Multispecies Biofilm Research Articles

Anti-Biofilm Formation of an Adhesive Containing Calcium Salts of Acidic Monomers against Oral Candida Related to Root Caries

The proportion of older people within the general population is expanding due to the decline of birth rate and the increase of life expectancy. Increasing elderly populations, which is retaining its teeth longer, leads to oral health problems, for example, root caries. The main etiological factor for the initiation and development of root caries is the appearance of cariogenic biofilm. The acid-producing and acid-tolerant bacteria and oral yeast, such as Candida albicans are the major contributors in root caries formation. A new generation of adhesives is developed with several modifications for the prevention of dental caries by incorporating antimicrobial components. The aim of this study was to investigate the antimicrobial effect of adhesive containing calcium salt of acidic monomers on the biofilm formation of oral Candida related to root caries. In the experiments, the flat-bottom surfaces of 96-well plate were painted with an adhesive containing calcium salts of 4-methacryloxyethyl trimellitic acid (CMET) and 10-methacryloyloxydecyl dihydrogen calcium phosphate (MDCP) (Bio-Coat, CA, Sun Medical, Japan). Then they were LED light-cured and coated with sterile saliva at 37 °C for 60 min. The biofilm formation was made by adding of Candida albicans (ATCC 10238 and two clinical strains) suspensions (107 colony forming unit/mL) and incubated at 37°C for 24 h. The amount of vital biofilm was determined by WST-8 Microbial Cell Counting Kit (Dojindo Molecular Technologies, USA). All experiments were done in triplicate and repeated three times. Statistical analysis was performed using Student’s t-test. The results clearly showed that adhesive could significantly inhibit biofilm formation of all tested Candida compared with a control. This suppressive effect was not different among the strains of Candida. The percentages of vital biofilm reduction were 25% to 40%. The ability of this adhesive to suppress biofilm of oral yeast may be its antimicrobial property of acidic monomer or the effect of calcium ion within the adhesive, which can alter Candida cell morphology, and influence their structures or process of biofilm formation. In conclusion, an adhesive containing calcium salts of acidic monomers could significantly inhibit biofilm formation of C. albicans. This adhesive could be effectively applied to exposed root surfaces to prevent or inhibit the progression of root caries. Further studies are necessary to clarify the effect on multispecies biofilm, on long-term activity, and in vivo conditions.

A novel chlorhexidine-hexametaphosphate coating for titanium with antibiofilm efficacy and stem cell cytocompatibility

Dental implants are an increasingly popular way to replace missing teeth. Whilst implant survival rates are high, a small number fail soon after placement, with various factors, including bacterial contamination, capable of disrupting osseointegration. This work describes the development of chlorhexidine-hexametaphosphate coatings for titanium that hydrolyse to release the antiseptic agent chlorhexidine. The aim was to develop a coating for titanium that released sufficient chlorhexidine to prevent biofilm formation, whilst simultaneously maintaining cytocompatibility with cells involved in osseointegration. The coatings were characterised with respect to physical properties, after which antibiofilm efficacy was investigated using a multispecies biofilm model, and cytocompatibility determined using human mesenchymal stem cells. The coatings exhibited similar physicochemical properties to some implant surfaces in clinical use, and significantly reduced formation of multispecies biofilm biomass up to 72 h. One coating had superior cytocompatibility, with mesenchymal stem cells able to perform normal functions and commence osteoblastic differentiation, although at a slower rate than those grown on uncoated titanium. With further refinement, these coatings may have application in the prevention of bacterial contamination of dental implants at the time of surgery. This could aid a reduction in rates of early implant failure.

Gardnerella Vaginalis Dominates Multi-Species Biofilms in both Pre-Conditioned and Competitive In Vitro Biofilm Formation Models.

Bacterial vaginosis (BV) is one of the most common bacterial vaginal infections worldwide. Despite its high prevalence, BV etiology is still unknown. Nevertheless, a hallmark of BV is the presence of a highly structured polymicrobial biofilm on the vaginal epithelium, formed primarily by Gardnerella spp. and other anaerobic species, of which co-colonization with Fannyhessea vaginae is considered an important diagnostic marker. We previously developed an in vitro biofilm model wherein Gardnerella was first allowed to establish an early biofilm that served as a scaffold for other species to adhere to. To better understand ecological interactions between BV-associated bacteria, we compared triple-species biofilms formed using two distinct models: a pre-conditioned (wherein Gardnerella vaginalis formed the early biofilm) model and a competitive (wherein all three bacteria were co-incubated together) model. Interestingly, synergistic growth interactions were more significant in the competitive model. Furthermore, the biofilm structure and species-specific distribution, as assessed by confocal laser scanning microscopy and using peptide nucleic acid fluorescence in situ hybridization method, revealed two very different triple-species morphotypes, suggesting that different interactions occur in the different models. Interestingly, independent of the model or triple-species consortium tested, we observed that G. vaginalis represented most of the biofilm bacterial composition, further highlighting the relevance of this taxon in BV.

Efficacy of alpha-mangostin for antimicrobial activity against endodontopathogenic microorganisms in a multi-species bacterial-fungal biofilm model

ObjectiveTo determine the activity of alpha-mangostin on preformed bacterial-fungal multi-species biofilms in vitro, and to ascertain the impact on metabolic activity, biofilm structure and viability. DesignInhibitory concentrations (ICs) for alpha-mangostin against planktonic cultures of Candida albicans, Enterococcus faecalis, Lactobacillus rhamnosus, and Streptococcus gordonii were determined using a standard broth microdilution method. Single and multi-species (all species 1:1:1:1) biofilms were grown on polystyrene coverslips in Roswell Park Memorial Institute Medium for 48 h. The biofilms were then exposed to 0.2% (w/v) alpha-mangostin for 24 h. These concentrations were selected based on pilot experiments and the solubility of these compounds. 2% (v/v) chlorhexidine was used as a positive control and Roswell Park Memorial Institute Medium as a negative control. The metabolic activity of the biofilms after exposure was measured using metabolic (XTT) assays. Biofilms were visualised and quantified using fluorescent BacLight™ LIVE/DEAD staining. The biofilms were assessed for cell viability by culture and colony counting (CFU/mL). Results8 mg/L of alpha-mangostin was cidal against planktonic bacteria and 1000 mg/L for Candida. Alpha-mangostin was most active against L. rhamonosus biofilms and least active against C. albicans biofilm (metabolism inhibited by 99% and 78%, respectively). Alpha-mangostin exposure reduced the number of viable cells in the biofilms. ConclusionAlpha-mangostin inhibited the metabolic activity of bacterial-fungal biofilms effectively. The anti-biofilm activity of alpha-mangostin was comparable to chlorhexidine and thus has potential as a novel agent for endodontic therapy.

Chitosan Enhances the Anti-Biofilm Activity of Biodentine against an Interkingdom Biofilm Model.

Endodontic infection is a biofilm disease that is difficult to irradicate with current treatment protocols, and as such, persistent micro-organisms may lead to ongoing or recurrent disease. The potential for the use of enhanced filling materials to modify biofilm regrowth is a promising strategy. This current study aimed to evaluate the anti-biofilm efficacy of calcium silicate cements modified with chitosan. The development of mono-species and multi-species biofilms on ProRoot MTA, Biodentine and bovine dentine discs were explored using quantitative microbiology analysis. The effect on regrowth of biofilms was assessed following the addition of chitosan to each cement. In comparison to a dentine substrate, both materials did not show the ability to inhibit biofilm regrowth. Biodentine incorporated with chitosan displayed a dose-dependent reduction in multi-species biofilm regrowth, unlike MTA. Notably, interkingdom biofilms were shown to enhance bacterial tolerance in the presence of chitosan. This study demonstrates the potential to enhance the antimicrobial properties of Biodentine. The findings highlight the need for appropriate model systems when exploring antimicrobial properties of materials in vitro so that interspecies and interkingdom interactions that modify tolerance are not overlooked while still supporting the development of innovative materials.

Influence of Bioinspired Lithium-Doped Titanium Implants on Gingival Fibroblast Bioactivity and Biofilm Adhesion.

Soft tissue integration (STI) at the transmucosal level around dental implants is crucial for the long-term success of dental implants. Surface modification of titanium dental implants could be an effective way to enhance peri-implant STI. The present study aimed to investigate the effect of bioinspired lithium (Li)-doped Ti surface on the behaviour of human gingival fibroblasts (HGFs) and oral biofilm in vitro. HGFs were cultured on various Ti surfaces—Li-doped Ti (Li_Ti), NaOH_Ti and micro-rough Ti (Control_Ti)—and were evaluated for viability, adhesion, extracellular matrix protein expression and cytokine secretion. Furthermore, single species bacteria (Staphylococcus aureus) and multi-species oral biofilms from saliva were cultured on each surface and assessed for viability and metabolic activity. The results show that both Li_Ti and NaOH_Ti significantly increased the proliferation of HGFs compared to the control. Fibroblast growth factor-2 (FGF-2) mRNA levels were significantly increased on Li_Ti and NaOH_Ti at day 7. Moreover, Li_Ti upregulated COL-I and fibronectin gene expression compared to the NaOH_Ti. A significant decrease in bacterial metabolic activity was detected for both the Li_Ti and NaOH_Ti surfaces. Together, these results suggest that bioinspired Li-doped Ti promotes HGF bioactivity while suppressing bacterial adhesion and growth. This is of clinical importance regarding STI improvement during the maintenance phase of the dental implant treatment.

Transcriptional Changes in Bifidobacterium bifidum Involved in Synergistic Multispecies Biofilms.

Bifidobacterium bifidum is part of the core microbiota of healthy infant guts where it may form biofilms on epithelial cells, mucosa, and food particles in the gut lumen. Little is known about transcriptional changes in B. bifidum engaged in synergistic multispecies biofilms with ecologically relevant species of the human gut. Recently, we reported prevalence of synergism in mixed-species biofilms formed by the human gut microbiota. This study represents a comparative gene expression analysis of B. bifidum when grown in a single-species biofilm and in two multispecies biofilm consortia withBifidobacterium longum subsp. infantis, Bacteroides ovatus, and Parabacteroides distasonis in order to identify genes involved in this adaptive process in mixed biofilms and the influence on its metabolic and functional traits. Changes up to 58% and 43% in its genome were found when it grew in three- and four-species biofilm consortia, respectively. Upregulation of genes of B. bifidum involved in carbohydrate metabolism (particularly the galE gene), quorum sensing (luxS and pfs), and amino acid metabolism (especially branched chain amino acids) in both multispecies biofilms, compared to single-species biofilms, suggest that they may be contributing factors for the observed synergistic biofilm production when B. bifidum coexists with other species in a biofilm.

Assessment of Disinfection Potential of Q-Switch Nd: YAG Laser on Contaminated Titanium Implant Surfaces.

Peri-implantitis (PI) is a relatively frequent pathology that compromises the overall survival of the dental implant. Adjunctive approaches for the conventional mechanical debridement are being suggested to optimize the treatment of PI. The goal of the study was the assessment of the disinfection potential of the Q-Switch Nd: YAG laser on contaminated titanium implant surfaces. A total of 72 sterile titanium discs were used and divided into three groups: 24 contaminated titanium discs treated with the laser (study Group L), 24 contaminated titanium discs with no treatment (control 1—Group C), and 24 sterile titanium discs with no treatment (control 2—Group S). Multi-species biofilm was used: Porphyromonas gingivalis, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans, Streptococcus mutans, Streptococcus sobrinus, and Prevotella intermedia. Commensal bacteria were included also: Actinomyces naeslundii, Actinomyces viscosus, Streptococcus cristatus, Streptococcus gordonii, Streptococcus mitis, Streptococcus oralis, Streptococcus sanguinis, Streptococcus parasanguinis, and Veillonella parvula. Parameters delivered per pulse on the targeted surfaces of the titanium discs were an energy density of 0.597 J/cm2 each pulse, a pulse power of 270 mW, a laser beam spot of 2.4 mm in diameter, and a rate of repetition of 10 Hertz (Hz) for a pulse duration of 6 nanoseconds (ns). The mode was no contact, and a distance of 500 micrometers was used with a total time of irradiation equal to 2 s (s). The collection of microbiological samples was made for all groups; colony-forming units (CFU) were identified by two different practitioners, and the average of their examinations was considered for each sample. The average of the TBC (CFU/mL) was calculated for each group. Values were 0.000 CFU/mL, 4767 CFU/mL, and 0.000 CFU/mL for Group L, Group C, and Group S, respectively. Therefore, the suggested treatment protocol was able to provoke a total disinfection of the contaminated titanium surfaces. A statistical difference was only found between Group L vs. Group C and between Group S vs. Group C. The difference was not significant between Group S and Group L. In conclusion, the present study confirmed that the Q-Switch Nd: YAG laser under our specific conditions can provide a total disinfection of the contaminated titanium surfaces.

Development of multi-species biofilm formed by thermophilic bacteria on stainless steel immerged in skimmed milk

Thermophilic bacteria, such as Bacillus licheniformis, Geobacillus stearothermophilus, Bacillus Subtilis and Anoxybacillus flavithermus, are detected frequently in milk powder products. Biofilms of those strains act as a major contamination to milk powder manufactures and pose potential risks in food safety. In this study, we explored the developing process of multi-species biofilm formed by the four thermophilic bacteria on stainless steel immerged in skimmed milk. The results showed that the thermophilic strains possessed strong capacities to decompose proteins and lactose in skimmed milk, and the spoilage effects were superimposed from multiple strains. B. licheniformis was the most predominant species in the mixed-species biofilm after 12-h incubation. From 24 h to 48 h, G. stearothermophilus occupied the highest proportion. Within the multi-species biofilm, competitive relation existed between B. licheniformis and G. stearothermophilus, while synergistic impacts were observed between B. licheniformis and A. flavithermus. The interspecies mutual influences on biofilm development provided important evidences for understanding colonization of the predominant thermophilic bacteria during milk powder processing.

Antimicrobial Mechanisms of Leucocyte- and Platelet Rich Fibrin Exudate Against Planktonic Porphyromonas gingivalis and Within Multi-Species Biofilm: A Pilot Study.

Leucocyte- and platelet rich fibrin (L-PRF) is an autologous biomaterial used in regenerative procedures. It has an antimicrobial activity against P. gingivalis although the mechanism is not fully understood. It was hypothesized that L-PRF exudate releases hydrogen peroxide and antimicrobial peptides that inhibit P. gingivalis growth. Agar plate and planktonic culture experiments showed that the antimicrobial effect of L-PRF exudate against P. gingivalis was supressed by peroxidase or pepsin exposure. In developing multi-species biofilms, the antimicrobial effect of L-PRF exudate was blocked only by peroxidase, increasing P. gingivalis growth with 1.3 log genome equivalents. However, no effect was shown on other bacteria. Pre-formed multi-species biofilm trials showed no antimicrobial effect of L-PRF exudate against P. gingivalis or other species. Our findings showed that L-PRF exudate may release peroxide and peptides, which may be responsible for its antimicrobial effect against P. gingivalis. In addition, L-PRF exudate had an antimicrobial effect against P. gingivalis in an in vitro developing multi-species biofilm.

In vitro inhibition of a multispecies oral cavity biofilm by Syzygium aromaticum essential oil.

To evaluate the anti-fungal activity of Syzygium aromaticum essential oil and its inhibition of a multispecies biofilm in patients with oral candidiasis. Inhibiting biofilm formation on the denture surface is an important practice for preventing denture stomatitis. The anti-fungal activity against Candida albicans and non-albicans Candida species was evaluated through the microdilution method to define Minimal Inhibitory (MIC) and Fungicidal (MFC) Concentrations. Time-kill assay assessed growth kinetics of C.albicans based on pre-determined time points (0, 1, 2, 4, 6 and 24hours). A multi-species biofilm was formed using human saliva from patients with oral candidiasis and anti-biofilm activity determined by Colony Forming Units per milliliter (CFU/mL) count, fluorescence microscopy with calcofluor white to observe yeast presence and structure, and metabolic activity by XTT (2,3-Bis-(2Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-carboxanilide) reduction assay. The essential oil showed an anti-fungal activity against allCandidaspecies (MIC 500-1000µg/mL, MFC 1000-2000µg/mL), and the time-kill assay showed that 2000µg/mL (from 2hours onward) and 1000µg/mL (from 4hours onward) concentrations had substantially lower yeast growth than the negative control. In the biofilm analysis, the essential oil had a lower CFU/mL count and a biofilm metabolic activity (91.4%) than seen with its negative control, and in both analyses, the essential oil was not significantly different from the positive control (chlorhexidine). Morphological analysis showed amorphous and fragmented cellular structures after treatment with the essential oil. Syzygium aromaticum essential oil had anti-fungal activities, reduced the Candida growth kinetics substantially and inhibited the multi-species biofilm formation.

Mapping of a Subgingival Dual-Species Biofilm Model Using Confocal Raman Microscopy.

Techniques for continuously monitoring the formation of subgingival biofilm, in relation to the determination of species and their accumulation over time in gingivitis and periodontitis, are limited. In recent years, advancements in the field of optical spectroscopic techniques have provided an alternative for analyzing three-dimensional microbiological structures, replacing the traditional destructive or biofilm staining techniques. In this work, we have demonstrated that the use of confocal Raman spectroscopy coupled with multivariate analysis provides an approach to spatially differentiate bacteria in an in vitro model simulating a subgingival dual-species biofilm. The present study establishes a workflow to evaluate and differentiate bacterial species in a dual-species in vitro biofilm model, using confocal Raman microscopy (CRM). Biofilm models of Actinomyces denticolens and Streptococcus oralis were cultured using the “Zürich in vitro model” and were analyzed using CRM. Cluster analysis was used to spatially differentiate and map the biofilm model over a specified area. To confirm the clustering of species in the cultured biofilm, confocal laser scanning microscopy (CLSM) was coupled with fluorescent in vitro hybridization (FISH). Additionally, dense bacteria interface area (DBIA) samples, as an imitation of the clusters in a biofilm, were used to test the developed multivariate differentiation model. This confirmed model was successfully used to differentiate species in a dual-species biofilm and is comparable to morphology. The results show that the developed workflow was able to identify main clusters of bacteria based on spectral “fingerprint region” information from CRM. Using this workflow, we have demonstrated that CRM can spatially analyze two-species in vitro biofilms, therefore providing an alternative technique to map oral multi-species biofilm models.

Reversal of Polymicrobial Biofilm Tolerance to Ciprofloxacin by Blue Light plus Carvacrol.

Chronic wound infections are often caused by multi-species biofilms and these biofilm-embedded bacteria exhibit remarkable tolerance to existing antibiotics, which presents huge challenges to control such infections in the wounds. In this investigation, we established a polymicrobial biofilm composed of P. aeruginosa, S. aureus, K. pneumoniae, and A. baumannii. We tested a cocktail therapy comprising 405-nm blue light (BL), carvacrol (Ca), and antibiotics on the multispecies biofilm. Despite the fact that all strains used to form the biofilm were susceptible to ciprofloxacin (CIP) in planktonic cultures, the biofilm was found to withstand ciprofloxacin as well as BL-Ca dual treatment, mainly because K. pneumoniae outgrew and became dominant in the biofilm after each treatment. Strikingly, when ciprofloxacin was combined with BL-Ca, the multispecies biofilms succumbed substantially and were eradicated at an efficacy of 99.9%. Mechanistically, BL-Ca treatment increased membrane permeability and potentiated the anti-biofilm activity of ciprofloxacin, probably by facilitating ciprofloxacin’s entrance of the bacteria, which is particularly significant for K. pneumoniae, a species that is refractory to either ciprofloxacin or BL-Ca dual treatment. The results suggest that bacterial membrane damage can be one of the pivotal strategies to subvert biofilm tolerance and combat the recalcitrant multispecies biofilms.

Biofilm mediated strategies to mitigate heavy metal pollution: A critical review in metal bioremediation

Mining and various industrial activities, along with generation and accumulation of heavy metals due to agricultural and domestic activities are languishing human health and the environment as a whole. Employment of conventional strategies in eradicating these toxic loads, are way expensive and concerned with the production of secondary wastes. Biofilm being a syntropic microbial consortium, through the mechanism of cell communication and signaling, quorum sensing and modulating strategies accomplish the removal of contaminant metals from environment. Such biofilm mediated bioremediation may be explicitly explained by implementing synthetic biology models and through unique mathematical modelling of mechanisms of multi species biofilm formation and survival strategies in toxic environments. Microbial metabolism for extracellular electron transfer (EET) and development of niche-specific microbial electrochemistry-driven biotechnologies, exploit the bio electrochemical system of electroactive biofilms (EABs) for removal of metal ions from environment. The multi omics platform predicts the pathways of modified metal-biofilm interaction and sequestration strategies as crucial mechanism to accelerate biofilm mediated bioremediation processes.

Actinobacillus pleuropneumoniae Surviving on Environmental Multi-Species Biofilms in Swine Farms.

Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, an important respiratory disease for the pig industry. A. pleuropneumoniae has traditionally been considered an obligate pig pathogen. However, its presence in the environment is starting to be known. Here, we report the A. pleuropneumoniae surviving in biofilms in samples of drinking water of swine farms from Mexico. Fourteen farms were studied. Twenty drinking water samples were positive to A. pleuropneumoniae distributed on three different farms. The bacteria in the drinking water samples showed the ability to form biofilms in vitro. Likewise, A. pleuropneumoniae biofilm formation in situ was observed on farm drinkers, where the biofilm formation was in the presence of other bacteria such as Escherichia coli, Stenotrophomonas maltophilia, and Acinetobacter schindleri. Our data suggest that A. pleuropneumoniae can inhabit aquatic environments using multi-species biofilms as a strategy to survive outside of their host.

Differences in chlorhexidine mouthrinses formulations influence the quantitative and qualitative changes in in-vitro oral biofilms.

Chlorhexidine mouthrinses are marketed in different formulations. This study aimed at investigating qualitative and quantitative changes in in-vitro multispecies oral biofilms, induced by different chlorhexidine-containing mouthrinses. Earlier studies comparing chlorhexidine mouthrinses are either clinical studies or in-vitro studies assessing the antimicrobial efficacy of the mouthrinses. However, no clear investigations are available regarding ecological impact of different chlorhexidine formulations on in-vitro multispecies oral biofilms after rinsing with different chlorhexidine formulations. Nine commercially available chlorhexidine mouthrinses were selected. Multispecies oral communities (14species) were grown for 48h in a Biostat-B Twin bioreactor. After that, they were used to develop biofilms on the surface of hydroxyapatite disks in 24-well pates for 48h. Biofilms were then rinsed once or multiple times with the corresponding mouthrinse. Biofilms were collected before starting the rinsing experiment and every 24h for 3days and vitality quantitative PCR was performed. The experiment was repeated 3 independent times on 3 different days and the results were analyzed using a linear mixed model. The mouthrinses provoked different effects in terms of change in total viable bacterial load (VBL), ecology, and community structure of the multispecies biofilms. There was no relation between chlorhexidine concentrations, presence, or absence of cetylpyridinium chloride and/or alcohol, and the observed effects. Some tested chlorhexidine mouthrinses (MC, HG, HH, and HI) strongly lowered the total VBL (≈1007 Geq/ml), but disrupted biofilm symbiosis (≥40% of the biofilms communities are pathobionts). On the other hand, other tested chlorhexidine mouthrinses (MD, ME, and HF) had limited impact on total VBL (≥1010 Geq/ml), but improved the biofilm ecology and community structure (≤10% of the biofilms communities are pathobionts). Not all chlorhexidine mouthrinses have the same effect on oral biofilms. Their effect seems to be strongly product dependent and vary according to their compositions and formulations.

Antibacterial Effect of Sodium Hypochlorite and EDTA in Combination with High-Purity Nisin on an Endodontic-like Biofilm Model.

Antimicrobial peptides have been proposed as antibiofilm agents. Therefore, we evaluated the effect of endodontic irrigants combined or not with the antimicrobial peptide nisin against an endodontic biofilm model composed of eleven bacterial species. Biofilms were grown on hydroxyapatite discs for 3, 15 and 21 days and treated with 1.5% sodium hypochlorite (NaOCl) or 17% EDTA followed by high-purity nisin (nisin ZP) or saline for 5 min each. Differences between groups were tested by two-way ANOVA and Tukey’s multiple comparisons test (p < 0.05). Treatment with 1.5% NaOCl completely eliminated 3-d and 15-d biofilms but did not eradicate 21-d biofilms. Treatment with 1.5% NaOCl and 17% EDTA was equally effective against 21-d biofilms, showing 5-log and 4-log cell reduction, respectively, compared to the untreated control (9 log10, p < 0.05). No significant difference was found between 1.5% NaOCl + nisin ZP and 1.5% NaOCl in 21-d biofilms (p > 0.05). Likewise, no significant difference was found between 17% EDTA + nisin ZP and 17% EDTA treatments (p > 0.05). In conclusion, 1.5% NaOCl or 17% EDTA were effective strategies to combat mature biofilms. The additional use of nisin did not improve the activity of conventional irrigants against multispecies biofilms.

Multilevel selection favors fragmentation modes that maintain cooperative interactions in multispecies communities.

Reproduction is one of the requirements for evolution and a defining feature of life. Yet, across the tree of life, organisms reproduce in many different ways. Groups of cells (e.g., multicellular organisms, colonial microbes, or multispecies biofilms) divide by releasing propagules that can be single-celled or multicellular. What conditions determine the number and size of reproductive propagules? In multicellular organisms, existing theory suggests that single-cell propagules prevent the accumulation of deleterious mutations (e.g., cheaters). However, groups of cells, such as biofilms, sometimes contain multiple metabolically interdependent species. This creates a reproductive dilemma: small daughter groups, which prevent the accumulation of cheaters, are also unlikely to contain the species diversity that is required for ecological success. Here, we developed an individual-based, multilevel selection model to investigate how such multi-species groups can resolve this dilemma. By tracking the dynamics of groups of cells that reproduce by fragmenting into smaller groups, we identified fragmentation modes that can maintain cooperative interactions. We systematically varied the fragmentation mode and calculated the maximum mutation rate that communities can withstand before being driven to extinction by the accumulation of cheaters. We find that for groups consisting of a single species, the optimal fragmentation mode consists of releasing single-cell propagules. For multi-species groups we find various optimal strategies. With migration between groups, single-cell propagules are favored. Without migration, larger propagules sizes are optimal; in this case, group-size dependent fissioning rates can prevent the accumulation of cheaters. Our work shows that multi-species groups can evolve reproductive strategies that allow them to maintain cooperative interactions.

Antibiofilm and cytotoxic effect of 3,3′-dihydroxycurcumin (DHC) as photosensitizer agent in antimicrobial photodynamic therapy for endodontic purposes

BackgroundCurcuminoids have been designed not only to improve chemical and metabolic stability of curcumin (CUR), but also to increase its antimicrobial activity, without effecting its ability as photosensitizer agent in antimicrobial photodynamic therapy (aPDT) with light emitting diode (LED). This study evaluated the antimicrobial and antibiofilm action of curcumin analog DHC (or 3,3′-dihydroxycurcumin), submitted or not to LED irradiation, on microorganisms of endodontic importance and its influence on fibroblasts viability. MethodsDHC was synthetized by modified Pablon's methodology and the experiments were conducted under irradiation or not with indium gallium nitride-based LED (440-480nm, 100 mW/cm2, 0.78 cm2,60 s). The antimicrobial activity of CUR and DHC were determined by the Minimum Inhibitory and Bactericidal Concentration assays against Gram-positive and Gram-negative bacteria and the effect of both compounds on fibroblast viability was tested using colorimetric assays. They were also evaluated on 72h and 7days single-species biofilms and on 14 days multispecies biofilms formed inside dentin tubules by bacterial colonies counts and confocal microscopy, respectively. Data were analyzed statistically considering p<0.05. ResultsDHC had bactericidal effect against all bacteria tested higher than CUR, in planktonic conditions. CUR and DHC (at 39 and 19 μg/mL, respectively) were cytocompatible and LED irradiation reduced fibroblast viability, regardless of compound. CUR and DHC reduced the growth of single-species biofilms and the effect of aPDT was bacteria dependent. DHC reduced more than 70% of microorganisms from multispecies biofilms, superior to CUR effect. ConclusionsDHC showed low cytotoxicity and antibiofilm effect similar to curcumin, when submitted or not to aPDT, and could be further explored as a bioactive compound for endodontic purposes.

Multiscale Modeling of Uranium Bioreduction in Porous Media by One-Dimensional Biofilms

We formulate a multiscale mathematical model that describes the bioreduction of uranium in porous media. On the mesoscale we describe the bioreduction of uranium [VI] to uranium [IV] using a multispecies one-dimensional biofilm model with suspended bacteria and thermodynamic growth inhibition. We upscale the mesoscopic (colony scale) model to the macroscale (reactor scale) and investigate the behavior of substrate utilization and production, attachment and detachment processes, and thermodynamic effects not usually considered in biofilm growth models. Simulation results of the reactor model indicate that thermodynamic inhibition quantitatively alters the dynamics of the model and neglecting thermodynamic effects may over- or underestimate chemical concentrations in the system. Furthermore, we numerically investigate uncertainties related to the specific choice of attachment and detachment rate coefficients and find that while increasing the attachment rate coefficient or decreasing the detachment rate coefficient leads to thicker biofilms, performance of the reactor remains largely unaffected.