Christine A. Kressirer

White-spot Lesions and Gingivitis Microbiotas in Orthodontic Patients:

White-spot lesions (WSL) associated with orthodontic appliances are a cosmetic problem and increase risk for cavities. We characterized the microbiota of WSL, accounting for confounding due to gingivitis. Participants were 60 children with fixed appliances, aged between 10 and 19 yrs, half with WSL. Plaque samples were assayed by a 16S rRNA-based microarray (HOMIM) and by PCR. Mean gingival index was positively associated with WSL (p = 0.018). Taxa associated with WSL by microarray included Granulicatella elegans (p = 0.01), Veillonellaceae sp. HOT 155 (p < 0.01), and Bifidobacterium Cluster 1 (p = 0.11), and by qPCR, Streptococcus mutans (p = 0.008) and Scardovia wiggsiae (p = 0.04) Taxa associated with gingivitis by microarray included: Gemella sanguinis (p = 0.002), Actinomyces sp. HOT 448 (p = 0.003), Prevotella cluster IV (p = 0.021), and Streptococcus sp. HOT 071/070 (p = 0.023); and levels of S. mutans (p = 0.02) and Bifidobacteriaceae (p = 0.012) by qPCR. Species’ associations with WSL were minimally changed with adjustment for gingivitis level. Partial least-squares discriminant analysis yielded good discrimination between children with and those without WSL. Granulicatella, Veillonellaceae and Bifidobacteriaceae, in addition to S. mutans and S. wiggsiae, were associated with the presence of WSL in adolescents undergoing orthodontic treatment. Many taxa showed a stronger association with gingivitis than with WSL.

Characterization and in vitro properties of oral lactobacilli in breastfed infants

BackgroundLactobacillus species can contribute positively to general and oral health and are frequently acquired by breastfeeding in infancy. The present study aimed to identify oral lactobacilli in breast and formula-fed 4 month-old infants and to evaluate potential probiotic properties of the dominant Lactobacillus species detected. Saliva and oral swab samples were collected from 133 infants who were enrolled in a longitudinal study (n=240) examining the effect of a new infant formula on child growth and development. Saliva was cultured and Lactobacillus isolates were identified from 16S rRNA gene sequences. Five L. gasseri isolates that differed in 16S rRNA sequence were tested for their ability to inhibit growth of selected oral bacteria and for adhesion to oral tissues. Oral swab samples were analyzed by qPCR for Lactobacillus gasseri.Results43 (32.3%) infants were breastfed and 90 (67.7%) were formula-fed with either a standard formula (43 out of 90) or formula supplemented with a milk fat globule membrane (MFGM) fraction (47 out of 90). Lactobacilli were cultured from saliva of 34.1% breastfed infants, but only in 4.7% of the standard and 9.3% of the MFGM supplemented formula-fed infants. L. gasseri was the most prevalent (88% of Lactobacillus positive infants) of six Lactobacillus species detected. L. gasseri isolates inhibited Streptococcus mutans binding to saliva-coated hydroxyapatite, and inhibited growth of S. mutans, Streptococcus sobrinus, Actinomyces naeslundii, Actinomyces oris, Candida albicans and Fusobacterium nucleatum in a concentration dependent fashion. L. gasseri isolates bound to parotid and submandibular saliva, salivary gp340 and MUC7, and purified MFGM, and adhered to epithelial cells. L. gasseri was detected by qPCR in 29.7% of the oral swabs. Breastfed infants had significantly higher mean DNA levels of L. gasseri (2.14 pg/uL) than infants fed the standard (0.363 pg/uL) or MFGM (0.697 pg/uL) formula.ConclusionsLactobacilli colonized the oral cavity of breastfed infants significantly more frequently than formula-fed infants. The dominant Lactobacillus was L. gasseri, which was detected at higher levels in breastfed than formula-fed infants and displayed probiotic traits in vitro.

Antimicrobial peptides in saliva of children with severe early childhood caries

OBJECTIVE Controversies exist regarding the relationship between the concentrations of antimicrobial peptides (AMPs) and presence of dental caries in children. Thus, the aim of this study was to examine levels of AMPs in saliva of caries-free (CF), early childhood caries (ECC) and severe early childhood caries (S-ECC) children to determine if the levels of these salivary peptides individually or in combinations were related to caries severity and mutans streptococci levels. DESIGN 36 to 60 month-old children were selected to participate in this study. Children were grouped into CF group (n=29), ECC group (n=25) and S-ECC group (n=29). Saliva was collected from children for microbiological analysis by culture. Salivary concentrations of cathelicidin LL-37, human β-defensin 2 (hBD-2), human β-defensin 3 (hBD-3) and histatin-5 (HTN-5) were determined by ELISA. RESULTS Salivary concentrations of AMPs did not differ among CF, ECC and S-ECC groups. Data showed positive correlations between mutans streptococci levels and salivary hBD-2 or HTN-5. Positive correlations were found between hBD-2, hBD-3, LL-37 and HTN-5. Combinations among AMPs, mainly LL-37, were positively associated with caries levels. CONCLUSIONS Salivary concentrations of AMPs individually were not associated with the severity of early childhood caries. The stimulus of caries appears to trigger a biological response, however, with a combination of these peptides.

The Caries Microbiome: Implications for Reversing Dysbiosis

The oral microbiome plays a critical role in maintaining oral health. Frequent dietary carbohydrate intake can lead to dysbiosis of the microbial community from overproduction of acid with selection for increases in acidogenic, acid-tolerant bacteria. Knowledge of the caries-associated microbiome is key in planning approaches to reverse the dysbiosis to achieve health. For risk assessment and treatment studies, it would be valuable to establish whether microbial monitoring requires assay of multiple species or whether selected key species would suffice. Early investigations of the oral microbiota relied on culture-based methods to determine the major bacteria in health and disease. Microbial monitoring using gene probes facilitated study of larger populations. DNA probe methods confirmed and expanded the importance of transmission of bacteria from mother to infant and association of preselected species, including mutans streptococci and lactobacilli with caries in larger populations. 16S ribosomal RNA (rRNA) probes confirmed the wide diversity of species in oral and caries microbiomes. Open-ended techniques provide tools for discovery of new species, particularly when strain/clone identification includes gene sequence data. Anaerobic culture highlighted the caries association of Actinomyces and related species. Scardovia wiggsiae, in the Actinomyces/Bifidobacterium family, and several Actinomyces species have the cariogenic traits of acid production and acid tolerance. Next-generation sequencing combined with polymerase chain reaction methods revealed a strong association with mutans streptococci in a high caries population with poor oral hygiene and limited access to care. A population with a lower caries experience generally had lower or no Streptococcus mutans detection but harbored other acidogenic taxa in the microbiome. Study of the microbiome suggests a role for the assay of selected putative cariogenic species in more aggressive diseases. For many populations with caries progression, however, assay of multiple species will likely be warranted to determine the caries profile of the population and/or individuals under study.

Oral microbial profiles of individuals with different levels of sugar intake

ABSTRACT The aim was to compare the oral microbial profiles in young adults with an intake of free sugars above or below the current recommendations by the WHO for sugar consumption. Seventy subjects completed a Quantitative Food Frequency Questionnaire to establish the proportion of free sugars in relation to the total energy intake (% E). Subjects with <5% E (n = 30) formed the low-sugar group, while those with ≥5% E (n = 40) were regarded as reference group. Saliva and plaque samples were analyzed by qPCR, and 52 of the plaque samples were assayed by HOMINGS. The HOMINGS analysis revealed a comparable core microbiota in plaque samples with Streptococcus, Leptotrichia, Actinobaculum, and Veillonella as predominant. No major differences between groups were revealed by α-diversity testing (p = 0.83), principal component analysis, or correspondence analysis. Higher relative abundance of Streptococcus sobrinus and Prevotella melaninogenica was observed in plaque samples in the reference group. By qPCR, Scardovia wiggsiae was associated with elevated sugar intake. The findings suggests that the amount of ingested sugars had a marginal influence on microbial profiles in dental plaque and saliva. However, some caries-associated species were less abundant in the dental plaque of the low sugar group.

Scardovia wiggsiae and its potential role as a caries pathogen

Background Streptococcus mutans has been strongly associated with dental caries but caries also occurs in its absence. Association of a new species, Scardovia wiggsiae with childhood caries suggests this could be a new caries pathogen. Highlight S. mutans is considered a caries pathogen based on its association with caries, and on its ability to produce acid, to survive low pH environments, and to induce caries in experimental animals. S. wiggsiae was significantly associated with severe-early childhood caries in the presence and absence of S. mutans. Further S. wiggsiae was elevated in initial carious lesions in adolescents with fixed orthodontic appliances. S. wiggsiae detection was enriched on a low pH agar suggesting acid-tolerance. S. wiggsiae isolates were acid tolerant and produced acid from several sugars at low initial pH values, and were not arginine deiminase positive, characteristics consistent with potential cariogenicity. Cariogenicity of S. wiggsiae was tested in a rat animal model in parallel with S. mutans. While S. wiggsiae by itself showed minimal caries induction, when co-inoculated with S. mutans, there was significant cavity production. Conclusion S. wiggsiae was associated with advanced and initial caries, is acid tolerant and produces acid to low pH at initial neutral and low pH conditions. In combination with S. mutans, S. wiggsiae was detected in caries in an animal model. Together, these data suggest that S. wiggsiae has many of the characteristics consistent with its being a caries-associated species.

Quantitative assessment of salivary oral bacteria according to the severity of dental caries in childhood

This study aimed to assess differences in selected oral bacteria in children according to the severity of dental caries. One hundred and thirty-six children, 36-60 months old were divided into three groups according to caries status: caries-free (CF) (n=47), early childhood caries (ECC) (n=40) and severe-early childhood caries (S-ECC) (n=49). Saliva was collected for detection and quantification of selected oral streptococci, Actinomyces naeslundii, Lactobacillus spp., Bifidobacterium spp., and Scardovia wiggsiae by quantitative-polymerase chain reaction. The results showed that the detection and quantitative levels of S. mutans, S. sobrinus, Bifidobacterium spp. and S. wiggsiae were significantly higher in S-ECC children compared to CF and ECC children, while for S. salivarius were significantly higher in CF compared to ECC and S-ECC children. There was no statistical difference among the clinical groups for S. mitis, S. oralis, A. naeslundii and Lactobacillus spp. levels and detection. S-ECC children had a lower monthly family income, started tooth brushing later and were breastfeed for a longer duration compared to CF children. S. mutans levels were positively correlated with S. wiggsiae and Bifidobacterium spp. levels, lower mothers education and child bottle-feeding before sleeping and negatively correlated with S. salivarius. It was concluded that in addition to S. mutans, other bacterial species, including bifidobacteria, Scardovia wiggsiae and S. sobrinus, are associated with severity of early childhood caries, although their role in the progress of dental caries remains unclear.

Functional profiles of coronal and dentin caries in children

ABSTRACT Background: Dental caries results from a dysbiosis of tooth-associated biofilms and frequently extends through enamel into dentin which has a different structure and composition. Objective: To evaluate the metatranscriptome of caries to determine the metabolic potential of caries communities compared with health. Design: Samples from children, caries-free (CF: n = 4) or with coronal (CC: n = 5) or dentin (DC: n = 5) caries were examined for gene expression potential. Functional profiling was performed using HUMAnN2 (HMP Unified Metabolic Analysis Network). Results: There was increased gene expression diversity in DC compared with CC and CF. Genes in CF included alcohol dehydrogenase from Neisseria sicca, methylenetetrahydrofolate reductase from Streptococcus sanguinis and choline kinase from streptococci. Genes in CC mapped mainly to Streptococcus mutans. Arginine deiminase in DC mapped to S. sanguinis and Actinomyces naeslundii. Glycerol kinase genes mapped to S. sanguinis in all groups whereas glycerol kinase in DC were from Rothia, Prevotella and streptococci. Uracil-DNA glycosylase in DC mapped to Prevotella denticola and Actinomyces. Repressor LexA in DC mapped to Scardovia wiggsiae, Dialister invisus and Veillonella parvula. Conclusions: Functional profiling revealed enzyme activities in both caries and caries-free communities and clarified marked differences between coronal and dentin caries in bacterial composition and potential gene expression.

Bacterial metatranscriptome of dentin caries

ABSTRACT Dental caries results from altered microbial community activity in disease compared to health. Caries advanced into dentin was compared with coronal caries and caries-free sites. Oral bacterial samples were obtained from young children, placed in RNAlater and stored frozen. mRNA was purified and sequenced on an Illumina NextSeq 500 platform. Gene sequences were aligned against taxa in HOMD and gene expression was compared between dentin and coronal caries and caries-free sites using GO terms. There were more genes expressed in caries than caries-free sites. Coronal samples grouped together but not with dentin caries. Compared with caries-free, coronal caries species with higher gene expression included S. mutans, Streptococcus and Actinomyces whereas in dentin expressed genes mapped principally to S. mutans and Scardovia wiggsiae. Higher gene expression mapped to S. wiggsiae and Dialister invisus in dentin compared with coronal caries. Dentin caries had greater number of over-represented activities from GO terms compared with health or coronal caries. Dentin caries had a high number of GO terms associated with sugar metabolism. We conclude that in the samples analyzed, dentin caries appeared more active than coronal lesions with greater sugar metabolism and that S. wiggsiae was a major player in lesions advanced into dentin.