Multispecies Bacterial Biofilm Research Articles

Oxygen enrichment mediated by calcium peroxide loaded gelatin methacrylate hydrogel eradicates periodontal biofilms

The low oxygen environment of the periodontal pocket favors pathogenic anaerobes' growth, biofilm formation, and quick recurrence after periodontal treatment. In contrast, oxygen is detrimental to anaerobes, such as Porphyromonas gingivalis (P. gingivalis), since they lack a complete anti-oxidation mechanism to detoxify the oxygen challenge. Therefore, consistently feeding pathogenic anaerobes with abundant oxygen would be an effective strategy to combat them. Here, we reported injectable oxygen-generating hydrogels as oxygen mediators to alleviate the local anaerobic environment and eliminate periodontal pathogens. Gelatin methacrylate (GelMA) hydrogels loaded with calcium peroxide (CPO) possessed excellent injectability and exhibited burst releases of oxygen within 24 h with a 40 % oxygen tension peak. CPO-GelMA hydrogels with CPO concentrations of 5, 10, and 15 % reduced 60, 99, and 89.9 % viable P. gingivalis, respectively. Five percentage CPO-GelMA hydrogel downregulated gingipain and fimA gene expression in P. gingivalis without resistance development. Moreover, the CPO-GelMA hydrogels remarkably prevented biofilm formation and eradicated both monospecies and multispecies bacterial biofilms. In conclusion, CPO-GelMA hydrogels exert remarkable antimicrobial and antibiofilm effects on subgingival biofilms, providing a promising strategy for periodontal treatment.

Small molecular adjuvants repurpose antibiotics towards Gram-negative bacterial infections and multispecies bacterial biofilms.

Gram-negative bacterial infections pose a significant challenge due to two major resistance elements, including the impermeability of the outer membrane and the overexpression of efflux pumps, which contribute to antibiotic resistance. Additionally, the coexistence of multispecies superbugs in mixed species biofilms further complicates treatment, as these infections are refractory to most antibiotics. To address this issue, combining obsolete antibiotics with non-antibiotic adjuvants that target bacterial membranes has shown promise in combating antibacterial resistance. However, the clinical translation of this cocktail therapy has been hindered by the toxicity associated with these membrane active adjuvants, mainly due to a limited understanding of their structure and mechanism of action. Towards this goal, herein, we have designed a small molecular adjuvant by tuning different structural parameters, such as the balance between hydrophilic and hydrophobic groups, spatial positioning of hydrophobicity and hydrogen bonding interactions, causing moderate membrane perturbation in bacterial cells without any toxicity to mammalian cells. Moderate membrane perturbation not only enhances the internalization of antibiotics, but also increases the intracellular concentration of drugs by hampering the efflux machinery. This revitalises the efficacy of various classes of antibiotics by 32-512 fold, without inducing toxicity. The leading combination not only exhibits potent bactericidal activity against A. baumannii biofilms but also effectively disrupts mature multispecies biofilms composed of A. baumannii and methicillin-resistant Staphylococcus aureus (MRSA), which is typically resistant to most antibiotics. Importantly, the combination therapy demonstrates good biocompatibility and excellent in vivo antibacterial efficacy (>99% reduction) in a skin infection model of A. baumannii. Interestingly, A. baumannii shows reduced susceptibility to develop resistance against the leading combination, underscoring its potential for treating multi-drug resistant infections.

Novel Antibacterial and Biocompatible Nanostructured Gels Based on One-step Synthesis as a Potential Disinfectant for Endodontic Infection Control

AimThe objective of this study was to develop nanostructured gels as biocompatible intracanal disinfectants by one-step microwave radiation-assisted synthesis. MethodsPolyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP) were used as a support network, and polyethylene glycol (PEG) was used as a reducing agent. The gels were characterized by measuring the swelling ratio (SR) and rheological properties and by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM). The antibacterial effects of each gel were evaluated against the endodontic clinical strain Enterococcus faecalis. Then, the viability of the 21-day mature multispecies bacterial biofilm was assessed using confocal microscopy in an ex vivo model, where the biofilm was exposed to the mix of nanogels. The cell proliferation, viability, and morphology of human periodontal ligament (HPDL) cells were quantified using a real-time IncuCyte® S3 Live-Cell System. Viability was measured by confocal microscopy using an ex vivo model exposing a 21-day mature multispecies bacterial biofilm to the mix of nanogels. ResultsThe antibacterial activity of the gels coincided with the superficial characterization and the solubility of the gel in the growth medium. Gels with higher viscosity (327.85-980.58 Pa s), higher dissolution (42-70%SR), and lower porosity (no porosity and 611.63 nm) showed excellent antibacterial activity against E. faecalis. Despite their physicochemical characteristics, CuNPs gels showed greater effectiveness against E. faecalis.These nanostructured gels with high PVA concentrations promote HPDL cells proliferation while still exerting antibacterial properties. Mix of nanogels showed an increase non-viable cells biomass from at of application. ConclusionsThe use of biocompatible polymers influences the physicochemical, bactericidal, and cytotoxic response, making these materials potential disinfectant agents against resistant bacteria with good biocompatibility and improved HPDL cells proliferation.

Discovery of a polymer resistant to bacterial biofilm, swarming, and encrustation.

Innovative approaches to prevent catheter-associated urinary tract infections (CAUTIs) are urgently required. Here, we describe the discovery of an acrylate copolymer capable of resisting single- and multispecies bacterial biofilm formation, swarming, encrustation, and host protein deposition, which are major challenges associated with preventing CAUTIs. After screening ~400 acrylate polymers, poly(tert-butyl cyclohexyl acrylate) was selected for its biofilm- and encrustation-resistant properties. When combined with the swarming inhibitory poly(2-hydroxy-3-phenoxypropyl acrylate), the copolymer retained the bioinstructive properties of the respective homopolymers when challenged with Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. Urinary tract catheterization causes the release of host proteins that are exploited by pathogens to colonize catheters. After preconditioning the copolymer with urine collected from patients before and after catheterization, reduced host fibrinogen deposition was observed, and resistance to diverse uropathogens was maintained. These data highlight the potential of the copolymer as a urinary catheter coating for preventing CAUTIs.

Surface properties and initial bacterial biofilm growth on 3D-printed oral appliances: a comparative in vitro study

ObjectivesTo investigate the initial bacterial adhesion on 3D-printed splint materials in relation to their surface properties.Materials and methodsSpecimens of five printable splint resins (SHERAprint-ortho plus UV, NextDent Ortho Rigid, LuxaPrint Ortho Plus, V-Print Splint, KeySplint Soft), one polymethylmethacrylate (PMMA) block for subtractive manufacturing (Astron CLEARsplint Disc), two conventional powder/liquid PMMA materials (FuturaGen, Astron CLEARsplint), and one polyethylene terephthalate glycol (PETG) thermoplastic sheet for vacuum forming (Erkodur Thermoforming Foil) were produced and finished. Surface roughness Ra was determined via contact profilometry. Surface morphology was examined under a scanning electron microscope. Multi-species bacterial biofilms were grown on entire splints. Total biofilm mass and viable bacterial counts (CFU/ml) within the biofilms were determined. Statistical analyses were performed with a one-way ANOVA, Tukey’s post hoc test, and Pearson’s test (p < 0.05).ResultsAstron CLEARsplint and KeySplint Soft specimens showed the highest surface roughness. The mean total biofilm mass on KeySplint Soft splints was higher compared to all other materials (p < 0.05). Colony-forming unit per milliliter on FuturaGen, Astron CLEARsplint, and KeySplint Soft splints was one log scale higher compared to all other materials. The other four printable resins displayed overall lower Ra, biofilm mass, and CFU/ml. A positive correlation was found between Ra and CFU/ml (r = 0.69, p = 0.04).ConclusionsThe 3D-printed splints showed overall favorable results regarding surface roughness and bacterial adhesion. Thermoplastic materials seem to display a higher surface roughness, making them more susceptible to microbial adhesion.Clinical relevanceThe development of caries and gingivitis in patients with oral appliances may be affected by the type of material.

Does Secondary Plant Metabolite Ursolic Acid Exhibit Antibacterial Activity against Uropathogenic Escherichia coli Living in Single- and Multispecies Biofilms?

Multispecies bacterial biofilms are the often cause of chronic recurrent urinary tract infections within the human population. Eradicating such a complex bacterial consortium with standard pharmacotherapy is often unsuccessful. Therefore, plant-derived compounds are currently being researched as an alternative strategy to antibiotic therapy for preventing bacterial biofilm formation and facilitating its eradication. Therefore, our research aimed to determine the effect of secondary plant metabolite ursolic acid (UA) on the growth and survival, the quantity of exopolysaccharides formed, metabolic activity, and morphology of uropathogenic Gram-negative rods living in single- and mixed-species biofilms at various stages of their development. Spectrophotometric methods were used for biofilm mass formation and metabolic activity determination. The survival of bacteria was established using the serial dilution assay. The decrease in survival and inhibition of biofilm creation, both single- and multispecies, as well as changes in the morphology of bacterial cells were noticed. As UA exhibited better activity against young biofilms, the use of UA-containing formulations, especially during the initial steps of urinary tract infection, seems to be reasonable. However, the future direction should be a thorough understanding of the mechanisms of UA activity as a bioactive substance.

Evaluation of photosensitizer-containing superhydrophobic surfaces for the antibacterial treatment of periodontal biofilms.

Antimicrobial photodynamic therapy (aPDT) is a promising approach to control biofilms involved in periodontal diseases. However, certain challenges, such as staining of teeth, preferential interaction of photosensitizer (PS) with Gram-positive versus Gram-negative bacteria, and insufficient oxygen in hypoxic periodontal pockets have presented barriers to its use in the clinic. To overcome these challenges, a novel superhydrophobic (SH) film that generates airborne singlet oxygen has been developed. The SH-aPDT approach isolates the PS onto a topologically rough solid SH film on which channels allow air to diffuse to the PS surface, thus ensuring sufficient oxygen supply. Upon illumination, gas phase singlet oxygen (1O2) is produced and diffuses from the SH surface to the underlying biofilm. The killing efficacy was assessed as a function of transmitted fluence (17.9-89.5J/cm2) and chorin e6 loading (96-1110nmol/cm2) by counting of colony forming units, biofilm metabolism by XTT and confocal microscopy. The decrease in viability of both Gram-positive and Gram-negative bacteria in a multi-species biofilm was found to be linearly dependent on the fluence as well as the loading of the PS up to 71.6J/cm2 when 1110 nmols/cm2 of chlorin e6 was used. A>4.6 log bacterial reduction was observed under these conditions (p<0.05). This novel SH-aPDT approach shows promise as an effective method to disinfect multi-species bacterial biofilms associated with periodontal disease and will be evaluated in animal models in future studies.

Light-Propelled Nanorobots for Facial Titanium Implants Biofilms Removal.

Titanium miniplates are biocompatible materials used in modern oral and maxillofacial surgery to treat facial bone fractures. However, plate removal is often required due to implant complications. Among them, a biofilm formation on an infected miniplate is associated with severe inflammation, which frequently results in implant failure. In light of this, new strategies to control or treat oral bacterial biofilm are of high interest. Herein, the authors exploit the ability of nanorobots against multispecies bacterial biofilm grown onto facial commercial titanium miniplate implants to simulate pathogenic conditions of the oral microenvironment. The strategy is based on the use of light-driven self-propelled tubular black-TiO2 /Ag nanorobots, that unlike traditional ones, exhibit an extended absorption and motion actuation from UV to the visible-light range. The motion analysis is performed separately over UV, blue, and green light irradiation and shows different motion behaviors, including a fast rotational motion that decreases with increasing wavelengths. The biomass reduction is monitored by evaluating LIVE/DEAD fluorescent and digital microscope images of bacterial biofilm treated with the nanorobots under motion/no-motion conditions. The current study and the obtained results can bring significant improvements for effective therapy of infected metallic miniplates by biofilm.

Preclinical Testing of Boron-Doped Diamond Electrodes for Root Canal Disinfection-A Series of Preliminary Studies.

While numerous approaches have meanwhile been described, sufficient disinfection of root canals is still challenging, mostly due to limited access and the porous structure of dentin. Instead of using different rinsing solutions and activated irrigation, the electrolysis of saline using boron-doped diamond (BDD) electrodes thereby producing reactive oxygen species may be an alternative approach. In a first step, experiments using extracted human teeth incubated with multispecies bacterial biofilm were conducted. The charge quantities required for electrochemical disinfection of root canals were determined, which were subsequently applied in an animal trial using an intraoral canine model. It could be shown that also under realistic clinical conditions, predictable disinfection of root canals could be achieved using BDD electrodes. The parameters required are in the range of 5.5 to 7.0 V and 9 to 38 mA, applied for 2.5 to 6.0 min with approximately 5 to 8 mL of saline. The direct generation of disinfective agents inside the root canal seems to be advantageous especially in situations with compromised access and limited canal sizes. The biologic effect with respect to the host reaction on BDD-mediated disinfection is yet to be examined.

Polymeric Nanoparticles Active against Dual-Species Bacterial Biofilms.

Biofilm infections are a global public health threat, necessitating new treatment strategies. Biofilm formation also contributes to the development and spread of multidrug-resistant (MDR) bacterial strains. Biofilm-associated chronic infections typically involve colonization by more than one bacterial species. The co-existence of multiple species of bacteria in biofilms exacerbates therapeutic challenges and can render traditional antibiotics ineffective. Polymeric nanoparticles offer alternative antimicrobial approaches to antibiotics, owing to their tunable physico-chemical properties. Here, we report the efficacy of poly(oxanorborneneimide) (PONI)-based antimicrobial polymeric nanoparticles (PNPs) against multi-species bacterial biofilms. PNPs showed good dual-species biofilm penetration profiles as confirmed by confocal laser scanning microscopy. Broad-spectrum antimicrobial activity was observed, with reduction in both bacterial viability and overall biofilm mass. Further, PNPs displayed minimal fibroblast toxicity and high antimicrobial activity in an in vitro co-culture model comprising fibroblast cells and dual-species biofilms of Escherichia coli and Pseudomonas aeruginosa. This study highlights a potential clinical application of the presented polymeric platform.

The anti-biofilm capability of nano-emodin-mediated sonodynamic therapy on multi-species biofilms produced by burn wound bacterial strains

BackgroundManagement of burn wound infections (BWIs) is difficult due to the emergence of multidrug-resistant microorganisms. This study aimed to explore the anti-biofilm efficacy of sonodynamic therapy (SDT) using nano-emodin (N-EMO) against multi-species bacterial biofilms containing Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii. MethodsFollowing synthesis and confirmation of N-EMO as a sonosensitizer, the anti-biofilm efficacy of SDT against multi-species bacterial biofilms was determined using minimum inhibitory concentrations (MIC), minimum biofilm inhibitory concentration (MBIC), and minimal biofilm eradication concentration (MBEC) of N-EMO. The reduction of multi-species bacterial biofilms was then evaluated following the treatments using Log reduction and crystal violet (CV) assays. In addition, the expression profiling of abaI, agrA, and lasI genes using SDT with sub-MIC, sub-MBIC, and sub-MBEC of N-EMO was assessed. ResultsSuccessful synthesis of N-EMO was confirmed through several characterization tests. As the results demonstrated, the MIC value of N-EMO for the multi-species bacterial suspension was 0.15 × 10–4 g/L, as well as, the MBEC value of N-EMO was 2.5 × 10–4 g/L, approximately 4-fold higher than that of MBIC (0.62 × 10–4 g/L). According to the CV assay, there were 57.8 %, 71.0 %, and 81.5 % reduction in the biofilm of multi-species bacterial growth following SDT using 1/128 MBEC, 1/16 MBIC, and 1/2 MIC of N-EMO, respectively. Log reductions analysis demonstrated that 1/2 MIC of N-EMO was more potent in inhibiting the biofilm growth of multi-species test bacteria by 5.725 ± 0.12 (99.9993 %). In this study, N-EMO-mediated SDT could obviously downregulate the gene expression of virulence factors (P < 0.05). The gene expression of lasI, agrA, and abaI were downregulated about 2.5-, 3.6-, and 5.5-fold; and 3.0-, 5.2-, and 7.4-fold following SDT with sub-MBIC and sub-MBEC of N-EMO, respectively. ConclusionThese results highlight the potential of N-EMO-mediated SDT in inhibition of biofilm formation, degradation of formed biofilms, and reduction of virulence factor associated with biofilms of multi-species bacterial biofilms in BWIs.

Honey Potential as Antibiofilm, Antiquorum Sensing and Dispersal Agent against Multispecies Bacterial Biofilm.

This study is first to test Pakistani honey bees, Apis dorsata and A. cerana honey samples as anti biofilm, anti quorum sensing (QS) and biofilm dispersal agents honey against multispecies biofilm of bacteria (obtained from obese patients). Briefly, five previously identified isolates Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Morganella morganii and Klebsiella pneumoniae (MT448672-MT448676) were selected. Antibiogram study of all five isolates was tested against three antibiotics viz., erythromycin (20 µg/mL), lincomycin (100 µg/mL) and rifampicin (100 µg/mL). In order to form multispecies biofilm, identified bacteria were grown in batch culture by mixing equal volumes (OD590nm = 0.1) of 2, 3 and 5 bacterial isolates. In total 11 groups (g1-g11) were made. Crystal violet (CV) staining method was used to evaluate the antibiofilm potential and biofilm dispersal potential of both honey samples. QS inhibition in P. aeruginosa was measured following culture supernatant method. Antibiogram study showed significant (p < 0.05) resistance by P. aeruginosa against tested antibiotics. E. coli, M. morganii and K. pneumoniae were significantly susceptible to erythromycin and S. aureus to lincomycin. Both honey samples at 2% and 5% concentrations showed significant (p < 0.05) inhibition potential of multispecies biofilm by all test groups (g1-g11). Though A. dorsata honey significantly inhibited biofilm formation at 2 and 5% against all groups but 2% concentration was highly significant against g2-g4 groups. Regarding A. cerana honey, 2% concentration was significantly effective against g1, g4-g7 and g9-g11 groups. Both honey samples significantly inhibited QS at 2 and 5%. The 5% concentration of A. dorsata honey significantly dispersed biofilm by all groups compared to 2% which showed dispersal potential only by g2 and g3 groups. Accordingly, honey samples showed significant antibiofilm, anti-QS and biofilm dispersal potentials thus can be considered as good alternative to antibiotics.

Optimization of Agent-Based Models Through Coarse-Graining: A Case Study in Microbial Ecology.

Optimization and control are important objectives across biology and biomedicine, and mathematical models are a key enabling technology. This paper reports a computational study of model-based multi-objective optimization in the setting of microbial ecology, using agent-based models. This modeling framework is well-suited to the field, but is not amenable to standard control-theoretic approaches. Furthermore, due to computational complexity, simulation-based optimization approaches are often challenging to implement. This paper presents the results of an approach that combines control-dependent coarse-graining with Pareto optimization, applied to two models of multi-species bacterial biofilms. It shows that this approach can be successful for models whose computational complexity prevents effective simulation-based optimization.

A multimodel regime for evaluating effectiveness of antimicrobial wound care products in microbial biofilms.

Microbial biofilms have become increasingly recognized as a cause of wound chronicity. There are several topical antimicrobial wound care products available for use; however, their effectiveness has routinely been demonstrated with planktonic microorganisms. There is no target reference value for antimicrobial effectiveness of wound care products in biofilm models. In addition, data on antimicrobial activity of products in biofilm models are scattered across many test methods in a variety of studies. The aim of this work is to directly compare commercial products containing the commonly used topical antimicrobial agents iodine, silver, polyhexamethylene biguanide, octenidine, hypochlorous acid, benzalkonium chloride, and a surfactant‐based topical containing poloxamer 188. Five different in vitro biofilm models of varied complexity were used, incorporating several bacterial pathogens such as Staphylococcus, Enterococcus, Streptococcus, Pseudomonas, Acinetobacter, Klebsiella, and Enterobacter. The fungal pathogens Candida albicans and Candida auris were also evaluated. A multispecies bacterial biofilm model was also used to evaluate the products. Additionally, C. albicans was used in combination with S. aureus and P. aeruginosa in a multikingdom version of the polymicrobial biofilm model. Statistically significant differences in antimicrobial performance were observed between treatments in each model and changing microbial growth conditions or combinations of organisms resulted in significant performance differences for some treatments. The iodine and benzalkonium chloride‐containing products were overall the most effective in vitro and were then selected for in vivo evaluation in an infected immunocompromised murine model. Unexpectedly, the iodine product was statistically (P > .05) no different than the untreated control, while the benzalkonium chloride containing product significantly (P < .05) reduced the biofilm compared to untreated control. This body of work demonstrates the importance of not only evaluating antimicrobial wound care products in biofilm models but also the importance of using several different models to gain a comprehensive understanding of products' effectiveness.

Potentiation effects of antimicrobial photodynamic therapy on quorum sensing genes expression: A promising treatment for multi-species bacterial biofilms in burn wound infections.

As the increase in the prevalence of antimicrobial resistant bacterial strains, development of adjuvant antimicrobial approach for the treatment of burn wound infection is important. Therefore, the aim of this study was to assess the effectiveness of antimicrobial photodynamic therapy (aPDT) using indocyanine green (ICG) in reducing the bacterial load and expression profiling of the quorum sensing (QS) system associated with the biofilm formation in multi-species bacterial biofilms. Multi-species bacterial suspension including Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus were photosensitized with ICG, which was excited at a wavelength of 810 nm. After evaluating the dose of ICG-aPDT contributing to the sub-significant reduction of colony forming unit (CFU)/mL, the gene expression levels of abaI, agrA, and lasI were assessed using real-time quantitative reverse transcription polymerase chain reaction. As well as, lipid peroxidation, superoxide radicals and reactive oxygen species (ROS) were measured and the morphological changes of multi-species bacteria were evaluated by scanning electron microscopy (SEM). According to the results, maximal sub-significant reduction dose of aPDT against CFUs/mL of multi-species bacterial cells was found with a combination of 31.2 μg/mL of ICG and irradiation by the diode laser for 1 min, with an estimated average output light energy 31.2 J/cm2. The expression levels of abaI, agrA, and lasI in A. baumannii, P. aeruginosa, and S. aureus were down-regulated to approximately 1.9-, 3.7-, and 4.9-fold, respectively. The amount of lipid peroxidation, superoxide radical production, and ROS generation significantly increased (P < 0.05). Also, the cell morphology under SEM showed that ICG-aPDT is a stressful condition for multi-species bacterial cells. ICG-aPDT with antimicrobial, anti-biofilm, and gene expression inhibitor characteristics, as well as, ROS generation can be used for the treatment of burn wound infections in vivo.

Lyso-Gb3 modulates the gut microbiota and decreases butyrate production

Fabry disease is a rare X-linked lysosomal storage disorder resulting from deficient activity of α-galactosidase A, leading to the accumulation of glycosphingolipids such as globotriaosylsphingosine (lyso-Gb3). The gastrointestinal symptoms of this disease may be disabling, and the life expectancy of affected patients is shortened by kidney and heart disease. Our hypothesis was that lyso-Gb3 may modify the gut microbiota. The impact of a clinically relevant concentration of lyso-Gb3 on mono- or multispecies bacterial biofilms were evaluated. A complex bacterial community from the simulated transverse colon microbiota was studied using quantitative PCR to estimate different bacterial group concentrations and a HPLC was used to estimate short-chain fatty acids concentrations. We found that lyso-Gb3 increased the biofilm-forming capacity of several individual bacteria, including Bacteroides fragilis and significantly increased the growth of B. fragilis in a multispecies biofilm. Lyso-Gb3 also modified the bacterial composition of the human colon microbiota suspension, increasing bacterial counts of B. fragilis, among others. Finally, lyso-Gb3 modified the formation of short-chain fatty acids, leading to a striking decrease in butyrate concentration. Lyso-Gb3 modifies the biology of gut bacteria, favoring the production of biofilms and altering the composition and short-chain fatty-acid profile of the gut microbiota.

Lactobacillus plantarum Lipoteichoic Acid Inhibits Oral Multispecies Biofilm

IntroductionApical 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. MethodsHighly 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. ResultsBiofilm 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. ConclusionsThese 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.

Soft, adhesive (+) alpha tocopherol phosphate planar bilayers that control oral biofilm growth through a substantive antimicrobial effect

‘Soft’ nanomaterials have the potential to produce substantive antibiofilm effects. The aim of this study was to understand the oral antimicrobial activity of soft nanomaterials generated from alpha-tocopherol (α-T) and alpha-tocopherol phosphate (α-TP). (+) α-TP formed planar bilayer islands (175 ± 21 nm, −14.9 ± 3.5 mV) in a Trizma® buffer, whereas (+) α-T formed spherical liposomes (563 ± 1 nm, −10.5 ± 0.2 mV). The (+) α-TP bilayers displayed superior Streptococcus oralis biofilm growth retardation, a more substantive action, generated a superior adsorption to hydroxyapatite and showed an enhanced inhibition of multi-species bacterial saliva biofilm growth (38 ± 7μm vs 58 ± 18 μm, P ˂ 0.05) compared to (+) α-T. Atomic force microscopy data indicated that the ability of the ‘soft’ α-TP nanomaterials to transition into planar bilayer structures upon contact with interfaces facilitated their adhesive properties and substantive antimicrobial effects.

Synergistic Interactions within a Multispecies Biofilm Enhance Individual Species Protection against Grazing by a Pelagic Protozoan.

Biofilm formation has been shown to confer protection against grazing, but little information is available on the effect of grazing on biofilm formation and protection in multispecies consortia. With most biofilms in nature being composed of multiple bacterial species, the interactions and dynamics of a multispecies bacterial biofilm subject to grazing by a pelagic protozoan predator were investigated. To this end, a mono and multispecies biofilms of four bacterial soil isolates, namely Xanthomonas retroflexus, Stenotrophomonas rhizophila, Microbacterium oxydans and Paenibacillus amylolyticus, were constructed and subjected to grazing by the ciliate Tetrahymena pyriformis. In monocultures, grazing strongly reduced planktonic cell numbers in P. amylolyticus and S. rhizophila and also X. retroflexus. At the same time, cell numbers in the underlying biofilms increased in S. rhizophila and X. retroflexus, but not in P. amylolyticus. This may be due to the fact that while grazing enhanced biofilm formation in the former two species, no biofilm was formed by P. amylolyticus in monoculture, either with or without grazing. In four-species biofilms, biofilm formation was higher than in the best monoculture, a strong biodiversity effect that was even more pronounced in the presence of grazing. While cell numbers of X. retroflexus, S. rhizophila, and P. amylolyticus in the planktonic fraction were greatly reduced in the presence of grazers, cell numbers of all three species strongly increased in the biofilm. Our results show that synergistic interactions between the four-species were important to induce biofilm formation, and suggest that bacterial members that produce more biofilm when exposed to the grazer not only protect themselves but also supported other members which are sensitive to grazing, thereby providing a “shared grazing protection” within the four-species biofilm model. Hence, complex interactions shape the dynamics of the biofilm and enhance overall community fitness under stressful conditions such as grazing. These emerging inter- and intra-species interactions could play a vital role in biofilm dynamics in natural environments like soil or aquatic systems.

Suppression of \u03b1v\u03b26 Integrin Expression by Polymicrobial Oral Biofilms in Gingival Epithelial Cells

Periodontal diseases manifest by the formation of deep pockets between the gingiva and teeth where multispecies bacterial biofilms flourish, causing inflammation and bone loss. Epithelial cell receptor αvβ6 integrin that regulates inflammation by activating the anti-inflammatory cytokine transforming growth factor-β1, is highly expressed in healthy junctional epithelium that connects the gingiva to the tooth enamel. However, its expression is attenuated in human periodontal disease. Moreover, Itgb6−/− mice display increased periodontal inflammation compared to wild-type mice. We hypothesized that bacterial biofilms present in the periodontal pockets suppress αvβ6 integrin levels in periodontal disease and that this change aggravates inflammation. To this end, we generated three-week-old multi-species oral biofilms in vitro and treated cultured gingival epithelial cells (GECs) with their extracts. The biofilm extracts caused suppression of β6 integrin expression and upregulation of pro-inflammatory cytokines, including interleukin-1β and -6. Furthermore, GECs with β6 integrin siRNA knockdown showed increased interleukin-1β expression, indicating that αvβ6 integrin-deficiency is associated with pro-inflammatory cytokine responsiveness. FSL-1, a synthetic bacterial lipopeptide, also suppressed β6 integrin expression in GECs. Therefore, biofilm components, including lipopeptides, may downregulate αvβ6 integrin expression in the pocket epithelium and thus promote epithelial cell-driven pro-inflammatory response in periodontal disease.