Patient-specific establishment of bacterial composition within the peri-implant microbiota during the earliest weeks after implant uncovering.

BACKGROUND AND OBJECTIVE: Dysbiosis, a loss of balance in the microbiota, is a potential factor of peri-implantitis. However, compositional change of the peri-implant microbiota soon after implant uncovering is still unknown. In this study, bacterial composition in the peri-implant sulcus was examined to understand the establishment of bacterial composition within the peri-implant microbiota during the earliest weeks after implant uncovering. METHODS: Microbiota samples were collected at weeks 1, 2, 4, and 6 after stage-two surgery. Bacterial DNA was isolated from the samples, and a 16S rRNA gene library was constructed. Sequence reads were obtained using a high-throughput sequencing platform and were taxonomically assigned at the phylum and genus levels. RESULTS: Alpha diversity indices, which did not include taxonomic information, were at similar levels throughout the four time points. At 1 and 2 weeks, the bacterial composition was similar among patients with the predominance of Firmicutes and Proteobacteria. However, the composition was diverse at 4 and 6 weeks and significantly dissimilar to the composition at 1 week. CONCLUSIONS: At 1 week, the peri-implant microbiota was already formed with alpha diversity as high as that at the later time points. However, the bacterial composition was not highly dissimilar among patients at 1 week. The composition changed over the passage of several weeks and was specific for each patient.

An Application of X-Ray Fluorescence as Process Analytical Technology (PAT) to Monitor Particle Coating Processes.

An attempt to apply X-Ray Fluorescence (XRF) analysis to evaluate small particle coating process as a Process Analytical Technologies (PAT) was made. The XRF analysis was used to monitor coating level in small particle coating process with at-line manner. The small particle coating process usually consists of multiple coating processes. This study was conducted by a simple coating particles prepared by first coating of a model compound (DL-methionine) and second coating by talc on spherical microcrystalline cellulose cores. The particles with two layered coating are enough to demonstrate the small particle coating process. From the result by the small particle coating process, it was found that the XRF signal played different roles, resulting that XRF signals by first coating (layering) and second coating (mask coating) could demonstrate the extent with different mechanisms for the coating process. Furthermore, the particle coating of the different particle size has also been investigated to evaluate size effect of these coating processes. From these results, it was concluded that the XRF could be used as a PAT in monitoring particle coating processes and become powerful tool in pharmaceutical manufacturing.

Predicting oral malodour based on the microbiota in saliva samples using a deep learning approach.

BACKGROUND: Oral malodour is mainly caused by volatile sulphur compounds produced by bacteria and bacterial interactions. It is difficult to predict the presence or absence of oral malodour based on the abundances of specific species and their combinations. This paper presents an effective way of deep learning approach to predicting the oral malodour from salivary microbiota. METHODS: The 16S rRNA genes from saliva samples of 90 subjects (45 had no or weak oral malodour, and 45 had marked oral malodour) were amplified, and gene sequence analysis was carried out. Deep learning classified oral malodour and healthy breath based on the resultant abundances of operational taxonomic units (OTUs) RESULTS: A discrimination classifier model was constructed by profiling OTUs and calculating their relative abundance in saliva samples from 90 subjects. Our deep learning model achieved a predictive accuracy of 97%, compared to the 79% obtained with a support vector machine. CONCLUSION: This approach is expected to be useful in screening the saliva for prediction of oral malodour before visits to specialist clinics.

The effects of cigarette smoking on the salivary and tongue microbiome.

OBJECTIVES: It has been suggested that smoking affects the oral microbiome, but its effects on sites other than the subgingival microbiome remain unclear. This study investigated the composition of the salivary and tongue bacterial communities of smokers and nonsmokers in periodontally healthy adults. METHODS: The study population included 50 healthy adults. The bacterial composition of resting saliva and the tongue coating was identified through barcoded pyrosequencing analysis of the 16S rRNA gene. The Brinkman index (BI) was used to calculate lifetime exposure to smoking. The richness and diversity of the microbiome were evaluated using the t-test. Differences in the proportions of bacterial genera between smokers and nonsmokers were evaluated using the Mann-Whitney U test. The quantitative relationship between the proportions of genera and the BI was evaluated using Pearson's correlation analysis. RESULTS: The richness and diversity of the oral microbiome differed significantly between saliva and the tongue but not between smokers and nonsmokers. The saliva samples from smokers were enriched with the genera Treponema and Selenomonas. The tongue samples from smokers were enriched with the genera Dialister and Atopobium. The genus Cardiobacterium in saliva, and the genus Granulicatella on the tongue, were negatively correlated with BI values. On the other hand, the genera Treponema, Oribacterium, Dialister, Filifactor, Veillonella, and Selenomonas in saliva and Dialister, Bifidobacterium, Megasphaera, Mitsuokella, and Cryptobacterium on the tongue were positively correlated with BI values. CONCLUSIONS: The saliva and tongue microbial profiles of smokers and nonsmokers differed in periodontally healthy adults. The genera associated with periodontitis and oral malodor accounted for high proportions in saliva and on the tongue of smokers without periodontitis and were positively correlated with lifetime exposure to smoking. The tongue might be a reservoir of pathogens associated with oral disease in smokers.

Comprehensive analysis of bacteriocins in Streptococcus mutans.

Streptococcus mutans produces bacteriocins that show antibacterial activity against several bacteria. However, comprehensive analysis of these bacteriocins has not been well done. In this study, we isolated 125 S. mutans strains from volunteers and determined their whole genome sequence. Based on the genome analysis, the distribution of each bacteriocin gene (mutacins I-IV, K8 and Smb) was investigated. We found 17, 5, and 2 strains showing 100% matches with mutacin I, mutacin II and mutacin III, respectively. Five mutacin III-positive strains had 2 mismatches compared to mature mutacin III. In 67 mutacin IV-positive strains, 38 strains showed 100% match with mutacin IV, while 29 strains showed some variations. In 23 mutacin K8- and 32 mutacin Smb-positive strains, all except one mutacin K8-positive strain showed 100% match with the mature peptides. Among 125 strains, 84 (65.1%), 26 (20.2%), and 5 (3.9%) strains were positive for one, two and three bacteriocin genes, respectively. Then, the antibacterial activity against oral streptococci and other oral bacterial species was investigated by using bacteriocin gene single-positive strains. Each bacteriocin gene-positive strain showed a different pattern of antibacterial activity. These results speculate that individual S. mutans strains may affect the bacterial composition of dental plaques.

Relationship between oral malodor and the global composition of indigenous bacterial populations in saliva.

Oral malodor develops mostly from the metabolic activities of indigenous bacterial populations within the oral cavity, but whether healthy or oral malodor-related patterns of the global bacterial composition exist remains unclear. In this study, the bacterial compositions in the saliva of 240 subjects complaining of oral malodor were divided into groups based on terminal-restriction fragment length polymorphism (T-RFLP) profiles using hierarchical cluster analysis, and the patterns of the microbial community composition of those exhibiting higher and lower malodor were explored. Four types of bacterial community compositions were detected (clusters I, II, III, and IV). Two parameters for measuring oral malodor intensity (the concentration of volatile sulfur compounds in mouth air and the organoleptic score) were noticeably lower in cluster I than in the other clusters. Using multivariate analysis, the differences in the levels of oral malodor were significant after adjustment for potential confounding factors such as total bacterial count, mean periodontal pocket depth, and tongue coating score (P < 0.001). Among the four clusters with different proportions of indigenous members, the T-RFLP profiles of cluster I were implicated as the bacterial populations with higher proportions of Streptococcus, Granulicatella, Rothia, and Treponema species than those of the other clusters. These results clearly correlate the global composition of indigenous bacterial populations with the severity of oral malodor.

Prediction of plausible bacterial composition based on terminal restriction fragment length polymorphisms using a Monte Carlo method.

We have developed a new approach for the estimation of bacterial proportional compositions in microbiota based on terminal restriction length polymorphism (T-RFLP) data and a Monte Carlo algorithm. This program estimates proportional compositions by minimizing distances between peak values and the relative abundance of each group, containing several species, estimated from peak areas of capillary electrophoresis for T-RFLP analysis. Oral bacteria in 36 saliva samples obtained from three individuals were analyzed using the program. Upon comparison, the estimated proportional composition obtained from one of the samples matched that from a clone library. Additionally, comparisons among the bacterial proportional compositions of saliva samples obtained from three individuals four times per day for 3 days revealed that the types of microbiota present in each individual did not change within each 24-h time period and were distinguishable from those in other individuals.

3-Chloro-DL-alanine resistance by L-methionine-alpha-deamino-gamma-mercaptomethane-lyase activity.

The antibacterial agent 3-chloro-DL-alanine (3CA) is an inhibitor of peptidoglycan synthesis. Fusobacterium nucleatum and Porphyromonas gingivalis, the bacteria responsible for oral malodor, are shown to be resistant to 1 mM 3CA, whereas Streptococcus mutans and Escherichia coli are sensitive to this antibacterial agent at the same concentration. We isolated the 3CA resistance gene from F. nucleatum and showed that the gene encodes an L-methionine-alpha-deamino-gamma-mercaptomethane-lyase that catalyzes the alpha,gamma-elimination of L-methionine to produce methyl mercaptan. The enzyme also exhibits 3CA chloride-lyase (deaminating) activity. This antibacterial agent is expected to be useful for specific selection of malodorous oral bacteria producing high amounts of methyl mercaptan.

Correlation between oral malodor and periodontal bacteria.

Volatile sulfur compounds (VSCs), including hydrogen sulfide, methyl mercaptan, and dimethyl sulfide, are primarily responsible for oral malodor. Recently, the mgl gene encoding L-methionine-alpha-deamino-gamma-mercaptomethane-lyase, which produces methyl mercaptan, was cloned from Porphyromonas gingivalis. This article discusses the mechanism and pathogenic role of the formation of VSCs by oral bacteria.

Cloning and characterization of the L-cysteine desulfhydrase gene of Fusobacterium nucleatum.

Hydrogen sulfide and methyl mercaptan are the two major compounds associated with oral malodor. These compounds are highly toxic, and are thought to play an important role in periodontal disease. Fusobacterium nucleatum, an oral bacterium, produces large amounts of hydrogen sulfide from L-cysteine by the enzymatic action of L-cysteine desulfhydrase. We cloned and sequenced the cdl gene encoding L-cysteine desulfhydrase from F. nucleatum ATCC 10953, and revealed that the structural cdl gene consists of 921 bp and encodes a 33.4-kDa protein. The cloned gene was inserted into an expression vector, pDEST17, and expressed in Escherichia coli as a fused protein. The purified enzyme was tested for substrate specificity using various SH-containing compounds. Only L-cysteine served as a substrate for L-cysteine desulfhydrase to produce hydrogen sulfide.

Evaluation of soft tissue morphologic changes after using the alar base cinch suture in Le Fort I osteotomy in mandibular prognathism with and without asymmetry.

PURPOSE: The purpose of this study was to investigate the effects of alar base cinch suture in Le Fort I osteotomy in mandibular prognathism with and without asymmetry. MATERIAL AND METHODS: Forty patients who underwent Le Fort I osteotomy were divided into an asymmetry group and a symmetry group (n = 20 each). Computed tomography (CT) was taken in all patients before and 1 year after surgery. The CT-3D volume rendering data with Aquarius Net (TeraRecon, Foster City, CA, USA) was reconstructed and the soft tissue was measured as follows, the distance between the bilateral alar base (Alar base width), the distance between the bilateral alar (Alar width), the angles between the FH plane and the line between the bilateral alar base (Alar base angle), the angle between the FH plane and the line between the bilateral alar (Alar angle), the angle between the FH plane and the line between the bilateral corners of the mouth (Lip angle), and the angle between the perpendicular line to the FH plane and the Philtrum (Philtrum angle). RESULTS: Significant differences were observed in the Alar width (P = 0.0448), the Alar angle (P = 0.0044), the Lip angle (P < 0.0001), and the Philtrum angle (P = 0.0057) between before and after surgery in the asymmetry group. CONCLUSIONS: This study suggested that the alar base cinch suture could prevent increases in the alar base width in both groups and help to improve the angle of soft tissue in the asymmetry group, although alar width significantly increases after surgery.

The time-course change in the lip closing force in Class III malocclusion after orthognathic surgery.

OBJECTIVES: The purpose of this study was to examine the changes with time in lip pressure after orthognathic surgery for skeletal Class III patients. STUDY DESIGN: The subjects consisted of 63 patients (32 female and 31 male) diagnosed with mandibular prognathism who underwent sagittal split ramus osteotomy with and without Le Fort I osteotomy. Maximum and minimum lip closing forces were measured with Lip De Cum(®). The changes with time were compared statistically. RESULTS: The maximum and minimum lip closing force increased time-dependently in men and women after surgery and there were significant differences between men and women with changes with time in the maximum lip closing force (p = 0.0086) and the minimum lip closing force (p = 0.0302). After 1 year, the Class III group maximum lip closing force was significantly smaller than the control group in both men (p < 0.0001) and women (p < 0.0001). CONCLUSIONS: This study suggests that there was significant difference in over time in the lip closing force between men and women in Class III patients. Although the maximum lip closing force increased with time, it did not reach the level of the control group after 1 year.

Supervised machine learning-based classification of oral malodor based on the microbiota in saliva samples.

OBJECTIVE: This study presents an effective method of classifying oral malodor from oral microbiota in saliva by using a support vector machine (SVM), an artificial neural network (ANN), and a decision tree. This approach uses concentrations of methyl mercaptan in mouth air as an indicator of oral malodor, and peak areas of terminal restriction fragment (T-RF) length polymorphisms (T-RFLPs) of the 16S rRNA gene as data for supervised machine-learning methods, without identifying specific species producing oral malodorous compounds. METHODS: 16S rRNA genes were amplified from saliva samples from 309 subjects, and T-RFLP analysis was carried out with the DNA fragments. T-RFLP analysis provides information on microbiota consisting of fragment lengths and peak areas corresponding to bacterial strains. The peak area is equivalent to the frequency of a specific fragment when one molecule is selected from terminal fragments. Another frequency is obtained by dividing the number of species-containing samples by the total number of samples. An SVM, an ANN, and a decision tree were trained based on these two frequencies in 308 samples and classified the presence or absence of methyl mercaptan in mouth air from the remaining subject. RESULTS: The proportion that trained SVM expressed as entropy achieved the highest classification accuracy, with a sensitivity of 51.1% and specificity of 95.0%. The ANN and decision tree provided lower classification accuracies, and only classification by the ANN was improved by weighting with entropy from the frequency of appearance in samples, which increased the accuracy to 81.9% with a sensitivity of 60.2% and a specificity of 90.5%. The decision tree showed low classification accuracy under all conditions. CONCLUSIONS: Using T-RF proportions and frequencies, models to classify the presence of methyl mercaptan, a volatile sulfur-containing compound that causes oral malodor, were developed. SVM classifiers successfully classified the presence of methyl mercaptan with high specificity, and this classification is expected to be useful for screening saliva for oral malodor before visits to specialist clinics. Classification by a SVM and an ANN does not require the identification of the oral microbiota species responsible for the malodor, and the ANN also does not require the proportions of T-RFs.

Evaluation of overlapped cortical bone area after modified plate fixation with bent plate in sagittal split ramus osteotomy.

PURPOSE: The purpose of this study was to evaluate bony change in an overlapped cortical bone area at the anterior site of the proximal segment after modified plate fixation with a bent plate in sagittal split ramus osteotomy (SSRO). SUBJECTS AND METHODS: The subjects were 66 patients (132 sides) who underwent bilateral SSRO setback surgery. After the surgery in SSRO, an overlapped area of cortical bone at the anterior site of the proximal segment was not removed to keep the contact area between the proximal and distal segments intact, and was fixed with a bent plate and 4 screws in each side of the mandible. At the posterior site, a 3-7 mm gap was maintained between the proximal and distal segments to prevent inward-rotation of the condylar long axis. Ramus width, lateral cortex width and lateral cortex step angle were assessed in a coronal image immediately after the surgery, and 1-year postoperative by computed tomography (CT). RESULTS: Ramus width after 1-year was significantly larger than that before surgery and smaller than that immediately after surgery in both sides (P < 0.0001). Lateral cortex width after 1-year was significantly larger than the preoperative value and smaller than that immediately after surgery in both sides (P < 0.0001). Lateral cortex angle after 1-year was significantly larger than the preoperative value in both sides (P < 0.0001). CONCLUSION: The above findings suggested that the overlapped cortical bone decreased thickness and the cortical bone step disappeared following favourable bone remodelling after 1-year, even though the cortical bone was not removed at the anterior site of the proximal segment.

Two mechanisms of oral malodor inhibition by zinc ions

Abstract Objectives The aim of this study was to reveal the mechanisms by which zinc ions inhibit oral malodor. Material and Methods The direct binding of zinc ions to gaseous hydrogen sulfide (H2S) was assessed in comparison with other metal ions. Nine metal chlorides and six metal acetates were examined. To understand the strength of H2S volatilization inhibition, the minimum concentration needed to inhibit H2S volatilization was determined using serial dilution methods. Subsequently, the inhibitory activities of zinc ions on the growth of six oral bacterial strains related to volatile sulfur compound (VSC) production and three strains not related to VSC production were evaluated. Results Aqueous solutions of ZnCl2, CdCl2, CuCl2, (CH3COO)2Zn, (CH3COO)2Cd, (CH3COO)2Cu, and CH3COOAg inhibited H2S volatilization almost entirely. The strengths of H2S volatilization inhibition were in the order Ag+ > Cd2+ > Cu2+ > Zn2+. The effect of zinc ions on the growth of oral bacteria was strain-dependent. Fusobacterium nucleatum ATCC 25586 was the most sensitive, as it was suppressed by medium containing 0.001% zinc ions. Conclusions Zinc ions have an inhibitory effect on oral malodor involving the two mechanisms of direct binding with gaseous H2S and suppressing the growth of VSC-producing oral bacteria.

Discrimination of the oral microbiota associated with high hydrogen sulfide and methyl mercaptan production.

Both hydrogen sulfide (H2S) and methyl mercaptan (CH(3)SH) are frequently detected in large amounts in malodorous mouth air. We investigated the bacterial composition of saliva of 30 subjects with severe oral malodor exhibiting extreme CH(3)SH/H(2)S ratios (high H(2)S but low CH(3)SH concentrations, n 5 14; high CH(3)SH but low H2S concentrations, n 5 16) and 13 subjects without malodor, using barcoded pyrosequencing analysis of the 16S rRNA gene. Phylogenetic community analysis with the UniFrac distance metric revealed a distinct bacterial community structure in each malodor group. The H2S group showed higher proportions of the genera Neisseria, Fusobacterium, Porphyromonas and SR1 than the other two groups, whereas the CH(3)SH group had higher proportions of the genera Prevotella, Veillonella,Atopobium, Megasphaera, and Selenomonas. Our results suggested that distinct bacterial populations in the oral microbiota are involved in production of high levels of H2S and CH3SH in the oral cavity.

The ecological proportion of indigenous bacterial populations in saliva is correlated with oral health status.

To obtain deeper insights into the etiology of oral disease, an understanding of the composition of the surrounding bacterial environments that lead to health or disease is required, which is attracting increasing attention. In this study, the bacterial compositions in the saliva of 200 subjects aged 15-40 years were depicted as peak patterns by terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes. The subjects were classified into three clusters by partitioning around medoids clustering based on their T-RFLP profiles, and the clinical oral health parameters of the clusters were compared. The clustering of the T-RFLP profiles in this study was mainly based on differences in the abundance distribution of the dominant terminal restriction fragments (TRFs) detected in most of the subjects. Predicted from the sizes of the TRFs, the characteristically more predominant members of each were Prevotella and Veillonella species in cluster I; Streptococcus species in cluster II and Neisseria, Haemophilus or Aggregatibacter species and Porphyromonas species in cluster III. The parameters associated with periodontal disease were significantly different among the clusters. Clusters I and II had a higher percentage of sites of periodontal pockets greater than 4 mm than cluster III, and cluster I contained sites exhibiting bleeding on probing more often than cluster II or III; no significant differences were observed in other parameters. These results suggest that the abundance distribution of commensal bacteria in saliva is correlated with periodontal health, and might be involved in the susceptibility of an individual to periodontal disease.

Quantitative analysis of multi-species oral biofilms by TaqMan Real-Time PCR.

Oral infectious diseases, including dental caries, various forms of periodontitis and oral malodor, are not caused by a single pathogen. The etiology of these diseases is known to be associated with bacterial accumulation and plaque composition on the hard and soft tissues of the oral cavity. Therefore, the quantitative, as well as qualitative, analysis of the microorganisms present in oral biofilms, namely dental plaque, subgingival plaque and tongue debris, is important for diagnosis and rational treatment decisions. The quantitative microbial analysis of oral multi-species biofilms also provides useful information for establishing the etiology of oral infectious diseases. Recently, a 5' fluorogenic, nuclease-based, real-time polymerase chain reaction (PCR) technique has been increasingly employed for the quantitative microbial assessment of the human oral cavity. We review the development and use of TaqMan real-time PCR for quantifying oral bacteria, its role in the diagnosis of oral infectious diseases and their microbial etiology.

Identification and characterization of an autolysin-encoding gene of Streptococcus mutans.

We identified a gene (atlA) encoding autolytic activity from Streptococcus mutans Xc. The AtlA protein predicted to be encoded by atlA is composed of 979 amino acids with a molecular weight of 107,279 and has a conserved beta-1,4-N-acetylmuramidase (lysozyme) domain in the C-terminal portion. Sodium dodecyl sulfate extracts of strain Xc showed two major bacteriolytic bands with molecular masses of 107 and 79 kDa, both of which were absent from a mutant with inactivated atlA. Western blot analysis revealed that the 79-kDa band was derived from the 107-kDa peptide by cleavage of its N-terminal portion. The inactivation of atlA resulted in a marked decrease in autolysis and the formation of very long chains of cells compared to the case for the parent strain. Although both the parent and mutant strains formed biofilms in the presence of sucrose, the biofilms formed by the mutant had a sponge-like architecture with large gaps and contained 30% less biomass than those formed by the parent strain. Furthermore, strain Xc formed glucose-dependent, loose biofilms in the absence of sucrose, but the mutant lost this ability. These results suggest that AtlA may play an important role in biofilm formation by S. mutans. The antibody produced against the C-terminal peptide containing the beta-1,4-N-acetylmuramidase domain drastically inhibited the autolytic activity of strain Xc. This inhibition was specific among the oral streptococci to S. mutans. These results indicate that the catalytic domain of AtlA is located at the C terminus, suggesting that further characterization of this domain may provide a means to control cariogenic dental plaque formation.

High production of methyl mercaptan by L-methionine-alpha-deamino-gamma-mercaptomethane lyase from Treponema denticola.

Methyl mercaptan is derived from l-methionine by the action of l-methionine-alpha-deamino-gamma-mercaptomethane lyase (METase) and is a major component of oral malodor. This compound is highly toxic and is thought to play an important role in periodontal disease. We found that Treponema denticola, a member of the subgingival biofilm at periodontal disease sites, produced a large amount of methyl mercaptan even at low concentration of l-methionine. METase activity in a cell-free extract from T. denticola was detected by two-dimensional electrophoresis under non-denaturing conditions, and the protein spot that exhibited high METase activity was identified using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. The identified gene produced a METase with a K(m) value for l-methionine (0.55mM) that is much lower than those of METases previously identified in the other organisms. This result suggests that T. denticola is an important producer of methyl mercaptan in the subgingival biofilm.