Antifungal Effect of Hydrogen Peroxide on Catalase …downloads.hindawi.com/journals/idog/1995/983197.pdftion of hydrogen peroxide. The catalase was ex-pressed as units, in which unit

Infectious Diseases in Obstetrics and Gynecology 3:73-78 (1995)(C) 1995 Wiley-Liss, Inc.

Antifungal Effect of Hydrogen Peroxide onCatalase-Producing Strains of Candida spp.

Bryan Larsen and Sandra WhiteDepartments of Obstetrics and Gynecology (B.L., S.W.) and Microbiology, Immunology, and Molecular

Genetics (B.L.), Marshall University School of Medicine, Huntington, WV

ABSTRACT

Objective: Clinical isolates of Candida were tested for the presence of catalase and susceptibility tohydrogen peroxide.

Methods: MIC was tested by broth dilution technique and catalase was determined by a spectro-photometric procedure.

Results: All 38 strains tested were inhibited by hydrogen peroxide in concentrations ranging from4.4 to 88 mM/l, with non-albicans isolates generally requiring higher concentrations of hydrogenperoxide for inhibition. Growth media consisting of glucose and protein diminished the antifungaleffectiveness of hydrogen peroxide, as did the presence of hemoglobin, in incubation mixtures.However, hydrogen peroxide exerted greater inhibition at pH 4 than at pH 7. Although all Candidaisolates tested possessed catalase, there was no apparent correlation between the catalase activity ofindividual isolates and the minimal antifungal concentration of hydrogen peroxide.

Conclusions: This study suggested that, despite the production of catalase by vaginal microorgan-isms, hydrogen peroxide may exert a regulating influence which may be further modified by theproteins found in the vaginal milieu. (C) 1995 Wiley-Liss, Inc.

KEY WORDS

Normal vaginal flora, vaginal bacteria, lactobacilli

or many years, lactobacilli have been consideredto be an important part of the vaginal microbial

ecosystem, often credited with maintaining vaginalhealth by inhibiting other microbial species. Clin-ical evidence indicates an association between thepresence of the symptoms of bacterial vaginosis,characterized by an altered vaginal flora with in-creased numbers of Gardnerella vaginalis and vari-ous anaerobic species, concomitant with decreasedlactobacillus colonization, obvious even on Gram-stained smears. 2 This apparently protective effectwas originally attributed to acid production by thelactobacilli, but even in the early part of this cen-

tury some investigators cast doubt on the role oflactobacilli in producing vaginal acidity. 3’4 In re-cent years, an alternative explanation has been setforth. Several investigators have indicated that some

strains of lactobacilli are able to generate hydrogenperoxide and that its production by these organismsaccounts for their beneficial effect. 5-8

Despite the apparent importance of lactobacillito the composition of the vaginal flora, the diver-sity of bacterial species at this site is well known,including, in addition to hydrogen-peroxide-pro-ducing lactobacilli, both catalase-producing andnon-catalase-producing organisms. 9 It is not clearwhy organisms lacking catalase persist in the pres-ence of hydrogen peroxide producers or why cata-

lase-negative bacteria persist in the presence of hy-drogen-peroxide-producing bacteria. Studies of thevaginal flora have indicated that lactobacilli andCandida are frequently coisolated. 10 Because Can-dida albicans contains a catalase, 11 the present studywas undertaken to determine its relationship to the

Address correspondence/reprint requests to Dr. Bryan Larsen, 1801 6th Avenue, Huntington, WV 25703.

Received January 23, 1995Basic Science Article Accepted June 5, 1995

ANTIFUNGAL EFFECT OF H202 ON CANDIDA SPP. LARSEN AND WHITE

hydrogen peroxide that may be present in the vagi-nal microenvironment.

MATERIALS AND METHODSMicroorganisms

Vaginal cultures were obtained from pregnant pa-tients at their initial prenatal visits. The specimenswere plated on BIGGY (BBL, Cockeysville, MD)agar lz and returned to the laboratory. Candida iso-lates from these vaginal specimens were prelimi-narily identified by brown colonies on the BIGGYagar. Individual isolates were verified as fungalorganisms by microscopic morphology. The abilityof each isolate to produce germ tubes was tested inhuman serum. Germ tubes were produced by 26strains and were presumptively considered to be C.albicans or C. stellatoidia; the remaining 12 weredescribed as non-albicans Candida. All isolates werepreserved and propagated by weekly subculture onSabouraud’s agar. All fungal strains in the culturecollection showed catalase activity evidenced bythe generation of bubbles when a colony from a

Sabouraud’s agar plate was placed in a drop of3% hydrogen peroxide (certified 3 %, Fisher, Fair-lawn, NJ).

Media Effect of Inhibition

The antifunal effect ofhydrogen peroxide was testedin phosphate buffered saline (PBS) or in Sabou-raud’s broth (2% glucose, 1% primatone peptone,Sigma, St. Louis, MO). Dilutions of hydrogenperoxide (ranging from 0 to 17 mM/1) in both PBSand Sabouraud’s broth were prepared. Two strainsof C. albicans were grown overnight in Sabou-raud’s broth and each culture was diluted 1:10 priorto use in inoculation. One milliliter of each hydro-gen peroxide dilution was inoculated with 10 I1 ofdiluted starter culture containing about 104 viableyeast cells verified by plate count. The dilutionswere incubated at 37C. After 2, 6, and 24 h ofincubation, aliquots were removed from each tubeand plated on Sabouraud’s agar to determine thenumber of viable cells in each dilution. Because ofthe size of the experiment, only 2 yeast strains wereevaluated.

Minimal Inhibitory Concentration (MIC)/MinimalCidal Concentration (MCC)

Hydrogen peroxide was added to Sabouraud’s brothproviding a range of concentrations from 4.4 to

176 mM/1 and inoculated with approximately 104

viable yeast cells, as described above. A controlcontaining only Sabouraud’s broth was also inocu-lated. After 24 h at 37C, all tubes were evaluatedby visual observation for turbidity, and the lowestconcentration of hydrogen peroxide that preventedthe development of turbidity was determined to bethe MIC. A 10-11 aliquot removed from the tubeshowing the MIC endpoint was inoculated onto a

Sabouraud’s agar plate. If no growth was foundon the plate, the MIC was considered to also bethe MCC.

Germ-tube production provides a simple pre-sumptive method of identifying C. albicans, al-though the less commonly isolated C. stellatoidiaalso germinates. 13 Germ-tube production was testedas described elsewhere 13 with the exception that itwas done in a small volume. Each isolate was grownon a Sabouraud’s agar plate. A single colony fromeach strain was combined with 100 I1 of humanserum in a microtiter well and incubated for 2-3 h.The content of each well was viewed microscopi-cally and evaluated for the presence or absence ofgerm tubes.

The catalase activity was determined as describedby Alcorn et al. 14 Briefly, a starter culture of yeastcells was grown on Sabouraud’s agar and a heavysuspension made in PBS. An aliquot was removedfor a plate count, and 100 I1 of cell suspension wasadded to 1.8 ml of a 17-mM/1 solution of hydrogenperoxide. The cells were allowed to stand for 15rain at room temperature and then centrifuged at

10,000g for min to stop the reaction. The result-ing supernatant was decanted into a quartz cuvette,and absorbance at 240 nm was determined andcompared with the absorbance of a 17-mM/1 solu-tion of hydrogen peroxide. The catalase was ex-pressed as units, in which unit of catalase con-sumes IM of hydrogen peroxide per minute atroom temperature per 106 viable yeast cells.

RESULTSTo test whether C. albicans is intrinsically resistantto hydrogen peroxide, we randomly selected 2 iso-lates from the culture collection and incubated theorganisms in dilutions of hydrogen peroxide inPBS ranging from 0 to 17 mM/1. Each dilutionwas inoculated with approximately 104 viable yeastcells and incubated at 37C. Plate counts were takenat 2, 6, and 24 h to determine the viability of the

74 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

ANTIFUNGAL EFFECT OF HeOz ON CANDIDA SPP. LARSEN AND WHITE

Controls (No Hydrogen Peroxide)1.00E+7.

1.00E+6-, PBS /1.00E+5,

1.00E+4

1.00E+3

1.00E+2

1.00E+I

1.00E+O0 2 6

Time (Hours)

Hydrogen Peroxide (0.85 mM)

.00E+7]

.00E+6-

.00E+5-

.00E+4-

.00E+3-

1.00E+2t’, OoOo :ot

0

Time (Hours)

Hydrogen Peroxide (1.7 mM)

0 2 6 24

Time (Hours)

Hydrogen Peroxide (3.4 mM)1.00E+7

1.00E+6.

1.00E+5.

1.00E+4

1.00E+3

o00E+2

1.00E+1

1.00E+00 2 6 24

Hydrogen Peroxide (8.5 mM)1.00E+7-1.00E+6-11.00E+5-I1.00E+4-I ====1.00E+3- -’-’---’----’1.00E+2

I-e- SAB1.00E+1-

1.00E+0-:0 2 6 24

Hydrogen Peroxide (17 mM)1.00E+71.00E+6-11.00E+5-11.00E+41.00E+3-11.00E+21.00E+

1.00E+0-:0 2 6 24

Time (Hours) Time (Hours) Time (Hours)

Fig. I. Time-dependent inhibition of a representative C.albicans strain in various concentrations of hydrogen perox-ide. Hydrogen peroxide dilutions were prepared in growthmedium (Sabouraud’s broth) or PBS and inoculated withapproximately 104 viable yeast cells. The number of

viable yeast cells at various sampling times is plotted on alogarithmic scale. Hydrogen peroxide in Sabouraud’s brothwas less effective in inhibiting yeast growth than equivalentconcentrations in PBS. This experiment was repeated with asecond yeast strain with similar results.

cells. The results obtained for one of the isolatestested is shown in Figure 1. Because complex me-

dia components may alter the effectiveness of hy-drogen peroxide on C. albicans, we simultaneouslyincubated the organisms in dilutions of hydrogenperoxide prepared in Sabouraud’s broth and countedthe viable yeast cells during the subsequent 24h.The addition of undefined media to the incuba-tion mixture mitigated but did not completely re-

verse the effect of the hydrogen peroxide (Fig. 1).We also noted that, despite the fact that hydrogenperoxide could be fungicidal, the effect was not

instantaneous.The results obtained from a second fungal strain

(results not illustrated to avoid redundancy) showedthe same trends of time-dependent susceptibility to

hydrogen peroxide and diminished antifungalactivity in the presence of growth media, althoughthis second organism was slightly less susceptible to

hydrogen peroxide, which suggested that individ-ual strains may vary in their sensitivity to hydrogenperoxide (range of MIC values reported inTable 1).

TABLE I. MIC data for all Candida isolates

MIC No. of(mM H2Oz) strains inhibited

Mean catalase activityas units/million

viable yeast cells(+_S.D.)

88 mM/I 10 0.0104 (0.0146)17 mM/I 26 0.0103 (0.0049)8.8 mM/I4.4 mM/I

To determine which component of Sabouraud’sbroth was responsible for the diminished inhibitoryeffect of hydrogen peroxide, we tested the antifun-gal effect of hydrogen peroxide on 4 strains of yeastin the presence of PBS, PBS with 1% glucose or

2% peptone (primatone enzymatic meat hydroly-sate, Sigma), or both. Peptone alone was muchmore effective than glucose in diminishing the in-hibitory effect of hydrogen peroxide.

Having determined that the growth of Candidacould be inhibited by sufficient concentrations ofhydrogen peroxide, we next determined whether

INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 75

ANTIFUNGAL EFFECT OF HeO2 ON CANDIDA SPP. LARSEN AND WHITE

100

rj 80

2O

albicans

n=12

non-albicans

Candida

Fig. 2. Susceptibility of 38 Candida strains to hydrogen per-oxide. The average MIC (S.D.) for C. albicans expressed asmM/I is shown to be lower than the MIC for non-albicansCandida.

the susceptibility to hydrogen peroxide was relatedto the catalase content of the organisms. As shownin Table 1, most isolates in our culture collectionwere susceptible to 17mM/1 of hydrogen peroxide.For each yeast strain tested, an aliquot from thetube corresponding to the MIC was plated ontoSabouraud’s agar to determine if the MIC was alsothe fungicidal endpoint. For most strains of Can-dida, a higher concentration of hydrogen peroxidewas needed to produce a fungicidal effect than was

required to prevent the development of turbidity.We also observed that the least susceptible organ-isms were the non-albicans Candida, as illustratedby Figure 2, which indicates that the average MICfor non-albicans Candida is nearly 4 times that forC. albicans.. There was no apparent relationshipbetween the amount of catalase measured and thesusceptibility of individual strains (Table 1).

Although we demonstrated that media compo-nents could diminish the effectiveness of hydrogenperoxide on yeast, we anticipated that hemoglobinmay have an even more profound effect on theantifungal capacity of hydrogen peroxide since it isconsidered to have a pseudocatalase activity. Al-though we anticipated that hemoglobin added tosolutions of hydrogen peroxide may diminish itsantifungal effect, we also wondered if growth of theorganism in the presence of hemoglobin might al-ter the organism’s susceptibility to hydrogen perox-ide. An experiment was conducted with 8 represen-tative yeast strains. Each test organism was grownovernight in either Sabouraud’s broth or Sabou-raud’s broth with 1% bovine hemoglobin. MIC

tests were prepared as described earlier and inocu-lated with the hemoglobin-adapted organisms or

the non-adapted organisms. In addition, a set ofhydrogen peroxide dilutions was prepared with 1%hemoglobin (final concentration) added and anotherset with 0.1% hemoglobin added. A 1% final con-centration completely abrogated inhibition, even at

88 raM/1 of hydrogen peroxide, but lesser concen-trations of hemoglobin had a minimal effect, as

shown in Table 2. When hemoglobin was added ina final concentration of 0.1%, the MIC of most

strains was unaltered. Growth of the organisms inthe presence of hemoglobin generally did not affecttheir susceptibility to hydrogen peroxide, as shownin Table 2.

All previous studies were performed in mediawith a neutral pH, whereas the pI-I of the vagina ismore acidic. To predict how the vaginal pI-I mightaffect the hydrogen peroxide, we compared the an-

tifungal activity of hydrogen peroxide against 4strains of yeast at pH 4 and pI-I 7.2. We selected 2yeast strains that had been shown to be quite sensi-tive to hydrogen peroxide and 2 that were lesssensitive. For this study, we used PBS at pI-I 7.2and PBS with the pH adjusted to 4.0. To thesewere added hydrogen peroxide in a final concentra-tion of 44 mM/1 for the least sensitive strains

(strains W23 and W26) and 2.2 mM/1 for the mostsensitive strains (strains M47 and $6921). Eachtube was inoculated with 104 viable yeast cellsand incubated for 24 h at 37C. Ten microliterswas plated and the viable cells were counted (Fig-ure 3), indicating that the antifungal effect ofhydrogen peroxide at pI-I 4 was greater than at

pH 7.2.

DISCUSSIONThe intricacies of the interactions among vaginalmicroorganisms and the underlying epithelium are

being addressed by current research, which pro-vides a partial explanation for the association oflactobacillus colonization and absence of vaginalsymptoms. The production of bacteriocin, perox-ide, and the elaboration of acidic metabolic end-products may serve to limit to ability of othervaginal organisms to proliferate unimpeded. Nev-ertheless, many bacteria commonly found as mem-bers of the vaginal flora produce catalase whichcould eliminate hydrogen peroxide from the intra-vaginal milieu. The present investigation was un-


ANTIFUNGAL EFFECT OF H202 ON CANDIDA SPP. LARSEN AND WHITE

TABLE 2. Effect of bovine hemoglobin on the susceptibility of 8 Candida isolates to hydrogen peroxide

Strain number oftest organism

Candida grown in Sabouraud’sbroth without hemoglobin (mM/I)

MIC of H202

without hemoglobinMIC of H2Oz with0. I% hemoglobin

Candida grown in Sabouraud’sbroth plus I% hemoglobin

(mM/I)MIC of HzO withoutadditional hemoglobin

47 88 88 88$6921 88 88 889324 320 88 888746 88 88 8836 88 88 88W26 8.8 8.8 17.68749 8.8 8.8 8.8W23 35 35 35

300

250- J’J Counts at pH 7

200- Counts at pH 4

150-

100

500 L_._W23 W26 M47 $6921

Yeast Strain Number

Fig. 3. Effect of pH on the susceptibility of 4 strains of C.albicans to hydrogen peroxide. Equal numbers of the testorganisms listed were added to PBS, with the pH adjusted topH 7.2 or pH 4, and hydrogen peroxide equal to one-half theMIC for each organism was added. After incubation for 24 hat 37C, a 10-11 aliquot was removed and plated on Sab-ouraud’s agar. The number of colonies from the 2 differentpH values were plotted and, as shown by the figure, indicatedthat a lower pH accentuates the inhibitory effect of hydro-gen peroxide.

dertaken to determine if a catalase-producing or-

ganism is intrinsically resistant to the antimicrobialeffect of hydrogen peroxide.We chose to study the effect of hydrogen perox-

ide on Candida because this organism is frequentlyfound in vaginal cultures and is positive for catalasedetected by the classical method of elaboration ofbubbles by organisms added to a drop of 3% hydro-gen peroxide. No correlation was found betweenthe intrinsic catalase activity (measured as units permillion viable yeast cells) and the concentration ofhydrogen peroxide required to inhibit fungal

growth in culture. Alcorn and coworkers 14 indi-cated that the susceptibility of Neisseria gonorrhoeaeto hydrogen peroxide was not solely due to its cata-lase content, a finding similar to the present obser-vations on Candida susceptibility.

From the small amount of hydrogen peroxidedegraded by intact yeast cells, we inferred that in-tact yeast cells may have sufficient catalase to dis-pose of internally generated peroxide, but the en-

zyme is unavailable to destroy substantial quantitiesof extracellular hydrogen peroxide. The intense re-lease of bubbles when a yeast colony is added to a

drop of hydrogen peroxide reagent may be due to a

disruption of the yeast cell integrity which releasessignificant catalase. This possibility was also sug-gested by a microscopic examination of the yeastcells exposed to 3% hydrogen peroxide (datanot shown) which was consistent with yeast-celldamage.

The implication of these findings for the notionthat peroxide-producing lactobacilli have a control-ling influence on the vaginal flora is apparent.While it is impossible to extrapolate to the condi-tions in vivo, it appears that catalase production byCandida may not exempt them from growth restric-tion by hydrogen peroxide. In addition, hydrogenperoxide produced by vaginal flora organisms couldexert antimicrobial effects through mechanismsother than the direct effect we examined in vitro.For example, Klebanoff and Smith 15 indicated thata hydrogen peroxide-peroxidase-halide system couldbe functional in the female genital tract. Otherconditions extant in the vaginal microenvironmentmay also have an influence on the antimicrobialactivity of hydrogen peroxide. For example, pro-

INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 77

ANTIFUNGAL EFFECT OF H202 ON CANDIDA SPP.

teins present in the vaginal milieu may moderatethe effect of hydrogen peroxide, and hemoglobinreleased during menstruation could significantlyalter its influence. It has been previously observedthat menstruation increases the prevalence of vagi-nal staphylococcal colonization 16 and the prevalenceof various organisms is increased immediately post-partum. Iv Although these observations could beexplained by the nutrient properties of blood, theycould also be explained by the destruction of hydro-gen peroxide by hemoglobin. Furthermore, whilesome conditions in the vagina could diminish theantimicrobial effect of hydrogen peroxide, otherssuch as low pH could stabilize its effect.

The composition and control of the vaginal floraare not adequately explained by one simple aspect ofthe microenvironment, but the present study indi-cates that catalase production by vaginal bacteriamay not preclude hydrogen peroxide from exertinga controlling influence on the normal flora.

REFERENCES

1. Redondo-Lopez V, Cook RL, Sobel JD: Emerging roleof lactobacilli in the control and maintenance of the vagi-nal bacterial flora. Rev Infect Dis 12:856-872, 1990.

2. Spiegel CA, Amsel R, Holmes KK (eds.): Diagnosis ofbacterial vaginosis by direct Gram stain of vaginal fluid. JClin Microbiol 18:170-177, 1983.

3. Weinstein L, Wawro NW, Worthington RV, Allen E:The influence of estrogenic hormone on the H-ion con-

centration and bacterial flora of the vagina of the imma-ture monkey. YaleJ Biol Med 11:141-148, 1938.

4. Weinstein L, Howard JH: The effect of estrogenic hor-mone on the H-ion concentration and the bacterial contentof the human vagina, with special reference to the Doder-lein bacillus. Am J Obstet Gynecol 37:698-703, 1939.

5. Klebanoff SJ, Hillier SL, Eschenbach DA, WaltersdorfAM: Control of the microbial of the vagina by H202generating lactobacilli. J Infect Dis 164:94-100, 1991.

LARSEN AND WHITE

6. Eschenbach DA, Davick PR, Williams BL, KlebanoffSJ, Young-Smith K, Critchlow CM, Holmes KK: Prev-alence of hydrogen peroxide-producing Lactobacillus spe-cies in normal women and women with bacterial vagino-sis. J Clin Microbiol 27:251-256, 1989.

7. Nagy E, Petterson M, Mardh P-A: Antibiosis betweenbacteria isolated from the vagina of women with andwithout signs of bacterial vaginosis. AP Microbiol Im-munol Scand 99:739-744, 1991.

8. Hillier SL, Krohn MA, Klebanoff SJ, Eschenbach DA:The relationship of hydrogen peroxide producing lactoba-cilli to bacterial vaginosis and genital microflora in preg-nant women. Obstet Gynecol 79:369-373, 1992.

9. Ohm JM, Galask RP: Bacterial flora of the cervix from100 prehysterectomy patients. Am J Obstet Gynecol 122:683-687, 1975.

10. Larsen B: Normal genital microflora. In Keith LG,Berger GS, Edelman (eds): Common Infections. Lan-caster: MTP Press, pp 3-32, 1985.

11. Tosado-Acevedo R, Toranzos GA, Alsina A: Extractionand purification of a catalase from Candida albicans. PRHealth Sci J 11:77-80, 1992.

12. Nickerson WJ: Reduction of inorganic substances by yeast:Extracellular reduction of sulfate by species of Candida. JInfect Dis 93:43-56, 1953.

13. Lenette EH, Balows A, I-Iausler WJ Jr, TruantJP: Man-ual of Clinical Microbiology. 3rd Ed. American Societyfor Microbiology, Washington DC, pp 562-576, 1980.

14. Alcorn TM, Zheng H-Y, Gunther MR, Hassett DJ,Cohen MS: Variation in hydrogen peroxide sensitivitybetween strains of Neisseria gonorrhoeae is dependent on

factors in addition to catalase activity. Infect Immun 62:2138-2140, 1994.

15. Klebanoff SJ, Smith DC: Peroxidase-mediated antibacte-rial effect of rat-uterine fluid. Obstet Gynecol Invest 1:21,1970.

16. Larsen B, Galask RP: Vaginal microbial flora: Composi-tion and influences of host physiology. Ann Intern Med96:926-930, 1982.

17. Goplerud CP, Ohm MJ, Galask RP: Aerobic and anaer-

obic flora of the cervix during pregnancy and the puerpe-rium. Am J Obstet Gynecol 126:858-868, 1976.


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Antifungal Effect of Hydrogen Peroxide on Catalase …downloads.hindawi.com/journals/idog/1995/983197.pdftion of hydrogen peroxide. The catalase was ex-pressed as units, in which unit