Changes in biofilm structure during the colonization of chalcopyrite by Acidithiobacillus thiooxidans

Biofilms of Acidithiobacillus thiooxidans were grown on the surface of massive chalcopyrite electrodes (MCE) where different secondary sulfur phases were previously formed by potentiostatic oxidation of MCE at 0.780?≤?E _an?≤?0.965 V (electrooxidized MCE, eMCE). The formation of mainly S⁰ and minor amounts of CuS and S _n ^2? were detected on eMCEs. The eMCEs were incubated with A. thiooxidans cells for 1, 12, 24, 48, and 120 h in order to temporally monitor changes in eMCE's secondary phases, biofilm structure, and extracellular polymeric substance (EPS) composition (lipids, proteins, and polysaccharides) using microscopic, spectroscopic, electrochemical, and biochemical techniques. The results show significant cell attachments with stratified biofilm structure since the first hour of incubation and EPS composition changes, the most important being production after 48–120 h when the highest amount of lipids and proteins were registered. During 120 h, periodic oxidation/formation of S⁰/S _n ^2? was recorded on biooxidized eMCEs, until a stable CuS composition was formed. In contrast, no evidence of CuS formation was observed on the eMCEs of the abiotic control, confirming that CuS formation results from microbial activity. The surface transformation of eMCE induces a structural transformation of the biofilm, evolving directly to a multilayered biofilm with more hydrophobic EPS and proteins after 120 h. Our results suggest that A. thiooxidans responded to the spatial and temporal distribution and chemical reactivity of the S _n ^2?/S⁰/CuS phases throughout 120 h. These results suggested a strong correlation between surface speciation, hydrophobic domains in EPS, and biofilm organization during chalcopyrite biooxidation by A. thiooxidans.     

Effects of Contact Time, Pressure, Percent Relative Humidity (%RH), and Material Type on Listeria Biofilm Adhesive Strength at a Cellular Level Using Atomic Force Microscopy (AFM)

Whereas the transfer of Listeria from surfaces to foods and vice versa has been well documented, little is known about the mechanism of bacterial transfer. The objective of this work is to gain a better understanding of the forces involved in listerial biofilms adhesion using atomic force microscopy (AFM). L. monocytogenes Scott A was grown as biofilms on stainless steel surfaces by inoculating stainless steel coupons with Listeria and incubating the coupons for 48 h at 32 °C with a diluted 1:20 tryptic soy broth. After growth, biofilms were equilibrated over saturated salt solutions at a constant relative humidity (%RH) before measurement of adhesion forces using AFM. The effects of contact time, loading force, and biofilm relative humidity (%RH) suggested that neither contact time, loading force nor biofilm %RH had a significant effect on biofilm adhesiveness at a cellular level (P > 0.05). In a second set of experiments, the influence of material type on biofilm adhesiveness was evaluated using two different colloidal probes (SiO₂ and polyethylene). Results showed that the maximum pull-off force and retraction work needed to retract the cantilever for glass (−85.42 nN and 1.610⁻¹⁵ J, respectively) were significantly lower than those of polyethylene (−113.38 nN and 2.7 × 10^–15 J, respectively; P < 0.001). The results of this study suggest that Listeria biofilms adhere more strongly to hydrophobic surfaces than hydrophilic surfaces when measured at a cellular level. These results provide important insights that could lead to new ways to remediate and avoid listerial biofilm formation in the food industry.     

Influence of the sulfur species reactivity on biofilm conformation during pyrite colonization by Acidithiobacillus thiooxidans

Massive pyrite (FeS₂) electrodes were potentiostatically modified by means of variable oxidation pulse to induce formation of diverse surface sulfur species (S _n ^2?, S⁰). The evolution of reactivity of the resulting surfaces considers transition from passive (e.g., Fe_1?x S₂) to active sulfur species (e.g., Fe_1?x S_2?y , S⁰). Selected modified pyrite surfaces were incubated with cells of sulfur-oxidizing Acidithiobacillus thiooxidans for 24 h in a specific culture medium (pH 2). Abiotic control experiments were also performed to compare chemical and biological oxidation. After incubation, the attached cells density and their exopolysaccharides were analyzed by confocal laser scanning microscopy (CLMS) and atomic force microscopy (AFM) on bio-oxidized surfaces; additionally, S _n ^2?/S⁰ speciation was carried out on bio-oxidized and abiotic pyrite surfaces using Raman spectroscopy. Our results indicate an important correlation between the evolution of S _n ^2?/S⁰ surface species ratio and biofilm formation. Hence, pyrite surfaces with mainly passive-sulfur species were less colonized by A. thiooxidans as compared to surfaces with active sulfur species. These results provide knowledge that may contribute to establishing interfacial conditions that enhance or delay metal sulfide (MS) dissolution, as a function of the biofilm formed by sulfur-oxidizing bacteria.     

Degradation of massive pyrite: Physical,chemical, and bacterial effects

Massive pyrite was shown to produce soluble iron, hydrogen, and sulfate ions on exposure to air and water. The rate of this process was directly proportional to the surface area of the mineral; it was unaffected by a drop in the pH and the presence of the ferrous and sulfate ions formed. Cupic ion had no effect but ferric ion accelerated pyrite degradation until all the ferric ion was consumed, in accordance with FeS₂ + 2Fe³⁺ —>‐3Fe²⁺ + 2S°. Thiobacillus ferrooxidans increased pyrite degradation considerably; the presence of Thiobacillus thiooxidans had no influence on pyrite degradation.     

Influence of the surface speciation on biofilm attachment to chalcopyrite by Acidithiobacillus thiooxidans

Surfaces of massive chalcopyrite (CuFeS₂) electrodes were modified by applying variable oxidation potential pulses under growth media in order to induce the formation of different secondary phases (e.g., copper-rich polysulfides, S _n ^2?; elemental sulfur, S⁰; and covellite, CuS). The evolution of reactivity (oxidation capacity) of the resulting chalcopyrite surfaces considers a transition from passive or inactive (containing CuS and S _n ^2?) to active (containing increasing amounts of S⁰) phases. Modified surfaces were incubated with cells of sulfur-oxidizing bacteria (Acidithiobacillus thiooxidans) for 24 h in a specific culture medium (pH 2). Abiotic control experiments were also performed to compare chemical and biological oxidation. After incubation, the density of cells attached to chalcopyrite surfaces, the structure of the formed biofilm, and their exopolysaccharides and nucleic acids were analyzed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy coupled to dispersive X-ray analysis (SEM-EDS). Additionally, CuS and S _n ^2?/S⁰ speciation, as well as secondary phase evolution, was carried out on biooxidized and abiotic chalcopyrite surfaces using Raman spectroscopy and SEM-EDS. Our results indicate that oxidized chalcopyrite surfaces initially containing inactive S _n ^2? and S _n ^2?/CuS phases were less colonized by A. thiooxidans as compared with surfaces containing active phases (mainly S⁰). Furthermore, it was observed that cells were partially covered by CuS and S⁰ phases during biooxidation, especially at highly oxidized chalcopyrite surfaces, suggesting the innocuous effect of CuS phases during A. thiooxidans performance. These results may contribute to understanding the effect of the concomitant formation of refractory secondary phases (as CuS and inactive S _n ^2?) during the biooxidation of chalcopyrite by sulfur-oxidizing microorganisms in bioleaching systems.     

Use of lectins to in situ visualize glycoconjugates of extracellular polymeric substances in acidophilic archaeal biofilms

Biofilm formation and the production of extracellular polymeric substances (EPS) by meso‐ and thermoacidophilic metal‐oxidizing archaea on relevant substrates have been studied to a limited extent. In order to investigate glycoconjugates, a major part of the EPS, during biofilm formation/bioleaching by archaea on pyrite, a screening with 75 commercially available lectins by fluorescence lectin‐binding analysis (FLBA) has been performed. Three representative archaeal species, Ferroplasma acidiphilum DSM 28986, Sulfolobus metallicus DSM 6482^T and a novel isolate Acidianus sp. DSM 29099 were used. In addition, Acidianus sp. DSM 29099 biofilms on elemental sulfur were studied. The results of FLBA indicate (i) 22 lectins bound to archaeal biofilms on pyrite and 21 lectins were binding to Acidianus sp. DSM 29099 biofilms on elemental sulfur; (ii) major binding patterns, e.g. tightly bound EPS and loosely bound EPS, were detected on both substrates; (iii) the three archaeal species produced various EPS glycoconjugates on pyrite surfaces. Additionally, the substratum induced different EPS glycoconjugates and biofilm structures of cells of Acidianus sp. DSM 29099. Our data provide new insights into interactions between acidophilic archaea on relevant surfaces and also indicate that FLBA is a valuable tool for in situ investigations on archaeal biofilms.     

Changes in biooxidation mechanism and transient biofilm characteristics by As(V) during arsenopyrite colonization with <Emphasis Type="Italic">Acidithiobacillus thiooxidans</Emphasis>

Chemical and surface analyses are carried out using Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM–EDS), atomic force microscopy (AFM), confocal laser scanning microscopy (CLSM), glow discharge spectroscopy (GDS) and extracellular surface protein quantification to thoroughly investigate the effect of supplementary As(V) during biooxidation of arsenopyrite by Acidithiobacillus thiooxidans. It is revealed that arsenic can enhance bacterial reactions during bioleaching, which can strongly influence its mobility. Biofilms occur as compact-flattened microcolonies, being progressively covered by a significant amount of secondary compounds (S _n ^2- , S⁰, pyrite-like). Biooxidation mechanism is modified in the presence of supplementary As(V), as indicated by spectroscopic and microscopic studies. GDS confirms significant variations between abiotic control and biooxidized arsenopyrite in terms of surface reactivity and amount of secondary compounds with and without As(V) (i.e. 6 μm depth). CLSM and protein analyses indicate a rapid modification in biofilm from hydrophilic to hydrophobic character (i.e. 1–12 h), in spite of the decrease in extracellular surface proteins in the presence of supplementary As(V) (i.e. stressed biofilms).     

Sulfur Transformation in Microbially Mediated Pyrite Oxidation by Acidithiobacillus ferrooxidans: Insights From X-ray Photoelectron Spectroscopy-Based Quantitative Depth Profiling

The oxidation of pyrite and other sulfides is responsible for the generation of acid mine drainage and acid rock drainage, which leads to further contamination of soil and water. In these processes, microbial oxidation usually prevails over chemical oxidation. To determine the mechanism of microbial oxidation of pyrite, the interaction of Acidithiobacillus ferrooxidans with pyrite was comprehensively studied, and the sulfur transformation in the interaction was disclosed using X-ray photoelectron spectroscopy (XPS) depth profiling. Abundant bacterial cells attach to pyrite surface and form biofilms, which greatly enhances surface corrosion and results in two types of etching pits: bacteria-driven rod-shaped and chemically driven hexagonal etching pits. The details of XPS depth profiles on a reacted pyrite surface reveal that the surface sulfur was first oxidized into elemental sulfur. Thereafter, elemental sulfur was further oxidized to intermediate species S₂O₃^2?, SO₃^2?, and ultimately to SO₄^2?. The oxidation sequence of sulfur is S₂^2?/S^2?→S_n^2?, S⁰→SO₃^2?, and S₂O₃^2?→SO₄^2?. Meanwhile, the remnant ferrous iron in the surface layer was released into solution and subsequently oxidized into Fe³⁺ by A. ferrooxidans and dissolved oxygen, which in turn enhanced the oxidation of sulfur. Fe³⁺, sulfate, and other ions (e.g., K⁺, Na⁺, NH₄⁺) in the solution precipitated as jarosite, hydroniumjarosite, and ammoniojarosite. On the basis of results, a three-staged model is proposed to interpret the kinetics of microbial oxidation of pyrite.     

Transformation of iron sulfide to greigite by nitrite produced by oil field bacteria

Nitrate, injected into oil fields, can oxidize sulfide formed by sulfate-reducing bacteria (SRB) through the action of nitrate-reducing sulfide-oxidizing bacteria (NR-SOB). When reservoir rock contains siderite (FeCO₃), the sulfide formed is immobilized as iron sulfide minerals, e.g. mackinawite (FeS). The aim of our study was to determine the extent to which oil field NR-SOB can oxidize or transform FeS. Because no NR-SOB capable of growth with FeS were isolated, the well-characterized oil field isolate Sulfurimonas sp. strain CVO was used. When strain CVO was presented with a mixture of chemically formed FeS and dissolved sulfide (HS⁻), it only oxidized the HS⁻. The FeS remained acid soluble and non-magnetic indicating that it was not transformed. In contrast, when the FeS was formed by adding FeCl₂ to a culture of SRB which gradually produced sulfide, precipitating FeS, and to which strain CVO and nitrate were subsequently added, transformation of the FeS to a magnetic, less acid-soluble form was observed. X-ray diffraction and energy-dispersive spectrometry indicated the transformed mineral to be greigite (Fe₃S₄). Addition of nitrite to cultures of SRB, containing microbially formed FeS, was similarly effective. Nitrite reacts chemically with HS⁻ to form polysulfide and sulfur (S⁰), which then transforms SRB-formed FeS to greigite, possibly via a sulfur addition pathway (3FeS + S⁰ → Fe₃S₄). Further chemical transformation to pyrite (FeS₂) is expected at higher temperatures (>60°C). Hence, nitrate injection into oil fields may lead to NR-SOB-mediated and chemical mineral transformations, increasing the sulfide-binding capacity of reservoir rock. Because of mineral volume decreases, these transformations may also increase reservoir injectivity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Studies on transfer processes in mixing vessels: effect of particles on gas-liquid mass transfer using modified Rushton turbine agitators

The influence of carbon dioxide concentration in liquid medium on elemental sulphur oxidation by Thiobacillus thiooxidans bacteria presented in this paper can be divided into 3 differing relationships. First relationship shows increase of sulphur biooxidation rate with increase of carbon dioxide concentration in liquid medium. Second one shows decrease of S⁰ oxidation rate with increase of CO₂ concentration in nutrient and in the third relationship there is no influence of carbon dioxide concentration on sulphur oxidation by Thiobacillus thiooxidans bacteria. The influence of carbon dioxide concentration in liquid nutrient on alive bacteria concentration in liquid medium is similar to those described above.     

The influence of topographic microstructures on the initial adhesion of L929 fibroblasts studied by single-cell force spectroscopy

Single-cell force spectroscopy was used to investigate the initial adhesion of L929 fibroblasts onto periodically grooved titanium microstructures (height ~6 μm, groove width 20 μm). The position-dependent local adhesion strength of the cells was correlated with their rheological behavior. Spherical cells exhibited a significantly lower Young’s modulus (<1 kPa) than that reported for spread cells, and their elastic properties can roughly be explained by the Hertz model for an elastic sphere. While in contact with the planar regions of the substrate, the cells started to adapt their shape through slight ventral flattening. The process was found to be independent of the applied contact force for values between 100 and 1,000 pN. The degree of flattening correlated with the adhesion strength during the first 60 s. Adhesion strength can be described by fast exponential kinetics as C₁ [ 1 - exp( - C₂ ·t ) ] C_{1} \left[ {1 - \exp \left( { - C_{2} \cdot t} \right)} \right] with C ₁ = 2.34 ± 0.19 nN and C ₂ = 0.09 ± 0.02 s⁻¹. A significant drop in the adhesion strength of up to 50% was found near the groove edges. The effect can be interpreted by the geometric decrease of the contact area, which indicates the inability of the fibroblasts to adapt to the shape of the substrate. Our results explain the role of the substrate’s topography in contact guidance and suggest that rheological cell properties must be considered in cell adhesion modeling.     

Bioleaching of chalcopyrite concentrate using Leptospirillum ferriphilum, Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans in a continuous bubble column reactor

To estimate the bioleaching performance of chalcopyrite for various hydraulic residence times (HRTs), laboratory-scale bioleaching of chalcopyrite concentrate was carried out in a continuous bubble column reactor with three different HRTs of 120, 80 and 40 h, respectively. An extraction rate and ratio of 0.578 g Cu l⁻¹ h⁻¹ and 39.7%, respectively, were achieved for an HRT of 80 h at a solids concentration of 10% (w/v). Lower bioleaching performances than this were obtained for a longer HRT of 120 h and a shorter HRT of 40 h. In addition, there was obvious competition between Leptospirillum ferriphilum and Acidithiobacillus ferrooxidans to oxidize ferrous iron, causing large compositional differences between the microbial communitys obtained for the different HRTs. Leptospirillum ferriphilum and Acidithiobacillus thiooxidans were found to be the dominant microbes for the longer HRT (120 h). Acidithiobacillus ferrooxidans became the dominant species when the HRT was decreased. The proportion of Acidithiobacillus thiooxidans was comparatively constant in the microbial community throughout the three process stages.     

Optimization of a chemically defined medium for mycelial growth and polysaccharide production by medicinal mushroom <Emphasis Type="Italic">Phellinus igniarius</Emphasis>

A chemically defined medium for mycelial growth and exopolysaccharide (EPS) production by submerged culture of Phellinus igniarius was investigated. The mainly defined medium compositions were optimized by using orthogonal matrix method. The optimal defined medium (per liter) was 40.0 g glucose, 4.0 g. glutamic acid, 4.0 g (NH₄)₂SO₄, and initial pH 6.0. Under the optimal medium, the maximal mycelial biomass and EPS production were 12.33 ± 0.89 and 1.21 ± 0.08 g l⁻¹ at 192 h in shake flask, while the maximal mycelial biomass and EPS production reached 13.86 ± 0.52 and 1.92 ± 0.07 g l⁻¹ at 168 h in 3 l fermenter, respectively. The molecular weights (g mol⁻¹) of four fractions isolated from EPS by gel permeation were about 6.4 × 10⁶, 3.3 × 10⁵, 2.7 × 10⁵ and 2.9 × 10³. This study should be widely applied to other secondary metabolites production from higher fungus in a chemically defined medium and quantitative regulation of the metabolic flux in polysaccharide biosynthesis.     

Relationship between the extracellular polymeric substances and surface characteristics of Rhodopseudomonas acidophila

The relationship between the extracellular polymeric substances (EPS) and surface characteristics of Rhodopseudomonas acidophila in its different growth phases was established. The equilibrium constant of partition (K _par) and the Gibbs energies of partition (△G _par) between hexadecane and aqueous phases were also calculated according to the microbial adhesion to hexadecane (MATH) testing. The EPS content decreased with cultivation time at the logarithmic phase, but kept almost unchanged around 22.9 mg g⁻¹ dry cell at the stationary phase. The EPS production of R. acidophila had a significant effect on its surface characteristics. The relative hydrophobicity and the K _par values of R. acidophila before EPS extraction were both lower than those after extraction. Both EPS content and ratio of proteins to carbohydrates had a negative effect on the water contact angle of the bacterium, but had a positive influence on the bacterial surface free energy and its polar component. On the other hand, the EPS were not related with MATH% or the Gibbs energy of partition between hexadecane and aqueous phase.     

Curdlan-like exopolysaccharide production by <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> UNP3 during growth on hydrocarbon substrates

Cellulomonas flavigena UNP3, a natural isolate from vegetable oil contaminated soil sample has been studied for growth associated exopolysaccharide (EPS) production during growth on glucose, groundnut oil and naphthalene. The EPS showed matrix formation surrounding the cells during scanning electron microscopy. Cell surface hydrophobicity and emulsifying activity studies confirmed the role of EPS as bioemulsifier. Emulsifying activity was found to increase with time (0.2 U/mg for 10 min to 0.27 U/mg for 30 min). Emulsification index, E₂₄ value increased with the increase in EPS concentration. Degradation of polyaromatic hydrocarbons was confirmed using gas chromatography analysis. FTIR analysis showed presence of characteristic absorbance at 895.10 cm⁻¹ for β-configuration of glucan. NMR studies also revealed EPS produced by C. flavigena UNP3 as a linear β-1, 3-d-glucan, and a curdlan like polysaccharide.     

Structural organization and dynamics of exopolysaccharide matrix and microcolonies formation by Streptococcus mutans in biofilms

Aims: To investigate the structural organization and dynamics of exopolysaccharides (EPS) matrix and microcolonies formation by Streptococcus mutans during the biofilm development process. Methods and Results: Biofilms of Strep. mutans were formed on saliva‐coated hydroxyapatite (sHA) discs in the presence of glucose or sucrose (alone or mixed with starch). At specific time points, biofilms were subjected to confocal fluorescence imaging and computational analysis. EPS matrix was steadily formed on sHA surface in the presence of sucrose during the first 8 h followed by a threefold biomass increase between 8 and 30 h of biofilm development. The initial formation and further development of three‐dimensional microcolony structure occurred concomitantly with EPS matrix synthesis. Tridimensional renderings showed EPS closely associated with microcolonies throughout the biofilm development process forming four distinct domains (i) between sHA surface and microcolonies, (ii) within, (iii) covering and (iv) filling the spaces between microcolonies. The combination of starch and sucrose resulted in rapid formation of elevated amounts of EPS matrix and faster assembly of microcolonies by Strep. mutans, which altered their structural organization and susceptibility of the biofilm to acid killing (vs sucrose‐grown biofilms; P < 0·05). Conclusions: Our data indicate that EPS modulate the development, sequence of assembly and spatial distribution of microcolonies by Strep. mutans. Significance and Impact of the Study: Simultaneous visualization and analysis of EPS matrix and microcolonies provide a more precise examination of the structural organization of biofilms than labelling bacteria alone, which could be a useful approach to elucidate the exact mechanisms by which Strep. mutans influences oral biofilm formation and possibly identify novel targets for effective antibiofilm therapies.     

Bioelectrochemical system for the biooxidation of a chalcopyrite concentrate by acidophilic bacteria coupled to energy current generation and cathodic copper recovery

Objectives

To develop a bioelectrochemical system (BES) to couple the biooxidation of chalcopyrite (CuFeS₂), bioelectrogenesis, and the cathodic Cu²⁺ reduction, bioanodes of acidophilic (pH < 2) and aerobic chemolithoautotrophic bacteria Acidithiobacillus thiooxidans (sulfur oxidizing) and Leptospirillum sp. (Fe²⁺ oxidizing) were used.

Results

CuFeS₂ biooxidation increases the charge transfer from the media due to the bioleaching of Cu and Fe. The biofilm on a graphite bar endows a more electropositive (anodic) character to the bioelectrode. By adding the bioleachate generated by both bacteria into the anodic chamber, the acidic bioleachate provides the faradaic intensity. The maximum current density was 0.86 ± 19 mA cm^?2 due to the low potential of the BES of 0.18 ± 0.02 V. Such low potential was sufficient for the cathodic deposit of Cu²⁺.

Conclusions

This work demonstrates a proof of concept for energy savings for mining industries: bioanodes of A. thiooxidans and Leptospirillum sp. are electroactive during the biooxidation of CuFeS₂.

     

Influence of stand density on soil CO2 efflux for a Pinus densiflora forest in Korea

We investigated the influence of stand density [938 tree ha⁻¹ for high stand density (HD), 600 tree ha⁻¹ for medium stand density (MD), and 375 tree ha⁻¹ for low stand density (LD)] on soil CO₂ efflux (R _S) in a 70-year-old natural Pinus densiflora S. et Z. forest in central Korea. Concurrent with R _S measurements, we measured litterfall, total belowground carbon allocation (TBCA), leaf area index (LAI), soil temperature (ST), soil water content (SWC), and soil nitrogen (N) concentration over a 2-year period. The R _S (t C ha⁻¹ year⁻¹) and leaf litterfall (t C ha⁻¹ year⁻¹) values varied with stand density: 6.21 and 2.03 for HD, 7.45 and 2.37 for MD, and 6.96 and 2.23 for LD, respectively. In addition, R _S was correlated with ST (R ² = 0.77–0.80, P < 0.001) and SWC (R ² = 0.31–0.35, P < 0.001). It appeared that stand density influenced R _S via changes in leaf litterfall, LAI and SWC. Leaf litterfall (R ² = 0.71), TBCA (R ² = 0.64–0.87), and total soil N contents in 2007 (R ² = 0.94) explained a significant amount of the variance in R _S (P < 0.01). The current study showed that stand density is one of the key factors influencing R _S due to the changing biophysical and environmental factors in P. densiflora.     

Cranberry Flavonoids Modulate Cariogenic Properties of Mixed-Species Biofilm through Exopolysaccharides-Matrix Disruption

The exopolysaccharides (EPS) produced by Streptococcus mutans-derived glucosyltransferases (Gtfs) are essential virulence factors associated with the initiation of cariogenic biofilms. EPS forms the core of the biofilm matrix-scaffold, providing mechanical stability while facilitating the creation of localized acidic microenvironments. Cranberry flavonoids, such as A-type proanthocyanidins (PACs) and myricetin, have been shown to inhibit the activity of Gtfs and EPS-mediated bacterial adhesion without killing the organisms. Here, we investigated whether a combination of cranberry flavonoids disrupts EPS accumulation and S. mutans survival using a mixed-species biofilm model under cariogenic conditions. We also assessed the impact of cranberry flavonoids on mechanical stability and the in situ pH at the biofilm-apatite interface. Topical application of an optimized combination of PACs oligomers (100–300 μM) with myricetin (2 mM) twice daily was used to simulate treatment regimen experienced clinically. Treatments with cranberry flavonoids effectively reduced the insoluble EPS content (>80% reduction vs. vehicle-control; p<0.001), while hindering S. mutans outgrowth within mixed-species biofilms. As a result, the 3D architecture of cranberry-treated biofilms was severely compromised, showing a defective EPS-matrix and failure to develop microcolonies on the saliva-coated hydroxyapatite (sHA) surface. Furthermore, topical applications of cranberry flavonoids significantly weaken the mechanical stability of the biofilms; nearly 90% of the biofilm was removed from sHA surface after exposure to a shear stress of 0.449 N/m² (vs. 36% removal in vehicle-treated biofilms). Importantly, in situ pH measurements in cranberry-treated biofilms showed significantly higher pH values (5.2 ± 0.1) at the biofilm-apatite interface vs. vehicle-treated biofilms (4.6 ± 0.1). Altogether, the data provide important insights on how cranberry flavonoids treatments modulate virulence properties by disrupting the biochemical and ecological changes associated with cariogenic biofilm development, which could lead to new alternative or adjunctive antibiofilm/anticaries chemotherapeutic formulations.     

Comparative Effect of Chlorhexidine and Some Mouthrinses on Bacterial Biofilm Formation on Titanium Surface

The aim of the present study was to evaluate the effectiveness of chlorhexidine digluconate (CHX) and commonly used mouthrinses to single- and poly-species biofilms by S. mutans, S. aureus and P. aeruginosa, on titanium discs of grade IV. The formation of single- and poly-species biofilms at 16.5, 40.5 and 64.5-h incubation on titanium surface was evaluated by plate count (CFU ml⁻¹) before and after exposure to CHX and four mouthrinses (Curasept, Listerine, Meridol and Buccagel) and expressed as percentage of Inhibitory Activity (IA%). The application of the different anti-plaque formulations on biofilm can reduce the adhesion of bacteria to titanium surface with different degrees. The higher efficacy was observed for Listerine that shows IA% = 100 on the biofilm formed by S. mutans at 16.5 h. Log count of CFU was dependent to culture time and four mouthrinses for S. mutans and S. aureus, whilst was not dependent to culture time but to mouthrinses for P. aeruginosa. In general, the efficacy was particularly lesser to poly-species biofilms; no statistical differences were evidenced between all the mouthrinses and CHX as control group. The tested mouthrinses, compared to reference CHX 0.2%, have demonstrated a significant lower antibacterial activity than Listerine towards the experimental biofilms. This “in vitro” biofilm model should prove extremely useful for pre-clinical testing of anti-plaque agents, which inhibit biofilm formation, can prevent subsequent implant failure.