STREPTOCOCCUS MUTANS AND DENTAL CARIES IN LONG-EVANS RATS WITH A NATURALLY-ACQUIRED ORAL FLORA H. G. HUXLEY* Faculty of Dentistry. University of Manitoba, Winnipeg, Manitoba R3E 0W3, Canada Summary-From each of 62 litters, one rat was killed 7, 14 and 21 days after weaning and being fed a cariogenic diet. From each rat, plaque was removed separately from 2 buccal and 2 fissure sites on the right lower first molar tooth. Caries was evaluated at the same sites and its penetration into the tooth was quantitated. Streptococcous mutans occurred in about 25 per cent of 726 plaque samples, and was distributed equally among the 4 tooth sites. Similar numbers of carious lesions occurred in rats with and without Strep. mutans, but the penetration of caries tended to be greater when Strep. mutans was present. In rats with Strep. mutans, there was no correlation between the penetration of caries and the number or proportion of Strep. mutans in plaque. Carries in the lower left teeth was assessed by a different method. In those rats harbouring Strep. mutans on the lower right first molar tooth, the incidence of cavitating fissure lesions and the extent of buccal caries tended to be higher in the lower left first and second molars. This investigation (1) indicates that caries initiation in the Long-Evans rat occurs equally with or without naturally-acquired Strep. mutans, (2) demonstrates that Strep. mutans accelerates the early progression of caries, (3) emphasizes the high caries incidence in rats lacking Strep. mutans, (4) suggests that some rat models may unduly accentuate the role of Strep. mutans in caries and (5) draws attention to differences between species of animal with respect to caries and Strep. mutqns.
INTRODUCT’ION
oral flora by means other than the feeding of a cariogenie diet, (3) to screen plaque for Strep. mutans at specific tooth sites and to score caries at the same sites, (4) to examine the plaque-caries relationship during the initiation and early progression of caries.
The precise role of Streptococcus mutans in the aetiology of human dental caries remains unknown. In man. this organism occurs preferentially in dental plaque overlying and within carious lesions (Shklair, Keene and Simonson, 1972; Rogers, 1973). Difficulty in diagnosing early caries has precluded a decision as to whether the organism initiates caries or is favoured ecologically by the environment within an existing lesion. Animal model systems have strongly implicated Strep. mutans as an initiator of caries. In most studies, large oral inocula of the organism were repeatedly administered to animals, often when a competing oral flora was limited through the use of gnotobiotic or specific pathogen-free animals, or through oral administration of antibiotics. By providing maximum opportunity for heavy colonization of teeth by Strep. mutuns, such experimental systems may have exaggerated its true role in caries. Elucidation of the role of naturally-acquired Strep. mutans in caries aetiology is pertinent in view of the many attempts to reduce dental caries through elimination or attenuation of this organism by vaccination and other means. An opportunity to study this role arose from the observation that some Long-Evans rats naturally harboured Strep. mutans whereas others from the same colony did not. To compare the caries incidence in the two subpopulations, experimental procedures were selected: (1) to promote rapid development of caries, (2) to avoid manipulation of the
MATERIALS
AND
METHODS
Animals 62 titters of Long-Evans rats were reared in stainless steel cages. A powdered starch-based diet, DDl (Huxley, 1977a), was fed to the dam during suckling and the young were weaned to cariogenic diet 2056 (Huxley, 1977a) when 22-24 days old. Diet 2056 is a modification of diet 2000 formulated by Keyes and Jordan (1964) and contains 56 per cent sucrose. At 7, 14 and 21 days after weaning, 1 rat was killed from each litter. Plaque collection and culturing After killing the rats by decapitation, lower jaws were dissected and kept moist with thioglycollate broth (Difco Laboratories, Detroit). Fas’t green was used as a disclosing agent and plaque was carefully removed from 4 sites of the lower right first molar tooth of each animal (Fig. 1) using a curette for the buccal sites and a fine scalpel for the fissures. To provide access to the central fissure, the tooth was split bucco-lingually (Huxley, 1972) but this was unnecessary with the wider, shallower distal fissure. Immediately after collection into thioglycollate broth, plaques were taken into an anaerobic chamber (Coy Mfg., Ann Arbor) and there homogenized for 30 s (Tissumizer@ lOOEN, Tekmar. Co., Cincinnati) and serially
* Present address: National Caries Program, NIDR. National Institutes of Health Bethesda, MD 20014, U.S.A 703
704
H. G Huxley
diluted lo-fold. Appropriate dilutions were plated on (1) mitis-salivarius agar (Difco), (2) MM10 agar containing 5 per cent sucrose but no blood (Loesche, Hackett and Syed, 1972), (3) Phenol red agar (Difco) containing 1 per cent mannitol. MM10 agar is a medium designed to culture a wide spectrum of oral microorganisms under anaerobic conditions. As a result of the high sucrose:nitrogen ratio, Strep. mutans produces distinctive heaped up, gummy colonies on MM10 agar. Phenol red-mannito1 agar favours a wide selection of oral microorganisms, and bacteria such as Strep. mutans that are capable of fermenting mannitok lower the pH locally and change the colour of the indicator. Thus, mannitol-fermenting organisms are readily recognisable as yellow colonies against a red background. All plates were incubated at 37°C for 7 days in the anaerobic chamber.
and severe caries. A more sensitive scoring method was therefore devised, based upon the penetration of caries into the tooth. This parameter has considerable significance in clinical dentistry. In rats, penetration is closely related to the surface area of carious enamel on buccal tooth surfaces and to the area of carious dentine in fissures (H. G. Huxley, unpublished). The right half-mandible was stained for 24 h in Alizarin red, which delineates carious enamel and dentine. Using a small abrasive stone mounted in a dental handpiece, the right lower first molar tooth was ground from the occlusal surface down to the level of the buccal carious lesions. Further successive increments were then carefully ground through the lesions. During this process, the penetration of caries from the enamel surface inwards was measured at x 20 magnification with a microscope eyepiece micrometer. The caries score for each lesion was the maximum penetration recorded, the unit of measurement being a micrometre unit. Caries in the central and distal fissures was revealed by grinding the remaining portion of the tooth successively from buccal to lingual. At these sites, the caries score was the maximum penetration of the lesion inwards from the enamel surface towards the pulp or pulp horn, and was again measured in micrometre units. (b) In the lower left first and second molar teeth. Plaque was not sampled from these teeth. The linear extent of buccal caries was estimated by the method of Keyes (1958), then the left molar teeth were sectioned and stained by the method of Kijnig et a[. (1958). In each rat the cumulative incidence of early, more advanced and cavitating fissure lesions was assessed according to the definition by Green and Hartles (1966) of these categories.
Detection of Strep. mutans
Statistical procedures
The strain of Strep. mutans occurring in the LongEvans rat produces distinctive colonies on all 3 media. To exclude the possibility that variant forms of Strep. mutans might occur with a smooth or otherwise non-distinctive colonial morphology, streptococci-fermenting mannitol were recovered from mannitol agar and further tested as follows: (1) fermentation of mannitol, sorbitol, melibiose, raffinose and starch, (2) hydrolysis of hippurate, (3) production of extracellular polysaccharide in 5 per cent sucrose broth culture, (4) sensitivity to sulphonamides, (5) Lancefield grouping. The methods described by Carlsson (1968) were followed in this testing. Enumeration of the total colony-forming units (CFU) on MM10 agar and the differential count of Strep. mutans CFU on this medium, permitted estimation of the proportion of Strep. mutans in plaque. Mitis-salivarius agar was used only to confirm the presence and approximate numbers of Strep. mutans.
The significance of the differences between caries penetration at sites with or without Strep. mutans was tested by Student’s t-test and association of caries penetration with the CFU or proportion of Strep. mutans by Pearson correlation. Frequency comparison of Strep. mutans and caries was by the Fisher test of exact significance or chi-square analysis.
Fig. 1. Tooth sites from which plaque was sampled and at which caries was evaluated. Sites 1 and 2 are on the buccal surface, site 3 is the distal fissure and site 4 the central fissure of the rat lower right first molar.
Caries evaluation (a) At sites on the lower right molar tooth from which plaque was sampled. In most studies u$ing rodents, the entire dentition or a selected group of teeth are used to evaluate caries incidence. Scoring methods in common use (KSnig, Marthaler and Miihlemann, 1958; Keyes, 1958) are either inapplicable to a discrete site on a single tooth, or at best provide only a crude differentiation between minimal, moderate
RESULTS
Results for both plaque and caries were available for 726 out of the total of 744 sites sampled; in the remainder, either the bacteriological plates were spoilt or a caries score could not be obtained. Mannitolfermenting streptococci other than Strep. mutans occurred in about half of the samples and accounted for only a small proportion of the total growth on mannitol agar. None produced adherent or soluble extracellular polysaccharide in 5 per cent sucrose broth, several hydrolysed hippurate and some fermented starch, characteristics not associated with Strep. mutans (Carlsson, 1968; Facklam, 1974; Perch, Kjems and Ravn, 1974). The frequency of Strep. mutans and caries, broken down by site and length of time, appears in Table 1. In only one instance did caries occur significantly more frequently at a site with Strep. mutans than without (Table 1). Strep. mutans was present at all 4 tooth sites in 70 per cent of rats harbouring the organism,
705
Strep. muruns and caries in rats
Table 1. Frequency and significance of caries and Slrep. mutans distribution at 4 sites of the right lower first molar tooth in rats fed cariogenic diet for 7, 14 or 21 days Days fed cariogenic diet 2056
Tooth site
7
:
7 7 7 14 14 14 14 21 21 21 21
3 4 I 2 3 4 1 2 3 4
Caries
-
Strep. mutans
+
-
+
-
-
+
+
11 3 0 2 16 6 0 0 16 8 0 0
41 49 46 50 30 42 40 46 29 38 39 41
2 2 1 0 0 1 0 0 3 2 0 0
Significance of frequency distribution
8 7 11 10 16 13 14 16 14 14 19 20
N.S. N.S. N.S. OY& N.S. N.S. N.S. N.S. N.S. N.S. N.S.
* N.S. = p > 0.05.
at 3 sites in 14 per cent and at 2 or single sites in 16 per cent of rats. There was no significant difference between different tooth sites in the frequency (Table 1) or in the proportion of Strep. mutans in plaque, but the organism tended to be present more frequently in rats fed a cariogenic diet for longer periods . . of time (x2 = 11.163p < 0001) Caries penetration was generally greater in rats harbouring Strep. mutans (Fig. 2) and in 4 out of 12 comparisons the difference was significant (JJ= 0.05 or less). No relationship was obvious in the correla-
tion between caries penetration and either the CFU or proportion of Strep. mutcms in plaque; a majority of the values of T were negative. Figure 3 shows the caries incidence in the sectioned teeth of the half-mandible not used for plaque sampling. Mean caries scores were generally slightly higher in rats harbouring Strep. mutuns. The only difference that reached a level of statistical significance (p < 0.05) was in the incidence of microcavities in rats fed diet 2056 for 14 days, although the incidence of buccal caries at 21 days just failed to reach this level of probability (0.1 > p > 0.05). DISCUSSION The bacteriological methods were designed to recover as many viable microorganisms as possible
Value 01 t s1g oft
clLr#s2056 Value oft
sag Of t
0.67 0 50
3.69 CO.OOl
1.35 0. I6
7
14
21
0.32 0 75
0.55 0 56
3 20 0 002
0.34 0 73
IO1
7 2 67 0 01
3.11 0003
0.63 0.53
14
21
I 67 010
I 76 008
Fig. 2. Mean (&SE) penetration of caries, in pm units, at 4 sites of the lower right first molar tooth in rats harbouring (O----O) or lacking (O---O) Strep. mutans in plaque. Graphs (a-d) refer to tooth sites 1-4, respectively. Littermate rats were fed cariogenic diet for 7, 14 or 21 days. The value and significance of t are reported for the comparison of caries penetration in rats harbouring or lacking Strep. mutons.
7
I4
21 Days
7
14
21
7
I4
21
2056
Fig. 3. Mean (&SE) incidence in lower left molar teeth of total fissure lesions (a), more advanced fissure lesions (b), fissure microcavities (c) and extent of buccal caries (d) in rats harbouring (O--+) or lacking (O----O) Strep. mutans in plaque on the lower right first molar tooth. Littermate rats were fed cariagenic diet for 7, 14 or 21 days.
706
H. c;. Huxley
from plaque. Nevertheless, with any set of recovery and culture conditions, some bacteria are lost. A disclosing solution was required to ensure the efficient removal of plaque from tooth surfaces: fast green was chosen because it is less inhibitory to oral bacteria than other agents (Caldwell and Hunt. 1969). Homogenization was preferred to sonication as a means of dispersion to avoid undue loss of Gam-negative bacteria (Robrish et al.. 1976). Anaerobic recovery and culture conditions are required for the satisfactory growth of many bacteria in plaque (Aranki er al.. 1969) though not for Strep. mutans. Omission of anaerobic techniques would have resulted in spuriously high proportions of Strep. mutans in plaque. Edwardsson (1968. 1970) described cariogenic varieties of Strep. mutans that had uncharacteristically smooth colonial morphology on mitis-salivarius agar and other high-sucrose media. It was essential to test for such strains, but none were found. The testing programme for mannitol-fermenting streptococci was designed on the basis of known biochemical properties of Stwp. rnutans and was not an attempt to classify other organisms. Enumeration of Strep. rnutarrs was on plates with 30-300 colonies. Detection of this organism was possible on crowded plates from lower dilutions of plaque because of its very characteristic colonial morphology on sucrose-containing media. Had Strep. mutans accounted for less than 0.3 per cent of the viable bacteria cultured, it would possibly have been missed. About one-quarter of the rats harboured Strep. mutans. If present at all. the organism generally occurred in all three members of a litter and in a majority of the tooth sites sampled, where it represented a substantial proportion of the total viable microbiota. Thus. Long-Evans litters either did not harbour Strep. mutans at all or tended to be heavily infected. It would be of interest to know whether such a distribution of Strep. mutans among the colony was the result of inadequate opportunity for transmission or inhibition by other microbial residents of plaque. The experimental time was short and covered the period of caries initiation and early progression. A majority of buccal sites and most fissures were carious at all times but only about one-quarter were infected with Strep. mutans. This organism is thus not essential for caries initiation in the Long-Evans rat. On the other hand. the progression of caries. measured by the extent of penetration of caries into the enamel and dentine, was often greater at sites infected with Strep. mutans. A longer experimental period may accentuate a difference in caries at sites harbouring or lacking Strep. mutc4n.s. Fewer sites had Strep. mutans earlier in the experrment. Possibly this organism occurred more frequently at an earlier stage but in numbers below the threshold of detection. The large number of litters used make it unlikely that systematic selection of more heavily-infected littermates occurred later in the experiment. Ideally, the same rat should be examined after different intervals, but methods for in-rlioo sampling of plaque and estimation of caries have yet to be propounded. A predilection by Strep. mutans for more advanced carious lesions (de Stoppelaar. van Houte and Backer Dirks, 1969; Loesche and Syed, 1973) makes it unlikely that false negative results
occurred as a result of the disappearance of the organism from an established lesion. Mean values for both the penetration and linear extent of caries on the buccal surface tended to decrease when the rats were fed the cariogenic diet for long periods. Further study has confirmed this trend and will be reported separately. Henry and Navia (1969) noted a similar effect, though Larson and Zickus (1972) did not. My investigation supports a large body of other evidence that Strrp. muruns plays a role in caries. Superimposing Strep. muta~s on a conventional oral Hora raises caries incidence particularly on the buccal surface and in the distal fissures of rat teeth (Konig. Guggenheim and Muhlemann. 1965; K&rig, Larson and Guggenheim. 1969: Ranke and Ranke, 1971; Regolati. Guggenheim and Muhlemann. 1972). A comparison between the results in the studies mentioned and mine is possible because experimental details such as the pre-weaning regimen, the cariogenie diet used and the duration were all similar, and because in all studies caries was evaluated by the method of Konig cr al. (I 958). The difference in caries incidence between rats infected with Strep. mutans and non-infected animals was much greater when the organism was deliberately introduced than when it was acquired by natural means. Whilst caution is required in extrapolating results from one strain of rat to another. it would seem that super-infecting with Strep. mutans may unduly emphasize its importance m caries aetiology. A lack of correlation between the total CFU or the relative proportion of Strep. mutans in plaque and caries is m accord with similar observations in the Sprague-Dawley rat (Huxley. 1977b). Cartes data from teeth that were sectioned and scored by the method of Konig er al. (1958) were included to facilitate comparison with the results of other Investigations. It was necessary to assume that, if Strep. mutanS occurred somewhere on the lower right first molar tooth. it ;Yould also be present on the left molars. Obviously. rt is more desirable to relate caries and plaque at the same sites than to extrapolate from one side of the mouth to the other and from one tooth site to another. For this reason. a caries assessment method based on penetration of the lesion at single sites was devised. There is substantial agreement between the two methods of scoring in the degree to which Strep. mutans augments caries. A relatively high level of caries in rats not infected with Strep. mutans corroborates similar findings in the Wistar rat (Huxley, 1975). It contrasts markedly with the hamster. where animals without Strep. mutans develop little caries (Fitzgerald and Keyes. 1960; Krasse, 1965: Zinner rt ul.. 1966). and possibly the macaque monkey too. because Bowen (I 969) found a dramatic rise in caries after inoculating monkeys with Strep. mutans. In man. a confused picture exists, some studies indicate that most carious lesions harbour Srrep. mutans (e.g. Littleton, Kakehashi and Fitzgerald, 1970; Shklair er al., 1972; Rogers, 1973; Ikeda, Sandham and Bradley, 1973). In others, substantial numbers of lesions are not associated with the organism (Loesche et al., 1975; Mikkelsen and Paulsen. 1976). Until it is decided whether there are two or more separate patterns of caries in man, a
Strep. mutans
rational decision cannot of various experimental
be made as to the suitability animal models.
Acknowledgernrnts-This investigation was supported by a grant from the Medical Research Council of Canada. I thank S. Chakrabarty. G. Young and J. Rzeszutek for their technical assistance. REFERENCES
Aranki A.. Syed S. A.. Kenney E. B. and Freter R. 1969. Isolation of anaerobic bacteria from human gingiva and mouse caecum by means of a simplified glove box procedure Appl. Microhiol. 17, 568-576. Bowen W. H. 1969. The induction of rampant dental caries in monkeys (Macaca irus). Caries Res. 3. 227-237. Caldwell R. C. and Hunt D. E. 1969. A comparison of the antimicrobial activity of disclosing agents. J. dent. Res. 48, 913-915. Carlsson J. 1968. A numerical taxonomic study of human oral streptococci. Odont. Rery 19. 137-160. de Stoppelaar J. D.. van Houte J. and Backer Dirks 0. 1969. The relationship between polysaccharide-producing streptococci and smooth surface caries in 13-year old children. Caries Res. 3, 190-199. Edwardsson S. 1968. Characteristics of caries-inducing human streptococci resembling Streptococcus mutans. Archs
oral Biol.
13, 631-646.
Edwardsson S. 1970. The caries-inducing property of variants of Srreptococcus mutans. Odonr. Rwy 21, 153-157. Facklam R. R. 1974. Characteristics of Streprococcus mutans Isolated from human dental plaque and blood. Int.
J. sysr. Bact. 24, 313-319.
Fitzgerald R. J. and Keyes P. 1960. Demonstration of the etiologic role of streptococci in experimental caries in the hamster. J. Am. denr. Ass. 61. 9-19. Green R. M. and Hartles R. L. 1966. The effects of differing high-carbohydrate diets on dental caries in the albino rat. Br. J. Nurr. 20, 317-323. Henry C. and Navia J. M. 1969. Sodium trimetaphosphate influence on the early development of rat caries and concurrent microbial changes. Caries Rex 3, 326-338. Huxley H. G. 1972. The recovery of microorganisms from the fissures of rat molar teeth. Archs oral Biol. 17, 1481-1485.
Huxley
H. G. 1975. An investigation
into the prevalence
of Streptococcus mutans in Wistar rats, its relationship to the initiation of dental caries. and the effect upon its presence of the diet fed before weaning. Archs oral Biol.
20, 351-357.
Huxley H. G. 1977a. The cariogenicity of dietary sucrose at various levels in two strains of rat under unrestricted and controlled-frequency feeding conditions. Caries. Res. 11. 237-242. Huxley H. G. 1977b. The relationship between plaque bacteria and dental caries at specific tooth sites in rats. In: Microbial Aspecrs of Dental Caries (Edited by Stiles H. M.. Loesche W. J. and O’Brien T. C.). Vol. 3. pp. 773-784. Information Retrieval Inc., Washington. DC. Ikeda T.. Sandham H. J. and Bradley E. L. 1973. Changes in Streptococcus murans and lactobacilli in plaque in relation to the initiation of caries in negro children. Archs oral Biol. 18, 555-566. Keys P. 1958. Dental caries in the molar teeth of rats. II. A method for diagnosing and scoring several types of lesions simultaneously. J. dent. Res. 37, 1088-1099.
707
an Id caries in rats
Keyes P. H. and Jordan H. V. 1964. Periodontal lesions in the Syrian hamster-III. Findings related to an infectious and transmissible component. Archs oral Biol. 9, 377-400.
Kiinig K. G., Marthaler T. M. and Miihlemann H. R. 1958. Methodik der Kurzfristig erzeugten RattenKaries. Dt. Zahn-Mund
u. Kieferheilk.
29. 99-127.
KGnig K. G.. Guggenheim B. and Miihlemann H. R. 1965. Modifications of the oral bacterial flora and their influence on caries in the rat. II. Inoculation of a cariogenie streptococcus and its effect in relation to varying time of depression of the indigenous flora. He/c. odont. Acta 9, 130-134.
KBnig K. G.. Larson R. H. and Guggenheim B. 1969. A strain-specific eating pattern as a factor limiting the transmissibility of caries activity in rats. Archs oral Biol. 14, 91-103. Krasse B. 1965. Effect of caries-inducing streptococci in hamsters fed diets w“h sucrose or glucose. Archv oral Biol. 10, 223-226. Larson R. H. and Zickus C. S. 1972. Patterns of dental caries in OsborneeMendel and NIH Black rats in relation to length of the caries test period. J. dent. Rex 51, 1375-1381. Littleton N. W., Kakehashi S. and Fitzgerald R. J. 1970. Recovery of specific “caries-inducing” streptococci from carious lesions in the teeth of children. Arch: (oral Biol. 15. 461463. Loesche W. J., Hackett R. N. and Syed S. A. 1972. The predominant cultivable flora of tooth surface plaque removed from institutionalized subjects. Archs orai Biol. 17, 131 l-1325. Loesche W. J. and Syed S. A. 1973. The predominant cultivable flora of carious plaque and carious dentine. Caries Rex 7, 201~216. Loesche W. J., Rowan J.. Straffon L. H. and Loos P. J. 1975. Association of Srreprococcus nwtuns with human dental decay. Infect. Immun. 11, 1252-1260. Mikkelsen L. and Poulsen S. 1976. Microbiological studies on plaque in relation to development of dental caries in man. Caries Res. 10, 178-188. Perch B., Kjems E. and Ravn T. 1974. Biochemical and serological properties of Streptococcus mutans from various human and animal sources. Acta path. micro&o/. stand. Section B 82. 357-370. Ranke B. and Ranke E. 1971. Untersuchungen zur Kariogenitlt extrazellullre Polysaccharide bildener Streptokokken aus menschlicher Zahnplaque. Dt. zahniir:t/. 2. 26, 29-36.
Regolati B.. Guggenheim B. and Miihlemann H. R. 1972. Synergisms and antagonisms of two bacterial strains superinfected in conventional Osborne-Mendel rats. He/u. odonr. Acta. 16, 84-88. Robrish S. A.. Grove S. B.. Bernstein R. S.. Marucha P. T.. Socranskv S. S. and Amdur B. 1976. Effect of sonic treatment on pure cultures and aggregates of bacteria. J. r/in. Microbial.
3, 474479.
Rogers A. H. 1973. The ecology of Srreptococcus mutans in carious lesions and on caries free surfaces of the same teeth. Aust. dent. J. 18, 226-228. Shklair I. L.. Keene H. J. and Simonson L. G. 1972. Distribution and frequency of Streptococcus mutans in cariesactive individuals. J.-dent. Rds. 51, 882. Zinner D. D.. Jablon J. M.. Aran A. P.. Saslaw M. S. and Fitzgerald R. J. 1966. Comparative pathogenicity of streptococci of human origin in hamster caries. Archs oral Biol. 11, 1419-1421.
INTRODUCT’ION
oral flora by means other than the feeding of a cariogenie diet, (3) to screen plaque for Strep. mutans at specific tooth sites and to score caries at the same sites, (4) to examine the plaque-caries relationship during the initiation and early progression of caries.
The precise role of Streptococcus mutans in the aetiology of human dental caries remains unknown. In man. this organism occurs preferentially in dental plaque overlying and within carious lesions (Shklair, Keene and Simonson, 1972; Rogers, 1973). Difficulty in diagnosing early caries has precluded a decision as to whether the organism initiates caries or is favoured ecologically by the environment within an existing lesion. Animal model systems have strongly implicated Strep. mutans as an initiator of caries. In most studies, large oral inocula of the organism were repeatedly administered to animals, often when a competing oral flora was limited through the use of gnotobiotic or specific pathogen-free animals, or through oral administration of antibiotics. By providing maximum opportunity for heavy colonization of teeth by Strep. mutuns, such experimental systems may have exaggerated its true role in caries. Elucidation of the role of naturally-acquired Strep. mutans in caries aetiology is pertinent in view of the many attempts to reduce dental caries through elimination or attenuation of this organism by vaccination and other means. An opportunity to study this role arose from the observation that some Long-Evans rats naturally harboured Strep. mutans whereas others from the same colony did not. To compare the caries incidence in the two subpopulations, experimental procedures were selected: (1) to promote rapid development of caries, (2) to avoid manipulation of the
MATERIALS
AND
METHODS
Animals 62 titters of Long-Evans rats were reared in stainless steel cages. A powdered starch-based diet, DDl (Huxley, 1977a), was fed to the dam during suckling and the young were weaned to cariogenic diet 2056 (Huxley, 1977a) when 22-24 days old. Diet 2056 is a modification of diet 2000 formulated by Keyes and Jordan (1964) and contains 56 per cent sucrose. At 7, 14 and 21 days after weaning, 1 rat was killed from each litter. Plaque collection and culturing After killing the rats by decapitation, lower jaws were dissected and kept moist with thioglycollate broth (Difco Laboratories, Detroit). Fas’t green was used as a disclosing agent and plaque was carefully removed from 4 sites of the lower right first molar tooth of each animal (Fig. 1) using a curette for the buccal sites and a fine scalpel for the fissures. To provide access to the central fissure, the tooth was split bucco-lingually (Huxley, 1972) but this was unnecessary with the wider, shallower distal fissure. Immediately after collection into thioglycollate broth, plaques were taken into an anaerobic chamber (Coy Mfg., Ann Arbor) and there homogenized for 30 s (Tissumizer@ lOOEN, Tekmar. Co., Cincinnati) and serially
* Present address: National Caries Program, NIDR. National Institutes of Health Bethesda, MD 20014, U.S.A 703
704
H. G Huxley
diluted lo-fold. Appropriate dilutions were plated on (1) mitis-salivarius agar (Difco), (2) MM10 agar containing 5 per cent sucrose but no blood (Loesche, Hackett and Syed, 1972), (3) Phenol red agar (Difco) containing 1 per cent mannitol. MM10 agar is a medium designed to culture a wide spectrum of oral microorganisms under anaerobic conditions. As a result of the high sucrose:nitrogen ratio, Strep. mutans produces distinctive heaped up, gummy colonies on MM10 agar. Phenol red-mannito1 agar favours a wide selection of oral microorganisms, and bacteria such as Strep. mutans that are capable of fermenting mannitok lower the pH locally and change the colour of the indicator. Thus, mannitol-fermenting organisms are readily recognisable as yellow colonies against a red background. All plates were incubated at 37°C for 7 days in the anaerobic chamber.
and severe caries. A more sensitive scoring method was therefore devised, based upon the penetration of caries into the tooth. This parameter has considerable significance in clinical dentistry. In rats, penetration is closely related to the surface area of carious enamel on buccal tooth surfaces and to the area of carious dentine in fissures (H. G. Huxley, unpublished). The right half-mandible was stained for 24 h in Alizarin red, which delineates carious enamel and dentine. Using a small abrasive stone mounted in a dental handpiece, the right lower first molar tooth was ground from the occlusal surface down to the level of the buccal carious lesions. Further successive increments were then carefully ground through the lesions. During this process, the penetration of caries from the enamel surface inwards was measured at x 20 magnification with a microscope eyepiece micrometer. The caries score for each lesion was the maximum penetration recorded, the unit of measurement being a micrometre unit. Caries in the central and distal fissures was revealed by grinding the remaining portion of the tooth successively from buccal to lingual. At these sites, the caries score was the maximum penetration of the lesion inwards from the enamel surface towards the pulp or pulp horn, and was again measured in micrometre units. (b) In the lower left first and second molar teeth. Plaque was not sampled from these teeth. The linear extent of buccal caries was estimated by the method of Keyes (1958), then the left molar teeth were sectioned and stained by the method of Kijnig et a[. (1958). In each rat the cumulative incidence of early, more advanced and cavitating fissure lesions was assessed according to the definition by Green and Hartles (1966) of these categories.
Detection of Strep. mutans
Statistical procedures
The strain of Strep. mutans occurring in the LongEvans rat produces distinctive colonies on all 3 media. To exclude the possibility that variant forms of Strep. mutans might occur with a smooth or otherwise non-distinctive colonial morphology, streptococci-fermenting mannitol were recovered from mannitol agar and further tested as follows: (1) fermentation of mannitol, sorbitol, melibiose, raffinose and starch, (2) hydrolysis of hippurate, (3) production of extracellular polysaccharide in 5 per cent sucrose broth culture, (4) sensitivity to sulphonamides, (5) Lancefield grouping. The methods described by Carlsson (1968) were followed in this testing. Enumeration of the total colony-forming units (CFU) on MM10 agar and the differential count of Strep. mutans CFU on this medium, permitted estimation of the proportion of Strep. mutans in plaque. Mitis-salivarius agar was used only to confirm the presence and approximate numbers of Strep. mutans.
The significance of the differences between caries penetration at sites with or without Strep. mutans was tested by Student’s t-test and association of caries penetration with the CFU or proportion of Strep. mutans by Pearson correlation. Frequency comparison of Strep. mutans and caries was by the Fisher test of exact significance or chi-square analysis.
Fig. 1. Tooth sites from which plaque was sampled and at which caries was evaluated. Sites 1 and 2 are on the buccal surface, site 3 is the distal fissure and site 4 the central fissure of the rat lower right first molar.
Caries evaluation (a) At sites on the lower right molar tooth from which plaque was sampled. In most studies u$ing rodents, the entire dentition or a selected group of teeth are used to evaluate caries incidence. Scoring methods in common use (KSnig, Marthaler and Miihlemann, 1958; Keyes, 1958) are either inapplicable to a discrete site on a single tooth, or at best provide only a crude differentiation between minimal, moderate
RESULTS
Results for both plaque and caries were available for 726 out of the total of 744 sites sampled; in the remainder, either the bacteriological plates were spoilt or a caries score could not be obtained. Mannitolfermenting streptococci other than Strep. mutans occurred in about half of the samples and accounted for only a small proportion of the total growth on mannitol agar. None produced adherent or soluble extracellular polysaccharide in 5 per cent sucrose broth, several hydrolysed hippurate and some fermented starch, characteristics not associated with Strep. mutans (Carlsson, 1968; Facklam, 1974; Perch, Kjems and Ravn, 1974). The frequency of Strep. mutans and caries, broken down by site and length of time, appears in Table 1. In only one instance did caries occur significantly more frequently at a site with Strep. mutans than without (Table 1). Strep. mutans was present at all 4 tooth sites in 70 per cent of rats harbouring the organism,
705
Strep. muruns and caries in rats
Table 1. Frequency and significance of caries and Slrep. mutans distribution at 4 sites of the right lower first molar tooth in rats fed cariogenic diet for 7, 14 or 21 days Days fed cariogenic diet 2056
Tooth site
7
:
7 7 7 14 14 14 14 21 21 21 21
3 4 I 2 3 4 1 2 3 4
Caries
-
Strep. mutans
+
-
+
-
-
+
+
11 3 0 2 16 6 0 0 16 8 0 0
41 49 46 50 30 42 40 46 29 38 39 41
2 2 1 0 0 1 0 0 3 2 0 0
Significance of frequency distribution
8 7 11 10 16 13 14 16 14 14 19 20
N.S. N.S. N.S. OY& N.S. N.S. N.S. N.S. N.S. N.S. N.S.
* N.S. = p > 0.05.
at 3 sites in 14 per cent and at 2 or single sites in 16 per cent of rats. There was no significant difference between different tooth sites in the frequency (Table 1) or in the proportion of Strep. mutans in plaque, but the organism tended to be present more frequently in rats fed a cariogenic diet for longer periods . . of time (x2 = 11.163p < 0001) Caries penetration was generally greater in rats harbouring Strep. mutans (Fig. 2) and in 4 out of 12 comparisons the difference was significant (JJ= 0.05 or less). No relationship was obvious in the correla-
tion between caries penetration and either the CFU or proportion of Strep. mutcms in plaque; a majority of the values of T were negative. Figure 3 shows the caries incidence in the sectioned teeth of the half-mandible not used for plaque sampling. Mean caries scores were generally slightly higher in rats harbouring Strep. mutuns. The only difference that reached a level of statistical significance (p < 0.05) was in the incidence of microcavities in rats fed diet 2056 for 14 days, although the incidence of buccal caries at 21 days just failed to reach this level of probability (0.1 > p > 0.05). DISCUSSION The bacteriological methods were designed to recover as many viable microorganisms as possible
Value 01 t s1g oft
clLr#s2056 Value oft
sag Of t
0.67 0 50
3.69 CO.OOl
1.35 0. I6
7
14
21
0.32 0 75
0.55 0 56
3 20 0 002
0.34 0 73
IO1
7 2 67 0 01
3.11 0003
0.63 0.53
14
21
I 67 010
I 76 008
Fig. 2. Mean (&SE) penetration of caries, in pm units, at 4 sites of the lower right first molar tooth in rats harbouring (O----O) or lacking (O---O) Strep. mutans in plaque. Graphs (a-d) refer to tooth sites 1-4, respectively. Littermate rats were fed cariogenic diet for 7, 14 or 21 days. The value and significance of t are reported for the comparison of caries penetration in rats harbouring or lacking Strep. mutons.
7
I4
21 Days
7
14
21
7
I4
21
2056
Fig. 3. Mean (&SE) incidence in lower left molar teeth of total fissure lesions (a), more advanced fissure lesions (b), fissure microcavities (c) and extent of buccal caries (d) in rats harbouring (O--+) or lacking (O----O) Strep. mutans in plaque on the lower right first molar tooth. Littermate rats were fed cariagenic diet for 7, 14 or 21 days.
706
H. c;. Huxley
from plaque. Nevertheless, with any set of recovery and culture conditions, some bacteria are lost. A disclosing solution was required to ensure the efficient removal of plaque from tooth surfaces: fast green was chosen because it is less inhibitory to oral bacteria than other agents (Caldwell and Hunt. 1969). Homogenization was preferred to sonication as a means of dispersion to avoid undue loss of Gam-negative bacteria (Robrish et al.. 1976). Anaerobic recovery and culture conditions are required for the satisfactory growth of many bacteria in plaque (Aranki er al.. 1969) though not for Strep. mutans. Omission of anaerobic techniques would have resulted in spuriously high proportions of Strep. mutans in plaque. Edwardsson (1968. 1970) described cariogenic varieties of Strep. mutans that had uncharacteristically smooth colonial morphology on mitis-salivarius agar and other high-sucrose media. It was essential to test for such strains, but none were found. The testing programme for mannitol-fermenting streptococci was designed on the basis of known biochemical properties of Stwp. rnutans and was not an attempt to classify other organisms. Enumeration of Strep. rnutarrs was on plates with 30-300 colonies. Detection of this organism was possible on crowded plates from lower dilutions of plaque because of its very characteristic colonial morphology on sucrose-containing media. Had Strep. mutans accounted for less than 0.3 per cent of the viable bacteria cultured, it would possibly have been missed. About one-quarter of the rats harboured Strep. mutans. If present at all. the organism generally occurred in all three members of a litter and in a majority of the tooth sites sampled, where it represented a substantial proportion of the total viable microbiota. Thus. Long-Evans litters either did not harbour Strep. mutans at all or tended to be heavily infected. It would be of interest to know whether such a distribution of Strep. mutans among the colony was the result of inadequate opportunity for transmission or inhibition by other microbial residents of plaque. The experimental time was short and covered the period of caries initiation and early progression. A majority of buccal sites and most fissures were carious at all times but only about one-quarter were infected with Strep. mutans. This organism is thus not essential for caries initiation in the Long-Evans rat. On the other hand. the progression of caries. measured by the extent of penetration of caries into the enamel and dentine, was often greater at sites infected with Strep. mutans. A longer experimental period may accentuate a difference in caries at sites harbouring or lacking Strep. mutc4n.s. Fewer sites had Strep. mutans earlier in the experrment. Possibly this organism occurred more frequently at an earlier stage but in numbers below the threshold of detection. The large number of litters used make it unlikely that systematic selection of more heavily-infected littermates occurred later in the experiment. Ideally, the same rat should be examined after different intervals, but methods for in-rlioo sampling of plaque and estimation of caries have yet to be propounded. A predilection by Strep. mutans for more advanced carious lesions (de Stoppelaar. van Houte and Backer Dirks, 1969; Loesche and Syed, 1973) makes it unlikely that false negative results
occurred as a result of the disappearance of the organism from an established lesion. Mean values for both the penetration and linear extent of caries on the buccal surface tended to decrease when the rats were fed the cariogenic diet for long periods. Further study has confirmed this trend and will be reported separately. Henry and Navia (1969) noted a similar effect, though Larson and Zickus (1972) did not. My investigation supports a large body of other evidence that Strrp. muruns plays a role in caries. Superimposing Strep. muta~s on a conventional oral Hora raises caries incidence particularly on the buccal surface and in the distal fissures of rat teeth (Konig. Guggenheim and Muhlemann. 1965; K&rig, Larson and Guggenheim. 1969: Ranke and Ranke, 1971; Regolati. Guggenheim and Muhlemann. 1972). A comparison between the results in the studies mentioned and mine is possible because experimental details such as the pre-weaning regimen, the cariogenie diet used and the duration were all similar, and because in all studies caries was evaluated by the method of Konig cr al. (I 958). The difference in caries incidence between rats infected with Strep. mutans and non-infected animals was much greater when the organism was deliberately introduced than when it was acquired by natural means. Whilst caution is required in extrapolating results from one strain of rat to another. it would seem that super-infecting with Strep. mutans may unduly emphasize its importance m caries aetiology. A lack of correlation between the total CFU or the relative proportion of Strep. mutans in plaque and caries is m accord with similar observations in the Sprague-Dawley rat (Huxley. 1977b). Cartes data from teeth that were sectioned and scored by the method of Konig er al. (1958) were included to facilitate comparison with the results of other Investigations. It was necessary to assume that, if Strep. mutanS occurred somewhere on the lower right first molar tooth. it ;Yould also be present on the left molars. Obviously. rt is more desirable to relate caries and plaque at the same sites than to extrapolate from one side of the mouth to the other and from one tooth site to another. For this reason. a caries assessment method based on penetration of the lesion at single sites was devised. There is substantial agreement between the two methods of scoring in the degree to which Strep. mutans augments caries. A relatively high level of caries in rats not infected with Strep. mutans corroborates similar findings in the Wistar rat (Huxley, 1975). It contrasts markedly with the hamster. where animals without Strep. mutans develop little caries (Fitzgerald and Keyes. 1960; Krasse, 1965: Zinner rt ul.. 1966). and possibly the macaque monkey too. because Bowen (I 969) found a dramatic rise in caries after inoculating monkeys with Strep. mutans. In man. a confused picture exists, some studies indicate that most carious lesions harbour Srrep. mutans (e.g. Littleton, Kakehashi and Fitzgerald, 1970; Shklair er al., 1972; Rogers, 1973; Ikeda, Sandham and Bradley, 1973). In others, substantial numbers of lesions are not associated with the organism (Loesche et al., 1975; Mikkelsen and Paulsen. 1976). Until it is decided whether there are two or more separate patterns of caries in man, a
Strep. mutans
rational decision cannot of various experimental
be made as to the suitability animal models.
Acknowledgernrnts-This investigation was supported by a grant from the Medical Research Council of Canada. I thank S. Chakrabarty. G. Young and J. Rzeszutek for their technical assistance. REFERENCES
Aranki A.. Syed S. A.. Kenney E. B. and Freter R. 1969. Isolation of anaerobic bacteria from human gingiva and mouse caecum by means of a simplified glove box procedure Appl. Microhiol. 17, 568-576. Bowen W. H. 1969. The induction of rampant dental caries in monkeys (Macaca irus). Caries Res. 3. 227-237. Caldwell R. C. and Hunt D. E. 1969. A comparison of the antimicrobial activity of disclosing agents. J. dent. Res. 48, 913-915. Carlsson J. 1968. A numerical taxonomic study of human oral streptococci. Odont. Rery 19. 137-160. de Stoppelaar J. D.. van Houte J. and Backer Dirks 0. 1969. The relationship between polysaccharide-producing streptococci and smooth surface caries in 13-year old children. Caries Res. 3, 190-199. Edwardsson S. 1968. Characteristics of caries-inducing human streptococci resembling Streptococcus mutans. Archs
oral Biol.
13, 631-646.
Edwardsson S. 1970. The caries-inducing property of variants of Srreptococcus mutans. Odonr. Rwy 21, 153-157. Facklam R. R. 1974. Characteristics of Streprococcus mutans Isolated from human dental plaque and blood. Int.
J. sysr. Bact. 24, 313-319.
Fitzgerald R. J. and Keyes P. 1960. Demonstration of the etiologic role of streptococci in experimental caries in the hamster. J. Am. denr. Ass. 61. 9-19. Green R. M. and Hartles R. L. 1966. The effects of differing high-carbohydrate diets on dental caries in the albino rat. Br. J. Nurr. 20, 317-323. Henry C. and Navia J. M. 1969. Sodium trimetaphosphate influence on the early development of rat caries and concurrent microbial changes. Caries Rex 3, 326-338. Huxley H. G. 1972. The recovery of microorganisms from the fissures of rat molar teeth. Archs oral Biol. 17, 1481-1485.
Huxley
H. G. 1975. An investigation
into the prevalence
of Streptococcus mutans in Wistar rats, its relationship to the initiation of dental caries. and the effect upon its presence of the diet fed before weaning. Archs oral Biol.
20, 351-357.
Huxley H. G. 1977a. The cariogenicity of dietary sucrose at various levels in two strains of rat under unrestricted and controlled-frequency feeding conditions. Caries. Res. 11. 237-242. Huxley H. G. 1977b. The relationship between plaque bacteria and dental caries at specific tooth sites in rats. In: Microbial Aspecrs of Dental Caries (Edited by Stiles H. M.. Loesche W. J. and O’Brien T. C.). Vol. 3. pp. 773-784. Information Retrieval Inc., Washington. DC. Ikeda T.. Sandham H. J. and Bradley E. L. 1973. Changes in Streptococcus murans and lactobacilli in plaque in relation to the initiation of caries in negro children. Archs oral Biol. 18, 555-566. Keys P. 1958. Dental caries in the molar teeth of rats. II. A method for diagnosing and scoring several types of lesions simultaneously. J. dent. Res. 37, 1088-1099.
707
an Id caries in rats
Keyes P. H. and Jordan H. V. 1964. Periodontal lesions in the Syrian hamster-III. Findings related to an infectious and transmissible component. Archs oral Biol. 9, 377-400.
Kiinig K. G., Marthaler T. M. and Miihlemann H. R. 1958. Methodik der Kurzfristig erzeugten RattenKaries. Dt. Zahn-Mund
u. Kieferheilk.
29. 99-127.
KGnig K. G.. Guggenheim B. and Miihlemann H. R. 1965. Modifications of the oral bacterial flora and their influence on caries in the rat. II. Inoculation of a cariogenie streptococcus and its effect in relation to varying time of depression of the indigenous flora. He/c. odont. Acta 9, 130-134.
KBnig K. G.. Larson R. H. and Guggenheim B. 1969. A strain-specific eating pattern as a factor limiting the transmissibility of caries activity in rats. Archs oral Biol. 14, 91-103. Krasse B. 1965. Effect of caries-inducing streptococci in hamsters fed diets w“h sucrose or glucose. Archv oral Biol. 10, 223-226. Larson R. H. and Zickus C. S. 1972. Patterns of dental caries in OsborneeMendel and NIH Black rats in relation to length of the caries test period. J. dent. Rex 51, 1375-1381. Littleton N. W., Kakehashi S. and Fitzgerald R. J. 1970. Recovery of specific “caries-inducing” streptococci from carious lesions in the teeth of children. Arch: (oral Biol. 15. 461463. Loesche W. J., Hackett R. N. and Syed S. A. 1972. The predominant cultivable flora of tooth surface plaque removed from institutionalized subjects. Archs orai Biol. 17, 131 l-1325. Loesche W. J. and Syed S. A. 1973. The predominant cultivable flora of carious plaque and carious dentine. Caries Rex 7, 201~216. Loesche W. J., Rowan J.. Straffon L. H. and Loos P. J. 1975. Association of Srreprococcus nwtuns with human dental decay. Infect. Immun. 11, 1252-1260. Mikkelsen L. and Poulsen S. 1976. Microbiological studies on plaque in relation to development of dental caries in man. Caries Res. 10, 178-188. Perch B., Kjems E. and Ravn T. 1974. Biochemical and serological properties of Streptococcus mutans from various human and animal sources. Acta path. micro&o/. stand. Section B 82. 357-370. Ranke B. and Ranke E. 1971. Untersuchungen zur Kariogenitlt extrazellullre Polysaccharide bildener Streptokokken aus menschlicher Zahnplaque. Dt. zahniir:t/. 2. 26, 29-36.
Regolati B.. Guggenheim B. and Miihlemann H. R. 1972. Synergisms and antagonisms of two bacterial strains superinfected in conventional Osborne-Mendel rats. He/u. odonr. Acta. 16, 84-88. Robrish S. A.. Grove S. B.. Bernstein R. S.. Marucha P. T.. Socranskv S. S. and Amdur B. 1976. Effect of sonic treatment on pure cultures and aggregates of bacteria. J. r/in. Microbial.
3, 474479.
Rogers A. H. 1973. The ecology of Srreptococcus mutans in carious lesions and on caries free surfaces of the same teeth. Aust. dent. J. 18, 226-228. Shklair I. L.. Keene H. J. and Simonson L. G. 1972. Distribution and frequency of Streptococcus mutans in cariesactive individuals. J.-dent. Rds. 51, 882. Zinner D. D.. Jablon J. M.. Aran A. P.. Saslaw M. S. and Fitzgerald R. J. 1966. Comparative pathogenicity of streptococci of human origin in hamster caries. Archs oral Biol. 11, 1419-1421.
