The effect of inoculating strains of Streptococcus mutans and Streptococcus sanguis upon caries incidence and bacterial content of plaque in rats

Arch oral Bid.

Vol.18,pp.1215-1226,

1973.

Pergamon Press. Printed in Great Britain.

THE EFFECT OF INOCULATING STRAINS OF STREPTOCOCCUS MUTANS AND STREPTOCOCCUS SANGUIS UPON CARIES INCIDENCE AND BACTERIAL CONTENT OF PLAQUE IN RATS H. G. HUXLEY University of Otago Dental School, P.O. Box 647, Dunedin, New Zealand Summary-In two experiments in which the oral flora had been depressed with antibiotic the inoculation of Streptococcus sunguis raised fissure-caries incidence above that of control rats, but significantly less than did the inoculation of Streptococcus mutans. Both organisms were shown to colonize preferentially on the teeth, with Strep. mutans accounting for a higher proportion of the bacteria in fissure and approximai plaque than Strep. sanguis. A weak positive correlation was noted between caries and both the total number of bacteria recoverable from plaque and the total recovery of Strep. mutans. In a third experiment, Strep. mutans and sanguis were inoculated into rats with an intact oral flora and a positive correlation was found between the total recovery of Strep. mutuns and caries, but a negative correlation between Strep. sanguis and caries. The content of Strep. sunguis in plaque tended to be inversely proportional to the content of Strep. mutans.

INTRODUCTION CONSIDERABLEevidence exists that caries may be a naturally transmissible disease among rodents (KEYES, 1960; KOENIG,MARTHALER and MUHLEMANN, 1963; LARSON, 1965; STRALFORS,CARLSSONand SUNDQVIST,1970).

The inoculation of Streptococcus

mutans into the mouths of experimental animals has repeatedly been shown to enhance

caries (reviewed by FITZGERALD and JORDAN, 1968; KOENIGand GUGGENHEIM, 1968; and JABLON, 1968; KEYES, 1968). The tooth is the preferred oral site of this organism in the hamster (KRASSE and EDWARDSSON, 1966) and in man (CARLSSON, 1967). KRASSEet al. (1968), DESTOPPELAAR, VANHOUTEand BACKERDIRKS(1969) and LITTLETON,KAKEHASHIand FITZGERALD(1970) noted a strong positive correlation between Strep. mutans and caries in man, although this does not necessarily indicate a causal relationship. However, ROGERS(1969) and DUANY,JABLONand ZINNER(1972) could not demonstrate such a relationship, whilst surveys of the prevalence of Strep. mutans have provided widely differing results (KRASSE et al., 1968; DE STOPPELAAR et al., 1969; JORDAN, ENGLANDER and LIM, 1969; ROGERS,1969; DUANYet al., 1972). Difficulty has also been experienced in demonstrating the establishment and transmission of Strep. mutans in man (KRASSE et al., 1967; JORDAN et al., 1972). Bacteria other than Strep. mutans can cause caries in otherwise germ-free animals (FITZGERALD,JORDANand ARCHARD,1966; ROSEN,LENNEYand O’MALLEY, 1968 ; KELSTRUPand GIBBONS,1970; DUANY, ZINNERand LANDY, 1971; LLORY, GUILLO ZINNER

1215

1216

H. G.

HUXLEY

and FRANK, 1971; MIKX et al., 1972). Of particular interest is Streptococcus sanguis, an extracellular polysaccharide-producing organism whose preferred habitat is the tooth (CARLSSON,1967; VAN Hour& GIBBONSand BANGHART,1970; VAN HOUTE, GIBBONSand PULKKINEN,1971). This organism appears to be more widespread among human populations than Strep. mutans (CARLSSON,1967; ROGERS,1969; DE STOPPELAARet al., 1969; VANHOUTEet al., 1970; VAN HOIJTEet al., 1971), but its cariogenic potential in animal test systems, although not as thoroughly tested as Strep. mutans, appears to be less (GUGGENHEIM,1968; RANKE and RANKE,1971; MIKX et al., 1972). In view of the diverse and sometimes conflicting evidence relating Strep. mutuns and Strep. sunguis to caries, this investigation aimed to study further this relationship in an albino strain of the Wistar rat. This rat was selected because both organisms occur from time to time in its oral flora, and it seemed logical to test first the cariogenic potential of indigenous strains of Strep. mutans and Strep. s’anguis. Techniques for assessing the caries potential of bacteria range from the inoculation of the organism to be tested into otherwise germ-free animals, through the inoculation into animals whose oral flora has been depressed with antibiotics, to inoculation into animals retaining an intact oral flora. Whilst the former two techniques enable an assessment to be made of the cariogenic potential of an organism in the absence or relative absence of competing bacteria, an intact flora is present in vivo and must, therefore, not be disregarded. Three experiments were performed, the object of the first being to investigate the cariogenic potential of inoculated strains of Strep. mutuns and Strep. $unguis and to determine their preferred oral site of colonization. Multiple antibiotics were used to suppress the oral flora of young rats before the eruption of their molar teeth; then, at the start of the experiment, antibiotic administration ceased and inoculation commenced. In this way, it was possible to avoid inoculating antibiotic-resistant strains of bacteria. A second experiment of similar design attempted to correlate more closely the bacterial content of fissures and caries incidence. In a third experiment, Strep. mutuns and Strep. sunguis were inoculated either singly or in various sequences into the mouths of rats retaining an intact oral flora and again an attempt was made to correlate bacteriological observations and caries incidence.

EXPERIMENTAL

METHODS

Maintenance of animals Litters of albino rats of the Wistar strain aged 10-12 days were obtained, together with their dams, from the University of Otago Animal Breeding Station. Each dam with her litter was housed in a stainless steel-mesh cage measuring 12 in. x 12 in. x 6 in. A non-cariogenic finely-powdered diet (PWl) (HUXLEY, 1973a) was fed. After weaning, and at the start of experiments, the young rats were distributed randomly (on a littermate basis) among treatment groups, using numbered individual containers and a table of random numbers. These rats were housed in cages similar to those used for rearing litters, except for the addition of a central partition, two rats being placed in each compartment. The cariogenic diet 2000H (HUXLEY, 1973b) was fed. Experiments were performed, at different times, in an experimental room with 12 hr artificial lighting daily and at 21-24°C. Cages comprising a single treatment were close together, but different treatment groups were widely spaced and disposable barriers were interspersed between them.

CARIESANDPLAQUEBACTERIA IN RATS

1217

Bacteria The strains of Strep. mutans and Strep. sanguis used were previously isolated from this strain of rat and characterized by HUXLEY(1972). Maintenance was by bi-weekly transfer in trypticase soy broth (B.B.L.). For inoculation, analiquot of the stock culture was grown overnight in 10 ml trypticase soy broth and the resultant heavy suspension was introduced into the mouth by means of an absorbent cotton-wool swab. Water bottles were withdrawn from the cages for a period of 1 hr after inoculations. Treatment of jaws In one experiment, the lower jaws of separate animals were used for the evaluation of caries and recovery of plaque bacteria whilst in two further experiments these procedures were carried out on separate half-mandibles from the same animal. Rats were decapitated and plaque was recovered from the freshly dissected jaws. Remaining jaws were stored in ethanol, stripped of soft tissue and allowed to dry. Assessment of caries Buccal and lingual caries (white decalcified areas) was assessed by examination of the dried intact jaws at x 15 magnification, following which jaws were sectioned, using an internal-rim rotary cutting machine, and stained with Schiff’s reagent. Caries occurring in the fissures and on the approximal surfaces of lower first and second molar teeth were classified as T-, B- or C-lesions according to the criteria of K~NIG, MARTHALERand MUHLEMANN(1958) and GREEN and HARTLES(1966). Recovery of bacteria Plaque was obtained from the central and distal fissures of lower first and second molar teeth and from the approximal surfaces of these teeth as described by HUXLEY(1973a). After combining plaques from these locations in 4 ml of brain-heart infusion broth (Oxoid) containing Tween 80, they were dispersed by shaking for 3 min followed by homogenization at 14,000 rev/min for 30 set in an MSE homogenizer. Buccal and lingual plaque was removed by means of a small curette and treated in the same manner. Bacteria were scraped from the dorsum of the tongue, the oral mucosa and the throat with a dental plastic carving instrument. Samples so obtained were shaken for 5 min in 4 ml of brain-heart infusion broth containing Tween 80. Preliminary work indicated that satisfactory dispersion of bacteria from these sites was achieved without the need for homogenization. A small sample of faecal material was taken from the caecum and treated in the same manner. Serial ten-fold dilutions of samples from all sites were made in brain-heart infusion broth and appropriate dilutions were plated in duplicate on mitis-salivarius agar (Oxoid) and trypticase soy agar (B.B.L.). Viable plate counts were performed on the growth after 48 hr incubation at 37°C in an atmosphere of 95 per cent nitrogen and 5 per cent carbon dioxide. S~aKWical treatment of results An analysis of variance was performed on results for caries incidence and the total recovery of viable bacteria, the levels of significance being determined by the F-test. In every instance, the variation attributable to the littermate blocks was separated from the residual error. The number of rats with approximal or buccal and lingual caries was examined by means of Fisher’s test of exact significance. A possible association between caries incidence and bacteriological findings was investigated by regression analysis. Carious lesions occurring in the mesial fissures of lower first molar teeth were not included in this test because bacterial samples were not taken from this site, but approximal lesions on the distal surface of the lower first molar and mesial surface of the lower second molar were. The significance of r was assessed by the t-test. Experiment 1 Eight litters of six young were used. Depression of the oral flora commenced at 16 days of age, shortly before the eruption of lower first molars, and was achieved by the inclusion in the drinking water of penicillin V (1 mg/ml), streptomycin (2 mg/ml), Polymyxin (0.1 mg/ml) and Nystatin (1 mg/ ml). The dam was removed when the young were l&days-old, but administration of antibiotic continued until 21 days, when the young were distributed among three treatment groups. Treatments were (1) control, no inoculation (2) inoculation of Strep. mutans (3) inoculation of Strep. sang&. Following the allocation of rats to treatment groups, the diet was changed to 2000H and the first of

1218

H. G.

HUXLEY

five consecutive daily inoculations of bacteria administered. One rat from each littermate pair in each treatment group was kept on diet 2000H for 42 days and its jaws were used for the assessment of caries. The other littermate in each treatment was killed at varying intervals after the start and its jaws were used for bacteriological sampling. A staggered series of killing dates was adopted, each involving one control rat and two rats inoculated with either Strep. mutans or Strep. sunguis. The control rat and one of the inoculated rats were used for bilateral fissure plaque examination, whereas the other inoculated rat was used for sampling fissure plaque, buccal and lingual plaque, tongue, oral mucosa, throat and faeces. Rats inoculated with Strep. mutans were examined 14, 25, 32 and 39 days after the start, while rats inoculated with Strep. sanguis were examined at 11, 23, 30 and 37 days.

Experiment 2 Twelve litters of six young were used. Penicillin V (1 mg/ml) was included in the drinking water between the ages of 12-18 days, after which the dam was removed and distilled water was given to drink. When the young rats were 22-days-old, they were distributed among three treatment groups, viz. (1) control (2) inoculation of Strep. mutans (3) inoculation of Strep. sanguis, and fed diet 2000H. Inoculation of bacteria occurred on three consecutive iaily occasions starting at 22 days. Diet 2000H was fed for a further 30 days after the last inoculation, making 33 days total. At the termination of the experiment, which occurred on two separate days to accommodate varying birth dates of the litters, one half-mandible from each rat was used for the assessment of caries whilst the other was used for the recovery of fissure plaque. The half-mandible from the same side was used for the same purpose throughout a littermate experimental block, but an alternating sequence of left and right was used for different blocks.

Experiment 3 Eight litters of six young were used and, upon weaning at 21-23 days old, they were distributed among six treatment groups. Treatments are shown in Table 1. The mixed inoculum of Strep. mutans and Strep. sanguis was obtained by pooling equal volumes of broth cultures of the single organisms exhibiting similar densities of growth. Diet 2000H was fed and daily inoculations commenced. Four treatment blocks were fed 2000H for 15 days and the remaining four for 30 days. From each rat onehalf mandible was used for the assessment of dental caries and the other for recovery of fissure plaque.

2

Treatment group

4

3

Inoculation days 1-3

Nil Strep. sanguis

Sfrep.

sanguis

Strep.

Inoculation days 4-6

Nil Nil

Strep.

mufans

Nil

mutans

6 Strep.

mumm

Strep.

sanguis

Strep.

sanguis

mUfanS Nil

+

Strep.

RESULTS

Experiment

1

The incidence of caries and the total number of bacteria recoverable from plaque appear in Table 2. Rats inoculated with Strep. sang& developed a significantly higher number of T-lesions than the controls (p < O-01) but significantly fewer T-lesions and B-lesions than rats inoculated with Strep. mutans (p < 0.05). There was no significant difference between treatments in the total number of viable bacteria recoverable from plaque after 42 days. No significant correlation was noted between caries and either the total recovery, or the percentage, of total growth accounted for by Strep. mutans or Strep, sanguis. The distribution of Strep. mutans and Strep. ganguis in various oral sites and in the faeces was expressed as the percentage of total growth, on trypticase soy agar, accounted for by these organisms and values obtained are presented in Table 3. Strep. sanguis was not recovered from control rats or rats inoculated with Strep. mutans, whilst Strep. mutans was not present in rats inoculated with Strep. sanguis but was recovered in small quantity from the plaques of three control animals.

CARIES AND

PLAQUE

BACTERIA

1219

IN RATS

TABLE 2. MEAN INCIDENCEOF TOTAL PISSUIU?LESIONS(T-LESIONS), MORE ADVANCED FISSURE LESIONS (B-LESIONS), NUMBEROP RATS W,TH APPROXIMALOR BUCCAL AND LINGUAL CARIES,TOOETHERWITH TOTAL.RECOVERYOP BACI’KUA PROMmssrnws IN EXPERIMENT1

T-lesions Standard deviation B-lesions Standard deviation No. of rats with approximal T-lesions (Total 8) No. of rats with buccal and lingual decalcification Total no. of bacteria recovered from plaque (X 106) Standard deviation

Control

Inoculated with Slrep. ?lZ”,Cllls

Inoculated with Slrep. san.euis

2.6 I.7 0.8 0.8

7.5 2.3 4.3 2.4

0 0

0’

t

1.97 1.78

3.48 1.46

4.09 4.70

5.0 1.4 2.9 1.9

Tns~i? 3. THE PERCENTAGEOF TOTAL OROWTH ACCOUNTEDFOR BY Strep. mutam AND Strep. sanguis M VAIUOUS ORAL SlTEs AND THE FAKES IN RATS INOCU‘A~D WITH ON!? OR OTmR ORGANLSMIN EXPERIMENT I

Site

No. of rats with Strep. nlu,ans (Total 4)

No. of rats with Slrep. sanguis (Total 4)

Proportion of swep. mutons

Proportion [of strep. sanguis

TClllgUe Oral muc0sa Buccal and lingual plaque Fissure plaque

4 :

5.5 8-3 45.8

1 :

7.8 4.9 23.5

Thyo;ttal 8 rats) FXCes

8 :

75.4 0.2 1.1

! 0

25.0 1.4 0.0

Experiment 2

One litter died before weaning and therefore results presented in Table 4 are derived from 11 litters. The incidence of T-lesions and B-lesions, and the total recovery of bacteria shown in Table 4 refer to half-mandibles only; therefore the values shown must be doubled to be comparable with those in Table 2. Rats inoculated with Strep. mutans or Strep. sunguis developed significantly more T-lesions than control rats (p < 0.01) and also more B-lesions. The total number of bacteria recoverable on trypticase soy did not differ significantly between control rats and rats inoculated with Strep. sanguis, but significantly more were recoverable from rats inoculated with Strep. mutans (p < 0.05). A statistically significant correlation was found between the total number of bacteria recoverable and T-lesions (r = +0*37, p < O*OS).A significant correlation was also observed between the total recovery of bacteria and B-lesions TABLE 4. THE MEAN INCIDENCEOF T-WIONS AND B-LESIONS, THE NUMBEROF RATS WITH APPROXLMALAND BUCCAL AND LINGUAL CARIES, THE TOTAL.NUMBEROF BACTXRIA RECOVERED FROM PISSURESAND THB PERCENTAGEOPGROWTH ACCOUNTED FOR BY Slrep. mufans AND Strep. sanguis IN EXPERIMENT2

Control T-lesions Standard deviation B-lesions No. of rats with approximal T-lesions (Total 11) No. of rats with buccal and lingual decalcification (Total 11) Total recovery of bacteria from fissure and approximal plaque ( x 106) Standard deviation Percentage of total growth accounted for by Strep. mwans Percentage of total growth accounted for by S:rep. sanguis

1.5 1.2 0.2 0 0 0.78 0.77 0 0

Inoculated with Strep.

muIL?nS 3.5 ::; :

;:g 76.3 0

Inoculated with Slrep. sanguis 2.8 0.8 1.8 2 2 1.32 1.26 2.8

1220

H. G. HUXLEY

(r = +0*47, p < 0.01). The correlation

between Strep. sanguis and caries was not significant, that between the total recovery of Strep. mutans and B-lesions was (r = j-O-65, p < 0.01).

Experiment 3

Data for caries incidence and bacteriological findings in plaque from half-mandibles are presented in Table 5. The prevalence of approximal, buccal and lingual caries, the total number of bacteria recovered on trypticase soy agar and the proportion of total growth accounted for by Strep. mutans did not differ significantly between different treatment groups. Significant differences found in caries incidence and the recovery of Strep. mutans are shown in Table 6. A possible association between caries and various parameters of plaque was examined by regression analysis and a summary of significant observations appears in Table 7.

Inoculated organism No. of rats T-lesions Standard deviation B-lesions Standard deviation No. of rats with approximal T-lesions No. of rats with smooth surfacx caries Total no. of bacteria recoverable x 106 Standard deviation No. of rats with Strep. sanguis Proportion of total growth accounted for by Strep. sanguis Proportion of growth on mitis salivarius accounted for by Strep. sanguis No. of rats with strep. mutans Proportion of total growth accounted for by Strep. mutans Proportion of growth on mitis salivarius accounted for by strep. mutanS

T-lesions 1. Un-inoculated 2. Un-inoculated 3. Un-inoculated B-lesions 1. Un-inoculated 2. Un-inoculated 15 days us 30 days Percentage

NolIe

strep. sanguis

strep. son&s A strep. mutnm

strep. m”tanS

II

I

i.3 0.8 4.4 1.4

4.3 0.8

i.1 1.1

i.9

$8

2.9 1.4

3.6 I .9

1.3

strep. tPI”t*llS c strep. sanguis :.9 1.2 4.1 18

Strep. sanguis + strep. MutanS

i:; 3.1 1.3

6

5

4

6

5

3

5

4

2

5

S

4

4.99 3.46

340 2.77

9.42 7.85

5.18 3.89

3.65 2.82

3

7

8

2

4

7

o-7

0.9

IO.5

IS.1

1.6

15.2

5.3

3.6

10.81 12.41

22.4

6.8

I.6

1.5

7

8

8

8

8

8

56.9

38.2

54.1

50.1

64.1

39.6

57.9

60.7

81.3

87.2

82,8

65.1

rats vs rats inoculated with Strep. smguis rats YS rats inoculated with Strep. sanguis followed by Strep. mutanS rats YSrats inoculated with Strep. sanguis and Strep. mutans simultaneously

p < 0.05 p < 0.01 p < 0.05

rats vs rats inoculated with Strep. sanguis followed by Strep. mutans rats YS rats inoculated with Strep. sanguis and Strep. mutmssimultaneously

p < 0.02s p < 0.05

of total growth accounted for by Strep. mutans

p < 0.05

Comparison

Value

Total recoveryof Strep.mutamvs T-lesions Total recoveryof Strep. smguis vs B-lesions Proportionof total growthaccountedfor by Strep. sanguis vs T-lesions Proportionof total growthaccountedfor by Strep. sanguis vs B-lesions Proportionof growthon mitissalivariusaccountedfor by Strep. mutm~ YS proportionof growthon mitissalivariusaccountedfor by Strep. sanguis

of I

‘-g::; -0.30 -0.32

Simificance p c 0.05 p < 0.01

-031

DISCUSSION

Depression

of the oral flora

Inoculation of bacteria under conditions of “relative gnotobiosis” (GUGGENHEIM, and M~~HLEMANN,1965) involves a continuous depression of the oral flora with antibiotic and the use of antibiotic-resistant strains. The technique described in this paper avoids the use of such organisms which do not thrive in competition with normal bacteria (FITZGERALD and KEYES,1963; KRASSEet al., 1967). GUGGENHEIM,K~NIG and REGOLATI(1969) criticized a similar technique because of the possibility of contamination, particularly of control animals. Whilst this possibility is real, it is easily detected using the plaque recovery method described and, moreover, can be avoided with careful experimentation. Starting antibiotic administration before the eruption of molar teeth ensured that suppression of the fissure bacteria was not hindered by poor diffusion of antibiotic through an established plaque. Using penicillin alone to suppress the oral flora avoided the diarrhoea that accompanied the use of multiple antibiotics. The loss of one litter before weaning was due to poor lactation on changing diets and was not connected with penicillin administration. In the second experiment, the inoculation of bacteria did not commence immediately after cessation of antibiotic administration, thereby reducing the risk of “carry-over” of antibiotic. No study was made of the flora remaining in fissures after the use of antibiotic, therefore the bacteria with which inoculated streptococci had to compete were unknown. K~NIG

Total recovery of plaque bacteria

The assumption that the total recovery of bacteria on trypticase soy agar is a reliable indication of the total microbial flora in the sites sampled is open to question. By the choice of one medium and one set of incubationconditions, certain oral bacteria will be excluded. Also, the technique for obtaining plaque is not claimed to give a total recovery, particularly from the approximal region. Deficiencies in the sampling method could account for the wide individual variation in the total recovery of bacteria recorded in all experiments. In order to relate caries incidence to the bacteriological findings, it was necessary to assume in Experiment 1 that both caries incidence and the plaque flora were similar within littermate animals. MARTHALER (1965) indicated that the caries experience of littermate rats is similar, but there appears to be no information on the similarity or otherwise of the plaque flora. In Experiments 2 and 3, therefore, the comparison was made within individual rats, since there is evidence to support the symmetry of caries A.O.B. 18/10--s

1222

H. G. HUXLEY

over a group of animals (WHEATCROFT et al., 1951; SHAWand SWEENEY, 1956; KINKEL et al., 1960), and also the fissure plaque flora (HUXLEY,1973a). Ideally, it would be desirable to recover bacteria from the same fissures that are assessed for caries; however, the sampling method described precludes the subsequent sectioning of teeth. The low correlation obtained in two experiments between caries and the total number of bacteria recoverable might be spurious and due to inadequate sampling methods, but might also indicate that one portion of the flora is of especial importance for caries. Effects of Strep. sanguis Streptococcus sanguis preferentially colonized the teeth when inoculated into the mouths of rats whose oral flora had been depressed with antibiotic. The explanation for this distribution might reside in a marked affinity by Strep. sanguis for cleaned tooth surfaces (VANHOUTEet al., 1970) and existing dental plaque (VANHOUTEet al., 1971). Its absence in faeces is in agreement with observations in man by VANHOUTE, JORDANand BELLACK(1971). Caries incidence was raised as a result of inoculation of Strep. sang&s, and the results of Experiment 2 in particular suggest that the reason for this lay in the increased number of bacteria colonizing the fissures and approximal surfaces. Although there was no significant correlation between Strep. sanguis and caries in the first two experiments, in both cases small numbers of animals were involved, whilst a higher value of r was obtained in the comparison between the total recovery of Strep. sanguis and caries than between the percentage of total growth accounted for by Strep. sanguis and caries. The observation that Strep. sanguis is capable of raising the incidence of caries is not in agreement with the results of FITZGERALD(1968) or KRASSEand CARLSSON(1970), but supports the work of GUGGENHEIM(1968), RANKEand RANKE(1971) and MIKX et al. (1972). In the third experiment, Strep. sunguis apparently played little part in the carious process and three statistically significant values of r pointed to an inverse relationship between this organism and caries. This confirms the observations of DE STOPPELAAR et al. (1969) who also noted an inverse relationship between Strep. sanguis and caries. Efects

of Strep. mutans

Inoculation of this organism raised the incidence of caries more than the inoculation of Strep. sanguis. Streptococcus mutans colonized teeth preferentially and regularly accounted for a high proportion of the total bacterial content of plaque. The observation that the fissures of teeth contained a higher proportion of Strep. mutans than the buccal or lingual surface is in agreement with the work in man of IKEDAand SANDHAM (1971). An association between Strep. mutans and caries appeared to be a reflection upon the increased number of bacteria in plaque as revealed by two significant correlations between its total recovery from plaque and caries. The evidence from the first two experiments suggests that Strep. mutans might have an important aetiological role in caries and, unlike Strep. sanguis, this evidence tends to be supported by observations in rats whose oral flora had not been suppressed. It was of interest that Strep. mutans apparently became more predominant in plaque with age. Although the comparison

CARIES

between animals,

1223

ANDPLAQUE BACTERIA IN RAB

15 and 30 days in Experiment its result supports unpublished

3 was not made within littermate pairs of observations in this strain of rat and also

observations in man by IKEDA and SANDHAM(1971). The occurrence of Strep. mutans in faeces agrees with the work of FITZGERALD and KEYES (1960) in the rat and KILIAN, THEILADE and SCHI~TT (1971) in man, and provides one possible explanation for the production of caries in rodents by inoculating faeces or caging various strains together. Serologically, distinct varieties of Strep. mutans occur (BRATHHALL, 1969; COYKENDALL, 1970) with apparently different distributions in man (DUANY, ZINNER and JABLON, 1970; BRATHHALL, 1972) and different cariogenic potentials when inoculated into animals (DUANY et al., 1971). It would be of interest to compare the cariogenie potential in the albino Wistar rat of its naturally occurring strain of Strep. mutans with that of other strains.

Interaction between plaque bacteria The inverse relationship noted in plaque between Strep. mutans and Strep. sanguis supports the evidence in man of DE STOPPELAAR et al. (1969) and DE STOPPELAAR, VAN HOUTE and BACKER DIRKS (1970). CARLSSON (1971) found that, on incubation of a mixture of Strep. mutans and Strep. sanguis, Strep. mutans became the predominant organism. However, HOLMBERG and HOLLANDER (1972) observed that Strep. sanguis inhibited the growth of Strep. mutans. KBNIG, GUGGENHEIM and M~~HLEMANN(1965) and VAN DER HOEVEN et al. (1972) found that simultaneously inoculating and other bacteria into the mouths of rodents reduced caries.

Strep. mutans

Bacterial specljicity of caries The present work gives some support to the idea that Strep. mutans is of some importance in the causation of dental caries in rats, whereas Strep. sanguis plays a lesser role. Streptococcus mutans seems to become predominant in plaque a few weeks after feeding a cariogenic diet whereas caries is initiated within a few days (HUXLEY, 1971), therefore this organism may not be associated with the initiation of caries but rather with its progression. Further work is in progress to clarify this matter. Whether any property of Strep. instance the formation their cariogenic

mutans or Strep. sanguis other than the production

potential

of intracellular

and extracellular

was not investigated

polysaccharides,

of acid, for enhances

in this study.

Acknowledgements-This research was supported by a grant from the Medical Research Council of New Zealand. I also gratefully acknowledge a grant from the New Zealand Dental Research Foundation and thank Miss C. BARROW for her technical

assistance.

R&urn&Au cows de deux exptkiences, pendant lesquelles la flore buccale a Ctt inhiWe par administration d’antibiotique, I’inoculation de Strep#ococc~s sanguis augmente la frequence carieuse des fissures; cette augmentation est cependant moins Clew% qu’aprks inoculation de Strepococcus mutans. Les deux types de bacttkies colonisent les surfaces dentaires de facon pr&f%entielle, avec un nombre plus Clew5 de Strep. mutans

1224

H. G. HUXLEY dans les plaques des fissures et des surfaces proximales. Une corr&lation positive faible est not& entre les caries et, g la fois, le nombre total de batteries cultivables & partir de la plaque et le nombre de Strep. mutans. Au tours d’une troisieme expkrience, des Strep. mutans et Strep. sanguis sont introduits dans la cavitC buccale de rats avec une flore buctale habituelle et une corr&ation positiveest observQ entre le nombre total de Strep. mutans et la carie dentaire: ce rapport est n6gatif pour Strep. sanguis. Le contenu en Strep. sanguis de la plaque tend B &tre inversement proportionnel au nombre de Strep. mutans.

Zusammenfassung-In zwei Versuchen, bie welchen die Mundflora durch Antibiotika zurfickgedrgngt worden war, steigerte die Inokulation von Streptococcus sanguis das Auftreten von Fissurenkaries iiber das bei den Kontroll-Ratten beobachtet MaD hinaus; das Ergebnis war jedoch signifikant kleiner als nach Inokulation mit Streptoccocus mutans. Beide Organismen wachsen vorzugsweise auf Zlhnen, wobei Strep. mutans den griil3eren Bakterienanteil in den Fissuren- und Approximalplaques als Strep. sanguis ausmacht. Zwischen der Karies einerseits und der Gesamtzahl der aus Plaques wiedergewinnbaren Bakterien sowie der Wiederaullindbarkeit von Strep. mutans andererseits war eine schwach positive Korrelation vorhanden. In eimen dritten Versuch wurden Strep. mutans und Strep. sanguis in Ratten mit intakter Mundflora inokuliert. Es fand sich eine positive Korrelation zwischen der Riickgewinnung von Strep. mutans und Karies, dagegen eine negative Korrelation zwischen Strep. sanguis und Karies. Der Anteil von Strep. sanguis in den Plaques verhielt sich annlhrend umgekehrt proportional zum Anteil von Strep. mutans.

BRATHHALL,D. 1969. Immunodiffusion Streptococcus

mutans.

Odont.

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