Association between dysbiotic perio-pathogens and inflammatory initiators and mediators in COVID-19 patients with diabetes.

It has been suggested that a corona virus infection is linked to chronic periodontitis (COVID-19). Our objectives were to look at the expression of angiotensin-converting enzyme-2 (ACE2) in periodontal compartments containing periodontal infections to determine if ACE2 is directly or indirectly responsible for the inflammation in periodontal tissues getting worse. In this study, six non-COVID-19 periodontitis patients without diabetes served as controls, and 23 hospitalized periodontitis patients were admitted with PCR-confirmed COVID-19 with diabetes mellitus (Group 1/G1, n = 10), and without diabetes (Group 2/G2, n = 13). We evaluated the mRNA expression of ACE2, IL-6, IL-8, complement C3, and LL-37, as well as the relative proportion of Porphyromonas gingivalis, Fusobacterium nucleatum, and Veillonella parvula to represent the dysbiosis condition in periodontal microenvironment using subgingival plaque and gingival crevicular fluids (GCF) samples and quantitative real time PCR (qPCR). Every analysis was done to ascertain how they related to one another. The area under the curve (AUC) and receiver operating characteristic (ROC) curve were used to determine the sensitivity and specificity of inflammatory indicators. All the grouped patients had ACE2 detected, according to our findings, but only the G1 patients had a positive correlation (p < 0.05) between ACE2 expression and the inflammatory markers. The combination of IL-6 and C3 mRNAs was found to be 0.78 and 0.55 for the G1 group and the G2 group, respectively, based on the ROC and AUC values. According to our research, the relationship between complement C3 and IL-6 may be able to predict the degree of periodontal inflammation in COVID-19 patients who also have diabetes.

The utility of salivary CRP and IL-6 as a non-invasive measurement evaluated in patients with COVID-19 with and without diabetes.

Background: The available evidence suggests that inflammatory responses, in both systemic and oral tissue, contribute to the pathology of COVID-19 disease. Hence, studies of inflammation biomarkers in oral fluids, such as saliva, might be useful to better specify COVID-19 features. Methods: In the current study, we performed quantitative real-time PCR to measure salivary levels of C-reactive protein (CRP) and interleukin-6 (IL-6) in saliva obtained from patients diagnosed with mild COVID-19, in a diabetic group (DG; n = 10) and a non-diabetic group (NDG; n = 13). All participants were diagnosed with periodontitis, while six participants with periodontitis but not diagnosed with COVID-19 were included as controls. Results: We found increases in salivary total protein levels in both the DG and NDG compared to control patients. In both groups, salivary CRP and IL-6 levels were comparable. Additionally, the levels of salivary CRP were significantly correlated with total proteins, in which a strong and moderate positive correlation was found between DG and NDG, respectively. A linear positive correlation was also noted in the relationship between salivary IL-6 level and total proteins, but the correlation was not significant. Interestingly, the association between salivary CRP and IL-6 levels was positive. However, a moderately significant correlation was only found in COVID-19 patients with diabetes, through which the association was validated by a receiver operating curve. Conclusions: These finding suggest that salivary CRP and IL-6 are particularly relevant as potential non-invasive biomarker for predicting diabetes risk in mild cases of COVID-19 accompanied with periodontitis.

Effect of hydrothermal treatment of titanium in high concentration of AgNO 3 solution on surface morphology and roughness.

Development of silver (Ag) modified titanium (Ti) as an antibacterial dental implant has recently been growing. Ag demonstrated an excellent antibacterial property without the risk of bacterial resistance. Hydrothermal treatment using AgNO 3 solution is one of the facile and promising methods to modify Ti surface with Ag. However, the effect of high AgNO 3 concentration and the absent of a toxic reduction agent has not been clearly studied. In this study, Ti surface was hydrothermally treated in 0.01 mol/L and 0.1 mol/L AgNO 3 solutions at 150 oC for 24 hours. Analysis of surface morphology using scanning electron microscopy with energy dispersive X-ray analysis suggested the formation of non-homogenous Ag coating with a tendency to be aggregated and thicken with the increase of AgNO 3 concentration. The Ag coating deposited on Ti surface were composed of mainly metallic and some oxide forms. Surface roughness of all AgNO 3 treated Ti surface was comparable based on the analysis of surface roughness parameter. In conclusion, hydrothermal treatment of Ti surface in solely AgNO 3 solution at high concentration produced non-homogenous Ag coating on its surface without significantly changed surface roughness. Keywords: Silver nitrate, titanium, hydrothermal, surface morphology, roughness.

Characterization of Oral Veillonella Species in Dental Biofilms in Healthy and Stunted Groups of Children Aged 6-7 Years in East Nusa Tenggara.

Impaired development that causes stunting is one of the most common health problems in Indonesia. In particular, the highest number of cases of stunting in Indonesia was reported in the East Nusa Tenggara (NTT) province. Previous studies have shown a tendency for deteriorating oral hygiene in children with a poor nutritional status. In addition, a higher proportion of oral Veillonella has been reported in children with poor oral hygiene. However, the relationship between populations of oral Veillonella and stunting has not been studied before. Therefore, this study aimed to analyze the oral Veillonella profile in the dental biofilms of healthy and stunted children aged 6-7 years. The participants were 60 elementary school students in the Nangapanda District, Ende, NTT, Indonesia. In this study, real-time polymerase chain reaction was used to examine dental biofilm samples from the healthy (n = 31) and stunted (n = 29) groups. The results revealed that seven oral Veillonella species were found in all groups. However, the number of four oral Veillonella species significantly differed between the healthy and stunted groups: V. denticariosi, V. infantium, V. rogosae, and V. tobetsuensis. This is the first study to demonstrate a potential association between oral Veillonella species and stunting in children.

ACE2 expression in saliva of patients with COVID-19 and its association with Candida albicans and Aggregatibacter actinomycetemcomitans.

Background: A relationship between oral microbiota and susceptibility to SARS-CoV-2 infection has been extensively studied. However, the relationship between oral commensal flora and expression of angiotensin-converting enzyme 2 ( ACE2) remains to be established. In this observational study, we collected saliva from patients with COVID-19 and evaluated the relationship between ACE2 expression and Candida albicans as well as with selected gram-negative bacteria ( Aggregatibacter actin o mycetemcomitans, Fusobacterium nucleatum, and Veillonella parvula). We investigated how this may be directly or indirectly involved in oral dysbiosis in patients with COVID-19. Methods: We included 23 hospitalized patients admitted to Universitas Indonesia Hospital with PCR-confirmed COVID-19, with six healthy participants serving as controls. Saliva and tongue surface swabs were collected from patients with diabetes (DG) and without diabetes (NDG) and subject controls. Using quantitative PCR (qPCR) we assessed the mRNA expression of ACE2, the abundance of C. albicans, and the transcription levels of its biofilm-associated genes, agglutinin-like protein 3 ( ALS3), hyphal wall protein 1 ( HWP1), and yeast-form wall protein 1 ( YWP1). We also counted the relative proportion of the three selected gram-negative oral bacteria in saliva. All analyses were performed to determine the relationship between ACE2 expression and C. albicans and gram-negative bacteria. Results: ACE2 mRNA expression was significantly higher in tongue swab samples than in saliva. However, no significant difference was observed between the patient groups. Conversely, DG patients had a significantly higher abundance of C. albicans in saliva compared to NDG patients and control group patients. The correlation and sensitivity/specificity relationship between ACE2 expression and C. albicans or the selected oral bacteria were also observed. Conclusions: The data show that ACE2 expression can be detected in saliva of patients with COVID-19 and its association with C. albicans and gram-negative oral bacteria might contribute toward developing an oral dysbiosis based predictor for prognosis of COVID-19 severity.

Novel Indigenous Probiotic Lactobacillus reuteri Strain Produces Anti-biofilm Reuterin against Pathogenic Periodontal Bacteria.

OBJECTIVE: The aim of this study was to evaluate the effect of reuterin produced by a novel probiotic strain of Lactobacillus reuteri against periodontal biofilms. MATERIALS AND METHODS: L. reuteri LC382415 (an indigenous Indonesian strain) was cultured in Man, Rogosa, and Sharpe (MRS) agar in anaerobic conditions for 24 hours. To isolate reuterin, L. reuteri was suspended in 300-mM glycerol in MRS broth and incubated under anaerobic conditions for 3 hours, and the supernatant fraction was filtered. The presence of reuterin was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and its concentration was determined. The effect of reuterin on Porphyromonas gingivalis ATCC 33277 and T. denticola ATCC 35405 biofilms was evaluated using biofilm assays. Biofilms were formed by incubating bacteria in 96-well microplates for 48 hours. A dose-dependent experiment was performed with reuterin concentrations of 12.5, 25, 50, and 100 µg/mL on biofilms. The inhibitory effect was measured at 1, 3, 6, and 24 hours. The biofilm masses were measured at 490 nm. Statistical analysis was using one-way ANOVA. RESULTS: The SDS-PAGE assay confirmed the presence of reuterin (52 kDa) in the culture supernatant of the L. reuteri strain. Reuterin in a concentration as low as 12.5 µg/mL significantly inhibited single- and mixed-species biofilms (p < 0.05). CONCLUSIONS: This is the first study to demonstrate the promising effect of reuterin isolated from L. reuteri LC382415 against periodontal bacteria. Further studies are warranted to explore the mechanism of this active component.

Casein phosphopeptide-amorphous calcium phosphate fluoride treatment enriches the symbiotic dental plaque microbiome in children.

OBJECTIVES: The dysbiotic oral microbiome plays a key role in the pathogenesis of caries in children. Topical application of casein phosphopeptide-amorphous calcium phosphate containing fluoride (CPP-ACP/F) is an effective treatment modality for children with caries (CC). Hitherto the mechanism by which CPP-ACP/F modules the oral microbiome in CC has not been investigated. The study aimed to examine the CPP-ACP/F effect on the dental plaque microbiome of children group with caries. METHODS: This preliminary prospective clinical cohort included 10 children with caries. The children received topical fluoride CPP-ACP/F once-a-week for one month. Plaque samples were collected before and after treatment and subjected to 16S rDNA-based next-generation-sequencing. Microbial composition, diversity and functional roles were analyzed in comparison to the clinical characteristics of cohort using standard bioinformatics tools. RESULTS: CPP-ACP/F treatment modulated dysbiotic oral microbiome towards healthier community as the higher proportion of Proteobacteria and certain microbial protective species were enriched following CPP-ACP/F treatment. Despite overall uniformity of community structure in children with caries between the groups, some bacterial species were differentially represented in a statistically significant manner between pre- and post- treatments. Three bacterial species were found to be predictive of strongly sensitive to the CPP-ACP/F treatment, marked by decreased abundance of Lautropia mirabalis and increased abundance of Gemella haemolysans and Schwartzia succinivorans. CONCLUSION: Within the limits of the current study, it could be concluded that the CPP-ACP/F varnish treatment modulated the microbial composition of the dental plaque microbiome towards symbiosis. These symbiotic changes may demonstrate the potential clinical significance of CPP-ACP/F varnish treatment.

Diversity of Oral Microbiome of Women From Urban and Rural Areas of Indonesia: A Pilot Study.

Objective: The studies on the influence of geographical and socio-economic factors on the oral microbiome remain underrepresented. The Indonesia basic health research (RISKESDAS) 2018, showed an increasing trend in non-communicable diseases compared with the previous report in 2013. The prevalence of diabetes, heart disease, hypertension, and obesity are reported to be higher in urban areas than in rural areas. Interestingly, non-communicable diseases were found to be more prevalent in women than men. This pilot study aimed to examine the oral health and oral microbiome derived from tongue samples of healthy Indonesian women from urban and rural areas. Methods: Twenty women aged 21-47 years old from West Jakarta, residents of DKI Jakarta (n = 10) as representative of the urban area, and residents of Ende, Nangapanda, East Nusa Tenggara (n = 10) as representative of the rural area were recruited for this pilot study. The participants were evaluated by the Simplified Oral Hygiene Index (OHI-S) according to the criteria of Greene and Vermillion and divided into three groups. High-throughput DNA sequencing was performed on an Illumina iSeq 100 platform. Results: The principal component analysis displayed a marked difference in the bacterial community profiles between the urban and rural localities. The presence of manifest was associated with increased diversity and an altered oral bacterial community profile in the urban women. Two bacterial taxa were present at significantly higher levels (adjusted p < 0.01) in the urban oral microflora (Genus Prevotella and Leptotricia) could account for this difference irrespective of the individual oral hygiene status. The linear discriminant analysis effect size (LEfSe) analysis revealed several distinct urban biomarkers. At the species level, Leptotrichia wadei, Prevotella melaninogenica, Prevotella jejuni, and P. histicola, show an excellent discriminatory potential for distinguishing the oral microflora in women between urban and rural areas. Further, using SparCC co-occurrence network analysis, the co-occurrence pattern in the dominant core oral microbiome assembly was observed to be specific to its ecological niche between two populations. Conclusions: This is the first pilot study demonstrating the characterization of the oral microbiome in Indonesian women in urban and rural areas. We found that the oral microbiome in women displays distinct patterns consistent with geographic locality. The specific characterization of the microbiota of Indonesian women is likely linked to geographical specific dietary habits, cultural habits, and socio-economic status or the population studied.

COVID-19 Vaccines in Indonesia: Knowledge, Attitudes, and Acceptance Among Dental Professionals.

Background: At the time of writing, the number of Coronavirus Disease 2019 (COVID-19) cases in Indonesia has exceeded 2 million. The COVID-19 pandemic has caused severe disruptions in and unprecedented challenges to healthcare systems, including the one in Indonesia. Healthcare professionals, especially dentists, have an increased risk of contracting the virus. Many dental professionals in Indonesia have been exposed to the virus through close contact with aerosols and droplets from the oral cavities of their patients and have subsequently become infected. The Indonesian government commenced its COVID-19 vaccination program in January 2021. It is necessary to achieve high COVID-19 vaccination coverage rates among health workers. However, immunizations are voluntary, and some healthcare workers may be reluctant to receive them. The aim of this study was to evaluate the knowledge, attitudes, and acceptance of dental professionals of COVID-19 vaccines. Materials and Methods: A cross-sectional study was conducted among dentists taking part in the Indonesia Dental Association webinar in June 2021. Google Docs was used to create an online questionnaire, which was disseminated to the participants at the end of the webinar. The questionnaire consisted of 15 questions developed after being reviewed by experts. The questionnaire collected demographic data (age, gender, location, and affiliation/institution) and data on the dental professionals' knowledge and acceptance of COVID-19 vaccines in Indonesia as well as their attitudes toward COVID-19 vaccines. The respondents selected one option (agree/neutral/disagree) in response to each statement in the questionnaire. For data analysis, the respondents were divided into three groups according to their professional activity/employment category: national hospital (GOV), private hospital (PRIVATE), or academic faculty in a dental school (ACADEMIC). The data were analyzed using descriptive statistics and expressed as frequencies and percentages. A chi-square test was used to investigate the association between professional activity and acceptance of COVID-19 vaccines. Results: In total, 779 dentists from 34 provinces in Indonesia completed the questionnaire. There were 646 (83%) females and 133 (17%) males, with an age range of 24-73 years. In terms of the distribution of professional affiliations, the respondents included 23 (3%) academics, 285 (36.5%) private hospital/private practice practitioners, and 471 (60.5%) national hospital practitioners. In the statistical analysis, unadjusted odds ratios (ORs) were calculated with their 95% confidence intervals (CIs). Conclusions: Acceptance of COVID-19 vaccines is an essential determinant of vaccine uptake and the likelihood of controlling the COVID-19 pandemic. There is agreement between dental professionals in private hospitals and academic faculties (dental school) regarding the need for COVID-19 vaccination. Developing strategies to reduce public hesitation and increase trust is vital for implementing vaccination programs, and dentists can play a role in increasing the uptake of COVID-19 vaccines.

COVID-19 Awareness Among Dental Professionals in Indonesia.

Background: The COVID-19 pandemic caused by SARS-CoV-2 has claimed thousands of lives worldwide. To enhance knowledge and awareness of COVID-19, considerable online resources have been made available, including aspects related to the dental profession. The study aim was to examine the knowledge, perception, and attitude of dental professionals in Indonesia toward COVID-19. We conducted a survey via a questionnaire created using Google docs and distributed to 632 members of the Indonesian Dental Association in the context of a webinar hosted by the Indonesian Oral Biology Association on first June, 2020. Materials and Methods: The questionnaire consisted of 17 items pertaining to demographic data, knowledge and virus identification, awareness regarding drugs commonly used in dentistry during pandemic and research opportunities. Participants were asked to complete the questionnaire after the webinar by choosing one answer to each question. For the analysis, participants were divided into three groups according to their professional background i.e., employment at national hospital, private hospital, or academic faculty. Data were analyzed using descriptive statistics and expressed as frequencies and percentages. The chi-square test was used to investigate the association between professional activity and the level of knowledge, perceptions, and attitudes about COVID-19. Results: Sixty percent of the participants correctly identified the pathogenesis of the disease. This knowledge was not associated with their professional affiliation (p = 0.95). Sixty-seven percentage had comprehensive knowledge about virus detection methods. This knowledge was not associated with their affiliation either (p = 0.54). Questions regarding drugs of choice, prevention, and the spread of COVID-19 were correctly answered by 89, 96, and 82% of the participants, respectively. Knowledge of these aspects were significantly associated with the professional affiliation (p < 0.05). All respondents were optimistic regarding research opportunities (p < 0.01). Respondents from academics were more interested in joining COVID-19-related research projects with governmental institutions (p < 0.01). Conclusion: Knowledge and awareness of COVID-19 among Indonesian dentists are reasonably good. However, further improvement would be beneficial to manage patients during this pandemic. As the number of COVID-19 cases continue to rise in Indonesia, it is important that dentists keep abreast of the updated knowledge on this moving field. Dentist knowledge on infection control should be strengthened through continuous educational programs.

Identification and phylogenetic analysis of oral Veillonella species isolated from the saliva of Japanese children.

Background: As the most frequent infectious disease among children worldwide, dental caries have a strong relationship with oral hygiene status, specifically in the development of infection. However, the study regarding the identification and distribution of oral Veillonella are limited. The oral Veillonella community may affected by the differences in geographical location, age, diet, lifestyle, socio-economic status and oral hygiene status. Here, we studied the oral hygiene status by examining the composition and proportion of oral Veillonella species in saliva of Japanese children. Methods: Microbial samples collected from 15 Japanese children divided into three oral hygiene groups were cultured under anaerobic conditions after homogenization and dilution, and inoculated onto brain heart infusion and selective medium Veillonella agar. Genomic DNA was extracted from each isolate. Veillonella species were detected by one-step PCR using rpoB species-specific primers. To analyse the phylogenetic properties of the unknown Veillonella strains, PCR amplification and sequence analysis of rpoB were conducted for 10 representative strains. Results: Although V. rogosae was found as the predominant species among all groups, its prevalence was significantly lower in the children with poor oral hygiene than in those with good oral hygiene. V. parvula was the prevalent species in the poor oral hygiene group. Approximately 10% of the isolated Veillonella strains were not classified to any established species; the phylogenetic analysis showed that they were most closely related to V.infantiumConclusions: This study demonstrates that the composition and proportion of oral Veillonella species in the saliva of Japanese children is correlated with different oral hygiene status. Changes in detection ratios of V. parvula and V. rogosae can be useful indicators of oral hygiene status. Furthermore, new strains closely related to V. infantium were isolated from the saliva of Japanese children.

Identification of Veillonella Species in the Tongue Biofilm by Using a Novel One-Step Polymerase Chain Reaction Method.

Six Veillonella species have been frequently isolated from human oral cavities including infectious sites. Recently, it was reported that diet, smoking, and possibly socioeconomic status can influence the bacterial profile in oral cavities. In addition, oral hygiene habits may also influence oral microbiota in terms of both numbers and diversity of microorganisms. In this study, the identification of Veillonella species in tongue biofilms of Thai children, divided into three groups dependent on their status of oral hygiene. For this, we used a novel one-step PCR method with species-specific primer sets based on sequences of the rpoB gene. As shown in the results, the number of isolates of Veillonella species was 101 strains from only 10 of 89 subjects. However, the total number of bacteria was high for all subjects. Since it was reported in previous studies that Veillonella species were easy to isolate in human tongue biofilms at high numbers, the results obtained in this study may suggest country- or age-specific differences. Moreover, Veillonella species were detected predominantly in subjects who had poor oral hygiene compared to those with good or moderate oral hygiene. From these results, there is a possibility that Veillonella species may be an index of oral hygiene status. Furthermore, V. rogosae was a predominant species in tongue biofilms of Thai children, whereas V. parvula and V. denticariosi were not isolated at all. These characteristics of the distribution and frequency of Veillonella species are similar to those reported in previous studies. Although further studies are needed in other countries, in this study, a successful novel one-step PCR method was established to detect Veillonella species in human oral cavities easily and effectively. Furthermore, this is the first report investigating the distribution and frequency of Veillonella species in tongue biofilms of Thai children.