Journal of Applied Bacteriology 1978,45,313-382
Interrelationships between Lactobacilli and Streptococci in Plaque Formation on a Tooth in an Artificial Mouth C. RUSSELL AND
F. I. K. AHMED*
Department of Bacteriology and Virology, and Dental School, University of Manchester, Manchester, England Received 1 6 February 1978 and accepted 30 May 19 78 Lactobacillus acidophilus, L . casei and L . fermentum could not form plaques alone, but could do so in conjunction with Streptococcus mutans or Strep. sanguis. As the plaques develop the proportions between lactobacilli and streptococci changed so that in a mixed plaque, which contained initially all the organisms, only L . cusei, L .fermentum and Strep. mutans were present after 96 h. It was concluded that the level of pH dictated which organisms disappeared and that it was the production of polysaccharide by the streptococci which facilitated plaque formation by the lactobacilli. The term 'patina' is suggested, to apply to non-visible colonization of the tooth surface.
As LACTOBACCILI ARE often found in considerable numbers in carious dentine they have been the subject of extensive investigation both in vivo and in vitro. Although considered at one time to be the causative organisms in dental caries the primary role of lactobacilli in the initiation of caries is now believed to be very doubtful. Van Houte et al. (1972) noted that lactobacilli introduced into the human mouth did not adhere to the tooth surface. Evidence also indicates that retentive areas, such as artificial appliances or existing carious cavities, assist in the establishment of lactobacilli (Shklair & Mazzarella 1961; Sakamaki & Bahn 1968). From a longitudinal clinical survey, Ikeda et al. (1973) concluded that caries frequently occurred in the absence of lactobacilli but never in the absence of Streptococcus mutans; the number of lactobacilli in the plaque increased considerably after the appearance of caries. In contrast, Huxley (1976) observed that in the rat carious lesions could occur from which Strep. mutans could not be isolated but lactobacilli could, though there was no evidence of a causal relationship between caries and lactobacilli. This study was designed to examine colonization and plaque formation on a tooth by oral lactobacilli and the interrelationships between these organisms and streptococci, in view of the importance attached to the latter in caries (Loesche & Syed 1973) and plaque formation (Ritz 1967).
Materials and Methods Apparatus f o r plaque formation The artificial mouth system used has been described by Russell & Coulter (1975). It permits simultaneous and continuous measurement of pH and Eh of the plaque which develops on a tooth and intermittent sampling for microbial analysis.
* Present address: Faculty of Dentistry, Al Azhar University, Cairo, Egypt. 002 1-8847/78/030373 + 10%01.OO/O 0 1978 The Society for Applied Bacteriology I3731
374
C. RUSSELL AND F. I. K. AHMED
Organisms The following organisms were obtained from the Manchester University Collection of Bacteria: Lactobacillus acidophilus strain 372, Lactobacillus fermentum strain 260 and Lactobacillus casei strain 259; Streptococcus sanguis (ATCC 10558) and Streptococcus mutans strain D282 (NCTC 10832) were provided by Dr D. B. Drucker, Department of Bacteriology and Virology, University of Manchester. Medium and inoculation The basal medium (Russell & Coulter 1975) contained beef extract, yeast extract, peptone and artificial saliva and was supplied to the developing plaque at a rate of 0.1 ml/min. Sucrose (1% w/v) was incorporated unless otherwise mentioned. Brain Heart Infusion Broth (Oxoid) was used to prepare overnight cultures of the organisms to be used as inocula. The culture was allowed to drip on to the tooth for 30 min at the rate of 0.1 ml/min while the chosen medium flowed simultaneously. When required, a mixture of organisms was obtained by combining overnight cultures in a suitable container before inoculation. Sampling procedure In some experiments the effluent was sampled with a loop as it fell off the tooth (Coulter & Russell 1976). In others, using mixed cultures, the plaque itself was sampled. With the minimum disturbance the tooth was carefully touched with a sterile Pasteur pipette and a minute amount of plaque withdrawn. The loopful of effluent was dispersed in a modified Ringers solution (Coulter & Russell 1976) in a bijou bottle by a Whirlimixer (Fisons Scientific Apparatus Ltd); the plaque samples were dispersed by sonication for 10 s using an ultrasonic probe (Soniprobe Type 7530A, Dawe Instruments Ltd). Tenfold dilutions in the Ringers solution were then prepared and plated out using the Miles & Misra (1938) technique. Isolation and identijication of organisms In experiments in which single cultures of lactobacilli were used they were plated out on to Rogosa SL Agar (Difco). This was also used when a mixed plaque was grown of a lactobacillus and a streptococcus; the latter organisms were isolated on Mitis-Salivarius Agar (Oxoid). When mixtures containing a number of different species of lactobacilli and streptococci were used, the streptococci were again differentiated on MitisSalivarius Agar (Oxoid). It was found possible to use this medium for the lactobacilli also. There was no inhibitory effect compared with Rogosa SL Agar (Difco). After 3 d incubation at 35 "C in a candle jar containing air enriched with CO,, L. casei colonies were 3-4 mm diam., convex, translucent, light brownish blue with a dark centre; L. fermenturn produced dark blue, smooth flat colonies, 4-5 mm diam.; L. acidophilus colonies were light blue, transparent, 1-2 mm diam. Further, it was found that L. acidophilus grew on Rogosa SL Agar (Difco) containing tetracycline (10 pg/ml) whereas the other organisms did not. Accordingly, L. acidophilus was enumerated on Rogosa SL Agar (Difco) with tetracycline and L. casei and L. fermentum on MitisSalivarius Agar (Oxoid). The sum of the counts on these two media was satisfactory compared with the total count of these organisms on Rogosa SL Agar (Difco) carried out at the same time.
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375
Results Plaque formation by lactobacilli The tooth was inoculated with a pure culture of either L. acidophilus, L. fermentum or L. casei and sucrose medium (pH 7 and Eh ca. 400 mV) allowed to flow simultaneously. No visible plaque was developed by L . acidophilus or L. fermentum even after 120 h incubation; staining was not used as an aid in detecting plaque. In the case of L. casei a small amount of creamy, soft non-tenacious plaque began to develop after 72 h. The recorded pH and Eh of the tooth surface showed no significant changes due to L. acidophilus; L. fermentum produced no change in the pH but Eh started to fall gradually to level off at ca. + 3 10 mV after 48 h; L. casei caused a gradual fall in the Eh, to + 190 mV over 120 h. In the last instances the pH remained unchanged for ca. 70 h and then started to drop, reaching pH 6 after 120 h. Each of the three organisms was continuously isolated, and in increasing numbers from the effluent dropping off the tooth (see Table 1). TABLE1
Parameters of colonization by lactobacilli Incubation (h)
Organisms/ml efiluent
pH
Eh, mV
7.1 6.6 6.0 7.1 7.0 6.9 7.1 7.0 7.0
250 210 190 3 70 310 3 10 380 370 370
~~~~
Lactobacillus casei fermentum acidophilus
~
24 72 120 24 72 120 24 72 120
1.8 x 8.6 x 6.7 x 6x 8x 6.4 x
105 lo6 10’ lo4
lo5 lo6
2.4 x 104 9.6 x 104 4.6 x 105
Mixed culture plaque by streptococci and lactobacilli As no plaque was formed by the lactobacilli de novo it was decided to determine whether a primary streptococcal plaque would allow the lactobacilli to colonize subsequently. Streptococcus sanguis and Strep. mutans were used for primary plaque formation for the following reasons: they form plaque in the artificial mouth (Russell & Coulter 1977); they assist the colonization of a non-plaque forming organism such as Strep. mitis, apparently by their extracellular polysaccharide (Russell & Coulter 1977) and they are the major streptococci in carious and non-carious plaque (Carlsson 1967; Loesche & Syed 1973). In a set of six different experiments the tooth was inoculated initially with either Strep. sanguis or Strep. mutans and the plaque was allowed to develop; 24 h later L. acidophilus, L. fermentum or L. casei was introduced to the developing plaque and incubated for a further 96 h. A rough white tenacious plaque always developed after inoculation with either of the streptococci. Microscopically, this plaque consisted of clumps of streptococci in a Gram negative amorphous material, presumably polysaccharide (Carlsson 1967). In 24 h the pH always dropped from pH 7 to ca. pH 6.1 in the case of Strep. sanguis, and to ca. pH 5.6 with Strep. mutans. The Eh showed
C. RUSSELL A N D F. I. K. AHMED
376
TABLE2
Mixed culture plaques of a streptococcus and a lactobacillus Mixed incubation
24 h Organisms
Strep. sanguis + L. casei Strep. sanguis t L. fermenturn Strep. sanguis iL . acidophilus Strep. mutans i L . casei Strep. rnutans + L. fermenturn Strep. rnutans t L. acidophilus
96 h A
A
r
\
> f
Streptococci*
Lactobacilli*
pH
Eh, mV
Streptococci*
Lactobacilli*
pH
Eh, mV
57.3
42.1
5.0
310
ND
100
4.0
200
55.8
44.2
4.8
290
ND
100
4.2
290
83.9
16.1
5.0
320
33.6
66.4
4.8
280
45.4
54.6
4.1
270
34.5
65.5
4.2
150
16.0
24.0
5.1
330
49.9
50.1
4.5
320
78.3
21.7
4.5
300
ND
4.3
240
100
* Percentage in plaque. ND, not detected little change (from ca. +380 mV to ca. +340 mV). After lactobacilli were introduced to the 24 h old plaque the p H gradually fell to between 4 - 8 and 4 after a further 96 h. The E h remained above +240 mV except with Strep. sanguis and L. casei (+200 mV) and Strep. mutans and L. casei (+ 150 mV). Samples were taken from the plaque at 48 h, i.e. 24 h after inoculation with the lactobacilli, and then every 24 h. Changes in the percentage viable counts of the organisms are shown in Table 2. By the end of the experiments, thick rough, white, sticky plaque was always present. Smears of such plaque showed either masses of streptococci and lactobacilli embedded in Gram negative amorphous material, or only streptococci or lactobacilli in accordance with the distribution of viable counts shown in Table 2. In view of the above results, the tooth was inoculated simultaneously with the five organisms. The pH fell to pH 4 after ca. 37 h and was maintained at this value; Eh, TABLE 3
Mixed culture plaque containing three lactobacilli and two streptococci
24 48 72 96
28.5 55.4 62.8 64.8
12.2 16.2 14.6 11.3
5.1 1.4 ND ND
17.0 ND ND ND
Table shows percentage each organism in plaque. ND, not detected.
36.6 26.8 22.6 23.9
4.5 4.0 4.0 4.0
320 250 240 240
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311
starting at 420 mV, levelled off at +240 mV. Little plaque was seen on the tooth by the end of the experiment, but it was rough, creamy and adherent. Smears of this plaque at 120 h showed chains of streptococci and lactobacilli in Gram negative material. The proportional viable counts of each organism in the plaque samples (Table 3) showed the predominance of L. casei and the absence of Strep. sanguis and L . acidophilus by the end of the experiment.
Colonization by lactobacilli The above results indicated that lactobacilli depended on the streptococci for plaque formation on teeth. It was not clear, however, whether the streptococcal cells or the extracellular polysaccharide provide the means of attachment for the lactobacilli. Colonization by Lactobacillus fermentum on teeth covered by extracellular polysaccharide-free plaque The aim of this experiment was to cover the tooth with a primary plaque which did not contain extracellular polysaccharide and examine whether such plaque would help the colonization of lactobacilli. A tooth was therefore inoculated with Strep. sanguis for 30 min in the artificial mouth and incubated in sucrose-free basal medium, as Strep. sanguis does not form extracellular polysaccharide in the absence of sucrose. No visible plaque was produced after 24 h incubation. A tooth was then inoculated with fresh saliva (F.I.K.A.) for 30 min and incubated in sucrose-free basal medium for 24 h. A smooth, creamy plaque developed on the tooth; no lactobacilli were isolated from the plaque sample. The tooth and the covering plaque was then reinoculated using a culture of L. fermentum and the incubation continued in basal medium. Lactobacillus fermentum was still not isolated from subsequent plaque samples. Colonization by Lactobacillus fermentum of teeth previously coated with extracellular polysaccharide As plaque made of bacterial cells devoid of extracellular polysaccharide failed to support colonization of lactobacilli, we examined the role of extracellular polysaccharide itself. Two methods were used to coat the tooth: the first was to allow Strep. sanguis to produce extracellular polysaccharide in situ on the tooth; the second was to cover the tooth with a polysaccharide suspension. Firstly, a tooth was inoculated with Strep. sanguis and incubated in the presence of basal medium containing 1% (w/v) sucrose. After 48 h a heavy, rough, white plaque covered the tooth which was then taken out of the artificial mouth, carefully washed with water, left to dry and then sterilized in ethylene oxide. The tooth with its dry coat was refitted into the artificial mouth and basal medium without sucrose was dropped on to the tooth to wet the plaque; it was then inoculated with L. fermentum. Further sampling of the plaque after 24 and 48 h incubation in basal medium showed the presence of L. fermentum in increasing numbers. Smears of the plaque showed masses of lactobacilli embedded in Gram negative extracellular material but streptococci could hardly be seen in the smear. Thus L . fermentum colonized the tooth already covered with a sterilized plaque of Strep. sanguis and extracellular polysaccharide. For the second experiment, Strep. sanguis was cultured in 100 ml of 5% (w/v) sucrose broth and incubated for 3 d at 35 O C . The suspension was then centrifuged and
378
C. RUSSELL AND F. I. K. AHMED
the supernatant treated with an equal part of ethanol to precipitate the extracellular polysaccharide (Carlsson 1967). The precipitate was collected by centrifuging, washed several times with water and finally suspended in 20 ml water and sterilized by ultrasonics and hydrogen peroxide (Ahmed & Russell 1975). The tooth was coated by dropping the sterilized suspension of the extracellular polysaccharide on to it continuously for 1 h at 0.1 ml/min. The tooth was then immediately inoculated with L. fermentum and incubated in basal medium. Lactobacillus fermentum was isolated continuously from the effluent, and after 4 d incubation a very light, smooth, yellowish plaque was seen in the occlusal grooves but not on the smooth surfaces of the tooth. Smears of the plaque consisted solely of lactobacilli and Gram negative material, presumably the extracellular polysaccharide. In a third experiment, a tooth was dipped into the polysaccharide suspension, left to dry and then fitted into the artificial mouth and the apparatus sterilized. The tooth was then inoculated with L. fermentum and incubated in basal medium. Subsequent streaking of the effluent on blood agar and Rogosa SL Agar (Difco) showed the presence of L.fermentum. After 4 d incubation a small amount of smooth, yellowish plaque was seen in the occlusal grooves of the tooth but not on the smooth surfaces. Smears of the plaque showed the presence of masses of lactobacilli embedded in a Gram negative substance. The total amount of plaque was judged visually to be more than in the second experiment above. It seems that coating the tooth with the polysaccharide and allowing it to dry resulted in a thicker layer of the polysaccharide, more plaque being formed subsequently. Both techniques were successful, however, in demonstrating that a layer of polysaccharide per se allowed the lactobacilli to colonize the tooth when subsequently growing in medium without sucrose.
Discussion The strains of Lactobacillus spp. tested produced virtually no plaque on their own, although they were isolated continuously from the effluent. Jordan & Keyes (1966) noted that two strains of lactobacilli (not identified) formed very light plaque on nichrome wire. On the other hand, a strain of lactobacillus did not form plaque on wire when tested by Gilmore & Bhaskar (1972). Sidaway (1970) found that neither L. casei nor L. fermentum developed any plaque on teeth in vitro. When a strain of lactobacillus was inoculated into germ-free rats no plaque was found on the teeth although fissue caries was present (Rosen et al. 1968). Such observations led the present authors to suggest the term ‘patina’ for the invisible film which must be present when organisms can be isolated from a tooth on which conventional plaque cannot be seen. Lactobacillus acidophilus and L. fermentum did not develop plaque and the pH on the tooth remained unchanged; when L. casei began visibly to colonize the tooth the pH dropped from 7 to level off at pH 6 . This pH value is much higher than those attained by compact layers of L. casei in contact with glucose solution (pH 3.8) reported by Mahler & Manly (1956). The difference lies in the technique itself, the concentration of the organism colonizing the tooth and rate of flow of the nutrient on to the tooth; if it exceeds the rate of acid production the acids will be washed off the tooth. In mixed plaques of Strep. sanguis and L. acidophilus they were both constantly
PLAQUE IN ARTIFICIAL MOUTH
379
isolated from the plaque; in contrast, Strep. sanguis eventually disappeared from the plaque mixed with either L. fermentum or L. casei. When Strep. mutans was used to provide a ground for the lactobacilli, L. acidophilus was totally depressed by the end of the experiment whereas L.fermentum and L. casei were present continuously. The data in Table 2 indicate that neither Strep. sanguis nor L. acidophilus tolerated a pH below ca. 4.5. Russell & Coulter (1977) suggest that the critical pH value for Strep. sanguis is 4.6; in mixed culture plaque of Strep. sanguis and Strep. mutans the pH was 4.4 and Strep. sanguis was not recovered. The lowest pH recorded (pH 4) was produced by L. casei after the depression of Strep. sanguis, i.e. when the plaque was composed entirely of L. casei. This pH value is lower than that reached by mixed L. casei and Strep. mutans plaque where both organisms were present. The same applied to L. fermentum which lowered the pH to 4.2 when it overgrew Strep. sanguis but maintained a higher pH (4.5) in mixed plaque with Strep. mutans. It may be noted that Stephan & Hemmens (1947) found that a mixed culture of unidentified oral streptococci and lactobacilli yielded a slightly higher pH than lactobacilli alone. In contrast with Strep. sanguis, Strep. mutans was able to survive in pH as low as 4.2; the work of Drucker (1970), Donoghue & Tyler (1975) and Russell & Coulter (1977) also showed that Strep. mutans was more acidogenic and aciduric than other oral streptococci. Following these results one would expect that if all the streptococcal and lactobacillus strains tested were mixed together the resulting plaque would eventually contain L. casei, L. fermentum and Strep. mutans. That this was indeed the case validated the earlier results. The sharp fall in the pH (4.5) in the first 24 h affected the growth of the plaque organisms so that Strep. sanguis totally disappeared from the plaque as pH fell below 4.5; L. acidophilus disappeared by the time pH was pH 4. Again, this observation confirms that neither Strep. sanguis nor L. acidophilus can tolerate pH 4.5 or below; it is highly unlikely that their disappearance from the mixed plaque is due to the Eh (240 mV) reached after 72 h, since both organisms can grow anaerobically. The rate at which one or other organism disappears or becomes predominant will depend on their concentrations. In other work, not reported here, plaque has been produced following inoculation with 12-0.1 ml saliva. Plaque parameters after 24 h were unaltered except following the lowest inoculum; organisms present in saliva at low concentrations could no longer be found in the plaque. The amount of plaque was small compared with that formed by initial inoculation by streptococci followed by lactobacilli. Miller & Kleinman (1 974) noticed that in a mixed culture of L. casei with Strep. mutans or Strep. sanguis the amount of plaque formed on a nichrome wire was much less than that formed by Strep. mutans or Strep. sanguis alone. They suggested that sucrose utilization became a limiting factor for the growth of, and dextran production by, the streptococci in mixed culture; they dismissed the breakdown of dextran by L. casei as they found that it could not degrade dextran. In the present work, however, the competition for sucrose utilization could not be a limiting factor as 1% (w/v) sucrose was provided continuously to the plaque in contrast with the batch culture technique used for plaque formation on wires by Miller & Kleinman (1974). It would be reasonable to assume that the steep fall of the pH to 4 is the limiting factor for both the growth of Strep. mutans and dextran production. Drucker (1970) and Komiyama & Kleinberg (1974) provided evidence that the growth rate and sugar utilization by oral streptococci was much slower at an acid pH.
380
C. RUSSELL AND F. I. K. AHMED
The results of this series of experiments support and provide explanation for the clinical findings of Shovlin & Gillis (1969), Loesche & Syed (1973) a d Edwardsson (1 974) who noticed that L. casei and Strep. mutans comprised almost all Lactobacillus and Streptococcus strains isolated from deep carious dentine and carious plaque. O n the one hand Strep. sanguis is inhibited by Strep. mutans (Carlsson 1971; Russell & Coulter 1977) and also by L. fermentum and L. casei; on the other hand L. acidophilus was inhibited by Strep. mutans; this would leave Strep. mutans, L. casei and L. fermenturn. That the last organism was found only in small proportions in the plaque of mixed streptococci and lactobacilli may be due to the inhibitory effect of the plaque acidity, pH 4, which is doubtful, or due to antagonism between L. casei, the predominant organism and L. fermentum, although Holmberg & Hallander (1972) found no interference between the different oral lactobacilli on solid media. Formation of a primary plaque by either Strep. sanguis or Strep. mutans assisted the colonization of lactobacilli. Saliva-induced plaque grown in basal medium consisted only of masses of bacterial cells (Coulter & Russell 1976); extracellular polysaccharide, particularly dextran and levan, is not synthesized in the absence of sucrose (Critchley et al. 1967). Such plaque did not support colonization by L. fermentum, showing that it is not the cell-to-cell adhesion or the so-called ‘corn cob’ adhesion mechanism (Jones 1972) which helped the colonization of lactobacilli. O n the other hand, the presence of extracellular polysaccharide either formed in situ on the tooth or added separately did support colonization by L. fermentum. This clearly demonstrated the dependence of lactobacilli on extracellular polysaccharide produced by other oral organisms, mainly streptococci, for colonization. Some members of the genus Lactobacillus eg L . pastorianus have been found to synthesize extracellular polysaccharide (Dunican & Seeley 1965); whether these strains will colonize the tooth and form plaque is yet to be determined. The localization of L. fermentum plaque in the occlusal grooves of teeth covered with polysaccharide is of particular interest. Clinical examination has shown that the numbers of oral lactobacilli can be increased dramatically by insertion of mechanical appliances in the mouth (Sakamaki & Bahn 1968); accordingly, van Houte et al. (1972) suggested that these appliances as well as carious cavities simply serve as mechanical retentive areas which help the colonization of lactobacilli. In the present work, however, retentive areas such as occlusal grooves, the hole in the tooth for the pH electrode, the rough edges of the polythene tube, etc. were always present but still lactobacilli did not colonize the tooth in the absence of extracellular polysaccharide. Therefore it would be reasonable to assume that the increase in the lactobacillus count in the retentive areas of the mouth, which are usually inaccessible to cleaning, is mainly due to more plaque accumulation, thus more extracellular polysaccharide formation and a greater chance for the lactobacilli to colonize. These experiments on colonization by lactobacilli support the conclusion made by Ikeda et al. (1973), based on clinical studies, that lactobacilli were associated only with the progression but not with the initiation of caries. In their subjects superficial carious lesions were formed in the absence of lactobacilli but not in the absence of Strep. mutans. The present work indicates that as lactobacilli cannot form plaque on the tooth on their own, it is unlikely that they can be responsible for the initiation of dental caries unless other extracellular polysaccharide-producing streptococci were already present in high numbers. Once the lactobacilli establish themselves they will certainly participate
PLAQUE IN ARTIFICIAL MOUTH
38 1
in the formation of dental caries and they can eventually be totally responsible for the progress of these lesions.
References AHMED,F. I. K. & RUSSELL,C. 1975 Synergism between ultrasonic waves and hydrogen peroxide in the killing of micro-organisms. Journal of Applied Bacteriology 39,3 1-40. CARLSSON, J. 1967 Presence of various types of non-haemolytic streptococci in dental plaque and in other sites of the oral cavity in man. Odontologisk Revy 18,55-74. CARLSSON,J. 1971 Growth of Streptococcus mutans and Streptococcus sanguis in mixed culture. Archives of Oral Biology 16,963-965. COULTER, W. A. & RUSSELL,C. 1976 pH and Eh in single and mixed culture bacterial plaque in an artificial mouth. Journal of Applied Bacteriology 40,73-87. CRITCHLEY, P., WOOD,J. M., SAXTON,C. A. & LEACH,S. A. 1967 The polymerization of dietary sugars by dental plaque. Caries Research 1, 112-129. DONOGHUE, H. D. & TYLER,J. E. 1975 Antagonisms amongst streptococci isolated from the human oral cavity. Archives of Oral Biology 20,381-387. DRUCKER, D. B. 1970 Optimum pH values for growth of various plaque streptococci in vitro. In Dental Plaque ed. McHugh, W. D. pp. 241-245, Edinburgh: Livingstone. DUNICAN, L. K. & SEELEY,H. W. 1965 Extracellular polysaccharide synthesis by members of the genus Lactobacillus: conditions for formation and accumulation. Journal of General Microbiology 40,297-308. EDWARDSSON, S. 1974 Bacteriological studies on deep carious areas of carious dentine. Odontologisk Revy 25, (Suppl. 32) 1-143. GILMORE,E. L. & BHASKAR,S. N. 1972 Effect of tongue brushing on bacteria and plaque formed in vitro. Journal of Periodontology 43,4 18-422. HOLMBERG,K. & HALLANDER,H. 0. 1972 Interference between Gram positive microorganisms in dental plaque. Journal of Dental Research 5 1,588-595. HUXLEY,G. G. 1976 The relationship between plaque bacteria and dental caries at specific tooth sites in rats. In Proceedings, Microbial Aspects of Dental Caries ed. Stiles, H. M., Loesche, W. J. & O’Brien, T. C. Sp. Suppl. Microbiology Abstracts, Vol. 111, pp. 773-784. IKEDA,T., SANDHAM, H. J. & BRADLEY,E. L. 1973 Changes in Streptococcus mutans and lactobacilli in plaque in relation to the initiation of dental caries in negro children. Archives of Oral Biology 18,555-566. JONES,S. J. 1972 A special relationship between spherical and filamentous micro-organisms in mature human dental plaque. Archives of Oral Biology 17,613-616. JORDAN,H. V. & KEYES,P. H. 1966. In vitro methods for the study of plaque formation and carious lesions. Archives of Oral Biology 11,793-801. KOMIYAMA, K. & KLEINBERG, I. 1974 Comparison of glucose utilization and acid formation by S. rnutans and S. sanguis at different pH. Journal of Dental Research 53, 241, abstract 746. LOESCHE,W. J. & SYED,S. A. 1973 The predominant cultivable flora of carious plaque and carious dentine. Caries Research 7,201-216. MAHLER,I. R. & MANLY,R. S. 1956 The pH levels attained by compact layers of oral microorganisms in contact with glucose solutions. Journal of Dental Research 35,226-232. MILES,A. A. & MISRA,S. A. 1938 The estimation of the bactericidal power of the blood. Journal of Hygiene, Cambridge 38,732-749. MILLER,C. H. & KLEINMAN, J. C. 1974 Effect of microbial interactions on in vitro plaque formation by Streptococcus mutans. Journal of Dental Research 53,427-434. RITZ, H. L. 1967 Microbial population shifts in developing human plaque. Archives of Oral Biology 12, 1561-1568. ROSEN,S., LENNY,W. S. & O’MALLEY,J. E. 1968 Dental caries in gnotobiotic rats inoculated with Lactobacillus casei. Journal of Dental Research 47,358-363. RUSSELL,C. & COULTER, W. A. 1975 Continuous monitoring of pH and Eh in bacterial plaque grown on a tooth in an artificial mouth. Applied Microbiology 29, 141-144.
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RUSSELL,C. & COULTER,W. A. 1977 Plaque formation by streptococci in an artificial mouth and factors influencing colonization. Journal of Applied Bacteriology 42,337-344. SAKAMAKI, S . T. & BAHN, A. N. 1968 Effect of orthodontic banding on localized oral lactobacilli. Journal of Dental Research 47,275-279. SHKLAIR, I. L. & MAZZARELLA, M. H. 1961 Effect of full-mouth extraction on oral microbiota. Dental Progress 1,275-280. F. E. & GILLIS,R. E. 1969 Biochemical and antigenic studies of lactobacilli isolated SHOVLIN, from deep dental caries. I. Biochemical aspects. Journal of Dental Research 48,356-360. SIDAWAY, D. A. 1970 The bacterial composition of natural plaque and the in uitro production of artificial plaque. In Dental Plaque ed. McHugh, W. D. pp. 225-240, Edinburgh: Livingstone. E. S. 1947 Studies of changes in pH produced by pure cultures of STEPHAN, R. M. & HEMMENS, oral micro-organisms. Journal of Dental Research 26, 15-4 1. VAN HOUTE,J., GIBBONS, R. J. & PULKINNEN, A. J. 1972 Ecology of human oral lactobacilli. Infection and Immunity 6,123-729.
Interrelationships between Lactobacilli and Streptococci in Plaque Formation on a Tooth in an Artificial Mouth C. RUSSELL AND
F. I. K. AHMED*
Department of Bacteriology and Virology, and Dental School, University of Manchester, Manchester, England Received 1 6 February 1978 and accepted 30 May 19 78 Lactobacillus acidophilus, L . casei and L . fermentum could not form plaques alone, but could do so in conjunction with Streptococcus mutans or Strep. sanguis. As the plaques develop the proportions between lactobacilli and streptococci changed so that in a mixed plaque, which contained initially all the organisms, only L . cusei, L .fermentum and Strep. mutans were present after 96 h. It was concluded that the level of pH dictated which organisms disappeared and that it was the production of polysaccharide by the streptococci which facilitated plaque formation by the lactobacilli. The term 'patina' is suggested, to apply to non-visible colonization of the tooth surface.
As LACTOBACCILI ARE often found in considerable numbers in carious dentine they have been the subject of extensive investigation both in vivo and in vitro. Although considered at one time to be the causative organisms in dental caries the primary role of lactobacilli in the initiation of caries is now believed to be very doubtful. Van Houte et al. (1972) noted that lactobacilli introduced into the human mouth did not adhere to the tooth surface. Evidence also indicates that retentive areas, such as artificial appliances or existing carious cavities, assist in the establishment of lactobacilli (Shklair & Mazzarella 1961; Sakamaki & Bahn 1968). From a longitudinal clinical survey, Ikeda et al. (1973) concluded that caries frequently occurred in the absence of lactobacilli but never in the absence of Streptococcus mutans; the number of lactobacilli in the plaque increased considerably after the appearance of caries. In contrast, Huxley (1976) observed that in the rat carious lesions could occur from which Strep. mutans could not be isolated but lactobacilli could, though there was no evidence of a causal relationship between caries and lactobacilli. This study was designed to examine colonization and plaque formation on a tooth by oral lactobacilli and the interrelationships between these organisms and streptococci, in view of the importance attached to the latter in caries (Loesche & Syed 1973) and plaque formation (Ritz 1967).
Materials and Methods Apparatus f o r plaque formation The artificial mouth system used has been described by Russell & Coulter (1975). It permits simultaneous and continuous measurement of pH and Eh of the plaque which develops on a tooth and intermittent sampling for microbial analysis.
* Present address: Faculty of Dentistry, Al Azhar University, Cairo, Egypt. 002 1-8847/78/030373 + 10%01.OO/O 0 1978 The Society for Applied Bacteriology I3731
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Organisms The following organisms were obtained from the Manchester University Collection of Bacteria: Lactobacillus acidophilus strain 372, Lactobacillus fermentum strain 260 and Lactobacillus casei strain 259; Streptococcus sanguis (ATCC 10558) and Streptococcus mutans strain D282 (NCTC 10832) were provided by Dr D. B. Drucker, Department of Bacteriology and Virology, University of Manchester. Medium and inoculation The basal medium (Russell & Coulter 1975) contained beef extract, yeast extract, peptone and artificial saliva and was supplied to the developing plaque at a rate of 0.1 ml/min. Sucrose (1% w/v) was incorporated unless otherwise mentioned. Brain Heart Infusion Broth (Oxoid) was used to prepare overnight cultures of the organisms to be used as inocula. The culture was allowed to drip on to the tooth for 30 min at the rate of 0.1 ml/min while the chosen medium flowed simultaneously. When required, a mixture of organisms was obtained by combining overnight cultures in a suitable container before inoculation. Sampling procedure In some experiments the effluent was sampled with a loop as it fell off the tooth (Coulter & Russell 1976). In others, using mixed cultures, the plaque itself was sampled. With the minimum disturbance the tooth was carefully touched with a sterile Pasteur pipette and a minute amount of plaque withdrawn. The loopful of effluent was dispersed in a modified Ringers solution (Coulter & Russell 1976) in a bijou bottle by a Whirlimixer (Fisons Scientific Apparatus Ltd); the plaque samples were dispersed by sonication for 10 s using an ultrasonic probe (Soniprobe Type 7530A, Dawe Instruments Ltd). Tenfold dilutions in the Ringers solution were then prepared and plated out using the Miles & Misra (1938) technique. Isolation and identijication of organisms In experiments in which single cultures of lactobacilli were used they were plated out on to Rogosa SL Agar (Difco). This was also used when a mixed plaque was grown of a lactobacillus and a streptococcus; the latter organisms were isolated on Mitis-Salivarius Agar (Oxoid). When mixtures containing a number of different species of lactobacilli and streptococci were used, the streptococci were again differentiated on MitisSalivarius Agar (Oxoid). It was found possible to use this medium for the lactobacilli also. There was no inhibitory effect compared with Rogosa SL Agar (Difco). After 3 d incubation at 35 "C in a candle jar containing air enriched with CO,, L. casei colonies were 3-4 mm diam., convex, translucent, light brownish blue with a dark centre; L. fermenturn produced dark blue, smooth flat colonies, 4-5 mm diam.; L. acidophilus colonies were light blue, transparent, 1-2 mm diam. Further, it was found that L. acidophilus grew on Rogosa SL Agar (Difco) containing tetracycline (10 pg/ml) whereas the other organisms did not. Accordingly, L. acidophilus was enumerated on Rogosa SL Agar (Difco) with tetracycline and L. casei and L. fermentum on MitisSalivarius Agar (Oxoid). The sum of the counts on these two media was satisfactory compared with the total count of these organisms on Rogosa SL Agar (Difco) carried out at the same time.
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Results Plaque formation by lactobacilli The tooth was inoculated with a pure culture of either L. acidophilus, L. fermentum or L. casei and sucrose medium (pH 7 and Eh ca. 400 mV) allowed to flow simultaneously. No visible plaque was developed by L . acidophilus or L. fermentum even after 120 h incubation; staining was not used as an aid in detecting plaque. In the case of L. casei a small amount of creamy, soft non-tenacious plaque began to develop after 72 h. The recorded pH and Eh of the tooth surface showed no significant changes due to L. acidophilus; L. fermentum produced no change in the pH but Eh started to fall gradually to level off at ca. + 3 10 mV after 48 h; L. casei caused a gradual fall in the Eh, to + 190 mV over 120 h. In the last instances the pH remained unchanged for ca. 70 h and then started to drop, reaching pH 6 after 120 h. Each of the three organisms was continuously isolated, and in increasing numbers from the effluent dropping off the tooth (see Table 1). TABLE1
Parameters of colonization by lactobacilli Incubation (h)
Organisms/ml efiluent
pH
Eh, mV
7.1 6.6 6.0 7.1 7.0 6.9 7.1 7.0 7.0
250 210 190 3 70 310 3 10 380 370 370
~~~~
Lactobacillus casei fermentum acidophilus
~
24 72 120 24 72 120 24 72 120
1.8 x 8.6 x 6.7 x 6x 8x 6.4 x
105 lo6 10’ lo4
lo5 lo6
2.4 x 104 9.6 x 104 4.6 x 105
Mixed culture plaque by streptococci and lactobacilli As no plaque was formed by the lactobacilli de novo it was decided to determine whether a primary streptococcal plaque would allow the lactobacilli to colonize subsequently. Streptococcus sanguis and Strep. mutans were used for primary plaque formation for the following reasons: they form plaque in the artificial mouth (Russell & Coulter 1977); they assist the colonization of a non-plaque forming organism such as Strep. mitis, apparently by their extracellular polysaccharide (Russell & Coulter 1977) and they are the major streptococci in carious and non-carious plaque (Carlsson 1967; Loesche & Syed 1973). In a set of six different experiments the tooth was inoculated initially with either Strep. sanguis or Strep. mutans and the plaque was allowed to develop; 24 h later L. acidophilus, L. fermentum or L. casei was introduced to the developing plaque and incubated for a further 96 h. A rough white tenacious plaque always developed after inoculation with either of the streptococci. Microscopically, this plaque consisted of clumps of streptococci in a Gram negative amorphous material, presumably polysaccharide (Carlsson 1967). In 24 h the pH always dropped from pH 7 to ca. pH 6.1 in the case of Strep. sanguis, and to ca. pH 5.6 with Strep. mutans. The Eh showed
C. RUSSELL A N D F. I. K. AHMED
376
TABLE2
Mixed culture plaques of a streptococcus and a lactobacillus Mixed incubation
24 h Organisms
Strep. sanguis + L. casei Strep. sanguis t L. fermenturn Strep. sanguis iL . acidophilus Strep. mutans i L . casei Strep. rnutans + L. fermenturn Strep. rnutans t L. acidophilus
96 h A
A
r
\
> f
Streptococci*
Lactobacilli*
pH
Eh, mV
Streptococci*
Lactobacilli*
pH
Eh, mV
57.3
42.1
5.0
310
ND
100
4.0
200
55.8
44.2
4.8
290
ND
100
4.2
290
83.9
16.1
5.0
320
33.6
66.4
4.8
280
45.4
54.6
4.1
270
34.5
65.5
4.2
150
16.0
24.0
5.1
330
49.9
50.1
4.5
320
78.3
21.7
4.5
300
ND
4.3
240
100
* Percentage in plaque. ND, not detected little change (from ca. +380 mV to ca. +340 mV). After lactobacilli were introduced to the 24 h old plaque the p H gradually fell to between 4 - 8 and 4 after a further 96 h. The E h remained above +240 mV except with Strep. sanguis and L. casei (+200 mV) and Strep. mutans and L. casei (+ 150 mV). Samples were taken from the plaque at 48 h, i.e. 24 h after inoculation with the lactobacilli, and then every 24 h. Changes in the percentage viable counts of the organisms are shown in Table 2. By the end of the experiments, thick rough, white, sticky plaque was always present. Smears of such plaque showed either masses of streptococci and lactobacilli embedded in Gram negative amorphous material, or only streptococci or lactobacilli in accordance with the distribution of viable counts shown in Table 2. In view of the above results, the tooth was inoculated simultaneously with the five organisms. The pH fell to pH 4 after ca. 37 h and was maintained at this value; Eh, TABLE 3
Mixed culture plaque containing three lactobacilli and two streptococci
24 48 72 96
28.5 55.4 62.8 64.8
12.2 16.2 14.6 11.3
5.1 1.4 ND ND
17.0 ND ND ND
Table shows percentage each organism in plaque. ND, not detected.
36.6 26.8 22.6 23.9
4.5 4.0 4.0 4.0
320 250 240 240
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311
starting at 420 mV, levelled off at +240 mV. Little plaque was seen on the tooth by the end of the experiment, but it was rough, creamy and adherent. Smears of this plaque at 120 h showed chains of streptococci and lactobacilli in Gram negative material. The proportional viable counts of each organism in the plaque samples (Table 3) showed the predominance of L. casei and the absence of Strep. sanguis and L . acidophilus by the end of the experiment.
Colonization by lactobacilli The above results indicated that lactobacilli depended on the streptococci for plaque formation on teeth. It was not clear, however, whether the streptococcal cells or the extracellular polysaccharide provide the means of attachment for the lactobacilli. Colonization by Lactobacillus fermentum on teeth covered by extracellular polysaccharide-free plaque The aim of this experiment was to cover the tooth with a primary plaque which did not contain extracellular polysaccharide and examine whether such plaque would help the colonization of lactobacilli. A tooth was therefore inoculated with Strep. sanguis for 30 min in the artificial mouth and incubated in sucrose-free basal medium, as Strep. sanguis does not form extracellular polysaccharide in the absence of sucrose. No visible plaque was produced after 24 h incubation. A tooth was then inoculated with fresh saliva (F.I.K.A.) for 30 min and incubated in sucrose-free basal medium for 24 h. A smooth, creamy plaque developed on the tooth; no lactobacilli were isolated from the plaque sample. The tooth and the covering plaque was then reinoculated using a culture of L. fermentum and the incubation continued in basal medium. Lactobacillus fermentum was still not isolated from subsequent plaque samples. Colonization by Lactobacillus fermentum of teeth previously coated with extracellular polysaccharide As plaque made of bacterial cells devoid of extracellular polysaccharide failed to support colonization of lactobacilli, we examined the role of extracellular polysaccharide itself. Two methods were used to coat the tooth: the first was to allow Strep. sanguis to produce extracellular polysaccharide in situ on the tooth; the second was to cover the tooth with a polysaccharide suspension. Firstly, a tooth was inoculated with Strep. sanguis and incubated in the presence of basal medium containing 1% (w/v) sucrose. After 48 h a heavy, rough, white plaque covered the tooth which was then taken out of the artificial mouth, carefully washed with water, left to dry and then sterilized in ethylene oxide. The tooth with its dry coat was refitted into the artificial mouth and basal medium without sucrose was dropped on to the tooth to wet the plaque; it was then inoculated with L. fermentum. Further sampling of the plaque after 24 and 48 h incubation in basal medium showed the presence of L. fermentum in increasing numbers. Smears of the plaque showed masses of lactobacilli embedded in Gram negative extracellular material but streptococci could hardly be seen in the smear. Thus L . fermentum colonized the tooth already covered with a sterilized plaque of Strep. sanguis and extracellular polysaccharide. For the second experiment, Strep. sanguis was cultured in 100 ml of 5% (w/v) sucrose broth and incubated for 3 d at 35 O C . The suspension was then centrifuged and
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C. RUSSELL AND F. I. K. AHMED
the supernatant treated with an equal part of ethanol to precipitate the extracellular polysaccharide (Carlsson 1967). The precipitate was collected by centrifuging, washed several times with water and finally suspended in 20 ml water and sterilized by ultrasonics and hydrogen peroxide (Ahmed & Russell 1975). The tooth was coated by dropping the sterilized suspension of the extracellular polysaccharide on to it continuously for 1 h at 0.1 ml/min. The tooth was then immediately inoculated with L. fermentum and incubated in basal medium. Lactobacillus fermentum was isolated continuously from the effluent, and after 4 d incubation a very light, smooth, yellowish plaque was seen in the occlusal grooves but not on the smooth surfaces of the tooth. Smears of the plaque consisted solely of lactobacilli and Gram negative material, presumably the extracellular polysaccharide. In a third experiment, a tooth was dipped into the polysaccharide suspension, left to dry and then fitted into the artificial mouth and the apparatus sterilized. The tooth was then inoculated with L. fermentum and incubated in basal medium. Subsequent streaking of the effluent on blood agar and Rogosa SL Agar (Difco) showed the presence of L.fermentum. After 4 d incubation a small amount of smooth, yellowish plaque was seen in the occlusal grooves of the tooth but not on the smooth surfaces. Smears of the plaque showed the presence of masses of lactobacilli embedded in a Gram negative substance. The total amount of plaque was judged visually to be more than in the second experiment above. It seems that coating the tooth with the polysaccharide and allowing it to dry resulted in a thicker layer of the polysaccharide, more plaque being formed subsequently. Both techniques were successful, however, in demonstrating that a layer of polysaccharide per se allowed the lactobacilli to colonize the tooth when subsequently growing in medium without sucrose.
Discussion The strains of Lactobacillus spp. tested produced virtually no plaque on their own, although they were isolated continuously from the effluent. Jordan & Keyes (1966) noted that two strains of lactobacilli (not identified) formed very light plaque on nichrome wire. On the other hand, a strain of lactobacillus did not form plaque on wire when tested by Gilmore & Bhaskar (1972). Sidaway (1970) found that neither L. casei nor L. fermentum developed any plaque on teeth in vitro. When a strain of lactobacillus was inoculated into germ-free rats no plaque was found on the teeth although fissue caries was present (Rosen et al. 1968). Such observations led the present authors to suggest the term ‘patina’ for the invisible film which must be present when organisms can be isolated from a tooth on which conventional plaque cannot be seen. Lactobacillus acidophilus and L. fermentum did not develop plaque and the pH on the tooth remained unchanged; when L. casei began visibly to colonize the tooth the pH dropped from 7 to level off at pH 6 . This pH value is much higher than those attained by compact layers of L. casei in contact with glucose solution (pH 3.8) reported by Mahler & Manly (1956). The difference lies in the technique itself, the concentration of the organism colonizing the tooth and rate of flow of the nutrient on to the tooth; if it exceeds the rate of acid production the acids will be washed off the tooth. In mixed plaques of Strep. sanguis and L. acidophilus they were both constantly
PLAQUE IN ARTIFICIAL MOUTH
379
isolated from the plaque; in contrast, Strep. sanguis eventually disappeared from the plaque mixed with either L. fermentum or L. casei. When Strep. mutans was used to provide a ground for the lactobacilli, L. acidophilus was totally depressed by the end of the experiment whereas L.fermentum and L. casei were present continuously. The data in Table 2 indicate that neither Strep. sanguis nor L. acidophilus tolerated a pH below ca. 4.5. Russell & Coulter (1977) suggest that the critical pH value for Strep. sanguis is 4.6; in mixed culture plaque of Strep. sanguis and Strep. mutans the pH was 4.4 and Strep. sanguis was not recovered. The lowest pH recorded (pH 4) was produced by L. casei after the depression of Strep. sanguis, i.e. when the plaque was composed entirely of L. casei. This pH value is lower than that reached by mixed L. casei and Strep. mutans plaque where both organisms were present. The same applied to L. fermentum which lowered the pH to 4.2 when it overgrew Strep. sanguis but maintained a higher pH (4.5) in mixed plaque with Strep. mutans. It may be noted that Stephan & Hemmens (1947) found that a mixed culture of unidentified oral streptococci and lactobacilli yielded a slightly higher pH than lactobacilli alone. In contrast with Strep. sanguis, Strep. mutans was able to survive in pH as low as 4.2; the work of Drucker (1970), Donoghue & Tyler (1975) and Russell & Coulter (1977) also showed that Strep. mutans was more acidogenic and aciduric than other oral streptococci. Following these results one would expect that if all the streptococcal and lactobacillus strains tested were mixed together the resulting plaque would eventually contain L. casei, L. fermentum and Strep. mutans. That this was indeed the case validated the earlier results. The sharp fall in the pH (4.5) in the first 24 h affected the growth of the plaque organisms so that Strep. sanguis totally disappeared from the plaque as pH fell below 4.5; L. acidophilus disappeared by the time pH was pH 4. Again, this observation confirms that neither Strep. sanguis nor L. acidophilus can tolerate pH 4.5 or below; it is highly unlikely that their disappearance from the mixed plaque is due to the Eh (240 mV) reached after 72 h, since both organisms can grow anaerobically. The rate at which one or other organism disappears or becomes predominant will depend on their concentrations. In other work, not reported here, plaque has been produced following inoculation with 12-0.1 ml saliva. Plaque parameters after 24 h were unaltered except following the lowest inoculum; organisms present in saliva at low concentrations could no longer be found in the plaque. The amount of plaque was small compared with that formed by initial inoculation by streptococci followed by lactobacilli. Miller & Kleinman (1 974) noticed that in a mixed culture of L. casei with Strep. mutans or Strep. sanguis the amount of plaque formed on a nichrome wire was much less than that formed by Strep. mutans or Strep. sanguis alone. They suggested that sucrose utilization became a limiting factor for the growth of, and dextran production by, the streptococci in mixed culture; they dismissed the breakdown of dextran by L. casei as they found that it could not degrade dextran. In the present work, however, the competition for sucrose utilization could not be a limiting factor as 1% (w/v) sucrose was provided continuously to the plaque in contrast with the batch culture technique used for plaque formation on wires by Miller & Kleinman (1974). It would be reasonable to assume that the steep fall of the pH to 4 is the limiting factor for both the growth of Strep. mutans and dextran production. Drucker (1970) and Komiyama & Kleinberg (1974) provided evidence that the growth rate and sugar utilization by oral streptococci was much slower at an acid pH.
380
C. RUSSELL AND F. I. K. AHMED
The results of this series of experiments support and provide explanation for the clinical findings of Shovlin & Gillis (1969), Loesche & Syed (1973) a d Edwardsson (1 974) who noticed that L. casei and Strep. mutans comprised almost all Lactobacillus and Streptococcus strains isolated from deep carious dentine and carious plaque. O n the one hand Strep. sanguis is inhibited by Strep. mutans (Carlsson 1971; Russell & Coulter 1977) and also by L. fermentum and L. casei; on the other hand L. acidophilus was inhibited by Strep. mutans; this would leave Strep. mutans, L. casei and L. fermenturn. That the last organism was found only in small proportions in the plaque of mixed streptococci and lactobacilli may be due to the inhibitory effect of the plaque acidity, pH 4, which is doubtful, or due to antagonism between L. casei, the predominant organism and L. fermentum, although Holmberg & Hallander (1972) found no interference between the different oral lactobacilli on solid media. Formation of a primary plaque by either Strep. sanguis or Strep. mutans assisted the colonization of lactobacilli. Saliva-induced plaque grown in basal medium consisted only of masses of bacterial cells (Coulter & Russell 1976); extracellular polysaccharide, particularly dextran and levan, is not synthesized in the absence of sucrose (Critchley et al. 1967). Such plaque did not support colonization by L. fermentum, showing that it is not the cell-to-cell adhesion or the so-called ‘corn cob’ adhesion mechanism (Jones 1972) which helped the colonization of lactobacilli. O n the other hand, the presence of extracellular polysaccharide either formed in situ on the tooth or added separately did support colonization by L. fermentum. This clearly demonstrated the dependence of lactobacilli on extracellular polysaccharide produced by other oral organisms, mainly streptococci, for colonization. Some members of the genus Lactobacillus eg L . pastorianus have been found to synthesize extracellular polysaccharide (Dunican & Seeley 1965); whether these strains will colonize the tooth and form plaque is yet to be determined. The localization of L. fermentum plaque in the occlusal grooves of teeth covered with polysaccharide is of particular interest. Clinical examination has shown that the numbers of oral lactobacilli can be increased dramatically by insertion of mechanical appliances in the mouth (Sakamaki & Bahn 1968); accordingly, van Houte et al. (1972) suggested that these appliances as well as carious cavities simply serve as mechanical retentive areas which help the colonization of lactobacilli. In the present work, however, retentive areas such as occlusal grooves, the hole in the tooth for the pH electrode, the rough edges of the polythene tube, etc. were always present but still lactobacilli did not colonize the tooth in the absence of extracellular polysaccharide. Therefore it would be reasonable to assume that the increase in the lactobacillus count in the retentive areas of the mouth, which are usually inaccessible to cleaning, is mainly due to more plaque accumulation, thus more extracellular polysaccharide formation and a greater chance for the lactobacilli to colonize. These experiments on colonization by lactobacilli support the conclusion made by Ikeda et al. (1973), based on clinical studies, that lactobacilli were associated only with the progression but not with the initiation of caries. In their subjects superficial carious lesions were formed in the absence of lactobacilli but not in the absence of Strep. mutans. The present work indicates that as lactobacilli cannot form plaque on the tooth on their own, it is unlikely that they can be responsible for the initiation of dental caries unless other extracellular polysaccharide-producing streptococci were already present in high numbers. Once the lactobacilli establish themselves they will certainly participate
PLAQUE IN ARTIFICIAL MOUTH
38 1
in the formation of dental caries and they can eventually be totally responsible for the progress of these lesions.
References AHMED,F. I. K. & RUSSELL,C. 1975 Synergism between ultrasonic waves and hydrogen peroxide in the killing of micro-organisms. Journal of Applied Bacteriology 39,3 1-40. CARLSSON, J. 1967 Presence of various types of non-haemolytic streptococci in dental plaque and in other sites of the oral cavity in man. Odontologisk Revy 18,55-74. CARLSSON,J. 1971 Growth of Streptococcus mutans and Streptococcus sanguis in mixed culture. Archives of Oral Biology 16,963-965. COULTER, W. A. & RUSSELL,C. 1976 pH and Eh in single and mixed culture bacterial plaque in an artificial mouth. Journal of Applied Bacteriology 40,73-87. CRITCHLEY, P., WOOD,J. M., SAXTON,C. A. & LEACH,S. A. 1967 The polymerization of dietary sugars by dental plaque. Caries Research 1, 112-129. DONOGHUE, H. D. & TYLER,J. E. 1975 Antagonisms amongst streptococci isolated from the human oral cavity. Archives of Oral Biology 20,381-387. DRUCKER, D. B. 1970 Optimum pH values for growth of various plaque streptococci in vitro. In Dental Plaque ed. McHugh, W. D. pp. 241-245, Edinburgh: Livingstone. DUNICAN, L. K. & SEELEY,H. W. 1965 Extracellular polysaccharide synthesis by members of the genus Lactobacillus: conditions for formation and accumulation. Journal of General Microbiology 40,297-308. EDWARDSSON, S. 1974 Bacteriological studies on deep carious areas of carious dentine. Odontologisk Revy 25, (Suppl. 32) 1-143. GILMORE,E. L. & BHASKAR,S. N. 1972 Effect of tongue brushing on bacteria and plaque formed in vitro. Journal of Periodontology 43,4 18-422. HOLMBERG,K. & HALLANDER,H. 0. 1972 Interference between Gram positive microorganisms in dental plaque. Journal of Dental Research 5 1,588-595. HUXLEY,G. G. 1976 The relationship between plaque bacteria and dental caries at specific tooth sites in rats. In Proceedings, Microbial Aspects of Dental Caries ed. Stiles, H. M., Loesche, W. J. & O’Brien, T. C. Sp. Suppl. Microbiology Abstracts, Vol. 111, pp. 773-784. IKEDA,T., SANDHAM, H. J. & BRADLEY,E. L. 1973 Changes in Streptococcus mutans and lactobacilli in plaque in relation to the initiation of dental caries in negro children. Archives of Oral Biology 18,555-566. JONES,S. J. 1972 A special relationship between spherical and filamentous micro-organisms in mature human dental plaque. Archives of Oral Biology 17,613-616. JORDAN,H. V. & KEYES,P. H. 1966. In vitro methods for the study of plaque formation and carious lesions. Archives of Oral Biology 11,793-801. KOMIYAMA, K. & KLEINBERG, I. 1974 Comparison of glucose utilization and acid formation by S. rnutans and S. sanguis at different pH. Journal of Dental Research 53, 241, abstract 746. LOESCHE,W. J. & SYED,S. A. 1973 The predominant cultivable flora of carious plaque and carious dentine. Caries Research 7,201-216. MAHLER,I. R. & MANLY,R. S. 1956 The pH levels attained by compact layers of oral microorganisms in contact with glucose solutions. Journal of Dental Research 35,226-232. MILES,A. A. & MISRA,S. A. 1938 The estimation of the bactericidal power of the blood. Journal of Hygiene, Cambridge 38,732-749. MILLER,C. H. & KLEINMAN, J. C. 1974 Effect of microbial interactions on in vitro plaque formation by Streptococcus mutans. Journal of Dental Research 53,427-434. RITZ, H. L. 1967 Microbial population shifts in developing human plaque. Archives of Oral Biology 12, 1561-1568. ROSEN,S., LENNY,W. S. & O’MALLEY,J. E. 1968 Dental caries in gnotobiotic rats inoculated with Lactobacillus casei. Journal of Dental Research 47,358-363. RUSSELL,C. & COULTER, W. A. 1975 Continuous monitoring of pH and Eh in bacterial plaque grown on a tooth in an artificial mouth. Applied Microbiology 29, 141-144.
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RUSSELL,C. & COULTER,W. A. 1977 Plaque formation by streptococci in an artificial mouth and factors influencing colonization. Journal of Applied Bacteriology 42,337-344. SAKAMAKI, S . T. & BAHN, A. N. 1968 Effect of orthodontic banding on localized oral lactobacilli. Journal of Dental Research 47,275-279. SHKLAIR, I. L. & MAZZARELLA, M. H. 1961 Effect of full-mouth extraction on oral microbiota. Dental Progress 1,275-280. F. E. & GILLIS,R. E. 1969 Biochemical and antigenic studies of lactobacilli isolated SHOVLIN, from deep dental caries. I. Biochemical aspects. Journal of Dental Research 48,356-360. SIDAWAY, D. A. 1970 The bacterial composition of natural plaque and the in uitro production of artificial plaque. In Dental Plaque ed. McHugh, W. D. pp. 225-240, Edinburgh: Livingstone. E. S. 1947 Studies of changes in pH produced by pure cultures of STEPHAN, R. M. & HEMMENS, oral micro-organisms. Journal of Dental Research 26, 15-4 1. VAN HOUTE,J., GIBBONS, R. J. & PULKINNEN, A. J. 1972 Ecology of human oral lactobacilli. Infection and Immunity 6,123-729.
