Brazilian Journal of Oral Sciences
Piracicaba Dental School - UNICAMP
Vol. 3, Num. 8, 2004, pp. 384-389
Brazilian Journal of Oral Sciences, Vol. 3, No. 8, Jan/Mar. 2004, pp.
Salivary biotypes of mutans Streptococci
levels in school children aging 6-8 year
old having a socioeconomic base
Denise Madalena Palomari Spolidorio1, José Francisco Höfling2, Edvaldo
Antônio Ribeiro Rosa3, Cássio Vicente Pereira4, Daniella Moreira5, Reginaldo Bruno Gonçalves6
1Professor of Pathology area, AraraquaraDental School
of Microbiology and Immunologyarea, Piracicaba Dental School / UNICAMP .3Professor
of Microbiology and Immunologyarea, Paraná / PUC .4Professor
of Microbiology and Immunologyarea, Lavras University Center - UNILAVRAS/MG.5Dental Surgery , PhD.6Professor
of Microbiology and Immunologyarea, Piracicaba Dental School / UNICAMP.
Correspondence to: José Francisco Hofling Faculdade de Odontologia
de Piracicaba - UNICAMP Área de Microbiologia e Imunologia Avenida Limeira,
901, Areião CEP 13141-900, Piracicaba, SP, Brazil, Fone: 55-19-3412
5322 Fax: 55-19-3412-5218 e-mail:
Received for publication: June 15, 2003
Code Number: os04002
Accepted: August 25, 2003
There is still little information on the mutans Streptococci biotypes in
South America, specifically in Brazil, including regional and local information,
associated to different socioeconomic categories. In this
paper, the Streptococcus mutans levels and biotypes in Brazilian children
aged 6-8 year old having a socioeconomic base was investigated.
The sampling consisted of 200 selected children belonging to five
different socioeconomic categories. Bacterial examination was made
using children saliva diluted and cultivated in Bacitracin sucrose agar
(SB-20). The plates were incubated by 48 hours/37ºC at 10% CO2 in
anaerobiose and after colonies growth samples identification was performed
by biochemical tests. Results showed that 78% of the 491
tested samples harbored S. mutans, followed by S. sobrinus (11.6%),
S. rattus (4.69%), S. mutans V (2.65%), S. cricetus (1.83%) and S.
ferus (1.22%). The prevalence of Streptococcus mutans was found in
every socioeconomic level. Among children harboring multiple species,
the most frequent association detected was S. mutans/ S. sobrinus (17%).
Key Words: mutans Streptococci, distribution, children, socioeconomic base.
Several epidemiological studies have been made about the prevalence, level and
geographical distribution of the mutans Streptococci1-11.
These microorganisms are subdivided in seven different species and eight serotypes12-14,
having already been demonstrated that S. mutans, S. sobrinus, S.
rattus, S. cricetus and S. ferus are cariogenic for experimental
Many studies showed variations in the distribution
of those microorganisms. In Europe and North America, serotypes c (80%) and
with smaller frequency, d and a23 were found. In Egypt, the predominant
were a and b serotypes24. Therefore, there is little information
on the mutans Streptococci serotypes distribution in South America,
specifically in Brazil, including regional and local information, associated
to a different socioeconomic categories. The aim of this study was to isolate
and identify salivary biotypes of mutans Streptococci levels
in schoolchildren aged 6 to 8 year old having a socioeconomic base.
Material and Methods
Two hundred children aging 6 to 8 year old without race or sex distinction,
with different dental decay indexes without using antibiotics at least six
months were used in this study. Children of five different socioeconomic categories
(A - E), according to the ABA/ABIPEME criteria, from Piracicaba, SP, Brasil
urban perimeter were selected.
2. Bacterial isolates
A gum-base piece of 1.5 grams was supplied to children chewed it for 30 seconds.
The stimulated saliva was despised and starting from this moment it was marked
the time of 3 to 5 minutes and then collected it in tubes approximately of
3ml. The collected samples were conserved in ice during collection and transported
to the Microbiology and Immunology laboratory. The time elapsed from the samples
collection did not exceed 4 hours5. Samples were mixed for 30 seconds
and the saliva samples were diluted (10-1 to 10-4) in
0.05M Phosphate buffered/solution (PBS) pH7.3. Aliquots of 25mL of each dilution
were inoculated in Bacitracin Sucrose Agar/SB-2025 medium
and then incubated at 37ºC for 48 hours in anaerobic water-jacketed CO2 incubator
At least three typical colonies with morphological characteristics of S.
mutans according to Van Palenstein-Helderman et al.26 and
Torres et al.27 were transferred to 4ml of Brain Heart Infusion
(BHI) medium, incubated at 37ºC for 24 hours and after growth stored
at -70ºC for further biochemical identification tests28.
Among the 491 mutans Streptococci isolated, S. mutans (78%) was
predominant. S. sobrinus and S. rattus were 11.61% and 4.69%
respectively, for all the socioeconomic categories as shown in Table
2 shows the distribution and frequency (%) of children harboring different
species of Streptococcus group mutans according to the socioeconomic
categories A, B, C, D and E. Most of the individuals (59%) were carriers
mutans followed by the association of S. mutans + S. sobrinus (17%)
and S. mutans + S. rattus (7.5%). Other species showed less than 5.0%
The identification of the microorganisms (491 samples) performed through the
morphological and biochemical characteristics, according to the outline of
Hardie28 allowed the identification of multiple types of the bacterium
Streptococcus group mutans concerning to the different socioeconomic categories
(A to E). Most of the isolates were identified as being S. mutans (78%)
and S. sobrinus (11.6%). These results are in agreement with the data
of Marfort et al.2, Farghaly et al.29, Torres30 and
Saarela et al.31, which found the serotype c (biotype I) as the
prevalent specie in the population studied. In our study, the S. mutans serotype
c (biotype I) was also the prevalent specie among all the population concerning
to the five socioeconomic categories, followed by S. sobrinus serotype
d (biotype IV), S. rattus serotype b (biotype II), S. mutans V serotype
e (biotype V), S. cricetus serotype a (biotype III) and S.
c). These data suggest a possible predominance of the serotype c (biotype
I) in our region.
The frequency of S. sobrinus (serotype d
- biotype IV) was the second specie after the serotype c, being in agreement
with the findings of Nyvad and Kilian32. Our results
also showed that this specie was detected in association with the serotype
c - biotypes I and V - (Table
2) being also found in association with other
serotypes33-35. These authors still relate that
the decay index was larger in the children with the combination of S.
mutans and S. sobrinus species when compared to those
harboring only S. mutans.
The species S. rattus(serotype
b - biotype II), was detected and isolated in low proportion: at the socioeconomic
category B and C or in association with other biotypes (Table
to Farghaly et al.29, Nyvad and Kilian32 and
Beighton et al.36 this serotype was not detected
individually neither in association with other biotypes, as also found by
Marfort2 and Bratthal24.
According to Torres et al.27, the detection of the
serotype b (biotype II) may be associated to the socioeconomic status of
the population, to the lifestyle (residents in the periphery) of the individuals
and/or to the absence of provisioning water fluoretation. Our results showed
this microorganism individually in the socioeconomic categories B and
C and in association with other serotypes (c and d) in all the socioeconomic
categories, including the higher socioeconomic category A and B, residents
in the central area of the city, provisioned by water fluoretation. The socioeconomic
status of any population, may not necessarily be a decisive factor in the
detection of this biotype. This kind of association is still less found in
the literature, and being taken into account that the isolation of such serotype
has been detected in low frequency in the population sampled, statements
of such nature, need larger investigations.
The detection of the S. mutans V (serotype e - biotype V), differs of
the biotype I, by the melibiose use. In our sample, was present in most, in
association with other serotypes (c and d), and only two individuals (socioeconomic
categories A and D) showed this specie individually. These results are in agreement
with the data obtained by Thomson et al.37 and Miranda et al.38,
which also found such species in association with other serotypes. These authors
also showed that this biotype is not related to high dental decay indexes even
when associated with other biotype, not subject of our study. Concerning to
the frequency of multiple serotypes of mutans Streptococci, the most frequent
association founded was S. mutans/S. sobrinus (17%) approaching to the
results obtained by Shklair and Keene39 and Fracalanzza40,
which showed 27.6% and 21.4% respectively, followed by the association S.
mutans/S. rattus (7.5%) and S. mutans/S. mutans V (3.5%) as shown
in Table 2. Azevedo41, described the combination S. mutans/S.
sobrinus in a smaller percentage (2.78%), followed by S. mutans/S.
mutans V (1.85%), being the most frequent combination S. mutans/S. rattus (19.44%).
Fracalanzza3 and Shklair and Keene38 showed, for the
combination S. mutans/S. mutans V respectively, 28.6%, 1.85% and for
the triple association this last author showed S. mutans/S. sobrinus/S.
mutans V (71.0%), much higher than our results (1.0%).
This supposed disagreement
concerning to the multiple types combination, may be due to the saliva sampling
performed in this research, instead of "pool of plaque", as described
and preconized by Masuda et al.42,
showing also that 68% of the individuals harbored two or more serotypes of
those microorganisms. These diversities in the multiple types of mutans Streptococci isolations,
shows that geographical variations may be a limiting factor in the prevalence
of certain species. The association of the mentioned biotypes may not be determined
by mechanisms or well-known processes, happening therefore, by chance.
the statement of Beighton et al.5 and Thibodeau et al.43,
in relation to the need of more researches to elucidate the mechanisms by
which certain species of mutans Streptococci tend settling down, to
prevail and to colonize the buccal cavity, individually or in association
species must be take into account. Thus, new researches, seeking to elucidate
such variability, should be done by oral researchers looking for its meaning
in relation to dental decay.
- Seventy-eight percent of the 491 tested samples are colonies of S. mutans,
followed by S. sobrinus, S. rattus, S. mutans V, cricetus, and S. ferus;
- The predominance of S. mutans is find in all socioeconomic categories;
- Among individuals harboring multiple species, the most frequent association
is S. mutans/S.sobrinus.
We are grateful to the laboratory technicians Anderson L. Teixeira, Wilma C.
Ferraz and Elza Thomazini for their technical support. This project was funded
by Faep/ UNICAMP, CNPq and Fapesp Proc. 94/0908-0 to all we are deeply thankful.
- Carlsson P, Olsson B, Bratthall D. The relationship between bacterium Streptococcus
mutans in saliva and caries in children in Mozambique. Arch Oral Biol
1985; 30: 265-8.
- Marfort RA, Borgarelli MI, Piovano SH, Cazabat MC, Brizuela LE, Manto MDC. ldentificación
morfológica, bioquímica y serológica de S. mutans en
placa dental de ninõs. Rev Asoc Odont Argent 1985; 73: 40-4.
- Fujiwara T, Sasad E, Mima N, Ooshima T. Caries prevalence and salivary mutans
streptococci in 0-2 year-old children of Japan. Community Dent Oral Epidemiol
1991; 19: 151-4.
- Alaluusua S, Mättö J, Grönroos L, Innilä S. Oral colonization
by more than one clonal type of mutans Streptococcus in children with
nursing-bottle dental caries. Arch Oral Biol 1996; 41: 167-73,
- Bentley C, Rippon HR, Thomas HEC. The distribution of S. mutans serotypes
and dental caries in a group of 5 to 8 year old Hampshire schoolchildren.
Br Dent J 1987; 162:103-6.
- De Saet JJ, Holbrook WP, Van Amerongen WE, Schipper E, Homburg CH, De Graaff
J. Prevalence of Streptococcus sobrinus in relation to dental caries in children
from Iceland and the hetherlands. ASDC J Dent child
1990; 57: 37-42.
- Carlsson J. A numerical taxonomic study of human oral Streptococci.
Odont Revy 1968; 19: 137-60.
- Emilson CG. Prevalence of S. mutans with different
colonial morphologies in human plaque and saliva. Scand J Dent Res 1983;
- Babaahmady KG, Challacombe SJ, Marsh PD, Newman HN. Ecological
study of Streptococcus mutans, Streptococcus sobrinus and Lactobaccilus
spp. At sub-sites from aproximal
dental plaque. Caries Res 1998; 32: 51-8.
- Kohler B, Bjarnason S, Finnbogason
SY, Holbrook WP. Mutans Strptococci, Lactobacilli and caries experience
in 12-year-old Iceland urban children, 1984 and 1991.
Community Dent Oral Epidemiol 1995; 23: 65-8.
- Emanuelsson IM. Mutans Streptococci
- in families and tooth sites studies on the distribution, acquisition
and persistence DNA fingerprint. Swed Dent
J Suppl 2001; 148: 1-66
- Marsh P, Martin M. Oral microbiology. 3rd ed.
New York: Chapman & Hall,
- Bratthall D, Köhler B. Streptococcus mutans serotypes: some aspects
of their identification, distribution, antigenic shifts and relationship
to caries. J Dent Res1976; 55(special issue C): C15-21.
- Balakrishnam M, Simmonds
RS, Kilian M. Different bacteriocin activities of S.
mutans reflect distinct phylogenetic lineages. J Med Microbiol 2002;
- Loesche WJ. Ecology of S. mutans in the human mouth and evidence for
its role in carie initiation. In: Simposium on Current Topics in Dental
Caries. Proceedings, Matsudo, Japan; 1982. p.180-97.
- Fitzgerald DB, Fitzgerald
RJ, Adams BO, Morhart RE. Prevalence, distribution of serotypes and cariogenic
potencial in hamsters of mutans streptococci
from elderly individuals. Infect Immun 1983; 54: 288-90.
- Bowen WH, Ostron
CA, Konlonrides T. Establishment of potentially cariogenic in a experimental
human plaque: Strptococcus mutans. J Dent Res 1980; 59:
- Beighton D, Mayday H, Russel RRB, Whiley RA. S. macacae sp now
from dental plaque of monkeys. Inst J Syst Bact 1984; 34: 332-5
WH. The introduction of rampant dental caries in monkeys (Macaca irus).
Caries Res, Basel 1969; 3: 227-37
- Fitzgerald RJ, Keyes PH. Demonstration
of the etiologic role of Streptococci in experimental caries in the hamster.
J Am Dent Assoc 1960; 61: 9-19.
- Hilman JD, Yaphe BJ, Johnson KP. Colonization
of the human oral cavity by a strain of Streptococcus mutans. J
Dent Res 1985; 64: 1272-4.
- Lindquist B, Emilson CG. Dental location of
S. mutans and S. sobrinus in humans harboring both species. Caries Res
- Walter RG, Shklair IL. S. mutans in caries-free and caries active naval
recruits. J Dent Res 1982; 16: 1229-32.
- Bratthall D. lmmunofluorescent
identification of S. mutans. Odontol Rev
- Davey AL, Rogers AH. Multiple types of the bacterium mutans e seus biotipos
de placa dental de criancas da cidade do Streptococcus mutans in
the human mouth and their intra-Rio de Janeiro [dissertation]. Rio de Janeiro:
lnstituto de family transmission. Arch Oral Biol 1984; 29: 453-60. Microbiologia,
Universidade Federal do Rio de Janeiro; 1979.
- Van Palenstein-Helderman WH,
Ijsseldijk M, Huis In't Veld 41. Azevedo
RVP. O emprego da bacteriocinotipagem JHJ. A selective medium for the two
major subgroups of the (mutacinotipagem) no rastreamento epidemiológico
de bacterium Streptococcus mutans isolated from humam dental estreptococos
do "grupo mutans"[thesis]. São Paulo: Instituto plaque
and saliva. Arch Oral Biol 1983; 28: 599-603. de Ciências Biomédicas,
Universidade de São Paulo;1988.
- Torres AS, Pizzolitto AC, Ellias AM,
Ito IY. Estreptococos do 42. Masuda N, Shimamoto T, Kitamura K, Sobue S,
Hamada S. grupo mutans: Avaliação
do ágar SB-20 e MSB na determinação Transmission of Streptococcus
mutans in some selected de UFC na saliva e na placa dental de adolescentes.
Rev Bras families. Microbios 1985; 44: 223-32. Odontol 1993, 50: 18-21.
- Hardie JM. Oral streptococci. In: Holt JA. Bergey’s manual of
sistematic bacteriology. Baltimore: Williams & Wilkins; 1986.
vol.2, p. 1054-63.
- Farghaly MM, Eklund S, Loesche WJ. Streptococcus mutans levels and
biotypes in Egyptian Saudi Arabian students during the first mouths of residency
in the United States. J Dent Res 1984; 63: 52-5.
- Torres SA. Avaliação do agar SB-20 e MSB na contagem de estreptococos
do grupo mutans na saliva e na placa dental de adolescentes [thesis]. Araraquara:
Faculdade de Ciências Farmacêuticas de Araraquara, Universidade
Estadual Paulista; 1991.
- Saarela M, Hannula J, Matto J, Asikainen S, Alaluusua
S. Typing of mutans streptococci by arbitrarily polymerase chain reation.
Arch Oral Biol 1996; 41: 821-6.
- Nyvad B, Kilian M. Comparison of the initial
streptococcal microflora on dental enamel in caries-active and caries-inactive
individuals. Caries Res 1990,
- Kohler B, Bjarnason S. Mutans streptococci, lactobacilli and caries prevalence
in the 11 and 12 year old Iceland children. Community Dent Oral Epidemiol
1987; 15: 332-5.
- Höfling JF, Spolidorio DMP, Pereira CV, Rosa EAR, Moreira D. Presença
de Streptococcus mutans e Streptococcus mutans associado a Streptococcus
sobrinus em escolares de diferentes classes sócio-econômicas
e sua relação com a atividade cariogênica dessas populações.
Rev Odontol USP 1999; 13: 173-80.
- 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 1993; 27: 292-7.
- Beighton D, Crawford JG, Broderius
CA. Analytical and physiological variability of salivary microbial
counts. J Dent Res 1988; 67: 1409-13.
- Thomson LA, Little WA, Bowden WH, Sierra
LI, Aguirrer M, Gillespie G. Prevalence of S. mutans serotypes, actinomyces and
other bacteria in the plaque of children. J Dent Res 1980;59: 1581-9.
VC, Pizzolitto AC, Vertuan V. Meio de cultura para isolamento de S.
mutans. Rev Assoc Paul Cir Dent 1982, 36: 618-23.
- Shklair IL, Keene
HJ. Biochemical characterization and distribution of S. mutans in
three diverse populations. In: Stiles HM, Loesche WJ, O'Brien TC. eds.
Washington: Information Retrieval; 1976; p.201-10.
- Fracalanzza SEL. lsolamento
e caracterização de Streptococcus mutans e seus biotipos
de placa dental de criancas da cidade do
Rio de Janeiro [dissertation]. Rio de Janeiro: lnstituto de
Microbiologia, Universidade Federal do Rio de Janeiro; 1979.
- Azevedo RVP. O emprego da bacteriocinotipagem
(mutacinotipagem) no rastreamento epidemiológico de
estreptococos do “grupo mutans” [thesis]. São Paulo: Instituto
de Ciências Biomédicas, Universidade de São Paulo; 1988.
- Masuda N, Shimamoto T, Kitamura K, Sobue S, Hamada S.
Transmission of Streptococcus mutans in some selected families.
Microbios 1985; 44: 223-32.
- Thibodeau EA, O’sullivan DM, Tinanoff N. Mutans streptococci
and caries prevalence in pre-school children. Community Dent
Oral Epidemiol 1993, 21: 288-91.
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