Influence of keratinized mucosa width on the resolution of peri-implant mucositis: A prospective cohort study.

BACKGROUND: The prevalence of peri-implant diseases, driven by biofilm accumulation and influenced by factors such as the width of keratinized mucosa (KM), underscores the need for understanding their etiology and management. PURPOSE: To evaluate the association between the KM width and the clinical resolution of peri-implant mucositis after mechanical therapy. MATERIALS AND METHODS: Patients with an implant diagnosed with peri-implant mucositis were allocated to two groups: wide band of KM (WKM ≥ 2 mm) and narrow/no band of KM (NKM < 2 mm). Data and submucosa biofilm were collected at baseline and at 8, 12, and 24 weeks after nonsurgical therapy. A Brunner-Langer model was estimated for longitudinal data to evaluate and compare changes in any clinical parameter throughout follow-up between both groups. Furthermore, the microbial profiles were evaluated by 16S rRNA gene sequencing. RESULTS: A total of 38 implants were analyzed. At 24 weeks, bleeding on probing was substantially reduced in both groups, reaching statistical significance (p < 0.001). Treatment resulted in 23.9% less effective in achieving success for NKM. As such, NKM reduced the odds of disease resolution by 80% compared to WKM. The rest of the explored clinical parameters yielded more favorable outcomes for WKM versus NKM. Neither the alpha nor the beta diversity of the microbial profiles were significantly modulated by KM. CONCLUSIONS: KM width influences the clinical resolution of peri-implant mucositis after mechanical therapy (https://clinicaltrials.gov/study/NCT04874467?cond=keratinized%20mucosa&rank=8, NCT04874467, 04/30/2021).

Oral microbiota of adolescents with dental caries: A systematic review.

OBJECTIVE: This systematic review summarizes the current knowledge on the association between the oral microbiota and dental caries in adolescents. DESIGN: An electronic search was carried out across five databases. Studies were included if they conducted research on generally healthy adolescents, applied molecular-based microbiological analyses and assessed caries status. Data extraction was performed by two reviewers and the Newcastle-Ottawa Scale was applied for quality assessment. RESULTS: In total, 3935 records were reviewed which resulted in a selection of 20 cross-sectional studies (published 2005-2022) with a sample size ranging from 11 to 614 participants including adolescents between 11 and 19 years. The studies analyzed saliva, dental biofilm or tongue swabs with Checkerboard DNA-DNA hybridization, (q)PCR or Next-Generation Sequencing methods. Prevotella denticola, Scardoviae Wiggsiae, Streptococcus sobrinus and Streptococcus mutans were the most frequently reported species presenting higher abundance in adolescents with caries. The majority of the studies reported that the microbial diversity was similar between participants with and without dental caries. CONCLUSION: This systematic review is the first that shows how the oral microbiota composition in adolescents appears to differ between those with and without dental caries, suggesting certain taxa may be associated with increased caries risk. However, there is a need to replicate and expand these findings in larger, longitudinal studies that also focus on caries severity and take adolescent-specific factors into account.

Whole Genome Sequencing and Phenotypic Analysis of Antibiotic Resistance in Filifactor alocis Isolates.

There is scarce knowledge regarding the antimicrobial resistance profile of F. alocis. Therefore, the objective of this research was to assess antimicrobial resistance in recently obtained F. alocis clinical isolates and to identify the presence of antimicrobial resistance genes. Isolates were obtained from patients with periodontal or peri-implant diseases and confirmed by sequencing their 16S rRNA gene. Confirmed isolates had their genome sequenced by whole genome sequencing and their phenotypical resistance to nine antibiotics (amoxicillin clavulanate, amoxicillin, azithromycin, clindamycin, ciprofloxacin, doxycycline, minocycline, metronidazole, and tetracycline) tested by E-test strips. Antimicrobial resistance genes were detected in six of the eight isolates analyzed, of which five carried tet(32) and one erm(B). Overall, susceptibility to the nine antibiotics tested was high except for azithromycin in the isolate that carried erm(B). Moreover, susceptibility to tetracycline, doxycycline, and minocycline was lower in those isolates that carried tet(32). The genetic surroundings of the detected genes suggested their inclusion in mobile genetic elements that might be transferrable to other bacteria. These findings suggest that, despite showing high susceptibility to several antibiotics, F. alocis might obtain new antimicrobial resistance traits due to its acceptance of mobile genetic elements with antibiotic resistance genes in their genome.

Association of nine pathobionts with periodontitis in four South American and European countries.

Aim: Our aim was to compare the prevalence and load of nine pathobionts in subgingival samples of healthy individuals and periodontitis patients from four different countries. Methods: Five hundred and seven subgingival biofilm samples were collected from healthy subjects and periodontitis patients in Belgium, Chile, Peru and Spain. The prevalence and load of Eubacterium brachy, Filifactor alocis, Fretibacterium fastidiosum, Porphyromonas endodontalis, Porphyromonas gingivalis, Selenomonas sputigena, Treponema denticola, Tannerella forsythia and Treponema socranskii were measured by quantitative PCR. Results: The association with periodontitis of all species, except for T. socranskii, was confirmed in all countries but Peru, where only P. endodontalis, P. gingivalis and T. denticola were found to be significantly associated. Moreover, most species showed higher loads at greater CAL and PPD, but not where there was BOP. Through Principal Component Analysis, samples showed clearly different distributions by diagnosis, despite observing a smaller separation in Peruvian samples. Conclusions: Unlike prevalence, relative load was found to be a reliable variable to discriminate the association of the species with periodontitis. Based on this, F. alocis, P. endodontalis, P. gingivalis, T. denticola and T. forsythia may be biomarkers of disease in Belgium, Chile and Spain, due to their significantly higher abundance in periodontitis patients.

The quorum quenching enzyme Aii20J modifies in vitro periodontal biofilm formation.

Introduction: Recent studies have revealed the presence of N-acyl-homoserine lactones (AHLs) quorum sensing (QS) signals in the oral environment. Yet, their role in oral biofilm development remains scarcely investigated. The use of quorum quenching (QQ) strategies targeting AHLs has been described as efficient for the control of pathogenic biofilms. Here, we evaluate the use of a highly active AHL-targeting QQ enzyme, Aii20J, to modulate oral biofilm formation in vitro. Methods: The effect of the QQ enzyme was studied in in vitro multispecies biofilms generated from oral samples taken from healthy donors and patients with periodontal disease. Subgingival samples were used as inocula, aiming to select members of the microbiota of the periodontal pocket niche in the in vitro biofilms. Biofilm formation abilities and microbial composition were studied upon treating the biofilms with the QQ enzyme Aii20J. Results and Discussion: The addition of the enzyme resulted in significant biofilm mass reductions in 30 - 60% of the subgingival-derived biofilms, although standard AHLs could not be found in the supernatants of the cultured biofilms. Changes in biofilm mass were not accompanied by significant alterations of bacterial relative abundance at the genus level. The investigation of 125 oral supragingival metagenomes and a synthetic subgingival metagenome revealed a surprisingly high abundance and broad distribution of homologous of the AHL synthase HdtS and several protein families of AHL receptors, as well as an enormous presence of QQ enzymes, pointing to the existence of an intricate signaling network in oral biofilms that has been so far unreported, and should be further investigated. Together, our findings support the use of Aii20J to modulate polymicrobial biofilm formation without changing the microbiome structure of the biofilm. Results in this study suggest that AHLs or AHL-like molecules affect oral biofilm formation, encouraging the application of QQ strategies for oral health improvement, and reinforcing the importance of personalized approaches to oral biofilm control.

Impact of smoking on peri-implant bleeding on probing.

BACKGROUND: Studies around natural dentition demonstrated that smoking can reduce the tendency of inflamed tissue to bleed upon probing after controlling for possible confounders. In addition, previous research suggested that smokers may present alterations of the peri-implant microbiome. AIM: This study aimed at investigating the impact of smoking on: (1) peri-implant bleeding on probing (BOP; primary objective); (2) the association between BOP/bone loss and BOP/visible gingival inflammation; (3) peri-implant microbiome. METHODS: Partially edentulous patients with implants restored with a single crowns were included in this study. Subjects were either smokers (≥1 cigarettes per day) or nonsmokers (never smokers). The primary outcome of this cross-sectional study was BOP and secondary outcomes included: Probing pocket depth (PPD), Modified gingival Index (mGI) and Progressive Marginal Bone Loss. In addition, microbial profiles of the subjects were assessed through sequencing of the 16S rRNA gene. Univariate and multilevel multivariate analyses by means of Generalized Estimating Equations were conducted to analyze the association between smoking and peri-implant BOP. RESULTS: Overall, 27 nonsmokers and 27 smokers were included and 96.3% and 77.78% of patients presented peri-implant BOP in the nonsmoker and smoker group, respectively (p = 0.046). Smoking was inversely associated with BOP in the multivariate multilevel analysis (OR = 0.356; 95% CI: 0.193-0.660; p = 0.001) whereas a positive correlation was demonstrated for mGI > 0 (OR = 3.289; 95% CI: 2.014-5.371; p < 0.001); PPD (OR = 1.692; 95% CI: 0.263-0.883; p = 0.039) and gender (OR = 2.323; 95% CI: 1.310-4.120 p = 0.004). A decrease of BOP sensitivity in detecting visible gingival inflammation (mGI > 0) was observed in smokers. Besides, taxonomic and changes in diversity regarding the peri-implant microbiota were detected comparing the two groups. Significantly higher richness of the microbiota was demonstrated in the smoker group when implants affected by peri-implantitis were compared to either healthy implants or implants presenting mucositis. CONCLUSIONS: Smoking is a potential modifier of BOP and peri-implant microbiota.

Resistance to ß-lactams and distribution of ß-lactam resistance genes in subgingival microbiota from Spanish patients with periodontitis.

OBJECTIVES: The aim of this study was to analyze the distribution of ß-lactamase genes and the multidrug resistance profiles in ß-lactam-resistant subgingival bacteria from patients with periodontitis. MATERIALS AND METHODS: Subgingival samples were obtained from 130 Spanish patients with generalized periodontitis stage III or IV. Samples were grown on agar plates with amoxicillin or cefotaxime and incubated in anaerobic and microaerophilic conditions. Isolates were identified to the species level by the sequencing of their 16S rRNA gene. A screening for the following ß-lactamase genes was performed by the polymerase chain reaction (PCR) technique: blaTEM, blaSHV, blaCTX-M, blaCfxA, blaCepA, blaCblA, and blaampC. Additionally, multidrug resistance to tetracycline, chloramphenicol, streptomycin, erythromycin, and kanamycin was assessed, growing the isolates on agar plates with breakpoint concentrations of each antimicrobial. RESULTS: ß-lactam-resistant isolates were found in 83% of the patients. Seven hundred and thirty-seven isolates from 35 different genera were obtained, with Prevotella and Streptococcus being the most identified genera. blaCfxA was the gene most detected, being observed in 24.8% of the isolates, followed by blaTEM (12.9%). Most of the isolates (81.3%) were multidrug-resistant. CONCLUSIONS: This study shows that ß-lactam resistance is widespread among Spanish patients with periodontitis. Furthermore, it suggests that the subgingival commensal microbiota might be a reservoir of multidrug resistance and ß-lactamase genes. CLINICAL RELEVANCE: Most of the samples yielded ß-lactam-resistant isolates, and 4 different groups of bla genes were detected among the isolates. Most of the isolates were also multidrug-resistant. The results show that, although ß-lactams may still be effective, their future might be hindered by the presence of ß-lactam-resistant bacteria and the presence of transferable bla genes.

Tetracycline and multidrug resistance in the oral microbiota: differences between healthy subjects and patients with periodontitis in Spain.

Introduction: Antibiotic resistance is widely found even among bacterial populations not having been exposed to selective pressure by antibiotics, such as tetracycline. In this study we analyzed the tetracycline-resistant subgingival microbiota of healthy subjects and of patients with periodontitis, comparing the prevalence of tet genes and their multidrug resistance profiles. Methods: Samples from 259 volunteers were analyzed, obtaining 813 tetracycline-resistant isolates. The prevalence of 12 antibiotic resistance genes was assessed, and multidrug profiles were built. Each isolate was identified by 16S rRNA sequencing. Differences in qualitative data and quantitative data were evaluated using the chi-square test and the Mann-Whitney-U test, respectively. Results: tet(M) was the most frequently detected tet gene (52.03%). We observed significant differences between the prevalence of tet(M), tet(W), tet(O), tet(32) and tet(L) in both populations studied. Multidrug resistance was largely observed, with resistance to kanamycin being the most detected (83.64%). There were significant differences between the populations in the prevalence of kanamycin, chloramphenicol, and cefotaxime resistance. Resistant isolates showed significantly different prevalence between the two studied groups. Conclusion: The high prevalence of multidrug resistance and tetracycline resistance genes found in the subgingival microbiota, highlights the importance of performing wider and more in-depth analysis of antibiotic resistance in the oral microbiota.

Detection and expression analysis of tet(B) in Streptococcus oralis.

Tetracycline resistance can be achieved through tet genes, which code for efflux pumps, ribosomal protection proteins and inactivation enzymes. Some of these genes have only been described in either Gram-positive or Gram-negative bacteria. This is the case of tet(B), which codes for an efflux pump and, so far, had only been found in Gram-negative bacteria. In this study, tet(B) was detected in two clinical Streptococcus oralis strains isolated from the gingival sulci of two subjects. In both cases, the gene was completely sequenced, yielding 100% shared identity and coverage with other previously published sequences of tet(B). Moreover, we studied the expression of tet(B) using RT-qPCR in the isolates grown with and without tetracycline, detecting constitutive expression in only one of the isolates, with no signs of expression in the other one. This is the first time that the presence and expression of the tet(B) gene has been confirmed in Gram-positive bacteria, which highlights the potential of the genus Streptococcus to become a reservoir and a disseminator of antibiotic resistance genes in an environment so prone to horizontal gene transfer as is the oral biofilm.

Azithromycin and erythromycin susceptibility and macrolide resistance genes in Prevotella from patients with periodontal disease.

OBJECTIVES: To study oral Prevotella spp. isolated from patients with chronic periodontitis, to determine their susceptibility to azithromycin and erythromycin and to screen the presence of macrolide resistance genes therein. MATERIAL AND METHODS: Isolates with a Prevotella-like morphology were obtained from subgingival samples of 52 patients with chronic periodontitis. Each isolate was identified to the species level by sequencing of the 16S rRNA gene. In 100 Prevotella spp. isolates, azithromycin and erythromycin susceptibility was determined using the E test method, and the screening of erm(A), erm(B), erm(C), erm(F), erm(G), erm(Q) and mef(A) genes was done by PCR. RESULTS: Prevotella intermedia and Prevotella nigrescens were the most identified species (33% each). Minimum inhibitory concentrations (MICs) ranges for both antibiotics were 0.016/0.032 to >256 µg/ml. MIC50 values for azithromycin and erythromycin were 1.5 and 1 µg/ml, respectively, and MIC90 values were >256 µg/ml for both antibiotics. Nineteen per cent of the isolates carried erm(B), and 51% carried erm(F). CONCLUSIONS: The MIC values found were high compared to previous studies. erm(F) was greatly prevalent, and we describe for the first time the erm(B) gene in Prevotella spp. The presence of either of the genes seems to be associated with a higher degree of resistance to azithromycin and erythromycin.

Periodontal pathogens and tetracycline resistance genes in subgingival biofilm of periodontally healthy and diseased Dominican adults.

OBJECTIVE: The objective of this study was to compare the periodontopathogen prevalence and tetracycline resistance genes in Dominican patients with different periodontal conditions. METHODS: Seventy-seven samples were collected from healthy, gingivitis, chronic (CP) and aggressive (AgP) periodontitis patients. Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Prevotella intermedia, Parvimonas micra, Eikenella corrodens and Dialister pneumosintes and 11 resistance genes were studied by PCR. P. gingivalis fimA genotype was determined. RESULTS: In healthy patients, P. micra and P. intermedia were the most and least frequently detected, respectively. T. forsythia and E. corrodens appeared in 100% of gingivitis patients. Red complex, D. pneumosintes and E. corrodens were significantly more prevalent in CP compared to healthy patients. F. nucleatum and T. denticola were detected more frequently in AgP. A. actinomycetemcomitans was the most rarely observed in all groups. The fimA II genotype was the most prevalent in periodontitis patients. Seven tetracycline-resistant genes were detected. tet(Q), tet(32) and tet(W) showed the greatest prevalence. tet(32) was significantly more prevalent in CP than in healthy patients. CONCLUSIONS: Red complex bacteria and D. pneumosintes were significantly the most prevalent species among periodontitis patients. T. forsythia was the most frequently detected in this population. To our knowledge, this is the first study describing the tet(32) gene in subgingival biofilm from healthy and periodontally diseased subjects. CLINICAL RELEVANCE: This study contributes to the knowledge on the subgingival microbiota and its resistance genes of a scarcely studied world region. Knowing the prevalence of resistance genes could impact on their clinical prescription and could raise awareness to the appropriate use of antibiotics.