In vitro bacterial plaque suppression and recolonization by S. mutans and S. sobrinus

Supressão e recolonização de placa bacteriana por S. mutans e S. sobrinus in vitro

MEDICAL MICROBIOLOGY

In vitro bacterial plaque suppression and recolonization by S. mutans and S. sobrinus

Supressão e recolonização de placa bacteriana por S. mutans e S. sobrinus in vitro

Cássio Vicente Pereira^I; Luciano José Pereira^II; Reginaldo Bruno Gonçalves^III; José Francisco Höfling^III,*

^IDepartamento de Biologia Oral, Universidade Sagrado Coração, Bauru, SP, Brasil

^IIÁrea de Diagnóstico Bucal, Universidade Vale do Rio Verde, Três Corações, MG, Brasil

^IIILaboratório de Microbiologia e Imunologia, Faculdade de Odontologia de Piracicaba, Universidade Estadual de Campinas, Piracicaba, SP, Brasil

ABSTRACT

The in vitro study of the interactions between S. mutans and S. sobrinus is important to determine the role of these microorganisms in the formation of biofilms on dental structures and their potential to induce carious lesions. The objective of this research was to study the suppression of bacterial plaque formation and its recolonization by rifampycin-resistant S.mutans and streptomycin-resistant S. sobrinus. To study the competitive relationship between these species, previously standardized strains were incubated in media containing different fermentable carbohydrates. At determined time intervals, samples were collected from mixed cultures of S. mutans and S. sobrinus, diluted and plated on BHI-agar containing rifampycin or streptomycin to determine the number of viable cells of each species by counting colony-forming units. In order to study the bacterial colonization process and in vitro recolonization of bacterial plaque, three experiments were performed: I - co-cultivation of S. mutans and S. sobrinus; II - inoculation of bacterial plaque pre formed by S. sobrinus with S. mutans; and III - bacterial plaque pre formed by S. mutans dispersed and plated on BHI- agar containing streptomycin or rifampicin to determine the number of viable cells for each species. The results indicated a predominance of S. mutans in relation to S. sobrinus, demonstrating the capacity of S. mutans to inhibit plaque formation by S. sobrinus and recolonize the surfaces.

Key words: Streptococcus mutans, Streptococcus sobrinus, suppression

RESUMO

O estudo in vitro das interações entre S. mutans e S. sobrinus pode ser importante na determinação do papel desses microrganismos na formação de biofilmes nas estruturas dentais e seu potencial em induzir lesões cariosas. O objetivo da presente pesquisa foi estudar a supressão da formação da placa dental e sua recolonização por S. mutans rifampicina-resistentes e S. sobrinus estreptomicina-resistentes in vitro. Para avaliar as relações de competitividade entre essas espécies, cepas que foram previamente padronizadas foram incubadas em meio de cultura contendo diferentes carboidratos fermentáveis. Em intervalos de tempo determinados, amostras de S. mutans e S. sobrinus foram coletadas a partir de culturas mistas, diluídas e semeadas em placas com meio BHI-ágar contendo rifampicina ou estreptomicina para determinação do número de células viáveis de cada espécie por contagem de unidades formadoras de colônia. Para a avaliação da colonização bacteriana e recolonização da placa bacteriana in vitro, três experimentos foram realizados: I - co-cultivo de S. mutans e S. sobrinus; II - inoculação de S. mutans em placa bacteriana pré-formada por S. sobrinus; e III - placa bacteriana pré-formada por S. mutans dispersada e plaqueada em meio BHI-ágar contendo estreptomicina ou rifampicina para determinação do número de células viáveis para cada espécie. Os resultados indicaram uma predominância de S. mutans em relação ao S. sobrinus, demonstrando a capacidade do S. mutans em inibir a formação de placa por S. sobrinus e recolonizar a superfície dentária.

Palavras-chave: Streptococcus mutan, Streptococcus sobrinus, supressão

INTRODUCTION

Streptococci of the mutans group are closely associated with dental caries, mainly those involving smooth surfaces. Production of acid and extracellular polysaccharides due to hydrolysis of sucrose facilitates their adhesion to tooth surfaces (5,21).

In relation to the importance of the mouth microbiota in the decay process, S. mutans and S. sobrinus are the most frequent species from the mutans group encountered in saliva. Some authors have reported (1,8) that these bacterial species present a high caries-inducing potential, enhancing the infection process when combined. The beginning and development of caries are determined by a host-parasite relationship, which can be altered by several environmental factors as demonstrated by in vitro models simulating the in vivo phenomenon in the mouth (9,20). The in vitro studies of the bacterial relationship based on the action of these microorganisms in the plaque formation on tooth surfaces and their potential to induce caries are in agreement with several results reported in the literature (3,8-10,14,15).

The aim of the present study was to evaluate the suppression of bacterial plaque formation and recolonization by S. mutans and S. sobrinus, through in vitro tests using streptomycin- and rifampicin-resistant strains, cultivated separately and in combination.

MATERIALS AND METHODS

Suppression of bacterial plaque formation

Fresh culture samples of rifampicin-resistant S. mutans 32K and streptomycin-resistant S. sobrinus 6715 previously standardized by Mc Farland's scale (tube 2) in BHI medium were used. The samples were donated by the Microbiology Laboratory of Chiba University - Japan. A 0.2 mL sample from each culture was added to a tube containing 5 mL of BHI medium supplemented with 10% fermentable carbohydrate, either sucrose, glucose, fructose or glucose (5%) + fructose (5%), and incubated at 37ºC for 24 h in a 10% CO₂ atmosphere (10). After 3, 6, 12 and 24 h, 0.1 mL of the mixture was submitted to decimal dilution from 10^-1 to 10^-4 in sterile saline (0.9% NaCl), and plated in BHI-agar medium with rifampicin (0.1 mg/mL) or streptomycin (1.0 mg/mL). The plates were incubated for 24 h at 37ºC and the number of colony-forming units for each species was determined.

In vitro bacterial plaque colonization and recolonization

Fresh cultures of rifampicin-resistant S. mutans 32K and streptomycin-resistant S. sobrinus 6715 were standardized by the Mc Farland scale (tube 2) in BHI medium and used in the in vitro study of bacterial plaque colonization and recolonization. The tubes containing BHI medium supplemented with 10% fermentable carbohydrates - sucrose, glucose, fructose or glucose (5%) + fructose (5%) - were inoculated with S. mutans and S. sobrinus samples. A capillary tube, with one flame-closed end, was introduced into the tube, and the other end was modified (19) to promote the development of bacterial plaque. To facilitate the evaluation of the interaction between these species and to determine the colonization and recolonization potential (10) the procedures were divided in three distinct experiments.

Experiment I - S. mutans and S. sobrinus, 0.2 mL each, were transferred to a tube containing 5.0 mL BHI medium with 10% fermentable carbohydrate - sucrose, glucose, fructose or glucose (5%) + fructose (5%) - and the capillary tube. The tubes were incubated for 48 h at 37ºC in 10% CO₂.

Experiment II - The capillary tube containing the bacterial plaque formed by S. sobrinus during incubation in BHI medium as described above was transferred to a new tube containing fresh culture medium supplemented with 10% fermentable carbohydrate [sucrose, glucose, fructose or glucose (5%) + fructose (5%)]. S. mutans was then added and the culture tubes incubated for 48 h at 37ºC in 10% CO₂.

Experiment III -The procedure was similar to Experiment II: the capillary tube containing the plaque formed by S. mutans was transferred to a new culture tube containing fresh medium supplemented with 10% fermentable carboydrate [sucrose, glucose, fructose or glucose (5%) + fructose (5%)] and incubated further in the presence of added S. sobrinus for 48 h at 37ºC in 10% CO₂.

At the end of the experiments, the bacterial plaques were dispersed in sterile saline (0.9% NaCl) by means of a vibrator (Thornton - Marconi Ultrasound), then plated on BHI -agar medium containing streptomycin or rifampicin, and incubated for 24 h at 37ºC in 10% CO₂. The number of colony forming units per ml for each species was determined.

RESULTS

Fig. 1 shows that after 3h of incubation the number of S. sobrinus in the mixed colonies was greater than that of S. mutans. Afterwards, the number of S. sobrinus colonies decreased, and was lower than the number of CFU/mL of S. mutans. After 12 h of incubation, no viable S. sobrinus cells were detected.

As shown in Figs. 2, 3 and 4, the growth of S. mutans and S. sobrinus was similar regardless the substrate (glucose, fructose or glucose + fructose) added to the culture medium. The number of CFU/mL of S. mutans was greater than that of S. sobrinus for all incubation periods, in all culture media. After 12h of incubation, the samples obtained from the mixed culture of S. mutans and S. sobrinus in medium containing glucose, fructose or glucose + fructose, when plated on BHI - agar containing streptomycin, did not result in growth of S. sobrinus.

The growth interactions between S. mutans 32K and S. sobrinus 6715 in the in vitro colonization and recolonization process using media with different carbohydrate substrates added (Experiments I, II and III) are presented in Figs. 5, 6 and 7. According to these results, viable cells of S. mutans were shown to be present at the end of the incubation period while S. sobrinus decreased in number or was eliminated.

DISCUSSION

In the development of caries and formation of biofilms there are microbiotic interactions with the dental surface and among bacteria themselves and ecological changes caused by diet. Relevant physico-chemical characteristics and the composition of the plaque act together to initiate this pathology. These bacterial relationships that exist in the biofilm on the enamel surface are regulated by several factors such as the production of bacteriocins (antibacterial proteins that can interfere with the growth of other microorganisms, generally closely related bacteria). The bacteriocins produced by S. mutans are called mutacins, and these proteins are important for the establishment and balance of this species in dental plaque (7,15).

The results obtained from the bacterial suppression experiment shown in Figs. 1, 2 and 3 demonstrated similar behavior for S. mutans and S. sobrinus derived from mixed cultures during the incubation period, even though they showed a higher number of CFU/mL in medium with sucrose added when compared to the other carbohydrates. These data confirm other studies such as those of Ashley and Wilson (2), Brecx et al. (4), Macpherson and Dawes (12), Gibbons and Van Houte (6) and Margolis et al. (13), which determined sucrose to be the main substrate of the streptococcus mutans group. After a short incubation period (3 h), the mixed culture in medium containing sucrose or glucose (10%), fructose (10%) or glucose (5%) + fructose (5%) showed a greater number of CFU/mL of S. sobrinus compared to S. mutans. After a period of 12 h, there was no detection of viable cells of S. sobrinus in any of the culture media tested. These results are in agreement with Ikeda et al. (10), who showed that S. sobrinus was eliminated in mixed in vitro cultures by bacteriocins produced by S. mutans.

The data regarding bacterial plaque colonization and recolonization capacity of S. mutans and S. sobrinus strains are shown in Figs. 5, 6 and 7. These values indicate a higher number of viable cells of S. mutans in agreement with the results of Ikeda et al. (10) demonstrating the capacity of S. mutans to produce bacteriocins that inhibit the growth of other streptococcus of the mutans group and to recolonize the pre-formed bacterial plaque. These data suggest that S. mutans bacteriocins (mutacin) are able to eliminate sensitive plaque bacteria, resulting in greater colonization in vivo. Strains of the same S. mutans genotype show different mutacin profile, suggesting a high degree of interstrain diversity. Mutacin production seems to be of clinical importance in the colonization of S. mutans and is highly diversified in the S. mutans species (11-13).

In conclusion, Streptoccoccus mutans showed a higher number of cells after short or long incubation periods when cultivated with Streptococcus sobrinus indicating its inhibition and recolonization capacity in vitro.

Submitted: April 27, 2004; Returned to authors for corrections: August 23, 2004; Approved: February 15, 2006

• 1. Ahmady, K.; Marsh, P.D.; Newman, H.N.; Bulman, J.S. Distribution of S. mutans and S. sobrinus at sub-sites in human approximal dental plaque. Caries Res., 27, 135-139, 1993.
• 2. Ashley, F.P.; Wilson, R.F. The relationship between dietary sugar experience and the quantity and biochemical composition of plaque in man. Archs. Oral Biol., 22, 409-414, 1977.
• 3. Babaahmady, K.G.; Challacombe, S.J.; Marsh, P.D.; Newman, H.N. Ecological study of Streptococcus mutans, Streptococcus sobrinus and Lactobacillus spp. At sub-sites from aproximal dental plaque. Caries Res, 32, 51-58, 1998.
• 4. Drecx, M.; Theilade, J.; Attstrom, R. Ultrastructural estimation of the effect of sucrose and glucose rinses on early dental plaque formed on plastic films. Scand. J. Dent. Res., 89, 157-164, 1981.
• 5. Donoghue, H.D.; Perrons, C.J. Effect of nutrients on defined bacterial plaques and Streptococcus C67-1 implantation in a model mouth. Caries Res., 25, 108-115, 1991.
• 6. Gibbons, R.J.; Cohen, L.; Hay, D.I. Strains of Streptococcus mutans and Streptococcus sobrinus attach to different pellicle receptors. Infect. Immun., 52, 555-561, 1986.
• 7. Grönrooss, L.; Saarela, M.; Mätto, J.; Tanner-Salo, U.; Vurela, A.; Alaluusua, S. Mutacin production by Streptococcus mutans may promote transmission of bacteria from mother to child. Infect. Immun., 66, 2595-2600, 1998.
• 8. Hirose, H.; Hirose, K.; Isogai, E.; Miura, H.; Ueda, I. Close association between Streptococcus sobrinus in the saliva of young children and smooth-surface caries increment. Caries Res., 27, 292-297, 1993.
• 9. Ikeda, T.; Kurita, T.; Hidaka, H.; Michalek, S.M.; Hirasawa, M. Low-cariogenicity of the tetrasaccharide nystose. Gen. Pharmac., 21,175-179, 1990.
• 10. Ikeda, T.; Kurita, T.; Hirasawa, M. Suppression of Streptococcus sobrinus 6715 (9) in plaques by Streptococcus mutans 32K(c). J. Oral Path., 17, 471-474, 1988.
• 11. Kamiya, R.U.; Napimoga, R.H.; Höfling, J.F.; Gonçalves, R.B. Frequency of four different mutacin genes in Streptococcus mutans genotypes isolated from caries-free and caries-active individuals. J. Med. Microbiol., 54, 599-604, 2005.
• 12. Kamiya, R.U.; Napimoga, R.H.; Rosa, R.T.; Höfling, J.F.; Gonçalves, R.B. Mutacin production in Streptococcus mutans genotypes isolated from caries-affected and caries-free individuals. Oral Microbiol. Immunol., 20, 20-24, 2005.
• 13. Klein, M.I.; Florio, F.M.; Pereira, A.C.; Höfling, J.F.; Gonçalves, R.B. Longitudinal study of transmission, diversity, and stability of Streptococcus mutans and Streptococcus sobrinus genotypes in Brazilian nursey children. J. Clin. Microbiol., 42, 4620-4626, 2004.
• 14. Lindiquist, B.; Emilson, C.G. Dental location of S. mutans and S. sobrinus in humans harboring both species. Caries Res., 25, 146-152, 1991.
• 15. Lindiquist, B.; Emilson, C.G. Colonization of S. mutans and S. sobrinus genotypes and caries development in children to mothers harboring both species. Caries Res., 38, 95-103, 2004.
• 16. Macpherson, L.M.D.; Dawes, C. Effects of salivary film velocity on pH changes in an artificial plaque containing Streptococcus oralis, after exposure to sucrose. J. Dent. Res, 70, 1230-1234, 1991.
• 17. Margolis, H.C.; Zhang, Y.P.; Van Houte, J.; Moreno, E.C. Effect of sucrose concentration on the cariogenic potential of pooled plaque fluid from caries-free and caries-positive individuals. Caries Res., 27, 467-473, 1993.
• 18. Oho, T.; Yamashita, Y.; Shimazaki, Y.; Kushiyama, M.; Koga, T. Simple and rapid detection of Streptococcus mutans and Streptococcus sobrinus in human saliva by polymerase chain reaction. Oral Microbiol. Immunol., 15, 258-262, 2000.
• 19. Oliveira, C.M. Isolamento e caracterização de Streptococcus de placa dental Rio de Janeiro, 1974, 291p. (Ph.D. Thesis. Instituto de Microbiologia. Universidade Federal do Rio de Janeiro.
• 20. Olsson, J.; Van Der Hijde, Y.; Holmberg, K. Plaque formation in vivo and bacterial attachment in vitro on permanently hydrophobic and hydrophilic surfaces. Caries Res., 26, 428-433, 1992.
• 21. Wennerholm, K.; Birkhed, D.; Emilson, C.G. Effects of sugar restriction on Streptococcus sobrinus in saliva and dental plaque. Caries Res, 29, 54-56, 1995.

Publication Dates

• Publication in this collection
  19 May 2006
• Date of issue
  Mar 2006

History

• Accepted
  15 Feb 2006
• Received
  27 Apr 2004
• Reviewed
  23 Aug 2004