives. This correlated well with the inhibi-tion-reversal data obtained by using similar com-pounds with antifolate drugs plus or minus HPO.Taken together, such data suggest the existenceof HPO-labile sites in folate biosynthesis and insubsequent reactions involving folate derivatives.A report that unresolved mixtures of amino

acids reversed HPO toxicity for an Achromobacterspecies (9) supports the data presented here.From Fig. 1, it appears that serine or methionine,or both, could have specifically been responsiblesince reducing substances in the Achromobactermedium were reported not to be involved. Thia-mine has also been reported to prevent the toxiceffects of 02 on the metabolism of Staphylococcusaureus (22). The data presented here showed thata mixture of thiamine and pantothenate reversed02 toxicity for a pseudomonad. Further, thecurrent observation that adenine supplementationwas protective against HPO toxicity is supportedby reports that adenosine triphosphate decreasesin respiring mammalian tissue slices and ho-mogenates exposed to HPO (13, 17). A similareffect might be expected in bacteria if the mecha-nism postulated by Haugaard (13) was operative,i.e., a toxic action of HPO on pyruvate metabo-lism resulting from oxidation of the sulfhydrylgroup of coenzyme A. Other possible toxic effectshave been reviewed elsewhere (21). It is thus notnecessary to assume that HPO-induced depres-sion of cellular adenosine triphosphate resultsfrom a single metabolic block, just as it is un-likely that there is one mechanism of 02 poison-ing (13). Because adenine was the most protectiveof all additives employed, its metabolism may bevery closely related to HPO toxicity. Althoughmore than one aspect of such metabolism maybe involved, an inhibition of folate metabolismby HPO could indirectly influence adenine andadenosine triphosphate concentrations. Sinceadenine is vital for nucleic acid biosynthesis andwas found most effective in overcoming the bac-tericidal effects of HPO, alterations in nuclearas well as other structures might be expected toresult from HPO toxicity. This has not yet beenstudied in bacteria, although cells in organ cul-

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tures of rat liver slices show such structuralchanges (6, 19).

In vitro versus in vivo HPO effects. The reversalof HPO toxicity per se by the noncompetitivereversing mixture (Fig. 1) suggests an explana-tion for the report of Bornside and Nance (3).These authors indicated a lack of in vivo HPO-antimicrobial enhancement in the treatment ofburn-wound sepsis of rats as produced by thesame organism used in this work. The aminoacids, heterocyclic bases, and vitamins, used toreverse 02 toxicity in vitro (Fig. 1), are found inburn-wound exudates. Mammalian cell lysis re-leases noncompetitive antagonists of sulfonamideactivity, such as the heterocyclic purine and py-rimidine bases, which are not present in normalbody fluids. The presence of these compounds inexudates has been used to explain the in vivolack of activity of classical sulfonamides other-wise active in vitro (8). The current study indi-cated that a mixture of such bases, or adeninealone, could best overcome the inhibitory effectsof HPO. It now appears that possibly for the samereason HPO did not enhance the in vivo activityof mafenide or polymyxin B with pseudomonads(1, 3). The in vitro findings reported here suggestthese antagonists could substantially reduce theinhibitory effects of HPO per se and subsequentlyits ability to enhance the activity of an antimicro-bial drug. Such results were specifically foundwith mafenide in vitro (Tables 4 and 5). This sug-gestion is further supported by in vivo observa-tions which indicated HPO alone failed to eradi-cate pseudomonads in experimental wound in-fections (16, 23). In these cases the effects ofHPO may also have been nutritionally mitigatedby the immediate environment of the organismswhich consisted of wound exudate fluids.Based on reversals of inhibition by specific,

nutritional supplementation, this study indicatedthat aspects of folate metabolism may be inhibitedby HPO. Confirmation is needed at the enzymaticlevel. Such a concept has the advantage of pro-viding the simplest, unified explanation for theobserved HPO-enhancement of antimicrobialsin vitro, as well as the reported (3) in vivo lackof enhancement encountered during the treat-ment of burn-wound sepsis in rats.

ACKNOWLEDGMENTS

This study was initiated in the Department of Microbiology ofthe Jefferson Medical College, Philadelphia, Pa. The support of thelatter institution as well as that of Temple University is gratefullyacknowledged.

LITERATURE CITED

l. Argamaso, R. V., and G. M. Wiseman. 1967. The use of com-bined hyperbaric oxygen and polymyxin B in the treatment

of Pseudomonas infections of mice. Plast. Reconstruct.Surg. 40:81-85.

2. Bornside, G. H. 1967. Exposure of Pseudomonas aeruginosato hyperbaric oxygen: inhibited growth and enhanced ac-

tivity of polymyxin B. Proc. Soc. Exp. Biol. Med. 125:1152-1156.

3. Bormside, G. H., and F. C. Nance. 1969. High-pressure oxygen

combined with antibiotics in the therapy of experimentalburn wounds. Antimicrob. Ag. Chemother. 1968, p. 497-500.

4. Brown, G. M. 1962. The biosynthesis of folic acid. II. Inhibi-tion by sulfonamides. J. Biol. Chem. 237:536-540.

5. Brown, 0. R., R. G. Silverberg, and D. 0. Huggett. 1968.Synergism between hyperoxia and antibiotics for Pseudo-monas aeruginosa. Appl. Microbiol. 16:260-262.

6. Coupland, R. E., and J. D. B. MacDougall. 1968. The effect ofhyperbaric oxygen on rat liver cells in organ culture: a

light- and electron microscope study. J. Pathol. Bacteriol.96:149-159.

7. Davis, B. D. 1958. Principles of chemotherapy: drug-parasiteinteractions, p. 671-693. In R. J. Dubos (ed.), Bacterial andmycotic infections of man, 3rd ed. J. B. Lippincott Co.,Philadelphia.

8. Davis, B. D., R. Dulbecco, N. H. Eisen, H. S. Ginsberg, andW. B. Wood. 1967. Microbiology, p. 307-308. Harper andRow, New York.

9. Gottlieb, S. F. 1966. Bacterial nutritional approach to mecha-nisms of oxygen toxicity. J. Bacteriol. 92:1021-1027.

10. Gottlieb, S. F., and L. M. Pakman. 1968. Effect of high oxygen

tensions on the growth of selected, aerobic, gram-negative,pathogenic bacteria. J. Bacteriol. 95:1003-1010.

11. Gottlieb, S. F., N. R. Rose, J. Maurizi, and E. A. Lanphier.1964. Oxygen inhibition of growth of Mycobacterium tuber-culosis. J. Bacteriol. 87:838-843.

12. Grollman, A., and E. F. Grollman. 1970. Pharmacology andtherapeutics, 7th ed., p. 566-579. Lea and Febiger, Phila-delphia.

13. Haugaard, N. 1965. Poisoning of cellular reactions by oxygen.

Ann. N. Y. Acad. Sci. 117:736-744.14. Hitchings, G. H., and J. J. Burchall. 1965. Inhibition of folate

biosynthesis as a basis for chemotherapy. Advan. Enzymol.27:417-468.

15. Huges, D. E., and J. W. T. Wimpenny. 1969. Oxygen metabo-lism by micro-organisms, p. 197-232. In A. H. Rose, andJ. F. Wilkinson (ed.), Advances in microbial physiology,vol 3. Academic Press Inc., New York.

16. Irvin, T. T., J. N. Norman, A. Suwanagul, and G. Smith. 1966.Hyperbaric oxygen in the treatment of infections by aerobicmicroorganisms. Lancet 1:392-394.

17. Joanny, P., and J. Corriol. 1970. Hyperbaric oxygen: effects onmetabolism and ionic movement in cerebral cortex slices.Science 167:1508-1510.

18. Lampen, J. O., and M. J. Jones. 1946. The antagonism ofsulfonamide inhibition of certain lactobacilli and enterococciby pteroylglutamic acid and related compounds. J. Biol.Chem. 166:435-448.

19. Noguchi, T. T., F. F. Strasser, and H. Nichihara. 1967. Hyper-baric oxygen toxicity on tissue-culture cells. I. Light-micro-scope study. Amer. J. Pathol. 51:243-257.

20. Stonehill, E. H., and D. J. Hutchinson. 1966. Chromosomalmapping by means of mutational induction in synchronouspopulations of Streptococcus faecalis. J. Bacteriol. 92:136-143.

21. Wimpenny, J. W. T. 1969. Oxygen and carbon dioxide as

regulators of microbial growth and metabolism, p. 161-197.In P. Meadow and S. J. Pirt (ed.), Nineteenth Symposiumof The Society for General Microbiology. Cambridge Uni-versity Press, London.

22. Wolin, M. J., J. B. Evans, and C. F. Niven, Jr. 1956. A study ofthe methylene blue and oxygen inhibition of pyruvate oxida-tion by Micrococcus pyogenes var. aureus. Arch. Biochem.Biophys. 63:356-364.

23. Zaroff, L. I., H. L. Walker, E. Lowenstein, B. W. Evans, andL. S. Kroos. 1965. Hyperbaric oxygenation in aerobic infec-tion. Arch. Surg. 91:586-588.

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