Intraspecies interactions of Streptococcus mutans impact biofilm architecture and virulence determinants in childhood dental caries.

Early childhood dental caries (ECC) is the most common chronic disease among children, especially among low socioeconomic populations. Streptococcus mutans is most frequently associated with initiation of ECC. Although many studies report children with multiple S. mutans strains (i.e., genotypes) have greater odds of developing ECC, studies investigating intraspecies interactions in dental caries are lacking. This study investigates the impact of intraspecies interactions on cariogenic and fitness traits of clinical S. mutans isolates using in vitro and in vivo approaches. Association analysis evaluated if presence of multiple S. mutans genotypes within the first year of colonization was associated with caries. Initially, clinical S. mutans isolates from 10 children were evaluated. S. mutans strains (G09 and G18, most prevalent) isolated from one child were used for subsequent analysis. Biofilm analysis was performed for single and mixed cultures to assess cariogenic traits, including biofilm biomass, intra-polysaccharide, pH, and glucan. Confocal laser scanning microscopy (CLSM) and time-lapse imaging were used to evaluate spatial and temporal biofilm dynamics, respectively. A Drosophila model was used to assess colonization in vivo. Results showed the mean biofilm pH was significantly lower in co-cultured biofilms versus monoculture. Doubling of S. mutans biofilms was observed by CLSM and in vivo colonization in Drosophila for co-cultured S. mutans. Individual strains occupied specific domains in co-culture and G09 contributed most to increased co-culture biofilm thickness and colonization in Drosophila. Biofilm formation and acid production displayed distinct signatures in time-lapsed experiments. This study illuminates that intraspecies interactions of S. mutans significantly impacts biofilm acidity, architecture, and colonization.IMPORTANCEThis study sheds light on the complex dynamics of a key contributor to early childhood dental caries (ECC) by exploring intraspecies interactions of different S. mutans strains and their impact on cariogenic traits. Utilizing clinical isolates from children with ECC, the research highlights significant differences in biofilm architecture and acid production in mixed versus single genotype cultures. The findings reveal that co-cultured S. mutans strains exhibit increased cell density and acidity, with individual strains occupying distinct domains. These insights, enhanced by use of time-lapsed confocal laser scanning microscopy and a Drosophila model, offer a deeper understanding of ECC pathogenesis and potential avenues for targeted interventions.

Intraspecies interactions of Streptococcus mutans impact biofilm architecture and virulence determinants in childhood dental caries.

Early childhood dental caries (ECC) is the most common chronic disease among children with a heavy disease burden among low socioeconomic populations. Streptococcus mutans is most frequently associated with initiation of ECC. Many studies report children with multiple S. mutans strains (i.e., genotypes) having greater odds of developing ECC, studies investigating intraspecies interactions in dental caries are lacking. In this study, the impact of intraspecies interactions on cariogenic and fitness traits of clinical S. mutans isolates are investigated using in-vitro and in-vivo approaches. Initially clinical S. mutans isolates of 10 children from a longitudinal epidemiological study were evaluated. S. mutans strains (G09 and G18, most prevalent) isolated from one child were used for subsequent analysis. Association analysis was used to determine if presence of multiple S. mutans genotypes within the first-year of colonization was associated with caries. Biofilm analysis was performed for single and mixed cultures to assess cariogenic traits, including biofilm biomass, intra-polysaccharide, pH, and glucan. Confocal Laser Scanning Microscopy (CLSM) and time-lapse imaging were used to evaluate spatial and temporal biofilm dynamics, respectively. A Drosophila model was used to assess colonization in-vivo. Mean biofilm pH was significantly lower in co-cultured biofilms as compared with monoculture biofilms. Doubling of S. mutans in-vitro biofilms was observed by CLSM and in-vivo colonization in Drosophila for co-cultured S. mutans. Individual strains occupied specific domains in co-culture and G09 contributed most to increased co-culture biofilm thickness and colonization in Drosophila. Biofilm formation and acid production displayed distinct signatures in time-lapsed experiments.

Streptococcus mutans serotypes and collagen-binding proteins Cnm/Cbm in children with caries analysed by PCR.

Streptococcus mutans, a primary bacterium associated with dental caries, has four known clinical serotypes (c, e, fand k). Certain serotypes, the presence of multiple serotypes and strains with collagen-binding proteins (CBP, Cnm and Cbm) have been linked with systemic disease. Evaluation of S mutans serotype distribution and caries association is needed in the United States. The purpose of this study was to evaluate the prevalence of S mutans serotypes from two cohorts of African-American children in rural Alabama using three sample types (saliva, plaque and individual S mutans isolates) by PCR detection for association with caries. Detection of CBP was also performed by PCR. In total, 129 children were evaluated and overall prevalence of serotypes were: serotype c(98%), e(26%), f(7%) and k(52%). Serotype c was statistically associated with higher caries scores in older children (P < 0.001) and serotype k was statistically more likely in females (P = 0.004). Fourteen per cent of children had CBP. Thirteen S mutans isolates from five children tested positive for both CBP. This study is the first to report on the prevalence of S mutans serotypes in a US population using the PCR-based approach. The frequency of serotype k in this study is the highest reported in any population, illustrating the need for further study to determine the prevalence of this clinically relevant serotype in the US. This is the first study to report S mutans isolates with both Cnm and Cbm in the same strain, and further analysis is needed to determine the clinical significance of these strains.

Transmission patterns of Streptococcus mutans demonstrated by a combined rep-PCR and MLST approach.

OBJECTIVE: Clinical typing methods of the oral pathogen Streptococcus mutans with molecular analysis can be very specific, but expensive. Repetitive extragenic palindromic PCR (rep-PCR) is a relatively inexpensive pre-screening alternative for isolate selection for additional analyses. This study evaluated the prediction accuracy of using rep-PCR to identify S. mutans multilocus sequence typing (MLST) sequence types (ST) among children and their family members. Potential S. mutans strain sources were evaluated for evidence of transmission. MATERIAL AND METHODS: Ten dendrograms (rep-PCR), with 20 isolates each of the 10 most common S. mutans genotypes, were generated from different subjects. Using a cut-off of 98% similarity, 7-11 isolates of each genotype were selected for MLST analysis to determine ST match/no-match. RESULTS: Overall, rep-PCR was 75% effective at determining MLST ST match/no-match and 90% effective when applied to related individuals. Most genotypes were further differentiated by MLST. MLST ST diversity was greatest for one genotype (genotype 12, G12) and evidence of transmission among children and their family members was identified by rep-PCR and MLST. Younger children (6 months to 4 years old) shared ST with their mothers but 50% of older children (5-9 years old) had ST not identified in their mother. Six ST were shared between different families and probable source members were identified. CONCLUSION: This study confirms that rep-PCR offers an affordable option to predict diverse isolates for downstream applications. CLINICAL RELEVANCE: Using a combined rep-PCR and MLST approach, it is possible to track probable transmission and strain sources for S. mutans genotypes.

Association Between Early Childhood Caries and Colonization with Streptococcus mutans Genotypes From Mothers.

PURPOSE: The purpose of this study was to evaluate Streptococcus mutans genotypes (GT) between mother and child (M-C) in a high caries risk cohort to explore the association with early childhood caries (ECC). METHODS: Sixty-nine infants (each approximately one year old) had periodic oral examinations (dmfs) and microbial samples collected from dental plaque, saliva, and other oral surfaces. Their mothers had an examination and plaque collected. S mutans isolates were genotyped using repetitive extragenic palindromic-PCR (rep-PCR). Statistical analyses were conducted for associations of S mutans in M-C dyads with caries outcomes. RESULTS: Twenty-seven S mutans genotypes (GT) from 3,414 isolates were identified. M-C were categorized as GT match (n equals 40) or no-match (n equals 29). When modeling the severity of ECC at 36 months (approximately four years old), the estimated dmfs in the match group was 2.61 times that of the no-match group (P=.014). CONCLUSIONS: Colonization of children with Streptococcus mutans genotypes that matched with mothers was shown to be highly associated with early childhood caries. Although the data suggest vertical transmission of S mutans in 40 of 69 children that shared GT with their mother, it is possible that other individuals transmitted the S mutans. Nonetheless, these findings support the importance of the mother's oral microbial status as a contributing influence to their children's oral health.

Genetic Diversity and Evidence for Transmission of Streptococcus mutans by DiversiLab rep-PCR.

This two-part study investigated the genetic diversity and transmission of Streptococcus mutans using the DiversiLab repetitive extragenic palindromic PCR (rep-PCR) approach. For children with S. mutans and participating household members, analysis for evidence of unrelated child-to-child as well as intra-familial transmission was evaluated based on commonality of genotypes. A total of 169 index children and 425 household family members from Uniontown, Alabama were evaluated for genetic diversity using rep-PCR. Thirty-four unique rep-PCR genotypes were observed for 13,906 S. mutans isolates. For transmission, 117 child and household isolates were evaluated for shared genotype (by child and by genotype cases, multiple matches possible for each child). Overall, children had 1-9 genotypes and those with multiple genotypes were 2.3 times more likely to have caries experience (decayed, missing and filled teeth/surfaces>0). Only 28% of children shared all genotypes within the household, while 72% had at least 1 genotype not shared with anyone in the household. Children had genotype(s) not shared with any household members in 157 cases. In 158 cases children and household members shared a genotype in which 55% (87/158 cases) were shared with more than one family member. Children most frequently shared genotypes with their mothers (54%; 85/158), siblings (46%; 72/158) and cousins (23%; 37/158). A reference library for S. mutans for epidemiological surveillance using the DiversiLab rep-PCR approach is detailed. The genetic diversity of S. mutans in this population demonstrated frequent commonality of genotypes. Evidence for both child-to-child and intra-familial transmission of S. mutans was observed by rep-PCR.

The pH of beverages in the United States.

BACKGROUND: Dental erosion is the chemical dissolution of tooth structure in the absence of bacteria when the environment is acidic (pH < 4.0). Research indicates that low pH is the primary determinant of a beverage's erosive potential. In addition, citrate chelation of calcium ions may contribute to erosion at higher pH. The authors of this study determined the erosive potential measured by the pH of commercially available beverages in the United States. METHODS: The authors purchased 379 beverages from stores in Birmingham, Alabama, and categorized them (for example, juices, sodas, flavored waters, teas, and energy drinks) and assessed their pH. They used a pH meter to measure the pH of each beverage in triplicate immediately after it was opened at a temperature of 25°C. The authors recorded the pH data as mean (standard deviation). RESULTS: Most (93%, 354 of 379) beverages had a pH of less than 4.0, and 7% (25 of 379) had a pH of 4.0 or more. Relative beverage erosivity zones based on studies of apatite solubility in acid indicated that 39% (149 of 379) of the beverages tested in this study were considered extremely erosive (pH < 3.0), 54% (205 of 379) were considered erosive (pH 3.0 to 3.99), and 7% (25 of 379) were considered minimally erosive (pH ≥ 4.0). CONCLUSIONS: This comprehensive pH assessment of commercially available beverages in the United States found that most are potentially erosive to the dentition. PRACTICAL IMPLICATIONS: This study's findings provide dental clinicians and auxiliaries with information regarding the erosive potential of commercially available beverages. Specific dietary recommendations for the prevention of dental erosion may now be developed based on the patient's history of beverage consumption.

Assessment of clonality and serotypes of Streptococcus mutans among children by multilocus sequence typing.

Studies using multilocus sequence typing (MLST) have demonstrated that Streptococcus mutans isolates are genetically diverse. Our laboratory previously demonstrated clonality of S. mutans using MLST but could not discount the possibility of sampling bias. In this study, the clonality of randomly selected S. mutans plaque isolates from African-American children was examined using MLST. Serotype and the presence of collagen-binding proteins (CBPs) encoded by cnm/cbm were also assessed. One-hundred S. mutans isolates were randomly selected for MLST analysis. Sequence analysis was performed and phylogenetic trees were generated using start2 and mega. Thirty-four sequence types were identified, of which 27 were unique to this population. Seventy-five per cent of the isolates clustered into 16 clonal groups. The serotypes observed were c (n = 84), e (n = 3), and k (n = 11). The prevalence of S. mutans isolates of serotype k was notably high, at 17.5%. All isolates were cnm/cbm negative. The clonality of S. mutans demonstrated in this study illustrates the importance of localized population studies and are consistent with transmission. The prevalence of serotype k, a recently proposed systemic pathogen, observed in this study, is higher than reported in most populations and is the first report of S. mutans serotype k in a United States population.

Assessment of two multilocus sequence typing (MLST) schemes available for Streptococcus mutans.

OBJECTIVE: Two multilocus sequencing typing (MLST) schemes are currently available for Streptococcus mutans. The first, introduced by Nakano et al. in 2007, consists of 8 conserved housekeeping genes. The second, introduced in 2010 by Do et al., includes 6 housekeeping genes and 2 putative virulence genes. The purpose of the current study was to compare the two MLST schemes for use in validating repetitive extragenic palindromic polymerase chain reaction (rep-PCR) genotypes. DESIGN: Thirty-three S. mutans isolates, representing the 11 most commonly occurring rep-PCR genotype groups, were selected for MLST. MLST was performed with SYBR Green™ PCR with published primers for both MLST schemes. Amplicons were purified, sequenced, and data checked against the www.PubMLST.org database for allelic and sequence type (ST) assignment. Discriminatory power, congruence, and convenience criteria were evaluated. Concatenated sequences for each scheme were analyzed using MEGA to generate phylogenetic trees using minimum evolution with bootstrap. RESULTS: No significant difference in discriminatory power was observed between the two MLST schemes for S. mutans. Clonal clusters were consistent for both schemes. Overall, MLST demonstrated marginally greater discriminatory power than rep-PCR; however all methods were found to be congruent. New alleles and ST are reported for each scheme and added to the PubMLST database. CONCLUSIONS: Clonality, supported by both methods and rep-PCR, indicates S. mutans genotypes are shared between unrelated subjects. Both Nakano and Do schemes demonstrates similar genotype discrimination for S. mutans isolates suggesting each are well designed and may be used to verify rep-PCR genotypes.

Isolation of Raoultella planticola from refillable antimicrobial liquid soap dispensers in a dental setting.

BACKGROUND: Liquid antimicrobial soaps are commonly used in the dental health care setting for hand washing to minimize the potential spread of infectious agents to health care workers and patients. The purpose of the current study was to evaluate possible bacterial contamination of antimicrobial liquid soap dispensers located in 2 institutional comprehensive dental care clinics. METHODS: Fourteen soap dispensers and 16 original stock containers were sampled. A 1-milliliter aliquot was diluted in 10 mL of phosphate buffer (Tween-80; Acros). Serial dilutions were plated in duplicate on neutralizing agar and incubated for 7 days. Molecular identification was performed using 500 base pair comparisons of 16S ribosomal ribonucleic acid sequencing. Taq polymerase chain reaction was performed with sequence-specific primers for Raoultella species. RESULTS: Bacterial growth was observed at 18 hours for 57% (8 of 14) of soap dispenser samples. Bacterial densities ranged from 4 × 10(2) to 6 × 10(9) colony-forming units per milliliter. Original commercial containers exhibited no growth. Isolates were identified as Raoultella (Klebsiella) planticola. CONCLUSIONS: This is the first study to the authors' knowledge indicating recovery of R. planticola from antimicrobial liquid soap dispensers. R. planticola is a recognized environmental opportunistic pathogen that potentially poses a health concern. PRACTICAL IMPLICATIONS: These findings indicate compliance problems with infection prevention recommendations and support the US Centers for Disease Control and Prevention's recommendation that dispensers should not be topped off. High bacterial loads of R. planticola are inconsistent with infection control practices and are a concern because transmission and possible infection to the health care worker or the patient may occur.

Mutans streptococci enumeration and genotype selection using different bacitracin-containing media.

The primary etiological agents associated with dental caries include the mutans streptococci (MS) comprised of Streptococcus mutans and Streptococcus sobrinus. The effective cultivation and isolation of MS are necessary for the study of MS, including their proper clinical assessment in the epidemiological study of dental caries. Several selective media have been developed for the isolation, enumeration, and characterization of MS. However, inhibition of MS may occur, reducing counts and perhaps limiting selection of some strains. The purpose of this study was to compare five culture media containing bacitracin recommended for the isolation of MS. Five commonly used bacitracin-containing media (MSB, MSKB, GTSB, TYS20B, and TYCSB) used for MS isolation were quantitatively evaluated. Standard plate counts were performed in duplicate for 2 prototype MS strains (S. mutans UA159 and S. sobrinus 6715) and for MS isolates from clinical saliva samples obtained from 16 children (approximate age 5years) to determine total plate counts, and total S. mutans counts. Selected isolates (n=249) from all five media for 5 saliva samples were further confirmed as S. mutans with real-time PCR then subsequently evaluated qualitatively with rep-PCR for genotype determination. All media resulted in variable enumeration with no significant difference in MS counts. MS prototype strains grew well on all five media; clinical isolates demonstrated more variability in counts but no overall significant differences were found. MSB demonstrated comparable ability to grow S. mutans but allowed for more non-S. mutans growth. All 5 media identified a consistent predominant genotype by rep-PCR. Recovery of minor genotypes was not inhibited by media type.

Characteristics of Streptococcus mutans genotypes and dental caries in children.

This longitudinal cohort study evaluated the diversity, commonality, and stability of Streptococcus mutans genotypes associated with dental caries history. Sixty-seven 5- and 6-yr-old children, considered as being at high caries risk, had plaque collected from baseline through 36 months for S. mutans isolation and genotyping using repetitive extragenic palindromic-PCR (4,392 total isolates). Decayed, missing, or filled surfaces (dmfs (primary teeth)/DMFS (secondary teeth)) for each child were recorded at baseline. At baseline, 18 distinct genotypes were found among 911 S. mutans isolates from 67 children (diversity), and 13 genotypes were shared by at least two children (commonality). The number of genotypes per individual was positively associated with the proportion of decayed surfaces (p-ds) at baseline. Twenty-four of the 39 children who were available at follow-up visits maintained a predominant genotype for the follow-up periods (stability) and this was negatively associated with the p-ds. The observed diversity, commonality, and stability of S. mutans genotypes represent a pattern of dental caries epidemiology in this high-caries-risk community, which suggests that fewer decayed surfaces are significantly associated with lower diversity and higher stability of S. mutans genotypes.

Evaluation of in-office dental unit waterline testing.

In-office dental unit waterline (DUWL) testing systems are commercially available for monitoring DUWL bacteria. The current study compared Aquasafe, Petrifilm, and Heterotrophic Plate Count Sampler (HPCS) with R2A plating methodology, considered the gold standard for enumerating heterotrophic bacteria in potable water. Samples were collected from 20 dental units. Heterotrophic bacterial counts of ≤500 CFUs/mL were used as the cut-off for assessing in-office testing compared to R2A laboratory plating. Validity was assessed using sensitivity and specificity, along with positive and negative predictive values. Results were also compared using concordance and kappa statistics. All in-office tests demonstrated 100% specificity and positive predictive values, while sensitivity and negative predictive values were low (Petrifilm, 57%/50%; HPCS, 50%/46%; Aquasafe, 21%/35%). Concordance and kappa values for agreement with R2A plating were as follows: Petrifilm 70% (κ = 0.44), HPCS 65% (κ = 0.38), and Aquasafe 45% (κ = 0.14). In-office DUWL testing with Aquasafe, Petrifilm, and HPCS agreed poorly with R2A plating methodology and is not valid or reliable as a means of accurately monitoring bacterial density in DUWL. These in-office test systems should not be used for assessing compliance with the ADA and CDC standard for acceptable heterotrophic bacterial counts in DUWLs (≤500 CFUs/mL).

Real-time quantitative polymerase chain reaction for enumeration of Streptococcus mutans from oral samples.

This study compared SYBR Green real-time quantitative PCR (qPCR) with standard plate counting for the enumeration of Streptococcus mutans in oral samples. Oral samples (n = 710) were collected from high-caries-risk children for quantification of S. mutans by qPCR using primer pairs. The S. mutans copy number was calculated with reference to a qPCR quantification cycle (Cq) standard curve and compared with the absorbance value at 600 nm of a standard suspension of S. mutans UA159. The S. mutans copy number results were evaluated in relation to standard plate count (SPC) results obtained from each sample following culture on Petri plates containing S. mutans selective media and reported as colony-forming units (CFUs). The mean S. mutans copy number calculated from qPCR was higher than the SPC CFUs (1.3 × 10(6) and 1.5 × 10(5) CFUs, respectively). The qPCR values were usually higher in individual samples and qPCR detected the presence of S. mutans 84% (231/276) of the time that the SPC did not, compared with 33% (4/12) of the time when qPCR failed to detect S. mutans and the SPC did. The qPCR technique was found to be more sensitive for detection of S. mutans from oral samples, a method that is not dependent on the viability of the sample taken and therefore is proposed as a more reliable and efficient means of quantification of S. mutans.