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Objective: This study aimed to standardize a quantitative polymerase chain reaction (qPCR)-based test to identify and quantify the uncultivable bacteria associated with periodontitis., Methods: The standardization of qPCR, the curves for the quantification of Eubacterium saphenum, Eubacterium brachy, Desulfobulbus oralis, and Filifactor alocis were developed by cloning the 16 S rRNA target gene fragment, using the GEMTEasy vector. The qPCRs were validated in 55 subgingival biofilm clinical samples, from different stages of periodontitis and from periodontally healthy/gingivitis individuals, which were previously evaluated by next-generation sequencing (NGS). The results obtained by the two methods were compared by the concordance of Cohen's Kappa index, and sensitivity, specificity, receiver operating characteristic (ROC) curve, and predictive values were established., Results: obtained by the two methods were compared using the concordance of Cohen's Kappa index, and sensitivity, specificity, predictive values, and ROC curves were generated. The qPCR test was standardized with efficiencies between 90% and 100% and R 2 : 0.997-0.999. Concordance between the qPCR and NSG was moderate to F. alocis (agreement 78.2%; kappa 0.56, p < 0.05) and fair to the other microorganisms (agreement 67.27%-72.73; kappa 0.37-0.38, p < 0.05). qPCR exhibited a high sensitivity (82.2-100%) and specificity (100%) for E. brachy, E. saphenum, and F. alocis. Sensitivity was lower to D. oralis. Conversely, qPCR demonstrated higher sensitivity to E. saphenum than NSG (100 vs. 68.1)., Conclusions: The uncultivable microorganisms associated with periodontitis, D. oralis, E. brachy, E. saphenum, and F. alocis can be detected and quantified with the newly developed and validates qPCR test., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Hypochlorous acid (HOCl) is an antimicrobial agent with high affinity to Gram-negative bacteria of the subgingival biofilm. It could have an equivalent or no inferiority effect to chlorhexidine (CHX) to avoid recolonization of these microorganisms after the post-surgical period., Objective: The objective is to compare the reduction of plaque index (PI), gingival index (GI), pocket depth (PD), gain of clinical attachment level (CAL), and bacterial recolonization of periodontopathic microorganisms in subgingival biofilm at 7, 21, and 90 days after Open Flap Debridement (OFD) under two antimicrobial protocols: (A) HOCl 0.05% followed by HOCl 0.025% and (B) CHX 0.2%/CHX 0.12% used per 21 days without regular oral hygiene during the post-surgical period., Material and Methods: A no-inferiority randomized controlled trial was carried out. Thirty-two patients were randomly divided to receive each antiplaque protocol after OFD in patients with periodontitis. Clinical indexes and bacterial recolonization were assessed using qPCR for up to 90 days. Data were analyzed using repeated measures ANOVA, mixed effects models adjusted for treatment, time, and the Chi-squared/Fisher test. A no-inferiority analysis was also performed using the Hodges-Lehmann hypothesis test for non-inferiority., Results: HOCl was not inferior to CHX in reducing PI. Both groups showed a comparable reduction of recolonization for Porphyromonas gingivalis , Tannerella forsythia , and Eubacterium nodatum . However, the HOCl protocol was non-inferior to the CHX protocol for Treponema denticola and Aggregatibacter actinomicetemcomitans ., Conclusions: HOCl improved periodontal healing. HOCl showed an impact in reducing the recolonization of periodontopathic bacteria in the postoperative period.

Objectives: Culturable and unculturable microorganisms have been associated with periodontitis. Their differential proportions and composition have not been evaluated by their severity and complexity defined by stages in the 2018 AAP-EEP classification., Methods: One hundred eighty subgingival biofilm samples were collected in Spain and Colombia from subjects categorized as health/gingivitis: periodontitis stages I/II periodontitis stages III/IV. Target culturable microorganisms (Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Treponema denticola, and Eubacterium nodatum) and target unculturable microorganisms (Filifactor alocis, Eubacterium saphenum, Eubacterium brachy, Desulfobulbus oralis) were evaluated by quantitative PCR analysis. In addition, their differences and association with periodontal status were analyzed by ANCOVA and logistic regression models once adjusted to age, current smoking, and country., Results: P. gingivalis was significantly associated with periodontitis stages I/II, OR 2.44 (CI 95% 1.08-5.47) and stages III/V, OR 6.43 (CI 95% 2.43-16.9). T forsythia, OR 7.53 (CI 95% 2.07-27.4); D. oralis, OR 5.99 (CI 95% 2.71-13.23); F. alocis, OR 10.9 (CI 95% 4.56-23.2); E. brachy, 3.57 (CI 95% 1.40-9.11); and E. saphenum, 4.85 (CI 95% 1.99-11.7) were significantly associated only with stages III/IV periodontitis. P. gingivalis evidenced significant differences with the increase in the severity of the periodontal lesion: 2.97 colony forming unit (CFU)/μL (CI 95% 2.32-3.54) health/gingivitis, and 4.66 CFU/μL (CI 95% 4.03-5.30) and 5.90 CFU/μL (CI 95% 5.20-6.48) in stages I/II and III/IV respectively (p < 0.0001). Unculturable microorganisms only evidenced differences in concentration in stages III/IV compared with health-gingivitis (p ≤ 0.001)., Conclusion: Culturable and unculturable are strongly associated with stages III/IV periodontitis. Classic culturable microorganisms are more sensitive to differentiate between stages of periodontitis in the quantitative analysis., Clinical Relevance: Future interventional studies of periodontal disease should include Filifactor alocis, Eubacterium saphenum, Eubacterium brachy, and Desulfobulbus oralis as possible markers of therapy response and as indicators of progressive disease., (© 2023. The Author(s).)

Porphyromonas gingivalis secretes virulence factors like Arg-gingipains and peptidyl arginine deiminase (PPAD), that are associated with rheumatoid arthritis (RA) pathogenesis. However, there is no information regarding the antibody titers for these bacterial enzymes as systemic indicators or biomarkers in RA. In this cross-sectional study, 255 individuals were evaluated: 143 were diagnosed with RA, and 112 were without RA. Logistic regression models adjusted for age, sex, basal metabolic index, smoking, and periodontitis severity were used to evaluate the association of RA with rheumatoid factor (RF), anti-citrullinated protein antibodies (ACPAs), erythrocyte sedimentation rate, high sensitivity C-reactive protein, anti-RgpA, anti-PPAD, and double positive anti-RgpA/anti-PPAD. It was found that RF (odds ratio [OR] 10.6; 95% confidence interval [CI] 4.4-25), ACPAs (OR 13.7; 95% CI 5.1-35), and anti-RgpA/anti-PPAD double positivity (OR 6.63; 95% CI 1.61-27) were associated with RA diagnoses. Anti-RgpA was also associated with RA (OR 4.09; 95% CI 1.2-13.9). The combination of anti-RgpA/anti-PPAD showed a high specificity of 93.7% and 82.5% PPV in identifying individuals with RA. RgpA antibodies were associated with the periodontal inflammatory index in RA individuals ( p < 0.05). The double positivity of the anti-RgpA/anti-PPAD antibodies enhanced the diagnosis of RA. Therefore, RgpA antibodies and anti-RgpA/anti-PPAD may be biomarkers for RA.

Adipokines are associated with the pathogenesis of rheumatoid arthritis (RA) and are potential biomarkers of disease activity, periodontitis, and obesity. The aim of this was to establish the association between adipokine profile, RA disease activity, body mass index, and periodontal infection. This study evaluated 51 patients with early-RA and 51 controls including serum rheumatological markers, adipokine levels, detection of Porphyromonas gingivalis and serum anti- Porphyromonas gingivalis antibodies, clinical and periodontal measurements. Statistical analyses were run with SPSS ® V26, with a logistic regression model to confirm associations. The results show high levels of leptin were more frequent in patients ( p = 0.001) who simultaneously showed a higher frequency of Porphyromonas gingivalis ( p = 0.004). Patients with concomitant presence of Porphyromonas gingivalis , high clinical activity score, and overweight were correlated with high levels of leptin (OR, 7.20; 95% CI, 2.68-19.33; p = 0.0001) and adipsin (OR, 2.69; 95% CI, 1.00-7.28; p = 0.005). The conclusion is that high levels of leptin and adipsin are associated with greater clinical activity in early-RA patients with overweight and periodontal infection, whereby overweight and Porphyromonas gingivalis may enhance RA activity. This may represent a pathological mechanism between these conditions, where adipokines seem to have a key role.

Introduction: Purification of native gingipains is challenging because these proteases are frequently associated with the cell surface, which affects yield. This study aimed to purify native Arg-gingipain (RgpA) from Porphyromonas gingivalis Outer Membrane Vesicles (OMV)., Methods: Native RgpA was purified from P. gingivalis strain ATCC33277 OMV using a strategy including ultracentrifugation, sonication, and successive anionic and cationic fast protein liquid chromatography (FPLC). The presence and purity of the protease were confirmed by SDS-PAGE and detection of protease activity using fluorogenic substrates. Rat antibodies produced against the unique adhesin hemagglutinin (H1) domain of RgpA (amino acids 719-865) were titrated by ELISA at a 1:100 dilution using whole P. gingivalis lysate as an antigen and western blotting to detect a 75 kDa band corresponding to RgpA., Results: Double anionic-cationic FLPC yielded prominent peaks with evident amidolytic gingipain activity of the appropriate molecular weight, as confirmed by western blotting. The final RgpA yield from 1 L of bacterial culture with colony forming unit (CFU) (Log 10 ) 7.4 ± 0.08/mL was of 12.6% (2 mg/mL), with 3.2 FU/μg of amidolytic activity., Conclusions: This protocol allows purification of native RgpA from OMV that retains protease activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The Prevotella genus is a normal constituent of the oral microbiota, and is commonly isolated from mechanically treated polymicrobial infections. However, antibiotic treatment is necessary for some patients. This study compared the antibiotic susceptibility and the presence of resistance genes in clinical oral isolates of P. intermedia , P. nigrescens , and P. melaninogenica . Antibiotic susceptibility was assessed using the agar dilution method. PCR confirmed the species and resistance gene frequency in the Prevotella species. The frequencies of species P. intermedia , P. nigrescens , and P. melaninogenica were 30.2%, 45.7%, and 24.1%, respectively. No isolates of P. intermedia were resistant to amoxicillin/clavulanic acid, tetracycline, or clindamycin. P. nigrescens and P. melaninogenica were resistant to amoxicillin/clavulanic acid and tetracycline at frequencies of 40% and 20%, respectively. P. intermedia was resistant to metronidazole at a frequency of 30%, P. nigrescens at 20%, and P. melaninogenica at 40%. P. nigrescens and P. melaninogenica were resistant to 50% and 10% clindamycin, respectively. The gene most frequently detected was tetQ , at 43.3%, followed by tetM at 36.6%, bla TEM at 26.6%, ermF at 20%, cfxA , cfxA 2 , and nimAB at 16.6%, and nimAEFI at 3.3%. P. nigrescens was the species with the highest resistance to antibiotics such as amoxicillin/clavulanic acid, amoxicillin, and clindamycin, in addition to being the species with the largest number of genes compared to P. intermedia and P. melaninogenica.

This study investigated the effect of hypochlorous acid (HOCl) rinses and chlorhexidine (CHX) on the bacterial viability of S. mutans, A. israelii, P. gingivalis, A. actinomycetemcomitans, E. corrodens, C. rectus, K. oxytoca, K. pneumoniae and E. cloacae. The percentage of live bacteria was tested by fluorescence method using Live/Dead kit(r) and BacLight (Molecular Probes(r)) and compared between groups by the Kruskal-Wallis and U Mann-Whitney tests with Bonferroni correction (p value<0.012). The effect of HOCl and CHX on total proteins of P. gingivalis and S. mutans was determined by SDS-PAGE. CHX showed a higher efficacy than HOCl against S. mutans, A. israelii, E. corrodens and E. cloacae (p<0.001) while HOCl was more effective than CHX against P. gingivalis, A. actinomycetemcomitans, C. rectus and K. oxytoca (p=0.001). CHX and HOCl had similar efficacy against K. pneumoniae. Proteins of P. gingivalis and S. mutans were affected similarly by HOCl and CHX. HOCl reduced the bacterial viability especially in periodontopathic bacteria, which may support its use in the control of subgingival biofilm in periodontal patients.