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.     

Exopolysaccharides Produced by Streptococcus mutans Glucosyltransferases Modulate the Establishment of Microcolonies within Multispecies Biofilms

Streptococcus mutans is a key contributor to the formation of the extracellular polysaccharide (EPS) matrix in dental biofilms. The exopolysaccharides, which are mostly glucans synthesized by streptococcal glucosyltransferases (Gtfs), provide binding sites that promote accumulation of microorganisms on the tooth surface and further establishment of pathogenic biofilms. This study explored (i) the role of S. mutans Gtfs in the development of the EPS matrix and microcolonies in biofilms, (ii) the influence of exopolysaccharides on formation of microcolonies, and (iii) establishment of S. mutans in a multispecies biofilm in vitro using a novel fluorescence labeling technique. Our data show that the ability of S. mutans strains defective in the gtfB gene or the gtfB and gtfC genes to form microcolonies on saliva-coated hydroxyapatite surfaces was markedly disrupted. However, deletion of both gtfB (associated with insoluble glucan synthesis) and gtfC (associated with insoluble and soluble glucan synthesis) is required for the maximum reduction in EPS matrix and biofilm formation. S. mutans grown with sucrose in the presence of Streptococcus oralis and Actinomyces naeslundii steadily formed exopolysaccharides, which allowed the initial clustering of bacterial cells and further development into highly structured microcolonies. Concomitantly, S. mutans became the major species in the mature biofilm. Neither the EPS matrix nor microcolonies were formed in the presence of glucose in the multispecies biofilm. Our data show that GtfB and GtfC are essential for establishment of the EPS matrix, but GtfB appears to be responsible for formation of microcolonies by S. mutans; these Gtf-mediated processes may enhance the competitiveness of S. mutans in the multispecies environment in biofilms on tooth surfaces.Oral diseases related to dental biofilms afflict the majority of the world''s population, and dental caries is still the single most prevalent and costly oral infectious disease (12, 32). Dental caries results from the interaction of specific bacteria with constituents of the diet within a biofilm formed on the tooth surface known as plaque (5, 36). Streptococcus mutans is a key contributor to the formation of biofilms associated with dental caries disease, although other microorganisms may also be involved (3); S. mutans (i) effectively utilizes dietary sucrose (and possibly starch) to rapidly synthesize exopolysaccharides (EPS) using glucosyltransferases and a fructosyltransferase that adsorb to surfaces, (ii) adheres tenaciously to glucan-coated surfaces, and (iii) is acidogenic and acid tolerant (5, 30).In general, biofilms develop after initial attachment of microbes to a surface, followed by formation of highly structured cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which are in a complex extracellular matrix (6, 49). The majority of biofilm matrices contain exopolysaccharides, and dental biofilms are no exception; up to 40% of the dry weight of dental plaque is composed of polysaccharides (depending on the type of carbohydrate consumption and the time of plaque collection), which are mostly glucans synthesized by microbial glucosyltransferases (Gtfs) (for a review, see reference 36). S. mutans plays a major role in the development and establishment of the EPS matrix in dental biofilms. This bacterium produces at least three Gtfs, which are products of the gtfB, gtfC, and gtfD genes; GtfB synthesizes mostly insoluble glucans containing elevated amounts of α-1,3-linked glucose, GtfC synthesizes a mixture of insoluble and soluble glucans (rich in α-1,6-linked glucose), and GtfD synthesizes predominantly soluble glucans (for reviews, see references 30 and 36). The Gtfs secreted by S. mutans bind avidly to the pellicle formed on the tooth surface and to bacterial surfaces and are enzymatically active; when they are exposed to sucrose, glucans are formed in situ within minutes (17, 33, 38, 40, 46). It is noteworthy that most nonstreptococcal oral bacteria (e.g., Actinomyces and Veillonella spp.) do not produce glucans unless Gtfs are adsorbed on their surfaces (33, 46). The glucans synthesized in situ provide binding sites for colonization and accumulation of S. mutans on the apatitic surface and for binding to each other through interactions with several membrane-associated glucan-binding proteins and surface glucans (8, 39, 47). The exopolymers also contribute to the bulk and physical integrity and stability of the biofilm matrix (for a review, see reference 36). The glucan-mediated processes promote tight adherence and coherence of bacterial cells bound to each other and to the apatitic surface, which leads to the formation of microcolonies by S. mutans and thereby modulates the initial steps of cariogenic biofilm development.When dietary sucrose is consumed frequently, S. mutans, as a member of the oral biofilm community, continues to synthesize polysaccharides and metabolize this sugar to form organic acids. The elevated amounts of EPS, which may involve upregulation of gtf genes in response to pH and carbohydrate availability (29), increase the virulence of the biofilms (42, 51). In addition, the ability of S. mutans to utilize some extra- and intracellular polysaccharides as short-term storage compounds provides an additional ecological benefit and simultaneously increases the amount of acid produced and the extent of acidification within the biofilm (5, 7). The persistence of this aciduric environment leads to selection and dominance of highly acid-tolerant (and acidogenic) organisms, such as S. mutans (32, 37); the low-pH environment in the biofilm matrix results in dissolution of enamel, thus initiating the pathogenesis of dental caries (32, 36).Recently, we have shown that EPS produced by S. mutans Gtfs modulate the initial formation, sequence of assembly, and structural organization of microcolonies by this bacterium on apatitic surfaces (50). However, it was unclear which of the Gtf enzymes were associated with these processes. Furthermore, the polysaccharides may also modulate the formation of microcolonies by complex ecological interactions in a multispecies system. In this study, we investigated (i) the role of each of the S. mutans gtf genes in EPS matrix and microcolony development on a saliva-coated hydroxyapatite (sHA) surface and (ii) the influence of exopolysaccharides on establishment of microcolonies at distinct developmental phases during formation of biofilms by S. mutans in the presence of Streptococcus oralis and Actinomyces naeslundii.(This study was presented at 5th ASM Conference on Biofilms, Cancun, Mexico, 15 to 19 November 2009.)     

Influences of naturally occurring agents in combination with fluoride on gene expression and structural organization of Streptococcus mutans in biofilms

Background  

The association of specific bioactive flavonoids and terpenoids with fluoride can modulate the development of cariogenic biofilms by simultaneously affecting the synthesis of exopolysaccharides (EPS) and acid production by Streptococcus mutans, which enhanced the cariostatic effectiveness of fluoride in vivo. In the present study, we further investigated whether the biological actions of combinations of myricetin (flavonoid), tt-farnesol (terpenoid) and fluoride can influence the expression of specific genes of S. mutans within biofilms and their structural organization using real-time PCR and confocal fluorescence microscopy.     

The Exopolysaccharide Matrix Modulates the Interaction between 3D Architecture and Virulence of a Mixed-Species Oral Biofilm

Virulent biofilms are responsible for a range of infections, including oral diseases. All biofilms harbor a microbial-derived extracellular-matrix. The exopolysaccharides (EPS) formed on tooth-pellicle and bacterial surfaces provide binding sites for microorganisms; eventually the accumulated EPS enmeshes microbial cells. The metabolic activity of the bacteria within this matrix leads to acidification of the milieu. We explored the mechanisms through which the Streptococcus mutans-produced EPS-matrix modulates the three-dimensional (3D) architecture and the population shifts during morphogenesis of biofilms on a saliva-coated-apatitic surface using a mixed-bacterial species system. Concomitantly, we examined whether the matrix influences the development of pH-microenvironments within intact-biofilms using a novel 3D in situ pH-mapping technique. Data reveal that the production of the EPS-matrix helps to create spatial heterogeneities by forming an intricate network of exopolysaccharide-enmeshed bacterial-islets (microcolonies) through localized cell-to-matrix interactions. This complex 3D architecture creates compartmentalized acidic and EPS-rich microenvironments throughout the biofilm, which triggers the dominance of pathogenic S. mutans within a mixed-species system. The establishment of a 3D-matrix and EPS-enmeshed microcolonies were largely mediated by the S. mutans gtfB/gtfC genes, expression of which was enhanced in the presence of Actinomyces naeslundii and Streptococcus oralis. Acidic pockets were found only in the interiors of bacterial-islets that are protected by EPS, which impedes rapid neutralization by buffer (pH 7.0). As a result, regions of low pH (<5.5) were detected at specific locations along the surface of attachment. Resistance to chlorhexidine was enhanced in cells within EPS-microcolony complexes compared to those outside such structures within the biofilm. Our results illustrate the critical interaction between matrix architecture and pH heterogeneity in the 3D environment. The formation of structured acidic-microenvironments in close proximity to the apatite-surface is an essential factor associated with virulence in cariogenic-biofilms. These observations may have relevance beyond the mouth, as matrix is inherent to all biofilms.     

Streptococcus mutans Protein Synthesis during Mixed-Species Biofilm Development by High-Throughput Quantitative Proteomics

Biofilms formed on tooth surfaces are comprised of mixed microbiota enmeshed in an extracellular matrix. Oral biofilms are constantly exposed to environmental changes, which influence the microbial composition, matrix formation and expression of virulence. Streptococcus mutans and sucrose are key modulators associated with the evolution of virulent-cariogenic biofilms. In this study, we used a high-throughput quantitative proteomics approach to examine how S. mutans produces relevant proteins that facilitate its establishment and optimal survival during mixed-species biofilms development induced by sucrose. Biofilms of S. mutans, alone or mixed with Actinomyces naeslundii and Streptococcus oralis, were initially formed onto saliva-coated hydroxyapatite surface under carbohydrate-limiting condition. Sucrose (1%, w/v) was then introduced to cause environmental changes, and to induce biofilm accumulation. Multidimensional protein identification technology (MudPIT) approach detected up to 60% of proteins encoded by S. mutans within biofilms. Specific proteins associated with exopolysaccharide matrix assembly, metabolic and stress adaptation processes were highly abundant as the biofilm transit from earlier to later developmental stages following sucrose introduction. Our results indicate that S. mutans within a mixed-species biofilm community increases the expression of specific genes associated with glucan synthesis and remodeling (gtfBC, dexA) and glucan-binding (gbpB) during this transition (P<0.05). Furthermore, S. mutans up-regulates specific adaptation mechanisms to cope with acidic environments (F1F0-ATPase system, fatty acid biosynthesis, branched chain amino acids metabolism), and molecular chaperones (GroEL). Interestingly, the protein levels and gene expression are in general augmented when S. mutans form mixed-species biofilms (vs. single-species biofilms) demonstrating fundamental differences in the matrix assembly, survival and biofilm maintenance in the presence of other organisms. Our data provide insights about how S. mutans optimizes its metabolism and adapts/survives within the mixed-species community in response to a dynamically changing environment. This reflects the intricate physiological processes linked to expression of virulence by this bacterium within complex biofilms.     

Presence of Extracellular DNA in the <Emphasis Type="Italic">Candida albicans</Emphasis> Biofilm Matrix and its Contribution to Biofilms

DNA has been described as a structural component of the extracellular matrix (ECM) in bacterial biofilms. In Candida albicans, there is a scarce knowledge concerning the contribution of extracellular DNA (eDNA) to biofilm matrix and overall structure. This work examined the presence and quantified the amount of eDNA in C. albicans biofilm ECM and the effect of DNase treatment and the addition of exogenous DNA on C. albicans biofilm development as indicators of a role for eDNA in biofilm development. We were able to detect the accumulation of eDNA in biofilm ECM extracted from C. albicans biofilms formed under conditions of flow, although the quantity of eDNA detected differed according to growth conditions, in particular with regards to the medium used to grow the biofilms. Experiments with C. albicans biofilms formed statically using a microtiter plate model indicated that the addition of exogenous DNA (>160 ng/ml) increases biofilm biomass and, conversely, DNase treatment (>0.03 mg/ml) decreases biofilm biomass at later time points of biofilm development. We present evidence for the role of eDNA in C. albicans biofilm structure and formation, consistent with eDNA being a key element of the ECM in mature C. albicans biofilms and playing a predominant role in biofilm structural integrity and maintenance.     

α-Mangostin Disrupts the Development of Streptococcus mutans Biofilms and Facilitates Its Mechanical Removal

α-Mangostin (αMG) has been reported to be an effective antimicrobial agent against planktonic cells of Streptococcus mutans, a biofilm-forming and acid-producing cariogenic organism. However, its anti-biofilm activity remains to be determined. We examined whether αMG, a xanthone purified from Garcinia mangostana L grown in Vietnam, disrupts the development, acidogenicity, and/or the mechanical stability of S. mutans biofilms. Treatment regimens simulating those experienced clinically (twice-daily, 60 s exposure each) were used to assess the bioactivity of αMG using a saliva-coated hydroxyapatite (sHA) biofilm model. Topical applications of early-formed biofilms with αMG (150 µM) effectively reduced further biomass accumulation and disrupted the 3D architecture of S. mutans biofilms. Biofilms treated with αMG had lower amounts of extracellular insoluble and intracellular iodophilic polysaccharides (30–45%) than those treated with vehicle control (P<0.05), while the number of viable bacterial counts was unaffected. Furthermore, αMG treatments significantly compromised the mechanical stability of the biofilm, facilitating its removal from the sHA surface when subjected to a constant shear stress of 0.809 N/m² (>3-fold biofilm detachment from sHA vs. vehicle-treated biofilms; P<0.05). Moreover, acid production by S. mutans biofilms was disrupted following αMG treatments (vs. vehicle-control, P<0.05). The activity of enzymes associated with glucan synthesis, acid production, and acid tolerance (glucosyltransferases B and C, phosphotransferase-PTS system, and F₁F₀-ATPase) were significantly inhibited by αMG. The expression of manL, encoding a key component of the mannose PTS, and gtfB were slightly repressed by αMG treatment (P<0.05), while the expression of atpD (encoding F-ATPase) and gtfC genes was unaffected. Hence, this study reveals that brief exposures to αMG can disrupt the development and structural integrity of S. mutans biofilms, at least in part via inhibition of key enzymatic systems associated with exopolysaccharide synthesis and acidogenicity. αMG could be an effective anti-virulence additive for the control and/or removal of cariogenic biofilms.     

Mechanisms of Bacillus cereus biofilm formation: an investigation of the physicochemical characteristics of cell surfaces and extracellular proteins

Microbial biofilms contribute to biofouling in a wide range of processes from medical implants to processed food. The extracellular polymeric substances (EPS) are implicated in imparting biofilms with structural stability and resistance to cleaning products. Still, very little is known about the structural role of the EPS in Gram-positive systems. Here, we have compared the cell surface and EPS of surface-attached (biofilm) and free-floating (planktonic) cells of Bacillus cereus, an organism routinely isolated from within biofilms on different surfaces. Our results indicate that the surface properties of cells change during biofilm formation and that the EPS proteins function as non-specific adhesions during biofilm formation. The physicochemical traits of the cell surface and the EPS proteins give us an insight into the forces that drive biofilm formation and maintenance in B. cereus.     

Exopolymer Diversity and the Role of Levan in Bacillus subtilis Biofilms

Exopolymeric substances (EPS) are important for biofilm formation and their chemical composition may influence biofilm properties. To explore these relationships the chemical composition of EPS from Bacillus subtilis NCIB 3610 biofilms grown in sucrose-rich (SYM) and sucrose-poor (MSgg and Czapek) media was studied. We observed marked differences in composition of EPS polymers isolated from all three biofilms or from spent media below the biofilms. The polysaccharide levan dominated the EPS of SYM grown biofilms, while EPS from biofilms grown in sucrose-poor media contained significant amounts of proteins and DNA in addition to polysaccharides. The EPS polymers differed also in size with very large polymers (Mw>2000 kDa) found only in biofilms, while small polymers (Mw<200 kD) dominated in the EPS isolated from spent media. Biofilms of the eps knockout were significantly thinner than those of the tasA knockout in all media. The biofilm defective phenotypes of tasA and eps mutants were, however, partially compensated in the sucrose-rich SYM medium. Sucrose supplementation of Czapek and MSgg media increased the thickness and stability of biofilms compared to non-supplemented controls. Since sucrose is essential for synthesis of levan and the presence of levan was confirmed in all biofilms grown in media containing sucrose, this study for the first time shows that levan, although not essential for biofilm formation, can be a structural and possibly stabilizing component of B. subtilis floating biofilms. In addition, we propose that this polysaccharide, when incorporated into the biofilm EPS, may also serve as a nutritional reserve.     

Effects of extracellular DNA and DNA‐binding protein on the development of a Streptococcus intermedius biofilm

Aims

The aim of this study was to clarify the effects of homologous and heterologous extracellular DNAs (eDNAs) and histone‐like DNA‐binding protein (HLP) on Streptococcus intermedius biofilm development and rigidity.

Methods and Results

Formed biofilm mass was measured with 0·1% crystal violet staining method and observed with a scanning electron microscope. The localizations of eDNA and extracellular HLP (eHLP) in formed biofilm were detected by staining with 7‐hydoxyl‐9H‐(1,3‐dichloro‐9,9‐dimethylacridin‐2‐one) and anti‐HLP antibody without fixation, respectively. DNase I treatment (200 U ml^?1) markedly decreased biofilm formation and cell density in biofilms. Colocalization of eHLP and eDNA in biofilm was confirmed. The addition of eDNA (up to 1 μg ml^?1) purified from Strep. intermedius, other Gram‐positive bacteria, Gram‐negative bacteria, or human KB cells into the Strep. intermedius culture increased the biofilm mass of all tested strains of Strep. intermedius, wild‐type, HLP‐downregulated strain and control strains. In contrast, the addition of eDNA (>1 μg ml^?1) decreased the biofilm mass of all Strep. intermedius strains.

Conclusions

These findings demonstrated that eDNA and eHLP play crucial roles in biofilm development and its rigidity.

Significance and Impact of the Study

eDNA‐ and HLP‐targeting strategies may be applicable to novel treatments for bacterial biofilm‐related infectious diseases.     

Lipopolysaccharide O-Chain Core Region Required for Cellular Cohesion and Compaction of In Vitro and Root Biofilms Developed by Rhizobium leguminosarum

The formation of biofilms is an important survival strategy allowing rhizobia to live on soil particles and plant roots. Within the microcolonies of the biofilm developed by Rhizobium leguminosarum, rhizobial cells interact tightly through lateral and polar connections, forming organized and compact cell aggregates. These microcolonies are embedded in a biofilm matrix, whose main component is the acidic exopolysaccharide (EPS). Our work shows that the O-chain core region of the R. leguminosarum lipopolysaccharide (LPS) (which stretches out of the cell surface) strongly influences bacterial adhesive properties and cell-cell cohesion. Mutants defective in the O chain or O-chain core moiety developed premature microcolonies in which lateral bacterial contacts were greatly reduced. Furthermore, cell-cell interactions within the microcolonies of the LPS mutants were mediated mostly through their poles, resulting in a biofilm with an altered three-dimensional structure and increased thickness. In addition, on the root epidermis and on root hairs, O-antigen core-defective strains showed altered biofilm patterns with the typical microcolony compaction impaired. Taken together, these results indicate that the surface-exposed moiety of the LPS is crucial for proper cell-to-cell interactions and for the formation of robust biofilms on different surfaces.     

A three-species biofilm model for the evaluation of enamel and dentin demineralization

Although Streptococcus mutans biofilms have been useful for evaluating the cariogenic potential of dietary carbohydrates and the effects of fluoride on dental demineralization, a more appropriate biofilm should be developed to demonstrate the influence of other oral bacteria on cariogenic biofilms. This study describes the development and validation of a three-species biofilm model comprising Streptococcus mutans, Actinomyces naeslundii, and Streptococcus gordonii for the evaluation of enamel and dentin demineralization after cariogenic challenges and fluoride exposure. Single- or three-species biofilms were developed on dental substrata for 96?h, and biofilms were exposed to feast and famine episodes. The three-species biofilm model produced a large biomass, mostly comprising S. mutans (41%) and S. gordonii (44%), and produced significant demineralization in the dental substrata, although enamel demineralization was decreased by fluoride treatment. The findings indicate that the three-species biofilm model may be useful for evaluating the cariogenic potential of dietary carbohydrates other than sucrose and determining the effects of fluoride on dental substrata.     

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.     

Extracellular DNA and lipoteichoic acids interact with exopolysaccharides in the extracellular matrix of Streptococcus mutans biofilms

Streptococcus mutans-derived exopolysaccharides are virulence determinants in the matrix of biofilms that cause caries. Extracellular DNA (eDNA) and lipoteichoic acid (LTA) are found in cariogenic biofilms, but their functions are unclear. Therefore, strains of S. mutans carrying single deletions that would modulate matrix components were used: eDNA – ?lytS and ?lytT; LTA – ?dltA and ?dltD; and insoluble exopolysaccharide – ΔgtfB. Single-species (parental strain S. mutans UA159 or individual mutant strains) and mixed-species (UA159 or mutant strain, Actinomyces naeslundii and Streptococcus gordonii) biofilms were evaluated. Distinct amounts of matrix components were detected, depending on the inactivated gene. eDNA was found to be cooperative with exopolysaccharide in early phases, while LTA played a larger role in the later phases of biofilm development. The architecture of mutant strains biofilms was distinct (vs UA159), demonstrating that eDNA and LTA influence exopolysaccharide distribution and microcolony organization. Thus, eDNA and LTA may shape exopolysaccharide structure, affecting strategies for controlling pathogenic biofilms.     

Architecture of a Nascent Sphingomonas sp. Biofilm under Varied Hydrodynamic Conditions

The architecture of a Sphingomonas biofilm was studied during early phases of its formation, using strain L138, a gfp-tagged derivative of Sphingomonas sp. strain LB126, as a model organism and flow cells and confocal laser scanning microscopy as experimental tools. Spatial and temporal distribution of cells and exopolymer secretions (EPS) within the biofilm, development of microcolonies under flow conditions representing varied Reynolds numbers, and changes in diffusion length with reference to EPS production were studied by sequential sacrificing of biofilms grown in multichannel flow cells and by time-lapse confocal imaging. The area of biofilm in terms of microscopic images required to ensure representative sampling varied by an order of magnitude when area of cell coverage (2 × 10⁵ μm²) or microcolony size (1 × 10⁶ μm²) was the biofilm parameter under investigation. Hence, it is necessary to establish the inherent variability of any biofilm metric one is attempting to quantify. Sphingomonas sp. strain L138 biofilm architecture consisted of microcolonies and extensive water channels. Biomass and EPS distribution were maximal at 8 to 9 μm above the substratum, with a high void fraction near the substratum. Time-lapse confocal imaging and digital image analysis showed that growth of the microcolonies was not uniform: adjacently located colonies registered significant growth or no growth at all. Microcolonies in the biofilm had the ability to move across the attachment surface as a unit, irrespective of fluid flow direction, indicating that movement of microcolonies is an inherent property of the biofilm. Width of water channels decreased as EPS production increased, resulting in increased diffusion distances in the biofilm. Changing hydrodynamic conditions (Reynolds numbers of 0.07, 52, and 87) had no discernible influence on the characteristics of microcolonies (size, shape, or orientation with respect to flow) during the first 24 h of biofilm development. Inherent factors appear to have overriding influence, vis-à-vis environmental factors, on early stages of microcolony development under these laminar flow conditions.     

Inhibitory effect of methyl gallate and gallic acid on oral bacteria

This study examined the ability of methyl gallate (MG) and gallic acid (GA), the main compounds of gallo-tannins in Galla Rhois, to inhibit the proliferation of oral bacterial and the in vitro formation of Streptococcus mutans biofilms. The antimicrobial activities of these compounds were evaluated in vitro using the broth microdilution method and a beaker-wire test. Both MG and GA had inhibitory effects on the growth of cariogenic (MIC<8 mg/ml) and periodontopathic bacteria (MIC=1 mg/ml). Moreover, these compounds significantly inhibited the in vitro formation of S. mutans biofilms (MG, 1 mg/ml; GA, 4 mg/ml; P<0.05). MG was more effective in inhibiting bacterial growth and the formation of S. mutans biofilm than GA. In conclusion, MG and GA can inhibit the growth of oral pathogens and S. mutans biofilm formation, and may be used to prevent the formation of oral biofilms.     

Escherichia coli biofilm: development and therapeutic strategies

Escherichia coli biofilm consists of a bacterial colony embedded in a matrix of extracellular polymeric substances (EPS) which protects the microbes from adverse environmental conditions and results in infection. Besides being the major causative agent for recurrent urinary tract infections, E. coli biofilm is also responsible for indwelling medical device‐related infectivity. The cell‐to‐cell communication within the biofilm occurs due to quorum sensors that can modulate the key biochemical players enabling the bacteria to proliferate and intensify the resultant infections. The diversity in structural components of biofilm gets compounded due to the development of antibiotic resistance, hampering its eradication. Conventionally used antimicrobial agents have a restricted range of cellular targets and limited efficacy on biofilms. This emphasizes the need to explore the alternate therapeuticals like anti‐adhesion compounds, phytochemicals, nanomaterials for effective drug delivery to restrict the growth of biofilm. The current review focuses on various aspects of E. coli biofilm development and the possible therapeutic approaches for prevention and treatment of biofilm‐related infections.