Oral Colonization of Mutans Streptococci in Young Children - Research Paper Example

DETAILED OUTLINE WITH ABSTRACT Detailed outline of: Oral colonization of mutans streptococci (MS) in young children ABSTRACT The aim of this study was to evaluate influence of host and bacterial factors on oral colonization by MS in young children. It was observed during the course of investigation that children acquired MS by horizontal as well as vertical transmission. More than one genotype of the mutans was harbored by the same child and more the mother’s level of MS more chances of transmission to child expected. The bacterial virulence factors viz. mutacin production and biofilm formation wee vital for its colonization. The infection occurred in predentate children also however, teeth eruption, whether primary or permanent, made young children most vulnerable to MS colonization. Since at both teeth eruptions, the infection rates were highest. Early infection by MS led to more chances with MS colonization and caries experiences and higher numbers in oral cavity. Nonmutans possibly attenuate colonization and infectivity by MS. The paper concludes with suggestions of further studies on MS colonization and also indicates some strategies for its suppression viz. by probiotic treatment and reducing mother’s load of oral MS. Key words: Mutan Streptococci, dental caries, dental plaque, Streptococcus mutans, Streptococcus sobrinus, oral streptococci Various surveys in many countries indicated presence of a type of dental caries, early childhood caries (ECC). These decays of primary teeth lead to dental abscesses and toothache which often requires anesthesia for treatment. Colonization by mutans streptococci (MS), particularly S. mutans and S. sobrinus, is the major cause of ECC in young children. There are disputes regarding time of entry of oral MS in young children and whether these are part of normal oral microflora or not (Law et al 99). Since colonization by MS could be targeted to relieve young children from experiences of ECC. The paper would critically discuss host and microbial factors important for transmission and colonization of these bacteria. The introduction and subsequent research would focus on: Time of colonization of MS in young mouths Whether diet and bottle feeding behavior of children affect MS colonization The bacterial properties (virulence factors) of these organisms favorable for colonization Can acquiring MS at early age influence incidences of dental caries in later years ? MS transmission routes. Whether nonmutans in oral flora affect colonization and or action of Mutans?. Research question: what are the host and microbe factors influencing oral colonization by MS in young children. Materials and Methods: In the course of research paper preparation the microbiological as well as molecular biological approaches taken by the researchers would be presented. These are used to differentiate between mutans and nonmutans from oral cavity, production of mutacin and glucan, identification of different MS genotypes etc. The methods of longitudinal surveys used in various studies are also part of the intended research. Results and discussion: The results obtained in the chosen host and bacterial factors responsible for MS colonization would be presented. For the convenience of explanation of results the results and discussion are combined in this paper. The manuscript would contain at least a total of 7-8 tables and figures. These are integrated in the text and attached as per the instructions. The title page with author’s (of this paper) name and affiliation, abstract, acknowledgement and a bibliography of all sources of literature also to be included in the paper. FULL PAPER Author (s): Affiliation(s): Oral colonization of mutans streptococci (MS) in young children ABSTRACT The aim of this study was to evaluate influence of host and bacterial factors on oral colonization by MS in young children. It was observed during the course of investigation that children acquired MS by horizontal as well as vertical transmission. More than one genotype of the mutans was harbored by the same child and more the mother’s level of MS more chances of transmission to child expected. The bacterial virulence factors viz. mutacin production and biofilm formation wee vital for its colonization. The infection occurred in predentate children also however, teeth eruption, whether primary or permanent, made young children most vulnerable to MS colonization. Since at both teeth eruptions, the infection rates were highest. Early infection by MS led to more chances with MS colonization and caries experiences and higher numbers in oral cavity. Nonmutans possibly attenuate colonization and infectivity by MS. The paper concludes with suggestions of further studies on MS colonization and also indicates some strategies for its suppression viz. by probiotic treatment and reducing mother’s load of oral MS. Key words: Mutan Streptococci, dental caries, dental plaque, Streptococcus mutans, Streptococcus sobrinus, oral streptococci Oral colonization of mutans streptococci (MS) in young children Various surveys, in many countries, have indicated presence of a type of dental caries, early childhood caries (ECC). These decays of primary teeth lead to dental abscesses and toothache which often requires anesthesia for treatment. Colonization by mutans streptococci (MS), particularly S. mutans and S. sobrinus, is the major cause of ECC in young children. There are disputes regarding time of entry of oral MS in young children and whether these are part of normal oral microflora or not (Law et al 99). Since colonization by MS could be targeted to relieve young children from experiences of ECC, the paper would critically discuss host and microbial factors important for transmission and colonization of these bacteria. To achieve this objective the discussion would focus on: Time of colonization of MS in young mouths: S. mutans was also detected in predentate children, despite a low caries rate. Together, these reports suggest that children may be colonized by S. mutans before the "window of infectivity" opens. A predentate infant is not likely to have MS in salivary flow since swallowing occurs fast while bacterial divisions are only 2-4 times. MS could instead persist by forming adherent colonies on mucosal surfaces. Tanner and coworkers have demonstrated that the fur­rows of the tongue appear to be an important ecological niche for oral MS (as cited in Berkowitz 106-107). It is contrary to the prior observations that MS colonization occurs only at the time of primary teeth eruption. In A study by Tanner et al (53-56) predentate children showed S. mutans though at much lower levels than children with teeth eruption. They found 28% of the 6- to 18-month-old children had caries and a 55% S. mutans detection rate from tooth samples, which rose to 72% caries and 75% S. mutans detection in the 19- to 36-month-old children. Earlier research pointed towards a "window of infectivity" for mutans streptococci at an early age and after which colonization was unlikely. Later study indicates that the acquisition of MS, may have two "window of infectivity" periods, the time of primary and permanent dentition, respectively Straetemans (1853-1854). Does acquiring MS at early age influence incidences of dental caries in later years? Children with low or non-detectable levels of MS at an early age are at lower risk for caries in later years. Many studies have confirmed that late- or non-colonized children have a significantly lower caries experience than children colonized before the age of 5 (Straetemans et al 1854). In a more conclusive observation by Kohler et al 89% of children with MS colonization by 2 years of age experienced dental caries by 4 years of age with a mean dfs score of 5.0. In contrast, only 25% of children not infected with MS prior to 2 years of age had caries by 4 years of age (mean dfs score=0.3) (as cited in Berkowitz 107). MS transmission routes: VERTICAL TRANS MISSSION: Vertical transmission is the transmission of microbes from caregiver (usually the mother) to child. Mother is the major reservoir from which infants acquire MS. Successful infant colonization of maternally transmitted MS cells may be related to microbial level of the inoculums, frequency of inoculations and a minimum infective dose. Berkowitz and coworkers reported that, when mothers harbored greater than 10 5 colony forming units (cfu) of MS per mL of saliva, the infant infection was 58%. While at 103 cfu the frequency of infant infection dropped to 6%. Thus mothers with dense salivary levels of MS are at high risk for infecting their infants early in life age (as referred in Berkowitz 107- 108). Klein et al found that among the 195, S. mutans strains isolated from the mothers' oral cavities, 36 distinct genotypes were found, 16 of which were transmitted to their children. Contrary to other studies, Roeters et al (277-278) did not find correlation between MS levels in mother and child except for the children with caries experience who showed significant correlation with the levels of mother. Mother’s education level correlates with MS colonization in child probably due to low awareness about oral health. HORIZONTAL TRANSMISSION: Horizontal transmission is the transmission of microbes be­tween family members of a similar age or students in a classroom. Mattos-Graner et al isolated identical Strep­tococcus mutans strains from subjects from Brazilian nursery school children of 12 to 30 months of age. When genotyped by arbitrarily primed polymerase chain reaction (AP-PCR) and restriction fragment length polymorphism analysis, 2-5 matching genotypes were observed between children of same school but from unrelated families. The finding strongly suggests horizontal transmission (as cited Berkowitz 107). Mattos-Graner further hypothesized that MS could be laterally transmitted among nursery cohorts with long exposure to an environment that favors the transmission of bacteria and shorter periods of contact with mothers. Klein et al (4620-23) also found that the presence of genotypes in children that were different from their mothers’ indicated horizontal transmission probably in nursery or day care centers. In their study among the 773 S. mutans strains isolated from the children's mouth, 52 distinct genotypes were detected. Of these 30 genotypes detected at the time of initial acquisition and 22 during the follow-up period. Whether diet and bottle feeding behavior of children affect MS colonization: Kreulen et al. (109-110) advised great caution while explaining MS colonization. For nursing bottle carriers, whether they used sweetened and unsweetened milk bottles should be clearly pointed out. They reported that nursing bottle carriers’ teeth were already browned at the time of eruption indicating early infection. These children may colonize cariogenic flora much earlier. They have also observed that high CFU mothers had children with different MS colonization levels thus other factors viz. age of first teeth eruption and child’s immunity levels may decide colonization. Helderman et al (535-539) reviewed the salivary MS levels, dietary patterns and caries incidents among children of Africa, North America and Europe. The major MS species is s . mutans with some incidents of S. sobrinus but other MS species were negligible. The normal dietary conditions do not have much impact on MS counts in young children. Surprisingly even the sugar consumption shows rather weak correlation with the oral MS counts. However the cariogenicity is determined by diet without affecting the number of mutans streptococci. Thus it is not useful to consider the latter as indicator of caries. On the other hand MS are ubiquitous in young children so these are basically the part of indigenous microflora of mouth. The bacterial properties (virulence factors) of MS favorable for colonization: Transmission was probable in mother-child pairs on the basis of the presence of identical strains, as determined by ribotyping and bacteriocin (mutacin) typing. S. mutans strains shared between a mother and her child showed a broader spectrum of inhibitory activity than did nontransmitted strains. In conclusion, the mutacin activity of clinical isolates is reasonably stable, and this virulence factor seems to be of clinical importance in transmittance and early colonization by S. mutans (Gronroos et al 2595-96); Kuramitsu and Wang 2-4 ). The biofilm formation from sucrose which is major cause of dental plaque is yet another virulence property effective in colonization (kuramitsu and Wang 2-4). This sucrose dependent decay leading to caries is a result of formation of water insoluble glucan (WIG)by S. mutans. The WIG synthesis was higher in isolates from caries-active children, even when children with the same levels of MS were compared (Mattos-Graner 1371-73). Do nonmutans in oral microflora affect colonization and or action of Mutans?: S. gordonii challis (nonmutan) attenuates sucrose biofilm formation, bacteriocin production and genetic transformation of S. mutans GS5. It is assumed that other nonmutans such as S. sanguis, S. mitis, and S. oralis can also have same attenuation effects on S. mutan’ virulence properties . The S. gordonii challis attenuated genetic transformation in S.mutans. As a result the latter was restricted in expanding its virulent properties besides endangering its survival under nutrient limitation as use of purine and pyrimidine nutrients was affected negatively (Kuramitsu and Wang 2-4). However, considerable work is required in this direction as there are large numbers of genotypes of S. mutans found in oral cavities (Klein et al 4620). Materials and methods: Laboratory methods: Microbiological methods include isolation of MS from saliva, tongue dorsum, alveolar ridge mucosa, and dental plaque from children or mother- child for cultivation in appropriate media (Kuramitsu and Wang 2-4; Klein et al 4623; Mattos- Graner et al 2314-15). The mutans and non mutans were differentiated on erythromycin containing Mitis salivarius agar The biofilm formation by mutans, S. mutans and non mutan, S. gordonii challis used in the study, was carried out in polystyrene microtitre plates using Todd-Hewitt broth (THB) containing sucrose or glucose (Kuramitsu and Wang 2-3 ; Klein et al 4622 ). Molecular biology approach was also used for genotype characterization. Saliva samples from mother and child were used for arbitrarily primed PCR (AP-PCR) for S. mutans (968 isolates) and S. sobrinus (111 isolates). The AP-PCR fingerprinting was performed with two primers, OPA-02 (5'-TGCCGAGCTG-3') and OPA-13 (5'-CAGCACCCAC-3'). Mattos- Graner et al also used former primer for oral samples from 35 MS-infected children between 12 and 30 months. A sample of children 6- 36 months of age was used by Klein et al for MS identification by checkerboard hybridization method Mattos –Graner et al (1372) isolated MS components From caries free and caries- active children on SDS-PAGE and incubated in solution of Lubrol, sucrose in sodium phosphate buffer (pH 6.5) to observe opaque white bands of water-insoluble glucans. For mutacin production study, Gronroos et al (2596) overlaid culture of mutans with indicator organism in Trypticase soy agar (TSA). The diameter of inhibition zone was measured. Longitudinal survey methods: in a group of 109 children who had participated in an earlier longitudinal survey stimulated saliva was obtained from 43 children and plated on TSY20B agar.The children had not used any antibiotic prior to sampling (Straetemans et al 1851-52). The subjects of Klein et al chosen were 16 mother-child pairs who were monitored for 20 months. Roeters et al (272-273) took 193 children between age 1.9- 2.8 (Average age 2.3 years) for a three years study. Besides age the SES and education level of parents was also considered. The plaque and saliva samples were taken for microbiological examination and caries were scored visually during this study. The survey data were explained by appropriate statistical tests viz. Spearman Correlation Coefficients (r), Mann-Whitney etc. RESULTS AND DISCUSSION: Tanner and coworkers have demonstrated that the fur­rows of the tongue appear to be an important ecological niche (as cited in Berkowitz (107). Tanner et al (53-54) observed oral microbiota of 176 children in the age range 6- 36 months and found 38 oral microbial species. The tongue samples in children under 18 months showed: S. mutans 70%, S. sobrinus 72%, P. gingivalis 23%, B. forsythus 11%, and A. actinomycete comitans 30%, with similar detection frequencies in children over 18 months. Thus, S. mutans and the periodontal pathogens, P. gingivalis and B. forsythus, were detected even in the youngest subjects. In both age groups, 6-18-month-old and 19- to 36-month-old children S. mutans detection was highest in tooth and tongue samples of children with more than two (> 2) carious teeth (Fig. 1). This association was significant in the 19- to 36-month-old children. In the study, 28% of the 6- to 18-month-old children had caries and a 55% S. mutans detection rate from tooth samples, which rose to 72% caries and 75% S. mutans detection in the 19- to 36-month-old children (p 56) . The finding suggests that children may be colonized by S. mutans before the "window of infectivity" opens. The individual information about the bacteriological examination was not possible as children entered at different ages in the survey so average MS examination was carried out in another study. Microorganisms in plaque or saliva could be absent, decrease or increase during research (Fig 2) MS were more frequently detected in saliva (72%) than in plaques (56% of Samples). Children with initial high levels maintained high levels while fluctuation occurred in children with low initial levels. More MS in saliva indicate localized establishment of these organisms (Roeters et al 274, 276). At the time of acquisition of the strains, children carried one to four distinct genotypes of S. mutans and only one genotype of S. sobrinus. Despite higher S. mutans incidence and genotypic diversity compared to S. sobrinus, the mother-child pairs harbored identical genotypes of S. mutans and S. sobrinus was in 81.25 and 83.33% cases, respectively (Klein et al 4623). This longitudinal study showed an increase in genotypic diversity of S. mutans in the oral cavity during the 20 month-follow-up period. Most genotypes transmitted by the mother persisted and only some were lost while some new strains were also acquired during follow-up. At the time of initial acquisition, or during the follow up period, Child received one to three S. mutans genotypes from the mother (Tables 1). The children harbored one to four distinct genotypes of S. mutans. The child in pair 18 carried four distinct genotypes none of which were acquired from the mother. Seven children acquired only one S. mutans genotype initially while five acquired two genotypes, and three children acquired three genotypes (Table1). Only one S. sobrinus genotype was detected at the time of initial acquisition. The identification of the source of MS transmission is essential to the development of strategies for the prevention of dental caries. The presence of matching genotypes of S. mutans and S. sobrinus in mother-child pairs suggest vertical transmission. Presence of different genotypes indicates other types of transmission as in nursery or day care centers (Klein et al 4623 -25). The fluctuations in MS may reflect variation due to sampling method including amount of plaque collected. Contrary to other studies, the correlation between MS Levels in mother and child was not significant except for the children with caries experience who showed significant correlation the levels of mother. Sugar intake did not affect MS levels much. Mother’s education level correlates with MS colonization child probably due to low awareness about oral health (Roeters 277-278). Of the 43 children in study, 30 have been MS-free until 5 years of age (group 1) while 13 had always been positive for MS since the age of 2 (group 2). For the first group, the mfs and MFS values at 11 years of age were found to be much lower than those of 11-year-old children of group 2. From the 30 children of group 1, 22 had acquired MS at 11 years of age, though in significantly lower counts compared to second group. In Table 2, mfs and MFS values are related to the colonization status at the age of 5 (Straetemans et al 1653). The children who were colonized before the age of 5 always had more caries lesions in the primary and permanent dentition (55% and 34% respectively). No colonization occurred in eight out of 30 children, without detectable MS at 5 years of age. All 13 children positive for MS at the age of 5 were still MS-positive (Straetemans et al 1653) Mattos-Graner took children attending nursery schools in the city of Piracicaba, São Paulo, Brazil as the subjects in their study. These children received total of four sucrose-rich meals daily in the school. It is hypothesized that MS could be laterally transmitted among nursery cohorts with prolonged exposure to an environment that favors the spread of infectious agents and shorter periods of contact with mothers. Such horizontal transmission is suggested from the present study, since two children attending the same nursery carried the same S. mutans genotype (Fig. 3). This is probably the first report of S. mutans matching genotypes in children from unrelated families that had no contact beyond day care. MS obtained by the tongue-blade sampling method, and the presence of visible plaque on upper incisors, were measured in 101 children from 12- to 30-month age group. The ability to synthesize Water insoluble glucan (WIG) is an important virulence factor in initial caries development. It increases MS adherence and accumulation in the plaque of young children Mattos-Graner et al(1371). There was a positive relationship between WIG synthesis and caries incidence and oral levels of MS by Spearman rank correlation analysis. The GTF activity ratio was positively associated with caries incidence and oral levels of MS (Table 3). The caries incidence among heavily infected children was significantly higher when compared with the children having undetectable or low levels of MS colonization . Gronroos et al (2596-7) the mutacin The inhibition zone sizes for producer strains varied from 4 to 26 mm in diameter. S. mutans isolates harbored by the mothers represented 35 distinct types, determined by ribotyping and mutacin typing (Table 4). Eight of these had been transmitted to the child. The transmitted strains inhibited a mean of 10.6 indicator strains and nontransmitted types inhibited a mean of 7.0 indicator strains the difference was statistically significant (equality of medians tested by Fisher’s exact test, P < 0.002). Thus mutacin production capacity could be considered an important factor for transmissions and persistence of MS strain. Nonmutans inhibited Biofilm formation and genetic transformation by mutans (Table 4). The non mutan S. gordonii challis produces a protease, challisin that degrade competence stimulating peptide (CSP) required by S. mutans GS5 for bacteriocin production and genetic transformation (Kuramitsu et al 2-4). The research indicates that nonmutans attenuate virulent properties of mutans streptococci in vitro. It would be interesting to know whether same occurs in vivo as well. Besides, the work provides developing antagonists to CSP or taking a probiotic approach for treatment. Conclusion and recommendation: The study revealed that oral colonization of MS in young children has, in fact, two ‘windows of infectivity’. The dentition, both ,primary as well as permanent makes children vulnerable to infection by these microbes. There are contradictory results regarding correlation between MS load and dental caries experiences of children. Besides, mother is considered major reservoir of MS for very young children but the MS load in different children of same mother varies greatly (Kreulen et al 109-111). The microbial virulence factors favoring colonization are considered to be mutacin production, genetic recombination and sucrose biofilm formation. It is surprising, yet, logical that diet is basically the cariogenic factor rather than the MS load in young children (Helderman et al 535-538). For conclusive outcomes, more than one aspect needs to be considered. The inhibition of competence stimulating factor, probiotic approach for treatment may provide further research. MS suppression of oral MS reservoirs in highly infected mothers by Xylitol chewing gum consump­tion is shown to be associated with a statistically significant reduction of the proability of vertical transmission of MS at 2 years of age (as referred in Berkowitz 107- 108). Besides, S. Mutans is shown to have many genotypes, the studies must focus on whether it is a strategy for better survival. Methods for collection and analysis of samples also needs to be developed since the fluctuations in MS may reflect variation due to sampling method including amount of plaque, saliva collected (Roeters et al 277). Acknowledgements: The sources of scientific literature used in this paper are duly acknowledged. References: TABLES TABLE 1. Distribution and number of S. mutans genotypes isolated from oral cavities of 16 mother-child pairs at time of S. mutans initial acquisition and during follow-up (source: Klein et al 4622 ) Group (%)a Pair no. (gender)b S. mutans genotype(s) in childreng S. mutans genotype(s) in mothersg Initial acquisition, ageh Follow upi Matching (81.25) 2 (F)c 3 (A,B,C), 15.4 2 (A*, D) 1 (A) 3 (F) 3 (A, B, C), 16.8 1 (B*, D) 1 (A) 4 (F) 1 (A), 17.6 1 (A*) 4 (A, B, C, D) 5 (F) 2 (A, B), 14.2 2 (A*, B*) 2 (A, B) 7 (M)d 1 (C), 17.8 2 (A, D, E) 2 (A, B) 8 (F)ef 2 (A, B), 12.8 5 (A*, B*, C, D, E) 3 (A, B, C) 10 (M)d 1 (C), 11.6 2 (A, D) 2 (A, B) 12 (M) 1 (A), 16.6 1 (E) 4 (A, B, C, D) 13 (F) 1 (A), 14.5 1 (A*) 2 (A, B) 17 (F) 1 (A), 16.6 — 1 (A) 20 (F) 2 (A, C), 13.4 2 (A*, C) 2 (A, B) 23 (F)f 2 (A, B), 16.1 4 (C, D, E, F) 2 (A, B) 24 (M) 2 (A, C), 18 — 1 (A) Nonmatching (18.75) 18 (M) 4 (F, G, H, J), 14.6 2 (I, L) 5 (A, B, C, D, E) 28 (F) 1 (C), 18.4 5 (D, E, F, G, H) 2 (A, B) 32 (M) 3 (C, D, E), 12.6 2 (D*, E*) 2 (A, B) Total of distinct genotypes detected 30 22 36 Total of genotypes transmitted 13 3 16 Explanations (Table 1): a Fisher's exact test showed statistical difference among the number of children who acquired or did not acquire S. mutans genotypes from their mothers (P = 0.0011).b Number of pairs = 16. Gender, gender of child; M, male; F, female. c The child in pair 2 is the only child who showed incipient caries on the buccal surfaces of the upper anterior incisors. No other child showed caries lesions during this study. d The children from pairs 7 and 10 did not acquire genotypes from their mothers at the time of initial acquisition. However, these children acquired S. mutans from their mothers in later samplings. e The child in pair 8 acquired one more genotype from his mother in later samplings, in addition to genotypes acquired the at the time of initial acquisition. f The children from pairs 8 and 23 acquired two genotypes from their mothers at the time of initial acquisition. g Letters in parentheses indicate genotypes detected in the subjects. Genotypes indicated by the same letters are identical only within that mother-child pair and bear no relation to types indicated by the same letter in any other mother-child pair. h Age is indicated in months. i Asterisks indicate S. mutans genotypes considered to be stable genotypes. [—], S. mutans was not isolated in posterior samplings. Table 2. The mean (± SD) and median number of missed and filled surfaces (mfs) of the children at the age of 5 and the increment, the additional number of caries lesions, from 5 until 11 years of age, plus the number and percentage of caries-free (CF) individuals in both dentitions at 5 and 11 years of age (Straetemans 1852) Caries scores At the age of 5 years Increment from 5 to 11 years No MS before MS before 5 No MS Before MS before 5 5 years years 5 years years mfs Mean 0.10 ± 0.55a 2.12 ± 3.35a 1.12 ± 2.81b 3.10 ± 3.43b Median 0 1 0 2 MFS Mean - - 0.44 ± 0.88c 1.20 ± 1.91c Median - - 0 0 CF 55 (95%) 25 (49%) 34 (59%) 12 (24%) ap < 0.0001, Mann-Whitney (unpaired, two-tailed). bp = 0.0007, Mann-Whitney (unpaired, two-tailed). cp =p = 0.0374, Mann-Whitney (unpaired, two-tailed). Table 3. Correlation of Water-insoluble Glucan (WIG) Synthesis, Glucosyltransferase (GTF) Activity Ratio, Percentage of Cells Adhering to Glass in the Presence of Sucrose, and Final pH in 20 MS Strains Isolated from Caries-active and Caries-free Children with Caries Incidence in a One-year Follow-up (Mattos-graner 1371). Group of Children Spearman Correlation Coefficients (r) Caries-active Caries-free Caries Incidence MS Oral Levels Virulence factors (n =9) (n = 10) r p Value r p Value WIG synthesis b 0.90± 0.30a 0.54 ± 0.30 0.68 0.004 0.80 0.000 TF activity ratioc 0.55± 0.16 0.45 ± 0.29 0.52 0.024 0.56 0.014 % of adherence 28.9 ± 5.98 27.8 ± 13.60 0.34 0.144 0.63 0.006 Final pH 4.07 ± 0.10 4.03 ± 0.10 0.04 0.856 0.32 0.160 a Mean ± SD. b Intensities of WIG glucan bands measured by scanning densitometry. C Intensities of WIG bands normalized by intensities of the corresponding protein-stained GTF bands. d Proportion of spectrophotometric measurement (550 nm) of cells adhering to glass, by total cell density. Mann-Whitney U test: p < 0.05 Table 4. S. mutans GS5 biofilm formation on preformed S. gordonii biofilms.. Total cell numbers of S. mutans S. gordonii (x105) were determined following dilution of the biofilm cells to three different levels. S. mutans LT11,S. gordonii Challis, or their mixtures were transformed with pPGS749 in either broth cultures. Data are the mean ± standard deviation of duplicate platings from one of two reproducible experiments. Dilutiona GS5/Challis Transformants (107 cells)b x 1000 11.34 ± 2.46/26.54 ± 4.56 S. mutans 80.7 +/-44.5 x 2000 1.04 ± 0.18/36.25 ± 5.82 S. gordonii 1273.8 +/-402.3 x 4000 0.26 ± 0.04/41.5 ± 4.96 S. mutans + S. gordonii 4.3 +/-5.4/1017.2 +/-261.1a Kuramitsu and Wang BMC Oral Health 2006 6(Suppl 1):S11   doi:10.1186/1472-6831-6-S1-S11 a Three dilutions used only for determination of biofilm formation. bTransformants are presented in undiluted samples. Legends to figures Fig 1. Detection frequency of S. mutans and caries level. The Fig. illustrates a positive association by Mantel-Haenszel chi-square test (p < 0.05) only in the older children, between detection of S. mutans and caries level in both tooth and tongue samples. (Source: Tanner et al (56) Fig 2. Variations in numbers of mutans streptococci (MS, in plaque and saliva) and Lacobacilli (LBC, in saliva) In children during experimental period (3 years) (Roeters et al 276) FIG. 3. DNA fingerprinting profiles of S. mutans strains isolated from child F (strains F1 and F2) and child G (strains G1 and G2), both of whom attended the same nursery school. AP-PCR (A) and chromosomal DNA RFLP obtained by digestion with HaeIII (B) indicated that these two children harbored the same genotype of S. mutans (Matos –Graner et al 2315) Fig 1. Fig 2. Fig 3. Read More