Prevalence of Streptococcus sanguis and Streptococcus mutans in the mouth of persons wearing full-dentures

ArchsoralBiol. Vol. 14,pp.243-249,1969.Pergamon Press.Printedin Gt. Britain.

OF STREPTOCOCCUS SANGUIS AND STREPTOCOCCUS MUTANS IN THE MOUTH

PREVALENCE

OF PERSONS J.

WEARING

CARLSSON,*

FULL-DENTURES

G. S~~DERHOLM and

I.

ALMFELDT

Departments of Oral Microbiology and Periodontology, University of Lund, School of Dentistry, Malm(i, Sweden Summary-In 20 edentulous persons wearing full dentures the prevalence of Streptococcus sanguis, Streptococcus mutans and Streptococcus salivarius was studied in samples of the bacterial plaque on the dentures and in samples of the saliva. Streptococcus sanguis could be recovered from 17 and Streptococcus mutans from 14 of the subjects. These organisms constituted a higher percentage of the streptococcal flora in the denture plaque than in saliva. Seven subjects refrained from using their dentures for 2 days after which the number of these streptococci was so few in saliva that they could not be recovered with the methods used. Streptococcus salivarius was present in all 20 subjects and constituted a higher percentage of the streptococcal flora in the saliva than in the denture plaque. The composition of the streptococcal flora on the dentures was similar to that previously found on teeth. The accumulated evidence on the ecology of Streptococcus sanguis and Streptococcus mutans indicate that these streptococci can only be established in the mouth if solid surfaces such as on teeth and dentures are present. INTRODUCTION AMONG

the predominant bacteria in the dense microbial community on the teeth are

Streptococcus sunguis and Streptococcus mutans (CARLSSON,1965, 1967a). The pre-

valence of these streptococci in other sites within the oral cavity and changes related to the cleanliness of the teeth indicate that the teeth are their most favourable, if not their only, habitat in the mouth (CARLSSON, 1967a, b). The aim of the present investigation was to determine whether these streptococci are present in the mouth of edentulous persons and if their prevalence was influenced by the dentures worn by these persons. MATERIAL

AND

METHODS

Twenty edentulous persons wearing acrylic full-dentures were the subjects of this study. The dentures were taken out of the mouth and a specimen of the bacterial flora on the denture (denture plaque) was obtained by streaking a dry cotton swab over the teeth and base plate. The swab was immediately put into a test-tube containing 5 ml of a storage medium (VMG II, MILLER, 1966). About 1 ml of saliva (not specially stimulated) was at the same time collected into another test-tube containing the same medium. Each tube was treated in a Whirlimixer (Scientific Industries Ltd., England) for 30 sec. * Present address: School of Dentistry,

University of Ume&, Urn& 6, Sweden. 243

J. CARLSSON, G. SBDERHOLM AND I. ALMFELDI

244

Each sample was then diluted in IO-fold steps. From the dilutions (103-10s), 0.1 ml samples were taken and cultured on the surface of Mitis Salivarius Agar plates (Difco Laboratories, Detroit, Mich., U.S.A.). From each dilution one plate was incubated aerobically at 37°C for 48 hr, and another plate was incubated in a jar at 37°C under an atmosphere of 95 per cent Nz and 5 per cent CO, for 24 hr followed by aerobic storage at room temperature for 24 hr before examination (JORDAN,KRASSEand MILLER, 1968). The total number of colonies on each plate was counted, and the proportion of colonies resembling those of Streptococcus sang&, Streptococcus mutans and Streptococcus saharius (Group I, II and III, respectively, according to CARLSSON, 1967a, 1968a) was determined. One colony of each of those types which resembled Streptococcus sanguis and Streptococcus mutans was isolated from each person. The isolated strains were then tested for physiological characteristics as described by CARWON (1968a). Seven persons volunteered to refrain from using their dentures for 2 days. The bacterial specimens from these persons were cultured in addition to the Mitis Salivarius Agar also on MC-agar. This medium permits the isolation of Streptococcus mutans when it occurs in small numbers compared to other streptococci in the sample (CARLSSON, 1967c). During this 2-days period one or both of the dentures were relined, i.e. a new denture base material was added to the existing base, in order to get it refitted to the oral tissues and the opposing denture. The dentures were stored in water when not treated during this period. After the 2 days without dentures in their mouths the subjects supplied new

0 Strep.

sanguis Strep. mutans + Strep. salivarius

l

% 100

OB 0

z

O

P

,

+

g50: 4 +

+

0

.

a

z

++

00”

2

00

00

0

.

0

+

0

+

50 in saliva

FIG. 1. The percentage of Strep. sanguis, Strep. mutans and Strep. saIivarius in the total count of streptococci in samples of denture plaque and saliva from 20 persons wearing full-dentures.

PREVALENCE

sanguis

OF StreptOCOCCUS

AND

StfYptOCOCCUS

t?IUtatIS

IN THE

MOUTH

245

samples of saliva. The dentures were inserted and, after another 2-4 days, the streptococcal flora in saliva and on the dentures were studied as previously described. RESULTS

The prevalence of Streptococcus sang& Streptococcus mutans and Streptococcus salivarius in denture plaque and in saliva is shown in Fig. 1. The physiological characteristics of strains, considered to be Streptococcus sanguis and Streptococcus mutans, are given in Table 1. There were higher percentages of Streptococcus sanguis and Streptococcus mutans in the total account of streptococci in samples of denture plaque than in samples of saliva, the opposite being true for the prevalence of Streptococcus salivarius. Streptococcus salivarius was present in all 20 persons, while Streptococcus sanguis could be recovered from 17 and Streptococcus mutans from 14 persons. 0 Strep. a Strep. + Strep.

% 100

sanguis mutans salivarius + +

+ P 0

-

8

$

+

Y

0

0.1

l

/

0

without dentures

2

with 4-6

days

dentures

FIG. 2. Changes in percentageof Strep. sanguis, Strep. mutans and Strep. salivarius in the total count of streptococci in samples of saliva from seven persons refraining from wearing their dentures for 2 days and after replacement of the dentures. The method did not permit the recognition of Strep. sangius in lower percentage than 0.5 of the total streptococcal flora and Strep. mutans in lower percentage than O-01.

246

J. CARLSSON,G. S~DERHOLMAND I. ALMFELDT

TABLE 1. ~HYSIOUX~ICAL c~~a~cr~arsr~s OF Strep. sanguis AND Strep. mutans MOUTHOF 17 EDENTULOUS PERSONS WJWRING FULL DENTURES

Character

Haemolysis in blood pour-plate

Hydrogen peroxide formed Final pH in broth containing glucose

Group* No. of strains

alpha beta gamma 54.1 4.2 - 4.4 4.5 _(

Strep. sanguis 1:A 1:B 24 10

ISOLATED

FROMTHE

Strep. mutans II 42 5

0

0

16 26 2 24 18 0

0 5 5 5 0 0

0 0 24 24 4 4 23 17 21 24 5

42 41

:: 37 0 0

5 0 5 5 0 0 3 2 5 0 2

0 23 24 0

29 42 42 42

3 5 5 5

2 9 10 24 L-tyrosine

0 0

1 5

10 0 0 10 1 2 7

24 0 0 24 0 2 22

0 0 4 10 2 6 1 0 0 0 1 0 0 8 1

Final pH c 5.6 in broth containing D-IDaMitOl

n-sorbitol Trehalose Lactose Melibiose Rathnose Salicin Esculin Inulin Ammonia produced from L-arginine HCl Growth at 45°C Growth in broth containing Sodium chloride 6.5 % Sodium chloride 4 *O% Sodium chloride 3.0 % Growth at pH 5.2, acetate buffer Growth inhibited by Bacitracin (5 units) Sulphafurazol (100 pg) No strain contained catalase, fermented starch, or decarboxylated

$ 36 36

* According to CAIUWN (1968a).

In the seven subjects who refrained from wearing their dentures for 2 days, Streptococcus sang& could not be detected in the saliva and Streptococcus mutans

was found in only one of the subjects. When the dentures had been used again for 2-4 days, Streptococcus sanguis was found in all the subjects and Streptococcus mutans in five of them (Fig. 2). The prevalence of Streptococcus salivarius was not affected by the dentures. DISCUSSION

The composition of the streptococcal flora in the microbial aggregation on the teeth is unique within the oral cavity and is characterized by the predominance of Streptococcus sanguis and the presence of Streptococcus mutans (CARLSSON, 1965, 1967a).

PREVALENCE

OF

Streptococcus sanguis AND Streptococcus mutans m -rm

MOUTH

247

In the present study a streptococcal flora of similar composition was found on the surface of acrylic dentures worn by edentulous persons. Similar physiological varieties of Streptococcus sanguis were found on the dentures as previously described from dental plaque (CARLSSON,1965, 1967a, 1968a). This was also the case with the majority of the isolated strains of Streptococcus mutans. However, five isolated strains with cultural characteristics on Mitis Salivarius Agar as Streptococcus mutans were sensitive to sulphonamide and did not ferment sorbitol. Further work will show if these strains can be included in the Streptococcus mutans group. The specially composed medium (MC-agar, CARLSSON,1967c) for isolation of Streptococcus mutans proved to be a valuable requisite in the present study of the ecology of these streptococci. They could be recovered when present in very low numbers and were often the only bacteria growing on this medium. Both the teeth and acrylic dentures seem to provide an environment suitable for the metabolism of Streptococcus sanguis and Streptococcus mutans. The disappearance of these streptococci in detectable numbers from the mouth of the edentulous persons when refraining from using their dentures in the present study and the small number of these streptococci in saliva after cleansing of the teeth (CARLSSON,1967b) strongly suggest that teeth and dentures are the only habitat for these streptococci in the mouth. The prevalence of Streptococcus salivarius was not influenced by the dentures. This confirms previous findings that the preferable habitat for this organism is the tongue (KRASSE,1954; GIBBONS,KAPSIMALI.S and SOCRANSKY, 1964; CARLSSON,1967a). SHKLAIRand MAZZARELLA (1961) studied the effect of full mouth extraction on the oral microbiota. Lactobacilli and yeast disappeared after extraction of the teeth while the prevalence of Streptococcus salivarius was unaffected or showed some increase in individual cases. Five weeks after denture placement the incidence of these microorganisms was stabilized at pre-extraction levels. These findings, together with the results of the present study, raise the question of whether Streptococcus sanguis and Streptococcus mutans occupy similar ecological niches in the mouth as lactobacilli and yeasts. However, our knowledge on the metabolism and ecology of these organisms is too limited for any conclusion on their niches in the mouth. The lactobacilli have been found in large numbers in carious lesions (BOWEN,1967). They increase in number in the mouth after application of orthodontic appliances (OWEN, 1949; BLOOMand BROWN, 1964) and experimentally-applied plastic plates to the palate (ONISIand KONDO,1956). They decrease in number after removal of these devices and after restoration of the carious teeth (SHKLAIRet al., 1956; GREEN, WEISENSTEIN and PERMAN,1957). The carious lesions, the orthodontic appliances and the plastic plates may be means of food entrapment and in that way favour the establishment and growth of lactobacilli. On the other hand Streptococcus sanguis and Streptococcus mutans have been found to grow on cleaned smooth surfaces of the teeth (CARLSSON,1967a). It is possible that this colonization on the cleaned tooth surfaces is a parallel to what happens on solid surfaces in waters. Organic matter is adsorbed to the solid surface and in this bacteria

3. CARLSSON, G. S~DERHOLM ANDI. ALMFELDT

248

start to grow and multiply (ZOBELL, 1943 : BROCK, 1966). In addition to a concentration of the nutrients by the adsorption chemical

configuration

to the surface, this may also involve a change in

of the adsorbed

material (CUMPER and ALEXANDER, 1950) in

such a way that it can be utilized by the bacteria. The deposit formed glycoproteins

on cleaned tooth surfaces has a composition

in saliva (ARMSTRONG, 1967). It is possible

similar to the

that Streptococcus sanguis

and Streptococcus mutuns have the capacity of utilizing this deposit and that a similar deposit is formed on the dentures thereby providing nutrients to these organisms in the edentulous mouth. Bearing in mind the possible role of Streptococcus sanguis and Streptococcus mutans in the pathogenesis of caries (see CARLSSON, 1968b), it will be of interest to elucidate why these streptococci colonize on the teeth. The fact that they grow on dentures as well as on teeth may mean that no essential nutrients are provided thus for the study of the morphology,

bacteriology

by the teeth and

and chemica1 composition

of the

early deposit formed on solid surfaces in the mouth, acrylic plates instead of teeth can be used. This will in many respects facilitate such studies. R&m&-Chez 20 &dent&, portant des protheses completes, la frequence du Streptococcus sang&, du Streptococcus mutans et du Streptococcus salivarius est ttudiee au niveau des dentiers et dans la salive. Le Streptococcus sanguis et le Streptococcus mutans ont Bte retrouves respectivement chez 17 et 14 sujets. Ces microorganismes sont plus frequents au niveau des dentiers que dans la salive. Sept de ces sujets n’ont pas utilise leurs dentiers pendant deux jours et, darts ces cas, Ia salive est pratiquement depourvue de ces streptocoques. Le Streptococcus salivarius est retrouve chez tous les vingt sujets et constitue une proportion plus &levee des streptocoques dans la salive que sur les dentiers. La distribution des streptocoques, au niveau des dentiers, parait identique a celle rencontr& au niveau des dents. 11 semble que le Streptococcus sanguis et le Streptococcus mutans n’apparaissent en bouche que lorsque des surfaces solides telles que des dents ou des dentiers s’y trouvent. Zusammenfassung-Bei 20 zahnlosen Personen mit totalen Prothesen wurde die H&ttigkeit von Streptococcus sanguis, Streptococcus mutans und Streptococcus salivarius in Bakterienplaqueproben auf den Zahnersatzstiicken und in Speichelproben untersucht. Streptococcus sanguis konnte bei 17, Streptococcus mutans bei 14 der untersuchten Personen gewonnen werden. Diese Mikroorganismen bildeten einen hoheren Prozentsatz der Streptococcenllora in der Prothesenplaque als im Speichel. Sieben Personen trugen ihre Zahnersatzstiicke 2 Tage lang nicht. Danach war die Anzahl dieser Streptococcen im Speichel so gering, daR sie mit Hilfe der angewandten Methoden nicht festgestellt werden konnten. Streptococcus salivarius war bei allen 20 Personen vorhanden und bildete im Speichel einen hiiheren Prozentsatz der Streptococcenflora als in der Prothesenplaque. Die Zusammensetzung der Streptococcenllora auf den Prothesen war der friiher auf Ziihnen gefundenen Bhnlich. Es scheint so zu sein, da8 Streptococcus sanguis und Streptococcus mutans nur dann in der Mundhiile festgestellt werden k&men, wenn solide ObertXchen wie die auf Z&l-men oder Prothesen vorhanden sind. REFERENCES ARMSTRONG, W. G. 1967. The composition of organic films formed on human teeth. Caries Res. 1, 89-103. BLOOM, R. H. and BROWN,L. R., JR. 1964. A study of the effects of orthodontic appliances on the oral microbial flora. Oral Surg. 17,658-667. BOWEN,W. H. 1967. The lactobacilli in the saliva, plaque and carious dentine in Macaca irus. J. Path. Bact. 94,55-61.

PREVALENCE OF StiVptOCOCCUS SangUiSAND StreptOCOCCUS WU4taFlSINTHEMOUTH BROCK, T. D. 1966. Principles of Microbial Ecology. Prentice-Hall,

249

Englewood Cliffs, New Jersey. J. 1965. Zooglea-forming streptococci, resembling Streptococcus sanguis, isolated from dental plaque in man, Odont. Revy 16, 348-358. CARLSON, J. 1967a. Presence of various types of non-haemolytic streptococci in dental plaque and in other sites of the oral cavity in man. Odont. Revy 18, 55-74. CARLSSON,J. 1967b. Dental plaque as a source of salivary streptococci. Odont. Revy 18, 173-178. CARLSSON,J. 1967~. A medium for isolation of Streptococcus mutans. Archs oral Biol. 12, 1657-1658. CARLSSON,J. 1968a. A numerical taxonomic study of human oral streptococci. Odont. Revy 19, 137-160. CARLSSON,J. 1968b. Plaque formation and streptococcal colonization on teeth. Odont. Revy 19, suppl. 14. CUMPER,C. W. N. and ALEXANDER,A. E. 1950. The surface chemistry of proteins, Trans. Farad. Sot. 46,235-253. GIBBONS,R. J., KAPSIMALIS,B. and SOCRANSKY,S. S. 1964. The source of salivary bacteria. Archs oral Biol. 9, 101-103. GREEN,G. E., WEISENSTEIN, P. R. and PFRMAN,D. 1957. Studies on salivary lactobacilli and dental caries in children during a dentifrice test program. J. dent. Res. 36, 828-838. JORDAN,H. V., KRASSE,B. and MILLER, A. 1968. A method of sampling human dental plaque for certain “caries-inducing” streptococci. Archs oral Biol. 13, 919-927. KRASSE,B. 1954. The proportional distribution of Streptococcus salivarius and other streptococci in various parts of the mouth. Odont. Revy 5,203-211. MILLER, A. J. R. 1966. Microbiological examination of root canals and periapical tissues of human teeth. Odont Tidrbr. 74, No. 516, Special article. ONISI, M. and KONDO, W. 1956. Establishing an environment for growth of aciduric bacteria in the oral cavity. J. dent. Res. 35, 596-602. OWEN,0. W. 1949. A study of bacterial counts (lactobacilli) in saliva related to orthodontic appliances. Am. J. Orthodont. 35,672-678. SHKLAIR,I. L., ENGLANDER,H. R., STEIN, L. M. and KESEL, R. G. 1956. Preliminary report on the effect of complete mouth rehabilitation on oral lactobacilli counts. J. Am. dent. Ass. 53,155-158. SHKLAIR,I. L. and MAZZARELLA,M. A. 1961. Effects of full mouth extraction on oral microbiota. Dent. Prog. 1, 275-280. ZOBELL, C. E. 1943. The effect of solid surfaces upon bacterial activity. J. Bact. 46, 39-56. C-N,