Contribution of plaque polysaccharides to growth of cariogenic microorganisms

Archs oral Bid

Vol. 16, pp. 855-862.

1971. Pergamon Press. Printed inGreatBritain.

CONTRIBUTION OF PLAQUE POLYSACCHARIDES GROWTH OF CARIOGENIC MICROORGANISMS

TO

R. B. PARKERand H. R. CREAMER Department of Microbiology, University of Oregon Dental School, Portland, Oregon 97201, U.S.A. Summary-The possible role of microbial polysaccharides (levans, dextrans, and amylopectin) in the nutrition of pure cultures of oral bacteria was investigated. All polysaccharides tested were utilized to some degree by one or more of the individual oral species employed in the study. Certain polysaccharides, namely a levan produced by a non-cariogenic steptococcus and amylopectin from Neisseriu, were utilized more readily by streptococci and a lactobacillus than was sucrose. Data also suggest that the synthesis of polysaccharides by non-cariogenic and even non-acid producing oral bacteria may serve as substrates for other species, and may play a role in the development and maintenance of dental plaque and in the production of dental decay.

THE ROLE of

a gelatinous substance (possibly polysaccharide) in development of the dental plaque was suggested as early as 1890 by MILLER. SNYDERet al. (1955) demonstrated synthesis of levan by plaque microorganisms. Two genera were considered responsible for synthesis of most oral polysaccharides, namely Streptococcus and Neisseria. This synthesis was presumed to occur from sucrose but not monosaccharides or invert sugars through non-phosphorylative mechanisms as outlined by HEHRE(1946). The significance of reserve polysaccharides to the endogenous metabolism of streptococci has been documented by many, including GIBBONS and SOCRANSKY (1962). SNYDER et al. (1955) showed that levan synthesis occurred rapidly following application of a source of sucrose to oral streptococci and Neisseria. They suggested that “localized synthesis occurs naturally following ingestion of sucrose”. MANLY(1961) suggested that microorganisms of plaque could store and perhaps utilize carbohydrates from sucrose solution in some non-diffusable form such as polysaccharide, mucoprotein or phosphate esters. MCDOUGALL(1964) found levan synthesis within the plaque and WOOD(1964) reported that dental plaque suspensions were able to utilize levans from Streptococcus salivarius. PARKERet al. (unpublished) found that amylopectin was utilized whereas dextran did not serve as a carbohydrate source for growth of plaque streptococci. Other bacterial polysaccharides (levans) were intermediate with respect to dextran and amylopectin. GIBBONSand BANGHART(1967) and DE COSTA and GIBBONS (1968) and VANHOUTE and JANSEN(1968) have investigated synthesis and degradation of dextrans and levans by the mixed oral bacteria of human dental plaque and saliva. More recently CREAMER,SANDERSand PARKER(1969) and LEACHet al. (1969) found that levan fractions of the plaque serve as carbohydrate sources for growth of streptococci and thus may contribute to the caries process. 855

856

R. B. PARKERAND

H. R. CREAMER

It was the attempt of this investigation to further detail the role of various bacterial species in their contribution to the overall ecology of the plaque, specifically with regard to interconversion of carbohydrate sources. MATERIAL

AND

METHODS

(1) Bacterial species Streptococcal strains included in the study were strain Ri a levan-producing non-cariogenic strain resembling Strep. salivarius, strain E 49 a dextran-producing cariogenic strain resembling Strep. mutans, strain SL 1, a dextran-producing cariogenic streptococcus and strain FA I a cariogenic streptococcus producing dextran and resembling Strep. mutans. In addition to the above streptococci, we employed Lactobacillus casei variety rhamnosus (UODS), Actinomyces (formerly Odontomyces) viscosus strain T6 ATCC 15987, Corynebacterium Javidum, Bacterionema matruchotii ATCC 14266 and Neisseria perflava UODS NI. (2) Medium The basal medium for use in all growth studies had the following composition: Trypticase (BBL) 2-O per cent, K2HP04 0.4 per cent, KH2P04 0.2 per cent, NaCl O-2 per cent, 0.001 M MgSO, and O*OOOl M MnSO,. Preliminary studies had indicated that streptococci and most other species would grow in the medium employed providing a metabolizable carbohydrate such as sucrose or glucose was added. We therefore supplemented this basal medium with 0.5 per cent sucrose as a readily available carbohydrate and compared growth obtained on this carbon source with populations obtained from addition of O-5 per cent bacterial polysaccharides. Inocula were washed-cell preparations obtained from the above medium containing O-5 per cent sucrose. (3) Bacterial counts Streptococci, the LactobaciIlus and Act. viscosus were grown under 95 per cent N2 and 5 per cent COZ tension on trypticase soy agar, while Nei. perjlava, Bact. matruchotii and Cory. flavidum were grown on the same medium aerobically. (4) Preparation of the polysaccharide Cultures were grown in the above basal medium containing 10 per cent sucrose. Ten millilitres of an 18-hr culture was inoculated in 2 litres of the medium in a 2500 ml flask. For maximum polysaccharide yield, the inoculated Basks were incubated at 37°C. for 24 hr and then held an additional day at 21°C. Bacterial cells and particulate matter were then removed by centrifugation and the polysaccharide precipitated from the medium by adding ethanol to 70percent v/v concentration. After precipitation, the crude polysaccharide was again resuspended in water and purified with chloroform extraction according to HEHREet aI. (1946). A grey cloudy emulsion tended to form between the chloroform layer and the aqueous layer when shaken in a separatory funnel. When this layer was distinctly formed (usually less than 2 hr), the aqueous layer was withdrawn and chloroform and emulsion layers discarded. After an additional two or three such precipitations in alcohol and m-extractions with chloroform, the above emulsion layer was not present and theremaininglayem were clear. At this point, the polysaccharide present in the aqueous phase was biuret-negative and it was lyophiiized for future use. Paper chromatography was used to identify the individual sugar moieties of each of these fractions and to ensure purity.

RESULTS

and Nei. pet-Java were not influenced by the addition of any of the carbohydrates tested, including sucrose. Therefore no data are presented for these species. Growth responses of the six other organisms on each carbohydrate source are shown in Figs. l-6. Each figure depicts the growth response of one organism on sucrose and on extracellular polysaccharides isolated from Nei. perflaua, and from streptococcal strains Ri, E 49, SL-I and FA 1. Population

levels

of Cory. flavidum

1o,

!

FIG.

Time (hours)

STREPTOCCUS

1. Growth of SL 1.

Ri Basal SL-I E49 FAI

* w

per

sucrose

N.

OF CARlOGENlC

-

GROWTH

SL-I I

IO’ r

FIG.

2. Growth

of E 49.

STREPTOCCUS

Time (hours)

t 12

I 8

I

Sucrose Ri Basal SL-I

4

N. per

u -

OF CARIOGENIC

-

GROWTH

E49

I 24

R. B. PARKERAND H. R. CREAMER

858

“0 _

UOJJDlndOd

2 Uq,JD//ndOd

I

Time

12

(hours)

J 24

IO’

FIG. 6. Growth

I

8

I

24

of Bacterionema matruchotii.

Time

1

I

1hours)

N. par Sucrose Ri Basal SL-I E49

I2

H b-d -

-

OF B. MATRUCHOTII

4

GROWTH

FIGS. 5 and 6. Growth of Actinmtyces viscosus (Fig. 5) and Bacterionema matruchotii (Fig. 6) on polysaccharides isolated from selected oral microorganisms. Each growth curve line represents populations obtained on a single polysaccharide.

FIG. 5. Growth of Actinomyces viscosus.

I

8

I

SL-I E49

&s0l

N. per Sucrose Ri

OF A. VISCOSUS

4

D--Q

-

-

-

-

GROWTH

860

R. B. PARKER AND H. R. CREAMER

Figure 1 presents the results of comparative growth of human cariogenic strain SL-I on polymers isolated from five other bacterial species. It may be noted that this organism grew on its own polymer to an average of about two replications within four hours, and then continued to develop at about the same rate observed in the basal medium. It is possible that some minor stimulation was provided by contaminating substances in the polysaccharide which carried through the isolation procedure. However, reference to growth of other species on this material suggested that such is not the case as polysaccharide from SL-1 did not show this pattern elsewhere. Most notably, growth of SL-1 on neisserial polysaccharide exceeded growth obtained on sucrose. Growth of this cariogenic species was, thus, some 50-fold greater on polysaccharide isolated from Neisseriu than on polysaccharides isolated from streptococci. Data obtained with cariogenic streptococcus E-49 on isolated polysaccharides are recorded in Fig. 2. As was observed with dextran-forming Strep. SL-1, strain E-49 grows more rapidly on polysaccharide isolated from Neisseria than on sucrose. The other polymer metabolized rapidly was the levan formed by Strep. strain Ri. By contrast, dextrans were not metabolized at any appreciable rate. Figure 3 presents the results of growth of a non-cariogenic Strep. strain Ri on the assorted polymers. It may be seen that the streptococcal levan and the dextrans from the cariogenic strains were metabolized to some extent. Again, we may observe that amylopectin was metabolized at a high rate, although in this instance less rapidly than sucrose. The pattern of growth of Lat. casei on isolated polysaccharides may be noted in Fig. 4. In this instance, levan isolated from Strep. strain Ri permitted markedly increased growth over that of any other carbohydrate source including sucrose. Growth of Act. viscosus strain on polysaccharide may be seen on Fig. 5. In general, populations of this organism on carbohydrates other than sucrose were minimal. In two trials with this organism, we observed an apparent inhibition or death of strain T-6 growing on an amylopectin isolated from Neisseria. Thus, at 8 and 12 hr there is apparent death of about half the cells present. This phenomenon was observed in both trials, and after 12 hr the negative phase was reversed with fairly active growth of Act. viscosus occurring between 12 and 24 hr. It is not normal for this organism to die out over such a period of time as may be noted in figures showing slight but consistent growth of Act. viscosus in the basal medium. Comparative growth of Bact. matruchotii is given in Fig. 6. The only significant utilization of a carbohydrate source appeared in growth on neisserial polysaccharide. Other growth rates approximated that obtained on the basal medium. DISCUSSION

The data are in agreement with earlier reports stating that levans produced by streptococci are metabolized more readily than the dextrans of this genus. In certain instances, such as those noted with Lat. casei, the levan from streptococcus Ri is metabolized much more rapidly than even sucrose. The outstanding finding of this study was that polysaccharide from Neisseria served as a remarkable source of carbohydrate for every organism tested with the

PLAQUE

POLYSACCHARIDES

TO GROWTH

OF CARIOGENIC

MICROORGANISMS

861

possible exception of Act. viscosus. Such observations suggest the possible contribution of starch to the cariogenic process. LEACH et al. (1969) have questioned the reason for observations that starch is cariogenic in animal studies. We have observed that cultures of Nei. perfava grown on starch produce an extracellular polysaccharide intermediate in iodophilic reactions between glycogen and starch. The fact that this material is (a) extracellular, (b) produced on substrates other than sucrose and (c) is highly metabolizable by cariogenic organisms, suggests that bacterial interactions of carbohydrate synthesis and utilization may be involved in caries. There is, thus, evidence for a sequence of events as follows: Starch -+ metabolized by Neisseria + neisserial polysaccharide +- plaque matrix -+ glycolysis by cariogenic streptococci --f decay processes. RITZ (1969) has observed the formation of matrix material around numerous plaque Neisseria outlined by fluorescent antibody techniques. Further, the fact that both levans and amylopectins are utilized-in some instances rapidly-by cariogenic streptococci, suggest that the more metabolizable fractions of the plaque may indeed be critical to the cariogenic process. They may thus serve as a continuous source of available carbohydrate for cariogenic streptococci. These results also suggest that synthesis of plaque components by non-cariogenic, even non-acidogenic microorganisms, may result in bacterial interactions contributing to the caries process. Acknowledgements-The expert technical assistance of Mrs. SHELBYWEXOTT was critical to the success of this project and is gratefully acknowledged. This work was supported in part by Public Health Service Grant DE01784.

R&runt&Le role eventuel des polysaccharides microbiens (levanes, dextranes et amylopectine) sur le metabolisme de cultures pures de batteries buccales est btudie. TOW les polysaccharidcs testes sont utilisb par l’une ou plusieurs des esp&ces buccales test&s. Certains polysaccharides, en particulier un levane produit par un streptocoque non-cariog&ne et l’amylopectine de Neisseria, sont utili&s plus facilement par des strcptocoques et un lactobacille que le sac&arose. Ces observations montrent que la synth&se de polysaccharides par des bact6ries buccalcs non-cario&res, et meme ne produisant pas d’acide, pcut servir de substrats par d’autrcs esp&ces et joue un r81e dans le developpement et la survie dune plaque dentaire et la production de caries. Zusammenfassung--Es wurde die mogliche Rolle bakterieller Polysaccharide &Wane, Dextrane und Amylopektin) als Nahnmgssubstanxen ftir Reinkulturen von Mundbakterien untersucht. Alle untersuchten Polysaccharide wurden in gewissem AusmaD von einem oder mehreren der in dieser Untersuchung verwandten Mundmikroorganismen abgebaut. Restimmte Polysaccharide, namentlich ein von einem nichtkariogenen Streptokokkus gebildetes L&van und Amylopektin von Neisseria, wurden von Streptokokken und einem Lactobacillus leichter abgebaut als Rohrxuckcr. Die Ergebnisse legen such nahe dal3 die von nichtkariogenen und selbst von nicht-s&rreproduxierenden Mundhiihlenbakterien synthetisierten Polysaccharide als Substrate ftir andere Arten dienen k&men, und da0 6ie eine Rolle bei der Entwicklung wie such behn Restehenbleiben der Zahnplaque und bei der Zahnkaries spielen k&men.

862

R. B. PARKERAND H. R. CREAMER REFERENCES

CREAMER, H.

R., SANDERS,S., and PARKER,R. B. 1969. The utilization of extracellular levans. Internat. Ass. for Dent. Res. Preprinted abstracts, 47th General Meeting, Abstract 281. DE COSTA, T. and GIBBONS, R. S. 1968. Hydrolysis of levan by human plaque streptococci. Archs oral Biol. 13, 609-617. GIBBONS, R. J. and BANGHART,S. B. 1967. Synthesis of extracellular dextran by cariogenic bacteria and its presence in human dental plaque. Archs oral Biol. 12, 1l-24. GIBBONS, R. J. and SOCRANSKY,S. S. 1962. Intracellular polysaccharide storage by organisms in dental plaque. Its relation to dental caries and microbial ecology of the oral cavity. Archs oral Biol. 7, 73-80. HEHRE, E. J. 1946. Studies on the enzymatic synthesis of dextran from sucrose. J. biol. Chem. 163, 221-226. HEHRE, E. J., GENGHOFF,D. S. and NEILL, J. M. 1946. Serological reactions of two bacterial levans. J. Immunol. 51, 5. LEACH,S. A., GREEN, R. M., HAYES,M. L. and DADA, 0. A. 1969. Biochemical studies on the formation and composition of dental plaque in relation to dental caries: extracellular polysaccharides. J. dent. Res. 48, 811-817. MANLY, R. S. 1961. Retention of carbohydrates from sugar solutions by salivary sediment. J. dent. Res. 40, 379-384. MCDOUGAL,W. A. 1964. Studies on the dental plaque: IV Levans and the dental plaque. Aust. dent. J. 9, l-5. MILLER,W. D. 1890. The Microorganisms of the Human Mouth. S. S. White, Dental Manufacturing Company, Philadelphia, 22. Rtrz, H. L. 1969. Fluorescent antibody staining of Neisseria, Streptococcus and Veilionella in frozen sections of dental plaque. Archs oral Biol. 14, 1073-1083. _ SNYDER.M. L.. HACKEDORN.H. M.. MARTIN. D. 0. and JOHNSON.D. D. 1955. The svnthesis of muscinous’polysaccharides from’sucrose by oral bacteria. J. den;. Res. 3$. 368-379. - ~~VAN Hours, J. and JANSEN,H. M. 1968. Levan degradation by streptococct Isolated from human dental plaque. Archs oral Biol. 13, 827-830. WOOD, J. M. 1964. Polysaccharide synthesis and utilization by dental plaque. J. dent. Res. 43,955. Abstract.