Porphyromonas Gingivalis Strains Research Articles

Frequency of Fimbrial Gene Types I, Ib, and II in Clinical Strains of Porphyromonas gingivalis Characterized From Periodontitis Patients.

Objective Porphyromonas gingivalis (P. gingivalis) is considered the predominant pathogen in association with different stages of periodontitis, and fim genes play a vital role in adherence and colonization. This study is thus aimed to detect the prevalence of P. gingivalis and the frequency of fim gene types among the clinical strains isolated from periodontitis patients. Methods Plaque samples (N = 45) were collected from patients with three different stages of periodontitis (n = 15 in each group). All the samples were inoculated onto sterile anaerobic blood agar and were processed anaerobically using a GasPak system at 37°C for five to sevendays. Standard microbiological techniques were used to identify P. gingivalis. Genomic DNA was extracted, and polymerase chain reaction (PCR) was carried out to detect the frequency of three fim gene types, using specific primers. Results P. gingivalis was more prevalent in Group III (93.3%), followed by 26.7% in Group II, and 13.3% in Group I. Maximum isolates were seen in the age group of 40-50, with no significance within the genders. fim type I was frequent in Group III (78.5% (n = 11)), followed by 0.25% (n = 1) under Group II, with no other fim types in the other groups. Conclusion Prevalence of P. gingivalis and frequency of fim genes, in association with its virulence, were observed. Periodical monitoring of such virulence genes would aid in the theranostic approach to combat the complications caused by P. gingivalis in periodontitis cases.

Complete genome sequences of three Porphyromonas gingivalis strains, isolated from patient with esophageal squamous cell carcinoma and healthy individual in China.

Here, we report the complete genome sequences of three Porphyromonas gingivalis, one from patient with esophageal cancer (LyEC01), and the other two from periodontally healthy individuals (LyG-1 and LyG-2) in 2021 and 2023. The genome sizes of LyEC01, LyG-1, and LyG-2 were 2,408,275, 2,411,440, and 2,411,481 bp, respectively.

Differential Response of Human Dendritic Cells upon Stimulation with Encapsulated or Non-Encapsulated Isogenic Strains of Porphyromonas gingivalis.

During periodontitis, the extracellular capsule of Porphyromonas gingivalis favors alveolar bone loss by inducing Th1 and Th17 patterns of lymphocyte response in the infected periodontium. Dendritic cells recognize bacterial antigens and present them to T lymphocytes, defining their activation and polarization. Thus, dendritic cells could be involved in the Th1 and Th17 response induced against the P. gingivalis capsule. Herein, monocyte-derived dendritic cells were obtained from healthy individuals and then stimulated with different encapsulated strains of P. gingivalis or two non-encapsulated isogenic mutants. Dendritic cell differentiation and maturation were analyzed by flow cytometry. The mRNA expression levels for distinct Th1-, Th17-, or T-regulatory-related cytokines and transcription factors, as well as TLR2 and TLR4, were assessed by qPCR. In addition, the production of IL-1β, IL-6, IL-23, and TNF-α was analyzed by ELISA. The encapsulated strains and non-encapsulated mutants of P. gingivalis induced dendritic cell maturation to a similar extent; however, the pattern of dendritic cell response was different. In particular, the encapsulated strains of P. gingivalis induced higher expression of IRF4 and NOTCH2 and production of IL-1β, IL-6, IL-23, and TNF-α compared with the non-encapsulated mutants, and thus, they showed an increased capacity to trigger Th1 and Th17-type responses in human dendritic cells.

Defining Porphyromonas gingivalis strains associated with periodontal disease

Porphyromonas gingivalis, a Gram-negative anaerobic bacterium commonly found in human subgingival plaque, is a major etiologic agent for periodontitis and has been associated with multiple systemic pathologies. Many P. gingivalis strains have been identified and different strains possess different virulence factors. Current oral microbiome approaches (16S or shotgun) have been unable to differentiate P. gingivalis strains. This study presents a new approach that aims to improve the accuracy of strain identification, using a detection method based on sequencing of the intergenic spacer region (ISR) which is variable between P. gingivalis strains. Our approach uses two-step PCR to amplify only the P. gingivalis ISR region. Samples are then sequenced with an Illumina sequencer and mapped to specific strains. Our approach was validated by examining subgingival plaque from 153 participants with and without periodontal disease. We identified the avirulent strain ATCC33277/381 as the most abundant strain across all sample types. The W83/W50 strain was significantly enriched in periodontitis, with 13% of participants harboring that strain. Overall, this approach can have significant implications not only for the diagnosis and treatment of periodontal disease but also for other diseases where P. gingivalis or its toxins have been implicated, such as Alzheimer's disease.

Comparative analysis of Porphyromonas gingivalis A7436 and ATCC 33277 strains reveals differences in the expression of heme acquisition systems.

Periodontitis belongs to a group of multifactorial diseases, characterized by inflammation and destruction of tooth-supporting tissues. P. gingivalis is one of the most important microbial factors involved in the initiation and progression of periodontitis. To survive in the host, the bacterium must acquire heme as a source of iron and protoporphyrin IX. P. gingivalis strains respond differently to changing iron and heme concentrations, which may be due to differences in the expression of systems involved in iron and heme acquisition. The ability to accumulate iron intracellularly, being different in more and less virulent P. gingivalis strains, may influence their phenotypes, production of virulence factors (including proteins engaged in heme acquisition), and virulence potential of this bacterium.

Assessment of Minimum Inhibitory Concentration and Anti-biofilm Activity of Plectranthus Amboinicus Solvent Extract against Pure Strains of Putative Periodontal Pathogens: An In-vitro Study

Introduction: Antiseptic agents used in periodontics as anti-plaque and anti-gingivitis agents are primarily chemical substances such as Bis-biguanide derivatives (chlorhexidine) or essential oils. Herbal derivatives have gained prominence in the recent past due to their activity against putative periodontal pathogens; however, only a few have achieved commercialisation. This study focusses on determining the efficacy of an extract from a widely available herb, Indian mintPlectranthus Amboinicus Methanolic extract (PAM), which has known anti-microbial and anti-inflammatory properties against periodontal pathogens in-vitro. Aim: To assess the Minimum Inhibitory Concentration (MIC) and anti-biofilm property of PAM solvent extract against pure strains of putative periodontal pathogens, namely Porphyromonas gingivalis (American Type Culture Collection-ATCC 33277), Fusobacterium nucleatum (ATCC 25586), and Aggregatibacter actinomycetemcomitans (ATCC 43718). Materials and Methods: The extract of PA was prepared using methanol and a Soxhlet extractor. An in-vitro analysis of the MIC and anti-biofilm efficacy of the extract was performed against standard strains of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum using the broth dilution method and microtitre-crystal violet assay, respectively. The MIC activity of the PAM extracts was compared with Chlorhexidine as a standard. Results: The MIC value of P. amboinicus extract was nearly similar to Chlorhexidine as assessed by the broth dilution method. The MIC of P. amboinicus extract for A.a and P.g was 0.4 ug/mL, F.n was 0.8 ug/mL, and the Chlorhexidine values against all three periodontal pathogens were 0.2 ug/mL. The anti-biofilm activity of The extract of PAMwas evaluated using the microtitrecrystal violet assay, and the Optical Density (OD) values were reduced after exposure to the extract, with a significant reduction (p<0.001) of the biofilm-forming bacteria observed. Conclusion: The methanol extract of PAM demonstrated a noteworthy MIC, exhibiting effectiveness at a low concentration of 0.4 μg/mL against Aggregatibacter actinomycetemcomitans in three repeated trials. Moreover, this extract displayed significant inhibitory effects on the biofilm formation of periodontal pathogens Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum (Pg, Aa, Fn), suggesting its potential as an alternative to conventional chemical anti-microbials.

Further Preclinical Development of a Bio‐therapeutic Against Porphyromonas gingivalis for the control of Systemic and Neuro‐inflammation related Dementia and Alzheimer’s Disease

AbstractAddressing novel mechanisms and effective therapeutic treatments for cognitive decline, Dementia and Alzheimer’s Disease is a major public health need. Keystone Bio have identified that specific virulent strains of Porphyromonas gingivalis (Pg) and the release of specific virulence factors/toxins in the oral cavity as the primary driver/causation of systemic/neuro‐vascular inflammation/degenerative diseases i.e., cognitive decline/dementias/Alzheimer’s disease (Nara et. al 2021). A recent landmark finding was reported in a sub‐cohort analysis from a Phase 2/3 GAIN trial (n = 238) demonstrated that lowering the load of Pg in the mouth leads to a significant improvement of cognitive slowing at both 24 and 48 weeks in participants with mild to moderate AD. Another study showed lowering the load of Pg in the mouth had a favorable effect on AD‐related brain atrophy (https://doi.org/10.1002/alz.12378). Pg OMVs and systemic system diseases has many well‐defined examples such as: cardiometabolic diseases‐ Pg OMVs attenuate insulin induced Akt/GSK‐3_ signaling in hepatic HepG2 cells, thereby causing changes in glucose metabolism in the liver and promoting the development of diabetes and increase vascular permeability by cleaving endothelial cell connexins such as PECAM‐1, thereby promoting cardiovascular diseases.Keystone Bio has further developed both a companion diagnostic (CDx) and a clinical, proof‐of‐concept tested, first generation, safe, efficacious, precision, bio‐therapeutic murine monoclonal antibody (KB‐001) for the diagnosis, treatment and monitoring against the oral bacteria and major virulent factor/toxin of Pg. The antibody engagement is with the later stages of the complex virulent factor/toxin secretion containing outer membrane vesicles from the bacteria thereby interfering/stopping all necessary metabolic, host defense, energy‐producing sources, adherence and biofilm formation and integrity (Nara et. al 2021). The talk will review what now seems like a solid case for causation in the role of virulent/toxin secreting strains of in neuro‐inflammation leading to cognitive decline, dementia, Sporadic Alzheimer’s disease and possibly Parkinson’s.

In-vitro polymicrobial oral biofilm model represents clinical microbial profile and disease progression during implant-related infections.

Clinically relevant in-vitro biofilm models are essential and valuable tools for mechanistically dissecting the etiopathogenesis of infectious diseases and test new antimicrobial therapies. Thus, the aim of this study was to develop and test a clinically relevant in-vitro oral polymicrobial biofilm model that mimics implant-related infections in terms of microbial profile. For this purpose, 24-well plate system was used to model oral biofilms, using three different microbial inoculums to grow in-vitro biofilms: (1) human saliva from periodontally healthy patients; (2) saliva as in inoculum 1+Porphyromonas gingivalis strain; and (3) supra and subgingival biofilm collected from peri-implant sites of patients diagnosed with peri-implantitis. Biofilms were grown to represent the dynamic transition from an aerobic to anaerobic community profile. Subsequently, biofilms were collected after each phase and evaluated for microbiological composition, microbial counts, biofilm biomass, structure, and susceptibility to chlorhexidine (CHX). Results showed higher live cell count (P<.05) for biofilms developed from patients' biofilm inoculum, but biomass volume, dry weight, and microbiological composition were similar among groups (P>.05). Interestingly, according to the checkerboard DNA-DNA hybridization results, the biofilm developed from stimulated human saliva exhibited a microbial composition more similar to the clinical subgingival biofilm of patients with peri-implantitis, with proportions of the main pathogens closer to those found in the disease. In addition, biofilm developed using saliva as inoculum was shown to be susceptible to CHX with significant reduction in bacteria compared with biofilms without exposure to CHX (P<.05). The findings suggested that the in-vitro polymicrobial biofilm developed from human saliva as inoculum is a suitable model and clinically relevant tool for mimicking the microbial composition of implant-related infections.

Corydalis saxicolaBunting total alkaloid eliminates Porphyromonas gingivalis strain 33277 internalized into macrophages by inhibition of TLR2

ObjectiveThis study aimed to investigate the impact of Corydalis Saxicola Bunting Total Alkaloid (CSBTA) on Porphyromonas gingivalis internalization within macrophages and explore the potential role of Toll-Like Receptor 2 (TLR2) in this process. MethodsWe established a P. gingivalis internalization model in macrophages by treating P. gingivalis-infected macrophages (MOI=100:1) with 200 μg/mL metronidazole and 300 μg/mL gentamicin for 1 h. Subsequently, the model was exposed to CSBTA at concentrations of 0.02 g/L or 1 μg/mL Pam3CSK4. After a 6 h treatment, cell lysis was performed with sterile water to quantify bacterial colonies. The mRNA expressions of TLR2 and interleukin-8 (IL-8) in macrophages were analyzed using RT-qPCR, while their protein levels were assessed via Western blot and ELISA respectively. ResultsP. gingivalis could internalize into macrophages and enhance the expression of TLR2 and IL-8. Activation of TLR2 by Pam3CSK4 contributed to P. gingivalis survival within macrophages and increased TLR2 and IL-8 expression. Conversely, 0.02 g/L CSBTA effectively cleared intracellular P. gingivalis, achieving a 90 % clearance rate after 6 h. Moreover, it downregulated the expression of TLR2 and IL-8 induced by P. gingivalis. However, the inhibitory effect of CSBTA on the internalized P. gingivalis model was attenuated by Pam3CSK4. ConclusionCSBTA exhibited the ability to reduce the presence of live intracellular P. gingivalis and lower IL-8 expression in macrophages, possibly by modulating TLR2 activity.

Isolation, characterization, and fibroblast uptake of bacterial extracellular vesicles from Porphyromonas gingivalis strains.

Periodontitis is an inflammatory condition caused by bacteria and represents a serious health problem worldwide as the inflammation damages the supporting tissues of the teeth and may predispose to systemic diseases. Porphyromonas gingivalis is considered a keystone periodontal pathogen that releases bacterial extracellular vesicles (bEVs) containing virulence factors, such as gingipains, that may contribute to the pathogenesis of periodontitis. This study aimed to isolate and characterize bEVs from three strains of P. gingivalis, investigate putative bEV uptake into human oral fibroblasts, and determine the gingipain activity of the bEVs. bEVs from three bacterial strains, ATCC 33277, A7A1-28, and W83, were isolated through ultrafiltration and size-exclusion chromatography. Vesicle size distribution was measured by nano-tracking analysis (NTA). Transmission electron microscopy was used for bEV visualization. Flow cytometry was used to detect bEVs and gingipain activity was measured with an enzyme assay using a substrate specific for arg-gingipain. The uptake of bEVs into oral fibroblasts was visualized using confocal microscopy. NTA showed bEV concentrations from 108 to 1011 particles/mL and bEV diameters from 42 to 356 nm. TEM pictures demonstrated vesicle-like structures. bEV-gingipains were detected both by flow cytometry and enzyme assay. Fibroblasts incubated with bEVs labeled with fluorescent dye displayed intracellular localization consistent with bEV internalization. In conclusion, bEVs from P. gingivalis were successfully isolated and characterized, and their uptake into human oral fibroblasts was documented. The bEVs displayed active gingipains demonstrating their origin from P. gingivalis and the potential role of bEVs in periodontitis.

Systematic analysis of prophages carried by Porphyromonas gingivalis.

To systematically investigate the prophages carrying in Porphyromonas gingivalis (P. gingivalis) strains, analyze potential antibiotic resistance genes (ARGs) and virulence genes in these prophages. We collected 90 whole genome sequences of P. gingivalis from NCBI and utilized the Prophage Hunter online software to predict prophages; Comprehensive antibiotic research database (CARD) and virulence factors database (VFDB) were adopted to analyze the ARGs and virulence factors (VFs) carried by the prophages. Sixty-nine prophages were identified among 24/90 P. gingivalis strains, including 17 active prophages (18.9%) and 52 ambiguous prophages (57.8%). The proportion of prophages carried by each P. gingivalis genome ranged from 0.5% to 6.7%. A total of 188 antibiotic resistance genes belonging to 25 phenotypes and 46 different families with six mechanisms of antibiotic resistance were identified in the 17 active prophages. Three active prophages encoded 4 virulence genes belonging to type III and type VI secretion systems. The potential hosts of these virulence genes included Escherichia coli, Shigella sonnei, Salmonella typhi, and Klebsiella pneumoniae. In conclusion, 26.7% P. gingivalis strains carry prophages, while the proportion of prophage genes in the P. gingivalis genome is relatively low. In addition, approximately 39.7% of the P. gingivalis prophage genes have ARGs identified, mainly against streptogramin, peptides, and aminoglycosides. Only a few prophages carry virulence genes. Prophages may play an important role in the acquisition, dissemination of antibiotic resistance genes, and pathogenicity evolution in P. gingivalis.

Antimicrobial activity, viability, and physicochemical properties of an MTA-type cement with different concentrations of bismuth trioxide

Abstract In medicine, bismuth is used as an antimicrobial agent. In dentistry, it is used primarily to increase radiopacity in some endodontic materials. The objective is to evaluate the antimicrobial activity, cell viability, pH, solubility, film thickness, and setting time of a mineral trioxide aggregated (MTA)-types of cement with different concentrations of bismuth trioxide. Three experimental MTA-types of cement with a bismuth trioxide (Bi2O3) concentration of 15 wt%, 20 wt%, and 25 wt% were used. The antimicrobial activity test was conducted on Streptococcus mutans and Porphyromonas gingivalis strains. Cell viability was measured by the quantitative colorimetric assay using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) assay on a mouse fibroblast cell line (L929). Solubility, film thickness, and setting time were performed according to ISO 6876. The lowest Bi2O3 concentrations showed the best antimicrobial activity and cell viability. pH, solubility, setting time, and film thickness did not show statistically significant differences between the different Bi2O3 concentrations tested.

Circulating IgG antibodies to periodontal bacteria and lung cancer risk in the CLUE cohorts.

Oral health is a key indicator of overall health, well-being, and quality of life. Several studies have provided new evidence about the role of oral diseases, specifically periodontitis, in generating risk for various forms of cancers, including lung, colorectal, and pancreatic cancers. Incident lung cancer cases (n = 192) and matched controls (n = 192) were selected from participants of the CLUE I and CLUE II cohorts. Archived serum samples collected from participants in 1974 (in CLUE I) were analyzed using immunoblotting for immunoglobulin G (IgG) antibody levels to 13 bacteria of the periodontium. Associations between antibody levels and lung cancer were estimated using conditional logistic regression. Most of the periodontal bacterial antibodies measured were inversely associated with lung cancer risk; of these, 3 were statistically significant (Prevotellaintermedia, Actinomyces naeslundii, and Veillonella parvula). A statistically significant positive association was observed for one of the Porphyromonas gingivalis strains after adjusting for P. intermedia. The sum of the logarithm of antibodies against the 13 measured bacteria was inversely associated with risk of lung cancer when the analysis was restricted to a longer follow-up (31-44 years after blood collection, highest vs lowest quartile: odds ratio = 0.26, 95% confidence interval = 0.08 to 0.84). Findings from this study highlight the complexity of using serum IgG antibodies to periodontal bacteria to identify associations between oral pathogens and risk of lung cancer. The inverse associations observed for antibodies to periodontal bacteria suggest that these may represent markers of immunity that provide some advantage in reducing the development of lung cancer.

High-fat diet and oral infection induced type 2 diabetes and obesity development under different genetic backgrounds.

Type 2 diabetes (T2D) is an adult-onset and obese form of diabetes caused by an interplay between genetic, epigenetic, and environmental components. Here, we have assessed a cohort of 11 genetically different collaborative cross (CC) mouse lines comprised of both sexes for T2D and obesity developments in response to oral infection and high-fat diet (HFD) challenges. Mice were fed with either the HFD or the standard chow diet (control group) for 12 weeks starting at the age of 8 weeks. At week 5 of the experiment, half of the mice of each diet group were infected with Porphyromonas gingivalis and Fusobacterium nucleatum bacteria strains. Throughout the 12-week experimental period, body weight (BW) was recorded biweekly, and intraperitoneal glucose tolerance tests were performed at weeks 6 and 12 of the experiment to evaluate the glucose tolerance status of mice. Statistical analysis has shown the significance of phenotypic variations between the CC lines, which have different genetic backgrounds and sex effects in different experimental groups. The heritability of the studied phenotypes was estimated and ranged between 0.45 and 0.85. We applied machine learning methods to make an early call for T2D and its prognosis. The results showed that classification with random forest could reach the highest accuracy classification(ACC=0.91) when all the attributes were used. Using sex, diet, infection status, initial BW, and area under the curve (AUC) at week 6, we could classify the final phenotypes/outcomes at the end stage of the experiment (at 12 weeks).

Intestinal cancer development in response to oral infection with high-fat diet-induced Type 2 diabetes (T2D) in collaborative cross mice under different host genetic background effects.

Type 2 diabetes (T2D) is a metabolic disease with an imbalance in blood glucose concentration. There are significant studies currently showing association between T2D and intestinal cancer developments. High-fat diet (HFD) plays part in the disease development of T2D, intestinal cancer and infectious diseases through many biological mechanisms, including but not limited to inflammation. Understanding the system genetics of the multimorbidity of these diseases will provide an important knowledge and platform for dissecting the complexity of these diseases. Furthermore, in this study we used some machine learning (ML) models to explore more aspects of diabetes mellitus. The ultimate aim of this project is to study the genetic factors, which underline T2D development, associated with intestinal cancer in response to a HFD consumption and oral coinfection, jointly or separately, on the same host genetic background. A cohort of 307 mice of eight different CC mouse lines in the four experimental groups was assessed. The mice were maintained on either HFD or chow diet (CHD) for 12-week period, while half of each dietary group was either coinfected with oral bacteria or uninfected. Host response to a glucose load and clearance was assessed using intraperitoneal glucose tolerance test (IPGTT) at two time points (weeks 6 and 12) during the experiment period and, subsequently, was translated to area under curve (AUC) values. At week 5 of the experiment, mice of group two and four were coinfected with Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn) strains, three times a week, while keeping the other uninfected mice as a control group. At week 12, mice were killed, small intestines and colon were extracted, and subsequently, the polyp counts were assessed; as well, the intestine lengths and size were measured. Our results have shown that there is a significant variation in polyp's number in different CC lines, with a spectrum between 2.5 and 12.8 total polyps on average. There was a significant correlation between area under curve (AUC) and intestine measurements, including polyp counts, length and size. In addition, our results have shown a significant sex effect on polyp development and glucose tolerance ability with males more susceptible to HFD than females by showing higher AUC in the glucose tolerance test. The ML results showed that classification with random forest could reach the highest accuracy when all the attributes were used. These results provide an excellent platform for proceeding toward understanding the nature of the genes involved in resistance and rate of development of intestinal cancer and T2D induced by HFD and oral coinfection. Once obtained, such data can be used to predict individual risk for developing these diseases and to establish the genetically based strategy for their prevention and treatment.

Comparative Evaluation on the effect of Herbal mouthwash on Putative Periodontal Pathogens – In vitro study

Introduction: The architecture of dental plaque biofilms provides a resistant environment for bacteria. Chemical plaque control agents containing antimicrobials (chlorhexidine, herbal mouthwash) are adjunctively used to halt the growth of dental plaque. In order to overcome the adverse effects that result due to the extensive use of synthetic formulation, several herbal preparations have been tested for their effectiveness. In the current study, the effects of chlorhexidine (CHX) and herbal mouthwash in vitro on the planktonic and sessile phases of selected strains of putative periodontal pathogens are compared. Methods: The standard strains of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Fusobacterium nucleatum were used for assessing the antimicrobial properties of the two mouthwashes. The methods of broth dilution and agar diffusion were used for assessing the minimum inhibitory concentration (MIC) in the planktonic phase. The antimicrobial effects in the sessile phase were determined by crystal violet microtiter assay. Results: The broth dilution method showed no statistical significant difference in MIC between the two mouthwashes. The agar diffusion method showed a statistically significant difference between the two types of mouthwashes against the F. nucleatum. The crystal violet microtiter assay suggested that the herbal mouthwash exhibited an antimicrobial effect even after 24 hrs on a mixed biofilm. Conclusion: Antimicrobial action exerted by the herbal mouthwash after 24 hrs was higher than that of CHX, against a biofilm constituting the three putative periodontal pathogens.

Antimicrobial and Antibiofilm Effect of Brazilian Green Propolis Aqueous Extract against Dental Anaerobic Bacteria.

Green propolis may represent a promising therapeutic alternative against dental anaerobic pathogens because of its antimicrobial action. This study aimed to evaluate the antimicrobial and antibiofilm actions of Brazilian green propolis aqueous extract (BGP-AqExt) against dental anaerobic bacteria. The minimum inhibitory concentration (MIC) and minimum microbicide concentration (MMC) of the extract were determined against the standard strains (ATCC) of Fusobacterium nucleatum, Parvimonas micra, Prevotella intermedia, Porphyromonas gingivalis and Porphyromonas endodontalis. BGP-AqExt was chemically characterized by high-performance liquid chromatography with diode-array detection (HPLC-DAD) analysis. Antibiofilm action was measured by MTT and crystal violet tests. The data were statistically analyzed by ANOVA and Tukey (5%) tests. The extract had antimicrobial action against all tested anaerobic bacteria, with an MIC value of 55 mg/mL for all bacteria, an MMC of 27.5 mg/mL for F. nucleatum and P. micra and 55 mg/mL for P. intermedia. Chemically, BGP-AqExt is composed of quercetin, gallic acid, caffeic and p-coumaric acid, drupani, kaempferol and Artepillin C. Significant reductions in biomass and metabolic action of biofilms were found after BGP-AqExt application. Therefore, BGP-AqExt has an antimicrobial and antibiofilm effect against dental anaerobic bacteria.

Cortisol Promotes Surface Translocation of Porphyromonas gingivalis.

Studies are showing that the stress hormone cortisol can reach high levels in the gingival sulcus and induce shifts in the metatranscriptome of the oral microbiome. Interestingly, it has also been shown that cortisol can influence expression levels of Type IX Secretion System (T9SS) genes involved in gliding motility in bacteria belonging to the phylum Bacteroidota. The objective of this study was to determine if cortisol impacts gene expression and surface translocation of Porphyromonas gingivalis strain W50. To conduct these experiments, P. gingivalis was stabbed to the bottom of soft agar plates containing varying cortisol concentrations (0 μM, 0.13 μM, 1.3 μM, and 13 μM), and surface translocation on the subsurface was observed after 48 h of incubation. The results show that when grown with certain nutrients, i.e., in rich medium with the addition of sheep blood, lactate, or pyruvate, cortisol promotes migration of P. gingivalis in a concentration-dependent manner. To begin to examine the underlying mechanisms, quantitative PCR was used to evaluate differential expression of genes when P. gingivalis was exposed to cortisol. In particular, we focused on differential expression of T9SS-associated genes, including mfa5, since it was previously shown that Mfa5 is required for cell movement and cell-to-cell interactions. The data show that mfa5 is significantly up-regulated in the presence of cortisol. Moreover, an mfa5 deletion mutant showed less surface translocation compared to the wild-type P. gingivalis in the presence of cortisol, and the defects of the mfa5 deletion mutant were restored by complementation. Overall, cortisol can stimulate P. gingivalis surface translocation and this coincides with higher expression levels of T9SS-associated genes, which are known to be essential to gliding motility. Our findings support a high possibility that the stress hormone cortisol from the host can promote surface translocation and potentially virulence of P. gingivalis.

An in vitro study on the degradation of multispecies biofilm of periodontitis-related microorganisms by bovine trypsin.

To investigate the degradation effect of bovine trypsin on multispecies biofilm of periodontitis-related bacteria and to provide an experimental reference for exploring new methods for controlling biofilms of periodontitis-related microorganisms, the multispecies biofilm of periodontitis-related microorganisms was established. Standard strains of Porphyromonas gingivalis, Fusobacterium nucleatum subsp. polymorpha, Actinomyces viscosus, and Aggregatibacter actinomycetemcomitans were co-cultured to form the biofilm. The experimental groups were treated with bovine trypsin, distilled water was applied as the blank control group, and phosphate saline buffer (pH = 7.4) as the negative control group. Morphological observation and quantitative analysis of extracellular polymeric substances (EPS), live bacteria, and dead bacteria were conducted using a laser confocal microscope. The morphological changes of EPS and bacteria were also observed using a scanning electron microscope. The results of morphological observations of modeling were as follows. EPS aggregated as agglomerates, and bacteria flora were wrapped by them, showing a three-dimensional network structure, and channel-like structures were inside the biofilm. Live bacteria were distributed on the surface of the EPS or embedded in them, dead bacteria aggregated between live flora and the bottom layer of biofilms. After being treated with bovine trypsin, the three-dimensional network structure and the channel-like structure disappeared, and the EPS and live and dead bacteria decreased. Quantitative analysis results are as follows. When biofilm was treated for 30 s, 1 min, and 3 min, the minimum effective concentrations of bovine trypsin to reduce EPS were 2 mg/ml (P < 0.05), 0.5 mg/ml (P < 0.05), and 0.25 mg/ml (P < 0.05), respectively. The minimum effective concentrations of bovine trypsin to reduce the live or dead bacteria were 2 mg/ml (P < 0.05), 0.5 mg/ml (P < 0.05), and 0.5 mg/ml (P < 0.05), respectively. There was no significant difference in the ratio of live/dead bacteria after the biofilm was treated for 30 s with bovine trypsin at the concentration of 0.25, 0.5, 1, and 2 mg/ml (P > 0.05), and the minimum effective concentration to reduce the ratio of live bacteria/dead bacteria was 0.25 mg/ml (P < 0.05) after treatment for 1 min and 3 min. Therefore, bovine trypsin can destroy biofilm structure, disperse biofilm and bacteria flora, and reduce the EPS and bacterial biomass, which are positively correlated with the application time and concentration.

Polymicrobial synergy stimulates Porphyromonas gingivalis survival and gingipain expression in a multi-species subgingival community

BackgroundDysbiosis in subgingival microbial communities, resulting from increased inflammatory transudate from the gingival tissues, is an important factor in initiation and development of periodontitis. Dysbiotic communities are characterized by increased numbers of bacteria that exploit the serum-like transudate for nutrients, giving rise to a proteolytic community phenotype. Here we investigate the contribution of interactions between members of a sub-gingival community to survival and development of virulence in a serum environment—modelling that in the subgingival pocket.MethodsGrowth and proteolytic activity of three Porphyromonas gingivalis strains in nutrient broth or a serum environment were assessed using A600 and a fluorescent protease substrate, respectively. Adherence of P. gingivalis strains to serum-coated surfaces was studied with confocal microscopy and 2D-gel electrophoresis of bacterial supernatants used to investigate extracellular proteins. A model multi-species sub-gingival community containing Fusobacterium nucleatum, Streptococcus constellatus, Parvimonas micra with wild type or isogenic mutants of P. gingivalis was then created and growth and proteolytic activity in serum assessed as above. Community composition over time was monitored using culture techniques and qPCR.ResultsThe P. gingivalis strains showed different growth rates in nutrient broth related to the level of proteolytic activity (largely gingipains) in the cultures. Despite being able to adhere to serum-coated surfaces, none of the strains was able to grow alone in a serum environment. Together in the subgingival consortium however, all the included species were able to grow in the serum environment and the community adopted a proteolytic phenotype. Inclusion of P. gingivalis strains lacking gingipains in the consortium revealed that community growth was facilitated by Rgp gingipain from P. gingivalis.ConclusionsIn the multi-species consortium, growth was facilitated by the wild-type and Rgp-expressing strains of P. gingivalis, suggesting that Rgp is involved in delivery of nutrients to the whole community through degradation of complex protein substrates in serum. Whereas they are constitutively expressed by P. gingivalis in nutrient broth, gingipain expression in the model periodontal pocket environment (serum) appeared to be orchestrated through signaling to P. gingivalis from other members of the community, a phenomenon which then promoted growth of the whole community.