Australian Dental Journal, August, 1979

Volume 24, No. 4

23 1

Dental plaque ecology related to caries and periodontal diseases*

Ernest Newbrun, B.D.S., M.S., D.M.D., Ph.D.

Professor of Oral Biology, University of California San Francisco

ABSTRACT-Dental plaque consists of a dynamic microbiota which responds to ecological changes. There are major technical difficulties in obtaining representative plaque samples and in dispersing, cultivating, identifying and quantifying the microbial components. Cumulative findings on plaque microbiota associated with dental caries and different forms of periodontal disease support the specific plaque hypo- thesis. However, the data do not permit designation of any single organism as the distinct aetiological agent. The characteriskic pattern is autogenic succession, in which one or more microbial species alter the plaque environment and are replaced or succeeded by other species.

(Received for publication April, 1979)

Introduction Dental plaque is a prerequisite for the development of

both caries and periodontal diseases, but it is not the same type of plaque which causes both types of disease. Two prevailing philosophies exist concerning prophyl- axis and therapy of plaque-related dental disease.

They are: - (1) nonspecific plaque hypothesis (NSPH) and (2) specific plaque hypothesis (SPH). The NSPH states that caries and periodontal disease

result from elaboration of noxious products by the entire plaque flora. It assumes there is a host threshold for these products. Amounts of irritants below this threshold value can be overcome by host defences such as salivary buffering, detoxification, and immune responses. In essence, this theory emphasizes the quantity rather than the quality of the plaque flora.

* Presented at Workshop on Oral Microbiology, 18th Annual Meeting, Australian Society of Microbiology, Adelaide, May 16, 1979.

Loesche, W. J.-Chemotherapy of dental plaque infections. Oral Sci. Rev., 9 : 65-107, 1976.

The SPH states that only certain plaques cause infections, owing to the presence of either specific pathogens or a relative increase in the levels of certain indigenous plaque organisms or both of these factors. Plaque is a mixture of many microorganisms, never a pure culture. An organism need not predominate to be a pathogen; however, it should be found consistently in plaquks associated with the particular disease and its numbers should increase relative to “normal” plaque which is not associated with the disease.

The SPH is difficult to prove because plaque consists of a mtlange of organisms which varies depending not only on the site and customary diet, but also on how much time the plaque has had to “mature”. Because of the major technical difficulties in obtaining representative plaque samples and in dispersing, cultivating, identifying and quantifying the microbial components, Socransky* has facetiously enunciated the First Law of Oral Biology: “A plaque sample will not satisfy anybody; it is either pooled or too small, unrepresentative, too young or too old”.

Socransky, S. S.-Personal communication, 1974.

232

S.mPW procedure

Since the oral cavity consists of a number of ecological niches, the flora on the tongue, tonsils, occlusal fissures, smooth surfaces of teeth, and subgingival sulcus are each unique. Even a single tooth comprises several distinct microenvironments. The practice of using pooled plaque (combined samples from several teeth) obscures im- portant site variations. Using dental explorers, scalers, abrasive strips, floss, “spoons” of metal or plastic, fine probes, wire, or needles makes it possible to sample specific sites on the tooth or gingival sulcus.

Once collected, the plaque samples must be trans- ported in a suitable media from the clinic to the laboratory for processing. Anaerobic storage in plastic bags that are sealable and contain an anaerobic generator enhances survival of plaque flora between collection and culture. The sooner a sample is processed, the greater the viable recovery.

Australian Dental Journal, August, 1979

Identification and qunntification Several genera of plaque microorganisms, such as

Streptococcus and Acrinomyces, are well defined, but many plaque isolates cannot readily be identified by current criteria.

Dispersion For plaque microbiota to be separated and quantified,

the cells should be uniformly suspended, thus allowing representative dilutions and platings. Physical methods have usually been employed to disperse the zoogleal plaque clusters. These methods include sonic oscillation, vortex mixing, grinding or homogenizing, shaking with glass beads, and repeated forceful expulsion from hypo- dermic syringes.

Different types of plaque require different treatments. For example, subgingival plaque has a looser structure (“soup”) and can be dispersed with mild force. More vigorous efforts are needed to separate the tightly packed supragingival plaque, in which many of the cells are stuck together by insoluble extracellular polysaccharides. Ultimately, the treatments used are a compromise aimed at maximizing viable recovery.

Isolation and culture conditions Anaerobic procedures can more than double the

survival and recovery of the total plaque flora. When anaerobic conditions and several culture media are used, 70 to 75 per cent of the organisms seen in a smear can be recovered in ~ul ture .~. The remaining 25 to 30 per cent of the flora is either dead or cannot grow on the available media. In this way we may lose important plaque components. For example, spirochetes, an important organism in plaque of all types of periodontal disease, are mostly lost Oh culture.

Gordon, D., Stutman, M., and Loesche, W. J.-Improved isolation of anaerobic bacteria from the gingival crevice area of man. Appl. Microbiol., 21 : 1W1050 (June) 1971.

Manganiello, A. D., Socransky, S. S., Smith, C., Propas, D., Oram, V. , and Dogon, 1. L.-Attempts to increase viable count recovery ofhuman supragingival plaque. J. Periodont. Res., 12: 2, 107-119 (Mar.) 1977.

Streptococcus mrttllns and dental caries In any current dental journal one can read statements

such as: “Considerable evidence points to an important role played by S. mutuns in the aetiology of dental ~ a r i e s ” ~ ; “S. mutuns is one of the most important cariogenic organisms in man”6; “Studies carried out on rodents, monkeys and man suggest an important role for S. muruns in the pathogenesis of dental caries”’. The collective data implicating S. mutuns as an important pathogen and the role of bacterial specificity in human dental caries is discussed in detail elsewhere.*.

In cross-sectional studies, plaque associated with the development of incipient smooth-surface carious lesions contains significantly higher proportions of S. mutuns than plaque covering clinically sound enamel immediate- ly adjacent to the lesion. However, S. mutuns rarely predominates among the other plaque bacteria on smooth surfaces, often constituting less than 1 per cent of the cultivable flora.’ In carious fissures, it has been found to constitute 10 to 35 per cent of the flora.I0

Several serotypes of S. mutuns may be responsible for the initiation of dental caries in humans, but it is not clear whether one serotype is more virulent than another. However, when compared with other plaque organisms, all the serotypes of S. mutuns are usually more cariogenic in test animals receiving sucrose diets.

Thus, the principal evidence supporting S. muruns as the causative organism in human dental caries is derived from epidemiological cross-sectional studies in humans and pathogenicity studies in animals. In the compara- tively fewer longitudinal studies involving repeated examination of the plaque flora on select tooth surfaces, the findings have not been clear-cut. S. mutuns may

Bratthall, D., Gahnberg, L., and Krasse, B.-Method for detecting IgA antibodies to Sfreprococcus mutans serotypes in parotid saliva. Arch. Oral Biol., 23: 10, 843-849, 1978.

Ivanyi, I., and Lehner, T.-The relationship between caries index and stimulation of lymphocytes bv StreDrococcus mufans in mothers and thecr neonates. Arch. Oral Biol., 23: 10, 851-856, 1978.

’ Duchin, S., and van Houte, J.-The relationship of S. mufuss and lactobacillus to incipient smooth-surface dental caries. Arch. Oral Biol., 23: 9, 779-786, 1978.

Gibbons, R. J . , and van Houte, J.-Bacteriology of dental caries. In, Textbook of Oral Biology. Edits. Shaw, J . H., Sweeney, E. A., Cappuccino, C. C., and Meller, S. Phila- delphia, W. B. Saunders and Company, 1978 (pp. 975-991).

Newbrun, E. -Cariology. Baltimore, Williams and Wilkins Company, 1978 (pp. 44-75).

l o Loesche, W. J., Rowan, J . , StrafTron, L. H., and Loos, P. J . - Associations of Sfreprococcus mufons with human dental decay. Infect. and Immun., 1 I : 6, 1252-1260 (June) 1975.

Australian Dental Journal, August, 1979 233

TABLE 1 Organisms of probable aetiological significance in various

types of periodontal disease

Disease Gingivitis

Acute necrotising ulcerative gingivitis

Chronic inflammatory periodontitis

Rapidly destructive periodontitis

Juvenile periodontitis

Organisms Actinomyces sp. Campylobacter Fusobacteriwn Spirochetes Veillonella Spirochetes (large) with 2 20 axial

fibrils Fusobacterium Actinomyces sp. Bacteroides sp. Spirochetes Capnophilic rods Bacteroides melaninogenicus Bacteroides sp. (corroding, gelatin-

Spirochetes Eikenella corrodens Fusobacterium nucleatum Selenomonus Campylobacter Veillonella Gram-negative anaerobic rods Fusobacterium Capnocytophaga Bacteroides sp. Spirochetes

hydrolyzing)

increase in number but need not predominate for a lesion to develop.l1-l3

Since dental caries is a multifactorial disease, no single unfavourable factor (host, microflora, or diet) will assure a high risk of caries. Both sucrose and S. mufuns correlate strongly with caries, but there are individuals who have high numbers of S. mutuns and little if any caries.

There may also be the exceptional patient who eats sticky sweets frequently yet develops no cavities. Such persons prove that neither high numbers of S. mutuns nor frequent ingestion of sucrose alone produces caries. Instead, caries could fit the hypothetical formula: S. mutuns x sucrose=caries. The product, however, is not an absolute value: some persons get very few lesions whereas others develop rampant caries. The varied

results might depend on differences in the caries-inducing factors.14

Specific bacteria and human periodontal diseases The relationship between specific plaque bacteria and

periodontal diseases is even more complicated than the relationship between specific bacteria and caries, in part because the clinical criteria used to distinguish various forms of periodontal disease are somewhat arbitrary. Furthermore, because chronic inflammatory periodontal disease undergoes periods of exacerbation and remission, it is difficult to know whether the disease is active or arrested.

Nevertheless, evidence suggesting bacterial specificity in periodontal diseases has emerged from experimental animal models and from examination of plaque samples from humans. These findings are reviewed in detail elsewhere' -' ' and are summarized in Table 1.

I ' Bowden, G. H., Hardie, J. M., McKee, A. S., Marsh, P. D., Phillery, E. D., and Slack, G. L.-The microflora associated with developing carious lesions of the distal surfaces on the upper first premolars in 13-14 year old children. In, Micro- bial Aspects of Dental Caries. Edits. Stiles, H. M., Loesche, W. J., and OBriep, T. E. Sp. Supp. Microbiology Abstracts, I : 223-242, 1976.

I 2 Mikkelsen, L., and Poulsen, S.-Microbiological studies on plaque in relation to development of dental caries in man. Caries Res., 10: 3, 178-188, 1976.

l 3 Swenson, J. I., Liljemark, W. F., and Schuman, L. M.-A longitudinal epidemiological evaluation of the association between the detection of plaque streptococci and dixelop- ment of dental caries in children. In, Microbial Aspects of Dental Caries. Op. cit., pp. 21 1-222.

l4 Krasse, B., and Newbrun, E.-Objective methods for evaluating caries activity and their application. In, Pediatric dentistry: Scientific foundations and current practice. Edit. Wei, S . H. Y., St. Louis, The C. V. Mosby Company, 1979 (in press).

lJ Gibbons, R. J., and van Houte, J.-Bacteriology of perio- dontal disease. In, Textbook of oral biology. Op. cif . , pp. 1032-1 047.

I6 Socransky, S. S.-Microbiology of periodontal disease- present status and future considerations. J. Periodontol., 48: 9, 497-504 (Sept.) 1977.

Tanzer, J.-Current status of the microbiology of periodontal disease. In, International conference on research in biology of periodontal disease. Chicago, University of Illinois, June, 1977 (in press).

234 Australian Dental Journal, August, 1979

and other organisms in the deep lesion is an example of autogenic succession. This occurs when the resident microbial species alter the environment to such a degree that they are replaced by other species more suited to the modified environment. In this case, the more aciduric lactobacilli take over in the deepest part of the lesion where the pH is lowest.z1 Similar examples of autogenic succession occur in the gingival sulcus in plaque associ- ated with gingivitis and periodontal disease. Experiment- al gingivitis in humans is associated with a change from a plaque dominated by streptococci to a plaque dominated by Actinomyces species. The proportions of A. viscosus, Bacteroides melaninogenicus, and Campylo- bacter sputorum increase greatly.zz When plaque forms on a cleaned smooth surface the Eh falls, as shown by oxidation-reduction indicatorsz3* z 4 or direct potentio- metric measurement^.^^^ z6 Lowering of the Eh contri- butes to successional changes in the flora of the gingival sulcus. In juvenile periodontitis, for example, surface translocating gram-negative anaerobes and microaero- philic rods become pred~minant,~’. z8 and in chronic inflammatory periodontitis the characteristic microbiota contains many anaerobic rods and spirochetes.’*. 1 9 . z9

We have studied ulcerative periodontal disease associated with Shigella j7exneri in Macaques.3o. 31

Xjlexneri is not the predominant organism found in the plaque of monkeys with this form of periodontal disease. We hypothesize that S.@exneri initially causes ulceration of the gingivae by invading or damaging the epithelial cells. This creates an anaerobic environment and provides haemin (ulcerative bleeding). These sites then favour colonization and proliferation of Bacillus melaninogenicus, other anaerobic gram-negative rods, vibrios, and spirochetes. Thus, this primate model provides another example of autogenic succession. These examples of aut,agenic succession demonstrate

the dynamic nature of the plaque microbiota. They also illustrate the difficulty in proving the specific plaque hypothesis but in no way detract from it. Studies of the plaque microbiota over a longitudinal period give a perspective of the stage of the disease, and allow for a more logical interpretation than when cross-sectional samples are examined. A better understanding of the factors responsible for these successional changes would allow appropriate intervention by the clinician to prevent the development of caries and onset of periodontal diseases.

Examining direct smears of subgingival plaque by darkfield microscopy can be most useful in detecting motile organisms and spirochetes. Quantitative changes in the composition of such smears can be used clinically to evaluate the success of therapy and home care.18. 19

Autogenic succession Although coronal caries starts in the enamel surface,

it eventually progresses into the dentine. Because the environment in a deep dentinal lesion is different from that in the initial lesion, the flora here is also different. The predominant organism is Luctobacillus. Frequently isolated gram-positive anaerobic rods and filaments are Arachnia, Bijidobacteria, Eubacteria and Propionibac- teria. Actinomyces, Rothia, and Bacillus also occur in the forefront of deep dentinal lesions.z0

The changes in plaque flora from predominantly streptococci at the surface to predominantly lactobacilli

I s Listgarten, M. A,, and Helldtn, L.-Relative distribution of bacteria at clinically healthy and periodontally diseased sites in humans. J. Clin. Periodon., 5 : 2, 115-132 (May) 1978.

I9 Keyes, P. H., Wright, W. E.. and Howard, S. A.-The use of phase-contrast microscopy and chemotherapy in the diagnosis and treatment of periodontal lesions-an initial report. Quintessence Internat., 9 : I , 51-56 (Jan.) 1978.

z o Edwardsson, S.-Bacteriological studies on deep areas of carious dentine. Odontol. Revy, 25: Suppl. 32, 1974.

2 1 Dirksen, T. R., Little, M. F., and Bibby, B. G.-The pH of carious cavities-11. The pH at different depths in isolated cavities. Arch. Oral Biol., 8: 91-97 (MarJApr.) 1963.

z 2 Loesche, W. J., and Syed, S. A.-Bacteriology of Human Experimental Gingivitis : Effect of Plaque and Gingivitis Score. Infect. and Immun. 21 : 3, 830-839 (Sept.) 1978.

z3 Katayama, T., Susuki, T., and Okada, S.-Clinical observa- tion of dental plaque maturation. Application of oxidation- reduction indicator dyes. J. Periodontol., 46: 10, 610-613 (Oct.) 1975.

z 4 Socransky, S. S., and Manganiello, A. D.-Mechanism of Eh reduction in dental plaque. J. Dent. Res., 54: Sp. Iss. A, Abstract 118, 74 (Feb.) 1975.

zs Kenney, E. B., and Ash, M. M.-Oxidation reduction potential of developing plaque, periodontal pockets and gingival sulci. J. Periodontol., 40: 630-632 (Nov.) 1969.

26 Russell, C.. and Coulter, W. A.-Continuous monitoring of pH and Eh in bacterial plaque grown on an artificial mouth. Appl. Microbiol., 29: 2. 141-144 (Feb.) 1975.

2 7 Newman, M. G., and Socransky, S. -Predominant microbiota in periodontosis. J. Periodont. Res., 12: 2, 12&128 (Mar.) 1977.

’* Slots, J.-The predominant cultivable organisms in juvenile periodontitis. Scand. J. Dent. Res., 84: 1, 1-10 (Jan.) 1976.

2 9 Slots, J. -The predominant cultivable microflora of advanced periodontitis. &and. J. Dent. Res., 85: 2, 114-121 (Jan.- Feb.) 1971.

30 Armitage, G. C., Newbrun, E., Polando, V., Lodberg, P., and Anderson, J.-Oral shigellosis and periodontal disease in Macaques. I. Clinical and ultrastructural observations. J. Dent. Res., 57: Sp. Iss. A, Abstract 596, 223 (Jan.) 1978.

3 1 Newbrun, E., Hoover, C., Armitage, G.. and Anderson, J. - Oral shigellosis and periodontal disease in Macaques. 11. Microbiological findings. J. Dent. Res., 57: Sp. Iss. A, Abstract 1108, 351 (Jan.) 1978.

Department of Oral Medicine and Hospital Dentistry,

University of California San Francisco, California, 94143, U.S.A.