Sucrose metabolism in situ by dental plaque in appliance-borne bovine enamel tooth fissure inserts in man

ArchsoralBid.Vol. 29,No. 6,pp.467-471,1984

0003-9969/84 $3.00+0.00

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SUCROSE METABOLISM IN SITU BY DENTAL PLAQUE IN APPLIANCE-BORNE BOVINE ENAMEL TOOTH FISSURE INSERTS IN MAN G. E. MINAH and N. CHU Baltimore College of Dental Surgery, Dental School, University of Maryland at Baltimore, 666 W. Baltimore Street, Baltimore, MD 21201, U.S.A. metabolism of dental plaque which accumulated in fissure-like spaces between small bovine enamel cubes on removable U-shaped appliances was analysed using radiolabelled techniques. The appliances were worn by 8 dental students for 21 days. Half of the fissural inserts (3) on one wing of the appliance were exposed to a 10 per cent sucrose solution in normal saline and half (3) to normal saline as a control on the opposite wing, 8 x /day. At 21 days [“‘Cl-sucrose (U) was applied directly to sucrose and saline-exposed inserts extra-orally for 60 min at 37°C (non-dispersed plaques, NDP). Adjacent inserts on both sides of the appliances were removed. The plaque was dispersed in buffer by sonication, cultured and was exposed to radiolabelled sucrose for the same incubation period (dispersed plaques, DP). The following observations were made: (1) quantitative values for sucrose consumption and lactic acid, cell-bound and insoluble product, soluble polysaccharide, carbon dioxide and total volatile acid formation did not differ between sucrose and saline exposed DP or sucrose- and saline-exposed NDP: (21 all DP consumed more sucrose and formed higher quantitative levels of all products than NDP counterparts, p -K0.001; (3) sucrose-exposed DP and sucrose exposed NDP formed proportionally more lactic acid than saline-exposed counterparts, p < 0.05; (4) sucrose-exposed NDP formed proportionally more lactic acid and less soluble polysaccharide than sucrose exposed DP, p < 0.025; (5) concentrations of lactobacilli were higher in sucrose exposed plaques, p < 0.049.Summary-Sucrose

INTRODUCTION

Dental caries is most frequent beneath dental plaque in occlusal pits and fissures or buccal and lingual pits. Although the microbial composition of fissural plaque has been analysed by using bovine enamel inserts worn in removable appliances (Minah, Lovekin and Chen, 1981a), Mylar fissures worn in molar restorations (Svanberg and Loesche, 1977, 1978) and segments of autogenous third molars worn in occlusal restorations (Theilade et al., 1978, 1982), little is known of the physiology of plaque in fissures. There are indications that cells absorbed on surfaces or on adjacent cells, such as would occur in fissures, express different glycolytic or growth activity from when dispersed in fluid (Zobell, 1943; Tschapek and Giambiage, 1956; Berry and Henry, 1977). Our aim was to examine sucrose metabolism of sucrose-exposed dental plaques which developed in fissure-like spaces created between two cubes of bovine enamel. MATERIALS AND METHODS Fissural model system

The fissural inserts (Minah et al., 1981a) are modified versions of devices designed by Koulourides (Ostrom and Koulourides, 1976). A U-shaped plate of acrylic resin was made to fit the palate of each subject; 3 fissural inserts were placed in a row on each side close to the midline. The fissure-like spaces in the inserts were created by cementing 2 enamel cubes (each 3.0 mm3) separated by a Mylar strip 0.2 mm thick with cyanoacrylic cement (Elmer’s Wonder Bond) to the bases of depressions cut in the acrylic resin. Base plate wax was applied to fill void around the enamel insert. When the Mylar was removed, fissure-like space was left in the cubes. Bovine rather 467

than human enamel was used in order to avoid variations in mineral composition. Bovine enamel has properties similar to human (Feagin, Koulourides and Pigman, 1969). Experimental design

Eight student volunteers at the University of Maryland Dental School wore the appliances for 3 weeks. Appliances were removed only for exposure of the experimental plaques to substrates 8 times/day for 20min each. During substrate exposure, half of the enamel inserts were immersed in a 10.0 per cent (arbitrarily selected) solution of peppermintflavoured candy (Lifesaver Inc.) in normal saline; candies consisted of compacted sucrose with peppermint flavouring. The other half were simultaneously treated with normal saline. The choice of peppermint Lifesavers was dictated by a previous cultural study of natural plaque in which the same volunteers consumed a package (12 candies) of this product daily for 3 weeks. At the end of the third week, appliances were divided in half with Carborundum disks. A sucroseand a saline-exposed insert on each portion were isolated with small rim of wax. A drop (50~1) of uniformly-labelled [‘4C]-sucrose (New England Nuclear, 4.OpCi) made 2.0 per cent wt/vol with unlabelled sucrose was applied to each fissural insert. These non-dispersed plaques (NDP) were incubated at 37°C for 60 min. After the incubation, the radioactive substrates were removed with 20 ~1 capillary pipettes and introduced into 200 ~1 of reduced transport fluid (RTF; Loesche, Hackett and Syed, 1972) in a l-dram screw-capped vial in crushed ice. Fissural inserts were then washed with 50~1 of RTF which was placed into the same container. Inserts and

468

G. E.

MINAH

plaque were then placed in vials where the plaque was dispersed by sonication for 10s (Kontes Cell Disruptor). Products of sucrose metabolism were isolated and quantitated (see Metabolic Procedures below). During the same period, a sucrose- and a salineexposed fissural insert adjacent to those described above were removed from the appliances and placed in 1.Oml of RTF in l-dram vials. After sonication for 10 s to disperse the plaques (dispersed plaques DP), 100 p 1from each was introduced into another l-dram vial containing 200 ~1 of [‘4C]-sucrose (8.0 pCi). Enough unlabelled sucrose was added to make the final concentration 2.0 per cent wt/vol. After incubation for 60min at 37°C the vials were placed in crushed ice. Metabolic products were isolated and quantitated (see Metabolic Procedures below) and compared to those of NDP. The contents of the original vial were cultured (see below). Metabolic procedures

The metabolic procedures were similar to those reported earlier (Minah, Matheus and Finney, 198 1b; Minah, Lovekin and Finney, 1981~). Values for sucrose catabolism and formation of lactic acid, total volatile products, carbon dioxide (CO,), total cellbound and insoluble products and soluble polysaccharide were expressed both quantitatively as pmoles sucrose equivalents/6.0 x 10’ bacteria in the sample/60 min of incubation and proportionally as percentage of sucrose consumed. Microbiological procedures

The DP specimens in the original collection vials were serially diluted in RTF. Samples of appropriate dilutions were plated on the following non-selective and selective media. (1) MM10 sucrose blood agar for anaerobic incubation (MMlO) anaerobic; Loesche et al., 1972). Counts of colony-forming units on this non-selective media served as a general reference for the number of bacteria present in the specimen. Quantities of specific bacteria or groups of bacteria (i.e. Gramstain types) on this medium or on each of the selective media were expressed as a proportion of the total colony count on the MM10 anaerobic plates. Distinctive colony morphology types of Streptococcus mutans, Streptococcus salivarius, Streptococcus sanguis, black-pigmented Bacteroides, Fusobacterium nucleatum, Veillonella species and Actinomyces odontolyticus were detected on this

medium. Hydrogen peroxide (3.0 per cent) was applied to each colony with a 10~1 pipette to determine catalase activity. Actinomyces viscosus and one Veillonella species can be presumptively identified by this method. All unidentified colonies were Gramstained. Incubation time-7 days at 37°C in the anerobic chamber. (2) MM 10 sucrose blood agar for incubation in 10 per cent CO, in air (MM10 CO*). The same procedures conducted with MM 10 anaerobic plates were followed using this non-selective medium. The total colony counts divided by the total colony counts of MM10 anaerobic plates represented the CO,/ anaerobic ratio, an approximate indication of the concentrations of obligate anaerobes and facultative

and N.

CHU

anaerobes plus aerobes in the specimen. Incubation time-7 days at 37°C in 10 per cent COz and air (Bellco Glass Co. CO, Incubator) (3) Mitis-salivarius bacitracin sucrose agar (Gold, Jordan and Van Houte, 1973) for Strep. mutans. Incubation time-3 days at 37°C anaerobically. (4) Actinomyces agar (Zybler and Jordan, 1982). Hydrogen peroxide was added to each colony to aid in the presumptive identification of A. viscosus (cataIase positive). Incubation time-3 days at 37°C anaerobically. (5) Neisseria agar (Ritz, 1967). Catalase and oxidase activities of each colony were determined (p-aminodimethylanilline from a moistened Taxo N disc, BBL, containing this reagent were applied to each colony. A darkened colony indicated a positive oxidase reaction). Incubation time-3 days a 37°C aerobically. (6) Rogosa SL agar (Difco) for lactobacilli. Colonies of Lactobacillus casei subspecies are distinctive on this medium. Incubation time-3 days at 37°C in 10 per cent CO*. (7) Veillonella Agar (Difco). Incubation time-3 days at 37°C anaerobically. (8) MM10 sucrose blood agar containing 50 pg/ml kanamycin sulphate (S.A. Syed, personal communication) for Gram-negative anaerobic rods. Incubation time-7 days at 37°C anaerobically. (9) Crystal violet erythromycin agar (Walker et al., 1979) for Fusobacterium species. Incubation time4 days at 37°C anaerobically. (10) Sabouraud dextrose agar (BBL) for yeasts. Incubation time-3 days at 37°C in 10 per cent CO*. (11) Mannitol salt agar (BBL) for staphylococci. Staphylococcus aureus colonies were differentiated from Staphylococcus epidermidis colonies. Incubation time-3 days at 37°C in 10 per cent CO,. (12) Pseudocel agar (BBL) for Pseudomonas aeruginosa. Incubation time-3 days at 37°C in 10 per cent CO*. (13) Desoxycholate agar (Difco) for members of the family Enterobacteriacaeae. Incubation time-3 days at 37°C in 10 per cent COz. Representative colony-types from MM 10 anaerobic and MM10 CO* appearing in concentrations greater than 5 per cent of the total recoverable flora which could not be accounted for by colonial identification, were subcultured on anaerobic bloodagar plates containing 0.1 per cent glucose as the sole carbohydrate source. These were incubated from one to three days, either anaerobically, or in 10 per cent CO1 in air. Single colonies were selected and identified using either the Minitek Anaerobic II (BBL), or the API 20A (Analytab Products, Ayerst Laboratories) minaturized identification systems. Each colony type appearing on selective media were Gram-stained for verification purposes. Colony types, appearing in greater than 5 per cent of the isolates which could not be identified by colony morphology were subcultured for speciation by the rapid identification systems. Normalization of the data

All metabolic data were normalized on the basis of (1) radioactivity levels in each reaction mixture, (2) the number of bacteria recovered from DP specimens

Sucrose metabolism in experimental dental plaque

469

Table 1. Sucrose consumption and metabolic product formation by dispersed and non-dispersed experimental dental plaques (quantitative values) pmol of [“‘Cl-sucrose equivalents/6 x 10’ c.f.u.*/60 min incubation Cell-bound Plaque type/ substrate DPt/sucrose NDP 11 /sucrose DP/saline NDP/saline

172.9: (+ 35.4)9 28.1 (k 13.3) 211.6 (k92.4) 31.6 (k23.7)

*c f.u.-Colonv-formine

Volatile

Lactic acid

and insoluble products

Soluble polysaccharide

63.6 (k 17.4) 12.2(*4.37) 66.6 (k 66.6) 8.51 (k5.48)

4.14(+2.51) 0.74 (* 0.74) 6.66(*9.62) OkI(kO.52)

51.8 (+ 17.0) 5.92(+3.7) 74.0 (k 74.0) 4.74(&4.44)

Sucrose utilized

products except CO, 50.0 (* 17.0) 9.18(&5.62) 62.9 (251.0) 17.4 (k20.2)

CO, 3.4 (k4.4) 0.30(+0.27) 1.48(+2.22) 0.52(*0.37)

units on MM10 sucrose blood agar incubated anaerobically 7 days at 37°C. tDP-dispersed

plaques. &lean valie. §Standard deviation. IINDP-n&-dispersed sucrose blood agar plates (Loesche et al., 1972) incubated for 7 days in a Coy anaerobic chamber (Aranki et al., 1969) and (3) the dilution factor based upon the volume of the sample employed in the procedure relative to the total volume of the specimen. The number of bacteria in NDP that were not cultured were estimated to be similar to the adjacent dispersed plaques from fissural inserts of the same dimensions. Mean cultural values of dispersed plaques were normally distributed and varied by approx. 10 per cent. Counts of the specimens were corrected for quenching and technique efficiency (Minah and Loesche, 1976, 1977). on MM10

Statistical analysis

Significance of differences between sucrose- and saline-exposed plaques and DP and NDP were tested using a paired-t-test statistical program in a Texas Instruments SR-60 Programmable Computer. With quantitative and proportional values for each metabolic activity, the following plaques were compared: (1) sucrose-exposed DP versus NDP, (2) salineexposed DP versus NDP, (3) sucrose versus salineexposed DP, (4) sucrose versus saline-exposed NDP. Proportions of selected microbial isolates in sucrose and saline-exposed DP were compared using the paired t-test. RESULTS

Quantitative values for metabolic measurements of DP and NDP which were exposed to sucrose or saline

fcr 3 weeks are presented in Table 1. Sucrose consL-mption and quantities of all metabolic by-products wzre significantly greater in both sucrose and salineexposed DP (p < 0.001, paired t-test). Saline-exposed DP consumed higher mean quantities of sucrose and fcrmed more lactic acid, cell-bound and insoluble

plaques.

products, volatile products, CO2 and soluble polysaccharides than sucrose-exposed counterparts. Sucrose-exposed NDP formed more lactic acid, cellbound and insoluble products and soluble polysaccharide than saline-exposed counterparts. Higher volatile acids and CO2 production in saline-exposed NDP accounted for their higher rate of sucrose consumption. Differences between sucrose versus saline-exposed DP and sucrose versus saline-exposed NDP were not significant. The proportional values of all by-products based on levels of sucrose consumed are presented in Table 2, with statistical analysis in Table 3. Proportions of lactic acid, soluble polysaccharides and CO? differed significantly between various experimental plaques. DP and NDP sucrose-exposed plaques contained significantly more lactic acid than saline-exposed counter parts. This represented the only significant difference attributable to substrate exposure. Significant proportional differences were found when NDP and DP were compared. Sucrose-exposed NDP formed more lactic acid than DP whereas DP formed more soluble polysaccharides. Saline-exposed NDP also formed higher proportions of lactic acid than DP, but the difference was not significant. Salineexposed NDP formed significantly higher levels of soluble polysaccharides and CO, than saline-exposed DP. The only microbial category to differ significantly in plaques exposed frequently to either sucrose or saline for 3 weeks was lactobacilli concentrations (Table 4). Lactobacilli comprised only 0.8 ( f 1.6) per cent of the total recoverable flora on selective media and 0.5 (f 1.6) on non-selective media in salineexposed plaques, but ascended to 6.9 (&- 11.0) per cent (selective media) and 16.3 (+ 19.6) per cent (non-selective media) in sucrose-exposed plaques. L. casei subspecies casei, and L. casei subspecies

Table 2. Metabolic product formation from sucrose by dispersed and non-dispersed experimental dental plaques (proportional values) Percentage of 1’4C1-sucroseconsumed/6 x 10’ c.f.u.*/60 min incubation Plaque type/ substrate DPt/sucrose NDP]l/sucrose DP/saline NDP/saline

Lactic acid

Cell-bound and insolubule products

36.02 ( f 5.96)§ 45.0 (+ 6.5) 28.4(*8.4) 33.8 (+ 14.2)

2.5 (+ 1.9) 2.2(*1.7) 3.2(+3.2) 1.5(f0.7)

Soluble polysaccharide

Volatile products (except CO,)

30.0(+8.7) 21.4(+9.3) 32.6 (k 14.6) 20.8 (+ 13.4)

28.4 (k4.79) 30.0(fll.l) 35.O(k 16.1) 43.0(+20.7)

CO* 2.0 (k2.89) 1.0 (kO.66) 0.67 (kO.29) 1.7 (kO.9)

*c.f.u.-Colony-forming units on MM10 sucrose blood agar incubated 7 days at 37°C. tDP-dispersed plaques. fMean value. §Standard deviation. JINDP-non-dispersed plaques.

G. E. MINAH and N. CHU

410

Table 3. Statistical evaluation of proportional differences in product formation by sucrose and salineexposed dispersed (DP) and non-dispersed (NDP) experimental plaques Level of significance* Plaque type/ substrate DP versus NDP (sucrose) DP versus NDP (saline) Sucrose versus saline (DP) Sucrose versus saline (NDP) *Paired-t-test.

tNS-not

Lactic acid

Cell-bound and insoluble products

Soluble polysaccharide

Volatile products (except CO,)

CO*

0.025

NW

0.025

NS

NS

NS

NS

0.05

NS

0.025

0.005

NS

NS

NS

NS

0.05

NS

NS

NS

NS

significant.

rhamnosus were the only Lactobacillus species identified in the plaques. Strep. mutans did not reach prominence in either the sucrose or saline-exposed plaques although its mean level was higher in sucroseexposed 3.1 ( f 4.7) per cent, versus saline-exposed plaques, 0.8 (+ 2.6) per cent (non-selective media). This difference was not statistically significant. DISCUSSION

The ecological effect of the sucrose solution on the microbial composition of the experimental dental plaques was seen in the sharp increase in lactobacilli concentrations. Although Strep. mutans appeared to shift to higher levels in sucrose versus saline-exposed plaques, the shift was not significant. Similar recovery of these two types of microorganisms has been reported in a study of plaque from human-carious fissures by Loesche and Staffon (1979). However, the more common findings in their study were low levels or the absence of lactobacilli and high levels of Strep. mutans. As previous studies in this laboratory with experimental dental plaques on removable palatal appliances (Minah et al., 198lb, 1981~) revealed a microbial pattern similar to that observed in the

present study, such a pattern might be unique to this type of experimental system. The metabolic behaviour of the plaques was also similar to that observed in previous appliance studies. The quantitative values for the metabolic categories did not differ significantly in sucrose and salineexposed plaques; but sucrose-exposed plaques transformed significantly more of the catobilized sucrose into lactic acid versus control plaques. These findings, evident in both DP and NDP, might be a characteristic of lactobacilli-dominated versus deficient plaques. The most explanation for the obvious substantially-increased sucrose consumption by DP versus NDP is greater accessibility to the substrate by DP. However, proportional formation of various products were quite different in the two types of plaques, suggesting that other explanations be pursued. Evidence from partially-related experimentation indicates that surface adsorption by bacteria as would exist in NDP, affects their metabolism. Enteric bacilli showed more active metabolism when adsorbed to glass surfaces than when free in water (Hendricks, 1974), and strains of Strep. mutans and Strep. sanguis produced greater quantities of lactic

Table 4. Microbial composition of plaque in fissural inserts after frequent exposure for 21 days to either sucrose or saline Bacterial categories Selective media Strep. mutans

Lactobacilli Veillonella Actinomyces sp. Actino. (cat. + ) Bacteroides sp. Neisseria sp.

Yeasts

Sucroseexposed

Salineexposed

0.7*(*1.5)t 0.05(+0.1) 6.9(*11.0) 0.8(+1.6) 26.9 (+ 23.7) 33.7 (k 27.3) 3.6(+3.7) 8.O(k 10.0) 1.7(+2.2) 2.4 (k 5.3) 6.1 (k9.2) 4.9 (+7.2) 2.4(+4.7) 2.2(&2.2) 0.0 0.05 (+ 0.2)

Significance (paired-t-test)

NSS

p < 0.049

NS NS NS NS NS NS

Non -selective media

Gram Gram Gram Gram Gram

+ + + -

cocci cocci rods (Actino) rods (lacto.) rods

Strep. mutans Strep. sanguis

19.1 (+ 15.0) 29.9(* 13.9) 35.0(*16.3) 16.3(+19.6) 7.7(*7.5) 3.1 (k4.7) 2.1 (If. 2.7)

15.5 (k 18.9) 32.5 (k28.4) 39.2(+31.9) O.S(kl.6) 11.4(_f24.1) 0.8 (k2.6) 1.4(&4.1)

NS NS NS p < 0.015

NS NS NS

*Mean of colony-forming units/total viable count on MMlO, anaerobic incubation. tstandard deviation. $NS-not significant.

Sucrose metabolism in exrrerimental dental plaque acid when adsorbed on hydroxyapatite to than when in fluid culture (Berry and Henry, 1977). Convincing explanations for cohesion or adhesion influences on metabolism have not been established. One hypothesis is increased substrate availability to the adsorbed following nutrient concentration by the solid surface. Another hypothesis is decreased local acidity from buffering action by the adsorbent. Berry and Henry (1977) however, observed no influence on metabolic activity when adsorbed cells were placed in buffered or unbuffered media. In the present study, where the adsorbed bacteria (NDP) were not permitted equal access to the substrate as the non-adsorbed bacteria (DP), fair determination of quantitative metabolic differences between the two types of specimens would be difficult. The preferential lactic-acid formation by NDP, however, may have been influenced by the environmental pH which, due to stagnation of acidic by-products, would theoretically reach a lower level in NDP. We have observed an increase in lactic-acid formation in experimental plaques (dispersed) comprised of high levels of lactobacilli when the pH of the reaction mixture was reduced from 7.0 to 5.0 (Minah ei al., 1981b). Another characteristic of NPD, that of reduced soluble extracellular polysaccharide production, might have been the result of limited access of the glycosyl and fructosyl transferases to sucrose. Although these data do not elucidate various mechanisms involved in the metabolic activities observed, they do indicate that unique metabolic characteristics of retention site plaque might exist and thereby should be considered in investigations of plaque formation. acknowledgement-This project was supported by National Institute of Dental Research Grant No. DE-04795.

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