BACKGROUND OF THE INVENTION Dental caries is a chronic infectious disease that affects three billion people with non-treated caries worldwide (1-3). The global economic burden of dental diseases amounted to 442 billion USD in 2010 and averaged 4.6 % of global health expenditure (4). In Western populations of lower caries prevalence, caries is unevenly distributed with a 15-20 % high-risk group with a non-responder behavior to traditional prevention including fluoride (5). Causative lifestyle (e.g. oral hygiene, sugar consumption), aciduric bacteria (e.g. Streptococcus mutans and Lactobacillus) and saliva factors are poor predictors of caries (1 , 6). This questions the role of lifestyle as the major causative factor in caries (2, 7, 8).

Dental caries is traditionally grouped into subtypes based on affected surfaces (e.g. root and coronal caries), dentition (i.e. primary, permanent) and age group (e.g. early childhood, adolescents, elderly) (1). Twin (9) and association studies suggest that genetic traits may influence the development of caries (10-14). Polymorphic saliva proteins (15) associated with caries are acidic and basic proline-rich proteins (PRPs), encoded by PRHI -2 and PRB1-4 respectively, and DMBTl (11 , 12, 16).

Bacterial adhesion to salivary pellicles mediates colonization of clean tooth surfaces by oral indigenous Actinomyces-Streptococcus microbiota and S. mutans associated with caries (17), whereas coadhesion between bacteria promotes subsequent plaque accumulation and maturation associated with periodontitis. Acidic PRPs are primary receptors for the indigenous microbiota (17, 18) and co-receptors for S. mutans (19) in the salivary pellicles on teeth, as well as mediate pellicle barrier properties against bacterial acids and release biofilm modulating peptides upon bacterial and endogenous cleavage (13, 20, 22). The acidic PRP allelic variants PRPl, PRP2 (PRHI) and Pa, Db, PIF (PRHI) and their post-translational variants generate a set of mixed acidic PRP phenotypes in saliva (21 , 22). The multidomain DMBTl (Deleted in Malignant Brain Tumour 1) protein interacts with many microorganisms. Basic PRPs, which neutralize pathogens (20, 23, 24) and salivary DMBTl , are surface (adhesion) and solution (aggregation) receptors for S. mutans with adhesin Agl/II (25-27). DMBTl entraps bacterial ligands with anti-microbial proteins (e.g. S-IgA, protein surfactants D and A) and evokes innate and adaptive immunity (16). In contrast to Db-negative phenotypes associated with caries resistance and adhesion of indigenous

actinomycetes (11 , 12, 19) both DMBTl size variant I and acidic PRP Db-positive phenotypes coincide with caries susceptibility and adhesion of S. mutans. US 2007/0292846 Al discloses a method for detecting bacteria of the genus Staphylococcus.

OBJECTS OF THE INVENTION

It is an object of the invention to provide a method for assessing the risk of a person developing dental caries.

Another object of the invention is to provide a means for carrying out the method.

Further objects of the invention will become evident from the following short description of the invention and of a number of preferred embodiments thereof, and of the appended claims.

SUMMARY OF THE INVENTION The present invention is based on the insight that a person can be genetically predisposed to develop dental caries or be not predisposed and even predisposed against developing dental caries under identical environmental, nutritional and oral hygiene conditions. The development of dental caries in a person thus can be predominantly or exclusively a consequence of genetic predisposition or caused by a caries promoting life style.

A genetically predisposed person cannot avoid or substantially reduce the development of caries by adopting a life style that, in a genetically not predisposed person, would substantially suffice to keep dental caries at bay. However, adopting a life style comprising good oral hygiene and keeping consumption of sweets and candies at a minimum will at least ensure that the rate of caries development is only controlled or substantially controlled by genetic disposition. This concept provides a rationale for subtyping of mechanistically different types of caries to identify individuals being at high, intermediate, or low risk.

More specifically the present invention is based on the insight that the three major PRHI, PRH2 acidic PRP phenotypes of high (P4a), moderate (P6), and low (PI) caries levels identified in a cohort of Swedish children are indicative of high (P4a) caries susceptible, moderate (P6) caries susceptible, and caries resistant (PI) phenotypes. The caries susceptible P4a phenotype (Db, PIF, PRPI2) of 19 % prevalence has a risk of developing caries increased by a factor of from 1.4 to 1.6 compared to P4a-negative and resistant PI children. In P4a children caries prevails despite extra fluoride treatment, thus exhibiting a non-responder behavior to traditional caries prevention. In other words, P4a subjects are at high risk of developing dental caries substantially independent of oral hygiene. Naturally resistant PI subjects (28 % prevalence) coincide significantly with poor oral hygiene (p = 0.007), as opposed to the susceptibility phenotypes (p≥ 0.48). Thus, caries in PI subjects arises mainly or exclusively from enrichment of aciduric S. mutans and lactobacilli by a life style neglecting oral hygiene including consumption of sugar while, in P4a and P6 subjects, it arises additionally or predominantly from innate immunity factors affecting saliva composition.

It is evident that conventionally oral hygiene is beneficial in avoiding caries or at least reduces the rate of caries development in all subjects. However, for caries susceptible P4a individuals maintaining good oral hygiene is insufficient for preventing caries development; they will thus substantially benefit from preventive strategies, including administration of probiotics aiming at substituting their cariogenic microflora. Conventional oral hygiene, on the other hand, will assist genetically caries resistant PI individuals to remain substantially free from caries.

According to the present invention is disclosed a method for assessing the risk of a person developing dental caries by analysis of a sample of said person's saliva, buccal scraping or soft tissue, the method comprising: measuring in said saliva sample allelic phenotypes of acidic proline rich proteins (PRP's) and/or measuring in said tissue sample genotypes corresponding to said allelic phenotypes of acidic PRP's; grouping measured allelic phenotypes or a majority thereof into one of: P4a (Db, PIF, PRP1 ₂ ), PI (PIF ₂ , PRP1 ₂ ) and P6 (Pa, PIF, PRP1 , PRP2) saliva phenotypes; assigning a high risk to a P4 genotype person producing saliva of P4a phenotype, a moderate risk to a P6 genotype person producing saliva of P6 phenotype, and a low risk to a genotype PI person producing saliva of PI phenotype. While a P4a or P6 genotype person is genetically caries susceptible a PI genotype person is not genetically caries susceptible, thus only life-style caries susceptible.

According to a preferred aspect the method of the invention additionally comprises grouping of measured allelic phenotypes into one or more of P5 (PIF2, PRP1, PRP2), P7 (Db, Pa, PRP1, PRP2), P8 (Db, PIF, PRP1, PRP2), P10 (Pa, Pa, PRP2, PRP2).

Due to a risk of developing dental caries being based, i.a., on genetic factors the risk can vary between ethnic groups.

According to the present invention, the risk of a person developing dental caries in addition to be dependent on genetic factors is dependent on the person's oral bacterial flora.

Caries is a mixed species infection from aciduric and acidogenic bacteria of which Streptococcus mutans is a prominent member. S. mutans is a genetically diverse organism that colonized about half of the Swedish population with one or a few genotypes transmitted from parent to child through saliva (28, 29). S. mutans causes caries in a further subset of individuals as a consequence of differences in susceptibility (10, 11), bacterial virulence (30, 31), and life style (1).

Thus, according to a particularly preferred embodiment of the invention, the predictive precision of the method of the invention for assessing the risk of a person developing dental caries by measuring allelic phenotypes of acidic proline rich proteins (PRP's) in a saliva sample is enhanced by measuring Streptococcus mutans adhesion phenotypes. Presence and type of 5. mutans detennine the cariogenicity of oral biofilms. The presence in saliva of Agl/II Bi and Cnm phenotypes indicates formation of dental biofilms of increased cariogenicity.

In particular, presence of Agl/II Bi - or Cnm-positive S. mutans phenotypes in saliva increases caries, such as by a factor of 1.5 in respect of Agl/II A-positive phenotypes. In particular, the presence of S. mutans Agl II Bi in saliva is predicative of a 1.8-fold increased 5-year caries increment in a subset (16 %) of high-risk subjects, whereas S. mwians-negative saliva is predicative of low caries levels.

The method of the invention provides for a grouping result comprising or consisting of P4a, Cnm and C6-like 1 to identifiy a person of particularly high risk of developing dental caries. Furthermore, the method of the invention provides for a grouping result comprising or consisting of PI , Agl/II A and CI to identify a person of particularly low risk of developing dental caries.

The role of the Db allele in caries development and S. mutans adhesion, while prior known (1 1 , 12), is unclear. According to the present invention its role has been clarified by resolving the mixed Db, PIF, PRPh susceptibility phenotype P4a in a larger and more representative sample of 452 Swedish subjects. PRH1, PRH2 genotyping resolved the initially explored protein phenotype P4 (Db, PIF/PRPh from native alkaline electrophoresis) into high caries P4a (Db, PIF, PRPh, n=75 subjects) and low caries P4b (n=9 subjects) phenotypes.

Accordingly, high caries activity coincides with PRPh (at PRH2) and heterozygosity at PRHl (Db, PIF), whereas low caries activity of P4b (PRPh, Db ₂ ) and PI (PRPli, PIFi) coincides with homozygosity at both loci. All Db, PIF, Pa, PRP1, and PRP2 alleles had a highly conserved pattern of the 40 SNPs used to resolve the mixed PRHl, PRH2 genotypes; accordingly three protein coding single nucleotide polymorphisms (SNPs) generated essentially identical caries- related outcomes in Taqman typing. The majority of SNPs are localized in intron and non-coding regions, emphasizing both a direct protein effector mechanism as well as regulatory and micro RNA or PRH-PRB haplotype mechanism in caries development.

In children and young adults new caries-vulnerable sites are often introduced by orthodontic treatment with dental brackets. Dental brackets increase 4-fold a 5-year caries increment in P4a (Db, PIF, PRPh) versus PI children, thus supporting a direct etiological role of PRHl, PRH2 in caries development. Caries susceptible P4a children reach their caries incidence peak and thus lesion saturation at caries predilection sites earlier than caries resistant PI children, which however may also develop caries at these sites later as a consequence of life style changes in adolescence. Sucrose-mediated co-adhesion of S. mutans can drive ecology changes in mature plaque and thus life style dependent caries in PI children.

According to an important aspect of the invention, P4a and P6 genotype persons are genetically caries susceptible. According to another important aspect of the invention a PI genotype person is not genetically caries susceptible, thus only life-style caries susceptible.

According to the present invention presence and type of S. mutans determine cariogenicity of oral biofilms. Subjects with S. mutans Agl/II Bi - or Cnm-positive salivas have 1.5-fold more caries than children with Agl II A-positive salivas; presence and type but not numbers of S. mutans predict their 5-year caries increment in the order of: agl/IIBi and cnm > S. mwto/js-positive > agl/IIA > S. mi/to/is-negative. S. mutans and Agl/II Bi and Cnm phenotypes specify mixed species biofilms of increased cariogenicity. Agl/II Bi and Cnm strains of S. mutans isolated from children have increased adhesion to saliva and DMBTl as well as acid tolerance; they are thus capable to promote biofilm formation and cariogenicity. Collagen binding of Cnm isolates and their acid tolerance under anaerobic conditions promotes deep infections in dentine and tooth tissues, cim-positive subjects display low caries levels; this supports a differential role of cnm and cbm in human infections and that collagen binding does co-operate with additional traits in infections.

In a saliva or buccal scraping sample can be present S. mutans clones of high and/or low cariogenicity. The isoadhesin and clonal nature of S. mutans predicts a 5-year caries increment in an even wider range than presence versus absence of S. mutans, that is, in the order of

C6-like 1 > agl/II Bi and cnm > S. mutons-positive > agl/II A > S. mutans negative and CI isolates or salivas.

According to a preferred aspect of the invention determination of whether i) a saliva sample comprises S. mutans expressing Agl/II Bi and/orCnm adhesin, ii) is S. mu tons-positive but does not express Agl/II Bi and/or Cnm adhesin, iii) comprises S. mutans of agl/II A genotype, or iv) is S. WMto/w-negative, is useful for assessing the risk of a person developing dental caries. S. mutans Agl/II Bi -positive saliva predicts a 1.8 -fold increased risk of developing caries in respect of a person having a S. mwtons-negative saliva.

While not wishing to be bound by theory it is postulated that receptor binding avidity is indicative of cariogenic mechanisms of S. mutans Agl/II Bi and Cnm phenotypes.

Both clustered (Agl/II A, Bi, B2) and single (Lys/Gln ⁶³⁵ and Ser/Asn ¹²⁴⁶ ) amino acid substitutions in the receptor-binding V- and C-domains coincide with increased host caries and DMBTl binding levels (Table 5), whereas binding avidity of Cnm isolates to both DMBTl and collagen correlates positively with individual caries activity. It is furthermore postulated that high avidity binding to DMBTl and collagen assists S. mutans in adhering to, colonizing and invading tooth tissues as well as in evading DMBTl -mediated aggregation or neutralization by anti-microbial factors in saliva.

Thus the method of the invention comprises measuring in a saliva or buccal scraping sample one or more Streptococcus mutans Agl II A, Bi , and B2 isoadhesins and/or Cnm adhesin; grouping the measured isoadhesins and Cnm adhesin or a majority thereof into one of: Agl/II Bi and Cnm phenotypes of high caries susceptibility and Agl/II A phenotypes of low caries susceptibility. It is preferred to group S. mutans phenotypes into corresponding CI - and/or C6-like 1 genotypes. It is preferred to additionally group S. mutans phenotypes into corresponding CI - and/or C6-like 1 genotypes. A grouping result comprising or consisting of P4a, Cnm and C6-like 1 identifies a person of particularly high risk of developing dental caries. A grouping result comprising or consisting of PI , Agl/II A and CI identifies a person of particularly low risk of developing dental caries. In particular, a grouping result comprising or consisting of P4a, Cnm and C6-like 1 identifies a person of particularly high risk of developing dental caries whereas a grouping result comprising or consisting of PI , Agl/II A and CI identifies a person of particularly low risk of developing dental caries.

A measurement by the method of the invention comprises, for instance, measuring

SNP variation in DNA such as by array techniques and/or DNA sequencing; PCR; determining protein variation such as by electrophoresis, HPLC, antibody binding and/or mass spectrometry.

According to the present invention is furthermore disclosed an analytic kit for determining the risk of a person developing dental caries, comprising a means for measuring a saliva sample of said person allelic phenotypes of acidic proline rich proteins (PRP's) and/or a means for measuring in a soft tissue sample of said person genotypes corresponding to said allelic phenotypes of acidic PRP's, further comprising a means for grouping said allelic phenotypes of acidic PRP's into one of P4a (Db, PIF, PRP1 ₂ ), PI (PIF2, PRPI2) and P6 (Pa, PIF, PRP1 , PRP2) saliva phenotype; optionally comprising a means for procuring a saliva, buccal scraping and/or tissue sample. The kit may additionally comprise a means for measuring in said saliva sample one or more Agl/II A, Bi, B2 isoadhesins and/or Cnm adhesin. It is preferred for the measuring means to comprise one more apparatus or device for performing array techniques; PCR; DNA sequencing; determining protein variation, such as apparatus or device for electrophoresis, antibody binding, HPLC or mass spectrometry. Means for procuring saliva samples include medical swabs; means of procuring a buccal scraping or smear or a tissue sample include medial spatulas.

A preferred amplification primer for use in the method comprises at least 15 or at least 18 nucleotide motifs of any of SEQ ID Nos. 1 -6 or, most preferred, is one of SEQ ED Nos. 1-6.

A preferred pair of amplification primers for use in the method is selected from the group consisting of: SEQ ID No. 1 and 2; SEQ ID Nos. 3 and 4; SEQ ID Nos. 5 and 6; at least 15 or 18 nucleotide motifs of each pair of primers. Also disclosed is a kit for diagnosing a dental caries-promoting strain of Staphylococcus mutans, comprising such primer or pair of primers. Also disclosed is the use of such primer or pair of primers for diagnosing a dental caries-promoting strain of Staphylococcus mutans in a saliva or buccal scrap sample of a subject. DESCRIPTION OF PREFERRED EMBODIMENTS

MATERIALS, METHODS AND EXPERIMENTS Study participants, recordings and biological samples. A total of 452 12-year-old children were recruited from five Public Dental Service Clinics in the Vasterbotten county Sweden. A first sample (218 children born in 1996) was pre-selected in a 1 : 1 ratio of children without and with caries lesions (>1 decayed, filled surfaces, DFS, lesions) and collected in 2008. A second sample (234 children born in 1998) was pre-selected in a 1 : 1 ratio of children without or with caries lesions (>2 DFS lesions) and collected in 2010. Both samples and a total of 390 children were re-examined after five years (mean 5.5 years) with a 14% drop-out (62/552) as 20 children had moved out of the area and 42 missed the examination despite reminders. Using a mirror and probe and two bitewing radiographs caries was recorded as numbers of decayed (enamel caries included), filled surfaces in the permanent dentition (DeFS index) by two calibrated dentists (intra- and inter-examiner kappa > 0.979) (5, 6). The 5-year caries increment (ADeFS) was calculated by subtracting latest from earliest DeFS and dividing it with the number of observed years and multiplied by 5. Treatment by orthodontic appliances (i.e. multibrackets) between 12 to 17 years was determined from the dental records. Parotid and whole saliva secretions were collected and measured (PS and WS, mL/min) (6, 11). Plaque was collected by pelleting from the buccal surfaces of premolars and the first molar of the left lower jaw. Fresh whole saliva and plaque (pi) samples were cultured on selective substrates to establish children colonization-positive or negative for mutans streptococci (ms, i.e. S. mutans) and lactobacilli (lbc) as well as their numbers and the proportions of ms out of oral streptococci (strept) in saliva (% ms) and plaque (% ms pi) (6). Sociodemographic data (sex and ethnicity) were collected from parents via pre-posted questionnaire, and oral-behavior data (oral hygiene, intake frequency of sweets and sugary drinks, use of extra fluoride) collected from the children at the clinic with a questionnaire, previously used in a cohort of 3,400 12-year-old children (5). Swedish ethnicity was defined by both parents reporting Swedish ethnicity. Oral hygiene was measured as how often the child brushes their teeth: irregularly, once per day, twice per day, more than twice per day. Intake frequency of sweets (cookies, biscuits, ice cream, or dried fruit) or sweetened drinks was measured (never, once per month, once per week, several times per week, once per day, several times per day). Extra fluoride treatment in addition to fluoride in toothpaste was recorded (none, fluoride mouth rinsing more than once per month, fluoride-containing chewing gum 1-3 pieces per day, or 1-3 fluoride tablets per day). Native alkaline electrophoresis of acidic PRPs. Acidic PRPs in parotid saliva were typed on 7.0% (w/w) polyacrylamide (27:1 bisacrylamide w/w) gels (PROTEAN Ilxi, BioRad) and quantified via densitometry (optical density x mm ² ) (19). Samples (n =218) were analyzed in a double-blind fashion, the vast majority typable (n=213) and representatives of each PRP phenotype were validated using high-performance liquid chromatography (22).

Slot-blot measurements ofDMBTl in saliva. DMBTl in parotid saliva was quantified in a slot-blot assay using anti-DMBTl monoclonal antibody mAbl 43 and densitometry (1 1 , 19).

Bacterial adhesion of indigenous and pathogenic bacteria. Adhesion of ³⁵ S-metabolically labeled bacteria to saliva-coated hydroxyapatite beads was measured in 96-well plates (19), and expressed as the portion of bound bacteria (adh%) or total radioactivity counts (adhtot) after washing away unbound bacteria. Indigenous reference strains representative of clinical isolates were

Streptococcus gordonii strain SKI 2 and Actinomyces naeslundii strains LY7 and T14V with types 1 and 2 pili and its mutant T14Vm without type-2 pili (18, 27). Pathogen strains representative of clinical isolates were S. mutans Ingbritt and Streptococcus pyogenes A8173 (19, 27) (Table 2). PRH1, PRH2 and PRB1-4 genotyping. Buccal epithelia were collected with swabs (Catch-Ail™ Sample Collection Swab, Epicentre Biotechnologies) and genomic DNA was isolated using the QIAamp DNA Micro Kit (QIAGEN). The genomic DNA was whole genome amplified (Illustra Ready-To-Go™ GenomiPhi V3 DNA kit, GE Healthcare) and genotyped using 6 tag and 39 additional single nucleotide polymorphisms (SNPs) with a allele frequency generally above 3-5% of PRH1, PRH2 and 16 tag SNPs oiPRBl-4 in an Illumina Golden Gate array at the SNP&SEQ Technology Platform in Uppsala (Table SI ). Five PRH1, PRH2 SNPs and one PRB3 SNP of no call frequencies were excluded. Mixed PRH1, PRH2 genotypes were resolved in the vast majority of subjects (n=441) by principal component analyses of the remaining PRH1, PRH2 SNPs (n=40). All Db, PIF, Pa, PRP1, and PRP2 alleles had a highly conserved SNP patterns. Illumina and protein typing as well as Taqman typing using protein coding SNPs rs2923234, rs 10491 17 and rs 10491 12 showed 98-100 % typing congruence and generated accordingly essentially identical caries-related outcome results.

Saliva A, Bj and B2 genotyping by quantitative polymerase chain reaction (qPCR). Plaque or bacterial DNA was prepared from whole saliva with the GenElute™ bacterial genome DNA kit (Sigma Aldrich) and genotyped as cnm or cbm and as agl/II A, Bi or B2 by qPCR using the KAPA Sybr Fast Universal qPCR kit. The primers for cnm and cbm were as described (27). The primers for agl/II A, B _\ , and B2 were designed from the agl/II full gene sequences: A-F 5 ^' - GGAGCTTTATGGGAATTTTGG-3 ^' and A-R 5 '-GTATTGTCCTTACCAGGAACAACAG-3 ', B,-F 5 -TGGTGATAAAGCTGGCTGGT-3 ' and Bi-R 5 ^' -GACCAGACATGCGGATAGCA-3 ', B ₂ -F 5 -TGTGGATAACGCATTTAAGCAAGA-3 ^' and B ₂ -R 5 -CAGTGGCTCCTGCGTTTTCT- 3 ^' . The genotyping used internal standards and quantitative calibration curves based on dilutions of DNA purified from a reference genotype and cut-off values for A (3,000 pg), Bi (3,000 pg), B2 (6,000 pg), cnm (3,000 pg), and cbm (1 ,000 pg).The primers used for S. mutans agl/IIA, Bj, or B2 genotyping were selected from prior testing of several forward and reverse primers designed from the agl/II full gene sequences of each genotype, and they did not amplify DNA from oral commensal streptococci with agl/II analogs (data not shown). Similarly, cnm and cbm primers did not cross-react in between or with other's templates.

Quantification of S. mutans in whole saliva samples. S. mutans in whole saliva were quantified by culturing and qPCR (32). Serial dilutions of whole saliva were cultured on MSB agar plates and colony forming units of S. mutans, ms, were counted. Plaque DNA purified from whole saliva samples using the GenElute Bacterial Genomic DNA Kit (Sigma) was used to measure S. mutans by qPCR using the KAPA SYBR FAST Universal qPCR kit (Corbett Rotor-Gene 6000) and S. mutans-specific primers (32). Quantitative calibration curves from DNA prepared from serial dilutions of an S. mutans reference strain were used to transform the qPCR responses into colony- forming units.

S. mutans clonal complexes in the major acidic PRP phenotypes. A single isolates of S. mutans from 36 colonization-positive P4a, PI or P6 children (and 37 additional isolates from 14 of the same children; range 1-8 isolates/child), were isolated from teeth 34-36 of caries-free subjects and from caries lesions of decayed subjects. The isolates were subjected to sequencing of the agl/II full gene and housekeeping gene segments {map, sod, rpoB, ppaC, pfl, pyk, tuj) as described (19). The sequences defined clonal complexes (C1-C9) that shared at least 6 out of 8 alleles with at least one other member in the complex using the eBURST software (33). The sequences from additional isolates verified a dominant genotype in each child (19), data not shown).

Statistical analyses. Multivariate partial least squares (PLS) models, which relates two data matrices (Jf and Y) to each other, were generated using Simca ⁺ P12.01 (34). The PLS models show the ability of the to explain (R ² ) or predict (Q ² ) the variation in Y and the relative variable importance in the projection is given by the VIP -values (VIP > 1.0 marks influential x variables). The predictive ability (Q ² ) is estimated by cross-validation via step-wise exclusion of one seventh of the data in the model which is used to predict the left out data. Data were log transformed and auto-scaled to unit variance before PLS models were generated. Univariate statistical analyses were done using the Chi-squared test, Fischer's exact test, Mann- Whitney U-test, or Spearman's rank correlation (SPSS or GraphPad softwares) with p values < 0.05 considered significant.

Quantification of S. mutans in whole saliva samples. S. mutans in whole saliva were quantified by culturing and qPCR (32). Serial dilutions of whole saliva were cultured on MSB agar plates and colony forming units of S. mutans, ms, were counted. Plaque DNA purified from whole saliva samples was measured by qPCR using the APA SYBR FAST Universal qPCR kit and Corbett Rotor-Gene 6000 and S. mutans-specific primers (32). Quantitative calibration curves from DNA prepared from serial dilutions of an S. mutans reference strain were used to transform the qPCR responses into colony-forming units.

S. mutans cnm full gene sequencing. The full-length cnm gene was amplified and sequenced from purified DNA from S. mutans isolates using iProof DNA polymerase (Invitrogen) and the cwm-lF and cnm- 1R primers (31). The full gene sequences were analyzed with MEGA5 and DnaSP (35, 36).

Multilocus sequence typing. Multilocus sequence typing (MLST) of S. mutans isolates used housekeeping gene segments of map, sod, rpoB, ppaC, pfl, pyk, and tuf and the agl/II full gene sequence (19, 37). Sequence characteristics and sequence types (STs) were generated by DnaSP (36). The S. mutans genetic structure was resolved by the neighboring joining method (which categorizes isolates based on quantitative nucleotide diversity) using MEGA5 (35). The neighboring joining tree of the concatenated agl/II or housekeeping gene sequences was more stable (upon bootstrapping) and tended to cluster isolates with caries-free or decayed children better than when agl/II or housekeeping gene sequences were analyzed separately (data not shown). The clonal complex method, which categorizes isolates based on qualitative sharing of identical alleles, was applied using eBURST (38). Isolates were grouped into clonal complexes when at least six out of eight alleles were shared with at least one other member in the group. cnm and cbm genotyping of S. mutans isolates. Isolates of S. mutans were genotyped as cnm or cbm by PCR using pure DNA and the KAPA Sybr Fast Universal qPCR kit and Corbett Rotor-Gene 6000 and cnm and cbm specific primers (39).

Serotyping. Serotyping of S. mutans isolates was done by PCR (40) using purified DNA and the Ready-To-Go PCR polymerase kit (GE Healthcare). RESULTS

Acidic PRPs specify high, moderate and low caries phenotypes. The role of allelic and mixed PRH1, PRH2 phenotypes in caries, parotid saliva from 218 Swedish 12-year-old children was explored by measuring allelic types of acidic PRPs by native alkaline electrophoresis. The mixed allelic composition grouped the children into three major P4 (Db, PIF/PRPh), PI (PIF ₂ , PRPh) and P6 (Pa, PIF/PRPl , PRP2) and five minor acidic PRP saliva phenotypes. Caries prediction modeling by PLS (R ² = 25%, Q ² = 18%) of mixed allelic acidic PRP phenotypes among traditional factors verified life style and bacteria risk factors and indicated a differential association of the major PRP phenotypes with caries; a positive association for P4 (VIP = 1.1 1 ), a negative for P 1 , and a non-influential association for P6, phenotypes with caries. Accordingly, the numbers of surfaces affected by caries were high in P4 (mean 2.75 DeFS ± 3.07 SD), moderate in P6 (1.91 ± 2.41) and low in PI (1.66 ± 2.26) children, and P4 children had 1.6-fold more caries than PI children (2.75 vs.l .66, p=0.049). The mixed P4 phenotype coincided more strongly with caries than Db or other allelic acidic PRP phenotypes. Caries phenotypes differ in risk factor profiles. By exploring the risk factor and bacterial adhesion profiles of the P4, PI and P6 phenotypes by PLS modeling against caries it was found that the intra-type caries prediction models were stronger (P4) or equally strong (PI , P6) as the model for the whole sample (Table 1) and displayed differential risk factor profiles even though they all had highly influential S. mutans and lactobacilli risk factors. The low-caries PI phenotype had influential oral hygiene and sweet consumption risk factors (VIPs = 1.3-1.5), while the high- and moderate-caries P4 and P6 phenotypes were not influenced by these factors (VIPs « 1.0). In the P4 phenotype and reminiscent of our previous notion in extremes of caries cases-controls (11, 12), caries correlated positively with adhesion of S. mutans IB, while negatively with adhesion of indigenous . naeslundii LY7. However, both adhesion types coincided positively in PI , and negatively in P6, phenotypes with caries. Extra fluoride treatment was an influential factor in P4 phenotypes alone (VIP > 1.0), and thus a marker for children identified as high-risk subjects at the clinics.

Replication of high (P4a), moderate (P6) and low (PI) caries phenotypes by PRH1 and PRH2 genotyping. For verification of the afore described findings of three major caries phenotypes as a consequence of PRH1 and PRH2 variation a second sample of 234 children was recruited and the pooled sample of 452 children analyzed by PRH1 and PRH2 genotyping related to caries at baseline and 5-year prospective follow up (Table 2). lllumina array genotyping of 45 SNPs in the 452 children of mixed ethnicity resolved phenotype P4 (Db-PIF/PRP , n = 84) into high-caries P4a (Db-PIF-PRP1 ₂ , n = 75) and low-caries P4b {Dbi-PIF-PRPI, n = 9) phenotypes (mean 3.08 and 1.44 DeFS respectively) (Tables 1 , 2 and 4). The P4a phenotype had 1.6- to 1.7-fold more caries than PI at baseline and 5-year prospective follow up (p = 0.008, p = 0.004), as well as 1.4-fold more caries than P4a-negative children (p = 0.024, p = 0.06). Moreover, the P4a, P6, and PI phenotypes consistently had high, moderate and low caries regardless of sample or caries measure being used (Table 2). In addition, the dependence of baseline caries on oral hygiene in PI and P4a ^" (all p<0.007), but not in Ρ , P4a or P6 (all p>0.12), phenotypes was replicated by univariate analyses in the 452 sample (Table 1).

About 15 % of the children were subject to orthodontic treatment with multibrackets, a strong caries risk factor, between 12 to 17 years, and the caries incidence peak (and lesion saturation at caries predilection sites) occurred earlier in high- compared to low-caries children; that is, numbers of DeFS lesions at 12 and 17 years correlated inversely (r = - 0.85, p = 0.004). Accordingly, the introduction of new caries-vulnerable sites by orthodontic multibrackets increased the 5-year caries increment 4-fold in P4a compared to PI children (5.5 vs. 1.35, p = 0.021), whereas the remaining non-orthodontic treatment and whole sample groupings displayed less pronounced differences in caries between P4a and PI children (Table 4).

Caries phenotypes differ in innate immunity behavior. To ascertain whether ecological changes from poor life style promote the presence of S. mutans and caries in low-caries PI children, and whether the high-caries P4a and moderate-caries P6 phenotypes and their differential S. mutans and adhesion risk factor profiles are derived from misbehaving saliva innate immunity the major P4, PI and P6 phenotypes were explored for salivary differences in S. mutans versus indigenous adhesion and S. mutans receptor DMBTl and basic PRP innate immunity mechanisms. Upon PLS prediction modeling, saliva adhesion of indigenous A. naeslundii and S. gordonii (both with acidic PRP receptors) correlated positively with phenotype P6, Pa, PRP2 and acidic PRP amount and negatively with phenotypes P4 and PI and PRPl/PIF whereas Db was non-influential (Table 1). Inversely, adhesion of S. mutans (with DMBTl receptors and acidic PRP co-receptors) was positively associated with phenotype P4 and Db and negatively with phenotype P6 (Tables 1 and 3). Although adhesion of both indigenous species was nearly linearly predicted by acidic PRP composition (results not shown), neither adhesion of S. mutans nor of indigenous A. naeslundii correlated with caries in between the P1-P10 phenotypes. This verified the finding of a unique high S. mutans versus low A. naeslundii adhesion profile in high-caries P4a children.

To investigate whether the S. mutans DMBTl receptor behaves differently in the PRH1, PRH2 background of P4a children modulation by saliva DMBTl amount (measured by mAbl43) of caries and adhesion of S. mutans in the P4a, PI and P6 phenotypes was investigated. It was found (Table 1) that the amount of DMBTl correlated with the development of caries only in P4a phenotype, but in all three phenotypes with surface adhesion of S. mutans.

It was further investigated whether basic PRP variation modulates caries differently in the PRH1, PRH2 backgrounds of P4a, P6 and PI phenotypes; to this end tag SNPs ofPRBl-4 were screened against caries (Table 1). Two PRB4, but none PRB1-3, SNPs coincided with caries in the overall sample (n=452) and exclusively in the P6 phenotype. Accordingly, heterozygosity of two noncoding PRB4 SNPs, although more prevalent in phenotype P4a, increases caries up to 2-fold in phenotype P6 (p=0.001) but not in other phenotypes. Selection of different S. mutans clonal complexes by PRH1 and PRH2 background. Children are colonized by a predominant clonal type of S. mutans (21 , 34). P4a, PI and P6 children

colonization-positive for 5. mutans was found not to differ in overall saliva and plaque numbers of the organism (results not shown). To verify that P4a children may carry particular S. mutans clonal types single strains of S. mutans from each of 36 colonization-positive P4a, PI , and P6 children were isolated and genetically grouped into clonal complexes by sequencing of the agl/II adhesin full gene and seven housekeeping gene segments (Table 1). The clonal complexes CI to C9, which each share at least six out of eight alleles with any other member of the group, appeared to be unevenly distributed among P4a, PI , and P6 children; C5, C7, and C8 were especially prominent in P4a children, C2, C3, and C9 in PI and P6 children and complexes CI and C4 in all three phenotypes.

Children with S. mutans agl/II Bl- and cnm-positive saliva display increased caries levels.

To explore the prevalence and relative cariogenicity of S. mutans and its adhesin genotypes (agl/II A, Bl , B2, cnm, cbm), the predominant genotype in whole salivas was measured in 452 children using qPCR and related to numbers of caries DeFS lesions. Half (48 %) of the children was colonized by S. mutans and prevalence of genotypes was agl/II A (26 %), Bl (16 %), B2 (4.5 %), cnm (6 %), and cbm (1.5 %). The children generally had a dominant agl/II A, Bl, or B2 genotype (91 %) and rarely (9 %) a mixture of two agl/II genotypes and none a mixture of cnm and cbm genotypes.

Children colonized by S. mutans (48 %) had 2.1 -fold more caries than those being negative (3.6 vs. 1.7 DeFS, p < 0.0001 ; Table 5). Children with agl/II Bl and cnm saliva had 1.5-fold more caries than those with genotype A saliva (p =0.034, 0.012) or cbm and 1.8 to 1.9-fold more caries than those with agl/II B 1 - and cnm- negative saliva (p < 0.0001 ) (Table 5). However, salivary numbers of S. mutans or genotypes (e.g. agl/II Bl) did not distinguish high versus low caries individuals, and the saliva genotypes did not differ in numbers of S. mutans except for lower numbers in cbm saliva. Thus, presence and genotype but not numbers of S. mutans specify cariogenicity of plaque biofilms.

Children with S. mutans- and S. mutans agl/II Bl -positive saliva show increased

5-year caries increments (Table 5). To explore if children with S. mutans-, agl/II Bl - and cnm- positive saliva had more caries also prospectively, we re-examined the children for caries at 17 years and for 5 -year caries increments. Children colonized by agl/II Bl or cnm genotypes consistently had more caries than those colonized by agl II A, B2 or cbm genotypes regardless of caries at 17 years or caries increment. Accordingly, children colonization-positive for S. mutans (48 %) versus negative at baseline had 1.8-fold increased 5-year caries increments (5.5 vs. 3.1 DeFS, p = 0.00003) and children positive for agl/II Bl (16 %) versus negative had 1.8-fold increased 5-year caries increments (6.7 vs. 3.8 DeFS, p = 0.0008). However, the differences were less pronounced for 5-year caries increments since the caries incidence peak (and lesion saturation at caries predilection sites) occurred earlier in high- compared to low-caries children; that is, numbers of DeFS lesions at 12 and 17 years correlated inversely (r = - 0.85, p = 0.004).

Presence of S. mutans serotype f /k and collagen-binding cnm/cbm isolates of systemic virulence potential in the children. To explore the dominant S. mutans phenot pes in the children for potential of systemic virulence a total of 214 strains of S. mutans was isolated from most of the 234 children colonized by the organism (48 %). The strains were sero- and genotyped and the isolates were tested for collagen binding. The isolates generally showed the same agl/II A, Bl or B2 character as the saliva donor (mean concordance 91 %) except for in cnm-positive children (n = 26) where twelve children had cnm-positive and 11 children cnm-negative isolates and 3 children lacked isolates (concordance 52 %). Isolates were serotype c (n = 192), e (n = 18), f (n = 2) or k (n = 2) and collagen-binding cnm or cbm status occurred in one serotype f and two k isolates, respectively, but at low frequencies in serotypes c (4 %) and e (1 1 %) isolates.

Receptor binding avidity of the Cnm phenotype correlates with caries activity. To explore the relationship between cnm-related strain cariogenicity of S. mutans and binding to saliva and tissue receptors, the twelve cnm isolates were tested for binding to collagen, DMBTl and saliva and sequence for their cnm gene. The cnm isolates bound more avidly to collagen, DMBTl, and saliva than cnm-negative isolates, and avidity of binding varied between isolates. Binding avidity of the cnm isolates to collagen and DMBTl also correlated positively with caries activity in their human carriers. Eight isolates had Cnm proteins that varied by amino acid substitutions in the collagen- binding domain and by copy numbers of β-repeats (range 8 to 23), whereas four isolates had truncated Cnm protein sequences devoid of the collagen binding domain but with weak binding to DMBTl compared to the isolates with full size proteins.

Receptor binding avidity of Agl/II phenotypes coincides with caries activity. The role of DMBTl binding was also explored in respect of agl/II-related cariogenicity. To this end a subset of 70 isolates from 35 extremes each of caries cases and nearly caries-free children was agl/II full gene sequenced; binding to DMBTl and saliva was investigated. All isolates had Agl/II isoforms A, Bl or B2 (specified by clustered amino acid substitutions along opposite sides of a potential receptor binding pocket in the V domain) and single Gln/Lys635 and Ser/Asnl246 substitutions (in the receptor binding V and C domains respectively). Accordingly, all isolates bound to DMBTl and saliva to varying degrees. The Agl/II Bl and Lys635 and Asnl246 substitutions coincide positively with caries and binding to DMBTl or saliva upon multi(PLS)- and univariate analyses. By contrast, the Agl/Π A and Gln635 and Serl246 substitutions behave in the opposite way. Additional amino acid substitutions present in Agl/II coincide neither with caries nor with adhesion or occurred only in single strains/children (data not shown).

Overall S. mutans genetic structure in agl/II- and cnm-related cariogenicity. The overall genetic structure in agl/II- and cnm-related cariogenicity was explored by subjecting the 70 isolates of

5. mutans from human extremes to multilocus sequence typing using agl/II and seven

housekeeping genes related to host caries, adhesion and acid tolerance. The agl/II A, Bl and B2 genotypes overlapped completely with three genetic lineages A, B 1 and B2 with unique clonal complexes of distinct allelic compositions; Cl-3, C5, and C8 in lineage A; C6, C7, and C9 in lineage B 1 ; and C4 in lineage B2. The clonal complexes and associated clusters of isolates ranged more widely than their corresponding genetic lineages A and Bl in terms of caries and adhesion. The clonal complexes C6 and C7 coincided positively with caries and adhesion, whereas CI and C2 behaved in the opposite way upon multi(PLS) and univariate analyses. Isolates that clustered around C6, i.e. C6-like 1, coincided with significantly increased baseline caries and 5-year caries increments and DMBT1 and saliva binding compared to CI isolates that coincided with caries levels equal to that of children being S. mutans-neg&\i\e (Table 6). By contrast, the cnm isolates distributed over the S. mutans genetic lineages regardless of the lineages A, Bl and B2.

The clonal complexes and associated clusters of the 70 isolates ranged also more widely than their corresponding genetic lineages A and Bl in acid tolerance. The agl/II Bl isolates had higher relative acid tolerance than the agl/II A (p = 0.004) and cnm genotypes when tested for acid tolerance by growth at low pH under aerobic conditions. By contrast, the cnm isolates had lower acid tolerance than cnm-negative isolates under anaerobic conditions.

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40. Nomura R et al. J Med Microbiol 58 (2009) 469-475. Table 1. Summary of genetic and life style dependent types of caries defined by PRH1, PRH2 variation

PRP genotype

P4a (P4 ^a ) P6 PI

Caries type Genetic Genetic Life style

Genetic susceptibility Susceptible Susceptible Resistent

Prevalence 19% 32% 28%

Composition PRH1 Db, PIF Pa, PIF PIF, PIF

PRH2 PRP1, PRP1 PRP1, PRP2 PRP1, PRP1

Caries DeFS lesions ^b High Moderate Low

Baseline (mean DeFS) 3.08 (p=0.008) 2.34 1.93 (Ref)

5-year follow up (DDeFS) 7.46 (p=0.009) 5.64 4.77 (Ref)

Orthodontic brackets (DDeFS) 5.54 (p=0.021) 2.68 1.35 (Ref)

Caries PLS prediction ^{11, c}

PLS models (R ² , Q ² ) 42%, 23% 33%, 20% 36%, 16%

Life style dependency Weak Weak Strong

S.mutans adhesion + - +

A.naeslundii adhesion - - +

Bacterial adhesion % S. mutans 40% 33% 37%

A. naeslundii 16% 26% 15%

DMBT1 correlation Adhesion 0.62 ^d 0.71 ^d 0.70 ^d

Caries 0.50 ^d 0.17 0.08

Caries by PRB4 SNP heterozygosity - + 0=0.004)

Possible unique S. mutans clonal

complexes

a P4 protein phenotype; ^b DeFS, Decayed, enamel included, Filled Surfaces; ^c PLS, Partial Least Squares intra type risk factor profiles; ^d p = < 0.003.

Table 2. Association of mixed PRHl, PRH2 genotypes with caries in 452 Swedish children.

Baseline 5 year follow up

Human

Allelic structure' DeFS ^b DeFS" ADeFS ^c

n meaniSD -value ^d n mean±SD ?-value ^d n mean-fcSD p-value ^d

P4a Db-PIF-PRPl-PRPl 84 3.23±3.26 0.030 71 8.06±8.29 0.031 71 4.75±6.63 0.292

P6 Pa-PIF-PRP1-PRP2 123 2.49±2.55 0.426 112 6.61±7.63 0.288 112 3.97±5.58 0.483

PI PIF-PIF-PRP1-PRP1 143 2.19±2.30 Ref. 118 5.74±6.52 Ref. 118 3.57±5.10 Ref.

P4 Db-PIF/PRPl-PRPl ³ 95 3.01±3.19 0.105 82 7.52±7.92 0.066 82 4.46±6.26 0.344

P4b Db-Db-PRPl -PRPl 11 1.36±2.11 n.t. 11 4.09±3.42 n.t. 11 2.58±2.31 n.t.

P4a ^" 348 2.42±2.53 0.060 302 6.43±7.10 0.119 302 3.92±5.58 0.496 ^a Mixed acidic PRP phenotypes from PRHl, PRHl genotyping. In the P4 phenotype PIF/PRPl are not resolved.

b DeFS, Decayed, enamel included, Filled Surfaces

c ADeFS = 5 year caries increment.

dp- values from Mann-Whitney U-test compared with the reference (Ref.) and P4a vs. P4a ^" . n.t. = not tested.

Table 3. Indigenous and S. mutans adhesion, amount of acidic PRP, and caries levels according to PRHl and PRH2 saliva phenotypes

Adhesion and acidic PRP amount

Acidic

PRP Allelic structure A. naeslundii* acidic PRP

S. mutans* DeFS score ⁰ amount ^b

Protein typing Genotyping n mean (95% CI) mean (95% CI) mean (95% CI) n mean P

P4 Db-PIF/PRPl-PRPl Db-PIF PRPl-PRPl 42 15.7 (14.0-17.4) 40.5 (35.8-45.3) 28.6 (24.7-32.5) 95 3.01 0.105

P4a Db-PIF-PRPl-PRPl 35 16.0 (14.1-17.9) 40.2 (35.4-45.0) 29.1 (25.0-33.3) 84 3.23 0.030

P4b Db-Db-PRPl-PRPl 7 14.2 (10.1-18.4) 42.1 (21.8-62.5) 25.9 (11.8-40.0) 11 1.36 0.174

P6 Pa-PIF/PRP1-PRP2 Pa-PIF-PRP1-PRP2 65 24.5 (21.8-27.2) 32.6 (28.7-36.5) 31.1 (26.9-35.4) 123 2.49 0.426

PI PIF/PRPl-PR l PIF-PIF-PRPl-PRPl 70 15.1 (13.8-16.5) 36.9 (33.1-40.7) 23.7 (21.1-26.3) 143 2.19 Ref.

P5 PIF/PRP1-PRP2 PIF-PIF-PRP 1 -PRP2 4 13.0 (4.9-21.1) 38.9 (11.4-66.5) 18.9 (16.0-21.8) 8 4.13 0.027

P7 Db-Pa-PRP1-PRP2 Db-Pa-PRP1-PRP2 17 26.3 (20.7-31.9) 38.3 (29.5-47.0) 38.9 (29.1-48.7) 33 2.55 0.663

P8 Db-PIF/PRP1-PRP2 Db-PIF-PRP1-PRP2 6 19.0 (11.2-26.8) 23.3 (10.0-36.6) 25.1 (13.2-37.1) 11 2.73 0.957

P10 Pa-Pa-PRP2-PRP2 Pa-Pa-PRP2-PRP2 7 32.1 (22.5-41.7) 35.1 (21.6-62.5) 21.6 (12.1-31.1) 19 3.16 0.131

"Adhesion of A. naeslundii LY7 and S. mutans to salivary pellicles formed from parotid saliva from 218 children; CI, confidence interval.

^■ Total amount of saliva acidic PRP from gel densitometry of samples from 218 children.

cDeFS score from 452 children; P-values from the Mann- Whitney test versus the reference (Ref).

Table 4. Association of mixed PRHl, PRH2 genotypes with caries in children with orthodontic multibrackets

Baseline 5 year follow up

Human genotype' DeFS ^b DeFS ^b DDeFS ^c

meaniSD /?-value ^d n meaniSD ?-value ^d n mean±SD p-v& ie*

Orthodontic multibrackets

P4a 11 4.55±3.91 0.032 11 10.55±5.96 0.002 1 1 5.54±5.00 0.021

P6 14 2.36±2.37 0.382 14 5.57±8.17 0.631 14 2.68±5.40 0.923

PI 20 1.90±2.38 Ref. 18 3.33±3.09 Ref. 18 1.35±2.24 Ref.

P4a- 47 2.15±2.25 0.040 43 5.21±5.97 0.005 43 2.72±4.43 0.044

Non-Orthodontic multibrackets

P4a 64 2.83±2.93 0.053 54 7.57±8.60 0.098 54 4.58±6.91 0.512

P6 88 2.34±2.57 0.396 79 6.22±7.66 0.284 79 3.82±5.83 0.578

PI 95 1.94±2.25 Ref. 78 5.27±6.08 Ref. 78 3.43±4.97 Ref.

Whole sample

P4a 75 3.08±3.12 0.008 65 8.08±8.25 0.004 65 4.74±6.61 0.102 P6 102 2.34±2.54 0.285 93 6.12±6.70 0.209 93 3.64±5.75 0.477 PI 115 1.93±2.25 Ref. 96 4.91±5.67 Ref. 96 3.04±4.65 Ref. ^a Swedish ethnicity sample. Virtually identical results were obtained for P4a (13 children) vs. PI (23 children) in the whole sample; 5.34±4.82 vs. 1.33±2.16, p =0.016.

b DeFS, Decayed, enamel included, Filled Surfaces.

c DDeFS = 5 year caries increment.

dp values from Mann- Whitney U-test compared with the reference (Ref).

Table 5. Association of S. mutans cnm and agl/II plaque genotypes with caries in the 452 children

452 children

Plaque genBaseline DeFS ^b DeFS (5y) ^c ADeFS (5y) ^d otypes ³

n mean ± SD n mean ± SD n mean ± SD

Qualitative

S. mutans positive ⁶ 217 3.6 ±3.0 8xl0 ¹³ 185 9.2 ±8.8 4xl0 ^"8 185 5.5 ±7.1 3x10 ^"5

S. mutans negative 234 1.7 ± 2.1 204 4.9 ± 5.7 204 3.1 ±4.9 agl/II-A 111 3.0 ±2.8 Ref. 96 8.4 ± 8.2 Ref. 96 5.2 ±7.1 Ref. agl/II-B, 71 4.2 ±3.5 0.034 55 11.4± 11.1 0.045 55 6.7 ±8.8 0.110 agI/II-B ₂ 19 3.3 ± 1.8 0.365 18 7.5 ±4.8 0.746 18 4.3 ±4.1 0.929 cnm 26 4.4 ± 2.6 0.012 20 10.4 ±8.7 0.182 20 6.0 ±7.2 0.460 cbm 7 2.9 ±3.7 0.631 5 3.8 ±2.8 0.201 5 2.4 ± 1.6 0.599 agI/II-B, ⁺ 71 4.2 ±3.5 3xl0 ^"6 55 11.4 ± 11.1 5xl0 ^"4 55 6.7 ±8.8 8xl0 ^"4 agI/II-Bi ⁺ + mixed 88 4.2 ±3.4 lxlO ^"7 70 10.9 ± 10.6 2x10- ⁶ 70 6.4 ± 8.3 0.001 agl/II-Bf 363 2.2 ±2.4 Ref. 319 6.0 ± 6.5 Ref. 319 3.8 ±5.4 Ref. cnm* 26 4.4 ±2.6 lxlO ^"4 20 10.4 ±8.7 0.001 20 6.0 ±7.2 0.146 cnm ^' 425 2.5 ±2.7 369 6.7 ±7.5 369 4.2 ± 6.0 agI/II-A ⁺ 111 3.0 ±2.8 0.030 96 8.4 ±8.2 0.023 96 5.2 ±7.1 0.161 agl/II-A- 340 2.5 ±2.7 292 6.4 ± 7.2 293 3.9 ±5.7

Quantitative

High >75000 35 4.7 ±3.7 0.205 28 11.8 ± 11.1 0.939 28 6.9±8.7 0.966 Low <75000 36 3.6 ±3.2 27 10.9 ± 11.3 27 6.6 ±9.0

S. mutans

High >250000 49 3.9 ±3.0 0.284 43 10.3 ±9.8 0.544 43 6.4 ±7.8 0.502 Low <250000 168 3.5 ±3.0 142 8.8 ± 8.5 142 5.3 ±6.8 ^a Genotyped by qPCR in the 452 sample

b Baseline DeFS (Decayed, enamel included, Filled Surfaces)

c ADeFS = DeFS at five year follow up - DeFS at baseline

d 2-sided ?-value from Mann- Whitney U-test of Caries DeFS lesions compared to Agl/II genotype A.

e cut off value of 10000.

Table 6. Association of genetic types of S. mutans with caries in the 35 most decayed and 35 healthy children

Agl/II Clonal Baseline DeFS" ADeFS (5y) ^b genotype complex n mean ± SD P ^c n mean ± SD P ^c

Collagen binding (cnm, cbm) ^A 5 6.6 ± 5.3

Genetic lineage A A 39 3.2 ± 4.2

A CI 10 2.4 ± 3.9 Ref. 9 2.9 ± 5.0 Ref.

Genetic lineage Bi B, 27 3.8 ± 4.4

Bi C6 2 7.0 ± 2.8 0.1 19 2 18.2 ± 0.1 0.029

B, C6-likel 6 6.7 ± 3.9 0.041 4 12.4 ± 7.2 0.019

Genetic lineage B ₂ B ₂ 4 2.5 ± 2.5

Single amino acid substitutions

Gin ⁶³⁵ 8 2.6 ± 4.0 Ref.

Lys ⁶³⁵ 62 3.5 ± 4.2 0.667

Asn ¹²⁴⁶ 52 3.2 ± 4.2 Ref.

Ser ¹²⁴⁶ 18 4.1 ± 4.1 0.268

a DeFS (Decayed, enamel included, Filled Surfaces)

b ADeFS = DeFS at five year follow up - DeFS at baseline

c 2-sided p- value from Mann- Whitney U-test of Caries DeFS lesions compared to Clonal complex CI ^d Collagen binding cnm (n=4) and cbm (n=l) isolates

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