streptococcus mutans is generally considered to be the principal aetiological agent for dental caries [ 1 , 2 ] , which possesses a variety of mechanisms to colonize tooth surfaces . s. mutans species , under certain conditions , is numerically significant in cariogenic biofilms and forms biofilms with other organisms in the oral cavity   after the eruption and colonization of primary teeth . furthermore , epidemiologic surveys have confirmed that higher levels of s. mutans organisms in children are associated with a higher incidence of decayed , missing , and filled ( dmf ) teeth [ 2 , 6 ] . conversely , it can be found in populations with no caries or with low caries experience [ 7 , 8 ] . one possible explanation for their presence in subjects with low caries experience is that s. mutans virulence factors can differ between populations with contrasting caries prevalence . bowden   pointed out the necessity for understanding the clonality patterns of s. mutans in the caries - free subjects where it is important to ascertain whether s. mutans populations in subjects free of caries exhibit the same clonal diversity of caries - active groups or not . several studies have showed genetic heterogeneity among streptococcus mutans strains [ 1116 ] ; however , the relationship between caries activity and the genetic diversity of s. mutans is still controversial . suggested that caries - active children with high sucrose consumption carried greater ribotype diversity of s. mutans compared with caries - free children . .   found that caries - active subjects have more genotypes than caries - free subjects . on the other hand , kreulen et al the aim of the present paper was to evaluate the genotypic diversity of s. mutans in caries - free and caries - active preschool children in brazil . study participants consisted of 28 preschool children aged between 4 and 5 years old from low socioeconomic level families . the subjects were selected from a group of children attending a nursery located in a medium - sized city from southern brazil . all of them were from the day nursery , staying in the nursery for 5 days per week , 8 hours per day .   during the sample selection , subjects who had any chronic disease and were using antibiotic in the last 3 months were excluded . the aim and details of the experiments were explained , and the informed consent was obtained from parents and guardians prior to the beginning of the research procedures . experimental procedures were approved by the ethical committee of the university of north of parana school of dentistry . diagnosis was visual , using a mouth mirror and cotton rolls to assist visibility and a periodontal probe to remove any plaque or debris when necessary . caries experience was measured by the dmft ( decayed , missing , and filled teeth ) index , according to the world health organization . the caries experience was dichotomized into two groups : caries - free ( dmft = 0 ) and dental caries children ( dmft > 0 ) . the clinical examination was performed by the same examiner ( f.j.s.p . ) . streptococcus mutans clinical isolates were obtained from mitis - salivarius agar with bacitracin and potassium telurite . about 10 colonies resembling s. mutans from each child were transferred to brain heart infusion broth bhi ( difco , detroit , usa ) and incubated at 37c for 48 hours in an anaerobic jar . dna from 280 isolates were extracted by using a simple dna preparation in which the cells were washed and boiled for 10 minutes with te buffer ( 10  mm tris / hcl , 1  mm edta , ph 8.0 ) modified from saarela et al .   and welsh and mcclelland . the debris were pelleted and the supernatants were stored in a freezer at 20c until use . isolates were confirmed for species identity in pcr reactions with primers specific for gtfb , encoding glucosyltransferase   5act aca ctt tcg ggt ggc ttgg3 and 5cag tat aag cgc cag ttt catc3(invitrogen ) , yielding an amplicon of 517  pb for s. mutans   gtfb gene . each reaction consisted of 5  l template dna , 1  m of each primer , 200  m of each dntp , 5  l 1x pcr buffer , 1.5  mm mgcl2 , and 1  u taq dna polymerase ( invitrogen , so paulo , brazil ) in a total volume of 25  l . the amplification reaction was performed in 30 cycles as follows : denaturation 95c for 30 seconds , annealing at 59c for 30 seconds , and extension at 72c for 1 minute . one reference strain ( atcc 25175 ) was used as a positive control of s. mutans and distilled water was used as a negative control . amplification products were analysed electrophoretically in 1% agarose gels using tbe buffer ( 89  mmol  l tris borate , 89  mmol  l boric acid , 2  mmol  l edta ; ph 8) , stained with ethidium bromide and observed under uv light . a 100  bp dna ladder served as molecular - size marker in each gel . the sequences of the primers opa 02 ( 5tgccgagctg3 ) and opa 13   ( 5cagcacccac3 ) were used . the pcr reactions were performed as follows : 1x pcr buffer ( 200  mmol  l tris - hcl ph 8.4 ; 500  mmol  l kcl ) with 3.5  mm of mgcl2 , 0.2  mm of each dntps , 0.4  mm of primers , 2.5  u of taq dna polymerase , and 2.5  l of dna sample . the pcr conditions included 35 cycles of denaturation at 94c for 1 minute , annealing at 36c for 2 minutes , extension at 72c for 2 minutes , with initial denaturation at 94c for 5 minutes , and a final extension at 72c for 5 minutes . the eletrophoresis was carried out as described previously ; however amplification products were analysed in 2% agarose gel . individual ap - pcr amplicons were marked , and the individual bands were analyzed by using the dice coefficient ( > 95% ) following mitchell et al . . a dendrogram was constructed using the upgma cluster analysis with the aid of numerical taxonomy and multivariate analysis system ( ntsys ) program ( exeter software , setauket , ny ) . the differences between the genotypic diversity and dmft / caries experience were evaluated by  test and the spearman 's coefficient of correlation . the software statistical package for social science , v. 11.5 ( ssps , chicago , il , usa ) was used for the data analysis . a total of 140 isolates of the preschool children with dental caries and 140 isolates of the caries - free preschool children were analyzed by ap - pcr , and 62 different amplitypes were identified . figure 1 illustrates the ap - pcr patterns performed with opa-02 and opa-13 , with each of these primers generating a different spectrum of amplicons , indicative of genetic polymorphism . no significant correlation of s. mutans was found between genotypic diversity of s. mutans and gender . the spearman correlation test showed a strong association between genotypic diversity and caries experience ( r = 0.72 ;  p < .001 ) . there were more s. mutans genotypes in the group of preschool children with dental caries , compared with the caries - free group . among the children with more than 1 genotype , 13 had dental caries ( 2 to 5 genotypes ) and 4 were caries - free ( only 2 genotypes ) .   considering the whole population , some of the preschool children harbored just one genotype whereas others exhibited until five genotypes   ( figure 2 ) . the dental biofilm consists of a complex bacterial community , and the ability of specific strains of streptococcus mutans to compete with other strains may be essential for colonization . studies of s. mutans virulence factors and their correlation with other species are fundamental to understand the role played by colonization of different genotypes in the same individual . the knowledge of genotypic diversity of s. mutans may help in the development of new treatment strategies for caries , so as to prevent disease and promote health in addition to standard prevention treatments . although the findings of kreulen et al .   have demonstrated a negative relationship between caries activity and genotype diversity and the results of   lembo et al .   have shown no significant differences in the number of genotypes detected in caries - free and caries - active children , the findings of the present study showed a positive relationship between caries activity and the genetic diversity of s. mutans . the preschool children with dental caries have more genotypes than the caries - free children , which is consistent with earlier reports [ 17 , 18 , 28 , 29 ] . the existence of several genotypes in the biofilm could merely be a consequence of favorable circumstances for s. mutans . moreover , it is possible that the simultaneous action of different genotypes , with distinct virulence potential , further increases the risk of caries . in studies with young adults , emanuelsson et al .   found a maximum of seven genotypes in subjects who had previously experienced dental caries . napimoga et al .   also found a maximum of eight genotypes in caries - active subjects using ap - pcr . however , it has been observed that children harbor only one to five distinct genotypes of s. mutans   [ 3 , 11 , 1517 , 19 , 30 ] . it was observed that in the caries - free group , 10 preschool children had only one genotype . on the other hand , in the dental caries group , 13 children had more than one genotype . this may be attributed to heavy colonization and growth of multiple genotypes in the same oral cavity is likely to be consequences of frequent consumption of fermentable carbohydrates . different clonal types of s. mutans detected within the oral cavity of one subject may exhibit different phenotypic and genetic properties . in addition , the high clonal diversity of s. mutans can result in colonization by clones with different virulence attributes . our results support the previous findings of genetic diversity of s. mutans in preschool children being associated with dental caries . the investigation of such populations may be important for directing the development of programs for caries prevention worldwide .
aim . 
 the aim of the present paper was to evaluate the 
                   genotypic diversity of s. 
                   mutans in caries - free and caries - active 
                   preschool children in brazil . 
 design . 
 twenty - eight preschool 
                   children were examined regarding caries 
                   experience by the dmft index . 
 dna from 280 
                   isolates of s. mutans was 
                   extracted . 
 s. mutans evaluated 
                   using to the pcr method , with primers for the 
                   glucosyltransferase gene . 
 the genetic diversity 
                   of s. mutans isolates was 
                   analyzed by arbitrary primed - pcr ( ap - pcr ) 
                   reactions . 
 the differences between the diversity 
                   genotypic and dmft / caries experience were 
                   evaluated by 
                   2 
                   test and spearman 's correlation . 
                   results . 
 the spearman 
                   correlation test showed a strong association 
                   between genotypic diversity and caries 
                   experience ( r = 0.72 ; 
                   p < .001 ) . 
 there were more s. mutans 
                   genotypes in the group of preschool children 
                   with dental caries , compared with the 
                   caries - free group . among the children with more 
                   than 1 genotype , 13 had dental caries ( 2 to 5 
                   genotypes ) and 4 were caries - free ( only 2 
                   genotypes ) 
 . conclusion . 
 our 
                   results support the previous findings of genetic 
                   diversity of s. mutans in 
                   preschool children being associated with dental 
                   caries . 
 the investigation of such populations 
                   may be important for directing the development 
                   of programs for caries prevention 
                   worldwide .