Insights into oral microbiome and colorectal cancer - on the way of searching new perspectives.

Microbiome is a keystone polymicrobial community that coexist with human body in a beneficial relationship. These microorganisms enable the human body to maintain homeostasis and take part in mechanisms of defense against infection and in the absorption of nutrients. Even though microbiome is involved in physiologic processes that are beneficial to host health, it may also cause serious detrimental issues. Additionally, it has been proven that bacteria can migrate to other human body compartments and colonize them even although significant structural differences with the area of origin exist. Such migrations have been clearly observed when the causes of genesis and progression of colorectal cancer (CRC) have been investigated. It has been demonstrated that the oral microbiome is capable of penetrating into the large intestine and cause impairments leading to dysbiosis and stimulation of cancerogenic processes. The main actors of such events seem to be oral pathogenic bacteria belonging to the red and orange complex (regarding classification of bacteria in the context of periodontal diseases), such as Porphyromonas gingivalis and Fusobacterium nucleatum respectively, which are characterized by significant amount of cancerogenic virulence factors. Further examination of oral microbiome and its impact on CRC may be crucial on early detection of this disease and would allow its use as a precise non-invasive biomarker.

Diferencia en la expresión del ligando de receptor activador para el factor nuclear K B, en tejido tumoral frente a la infección por diferentes patógenos periodontales / Difference in receptor activator of nuclear factor K B expression in tumoral tissue by different periodontal pathogens

La enfermedad periodontal es una de las principales causas de pérdida dentaria. Clínicamente, esta patología, mediada por la desregulación del sistema inmune producto de una disbiosis ocurrida en el surco gingival, inicia con la inflamación de la encía y evoluciona con el daño irreversible de los tejidos que rodean el diente. El hueso alveolar es uno de los tejidos afectados esta patología, esto debido a la activación de osteoclastos por la sobreexpresión de la proteína RANKL en el huésped. El propósito de este trabajo es determinar el nivel de sobreexpresión de RANKL, en un modelo de células tumorales U2OS, frente a la infección con Porphyromonas gingivalis y Prevotella intermedia. Para identificar el nivel de RANKL, se definieron cuatro grupos: Un grupo control, no tratado; Grupo PG, tratado con P. gingivalis; Grupo PI, tratado con P. Intermedia; y un grupo PG+PI, tratado con ambas bacterias. El nivel relativo de la proteína RANKL fue determinado en el sobrenadante y en los extractos celulares de manera independiente, mediante la técnica Western blot. En sobrenadantes, el grupo PG mostró mayores niveles de RANKL comparados con PI (p < 0,05). En extractos celulares los niveles fueron mayores en el grupo PG+PI (p < 0,05). El grupo PI mostró los niveles más bajos de RANKL. La infección polimicrobiana resulta en una mayor expresión de RANKL en células tumorales U2OS, mientras que frente a la infección P. gingivalis, se observó mayor cantidad de RANKL soluble.

SUMMARY: Periodontal disease is one of the main causes of tooth loss. Clinically, this pathology, mediated by the deregulation of the immune system due to a dysbiosis occurred in the gingival sulcus, begins with the inflammation of the gum and evolves with the irreversible damage of the tissues that surround the tooth. Alveolar bone is one of the most affected tissues by this disease, due to the activation of osteoclasts by the upregulation of RANKL in the host. The aim of this study is to determine the increase of RANKL, in a U2OS tumor cells model, inoculated with Porphyromonas gingivalis and Prevotella intermedia. To identify the level of RANKL, four groups were defined: A control group, not treated; PG group, treated with P.gingivalis; PI group, treated with P. intermedia; and a PG+PI group, treated with both bacteria. The relative level of RANKL was determined in the supernatant and cell extracts independently, using the Western blot technique. In supernatants, the PG group showed higher RANKL levels compared to PI (p < 0.05). In cell extracts the levels were higher in the PG+PI group (p < 0.05.). The PI group showed the lowest levels of RANKL.Polymicrobial infection results in a greater expression of of soluble RANKL was observed.

Can metagenomics unravel the impact of oral bacteriome in human diseases?

Oral microbial ecosystems are vital in maintaining the health of the oral cavity and the entire body. Oral microbiota is associated with the progression of oral diseases such as dental caries, periodontal diseases, head and neck cancer, and several systemic diseases such as cardiovascular disease, rheumatoid arthritis, adverse pregnancy outcomes, diabetes, lung infection, colorectal cancer, and pancreatic cancer. Buccal mucosa, tongue dorsum, hard palate, saliva, palatine tonsils, throat, keratinized gingiva, supra-gingival plaque, subgingival plaque, dentures, and lips are microbial habitats of the oral cavity. Porphyromonas gingivalis may have a role in the development of periodontal diseases, oral cancer, diabetes, and atherosclerotic disease. Fusobacterium nucleatum showed a higher abundance in periodontal diseases, oral and colon cancer, adverse pregnancy outcomes, diabetes, and rheumatoid arthritis. The higher abundance of Prevotella intermedia is typical in periodontal diseases, rheumatoid arthritis, and adverse pregnancy outcome. S. salivarius displayed higher abundance in both dental caries and OSCC. Oral bacteria may influence systemic diseases through inflammation by releasing pro inflammatory cytokines. Identification of oral bacteria using culture-dependent approaches and next-generation sequencing-based metagenomic approaches is believed to significantly identify the therapeutic targets and non-invasive diagnostic indicators in different human diseases. Oral bacteria in saliva could be exploited as a non-invasive diagnostic indicator for the early detection of oral and systemic disorders. Other therapeutic approaches such as the use of probiotics, green tea polyphenol, cold atmospheric plasma (CAP) therapy, antimicrobial photodynamic therapy, and antimicrobial peptides are used to inhibit the growth of biofilm formation by oral bacteria.

Porphyromonas gingivalis may have a role in the development of periodontal diseases, oral cancer, diabetes, and atherosclerotic diseaseFusobacterium nucleatum showed a higher abundance in periodontal diseases, oral and colon cancer, adverse pregnancy outcomes, diabetes, and rheumatoid arthritisOral bacteria may influence systemic diseases through inflammation by releasing pro inflammatory cytokines.Identification of oral bacteria in saliva may be used as a non-invasive diagnostic indicator for the early detection of oral and systemic disorders.

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

Prevalencia de Entamoeba gingivalis y Trichomonas tenax en adolescentes de edad escolar / Prevalence of Entamoeba gingivalis and Trichomonas tenax in school-age adolescents

Entamoeba gingivalis y Trichomonas tenaxson protozoos que generalmente se encuentran en la cavidad oral de los humanos. E. gingivalis vive en la superficie de los dientes y las encías, bolsas gingivales cerca de la base de los dientes y rara vez en las criptas de las amígdalas. Por lo general, se transmiten por contacto directo de una persona a otra al besarse, rociarse con gotas o compartir utensilios para comer. Se sabe que hasta el 95% de las personas con poca higiene bucal pueden estar infectadas con esta ameba. Por otro lado, Trichomonas tenax es una pequeña tricomona que generalmente se encuentra en la cavidad oral del 5 al 10% de los humanos. Aunque se consideran comensales anaeróbicos no patógenos, el refugio de este protozoo es más común entre personas con mala higiene bucal o enfermedades. Los informes epidemiológicos demostraron variables tasas de prevalencia de la infección por E. gingivalis entre los niños, y su ocurrencia puede variar de acuerdo a la edad, presencia de gingivitis, periodontitis y condiciones inmunosupresoras. Los niños y adolescentes en edad escolar son extremadamente susceptibles a una amplia gama de infecciones gingivales como bacterias y protozoos. El objetivo de este trabajo es determinar la prevalencia de los protozoarios E. gingivalis y T. tenax en adolescentes con edades comprendidas entre 10 y 19 años en una población de 185 pacientes de la ciudad peruana de Huancayo, Perú. La prevalencia fue mayor en la población femenina (14,05%) que en la población masculina (11,35%), con un mayor porcentaje de prevalencia entre las edades de 17 a 19 años (12,43%), pH salival menor a 6,7 y con enfermedades periodentales (16,22%). Los resultados estadísticos demostraron que la edad (p=0,001), pH salival (p=0,024) y diagnóstico de otras patologías periodentales (p<0,001) fueron estadísticamente significativas(AU)

Entamoeba gingivalis and Trichomonas tenax are protozoa that are usually found in the oral cavity of humans. E. gingivalis lives on the surface of the teeth and gums, gum pockets near the base of the teeth, and rarely in the crypts of the tonsils. They are usually spread by direct person-to-person contact through kissing, dousing, or sharing eating utensils. It is known that up to 95% of people with poor oral hygiene may be infected with this amoeba. On the other hand, Trichomonas tenax is a small trichomona that is usually found in the oral cavity of 5-10% of humans. Although they are considered non-pathogenic anaerobic commensals, the harborage of this protozoan is more common among people with poor oral hygiene or disease. Epidemiological reports have shown variable prevalence rates of E. gingivalis infection among children, and its occurrence may vary according to age, presence of gingivitis, periodontitis, and immunosuppressive conditions. School-age children and adolescents are extremely susceptible to a wide range of gingival infections including bacteria and protozoa. The objective of this work is to determine the prevalence of the protozoa E. gingivalis and T. tenax in adolescents between the ages of 10 and 19 in a population of 185 patients from the Peruvian city of Huancayo, Peru. The prevalence was higher in the female population (14.05%) than in the male population (11.35%), with a higher percentage of prevalence between the ages of 17 to 19 years (12.43%), lower salivary pH to 6.7 and with periodontal diseases (16.22%). The statistical results showed that age (p=0.001), salivary pH (p=0.024) and diagnosis of other periodontal pathologies (p<0.001) were statistically significant(AU)

A Narrative Review on Oral and Periodontal Bacteria Microbiota Photobiomodulation, through Visible and Near-Infrared Light: From the Origins to Modern Therapies.

Photobiomodulation (PBM) consists of a photon energy transfer to the cell, employing non-ionizing light sources belonging to the visible and infrared spectrum. PBM acts on some intrinsic properties of molecules, energizing them through specific light wavelengths. During the evolution of life, semiconducting minerals were energized by sun radiation. The molecules that followed became photoacceptors and were expressed into the first proto-cells and prokaryote membranes. Afterward, the components of the mitochondria electron transport chain influenced the eukaryotic cell physiology. Therefore, although many organisms have not utilized light as an energy source, many of the molecules involved in their physiology have retained their primordial photoacceptive properties. Thus, in this review, we discuss how PBM can affect the oral microbiota through photo-energization and the non-thermal effect of light on photoacceptors (i.e., cytochromes, flavins, and iron-proteins). Sometimes, the interaction of photons with pigments of an endogenous nature is followed by thermal or photodynamic-like effects. However, the preliminary data do not allow determining reliable therapies but stress the need for further knowledge on light-bacteria interactions and microbiota management in the health and illness of patients through PBM.

Effect of Twinlight Laser on the Attachment of Human Gingival Fibroblasts to the Root Surface In Vitro.

BACKGROUND This study aimed to compare the effectiveness of subgingival scaling and root planing with the Twinlight laser, Er: YAG laser, and hand instrumentation on the removal of endotoxin and attachment of human gingival fibroblasts (HGFs) to cementum surfaces in vitro. MATERIAL AND METHODS Single-rooted teeth extracted for periodontal disease were collected and divided into 3 groups: group A, root planing with Gracey curet no. 5/6; group B, irradiation with Er: YAG laser; group C, irradiation with Er: YAG laser and Nd: YAG laser. Endotoxins were determined by the limulus amebocyte lysate test. Cell attachment and proliferation of HGFs on root specimens were evaluated by cell counting kit-8 assay. The root surface and cell morphology were observed by scanning electron microscope. RESULTS A flat root surface with scratches was found in group A, Group B had a homogeneous rough morphology without carbonization, and group C had a non-homogeneous rough morphology with ablation. The endotoxin concentration was highest in group A (P<0.05) and lowest in group C (P>0.05). HGFs cultured in group B showed significantly increased adhesion and proliferation compared with groups A and C (P<0.05). HGFs in group B were well attached, covered densely by pseudopodia. HGFs in group A were round with poor extension and short pseudopodia, while the cells in the group C were in narrow, triangular, or polygonal shapes. CONCLUSIONS Twinlight laser-assisted periodontal treatment effectively improved the biocompatibility of root surface and promoted the attachment and proliferation of fibroblasts by removing calculus and reducing the concentration of endotoxins.

Molecular Mechanisms Leading from Periodontal Disease to Cancer.

Periodontitis is prevalent in half of the adult population and raises critical health concerns as it has been recently associated with an increased risk of cancer. While information about the topic remains somewhat scarce, a deeper understanding of the underlying mechanistic pathways promoting neoplasia in periodontitis patients is of fundamental importance. This manuscript presents the literature as well as a panel of tables and figures on the molecular mechanisms of Porphyromonas gingivalis and Fusobacterium nucleatum, two main oral pathogens in periodontitis pathology, involved in instigating tumorigenesis. We also present evidence for potential links between the RANKL-RANK signaling axis as well as circulating cytokines/leukocytes and carcinogenesis. Due to the nonconclusive data associating periodontitis and cancer reported in the case and cohort studies, we examine clinical trials relevant to the topic and summarize their outcome.

Cigarette Smoking and Opium Use in Relation to the Oral Microbiota in Iran.

Cigarettes and opium contain chemicals and particulate matter that may modify the oral microbiota. This study aimed to investigate the association between cigarette and opium use with the oral microbiota. A total of 558 participants were recruited from Iran between 2011 and 2015. Individuals were categorized as never cigarette nor opium users, ever cigarette-only smokers, ever opium-only users, and ever both cigarette and opium users. Participants provided saliva samples for 16S rRNA gene sequencing. Logistic regression, microbiome regression-based kernel association test (MiRKAT), and zero-inflated beta regression models were calculated. For every increase in 10 observed amplicon sequence variants (ASVs), the odds for being a cigarette-only smoker, opium-only user, and both user compared to never users decreased by 9% (odds ratio [OR] = 0.91; 95% confidence interval [95% CI] = 0.86 to 0.97), 13% (OR = 0.87; 95% CI = 0.75 to 1.01), and 12% (OR = 0.88; 95% CI = 0.80 to 0.96), respectively. The microbial communities differed by cigarette and opium use as indicated by MiRKAT models testing the three beta-diversity matrices (P < 0.05 for all). Three genera were less likely and one genus was more likely to be detected in cigarette-only smokers or opium-only users than in never users. The relative abundance of the phylum Actinobacteria (never, 14.78%; both, 21.20%) was higher and the phyla Bacteroidetes (never, 17.63%; both, 11.62%) and Proteobacteria (never, 9.06%; both, 3.70%) were lower in users of both cigarettes and opium, while the phylum Firmicutes (never, 54.29%; opium, 65.49%) was higher in opium-only users. Cigarette and opium use was associated with lower alpha-diversity, overall oral microbiota community composition, and both the presence and relative abundance of multiple taxa. IMPORTANCE Cigarette smoking and opium use are associated with periodontal disease caused by specific bacteria such as Porphyromonas gingivalis, which suggests a link between cigarette smoking and opium use and the oral microbiota. Alterations of the oral microbiota in cigarette smokers compared to nonsmokers have been reported, but this has not been studied across diverse populations. Additionally, the association of opium use with the oral microbiota has not been investigated to date. We conducted this study to investigate differences in the oral microbiota between ever users of cigarettes only, opium only, and both cigarettes and opium and never users of cigarettes and opium in Iran. Lower alpha-diversity, distinct overall oral microbial communities, and the presence and relative abundance of multiple taxa have been found for users of cigarettes and/or opium.

Fusobacterium nucleatum - Friend or foe?

Fusobacterium nucleatum (F. nucleatum) is one of the most abundant Gram-negative anaerobic bacteria, part of the gut, and oral commensal flora, generally found in human dental plaque. Its presence could be associated with various human diseases, including, e.g., periodontal, angina, lung and gynecological abscesses. This bacteria can enter the blood circulation as a result of periodontal infection. It was proven that F. nucleatum migrates from its primary site of colonization in the oral cavity to other parts of the body. It could cause numerous diseases, including cancers. On the other hand, it was shown that Fusobacterium produces significant amounts of butyric acid, which is a great source of energy for colonocytes (anti-inflammatory cells). Therefore, it is very interesting to get to know the two faces of F. nucleatum.

A cocoa (Theobroma cacao L.) extract impairs the growth, virulence properties, and inflammatory potential of Fusobacterium nucleatum and improves oral epithelial barrier function.

Fusobacterium nucleatum is associated with many conditions and diseases, including periodontal diseases that affect tooth-supporting tissues. The aim of the present study was to investigate the effects of a cocoa extract (Theobroma cacao L.) on F. nucleatum with respect to growth, biofilm formation, adherence, and hydrogen sulfide (H2S) production. The anti-inflammatory properties and the effect on epithelial barrier function of the cocoa extract were also assessed. The cocoa extract, whose major phenolic compound is epicatechin, dose-dependently inhibited the growth, biofilm formation, adherence properties (basement membrane matrix, oral epithelial cells), and H2S production of F. nucleatum. It also decreased IL-6 and IL-8 production by F. nucleatum-stimulated oral epithelial cells and inhibited F. nucleatum-induced NF-κB activation in monocytes. Lastly, the cocoa extract enhanced the barrier function of an oral epithelial model by increasing the transepithelial electrical resistance. We provide evidence that the beneficial properties of an epicatechin-rich cocoa extract may be useful for preventing and/or treating periodontal diseases.

A bacterial tyrosine phosphatase modulates cell proliferation through targeting RGCC.

Tyrosine phosphatases are often weaponized by bacteria colonizing mucosal barriers to manipulate host cell signal transduction pathways. Porphyromonas gingivalis is a periodontal pathogen and emerging oncopathogen which interferes with gingival epithelial cell proliferation and migration, and induces a partial epithelial mesenchymal transition. P. gingivalis produces two tyrosine phosphatases, and we show here that the low molecular weight tyrosine phosphatase, Ltp1, is secreted within gingival epithelial cells and translocates to the nucleus. An ltp1 mutant of P. gingivalis showed a diminished ability to induce epithelial cell migration and proliferation. Ltp1 was also required for the transcriptional upregulation of Regulator of Growth and Cell Cycle (RGCC), one of the most differentially expressed genes in epithelial cells resulting from P. gingivalis infection. A phosphoarray and siRNA showed that P. gingivalis controlled RGCC expression through Akt, which was activated by phosphorylation on S473. Akt activation is opposed by PTEN, and P. gingivalis decreased the amount of PTEN in epithelial cells. Ectopically expressed Ltp1 bound to PTEN, and reduced phosphorylation of PTEN at Y336 which controls proteasomal degradation. Ltp-1 induced loss of PTEN stability was prevented by chemical inhibition of the proteasome. Knockdown of RGCC suppressed upregulation of Zeb2 and mesenchymal markers by P. gingivalis. RGCC inhibition was also accompanied by a reduction in production of the proinflammatory cytokine IL-6 in response to P. gingivalis. Elevated IL-6 levels can contribute to periodontal destruction, and the ltp1 mutant of P. gingivalis incited less bone loss compared to the parental strain in a murine model of periodontal disease. These results show that P. gingivalis can deliver Ltp1 within gingival epithelial cells, and establish PTEN as the target for Ltp1 phosphatase activity. Disruption of the Akt1/RGCC signaling axis by Ltp1 facilitates P. gingivalis-induced increases in epithelial cell migration, proliferation, EMT and inflammatory cytokine production.

Heat-killed Lactobacillus acidophilus mediates Fusobacterium nucleatum induced pro-inflammatory responses in epithelial cells.

Probiotics is widespreadly used nowadays. However, the safety issue with the use of live probiotics is still a matter of contention. In recent years, an expanding body of evidence supports the beneficial role of heat-killed probiotics in the maintenance of systemic health, whereas the role of these heat-killed bacteria on periodontal health remains unclear. This study aimed to evaluate the effects of heat-killed probiotics on periodontal pathogen virulence and associated mechanisms. We demonstrated that heat-killed Lactobacillus acidophilus was able to coaggregate with Fusobacterium nucleatum, the bridging bacteria of oral biofilm, and inhibit the adhesion and invasion of F. nucleatum, leading to a subsequent elimination of pro-inflammatory cytokine production in oral epithelial cells. This coaggregation further caused a suppression of the virulence gene fap2 expression in F. nucleatum. Therefore, heat-killed L. acidophilus might downregulate the pro-inflammatory cytokine expression in epithelial cells via coaggregation with F. nucleatum and suppression of F. nucleatum fap2 expression, which was the first demonstration that heat-killed probiotics modulate periodontal disease pathogenesis via coaggregation. Collectively, this finding provides new evidence that heat-killed probiotics might exert beneficial effects to periodontal health by coaggregating with periodontal pathogens and modulating their virulence.

Local and systemic mechanisms linking periodontal disease and inflammatory comorbidities.

Periodontitis, a major inflammatory disease of the oral mucosa, is epidemiologically associated with other chronic inflammation-driven disorders, including cardio-metabolic, neurodegenerative and autoimmune diseases and cancer. Emerging evidence from interventional studies indicates that local treatment of periodontitis ameliorates surrogate markers of comorbid conditions. The potential causal link between periodontitis and its comorbidities is further strengthened by recent experimental animal studies establishing biologically plausible and clinically consistent mechanisms whereby periodontitis could initiate or aggravate a comorbid condition. This multi-faceted 'mechanistic causality' aspect of the link between periodontitis and comorbidities is the focus of this Review. Understanding how certain extra-oral pathologies are affected by disseminated periodontal pathogens and periodontitis-associated systemic inflammation, including adaptation of bone marrow haematopoietic progenitors, may provide new therapeutic options to reduce the risk of periodontitis-associated comorbidities.

Molecular Mechanisms of Periodontal Disease.

Periodontal disease, one of the most prevalent human infectious diseases, is characterized by chronic inflammatory tissue destruction of the alveolar bone and the connective tissues supporting the tooth [...].

Innate Phagocyte Polarization in the Oral Cavity.

The oral cavity is a complex environment constantly exposed to antigens from food and the oral microbiota. Innate immune cells play an essential role in maintaining health and homeostasis in the oral environment. However, these cells also play a significant role in disease progression. This review will focus on two innate phagocytes in the oral cavity: macrophages and neutrophils, and examine their roles during homeostasis and disease development, with a focus on periodontal disease and cancer. Macrophages have a well-known ability to polarize and be activated towards a variety of phenotypes. Several studies have found that macrophages' polarization changes can play an essential role in maintaining health in the oral cavity and contribute to disease. Recent data also finds that neutrophils display phenotypic heterogeneity in the oral cavity. In both cases, we focus on what is known about how these cellular changes alter these immune cells' interactions with the oral microbiota, including how such changes can lead to worsening, rather than improving, disease states.

Construction of a Gene-Deletion Mutant in Tannerella forsythia.

Tannerella forsythia, a gram-negative anaerobic bacterium, is one of the most important pathogens in periodontal disease. However, it has been difficult to construct a gene-deletion mutant in this organism, which may serve as a useful tool in microbiological research. We reported a highly efficient method to construct a gene-deletion mutant of T. forsythia in 2007, and it was accomplished by preparing competent cells from a colony grown on an agar medium instead of a broth culture. Here, we describe the same method with some improvements.

Invasion of Gingival Epithelial Cells by Porphyromonas gingivalis.

Porphyromonas gingivalis is a major pathogen responsible for severe and chronic manifestations of periodontal disease, which is one of the most common infectious disorders of humans. Although human gingival epithelium prevents intrusions by periodontal bacteria, P. gingivalis is able to invade gingival epithelial cells. To study the dynamics and the fate of intracellular P. gingivalis, confocal laser scanning microscopy (CLSM) is a method of choice. Information gained with CLSM contains not only the number of P. gingivalis associated with gingival epithelial cells but also the bacterial localization on/inside the host cells, morphological change of host cells, and physical interaction between the bacteria and host organelle. In this chapter, we describe the protocols for microscopy techniques to morphologically study gingival epithelial cells infected by P. gingivalis.

Alteration of oral flora in betel quid chewers in Sri Lanka.

BACKGROUND: Betel quid chewing is known as a crucial risk factor for oral diseases such as periodontal diseases, oral cancer, and precancerous lesions in Southeast Asian countries. Although abnormal oral bacterial flora may be linked to betel quid related-oral diseases such as oral cancer, precancerous lesions, and periodontal diseases, little information is available on alterations of their oral flora thus far. To identify these alterations, we analyzed the oral flora in betel quid chewers (BQC) and non-chewers (NC) in Sri Lanka. METHODS: Samples obtained from buccal swabs of BQC and NC were analyzed with a next generation sequencer. Data were processed and analyzed using the QIIME software package. Mann-Whitney U test and Permutational multivariate analysis of variance were used for statistical analyses. P values < 0.05 were considered to be statistically significant. RESULTS: In BQC, the proportion of periodontal pathogens including Actinomyces, Tannerella, and Prevotella was higher than that in NC (P < 0.05), while the proportion of cariogenic pathogens including Streptococcus, Lautropia, and Actinobacillus was lower than that in NC (P < 0.05). A statistically significant difference in Shannon index and PD Whole tree was observed between BQC and NC (P < 0.05). PCoA analysis detected different clusters in BQC and NC (P < 0.05). CONCLUSION: The results suggested that betel quid chewing significantly altered oral flora. Adequate oral health care may help prevent BQC from developing bacterial pathogen-related oral diseases.

Porphyromonas gingivalis gingipains potentially affect MUC5AC gene expression and protein levels in respiratory epithelial cells.

Porphyromonas gingivalis (Pg) is a periodontopathic pathogen that may affect MUC5AC-related mucus hypersecretion along airway epithelial cells. Here, we attempted to establish whether Pg virulence factors (lipopolysaccharide, FimA fimbriae, gingipains) affect MUC5AC in immortalized and primary bronchial cells. We report that MUC5AC gene expression and protein levels are affected by Pg culture supernatant, but not by lipopolysaccharide or FimA fimbriae. Cells treated with either Pg single (Kgp or Rgp) or double (Kgp/Rgp) mutants had altered levels of MUC5AC gene expression and protein levels, and MUC5AC staining of double mutant-treated mouse lung cells showed that MUC5AC protein levels were unaffected. Taken together, we propose that Pg gingipains may be the primary virulence factor that influences both MUC5AC gene expression and protein levels.