Production and characterization of the lipopeptide with anti-adhesion for oral biofilm on the surface of titanium for dental implants.

Titanium implants are subject to bacterial adhesion and peri-implantitis induction, and biosurfactants bring a new alternative to the fight against infections. This work aimed to produce and characterize the biosurfactant from Bacillus subtilis ATCC 19,659, its anti-adhesion and antimicrobial activity, and cell viability. Anti-adhesion studies were carried out against Streptococcus sanguinis, Staphylococcus aureus, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Proteus mirabilis as the minimum inhibitory concentration and the minimum bactericidal concentration. Cell viability was measured against osteoblast and fibroblast cells. The biosurfactant was classified as lipopeptide, with critical micelle concentration at 40 µg mL- 1, and made the titanium surface less hydrophobic. The anti-adhesion effect was observed for Staphylococcus aureus and Streptococcus sanguinis with 54% growth inhibition and presented a minimum inhibitory concentration of 15.7 µg mL- 1 for Streptococcus sanguinis and Aggregatibacter actinomycetemcomitans. The lipopeptide had no cytotoxic effect and demonstrated high potential application against bacterial biofilms.

Phyllanthus emblica fruit extract alleviates halitosis and reduces the inflammatory response to oral bacteria.

OBJECTIVE: To assess the efficacy of Phyllanthus emblica extract in alleviating halitosis and reducing the inflammatory response to halitosis-related bacteria. METHODOLOGY: This investigation, using Phyllanthus emblica fruit extract (PE), involved four aspects. First, we evaluated the effect on growth and aggregation of halitosis-related bacteria, including Fusobacterium nucleatum, Porphyromonas gingivalis, and Solobacterium moorei, using a microdilution assay and scanning electron microscopy. Second, volatile sulfur compound (VSC) levels were measured on individuals with halitosis in randomized short-term (26 participants) and double-blind randomized long-term trials (18 participants in each group) after rinsing with PE for 3, 6, and 12 h, and 28 days. Third, we analyzed pro-inflammatory cytokine expression in TR146 cells using quantitative real-time PCR and enzyme-linked immunosorbent assays. Lastly, we assessed pro-inflammatory cytokine secretion and Toll-like receptor (TLR) 2 mRNA expression via the same experimental methods in a three-dimensional oral mucosal epithelial model (3D OMEM). RESULTS: PE extract dose-dependently inhibited the growth of F. nucleatum (50% inhibition concentration [IC50]=0.079%), P. gingivalis (IC50=0.65%), and S. moorei (IC50=0.07%) and effectively prevented bacterial aggregation. Furthermore, VSC contents decreased significantly at 3, 6, and 12 h after rinsing with 5% PE compared with those in the control. Long-term use of mouthwash containing 5% PE for 28 days led to a significant decrease in VSC contents. PE attenuated the F. nucleatum- or P. gingivalis-stimulated mRNA expression and protein release of interleukin (IL)-6 and IL-8 in TR146 cells. It also suppressed IL-8 and prostaglandin E2 secretion and TLR2 mRNA expression in F. nucleatum-induced OMEMs. CONCLUSION: Our findings support the use of PE in oral care products to alleviate halitosis and it may reduce inflammation.

Nanotopography and oral bacterial adhesion on titanium surfaces: in vitro and in vivo studies.

The present study aimed to evaluate the influence of titanium surface nanotopography on the initial bacterial adhesion process by in vivo and in vitro study models. Titanium disks were produced and characterized according to their surface topography: machined (Ti-M), microtopography (Ti-Micro), and nanotopography (Ti-Nano). For the in vivo study, 18 subjects wore oral acrylic splints containing 2 disks from each group for 24 h (n = 36). After this period, the disks were removed from the splints and evaluated by microbial culture method, scanning electron microscopy (SEM), and qPCR for quantification of Streptococcus oralis, Actinomyces naeslundii, Fusobacterium nucleatum, as well as total bacteria. For the in vitro study, adhesion tests were performed with the species S. oralis and A. naeslundii for 24 h. Data were compared by ANOVA, with Tukey's post-test. Regarding the in vivo study, both the total aerobic and total anaerobic bacteria counts were similar among groups (p > 0.05). In qPCR, there was no difference among groups of bacteria adhered to the disks (p > 0.05), except for A. naeslundii, which was found in lower proportions in the Ti-Nano group (p < 0.05). In the SEM analysis, the groups had a similar bacterial distribution, with a predominance of cocci and few bacilli. In the in vitro study, there was no difference in the adhesion profile for S. oralis and A. naeslundii after 24 h of biofilm formation (p > 0.05). Thus, we conclude that micro- and nanotopography do not affect bacterial adhesion, considering an initial period of biofilm formation.

Antineoplastic therapy in childhood cancer patients presents a negative impact in the periodontal tissues: a cohort study.

OBJECTIVES: To investigate the effect of antineoplastic therapy (AT) in the periodontal tissues of childhood cancer (CC) patients. MATERIALS AND METHODS: Seventy-two individuals were divided into CC (n=36) and healthy individuals (control group-CG, n=36). Demographics, hygiene habits, CC type, and AT were collected. Salivary flow and the presence and concentration of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, and Fusobacterium nucleatum were analyzed. Clinical evaluation included plaque (PI) and gingival indexes (GI), periodontal probing depth (PPD), and clinical attachment level (CAL). Patients were classified into periodontal health, gingivitis, or periodontitis. Descriptive statistics, T test, Mann-Whitney test, chi-square, Fisher's exact test, and two-way analysis of variance were used (p<0.05). RESULTS: The mean age of the patients was similar (CC 12.0±3.9 years and CG 12.0±4.0 years). In the CC group, all patients underwent chemotherapy and nine radiotherapy. Color/race, income, and family education showed significant differences between groups. There was no difference between groups in salivary flow. Higher levels of Fusobacterium nucleatum were seen in CC (p=0.02). Significant difference between groups was found for PI (CC: 30.5%, CG: 22.6%), GI (CC: 28.8%, CG: 17.3%), PPD (CC: 1.77 mm, CG: 1.61 mm), and CAL (CC: 1.77 mm, CG: 1.57 mm), periodontal health (CC: 3, CG: 7), gingivitis (CC: 16, CG: 24), or periodontitis (CC: 17, CG: 5). CONCLUSION: AT in CC patients presents a negative impact in the periodontal and microbiological parameters. CLINICAL RELEVANCE: Childhood cancer individuals showed worse periodontal parameters and higher levels of Fusobacterium nucleatum in the saliva when compared to healthy individuals.

Fusobacterium nucleatum and Prevotella in women with periodontitis and preterm birth.

BACKGROUND: Studies try to explain the hypothesis that maternal periodontitis may be associated with preterm birth. MATERIAL AND METHODS: This is a case-control study with 120, 40 cases (gestational age <37 weeks) and 80 controls (gestational age ≥37 weeks), that were submitted to the clinical periodontal examination and subgingival biofilm collection. Bacterial DNA of subgingival biofilm was performed and processed by qPCR. RESULTS: Periodontitis was statistically significant in the Case group (35%) when compared to the Control group (11.2%) and Gingival Bleeding Index (GBI), sites with PS ≥ 4mm and sites with CAL ≥ 5mm were statistically higher in the Case group (p < 0.05). The proportions of Pi (p = 0.026) and Fn (p = 0.041) of subgingival biofilm were higher in the Case group. A greater number of sites with PS ≥ 4mm (r = -0.202; p = 0.026) and CAL ≥ 5mm (r = -0.322; p < 0.001) were correlated to lower gestational age. CONCLUSIONS: Periodontitis, preterm delivery, and/or low birth weight may have a possible relationship based on clinical parameters and the ratio of Pi and Fn at periodontal sites.

Salivary pellicle modulates biofilm formation on titanium surfaces.

OBJECTIVES: The present study aimed to evaluate the potential of the salivary pellicle (SP) formed on titanium (Ti) surfaces to modulate the formation of a biofilm composed of Streptococcus gordonii, Actinomyces naeslundii, Fusobacterium nucleatum, and Porphyromonas gingivalis. MATERIALS AND METHODS: Ti substrates were incubated for 2 h with a pool of saliva samples obtained from 10 systemically and periodontally healthy subjects. Enamel substrates were included as a biological reference. Scanning electron microscopy (SEM) and Raman spectroscopy analysis were used to analyze the formation of the salivary pellicle. After the SP formation, the surfaces were incubated for 12 h with a mix of Streptococcus gordonii, Actinomyces naeslundii, Fusobacterium nucleatum, and Porphyromonas gingivalis. The number of bacterial cells attached to each surface was determined by the XTT assay while bacterial viability was analyzed by fluorescence microscopy using the LIVE/DEAD® BacLightTM kit. RESULTS: The SEM and Raman spectroscopy analysis confirmed the presence of a salivary pellicle formed on the tested surfaces. Regarding the biofilm formation, the presence of the SP decreases the number of the bacterial cells detected in the test surfaces, compared with the uncover substrates. Even more, the SP-covered substrates showed similar bacterial counts in both Ti and enamel surfaces, meaning that the physicochemical differences of the substrates were less determinant than the presence of the SP. While on the SP-uncover substrates, differences in the bacterial adhesion patterns were directly related to the physicochemical nature of the substrates. CONCLUSIONS: The salivary pellicle was the main modulator in the development of the biofilm consisting of representative oral bacteria on the Ti substrates. CLINICAL RELEVANCE: The results of this study provide valuable information on the modulatory effect of the salivary pellicle on biofilm formation; such information allows us to understand better the events involved in the formation of oral biofilms on Ti dental implants.

Comparative proteomics of saliva of healthy and gingivitis individuals from Rio de Janeiro.

PURPOSE: In this work, we identified human and bacterial proteomes in the saliva from volunteers with gingivitis or healthy. EXPERIMENTAL DESIGN: The reported population consisted of 18 volunteers (six with gingivitis and 12 healthy controls). Proteomics characterization was performed using a quantitative mass spectrometry method. RESULTS: A total of 74 human and 116 bacterial proteins were identified in saliva. The major functional category that was modified in the human proteome was the immune response, followed by transport and protease inhibition. In the bacterial proteome, most of the proteins identified were from the Fusobacteria phylum, followed by Chlamydiae and Spirochaetes. CONCLUSIONS AND CLINICAL RELEVANCE: We observed statistically relevant differences in the data between the groups. The 15 most important human proteins affecting the variation between case and control groups included cystatin S, alpha amylase, lactotransferrin, and negative elongation factor E. We found that bacterial proteins from Porphyromonas gingivalis and Fusobacterium nucleatum subsp. nucleatum related to the red and orange complexes were closely correlated with the occurrence of periodontal diseases.

Interação entre Lactobacillus reuteri e bactérias periodontopatogênicas: estudo in vitro e em modelo de invertebrado / Interaction of Lactobacillus reuteri and periodontopathogenic bacteria: an in vitro study and on invertebrate model

A doença periodontal, afecção crônica inflamatória multifatorial, está entre as principais doenças bucais que afetam a população mundial. Entre as bactérias associadas à periodontite, estão Fusobacterium nucleatum e Aggregactibacter actinomycetemcomitans. Novas terapias adjuntas ao tratamento convencional têm sido propostas para a doença periodontal, entre elas o uso de probióticos. Porém, seu uso não está isento de riscos, e uma alternativa para minimizá-los é inativar os microorganismos, mantendo suas propriedades benéficas, o que ocorre com os chamados paraprobióticos. Desse modo, são objetivos deste estudo avaliar os efeitos antimicrobianos de L. reuteri vivo, inativado pelo calor e seus produtos sobre F. nucleatum, A. actinomycetemcomitans e sobre as bactérias comensais, Streptococcus mitis e Streptococcus salivarius, além de estudar os efeitos da interação das preparações e periodontopatógenos em modelo de invertebrado. A atividade antimicrobiana in vitro foi avaliada associando-se as bactérias patogênicas ou comensais a L. reuteri vivo, inativado ou sobrenadante. Após interação, as bactérias foram cultivadas em meio seletivo para contagem de unidades formadoras de colônias (UFC). No estudo em Galleria mellonella, após a infecção com as bactérias patogênicas e as preparações de L. reuteri, foi avaliada a curva de sobrevivência e densidade hemocitária. Os dados foram analisados com o teste estatístico apropriado, ao nível de 5%. Após interação bacteriana in vitro, S. salivarius reduziu o número de UFC de L. reuteri, enquanto que não houve interferência das outras bactérias. O probiótico foi mais eficiente em reduzir o crescimento das bactérias periodontopatogênicas e comensais, exceto para F. nucleatum onde a preparação do sobrenadante foi melhor. In vivo, o sobrenadante foi mais eficiente em aumentar a sobrevivência das lagartas quando infectadas por F. nucleatum. Entretanto, quando infectadas por A. actinomycetemcomitans, nenhuma das preparações aumentou a sobrevivência. Da mesma maneira, nenhuma das preparações aumentaram o número de hemócitos das lagartas após infecção por F. nucleatum e A. actinomycetemcomitans, apesar de todas resultarem em redução na contagem de colônias das bactérias periodontopatogênicas. Dessa forma, concluiu-se que tanto o probiótico quanto os produtos dele derivados apresentaram efeitos antimicrobianos e aumentaram a sobrevivência de G. mellonella quando infectada por F. nucleatum (AU)

Periodontal disease, a chronic multifactorial inflammatory disease, is among the major oral diseases that affects the worldwide population. Among the bacteria associated with periodontitis are Fusobacterium nucleatum and Aggregactibacter actinomycetemcomitans. New therapies have been proposed for periodontal disease as adjunct to conventional treatment, including the use of probiotics. However, their use isn't risk-free and an alternative to that is the inactivation of the microorganisms, maintaining their beneficial properties, which occurs with the paraprobiotics. Thus, the objective of this study is to evaluate the antimicrobial effects of living and heat-killed L. reuteri, and its products on F. nucleatum, A. actinomycetemcomitans and on the commensal bacteria, Streptococcus mitis and Streptococcus salivarius, as well as to study the interaction of the preparations and periodontopathogens in an invertebrate model. In vitro antimicrobial activity was evaluated by associating the pathogenic or commensal bacteria to live, heat killed or L. reuteri supernatant. After interaction, the bacteria were cultured in a selective medium for colony-forming unit (CFU) count. In the study with Galleria mellonella, after infection with pathogenic bacteria and L. reuteri preparations, the survival curve and hemocyte density were evaluated. The data were analyzed with the appropriate statistical test at the 5% level. After bacterial interaction in vitro, S. salivarius reduced the number of CFU of L. reuteri, while there was no interference of the other bacteria. The probiotic was more efficient in reducing the growth of periodontopathogenic and commensal bacteria, except for F. nucleatum. In this case, the supernatant presented better results. In vivo, the supernatant was more efficient in increasing the larvae survival when infected by F. nucleatum. However, when infected by A. actinomycetemcomitans, none of the preparations increased the survival. Likewise, none of the preparations increased the number of hemocytes of the larvae after infection by F. nucleatum and A. actinomycetemcomitans, although all resulted in reduction in the CFU of the periodontopathogenic bacteria. Thus, it was concluded that the probiotic and the products derived therefrom presented antimicrobial effects and increased the survival of G. mellonella when infected by F. nucleatum.(AU)

Nicotine is a potent extracellular polysaccharide inducer in Fusobacterium nucleatum biofilms

The purpose of this in vitro study was to analyze the influence of nicotine on the extracellular polysaccharides in Fusobacterium nucleatum biofilm. Methods: F. nucleatum (ATCC 10953) biofilms supplemented with different concentrations of nicotine (0, 0.5, 1, 2, 4, and 8 mg/mL) were grown in two different BHI broth conditions [no sucrose and 1% sucrose]. Extracellular polysaccharides assay, pH measurements, and a spectrophotometric assay were performed. Data were submitted for ANOVA and Tukey honestly significant difference analyses (HSD) tests (α =.05). Results: Extracellular polysaccharides synthesis was influenced by an interaction between nicotine concentrations and growth medium solution containing sucrose (P<.05). The pH values declined in the sucrose-exposed biofilm were greater than in the group exposed only to nicotine (P<.05). The biofilm exposed to sucrose and nicotine had a higher total biofilm growth (P<.05) than the nicotine-treated biofilm without sucrose. Conclusions: Regardless of sucrose exposure, biofilms exposed to different nicotine concentrations influenced the amount of extracellular polysaccharides

Antibiotic Resistance in Patients with Peri-Implantitis: A Systematic Scoping Review.

The implementation of adjunctive antibiotics has been recommended for the therapy of peri-implantitis (PI). In this review, antibiotic resistance patterns in PI patients were assessed. A systematic scoping review of observational studies and trials was established in conjunction with the PRISMA extension for scoping reviews. The SCOPUS, PubMed/MEDLINE, EMBASE, SCIELO, Web of Science, and LILACS databases were reviewed along with the gray literature. The primary electronic examination produced 139 investigations. Finally, four observational studies met the selection criteria. These studies evaluated 214 implants in 168 patients. Porphyromonas gingivalis and Fusobacterium nucleatum mainly presented high resistance to tetracycline, metronidazole, and erythromycin in PI patients. Similarly, Aggregatibacter actinomycetemcomitans was also highly resistant to clindamycin and doxycycline. Other microorganisms such as Tannerella forsythia, Parvimonas micra, and Prevotella intermedia/nigrescens also presented significant levels of resistance to other antibiotics including amoxicillin, azithromycin, and moxifloxacin. However, most microorganisms did not show resistance to the combination amoxicillin metronidazole. Although the management of adjunctive antimicrobials in the therapy of PI is controversial, in this review, the resistance of relevant microorganisms to antibiotics used to treat PI, and usually prescribed in dentistry, was observed. Clinicians should consider the antibiotic resistance demonstrated in the treatment of PI patients and its public health consequences.

Higher frequency of specific periodontopathogens in hypertensive patients. A pilot study.

Periodontitis and arterial hypertension are two of the pathologies with the highest global prevalence; evidence reported so far has been favorable to an association between them. This cross-sectional study aimed to evaluate and compare the microbiological counts of hypertensive and normotensive patients with periodontitis. Sociodemographic, behavioral, systemic health data and periodontal clinical parameters were assessed. Counts of A. actinomycetemcomitans, P. intermedia, P. gingivalis and F. nucleatum were performed by real-time polymerase chain reaction using subgingival biofilm samples. Thirty-eight patients were included in this preliminary analysis, divided into two groups: Normotensive Group (NG) (n = 14) and Hypertensive Group (HG) (n = 24). Patients diagnosed with periodontitis composed both groups. Data analysis was performed with significance level of 5%. There was no significant difference between groups for clinical periodontitis diagnosis. In addition, hypertensive individuals had higher P. intermedia, P. gingivalis, and F. nucleatum counts when compared to normotensive individuals. The parameters probing pocket depth, bleeding on probing, and A. actinomycetemcomitans count did not presented statistical differences between groups. With these preliminary results, it can be concluded that the presence of arterial hypertension may be associated with a greater quantity of periodontopathogenic bacterial of some species in individuals with periodontitis.

Association of Fusobacterium nucleatum infection and colorectal cancer: A Mexican study.

INTRODUCTION AND AIMS: Colorectal cancer (CRC) is the third most prevalent cancer worldwide. Many risk factors are involved, and current evidence links the gut microbiota and colorectal carcinogenesis. Fusobacterium nucleatum (F. nucleatum) is proposed as one of the risk factors at the onset and during the progression of CRC, due to immune system and inflammatory modulation. MATERIALS AND METHODS: Ninety samples from three different regions of the colon were collected through colonoscopy in patients with CRC, and qPCR TagMan® was conducted to detect F. nucleatum and cytokines (IL-17, IL-23, and IL-10) in tumor, peritumor, and normal samples. The differences between them were analyzed and correlated. RESULTS: The abundance of F. nucleatum determined through the 2-ΔΔCt method in CRC (7.750 [5.790-10.469]) was significantly higher than in the normal control (0.409 [0.251-0.817]) (p < 0.05). There was no significant association between F. nucleatum and the cytokines (p > 0.05). CONCLUSIONS: CRC is a heterogeneous disease that presents and progresses in a complex microenvironment, partially due to gut microbiome imbalance. F. nucleatum was enriched in CRC tissue, but whether that is a cause of the pathology or a consequence, has not yet been clearly defined.

Efeito antimicrobiano e anti-inflamatório de ácidos fenólicos isolados, combinados e incorporados em hidrogéis de quitosana para aplicação endodôntica / Antimicrobial and anti-inflammatory effect of phenolic acids, alone, combined and incorporated in chitosan hydrogels for endodontic application

O objetivo do tratamento endodôntico é manter a integridade da raiz, bem como, prevenir ou resolver doenças periapicais, pela erradicação dos microrganismos e de suas fontes de nutrientes provenientes do sistema de canais radiculares. A complexidade da anatomia dos canais radiculares e dos biofilmes multiespécies aumenta a dificuldade em eliminar os microrganismos e controlar a inflamação por procedimentos químico-mecânicos convencionais, o que justifica o uso de medicações intracanais. Novos compostos com amplo efeito antimicrobiano e potencial antiinflamatório, como os ácidos fenólicos, poderiam ser explorados como princípios ativos de medicamentos intracanais. Entretanto, para aumentar a solubilidade, controlar a liberação e estender os efeitos biológicos dos ácidos fenólicos, seria interessante incorporá-los em carreadores de medicamentos como os hidrogéis de quitosana. Este estudo foi dividido em dois capítulos que apresentaram como objetivos: 1) avaliar as atividades antimicrobiana, antibiofilme e antiinflamatória e a citotoxicidade do ácido cinâmico e seus derivados; 2) sintetizar e caracterizar as propriedades químicas e físico-mecânicas de hidrogéis termossensíveis de quitosana e poloxamer contendo ácidos fenólicos, e avaliar o efeito desses hidrogéis sobre biofilmes multiespécies e na viabilidade de macrófagos e fibroblastos. No capítulo 1, a atividade antimicrobiana do ácido cinâmico (CI) e seus derivados ácido cumárico (CO), ácido cafeico (CA), ácido ferúlico (FE) e ácido sinápico (SI) foi avaliada pela determinação da concentração inibitória e bactericida mínima (CIM/CBM) e Concentração Inibitória Fracionada (CIF) sobre Enterococcus faecalis, Streptococcus mutans, Lactobacillus casei, Actinomyces israelii e Fusobacterium nucleatum. Os ácidos fenólicos foram selecionados e seu efeito em biofilmes dual-espécies e multiespécies com as mesmas cepas padrão ou cepas clínicas foram avaliados por contagem bacteriana, microscopia eletrônica de varredura e microscopia confocal. A viabilidade de fibroblastos L929 e macrófagos RAW 264.7 na presença desses ácidos fenólicos foi avaliada por ensaios de resazurina. Além disso, os níveis de mRNA dos marcadores pró-inflamatórios TNF-α, IL-1ß, iNOS e COX-2 foram determinados por PCR quantitativo TaqMan após exposição de macrófagos aos ácidos fenólicos e ao lipopolissacarídeo (LPS). No capítulo 2, foi realizada a síntese e caracterização físicomecânica de hidrogéis de quitosana-poloxamer (CPH) contendo ácidos fenólicos e avaliado seus efeitos no biofilme multiespécies e na viabilidade de macrófagos e fibroblastos. Os dados foram analisados estatisticamente considerando p< 0,05. O ácido cinâmico e o ácido cafeico apresentaram efeito inibitório e bactericida contra todas as espécies bacterianas testadas, com os menores valores de CIM e CBM. Entretanto, não houve efeito sinérgico entre eles (FICI> 0,5). Ambos os compostos (5x a CIM mais alta) foram eficazes na eliminação de biofilmes dual-espécies e na redução significativa de biofilmes multiespécies, especialmente o ácido cinâmico. O ácido cinâmico causou toxicidade mínima para ambas as culturas celulares nas concentrações de CIM e o ácido cafeico não foi citotóxico em concentrações abaixo de 0,125 mg/mL. Ambos os compostos reduziram significativamente TNF-α, IL-1ß, iNOS e COX-2, de maneira dose-dependente. Os CPH foram caracterizados como termorreversíveis e com propriedades mecânicas e bioadesivas desejáveis. O efeito dos hidrogéis CPH+CA (77,8%) e CPH+CI (73,2%) em reduzir os biofilmes multiespécies foi superior ao CPH+ hidroxido de cálcio (CH) (53,6%) e CPH+ clorexidina (CHX) (39,9%). Em geral, CPH + CI causou menor citotoxicidade quando comparado a CPH + CA, para ambas as linhagens celulares. Conclui-se que o ácido cinâmico e ácido cafeico apresentaram efeito bactericida e contra biofilmes formados por bactérias associadas com infecções endodônticas, causando baixa citotoxicidade. Ambos os compostos apresentaram efeito antiinflamatório, inibindo a expressão de marcadores próinflamatórios em macrófagos estimulados por LPS. Os hidrogéis de quitosana-poloxamer foram termorreversíveis e apresentaram adequadas propriedades mecânicas e adesivas para aplicação clínica, e quando combinados principalmente com ácido cinâmico, promoveram a redução de biofilmes multiespécies formados nos túbulos dentinários, causando baixa toxicidade em fibroblastos e macrófagos(AU)

The objective of endodontic treatment is to maintain the integrity of the root, as well as to prevent or resolve periapical diseases, by eradicating microorganisms and their sources of nutrients from the root canal system. The complexity of root canal anatomy and multispecies biofilms increases the difficulty in eliminating microorganisms and controlling inflammation by conventional chemical-mechanical procedures, which justifies the use of intracanal medications. New compounds with broad antimicrobial effect and anti-inflammatory potential, such as phenolic acids, could be explored as active principles of intracanal medications. However, to increase the solubility, control the release and extend the biological effects of phenolic acids, it would be interesting to incorporate them into drug carriers such as chitosan hydrogels. This study was divided into two chapters with the following objectives: 1) to evaluate the antimicrobial, antibiofilm and anti-inflammatory activities and the cytotoxicity of cinnamic acid and its derivatives; 2) synthesize and characterize chemical and physicomecanical properties of thermosensitive chitosan and poloxamer hydrogels containing phenolic acids and evaluate the effect of these hydrogels on multispecies biofilms and on the viability of macrophages and fibroblasts. In chapter 1, the antimicrobial activity of cinnamic acid (CI) and its derivatives coumaric acid (CO), caffeic acid (CA), ferulic acid (FE) and sinapic acid (SI) was evaluated by determining the minimum inhibitory and bactericidal concentration (MIC/MBC) and Fractional Inhibitory Concentration (FIC) on Enterococcus faecalis, Streptococcus mutans, Lactobacillus casei, Actinomyces israelii and Fusobacterium nucleatum. Phenolic acids were selected and their effect on bispecies and multispecies biofilms with the same standard or clinical strains were evaluated by bacterial counts, scanning electron microscopy and confocal microscopy. The viability of L929 fibroblasts and RAW 264.7 macrophages in the presence of these phenolic acids was evaluated by resazurin assays. In addition, mRNA levels of the proinflammatory markers TNF-α, IL-1ß, iNOS and COX-2 were determined by quantitative TaqMan PCR after macrophage exposure to phenolic acids and lipopolysaccharide (LPS). In chapter 2, the synthesis and physical-mechanical characterization of chitosanpoloxamer (CPH) hydrogels containing phenolic acids were performed and their effects on multispecies biofilm and on the viability of macrophages and fibroblasts were evaluated. Data were statistically analyzed considering p< 0.05. Cinnamic acid and caffeic acid showed an inhibitory and bactericidal effect against all bacterial species tested, with the lowest MIC and MBC values. However, no synergistic effect was observed between the compounds (FICI> 0.5). Both compounds (at 5x the highest MIC) were effective in eliminating dual-species biofilms and significantly decreasing multispecies biofilms, especially cinnamic acid. Cinnamic acid caused minimal toxicity to both cell cultures at MIC concentrations and caffeic acid was not cytotoxic at concentrations below 0.125 mg/mL. Both compounds significantly reduced TNF-α, IL1ß, iNOS and COX-2, in a dose-dependent manner. CPH were characterized as thermoreversible and with adequate mechanical and bioadhesive properties. The effect of CPH+CA (77.8%) and CPH+CI (73.2%) hydrogels against multispecies biofilms was superior to CPH + calcium hydroxide (CH) (53.6%) and CPH + chlorhexidine (CHX) (39.9%). In general, CPH + CI caused less cytotoxicity when compared to CPH + CA, for both cell lines. In conclusion, cinnamic acid and caffeic acid showed bactericidal effect and against biofilms of bacteria associated with endodontic infections, causing minimal cytotoxicity. In addition, both compounds showed an anti-inflammatory effect, inhibiting the expression of pro-inflammatory markers in LPS-stimulated macrophages. The chitosan-poloxamer hydrogels were thermoreversible and presented adequate mechanical and bioadhesive properties for clinical application, and when combined specially with cinnamic acid, they promoted the reduction of multispecies biofilms formed in the dentinal tubules, causing low toxicity to fibroblasts and macrophages(AU)

Nivel de higiene y prevalencia de Porphyromona gingivalis y Fusobacterium nucleatum en pacientes recuperados de SARS-CoV-2 / Hygiene level and prevalence of Porphyromona gingivalis and Fusobacterium nucleatum in recovered SARS-CoV-2 patients

Introducción: El SARS-CoV-2 afecta el sistema respiratorio en diferentes grados. La cavidad oral es el lugar más colonizado por bacterias, por lo tanto, al no tener una adecuada higiene pueden presentarse diferentes enfermedades secundarias, lo que ha causado alerta en el gremio odontológico, ya que puede contribuir a complicaciones posteriores en los pacientes. Material y métodos: El estudio fue conformado por 47 pacientes voluntarios recuperados de SARS-CoV-2, residentes de Montemorelos, Nuevo León, México, donde fueron atendidos en Bucalia Dent, consultorio dental. Después del consentimiento informado de cada paciente, se realizó una historia clínica para conocer los síntomas, enfermedades sistémicas, ausencia de dientes y nivel de inflamación gingival de acuerdo al índice de Loe y Silness. A continuación, se tomó una muestra de biofilm microbiano (placa dentobacteriana), la cual se suspendió en una solución buffer de fosfato, posteriormente fue llevada al Centro de Investigación y Desarrollo en Ciencias de la Salud (CIDICS), Monterrey, N.L, México. Se extrajo DNA y se purificó, después se realizó PCR para detectar los patógenos orales; la PCR se visualizó en gel de agarosa (1.5%) por tinción de bromuro de etidio. Resultados: Se detectó 80.85% Porphyromona gingivalis y 68.09% Fusobacterium nucleatum en pacientes recuperados de SARS-CoV-2; 23.4% presentaron inflamación leve de acuerdo al índice de Loe y Silness, 54.5% fueron masculinos y 45.5% femeninos. Por otro lado, 36.4% de los pacientes con inflamación leve tenían de cuatro a seis dientes ausentes. En estos pacientes se detectó 18.18% únicamente con Fusobacterium nucleatum y 27.27% sólo con Porphyromona gingivalis; el sexo masculino tuvo predisposición en 66.6% y el femenino en 33.33%. Se observó infección con los dos patógenos presentes en 45.45%; y 60% de estos pacientes fueron masculinos. Conclusiones: Los pacientes recuperados de SARSCoV- 2 analizados en esta investigación mostraron mala higiene oral y alta prevalencia de los patógenos mencionados altamente relacionados a inflamación gingival o enfermedad periodontal, lo que nos indica que es indispensable la intervención del odontólogo al finalizar el periodo de infección de cada paciente (AU)

Introduction: SARS-CoV-2 affects the respiratory system to different degrees. The oral cavity is a colonized place by bacterias, therefore, by not having good hygiene, different secondary diseases can occur; this has caused an alert in the dental industry, since it can contribute to later complications in patients. Material and methods: The study was conducted in 47 SARS-CoV-2 recovered volunteers from the Montemorelos city of the Nuevo León state, Mexico, who were attended at the Bucalia Dent dental clinic. An informed consent was obtained from each of the patients, then their clinical history was documented in order to know the symptoms, previous systemic diseases, absence of teeth and degree of gingival inflammation, as suggested by Loe and Silness. Subsequently, a dental plaque sample was taken from all patients, which was suspended in a phosphate buffered solution and shipped to The Center for Research and Development in Health Sciences (CIDICS), Monterrey, NL, Mexico for storage. DNA extraction and purification was performed and PCR was carried out for the oral pathogens detection. All PCR products were visualized on 1.5% agarose gel by ethidium bromide staining. Results: Porphyromona gingivalis and Fusobacterium nucleatum were detected in 80.85% and 68.09% of SARS-CoV-2 recovered patients, respectively. 23.4% showed mild inflammation based on the Loe and Silness criteria, 54.5% were male and 45.5% female. On the other hand, 36.4% of patients with mild inflammation had between 4 to 6 missing teeth. A single infection by Fusobacterium nucleatum was detected in 18.18% and by Porphyromona gingivalis in 27.27%; the male sex had a predisposition with 66.66% and 33.33% female; coinfection of both pathogens was observed in 45.45% where 60% were male. Conclusions: SARS-CoV-2 recovered patients show poor oral hygiene and a high prevalence of oral pathogens related to the development of inflammatory gingival or periodontal disease, this suggests the need for an odontological clinical intervention at the end of the course of infection or disease caused by SARS-CoV-2 (AU)

Cytocompatibility and Synergy of EGCG and Cationic Peptides Against Bacteria Related to Endodontic Infections, in Planktonic and Biofilm Conditions.

This study evaluated the cytocompatibility and antimicrobial/antibiofilm effects of epigallocatechin-3-gallate (EGCG) associated with peptide LL-37 and its analogue KR-12-a5 against oral pathogens. The effect of the compounds on metabolism of fibroblasts was evaluated by methyltetrazolium assays. Antimicrobial activity of the compounds was evaluated on Streptococcus mutans, Enterococcus faecalis, Actinomyces israelii, and Fusobacterium nucleatum under planktonic conditions, on single- and dual-species biofilms and E. faecalis biofilms in dentinal tubules and analyzed by bacterial counts and confocal microscopy. Data were statistically analyzed considering p < 0.05. EGCG and peptide combinations were not toxic to fibroblasts. KR-12-a5 showed synergistic or addictive effects with EGCG and LL-37 against all bacteria tested. However, EGCG associated with KR-12-a5 demonstrated the highest bactericidal activity on all bacteria tested, at lower concentrations. In single-species biofilms, EGCG + KR-12-a5 eliminated S. mutans and A. israelii and reduced E. faecalis and F. nucleatum counts around 5 log CFU/mL. EGCG + KR-12-a5 reduced E. faecalis (-3.93 log CFU/mL) and eliminated S. mutans in dual-species biofilms. No growth of E. faecalis and significant reduction in A. israelii (-6.24 log CFU/mL) and F. nucleatum (-4.62 log CFU/mL) counts were detected in dual-species biofilms. The combination of EGCG and KR-12-a5 led to 88% of E. faecalis dead cells inside dentin tubules. The association of EGCG and KR-12-a5 was cytocompatible and promoted synergistic effect against biofilms of bacteria associated with endodontic infections.

Pulmonary and thoracic infection by Fusobacterium nucleatum. / Infección pulmonar y torácica por Fusobacterium nucleatum.

INTRODUCTION: Fusobacterium nucleatum is an anaerobic bacillus that is part of the oral microbiota and dental pla que. This can cause local and potentially remote infections, which are exceptional in pediatrics. Ob jective: To present the case of a patient with lung injury with chest wall invasion by Fusobacterium nucleatum. CLINICAL CASE: An 11-year-old female immunocompetent patient who consulted due to a two-week history of cough, night sweats, without fever or weight loss, and increased volume at the left spleen thoracic level. There was no history of chest wall trauma or travel outside the country. Two weeks before the onset of symptoms, she was treated for dental caries. Imaging studies and CT scan showed left spleen pneumonia, which invades the pleura and the chest wall. A minimal thoracotomy was performed, releasing a thick, foul-smelling liquid. The studies for common germs and tubercu losis were negative. Hematology ruled out tumor lesions. The anaerobic study reported the develo pment of Fusobacterium nucleatum. The patient was treated with penicillin followed by amoxicillin presenting good clinical and radiological responses. The dental procedure was suspected as the cause of infection. CONCLUSIONS: Fusobacterium nucleatum can occasionally cause remote or extra-oral in fections in immunocompetent patients, such as pneumonia with chest wall invasion, therefore it is necessary to bear it in mind.

Identification of Fusobacterium nucleatum in primary and secondary endodontic infections and its association with clinical features by using two different methods.

OBJECTIVE: Fusobacterium nucleatum is an important oral pathogen involved in endodontic infections. This study aimed to assess the frequency of Fusobacterium nucleatum in primary and secondary endodontic infections and its associations with the clinical features in a Brazilian population by using both culture and nested PCR methods. METHODS: A total of 100 microbial samples from patients with primary (n=50) and secondary endodontic infections (n=50) were analyzed by using culture and nested PCR methods. Strict anaerobic techniques were used for culture and identification of F. nucleatum. The DNA extracted from the samples was analyzed for the presence of target species by using species-specific primers. RESULTS: Culture and nested PCR methods detected F. nucleatum, respectively, in 11/100 and 82/100 root canals. F. nucleatum was isolated by culture from 10/50 (20%) root canals with primary infections and from 1/50 (2%) root canal with secondary/persistent infections. Nested PCR detected F. nucleatum in 42/50 (84%) root canals with primary infections and in 40/50 (80%) root canals with secondary/persistent endodontic infections. F. nucleatum was associated with spontaneous pain, tenderness to percussion, pain on palpation, swelling, tooth mobility, wet root canals, hemorrhagic exudate, tooth decay, inadequate restoration, and poor endodontic filling. CONCLUSION: F. nucleatum was found in more cases of primary endodontic infections than in cases of secondary/persistent ones. A higher prevalence of F. nucleatum was detected by using the nested PCR method than by using culture. The presence of F. nucleatum in the root canals was associated with several clinical features. CLINICAL RELEVANCE: The high prevalence of F. nucleatum in the root canals detected by molecular methods, and its association with several clinical features reveals the importance of these species in the development of apical pathologies and reinforces the need of an endodontic treatment directed to bacterial elimination.

The Role of Fusobacterium nucleatum in Colorectal Carcinogenesis.

Colorectal cancer (CRC) is one of the most frequent and deadly neoplasms worldwide. Genetic factors, lifestyle habits, and inflammation are important risk factors associated with CRC development. In recent years, growing evidence has supporting the significant role of the intestinal microbiome in CRC carcinogenesis. Disturbances in the healthy microbial balance, known as dysbiosis, are frequently observed in these patients. Pathogenic microorganisms that induce intestinal dysbiosis have become an important target to determine the role of bacterial infection in tumorigenesis. Interestingly, the presence of different bacterial strains, such as Fusobacterium nucleatum, has been detected in tissue and stool from patients with CRC and associated with substantial clinical and molecular features, as well as with patient therapy response. Therefore, understanding how the presence and levels of F. nucleatumstrains in the gut affect the risk of CRC onset and progression may inform suitable candidates for interventions focused on modulation of this bacteria. Here we review new insights into the role of gut microbiota in CRC carcinogenesis and the clinical utility of using the detection of F. nucleatum in different settings such as screening, prognosis, and microbiota modulation as a means to prevent cancer, augment therapies, and reduce adverse effects of treatment.

Identification of Proteins Associated with the Formation of Oral Biofilms

ABSTRACT Objective: To identify proteins associated with the formation of Streptococcus gordonii and Fusobacterium nucleatum biofilms. Material and Methods: Biofilms composed of two bacterial species, S. gordonii and F. nucleatum, were cultured for 1, 4, 7, and 10 days. The presence of both species was confirmed via amplification of the srtA and radD genes using real-time PCR. The concentrations of proteins associated with the biofilms and individual species were quantified using Western blotting. Results: The protein profiles of S. gordonii and F. nucleatum from individual cultures determined using one-dimensional electrophoresis revealed proteins found in S. gordonii and in F. nucleatum. Ct and reciprocal Ct values were determined for the exposed S. gordonii and F. nucleatum biofilms. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein was detected in biofilms and F. nucleatum, whereas HSP40 protein was present only in biofilms after 7 and 10 days of formation. Conclusion: HSP40 was detected only in the formed biofilms; thus, HSP40 is an essential proteins for adhesion.