[Show abstract][Hide abstract] ABSTRACT: Porphyromonas strains, including Porphyromonas-like strains, have been isolated from oral and various other systemic infections. The characterization of such strains is a crucial issue, because such information contributes to both the taxonomy of anaerobic bacteria and the clinical aspects of infectious diseases. We previously isolated four Porphyromonas-like strains from intraoperative bronchial fluids of a patient with non-small cell lung cancer. This study aimed to characterize the genetic, biochemical and chemotaxonomic aspects of these isolates. Each strain only grew under anaerobic conditions and their colony morphology was convex, 0.1-1.0 mm in diameter, light gray, and slightly glistening colony, with no black or brown pigmentation on blood agar plates after five-day incubation. The pigmentation was helpful to differentiate the isolates from other Porphyromonas, as most of Porphyromonas species show the pigmentation. In the 16S rRNA gene phylogenetic analysis (98% sequence identity of isolates indicates the same species), the four isolates were closely related to one another (99.7-100.0%), but not related to Porphyromonas (P.) catoniae, the closest species (96.9%). In addition, the DNA-DNA hybridization data revealed less than 16% similarity values between a representative isolate and the P. catoniae, indicating that the strains were genetically independent. Biochemically, the isolates could be differentiated from closely related species, i.e., P. catoniae, P. gingivalis, P. gulae, and P. pogonae, with trypsin activity (negative only in the isolates) and leucine arylamidase activity (positive only in the isolates). We therefore propose a new species to include these isolates: Porphyromonas bronchialis sp. nov.
The Tohoku Journal of Experimental Medicine 08/2015; 237(1):31-7. DOI:10.1620/tjem.237.31 · 1.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Both Streptococcus and Actinomyces can produce acids from dietary sugars and are frequently found in caries lesions. In the oral cavity, nitrogenous compounds, such as peptides and aminoacids, are provided continuously from saliva and crevicular gingival fluid. Since these bacteria can also utilize nitrogen compounds for their growth, it was hypothesized that nitrogenous compounds might influence their acid production; however, no previous studies have examined this topic. Therefore, the present study aimed to assess the effects of nitrogenous compounds (tryptone and glutamate) on glucose-derived acid production by Streptococcus and Actinomyces. Acid production was evaluated using pH-stat method under anaerobic conditions, while the levels of metabolic end-products were quantified using high performance liquid chromatography. Tryptone enhanced glucose-derived acid production by up to 2.68-fold, while glutamate enhanced Streptococcus species only. However, neither tryptone nor glutamate altered the end-product profiles, indicating that the nitrogenous compounds stimulate the whole metabolic pathways involving in acid production from glucose, but are not actively metabolized nor alter metabolic pathways. These results suggest that nitrogenous compounds available in the oral cavity promote acid production by Streptococcus and Actinomyces in vivo.
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Microbiology and Immunology 07/2015; 59(9). DOI:10.1111/1348-0421.12283 · 1.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Polymethyl methacrylate (PMMA)-made prostheses used in the oral cavity were evaluated by multimodal assessment in order to elucidate the biodeterioration of PMMA. In used dentures (UD), the micro-Vickers hardness of the polished denture surface and denture basal surface was lower than that of the torn surface (p<0.05), whereas the shaved surface approximately 100 µm from the polished surface showed a similar value to the torn surface. By contrast, there were no differences among these surfaces in new resin (NR). The volatile content of UD was higher than that of NR (p<0.05). Component analysis by ATR-FTIR showed specific spectra (1,700-1,400 cm(-1)) only in UD. This study revealed that PMMA deteriorated during long-term use in the oral cavity in terms of hardness and volatile content with component alteration, and suggests the involvement of biodeterioration, possibly due to saliva and oral microbiota.
[Show abstract][Hide abstract] ABSTRACT: Objectives The source of the bacteria involved in silent aspiration remains to be completely defined. This study aimed to obtain reliable evidence on silent aspiration of oral bacteria in elderly patients. Methods After obtaining informed consent, the cough and swallowing reflexes of patients were assessed. Bronchial fluids from patients undergoing lung resections were collected with a micro-sampling probe, and α-amylase activity of bronchial fluids was measured to estimate the degree of silent aspiration. The bronchial fluids were cultured aerobically and anaerobically on blood agar plates, and colonies were identified by 16S rRNA gene sequencing. Additionally, whole saliva bacterial amounts and composition were analyzed. Results Six patients (72.2±5.8 years) exhibited an impaired swallowing reflex and 5 (75.4±7.9 years) had a normal swallowing reflex, while all patients had a normal cough reflex. α-Amylase activity was detected in bronchial fluids of both the impaired and normal reflex groups. The amount of anaerobic bacteria in bronchial fluids in the impaired reflex group [(3.0±3.5)×104] was higher than in the normal reflex group [(2.5±5.3)×104sup], although the difference was not significant. Actinomyces, Gemella, Streptococcus, Rothia, Mogibacterium, and Campylobacter were the predominant bacterial species in bronchial fluids of the impaired reflex group, while Streptococcus, Lactobacillus, Veillonella, and Actinomyces were predominant in the normal reflex group. Conclusions Our results suggest that bacteria in bronchial fluids associated with silent aspiration are derived from saliva, and that the bronchial fluids of elderly patients with an impaired swallowing reflex may have a characteristic microbiota.
Journal of Oral Biosciences 12/2014; 57(2). DOI:10.1016/j.job.2014.11.001
[Show abstract][Hide abstract] ABSTRACT: Ketone bodies including acetone are disease biomarkers for diabetes that sometimes causes severe ketoacidosis. The present study was undertaken to clarify the significance of exhaled acetone and plasma ketone bodies at bedside in a clinical setting. The oral glucose tolerance test (OGTT) was performed in 10 healthy Japanese volunteers (five females and five males). Exhaled breath acetone and volatile sulfide compounds (VSCs) in mouth air were measured simultaneously with blood sampling during the OGTT using a portable gas chromatograph equipped with an In2O3 thick-film type gas sensor and a VSC monitor. Acetone, β-hydroxybutyrate (β-OHB) and acetoacetate (AcAc) in blood plasma as well as glucose and insulin were examined. Oral conditions were examined based on the Community Periodontal Index (CPI) by one dentist. In addition, the same type of analysis was applied to two uncontrolled type 2 diabetes mellitus patients hospitalized at Tohoku University Hospital. Exhaled acetone was measured at the same time as blood withdrawal in the morning before breakfast and at night before bed at the beginning, the middle, and the end of hospitalization. All volunteers showed normal OGTT patterns with no ketonuria and periodontitis; however, there were significant correlations between breath acetone and plasma β-ΟΗΒ and between breath acetone and plasma AcAc under fasting conditions. Breath acetone of the type 2 diabetes mellitus patients showed positive correlations with plasma glucose when the level of plasma glucose tended to decrease during hospitalization. In spite of a very limited number of cases, our results support the idea that exhaled breath acetone may be related to plasma β-OHB and AcAc, which reflect glucose metabolism in the body.
Journal of Breath Research 12/2014; 8(4):046008. DOI:10.1088/1752-7155/8/4/046008 · 4.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A complex of species has been associated with dental caries under the ecological hypothesis. This study aimed to develop a rapid, sensitive PCR-dipstick DNA chromatography assay that could be read by eye for multiplex and semiquantitative analysis of plaque bacteria. Parallel oligonucleotides were immobilized on a dipstick strip for multiplex analysis of target DNA sequences of the caries-associated bacteria,
. Streptavidin-coated blue-colored latex microspheres were to generate signal. Target DNA amplicons with an oligonucleotide-tagged terminus and a biotinylated terminus were coupled with latex beads through a streptavidin-biotin interaction and then hybridized with complementary oligonucleotides on the strip. The accumulation of captured latex beads on the test and control lines produced blue bands, enabling visual detection with the naked eye. The PCR-dipstick DNA chromatography detected quantities as low as 100 pg of DNA amplicons and demonstrated 10- to 1000-fold higher sensitivity than PCR-agarose gel electrophoresis, depending on the target bacterial species. Semiquantification of bacteria was performed by obtaining a series of chromatograms using serial 10-fold dilution of PCR-amplified DNA extracted from dental plaque samples. The assay time was less than 3 h. The semiquantification procedure revealed the relative amounts of each test species in dental plaque samples, indicating that this disposable device has great potential in analysis of microbial composition in the oral cavity and intestinal tract, as well as in point-of-care diagnosis of microbiota-associated diseases.
[Show abstract][Hide abstract] ABSTRACT: Indigenous oral bacteria in the tongue coating such as Veillonella have been identified as the main producers of hydrogen sulfide (H2S), one of the major components of oral malodor. However, there is little information on the physiological properties of H2S production by oral Veillonella such as metabolic activity and oral environmental factors which may affect H2S production. Thus, in the present study, the H2S-producing activity of growing cells, resting cells and cell extracts of oral Veillonella species and the effects of oral environmental factors including pH and lactate were investigated. Type strains of Veillonella atypica, Veilonella dispar and Veillonella parvula, were used. These Veillonella species produced H2S during growth in the presence of L-cysteine. Resting cells of these bacteria produced H2S from L-cysteine, and the cell extracts showed enzymatic activity to convert L-cysteine to H2S. H2S production by resting cells was higher at pH 6 - 7 and lower at pH 5. The presence of lactate markedly increased H2S production by resting cells (4.5 - 23.7-fold), while lactate had no effect on enzymatic activity in cell extracts. In addition to H2S, ammonia was produced in cell extracts of all the strains, indicating that H2S was produced by the catalysis of cystathionine γ-lyase (EC 184.108.40.206). Serine was also produced in cell extracts of V. atypica and V. parvula, suggesting the involvement of cystathionine β-synthase lyase (EC 220.127.116.11) in these strains. This study indicates that Veillonella produce H2S from L-cysteine and their H2S production can be regulated by oral environmental factors, pH and lactate.
[Show abstract][Hide abstract] ABSTRACT: Postoperative pneumonia may occur when protective reflexes in the upper respiratory tract such as cough and/or swallowing reflexes are impaired; thus, silent aspiration of oral bacteria is possibly involved in the postoperative pneumonia. This study aimed to quantify and identify bacteria existing in intraoperative bronchial fluids, and evaluate the relationship between impairment of cough/swallowing reflexes and silent aspiration of oral bacteria in elderly patients. After obtaining informed consent, cough and swallowing reflexes were measured using an ultrasonic nebuliser and a nasal catheter, respectively. The intraoperative bronchial fluids from 9 subjects with pulmonary carcinoma were collected with the micro-sampling probe, and were cultured anaerobically on blood agar plates. After 7 days, colony forming units (CFU) were counted and isolated bacteria were identified by 16S rRNA gene sequencing. Four subjects (71.0 ± 8.4 years) showed impaired swallowing reflex with normal cough reflex, while 5 subjects (73.6 ± 6.5 years) showed normal cough and swallowing reflexes. The bacterial counts (mean CFU ± SD) in intraoperative bronchial fluids of subjects with impaired swallowing reflex [(5.1 ± 7.7) × 105] were higher than those with normal one [(1.2 ± 1.9) × 105], although the differences were not statistically significant. Predominant isolates from intraoperative bronchial fluids were Streptococcus (41.8%), Veillonella (11.4%), Gemella (8.9%), Porphyromonas (7.6%), Olsenella (6.3%) and Eikenella (6.3%). These results indicate that intraoperative bronchial fluids contains bacteria, probably derived from the oral microbiota, and suggest that silent aspiration of oral bacteria occurs in elderly patients irrespective of impairment of swallowing reflex.
Microbiology and Immunology 05/2014; 58(7). DOI:10.1111/1348-0421.12157 · 1.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Titanium-silver (Ti-Ag) alloy has been improved for machinability and mechanical properties, but its anti-biofilm properties have not been elucidated yet. Thus, this study aimed to evaluate the effects of Ti-Ag alloy on biofilm formation and bacterial viability in comparison with pure Ti, pure Ag and silver-palladium (Ag-Pd) alloy. Biofilm formation on the metal plates was evaluated by growing Streptococcus mutans and Streptococcus sobrinus in the presence of metal plates. Bactericidal activity was evaluated using a film contact method. There were no significant differences in biofilm formation between pure Ti, pure Ag and Ag-Pd alloy, while biofilm amounts on Ti-20% Ag and Ti-25% Ag alloys were significantly lower (p<0.05). In addition, Ti-Ag alloys and pure Ti were not bactericidal, although pure Ag and Ag-Pd alloy killed bacteria. These results suggest that Ti-20% Ag and Ti-25% Ag alloys are suitable for dental material that suppresses biofilm formation without disturbing healthy oral microflora.
[Show abstract][Hide abstract] ABSTRACT: Objective:
To characterize the metabolic system of oral squamous cell carcinoma (OSCC) by metabolome analysis.
The metabolome profiles, including the Embden-Meyerhof-Parnas pathway (EMPP), the pentose phosphate pathway, the tricarboxylic acid cycle (TCAC), and amino acids, were obtained from OSCC and its surrounding normal tissues (32 patients) using capillary electrophoresis and a time-of-flight mass spectrometer.
Enhancement of glucose consumption and lactate production (Warburg effect) was observed in OSCC tissues. The decrease of glucose along with the decrease of the downstream intermediates in the EMPP suggests that incorporated glucose is mainly consumed for biosynthesis. Glutamine consumption with the increase of the intermediates in the last half of the TCAC suggests the involvement of glutaminolysis, in which glutamine is converted to lactate via the last half of the TCAC.
It is suggested that OSCC tissues show the Warburg effect, which stems from the combined enhancement of glucose consumption and glutaminolysis.
[Show abstract][Hide abstract] ABSTRACT: Inhibition of bacterial acid production by dental restorative materials is one of the strategies for secondary caries prevention. This study aimed to evaluate the effect of fluoride-releasing restorative materials on bacteria-induced pH fall at the bacteria-material interface.
Four fluoride-releasing restorative materials, glass-ionomer cement (GIC), resin-modified glass-ionomer cement (RMGIC), resin composites (RC) and flowable resin composite (FRC) were used. Each specimen was immersed in potassium phosphate buffer at pH 7.0 for 10min and 4 weeks, and in potassium acetate buffer at pH 5.5 for 4 weeks. An experimental apparatus was made of polymethyl methacrylate and had a well with restorative materials or polymethyl methacrylate (control) at the bottom. The well was packed with cells of Streptococcus mutans, and the pH at the interface between cells and materials was monitored using a miniature pH electrode after the addition of 1% glucose for 90min, and the fluoride released into the well was quantified using a fluoride ion electrode.
The pH of GIC (4.98-5.18), RMGIC (4.77-4.99), RC (4.62-4.75) and FRC (4.54-4.84) at 90min were higher than that of control (4.31-4.49). The fluoride amounts released from GIC were the highest, followed by RMGIC, RC and FRC, irrespective of immersion conditions. Saliva coating on materials had no significant effect.
The fluoride-releasing restorative materials inhibited pH fall at the bacteria-material interface. The degree of inhibition of pH fall seemed to correspond to the amount of fluoride detected, suggesting that the inhibition was due to the fluoride released from these materials.
A little amount of fluoride actually released from the fluoride-releasing materials may have caries preventive potential for oral bacteria.
Journal of dentistry 11/2013; 42(1). DOI:10.1016/j.jdent.2013.11.006 · 2.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Actinomyces are predominant oral bacteria; however, its cariogenic potential, such as acid production and fluoride sensitivity, has not been elucidated in details and compared with the other caries-associated oral bacteria, such as Streptococcus. Therefore, this study aimed to elucidate and compare the acid production and growth of Actinomyces and Streptococcus, considering the presence of bicarbonate and fluoride, mimicking the oral cavity. The acid production from glucose was measured by pH-stat at pH 5.5 and 7.0 under anaerobic conditions. The growth rate was assessed by optical density in anaerobic culture. Although the acid production rate by Actinomyces was lower than that of Streptococcus, the acid production of Actinomyces was more tolerant to fluoride (IDacid production 50 = 110-170 ppm at pH 7.0 and 10-13 ppm at pH 5.5) than that of Streptococcus (IDacid production 50 = 36-53 ppm at pH 7.0 and 6.3-6.5 ppm at pH 5.5). Bicarbonate increased the acid production of Actinomyces with prominent succinate production, and enhanced the fluoride tolerance (IDacid production 50 = 220-320 ppm at pH 7.0 and 33-52 ppm at pH 5.5). Bicarbonate had no effect on Streptococcus. In addition, although the growth rate of Actinomyces was lower than that of Streptococcus, Actinomyces growth was more tolerant to fluoride (IDgrowth 50 = 130-160 ppm) than Streptococcus (IDgrowth 50 = 27-36 ppm). These results indicate that oral Actinomyces are more tolerant to fluoride than oral Streptococcus, and bicarbonate enhances the fluoride tolerance of oral Actinomyces. Further study is needed to generalize to the genus level due to the limited number of species tested.
Microbiology and Immunology 09/2013; 57(12). DOI:10.1111/1348-0421.12098 · 1.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oral bacteria adhered to dental material surfaces are known to cause various oral diseases. This study aimed to develop a highsensitive and non-radioisotopic fluorescence dye method for quantification of oral bacteria (Streptococcus, Actinomyces and Veillonella) adhered to denture material surfaces. The amount of adhered bacteria was estimated from the fluorescence intensity derived from resazurin, which is reduced by bacterial metabolic reactions. The addition of bacterial metabolic substrates (glucose for Streptococcus and Actinomyces and sodium lactate for Veillonella) to the reaction mixture increased the fluorescence intensity by 2.3-110 times, subsequently improved the sensitivity. Furthermore, an experimental device having silicon wells containing test material was carefully designed for accurate quantification of bacteria adhered to test materials. The improved resazurin method using a new experimental device successfully enabled the quantification of bacterial adhesion to polymethyl methacrylate and other three conventional denture materials.
[Show abstract][Hide abstract] ABSTRACT: One preventive effect of topical fluoride application is derived from the fact that fluoride can inhibit bacterial acid production. Furthermore, divalent cations such as Ca(2+) and Mg(2+) increase the binding of fluoride to bacterial cells. These findings suggest that exposure of oral bacteria to fluoride in the presence of divalent cations increases fluoride binding to bacterial cells and subsequently enhances fluoride-induced inhibition of bacterial acid production. This study investigated the effects of fluoride exposure (0-20,000 ppm F) in the presence of Ca(2+) or Mg(2+) prior to glucose challenge on pH fall ability by bacterial sugar fermentation, as well as fluoride binding to bacterial cells by exposure to fluoride, and fluoride release from bacterial cells during bacterial sugar fermentation, using caries-related bacteria, Streptococcus mutans and Streptococcus sanguinis. The pH fall by both streptococci was inhibited by exposure to over 250 ppm F in the presence of Ca(2+) (p < 0.01), whereas in the presence of Mg(2+), the pH fall by S. mutans and S. sanguinis was inhibited after exposure to over 250 and 950 ppm F, respectively (p < 0.05). The amounts of fluoride binding to and released from streptococcal cells increased with the concentration of fluoride the cells were exposed to in the presence of Mg(2+), but were high enough even after 250 ppm F exposure in the presence of Ca(2+). The enhanced inhibition of acid production in the presence of divalent cations is probably due to the improved efficiency of fluoride binding to bacterial cells being improved via these divalent cations.
Caries Research 11/2012; 47(2):141-149. DOI:10.1159/000344014 · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to evaluate the ability of a coating material containing the surface pre-reacted glass-ionomer (S-PRG) filler to protect the root from demineralization in vitro. The proprietary coating resin containing the S-PRG filler (PRG Barrier Coat) was applied to human root dentin and immersed in acid buffer at pH 4.5 for 3 d. Demineralization was evaluated by micro-CT scanning and the dentin-material interface observed by scanning electron microscopy. The ability of the coating resin to modify acid production by Streptococcus mutans was investigated by monitoring pH using an ion-sensitive field-effect transistor pH electrode. Application of PRG Barrier Coat produced a coating layer with the thickness of approximately 200 µm and completely inhibited demineralization. The bacteria-induced pH fall at the material surface was significantly inhibited. We conclude that S-PRG fillercontaining coating resin may be an effective material for protecting exposed root from both chemical and biological challenges.
[Show abstract][Hide abstract] ABSTRACT: Research approaches to biofilm are stratified by a series of analyses: (1) microbial number and species; (2) microbial proteins, such as enzymes; and (3) microbial activity, such as metabolic activity. On the other hand, the hierarchical structure of biology includes the genome, proteome, and metabolome, in which the metabolome is the final output of biological function. Metabolome analysis is the comprehensive analysis of the metabolome, a new strategy for biological research in the 21st century. The stratified structure of biofilm research corresponds to the biological hierarchy, and the analysis of microbial activity, especially metabolic activity, is comparable to metabolome analysis; however, oral biofilm samples are too small to analyze the metabolome by conventional methods. Recently, a new device involving capillary electrophoresis (CE) and time-of-flight mass spectrometry (MS) has been developed, facilitating metabolomic investigation of the central carbon metabolic pathways (i.e., the EMP pathway, pentose phosphate pathway, and TCA cycle) in oral biofilm. Using CE–MS, we analyzed metabolome profiles of oral biofilm after oral rinsing with glucose in vivo and evaluated the effects of oral rinsing with fluoride and xylitol on the metabolome profiles of oral biofilm. The results were somewhat consistent with previous in vitro data obtained from single bacterial strains, namely, Streptococcus and Actinomyces; however, new information describing the metabolic properties of oral biofilm was also obtained. This metabolomic approach will reveal the functional characteristics of oral biofilm in vivo, potentially providing new insights into the nature of oral biofilm in health and disease.
Journal of Oral Biosciences 08/2012; 54(3):138–143. DOI:10.1016/j.job.2012.02.005
[Show abstract][Hide abstract] ABSTRACT: To quantify and identify bacteria detected in acrylic resin dentures and dento-maxillary obturator-prostheses after long-term use.
The internal layer of denture bases from 13 daily-use removable acrylic resin dentures was sampled, while the inner fluid samples/no-fluid samples of obturators were collected from 11 in-use acrylic resin dento-maxillary obturator-prostheses. Samples were cultured, and isolated bacteria were counted and identified by molecular biological methods.
Bacteria were detected in five (38.5%) acrylic resin dentures and six (54.5%) acrylic resin obturators. Four Lactobacillus species and one Propionibacterium species were isolated from three repaired denture bases, and from two non-repaired dentures, two Actinomyces species and Streptococcus mutans were isolated. On the other hand, 17 bacterial species, belonging to the family and genera of Olsenella, Bacillus, Citrobacter, Enterobacteriaceae, Lactobacillus, Pantoea, Peptoniphilus, Klebsiella and Pseudomonas, were isolated from obturators. Several species of viable bacteria were detected in acrylic resin denture bases and obturators.
American journal of dentistry 06/2012; 25(3):171-5. · 0.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective. The bacterial examination has been performed during the course of the root canal treatment. In the present pilot study, the new developed method, using fluorescence reagents and a membrane filter, was applied to the detection and quantification of bacteria in infected root canals, in order to evaluate the outcomes of the treatment. Methods. Six infected root canals with periapical lesions from 5 subjects were included. Informed consent was obtained from all subjects (age ranges, 23-79 years). Samples from infected root canals were collected at the beginning of the treatment (termed #25 First), the end of the first day of treatment (termed #55 First), and the next appointment day (termed #55 Second). Then, the bacterial count (CFU) was measured using fluorescence reagents (4',6'-diamidino-2-phenylindole and propidium iodide) and the polycarbonate membrane filter by Bioplorer. Results. The mean ± SD of CFU in the sample of "#25 First" was (1.0 ± 1.4) × 10(5). As the root canal treatment progressed, the CFU decreased as 7.9 × 10(3) (#55 First) and 4.3 × 10(2) (#55 Second). Conclusion. In the present pilot study, rapid detection and quantification of bacteria in infected root canals were found to be successfully performed using fluorescence reagents and a membrane filter (Bioplorer analysis).
International Journal of Dentistry 05/2012; 2012:172935. DOI:10.1155/2012/172935