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Brazilian Journal of Oral Sciences
Piracicaba Dental School - UNICAMP
EISSN: 1677-3225
Vol. 3, Num. 8, 2004, pp. 384-389
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Brazilian Journal of Oral Sciences, Vol. 3, No. 8, Jan/Mar. 2004, pp.
390-394
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
/ UNESP.2Chairperson
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:
hofling@fop.unicamp.br
Received for publication: June 15, 2003
Accepted: August 25, 2003
Code Number: os04002
Abstract
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.
Introduction
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
models14-22.
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
1. Subjects
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
(Cole-Parmer Instruments).
3. Biotyping
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.
Results
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
1.
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
of S.
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%
of association.
Discussion
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.
ferus (serotype
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
2). According
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.
However,
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
with another
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.
Conclusion
- 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.
Acknowledgements
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.
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