Polybacterial Infection Research Articles

Metagenomic analysis of healthy and diseased peri-implant microbiome under different periodontal conditions: a cross-sectional study

BackgroundPeri-implantitis is a polybacterial infection that can lead to the failure of dental implant rehabilitation. This study aimed to profile the microbiome of the peri-implant plaque and estimate the effect of periodontitis on it among 40 Chinese participants with dental implant prostheses and presenting with varying peri-implant and periodontal health states.MethodsSubmucosal plaque samples were collected from four distinct clinical categories based on both their implant and periodontal health status at sampling point. Clinical examinations of dental implant and remaining teeth were carried out. Metagenomic analysis was then performed.ResultsThe microbiome of the peri-implantitis sites differed from that of healthy implant sites, both taxonomically and functionally. Moreover, the predominant species in peri-implantitis sites were slightly affected by the presence of periodontitis. T. forsythia, P. gingivalis, T. denticola, and P. endodontalis were consistently associated with peri-implantitis and inflammatory clinical parameters regardless of the presence of periodontitis. Prevotella spp. and P. endodontalis showed significant differences in the peri-implantitis cohorts under different periodontal conditions. The most distinguishing function between diseased and healthy implants is related to flagellar assembly, which plays an important role in epithelial cell invasion.ConclusionsThe composition of the peri-implant microbiome varied in the diseased and healthy states of implants and is affected by individual periodontal conditions. Based on their correlations with clinical parameters, certain species are associated with disease and healthy implants. Flagellar assembly may play a vital role in the process of peri-implantitis.

Bacteriophage entrapped chitosan microgel for the treatment of biofilm-mediated polybacterial infection in burn wounds

Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) bacteria are most commonly present in burn wound infections. Multidrug resistance (MDR) and biofilm formation make it difficult to treat these infections. Bacteriophages (BPs) are proven as an effective therapy against MDR as well as biofilm-associated wound infections. In the present work, a naturally inspired bacteriophage cocktail loaded chitosan microparticles-laden topical gel has been developed for the effective treatment of these infections. Bacteriophages against MDR S. aureus (BPSAФ1) and P. aeruginosa (BPPAФ1) were isolated and loaded separately and in combination into the chitosan microparticles (BPSAФ1-CHMPs, BPPAФ1-CHMPs, and MBP-CHMPs), which were later incorporated into the SEPINEO™ P 600 gel (BPSAФ1-CHMPs-gel, BPPAФ1-CHMPs-gel, and MBP-CHMPs-gel). BPs were characterized for their morphology, lytic activity, burst size, and hemocompatibility, and BPs belongs to Caudoviricetes class. Furthermore, BPSAФ1-CHMPs, BPPAФ1-CHMPs, and MBP-CHMPs had an average particle size of 1.19 ± 0.11, 1.42 ± 0.21, and 2.84 ± 0.28 μm, respectively, and expressed promising in vitro antibiofilm eradication potency. The ultrasound and photoacoustic imaging in infected burn wounds demonstrated improved wound healing reduced inflammation and increased oxygen saturation following treatment with BPs formulations. The obtained results suggested that the incorporation of the BPs in the MP-gel protected the BPs, sustained the BPs release, and improved the antibacterial activity.

Global Noncoding microRNA Profiling in Mice Infected with Partial Human Mouth Microbes (PAHMM) Using an Ecological Time-Sequential Polybacterial Periodontal Infection (ETSPPI) Model Reveals Sex-Specific Differential microRNA Expression.

Periodontitis (PD) is a polymicrobial dysbiotic immuno-inflammatory disease. It is more prevalent in males and has poorly understood pathogenic molecular mechanisms. Our primary objective was to characterize alterations in sex-specific microRNA (miRNA, miR) after periodontal bacterial infection. Using partial human mouth microbes (PAHMM) (Streptococcus gordonii, Fusobacterium nucleatum, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia) in an ecological time-sequential polybacterial periodontal infection (ETSPPI) mouse model, we evaluated differential mandibular miRNA profiles by using high-throughput Nanostring nCounter® miRNA expression panels. All PAHMM mice showed bacterial colonization (100%) in the gingival surface, an increase in alveolar bone resorption (p < 0.0001), and the induction of a specific immunoglobin G antibody immune response (p < 0.001). Sex-specific differences in distal organ bacterial dissemination were observed in the heart (82% male vs. 28% female) and lungs (2% male vs. 68% female). Moreover, sex-specific differential expression (DE) of miRNA was identified in PAHMM mice. Out of 378 differentially expressed miRNAs, we identified seven miRNAs (miR-9, miR-148a, miR-669a, miR-199a-3p, miR-1274a, miR-377, and miR-690) in both sexes that may be implicated in the pathogenesis of periodontitis. A strong relationship was found between male-specific miR-377 upregulation and bacterial dissemination to the heart. This study demonstrates sex-specific differences in bacterial dissemination and in miRNA differential expression. A novel PAHMM mouse and ETSPPI model that replicates human pathobiology can be used to identify miRNA biomarkers in periodontitis.

Severe polymicrobial and fungal periprosthetic osteomyelitis persisting after hip disarticulations treated with caspofungin in risk patients: a case series

BackgroundPeriprosthetic fungal infections are considered rare and opportunistic infections. Treatment is difficult, and established standards do not yet exist. The choice of the appropriate antifungal drug might affect the patient outcome.CasesAll the three cases presented showed polybacterial recurrent infection of the revision hip arthroplasty. All patients were of younger age, had multiple revisions of the endoprosthesis, each had a large partial femoral replacement greater than 40% of the femoral length, gentamycin-loaded cement, and a long anchoring distance of the used intramedullary stem. Due to the severe life-threatening infection with deep osteomyelitis, an amputation had to be performed. However, despite surgical intervention, the fungal dominated infection persisted. Finally, only the use of caspofungin allowed permanent infection control.ConclusionThe polybacterial infection is driven by the symbiosis between fungi and bacteria. Therefore, eradication of the fungus is required to achieve elimination of the bacteria. Antimycotics of the echinocandin-class, such as caspofungin, may be considered as initial treatment.

Molecular characterization of bacterial pathogens-causing snuffles in rabbits in terms of prevalence and pathogenicity

Respiratory disease or snuffling is a major problem associated with serious economic losses in the rabbit industry. The disease in rabbit is a polybacterial infection in nature, resulting from co-infection with more than one bacterial pathogen. To determine the involved bacterial pathogens in respiratory infections in terms of prevalence and pathogenicity, different specimens from rabbits with typically respiratory disorders are subjected to the bacteriological examination followed by molecular verification through RT-PCR or PCR followed by sequencing the PCR-product. The results identified Mycoplasma sp. as a major partner in mixed infection with virulent strains of P. multocida or S. aureus, suggesting that, initial infection with Mycoplasma sp creates vulnerability to subsequent infection with P. multocida or S. aureus. Gross and microscopic analysis were also applied to demonstrate lesions accompanied by the detected bacterial pathogens. In conclusion, this study described the prevalence and characteristic of respiratory illness in rabbits domesticated in Al-Sharkia Governorate, Egypt and highlighted for the first time the role of Mycoplasma sp in increasing the severity of rabbit-respiratory infections.

Characterization of Polybacterial versus Monobacterial Conjunctivitis Infections in Pediatric Subjects Across Multiple Studies and Microbiological Outcomes with Besifloxacin Ophthalmic Suspension 0.6.

IntroductionThe choice of empiric therapy for bacterial conjunctivitis should be guided by an awareness of typical causative pathogen distributions. Bacterial conjunctivitis can be polybacterial, although pediatric-specific data are lacking.MethodsThis was a post-hoc analysis of data in pediatric subjects (1–17 years) from five bacterial conjunctivitis trials evaluating besifloxacin ophthalmic solution 0.6%.ResultsOf the 730 pediatric subjects with culture-confirmed conjunctivitis, nearly one-fourth (23.6%) had polybacterial infections and three-fourths (76.4%) had monobacterial infections at baseline. In both polybacterial and monobacterial infections, the most prevalent organisms were Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus, and Streptococcus mitis/S. mitis group. In polybacterial versus monobacterial infections, S. mitis/S. mitis group (8.7% vs 4.3%; P=0.032) and Moraxella catarrhalis (4.7% vs 0.5%; P<0.001) were identified more frequently, whereas S. pneumoniae (14.0% vs 28.1%; P<0.001) was identified less frequently, as the dominant infecting species. MICs for individual species were similar for tested antibiotics regardless of polybacterial or monobacterial infection, except Staphylococcus epidermidis for which fluoroquinolone MICs were ≥3 dilutions higher for isolates of this species sourced from polybacterial compared to monobacterial infections. Treatment with besifloxacin resulted in microbial eradication in 79.1% of polybacterial and 92.3% of monobacterial infections (P≤0.005 vs vehicle).DiscussionOne in four pediatric bacterial conjunctivitis infections is polybacterial, highlighting the need for a broad-spectrum antibiotic when choosing empiric therapy.

Robust rapid-setting antibacterial liquid bandages

Bandaging is a steadfast but time-consuming component of wound care with limited technical advancements to date. Bandages must be changed and infection risk managed. Rapid-set liquid bandages are efficient alternatives but lack durability or inherent infection control. We show here that antibacterial zinc (Zn) and copper (Cu) species greatly enhance the barrier properties of the natural, waterproof, bio-adhesive polymer, shellac. The material demonstrated marked antibacterial contact properties and, in ex-vivo studies, effectively locked-in pre-applied therapeutics. When challenged in vivo with the polybacterial bovine wound infection ‘digital dermatitis’, Zn/Cu-shellac adhered rapidly and robustly over pre-applied antibiotic. The bandage self-degraded, appropriately, over 7 days despite extreme conditions (faecal slurry). Treatment was well-tolerated and clinical improvement was observed in animal mobility. This new class of bandage has promise for challenging topical situations in humans and other animals, especially away from controlled, sterile clinical settings where wounds urgently require protection from environmental and bacterial contamination.

238. Direct identification of Bacterial Species with MinION Nanopore Sequencer In Clinical Specimens Suspected of Polybacterial Infection

BackgroundConventional culture tests usually identify only a few bacterial species, which can grow well in the culture system, in the cases of polybacterial infection. 16S rRNA gene nanopore sequencing enables semi-quantitative identification of bacterial genetic materials. We aimed to evaluate usefulness of 16s rRNA gene nanopore sequencing in the cases suspected of polybacterial infection.MethodsThe research was conducted in a single university hospital for one year. Conventional bacterial culture identification and nanopore sequencing of 16s rRNA gene were carried out simultaneously for cases where polybacterial infection is strongly suspected. Blood agar plate was used for conventional culture, and Microscan (Beckman Coulter, United States) and Vitek 2 (Biomerieux, FR) automated systems were used for identification. For nanopore sequencing, 16S rRNA gene PCR was performed from the clinical specimens, and sequencing libraries were generated from the PCR products using the rapid barcoding sequencing kit (Oxford nanopore technologies, UK). MinION sequencing was performed for 1–3 hours and the generated reads were analyzed using the EPI2ME 16S BLAST workflow.ResultsSpecimens were obtained from 15 patients; 6 liver abscess, 2 psoas abscess, 2 thigh abcess, 1 paraspinal abscess, 1 mycotic aneurysm, 1 necrotizing fasciitis, 1 fingertip gangrene and 1 abscess in coccyx area. 16s rRNA gene nanopore sequencing showed monobacterial organism in 8 (53.3%) specimens and polybacterial organisms in 7 (46.6%) specimens. In three (37.5%) cases of 8 cases with monobacterial infections identified by 16s rRNA gene sequencing, no organism was grown in conventional culture, possibly due to previous antibiotic administration. Notably, among 8 cases with polybacterial infection by 16s rRNA gene nanopore sequencing test, traditional culture test showed polybacterial infection in only two (25%) cases and single bacterial organism was identified in the other 6 (75%) cases.ConclusionNanopore sequencing of 16s rRNA gene using the MinION sequencer may be useful for identification of causing microorganism and differentiation between monobacterial and polybacterial infection when polybacterial infection is suspected.Disclosures All authors: No reported disclosures.

Fournier’s Gangrene without Organ Perforation Following Docetaxel: A Rare Urological Emergency Occurrence in a Patient with Metastatic Prostate Cancer

Fournier’s gangrene (FG) which is known as necrotizing fasciitis of urogenital regions, is a rapidly progressive polybacterial infection, involving the subcutaneous and deep fascias in the urogenital region. The most common reasons are localized infections caused by colorectal, genitourinary and dermatological factors. On the other hand, its development following chemotherapy application is rare. Emergency and repetitive radical surgical debridement, wound drainage and parenteral antibiotherapy are mandatory because of high mortality rates in FG.

Abstract 2824: Modulation of oral cancer cells survival and invasiveness by fusobacteria

Abstract Oral cancer represents a significant health burden worldwide while periodontitis is a common chronic infection of the periodontium. Many studies have suggested the association between periodontal disease and oral cancer. Studies have shown that biofilms on oral cancer lesions are rich in anaerobic bacteria, known to be associated with periodontal disease. This establishes that periodontal bacteria are present in the oral cancer microenvironment and likely directly interact with cancer cells. Therefore, it is vital to understand the interactions between the oral bacteria and cancer cells in order to improve treatment outcomes. The aim of this study was to investigate the effect of periodontal bacteria on oral cancer cells. Three oral cancer cell lines (OQ01, BHY and HN) were polyinfected with 4 bacteria associated with chronic periodontitis (Tanneralla forsynthia, Treponema denticola, Porphyromonas gingivalis and Fusobacterium nucleatum). Likely because BHY and HN already have a high baseline level of IL-8, OQ01 alone showed significantly enhanced IL-8 secretion (P&amp;lt; 0.05) but enhanced TFG-β secretion was detected in all cell lines tested. Polybacterial infection of oral cancer cells also upregulated mRNA for MMP1 (40 fold) and MMP9 (more than 60 fold), which are known to enhance cancer cell invasiveness. In addition, the expression of ZEB1 (4 fold higher), an oncogene known to induce epithelial mesenchymal transition in cancer cells, and MYC, JAK1 and STAT3, oncogenes involved in cell survival, were all significantly enhanced in polybacterial infected cancer cells. Further analysis using OQ01 cells in a single bacterial infection showed that F. nucleatum alone had the same or greater effect (4 fold higher IL-8 secretion) as the polybacterial infection with all 4 bacteria. All three strains of F. nucleatum tested enhanced the invasiveness of the oral cancer cells in vitro. Interestingly, Fusobacterial supernatant alone was enough to induce the same invasive phenotype as live bacteria in OQ01. These results suggest that F. nucleatum is the main periodontal pathogen responsible for inducing an invasive phenotype in these oral cancer cells. For in vivo studies, we used a mouse model of infection-associated oral tumorigenesis, which included the induction of chronic periodontitis by P. gingivalis and F. nucleatum and the administration of an oral carcinogen 4-nitroquinoline-1-oxide (4NQO). The results demonstrated that infection with Fusobacteria and P. gingivalis promoted oral cancer progression and enhanced invasion compared to controls. Our data also showed that Fusobacteria enhanced both survival and invasiveness of oral cancer cells. Collectively, this is the first study revealing the interaction between oral cancer cells and multiple periodontal bacteria. The modulation of the tumor microenvironment by oral bacteria has the potential to identify target genes for cancer treatment and improve patients’ prognosis. Citation Format: Amani Harrandah, Sasanka S. Chukkapalli, William JR Dunn, Ann Progulske-Fox, Edward K. Chan. Modulation of oral cancer cells survival and invasiveness by fusobacteria [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2824.

Noma (cancrum oris): An unresolved global challenge.

Noma (canrum oris) is a mutilating necrotizing disease of uncertain etiology, but it is accepted that it is caused primarily by a polybacterial infection with secondary ischemia. The consequent necrotizing fasciitis, myonecrosis, and osteonecrosis results in destruction of facial structures with severe functional impairment and disfigurement. It most frequently affects children, particularly in sub‐Saharan Africa, who are malnourished or debilitated by systemic conditions including but not limited to malaria, measles, and tuberculosis; and less frequently debilitated HIV‐seropositive subjects. In the vast majority of cases, in susceptible subjects, noma is preceded by necrotizing stomatitis. However, it has been reported, albeit rarely, that noma can arise without any preceding oral lesions being observed. Noma is not recurrent and is not transmissible.

Impaired innate immune signaling due to combined Toll-like receptor 2 and 4 deficiency affects both periodontitis and atherosclerosis in response to polybacterial infection.

Plasma membrane-associated Toll-like receptor (TLR2 and TLR4) signaling contributes to oral microbe infection-induced periodontitis and atherosclerosis. We recently reported that either TLR2 or TLR4 receptor deficiency alters recognition of a consortium of oral pathogens, modifying host responses in periodontitis and atherosclerosis. We evaluated the effects of combined TLR2-/-TLR4-/- double knockout mice on innate immune signaling and induction of periodontitis and atherosclerosis after polybacterial infection with Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia and Fusobacterium nucleatum in a mouse model. Multispecies infections established gingival colonization in all TLR2-/-TLR4-/- mice and induced production of bacterial-specific IgG antibodies. In combined TLR2-/-TLR4-/- deficiency there was, however, reduced alveolar bone resorption and mild gingival inflammation with minimal migration of junctional epithelium and infiltration of inflammatory cells. This indicates a central role for TLR2 and TLR4 in periodontitis. Atherosclerotic plaque progression was markedly reduced in infected TLR2-/-TLR4-/- mice or in heterozygotes indicating a profound effect on plaque growth. However, bacterial genomic DNA was detected in multiple organs in TLR2-/-TLR4-/- mice indicating an intravascular dissemination from gingival tissue to heart, aorta, kidney and lungs. TRL2 and TLR4 were dispensable for systemic spread after polybacterial infections but TLR2 and 4 deficiency markedly reduces atherosclerosis induced by oral bacteria.

Abstract 5139: F. nucleatum induces invasive phenotypes in oral cancer cells in vitro

Abstract Oral cancer is one of the most common cancers that affect public health with 500,000 new cases diagnosed yearly worldwide. The behavior of oral cancer might be affected by bacterial pathogens present in the oral cavity. The association between periodontitis, a chronic infection that affect the periodontium, and oral cancer have been reported in many studies. However, the effect of interaction between periodontal bacteria and oral cancer cells on cancer progression and aggressiveness is not well studied. Therefore, we aim to investigate the effect of four major periodontal bacteria on cancer cell pathology and treatment response. Three oral cancer cell lines OQ01, BHY and HN were used. Periodontal infection was represented by four periodontal bacteria Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum. These bacteria were used to infect the oral cancer cells as polybacterial infection. Further analysis was done on OQ01 with monobacterial infection. The level of different cytokines (IL8 and TGF-β) were quantified in the culture supernatants after 6 and 24 h using ELISA. After RNA isolation from cell lysate, the relative changes in expression of six different oncogenes (MMP1, MMP9, MYC, JAk1, ZEB1, and STAT3) were measured using RT-PCR. To further investigate the role of F. nucleatum in inducing autophagy, three strains of F. nucleatum (Vincinti, Polymorphum and Periodontium) were used and the levels of autophagy markers (ATG7, ATG12 and ULK1) were measured after 6 and 24 hours. The effect of F. nucleatum on cancer cell invasion was was done starting from 4 h after infection. Statistical analysis was done using two-tailed Student's t-test OQ01 alone showed significantly enhance IL-8 secretion after polyinfection (P= 0.0003); however, enhanced TFG-β secretion was detected in all cell lines tested (P= 0.004). Polybacterial infection of oral cancer cell lines also upregulated MMP1 and MMP9 (P= 0.002) (known to enhance cancer cell invasiveness), ZEB1 (known to induce epithelial mesenchymal transition in cancer cells), and MYC, JAK1 and STAT3 (oncogenes involved in cell survival). Further analysis using OQ01 cells in monobacterial infection showed that F. nucleatum alone had the same or greater effect as polybacterial infection (P= 0.001). These results showed that F. nucleatum was the main periodontal bacteria responsible for inducing invasive phenotype in these oral cancer cells. In addition, F. nucleatum was able to enhance cancer cell invasion in vitro. Furthermore, F. nucleatum appear to upregulate autophagy markers in vitro which can lead to enhanced cancer cell survival and chemoresistance. This study demonstrated that the interaction between oral cancer cells and periodontal bacteria might be both cancer cell- and bacteria-specific. Our study can be highly useful in examining how modulating oral cancer environment can improve treatment outcome. Citation Format: Amani Harrandah, Sasanka S. Chukkapalli, Ann Progulske-Fox, William Dunn, Kesavalu Lakshmyya, Edward K. Chan. F. nucleatum induces invasive phenotypes in oral cancer cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5139.

Polybacterial stimulation suggests discrete IL-6/IL-6R signaling in human fetal membranes: Potential implications on IL-6 bioactivity

The polybacterial invasion of the amniotic cavity and risk of preterm birth is often due to cervicovaginal bacteria such as genital mycoplasmas (Mycoplasma hominis and Ureaplasma urealyticum) and Gardnerella vaginalis. The most studied biomarker associated with preterm birth is interleukin-6 (IL-6), a pleiotropic cytokine that performs different functions based on classical or trans-signaling mechanisms. This study evaluated the changes in IL-6 and IL-6 function associated accessory molecules by human fetal membranes to determine the functional availability of IL-6 assessment in an in vitro model of polybacterial infection. Fetal membranes were treated with LPS or heat-inactivated genital mycoplasmas and G. vaginalis alone or in combination. IL-6 and its soluble receptors (sgp130, sIL-6R) were assessed in conditioned medium by immunoassays and membrane-bound receptors were evaluated in the tissue using immunohistochemistry and RT-PCR. Data from protein and gene expression were evaluated using linear mixed effects models. Data from immunohistochemistry were evaluated using one-way analysis of variance followed by the Tukey test. Genital mycoplasmas alone, or in combination, inhibited IL-6 trans-signaling with increased sgp130 production. G. vaginalis activated the classical IL-6 signaling pathway, as did LPS. Polybacterial treatment resulted in a balanced response with neither pathway being favored. The increase in IL-6 production by fetal membranes in response to infection is likely a non-specific innate response and not an indicator of a functional mediator of any labor-inducing pathways. This suggests that correlating the risk of adverse pregnancy outcomes and designing interventions based on IL-6 levels without considering soluble receptors may be an ineffective strategy.

Abstract 34: Predominant Periodontal Bacteria Augments Ischemic Stroke Outcomes

Background: Chronic infection and persistent systemic inflammation are important risk factors for poor stroke outcomes. Chronic periodontitis (PD) are among the most common chronic immunoinflammatory infections of humans. Interestingly, in 2012, the American Heart Association reported that observational studies support an association between PD and atherosclerotic vascular disease. The association between PD and the occurrence of stroke has been explored clinically in retrospective studies. However, role of PD bacteria in hemorrhagic and ischemic stroke outcomes has not been investigated in preclinical animal models. Here we tested the hypothesis whether PD bacteria augments stroke outcomes. Methods: Ten-week-old male C57BL/6NHsd mice were randomly assigned to the infected (PD) and the sham-infected groups. Mice were infected by oral lavage with 4 periodontal bacteria ( Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum ) as polybacterial infection for 12wks (inoculating 4 times per week every third week) to induce periodontitis. Sham-infected mice was inoculated with vehicle. At the end of the 12wks infection period, mice were subjected to 60min of transient ischemia. Mice were tested for PD [bacterial infection, periodontal inflammation, alveolar bone resorption (ABR)] and functional and anatomical stroke outcomes at 48h post-stroke. Results: All bacteria were colonized/infected in the mice gingival surface (83-100%). Polybacterial-infected mice developed mild periodontal inflammation with a trend to increased ABR. We also observed that the neurological deficit score in PD group increased by 32.40±18.56% (p&lt;0.01). Similarly, the infarction volume in the infected mice (n=7) also increased significantly by 44.26±26.04% (p&lt;0.05) as compared with controls (n=8). We also observed a significant difference in parenchymal bleeding in infected mice as compared with the sham-infected mice (30% vs 11%; p&lt;0.001). Conclusion: This is the first report demonstrating that oral bacteria induced significant neurological deficits, infarction volume, and parenchymal bleeding resulting in enhanced ischemic stroke damages.

Global TLR2 and 4 deficiency in mice impacts bone resorption, inflammatory markers and atherosclerosis to polymicrobial infection

Toll-like-receptors (TLRs) play a significant role in the generation of a specific innate immune response against invading pathogens. TLR2 andTLR4 signaling contributes to infection-inducedinflammation in periodontal disease (PD) and atherosclerosis. Observational studies point towards a relationship between PD and atherosclerosis, but the role of TLR2 and TLR4 in the recognition of multiple oral pathogens and their modulation of host response leading to atherosclerosis are not clear. We evaluated the role of TLR2 and TLR4 signaling in the induction of both PD and atherosclerosis in TLR2-/- and TLR4-/- mice to polymicrobial infection with periodontal pathogens Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum. Polybacterial infections have established gingival colonization in TLR2-/- and TLR4-/- mice and induction of a pathogen-specific immunoglobulin G immune response. But TLR deficiency dampened accelerated alveolar bone resorption and intrabony defects, indicating a central role in infection-induced PD. Periodontal bacteria disseminated from gingival tissue to the heart and aorta through intravascular dissemination; however, there was no increase in atherosclerosis progression in the aortic arch. Polybacterial infection does not alter levels of serum risk factors such as oxidized low-density lipoprotein, nitric oxide, and lipid fractions in both mice. Polymicrobial-infected TLR2-/- mice demonstrated significant levels (P<0.05 to P<0.01) of T helper type 2 [transforming growth factor-β1 , macrophage inflammatory protein-3α, interleukin-13 (IL-13)] and T helper type 17 (IL-17, IL-21, IL-22, IL-23) splenic T-cell cytokine responses. Increased heat-shock protein expression, hspa1a for Hsp 70, was observed for both TLR2-/- and TLR4-/- mice. This study supports a role for TLR2 and TLR4 in PD and atherosclerosis, corroborating an intricate association between two inflammatory diseases.

Are viruses just the bystanders for Periodontal diseases?: A review

Periodontal disease is multifactorial in nature. To meet long lasting therapeutic end points in treatment of it, a solid understanding of the etiology of periodontitis is need of an hour. Current research has shifted its focus from periodontitis being polybacterial infection to involvement of various viruses in the etiopathogeny of destructive periodontal disease. Discovery of the high copy counts of Epstein–Barr virus and cytomegalovirus in aggressive and chronic periodontitis, it is unlikely that these pathogenic viruses are acting merely as innocuous bystanders in pathogenesis of the periodontal diseases. These viruses probably might not act as primary periodontopathic agents but they might cooperate the specific bacteria in periodontal disease progression. The periodontal research delving more into role of virus as a causative agent for periodontal diseases can open up a whole new era in therapeutic approaches and prove to be a paradigm shift. The purpose of this review is to evaluate the evidence supporting the viral hypothesis of periodontal eitopathogenesis.

Polymicrobial Oral Infection with Four Periodontal Bacteria Orchestrates a Distinct Inflammatory Response and Atherosclerosis in ApoE null Mice.

Periodontal disease (PD) develops from a synergy of complex subgingival oral microbiome, and is linked to systemic inflammatory atherosclerotic vascular disease (ASVD). To investigate how a polybacterial microbiome infection influences atherosclerotic plaque progression, we infected the oral cavity of ApoEnull mice with a polybacterial consortium of 4 well-characterized periodontal pathogens, Porphyromonas gingivalis, Treponema denticola, Tannerealla forsythia and Fusobacterium nucleatum, that have been identified in human atherosclerotic plaque by DNA screening. We assessed periodontal disease characteristics, hematogenous dissemination of bacteria, peripheral T cell response, serum inflammatory cytokines, atherosclerosis risk factors, atherosclerotic plaque development, and alteration of aortic gene expression. Polybacterial infections have established gingival colonization in ApoEnull hyperlipidemic mice and displayed invasive characteristics with hematogenous dissemination into cardiovascular tissues such as the heart and aorta. Polybacterial infection induced significantly higher levels of serum risk factors oxidized LDL (p < 0.05), nitric oxide (p < 0.01), altered lipid profiles (cholesterol, triglycerides, Chylomicrons, VLDL) (p < 0.05) as well as accelerated aortic plaque formation in ApoEnull mice (p < 0.05). Periodontal microbiome infection is associated with significant decreases in Apoa1, Apob, Birc3, Fga, FgB genes that are associated with atherosclerosis. Periodontal infection for 12 weeks had modified levels of inflammatory molecules, with decreased Fas ligand, IL-13, SDF-1 and increased chemokine RANTES. In contrast, 24 weeks of infection induced new changes in other inflammatory molecules with reduced KC, MCSF, enhancing GM-CSF, IFNγ, IL-1β, IL-13, IL-4, IL-13, lymphotactin, RANTES, and also an increase in select inflammatory molecules. This study demonstrates unique differences in the host immune response to a polybacterial periodontal infection with atherosclerotic lesion progression in a mouse model.

Abstract 14157: Periodontal Microbiome Induces Inflammation and Atherosclerosis in Hyperlipidemic Atherogenic Apoe Null Mice

Introduction: Periodontal disease (PD) is a polymicrobial dysbiotic chronic inflammatory disease produced by a complex subgingival microbiome, and is linked to systemic inflammatory atherosclerotic vascular disease (ASVD). American Heart Association supported the association between PD and ASVD but not causal association. Prior reports investigated the associations between these two diseases PD and ASVD. But the complex signaling molecules and mechanisms establishing a causal association are not well defined. Methods: To investigate how polybacterial infections initiate atherosclerotic plaque growth, ApoE null mice were orally infected with a polybacterial consortium of 4 well-characterized periodontal pathogens Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia and Fusobacterium nucleatum . We assessed PD characteristics, hematogenous dissemination of bacteria, peripheral T cell response, serum inflammatory cytokines, atherosclerosis risk factors, atherosclerotic plaque development, and alteration of aortic gene expression. Results: Polybacterial infections have enhanced gingival colonization and significantly induced alveolar bone loss ApoE null mice. Polybacterial infections were found to be invasive with hematogenous dissemination into heart and aorta. Polybacterial infection-induced significantly higher levels of serum risk factors (oxidized LDL, nitric oxide), altered lipid profiles (cholesterol, triglycerides, chylomicrons, VLDL) as well as accelerated aortic plaque formation in ApoE null mice. Polybacterial infection is associated with significant decreases in Apoa1, Apob, Birc3, Fga, FgB genes that are associated with atherosclerosis. Gingival infections after 12 weeks had modified levels of inflammatory molecules, with decreased Fas ligand, IL-13, SDF-1 and increased chemokine RANTES. At 24 weeks of chronic infection additional changes in inflammatory molecules were induced with reduced KC, MCSF, and enhanced GM-CSF, IFNγ, IL-1β, IL-13, IL-4, IL-13, lymphotactin, and RANTES. Conclusions: This is the first study demonstrating differences in the host immune response to a polybacterial infection with atherosclerotic lesion progression in a mouse model.

Periodontal pathogens invade gingiva and aortic adventitia and elicit inflammasome activation in αvβ6 integrin-deficient mice.

The American Heart Association supports an association between periodontal diseases and atherosclerosis but not a causal association. This study explores the use of the integrin β6(-/-) mouse model to study the causality. We investigated the ability of a polymicrobial consortium of Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum to colonize the periodontium and induce local and systemic inflammatory responses. Polymicrobially infected Itgβ6(-/-) mice demonstrate greater susceptibility to gingival colonization/infection, with severe gingival inflammation, apical migration of the junctional epithelium, periodontal pocket formation, alveolar bone resorption, osteoclast activation, bacterial invasion of the gingiva, a greater propensity for the bacteria to disseminate hematogenously, and a strong splenic T cell cytokine response. Levels of atherosclerosis risk factors, including serum nitric oxide, oxidized low-density lipoprotein, serum amyloid A, and lipid peroxidation, were significantly altered by polybacterial infection, demonstrating an enhanced potential for atherosclerotic plaque progression. Aortic gene expression revealed significant alterations in specific Toll-like receptor (TLR) and nucleotide-binding domain- and leucine-rich-repeat-containing receptor (NLR) pathway genes in response to periodontal bacterial infection. Histomorphometry of the aorta demonstrated larger atherosclerotic plaques in Itgβ6(-/-) mice than in wild-type (WT) mice but no significant difference in atherosclerotic plaque size between mice with polybacterial infection and mice with sham infection. Fluorescence in situ hybridization demonstrated active invasion of the aortic adventitial layer by P. gingivalis. Our observations suggest that polybacterial infection elicits distinct aortic TLR and inflammasome signaling and significantly increases local aortic oxidative stress. These results are the first to demonstrate the mechanism of the host aortic inflammatory response induced by polymicrobial infection with well-characterized periodontal pathogens.