Archs oral Ewl. Vol. 37. Xo. 8. pp. 613-622. Printed in Great Britain. All rights reserved

1992 Cop>nght

0003.9969 92 $5 00 - 0.00 c 1992 Pergamon Press Ltd

THE INFLUENCE OF MORPHOLOGICAL VARIATION ON CANDIDA ALBICANS ADHESION TO DENTURE ACRYLIC IN VITRO A. VASILAS,’L. MOLINA,’M. HOFFMAN’and C. G. HAIDARIS!.:.* Departments of ’ Dental Research and :Microbiology and Immunology. University of Rochester School of Medicine and Dentistry, Rochester, NY 146-12.U.S.A. (Accepred 26 March 1992)

Summary-Using

denture acrylic pieces coated with either whole human stimulated saliva or oral streptococci, the binding ability of three different Candidu ulbicuns strains was investigated. The C. ulbicuns strains include a clinical isolate with the commonly observed, smooth, round colonial morphology (strain 613~). a morphological variant spontaneously derived from the clinical isolate strain 613~ (strain 613mlBK) and a clinical isolate from an oral lesion that was also a morphological variant upon primary isolation (strain 228). Levels of adhesion to the acrylic pieces were determined radiometrically using C. ulbicans cells metabolically labelled with [35S]-methionine. Whole stimulated saliva significantly increased the binding of all strains compared to uncoated acrylic. However, the level of binding of strain 613~ to saliva-coated acrylic was significantly greater than the levels observed for the morphological variant strain 613mlBK. Coating acrylic pieces with either Streprococcus sunguis NCTC 10904. Sfrep. mutans GS-5 or Strep. sobrinus ATCC 27352 instead of saliva resulted in significantly greater binding by strain 613~ compared to uncoated acrylic. Pre-coating the acrylic with the oral streptococci did not significantly increase the binding of morphological variant strains 613mlBK and 228 compared to uncoated acrylic. In general, preincubation of adherent streptococci with sucrose to induce the synthesis of extracellular carbohydrate polymers did not significantly increase the binding levels of the C. albicans strains above those observed using streptococci in buffer alone. Compared to its parental strain 613~. morphological variant strain 613mlBK adhered poorly to denture acrylic coated with either salivary constituents or oral streptococci, while strain 228 adhered to the same substrates at an intermediate level. Furthermore, physical disaggregation of clusters of the morphological variant strain 613mlBK did not appear to increase its binding capacity to saliva-coated denture acrylic. The effect of whole stimulated saliva on the adherence of C. ulbicuns 613~ to a variety of plastic substrates in addition to denture acrylic was examined. Overall, saliva pre-coating of the various plastics promoted C. albicuns 613~ adhesion. The adhesion of strain 613~ to denture acrylic coated with whole stimulated saliva from each of five different donors or with parotid and submandibular/sublingual saliva from each of two donors was also examined. Regardless of donor, a coating of whole stimulated saliva significantly increased the binding of strain 613~ to denture acrylic compared to uncoated acrylic. In addition, a coating of parotid saliva significantly increased the binding of strain 613~ to denture acrylic compared to submandibular sublingual saliva. Key words: Cundidu ulbicuns, denture acrylic, saliva, adhesion, streptococci, morphological variation,

ISTRODUCTION Denture stomatitis is a localized inflammation of the soft tissues underlying removable dental prostheses. It usually affects the palatal epithelium covered by a denture (Arendorf and Walker, 1987). The aetiology of denture stomatitis is multifactorial (Renner er al., 1979; Arendorf and Walker, 1987). The most commonly described non-systemic factors predisposing to inflammation of the denture-bearing mucosa are: poor fit of the denture (especially in the maxilla) and/or unsatisfactory occlusion with consequent trauma, inadequate oral hygiene and the consumption of a carbohydrate-rich diet (Love, Goska and Mixson, 1967; Budtz-Jorgensen, 1974; Ambjbmsen, *To whom all correspondence should be addressed at the Department of Microbiology and Immunology. IA, index of adherence; PBS, phosphatebuffered saline.

Abbreciurions:

Am

37 5-B

1985; Samaranayake, 1986; Schou, Wright and Cumming, 1987). Although bacteria or other yeasts may act as pathogens in denture stomatitis (Gusberti et al., 1985) C. nlbicuns has been implicated as the major cause of microbial origin (Turrell, 1966; Budtz-Jorgensen, 1974; Renner er al., 1979; BudtzJorgensen, Theilade and Theilade, 1983; Theilade and Budtz-Jorgensen, 1988). Newly formed plaque on the undersurface of maxillary dentures acts as the substrate that mediates colonization by C. albicans (Theilade, Budtz-Jorgensen and Theilade, 1983; Morris et al., 1987). Although the prosthesis is the main reservoir of yeasts in the mouths of edentulous people (BudtzJorgensen and Theilade, 1983), few studies have dealt with factors that may promote C. albicans adherence to these devices. Moreover. the role of oral microorganisms and salivary molecules in promoting the colonization of denture acrylic by C. albicans is poorly understood (Samaranayake and MacFarlane, 1990).

613

A. VASILAS et al.

611

An in vitro system was therefore developed for assaying the adherence of C. albicans to denture acrylic in conjunction with other components of the oral environment. Whole stimulated, parotid and submandibular/sublingual salivas from various donors, as well as oral streptococci, were examined for their ability to mediate adherence of C. a&cans. Several C. albicans strains displaying different morphological characteristics were compared for their ability to bind to coated and uncoated denture acrylic. The effect of coating various plastic substrates other than denture acrylic with whole stimulated saliva on the subsequent adherence of C. albicans, compared to uncoated substrates, was also determined. MATERIALS

AND METHODS

Culture media and experimental micro -organisms C. albicuns strains were maintained on yeast extract peptone dextrose (YEPD) agar plates [l% yeast extract (Difco, Laboratories, Detroit, MI, U.S.A.), 2% Bacto-Peptone (Difco), 2% dextrose (EM Science, Gibbstown, NJ, U.S.A.) and 2% agar (Difco)] and transferred monthly. C. ulbicans strains were grown as blastospores in yeast nitrogen base-galactose (YNB-gal) liquid growth medium [0.67% yeast nitrogen base (Difco) and 277 mM galactose (JT Baker Inc., Phillipsburg, NJ, U.S.A.)]. Medium 199 with Earle’s salts, pH 6.7 (Sigma Chemical Co., St Louis, MO, U.S.A.), was used to promote germ tube formation from yeast cells. C. albicuns 613~ (Meitner, Bowen and Haidaris, 1990) was isolated from a cutaneous lesion at the Department of Clinical Microbiology, Strong Memorial Hospital, Rochester, NY, U.S.A. On YEPD agar plates, C. ulbicuns 613~ formed white, smooth, round colonies and grew predominantly as singlet blastospores in YNB-gal liquid broth, with less than 1% as pseudohyphae. In medium 199 at 37’C, C. ulbicuns 613~ produced germ tubes at a high conversion rate (> 90%). C. albicuns 613m1, a spontaneous morphological mutant of C. albicuns 613p, was isolated in the Department of Microbiology and Immunology (University of Rochester, Rochester, NY, U.S.A.) from colonies of C. albicuns 613~ plated on YEPD agar. C. ulbicuns 613ml differed from the parental strain C. albicans 613~ in colony morphology. Strain 613mlBK was derived from 613ml after its passage through a surgically desalivated rat (Meitner et al., 1990). On YEPD agar, C. ulbicans 613mlBK produced rough colonies, with an irregular perimeter and wavy prominences radially distributed from the centre of the colony. Microscopically, C. albicuns 613mlBK appeared in aggregated blastospores of 20-50 closely packed spheroid cells, with occasional thin long pseudohyphae. In M 199 medium, C. albicuns 613mlBK converted to germ tubes poorly (>S%). C. ulbicuns 228 was isolated in the Department of Clinical Microbiology, Strong Memorial Hospital, Rochester, NY, U.S.A. from an oral lesion of a human immunodeficiency virus-positive patient. C. ulbicuns 228 also exhibited morphological variation compared to C. ulbicans 613~ directly upon primary plating, with colonies that had a smooth

perimeter with an elevated volcano-like centre and radial prominences. &licroscopically, C. a&cans 228 grew in YNB-gal as single, spheroid cells or in small aggregates of 2-10 blastospores. and formed short germ tubes abundantly (>90%) in Ml99 medium. In summary, strain 613~ resembled the more frequently observed colonial and cell morphology of C. afbicans isolates. while strains 613m I BK and 228 were morphological variants. C. ulbicuns strains 613p, 613mlBK and 228 were all agglutinated by two commercially available C. ulbicuns antisera (Difco: and Iatron Laboratories Inc., Tokyo, Japan). Strep. sunguis NCTC 10904 (Hamada et al.. 1980) and Strep. mutans GS-5 (Bratthall. 1970) were obtained from the Department of hlicrobiology and Immunology, University of Rochester. Rochester, NY, U.S.A. (courtesy of Dr Robert Marquis). Strep. sobrinus ATCC 27352 (Spine11 and Gibbons, 1974) was passaged through a desalivated rat and obtained from the Department of Dental Research. University of Rochester, Rochester, NY, U.S.A. (courtesy of Dr William H. Bovven). Streptococci were maintained frozen in tryptic soy broth with 15% glycerol (JT Baker) at -7O’C until use. Culture conditions and preparation of cells C. ulbicuns was cultured overnight at 37-C in YNB-gal and metabolically radiolabelled with 5 pCi/ml r-[35S]-methionine (IO mCi/ml; > 1000 Ci mmol; Amersham Corp., Arlington Heights, IL. U.S.A.). Washed C. ulbicans cells were resuspended in PBS and sonicated (Branson Sonifier 250: Branson Ultrasonic Corp., Danbury, CT. U.S.A.) at 35 W in an ice-chilled polypropylene container for a total of 30 s (in two 15-s pulses with a minimum interval of 5 s between). During the wash, I OO-\lI portions of cell suspension or supernatant were removed and processed for liquid scintillation counting. The values obtained were used to evaluate uptake of radiolabel by the C. albicans cells and the efficiency of the washes in removing unbound radiolabel. Sonication did not significantly affect the viability of C. ulbicuns cells as determined by plating on YEPD agar plates. The cell concentration was measured spectrophotometrically (Spectronic 1001 Plus: Milton Roy Company, Rochester, NY. U.S.A.) at 600 nm, as two of the strains grew as aggregates. A C. albicuns 613~ concentration of 2 x lO”cells/ml corresponded to 0.D .MxI = 0.05. A frozen stock of streptococci was grown overnight in tryptic soy broth (Difco) supplemented with 0.5% yeast extract (Difco) and on tryptic soy agar plates (Difco). Streptococci inoculated from a stationary-phase liquid culture lvere metabolically radiolabelled with 4 pCi/ml [methyl-3H]-thymidine (1 mCi/ml; 70-85 Ci mmol; Amersham). Washed streptococcal cells were sonicated as described for C. ulbicans to disperse aggregates; sonication did not adversely affect cell viability. During the procedure, lOO-,nl samples of cell suspension or supernatant were removed and processed for liquid scintillation counting. Human saliru collection

Human whole saliva was collected during masticatory stimulation with Parafilm M (American

Candida

adhesion to denture acrylic

Can Co.. Greenwich. CT, U.S.A.) in an ice-chilled polypropylene tube and clarified by centrifugation at 10,OOOg for 10min at 4’C (Gibbons. Cohen and Hay, 1986). For uniformity, each saliva sample was collected at the same time of day and the volume limited to 50 ml per collection period. The supernatant was removed and frozen at -2O’C until used. Unless otherwise noted, experiments were conducted using whole stimulated saliva from one donor (AV). All donors of whole saliva were male adults. aged between 27-35, who had not received any medications known to affect salivary composition and flow. We did not determine whether the donors harboured C. albicans in their mouths or Lvhether their salivas contained C. albicans -specific antibodies. Human parotid saliva was collected using a Lashley device. The parotid saliva was lyophilized and reconstituted with parotid saliva buffer (Bennick and Cannon, 1978) at 2 mg of dry weight/ml. Submandibular/sublingual saliva was collected in an ice-chilled polypropylene tube using a collection device (Block and Brottman, 1962) adapted to the floor of the mouth with a vinyl polysiloxane impression material (Reprosil Cartridge System. Type 1 medium viscosity; LD Caulk Div. Dentsply International Inc., Milford, DE1 U.S.A.). This saliva was iyophilized and reconstituted with submandibular#sublingual saliva buffer (Bennick and Cannon 1978) at 2mg of dry weight ml. A mucin-rich fraction of pooled submandibular/sublingual saliva, a generous gift of Dr L. A. Tabak, University of Rochester, was obtained and prepared as described by Prakobphol et al. (1982). The lyophilized mucinrich fraction (fraction A) was reconstituted with submandibular/sublingual saliva buffer (Bennick and Cannon, 1978) at 2mg of dry vveight/ml. All ductal salivas and saliva fractions were frozen at -2O’C until use. Fabrication of acrylic pieces

Heat-cured denture acrylic sheets were fabricated according to conventional prosthodontic techniques: a flat piece of polystyrene (75 x 65 x 1.6 mm) was invested in a maxillary denture-processing flask (Varsity, Hanau Engineering Co. Inc.. Buffalo, NY, U.S.A.) using Die-stone (Modern Materials, Columbus Dental, St Louis, MO, U.S.A.). The wax was removed, the exposed surfaces were washed with liquid detergent (Procter and Gamble, Cincinnati, OH, U.S.A.), thoroughly rinsed with boiling tap tvater and air-dried. Two coats of separating medium (Alcote. L.D. Caulk Co., Milford, DE. U.S.A.) were applied to the warm stone, and allowed to dry and cool. Denture acrylic polv(methylmethacrylate) powder (Hy-Pro Lucitone, pmk; Dentsply, York, PA) and monomer liquid (Lucitone, Standard Liquid; Dentsply) were mixed according to the manufacturer’s recommendations. The mixture was packed into the flask, allowed to bench-cure for 1 h and then processed in a water tank (Hanau Engineering Co. Inc.) at 74’C (165’F) for 9 h. The processed acrylic sheet was rinsed and stored for a minimum of 30 days in distilled water. Each acrylic sheet was cut into 42 square pieces, 9 x 9 x 1.6 mm. The top surface was ground on a 320-grit silicon carbide sandpaper (02004; 413Q Wetordry Tri-M-ite

615

Paper EF. Grit 320-A, 3M Household Products Division. St Paul, MN, U.S.A.). Molloplast-B (Regneri GmbH & Co. KG, Karlsruhe, Germany), a heat-cured silicone denture liner, was processed according to manufacturer’s recommendations: the uncured material was packed in a stone mould, the flask was placed in a curing tank and gradually brought to 1OO’C (212’F) over 9 h. After boiling for 3 h. it was allowed to cool. The sheet of Molloplast-B was cut into pieces with a sharp scalpel blade. Petri dish polystyrene pieces were cut from disposable Petri dishes (Falcon, Becton Dickinson Labware. Lincoln Park, NJ, U.S.A.). Tissue-culture flask polystyrene pieces were cut from polystyrene disposable tissue-culture flasks (Corning Glass Works). Both were cut similarily to the acrylic resin pieces into pieces 9 x 9 x 1.6 mm; some were sanded similarly to the acrylic pieces. All pieces were thoroughly rinsed and ultrasonically cleaned (Model LU; L&R Ultrasonic. Kearny, NJ. U.S.A.) twice for 15 min in distilled water and stored in polypropylene tubes with distilled water. Before each experiment, the pieces were stirred overnight in a glass flask with distilled water. Adhesion nssay

The acrylic pieces were incubated individually with saliva or PBS in ZCmultiwell polystyrene tissueculture plates (Falcon) with the 320 grit-treated surface facing up at 37’C for 2 h. Unless otherwise noted, the pieces were incubated with 750 111of saliva. After incubation, each piece was washed in distilled Lvater. For experiments examining the adherence of C. albicam to denture acrylic precoated with streptococci, 900 ~1 of the streptococcal suspension in buffer (approx. lO’cells/ml: O.D.,, = 0.7) were added to each acrylic piece in the wells. Next. 100 /II of either PBS alone or PBS containing 1.5 M sucrose (JT Baker) and 200mM dextran (mol. wt 9000; Sigma), pH 7.5, were added to each well. The solution containing 0.15 M sucrose and 20 mM dextran is referred to as BSD; dextran was added because it has been reported to act as a primer for the synthesis of extracellular polysaccharides by streptococci (Chassy et al., 1976: Ciardi, 1983). The 24-well plates Lvere placed on an orbital shaker (60 revjmin) at 37’C for 4 h. The pieces were washed thoroughly in distilled water and allowed to dry. Ml99 preheated to 37’C and C. nlbicans cells at a concentration of O.D., = 0.05 (2 x lo6 cells,‘ml for C. albicans 613~) were placed stationary at 37’C for 2 h to allow for adhesion of C. albicans. The acrylic pieces were rinsed with distilled water to remove unbound and loosely bound C. albicans cells. The acrylic pieces with adherent micro-organisms were allowed to dry, processed for liquid scintillation counting or stained for microscopic observation. The activity of the radioactive samples was counted in a liquid scintillation counter (LS 1801; Beckman Instruments, Inc., Irvine, CA, U.S.A.). For experiments examining the effect of disaggregating C. albicans 613mlBK cell aggregates, C. albicans 613m 1BK cells were additionally vortexed with glass beads (Sigma Chemical Corp., Arlington Heights. IL, U.S.A.). Four ml of cells and 1 ml of

glass beads were vortexed at maximum power in a IS ml polypropylene centrifuge tube (Corning Glass Works) for 3 min, which resulted in aggregates of 2-3 C. albicans 613m 1BK cells. Cell viabihty, as examined microscopically with the trypan blue dye exclusion test, was not affected. C. albicans 613~ acted as a control for adherence and cell viability.

Coating n wss c3Streptococci/sucrose 0 Streptococci/buffer R Uncoated

3000

Analysis of results

IA 2000

Because there were differences in radiolabel uptake among different micro-organisms and experiments, the counts/min values obtained were normalized, as follows. Results are expressed as index of adherence (IA): IA =

1000 ” 613~ 613mlBK

counts/min,‘acrylic piece x 10” uptake (countsimin)

’

where, uptake is the counts/min of 100 ~1 of C. albicans cell suspension of O.D., = 1.000 or the counts/min of 100 ~1 of streptococcal cell suspension of O.D.m = 1.000. The uptake for each microbial strain examined was determined for each experiment. Mean IA values and sample SDS were calculated for experimental and control groups. Unless otherwise noted the number of samples in each group was 4. Results shown are from a single experiment, representative of duplicate experiments. Statistical analysis of the results was made by the Department of Biostatistics, School of Medicine and Dentistry, University of Rochester, NY, U.S.A. Both Tukey’s Studentized test and Sceffe’s test were used for the analysis (Snedecor and Cochran, 1980). RESULTS

Adherence of C. albicans to denture acrylic precoated with either whole stimulated saliva or streptococci

Coating the denture acrylic resin with this saliva significantly increased (p < 0.05) the subsequent adherence of the C. afbicans strains when compared to the adherence to acrylic coated with streptococci and to uncoated acrylic (Figs 1 and 2). This increase was more prominent for C. albicans strains 613~ and 228 than 613mlBK. Coating the denture acrylic resin with oral streptococci generally increased the subsequent adherence of C. albicans strains, compared to uncoated acrylic. Coating the acrylic with a primary oral colonizer, Strep. sanguis NCTC 10904 (Fig. l), or secondary oral colonizers, Strep. sobrinus ATCC 27352 (Fig. 2) and Strep. mutans GS-5 (data not shown), increased the number of adherent C. albicans to a similar degree. However, the increase in adherence of C. albicans to acrylic coated with streptococci compared to uncoated acrylic was statistically significant (p c 0.05) only for C. albicans 613p, and not for C. a/bicans 613mlBK and C. albicans 228.

Adding sucrose and dextran to PBS did not increase the adhesion of C. albicans 613~ compared to that on acrylic pieces coated with Strep. sanguis NCTC 10904 (Fig. 1) and Strep. mutans GS-5 in buffer alone (data not shown). In contrast, coating the acrylic pieces with Strep. sobrinus ATCC 27352 (Fig. 2) suspended in BSD resulted in a slight (not statistically significant) increase of IA for the adherent C. albicans 613~ compared to coating the pieces with the streptococci in PBS alone. It should

228

C. albicans

Fig. I. Adherence of C. albicans strains 613~. 613mlBK and 228 to denture acrylic pre-coated with whole stimulated saliva (WsS), denture acrylic pre-coated with &rep. sanguis NCTC 10904 in PBS (streptococci/buffer), denture acrylic pre-coated with Strep. sanguis NCTC 10904 in BSD (streptococci/sucrose) and uncoated (uncoated) denture acrylic. In each group of four adjacent bars, the two middle bars represent adherent C. albicans on acrylic pieces coated with streptococci. The insert figure represents the adherence of Strep. sanguis NCTC 10904 in PBS and Strep. sanguis NCTC 10904 in BSD (open bar) and Strep. sanguis NCTC 10904 in BSD (ruled bar) to the denture acrylic pieces used in the assay. IA, index of adherence. be noted, however, that about 50% fewer cells of S. sobrinus ATCC 27352 were attached to the acrylic when incubated in BSD compared to PBS alone (insert graph of Fig. 2). Therefore, the number of adherent C. albicans 6 13~ cells per streptococcal cell was doubled when S. sobrinus ATCC 27352 was incubated with the acrylic in BSD, compared to Coating n wss q Streptococci/buffer

0 Streptococci/sucrose EJUncoated

1000 IA 500

” ”

613~

613mlBK

226

C. albicans

Fig. 2. Adherence of C. albicans strains 613~. 613mlBK and 228 to denture acrylic pre-coated with whole stimulated saliva (WsS), denture acrylic pre-coated with Srrep. sobrinus ATCC 27352 in PBS (streptococci/buffer), denture acrylic pm-coated with Strep. sobrinus ATCC 27352 in BSD (strep tococci/sucrose) and uncoated (uncoated) denture acrylic. In each group of four adjacent bars, the two middle bars represent adherent C. albicanson acrylic pieces coated with streptococci. The insert figure represents the adherence of Strep. sobrinus ATCC 27352 in PBS and Strep. sobrinus ATCC 27352 in BSD (open bar) and Strep. sobrinus ATCC 27352 in BSD (ruled bar) to the denture acrylic pieces used in the assay. IA, index of adherence.

617

Cnndidu adhesion to denture acrylic S. sobrinus ATCC 27352 that adhered in PBS. Strep. sobrinus ATCC 27352 incubated with acrylic

pieces in BSD formed cell aggregates that were macroscopically visible. Strep. sobrinus ATCC 27352 incubated with acrylic pieces in PBS, and Strep. sanguis NCTC 10904 and Strep. mutans GS-5 incubated with acrylic pieces either in PBS or BSD did not form macroscopically visible aggregates.

0 WsS coated

3000

0 Uncoated t

2000

IA

1 1000 c

Effect of disaggregating C. albicans 613mIBK cell aggregates on their adherence to denture acrylic

We examined the effects of cell aggregation of C. albicans 613mlBK on its ability to adhere to whole stimulated saliva-coated and uncoated acrylic when compared to its parental strain C. albicans 613~. C. albicans 613mlBK normally grew as cell aggregates of 20-50 cells; vortexing strain 613mlBK with glass beads produced aggregates of 2-3 cells without affecting cell viability. Vortexing and disaggregating C. albicans 6 13ml BK did not result in a considerable difference in adherence to saliva-coated and uncoated acrylic, compared to C. albicans 613mlBK cells that were not vortexed with glass beads (Fig. 3). Both C. albicans 613mlBK (p ~0.012) and C. albicans 613~ (p < 0.009) adhered at significantly lower numbers to whole saliva-coated denture acrylic after the cells had been vortexed with glass beads. Eflect of whole stimulated saliva coating on the adherence of C. albicans to various plastic substrates

We examined whether coating various plastic substrates other than denture acrylic with whole stimulated saliva would modify their ability to bind C. albicans compared to the binding of C. albicans to the uncoated surfaces. Saliva coating of the various plastic substrates diminished the differences observed for the uncoated surfaces (Fig. 4). The most marked difference produced by this coating was in denture acrylic. As noted from all of our experiments, coating denture acrylic with whole stimulated saliva pro1500

T

t

q WsS coated 0 Uncoated

1000 IA 500

0

C. albicans

strain/treatment

Fig. 3. Adherence of C. albicunsstrains 613p, 613mlBK to whole stimulated saliva-coated denture acrylic (WsS coated) and to uncoated denture acrylic (uncoated). Cells were washed and sonicated only (613mlBK and 613p, respectively), or, in the remaining two groups (613mlBK/G and 613u!G, resoectivelv). the cells were washed. sonicated and additionally*vortexed. with glass beads to disrupt large cell aggregates. IA, index of adherence.

Plastic substrate

Fig. 4. Comparison of adherence of C. albicans 613~ to various plastic materials either uncoated (uncoated) or pre-coated with whole stimulated saliva (WsS). Plastic materials used: Petri dish polystyrene (PDP); tissue culture flash polystyrene (TCFP); PDP ground with 320-grit sandpaper (PDP-320); TCFP ground with 320-grit sandpaper (TCFP-320) Molloplast-B (Molloplast); denture acrylic (acrylic). [Acrylic pieces were also ground with 320-grit sandpaper (see Materials and Methods).] IA. index of adherence.

duced a multifold increase in adherent

C. albicans

613~.

The uncoated substrates varied in their ability to bind C. afbicans 613~. Tissue culture flask polystyrene and Molloplast-B s exhibited the highest affinity for C. albicans. Petri dish polystyrene, and surface-ground Petri dish and flask polystyrenes exhibited intermediate affinity, whereas denture acrylic had the lowest affinity. Coating the flask polystyrene with whole stimulated saliva did not significantly modify its binding affinity for C. albicans 613~ compared to that of uncoated flask polystyrene. Coating Molloplast-B 8 with whole stimulated saliva significantly (p < 0.0009) reduced its ability to bind C. albicans 613~. Coating Petri dish polystyrene (p < 0.0127) ground Petri dish polystyrene (p < 0.0003), ground flask polystyrene (p < 0.0036) and denture acrylic (p c 0.0001) with whole stimulated saliva significantly increased the number of adherent C. albicans 613~ compared to the uncoated substrates. Significantly higher numbers of C. albicans 613~ adhered to whole saliva-coated tissue-culture flask polystyrene, Petri dish polystyrene, and their ground counterparts compared to either whole saliva-coated Molloplast or denture acrylic (p < 0.05); however, there was no significant difference in adherence levels when whole saliva-coated dish and flask polystyrenes (ground and unground) were compared to each other. Similarly, there was no significant difference in adherence levels when whole saliva-coated Molloplast and denture acrylic were compared to each other. Adherence of C. albicans 613~ to denture acrylic coated with various types of saliva from different donors

We determined whether denture acrylic pieces coated with whole stimulated saliva from five donors

A. VASILASer al.

618

and ductal saliva from two donors differed in their ability to bind C. al&cans 613~ cells. Precoating denture acrylic tvith whole saliva from each of five young adult male donors who had not received any medication known to affect salivary composition and flow significantly enhanced (p < 0.00001) the subsequent binding of C. al&cans 613~ compared to binding to uncoated acrylic (data not shown). Although statistically significant differences were found among the whole stimulated salivas of various donors in their ability to bind C. albicans, there was overlap between donors. Overall, coating the acrylic with whole saliva from the five donors increased the adherence of C. albicans at similar levels compared to the adherence to uncoated acrylic. The coating of denture acrylic with whole or parotid saliva from donors AV and GW enhanced the subsequent adherence of C. afbicans 613~ at similar levels compared to uncoated acrylic (p < 0.0001) (Fig. 5). No statistically significant differences were observed betlveen these types of saliva from donors AV and GW. Coating the acrylic with submandibular/sublingual saliva from donor AV resulted in significantly (p < 0.05) lower numbers of adherent C. a&cans compared to whole and parotid saliva from AV. Similarly, coating the acrylic with submandibular ‘sublingual saliva from donor GW resulted in significantly lower (p < 0.0007) numbers of adherent C. albicuns compared to those salivas from GW. HoLvever, coating the acrylic with submandibular/sublingual saliva from donor AV and a mucin-rich fraction of saliva did not have a significant effect on the adherence of C. albicans 613~ compared to the adherence of C. dbicans 613~ to uncoated acrylic. In contrast, coating the acrylic with submandibular,‘sublingual saliva from donor GW significantly increased the adherence of C. albicans 613~ compared with the adherence of C. albicans 613~ to uncoated acrylic (p < 0.0007). DISCUSSION

An in vitro system was developed to investigate quantitatively the adherence of C. afbicans to denture acrylic in conjunction with other components of the oral environment. Some of the methodological aspects followed were different from those that had been conventionally used in the past. First, heat-cured denture acrylic resin was used because it is the material most commonly used for fabrication of dental prostheses. In previous studies on the adhesion of C. albicans to saliva-coated denture acrylic (Samaranayake and MacFarlane, 1980; Samaranayake, McCourtie and MacFarlane, 1980; McCourtie and Douglas, 1981; McCourtie, MacFarlane and Samaranayake, 1986), cold-cured acrylic had been used. Structural differences have been reported between heat-cured and cold-cured acrylic resin (Ellis and Faraj, 1980). We did not examine whether any differences in C. albicans adherence ocurred between cold- and heat-cured acrylic or whether different salivary compounds were bound by cold- and heat-cured acrylic. However, the use of the same material as that used for dentures clinically, having the same electrochemical surface characteristics and similar potential for adsorbing various

Coating wss 13 PS q

n SMSL q Mucin-rich

fraction

0 Uncoated IA 500

n AV

GW

Saliva donor Fig. 5. Adherence of C. albicans 613~ 10 denture acrylic pre-coated with: whole stimulated saliva (WsS), parotid saliva (PS) and submandibular sublingual saliva (SMSL) from two donors (AV, GW). a mucin-rich fraction of SMSL (mucin-rich fraction), and to uncoated denture acrylic (uncoated). IA. index of adherence. molecules (Sanju and Glantz, 1975: Minagi et al., 1985), justified the additional time and effort that fabrication of heat-cured acrylic required. Secondly, suspending the C. albicans cells in M 199 during the adhesion assay induced rhe formation of germ tubes. It has been reported that germ tubes adhere in larger numbers to solid substrates than blastospores (Rotrosen. Calderone and Edwards, 1986; Tronchin et al., 1988). With this observation in mind, Ml99 was used during the adhesion step throughout our experiments. However, comparison of the adherence of C. albicans 613~ suspended either in Ml99 or in YNB-gal to whole saliva-coated acrylic indicated that germ tubes and blastospores did not differ substantially in their ability to adhere to that substrate; blastospores adhere to salit-a-coated acrylic at levels approx. 40% of those observed with germ tubes (data not shown). It appears. therefore, that C. albicans binding sites for salivary molecules adsorbed to acrylic are present both on blastospores and germ tubes of strain 613~. However, additional experiments are needed before any definitive conclusion can be made about the structure and distribution of saliva-binding moieties on blastospores and germ tubes of C. albicans strain 613~. Thirdly, our use of a radiometric technique for assaying adherent micro-organisms allowed for the simultaneous determination of the levels of adherent C. albicans and streptococcal cells on each acrylic piece. Quantitation of adherent fungi by light microscopy can be misleading, particularly when the C. albicans cells form aggregates. The radiometric system enabled us to study a variety of factors that may play a part in C. albicans colonization of the acrylic denture in a reproducible manner. The role of salivary components in the colonization of teeth by oral bacteria has been well documented (Riilla, 1977; Gibbons, Etherden and Peros, 1985). In contrast, the possible importance of saliva in mediating adherence of C. albicans to denture acrylic has not been extensively studied (Samaranayake and MacFarlane, 1990, pp. 35-36). Studies by Samaranayake et al. (1980), McCourtie and Douglas (1981) and McCourtie et al. (1986) suggested that

Candida adhesion to denture acrylic

saliva either decreased or did not affect the adherence of C. &cans to denture acrylic. In contrast, we observed that salivary components enhanced C. albicans adherence to denture acrylic, suggesting that an acquired salivary pellicle may play an important part in the colonization of the acrylic denture by C. ulbicuns. The numbers of C. albicuns and streptococcal cells adherent to acrylic pieces examined microscopically appeared to reflect the IA values obtained from liquid scintillation counting throughout all the experiments (data not shown). Adherent streptococci appeared in aggregates that covered approx. 50% of the acrylic surface. Higher numbers were found to be attached in the grooves on that surface. The highest number of C. ulbicuns adherent to acrylic pieces covered only a small percentage of the surface (roughly no more than 2%). Because C. ulbicuns cells are larger than streptococcal cells, it was not possible to visualize C. ulbicuns cells directly adherent to underlying streptococci. Preliminary experiments showed that Strep. sunguis NCTC 10904, Strep. muruns GS-5 and Strep. sobrinus ATCC 27352 adhered poorly to whole saliva-coated acrylic. Considering the increase in adherent C. ulbicans that resulted from whole saliva coating of the acrylic, no conclusions could be drawn for groups of acrylic sequentially exposed to whole stimulated saliva, streptococci and C. ulbicuns (data not shown). As the bulk of whole stimulated saliva is composed of parotid saliva (Mandel and Wotman, 1976), it was not surprising that whole and parotid salivas enhanced C. ulbicuns adherence to denture acrylic to a similar degree. In contrast to whole stimulated saliva, most of the volume of whole unstimulated saliva is submandibular/sublingual (Malamud, 1985), which had a less prominent effect than either whole stimulated or parotid saliva in increasing C. ulbicuns adherence. The differences between our findings and those of Samaranayake, McCourtie and Douglas may also be attributed to differences in the type of acrylic used, in the C. ulbicans strains examined, and in the origin, treatment and storage conditions of the saliva (Tenovuo. 1989). The aforementioned investigators used either unstimulated whole saliva or stimulated parotid saliva, which they clarified by centrifugation at higher sedimentation rates and for longer time periods than we used. The coating of acrylic pieces with whole stimulated saliva from several different healthy donors resulted in similar levels of adherent C. ulbicuns 613~. Hence, the salivary constituents responsible for promoting the adherence of C. ulbicuns to denture acrylic appeared to be present in the whole stimulated salivas of all the donors examined. It is unclear ahether compositional changes in saliva may affect C. ulbicuns colonization on saliva-coated prosthetic surfaces and predispose individuals to denture stomatitis. Clinical sampling of saliva from healthy individuals and patients with denture stomatitis, and longitudinal studies on individuals who are candidates for dental prostheses would address this question. .A primary oral colonizer, Strep. sunguis NCTC 10904, as well as two different secondary colonizers,

619

Strep. mutans GS-5 and Strep. sobrinus ATCC 27352.

each consistently increased the adherence of C. ulbicans strains 613~. The increase that we noted (on average, sevenfold for C. u!bicuns 613~) was considerably higher than values reported by Verran and Motteram (1987). Therefore, our results indicate that oral streptococci may facilitate colonization of dental prostheses by C. u/bicans. Sucrose rinses initiate or aggravate denture stomatitis in humans (Olsen and Birkeland, 1976) and sucrose intake in experimental animals has been associated with the prevalence of C. ulbicuns (Bowen and Cornick, 1970; Bowen, 1974). Furthermore. the presence of sucrose during in citro incubation with streptococci has been found by several groups to promote the adherence of C. ulbicuns; bindmg of the yeasts to glucans produced by the streptococci was a suggested mechanism for this observation (Miller and Kleinman, 1974; Richards and Russell. 1987; Branting, Sund and Linder, 1989). Strep. sobrinus ATCC 27352 adhered in lower numbers to acrylic when it was incubated in BSD than in PBS alone. The large cell aggregates formed during the adhesion of Strep. sobrinus ATCC 27352 to acrylic in BSD may account for the decrease in adherent streptococci (Liljemark, Bloomquist and Germaine. 1981) in this case. Although we did not determine whether the streptococcal strains examined produced extracellular polysaccharide in the presence of sucrose, the aggregation of Strep. sobrinus ATCC 27352 provides indirect evidence that insoluble extracellular polysaccharides were produced during the adhesion of the streptococci to acrylic in BSD. Nonetheless, the apparent enhancement in the binding of C. albicuns 613~ to Strep. sobrinus ATCC 27352 incubated in BSD was not striking. In general, C. albicans 613~ adhered quite vvell to the three streptococcal strains tested, even in the absence of additional sucrose. C. ulbicuns 613~ grew as individual cells and C. ulbicuns 613mlBK grew in large aggregates. Liljemark et al. (1981) reported that streptococci in small aggregates adhered to saliva-coated hydroxyapatite in larger numbers than did streptococci m large aggregates. To determine whether the difference between C. ulbicuns 613~ and C. ulbicuns 613mlBK in adherence to saliva-coated acrylic was due to the formation of large cell aggregates by C. ulbicuns 613mlBK or to potential differences in the salivabinding moieties on the cell surfaces of the two strains, cell aggregates of C. ulbicuns 613mlBK were disrupted by vortexing with glass beads. After this treatment, both strains had significantly different binding capacities to whole stimulated saliva-coated acrylic, reflecting quantitative and/or qualitative differences in cell wall composition and structure between them. C. ulbicuns 613~ may possess either a larger number of adhesins or adhesins w-ith a higher affinity for whole saliva-coated acrylic than C. ulbicuns 613mlBK. C. albicans 613~ exhibited marked variability in its ability to adhere to the various uncoated plastic substrates tested. However, coating of the various plastic substrates with whole stimulated saliva appeared to mask the differences in the adherence of C. albicans 6 I3p that were observed when uncoated

620

A. VASILAS et al

substrates were used. This result was in agreement with those of Jendresen and Glantz (1981) who demonstrated that the adhesive properties of artificial surfaces were considered modified in the oral environment. owing to the acquired pellicle that rapidly forms on all surfaces. They concluded that surfaces which were originally different were quickly brought to the same state by the adsorption of a surface film (Jendresen and Glantz, 1981). Tissue-culture flask polystyrene has been treated by the manufacturer, altering its electrostatic surface properties and optimizing cell adhesion. Thus, it is not surprising that the I.4 of C. albicans to uncoated flask polystyrene was high. Although roughening this polystyrene with sandpaper increased the surface area available for C. aibicans binding, it appeared that the grinding changed its surface properties. This was reflected in a stgnificantly decreased IA (p < 0.05) compared to uncoated, untreated flask polystyrene. The difference between uncoated polystyrene and denture acrylic in binding C. ulbicans 613~ are in agreement with observations by Klotz, Drutz and Zajic (1985). Molloplast-Be, which accumulates C. &cans rapidly in vim (Mlkila and Hopsu-Havu, 1977). exhibited a high binding affinity for C. afbicuns in rim. Our results suggest that materials used in in ritro binding assays should be similar to those materials used in oral prostheses. Moreover, the results emphasize the importance of saliva coating in altering the binding properties of the materials investigated. As all surfaces and micro-organisms in the mouth are usually coated with saliva, it is important to consider saliva as a factor in in citro binding models for oral micro-organisms (Gibbons ef al., 1985. 1986). Variation in adhesive capacity has been reported among Cundidu spp. and C. ulbicuns strains (McCourtie and Douglas, 1984; McCourtie et al., 1986: Segal, Lehrman and Dayan, 1988). The C. u&cans strains that we examined showed marked variability in their adherence to acrylic coated and uncoated by whole stimulated saliva. C. ulbicans 613~ \vas the most adhesive strain, whereas the morphological variant strain derived from strain 613~. C. ulbicnns 613mlBK, had the lowest binding capacity. C. albicuns strain 228, a recent clinical isolate that was also a morphological variant, demonstrated an intermediate level of binding. Since the initial observation by Negroni (1935), spontaneously derived. morphological variants of C. ulbicuns have been reported by several investigators (BrownThomsen. 1968; Slutsky, Buff0 and Soll, 1985). DiMenna (1952) reported that a high frequency (13%) of C. nlbicuns isolates from the mouth of patients undergoing antibiotic therapy consisted of ‘rough’ colony forms, an observation that associated variant colony forms with clinical material for the first time. C. ulbicuns morphological variants from patients with systemic (Sol1 ef al., 1988) and vaginal (Sol1 et al., 1987, 1989) infections have also been characterized. We have obtained several C. ulbicuns morphological variants upon primary isolation from the oral mucosa of patients with human immunodeficiency virus infection. One of the strains, C. ulbicans 228, was used in this study. Despite the frequent occurrence of morphological variants in

primary isolates from patients, the relationship between morphological variation and Can&la infection in the mouth is poorly understood. C. albicans morphological variants should serve as a powerful tool for investigating the mechanisms by which the fungus colonizes prosthetic and mucosal surfaces in the mouth. Given the importance of the cell wall in attachment to these surfaces. it is reasonable to expect that a variant in cell wall morphology and function may differ from a parental strain in its ability to cause infection. Data supporting this expectation have been reported for variants of C. ulbicuns in animal models of systemic (Hubbard, Markie and Poulter, 1986) and vaginal (Sobel. Muller and Buckley, 1984) infection, where the variants were of diminished virulence. C. ulbicuns strains that cannot convert to germ tubes have also attracted interest as potentially less adherent and less virulent strains (Sobel er al., 1984; Hubbard et ul., 1986). experimental conditions used, Under the C. ulbicans 613ml BK was incapable of germ-tube formation. C. ulbicuns 613~ and C. ulbicuns 613mlBK also exhibited distinctly different phenotypes and differed considerably in their ability to adhere to denture acrylic when coated or not by whole stimulated saliva. Using a hyposalivatory rat model of oral candidosis (Mertner et a(., 1990). we have earlier shown that strain 613~ was transmitted significantly more rapidly (p < 0.05) from an infected donor animal to an uninfected recipient animal than morphological variant strain 613mlBK. The diminished capacity of C. ulbicum 613mlBK to colonize a recipient animal in this model may be a direct reflection of the diminished adhesive capacity observed in vitro. Adherence to salivary macromolecules is an important factor in the ability of C. ulbicuns to colonize prosthetic devices in the mouth. Further investigations at the biochemical level will both clarify the adhesin-receptor interactions involved in the adherence in r?tro of C. ulbicuns to acrylic coated with saliva and oral streptococci, and help to elucidate the mechanisms involved in C. ulbicuns colonization of dental prostheses in the mouth. Ackno~c,/ecigentents-This work was supported by USPHS Grants S7RROj303-28, DE07189 and DE07003, and by a gift from the Block Drug Co. The authors thank Dr Robert A. Burne for critical reading of the manuscript.

REFERENCES Ambjornsen E. (1985) An analytical epidemiological study of denture stomatitis in a group of Norwegian old-age prisoners. Gerodontics 1, 207-212. Arendorf T. M. and Walker D. M. (1987) Denture stomatitis: a review. J. oral Rehab. 14, 217-227. Bennick A. and Cannon M. (1978) Quantitative study of the interaction of salivary acidic proline-rich proteins with hydroxyapatite. Caries kes. 12,m159-169. Block P. L. and Brottman S. (1962) A method of submaxillary saliva collection without cannulization. N.Y. SI. dent. J. 28, 116-118. Bowen W. H. (1974), Effect of restrictine oral intake to invert sugar or casein on the microbiology of plaque in Macaca juscicttlaris (irus). Archs oral Biol. 19, 231-239.

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Bowen W. H. and Cornick D. E. (1970) The microbiology of gingival-dental plaque; recent findings from primate research. Dent. J. 20, 382-395. Branting C., Sund M.-L. and Linder L. E. (1989) The influence of Streptococcus mu~uns on adhesion of Candida albicans to acrylic surfaces in cirro. Archs oral BioL 34, 347-353.

Bratthall D. (1970) Demonstration of five serological groups of streptococcal strains resembling Srreprococcus muruns. Odont.

Rmy

21, 143-152.

Brown-Thomsen J. (1968) Variability in Candida aibicons (Robin) Berkout. 1. Studies on morphology and biochemical activitv. Hereditas 60. 355-398. Budtz-Jorgensen E.