Patogenesi molecolare delle osteomieliti associate all'impianto

L’osteomielite associata all’impianto è un processo infettivo a carico del tessuto osseo spesso accompagnato dalla distruzione dell’osso stesso. La patogenesi delle osteomieliti associate all’impianto si basa su due concetti fondamentali: l’internalizzazione del patogeno all’interno degli osteoblasti e la capacità dei batteri di formare il biofilm. Entrambi i meccanismi consentono infatti di prevenire l’eliminazione del batterio da parte delle difese immunitarie dell’ospite e di ostacolare l’azione della maggior parte degli antibiotici (che non penetrano e non agiscono pertanto su microrganismi intracellulari), così sostenendo ed alimentando l’infezione. Il saggio di invasione messo a punto su micropiastra ha consentito di investigare in modo approfondito e dettagliato il ruolo ed il peso dell’internalizzazione nella patogenesi delle infezioni ortopediche peri-protesiche causate da S. aureus, S. epidermidis, S. lugdunensis ed E. faecalis. Lo studio ha evidenziato che l’invasione delle cellule MG-63 non rappresenta un meccanismo patogenetico delle infezioni ortopediche associate all’impianto causate da S. epidermidis, S. lugdunensis ed E. faecalis; al contrario, in S. aureus la spiccata capacità invasiva rappresenta un’abile strategia patogenetica che consente al patogeno di sfuggire alla terapia sistemica e alla risposta immunitaria dell’ospite. È stato studiato inoltre il ruolo dell’immunità innata nella difesa contro il biofilm batterico. In seguito all’incubazione del biofilm opsonizzato di S. epidermidis con i PMN è stato possibile osservare la formazione delle NETs. Le NETs rappresentano ottime armi nella difesa contro il biofilm batterico, infatti le trappole sono in grado di limitare la diffusione batterica e quindi di confinare l’infezione. La comprensione del ruolo dell’internalizzazione nella patogenesi delle osteomieliti associate all’impianto e lo studio della risposta immunitaria innata a questo tipo di infezioni, spesso caratterizzate dalla presenza di biofilm, sono presupposti per identificare e affinare le migliori strategie terapeutiche necessarie ad eradicare l'infezione.

Development of a biorefinery scheme for the valorization of olive mill wastewaters and grape pomaces

In the Mediterranean area, olive mill wastewater (OMW) and grape pomace (GP) are among the major agro-industrial wastes produced. These two wastes have a high organic load and high phytotoxicity. Thus, their disposal in the environment can lead to negative effects. Second-generation biorefineries are dedicated to the valorization of biowaste by the production of goods from such residual biomasses. This approach can combine bioremediation approaches to the generation of noble molecules, biomaterials and energy. The main aim of this thesis work was to study the anaerobic digestion of OMW and GP under different operational conditions to produce volatile fatti acids (VFAs) (first stage aim) and CH4 (second stage aim). To this end, a packed-bed biofilm reactor (PBBR) was set up to perform the anaerobic acidogenic digestion of the liquid dephenolized stream of OMW (OMWdeph). In parallel, the solid stream of OMW (OMWsolid), previously separated in order to allow the solid phase extraction of polyphenols, was addressed to anaerobic methanogenic digestion to obtain CH4. The latter experiment was performed in 100ml Pyrex bottles which were maintained at different temperatures (55-45-37°C). Together with previous experiments, the anaerobic acidogenic digestion of fermented GP (GPfreshacid) and dephenolized and fermented GP (GPdephacid) was performed in 100ml Pyrex bottles to estimate the concentration of VFAs achievable from each aforementioned GPs. Finally, the same matrices of GP and not pre-treated GP (GPfresh) were digested under anaerobic methanogenic condition to produce CH4. Anaerobic acidogenic and methanogenic digestion processes of GPs lasted about 33 days. Instead, the anaerobic acidogenic and methanogenic digestion process of OMWs lasted about 121 and 60 days, respectively. Each experiment was periodically monitored by analysing volume and composition of produced biogas and VFA concentration. Results showed that VFAs were produced in higher concentrations in GP compared to OMWdeph. The overall concentration of VFAs from GPfreshacid was approximately 39.5 gCOD L-1, 29 gCOD L-1 from GPdephacid, and 8.7 gCOD L-1 from OMWdeph. Concerning the CH4 production, the OMWsolid reached a high biochemical methane potential (BMP) at a thermophilic temperature (55°) than at mesophlic ones (37-45°C). The value reached was about 358.7 mlCH4 gSVsub-1. In contrast, GPfresh got a high BMP but at a mesophilic temperature. The BMP was about 207.3 mlCH4 gSVsub-1, followed by GPfreshacid with about 192.6 mlCH4 gSVsub-1 and lastly GPdephacid with about 102.2 mlCH4 gSVsub-1. In summary, based on the gathered results, GP seems to be a better carbon source for acidogenic and methanogenic microrganism compared to OMW, because higher amount of VFAs and CH4 were produced in AD of GP than OMW. In addition to these products, polyphenols were extracted by means of a solid phase extraction (SPE) procedure by another research group, and VFAs were utilised for biopolymers production, in particular polyhydroxyalkanoates (PHAs), by the same research group in which I was involved.

Clinical and antibacterial effect of an anti-inflammatory toothpaste formulation with Scutellaria baicalensis extract on experimental gingivitis

It was the aim of the study to evaluate the clinical and antibacterial effect of a dentifrice containing an anti-inflammatory plant extract (SB) versus a placebo (PLA) using an experimental gingivitis model. Forty subjects (20 per group) discontinued all oral hygiene measures for four teeth for a period of 21 days using a shield (to generate a possible gingivitis) while they could brush the other teeth normally. After brushing, the shield was removed and teeth were treated with the randomly assigned toothpaste slurry for 1 min. Löe and Silness gingival index (GI), Silness and Löe plaque index (PI), and biofilm vitality (VF%) were assessed at days 0, 14, and 21, respectively. Subjects of the PLA group developed a GI of 0.82?±?0.342 (day 14) and 1.585?±?0.218 (day 21), while the data of the SB group were significantly reduced (0.355?±?0.243 and 0.934?±?0.342, p?

Pro-inflammatory biomarkers during experimental gingivitis in patients with type 1 diabetes mellitus: a proof-of-concept study

To compare gingival crevicular fluid (GCF) biomarker levels and microbial distribution in plaque biofilm (SP) samples for subjects with type 1 diabetes (T1DM) versus healthy subjects without diabetes during experimental gingivitis (EG).

Escherichia coli variants in periprosthetic joint infection: diagnostic challenges with sessile bacteria and sonication

The diagnostic yield of prosthetic joint-associated infection is hampered by the phenotypic change of bacteria into a sessile and resistant form, also called biofilm. With sonication, adherent bacteria can be dislodged from the prosthesis. Species identification may be difficult because of their variations in phenotypic appearance and biochemical reaction. We have studied the phenotypic, genotypic, and biochemical properties of Escherichia coli variants isolated from a periprosthetic joint infection. The strains were collected from synovial fluid, periprosthetic tissue, and fluid from the explanted and sonicated prosthesis. Isolates from synovial fluid revealed a normal phenotype, whereas a few variants from periprosthetic tissue and all isolates from sonication fluid showed different morphological features (including small-colony variants). All isolates from sonication fluid were beta-galactosidase negative and nonmotile; most were indole negative. Because of further variations in biochemical properties, species identification was false or not possible in 50% of the isolates included in this study. In contrast to normal phenotypes, variants were resistant to aminoglycosides. Typing of the isolates using pulsed-field gel electrophoresis yielded nonidentical banding patterns, but all strains were assigned to the same clonal origin when compared with 207 unrelated E. coli isolates. The bacteria were repeatedly passaged on culture media and reanalyzed. Thereafter, most variants reverted to normal phenotype and regained their motility and certain biochemical properties. In addition, some variants displayed aminoglycoside susceptibility after reversion. Sonication of an explanted prosthesis allows insight into the lifestyle of bacteria in biofilms. Since sonication fluid also reveals dislodged sessile forms, species identification of such variants may be misleading.

Saliva/pathogen biomarker signatures and periodontal disease progression

The purpose of this study was to determine the role of saliva-derived biomarkers and periodontal pathogens during periodontal disease progression (PDP). One hundred human participants were recruited into a 12-month investigation. They were seen bi-monthly for saliva and clinical measures and bi-annually for subtraction radiography, serum and plaque biofilm assessments. Saliva and serum were analyzed with protein arrays for 14 pro-inflammatory and bone turnover markers, while qPCR was used for detection of biofilm. A hierarchical clustering algorithm was used to group study participants based on clinical, microbiological, salivary/serum biomarkers, and PDP. Eighty-three individuals completed the six-month monitoring phase, with 39 [corrected] exhibiting PDP, while 44 [corrected] demonstrated stability. Participants assembled into three clusters based on periodontal pathogens, serum and salivary biomarkers. Cluster 1 members displayed high salivary biomarkers and biofilm; 71% [corrected] of these individuals were undergoing PDP. Cluster 2 members displayed low biofilm and biomarker levels; 76% [corrected] of these individuals were stable. Cluster 3 members were not discriminated by PDP status; however, cluster stratification followed groups 1 and 2 based on thresholds of salivary biomarkers and biofilm pathogens. The association of cluster membership to PDP was highly significant (p < 0.0007). [corrected] The use of salivary and biofilm biomarkers offers potential for the identification of PDP or stability (ClinicalTrials.gov number, CT00277745).

Tongue piercing: the effect of material on microbiological findings

Biofilms on oral piercings may serve as a bacterial reservoir and lead to systemic bacteremia or local transmission of pathogenic microbiota. The use of piercing materials which are less susceptible to biofilm accumulation could contribute to prevention of problems. The present study investigated whether there are microbiological differences in bacterial samples collected from tongue piercings made of different materials.

Characterization of vascular cavity coatings and microscopic tube-shaped structures from Upper Cretaceous dinosaur bone: evidence of a bacterial origin for dinosaur blood vessels""

Recent claims of blood vessels extracted from dinosaur fossils challenge classical views of soft-tissue preservation. Alternatively, these structures may represent postdepositional,diagenetic biofilms that grew on vascular cavity surfaces within the fossil. Similar red, hollow, tube-shaped structures were recovered from well-preserved and poorly-preserved (abraded, desiccated, exposed) Upper Cretaceous dinosaur fossils in this study. Integration of light microscopy, scanning electron microscopy, and energy dispersive x-ray spectroscopy was used to compare these vessel structures to the fossils from which they are derived. Vessel structures are typically 100-400 μm long, 0.5-1.5 μm thick, 10-40 μm in diameter and take on a wide range of straight, curved, andbranching morphologies. Interior surfaces vary from smooth to globular and typically contain spheres, rods, and fibrous structures (< 2 μm in diameter) incorporated into the surface. Exterior surfaces exhibit 2-μm-tall converging ridges, spaced 1-3 μm apart, that are sub-parallel to the long axis of the vessel structure. Fossil vascular cavities are typically coated with a smooth or grainy orange layer that shows a wide range of textures including smooth, globular, rough, ropy, and combinations thereof. Coatings tend to overlay secondary mineral crystals and framboids, confirming they are not primary structures of the fossil. For some cavity coatings, the surface that had been in contact with the bone exhibits a ridged texture, similar to that of vessel structures, having formed as a mold of the intravascular bone surface. Thus, vessel structures are interpreted as intact cavity coatings isolated after the fossil is demineralized. The presence of framboids and structures consistent in size and shape with bacteria cells, the abundance of iron in cavity coatings, and the growth of biofilms directly from the fossil that resemble respective cavity coatings support the hypothesis that vessel structures result from ironconsuming bacteria that form biofilms on the intravascular bone surfaces of fossil dinosaur bone. This also accounts for microstructures resembling osteocytes as some fossil lacunae are filled with the same iron oxide that comprises vessel structures andcoatings. Results of this study show that systematic, high-resolution SEM analyses of vertebrate fossils can provide improved insight on microtaphonomic processes, including the role of bacteria in diagenesis. These results conflict with earlier claims of dinosaurblood vessels and osteocytes.

An Investigation On The Use Of Microalgae For Biogas Enrichment

There is a need for biomethane capture and carbon dioxide sequestration to mitigate evident global climate change. This research work investigated the potential for microalgae to remove CO2 from biogas as a biotechnical method for upgrading the thermal value for subsequent compression, liquification, or introduction to natural gas pipelines. Because biogas is largely methane, the effect of high methane environments on mixed microalgae was explored and found that specific carbon utilization rates were not statistically different when microalgae were exposed to biogas environments (70% v/v CH4) , relative to high CO2 environment. The uses of conventional bubbled column photobioreactors (PBR) were assessed for CO2 removal and subsequent CH4 enrichment. A continuously-bubbled biogas PBR (cB-PBR5) and intermittently-bubbled biogas PBR (iB-PBR) experienced CO2 loading rates of about 1664 and 832 mg C/L*day and showed 30.0 and 60.1 % carbon removal, respectively. However, a lack of biogas enrichment and issues associated growth inhibition due to high CO2 environments as well as stripping the dissolved gases, namely oxygen and nitrogen, from the bulk liquid and introduction to the outlet gas prompted the consideration for gas/liquid separation using nonporous hollow-fiber (HF) membranes for CO2 transfer. The potential for two non-porous HF membrane materials [polydimethylsiloxane (PDMS) and composite polyurethane (PU)] were modeled along fiber length using a mechanistic model based on polymeric material transport properties (Gilmore et al., 2009). Based on a high CO2:CH4 permeability selectivity for PU of 76.2 the model predicted gas enrichment along an 8.5 cm fiber length. Because PDMS permeability selectivity is low (3.5), evident gas transfer was not predicated along a 34.3 cm length. Both of these HF materials were implemented in hollow-fiber membrane-carbonated biofilm (HFMcB) PBRs for microalgal-mediated biogas enrichment. Phototrophic biofilm colonization occurred on the membrane, where CO2 concentration was greatest. The presence of a biofilm demonstrated greater resiliency to high CO2 environments, compared to the conventional PBRs. However, as the PDMS model predicted, the PDMS HFMcBs did not demonstrate gas enrichment. These reactors received CO2 loading rates of 200 mg C/L*day based on PDMS permeability flux and showed approximately 65% removal of the total C transferred across the membrane. Thus, the HFMcBs demonstrated controlled carbonation of the bulk liquid via a nonporous HF membrane. Likewise, the experimental PU HFMcB did not show gas enrichment yet this result should be further explored due to the high permeability selectivity of the polymeric material. Chemical stratifications, namely pH and dissolved O2, present in a PDMS membrane-carbonated biofilm were analyzed using electrochemical microsensors. Results indicated that high DO (20 mg L-1) exists at surface of the biofilm where light availability is greatest and low pH microenvironments (pH=5.40) exist deep in the biofilm where the diffusive flux of CO2 drives transfer through the biofilm. The presence of a 400-600 ¿m liquid phase boundary layer was evident from microsensor profiles. Cryosectioning of the biofilm samples showed the biofilm to be approximately 1.17 ± 0.07 mm thick, suggesting that the high localized concentration of biomass associated with the phototrophic biofilm aided in overcoming inhibition in a microenvironment dominated by CO2(aq). Challenges of biofilm detachment and PBR fouling as well as microalgal growth inhibition in the presence of high CO2 content remain for applications of microalgae for biogas enrichment.

[Periodontitis and cardiovascular disease]

The prevalence of periodontitis and cardiovascular disease (CVD) is high. A mixed infectious biofilm etiology of periodontitis is known but not fully established in CVD. Cofactors; smoking habits, stress, ethnicity, genetics, socioeconomics and age contribute to both diseases. The objectives of this report are to summarize factors in regards to CVD and periodontitis that are clinically relevant. The hypothesis behind a relationship between the two conditions can be founded in (I) shared infections etiology, (II) shared inflammatory response, (III) epidemiological and case-control studies, and (IV) periodontal studies demonstrating improvements of CVD markers. Streptococcus species in the S. mitis group, and S. anginosus group have been identified in periodontitis and are known as pathogens in endocarditis possibly transported from the oral cavity to the heart through bacteremia during dental therapies, and tooth brushing. Other periodontal bacteria such as Porphyromonas gingivalis, Fusobacterium nucleatum and Parvimonas micra are beta-lactamase producing and may contribute to antibiotic resistance (extended spectrum beta-lactamases). Other bacteria in CVD and periodontitis include Staphylococcus aureus, and Pseudomonas aeruginosa. Chlamydia pneumoniae and P. gingivalis lipopolyysaccharide capsels share homology and induce heat-shock protein activity and a cascade of proinflammatory cytokines. Associations between periodontitis and CVD have been presented in many studies when controlling for confounders. Other studies have demonstrated that periodontal therapies increase brachial artery flow rate and reduce serum inflammatory cytokine levels. Thus, physicians caring for subjects at CVD risk should consult with dentists/periodontists. Dentists must improve their medical knowledge and also learn to consult with physicians when treating patients at CVD risk.

INVESTIGATING THE IMPACTS OF STREAMBED HABITAT HETEROGENEITY ON ECOSYSTEM STRUCTURE AND PROCESSES USING BASIC AND APPLIED PERSPECTIVES

Stream restoration often focuses on increasing habitat heterogeneity to reverse ecosystem degradation. However, the connection between heterogeneity and ecosystem structure and processes is poorly understood. We looked to investigate this interaction from both applied and basic science perspectives. For the applied study, we examined two culvert replacements designed to mimic natural stream channels, to see if they were better at maintaining ecosystem processes within as well as upstream and downstream of culverts compared to non-replaced culverts. We measured three ecosystem processes (nutrient uptake, hydrologic characteristics, and coarse particulate organic matter retention) and found that stream simulation culvert restoration improved organic matter retention within culverts, and that there were no differences in processes measured upstream and downstream of both restoration designs. Our results suggest that measurements of ecosystem processes are more likely to show a response to restoration if they match the scale of the restoration activity. For the basic science study, we quantified the longitudinal spatial heterogeneity of physical and biofilm characteristics at microhabitat to segment scales on streams with different streambed variability. We found that all physical characteristics and biofilm characteristics were spatially independent at the macro-habitat scale and greater. Together, these studies demonstrate the importance of scale in ecological interactions and the value of incorporating considerations of scale into restoration activities.

Identification of pathogen and host-response markers correlated with periodontal disease

BACKGROUND: Periodontitis is the major cause of tooth loss in adults and is linked to systemic illnesses, such as cardiovascular disease and stroke. The development of rapid point-of-care (POC) chairside diagnostics has the potential for the early detection of periodontal infection and progression to identify incipient disease and reduce health care costs. However, validation of effective diagnostics requires the identification and verification of biomarkers correlated with disease progression. This clinical study sought to determine the ability of putative host- and microbially derived biomarkers to identify periodontal disease status from whole saliva and plaque biofilm. METHODS: One hundred human subjects were equally recruited into a healthy/gingivitis group or a periodontitis population. Whole saliva was collected from all subjects and analyzed using antibody arrays to measure the levels of multiple proinflammatory cytokines and bone resorptive/turnover markers. RESULTS: Salivary biomarker data were correlated to comprehensive clinical, radiographic, and microbial plaque biofilm levels measured by quantitative polymerase chain reaction (qPCR) for the generation of models for periodontal disease identification. Significantly elevated levels of matrix metalloproteinase (MMP)-8 and -9 were found in subjects with advanced periodontitis with Random Forest importance scores of 7.1 and 5.1, respectively. The generation of receiver operating characteristic curves demonstrated that permutations of salivary biomarkers and pathogen biofilm values augmented the prediction of disease category. Multiple combinations of salivary biomarkers (especially MMP-8 and -9 and osteoprotegerin) combined with red-complex anaerobic periodontal pathogens (such as Porphyromonas gingivalis or Treponema denticola) provided highly accurate predictions of periodontal disease category. Elevated salivary MMP-8 and T. denticola biofilm levels displayed robust combinatorial characteristics in predicting periodontal disease severity (area under the curve = 0.88; odds ratio = 24.6; 95% confidence interval: 5.2 to 116.5). CONCLUSIONS: Using qPCR and sensitive immunoassays, we identified host- and bacterially derived biomarkers correlated with periodontal disease. This approach offers significant potential for the discovery of biomarker signatures useful in the development of rapid POC chairside diagnostics for oral and systemic diseases. Studies are ongoing to apply this approach to the longitudinal predictions of disease activity.

Clinical outcomes following subgingival application of a novel erythritol powder by means of air polishing in supportive periodontal therapy: a randomized, controlled clinical study

OBJECTIVES The aim of this prospective, randomized, controlled clinical study was to compare the clinical outcomes of the subgingival treatment with erythritol powder by means of an air-polishing (EPAP) device and of scaling and root planing (SRP) during supportive periodontal therapy (SPT). METHOD AND MATERIALS 40 patients enrolled in SPT were randomly assigned to two groups of equal size. Sites had to show signs of inflammation (bleeding on probing [BOP]-positive) and a probing pocket depth (PPD) of ≥ 4 mm, however, without presence of detectable subgingival calculus. During SPT, these sites were treated with EPAP or SRP, respectively. Full mouth and site-specific plaque indices, BOP, PPD, and clinical attachment level (CAL) were recorded at baseline (BL) and at 3 months, whereas the percentage of study sites positive for BOP (BOP+) was considered as primary outcome variable. Additionally, patient comfort using a visual analog scale (VAS) and the time needed to treat per site was evaluated. RESULTS At 3 months, mean BOP level measured 45.1% at test sites and 50.6% at control sites, respectively, without a statistically significant difference between the groups (P > .05). PPD and CAL slightly improved for both groups with comparable mean values at 3 months. Evaluation of patient tolerance showed statistically significantly better values among patients receiving the test treatment (mean VAS [0-10], 1.51) compared to SRP (mean VAS [0-10], 3.66; P = .0012). The treatment of test sites was set to 5 seconds per site. The treatment of control sites, on the other hand, lasted 85 seconds on average. CONCLUSION The new erythritol powder applied with an air-polishing device can be considered a promising modality for repeated instrumentation of residual pockets during SPT. CLINICAL RELEVANCE With regard to clinical outcomes during SPT, similar results can be expected irrespective of the two treatment approaches of hand instrumentation or subgingival application of erythritol powder with an air-polishing device in sites where only biofilm removal is required.

Effect of photoactivated disinfection with a light-emitting diode on bacterial species and biofilms associated with periodontitis and peri-implantitis

BACKGROUND To determine the effect of photoactivated disinfection (PAD) using toluidine blue and a light-emitting diode (LED) in the red spectrum (wave length at 625-635 nm) on species associated with periodontitis and peri-implantitis and bacteria within a periodontopathic biofilm. METHODS Sixteen single microbial species including 2 Porphyromonas gingivalis and 2 Aggregatibacter actinomycetemcomitans and a multispecies mixture consisting of 12 species suspended in saline without and with 25% human serum were exposed to PAD. Moreover, single-species biofilms consisting of 2 P. gingivalis and 2 A. actinomycetemcomitans strains and a multi-species biofilm on 24-well-plates, grown on titanium discs and in artificial periodontal pockets were exposed to PAD with and without pretreatment with 0.25% hydrogen peroxide. Changes in the viability were determined by counting the colony forming units (cfu). RESULTS PAD reduced the cfu counts in saline by 1.42 log₁₀ after LED application for 30s and by 1.99 log₁₀ after LED application for 60s compared with negative controls (each p<0.001). Serum did not inhibit the efficacy of PAD. PAD reduced statistically significantly (p<0.05) the cfu counts of the P. gingivalis biofilms. The viability of the A. actinomycetemcomitans biofilms and the multi-species biofilms was statistically significantly decreased when PAD was applied after a pretreatment with 0.25% hydrogen peroxide. The biofilm formed in artificial pockets was more sensitive to PAD with and without pretreatment with hydrogen peroxide compared with those formed on titanium discs. CONCLUSIONS PAD using a LED was effective against periodontopathic bacterial species and reduced viability in biofilms but was not able to completely destroy complex biofilms. The use of PAD following pretreatment with hydrogen peroxide resulted in an additional increase in the antimicrobial activity which may represent a new alternative to treat periodontal and peri-implant infections thus warranting further testing in clinical studies.

Further characterization of the epa gene cluster and Epa polysaccharides of Enterococcus faecalis.

We previously identified a gene cluster, epa (for enterocococcal polysaccharide antigen), involved in polysaccharide biosynthesis of Enterococcus faecalis and showed that disruption of epaB and epaE resulted in attenuation in translocation, biofilm formation, resistance to polymorphonuclear leukocyte (PMN) killing, and virulence in a mouse peritonitis model. Using five additional mutant disruptions in the 26-kb region between orfde2 and OG1RF_0163, we defined the epa locus as the area from epaA to epaR. Disruption of epaA, epaM, and epaN, like prior disruption of epaB and epaE, resulted in alteration in Epa polysaccharide content, more round cells versus oval cells with OG1RF, decreased biofilm formation, attenuation in a mouse peritonitis model, and resistance to lysis by the phage NPV-1 (known to lyse OG1RF), while mutants disrupted in orfde2 and OG1RF_163 (the epa locus flanking genes) behaved like OG1RF in those assays. Analysis of the purified Epa polysaccharide from OG1RF revealed the presence of rhamnose, glucose, galactose, GalNAc, and GlcNAc in this polysaccharide, while carbohydrate preparation from the epaB mutant did not contain rhamnose, suggesting that one or more of the glycosyl transferases encoded by the epaBCD operon are necessary to transfer rhamnose to the polysaccharide. In conclusion, the epa genes, uniformly present in E. faecalis strains and involved in biosynthesis of polysaccharide in OG1RF, are also important for OG1RF shape determination, biofilm formation, and NPV-1 replication/lysis, as well as for E. faecalis virulence in a mouse peritonitis model.