Technological advances in DNA sequencing have provided unprecedented insights into the
composition of the oral microbiome in health and disease, and RNA-sequencing and
metabolomics-related ...technologies are beginning to yield information on the activities of
these organisms. Importantly, progress in this area has brought the scientific community
closer to an understanding of what constitutes a health-associated microbiome and is
supporting the notion that the microbiota in healthy sites assumes an active role in
promoting health and suppressing the acquisition, persistence, and activities of overt and
opportunistic pathogens. It is also becoming clear that a significant impediment to
developing a conclusive body of evidence that defines a healthy microbiome and the
mechanisms by which beneficial bacteria promote health is that an inherent characteristic
of the most abundant members of the oral flora, those that potentially play the greatest
roles in health and disease, is intraspecies genomic diversity. In particular, individual
isolates of abundant commensal and pathogenic streptococci show tremendous variability in
gene content, and this variability manifests in tremendous phenotypic heterogeneity.
Analysis of the consequences of this diversity has been complicated by the exquisite
sensitivity these bacteria have evolved to environmental inputs, inducing rapid and
substantial fluctuations in behaviors, and often only within subpopulations of the
organisms. Thus, the conditions under which the oral microbiota is studied can produce
widely different results within and between species. Fortunately, continually diminishing
costs and ongoing refinements in sequencing and metabolomics are making it practical to
study the oral microbiome at a level that will create a sufficiently robust understanding
of the functions of individual organisms and reveal the complex interrelationships of
these microbes (“the known unknowns”) in a way that researchers will be able to engage in
the rational design of reliable and economical risk assessments and preventive
therapies.
Summary
Alkali production by oral bacteria via the arginine deiminase system (ADS) increases the pH of oral biofilms and reduces the risk for development of carious lesions. This study tested the ...hypothesis that increased availability of arginine in the oral environment through an exogenous source enhances the ADS activity levels in saliva and dental plaque. Saliva and supra‐gingival plaque samples were collected from 19 caries‐free (CF) individuals (DMFT = 0) and 19 caries‐active (CA) individuals (DMFT ≥ 2) before and after treatment, which comprised the use of a fluoride‐free toothpaste containing 1.5% arginine, or a regular fluoride‐containing toothpaste twice daily for 4 weeks. ADS activity was measured by quantification of ammonia produced from arginine by oral samples at baseline, after washout period, 4 weeks of treatment, and 2 weeks post‐treatment. Higher ADS activity levels were observed in plaque samples from CF compared to those of CA individuals (P = 0.048) at baseline. The use of the arginine toothpaste significantly increased ADS activity in plaque of CA individuals (P = 0.026). The plaque microbial profiles of CA treated with the arginine toothpaste showed a shift in bacterial composition to a healthier community, more similar to that of CF individuals. Thus, an anti‐caries effect may be expected from arginine‐containing formulations due in large part to the enhancement of ADS activity levels and potential favorable modification to the composition of the oral microbiome.
Arginine metabolism by oral bacteria via the arginine deiminase system (ADS) increases the local pH, which can neutralize the effects of acidification from sugar metabolism and reduce the ...cariogenicity of oral biofilms. To explore the relationship between oral arginine metabolism and dental caries experience in children, we measured ADS activity in oral samples from 100 children and correlated it with their caries status and type of dentition. Supragingival dental plaque was collected from tooth surfaces that were caries-lesion-free (PF) and from dentinal (PD) and enamel (PE) caries lesions. Regardless of children’s caries status or type of dentition, PF (378.6) had significantly higher ADS activity compared with PD (208.4; p < .001) and PE (194.8; p = .005). There was no significant difference in the salivary arginolytic activity among children with different caries status. Mixed-model analysis showed that plaque caries status is significantly associated with ADS activity despite children’s age, caries status, and dentition (p < .001), with healthy plaque predicting higher ADS activity compared with diseased plaque. Plaque arginine metabolism varies greatly among children and tooth sites, which may affect their susceptibility to caries.
Health-associated biofilms in the oral cavity are composed of a diverse group of microbial species that can foster an environment that is less favorable for the outgrowth of dental caries pathogens, ...like
A novel oral bacterium, designated
A12, was previously isolated from supragingival dental plaque of a caries-free individual and was shown to interfere potently with the growth and virulence properties of
In this study, we applied functional genomics to begin to identify molecular mechanisms used by A12 to antagonize, and to resist the antagonistic factors of,
Using bioinformatics, genes that could encode factors that enhance the ability of A12 to compete with
were identified. Selected genes, designated potential competitive factors (
), were deleted. Certain mutant derivatives showed a reduced capacity to compete with
compared to that of the parental strain. The A12
mutant lost the ability to inhibit
-
nducing
eptide (XIP) signaling by
, while mutants with changes in the
locus were impaired in sensing of, and were more sensitive to, the lantibiotic nisin. Loss of PcfV, annotated as a colicin V biosynthetic protein, resulted in diminished antagonism of
Collectively, the data provide new insights into the complexities and variety of factors that affect biofilm ecology and virulence. Continued exploration of the genomic and physiological factors that distinguish commensals from truly beneficial members of the oral microbiota will lead to a better understanding of the microbiome and new approaches to promote oral health.
Advances in defining the composition of health-associated biofilms have highlighted the important role of beneficial species in maintaining health. Comparatively little, however, has been done to address the genomic and physiological bases underlying the probiotic mechanisms of beneficial commensals. In this study, we explored the ability of a novel oral bacterial isolate,
A12, to compete with the dental pathogen
using various gene products with diverse functions. A12 displayed enhanced competitiveness by (i) disrupting intercellular communication pathways of
, (ii) sensing and resisting antimicrobial peptides, and (iii) producing factors involved in the production of a putative antimicrobial compound. Research on the probiotic mechanisms employed by
A12 is providing essential insights into how beneficial bacteria may help maintain oral health, which will aid in the development of biomarkers and therapeutics that can improve the practice of clinical dentistry.
Caries lesions develop when acid production from bacterial metabolism of dietary carbohydrates outweighs the various mechanisms that promote pH homeostasis, including bacterial alkali production. ...Therapies that provide arginine as a substrate for alkali production in supragingival oral biofilms have strong anticaries potential. The objective of this study was to investigate the metabolic profile of site-specific supragingival plaque in response to the use of arginine (Arg: 1.5% arginine, fluoride-free) or fluoride (F: 1,100 ppm F/NaF) toothpastes. Eighty-three adults of different caries status were recruited and assigned to treatment with Arg or F for 12 wk. Caries lesions were diagnosed using International Caries Detection and Assessment System II, and plaque samples were collected from caries-free and carious tooth surfaces. Taxonomic profiles were obtained by HOMINGS (Human Oral Microbe Identification using Next Generation Sequencing), and plaque metabolism was assessed by the levels of arginine catabolism via the arginine deiminase pathway (ADS), acidogenicity, and global metabolomics. Principal component analysis (PCA), partial least squares–discriminant analysis, analysis of variance, and random forest tests were used to distinguish metabolic profiles. Of the 509 active lesions diagnosed at baseline, 70 (14%) were inactive after 12 wk. Generalized linear model showed that enamel lesions were significantly more likely to become inactive compared to dentin lesions (P < 0.0001), but no difference was found when treatment with Arg was compared to F (P = 0.46). Arg significantly increased plaque ADS activity (P = 0.031) and plaque pH values after incubation with glucose (P = 0.001). F reduced plaque lactate production from endogenous sources (P = 0.02). PCA revealed differences between the metabolic profiles of plaque treated with Arg or F. Arg significantly affected the concentrations of 16 metabolites, including phenethylamine, agmatine, and glucosamine-6-phosphate (P < 0.05), while F affected the concentrations of 9 metabolites, including phenethylamine, N-methyl-glutamate, and agmatine (P < 0.05). The anticaries mechanisms of action of arginine and fluoride are distinct. Arginine metabolism promotes biofilm pH homeostasis, whereas fluoride is thought to enhance resistance of tooth minerals to low pH and reduce acid production by supragingival oral biofilms.
Tagging of bacteria with fluorescent proteins has become an essential component of modern microbiology. Fluorescent proteins can be used to monitor gene expression and biofilm growth and to visualize ...host-pathogen interactions. Here, we developed a collection of fluorescent protein reporter plasmids for
UA159 and other oral streptococci. Using superfolder green fluorescent protein (sfGFP) as a reporter for transcriptional activity, we were able to characterize four strong constitutive promoters in
These promoter-
fusions worked both for single-copy chromosomal integration and on a multicopy plasmid, with the latter being segregationally stable in the absence of selective pressure under the conditions tested. We successfully labeled
UA159,
DL1, and
sp. strain A12 with sfGFP, DsRed-Express2 (red), and citrine (yellow). To test these plasmids under more challenging conditions, we performed mixed-species biofilm experiments and separated fluorescent populations using fluorescence-activated cell sorting (FACS). This allowed us to visualize two streptococci at a time and quantify the amounts of each species simultaneously. These fluorescent reporter plasmids add to the genetic toolbox available for the study of oral streptococci.
Oral streptococci are the most abundant bacteria in the mouth and have a major influence on oral health and disease. In this study, we designed and optimized the expression of fluorescent proteins in
and other oral streptococci. We monitored the levels of expression and noise (the variability in fluorescence across the population). We then created several fluorescent protein delivery systems (green, yellow, and red) for use in oral streptococci. The data show that we can monitor bacterial growth and interactions
, differentiating between different bacteria growing in biofilms, the natural state of the organisms in the human mouth. These new tools will allow researchers to study these bacteria in novel ways to create more effective diagnostic and therapeutic tools for ubiquitous infectious diseases.
Microbial biofilms form on oral surfaces. These biofilms usually exist in dynamic equilibria with host defenses and are compatible with maintenance of the integrity of the target tissues. Disease ...occurs when the composition and the metabolic activities of complex communities in biofilms are perturbed. These ecologically driven changes in oral biofilms result in increases in the proportions of pathogenic micro-organisms, which possess enzymatic and structural determinants that may render them more virulent than organisms associated with oral health. This brief review focuses on key environmental influences, and genetic and physiologic aspects of bacteria associated with the formation of dental caries, and attempts to identify some areas of oral microbiology in which interdisciplinary efforts will be essential for dissection of the molecular events controlling the development and persistence of pathogenic plaques. The focus is on strategies to enhance fundamental knowledge of oral biofilm composition, structure, and activities, with the rationale that broadly effective therapeutic strategies targeted at plaque physiology, or at biofilm development and persistence, can arise from such knowledge.
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