Zbl. Bakt. Hyg., 1.Abt, Orig. A 253, 390-396 (1982)

Instirut fiir Med izinische Mikr obiologie und Immunologie der Universitat Bonn(Direkto r : Prof. Dr . H. Brandis)

Antibacterial Activities of Candida Yeasts. Partial Purification

and Characterization of the Active Substance of Candida

guilliermondii

Antibakterielle Aktivitaten bei Candida-Hefen. Partielle Reinigungund Charakterisierung der aktiven Substanz bei Candida guilliermondii

A. BUDAK\ RG. SAHL, and H. BRANDIS

With 1 Figure' Received Jul y 3, 1982

Abstract

Th e activity spectra of 25 Candida strains on strains of different species of bacteria wererecorded. Th e active substance pro duced by Candida guilliermo ndii stra in 848 was par­tially pur ified by cat ion exchange and chromatography gel filtration on Sephadex G-15.Th e substance is heat stable (80 °C, 10 min), not susceptible to treatment with proteasesand most probably of a molecular weight smaller than 3500 Dalton. At a concentrationof 50 A.U./ml it acts bacteriostat ically on a Staphylo coccus epidermidis strain (Fig. 1).

Zusammenfassung

Die Aktivirarsspckrren von 25 Candida-Stammen gegen Stamme vcrschiedener Bakrerien­arten wurden untersucht. Die aktive Substanz des Candida guilliermo ndii-Stammes 848wurde part iell gereinigt tiber Kationenaustauschchromatographie und Gelfilrrarion mit Se­phadex G-15. Sie ist hitzestabil, nieht Protease-empfindlich und kleiner als 3500 Dalton.Bei einer Konzentration von 50 A. U./ml wirkt sie bakteriostatisch gegen einen Staphylo­coccus epidermidis-Stamm (Fig. 1).

Introduction

The growth of Neisseria gonorrhoeae may be inhibited in vitro not only by dif­ferent bacteria isolated from genitourinary tract (5) but also by yeasts of Candidagenus (2).

1 Stipendate of the " Deutscher Akademischer Austauschdiensr" (DAAD).

Antibacterial Activities of Candida 391

This latter effect was also observed in vivo in a model of subcutaneous chambersin guinea pigs. The inhibitory effect of bacterial strains exerted upon N. gonorrhoeaeis usually attributed to production of different antibacterial substances by thesebacteria, with special regards to staphylococci isolated from vagina and cervix (7, 10).Recently Morris et al. (9) demonstrated direct inhibitory properties of staphylo­coccin Bac R1 against N. gonorrhoeae.

On the contrary to bacterial strains inhibiting N. gonorrhoeae no data in theliterature are available concerning similar properties of yeasts. Therefore, we under­took studies to characterize the antibacterial properties of Candida yeasts isolatedfrom genitourinary tract of patients of a venereal disease clinic. Moreover, an attemptwas done to isolate the antagonistic substance of one Candida strain.

Materials and Methods

Strains

Twenty five strains of yeasts belonging to the genus Candida isolated from vaginal andcervical swabs taken from patients treated in the venereal disease clinic of Krakow, Poland,were tested for their antibacterial properties against following bacterial strains: Strepto­coccus group B (10), Streptococcus group D (9), Staphylococcus aureus (8), Staphylococcusepidermidis (4), Neisseria gonorrhoeae (2), Escherichia coli (10).

These strains were isolated from clinical materials in the Institute of Medical Micro­biology and Immunology, University of Bonn. One C. guilliermondii strain (No. 848) waschosen on the base of its strong inhibitory action for further characterization of its anti­bacterial properties. Two indicator strains of S. epidermidis used throughout these studieswere isolated in the Institute of Medical Microbiology and Immunology, University ofBonn, too.

In vivo interference between Candida and bacteria

A method described by Kraus et a!. (8) was adopted and modified. Columbia BloodAgar Base (Difco) supplemented with sheep blood was used throughout except for studiesdone on N. gonorrhoeae strains which were cultivated on Thayer-Martin's Agar (Difco).Yeast test strains were applied with a loop to the central part of the blood agar plate. Thediameter of the yeast inoculation zone was 1.7 cm. After 48 h of incubation at 37°C theconfluent growth of the yeast was removed by swabbing and the rest of yeast cells waskilled by chloroform vapours. Then a suspension of a bacterial indicator strain was in­oculated by floating over the whole surface of the plate. The concentration of bacteria inthe inoculum was adjusted to about 108 colony forming units (CFU)1m!.

Diameters of inhibition zones were measured after incubation for 20-24 h at 37°C.

Isolation of antibacterial substance from Candida guilliermondii strain 848

This was accomplished by testing cultures and culture extracts against the indicatorstrains S. epidermidis 25 Sa and 5 using the agar overlay method described by Saigh et a!.(10). 10 fll of the substance to be tested were applied to the surface of the medium alreadyinoculated with the indicator strain and diameters of inhibition zones were measured afterincubation over night.

Following methods were applied to obtain the antibacterial substance for purification:a) Liquid media: Supernatants were obtained by centrifugation (5000 rpm, 10 min) of

24 hrs old cultures of C. guilliermondii 848. Following media were tested: Nutrient Brothsupplemented with 5% of sheep blood. Tryptone Soy Broth (Difco), Casein-Yeast Extract-

392 A.Budak, H. G. Sahl, and H. Brandis

Glucose Broth (CG), and Casein Hydrolysate, 0.5%, with glucose, 0.8%, and yeast extract,0.2%, in phosphate buffer 100 mM pH 7.0.

b) Solid media: Petri plates of 18 em diameter were filled with 100 ml of different agarmedia: CG solidified with 0.8% agar, Sabouraud Agar (Difco), Tryptone Soy Agar (Difco).After solidification the agar surface was covered with a sterile dialyzing membrane (Tho­mapor, Reichelt Chemie Technik, FRG) and inoculated with a suspension in saline of a24 h old culture of C. guilliermondii 848 grown on Sabouraud medium. The plates wereincubated at 37°C for 24 h, the yeast growth discarded and the agar homogenized afteraddition of equal volumes of 100 mM phosphate buffer, pH 7.0. Agar eluates were clearedby centrifugation at 5000 rpm for 10 min.

c) Extraction from whole cells: Cells of C. guilliermondii were cultivated using themembrane technique on media described above and additionally on Sephadex G-25 soakedwith CG medium (Seph.-CG). Cells were harvested and washed with phosphate buffers,pH 7.0 of different ionic strength. After centrifugation at 5000 rpm for 10 min the super­natants were filtrated through a membrane filter (Millipore, pore size 0.45,um) and testedagainst indicator organisms.

Purification of the cell washings

The washings were fractionated using a CM Sephadex C-25 column (2.0 x 20 em)equilibrated with 100 mM phosphate buffer, pH 7.0. For elution a linear KCl gradient(0-2 M) in 100 mM phosphate buffer, pH 7, was applied. After testing against the indicatorstrain the active fractions were pooled, evaporated to dryness and resuspended in distilledwater. Then the preparation was desalted by gel filtration on a Sephadex G-15 column(2 x 30 em) using distilled water as an eluant. The active fractions were pooled.

Activity testing

For activity testing twofold dilution series of the active solution were prepared. 10,u1of each dilution step was given on TSA plates already inoculated with the indicator strain.The highest dilution yielding clear inhibition zone was defined as to contain 1 arbitraryunit (A.D.) /m!'

Investigations on the mode of action

The CG medium was inoculated with an 18 h broth culture of indicator strain S. epider­midis 25 Sa. The ratio of medium to the inoculum was 1: 10. Then the culture was grownat 37°C with constant shaking to an optical density of approximately 1.0 at 600 nm.

After controling the number of colony forming units (CFD) by plating serial 10 folddilutions on TSA, the culture was divided into a control flask and several flasks to whichdifferent preparations of the active substance were added at a final concentration of50 A. D./m!' The control received phosphate buffer only. Samples were taken in 15 minintervals, rapidly diluted in icecold buffer (100 mM PO., pH 7) and plated on TSA.

Viable counrs/rnl of indicator cells were calculated after incubation at 37°C for 24 h.

Results

The results obtained are presented in Tables 1 and 2. The in vitro interferencebetween 25 Candida test strains and different indicator bacteria is shown in Table 1.The inhibitory properties of the yeasts were more pronounced against strains ofS. aureus and S. epidermidis than against streptococcal strains with the exceptionof streptococci of the serological group D which were resistant to the inhibitoryaction of the yeasts. None of the Candida strains tested was active against all indi­cator organisms.

Antibacterial Activities of Candida 393

Table 1. Bacterial strains inhibited by yeast strains tested

Yeast No. of indicator strains inhibitedstrains (Total numbers of test strains given in parentheses)

Strepto- Strepto- Staph. Staph. N. gonor- E. colicoccus B coccus D aureus epiderm. rhoeae (10)(10) (9) (8) (4) (2)

443 0 0 2 1 0 1726 1 0 1 1 2 1437 2 0 1 3 2 1108 0 0 2 2 2 1248 0 0 2 3 2 1525 0 0 1 1 0 0749 0 0 2 1 0 0

50 0 0 2 2 0 1529 0 0 3 3 1 1431 0 0 2 1 1 1107 0 0 1 0 1 2160 0 0 3 1 2 2

21 0 0 1 1 2 1205 6 0 3 2 2 1214 0 0 2 3 2 0363 1 0 3 2 2 2499 5 0 3 2 2 1

80 1 0 3 3 2 1418 1 0 3 1 1 1125 2 0 2 2 2 0641 1 0 1 1 0 142 1 0 3 2 1 1

848 5 0 3 3 2 1247 2 0 2 2 2 0685 1 0 1 1 2 1

Table 2. Inhibitory effect of various cell washings of strain Candida guilliermondii 848exerted upon test strain Staphylococcus epidermidis 25 Sa

Solvents used

10 mM PO. buffer, pH 7.0100 mM PO. buffer, pH 7.0100 mM PO. buffer + 1 M NaCl, pH 7.0

CGaCulture Media used

CGA b Seph/CGc

++++++

a CG, casein-glucose medium.b CGA, casein-glucose-agar.C Sephadex G-25 soaked with CG medium.No antibacterial effect could be found in cell washing with the following solvents:

3 M NaCl, pH 7.0; 3 M NaCl, pH 2.0; 3 M NaCl, pH 10.0; 50% methanol; 70% methanol,pH 7.0; 70% methanol, pH 2.0; 70% methanol, pH 10.0; undil. methanol; ethanol; ace­ton; ethyl ether; methylene chloride.

394 A.Budak, H. G. Sahl, and H. Brandis

On the basis of a wide spectrum of the antibacterial properties three Candidastrains were selected: C. albicans 363, C. albicans 205 and C. guilliermondii 848.After a quantitative comparison of their activity against the indicator strain S.epi­dermidis 25 Sa only the strain C. guilliermondii 848 was chosen for further identifi­cation of its antibacterial substance(s).

First identification of the antibacterial substance was done by exclusion of theeffect of pH change on inhibition of growth of the indicator strain. It appeared thatthe pH of test medium was lowered after the growth of the Candida test strainonly by 0.3 and therefore it had no effect on the growth of the indicator strain.

No antibacterial substances were found in supernatants of broth cultures of thedonor strain on different media. Also no activity was found in agar eluates of thedonor strain on different media. Therefore, the activity was attributed to the intactyeast cells since it was possible to elute it from cells by washing with different phos­phate buffers. Regardless of the eluates used the activity was only shown in washingsof cells cultivated on dialysis membranes placed on Sephadex C-25 soaked with CG.

The amount of antibacterial substance produced by this produce was fairly low.So, a total purification could not be achieved. Media components were removed bycation exchange on CM Sephadex C-25 and gel filtration on Sephadex G-15, thusyielding a considerably purified preparation.

By determination of the number of CFU of the indicator strain after addition ofpartially purified active substance it appeared that the cell number did not decreaseindicating a bacteriostatic rather than a bactericidal action at the concentrationapplied (Fig. 1). During purification procedure it could be stated that the substancepossessed a cationic character. It easily diffused into agar since application of agarblocks cut from the zones of the inhibition were also able to inhibit the growth ofthe indicator after application to the surface of a new test plate.

Furthermore the substance should have a molecular weight smaller than 3500,since it was able to diffuse through dialysis membranes of this exclusion limit.

Addition of trypsin and pronase (1mg/ml each) and also treatment at 80°C (10min)had no effect on the activity of the substance.

9

=>LLU

o 20 40 60 80min

Fig. 1. Action of the partially purified antibacterial substance produced by Candida guil­liermondii 848 on the indicator strain Staphylococcus epidermidis 25 Sa. The experimentwas performed as described in Methods.• ---. control culture0---0 culture in the presence of 50 A.D./ml antibacterial substance

Antibacterial Activities of Candida 395

Discussion

Findings of several authors (2, 3, 12, Budak and Heczko, in press) have shownthat some of Candida strains inhibit the growth of many strains of N. gonorrhoeae.In our previous study this proportion was estimated to amount to 20% of vaginalisolates of Candida spp, This proportion markedly exceeds a corresponding quan­tity of bacterial strains producing bacteriocins in non-selected and non-stimulatedpopulations (1). In this study it was demonstrated that the activity spectrum of theselected 25 Candida strains was rather large: it was not restricted to Gram-positivebacteria as staphylococci and streptococci but covered also E. coli strains as repre­sentatives of the Gram-negative bacteria. Only the growth of all tested Group Dstreptococci was unaffected by the test organism. Among the species of the sensitivebacteria not all the strains tested were equally susceptible to various Candida strainswhich brought about a variety of patterns of the inhibitory spectrum of Candidastrains. It may also indicate the presence of different inhibitory substances producedby Candida strains.

We found that the antibacterial substances are produced by representatives oftwo Candida species: C. albicans and C. guilliermondii.

Preliminary results of our experiments may suggest that the substance producedby C. guilliermondii 848 is cell-associated, being found only in cell washings butnot in fluid (liquid) culture media. The substance is probably of a low molecularweight because it can pass a dialysis membrane. Moreover, the inhibitory factorof Candida is heat-stable and not susceptible to cleaving by trypsin and pronase.Its action on susceptible bacterial cells is more bacteriostatic than bactericidal. Itmay be concluded, therefore, that the antibacterial substance produced by C. guil­liermondii 848 resembles antibiotic and bacteriocin-like substances of low molecularweight produced by different Gram-positive bacteria as staphylococci and strepto­cocci (4, 6, 11). However, further studies on optimalization of the production ofthis substance and on its physicochemical properties are needed to elucidate itsrelations to antibacterial substances.

References

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9. Morr iss, D. M ,]. W Lawt on , and M. Rogolsky : Effect of a staphylococcin on Neisseriagonorrhoeae. Antimicrob. Agents Chemother. 14 (1978) 218

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Dr. H.-C .Sahl, Professor Dr. H. Brandis, Institut fur Med. Mikrobiologie und Immuno­logie, D-5300 Bonn-Venusberg