The Caries Microbiome: Implications for Reversing Dysbiosis.

Acid-tolerant bacteria such as Streptococcus mutans, Acidobacterium capsulatum, Escherichia coli, and Propionibacterium acidipropionici have developed several survival mechanisms to sustain themselves in various acid stress conditions. Some bacteria survive by minor changes in the environmental pH. In contrast, few others adapt different acid tolerance mechanisms, including amino acid decarboxylase acid resistance systems, mainly glutamate-dependent acid resistance (GDAR) and arginine-dependent acid resistance (ADAR) systems. The cellular mechanisms of acid tolerance include cell membrane alteration in Acidithiobacillus thioxidans, proton elimination by F1-F0-ATPase in Streptococcus pyogenes, biofilm formation in Pseudomonas aeruginosa, cytoplasmic urease activity in Streptococcus mutans, synthesis of the protective cloud of ammonia, and protection or repair of macromolecules in Bacillus caldontenax. Apart from cellular mechanisms, there are several acid-tolerant genes such as gadA, gadB, adiA, adiC, cadA, cadB, cadC, speF, and potE that help the bacteria to tolerate the acidic environment. This acid tolerance behavior provides new and broad prospects for different industrial applications and the bioremediation of environmental pollutants. The development of engineered strains with acid-tolerant genes may improve the efficiency of the transgenic bacteria in the treatment of acidic industrial effluents. KEY POINTS: • Bacteria tolerate the acidic stress by methylating unsaturated phospholipid tail • The activity of decarboxylase systems for acid tolerance depends on pH • Genetic manipulation of acid-tolerant genes improves acid tolerance by the bacteria.

Secondary caries is one of the major reasons causing restoration failure; however, little is known of its microbial etiology, mainly because of the difficulties in eliminating bacterial contamination during collection and sample preparation. Therefore, the purpose of this study was to investigate the feasibility of immunofluorescent techniques and confocal laser scanning microscopy for identification and quantification of bacteria in secondary carious lesions. Thirty-six extracted human teeth, clinically diagnosed as having secondary caries, were used in the study. The teeth were sectioned in half across the secondary carious lesion. One half of each tooth was processed using the Brown and Hopps histologic staining method for bacterial detection. Sections (100 microns thick) were obtained from the other half of each tooth for immunofluorescence labeling to detect and identify mutans streptococci in the subsurface of the lesion using confocal imaging techniques. Mutans streptococci were detected in 88.9% of the samples analyzed with the immunofluorescent technique. The Brown and Hopps histopathologic examination demonstrated evidence of bacteria in 94.4% of the samples. In addition, quantification of bacteria was conducted by digitalization of confocal images. The results indicated that the immunofluorescence confocal laser scanning microscopy technique was sensitive and specific for detection and quantification of mutans streptococci in secondary carious lesions.

Scardovia wiggsiae has been detected from caries in children and adolescents and has been suggested to be a caries-associated microorganism. To investigate the cariogenic potential of S. wiggsiae, we examined carbohydrate metabolism and acid productivity, the fluoride sensitivity of carbohydrate metabolism and the mechanism by which fluoride inhibits carbohydrate metabolism, and the acid sensitivity of carbohydrate metabolism in this bacterium. S. wiggsiae metabolized glucose and reduced the environmental pH to 3.5. It mainly produced acetic acid from glucose, together with small amounts of lactic and formic acid. The 50% inhibitory concentration of fluoride for acid production was 8.0 mM at pH 7.0 and 1.5 mM at pH 5.5, which were much higher than those of representative caries-associated bacteria, such as Streptococcus mutans. Metabolomic profiles showed the accumulation of 3-phosphoglycerate and a marked reduction in the pyruvate concentration in the presence of fluoride, suggesting that fluoride inhibits the latter half of glycolysis, including enolase activity. Enolase activity was inhibited by fluoride in S. wiggsiae, but it was more fluoride-tolerant than the enolase activity of S. mutans. Unlike in S. mutans, lactic acid did not inhibit acid production by S. wiggsiae at acidic pH. These results indicate that S. wiggsiae exhibits high acid production and tolerance to fluoride and lactic acid. S. wiggsiae possesses a unique metabolic pathway, the F6PPK shunt, which might allow it to avoid the lactate-formate pathway, including fluoride-sensitive enolase activity, and enable metabolic flow to the fluoride-tolerant acetate pathway. The fluoride tolerance of S. wiggsiae’s enolase activity also increases the fluoride tolerance of its carbohydrate metabolism. The lactic acid tolerance of S. wiggsiae’s acid production might result in S. wiggsiae having high acidogenic and aciduric potential and make it ecologically competitive in acidic environments, such as caries lesions, where lactic acid predominates.

ObjectivesThe evaluation of Mutans streptococci (MS) is one of the index for caries risk. DentocultTM and CRTTM are commercial kits to detect and evaluate MS, conveniently. However, the evaluation of MS has also been carried out simply using an instruction manual. But the instruction manual is not easy to use for evaluation of MS. The aim of this study was to examine the utility of modified Mitis-Salivalius Bacitracin (MSB) agar medium compared with MSB agar medium and commercial kits, and to establish a convenient kit (mMSB-kit) using modified MSB agar.MethodsThe MS in stimulated saliva from 27 subjects were detected by MSB, modified MSB agar medium and commercial kits. Laboratory and clinically isolated strains of MS were similarly evaluated. The ratios of MS in detected bacteria were compared by ELISA.ResultsThe scores using an mMSB-kit on the basis of modified MSB agar medium were tabulated. Saliva samples showed different levels of MS between culture methods and the commercial kit. Some samples which were full of MS were not detected by the commercial kit. The detection of MS by modified MSB agar medium and mMSB-kit were significantly higher when compared with MSB agar medium,CRTTM, (P< .01) and Dentocult SMTM (P<.05).ConclusionThe sensitivity for detection of MS is higher for modified MSB agar medium when compared with MSB agar medium. The mMSB-kit can be used simply, and can be an important contributor for the evaluation of MS as a caries risk factor.

This research aims to develop a simple, yet effective assay for mutans streptococci (Streptococcus mutans and Streptococcus sobrinus) detection based on a combination of magnetic separation and selective filtration. Magnetic nanoparticles (MNPs) synthesized by solvothermal process were first grafted with poly(acrylic acid) (PAA) bearing active carboxyl groups, readily available for conjugation with cell wall binding domain (CWBD) of automutanolysin that is specific to mutans streptococci. The functionalized MNPs were characterized by XRD, FT-IR, TEM, DLS, and TGA. To determine the bacteria binding, the CWBD-conjugated MNPs were mixed with bacteria solution and then isolated by a magnet. The isolated bacteria bound CWBD-conjugated MNPs were re-suspended and then passed through a membrane (pore size 0.8 µm) by vacuum filtration. The intensity of the colored spots on the membrane was found to increase linearly with concentration of both targeted bacteria, Mutans streptococci in a concentration range of 1x102 - 1x107 CFU/mL. The CWBD-conjugated MNPs were more specific to S.mutans and S.sobrinus with capture efficiency of 77 and 69%, respectively than non-targeted bacteria, S. salivarius and S. sanguinis with capture efficiency of 15 and 38%, respectively. Taking a benefit of peroxidase-like activity of MNPs, an addition of TMB and H2O2 on the spots on the membrane could amplify the signal and thus improve the detection limit of the assay for S. mutans detection from 20 to 8 CFU/mL. With the ease of operation, high sensitivity and specificity as well as the quick response time, the colorimetric assay employing CWBD-conjugated MNPs is a promising detection tool for mutans streptococci detection that may be implemented in clinical setting.

Risk-specific caries prevention requires validated and simple parameters for a caries risk assessment. The aim of the study was to evaluate the validity of a site-specific chair-side mutans streptococci (MS) test for the prediction of caries incidence in fissures. In 230 6- to 7-yr-old children, occlusal plaque samples of teeth 16 and 36 were cultured with Dentocult SM tests at 37 degrees C for 24 hr. Caries (DMFS), initial caries, sealants, and a plaque index (QHI) were recorded and oral hygiene habits were assessed. Not erupted, carious, filled and sealed teeth were excluded from the analysis (n = 154). After 2 yr, the status of the fissures was re-examined, and a fluoride history was recorded with a questionnaire filled out by the children's parents. Sealed teeth were excluded again (n = 54). With a classification of MS score 0 or 1 as low and MS score 2 or 3 as high caries risk, 92% agreement was reached by two independently working examiners. The MS scores and caries incidence correlated significantly. Seventy-eight % of the caries progression in fissures was prognosed correctly. Sensitivity was 50%, specificity 82%, positive predictive value 29%, and negative predictive value 92%. Children with caries progression tended to have lower fluoride scores. Low MS scores were most likely to be associated with low caries incidence, while high mutans streptococci scores seem to be partially compensated by other parameters.

Dental caries are one of the chronic diseases caused by organic acids made from oral microbes. However, there was a lack of knowledge about the oral microbiome of Korean children. The aim of this study was to analyze the metagenome data of the oral microbiome obtained from Korean children and to discover bacteria highly related to dental caries with machine learning models. Saliva and plaque samples from 120 Korean children aged below 12 years were collected. Bacterial composition was identified using Illumina HiSeq sequencing based on the V3–V4 hypervariable region of the 16S rRNA gene. Ten major genera accounted for approximately 70% of the samples on average, including Streptococcus, Neisseria, Corynebacterium, and Fusobacterium. Differential abundant analyses revealed that Scardovia wiggsiae and Leptotrichia wadei were enriched in the caries samples, while Neisseria oralis was abundant in the non-caries samples of children aged below 6 years. The caries and non-caries samples of children aged 6–12 years were enriched in Streptococcus mutans and Corynebacterium durum, respectively. The machine learning models based on these differentially enriched taxa showed accuracies of up to 83%. These results confirmed significant alterations in the oral microbiome according to dental caries and age, and these differences can be used as diagnostic biomarkers.

Recent RNA sequencing studies have given us a deeper insight into the cariogenic impact of carbohydrate sources in the bacterium Streptococcus mutans, the principal microbial agent in dental caries etiopathogenesis. The process of dental caries development is facilitated by the ability of this bacterium to ferment some carbohydrates into organic acids contributing to a pH decrease in the oral cavity and the demineralization of the hard tissues of the tooth. Furthermore, in dental caries progression, biofilm formation, which starts and ends with free planktonic cells, plays an important role and has several unique properties called virulence factors. The most cariogenic carbohydrate is sucrose, an easily metabolizable source of energy that induces the acidification and synthesis of glucans, forming typical bacterial cell clumps. We used multifaceted methodological approaches to compare the transcriptomic and metabolomic profiles of S. mutans growing in planktonic culture on preferred and nonpreferred carbohydrates and in fasting conditions. Streptococcus mutans in a planktonic culture with lactose produced the same pH drop as glucose and sucrose. By contrast, xylitol and lactose showed high effectiveness in regulating intracellular polysaccharide metabolism, cell wall structure, and overall virulence involved in the initial phase of biofilm formation and structure but with an opposite pattern compared with sucrose and glucose. Our results confirmed the recent findings that xylitol and lactose play a vital role in biofilm structure. However, they do not reduce its formation, which is related to the creation of a cariogenic environment.

Limosilactobacillus reuteri inhibits the acid tolerance response in oral bacteria

BackgroundThoracic actinomycosis is an uncommon, chronic suppurative bacterial infection caused by actinomyces species, especially Actinomyces israelii.MethodsIt is usually seen in immunocompetent patients with respiratory disorders, poor oral hygiene, alcoholism and chronic debilitating diseases.ResultsWe illustrate the radiological manifestations of thoracic actinomycoses in various involved areas in the thorax.ConclusionThoracic actinomycosis can be radiologically divided into the parenchymal type, the airway type including bronchiectasis, the endobronchial form, and the mediastinum or chest wall involvement type.Teaching Points Important risk factors for thoracic actinomycosis are underlying respiratory disorders such as emphysema and chronic bronchitis. Different CT patterns can be distinguished in thoracic actinomycosis: parenchymal, bronchiectatic, endobronchial and extrapulmonary. Typical CT findings in the parenchymal pattern are a central low density within the parenchymal consolidation and adjacent pleural thickening.

Actinomyces species are gram-positive filamentous non-acid fast anaerobic to microaerophilic bacteria that belong to human oral, gastrointestinal, and urogenital tract flora. Cervicofacial, abdominopelvic, and pulmonary infections are the most common presentations.Hematogenous spread is extremely rare and has been associated with Actinomyces meyeri, Actinomyces israelii, and Actinomyces odontolyticus. It affects individuals with poor oral hygiene, heavy alcohol intake, immunosuppressed, and underlying pulmonary diseases typically between the second and sixth decades of life with the peak incidence being between the fourth and fifth decades. We present a case of A. odontolyticus bacteremia in a patient with uncontrolled diabetes mellitus and chronic sinusitis.

The effects of some oral hygiene measures on Streptococcus mutants and approximal dental caries were evaluated. One hundred and eighty-seven 13-year-old individuals with high levels of salivary S. mutans (greater than 10(6)/mL) were selected. They were randomly distributed into three groups. Group I initially received professional mechanical tooth-cleaning, tongue-scraping, chlorhexidine treatment, and oral hygiene instructions concentrated on the approximal surfaces most colonized by S. mutans. The treatment was given four times with intervals of two days, followed by one single treatment every six months throughout the experimental period. The initial treatment period for group II, also consisting of four visits, included the same oral hygiene instructions as for group I. The instructions were repeated every six months. Group III was maintained in the preventive program provided by the local Dental Health Office, based on mechanical plaque control and topical use of fluorides and chlorhexidine at individualized intervals. Group I showed a significant immediate reduction of S. mutans in saliva as well as an approximal tooth surfaces. After six months, there were no differences among the three groups regarding these variables. Compared with baseline, there was a significant reduction of S. mutans in all groups. There was no significant difference in caries progression among the three groups. However, the selected "high-risk" individuals in group I developed 0.25 new manifest caries lesions approximally/year, compared with 0.27 for all children of the same age group in the area. Seventeen individuals had approximal surfaces with consistently high or consistently low S. mutans levels. Forty-six percent of the surfaces with high values developed new or progressive caries, compared with 2% of the surfaces with low values.

Since its discovery in 1924 by J Clarke, Streptococcus mutans has been the focus of rigorous research efforts due to its involvement in caries initiation and progression. Its ability to ferment a range of dietary carbohydrates can rapidly drop the external environmental pH, thereby making dental plaque inhabitable to many competing species and can ultimately lead to tooth decay. Acid production by this oral pathogen would prove suicidal if not for its remarkable ability to withstand the acid onslaught by utilizing a wide variety of highly evolved acid-tolerance mechanisms. The elucidation of these mechanisms will be discussed, serving as the focus of this review.

The ability of Streptococcus mutans to produce and tolerate organic acids from carbohydrate metabolism represents a major virulence factor responsible for the formation of carious lesions. Pyruvate is a key metabolic intermediate that, when rerouted to other metabolic pathways such as amino acid biosynthesis, results in the alleviation of acid stress by reducing acid end products and aiding in maintenance of intracellular pH. Amino acid biosynthetic genes such as ilvC and ilvE were identified as being upregulated in a proteome analysis of Streptococcus mutans under acid stress conditions (A. C. Len, D. W. Harty, and N. A. Jacques, Microbiology 150:1353-1366, 2004). In Lactococcus lactis and Staphylococcus carnosus, the ilvE gene product is involved with biosynthesis and degradation of branched-chain amino acids, as well as in the production of branched-chain fatty acids (B. Ganesan and B. C. Weimer, Appl. Environ. Microbiol. 70:638-641, 2004; S. M. Madsen et al., Appl. Environ. Microbiol. 68:4007-4014, 2002; and M. Yvon, S. Thirouin, L. Rijnen, D. Fromentier, and J. C. Gripon, Appl. Environ. Microbiol. 63:414-419, 1997). Here we constructed and characterized an ilvE deletion mutant of S. mutans UA159. Growth experiments revealed that the ilvE mutant strain has a lag in growth when nutritionally limited for branched-chain amino acids. We further demonstrated that the loss of ilvE causes a decrease in acid tolerance. The ilvE strain exhibits a defect in F(1)-F(o) ATPase activity and has reduced catabolic activity for isoleucine and valine. Results from transcriptional studies showed that the ilvE promoter is upregulated during growth at low pH. Collectively, the results of this investigation show that amino acid metabolism is a component of the acid-adaptive repertoire of S. mutans.

Streptococcus mutans is highly sensitive to inhibitors of proton-pumping F-type ATPase (F-ATPase) under acidic conditions. Herein, we investigated the role of S. mutans F-ATPase in acid tolerance using a bacterium expressing the F-ATPase β subunit at lower levels than the wild-type strain. We generated a mutant S. mutans expressing the catalytic β subunit of F-ATPase at lower levels than the wild-type bacterium. The mutant cells exhibited a significantly slower growth rate at pH 5.30, whereas the rate was essentially the same as that of wild-type cells at pH 7.40. In addition, the colony-forming ability of the mutant was decreased at pH<4.30 but not at pH 7.40. Thus, the growth rate and survival of S. mutans expressing low levels of the β subunit were reduced under acidic conditions. Together with our previous observations, this study indicates that F-ATPase is involved in the acid tolerance mechanism of S. mutans by secreting protons from the cytoplasm.