Eur. J. Epidemiol. 0392-2990
EUROPEAN JOURNAL
May 1992, p. 350-355
Vol. 8, No. 3
OF EPIDEMIOLOGY
INHIBITION OF ADHERENCE OF CANDIDA ALBICANS TO ACRYLIC BY A CHITIN DERIVATIVE E. SEGAL .1, I. KREMER.2 and D. DAYAN** *Department o f H u m a n Microbiology - Sackler Faculty o f Medicine - Tel Aviv University R a m a t Aviv 69978 - Tel Aviv - Israel. **Section o f Oral Pathology a n d Oral Medicine - The Maurice and Gabriela Goldschleger School o f Dental Medicine - Tel Aviv University - Tel Aviv - Israel
Key words: Candida - Adherence - Acrylic The purpose of this study was to assess the effect of a chitin derivative (CSE) on the adherence of Candida albicans to acrylic. Fungal adherence to acrylic dentures is considered an essential step in the development of denture stomatitis. Adherence of C. albicans to acrylic pieces (5 x 5ram) was assessed microscopically using a calibrated ocular objective and expressed as number of adherent yeasts/mm 2 of acrylic. CSE was prepared from commercial chitin (crab shell) and from chitin isolated from C. albicans blastospores. The effect of both CSE types on the adherence of C. albicans to acrylic was examined in two experimental systems: CSE present during the adherence assay and acrylic pieces pretreated with CSE prior to the assay. Both CSE types exerted a significant inhibitory effect when tested in the two experimental systems. These findings are significant for possible prevention of denture stomatitis.
INTRODUCTION
Denture stomatitis, a disease of the oral mucosa, is caused mainly by yeast-like fungi of the genus Candida (12). The infection may be found in partial or complete denture wearers of both jaws. However, it is most frequently seen in those with complete maxillary dentures. The yeasts are generally observed on the palatal surface of the denture (13). Adherence of microorganisms to the host's surface is believed to be an initial and essential step in the development of infection (1). A number of studies have shown a correlation between in vitro adhesion to host cells and various clinical situations. The ability of 1 Corresponding author. 2 Based on a thesis submitted to the Department of Human Microbiology, Sackler Faculty of Medicine, Tel Aviv University, in partial fulfillment of the requirements for the M.Sc. degree.
Candida to adhere in vitro to mucosal cells and the correlation of adhesion to infection have been reviewed recently by Douglas (4) and Segal (17). In vitro studies on adhesion of various Candida species to buccal epithelial cells have revealed that the adherence ability of those yeast species was correlated to the epidemiologically known data regarding their relative pathogenicity (7). Candida albicans, the major pathogen, showed the highest adherence values. Several investigators have studied the in vitro adhesion of Candida to various inert surfaces, including acrylic (3, 11, 15). In a previous study (20), we compared the adherence in vitro of 17 isolates of various Candida species to acrylic and found that although all 17 Candida strains adhered to acrylic, there was variability in the degree of adherence. Generally, all C. albicans strains were more adherent than the strains of the other Candida species tested. The mechanism of microbial attachment may be elucidated by inhibition studies with analogs of the
350
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Adherence of C. albicans to acrylic
microbial or host cell surface components from which the nature of the ligand-receptor involved in the adhesion process can be deduced. In previous studies (5, 8, 18, 21), we showed that the Candida cell wall component chitin, its monomer - N- acetylglucosamine (NAG) or a chitin derivate - chitin soluble extract (CSE), inhibited in vitro adhesion of the fungus to various epithelial cells, including buccal cells (16). Pretreatment of animal with these substances prevented development of experimental murine vaginitis (8, 9, 19) or cutaneous infection in guinea pigs (unpublished data). In view of the efficacy of CSE in inhibiting adhesion and potentially preventing infection, it was the purpose of the present study to investigate the possibility to inhibit C. albicans adherence to acrylic by CSE. We examined the effect of CSE obtained from commercially purchased chitin (prepared from crab shells) and from chitin isolated from C. albicans cultures on the in vitro adhesion of the fungus to acrylic, tested under different experimental conditions.
running water for 3 h, and sealed and sterilized in petri dishes until ready to use (20).
Preparation of chitin from C. albicans - Candida chitin was prepared from Candida blastospores using the procedure described by Braun and Calderone (2). The major steps of this procedure are illustrated in Figure 1 and include harvesting by centrifugation of C. albicans cultures grown in liquid yeast-extract, and heat extraction by acid (1N HC1) and alkali (1N NaOH). The insoluble product containing chitin was identified by detection of amino sugars. The chitin was liophilized and stored until used.
C. albicans cultures (48 hrs, 280c)
J
1. centrifuge 2. wash 3 x saline
-. i cell 'peUet J 1. 1N HCL, 90 rnin, 1000c 2. centrifuge 3. wash 2 x saline
supernatant
P
MATERIALS AND METHODS
extract I
Candida isolate - Adherence to acrylic surfaces in vitro was tested with C. albicans CBS 562, the type
species, obtained from the yeast culture collection, Centraalbureau Voor Schimmel-Cultures, Delft, Holland. This C. albicans isolate was extensively investigated in previous studies (5, 8, 9, 16-21). In a few experiments, an additional C.albicans strain (designated S), isolated from a patient with denture stomatitis, was also used. Growth conditions - Yeast cultures were maintained on Sabouraud's dextrose agar (Difco, Detroit, MI, USA) with chloramphenicol at 4° C and periodically subcultured at 28° C. Growth conditions for adherence assays were as described in previous studies (20). Briefly, yeast inocula from fresh subcultures were grown overnight at 28° C in yeast extract broth under constant shaking and harvested during the logarithmic growth phase. The yeasts were collected by centrifugation, washed three times with 0.1 M phosphate buffered saline (PBS), pH 7.3, and resuspended in PBS to a concentration of l0 s organisms/ mL (established through counting in a hemacytometer).
Preparation of acrylic pieces - Self=polymerizing acrylic powder (Justis acryl, Ivoclar, Schaan, Switzerland) and monomer liquid were mixed in accordance to the manufacturer's instructions. After 2-3 min, the mixture was placed between two glass slides, 7 x 7 cm. The slides were secured at each end with clips, leaving a uniform distance of 0.4 mm between them. The acrylic was then polymerized at 50oC for about 30 rain. Subsequently, the transparent acrylic sheet formed was stripped from the slides and cut into 5 x 5 mm pieces, the average thickness being 0.4 mm. The pieces were immersed in distilled water for 2 weeks to leach the excess monomer, them washed in 351
1. 2. 3. 4. [ CHITIN
1N KOH, 90 min, 1000c centrifuge wash 2 - 3 x H20 adjust pH to 7.0 (detection of aminosugars Morgan-Elson test)
by
Figure 1 - Isolation of chitin from C. albicans.
Preparation of chitin soluble extract (CSE) - CSE was prepared from a 20% suspension of commercially obtained chitin (Fluka, Buchs, Switzerland) or from 2.5% suspension of the chitin isolated from C. albicans cells (C.A. chitin-CSE). The procedure for preparation of CSE of both types is illustrated in Figure 2 in accordance with previously reported methods (8, 19).
CHITIN 1. extract in H 0 (20% w / v or 2.5% w / v ) 2. shake for 52hr supernatant
I
1. repeat as above x 7
dialysis I
lyophilization
Figure 2. - Preparation of chitin soluble extract (CSE)
Segal E. et al.
Eur. J. Epidemiol.
in vitro adherence assay - Adherence of yeasts to shaking), the relationship between CSE concentration acrylic was measured by a modification of the and the percentage of inhibition was further examined technique described by Samaranayake and only under non-shaking conditions. Adherence assays MacFarlane (15) and Segal, Lehrman and Dayan (20). were carried out in the presence of various CSE Briefly, 20 tJL of the yeast suspension (108 cells/mL) concentrations (3.12-50 mg/mL). As shown in Figure was added to each acrylic piece placed horizontally on 4, in the presence of 6 mg/mL CSE a significant (p < a microscopic slide. After an incubation period of lh 0.05) inhibition (33% vs control) was recorded and at 37° C, the pieces were washed with PBS, fixed in almost total inhibition (96%) was obtained when the methanol, stained with gram-crystal violet, and CSE concentration was 50 mg/mL. examined under a microscope using a calibrated ocular objective at x400 magnification. Six fields in five different positions (four at the periphery and one MEAN NO. OF ADHERENT YEASTS/MM 2 in the center) were counted in each piece. The 11oo ~] 25 mg/ml CSE adherence values were expressed as the number of looo CONTROL adherent yeasts/mm2 of acrylic. 9o0 In part of the experiments, the adherence assay Boo was carried out under shaking. In these experiments, the acrylic pieces were put into wells of an 8-well 7oo tissue culture plate (LabTek, Nunc, Napperville, IL, 600 USA) containing 200 laL of C. albicans suspension (10~ ~oo 1 organisms/mL). The plates were incubated on a rotary 4oo I shaker at 37° C for i h. After incubation, the acrylic pieces were rinsed, stained and evaluated as described above. 3oo
J
200
Statistical analysis - Data were analyzed by means of the Student's t-test and the analysis of variance (ANOVA) test to compare two groups or more than two groups, respectively.
100 0
A
B
Figure 3. - Adherence of C. albicans to acrylic in presence of CSE A. Non-shaking system B. Shaking system.
RESULTS
Effect of CSE prepared from commercial chitin on adherence of C. albicans to acrylic - The effect of CSE on the adherence of the fungus to acrylic was evaluated in the following experimental systems: (a) the reaction of adhesion carried out in the presence of CSE and (b) the adherence test performed with acrylic pieces pretreated with CSE. a. Adherence of C. albicans to acrylic in the presence of CSE - Since it was previously shown (8, 18) that 25 mg/mL CSE had an inhibitory effect on the adherence of C. albicans to various epithelial cells, we initially assessed the effect of this CSE concentration on the adherence of the fungus to acrylic. The assay in the presence of CSE was performed under shaking and non-shaking conditions. Figure 3 presents mean adherence values obtained from 20 experiments with 67 acrylic pieces exposed to CSE and 81 controls of the strain CBS 562. The data indicated that under both experimental conditions, CSE inhibited C. albicans adherence to acrylic by 8183%. Statistical analysis by the Student's t-test revealed a p < 0.001 value in both experimental conditions. An experiment with C. albicans S (data not shown) carried out in a non-shaking system revealed that CSE inhibited the adhesion to acrylic of this strain, as well. Since a similar degree of inhibition was noted in both experimental conditions (shaking and non-
MEAN NO ADHERENT YEASTS/MI~ 900 800 700
[L
600 500 400 300 200 100 0
coil'.rol
3.12
(I.28
12.8
25
80
CSE CONCENTRATIONS(mg/ml) Figure 4. - Effect of presence of various CSE concentrations on adherence of C. albicans to acrylic.
b. Adherence of C. albicans to CSE-pretreated acrylic pieces - In a further series of experiments, the number of adherent yeasts to acrylic pieces treated with various CSE concentrations (12.5-100 mg/mL) for 1-3 h prior to the adherence assay, was examined. It should be noted that these CSE-pretreated acrylic pieces were rinsed with PBS prior to the adherence assay to remove excess CSE.
352
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Adherence of C. albicans to acrylic
CSE pretreatment for 1 h reduced the adherence significantly (p < 0.001) at all concentrations tested (three experiments, 3-4 acrylic pieces for each experiment for each CSE concentration; 3-4 control acrylic pieces in each experiment). A similar effect was obtained with acrylic pieces pretreated with CSE for 2 or 3 h (2-3 experiments, 3-4 acrylic pieces in each experiment for each CSE concentration; 3-4 control acrylic pieces in each experiment). Figure 5 shows the percentage of inhibition versus CSE concentrations at the various pretreatment periods. A linear correlation between CSE concentrations and percentage of inhibition was noted only when the acrylic pieces were pretreated for 1 h. When the pretreatment was longer (2-3 h), the maximum percentage of inhibition was noted at lower CSE concentrations and reached a plateau.
adherence. We then tested various higher concentrations of CA. chitin-CSE: 5, 10, 20, 50 rag/ mL (2-3 experiments; 3-5 acrylic pieces in each C.A. chitin-CSE concentration in each experiment; 4-6 control acrylic pieces in each experiment) (Fig. 6). MEAN NO ADHERENT YEASTS/MM2 1000 900 800 700 800 500 400 300 200
INHIBITION (%) 60,
j _ j ~ 4 D
+
3hr8
-e-- 2 hr,~
55
/
,50
4:9
/
45
100 O - -
1 hr
~ontrol
2
6
10
20
80
C.A, C H I T I N CSE C O N C E N T R A T I O N S ( m g / m l )
0
t.igure 6. - Effect of presence of various C.A. chitin-CSE concentrations on adherence of C. albicans to acrylic.
All C.A. chitin-CSE concentrations starting from 5 mg/mL reduced the number of adherent yeasts compared to controls. However, a significant reduction (p < 0.001) of 46O/owas obtained with 50 mg/mL.
40 35 30
2~ 0
I
t
I
I
I
I
I
12.5
25
3?.5
50
62.5
75
87.5
2__1 100
112.5
CSE CONCENTRATIONS (mg/ml)
Figure 5. - Effect of pretreatment of acrylic with various CSE concentrations for different time intervals on C. albicans adherence.
Effect of CSE prepared from chitin isolated from C. albicans cultures (C. albicans-CSE) on the adherence of the fungus to acrylic - Following the observations that CSE from commercial chitin exerted an inhibitory effect on the adherence of C. albicans to acrylic, we initiated experiments to test whether CSE prepared from chitin isolated from C. albicans blastospores would have a similar effect. For this purpose, we isolated chitin from C. albicans cultures, prepared CSE from the chitin (C.A. chitin-CSE) and assessed the activity of this substance in two experimental systems: (a) adhesion in the presence of C.A. chitin-CSE and (b) adhesion with C.A. chitinCSE pretreated acrylic pieces. a. Adherence of C. albicans to acrylic in presence of C. albicans CSE - Based on data from a previous study (9) that 2.5 mg/mL C.A. chitin-CSE significantly inhibited attachment of C. albicans to epithelial cells, we tested the effect of a similar concentration (2 mg/ mL) on adherence to acrylic. The results of two experiments with 4-5 acrylic pieces in each experiment showed no inhibitory effect of C.A. chitin-CSE on
353
b. Adherence of C. albicans to C. albicans-CSE pretreated acrylic pieces - To assess the effect of pretreatment of acrylic pieces with C.A. chitin-CSE on adhesion, the pieces were exposed for 1 h to 2, 5 and 10 mg/mL concentrations. Results of three such experiments (3-5 acrylic pieces in each experiment for each C.A. chitin-CSE concentration; 3-5 control acrylic pieces in each experiment) are presented in Figure 7. The data demonstrated that exposure to 5 or 10 mg/mL C.A. chitin-CSE inhibited adherence by 6070% (p < 0.01) as compared to the control. MEAN NO. ADHERENT YEASTS/MM2 700 600
m
oo°°°ooiiiiiiii ,oo 0
!o
~5 ~2
I
~NTROL
itiiiiiii
C.A, CHITIN CSE CONCENTRATIONS(mg/ml)
Figure 7. - Effect of pretreatment of acrylic with various C.A. chitin-CSE concentrations for 1 h on C. albicans adherence.
Segal E. et aL
Eur. J. EpidemioL
DISCUSSION
In the present study, we assessed the effect of a chitin derivative (CSE), prepared from commercially purchased chitin (prepared from crab shells) and from chitin isolated from C. albicans on the adherence of the yeast to acrylic. The data demonstrated that this substance significantly reduced the number of adherent yeasts when tested in various experimental systems. Moreover, in some experimental conditions, it was possible to almost block the entire binding of the fungus to acrylic. This study was initiated following previous investigations (5, 8, 18) in which it was shown that CSE significantly inhibited the adherence of C. albicans to various epithelial cells: buccal and vaginal mucosa, corneocytes and gastrointestinal epithelium. Furthermore, pretreatment of animals, with CSE prevented the development of experimental candidiasis (8, 9, 19). Based on the data from those studies, it was assumed that CSE may have acted as an adhesin-like substance through competitive binding to specific C. albicans receptors on the host cells, thereby covering these sites and blocking the attachment of the yeasts. This assumption was strengthened by the finding that CSE from C. albicans chitin was active at significantly lower concentrations than CSE isolated from commercial chitin (9). Partial analysis and chromatographic separation of CSE (9) indicated that the inhibitory activity was apparently associated with a fraction containing amino sugars. In addition, NAG, the monomer of chitin, also exerted an inhibitory effect, while other Candida cell wall polysaccharides (mannan, glucan) and their monomers had no effect (8, 21). Since acrylic is an inert surface, it is difficult to assume that the mechanism of adherence would be similar. Moreover, it is generally believed that in adherence to acrylic or other inert surfaces the major forces involved in the mechanism of adhesion are of hydrophobic or electrostatic nature (6, 11, 14). Thus, it is reasonable to assume that CSE may act by interfering with these binding forces. This hypothesis would explain the differences between the behavior of CSE in the systems with host cells vs acrylic, such as lower activity of C.A. chitin-CSE in the latter or very low inhibitory activity of NAG (data not shown). However, the exact mechanism of the interference of CSE in the binding of the yeasts to acrylic should be further elucidated. McCourtie, MacFarlane and Samaranayake (10) reported that chlorhexidine reduced the adherence of C. albicans to denture acrylic. It should, however, be noted that chlorhexidine is known to have adverse effects. Moreover, most recently (22), it was observed in in vitro studies that saliva significantly reduced the microbicidal activity of chlorhexidine against all microorganisms tested including C. albicans. In an experiment in which we assessed the activity of CSE on the adherence of C. albicans to acrylic in the presence of saliva (data not shown), we noted that
CSE had an inhibitory effect under these conditions, as well. As the adherence of C. albicans to acrylic denture is a vital step in the pathogenesis of denture stomatitis, it is suggested that blocking this step may be of major significance in the clinical prevention of this infection. This hypothesis has to be further clarified by experiments in in vivo systems with CSE, which are currently in progress in our laboratory. REFERENCES
1. Beachey E., Eisenstein B. and Ofek L (1982): Bacterial
adherence in infectious diseases In: Current Concepts. Upjohn Co, Kalamazoo, pp. 1-52. 2. Braun P.C. and Calderone R.A. (1978): Chitin synthesis in Candida albicans: Comparison of yeast and hyphal forms - J. Bacteriol. 133: 1472-1477. 3.
Critchley I.A. and Douglas L.J. (1985): Differential adhesion of pathogenic Candida species to epithelial and inert surfaces - Fed. Eur. Microbiol. Soc. Microbiol. Lett. 28: 199-203.
4. Douglas J. (1987): Adhesion to surfaces - In: Rose
A.H. and Harrison J.S., eds. The yeasts - Academic Press, London, pp. 239-280. 5. Kahane M., Segal E., Schewach-Millet M. and Gov Y. (1988): In vitro adherence of Candida albicans to
human corneocytes - Inhibition by chitin soluble extract - Acta. Dermato. Venereol. 68: 98-101. 6. Kennedy M.J. (1988): Adhesion and association mechanisms of Candida albicans - Curr. Top. Med. Mycol. 2: 73-169. 7. King R.D., Lee J.C. and Morris A.L. (1980): Adherence of Candida albicans and other Candida
species to mucosal epithelial cells - Infect. Immun. 27: 667-674. 8. Lehrer N., Segal E. and Barr-Nea L. (1983): In vitro and in vivo adherence of Candida albicans to mucosal surfaces - Ann. Microbiol. 134: 293-306. 9. Lehrer N., Segal E., Lis H. and Gov Y. (1988): Effect of Candida albicans cell wall components on the
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adhesion of the fungus to human and murine vaginal mucosa- Mycopathologia 102: 115-121. 10. McCourtie J., MacFarlane T.W. and Samaranayake L.P. (1985): Effect of chlorhexidine gluconate on the adherence of Candida species to denture acrylic - J. Med. Microbiol. 20: 97-104. 11. Minagi S., Miyake Y., lnagaki K., Tsura H. and Suginaka H. (1985): Hydrophobic interaction in Candida albicans and Candida tropicalis to various denture base resin materials - Infect. Immun. 47:1114. 12. Odds F..C. (1988): Candida and candidosis. 2nd ed. Bailliere Tindall, London.
Adherence of C. albicans to acrylic
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13. Regezzi J.A. and Sciubba J.J. (1989): Oral pathology: Clinical-pathologic correlations - W.B. Saunders Co., Philadelphia, pp. 110-116.
Development of the assay and tests of inhibitors - J. Med. Vet. Mycol. 24: 477-479.
14. Rotrosen D., Calderone R.A. and Edwards Jr J.E. (1986): Adherence of Candida species to host tissues and plastic surfaces - Rev. Infect. Dis. 8: 73-85.
19. Segal E., Gottfried L. and Lehrer N. (1988): Candidal vaginitis in hormone treated mice: Prevention by a chitin extract - Mycopathologia 102: 157-163.
15. Samaranayake L.P., McCourtie S. and MaeFarlane T.W. (1980): Factors affecting the in vitro adherence of Candida albicans to acrylic surfaces - Arch. Oral Biol. 25: 611-615.
20. Segal E., Lehrman O. and Dayan D. (1988): Adherence in vitro of various Candida species to acrylic surfaces - Oral Surg. Oral Med. Oral Pathol. 66: 670-673.
16. Segal E. (1985): Inhibition of attachment of Candida to animal cells and inert surfaces - J. Dent. Res. 64: 730, Abstr. No. 4.
21. Segal E., Lehrer N. and Ofek L (1982): Adherence of Candida albicans to h u m a n vaginal epithelial cells: inhibition by amino sugars - Exp. Cell. Biol. 50: 1317.
17. Segal E. (1987): Pathogenesis of h u m a n mycoses: Role of adhesion to host surfaces - Microbiol. Sci. 4: 344-347. 18. Segal E. and Savage D. (1986): Adhesion of Candida albicans to mouse intestinal mucosa in vitro:
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22. Spijkervet F.K.L., Van Saene J.J.M., Van Saene H.K.E, Panders A.K., Vermey A. and Fidler V. (1990): Chlorhexidine inactivation by saliva - Oral Surg. Oral Med. Oral Pathol. 69: 444-449.
EUROPEAN JOURNAL
May 1992, p. 350-355
Vol. 8, No. 3
OF EPIDEMIOLOGY
INHIBITION OF ADHERENCE OF CANDIDA ALBICANS TO ACRYLIC BY A CHITIN DERIVATIVE E. SEGAL .1, I. KREMER.2 and D. DAYAN** *Department o f H u m a n Microbiology - Sackler Faculty o f Medicine - Tel Aviv University R a m a t Aviv 69978 - Tel Aviv - Israel. **Section o f Oral Pathology a n d Oral Medicine - The Maurice and Gabriela Goldschleger School o f Dental Medicine - Tel Aviv University - Tel Aviv - Israel
Key words: Candida - Adherence - Acrylic The purpose of this study was to assess the effect of a chitin derivative (CSE) on the adherence of Candida albicans to acrylic. Fungal adherence to acrylic dentures is considered an essential step in the development of denture stomatitis. Adherence of C. albicans to acrylic pieces (5 x 5ram) was assessed microscopically using a calibrated ocular objective and expressed as number of adherent yeasts/mm 2 of acrylic. CSE was prepared from commercial chitin (crab shell) and from chitin isolated from C. albicans blastospores. The effect of both CSE types on the adherence of C. albicans to acrylic was examined in two experimental systems: CSE present during the adherence assay and acrylic pieces pretreated with CSE prior to the assay. Both CSE types exerted a significant inhibitory effect when tested in the two experimental systems. These findings are significant for possible prevention of denture stomatitis.
INTRODUCTION
Denture stomatitis, a disease of the oral mucosa, is caused mainly by yeast-like fungi of the genus Candida (12). The infection may be found in partial or complete denture wearers of both jaws. However, it is most frequently seen in those with complete maxillary dentures. The yeasts are generally observed on the palatal surface of the denture (13). Adherence of microorganisms to the host's surface is believed to be an initial and essential step in the development of infection (1). A number of studies have shown a correlation between in vitro adhesion to host cells and various clinical situations. The ability of 1 Corresponding author. 2 Based on a thesis submitted to the Department of Human Microbiology, Sackler Faculty of Medicine, Tel Aviv University, in partial fulfillment of the requirements for the M.Sc. degree.
Candida to adhere in vitro to mucosal cells and the correlation of adhesion to infection have been reviewed recently by Douglas (4) and Segal (17). In vitro studies on adhesion of various Candida species to buccal epithelial cells have revealed that the adherence ability of those yeast species was correlated to the epidemiologically known data regarding their relative pathogenicity (7). Candida albicans, the major pathogen, showed the highest adherence values. Several investigators have studied the in vitro adhesion of Candida to various inert surfaces, including acrylic (3, 11, 15). In a previous study (20), we compared the adherence in vitro of 17 isolates of various Candida species to acrylic and found that although all 17 Candida strains adhered to acrylic, there was variability in the degree of adherence. Generally, all C. albicans strains were more adherent than the strains of the other Candida species tested. The mechanism of microbial attachment may be elucidated by inhibition studies with analogs of the
350
Vol. 8, 1992
Adherence of C. albicans to acrylic
microbial or host cell surface components from which the nature of the ligand-receptor involved in the adhesion process can be deduced. In previous studies (5, 8, 18, 21), we showed that the Candida cell wall component chitin, its monomer - N- acetylglucosamine (NAG) or a chitin derivate - chitin soluble extract (CSE), inhibited in vitro adhesion of the fungus to various epithelial cells, including buccal cells (16). Pretreatment of animal with these substances prevented development of experimental murine vaginitis (8, 9, 19) or cutaneous infection in guinea pigs (unpublished data). In view of the efficacy of CSE in inhibiting adhesion and potentially preventing infection, it was the purpose of the present study to investigate the possibility to inhibit C. albicans adherence to acrylic by CSE. We examined the effect of CSE obtained from commercially purchased chitin (prepared from crab shells) and from chitin isolated from C. albicans cultures on the in vitro adhesion of the fungus to acrylic, tested under different experimental conditions.
running water for 3 h, and sealed and sterilized in petri dishes until ready to use (20).
Preparation of chitin from C. albicans - Candida chitin was prepared from Candida blastospores using the procedure described by Braun and Calderone (2). The major steps of this procedure are illustrated in Figure 1 and include harvesting by centrifugation of C. albicans cultures grown in liquid yeast-extract, and heat extraction by acid (1N HC1) and alkali (1N NaOH). The insoluble product containing chitin was identified by detection of amino sugars. The chitin was liophilized and stored until used.
C. albicans cultures (48 hrs, 280c)
J
1. centrifuge 2. wash 3 x saline
-. i cell 'peUet J 1. 1N HCL, 90 rnin, 1000c 2. centrifuge 3. wash 2 x saline
supernatant
P
MATERIALS AND METHODS
extract I
Candida isolate - Adherence to acrylic surfaces in vitro was tested with C. albicans CBS 562, the type
species, obtained from the yeast culture collection, Centraalbureau Voor Schimmel-Cultures, Delft, Holland. This C. albicans isolate was extensively investigated in previous studies (5, 8, 9, 16-21). In a few experiments, an additional C.albicans strain (designated S), isolated from a patient with denture stomatitis, was also used. Growth conditions - Yeast cultures were maintained on Sabouraud's dextrose agar (Difco, Detroit, MI, USA) with chloramphenicol at 4° C and periodically subcultured at 28° C. Growth conditions for adherence assays were as described in previous studies (20). Briefly, yeast inocula from fresh subcultures were grown overnight at 28° C in yeast extract broth under constant shaking and harvested during the logarithmic growth phase. The yeasts were collected by centrifugation, washed three times with 0.1 M phosphate buffered saline (PBS), pH 7.3, and resuspended in PBS to a concentration of l0 s organisms/ mL (established through counting in a hemacytometer).
Preparation of acrylic pieces - Self=polymerizing acrylic powder (Justis acryl, Ivoclar, Schaan, Switzerland) and monomer liquid were mixed in accordance to the manufacturer's instructions. After 2-3 min, the mixture was placed between two glass slides, 7 x 7 cm. The slides were secured at each end with clips, leaving a uniform distance of 0.4 mm between them. The acrylic was then polymerized at 50oC for about 30 rain. Subsequently, the transparent acrylic sheet formed was stripped from the slides and cut into 5 x 5 mm pieces, the average thickness being 0.4 mm. The pieces were immersed in distilled water for 2 weeks to leach the excess monomer, them washed in 351
1. 2. 3. 4. [ CHITIN
1N KOH, 90 min, 1000c centrifuge wash 2 - 3 x H20 adjust pH to 7.0 (detection of aminosugars Morgan-Elson test)
by
Figure 1 - Isolation of chitin from C. albicans.
Preparation of chitin soluble extract (CSE) - CSE was prepared from a 20% suspension of commercially obtained chitin (Fluka, Buchs, Switzerland) or from 2.5% suspension of the chitin isolated from C. albicans cells (C.A. chitin-CSE). The procedure for preparation of CSE of both types is illustrated in Figure 2 in accordance with previously reported methods (8, 19).
CHITIN 1. extract in H 0 (20% w / v or 2.5% w / v ) 2. shake for 52hr supernatant
I
1. repeat as above x 7
dialysis I
lyophilization
Figure 2. - Preparation of chitin soluble extract (CSE)
Segal E. et al.
Eur. J. Epidemiol.
in vitro adherence assay - Adherence of yeasts to shaking), the relationship between CSE concentration acrylic was measured by a modification of the and the percentage of inhibition was further examined technique described by Samaranayake and only under non-shaking conditions. Adherence assays MacFarlane (15) and Segal, Lehrman and Dayan (20). were carried out in the presence of various CSE Briefly, 20 tJL of the yeast suspension (108 cells/mL) concentrations (3.12-50 mg/mL). As shown in Figure was added to each acrylic piece placed horizontally on 4, in the presence of 6 mg/mL CSE a significant (p < a microscopic slide. After an incubation period of lh 0.05) inhibition (33% vs control) was recorded and at 37° C, the pieces were washed with PBS, fixed in almost total inhibition (96%) was obtained when the methanol, stained with gram-crystal violet, and CSE concentration was 50 mg/mL. examined under a microscope using a calibrated ocular objective at x400 magnification. Six fields in five different positions (four at the periphery and one MEAN NO. OF ADHERENT YEASTS/MM 2 in the center) were counted in each piece. The 11oo ~] 25 mg/ml CSE adherence values were expressed as the number of looo CONTROL adherent yeasts/mm2 of acrylic. 9o0 In part of the experiments, the adherence assay Boo was carried out under shaking. In these experiments, the acrylic pieces were put into wells of an 8-well 7oo tissue culture plate (LabTek, Nunc, Napperville, IL, 600 USA) containing 200 laL of C. albicans suspension (10~ ~oo 1 organisms/mL). The plates were incubated on a rotary 4oo I shaker at 37° C for i h. After incubation, the acrylic pieces were rinsed, stained and evaluated as described above. 3oo
J
200
Statistical analysis - Data were analyzed by means of the Student's t-test and the analysis of variance (ANOVA) test to compare two groups or more than two groups, respectively.
100 0
A
B
Figure 3. - Adherence of C. albicans to acrylic in presence of CSE A. Non-shaking system B. Shaking system.
RESULTS
Effect of CSE prepared from commercial chitin on adherence of C. albicans to acrylic - The effect of CSE on the adherence of the fungus to acrylic was evaluated in the following experimental systems: (a) the reaction of adhesion carried out in the presence of CSE and (b) the adherence test performed with acrylic pieces pretreated with CSE. a. Adherence of C. albicans to acrylic in the presence of CSE - Since it was previously shown (8, 18) that 25 mg/mL CSE had an inhibitory effect on the adherence of C. albicans to various epithelial cells, we initially assessed the effect of this CSE concentration on the adherence of the fungus to acrylic. The assay in the presence of CSE was performed under shaking and non-shaking conditions. Figure 3 presents mean adherence values obtained from 20 experiments with 67 acrylic pieces exposed to CSE and 81 controls of the strain CBS 562. The data indicated that under both experimental conditions, CSE inhibited C. albicans adherence to acrylic by 8183%. Statistical analysis by the Student's t-test revealed a p < 0.001 value in both experimental conditions. An experiment with C. albicans S (data not shown) carried out in a non-shaking system revealed that CSE inhibited the adhesion to acrylic of this strain, as well. Since a similar degree of inhibition was noted in both experimental conditions (shaking and non-
MEAN NO ADHERENT YEASTS/MI~ 900 800 700
[L
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CSE CONCENTRATIONS(mg/ml) Figure 4. - Effect of presence of various CSE concentrations on adherence of C. albicans to acrylic.
b. Adherence of C. albicans to CSE-pretreated acrylic pieces - In a further series of experiments, the number of adherent yeasts to acrylic pieces treated with various CSE concentrations (12.5-100 mg/mL) for 1-3 h prior to the adherence assay, was examined. It should be noted that these CSE-pretreated acrylic pieces were rinsed with PBS prior to the adherence assay to remove excess CSE.
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Adherence of C. albicans to acrylic
CSE pretreatment for 1 h reduced the adherence significantly (p < 0.001) at all concentrations tested (three experiments, 3-4 acrylic pieces for each experiment for each CSE concentration; 3-4 control acrylic pieces in each experiment). A similar effect was obtained with acrylic pieces pretreated with CSE for 2 or 3 h (2-3 experiments, 3-4 acrylic pieces in each experiment for each CSE concentration; 3-4 control acrylic pieces in each experiment). Figure 5 shows the percentage of inhibition versus CSE concentrations at the various pretreatment periods. A linear correlation between CSE concentrations and percentage of inhibition was noted only when the acrylic pieces were pretreated for 1 h. When the pretreatment was longer (2-3 h), the maximum percentage of inhibition was noted at lower CSE concentrations and reached a plateau.
adherence. We then tested various higher concentrations of CA. chitin-CSE: 5, 10, 20, 50 rag/ mL (2-3 experiments; 3-5 acrylic pieces in each C.A. chitin-CSE concentration in each experiment; 4-6 control acrylic pieces in each experiment) (Fig. 6). MEAN NO ADHERENT YEASTS/MM2 1000 900 800 700 800 500 400 300 200
INHIBITION (%) 60,
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C.A, C H I T I N CSE C O N C E N T R A T I O N S ( m g / m l )
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t.igure 6. - Effect of presence of various C.A. chitin-CSE concentrations on adherence of C. albicans to acrylic.
All C.A. chitin-CSE concentrations starting from 5 mg/mL reduced the number of adherent yeasts compared to controls. However, a significant reduction (p < 0.001) of 46O/owas obtained with 50 mg/mL.
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I
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CSE CONCENTRATIONS (mg/ml)
Figure 5. - Effect of pretreatment of acrylic with various CSE concentrations for different time intervals on C. albicans adherence.
Effect of CSE prepared from chitin isolated from C. albicans cultures (C. albicans-CSE) on the adherence of the fungus to acrylic - Following the observations that CSE from commercial chitin exerted an inhibitory effect on the adherence of C. albicans to acrylic, we initiated experiments to test whether CSE prepared from chitin isolated from C. albicans blastospores would have a similar effect. For this purpose, we isolated chitin from C. albicans cultures, prepared CSE from the chitin (C.A. chitin-CSE) and assessed the activity of this substance in two experimental systems: (a) adhesion in the presence of C.A. chitin-CSE and (b) adhesion with C.A. chitinCSE pretreated acrylic pieces. a. Adherence of C. albicans to acrylic in presence of C. albicans CSE - Based on data from a previous study (9) that 2.5 mg/mL C.A. chitin-CSE significantly inhibited attachment of C. albicans to epithelial cells, we tested the effect of a similar concentration (2 mg/ mL) on adherence to acrylic. The results of two experiments with 4-5 acrylic pieces in each experiment showed no inhibitory effect of C.A. chitin-CSE on
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b. Adherence of C. albicans to C. albicans-CSE pretreated acrylic pieces - To assess the effect of pretreatment of acrylic pieces with C.A. chitin-CSE on adhesion, the pieces were exposed for 1 h to 2, 5 and 10 mg/mL concentrations. Results of three such experiments (3-5 acrylic pieces in each experiment for each C.A. chitin-CSE concentration; 3-5 control acrylic pieces in each experiment) are presented in Figure 7. The data demonstrated that exposure to 5 or 10 mg/mL C.A. chitin-CSE inhibited adherence by 6070% (p < 0.01) as compared to the control. MEAN NO. ADHERENT YEASTS/MM2 700 600
m
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!o
~5 ~2
I
~NTROL
itiiiiiii
C.A, CHITIN CSE CONCENTRATIONS(mg/ml)
Figure 7. - Effect of pretreatment of acrylic with various C.A. chitin-CSE concentrations for 1 h on C. albicans adherence.
Segal E. et aL
Eur. J. EpidemioL
DISCUSSION
In the present study, we assessed the effect of a chitin derivative (CSE), prepared from commercially purchased chitin (prepared from crab shells) and from chitin isolated from C. albicans on the adherence of the yeast to acrylic. The data demonstrated that this substance significantly reduced the number of adherent yeasts when tested in various experimental systems. Moreover, in some experimental conditions, it was possible to almost block the entire binding of the fungus to acrylic. This study was initiated following previous investigations (5, 8, 18) in which it was shown that CSE significantly inhibited the adherence of C. albicans to various epithelial cells: buccal and vaginal mucosa, corneocytes and gastrointestinal epithelium. Furthermore, pretreatment of animals, with CSE prevented the development of experimental candidiasis (8, 9, 19). Based on the data from those studies, it was assumed that CSE may have acted as an adhesin-like substance through competitive binding to specific C. albicans receptors on the host cells, thereby covering these sites and blocking the attachment of the yeasts. This assumption was strengthened by the finding that CSE from C. albicans chitin was active at significantly lower concentrations than CSE isolated from commercial chitin (9). Partial analysis and chromatographic separation of CSE (9) indicated that the inhibitory activity was apparently associated with a fraction containing amino sugars. In addition, NAG, the monomer of chitin, also exerted an inhibitory effect, while other Candida cell wall polysaccharides (mannan, glucan) and their monomers had no effect (8, 21). Since acrylic is an inert surface, it is difficult to assume that the mechanism of adherence would be similar. Moreover, it is generally believed that in adherence to acrylic or other inert surfaces the major forces involved in the mechanism of adhesion are of hydrophobic or electrostatic nature (6, 11, 14). Thus, it is reasonable to assume that CSE may act by interfering with these binding forces. This hypothesis would explain the differences between the behavior of CSE in the systems with host cells vs acrylic, such as lower activity of C.A. chitin-CSE in the latter or very low inhibitory activity of NAG (data not shown). However, the exact mechanism of the interference of CSE in the binding of the yeasts to acrylic should be further elucidated. McCourtie, MacFarlane and Samaranayake (10) reported that chlorhexidine reduced the adherence of C. albicans to denture acrylic. It should, however, be noted that chlorhexidine is known to have adverse effects. Moreover, most recently (22), it was observed in in vitro studies that saliva significantly reduced the microbicidal activity of chlorhexidine against all microorganisms tested including C. albicans. In an experiment in which we assessed the activity of CSE on the adherence of C. albicans to acrylic in the presence of saliva (data not shown), we noted that
CSE had an inhibitory effect under these conditions, as well. As the adherence of C. albicans to acrylic denture is a vital step in the pathogenesis of denture stomatitis, it is suggested that blocking this step may be of major significance in the clinical prevention of this infection. This hypothesis has to be further clarified by experiments in in vivo systems with CSE, which are currently in progress in our laboratory. REFERENCES
1. Beachey E., Eisenstein B. and Ofek L (1982): Bacterial
adherence in infectious diseases In: Current Concepts. Upjohn Co, Kalamazoo, pp. 1-52. 2. Braun P.C. and Calderone R.A. (1978): Chitin synthesis in Candida albicans: Comparison of yeast and hyphal forms - J. Bacteriol. 133: 1472-1477. 3.
Critchley I.A. and Douglas L.J. (1985): Differential adhesion of pathogenic Candida species to epithelial and inert surfaces - Fed. Eur. Microbiol. Soc. Microbiol. Lett. 28: 199-203.
4. Douglas J. (1987): Adhesion to surfaces - In: Rose
A.H. and Harrison J.S., eds. The yeasts - Academic Press, London, pp. 239-280. 5. Kahane M., Segal E., Schewach-Millet M. and Gov Y. (1988): In vitro adherence of Candida albicans to
human corneocytes - Inhibition by chitin soluble extract - Acta. Dermato. Venereol. 68: 98-101. 6. Kennedy M.J. (1988): Adhesion and association mechanisms of Candida albicans - Curr. Top. Med. Mycol. 2: 73-169. 7. King R.D., Lee J.C. and Morris A.L. (1980): Adherence of Candida albicans and other Candida
species to mucosal epithelial cells - Infect. Immun. 27: 667-674. 8. Lehrer N., Segal E. and Barr-Nea L. (1983): In vitro and in vivo adherence of Candida albicans to mucosal surfaces - Ann. Microbiol. 134: 293-306. 9. Lehrer N., Segal E., Lis H. and Gov Y. (1988): Effect of Candida albicans cell wall components on the
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adhesion of the fungus to human and murine vaginal mucosa- Mycopathologia 102: 115-121. 10. McCourtie J., MacFarlane T.W. and Samaranayake L.P. (1985): Effect of chlorhexidine gluconate on the adherence of Candida species to denture acrylic - J. Med. Microbiol. 20: 97-104. 11. Minagi S., Miyake Y., lnagaki K., Tsura H. and Suginaka H. (1985): Hydrophobic interaction in Candida albicans and Candida tropicalis to various denture base resin materials - Infect. Immun. 47:1114. 12. Odds F..C. (1988): Candida and candidosis. 2nd ed. Bailliere Tindall, London.
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13. Regezzi J.A. and Sciubba J.J. (1989): Oral pathology: Clinical-pathologic correlations - W.B. Saunders Co., Philadelphia, pp. 110-116.
Development of the assay and tests of inhibitors - J. Med. Vet. Mycol. 24: 477-479.
14. Rotrosen D., Calderone R.A. and Edwards Jr J.E. (1986): Adherence of Candida species to host tissues and plastic surfaces - Rev. Infect. Dis. 8: 73-85.
19. Segal E., Gottfried L. and Lehrer N. (1988): Candidal vaginitis in hormone treated mice: Prevention by a chitin extract - Mycopathologia 102: 157-163.
15. Samaranayake L.P., McCourtie S. and MaeFarlane T.W. (1980): Factors affecting the in vitro adherence of Candida albicans to acrylic surfaces - Arch. Oral Biol. 25: 611-615.
20. Segal E., Lehrman O. and Dayan D. (1988): Adherence in vitro of various Candida species to acrylic surfaces - Oral Surg. Oral Med. Oral Pathol. 66: 670-673.
16. Segal E. (1985): Inhibition of attachment of Candida to animal cells and inert surfaces - J. Dent. Res. 64: 730, Abstr. No. 4.
21. Segal E., Lehrer N. and Ofek L (1982): Adherence of Candida albicans to h u m a n vaginal epithelial cells: inhibition by amino sugars - Exp. Cell. Biol. 50: 1317.
17. Segal E. (1987): Pathogenesis of h u m a n mycoses: Role of adhesion to host surfaces - Microbiol. Sci. 4: 344-347. 18. Segal E. and Savage D. (1986): Adhesion of Candida albicans to mouse intestinal mucosa in vitro:
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22. Spijkervet F.K.L., Van Saene J.J.M., Van Saene H.K.E, Panders A.K., Vermey A. and Fidler V. (1990): Chlorhexidine inactivation by saliva - Oral Surg. Oral Med. Oral Pathol. 69: 444-449.
