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Objectives: Coculture models are limited by bacteria rapidly outcompeting host mammalian cells for nutrients in vitro , resulting in mammalian cell death. The goal of this study was to develop a coculture model enabling survival of mammalian cells and oral bacterial species to assess their competition for growth on dental implant materials. Methods: Two early colonizing oral bacterial species, Streptococcus mutans or Actinomyces naeslundii , were grown in coculture with primary human macrophages or human gingival fibroblasts for up to 7 days on tissue-culture treated polystyrene or polished titanium and zirconia disks. Chloramphenicol was supplemented in cell culture medium at bacteriostatic concentrations to maintain stable bacterial inoculum size. Planktonic and adherent bacterial growth was assessed via spot plating while mammalian cell growth and attachment were evaluated using colorimetric metabolic assay and confocal fluorescence microscopy, respectively. Results: Macrophages and fibroblasts proliferated in the presence of S. mutans and maintained viability above 70% during coculture for up to 7 days on tissue-culture treated polystyrene and polished titanium and zirconia. In contrast, both mammalian cell types exhibited decreasing proliferation and surface coverage on titanium and zirconia over time in coculture with A. naeslundii versus control. S. mutans and A. naeslundii were maintained within an order of magnitude of seeding inoculum sizes throughout coculture. Significance: Cell culture medium supplemented with antibiotics at bacteriostatic concentrations can suppress bacterial overgrowth and facilitate mammalian cell viability in coculture model systems. Within the study's limitations, oral bacteria and mammalian cell growth in coculture are comparable on polished titanium and zirconia surfaces.

Objectives: The aim of this study was to investigate the difference in dental biofilm formation according to substratum direction, using an artificial biofilm model., Methods: A three-species biofilm, consisting of Streptococcus mutans, Streptococcus oralis, and Actinomyces naeslundii, was formed on saliva-coated hydroxyapatite (sHA) discs oriented in three directions: downward (the discs placed in the direction of gravity), vertical (the discs placed parallel to the direction of gravity), and upward (the discs placed in opposite direction of gravity). The biofilms at 22 h and 46 h of age were analyzed using microbiological and biochemical methods, fluorescence-based assays, and scanning electron microscopy to investigate difference in bacterial adhesion, early and mature biofilm formation., Results: The biofilms formed in the upward direction displayed the most complex structure, with the highest number and biovolume of bacteria, as well as the lowest pH conditions at both time points. The vertical and downward directions, however, had only scattered and small bacterial colonies. In the 22-h-old biofilms, the proportion of S. oralis was similar to, or slightly higher than, that of S. mutans in all directions of substratum surfaces. However, in the 46-h-old biofilms, S. mutans became the dominant bacteria in all directions, especially in the vertical and upward directions., Conclusions: The direction of the substratum surface could impact the proportion of bacteria and cariogenic properties of the multi-species biofilm. Biofilms in an upward direction may exhibit a higher cariogenic potential, followed by those in the vertical and downward directions, which could be related to gravity., Competing Interests: Declaration of Competing Interest The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Introduction: The extrusion of bacteria from infected root canals may lead to increase in symptoms, expansion of periapical lesions, and contribution to systemic diseases. The aim of this study is to investigate a potential proof-of-concept model to study the extent to which bacteria can escape from infected root canals under dynamic loading (simulated chewing)., Methods: The study was completed in 2 experiments performed at 2 institutions. Biofilms of Streptococcus intermedius in the first experiment and S. intermedius and Actinomyces naeslundii were allowed to grow in root canals of single-rooted extracted teeth for 3 weeks. The roots of the teeth were suspended in a small chamber containing dental transport medium and were mounted on a lower sample holder of a chewing simulator. In the experimental group, simulated chewing cycles equivalent to 1 year of function were conducted, and then bacterial migration was quantified and compared with stationary teeth., Results: All experimental samples of the loading group revealed bacterial penetration in both experiments. Several of the unloaded samples revealed no bacterial penetration. In the first experiment, a significantly higher number of bacteria were able to escape into the periapex of the loaded group compared with the unloaded group (P = .017). In the second experiment, there was no significant difference between the 2 bacterial species used in the amount of extruded bacteria; however, there was a highly significant effect for occlusal loading (P = .0001)., Conclusions: The potential for occlusal forces to enhance bacterial extrusion from infected root canals should be further explored., (Copyright © 2023 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Saccharibacteria (TM7) are obligate epibionts living on the surface of their host bacteria and are strongly correlated with dysbiotic microbiomes during periodontitis and other inflammatory diseases, suggesting they are putative pathogens. However, due to the recalcitrance of TM7 cultivation, causal research to investigate their role in inflammatory diseases is lacking. Here, we isolated multiple TM7 species on their host bacteria from periodontitis patients. These TM7 species reduce inflammation and consequential bone loss by modulating host bacterial pathogenicity in a mouse ligature-induced periodontitis model. Two host bacterial functions involved in collagen binding and utilization of eukaryotic sialic acid are required for inducing bone loss and are altered by TM7 association. This TM7-mediated downregulation of host bacterial pathogenicity is shown for multiple TM7/host bacteria pairs, suggesting that, in contrast to their suspected pathogenic role, TM7 could protect mammalian hosts from inflammatory damage induced by their host bacteria., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Aim: The inhibitory and antibiofilm effects of Thymus vulgaris (EOTv) and Hyptis spicigera essential oils (EOHs) on cariogenic microorganisms were evaluated. Materials & methods: The chemical characterization of EOTv was performed by gas chromatography/mass spectrometry. Streptococcus mutans , Streptococcus gordonii , Streptococcus sanguinis , Streptococcus mitis , Streptococcus sobrinus , Lactobacillus acidophilus and Actinomyces naeslundii were used for agar diffusion assays and determination of minimal inhibitory and minimal bactericide concentrations. In addition, 20 streptococci and lactobacilli clinical isolates were also tested. The effects of essential oil on microbial initial biofilm formation and on preformed microcosm biofilm formed from human saliva were studied. Results & conclusion: Both essential oils had inhibitory effects on the cariogenic species and reduced the bacterial adherence to dental enamel. Essential oils were able to disrupt preformed microcosm biofilms. Thymus vulgaris and Hyptis spicigera essential oils have potential to be used in the development of formulations to the control of cariogenic biofilms.

Background: Actinomyces oris is an early colonizer and has two types of fimbriae on its cell surface, type 1 fimbriae (FimP and FimQ) and type 2 fimbriae (FimA and FimB), which contribute to the attachment and coaggregation with other bacteria and the formation of biofilm on the tooth surface, respectively. Short-chain fatty acids (SCFAs) are metabolic products of oral bacteria including A. oris and regulate pH in dental plaques. To clarify the relationship between SCFAs and fimbrillins, effects of SCFAs on the initial attachment and colonization (INAC) assay using A. oris wild type and fimbriae mutants was investigated. INAC assays using A. oris MG1 strain cells were performed with SCFAs (acetic, butyric, propionic, valeric and lactic acids) or a mixture of them on human saliva-coated 6-well plates incubated in TSB with 0.25% sucrose for 1 h. The INAC was assessed by staining live and dead cells that were visualized with a confocal microscope., Results: Among the SCFAs, acetic, butyric and propionic acids and a mixture of acetic, butyric and propionic acids induced the type 1 and type 2 fimbriae-dependent and independent INAC by live A. oris, but these cells did not interact with streptococci. The main effects might be dependent on the levels of the non-ionized acid forms of the SCFAs in acidic stress conditions. GroEL was also found to be a contributor to the FimA-independent INAC by live A. oris cells stimulated with non-ionized acid., Conclusion: SCFAs affect the INAC-associated activities of the A. oris fimbrillins and non-fimbrillins during ionized and non-ionized acid formations in the form of co-culturing with other bacteria in the dental plaque but not impact the interaction of A. oris with streptococci.

Understanding the importance of oral microbiota in human health and disease also leads to an expansion of the knowledge on functional, metabolic, and molecular alterations directly contributing to oral and systemic pathologies. To date, a compelling number of studies have documented the crucial role of some oral cavity-occurring microbes in the initiation and progression of cancers. Although this effect was noted primarily for Fusobacterium spp., the potential impact of other oral microbes is also worthy of investigation. In this study, we aimed to assess the effect of Enterococcus faecalis , Actinomyces odontolyticus , and Propionibacterium acnes on the proliferation capability and mechanical features of gingival cells and cell lines derived from lung, breast, and ovarian cancers. For this purpose, we incubated selected cell lines with heat-inactivated bacteria and supernatants collected from biofilms, cultured in both anaerobic and aerobic conditions, in the presence of surgically removed teeth and human saliva. The effect of oral bacteria on cell population growth is variable, with the highest growth-promoting abilities observed for E. faecalis in relation to human primary gingival fibroblasts (HGF) and lung cancer A549 cells, and P. acnes in relation to breast cancer MCF-7 and ovarian cancer SKOV-3 cells. Notably, this effect seems to depend on a delicate balance between the pro-stimulatory and toxic effects of bacterial-derived products. Regardless of the diverse effect of bacterial products on cellular proliferation capability, we observed significant alterations in stiffness of gingival and lung cancer cells stimulated with E. faecalis bacteria and corresponding biofilm supernatants, suggesting a novel molecular mechanism involved in the pathogenesis of diseases in oral cavities and tooth tissues. Accordingly, it is proposed that analysis of cancerogenic features of oral cavity bacteria should be multivariable and should include investigation of potential alterations in cell mechanical properties. These findings corroborate the important role of oral hygiene and root canal treatment to assure the healthy stage of oral microbiota., Competing Interests: The authors declare no conflicts of interest.

The host-microbe relationship is pivotal for oral health as well as for peri-implant diseases. Peri-implant mucosa and commensal biofilm play important roles in the maintenance of host-microbe homeostasis, but little is known about how they interact. We have therefore investigated the early host-microbe interaction between commensal multispecies biofilm (Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar, Porphyromonas gingivalis) and organotypic peri-implant mucosa using our three-dimensional model. After 24 hr, biofilms induced weak inflammatory reaction in the peri-implant mucosa by upregulation of five genes related to immune response and increased secretion of IL-6 and CCL20. Biofilm volume was reduced which might be explained by secretion of β-Defensins-1, -2, and CCL20. The specific tissue reaction without intrinsic overreaction might contribute to intact mucosa. Thus, a relationship similar to homeostasis and oral health was established within the first 24 hr. In contrast, the mucosa was damaged and the bacterial distribution was altered after 48 hr. These were accompanied by an enhanced immune response with upregulation of additional inflammatory-related genes and increased cytokine secretion. Thus, the homeostasis-like relationship was disrupted. Such profound knowledge of the host-microbe interaction at the peri-implant site may provide the basis to improve strategies for prevention and therapy of peri-implant diseases., (© 2020 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.)

Periodontitis affects oral tissues and induces systemic inflammation, which increases the risk of cardiovascular disease and metabolic syndrome. Subgingival plaque accumulation is a trigger of periodontitis. Fusobacterium nucleatum (FN) contributes to subgingival biofilm complexity by intercalating with early and late bacterial colonizers on tooth surfaces. In addition, inflammatory responses to FN are associated with the progression of periodontitis. Nigella sativa Lin. seed, which is known as black cumin (BC), has been used as a herbal medicine to treat ailments such as asthma and infectious diseases. The current study examined the inhibitory effect of BC oil and its active constituents, thymol (TM) and thymoquinone (TQ), on FN‑associated biofilm and inflammation. FN‑containing biofilms were prepared by co‑cultivation with an early dental colonizer, Actinomyces naeslundii (AN). The stability and biomass of FN/AN dual species biofilms were significantly higher compared with FN alone. This effect was retained even with prefixed cells, indicating that FN/AN co‑aggregation is mediated by physicochemical interactions with cell surface molecules. FN/AN biofilm formation was significantly inhibited by 0.1% TM or TQ. Confocal laser scanning microscopy indicated that treatment of preformed FN/AN biofilm with 0.01% of BC, TM or TQ significantly reduced biofilm thickness, and TQ demonstrated a cleansing effect equivalent to that of isopropyl methylphenol. TQ dose‑dependently suppressed TNF‑α production from a human monocytic cell line, THP‑1 exposed to FN, yet showed no toxicity to THP‑1 cells. These results indicated that oral hygiene care using TQ could reduce FN‑associated biofilm and inflammation in gingival tissue.

Bacteria utilize chemical and mechanical mechanisms to sense their environment, to survive hostile conditions. In mechanical sensing, intra-bilayer pressure profiles change due to deformation induced by the adhesion forces bacteria experience on a surface. Emergent properties in mono-species Streptococcus mutans biofilms, such as extracellular matrix production, depend on the adhesion forces that streptococci sense. Here we determined whether and how salivary-conditioning film (SCF) adsorption and the multi-species nature of oral biofilm influence adhesion force sensing and associated gene expression by S. mutans. Hereto, Streptococcus oralis, Actinomyces naeslundii, and S. mutans were grown together on different surfaces in the absence and presence of an adsorbed SCF. Atomic force microscopy and RT-qPCR were used to measure S. mutans adhesion forces and gene expressions. Upon SCF adsorption, stationary adhesion forces decreased on a hydrophobic and increased on a hydrophilic surface to around 8 nN. Optical coherence tomography showed that triple-species biofilms on SCF-coated surfaces with dead S. oralis adhered weakly and often detached as a contiguous sheet. Concurrently, S. mutans displayed no differential adhesion force sensing on SCF-coated surfaces in the triple-species biofilms with dead S. oralis, but once live S. oralis were present S. mutans adhesion force sensing and gene expression ranked similar as on surfaces in the absence of an adsorbed SCF. Concluding, live S. oralis may enzymatically degrade SCF components to facilitate direct contact of biofilm inhabitants with surfaces and allow S. mutans adhesion force sensing of underlying surfaces to define its appropriate adaptive response. This represents a new function of initial colonizers in multi-species oral biofilms.

Background: Brain abscesses are the rare and most severe form of actinomycosis, which usually manifests as abscesses of the occipital or parietal lobe due to direct expansion from an adjacent area, the oral cavity. In the medical literature, there are only a few reported cases of brain abscess caused by Actinomyces meyeri. In this report, we present a 35-year-old male patient who experienced an insidious headache and left-sided weakness and was diagnosed with an Actinomyces meyeri brain abscess., Case Presentation: A 35-year-old Nepalese man came to our institute with the primary complaint of insidious onset of headache and left-sided weakness. His physical examination was remarkable for the left-sided weakness with power 2/5 on both upper and lower limbs, hypertonia, hyperreflexia and positive Babinski sign, with intact sensory function. Cardiac examination revealed systolic murmur with regular S1 and S2, and lung examination was normal. The patient had poor dental hygiene. Biochemistry and haematology panel were normal. Urinalysis, chest X-ray and electrocardiogram revealed no abnormality. A transthoracic echocardiogram revealed mitral regurgitation. However, there was no evidence of valvular vegetation. A magnetic resonance imaging (MRI) of the brain was performed, which showed a bi-lobed rim enhancing lesion with a conglomeration of two adjoining round lesions in the right parietal parasagittal region. Perilesional oedema resulting in mass effect over the right lateral ventricle and mid-right uncal herniation with midline shift was noted. Craniotomy was performed, and the lesion was excised. Gram staining of the extracted sample revealed gram variable filamentous rods. Creamy white, moist, confluent colonies were observed after performing anaerobic culture in chocolate agar. On the gram staining, they showed gram-positive filamentous rods. Actinomyces meyeri was identified based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) technology. Based on the susceptibilities, he was successfully treated with ampicillin-sulbactam., Conclusions: In conclusion, Actinomyces should be considered in the differential diagnosis of brain abscess in patients with poor dental hygiene, and early diagnosis and appropriate treatment can lead to better results.

Background and Objectives: Recent evidence suggests that oral bacteria can affect extra-oral diseases by modulating aspects of the gut environment such as the microbiome, metabolome, and immune profiles. However, differences in the effects of different types of oral bacteria, particularly periodontopathic and health-associated bacteria, remain elusive., Materials and Methods: Five-week-old germ-free mice were orally administered with either periodontopathic bacteria as oral pathobionts (Porphyromonas gingivalis, Filifactor alocis, and Fusobacterium nucleatum) or bacteria associated with periodontal health (Actinomyces naeslundii, Streptococcus mitis, and Veillonella rogosae) twice a week for five weeks. The presence of all bacterial species in the feces and the livers of the mice was analyzed via polymerase chain reaction (PCR), using specific primers for 16S rRNA genes. Alveolar bone resorption was evaluated histologically. The expression profiles of various genes in the liver and small intestine were analyzed using real-time PCR. Sera were analyzed to determine the levels of antibodies and endotoxin. The proportions of T helper 17 (Th17) and regulatory T (Treg) cells in mesenteric lymph nodes and Peyer's patches were analyzed using flow cytometry., Results: Neither of the types of bacteria administered in this experiment induced alveolar bone resorption. All bacteria elicited some degree of systemic antibody response in the mice, although the response to S. mitis was not obvious. The response to P. gingivalis and V. rogosae was strongest. Generally, the health-associated bacteria but not the periodontitis-associated bacteria were detected in fecal samples. Interestingly, only Fusobacterium nucleatum DNA was detected in the liver, despite that live Fusobacterium nucleatum were not detected in the liver. The levels of interleukin-17 in the intestine and genes related to lipid accumulation in the liver were significantly higher in the mice that received periodontitis-associated bacteria. In addition, expression of the gene associated with endoplasmic reticulum stress was higher and that of the gene controlling circadian rhythm was lower in the periodontitis group. There was no difference in serum endotoxin, T-cell phenotypes in the lymphatic tissues, or genes related to the gut barrier., Conclusion: Oral administration of periodontitis-associated bacteria can induce pathological changes in the liver and intestine that are implicated in the process of periodontitis. These findings further support the importance of the oral-gut connection., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Biofilm-associated diseases are one of the main causes of implant failure. Currently, the development of implant surface treatment goes beyond the osseointegration process and focuses on the creation of surfaces with antimicrobial action and with the possibility to be re-activated (i.e., light source activation). Titanium dioxide (TiO 2 ), an excellent photocatalyst used for photocatalytic antibacterial applications, could be a great alternative, but its efficiency is limited to the ultraviolet (UV) range of the electromagnetic spectrum. Since UV radiation has carcinogenic potential, we created a functional TiO 2 coating codoped with nitrogen and bismuth via the plasma electrolytic oxidation (PEO) of titanium to achieve an antibacterial effect under visible light with re-activation potential. A complex surface topography was demonstrated by scanning electron microscopy and three-dimensional confocal laser scanning microscopy. Additionally, PEO-treated surfaces showed greater hydrophilicity and albumin adsorption compared to control, untreated titanium. Bismuth incorporation shifted the band gap of TiO 2 to the visible region and facilitated higher degradation of methyl orange (MO) in the dark, with a greater reduction in the concentration of MO after visible-light irradiation even after 72 h of aging. These results were consistent with the in vitro antibacterial effect, where samples with nitrogen and bismuth in their composition showed the greatest bacterial reduction after 24 h of dual-species biofilm formation ( Streptococcus sanguinis and Actinomyces naeslundii) in darkness with a superior effect at 30 min of visible-light irradiation. In addition, such a coating presents reusable photocatalytic potential and good biocompatibility by presenting a noncytotoxicity effect on human gingival fibroblast cells. Therefore, nitrogen and bismuth incorporation into TiO 2 via PEO can be considered a promising alternative for dental implant application with antibacterial properties in darkness, with a stronger effect after visible-light application.

Microbial infection has been shown to involve in oral carcinogenesis; however, the underlying mechanisms remain poorly understood. The present study aimed to characterize the growth of oral microorganisms as both monospecies and polymicrobial biofilms and determine the effects of their products on oral keratinocytes. Candida albicans (ALC3), Actinomyces naeslundii (AN) and Streptococcus mutans (SM) biofilms or a combination of these (TRI) were grown in flow-cell system for 24 h. The biofilms were subjected to fluorescent in situ hybridization using species-specific probes and analysed using confocal laser scanning microscopy. The effluent derived from each biofilm was collected and incubated with malignant (H357) and normal (OKF6) oral keratinocytes to assess extracellular matrix adhesion, epithelial-mesenchymal transition (EMT) and cytokines expression. Incubation of OKF6 with ALC3 and TRI effluent significantly decreased adhesion of the oral keratinocyte to collagen I, whereas incubation of H357 with similar effluent increased adhesion of the oral keratinocyte to laminin I, significantly when compared with incubation with artificial saliva containing serum-free medium (NE; P < 0.05). In OKF6, changes in E-cadherin and vimentin expression were not consistent with EMT although there was evidence of a mesenchymal to epithelial transition in malignant oral keratinocytes incubated with AN and SM effluent. A significant increase of pro-inflammatory cytokines expression, particularly interleukin (IL)-6 and IL-8, was observed when H357 was incubated with all biofilm effluents after 2- and 24-h incubation when compared with NE (P < 0.05). In conclusion, C.albicans, A.naeslundii and S.mutans form polymicrobial biofilms which differentially modulate malignant phenotype of oral keratinocytes., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Introduction: Apical periodontitis is an inflammatory disease in the periradicular region of teeth that results from infection by multispecies bacterial biofilm residing in the root canal system. In this study, we investigated whether Lactobacillus plantarum lipoteichoic acid (Lp.LTA) could inhibit multispecies oral pathogenic bacterial biofilm formation., Methods: Highly pure and structurally intact Lp.LTA was purified from L. plantarum. Actinomyces naeslundii, Lactobacillus salivarius, Streptococcus mutans, and Enterococcus faecalis were co-cultured to form oral multispecies biofilm in the presence or absence of Lp.LTA on culture plates or human dentin slices. Preformed biofilm was treated with or without Lp.LTA, followed by additional treatment with intracanal medicaments such as calcium hydroxide or chlorhexidine digluconate. Confocal microscopy and crystal violet assay were performed to determine biofilm formation. Biofilm on human dentin slices was visualized with a scanning electron microscope., Results: Biofilm formation of multispecies bacteria on the culture dishes was dose-dependently reduced by Lp.LTA compared with the nontreatment control group. Lp.LTA also inhibited multispecies biofilm formation on the dentin slices in a dose-dependent manner. Interestingly, Lp.LTA was shown to reduce preformed multispecies biofilm compared with the nontreatment group. Moreover, Lp.LTA potentiated the effectiveness of the intracanal medicaments in the removal of preformed multispecies biofilm., Conclusions: These results suggest that Lp.LTA is a potential anti-biofilm agent for treatment or prevention of oral infectious disease, including apical periodontitis, which is mainly caused by multispecies bacterial biofilm., (Copyright © 2018 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

The search for environment-friendly, economical and healthy alternatives to agrochemicals tempted us to evaluate the potential of naturally occurring actinomycetes to improve soil properties, plant growth and photosynthesis, grain yield and chemical composition of economically important cereals (wheat, barley, oat, maize and sorghum). To this end, actinomycetes were isolated from soils of local cereals fields, then their biological activities, namely antibacterial, antiprotozoal, antioxidant, and phenolic and flavonoid contents were evaluated. The four most active isolates (9, 16, 24 and 26) were selected and used for enriching the soils until seed set. Each isolate was separately applied. Seeds of the selected cereals were grown in the actinomycete-enriched soils. The soils were analyzed for their electrical conductivity, pH values, total phenolics, organic matter and mineral content. At the vegetative stage, chlorophyll content and gas exchange rates were measured. Mature seeds were then harvested, the yield was evaluated and the seeds were analyzed for their primary and secondary metabolites. The selected isolates improved the grain yield in all tested cereals and most noticeably in barley and maize as compared to control counterparts. These positive effects were probably a result of increased carbon gain due to higher chlorophyll and photosynthetic rate. Isolate 26 showed the highest effect on grains composition profiles followed by the isolate 16. Phenolics and sugars of all grains increased by treatment with the tested isolates. Isolate 26 was the most effective in this regard. All isolates generally improved vitamins, amino acids and organic acids contents in grains. However, fatty acids profile showed a decrease in the content of all measured fatty acids by isolate 26 and an increase in the contents by isolate 16. These results emphasize the potential of actinomycete enrichment as an alternative to agrochemicals and strongly suggest that they can be used in organic farming., (Copyright © 2018. Published by Elsevier B.V.)

This report describes weekly repeated anesthesia in a 7-yr-old, 1,030 kg, female Eastern black rhinoceros ( Diceros bicornis michaeli), that was immobilized six times using a combination of 2 mg etorphine (0.002 mg/kg), 5 mg medetomidine (0.005 mg/kg), 25 mg midazolam (0.024 mg/kg), and 300 mg ketamine (0.29 mg/kg) delivered intramuscularly (IM) via remote dart to facilitate long-term medical care of a bilateral, obstructive Actinomyces sp. rhinitis. The drug combination described in this study resulted in reliable, rapid recumbency of the animal within 2-8 min after initial administration via dart and produced deep anesthesia for 34-78 min without supplemental anesthetic administration. Antagonist drugs (100 mg naltrexone [0.1 mg/kg] and 25 mg atipamezole [0.024 mg/kg] IM) produced reliable and uneventful recoveries in all the procedures. During each anesthetic procedure, the animal was intubated and provided intermittent positive pressure ventilation with a megavertebrate demand ventilator. Tachycardia and hypoxia noted after induction resolved after positive pressure ventilation with oxygen. This report provides useful information on a novel anesthetic protocol used repeatedly for intensive medical management in a black rhinoceros.

Around one-quarter of bacterial diversity comprises a single radiation with reduced genomes, known collectively as the Candidate Phyla Radiation. Recently, we coisolated TM7x, an ultrasmall strain of the Candidate Phyla Radiation phylum Saccharibacteria, with its bacterial host Actinomyces odontolyticu s strain XH001 from human oral cavity and stably maintained as a coculture. Our current work demonstrates that within the coculture, TM7x cells establish a long-term parasitic association with host cells by infecting only a subset of the population, which stay viable yet exhibit severely inhibited cell division. In contrast, exposure of a naïve A. odontolyticu s isolate, XH001n, to TM7x cells leads to high numbers of TM7x cells binding to each host cell, massive host cell death, and a host population crash. However, further passaging reveals that XH001n becomes less susceptible to TM7x over time and enters a long-term stable relationship similar to that of XH001. We show that this reduced susceptibility is driven by rapid host evolution that, in contrast to many forms of phage resistance, offers only partial protection. The result is a stalemate where infected hosts cannot shed their parasites; nevertheless, parasite load is sufficiently low that the host population persists. Finally, we show that TM7x can infect and form stable long-term relationships with other species in a single clade of Actinomyces , displaying a narrow host range. This system serves as a model to understand how parasitic bacteria with reduced genomes such as those of the Candidate Phyla Radiation have persisted with their hosts and ultimately expanded in their diversity., Competing Interests: Conflict of interest statement: W.S. is a part-time chief science officer of C3 Jian, Inc., which has licensed technologies from the University of California Regents that could be indirectly related to this research project., (Copyright © 2018 the Author(s). Published by PNAS.)

Apical periodontitis is frequently associated with the presence of bacteria biofilm, which has an indisputable impact on the prognosis of endodontic therapy due to the high resistance to adverse environmental conditions, chemicals, and antibiotic therapy that characterize bacteria within biofilm. The biofilm matrix acts as a protective shield over the encased microorganisms. The aim of this investigation was to identify the main biochemical components of biofilm matrix from endodontic mono- and dual-species biofilms. Enterococcus faecalis and Actinomyces naeslundii were cultured as mono- and dual-species biofilms for 14 days. Crude extracellular polymeric substances (EPSs) from biofilm matrices were extracted using chemical and physical methods. High-performance liquid chromatography, gas chromatography, and mass spectrometry were used to determine the carbohydrate, protein, and fatty acid components. Chemical analysis of the biofilm matrices revealed that they were mainly composed of stachyose, maltose, and mannose carbohydrates. The protein profile in all biofilm samples showed abundant oxidoreductases and chaperone proteins and some virulence- associated proteins mainly located in the membrane surface. High percentages of saturated and monounsaturated fatty acids were identified in all biofilm matrices, with a major prevalence of palmitic, stearic, and oleic acids. Based on the results, it was possible to obtain for the first time a general overview of the biochemical profile of endodontic biofilm matrices., Competing Interests: The authors have declared that no competing interests exist.

Actinomyces are nonmotile, filamentous, Gram-positive bacteria that cause actinomycosis in immunodeficiency patients. Although the prognosis of actinomycosis is good, the diagnosis of actinomycosis is quite difficult. Recent studies on actinomycosis have shown that Actinomyces play an important role in various biological and clinical processes, such as the formation of dental plaque and the degradation of organics in the gastrointestinal tract. Here, the distribution of Actinomyces in the digestive tract, and different biological effects of actinomycosis, and its clinical association with inflammatory diseases are discussed. Furthermore, an overview of the most commonly used treatment methods and drugs used to treat Actinomyces infected alimentary canal diseases is presented.

Diterpenes are an important class of plant metabolites that can be used in the search for new antibacterial agents. ent-Copalic acid (CA), the major diterpene in Copaifera species exudates, displays several pharmacological properties. This study evaluates the CA antibacterial potential against the anaerobic bacteria Peptostreptococcus anaerobius and Actinomyces naeslundii. Antimicrobial assays included time-kill and biofilm inhibition and eradication assays. Time-kill assays conducted for CA concentrations between 6.25 and 12.5 μg/mL evidenced bactericidal activity within 72 h. CA combined with chlorhexidine dihydrochloride (CHD) exhibited bactericidal action against P. anaerobius within 6 h of incubation. As for A. naeslundii, the same combination reduced the number of microorganisms by over 3 log10 at 24 h and exerted a bactericidal effect at 48 h of incubation. CA at 500 and 2000 μg/mL inhibited P. anaerobius and A. naeslundii biofilm formation by at least 50%, respectively. CA at 62.5 and 1.000 μg/mL eradicated 99.9% of pre-formed P. anaerobius and A. naeslundii biofilms, respectively. These results indicated that CA presents in vitro antibacterial activity and is a potential biofilm inhibitory agent. This diterpene may play an important role in the search for novel sources of agents that can act against anaerobic bacteria., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The present study investigated the compounds present in the low molecular mass fraction of Lentinus edodes mushroom (shiitake) extract and their anti-virulence activity against oral pathogens (reference and clinical Streptococcus mutans, Actinomyces naeslundii, and Prevotella intermedia strains). Oxalic, succinic, and quinic acids, and adenine, inosine, and uridine were identified by HPLC-DAD-ESI-MS/MS. Their anti-biofilm production and preformed biofilm disaggregation activities were studied using commercial standard compounds at different concentrations. As regards S. mutans, the highest activity was shown by adenine at 5 mg mL-1 both in the biofilm inhibition (BI 50%) and biofilm disaggregation tests (BD 20%). Considering A. naeslundii, BI values close to 80% were registered for oxalic acid at 1 mg mL-1 and 2 mg mL-1 and BD 50% for quinic acid at 3 mg mL-1. A weaker activity was found against P. intermedia. Furthermore, different mixtures of the commercial standards were tested showing that the activity of a compound can be strongly and sometimes negatively affected by the presence of the other compounds.

The ability of microorganisms to regulate gene expression is thought to be critical for survival and growth during the development of polymicrobial biofilms such as dental plaque. The commensal dental plaque colonizer, Streptococcus gordonii, responds to cell--cell contact (coaggregation) with Actinomyces oris by regulating >20 genes, including those involved in arginine biosynthesis. We hypothesized that an S. gordonii extracellular protease is critical for sensing by providing amino acids that modulate gene expression. S. gordonii coaggregated strongly with A. oris in buffer, saliva or chemically defined medium (CDM). In wild-type S. gordonii, expression of arginine biosynthesis genes argC and argG increased within two hours' growth in CDM in monocultures, but not following coaggregation with A. oris. By contrast, coaggregation of A. oris with an S. gordonii mutant lacking sgc, encoding the extracellular protease Challisin, resulted in increases in argC and argG gene expression that were similar to monocultures. Genetic complementation of sgc restored the ability of S. gordonii to sense coaggregation with A. oris. Coaggregation enabled growth of S. gordonii in low/no arginine and disruption of sgc did not affect this ability. We propose that extracellular bacterial proteases may be key mediators of cell--cell contact sensing by diverse microbial species.

Streptococcus mutans strongly influences the development of pathogenic biofilms associated with dental caries. Our understanding of S. mutans behaviour in biofilms is based on a few well-characterized laboratory strains; however, individual isolates vary widely in genome content and virulence-associated phenotypes, such as biofilm formation and environmental stress sensitivity. Using an ecological biofilm model, we assessed the impact of co-cultivation of several S. mutans isolates with Streptococcus oralis and Actinomyces naeslundii on biofilm composition following exposure to sucrose. The laboratory reference strain S. mutans UA159 and clinical isolates Smu44 (most aciduric), Smu56 (altered biofilm formation) and Smu81 (more sensitive to oxidative stress) were used. Our data revealed S. mutans isolates varied in their ability to compete and become dominant in the biofilm after the addition of sucrose, and this difference correlated with sensitivity to H 2 O 2 produced by S. oralis. Smu81 was particularly sensitive to H 2 O 2 and could not compete with S. oralis in mixed-species biofilm, despite forming robust biofilms on its own. Thus, diminished oxidative stress tolerance in S. mutans isolates can impair their ability to compete in complex biofilms, even in the presence of sucrose, which could influence the progression of a healthy biofilm community to one capable of causing disease., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Since there is no consensus about whether starch increases the cariogenic potential of sucrose, we used a validated 3-species biofilm model to evaluate if starch combined with sucrose provokes higher root dentine demineralization than sucrose alone. Biofilms (n = 18) composed by Streptococcus mutans (the most cariogenic bacteria), Actinomces naeslundii (which has amylolytic activity), and Streptococcus gordonii (which binds salivary amylase) were formed on root dentine slabs under exposure 8 ×/day to one of the following treatments: 0.9% NaCl, 1% starch, 10% sucrose, or a combination of 1% starch and 10% sucrose. Before each treatment, biofilms were pretreated with human whole saliva for 1 min. The pH of the culture medium was measured daily as an indicator of biofilm acidogenicity. After 96 h of growth, the biofilms were collected, and the biomass, bacteria viability, and polysaccharides were analyzed. Dentine demineralization was assessed by surface hardness loss (% SHL). Biofilm bioarchitecture was analyzed using confocal laser scanning microscopy. Treatment with a starch and sucrose combination provoked higher (p = 0.01) dentine demineralization than sucrose alone (% SHL = 53.2 ± 7.0 vs. 43.2 ± 8.7). This was supported by lower pH values (p = 0.007) of the culture medium after daily exposure to the starch and sucrose combination compared with sucrose (4.89 ± 0.29 vs. 5.19 ± 0.32). Microbiological and biochemical findings did not differ between biofilms treated with the combination of starch and sucrose and sucrose alone (p > 0.05). Our findings give support to the hypothesis that a starch and sucrose combination is more cariogenic for root dentine than sucrose alone., (© 2018 S. Karger AG, Basel.)

Removal of bacterial biofilm from the root canal system is essential for the management of endodontic disease. Here we evaluated the antibacterial effect of N-acetylcysteine (NAC), a potent antioxidant and mucolytic agent, against mature multispecies endodontic biofilms consisting of Actinomyces naeslundii, Lactobacillus salivarius, Streptococcus mutans and Enterococcus faecalis on sterile human dentin blocks. The biofilms were exposed to NAC (25, 50 and 100mg/mL), saturated calcium hydroxide or 2% chlorhexidine solution for 7 days, then examined by scanning electron microscopy. The biofilm viability was measured by viable cell counts and ATP-bioluminescence assay. NAC showed greater efficacy in biofilm cell removal and killing than the other root canal medicaments. Furthermore, 100mg/mL NAC disrupted the mature multispecies endodontic biofilms completely. These results demonstrate the potential use of NAC in root canal treatment., (Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

The Gram-positive actinobacteria Actinomyces spp. are key colonizers in the development of oral biofilms due to the inherent ability of Actinomyces to adhere to receptor polysaccharides on the surface of oral streptococci and host cells. This receptor-dependent bacterial interaction, or coaggregation, requires a unique sortase-catalyzed pilus consisting of the pilus shaft FimA and the coaggregation factor CafA forming the pilus tip. While the essential role of the sortase machine SrtC2 in pilus assembly, biofilm formation, and coaggregation has been established, little is known about trans -acting factors contributing to these processes. We report here a large-scale Tn 5 transposon screen for mutants defective in Actinomyces oris coaggregation with Streptococcus oralis We obtained 33 independent clones, 13 of which completely failed to aggregate with S. oralis , and the remainder of which exhibited a range of phenotypes from severely to weakly defective coaggregation. The former had Tn 5 insertions in fimA , cafA , or srtC2 , as expected; the latter were mapped to genes coding for uncharacterized proteins and various nuo genes encoding the NADH dehydrogenase subunits. Electron microscopy and biochemical analyses of mutants with nonpolar deletions of nuo genes and ubiE , a menaquinone C-methyltransferase-encoding gene downstream of the nuo locus, confirmed the pilus and coaggregation defects. Both nuoA and ubiE mutants were defective in oxidation of MdbA, the major oxidoreductase required for oxidative folding of pilus proteins. Furthermore, supplementation of the ubiE mutant with exogenous menaquinone-4 rescued the cell growth and pilus defects. Altogether, we propose that the A. oris electron transport chain is biochemically linked to pilus assembly via oxidative protein folding. IMPORTANCE The Gram-positive actinobacterium A. oris expresses adhesive pili, or fimbriae, that are essential to biofilm formation and Actinomyces interactions with other bacteria, termed coaggregation. While the critical role of the conserved sortase machine in pilus assembly and the disulfide bond-forming catalyst MdbA in oxidative folding of pilins has been established, little is known about other trans -acting factors involved in these processes. Using a Tn 5 transposon screen for mutants defective in coaggregation with Streptococcus oralis , we found that genetic disruption of the NADH dehydrogenase and menaquinone biosynthesis detrimentally alters pilus assembly. Further biochemical characterizations determined that menaquinone is important for reactivation of MdbA. This study supports the notion that the electron transport chain is biochemically linked to pilus assembly in A. oris via oxidative folding of pilin precursors., (Copyright © 2017 Sanchez et al.)

Posttranslocational protein folding in the Gram-positive biofilm-forming actinobacterium Actinomyces oris is mediated by a membrane-bound thiol-disulfide oxidoreductase named MdbA, which catalyzes oxidative folding of nascent polypeptides transported by the Sec translocon. Reoxidation of MdbA involves a bacterial v itamin K ep o xide r eductase (VKOR)-like protein that contains four cysteine residues, C93/C101 and C175/C178, with the latter forming a canonical CXXC thioredoxin-like motif; however, the mechanism of VKOR-mediated reoxidation of MdbA is not known. We present here a topological view of the A. oris membrane-spanning protein VKOR with these four exoplasmic cysteine residues that participate in MdbA reoxidation. Like deletion of the VKOR gene, alanine replacement of individual cysteine residues abrogated polymicrobial interactions and biofilm formation, concomitant with the failure to form adhesive pili on the bacterial surface. Intriguingly, the mutation of the cysteine at position 101 to alanine (C101A mutation) resulted in a high-molecular-weight complex that was positive for MdbA and VKOR by immunoblotting and was absent in other alanine substitution mutants and the C93A C101A double mutation and after treatment with the reducing agent β-mercaptoethanol. Consistent with this observation, affinity purification followed by immunoblotting confirmed this MdbA-VKOR complex in the C101A mutant. Furthermore, ectopic expression of the Mycobacterium tuberculosis VKOR analog in the A. oris VKOR deletion (ΔVKOR) mutant rescued its defects, in contrast to the expression of M. tuberculosis VKOR variants known to be nonfunctional in the disulfide relay that mediates reoxidation of the disulfide bond-forming catalyst DsbA in Escherichia coli Altogether, the results support a model of a disulfide relay, from its start with the pair C93/C101 to the C175-X-X-C178 motif, that is required for MdbA reoxidation and appears to be conserved in members of the class Actinobacteria IMPORTANCE It has recently been shown in the high-GC Gram-positive bacteria (or Actinobacteria ) Actinomyces oris and Corynebacterium diphtheriae that oxidative folding of nascent polypeptides transported by the Sec machinery is catalyzed by a membrane-anchored oxidoreductase named MdbA. In A. oris , reoxidation of MdbA requires a bacterial VKOR-like protein, and yet, how VKOR mediates MdbA reoxidation is unknown. We show here that the A. oris membrane-spanning protein VKOR employs two pairs of exoplasmic cysteine residues, including the canonical CXXC thioredoxinlike motif, to oxidize MdbA via a disulfide relay mechanism. This mechanism of disulfide relay is essential for pilus assembly, polymicrobial interactions, and biofilm formation and appears to be conserved in members of the class Actinobacteria , including Mycobacterium tuberculosis ., (Copyright © 2017 American Society for Microbiology.)

The fungus Candida albicans is carried orally and causes a range of superficial infections that may become systemic. Oral bacteria Actinomyces oris and Streptococcus oralis are abundant in early dental plaque and on oral mucosa. The aims of this study were to determine the mechanisms by which S. oralis and A. oris interact with each other and with C. albicans in biofilm development. Spatial distribution of microorganisms was visualized by confocal laser scanning microscopy of biofilms labeled by differential fluorescence or by fluorescence in situ hybridization (FISH). Actinomyces oris and S. oralis formed robust dual-species biofilms, or three-species biofilms with C. albicans. The bacterial components tended to dominate the lower levels of the biofilms while C. albicans occupied the upper levels. Non-fimbriated A. oris was compromised in biofilm formation in the absence or presence of streptococci, but was incorporated into upper biofilm layers through binding to C. albicans. Biofilm growth and hyphal filament production by C. albicans was enhanced by S. oralis. It is suggested that the interkingdom biofilms are metabolically coordinated to house all three components, and this study demonstrates that adhesive interactions between them determine spatial distribution and biofilm architecture. The physical and chemical communication processes occurring in these communities potentially augment C. albicans persistence at multiple oral cavity sites., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Oral biofilms comprise of extracellular polysaccharides and polymicrobial microorganisms. The objective of this study was to determine the effect of polymicrobial interactions of Candida albicans, Actinomyces naeslundii, and Streptococcus mutans on biofilm formation with the hypotheses that biofilm biomass and metabolic activity are both C. albicans strain and growth medium dependent. To study monospecific biofilms, C. albicans, A. naeslundii, and S. mutans were inoculated into artificial saliva medium (ASM) and RPMI-1640 in separate vials, whereas to study polymicrobial biofilm formation, the inoculum containing microorganisms was prepared in the same vial prior inoculation into a 96-well plate followed by 72 hours incubation. Finally, biofilm biomass and metabolic activity were measured using crystal violet and XTT assays, respectively. Our results showed variability of monospecies and polymicrobial biofilm biomass between C. albicans strains and growth medium. Based on cut-offs, out of 32, seven RPMI-grown biofilms had high biofilm biomass (HBB), whereas, in ASM-grown biofilms, 14 out of 32 were HBB. Of the 32 biofilms grown in RPMI-1640, 21 were high metabolic activity (HMA), whereas in ASM, there was no biofilm had HMA. Significant differences were observed between ASM and RPMI-grown biofilms with respect to metabolic activity (P <01). In conclusion, biofilm biomass and metabolic activity were both C. albicans strain and growth medium dependent., (© The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Unlabelled: This paper describes a high-throughput method that relies upon a microplate reader to score coaggregation 60 min postmixing, and use of a high-speed real-time imaging technology to describe the rate of coaggregation over time. The results of visual, microplate, and FlowCam(™) aggregation scores for oral bacteria Streptococcus gordonii, Streptococcus oralis, and Actinomyces oris, whose ability to coaggregate are well characterized, are compared. Following mixing of all possible pairs, the top fraction of the supernatant was added to a microplate to quantify cell-density. Pairs were also passed through a flow cell within a FlowCam(™) to quantify the rate of coaggregation of each pair. Results from both the microplate and FlowCam(™) approaches correlated with corresponding visual coaggregation scores and microscopic observations. The microplate-based assay enables high-throughput screening, whereas the FlowCam(™) -based assay validates and quantifies the extent that autoaggregation and coaggregation occur. Together these assays open the door for future in-depth studies of autoaggregation and coaggregation among large panels of test strains., Significance and Impact of the Study: Coaggregation between bacterial species is integral to multi-species biofilm development. Difficulties in rapidly and reproducibly identifying and quantifying coaggregation have limited mechanistic studies. This paper demonstrates two complementary quantitative methods to screen for coaggregation. The first approach uses a microplate-based high-throughput approach and the other uses a FlowCam(™) device. The microplate-based approach enables rapid detection of coaggregation between candidate coaggregating pairs of strains simultaneously while controlling for variation between replicates. The FlowCam(™) approach allows for in-depth analysis of the rates of coaggregation and size of aggregates formed., (© 2016 The Society for Applied Microbiology.)

Objective: To study the effect of poly(ethylene glycol)methacrylate(PEG)-arginine-glycine-aspartic(RGD)polymer brushes graft on bacterial adhesion and MC3T3 osteoblast cell attachment on titanium, and to investigate if the modification of titanium will enable the implant to be anti-fouling and promot osteointegration., Methods: PEG was tethered on titanium surface modified with 2-bromoisobutyryl bromide(denoted as Ti-Br)to form Ti-PEG brushes. Functionalization of the Ti-PEG surface with RGD was performed to form Ti-PEG-RGD brushes. The chemical composition of modified titanium surfaces was characterized by X-ray photoelectron spectroscopy(XPS). Changes in surface hydrophilicity and hydrophobicity were characterized by static water contact angle measurements. Streptococcus mutans(Sm), Actinomyces naeslundii(An)and osteoblast cell were cultured on pure titanium(Ti), Ti-PEG, Ti-PEG-RGD surfaces respectively. There were ten samples in each group. The bacterial adhesion ability and cell attachment were confirmed by fluorescence microscopy and scanning electron microscopy(SEM)., Results: The static water contact angle of Ti, Ti-Br, Ti-PEG, Ti-PEG-RGD was less than 10°, 80°, 45°, 55° respectively. XPS confirmed that PEG-RGD brushes were successfully tethered on titanium surfaces. Anti-bacterial test showed that on the pure-Ti, there were large amount of bacteria from both groups, however, in the Ti-PEG, Ti-PEG-RGD surfaces, both kind of bacteria were rare and distributed diffusely. Cell culture test showed that on the Ti-PEG-RGD surfaces, the number of cells was significantly more than that on the Ti and the Ti-PEG surfaces., Conclusions: PEG can inhibit both kind of bacteria adhesion and osteoblast cell attachment, and PEG-RGD brushes can not only inhibit bacterial adhesion but also promote osteoblast cell attachment.

Unlabelled: The Gram-positive bacterium Actinomyces oris, a key colonizer in the development of oral biofilms, contains 18 LPXTG motif-containing proteins, including fimbrillins that constitute two fimbrial types critical for adherence, biofilm formation, and polymicrobial interactions. Export of these protein precursors, which harbor a signal peptide, is thought to be mediated by the Sec machine and require cleavage of the signal peptide by type I signal peptidases (SPases). Like many Gram-positive bacteria, A. oris expresses two SPases, named LepB1 and LepB2. The latter has been linked to suppression of lethal "glyco-stress," caused by membrane accumulation of the LPXTG motif-containing glycoprotein GspA when the housekeeping sortase srtA is genetically disrupted. Consistent with this finding, we show here that a mutant lacking lepB2 and srtA was unable to produce high levels of glycosylated GspA and hence was viable. However, deletion of neither lepB1 nor lepB2 abrogated the signal peptide cleavage and glycosylation of GspA, indicating redundancy of SPases for GspA. In contrast, the lepB2 deletion mutant failed to assemble the wild-type levels of type 1 and 2 fimbriae, which are built by the shaft fimbrillins FimP and FimA, respectively; this phenotype was attributed to aberrant cleavage of the fimbrillin signal peptides. Furthermore, the lepB2 mutants, including the catalytically inactive S101A and K169A variants, exhibited significant defects in polymicrobial interactions and biofilm formation. Conversely, lepB1 was dispensable for the aforementioned processes. These results support the idea that LepB2 is specifically utilized for processing of fimbrial proteins, thus providing an experimental model with which to study the basis of type I SPase specificity., Importance: Sec-mediated translocation of bacterial protein precursors across the cytoplasmic membrane involves cleavage of their signal peptide by a signal peptidase (SPase). Like many Gram-positive bacteria, A. oris expresses two SPases, LepB1 and LepB2. The latter is a genetic suppressor of lethal "glyco-stress" caused by membrane accumulation of glycosylated GspA when the housekeeping sortase srtA is genetically disrupted. We show here that LepB1 and LepB2 are capable of processing GspA, whereas only LepB2 is required for cleavage of fimbrial signal peptides. This is the first example of a type I SPase dedicated to LPXTG motif-containing fimbrial proteins. Thus, A. oris provides an experimental model with which to investigate the specificity mechanism of type I SPases., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Objectives: This study aimed to analyze interactions between oral biofilms and a dental triethylene glycol dimethacrylate (TEGDMA)-based resin infiltration material on enamel., Methods: Demineralized enamel specimens (14 days, acidic buffer, pH 5.0) were either infiltrated with a commercial TEGDMA resin and subjected to a three-species biofilm (Streptococcus mutans UAB 159, Streptococcus oralis OMZ 607 and Actinomyces oris OMZ 745) (group 1), applied to the biofilm (group 2), or merely resin infiltrated (group 3). A control group received no treatment (4). Biofilm formation and metabolic activity of biofilms were measured for group (1) and (2) after 24h CFU and a resazurin assay. Resin biodegradation was measured for group (1) and (3) by high performance liquid chromatography (HPLC) coupled with mass spectrometry after 6 and 24h incubation. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) images were taken to study the biofilm and material's autofluorescence in groups (1-4) after 24h., Results: SEM and CLSM images showed reduced biofilm formation on resin-infiltrated specimens (group 1) compared to group 2, while no biofilm was detectable on groups 3 and 4. CFU data (log10 CFU per mL) of group 1 showed significantly reduced bacterial numbers (p<0.05) compared to group 2. However, HPLC analysis of TEGDMA leakage after 6h and 24h revealed no differences between group 1 and group 3., Conclusions: The results of the current study indicate that freshly resin-infiltrated enamel surfaces show a biofilm reducing effect, while monomer leakage was not affected by bacterial presence., Clinical Significance: Resin infiltrated enamel surfaces are constantly exposed to the oral microflora. Yet, it is not known how biofilms interact with enamel-penetrated resins and if and to which extent accessory alignments in oral hygiene are needed., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Dental biofilm formation is not only a precursor to tooth decay, but also induces more serious systematic health problems such as cardiovascular disease and diabetes. Understanding the conditions promoting colonization and subsequent biofilm development involving complex bacteria coaggregation is particularly important. In this paper, we report a high-throughput microfluidic 'artificial teeth' device offering controls of multiple microenvironmental factors (e.g. nutrients, growth factors, dissolved gases, and seeded cell populations) for quantitative characteristics of long-term dental bacteria growth and biofilm development. This 'artificial teeth' device contains multiple (up to 128) incubation chambers to perform parallel cultivation and analyses (e.g. biofilm thickness, viable-dead cell ratio, and spatial distribution of multiple bacterial species) of bacteria samples under a matrix of different combinations of microenvironmental factors, further revealing possible developmental mechanisms of dental biofilms. Specifically, we applied the 'artificial teeth' to investigate the growth of two key dental bacteria, Streptococci species and Fusobacterium nucleatum, in the biofilm under different dissolved gas conditions and sucrose concentrations. Together, this high-throughput microfluidic platform can provide extended applications for general biofilm research, including screening of the biofilm properties developing under combinations of specified growth parameters such as seeding bacteria populations, growth medium compositions, medium flow rates and dissolved gas levels.

The success of endodontic treatment depends on the eradication of microorganisms from the root canal system and the prevention of reinfection. The purpose of this investigation was to evaluate the antibacterial and antibiofilm efficacy of N-acetylcysteine (NAC), an antioxidant mucolytic agent, as an intracanal medicament against selected endodontic pathogens. Minimum inhibitory concentrations (MICs) of NAC for Actinomyces naeslundii, Lactobacillus salivarius, Streptococcus mutans, and Enterococcus faecalis were determined using the broth microdilution method. NAC showed antibacterial activity, with MIC values of 0.78-1.56 mg/ml. The effect of NAC on biofilm formation of each bacterium and a multispecies culture consisting of the four bacterial species was assessed by crystal violet staining. NAC significantly inhibited biofilm formation by all the monospecies and multispecies bacteria at minimum concentrations of 0.78-3.13 mg/ml. The efficacy of NAC for biofilm disruption was evaluated by scanning electron microscopy and ATP-bioluminescence quantification using mature multispecies biofilms. Preformed mature multispecies biofilms on saliva-coated hydroxyapatite disks were disrupted within 10 min by treatment with NAC at concentrations of 25 mg/ml or higher. After 24 h of treatment, the viability of mature biofilms was reduced by > 99% compared with the control. Moreover, the biofilm disrupting activity of NAC was significantly higher than that of saturated calcium hydroxide or 2% chlorhexidine solution. Within the limitations of this in vitro study, we conclude that NAC has excellent antibacterial and antibiofilm efficacy against endodontic pathogens and may be used as an alternative intracanal medicament in root canal therapies.

Plant diseases cause dramatic yield losses worldwide. Current disease control practices can be deleterious for the environment and human health, calling for alternative and sustainable management regimes. Soils harbour microorganisms that can efficiently suppress pathogens. Uncovering mediators driving their functioning in the field still remains challenging, but represents an essential step in order to develop strategies for increased soil health. We set up plant communities of varying richness to experimentally test the potential of soils differing in plant community history to suppress the pathogen Rhizoctonia solani. The results indicate that plant communities shape soil-disease suppression via changes in abiotic soil properties and the abundance of bacterial groups including species of the genera Actinomyces, Bacillus and Pseudomonas. Further, the results suggest that pairwise interactions between specific plant species strongly affect soil suppressiveness. Using structural equation modelling, we provide a pathway orientated framework showing how the complex interactions between plants, soil and microorganisms jointly shape soil suppressiveness. Our results stress the importance of plant community composition as a determinant of soil functioning, such as the disease suppressive potential of soils.

Despite many examples of obligate epibiotic symbiosis (one organism living on the surface of another) in nature, such an interaction has rarely been observed between two bacteria. Here, we further characterize a newly reported interaction between a human oral obligate parasitic bacterium TM7x (cultivated member of Candidatus Saccharimonas formerly Candidate Phylum TM7), and its basibiont Actinomyces odontolyticus species (XH001), providing a model system to study epiparasitic symbiosis in the domain Bacteria. Detailed microscopic studies indicate that both partners display extensive morphological changes during symbiotic growth. XH001 cells manifested as short rods in monoculture, but displayed elongated and hyphal morphology when physically associated with TM7x. Interestingly, these dramatic morphological changes in XH001 were also induced in oxygen-depleted conditions, even in the absence of TM7x. Targeted quantitative real-time PCR (qRT-PCR) analyses revealed that both the physical association with TM7x as well as oxygen depletion triggered up-regulation of key stress response genes in XH001, and in combination, these conditions act in an additive manner. TM7x and XH001 co-exist with relatively uniform cell morphologies under nutrient-replete conditions. However, upon nutrient depletion, TM7x-associated XH001 displayed a variety of cell morphologies, including swollen cell body, clubbed-ends, and even cell lysis, and a large portion of TM7x cells transformed from ultrasmall cocci into elongated cells. Our study demonstrates a highly dynamic interaction between epibiont TM7x and its basibiont XH001 in response to physical association or environmental cues such as oxygen level and nutritional status, as reflected by their morphological and physiological changes during symbiotic growth.

Streptococcus gordonii SspA and SspB proteins, members of the antigen I/II (AgI/II) family of Streptococcus adhesins, mediate adherence to cysteine-rich scavenger glycoprotein gp340 and cells of other oral microbial species. In this article we investigated further the mechanism of coaggregation between S. gordonii DL1 and Actinomyces oris T14V. Previous mutational analysis of S. gordonii suggested that SspB was necessary for coaggregation with A. oris T14V. We have confirmed this by showing that Lactococcus lactis surrogate host cells expressing SspB coaggregated with A. oris T14V and PK606 cells, while L. lactis cells expressing SspA did not. Coaggregation occurred independently of expression of A. oris type 1 (FimP) or type 2 (FimA) fimbriae. Polysaccharide was prepared from cells of A. oris T14V and found to contain 1,4-, 4,6- and 3,4-linked glucose, 1,4-linked mannose, and 2,4-linked galactose residues. When immobilized onto plastic wells this polysaccharide supported binding of L. lactis expressing SspB, but not binding of L. lactis expressing other AgI/II family proteins. Purified recombinant NAVP region of SspB, comprising amino acid (aa) residues 41-847, bound A. oris polysaccharide but the C-domain (932-1470 aa residues) did not. A site-directed deletion of 29 aa residues (Δ691-718) close to the predicted binding cleft within the SspB V-region ablated binding of the NAVP region to polysaccharide. These results infer that the V-region head of SspB recognizes an actinomyces polysaccharide ligand, so further characterizing a lectin-like coaggregation mechanism occurring between two important primary colonizers., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Aim: To investigate the relationship between protease production and the ability of Enterococcus faecalis strains to coexist in biofilms with other bacteria commonly recovered from infected root canals., Methodology: Biofilms with bacteria in mono-, dual- and four-species communities were developed in flow chambers. The organisms used were Lactobacillus salivarius, Streptococcus gordonii and Actinomyces naeslundii and E. faecalis strains, GUL1 and OG1RF. Biovolume and species distribution were examined using 16S rRNA fluorescence in situ hybridization in combination with confocal microscopy and image analysis. The full proteome of the E. faecalis strains was studied using two-dimensional gel electrophoresis. Spots of interest were identified using tandem mass spectroscopy and quantified using Delta 2D software., Results: All bacteria formed biofilms and an anova analysis revealed that the biofilm biomass increased significantly (P ≤ 0.01) between 6 and 24 h. L. salivarius, S. gordonii and A. naeslundii formed mutualistic biofilm communities, and this pattern was unchanged when E. faecalis GUL1 was included in the consortium. However, with OG1RF, L. salivarius and S. gordonii were outcompeted in a 24-h biofilm. Proteomic analysis revealed that OG1RF secreted higher levels of proteases, GelE (P = 0.02) and SprE (P = 0.002) and a previously unidentified serine protease (P = 0.05), than GUL1., Conclusions: Different strains of E. faecalis can interact synergistically or antagonistically with a consortium of root canal bacteria. A possible mechanism underlying this, as well as potential differences in virulence, is production of different levels of proteases, which can cause detachment of neighbouring bacteria and tissue damage., (© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Bisphosphonates-related osteonecrosis of the jaws (BRONJ) is a severe complication of BPs therapy with unknown pathogenesis. This study aimed to evaluate the impact of Actinomyces naeslundii (A. naeslundii) on the progression of BRONJ in ovariectomized (OVX) rat model with periodontal diseases. Sixty rats were randomly assigned into four groups. All rats underwent bilateral ovariectomy. Six weeks after surgery, animals with periodontitis induced by ligature placement were administrated with normal saline (NS), NS &A. naeslundii inoculation, zolecdronic acid (ZA) and ZA &A. naeslundii inoculation for 12 weeks, respectively. Loads of total bacteria and A. naeslundii in the mouth were assessed by real time PCR. After sacrifice, the mandibles were harvested for micro-computed tomography (micro-CT) and histological examination. Real-time PCR demonstrated that A. naeslundii was not routinely found in the rats and ZA treatment did not promote its accumulation. Micro-CT examination disclosed that ligature placement induced significant alveolar bone loss, which was greatly attenuated by ZA treatment and aggravated by A. naeslundii. Histological assessment demonstrated that ZA treatment increased the risk of developing BRONJ-like disease but this condition was not worsen with the presence of A. naeslundii. Our study suggested that oral A. naeslundii inoculation aggravated periodontal disease but not BRONJ in our animal model., (Copyright © 2015 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Export of cell surface pilins in Gram-positive bacteria likely occurs by the translocation of unfolded precursor polypeptides; however, how the unfolded pilins gain their native conformation is presently unknown. Here, we present physiological studies to demonstrate that the FimA pilin of Actinomyces oris contains two disulfide bonds. Alanine substitution of cysteine residues forming the C-terminal disulfide bridge abrogates pilus assembly, in turn eliminating biofilm formation and polymicrobial interaction. Transposon mutagenesis of A. oris yielded a mutant defective in adherence to Streptococcus oralis, and revealed the essential role of a vitamin K epoxide reductase (VKOR) gene in pilus assembly. Targeted deletion of vkor results in the same defects, which are rescued by ectopic expression of VKOR, but not a mutant containing an alanine substitution in its conserved CXXC motif. Depletion of mdbA, which encodes a membrane-bound thiol-disulfide oxidoreductase, abrogates pilus assembly and alters cell morphology. Remarkably, overexpression of MdbA or a counterpart from Corynebacterium diphtheriae, rescues the Δvkor mutant. By alkylation assays, we demonstrate that VKOR is required for MdbA reoxidation. Furthermore, crystallographic studies reveal that A. oris MdbA harbors a thioredoxin-like fold with the conserved CXXC active site. Consistently, each MdbA enzyme catalyzes proper disulfide bond formation within FimA in vitro that requires the catalytic CXXC motif. Because the majority of signal peptide-containing proteins encoded by A. oris possess multiple Cys residues, we propose that MdbA and VKOR constitute a major folding machine for the secretome of this organism. This oxidative protein folding pathway may be a common feature in Actinobacteria., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Microbial interactions are necessarily associated with the development of polymicrobial oral biofilms. The objective of this study was to determine the coaggregation of eight strains of Candida albicans with Actinomyces naeslundii and Streptococcus mutans. In autoaggregation assays, C. albicans strains were grown in RPMI-1640 and artificial saliva medium (ASM) whereas bacteria were grown in heart infusion broth. C. albicans, A. naeslundii and S. mutans were suspended to give 10(6), 10(7) and 10(8) cells mL(-1) respectively, in coaggregation buffer followed by a 1 h incubation. The absorbance difference at 620 nm (ΔAbs) between 0 h and 1 h was recorded. To study coaggregation, the same protocol was used, except combinations of microorganisms were incubated together. The mean ΔAbs% of autoaggregation of the majority of RPMI-1640-grown C. albicans was higher than in ASM grown. Coaggregation of C. albicans with A. naeslundii and/or S. mutans was variable among C. albicans strains. Scanning electron microscopy images showed that A. naeslundii and S. mutans coaggregated with C. albicans in dual- and triculture. In conclusion, the coaggregation of C. albicans, A. naeslundii and S. mutans is C. albicans strain dependent., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Actinomyces naeslundii has been recovered from traumatized permanent teeth diagnosed with necrotic pulps. In this work, a triple antibiotic paste (TAP)-mimic scaffold is proposed as a drug-delivery strategy to eliminate A. naeslundii dentin biofilm., Methods: Metronidazole, ciprofloxacin, and minocycline were added to a polydioxanone (PDS) polymer solution and spun into fibrous scaffolds. Fiber morphology, mechanical properties, and drug release were investigated by using scanning electron microscopy, microtensile testing, and high-performance liquid chromatography, respectively. Human dentin specimens (4 × 4 × 1 mm(3), n = 4/group) were inoculated with A. naeslundii (ATCC 43146) for 7 days for biofilm formation. The infected dentin specimens were exposed to TAP-mimic scaffolds, TAP solution (positive control), and pure PDS (drug-free scaffold). Dentin infected (7-day biofilm) specimens were used for comparison (negative control). Confocal laser scanning microscopy was done to determine bacterial viability., Results: Scaffolds displayed a submicron mean fiber diameter (PDS = 689 ± 312 nm and TAP-mimic = 718 ± 125 nm). Overall, TAP-mimic scaffolds showed significantly (P ≤ .040) lower mechanical properties than PDS. Within the first 24 hours, a burst release for all drugs was seen. A sustained maintenance of metronidazole and ciprofloxacin was observed over 4 weeks, but not for minocycline. Confocal laser scanning microscopy demonstrated complete elimination of all viable bacteria exposed to the TAP solution. Meanwhile, TAP-mimic scaffolds led to a significant (P < .05) reduction in the percentage of viable bacteria compared with the negative control and PDS., Conclusions: Our findings suggest that TAP-mimic scaffolds hold significant potential in the eradication/elimination of bacterial biofilm, a critical step in regenerative endodontics., (Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Background and Objective: The prevalence and severity of periodontal disease increase in patients with insulin-deficient or insulin-resistant forms of diabetes. A common characteristic of diabetes is the presence of hyperglycemia. A critical consequence of hyperglycemia is the nonenzymatic glycation and oxidation of proteins and lipids, resulting in the irreversible formation of advanced glycation end-products (AGEs). A central means by which AGEs are believed to impart their pathogenic effects is via interacting with specific cellular receptors; the best-characterized of these is receptor for AGE (RAGE). The major consequences of the AGE-RAGE interaction are the generation of enhanced cellular oxidant stress, hypersecretion of inflammatory mediators and altered subgingival flora. The aim of this study was to elucidate the influence of glycated albumin (G-alb), with or without lipopolysaccharide (LPS) isolated from periodontal pathogens, on the secretion of inflammatory cytokines by cultured monocytic cells and also to investigate the role of G-alb in adherence of bacteria to epithelial cells., Material and Methods: Activated THP-1 cells (1 × 10(6) cells) were incubated for 24 h with G-alb or normal albumin (N-alb), with or without LPS isolated from two periodontal pathogens. Supernatant fluids were collected and assayed for the cytokines interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) by ELISA. For bacterial adhesion assays, S-G epithelial cells were grown on cover slips and incubated with G-alb (10 μg/mL) or N-alb (control) for 30 min. The cover slips were rinsed and then incubated with bacteria for 2 h. The number of adherent bacteria was determined by counting 20 epithelial cells chosen randomly under a light microscope., Results: The secretion of IL-1β, TNF-α and IL-6 by THP-1 cells was greater in the presence of G-alb than in the presence of N-alb. The amounts of cytokines secreted were even greater when THP-1 cells were incubated with G-alb and LPS of periodontal pathogens. The effect of G-alb and LPS was reduced when RAGE was blocked by its antibody. Coating the cultured epithelial cells with G-alb resulted in increased bacterial adherence., Conclusion: This study demonstrated the role of G-alb in stimulating cultured monocytic cells to secrete inflammatory cytokines. The stimulation was found to be greater when cells were incubated with LPS in addition to G-alb. The over-expression of inflammatory cytokines as a result of the combined effects of G-alb and bacterial LPS may contribute to the severity of periodontal disease in diabetic subjects., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

This work presented a highly efficient antibacterial Ti-surface which was grafted with poly(N-hydroxyethylacrylamide) (PHEAA) brush and further decorated with triclosan (TCS). The modified surfaces were characterized using contact angle measurements, X-ray photoelectron spectroscopy, and attenuated total reflectance Fourier transform infrared. The antibacterial performance of the modified surfaces was evaluated using the Streptococcus mutans and Actinomyces naeslundii attachment test. The Ti surface with PHEAA brush (Ti-PHEAA) was able to resist the adhesion of the bacteria, while the TCS-decorated Ti surface (Ti-TCS) showed the capability of killing the bacteria adhered on the surface. As we coupled the TCS to the PHEAA brush, the surface showed highly efficient antibacterial performance due to the combination of the resistance to the bacteria adhesion and its activity of killing bacteria.

Actinomyces israelii has long been recognized as a causative agent of actinomycosis. During the past 3 decades, a large number of novel Actinomyces species have been described. Their detection and identification in clinical microbiology laboratories and recognition as pathogens in clinical settings can be challenging. With the introduction of advanced molecular methods, knowledge about their clinical relevance is gradually increasing, and the spectrum of diseases associated with Actinomyces and Actinomyces-like organisms is widening accordingly; for example, Actinomyces meyeri, Actinomyces neuii, and Actinomyces turicensis as well as Actinotignum (formerly Actinobaculum) schaalii are emerging as important causes of specific infections at various body sites. In the present review, we have gathered this information to provide a comprehensive and microbiologically consistent overview of the significance of Actinomyces and some closely related taxa in human infections., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Actinomyces naeslundii is an early colonizer with important roles in the development of the oral biofilm. The effects of butyric acid, one of short chain fatty acids in A. naeslundii biofilm formation was observed using a flow cell system with Tryptic soy broth without dextrose and with 0.25% sucrose (TSB sucrose). Significant biofilms were established involving live and dead cells in TSB sucrose with 60mM butyric acid but not in concentrations of 6, 30, 40, and 50mM. Biofilm formation failed in 60mM sodium butyrate but biofilm level in 60mM sodium butyrate (pH4.7) adjusted with hydrochloric acid as 60mM butyric media (pH4.7) was similar to biofilm levels in 60mM butyric acid. Therefore, butyric acid and low pH are required for significant biofilm formation in the flow cell. To determine the mechanism of biofilm formation, we investigated initial A. naeslundii colonization in various conditions and effects of anti-GroEL antibody. The initial colonization was observed in the 60mM butyric acid condition and anti-GroEL antibody inhibited the initial colonization. In conclusion, we established a new biofilm formation model in which butyric acid induces GroEL-dependent initial colonization of A. naeslundii resulting in significant biofilm formation in a flow system., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Statement of Problem: Bacterial plaque must be routinely removed from teeth, adjacent structures, and prostheses. However, the removal of this plaque can inadvertently increase the risk of future bacterial adhesion., Purpose: The purpose of this investigation was to assess the change in the surface roughness of 3 different surfaces after dental prophylactic instrumentation and how this influenced bacterial adhesion., Material and Methods: Forty specimens each of Type III gold alloy, lithium disilicate, and zirconia were fabricated in the same dimensions. The specimens were divided into 4 groups: ultrasonic scaler, stainless steel curette, prophylaxis cup, and control. Pretreatment surface roughness measurements were made with a profilometer. Surface treatments in each group were performed with a custom mechanical scaler. Posttreatment surface roughness values were measured. In turn, the specimens were inoculated with Streptococcus mutans, Lactobacillus acidophilus, and Actinomyces viscosus. Bacterial adhesion was assessed by rinsing the specimens with sterile saline to remove unattached cells. The specimens were then placed in sterile tubes with 1 mL of sterile saline. The solution was plated and quantified. Scanning electron microscopy was performed. The statistical analysis of surface roughness was completed by using repeated-measures single-factor ANOVA with a Bonferroni correction., Results: The surface roughness values for gold alloy specimens increased as a result of prophylaxis cup treatment (0.221 to 0.346 Ra) (P<.01) and stainless steel curette treatment (0.264 to 1.835 Ra) (P<.01). The results for bacterial adhesion to gold alloy proved inconclusive. A quantitative comparison indicated no statistically significant differences in pretreatment and posttreatment surface roughness values for lithium disilicate and zirconia specimens. In spite of these similarities, the overall bacterial adherence values for lithium disilicate were significantly greater than those recorded for gold alloy or zirconia (P<.05). Instrumentation of the lithium disilicate and zirconia with the stainless steel curette significantly increased bacterial adhesion compared with the control (P<.05)., Conclusions: The results of this investigation indicate that Type III gold alloy exhibited increased surface roughness values after stainless steel curette and prophylaxis cup treatments. Zirconia was less susceptible to bacterial adhesion than lithium disilicate, and greater bacterial adhesion was found for the stainless steel curette than the other instrumentation methods., (Copyright © 2014 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.)