[The role of the microbiome in lupus and antiphospholipid syndrome]. / Die Rolle des Mikrobioms bei Lupus und Antiphospholipidsyndrom.

Systemic lupus erythematosus (SLE) and antiphospholipid syndrome are related, systemic autoimmune diseases of unclear etiology. Genetically predisposing factors are known; however, these alone cannot be decisive for the onset and severity of these diseases. This article explains the role of the bacterial microbiome in the origin and progression of these rheumatic diseases. The most recent knowledge in the field of microbiome research based on animal experimental approaches, patient cohorts and human samples is summarized. Various commensal bacteria that promote autoimmunity, so-called pathobionts, which originate from the gut, the skin and the oral cavity, are described. Additionally, their different mechanisms of action are described: Enterococcus gallinarum and Limosilactobacillus reuteri induce adaptive autoimmunity and innate type I interferon pathways via translocation from the small intestine to the liver and spleen; Bacteroides thetaiotaomicron, Actinomyces massiliensis, Pseudopropionibacterium propionicum, Corynebacterium amycolatum, Ruminococcus gnavus and Roseburia intestinalis lead to the formation of pathogenic T­cell and autoantibody responses via the cross-reactivity with autoantigens (Ro60, dsDNA and ß2 glycoprotein I). Finally, potential future treatment approaches are also discussed, such as immunization against certain pathobionts or the targeted modulation of the microbiome via dietary approaches, which can successfully reduce autoimmune pathologies in animal models.

Malignant T Cell Activation by a Bacillus Species Isolated from Cutaneous T-Cell Lymphoma Lesions.

Cutaneous T-cell lymphoma (CTCL) is a life-debilitating malignancy of lymphocytes homing to the skin. Although CTCL is thought to arise from a combination of genetic, epigenetic, and environmental factors, specific triggers are unclear. The skin is colonized by a unique microbiota and is heavily influenced by its interactions. We hypothesized that adaptive immune responses to skin commensals lead to clonal T-cell proliferation and transformation in the appropriate genetic background. We therefore collected lesional and nonlesional skin microbiota from patients with CTCL to study T cell interactions using skin T cell explants and peripheral, skin-homing CD4+ T cells. By various methods, we identified Bacillus safensis in CTCL lesions, a rare human commensal in healthy skin, and showed that it can induce malignant T cell activation and cytokine secretion. Taken together, our data suggest microbial triggers in the skin microbiota of patients with CTCL as potential instigators of tumorigenesis.

Skin Deep: The Role of the Microbiota in Cutaneous Autoimmunity.

The skin microbiota is thought to possibly contribute to the pathogenesis of skin autoimmune diseases. The gut microbiota affects systemically the development and function of the immune system, thereby potentially influencing cutaneous autoimmunity as well. In this paper, we review the role of the gut and skin microbiota in cutaneous autoimmune diseases. Besides direct inflammatory effects at the skin barrier, microbiota may contribute to the pathogenesis of skin autoimmune diseases by metabolites, recall immune cell responses, and permeation of antigens to the subepidermal space. Skin and gut barrier dysfunction may represent a common pathophysiologic process allowing microbiota or its particles to promote autoimmune diseases at barrier surfaces.

Differential Response of Oral Mucosal and Gingival Cells to Corynebacterium durum, Streptococcus sanguinis, and Porphyromonas gingivalis Multispecies Biofilms.

Polymicrobial interactions with oral mucosal surfaces determine the health status of the host. While a homeostatic balance provides protection from oral disease, a dysbiotic polymicrobial community promotes tissue destruction and chronic oral diseases. How polymicrobial communities transition from a homeostatic to a dysbiotic state is an understudied process. Thus, we were interested to investigate this ecological transition by focusing on biofilm communities containing high abundance commensal species and low abundance pathobionts to characterize the host-microbiome interactions occurring during oral health. To this end, a multispecies biofilm model was examined using the commensal species Corynebacterium durum and Streptococcus sanguinis and the pathobiont Porphyromonas gingivalis. We compared how both single and multispecies biofilms interact with different oral mucosal and gingival cell types, including the well-studied oral keratinocyte cell lines OKF4/TERT-1and hTERT TIGKs as well as human primary periodontal ligament cells. While single species biofilms of C. durum, S. sanguinis, and P. gingivalis are all characterized by unique cytokine responses for each species, multispecies biofilms elicited a response resembling S. sanguinis single species biofilms. One notable exception is the influence of P. gingivalis upon TNF-α and Gro-α production in hTERT TIGKs cells, which was not affected by the presence of other species. This study is also the first to examine the host response to C. durum. Interestingly, C. durum yielded no notable inflammatory responses from any of the tested host cells, suggesting it functions as a true commensal species. Conversely, S. sanguinis was able to induce expression and secretion of the proinflammatory cytokines IL-6 and IL-8, demonstrating a much greater inflammatory potential, despite being health associated. Our study also demonstrates the variability of host cell responses between different cell lines, highlighting the importance of developing relevant in vitro models to study oral microbiome-host interactions.

Targeted Swabbing of Implant-Associated Biofilm Formation-A Staining-Guided Sampling Approach for Optimizing Routine Microbiological Diagnostics.

Background: Swabbing of implants removed from potentially infected sites represents a time saving and ubiquitously applicable alternative to sonication approaches. The latter bears an elevated risk of processing related contaminations due to the high number of handling steps. Since biofilms are usually invisible to the naked eye, adequate swabbing relies on the chance of hitting the colonized area on the implant. A targeted directed swabbing approach could overcome this detriment. Method: Three dyes were tested at different concentrations for their toxicity on biofilm-associated cells of S. epidermidis, the species most frequently identified as a causative agent of implant-associated infections. Results: Malachite green (0.2%) delivered the highest bacterial recovery rates combined with the best results in biofilm visualization. Its suitability for diagnostic approaches was demonstrated for smooth and rough implant surfaces. Biofilm-covered areas were successfully visualized. Conclusion: Subsequent targeted swab-sampling resulted in a significantly increased bacterial recovery rate compared to a dye-free "random swabbing" diagnostic approach.

Synergism between Corynebacterium and Streptococcus sanguinis reveals new interactions between oral commensals.

The oral microbiome engages in a diverse array of highly sophisticated ecological interactions that are crucial for maintaining symbiosis with the host. Streptococci and corynebacteria are among the most abundant oral commensals and their interactions are critical for normal biofilm development. In this study, we discovered that Streptococcus sanguinis specifically responds to the presence of Corynebacterium durum by dramatically altering its chain morphology and improving its overall fitness. By employing gas chromatography-mass spectrometry (GC-MS) analysis, specific fatty acids were identified in C. durum supernatants that are responsible for the observed effect. Membrane vesicles (MVs) containing these fatty acids were isolated from C. durum supernatants and were able to replicate the chain morphology phenotype in S. sanguinis, suggesting MV as a mediator of interspecies interactions. Furthermore, S. sanguinis responds to C. durum lipids by decreasing the expression of key FASII genes involved in fatty acid synthesis. Several of these genes are also essential for the chain elongation phenotype, which implicates a regulatory connection between lipid metabolism and chain elongation. In addition, C. durum was found to affect the growth, cell aggregation, and phagocytosis of S. sanguinis, revealing a complex association of these species that likely supports oral commensal colonization and survival.

Pyruvate secretion by oral streptococci modulates hydrogen peroxide dependent antagonism.

Many commensal oral streptococci generate H2O2 via pyruvate oxidase (SpxB) to inhibit the growth of competing bacteria like Streptococcus mutans, a major cariogenic species. In Streptococcus sanguinis SK36 (SK36) and Streptococcus gordonii DL1 (DL1), spxB expression and H2O2 release are subject to carbon catabolite repression by the catabolite control protein A (CcpA). Surprisingly, ccpA deletion mutants of SK36 and DL1 fail to inhibit S. mutans despite their production of otherwise inhibitory levels of H2O2. Using H2O2-deficient spxB deletion mutants of SK36 and DL1, it was subsequently discovered that both strains confer protection in trans to other bacteria when H2O2 is added exogenously. This protective effect depends on the direct detoxification of H2O2 by the release of pyruvate. The pyruvate dependent protective effect is also present in other spxB-encoding streptococci, such as the pneumococcus, but is missing from spxB-negative species like S. mutans. Targeted and transposon-based mutagenesis revealed Nox (putative H2O-forming NADH dehydrogenase) as an essential component required for pyruvate release and oxidative protection, while other genes such as sodA and dps play minor roles. Furthermore, pyruvate secretion is only detectable in aerobic growth conditions at biofilm-like cell densities and is responsive to CcpA-dependent catabolite control. This ability of spxB-encoding streptococci reveals a new facet of the competitive interactions between oral commensals and pathobionts and provides a mechanistic basis for the variable levels of inhibitory potential observed among H2O2-producing strains of commensal oral streptococci.

Antibiotics release from cement spacers used for two-stage treatment of implant-associated infections after total joint arthroplasty.

Two-stage revision arthroplasty is the treatment of choice for periprosthetic infection, a serious complication after knee or hip arthroplasty. Our prospective clinical trial aimed to investigate the concentrations of gentamicin and vancomycin in wound exudate and tissue in two-stage revision arthroplasty. Wound exudate and periprosthetic membrane samples were collected from 18 patients (10 hip and eight knee patients), who were due for two-stage treatment after a periprosthetic joint infection. Samples were taken during insertion of antibiotic-impregnated spacers and after their removal. The concentrations of gentamicin and vancomycin in wound exudates and adjacent tissue were analyzed using high-performance liquid chromatography mass spectrometry. Average time period of spacer implantation was 13.6 weeks (9.3-22.6 weeks). The concentration of vancomycin in wound exudate decreased from a median of 43.28 µg/mL (0.28-261.22) after implantation to 0.46 µg/mL (0.13-37.47) after the removal of the spacer. In the adjacent tissue, vancomycin concentration was mainly undetectable prior to spacer implantation (0.003 µg/g [0.003-0.261]) and increased to 0.318 µg/g [0.024-484.16] at the time of spacer removal. This was also observed for gentamicin in the tissue of patients who previously had cement-free implants (0.008 µg/g [0.008-0.087] vs. 0.164 µg/g [0.048-71.75]) while in the tissue of patients with previously cemented prosthesis, baseline concentration was already high (8.451 µg/g [0.152-42.926]). Despite the rapid decrease in antibiotics release from spacer cement observed in vitro, in vivo antibiotics are much longer detectable, especially in the adjacent soft tissue. © 2018 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials Published By Wiley Periodicals, Inc. J Biomed Mater Res B Part B, 2019. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1587-1597, 2019.

Live and let die: Hydrogen peroxide production by the commensal flora and its role in maintaining a symbiotic microbiome.

The majority of commensal oral streptococci are able to generate hydrogen peroxide (H2 O2 ) during aerobic growth, which can diffuse through the cell membrane and inhibit competing species in close proximity. Competing H2 O2 production is mainly dependent upon the pyruvate oxidase SpxB, and to a lesser extent the lactate oxidase LctO, both of which are important for energy generation in aerobic environments. Several studies point to a broad impact of H2 O2 production in the oral environment, including a potential role in biofilm homeostasis, signaling, and interspecies interactions. Here, we summarize the current research regarding oral streptococcal H2 O2 generation, resistance mechanisms, and the ecological impact of H2 O2 production. We also discuss the potential therapeutic utility of H2 O2 for the prevention/treatment of dysbiotic diseases as well as its potential role as a biomarker of oral health.

Distinct Regulatory Role of Carbon Catabolite Protein A (CcpA) in Oral Streptococcal spxB Expression.

Pyruvate oxidase (SpxB)-dependent H2O2 production is under the control of carbon catabolite protein A (CcpA) in the oral species Streptococcus sanguinis and Streptococcus gordonii Interestingly, both species react differently to the presence of the preferred carbohydrate source glucose. S. gordonii CcpA-dependent regulation of spxB follows classical carbon catabolite repression. Conversely, spxB expression in S. sanguinis is not influenced by glucose but is repressed by CcpA. Here, we constructed strains expressing the heterologous versions of CcpA or the spxB promoter region to learn if the distinct regulation of spxB expression is transferable from S. gordonii to S. sanguinis and vice versa. While cross-species binding of CcpA to the spxB promoter is conserved in vitro, we were unable to swap the species-specific regulation. This suggests that a regulatory mechanism upstream of CcpA most likely is responsible for the observed difference in spxB expression. Moreover, the overall ecological significance of differential spxB regulation in the presence of various glucose concentrations was tested with additional oral streptococcus isolates and demonstrated that carbohydrate-dependent and carbohydrate-independent mechanisms exist to control expression of spxB in the oral biofilm. Overall, our data demonstrate the unexpected finding that metabolic pathways between two closely related oral streptococcal species can be regulated differently despite an exceptionally high DNA sequence identity.IMPORTANCE Polymicrobial diseases are the result of interactions among the residential microbes, which can lead to a dysbiotic community. Streptococcus sanguinis and Streptococcus gordonii are considered commensal species that are present in the healthy dental biofilm. Both species are able to produce significant amounts of H2O2 via the enzymatic action of the pyruvate oxidase SpxB. H2O2 is able to inhibit species associated with oral diseases. SpxB and its gene-regulatory elements present in both species are highly conserved. Nonetheless, a differential response to the presence of glucose was observed. Here, we investigate the mechanisms that lead to this differential response. Detailed knowledge of the regulatory mechanisms will aid in a better understanding of oral disease development and how to prevent dysbiosis.

Plasticity of the Pyruvate Node Modulates Hydrogen Peroxide Production and Acid Tolerance in Multiple Oral Streptococci.

Commensal Streptococcus sanguinis and Streptococcus gordonii are pioneer oral biofilm colonizers. Characteristic for both is the SpxB-dependent production of H2O2, which is crucial for inhibiting competing biofilm members, especially the cariogenic species Streptococcus mutans H2O2 production is strongly affected by environmental conditions, but few mechanisms are known. Dental plaque pH is one of the key parameters dictating dental plaque ecology and ultimately oral health status. Therefore, the objective of the current study was to characterize the effects of environmental pH on H2O2 production by S. sanguinis and S. gordoniiS. sanguinis H2O2 production was not found to be affected by moderate changes in environmental pH, whereas S. gordonii H2O2 production declined markedly in response to lower pH. Further investigation into the pyruvate node, the central metabolic switch modulating H2O2 or lactic acid production, revealed increased lactic acid levels for S. gordonii at pH 6. The bias for lactic acid production at pH 6 resulted in concomitant improvement in the survival of S. gordonii at low pH and seems to constitute part of the acid tolerance response of S. gordonii Differential responses to pH similarly affect other oral streptococcal species, suggesting that the observed results are part of a larger phenomenon linking environmental pH, central metabolism, and the capacity to produce antagonistic amounts of H2O2IMPORTANCE Oral biofilms are subject to frequent and dramatic changes in pH. S. sanguinis and S. gordonii can compete with caries- and periodontitis-associated pathogens by generating H2O2 Therefore, it is crucial to understand how S. sanguinis and S. gordonii adapt to low pH and maintain their competitiveness under acid stress. The present study provides evidence that certain oral bacteria respond to environmental pH changes by tuning their metabolic output in favor of lactic acid production, to increase their acid survival, while others maintain their H2O2 production at a constant level. The differential control of H2O2 production provides important insights into the role of environmental conditions for growth competition of the oral flora.

Influence of Probiotic Culture Supernatants on In Vitro Biofilm Formation of Staphylococci.

BACKGROUND: The effects of cell-free culture supernatants of probiotic Lactobacillus rhamnosus GG and Streptococcus salivarius K12 on replication and biofilm forming of Staphylococcus aureus and S. epidermidis were assessed in vitro. METHODS: S. aureus and S. epidermidis strains were exposed to cell-free culture supernatants of L. rhamnosus GG and S. salivarius K12 at different concentrations starting at 0, 4, and 24 h after the onset of incubation. Bacterial amplification was measured on microplate readers, as well as biofilm growth after safranine staining. Scanning electron microscopy was performed for visualization of biofilm status. RESULTS: The S. salivarius K12 culture supernatant not only reduced or prevented the formation and maturation of fresh biofilms but even caused a reduction of preformed S. epidermidis biofilms. The L. rhamnosus GG culture supernatant did not show clear inhibitory effects regardless of concentration or time of addition of supernatant, and even concentration-depending promotional effects on the planktonic and biofilm growth of S. aureus and S. epidermidis were observed. CONCLUSION: In particular, the inhibitory effects of the S. salivarius K12 culture supernatant on the formation of staphylococcal biofilms are of potential relevance for biofilm-associated diseases and should be further assessed by in vivo infection models.

Murein Hydrolase LytF of Streptococcus sanguinis and the Ecological Consequences of Competence Development.

The overall health of the oral cavity is dependent on proper homeostasis between health-associated bacterial colonizers and bacteria known to promote dental caries. Streptococcus sanguinis is a health-associated commensal organism, a known early colonizer of the acquired tooth pellicle, and is naturally competent. We have shown that LytF, a competence-controlled murein hydrolase, is capable of inducing the release of extracellular DNA (eDNA) from oral bacteria. Precipitated LytF and purified LytF were used as treatments against planktonic cultures and biofilms. Larger amounts of eDNA were released from cultures treated with protein samples containing LytF. Additionally, LytF could affect biofilm formation and cellular morphology. Biofilm formation was significantly decreased in the lytF-complemented strain, in which increased amounts of LytF are present. The same strain also exhibited cell morphology defects in both planktonic cultures and biofilms. Furthermore, the LytF cell morphology phenotype was reproducible in wild-type cells using purified LytF protein. In sum, our findings demonstrate that LytF can induce the release of eDNA from oral bacteria, and they suggest that, without proper regulation of LytF, cells display morphological abnormalities that contribute to biofilm malformation. In the context of the oral biofilm, LytF may play important roles as part of the competence and biofilm development programs, as well as increasing the availability of eDNA.IMPORTANCEStreptococcus sanguinis, a commensal organism in the oral cavity and one of the pioneer colonizers of the tooth surface, is associated with the overall health of the oral environment. Our laboratory showed previously that, under aerobic conditions, S. sanguinis can produce H2O2 to inhibit the growth of bacterial species that promote dental caries. This production of H2O2 by S. sanguinis also induces the release of eDNA, which is essential for proper biofilm formation. Under anaerobic conditions, S. sanguinis does not produce H2O2 but DNA is still released. Determining how S. sanguinis releases DNA is thus essential to understand biofilm formation in the oral cavity.

Augmented recovery of microorganisms from swabs by homogenization: a novel standardizable high-throughput approach.

A new approach introducing a quantitative and standardizable step into sample processing was evaluated by homogenizing in vitro inoculated swab tips with Precellys 24 high-throughput homogenizer. Recovery of microorganisms from homogenized swab tips was significantly higher as compared to conventional processing methods. Thus, swab homogenization is a promising approach introducing a new quality in microbial analysis.

Improved microbiological diagnostic due to utilization of a high-throughput homogenizer for routine tissue processing.

Tissue specimens are valuable materials for microbiological diagnostics and require swift and accurate processing. Established processing methods are complex, labor intensive, hardly if at all standardizable, and prone to incorporate contaminants. To improve analyses from tissue samples in routine microbiological diagnostics, by facilitating, fastening, and standardizing processing as well as increasing the microbial yield, performance of Precellys 24 high-throughput tissue homogenizer was evaluated. Therefore, tissue samples were artificially inoculated with Staphylococcus aureus, Escherichia coli, and Candida albicans in 3 different ways on the surface and within the material. Microbial yield from homogenized samples was compared to direct plating method. Further, as proof of principle, routine tissue samples from knee and hip endoprosthesis infections were analyzed. The process of tissue homogenization with Precellys 24 homogenizer is easy and fast to perform and allows for a high degree of standardization. Microbial yield after homogenization was significantly higher as compared to conventional plating technique.

Influence of antimicrobial coatings of vacuum-assisted closure dressings on methicillin-resistant Staphylococcus aureus growth kinetics: an in vitro study.

BACKGROUND: Silver-containing negative-pressure wound therapy (NPWT) foam dressings should reduce the microbial load of infected surgical sites and thereby promote healing. The effects of silver and an experimental copper coating of NPWT dressings on the growth kinetics of methicillin-resistant Staphylococcus aureus (MRSA) were investigated with a focus on the importance of the initial bacterial load and the incubation time. METHODS: Commercially available foam samples with and without silver coating were inoculated in vitro with six MRSA suspensions differing in bacterial concentration (1.85×10(3) to 1.85×10(8) colony-forming units per milliliter [CFU/mL]). In a second series, uncoated, silver-containing and experimental copper-coated foam samples were inoculated with one MRSA suspension (1.85×10(6) CFU/mL). The MRSA viable counts in the entrapped fluid were evaluated statistically after 1, 3, 7, and 14 d incubation. RESULTS: Silver foam samples reduced MRSA counts by two decimal powers compared with the corresponding inocula. With respect to the uncoated samples, silver coating reduced MRSA concentrations by up to 7 logs, which was significant (p≤0.045) for all groups except the one with the highest MRSA concentration. The antibacterial effect of copper became apparent only after 7 d, but thereafter was far more pronounced than the effects of silver (p<0.01 after 14 d). CONCLUSIONS: Antimicrobial-coated foam dressings showed significant in vitro antibacterial properties and thus could be advantageous in the treatment of MRSA-infected incisions.

Heterologous expression of sahH reveals that biofilm formation is autoinducer-2-independent in Streptococcus sanguinis but is associated with an intact activated methionine cycle.

Numerous studies have claimed deleterious effects of LuxS mutation on many bacterial phenotypes, including bacterial biofilm formation. Genetic complementation mostly restored the observed mutant phenotypes to WT levels, leading to the postulation that quorum sensing via a family of molecules generically termed autoinducer-2 (AI-2) is essential for many phenotypes. Because LuxS mutation has dual effects, this hypothesis needs to be investigated into the details for each bacterial species. In this study we used S. sanguinis SK36 as a model biofilm bacterium and employed physiological characterization and transcriptome approaches on WT and luxS-deficient strains, in combination with chemical, luxS, and sahH complementation experiments. SahH enables a direct conversion of SAH to homocysteine and thereby restores the activated methionine cycle in a luxS-negative background without formation of the AI-2 precursor 4,5-dihydroxy-2,3-pentanedione. With this strategy we were able to dissect the individual contribution of LuxS and AI-2 activity in detail. Our data revealed that S. sanguinis biofilm formation is independent from AI-2 substance pools and is rather supported by an intact activated methyl cycle. Of 216 differentially transcribed genes in the luxS mutant, 209 were restored by complementation with a gene encoding the S-adenosylhomocysteine hydrolase. Only nine genes, mainly involved in natural competence, were directly affected by the AI-2 quorum-sensing substance pool. Cumulatively, this suggested that biofilm formation in S. sanguinis is not under control of AI-2. Our study suggests that previously evaluated LuxS mutants in other species need to be revisited to resolve the precise contribution of AI-2 substance pools and the methionine pathways.

A five-species transcriptome array for oral mixed-biofilm studies.

BACKGROUND: Oral polymicrobial interactions and biofilm formation are associated with initiation and progression of caries, gingivitis, and periodontitis. Transcriptome studies of such interactions, allowing a first mechanistic insight, are hampered by current single-species array designs. METHODOLOGY/PRINCIPAL FINDINGS: In this study we used 385 K NimbleGene™ technology for design and evaluation of an array covering the full genomes of 5 important physiological-, cariogenic-, and periodontitis-associated microorganisms (Streptococcus sanguinis, Streptococcus mutans, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis). Array hybridization was done with cDNA from cultures grown for 24 h anaerobically. Single species experiments identified cross-species hybridizing array probes. These probes could be neglected in a mixed-species experimental setting without the need to exclude the whole genes from the analysis. Between 69% and almost 99% of the genomes were actively transcribed under the mono-species planktonic, monolayer, and biofilm conditions. The influence of Streptococcus mitis (not represented on the array) on S. mutans gene transcription was determined as a test for a dual-species mixed biofilm setup. Phenotypically, under the influence of S. mitis an increase in S. mutans biofilm mass and a decrease in media pH-value were noticed, thereby confirming previously published data. Employing a stringent cut-off (2-fold, p<0.05), 19 S. mutans transcripts were identified with increased abundance, and 11 with decreased abundance compared to a S. mutans mono-species biofilm. Several of these genes have previously been found differentially regulated under general and acid stress, thereby confirming the value of this array. CONCLUSIONS/SIGNIFICANCE: This new array allows transcriptome studies on multi-species oral biofilm interactions. It may become an important asset in future oral biofilm and inhibitor/therapy studies.

Polyphenols from Myrothamnus flabellifolia Welw. inhibit in vitro adhesion of Porphyromonas gingivalis and exert anti-inflammatory cytoprotective effects in KB cells.

AIM: Identification of anti-adhesive plant extracts against cell surface binding of Porphyromonas gingivalis and underlying mechanisms; investigation of potential cytoprotective effects of anti-adhesive extract on KB cells. MATERIALS AND METHODS: Polyphenol-enriched extract, fully characterized concerning flavan-3-ols and oligomeric proanthocyanidins, from Myrothamnus flabellifolia (MF), traditionally used for periodontitis, was tested for inhibition of P. gingivalis-mediated adhesion to KB cells by flow cytometry, for influence on gingipain activity (protease assay), haemagglutination and by microarray analysis for effects on bacterial transcriptome. The influence of MF on P. gingivalis-induced cytokine gene expression was monitored by RT-PCR and IL-6 titres by ELISA. RESULTS: MF (100 µg/ml) reduced P. gingivalis adhesion/invasion about 50% by interacting with bacterial OMPs. As shown by RT-PCR, fimbrillin and Arg-gingipain encoding genes were up-regulated by MF. On the protein level, inhibition (70%) of Arg-gingipain activity was observed, while the corresponding Lys-gingipain was hardly influenced. MF also inhibited haemagglutination. While exposure to P. gingivalis resulted in an increased expression of inflammation-related genes in KB cells, pre-treatment of KB cells with MF evoked cytoprotective effects concerning IL-1ß, IL-6, IL-8 and TNF-α gene expression as well as IL-6 release rates. Compounds from the plant extract belonging to the class of proanthocyanidins were shown to be responsible for the observed effects and were characterized for their respective structural features. CONCLUSIONS: While being cytoprotective, MF exerts anti-adhesive effects against P. gingivalis. Thus, MF may be useful for the prevention of P. gingivalis-associated periodontal diseases.

Setup of an in vitro test system for basic studies on biofilm behavior of mixed-species cultures with dental and periodontal pathogens.

BACKGROUND: Caries and periodontitis are important human diseases associated with formation of multi-species biofilms. The involved bacteria are intensively studied to understand the molecular basis of the interactions in such biofilms. This study established a basic in vitro single and mixed-species culture model for oral bacteria combining three complimentary methods. The setup allows a rapid screening for effects in the mutual species interaction. Furthermore, it is easy to handle, inexpensive, and reproducible. METHODS: Streptococcus mitis, S. salivarius and S. sanguinis, typical inhabitants of the healthy oral cavity, S. mutans as main carriogenic species, and Porphyromonas gingivalis, Fusobacterium nucleatum, Parvimonas micra, S. intermedius and Aggregatibacter actinomycetemcomitans as periodontitis-associated bacteria, were investigated for their biofilm forming ability. Different liquid growth media were evaluated. Safranin-staining allowed monitoring of biofilm formation under the chosen conditions. Viable counts and microscopy permitted investigation of biofilm behavior in mixed-species and transwell setups. FINDINGS: S. mitis, F. nucleatum, P. gingivalis and P. micra failed to form biofilm structures. S. mutans, S. sanguinis, S. intermedius and S. salivarius established abundant biofilm masses in CDM/sucrose. A. actinomycetemcomitans formed patchy monolayers. For in depth analysis S. mitis, S. mutans and A. actinomycetemcomitans were chosen, because i) they are representatives of the physiological-, cariogenic and periodontitis-associated bacterial flora, respectively and ii) their difference in their biofilm forming ability. Microscopic analysis confirmed the results of safranin staining. Investigation of two species combinations of S. mitis with either S. mutans or A. actinomycetemcomitans revealed bacterial interactions influencing biofilm mass, biofilm structure and cell viability. CONCLUSIONS: This setup shows safranin staining, microscopic analysis and viable counts together are crucial for basic examination and evaluation of biofilms. Our experiment generated meaningful results, exemplified by the noted S. mitis influence, and allows a fast decision about the most important bacterial interactions which should be investigated in depth.