Biofilm Age Research Articles

Insight into mature biofilm quorum sensing in full-scale wastewater treatment plants

Quorum sensing (QS) has been thoroughly investigated during initial biofilm formation stages, while the role of QS in mature biofilms has received little research attention. This study assessed QS in 22 biofilm samples from full-scale wastewater treatment plants in China. Results showed that the concentration of acyl-homoserine lactones (AHLs) in various biofilm bound forms, ranged from 15.63 to 609.76 ng/g. The highest concentration of AHLs was found in the tightly bound biofilm fraction, while the lowest concentrations were observed in the surface biofilm fraction. Environmental variables, C/N ratio and temperature, were found to be significant factors influencing biofilm AHL distribution (p < 0.01). Higher C/N ratios (ranging from 3 to 12) and low temperatures contributed to the higher concentration of AHLs in biofilms. Dominant AHLs (C10-HSL and C12-HSL) were significantly associated with biofilm activity (R2 = 0.98/0.97, p < 0.05), with the tightly bound biofilm fraction (TB-biofilm) presenting the highest activity (ATP concentration). Biofilm aging and re-formation processes were more active in the surface biofilm layer (S-biofilm), while the stable structure of the TB-biofilm layer which is attached to the surface of bio-carriers ensures high biofilm activity. This study furthers our understanding of the roles of AHLs in the regulation of mature biofilm activities.

The effect of aging methods on the fracture toughness and physical stability of an oxirane/acrylate, ormocer, and Bis-GMA-based resin composites.

To determine the effect of aging methods on the fracture toughness of a conventional Bis-GMA-based resin composite (Filtek Supreme), an ormocer-based resin composite (Admira), and an experimental hydrophobic oxirane/acrylate interpenetrating network resin system (OASys)-based composite. A 25 × 5 × 2.8-mm stainless-steel mold with 2.5mm single-edge center notch, following ASTM standards [E399-90], was used to fabricate 135 specimens (n = 15) of the composite materials and randomly distributed into groups. For the baseline group, specimens were fabricated and then tested after 24-h storage in water. For the biofilm challenge, specimens were randomly placed in a six-well tissue culture plate and kept at 37°C with bacterial growth media (Brain Heart Infusion (BHI); Streptococcus mutans) changed daily for 15days. For the water storage challenge, specimens were kept in 5ml of deionized distilled autoclaved water for 30days at 37°C. μCT evaluation by scanning the specimens was performed before and after the proposed challenge. Fracture toughness (KIc) testing was carried out following the challenges. μCT surface area and volume analyses showed no significant changes regardless of the materials tested or the challenge. Filtek and Admira fracture toughness was significantly lower after the biofilm and water storage challenges. OASys mean fracture toughness values after water aging were significantly higher than that of baseline. Toughness values for OASys composites after biofilm aging were not statistically different when compared to either water or baseline values. The fracture toughness of Bis-GMA and ormocer-based dental resin composites significantly decreased under water and bacterial biofilm assault. However, such degradation in fracture toughness was not visible in OASys-based composites. Current commercial dental composites are affected by the oral environment, which might contribute to the long-term performance of these materials.

An in-situ technique for the direct structural characterization of biofouling in membrane filtration

In the present work, a convenient and direct technique which enables to characterize the intrinsic structure and the mechanical properties of the biofilm without altering its chemical and physical properties is proposed. By utilizing the Optical Coherence Tomography (OCT) as a structural imaging tool coupled with an advance mathematical framework, thickness, micro-porosity, normal stress-strain curve, bulk modulus and total permeability of the biofilm structures are determined. The accuracy of this mathematical technique for the in situ characterization is validated by analyzing two different membrane structures for porosity and permeability values against the mercury intrusion porosimetry method. Three-dimensional images of biofouling were obtained with high resolution aided to numerically analyze the intrinsic biofilm structure at microscale. Growth of biofilm in a dead-end filtration experimental setup was investigated by varying the feed flow rate which allowed uniform compression and decompression to compute normal stress-strain relation of the evolving biofilm structure. At early development of biofilm (day 3), the thickness and normal stress/strain curve showed that the biofilm structure behave similar to elastic material. However, hysteresis-like trend starts to appear with the growth of biofilm suggesting the deviation of biofilm properties to viscoelastic nature at day 8. The microstructure porosity increased from 0.214 (day 3) to 0.482 (day 8) at a feed flow rate of 15 mL/min. The total membrane/biofilm permeability decreased with biofilm age to reach 5.19 × 10−15 m2 at day 8 at the same flow rate, leading to a reduction of permeate flux over time. All the structural properties were found to be time dependent as the biofilm continuously evolved.

Controlling cold temperature partial nitritation in moving bed biofilm reactor

Mainstream partial nitritation was studied at 10 °C in a moving bed biofilm reactor treating synthetic wastewater containing both nitrogen (≈40 mg L−1) and organic carbon at COD/N ratio ranging from 1.3 to 2.2. Three different control strategies were investigated to achieve partial nitritation. Initially, biofilm age was controlled by incorporating a media replacement strategy. Next, separately from the media replacement, oxygen limited conditions were investigated and finally pH control was incorporated together with oxygen limitation. Successful partial nitritation was achieved only by combining oxygen limitation with pH control. The average NH4-N concentration was equal to 16.0 ± 1.6 mg L−1 and average NO2-N concentration was equal to 15.7 ± 2.4 mg L−1 during steady state partial nitritation. The average residual NO3-N concentration was equal to 2.6 ± 2.2 mg L−1. The results obtained from this study prove for the first time that partial nitritation can be successfully controlled in a biofilm reactor treating wastewater with low nitrogen concentration, relatively high COD/N ratio and at low temperature. An algorithm for dynamic process control of partial nitritation has been also developed.

Biofilm aging in full-scale aerobic bioreactors from perspectives of metabolic activity and microbial community

The objective of this study was to establish a quantitative response to biofilm aging from perspectives of metabolic activity and microbial community applied in distinct wastewater. Biofilms developed on soft, integrated and suspended biocarriers in three full-scale aerobic bioreactors under stable operations from municipal and industrial wastewater treatment plants, were tracked for more than 11 weeks for biofilm aging study. Results showed the first evidence of biofilm aging in full-scale aerobic bioreactors by adenylate energy charge (AEC) that biofilm needed to be reactivated when AEC was less than 0.6. Biofilm aged more easily on soft and integrated biocarriers and high salinity, high organic contents and high suspended sludge concentration accelerated the aging process of biofilm. Caldilinea and Acinetobacter predominated in aging biofilm on suspended biocarriers. This study offered a novel evaluation methodology of biofilm aging and was believed to provide important reference for biofilm regulation in biofilm-based wastewater treatment process.

P. aeruginosa flow-cell biofilms are enhanced by repeated phage treatments but can be eradicated by phage-ciprofloxacin combination.

Phage therapy has shown promising results in the treatment of Pseudomonas aeruginosa biofilm infections in animal studies and case reports. The aim of this study was to quantify effects of phage treatments on P. aeruginosa biofilm production and structure. Confocal scanning microscopy was used to follow the interaction between a cocktail of three virulent phages and P. aeruginosa flow-cell biofilms. The role of (i) biofilm age, (ii) repeated phage treatments, (iii) alginate production and (iv) the combination with sub-MIC levels of ciprofloxacin was investigated. Single phage treatment in the early biofilm stages significantly reduced P. aeruginosa PAO1 biovolume (85%-98% reduction). Repeated phage treatments increased the biovolume from 18.25 (untreated biofilm) to 22.24 and 31.07 µm3/µm2 for biofilms treated with phages twice and thrice, respectively. Alginate protected against the phage treatment as the live biovolume remained unaffected by the phage treatment in the mucoid biofilm (20.11 µm3/µm2 in untreated and 21.74 µm3/µm2 in phage-treated biofilm) but decreased in the PAO1 biofilm from 27.35 to 0.89 µm3/µm2. We show that the combination of phages with antibiotics at sub-MIC levels caused a ∼6 log units reduction in the abundance of P. aeruginosa cells in biofilms and that phage treatment increased the size of microcolonies in flow-cell system.

Regulation of Biofilm Aging and Dispersal in Bacillus subtilis by the Alternative Sigma Factor SigB.

Bacterial biofilms are important in natural settings, biotechnology, and medicine. However, regulation of biofilm development and its persistence in different niches is complex and only partially understood. One key step during the biofilm life cycle is dispersal, when motile cells abandon the mature biofilm to spread out and colonize new niches. Here, we show that in the model bacterium Bacillus subtilis the general stress transcription factor SigB is essential for halting detrimental overgrowth of mature biofilm and for triggering dispersal when nutrients become limited. Specifically, SigB-deficient biofilms were larger than wild-type biofilms but exhibited accelerated cell death, significantly greater sensitivity to different stresses, and reduced dispersal. Interestingly, the signal detected by SigB to limit biofilm growth was transduced through the RsbP-dependent metabolic arm of the SigB regulatory cascade, which in turn positively controlled expression of SinR, the master regulator of biofilm formation and cell motility. This novel SigB-SinR regulatory circuit might be important in controlling the fitness of biofilms (either beneficial or harmful) in diverse environments.IMPORTANCE Biofilms are crucial for bacterial survival, adaptation, and dissemination in natural, industrial, and medical systems. Sessile cells embedded in the self-produced extracellular matrix of the biofilm benefit from a division of labor and are protected from environmental insults. However, as the biofilm ages, cells become stressed because of overcrowding, starvation, and accumulation of waste products. How does the sessile biofilm community sense and respond to stressful conditions? Here, we show that in Bacillus subtilis, the transcription factors SigB and SinR control whether cells remain in or leave a biofilm when metabolic conditions become unfavorable. This novel SigB-SinR regulatory circuit might be important for controlling the fitness of biofilms (either beneficial or harmful) in diverse environments.

Resistance of L. monocytogenes and S. Typhimurium towards Cold Atmospheric Plasma as Function of Biofilm Age

The biofilm mode of growth protects bacterial cells against currently applied disinfection methods for abiotic (food) contact surfaces. Therefore, innovative methods, such as Cold Atmospheric Plasma (CAP), should be investigated for biofilm inactivation. However, more knowledge is required concerning the influence of the biofilm age on the inactivation efficacy in order to comment on a possible application of CAP in the (food) processing industry. L. monocytogenes and S. Typhimurium biofilms with five different ages (i.e., 1, 2, 3, 7, and 10 days) were developed. For the untreated biofilms, the total biofilm mass and the cell density were determined. To investigate the biofilm resistance towards CAP treatment, biofilms with different ages were treated for 10 min and the remaining cell density was determined. Finally, for the one-day old reference biofilms and the most resistant biofilm age, complete inactivation curves were developed to examine the influence of the biofilm age on the inactivation kinetics. For L. monocytogenes, an increased biofilm age resulted in (i) an increased biomass, (ii) a decreased cell density prior to CAP treatment, and (iii) an increased resistance towards CAP treatment. For S. Typhimurium, similar results were obtained, except for the biomass, which was here independent of the biofilm age.

Chemical biofilm removal capacity of endodontic irrigants as a function of biofilm structure: optical coherence tomography, confocal microscopy and viscoelasticity determination as integrated assessment tools.

To investigate the influence of biofilm structure on the biofilm removal capacity of endodontic irrigants and to study changes in the architecture of the remaining biofilms. Streptococcus oralis J22 and Actinomyces naeslundii T14V-J1 were cocultured under different growth conditions on saliva-coated hydroxyapatite discs. A constant depth film fermenter (CDFF) was used to grow steady-state 4-day biofilms. Biofilms were grown under static conditions for 4 and 10days within a confined space. Twenty microlitres of 2% NaOCl, 2% Chlorhexidine (CHX), 17% Ethylene-diamine-tetra-acetic acid (EDTA) and buffer were applied statically on the biofilms for 60s. Biofilm removal was evaluated with optical coherence tomography (OCT). Post-treated biofilms were assessed via low load compression testing (LLCT) and Confocal laser scanning microscopy (CLSM). Optical coherence tomography data were analysed through a two-way analysis of variance (ANOVA). Low load compression testing and CLSM data were analysed through one-way ANOVA and Dunnett's post hoc test. The level of significance was set at a<0.05. The initial biofilm structure affected the biofilm removal capacity of the irrigants. NaOCl demonstrated the greatest chemical efficacy against the biofilms and was significantly more effective on the static than the CDFF biofilms (P<0.001). CHX was ineffective and caused a rearrangement of the biofilm structure. Ethylene-diamine-tetra-acetic acid exhibited a distinct removal effect only on the CDFF biofilms. Biofilm age influenced the structure of the remaining biofilms. The 4-day grown remaining biofilms had a significantly different viscoelastic pattern compared to the respective 10-day grown biofilms (P≤0.01), especially in the NaOCl-treated group. Confocal laser scanning microscopy analysis confirmed the CHX-induced biofilm structural rearrangement. Biofilm structure is an influential factor on the chemical efficacy of endodontic irrigants. Optical coherence tomography allows biofilm removal characteristics to be studied. NaOCl should remain the primary irrigant. Ethylene-diamine-tetra-acetic acid was effective against cell-rich/EPS-poor biofilms. Chlorhexidine did not remove biofilm, but rather rearranged its structure.

Biofilm Production of Leptospira spp. Strains

Background: Leptospirosis is a zoonosis that affects many species of mammals and occurs endemically in Brazil. The biofilm matrix provides structure and protection to the biofilm cells working as a physical barrier to antibiotic agents, which are attached or consumed by the matrix components. However, this attribute varies according to the matrix, antimicrobial agent and biofilm age. Leptospira may change morphologically according to environmental conditions, including cell aggregation and biofilm formation. Leptospira can colonize the ducts of kidney from hosts for a long time, forming a biofilm, which is believed to be an important factor for their maintenance in animals and in the environment. Thus, the objective of this research was to determine the biofilm formation capacity of four strains of Leptospira interrogans.Materials, Methods &amp; Results: The strains were typified by WHO/FAO/OIE and National Collaborating Center for Reference and Research on Leptospirosis (Kit Biomedical Research, Amsterdam, Netherlands). Leptospira interrogans strains, two isolated from cattle and two isolated from dogs were biofilms tested for adhesion on polystyrene plates, extracellular matrix composition and confocal microscopy. In the plating adhesion test, the suspension was inoculated into 96-well sterile polystyrene microplates with flat bottom at a ratio of 1:200 in EMJH medium, followed by 24 h incubation at 28°C, with medium renewal after 12 h. After this period the wells were washed three times with sterile PBS and following incubation; the plates were dried in the oven at 60°C for 30 min and added 200 μL of 1% violet crystal for five min. Subsequently, the plates were washed with distilled water, after complete removal, 200 μL of acetic acid 33% was added and the readings were performed at 570 nm in the ELISA reader. The proteins and polysaccharides were quantified in a scraped pooled sample diluted in 0.85% sterile saline solution to achieve an optimal amount for testing used reagents of the BCA kit. The polysaccharide content was determined by adding into a tube, an aliquot of 0.5 mL from the pooled sample, 0.5 mL of phenol and then immediately 2.5 mL of sulfuric acid. The solution was homogenized and left to react for 15 min at room temperature. The reading was performed at 490 nm in ELISA reader. The strains were compared regarding polysaccharides and protein matrices using analysis of variance (ANOVA) and Tukey test. At confocal microscopy the strains were incubated with the tested polypropylene material for 24 h. The materials were washed with sterile phosphate buffer and stained with propidium iodide. The reading was performed using a Laser Scanning Confocal Microscope (Zeiss 710) with laser excitation (488 nm) and 580-680 nm emission filters for propidium iodide (red marking). All strains displayed strong adherence on microplate and the amount of polysaccharides in biofilm was not statistically different among the studied strains, but the amount of protein was significantly different in strain 4 (P &gt; 0.5). The confocal microscopy showed the adherence of the Leptospira spp. strains to polypropylene material after washing.Discussion: Biofilm production plays an important role in the maintenance of a chronic infection by Leptospira interrogans with renal colonization. The exopolysaccharide (EPS) has various functions, such as checking insolubility in water; giving the three-dimensional conformation of the biofilm; protecting cells from physical (mechanical action, irradiation and temperature variations), chemical.

Cold Plasma Inactivation of Escherichia coli O157:H7 Biofilms

Pathogenic bacteria associated with biofilms on foods and food contact surfaces are a challenge to inactivate with conventional sanitizers. Cold plasma is a novel nonthermal process with potential efficacy against these pathogens. Biofilms of Escherichia coli O157:H7 were grown for 24, 48 or 72 h on glass slides and exposed to atmospheric cold plasma, 23 kHz to 48 kHz, for 5, 10 or 15 s. Distance from emitter to biofilms was 5 cm or 7.5 cm. The temperature of the process was established using infrared digital imagery. Cold plasma at 5 cm reduced biofilms by up to 1.41, 2.57 or 3.29 log cfu ml-1 for 5, 10 or 15 s, respectively. Cold plasma at 7.5 cm had reduced maximal efficacy at 5 s and 10 s (0.96 and 2.13 log cfu ml-1, respectively), but was unchanged for 15 s (3.29 log cfu ml-1). Biofilm age was not significant factor influenceing cold plasma efficacy. All treatments were confirmed to be non-thermal. Cold plasma can be a sanitizing tool for food contact surfaces.

A Multi-scale Biophysical Approach to Develop Structure-Property Relationships in Oral Biofilms

Over the last 5–10 years, optical coherence tomography (OCT) and atomic force microscopy (AFM) have been individually applied to monitor the morphological and mechanical properties of various single-species biofilms respectively. This investigation looked to combine OCT and AFM as a multi-scale approach to understand the role sucrose concentration and age play in the morphological and mechanical properties of oral, microcosm biofilms, in-vitro. Biofilms with low (0.1% w/v) and high (5% w/v) sucrose concentrations were grown on hydroxyapatite (HAP) discs from pooled human saliva and incubated for 3 and 5 days. Distinct mesoscale features of biofilms such as regions of low and high extracellular polymeric substances (EPS) were identified through observations made by OCT. Mechanical analysis revealed increasing sucrose concentration decreased Young’s modulus and increased cantilever adhesion (p < 0.0001), relative to the biofilm. Increasing age was found to decrease adhesion only (p < 0.0001). This was due to mechanical interactions between the indenter and the biofilm increasing as a function of increased EPS content, due to increasing sucrose. An expected decrease in EPS cantilever contact decreased adhesion due to bacteria proliferation with biofilm age. The application OCT and AFM revealed new structure-property relationships in oral biofilms, unattainable if the techniques were used independently.

Antibiofilm Effect of DNase against Single and Mixed Species Biofilm.

Biofilms are aggregates of microorganisms that coexist in socially coordinated micro-niche in a self-produced polymeric matrix on pre-conditioned surfaces. The biofilm matrix reduces the efficacy of antibiofilm strategies. DNase degrades the extracellular DNA (e-DNA) present in the matrix, rendering the matrix weak and susceptible to antimicrobials. In the current study, the effect of DNase I was evaluated during biofilm formation (pre-treatment), on preformed biofilms (post-treatment) and both (dual treatment). The DNase I pre-treatment was optimized for P. aeruginosa PAO1 (model biofilm organism) at 10 µg/mL and post-treatment at 10 µg/mL with 15 min of contact duration. Inclusion of Mg2+ alongside DNase I post-treatment resulted in 90% reduction in biofilm within only 5 min of contact time (irrespective of age of biofilm). On extension of these findings, DNase I was found to be less effective against mixed species biofilm than individual biofilms. DNase I can be used as potent antibiofilm agent and with further optimization can be effectively used for biofilm prevention and reduction in situ.

Anti-bacterial and anti-biofilm activity of commercial organic acid products against Salmonella enterica isolates recovered from an egg farm environment

ABSTRACTThis study evaluated the antibacterial activity of commercially available organic acid water additives against Salmonella enterica isolates and examined the susceptibility of Salmonella Typhimurium biofilms to these products. Three commercial organic acid products (A, B, and C) were evaluated for minimum inhibitory and bactericidal concentrations against isolates of S. enterica serovars. Three- and five-day-old S. Typhimurium biofilms were formed at 22 ± 2°C using an MBEC™ assay system and exposed for 30 min or 90 min at 0.2% and 0.4% concentrations. No significant difference among serovars for inhibitory and bactericidal concentrations was detected. Two products (A and C) significantly reduced viable cells from biofilms of both ages in a dose- and time-dependent manner. Increased biofilm age did not enhance resistance towards organic acid treatments. None of the products completely eliminated biofilm cells at any concentration or exposure time. Product composition, exposure time, and concentration of organic acid products were important factors in reducing viable biofilm cells. This study has expanded our understanding about the susceptibility of Salmonella biofilms to commercial organic acid products. These findings have implications in the usage, development, and optimization of organic acid products.

Modeling Reveals the Role of Aging and Glucose Uptake Impairment in L1A1 Listeria monocytogenes Biofilm Life Cycle.

Listeria monocytogenes is a food-borne pathogen that can persist in food processing plants by forming biofilms on abiotic surfaces. The benefits that bacteria can gain from living in a biofilm, i.e., protection from environmental factors and tolerance to biocides, have been linked to the biofilm structure. Different L. monocytogenes strains build biofilms with diverse structures, and the underlying mechanisms for that diversity are not yet fully known. This work combines quantitative image analysis, cell counts, nutrient uptake data and mathematical modeling to provide a mechanistic insight into the dynamics of the structure of biofilms formed by L. monocytogenes L1A1 (serotype 1/2a) strain. Confocal laser scanning microscopy (CLSM) and quantitative image analysis were used to characterize the structure of L1A1 biofilms throughout time. L1A1 forms flat, thick structures; damaged or dead cells start appearing early in deep layers of the biofilm and rapidly and massively loss biomass after 4 days. We proposed several reaction-diffusion models to explain the system dynamics. Model candidates describe biomass and nutrients evolution including mechanisms of growth and cell spreading, nutrients diffusion and uptake and biofilm decay. Data fitting was used to estimate unknown model parameters and to choose the most appropriate candidate model. Remarkably, standard reaction-diffusion models could not describe the biofilm dynamics. The selected model reveals that biofilm aging and glucose impaired uptake play a critical role in L1A1 L. monocytogenes biofilm life cycle.

Biofilm forming ability of Sphingomonas paucimobilis isolated from community drinking water systems on plumbing materials used in water distribution.

Sphingomonas paucimobilis, an oligotroph, is well recognized for its potential for biofilm formation. The present study explored the biofilm forming ability of a strain isolated from municipal drinking water on plumbing materials. The intensity of biofilm formation of this strain on different plumbing materials was examined by using 1 × 1 cm2 pieces of six different pipe materials, i.e. polyvinyl chloride (PVC), polypropylene (PP), polyethylene (PE), aluminium (Al), copper (Cu) and rubber (R) and observing by staining with the chemical chromophore, Calcofluor. To understand whether biofilm formation occurs under flow through conditions, a laboratory-scale simulated distribution system, comprised of the above materials was fabricated. Biofilm samples were collected from the designed system at different biofilm ages (10, 40 and 90 hours old) and enumerated. The results indicated that the biofilm formation occurred on all plumbing materials with Cu and R as exceptions. The intensity of biofilm formation was found to be maximum on PVC followed by PP and PE. We also demonstrated the chemical chromophore (Calcofluor) successfully for rapid and easy visual detection of biofilms, validated by scanning electron microscope (SEM) analysis of the plumbing materials. Chlorination has little effect in preventing biofilm development.

Extracellular DNA Contributes to Dental Biofilm Stability

Extracellular DNA (eDNA) is a major matrix component of many bacterial biofilms. While the presence of eDNA and its role in biofilm stability have been demonstrated for several laboratory biofilms of oral bacteria, there is no data available on the presence and function of eDNA in in vivo grown dental biofilms. This study aimed to determine whether eDNA was part of the matrix in biofilms grown in situ in the absence of sucrose and whether treatment with DNase dispersed biofilms grown for 2.5, 5, 7.5, 16.5, or 24 h. Three hundred biofilms from 10 study participants were collected and treated with either DNase or heat-inactivated DNase for 1 h. The bacterial biovolume was determined with digital image analysis. Staining with TOTO®-1 allowed visualization of eDNA both on bacterial cell surfaces and, with a cloud-like appearance, in the intercellular space. DNase treatment strongly reduced the amount of biofilm in very early stages of growth (up to 7.5 h), but the treatment effect decreased with increasing biofilm age. This study proves the involvement of eDNA in dental biofilm formation and its importance for biofilm stability in the earliest stages. Further research is required to uncover the interplay of eDNA and other matrix components and to explore the therapeutic potential of DNase treatment for biofilm control.

A reproducible microcosm biofilm model of subgingival microbial communities.

To develop a reproducible subgingival microcosm biofilm model. Subgingival plaque samples were collected from four deep pockets (probing pocket depth ≥6mm) in each of seven patients with periodontitis and from shallow pockets (probing pocket depth ≤3mm) in two periodontally healthy donors. An active attachment model and a peptone medium (Thompson et. al., Appl Environ Microbiol 2015;81:8307-8314) supplemented with 30% serum was used. Biofilms were harvested at 2 and 4weeks. DNA of dead cells was blocked for amplification by propidium monoazide treatment. Composition was analyzed using 16S rRNA gene amplicon pyrosequencing. Similarities between the biofilm samples were assessed by non-metric multidimensional scaling using the Bray-Curtis similarity index and similarity percentage analysis. Data from duplicate experiments, different biofilm sources and different biofilm age were compared. The non-metric multidimensional scaling revealed a strong clustering by the inoculum source, the donor and their periodontal status. Statistically significant differences were found between the sources of inoculum (P=.0001) and biofilm age (P=.0016). Furthermore, periodontitis biofilms (P) were distinct in composition from health-derived biofilms (H) by genera: Porphyromonas (P=19%; H=0%), Filifactor (P=10%; H=0%), Anaeroglobus (P=3%; H=0%), Phocaeicola (P=1.5%; H=0%), Parvimonas (P=19%; H=14%), Fusobacterium (P=2%; H=26%), Peptostreptococcus (P=20%; H=30%), Veillonella (P=7%; H=8%) and 57 other genera. Similarity distances (Bray-Curtis) (mean 0.73, SD 0.15) and the Shannon diversity index (mean 2, SD 0.2) revealed no differences between duplicate experiments (P=.121). This biofilm model allows reproducible production of complex subgingival microbial communities.

Assessment of herbal drugs for promising anti-Candida activity

BackgroundMicrobial infections are diverse and cause serious human diseases. Candida albicans infections are serious healthcare-related infections that are complicated by its morphological switching from yeast to hyphae, resistant biofilm formation and mixed infections with bacteria. Due to the increase in drug resistance to currently used antimicrobial agents and the presence of undesirable side effects, the need for safe and effective novel therapies is important. Compounds derived from plants are known for their medicinal properties including antimicrobial activities. The purpose of the study was to compare and evaluate the anti-Candida activities of several medicinal plants in order for the selection of a herbal drug for human use as effective antimicrobial. The selection was taking into considerations two important parameters; parameters related to the selected drug including activity, stability, solubility and toxicity and parameters related to the pathogen including its different dynamic growth and its accompanied secondary bacterial infections.MethodsSeven different plants including Avicennia marina (Qurm), Fagonia indica (Shoka’a), Lawsania inermis (Henna), Portulaca oleracea (Baq’lah), Salvadora persica (Souwak), Ziziphus spina- Christi (Sidr) and Asphodelus tenuifolius (Kufer) were ground and extracted with ethanol. The ethanol extracts were evaporated and the residual extract dissolved in water prior to testing against Candida albicans in its different morphologies. The antibacterial and cytotoxic effects of the plants extracts were also tested.ResultsOut of the seven tested plants, L. inermis and P. oleracea showed significant anti-Candida activity with MIC ~10 μg/mL. Furthermore, both plant extracts were able to inhibit C. albicans growth at its dynamic growth phases including biofilm formation and age resistance. Accompanied secondary bacterial infections can complicate Candida pathogenesis. L. inermis and P. oleracea extracts showed effective antibacterial activities against S. aureus, P. aeruginosa, E. coli, and the multidrug resistant (MDR) A. baumannii and Klebsiella pneumoniae. Both extracts showed no toxicity when measured at their MIC on human erythrocytes.ConclusionThe results from this study suggested that L. inermis and P. oleracea extracts and/or their chemicals are likely to be promising drugs for human use against C. albicans and MDR bacteria.

Strain-Specific Impact of Fusobacterium nucleatum on Neutrophil Function.

Neutrophil function is critical for initiation and progression of infecto-inflammatory diseases. Key quorum-sensing plaque bacteria, such as Fusobacterium nucleatum, act as bridging species between early and late colonizer pathogens, such as Porphyromonas gingivalis, as the biofilm ages and periodontal inflammation increases. This study is designed to determine impact of different F. nucleatum strains on neutrophil function. Cells of human promyelocytic leukemia cell line-60 were differentiated into neutrophil-like cells and cultured with F. nucleatum strains of subspecies (ssp.) nucleatum ATCC 25586, ssp. polymorphum ATCC 10953, and ssp. vincentii ATCC 49256. Neutrophil phagocytosis of F. nucleatum strains and neutrophil apoptosis were analyzed by flow cytometry. Superoxide generation was measured by cytochrome C reduction in the presence and absence of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) (1 μM) stimulation. Proinflammatory cytokine release was determined after 2, 6, and 24 hours of culture in the presence/absence of different F. nucleatum strains. Expression of Toll-like receptor (TLR)2, TLR4, and nuclear factor (NF)-kappa B mRNA levels were analyzed using real-time quantitative polymerase chain reaction. Each experiment was repeated at least three times in triplicate. Data were analyzed using analysis of variance followed by post hoc Bonferroni correction. All strains of F. nucleatum significantly increased phagocytic capacity of neutrophils. Neutrophil phagocytosis of F. nucleatum ssp. polymorphum was significantly greater than that of F. nucleatum ssp. vincentii and ssp. nucleatum (P <0.001). F. nucleatum ssp. nucleatum and ssp. polymorphum significantly blocked fMLP-induced superoxide generation (P <0.001). Although F. nucleatum vincentii also reduced superoxide generation (25%), the impact was not as strong as that of ssp. nucleatum (83%) and ssp. polymorphum (100%). All F. nucleatum strains stimulated significant increase in neutrophil apoptosis compared with control (P <0.001) and significantly increased expression of NF-κB mRNA in neutrophils (P <0.05). Levels of interleukin-8 and tumor necrosis factor-α produced by neutrophils were significantly increased in all F. nucleatum groups compared with control (P <0.001). These findings suggest that different strains of F. nucleatum impact neutrophil function in different ways. Two of three subspecies blocked neutrophil superoxide generation in response to a secondary stimulus, preventing oxidative killing by neutrophils. The direct role of bridging species in pathogenesis of periodontitis may be greater than previously suspected in which they create a favorable environment for pathogenic transition of the dental ecosystem.