the development of dental caries is a multistep process that often begins with the formation of plaque substance , a structurally and functionally defined biofilm with microbial communities organized in a polysaccharide matrix . the formation of dental plaque is a normal event even in an otherwise healthy mouth with no caries , and the microbial communities colonizing them primarily exist as symbionts , some of which essentially behave as commensals to prevent the pathogenic species from gaining access to the tooth surface . in individuals with poorer oral hygiene the microbial population of the plaque shifts toward acidogenic and acid - tolerant species that consequently promotes active carious lesions . the two most widely studied cariogenic bacteria are mutans streptococci ( ms ) and lactobacillus , the levels of which have been found to correlate with the number of decayed teeth or decayed , missing or filled teeth ( dmft ) . more specifically , the association of streptococcus mutans of ms group with cariogenesis has been extensively characterized . at the time of biofilm formation , the s. mutans adheres to the tooth surface with its dextran glucose rich polysaccharide capsule that promotes an acid - tolerant biofilm environment . as s. mutans in biofilm begins to divide and form microcolonies , its metabolic process breaks down dietary sucrose to glucose and fructose . while fructose is fermented for energy , glucose is polymerized into an extracellular dextran , which cements s. mutans to tooth enamel to provide a matrix for dental plaque . in the absence of dietary sucrose , s. mutans depolymerizes the extracellular dextran polymer to glucose molecules again for carbon source , which results in the production of lactic acid within the biofilm that subsequently decalcifies enamel . decalcification of enamel produces white spots on the tooth surface and is often considered as a primary carious lesion . streptococcus cricetus ( serotype a ) , streptococcus rattus ( serotype b ) , s. mutans ( serotypes c , e , f and k ) , streptococcus sobrinus ( serotypes d and g ) , streptococcus downei ( serotype h ) , streptococcus ferus ( serotype c ) and streptococcus macacae ( serotype c ) based on the composition and linkages of cell wall polysaccharides , physiological reactions , serological specificity and 16s rrna homology . more recently , primers designed to amplify specific variant regions of rhamnose - glucose polymer ( rgp ) operon , which encodes for enzymes that catalyze the formation of serotype specific rgps in the cell wall are used to classify the four serotypes of s. mutans : c , e , f and k. while variations downstream of rgpf gene region are used to type c , e and f , variations within the rgpf is used to type serotype k. recent studies have indicated an association between cariogenic s. mutans serotypes and infective endocarditis ( ie ) , an inflammatory condition of endothelial cells of the heart . indeed s. mutans serotypes have been found in the heart valves and blood samples of patients with ie . these findings highlight the clinical significance of elucidating the prevalence of s. mutans serotypes in a population . investigation on the prevalence of s. mutans serotype c in children from india with or without caries identified its wider prevalence in both noncaries and caries group , as has been reported elsewhere . however , the prevalence of the other three serotypes : e , f and k in indian subjects with or without caries remains yet to be identified . in this study , we sought to investigate , ( 1 ) the prevalence of s. mutans in school going children from a defined location within the southern region of india , ( 2 ) the prevalence of the three s. mutans serotypes e , f and k in s. mutans positive subjects by serotype specific polymerase chain reaction ( pcr ) technique , and ( 3 ) the association of the occurrence of serotypes with caries . school children in the age group of 6 - 12 years hailing from the coastal region of state of tamil nadu , south india , were selected by random sampling and screened for dental caries . children with dmft ( permanent dentition)/dmft ( deciduous dentition ) index > 5 or < 1 but otherwise clinically healthy were included in the study , after obtaining informed consent from their respective parents . the dmft / dmft index of 5 was used as a cut - off to define caries activity in the community addressed in this study , but not that of the general population . sixty children with dmft / dmft index > 5 and 39 children with dmft / dmft index < 1 were selected for inclusion in the study . the sample size was calculated based on standard normal distribution with reference to the prevalence of s. mutans and its serotypes e , f and k in japanese population , as the three serotypes have been simultaneously studied only in these samples by serotype specific pcr . the study protocol was approved by the institutional ethics committee . after obtaining demographic details , 2 ml of unstimulated saliva was collected from each child in a 15 ml sterile tube ( cat # 546021 , tarsons , kolkata , india ) , which was stored at  20c until transported to the laboratory . at the time of dna extraction , the saliva samples stored in the freezer were thawed and bacterial cells were pelleted by spinning them at 3000 rpm for 10 min at room temperature . pelleted cells were resuspended in 100 l of 1x phosphate buffered saline , ph 7.2 containing 20 l of fresh lysozyme ( 5 mg / ml , cay # l6876 , sigma - aldrich , st . louis , mo , usa ) , and the cell suspension was incubated at 4c for 15 min . an equal volume of cell lysis buffer containing 0.1% sodium dodecyl sulfate , 25 mm edta , 75 g/100 l proteinase - k ( cat # p2308 , sigma - aldrich , st . louis , mo , usa ) and 200 mm tris - cl at ph 8 ( cat # t6664 , sigma - aldrich , st . louis , mo , usa ) was then added to the cell suspension and incubated at 55c for 30 min . following lysis , 2.5 volume of cold ethanol was added to the cell lysates to precipitate genomic dna . the tubes were then centrifuged at 12,000 rpm for 15 min at room temperature to pellet the genomic dna precipitate . subsequently , the pellet was washed twice with 0.25 ml of 70% ethanol and then dissolved in 25 l of double deionized water ( cat # w4502 , sigma - aldrich , st . pcr amplification was performed on 25 ng of dna samples with strain or serotype specific primers as described elsewhere . the amplification conditions were similar to those published earlier , except that 25 cycles or 35 cycles of amplification were employed for strain specific or serotype specific pcr , respectively . a negative control reaction without dna was run to confirm the specificity of amplification in experimental samples . chromosomal dna of s. mutans serotypes e , f , and k ( kind gift of kazuhiko nakano , department of pediatric dentistry , osaka university graduate school of dentistry , japan ) were used as a positive control for amplification reactions . school children in the age group of 6 - 12 years hailing from the coastal region of state of tamil nadu , south india , were selected by random sampling and screened for dental caries . children with dmft ( permanent dentition)/dmft ( deciduous dentition ) index > 5 or < 1 but otherwise clinically healthy were included in the study , after obtaining informed consent from their respective parents . the dmft / dmft index of 5 was used as a cut - off to define caries activity in the community addressed in this study , but not that of the general population . sixty children with dmft / dmft index > 5 and 39 children with dmft / dmft index < 1 were selected for inclusion in the study . the sample size was calculated based on standard normal distribution with reference to the prevalence of s. mutans and its serotypes e , f and k in japanese population , as the three serotypes have been simultaneously studied only in these samples by serotype specific pcr . after obtaining demographic details , 2 ml of unstimulated saliva was collected from each child in a 15 ml sterile tube ( cat # 546021 , tarsons , kolkata , india ) , which was stored at  20c until transported to the laboratory . at the time of dna extraction , the saliva samples stored in the freezer were thawed and bacterial cells were pelleted by spinning them at 3000 rpm for 10 min at room temperature . pelleted cells were resuspended in 100 l of 1x phosphate buffered saline , ph 7.2 containing 20 l of fresh lysozyme ( 5 mg / ml , cay # l6876 , sigma - aldrich , st . louis , mo , usa ) , and the cell suspension was incubated at 4c for 15 min . an equal volume of cell lysis buffer containing 0.1% sodium dodecyl sulfate , 25 mm edta , 75 g/100 l proteinase - k ( cat # p2308 , sigma - aldrich , st . louis , mo , usa ) and 200 mm tris - cl at ph 8 ( cat # t6664 , sigma - aldrich , st . louis , mo , usa ) was then added to the cell suspension and incubated at 55c for 30 min . following lysis , 2.5 volume of cold ethanol was added to the cell lysates to precipitate genomic dna . the tubes were then centrifuged at 12,000 rpm for 15 min at room temperature to pellet the genomic dna precipitate . subsequently , the pellet was washed twice with 0.25 ml of 70% ethanol and then dissolved in 25 l of double deionized water ( cat # w4502 , sigma - aldrich , st . pcr amplification was performed on 25 ng of dna samples with strain or serotype specific primers as described elsewhere . the amplification conditions were similar to those published earlier , except that 25 cycles or 35 cycles of amplification were employed for strain specific or serotype specific pcr , respectively . a negative control reaction without dna was run to confirm the specificity of amplification in experimental samples . chromosomal dna of s. mutans serotypes e , f , and k ( kind gift of kazuhiko nakano , department of pediatric dentistry , osaka university graduate school of dentistry , japan ) were used as a positive control for amplification reactions . in order to determine the prevalence of s. mutans serotypes e , f and k in children of age group 6 - 12 years , the oral cavity of 99 school children of afore mentioned age group were screened by a single examiner to select the caries active ( ca ) and caries free ( cf ) groups . the intra examiner reliability was assessed by screening twice on 25 children at an interval of 2 weeks and the data was assessed by reliability test and kappa value was found to be > 0.91 . those children with clinically identifiable dmft / dmft index > 5 were considered as ca , while those with dmft / dmft index < 1 were considered as cf . dmft / dmft was chosen instead of dmfs as dmft / dmft , ( 1 ) is a world health organization standardized criteria for clinical registration of dental caries , ( 2 ) provides a rapid way of screening a large number of subjects in field trips , and ( 3 ) is commonly used in epidemiological studies in school children worldwide . hence , based on the above criterion 60 and 39 children were selected for inclusion in ca and cf groups respectively . subsequently , unstimulated saliva samples were obtained from both groups of children and screened to first identify the prevalence of s. mutans by strain specific pcr amplification method . this method uses primers that anneal and amplify a segment of gtfb gene specifically from s. mutans and hence may be used to detect its overall prevalence . gel analysis of the pcr amplified samples showed s. mutans specific band in 80% ( 48/60 ) of ca and 43% ( 17/39 ) of cf groups , which indicated a statistically higher prevalence of s. mutans in the ca group ( p = 0.0001 ) . the pcr amplicons were also confirmed for specificity of amplification of s. mutans gtfb gene by direct sequencing . some of the samples that were analyzed showed a less intense amplification relative to others [ figure 1 , compare lanes among lanes in ca and cf groups ] , but nevertheless were included in the study as it only reflected differential count of s. mutans population among the tested samples . a representative gel image of polymerase chain reaction based detection of streptococcus mutans in caries active and caries free sample groups is shown . note that the intensity of s. mutans specific band varies among samples , which is a reflection of variation in the s. mutans population in each of the sample next , we sought to identify the prevalence of the three s. mutans serotypes e , f and k in samples that had tested positive for the presence of s. mutans to investigate the possible association of serotype prevalence and dental caries . serotype specific pcr was performed on the above samples with primers that specifically amplify either one of the three serotypes along with respective positive controls , as described earlier . gel analysis of the pcr amplification reaction showed serotype specific dna band with respective set of primers for e , f or k in both ca and cf groups , which were confirmed by their relative size of migration with reference to respective positive controls [ figure 2 ] and direct sequencing . the number of samples that tested positive for either one of the serotype e , f or k in ca group was 7 , 3 and 13 respectively , while the same in cf group was 1 , 2 and 4 respectively . the occurrence of k serotype was highest in both ca and cf groups , which was followed by e and f serotypes in ca group . the occurrence of these two serotypes , that is , e and f in cf group were too low to be analyzed for its order of prevalence . statistical analysis of the above data showed a nonsignificant trend toward positive association between caries and higher prevalence of either of the serotypes , especially that of k and e [ table 1 ] . there was also a nonsignificant trend towards positive association between caries and cumulative percent prevalence of single serotypes ( e , f or k ) [ table 1 ] . however , no such trend was observable with independent or cumulative percent prevalence of multiserotypes ( e and k , f and k or e , f and k ) [ table 1 ] . about 37.5% of ca and 29.4% of cf samples were not typeable for any of the three serotypes and hence were classified as undetermined serotype [ table 1 ] . a representative gel image of serotype specific polymerase chain reaction of caries active and caries free samples that had tested positive for streptococcus mutans is shown ( a ) serotype e , ( b ) serotype f , and ( c ) serotype k percent distribution of s. mutans and its serotypes , e , f and k in ca and cf groups in order to determine the prevalence of s. mutans serotypes e , f and k in children of age group 6 - 12 years , the oral cavity of 99 school children of afore mentioned age group were screened by a single examiner to select the caries active ( ca ) and caries free ( cf ) groups . the intra examiner reliability was assessed by screening twice on 25 children at an interval of 2 weeks and the data was assessed by reliability test and kappa value was found to be > 0.91 . those children with clinically identifiable dmft / dmft index > 5 were considered as ca , while those with dmft / dmft index < 1 were considered as cf . dmft / dmft was chosen instead of dmfs as dmft / dmft , ( 1 ) is a world health organization standardized criteria for clinical registration of dental caries , ( 2 ) provides a rapid way of screening a large number of subjects in field trips , and ( 3 ) is commonly used in epidemiological studies in school children worldwide . hence , based on the above criterion 60 and 39 children were selected for inclusion in ca and cf groups respectively . subsequently , unstimulated saliva samples were obtained from both groups of children and screened to first identify the prevalence of s. mutans by strain specific pcr amplification method . this method uses primers that anneal and amplify a segment of gtfb gene specifically from s. mutans and hence may be used to detect its overall prevalence . gel analysis of the pcr amplified samples showed s. mutans specific band in 80% ( 48/60 ) of ca and 43% ( 17/39 ) of cf groups , which indicated a statistically higher prevalence of s. mutans in the ca group ( p = 0.0001 ) . the pcr amplicons were also confirmed for specificity of amplification of s. mutans gtfb gene by direct sequencing . some of the samples that were analyzed showed a less intense amplification relative to others [ figure 1 , compare lanes among lanes in ca and cf groups ] , but nevertheless were included in the study as it only reflected differential count of s. mutans population among the tested samples . a representative gel image of polymerase chain reaction based detection of streptococcus mutans in caries active and caries free sample groups is shown . note that the intensity of s. mutans specific band varies among samples , which is a reflection of variation in the s. mutans population in each of the sample next , we sought to identify the prevalence of the three s. mutans serotypes e , f and k in samples that had tested positive for the presence of s. mutans to investigate the possible association of serotype prevalence and dental caries . serotype specific pcr was performed on the above samples with primers that specifically amplify either one of the three serotypes along with respective positive controls , as described earlier . gel analysis of the pcr amplification reaction showed serotype specific dna band with respective set of primers for e , f or k in both ca and cf groups , which were confirmed by their relative size of migration with reference to respective positive controls [ figure 2 ] and direct sequencing . the number of samples that tested positive for either one of the serotype e , f or k in ca group was 7 , 3 and 13 respectively , while the same in cf group was 1 , 2 and 4 respectively . the occurrence of k serotype was highest in both ca and cf groups , which was followed by e and f serotypes in ca group . the occurrence of these two serotypes , that is , e and f in cf group were too low to be analyzed for its order of prevalence . statistical analysis of the above data showed a nonsignificant trend toward positive association between caries and higher prevalence of either of the serotypes , especially that of k and e [ table 1 ] . there was also a nonsignificant trend towards positive association between caries and cumulative percent prevalence of single serotypes ( e , f or k ) [ table 1 ] . however , no such trend was observable with independent or cumulative percent prevalence of multiserotypes ( e and k , f and k or e , f and k ) [ table 1 ] . about 37.5% of ca and 29.4% of cf samples were not typeable for any of the three serotypes and hence were classified as undetermined serotype [ table 1 ] . a representative gel image of serotype specific polymerase chain reaction of caries active and caries free samples that had tested positive for streptococcus mutans is shown ( a ) serotype e , ( b ) serotype f , and ( c ) serotype k percent distribution of s. mutans and its serotypes , e , f and k in ca and cf groups serotypes of s. mutans have been implicated in the development of dental caries . in this study , we have analyzed the saliva samples of children with caries ( ca ) or without caries ( cf ) to investigate the prevalence of the three s. mutans serotypes : e , f and k. both group of samples were initially screened for the prevalence of s. mutans by a strain specific pcr amplification protocol , which showed s. mutans presence in 80% ( n = 48/60 ) and 43% ( 17/39 ) of saliva samples from ca and cf groups respectively . the prevalence status was found to be statistically significant ( p = 0.0001 ) in agreement with those published earlier . subsequent serotype specific pcr of those saliva samples that had tested positive for s. mutans showed a high prevalence of serotype k in both ca and cf groups [ table 1 ] , which were followed by e and f. most of the studies , however , have reported the occurrence of serotype e with highest prevalence that ranged from 2% to 30% followed by serotypes : f and k in the range of 1 - 25% and around 5% respectively [ table 2 ] . since the findings of our study are in contrary to those that have been observed earlier , we speculate that this could be due to a shift in s. mutans serotype in subjects from this region . such serotype shifts have been reported elsewhere in other strains such as streptoccocus agalactiae from group b streptococcus for example , where in s. agalactiae serotype ia was found to occur consistently with a higher prevalence in north american population relative to those from other geographic regions ( p < 0.005 ) . distribution of s. mutans serotypes e , f and k in the oral cavity of individuals from different population , and techniques used to identify them more recent case controlled studies have identified multiserotypes in up to 53.8% ( n = 21/39 ) of subjects . however , we observed multiserotypes only in 14.5% ( n = 7/48 ) and 29.4% ( n = 5/17 ) of ca and cf samples respectively . this contradictory observation may be due to the fact that up to 37.5% of ca and 29.4% of cf samples were nontypeable and hence may have contributed toward the lower prevalence of multiserotypes . in addition , inclusion of serotype c analysis and discerning of undetermined serotypes may have significantly increased the probability of multiserotype identification . it is important to note that most of the studies that reported s. mutans serotypes prevalence employed biochemical , immunodiffusion or immunofluorescence techniques to determine them . s. mutans serotype specific pcr is a relatively recent technical advent , which was invented and used successfully by shibata et al . and nakano et al . , to identify the prevalence in children including those with ie . this technique was employed in the present study as it : ( 1 ) is highly sensitive and is believed to be more effective in detecting the presence of target molecule ( of a strain or serotype ) relative to immunostaining and is capable of detecting s. mutans and its serotypes from 1 to 10 pg of template dna , and ( 2 ) provides a platform to screen large number of samples rapidly . based on these facts , we believe that the inclusion of other detection techniques such as biochemical , immunodiffusion or immunofluorescence methods in this study may not have contributed to an increase in the significance of percent prevalence of either single or multiple serotypes . hence , in the present scenario , the identification of a nonsignificant trend towards positive association between caries and prevalence of independent serotypes , especially that of k and e , may be expected to reach a significant trend when a larger sample size is analyzed . this study brings forth two observations : ( 1 ) a novel prevalence hierarchy of the three determined s. mutans serotypes : k , e and f in the saliva of school children from this region , and ( 2 ) a nonsignificant positive association between caries and serotypes k and e.
background : the development of dental caries has been associated with the oral prevalence of streptococcus mutans . 
 four serotypes of s. mutans have been reported , namely serotype c , e , f , and k that are classified based on the composition and linkages of cell wall polysaccharides , response to physiological reactions , sero - specificity and 16s rrna homology . although the oral prevalence of s. mutans serotype c in indian subjects with or without caries is known , the prevalence of the other three serotypes , e , f , and k are not known . hence in this study 
 , we have investigated the occurrence of the e , f , and k serotypes in children with or without caries within the age group of 6 - 12 years.materials and methods : genomic dna isolated from whole saliva of caries active ( ca ) and caries free ( cf ) groups were first screened for the presence of s. mutans by strain specific polymerase chain reaction ( pcr ) . those samples that tested positive for the presence of s. mutans 
 were further analyzed by serotype specific pcr to identify the prevalence of the serotypes.results:strain specific pcr indicated a higher prevalence of s. mutans in ca group ( 80% ) relative to cf group ( 43% ) . 
 further analysis of the s. mutans positive samples in both groups indicated a higher prevalence of serotype k and e , followed by serotype f in ca group.conclusion:the present data clearly establishes a novel s. mutans serotype prevalence hierarchy in children from this region , compared with those that have been reported elsewhere . 
 besides , the data are also clinically significant as the occurrence of serotype k has been associated with infective endocarditis .