Study on Distribution of Four Pseudomonas Species in Living Environment Using Multiplex PCR

Purpose: The genus Pseudomonas is a ubiquitous microorganism frequently detected from immunocompromised patients. The inherent resistance to numerous antimicrobial agents contributes to the opportunistic character of this pathogen exhaustive monitoring of this pathogen is considered of critical importance to public health organizations. The reliable identification method able to distinguish genetic close Pseudomonas species is needed, because these organisms are difficult to differentiate by phenotypic or biochemical methods. The purpose of the present study was to design species-specific primers in order to identify and detect four Pseudomonas species which are frequently detected from the human oral cavities, and to investigate the distribution of these organisms in the living environment using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the rpoD gene of four Pseudomonas species. Swab samples were collected from fifty washstands, and the distribution of Pseudomonas species was investigated using a conventional PCR at genus level and a multiplex PCR at species level. Results: Multiplex PCR method developed in this study was able to distinguish four Pseudomonas species clearly. The genus Pseudomonas was detected from all samples (100%), whereas P. putida, P, aeruginosa, P. stutzeri and P. fluorescens were detected at 44%, 8%, 4% and 2% in fifty swab samples, respectively. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and works without requiring DNA extraction. It was indicated that washstands were the uninhabitable environment for P. putida, P, aeruginosa, P. stutzeri and P. fluorescens.

Stabilization of an abutment under a rigidly fixed bridge by holographical-speckle interferometry

Objective: There are no detailed reports of three-dimensional measurement of abutment teeth in mastication, because it is knotty to observe the rotation in chewing directly, and inexact to estimate indirectly. This work studies the three-dimensional stability of rigidly fixed bridge under the stresses of distributed loads and concentrated loads by optical method that gives the tip angle and rotation angle calculated directly based on measurement data. Methods: The specimen, taken from a 25-year-old male, was a left mandible without the second premolars and the first molars. As abutments, first premolar and second molar have complete periodontium. The specimen was soaked in formaldehyde solution. The bridge was fixed between two abutment teeth (first premolars and second molars), and the mandible was cemented in a steel box. The load was increased from 0 kg to 23 kg. Laser holographic technique was used to measure the three-dimensional bit shift of the dens, both buccolingual bit shift and mesiodistal bit shift, and determine tip angle and rotation angle. Results: The effects of stress distribution on the rigidly fixed bridge were evaluated, and stabilization of the bridge under the stresses of distributed loads and concentrated loads, respectively, were analyzed. The results showed that the tips of two abutments were very similar, and no distinct difference was observed between the distributed load and the concentrated load. However, the maximum rotation angle for the distributed load was two to four times as large as that for the concentrated load. In the experiment, the tip angle of the abutment teeth was no more than 0.65 degree, and the rotation angle was no more than 0.60 degree. All maximum angles occurred in the second molar. Conclusion: The fixed bridge is considered to be safe. In addition, a method for measuring the rotation angle was provided effectively.

Investigation of Bacteria Species Most Involved in Peri-Implantitis

Purpose: Currently, bacteriological examinations of implant treatments target periodontopathic bacteria such as red complex bacteria, including Porphyromonas gingivalis, and detect them qualitatively or quantitatively. However, it seems that those examinations do not reflect the peri-implant tissue conditions precisely, because periodontopathic bacteria are also frequently detected from healthy peri-implant sites. The purpose of the present study was to investigate bacteria species most involved in peri-implantitis using a PCR method. Methods: Polymerase chain reaction (PCR) primers in this study were designed based on partial sequences of 16S rDNA of bacteria species involved in peri-implantitis that were described in numerous previous studies. Peri-implant sulcus fluid (PISF) samples were collected from thirty periodontally healthy patients with implants (HI) and thirty patients with peri-implantitis (PI). Each detection frequency of bacteria species in PISFs of both groups was investigated using a PCR method, and was compared using Fisher’s exact test. Results: In PI group, detection frequencies of Corynebacterium durum, Fretibacterium fastidiosum and Slackia exigua were significantly higher than those of HI group (p P. gingivalis and Tannerella forsythia belonging to red complex were frequently detected in the PISF samples of HI group (p > 0.05). Conclusion: It was suggested that monitoring C. durum and F. fastidiosum levels in PISF samples was useful as a clinical indicator for the evaluation of peri-implant tissue conditions.

Registration of the Seated Condylar Position (SCP/CR): Part I: Rationale

The purpose of this paper is to present the rationale for registration of the Seated Condylar Position (SCP)/Centric Relation (CR) position of the condyles.

Registration of the Seated Condylar Position (SCP/CR): Part II: Technique: The Two-Piece Wax Bite Registration

The purpose of this paper is to present the technique for registration of the Seated Condylar Position (SCP)/Centric Relation (CR) position of the condyles: a two-piece wax bite registration with deprogramming and no mandibular manipulation.

Isolation and Identification Methods for Oral Klebsiella pneumoniae Involved in Onset of Inflammatory Bowel Disease

Purpose: Recently, it was reported that Klebsiella pneumoniae is related to the onset of inflammatory bowel disease including the Crohn disease. It was frequently reported that K. pneumoniae was detected in human oral cavities. Regrettably, it currently remains unclear whether K. pneumoniae is part of the normal oral flora. The aim of this study was to establish the isolation and identification methods for K. pneumoniae from human oral cavities, and investigate its transmission pattern. Methods: A selective medium, OKPSM, for the isolation of K. pneumoniae from oral cavities was developed in this study. Also, PCR primer for the identification and detection at subspecies level of K. pneumoniae was designed. Results: OKPSM and PCR method using the primers designed in this study were useful for the isolation and identification of K. pneumoniae from human oral cavities. K. pneumoniae subsp. pneumoniae was detected at 10.0% in 30 saliva samples. On the other hand, K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis were detected from no sample. Moreover, K. pneumoniae subsp. pneumoniae isolates from same subject at 0 month and after 3 months showed same genotypes on AP-PCR using OPA-07 primer. Conclusion: These results indicated that human oral cavities were not suitable for the habitat of K. pneumoniae.

Study on Distribution of Acinetobacter baumannii Complex in Dental Hospital Using Multiplex PCR

Purpose: In recent years, multidrug-resistant Acinetobacter baumannii has appeared and caused outbreaks of hospital infections all over the world. Close monitoring of this pathogen and other A. baumanii complex species is considered of critical importance to public health organizations. The reliable identification method able to distinguish A. baumannii from genetically close Acinetobacter species is needed, because these species are unable to be differentiated by phenotypic or biochemical methods. The purpose of the present study was to design species-specific primers in order to identify and detect A. baumanii complex species, and Acinetobacter lwoffii which is frequently detected from the human specimens, and to investigate the distribution of these organisms in dental hospital using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 16S ribosomal RNA (16S rRNA) gene and DNA gyrase subunit B (gyrB) of each species of A. baumanii complex species. Swab samples were collected from ten dental spittoon units in dental hospital, and the distribution of A. baumanii complex species was investigated using a multiplex PCR. Results: These primers were able to distinguish each species of A. baumanii complex species clearly. A. baumanii and A. calcoaceticus were detected at 20.0% and 10.0% in ten swab samples, respectively. On the other hand, A. nosocomialis, A. lowffii, and A. pittii were detected from no sample. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.

One Step Multiplex PCR for Identification of Candida haemulonii complex and Candia auris

Purpose: Recently, Candida haemulonii complex (Candida haemulonii, Candida duobushaemulonii and Candida haemulonii var. vulnera) and two genetically close species (Candida pseudohaemulonii and Candida auris) have emerged as an opportunistic fungal pathogen associated with various infectious diseases of humans, and most of those isolates have displayed antifungal resistance. Because it is difficult to differentiate these microorganisms by a current technique, unfortunately, it is important to establish a method for identifying them accurately. The purpose of the present study was to design species-specific primers in order to identify and detect C. auris, C. pseudohaemulonii, and C. haemulonii complex, i.e. , C. haemulonii, C. duobushaemulonii and C. haemulonii var. vulnera using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 26S rRNA, 18S rRNA, and RPB1 genes and ITS region of five Candida species. Results: The multiplex PCR method developed in this study was able to distinguish five Candida species clearly. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and works without requiring DNA extraction.

Effect of restoration technique on resistance to fracture of endodontically treated anterior teeth with flared root canals

This study was designed to compare the impact of post and core systems on resistance to fracture of endodontically treated anterior teeth with flared root canals and to assess their fracture pattern. Sixty central incisors were cut horizontally 2 mm coronal to the cementoenamel junction(CEJ). After root canal therapy, teeth were assigned into 6 groups(n = 10 each) based on a post system and used as follows: Group C, non-flared root received size #1 glass fiber posts(Control); Group AP, flared root restored with anatomical post; Group RC, flared root restored with size #1 fiber post and cemented with thick layer of resin cement; Group CR, flared root restored with size #1 and reinforced with composite resin; Group CM, cast post-core; Group CP, CAD/CAM polymer-infiltrated ceramic post and core.Following post cementation, core build-up and crown insertion, the specimens were thermo-cycled up to 10,000 cycles(5 C/55 C; 30 seconds dwell time, 6 seconds transition time) and then statically loaded at 1 mm/minute crosshead speed using a universal testing machine. One-way ANOVA and Tukey HSD post hoc test(α= 0.05) were used for data analysis. Group C recorded significantly higher resistance to fracture values [(826.9±39.1) N] followed by group CP [(793.8±55.6) N] while group RC yielded the lowest fracture resistance values [(586.7±51.4) N]. The resistance to fracture of wide root canals can be enhanced by using one-piece CAM/CAM post and core as an alternative to the use of either glass fiber post, relined with composite resin increasing the thickness of luting cement or the use of cast post and core system. However, this was an in vitro investigation and further in vivo studies are necessary.

One-Step Multiplex PCR for Simultaneous Detection and Identification of Eight Medically Important <i>Candida</i>Species

Recently, the incidence of Candida infections has substantially increased. Conventional identification methods for Candida species are technically difficult to conduct and cannot accurately distinguish each species. The purpose of the present study was to design primers to identify and detect simultaneously eight medically important Candida species using one-step multiplex PCR. PCR primers were designed based on partial sequences of intergenic spacer (IGS) and internal transcribed spacer (ITS) genes of eight medically important Candida species. These primers were able to distinguish each Candida species and did not display cross-reactivity with representative Candida species other than the eight Candida species. Moreover, our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.

Evaluation of xerostomia closely associated with systemic diseases using a dental approach

Based on the findings of epidemiological surveys in western countries, the number of person with suspected xerostomia in Japan is estimated to be 30,000,000. Xerostomia is caused by: 1) Systemic diseases;2) Medications;3) Tumors or trauma;4) Radiotherapy;and 5) Neurological factors. Its symptoms can be alleviated, and its progression can be stopped by close cooperation between the dental and medical departments. However, at present, symptomatic treatment methods such as water drinking, oral rinsing with water, and administration of Kampo medicines or parasymptomimetic drugs are mainly used, and a standard treatment has not been established. On the other hand, previous studies on xerostomia have reported improvement in dry mouth symptoms using dental approaches or a relationship between the development of candidiasis and the salivary flow rate. Therefore, in this study, to establish a dental method useful for improving xerostomia associated with systemic diseases and medications for them, we restored occlusal function, and as a result, increased the salivary flow rate, and evaluated changes in oral findings.

Isolation and Identification Methods for Actinomyces israelii Involved in Actinomycosis

Purpose: Actinomyces israelii is known as the key species to cause classical actinomycosis. Although A. israelii is frequently isolated from human oral cavities, the distribution of this microorganism has been little reported. The purpose of the present study was to develop selective media (AISM) for the isolation of A. israelii and to assess the prevalence of this organism in the oral cavity. Methods: To examine the bacterial population in the oral cavity, a novel selective medium (AISM) was developed for isolating A. israelii. AISM consists of BHI, yeast extract, agar, ofloxacin, fosfomycin, colistin, and sodium fluoride. Results: A. israelii strains grew well on AISM. A. israelii was detected in all dental plaque samples collected from 20 subjects and the mean number of this organism in the samples was 7.9 × 104 CFU/ml. Conclusion: These results indicated that the selective medium was useful for the isolation of A. israelii and this organism was a part of the normal flora in the human oral cavity.

Screening Survey of Pain Intensity in Patients with Temporomandibular Disorders

Objective: Pain tends to be the chief complaint in patients suffering temporomandibular disorders (TMD). Previous studies on pain and psychosocial factors have reported on the relationship between presence of pain and mental disorders. To date, however, few studies have addressed the relationship between intensity of pain and psychosocial factors. In this study, we investigated the relationship between intensity of pain and age, gender, palpation scores (PPS), tendencies toward depression, anxiety, and somatization, and oral parafunctional habits. Methods: This screening survey encompassed 104 patients (70 women and 34 men;mean age of 46.1 ± 19.3) who visited our clinic. We gathered the following data: age;gender;PPS included in Axis I diagnosis;and characteristic pain intensity (CPI), depression, anxiety, somatization, and oral parafunctional habits (assessed by the Oral Behavior Checklist) included in Axis II diagnosis. Based on the results of CPI, we divided patients into two groups: those experiencing low pain intensity (LP group) and those experiencing high pain intensity (HP group). The statistically significant level was set to below 5%. IBM SPSS Statistics V25 was used to perform all statistical analyses. Results: We observed no gender differences between LP and HP groups. The HP group included significantly more patients with higher scores for depression, anxiety, somatization, and oral parafunctional habits than the LP group. While no gender differences were observed in CPI, depression, anxiety, somatization, and oral parafunctional habits were significantly more common in women than in men. We observed no differences in age or PPS between the LP and HP groups. However, scores for depression, anxiety, somatization, and oral parafunctional habits were significantly higher in the HP group than in the LP group. We performed multiple regression analysis using the CPI score as the dependent variable and scores for depression, anxiety, somatization, and oral parafunctional habits as independent variables in both the LP and the HP groups. We identified no significant predictors for the LP group, but extracted depression as a significant predictor in the HP group. On evaluating the correlation of PPS with depression, anxiety, somatization, and oral parafunctional habits in both the LP and the HP groups, we found no correlation between the PPS and the seven-item generalized anxiety disorder (GAD-7) scale in the LP group but identified a significant correlation between the PPS and GAD-7 scores in the HP group. Moreover, the correlation coefficient between the patient health questionnaire (PHQ)-9 and GAD-7 scores was higher in the HP group than in the LP group. Conclusion: In those reporting more intense pain, we found a stronger correlation among psychological factors in patients diagnosed with TMD. Greater tendency toward depression was directly associated with pain intensity. The results point to the need to consider differences in psychosocial factors associated with pain intensity when treating TMD.

Study on the Distribution at Species Level of Genus Candida in Human Oral Cavities, Using Culture and Multiplex PCR Methods

Purpose: Although the genus Candida is frequently isolated from human oral cavities, the distribution at the species level of these organisms has been little reported. The purpose of the present study was to assess the distribution at the species level of the genus Candida in human oral cavities. Methods: This study was performed using culture and Multiplex PCR methods. Moreover, the genotyping classification of C. albicans was analyzed with a PCR. Results: Of all subjects (n = 90), detection frequency of genus Candida was 42.2%. Genus Candida was not detected in the subjects between 0 to 9 years old, and there was no difference in the detection frequencies of this organism among each generation from 10s to 80s. C. albicans was the most dominant species, followed by C. parapsilosis, C. glabrata, and C. dubliniensis. Plural Candida species tended not to be detected in the individual sample. Genotype A was dominant in the C. albicans isolates. Conclusion: These results indicated that C. albicans of genotype A was dominant and that the genus Candida rarely coexists with other Candida species, in each individual oral cavity.

One Step Multiplex PCR for Identifications at Subspecies Level of Fusobacterium nucleatum and Fusobacterium necrophorum

Purpose: Fusobacterium nucleatum is an opportunistic pathogen involved in periodontal diseases, extraoral infections, and colorectal cancer. Fusobacterium necrophorum causes a variety of necrotic infections. F. nucleatum and F. necrophorum are classified into five and two subspecies, respectively. Conventional identification methods were technically hard to distinguish each subspecies of two Fusobacterium species accurately. The purpose of the present study was to design primers to identify two medically important Fusobacterium species at the subspecies level, using one-step multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 16S ribosomal RNA (16S rRNA) gene, RNA polymerase B (rpoB) gene, and DNA gyrase subunit B (gyrB) of each subspecies of F. nucleatum and F. necrophorum. Results: These primers were able to distinguish each subspecies of F. nucleatum and F. necrophorum and did not display cross-reactivity with representative Fusobacterium species other than F. nucleatum and F. necrophorum. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.

Origin of Candida albicans in Human Oral Cavity

Purpose: Candida albicans is regarded as a part of normal flora in the human oral cavity. However, it remains unclear whether the genus Candida, especially C. albicans, is an oral resident microorganism and causes marital infection or not. The purpose of the present study was to elucidate the origin of oral C. albicans by investigating the colonization and infection route to oral cavities of this organism with arbitrarily primed polymerase chain reaction (AP-PCR). Methods: After C. albicans was isolated from four subjects (average age: 42.2, range: 33 - 56), the isolations of this organism from them were performed six months later again. To investigate whether C. albicans is an oral resident microorganism, the genotype homology of each C. albicans isolates that were isolated twice from the same subjects was compared. Moreover, C. albicans was isolated from five pairs of married couples (average period of cohabitation: 12.4 years, range: 5 - 31). To investigate whether C. albicans causes marital infection, the genotype homology of C. albicans isolates that were isolated from each pair of married couples was compared. Results: AP-PCR patterns of C. albicans that were isolated from each subject at o month and after 6 months showed the identical genotypes among each individual. C. albicans isolates from five pairs of married couples showed the identical genotypes between a husband and wife of each pair on AP-PCR. Conclusion: These results indicated that C. albicans was an oral resident microorganism and caused the marital infection.

Effect of Disc Position for Acute Closed Lock of the Temporomandibular Joint

In this manuscript, the authors have studied the Disc Displacement (DD) status of patients with acute Closed Lock (CL) to determine differences between DD with Reduction (DDwR) and DD without Reduction (DDwoR). Among the acute CL patients who visited our hospital within 2 weeks of the onset of CL, we studied 10 patients whose CL was released (DDwR) and 13 patients whose CL was not released (DDwoR). The DDwoR group was significantly older than the DDwR group. Although the mouth opening distance was significantly greater in the DDwoR group than in the DDwR group, the two groups were identical in the duration of CL. Sagittal MRI images showed no significant differences between the two groups in disc length and disc thickness (anterior band, intermediate zone, and posterior band). Multisection sagittal and coronal images identified lateral DD in 7 of the 10 patients in the DDwR group, although no specific direction of the DD was observed in the DDwoR group. Furthermore, deformation of the medial disc was common in the DDwoR group but uncommon in the DDwR group.

Antimicrobial properties of dental cements modified with zein-coated magnesium oxide nanoparticles

The aim of this study was to test the antimicrobial properties of dental cements modified with magnesium oxide(MgO)nanoparticles.Zein-modified MgO nanoparticles(zMgO)in concentrations(0.0,0.3,0.5,and 1.0%)were mixed with dental cements(Fuji II,Rely X Temp E,Ionoglass Cem,Es Temp NE,and System P link).Eight discs were fabricated from each zMgO-cement pair for a total of 32 specimens for each cement.Characterization of the dental cements incorporating zMgO was done by X-ray Diffraction(XRD)and Field Emission Scanning Electron Microscopy(FESEM).The antimicrobial properties of the mixtures were tested using direct contact and agar diffusion assays against Streptococcus mutans,Staphylococcus aureus,Enterococcus faecalis,and Candida albicans.Data was analyzed using two-way analysis of variance and LSD post hoc test at 0.05 significance level.XRD spectra showed sharp peaks of zMgO indicating its high crystalline nature,while the amorphous dental cements with zMgO had broad peaks.FESEM analysis showed a uniform distribution of the zMgO nanoparticles in the cement.There were significant inhibition zone values associated with all concentrations of zMgO-cement mixtures tested compared to controls(p<0.001)with a dose-response recorded only with Fuji II.Optical density values were significantly lower in zMgO groups compared to controls for all microorganisms.The effect was most prominent with Rely X against C.albicans and S.aureus.Dental cements containing zMgO showed significant antimicrobial properties that were dependent on the specific initial cement substrate.

Purkait’s triangle revisited:role in sex and ancestry estimation

Identification of unknown remains recovered from marine and terrestrial locations is a significant humanitarian problem.This investigation proposes a simple method applicable to fragmentary femora for a more refined level of ancestry and/or sex estimation.To that end,we re-examined Purkait’s triangle which involves three inter-landmark distances between the traction epiphyses and the articular rim of femoral head.A large sample(n=584)from geographically diverse(Egyptian,Indian and Greek)populations was compiled.Additionally,shape(n=3)and trigonometrically derived variables and ratios(n=9 variables)were employed to detect any geographically-clustered morphological differences between these populations.Random forest modelling(RFM)and linear discriminant function analysis(LDA)were employed to create classification models in instances where sex was known or unknown.The sample was apportioned into training and test sets with a ratio 70/30.The classification accuracies were evaluated by means of k fold cross-validation procedure.In sex estimation,RFM showed similar performance to LDA.However,RFM outperformed LDA in ancestry estimation.Ancestry estimation was satisfactory in the Indian and Egyptian samples albeit the Greek sample was problematic.The Greek samples presented greater morphological overlap with the Indian sample due to high within-group variation.Test samples were accurately assigned to their ancestral category when sex was known.Generally,higher classification accuracies in the validation sample were obtained in the sex-specific model of females than in males.Using RFM and the linear variables,the overall accuracy reached 83%which is distributed as 95%,71%and 86%for the Egyptian,Indian and Greek females,respectively;whereas in males,the overall accuracy is 72%and is distributed as 58%,87%and 50%for the Egyptian,Indian and Greek males,respectively.Classification accuracies were also calculated per group in the test data using the 12 derived variables.For the females,the accuracies using the medians model was comparable to the linear model whereas in males the angles model outperformed the linear model for each group but with similar overall accuracy.The classification rates of male specific ancestry were 82%,78%and 56%for the Egyptian,Indian and Greek males,respectively.In conclusion,Purkait’s triangle has potential utility in ancestry and sex estimation albeit it is not possible to separate all groups successfully with the same efficiency.Intrapopulation variation may impact the accuracy of assigned group membership in forensic contexts.