Background and objectivePorphyromonas gingivalis is a keystone pathogen in the onset and progression of periodontitis. Its pathogenicity has been related to its presence and survival within the subgingival biofilm. The aim of the present study was to compare the genome-wide transcription activities of P. gingivalis in biofilm and in planktonic growth, using microarray technology.Material and methodsP. gingivalis ATCC 33277 was incubated in multi-well culture plates at 37°C for 96 hours under anaerobic conditions using an in vitro static model to develop both the planktonic and biofilm states (the latter over sterile ceramic calcium hydroxyapatite discs). The biofilm development was monitored by Confocal Laser Scanning Microscopy (CLSM) and Scanning Electron Microscopy (SEM). After incubation, the bacterial cells were harvested and total RNA was extracted and purified. Three biological replicates for each cell state were independently hybridized for transcriptomic comparisons. A linear model was used for determining differentially expressed genes and reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to confirm differential expression. The filtering criteria of ≥ ±2 change in gene expression and significance p-values of <0.05 were selected.ResultsA total of 92 out of 1,909 genes (4.8%) were differentially expressed by P. gingivalis growing in biofilm compared to planktonic. The 54 up-regulated genes in biofilm growth were mainly related to cell envelope, transport, and binding or outer membranes proteins. Thirty-eight showed decreased expression, mainly genes related to transposases or oxidative stress.ConclusionThe adaptive response of P. gingivalis in biofilm growth demonstrated a differential gene expression.

Background Previous research findings support an antimicrobial effect of polyphenols against a variety of pathogens, but there is no evidence of this effect against periodontal pathogens in complex biofilms. The purpose of this study was to evaluate the antimicrobial activity of red wine and oenological extracts, rich in polyphenols, against the periodontal pathogens Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum and total bacteria growing in an in vitro oral biofilm static model. Methods A previously validated biofilm model, including Streptococcus oralis, Actinomyces naeslundii , Veillonella parvula , F. nucleatum , P. gingivalis and A. actinomycetemcomitans was developed on sterile hydroxyapatite discs. Red wine (and dealcoholized wine), and two polyphenols-rich extracts (from wine and grape seeds) were applied to 72 h biofilms by dipping the discs during 1 and 5 min in the wine solutions and during 30 s and 1 min in the oenological extracts. Resulting biofilms were analyzed by confocal laser scanning microscopy and viable bacteria (colony forming units/mL) were measured by quantitative polymerase chain reaction combined with propidium monoazide. A generalized linear model was constructed to determine the effect of the tested products on the viable bacterial counts of A. actinomycetemcomitans , P. gingivalis and F. nucleatum , as well on the total number of viable bacteria. Results The results showed that red wine and dealcoholized red wine caused reduction in viability of total bacteria within the biofilm, with statistically significant reductions in the number of viable P. gingivalis after 1 min ( p = 0.008) and in A. actinomycetemcomitans after 5 min of exposure ( p = 0.011) with red wine. No evidence of relevant antibacterial effect was observed with the oenological extracts, with statistically significant reductions of F. nucleatum after 30 s of exposure to both oenological extracts ( p = 0.001). Conclusions Although moderate, the antimicrobial impact observed in the total bacterial counts and counts of A. actinomycetemcomitans, P. gingivalis and F. nucleatum, encourage further investigations on the potential use of these natural products in the prevention and treatment of periodontal diseases.

BackgroundPorphyromonas gingivalis, a microorganism residing in the oral cavity within complex multispecies biofilms, is one of the keystone pathogens in the onset and progression of periodontitis. In this in vitro study, using DNA microarray, we investigate the differential gene expression of Porphyromonas gingivalis ATCC 33277 when growing in the presence or in absence of its own monospecies biofilm.ResultsApproximately 1.5% of genes (28 out of 1909 genes, at 1.5 fold change or more, p-value < 0.05) were differentially expressed by P. gingivalis cells when in the presence of a biofilm. These genes were predominantly related to the metabolism of iron, bacterial adhesion, invasion, virulence and quorum-sensing system. The results from microarrays were consistent with those obtained by RT-qPCR.ConclusionThis study provides insight on the transcriptional changes of planktonic P. gingivalis cells when growing in the presence of a biofilm. The resulting phenotypes provide information on changes occurring in the gene expression of this pathogen.

Microbial biofilm modeling has improved in sophistication and scope, although only a limited number of standardized protocols are available. This review presents an example of a biofilm model, along with its evolution and application in studying periodontal and peri-implant diseases. In 2011, the ETEP (Etiology and Therapy of Periodontal and Peri-Implant Diseases) research group at the University Complutense of Madrid developed an in vitro biofilm static model using representative bacteria from the subgingival microbiota, demonstrating a pattern of bacterial colonization and maturation similar to in vivo subgingival biofilms. When the model and its methodology were standardized, the ETEP research group employed the validated in vitro biofilm model for testing in different applications. The evolution of this model is described in this manuscript, from the mere observation of biofilm growth and maturation on static models on hydroxyapatite or titanium discs, to the evaluation of the impact of dental implant surface composition and micro-structure using the dynamic biofilm model. This evolution was based on reproducing the ideal microenvironmental conditions for bacterial growth within a bioreactor and reaching the target surfaces using the fluid dynamics mimicking the salivary flow. The development of this relevant biofilm model has become a powerful tool to study the essential processes that regulate the formation and maturation of these important microbial communities, as well as their behavior when exposed to different antimicrobial compounds.

In search for natural products with antimicrobial properties for use in the prevention and treatment of periodontitis, the purpose of this investigation was to evaluate the antimicrobial activity of two omega-3 fatty acids, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), using an in vitro multi-species subgingival biofilm model including Streptococcus oralis, Actinomyces naeslundii, Veillonella parvula, Fusobacterium nucleatum, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans. The antimicrobial activities of EPA and DHA extracts (100 µM) and the respective controls were assessed on 72 h biofilms by their submersion onto discs for 60 s. Antimicrobial activity was evaluated by quantitative polymerase chain reaction (qPCR), confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). ANOVA with Bonferroni correction was used to evaluate the antimicrobial activity of each of the fatty acids. Both DHA and EPA significantly reduced (p < 0.001 in all cases) the bacterial strains used in this biofilm model. The results with CLSM were consistent with those reported with qPCR. Structural damage was evidenced by SEM in some of the observed bacteria. It was concluded that both DHA and EPA have significant antimicrobial activity against the six bacterial species included in this biofilm model.