Showing papers on "Biofilm matrix published in 2009"

Bacterial extracellular polysaccharides involved in biofilm formation.

Abstract: Extracellular polymeric substances (EPS) produced by microorganisms are a complex mixture of biopolymers primarily consisting of polysaccharides, as well as proteins, nucleic acids, lipids and humic substances. EPS make up the intercellular space of microbial aggregates and form the structure and architecture of the biofilm matrix. The key functions of EPS comprise the mediation of the initial attachment of cells to different substrata and protection against environmental stress and dehydration. The aim of this review is to present a summary of the current status of the research into the role of EPS in bacterial attachment followed by biofilm formation. The latter has a profound impact on an array of biomedical, biotechnology and industrial fields including pharmaceutical and surgical applications, food engineering, bioremediation and biohydrometallurgy. The diverse structural variations of EPS produced by bacteria of different taxonomic lineages, together with examples of biotechnological applications, are discussed. Finally, a range of novel techniques that can be used in studies involving biofilm-specific polysaccharides is discussed.

Signals, regulatory networks, and materials that build and break bacterial biofilms

Abstract: Biofilms are communities of microorganisms that live attached to surfaces. Biofilm formation has received much attention in the last decade, as it has become clear that virtually all types of bacteria can form biofilms and that this may be the preferred mode of bacterial existence in nature. Our current understanding of biofilm formation is based on numerous studies of myriad bacterial species. Here, we review a portion of this large body of work including the environmental signals and signaling pathways that regulate biofilm formation, the components of the biofilm matrix, and the mechanisms and regulation of biofilm dispersal.

Assembly and development of the Pseudomonas aeruginosa biofilm matrix.

Abstract: Virtually all cells living in multicellular structures such as tissues and organs are encased in an extracellular matrix. One of the most important features of a biofilm is the extracellular polymeric substance that functions as a matrix, holding bacterial cells together. Yet very little is known about how the matrix forms or how matrix components encase bacteria during biofilm development. Pseudomonas aeruginosa forms environmentally and clinically relevant biofilms and is a paradigm organism for the study of biofilms. The extracellular polymeric substance of P. aeruginosa biofilms is an ill-defined mix of polysaccharides, nucleic acids, and proteins. Here, we directly visualize the product of the polysaccharide synthesis locus (Psl exopolysaccharide) at different stages of biofilm development. During attachment, Psl is anchored on the cell surface in a helical pattern. This promotes cell–cell interactions and assembly of a matrix, which holds bacteria in the biofilm and on the surface. Chemical dissociation of Psl from the bacterial surface disrupted the Psl matrix as well as the biofilm structure. During biofilm maturation, Psl accumulates on the periphery of 3-D-structured microcolonies, resulting in a Psl matrix-free cavity in the microcolony center. At the dispersion stage, swimming cells appear in this matrix cavity. Dead cells and extracellular DNA (eDNA) are also concentrated in the Psl matrix-free area. Deletion of genes that control cell death and autolysis affects the formation of the matrix cavity and microcolony dispersion. These data provide a mechanism for how P. aeruginosa builds a matrix and subsequently a cavity to free a portion of cells for seeding dispersal. Direct visualization reveals that Psl is a key scaffolding matrix component and opens up avenues for therapeutics of biofilm-related complications.

Biofilms of non-Candida albicans Candida species: quantification, structure and matrix composition.

Abstract: Most cases of candidiasis have been attributed to C. albicans, but recently, non-Candida albicans Candida (NCAC) species have been identified as common pathogens. The ability of Candida species to form biofilms has important clinical repercussions due to their increased resistance to antifungal therapy and the ability of yeast cells within the biofilms to withstand host immune defenses. Given this clinical importance of the biofilm growth form, the aim of this study was to characterize biofilms produced by three NCAC species, namely C. parapsilosis, C. tropicalis and C. glabrata. The biofilm forming ability of clinical isolates of C. parapsilosis, C. tropicalis and C. glabrata recovered from different sources, was evaluated by crystal violet staining. The structure and morphological characteristics of the biofilms were also assessed by scanning electron microscopy and the biofilm matrix composition analyzed for protein and carbohydrate content. All NCAC species were able to form biofilms although these were less extensive for C. glabrata compared with C. parapsilosis and C. tropicalis. It was evident that C. parapsilosis biofilm production was highly strain dependent, a feature not evident with C. glabrata and C. tropicalis. Scanning electron microscopy revealed structural differences for biofilms with respect to cell morphology and spatial arrangement. Candida parapsilosis biofilm matrices had large amounts of carbohydrate with less protein. Conversely, matrices extracted from C. tropicalis biofilms had low amounts of carbohydrate and protein. Interestingly, C. glabrata biofilm matrix was high in both protein and carbohydrate content. The present work demonstrates that biofilm forming ability, structure and matrix composition are highly species dependent with additional strain variability occurring with C. parapsilosis.

Biofilm Matrix Regulation by Candida albicans Zap1

Abstract: A biofilm is a surface-associated population of microorganisms embedded in a matrix of extracellular polymeric substances. Biofilms are a major natural growth form of microorganisms and the cause of pervasive device-associated infection. This report focuses on the biofilm matrix of Candida albicans, the major fungal pathogen of humans. We report here that the C. albicans zinc-response transcription factor Zap1 is a negative regulator of a major matrix component, soluble β-1,3 glucan, in both in vitro and in vivo biofilm models. To understand the mechanistic relationship between Zap1 and matrix, we identified Zap1 target genes through expression profiling and full genome chromatin immunoprecipitation. On the basis of these results, we designed additional experiments showing that two glucoamylases, Gca1 and Gca2, have positive roles in matrix production and may function through hydrolysis of insoluble β-1,3 glucan chains. We also show that a group of alcohol dehydrogenases Adh5, Csh1, and Ifd6 have roles in matrix production: Adh5 acts positively, and Csh1 and Ifd6, negatively. We propose that these alcohol dehydrogenases generate quorum-sensing aryl and acyl alcohols that in turn govern multiple events in biofilm maturation. Our findings define a novel regulatory circuit and its mechanism of control of a process central to infection.

Protein A-mediated multicellular behavior in Staphylococcus aureus.

Abstract: The capacity of Staphylococcus aureus to form biofilms on host tissues and implanted medical devices is one of the major virulence traits underlying persistent and chronic infections. The matrix in which S. aureus cells are encased in a biofilm often consists of the polysaccharide intercellular adhesin (PIA) or poly-N-acetyl glucosamine (PNAG). However, surface proteins capable of promoting biofilm development in the absence of PIA/PNAG exopolysaccharide have been described. Here, we used two-dimensional nano-liquid chromatography and mass spectrometry to investigate the composition of a proteinaceous biofilm matrix and identified protein A (spa) as an essential component of the biofilm; protein A induced bacterial aggregation in liquid medium and biofilm formation under standing and flow conditions. Exogenous addition of synthetic protein A or supernatants containing secreted protein A to growth media induced biofilm development, indicating that protein A can promote biofilm development without being covalently anchored to the cell wall. Protein A-mediated biofilm formation was completely inhibited in a dose-dependent manner by addition of serum, purified immunoglobulin G, or anti-protein A-specific antibodies. A murine model of subcutaneous catheter infection unveiled a significant role for protein A in the development of biofilm-associated infections, as the amount of protein A-deficient bacteria recovered from the catheter was significantly lower than that of wild-type bacteria when both strains were used to coinfect the implanted medical device. Our results suggest a novel role for protein A complementary to its known capacity to interact with multiple immunologically important eukaryotic receptors.

The BLUF-EAL protein YcgF acts as a direct anti-repressor in a blue-light response of Escherichia coli

Abstract: The blue light using FAD (BLUF)-EAL protein YcgF is a known blue-light sensor of Escherichia coli, but its direct regulatory output and physiological function have remained unknown. Here, we demonstrate that unlike other EAL domain proteins, YcgF does not degrade the signaling molecule c-di-GMP, but directly binds to and releases the MerR-like repressor YcgE from its operator DNA upon blue-light irradiation. As a consequence, a distinct regulon of eight small proteins (of 71-126 amino acids) is strongly induced. These include YmgA and YmgB, which, via the RcsC/RcsD/RcsB two-component phosphorelay system, activate production of the biofilm matrix substance colanic acid as well as acid resistance genes and the biofilm-associated bdm gene and down-regulate adhesive curli fimbriae. Thus, small proteins under YcgF/YcgE control seem to act as "connectors" that provide additional signal input into a two-component signaling pathway. Moreover, we found ycgF and ycgE expression to be strongly activated at low temperature, and we elucidate how blue light, cold, and starvation signals are integrated in the expression and activity of the YcgF/YcgE/small protein signaling pathway. In conclusion, this pathway may modulate biofilm formation via the two-component network when E. coli has to survive in an extrahost aquatic environment.

A fratricidal mechanism is responsible for eDNA release and contributes to biofilm development of Enterococcus faecalis

Abstract: Extracellular DNA (eDNA), a by-product of cell lysis, was recently established as a critical structural component of the Enterococcus faecalis biofilm matrix. Here, we describe fratricide as the governing principle behind gelatinase (GelE)-mediated cell death and eDNA release. GFP reporter assays confirmed that GBAP (gelatinase biosynthesis-activating pheromone) quorum non-responders (GelE-SprE-) were a minority subpopulation of prey cells susceptible to the targeted fratricidal action of the quorum responsive predatorial majority (GelE+SprE+). The killing action is dependent on GelE, and the GelE producer population is protected from self-destruction by the co-production of SprE as an immunity protein. Targeted gene inactivation and protein interaction studies demonstrate that extracellular proteases execute their characteristic effects following downstream interactions with the primary autolysin, AtlA. Finally, we address a mechanism by which GelE and SprE may modify the cell wall affinity of proteolytically processed AtlA resulting in either a pro- or anti-lytic outcome.

Therapeutic potential of biofilm-dispersing enzymes.

Abstract: Surface-attached colonies of bacteria known as biofilms play a major role in the pathogenesis of medical device infections Biofilm colonies are notorious for their resistance to antibiotics and host defenses, which makes most device infections difficult or impossible to eradicate Bacterial cells in a biofilm are held together by an extracellular polymeric matrix that is synthesized by the bacteria themselves Enzymes that degrade biofilm matrix polymers have been shown to inhibit biofilm formation, detach established biofilm colonies, and render biofilm cells sensitive to killing by antimicrobial agents This review discusses the potential use of biofilm matrix-degrading enzymes as anti-biofilm agents for the treatment and prevention of device infections Two enzymes, deoxyribonuclease I and the glycoside hydrolase dispersin B, will be reviewed in detail In vitro and in vivo studies demonstrating the anti-biofilm activities of these two enzymes will be summarized, and the therapeutic potential and possible drawbacks of using these enzymes as clinical agents will be discussed

Relevant Role of Fibronectin-Binding Proteins in Staphylococcus aureus Biofilm-Associated Foreign-Body Infections

Abstract: Staphylococcus aureus can establish chronic infections on implanted medical devices due to its capacity to form biofilms. Analysis of the factors that assemble cells into a biofilm has revealed the occurrence of strains that produce either a polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG) exopolysaccharide- or a protein-dependent biofilm. Examination of the influence of matrix nature on the biofilm capacities of embedded bacteria has remained elusive, because a natural strain that readily converts between a polysaccharide- and a protein-based biofilm has not been studied. Here, we have investigated the clinical methicillin (meticillin)-resistant Staphylococcus aureus strain 132, which is able to alternate between a proteinaceous and an exopolysaccharidic biofilm matrix, depending on environmental conditions. Systematic disruption of each member of the LPXTG surface protein family identified fibronectin-binding proteins (FnBPs) as components of a proteinaceous biofilm formed in Trypticase soy broth-glucose, whereas a PIA/PNAG-dependent biofilm was produced under osmotic stress conditions. The induction of FnBP levels due to a spontaneous agr deficiency present in strain 132 and the activation of a LexA-dependent SOS response or FnBP overexpression from a multicopy plasmid enhanced biofilm development, suggesting a direct relationship between the FnBP levels and the strength of the multicellular phenotype. Scanning electron microscopy revealed that cells growing in the FnBP-mediated biofilm formed highly dense aggregates without any detectable extracellular matrix, whereas cells in a PIA/PNAG-dependent biofilm were embedded in an abundant extracellular material. Finally, studies of the contribution of each type of biofilm matrix to subcutaneous catheter colonization revealed that an FnBP mutant displayed a significantly lower capacity to develop biofilm on implanted catheters than the isogenic PIA/PNAG-deficient mutant.

Towards a nondestructive chemical characterization of biofilm matrix by Raman microscopy

Abstract: In this study, the applicability of Raman microscopy (RM) for nondestructive chemical analysis of biofilm matrix, including microbial constituents and extracellular polymeric substances (EPS), has been assessed. The examination of a wide range of reference samples such as biofilm-specific polysaccharides, proteins, microorganisms, and encapsulated bacteria revealed characteristic frequency regions and specific marker bands for different biofilm constituents. Based on received data, the assignment of Raman bands in spectra of multispecies biofilms was performed. The study of different multispecies biofilms showed that RM can correlate various structural appearances within the biofilm to variations in their chemical composition and provide chemical information about a complex biofilm matrix. The results of RM analysis of biofilms are in good agreement with data obtained by confocal laser scanning microscopy (CLSM). Thus, RM is a promising tool for a label-free chemical characterization of different biofilm constituents. Moreover, the combination of RM with CLSM analysis for the study of biofilms grown under different environmental conditions can provide new insights into the complex structure/function correlations in biofilms.

Evaluation of different methods for extracting extracellular DNA from the biofilm matrix.

Abstract: The occurrence of high concentrations of extracellular DNA (eDNA) in the extracellular matrices of biofilms plays an important role in biofilm formation and development and possibly in horizontal gene transfer through natural transformation. Studies have been conducted to characterize the nature of eDNA and its potential function in biofilm development, but it is difficult to extract eDNA from the extracellular matrices of biofilms without any contamination from genomic DNA released by cell lysis during the extraction process. In this report, we compared several different extraction methods in order to obtain highly pure eDNA from different biofilm samples. After different extraction methods were explored, it was concluded that using chemical treatment or enzymatic treatment of biofilm samples may obtain larger amounts of eDNA than using the simple filtration method. There was no detectable cell lysis when the enzymatic treatment methods were used, but substantial cell lysis was observed when the chemical treatment methods were used. These data suggest that eDNA may bind to other extracellular polymers in the biofilm matrix and that enzymatic treatment methods are effective and favorable for extracting eDNA from biofilm samples. Moreover, randomly amplified polymorphic DNA analysis of eDNA in Acinetobacter sp. biofilms and Acinetobacter sp. genomic DNA and DNA sequencing analysis revealed that eDNA originated from genomic DNA but was not structurally identical to the genomic DNA.

Interactions of DNA with Biofilm-Derived Membrane Vesicles

Abstract: The biofilm matrix contributes to the chemistry, structure, and function of biofilms. Biofilm-derived membrane vesicles (MVs) and DNA, both matrix components, demonstrated concentration-, pH-, and cation-dependent interactions. Furthermore, MV-DNA association influenced MV surface properties. This bears consequences for the reactivity and availability for interaction of matrix polymers and other constituents.

Survival of bacterial biofilms within neutrophil extracellular traps promotes nontypeable Haemophilus influenzae persistence in the chinchilla model for otitis media.

Abstract: Nontypeable Haemophilus influenzae (NTHi) is a leading cause of acute and chronic otitis media, which are a major public health problem worldwide. The persistence of NTHi during chronic and recurrent otitis media infections involves multicellular biofilm communities formed within the middle-ear chamber. Bacterial biofilms resist immune clearance and antibiotic therapy due in part to encasement within a polymeric matrix. In this study, the contribution of biofilms to bacterial persistence in vivo and composition of the NTHi biofilm matrix during experimental otitis media were investigated. The presence of biofilms within the chinchilla middle-ear chamber was significantly correlated with increased bacterial load in middle-ear effusions and tissue. Examination of thin sections revealed polymorphonuclear cells within a DNA lattice containing elastase and histones, which is consistent with the definition of neutrophil extracellular traps. Viable multicellular biofilm communities with biofilm phenotypes were found within the DNA lattice throughout the biofilm. Further, NTHi was resistant to both phagocytic and extracellular neutrophil killing in vitro by means of lipooligosaccharide moieties that promote biofilm formation. These data support the conclusion that NTHi subverts neutrophil extracellular traps to persist in vivo. These data also indicate that a more inclusive definition for biofilms may be warranted.

The Staphylococcus aureus LytSR Two-Component Regulatory System Affects Biofilm Formation

Abstract: Studies of the Staphylococcus aureus LytSR two-component regulatory system have led to the identification of the cid and lrg operons, which affect murein hydrolase activity, stationary-phase survival, antibiotic tolerance, and biofilm formation. The cid gene products enhance murein hydrolase activity and antibiotic tolerance whereas the lrg gene products inhibit these processes in a manner believed to be analogous to bacteriophage-encoded holins and antiholins, respectively. Importantly, these operons have been shown to play significant roles in biofilm development by controlling the release of genomic DNA, which then becomes an important structural component of the biofilm matrix. To determine the role of LytSR in biofilm development, a lytS knockout mutant was generated from a clinical S. aureus isolate (UAMS-1) and the effects on gene expression and biofilm formation were examined. As observed in laboratory isolates, LytSR was found to be required for lrgAB expression. Furthermore, the lytS mutant formed a more adherent biofilm than the wild-type and complemented strains. Consistent with previous findings, the increased adherence of the mutant was attributed to the increased prevalence of matrix-associated eDNA. Transcription profiling studies indicated that the lrgAB operon is the primary target of LytSR-mediated regulation but that this regulatory system also impacts expression of a wide variety of genes involved in basic metabolism. Overall, the results of these studies demonstrate that the LytSR two-component regulatory system plays an important role in S. aureus biofilm development, likely as a result of its direct influence on lrgAB expression.

Periodontitis: An Archetypical Biofilm Disease

Abstract: Background Periodontitis is a classic example of biofilm-mediated diseases. Methods The authors reviewed selected publications in English-language peer-reviewed journals with respect to microbial biofilms, focusing on representative works that provided a historical to a contemporary perspective on periodontal oral biofilms in the larger context of biofilm microbiology. Results Developments in advanced microscopy and molecular microbiology have allowed scientists to examine and characterize microbial biofilm-mediated diseases, such as periodontitis, more accurately than in the past. Conclusions Periodontitis, like other biofilm infections, is refractory to antibiotic agents and host defenses because the causative microbes live in complex communities that persist despite challenges that range from targeted antibiotic agents to phagocytosis. Clinical Implications The regular delivery of nontargeted antibiofilm agents may be an effective strategy for treating biofilms, especially if these agents include oxidative agents that dissolve the biofilm matrix.

DegU and Spo0A Jointly Control Transcription of Two Loci Required for Complex Colony Development by Bacillus subtilis

Abstract: Biofilm formation is an example of a multicellular process which depends on cooperative behavior and differentiation within a bacterial population. Our findings indicate that there is a complex feedback loop that maintains the stoichiometry of the extracellular matrix and other proteins required for complex colony development by Bacillus subtilis. Analysis of the transcriptional regulation of two DegU-activated genes that are required for complex colony development by B. subtilis revealed additional involvement of global regulators that are central to controlling biofilm formation. Activation of transcription from both the yvcA and yuaB promoters requires DegU approximately phosphate, but transcription is inhibited by direct AbrB binding to the promoter regions. Inhibition of transcription by AbrB is relieved when Spo0A approximately phosphate is generated due to its known role in inhibiting abrB expression. Deletion of SinR, a key coordinator of motility and biofilm formation, enhanced transcription from both loci; however, no evidence of a direct interaction with SinR for either the yvcA or yuaB promoter regions was observed. The enhanced transcription in the sinR mutant background was subsequently demonstrated to be dependent on biosynthesis of the polysaccharide component that forms the major constituent of the B. subtilis biofilm matrix. Together, these findings indicate that a genetic network dependent on activation of both DegU and Spo0A controls complex colony development by B. subtilis.

Biofilm structure and its influence on clogging in drip irrigation emitters distributing reclaimed wastewater.

Abstract: Using reclaimed wastewater for crop irrigation is a practical alternative to discharge wastewater treatment plant effluents into surface waters. However, biofouling has been identified as a major contributor to emitter clogging in drip irrigation systems distributing reclaimed wastewater. Little is known about the biofilm structure and its influence on clogging in the drip emitter flow path. This study was first to investigate the microbial characteristics of mature biofilms present in the emitters and the effect of flow path structures on the biofilm microbial communities. The analysis of biofilm matrix structure using a scanning electron microscopy (SEM) revealed that particles in the matrix of the biofilm coupled extracellular polysaccharides (EPS) and formed sediment in the emitter flow path. Analysis of biofilm mass including protein, polysaccharide, and phospholipid fatty acids (PLFAs) showed that emitter flow path style influenced biofilm community structure and diversity. The correlations of biofilm biomass and discharge reduction after 360 h irrigation were computed and suggest that PFLAs provide the best correlation coefficient. Comparatively, the emitter with the unsymmetrical dentate structure and shorter flow path (Emitter C) had the best anti-clogging capability. By optimizing the dentate structure, the internal flow pattern within the flow path could be enhanced as an important method to control the biofilm within emitter flow path. This study established electron microscope techniques and biochemical microbial analysis methods that may provide a framework for future emitter biofilm studies.

Continuous power generation and microbial community structure of the anode biofilms in a three-stage microbial fuel cell system

Abstract: A mediator-less three-stage two-chamber microbial fuel cell (MFC) system was developed and operated continuously for more than 1.5 years to evaluate continuous power generation while treating artificial wastewater containing glucose (10 mM) concurrently. A stable power density of 28 W/m(3) was attained with an anode hydraulic retention time of 4.5 h and phosphate buffer as the cathode electrolyte. An overall dissolved organic carbon removal ratio was about 85%, and coulombic efficiency was about 46% in this MFC system. We also analyzed the microbial community structure of anode biofilms in each MFC. Since the environment in each MFC was different due to passing on the products to the next MFC in series, the microbial community structure was different accordingly. The anode biofilm in the first MFC consisted mainly of bacteria belonging to the Gammaproteobacteria, identified as Aeromonas sp., while the Firmicutes dominated the anode biofilms in the second and third MFCs that were mainly fed with acetate. Cyclic voltammetric results supported the presence of a redox compound(s) associated with the anode biofilm matrix, rather than mobile (dissolved) forms, which could be responsible for the electron transfer to the anode. Scanning electron microscopy revealed that the anode biofilms were comprised of morphologically different cells that were firmly attached on the anode surface and interconnected each other with anchor-like filamentous appendages, which might support the results of cyclic voltammetry.

Identification of a calcium-controlled negative regulatory system affecting Vibrio cholerae biofilm formation

Abstract: Vibrio cholerae's capacity to cause outbreaks of cholera is linked to its survival and adaptability to changes in aquatic environments. One of the environmental conditions that can vary in V. cholerae's natural aquatic habitats is calcium (Ca(+2)). In this study, we investigated the response of V. cholerae to changes in extracellular Ca(2+) levels. Whole-genome expression profiling revealed that Ca(2+) decreased the expression of genes required for biofilm matrix production. Luria-Bertani (LB) medium supplemented with Ca(2+) (LBCa(2+)) caused V. cholerae to form biofilms with decreased thickness and increased roughness, as compared with biofilms formed in LB. Furthermore, addition of Ca(2+) led to dissolution in biofilms. Transcription of two genes encoding a two-component regulatory system pair, now termed calcium-regulated sensor (carS) and regulator (carR), was decreased in cells grown in LBCa(2+). Analysis of null and overexpression alleles of carS and carR revealed that expression of vps (Vibriopolysaccharide) genes and biofilm formation are negatively regulated by the CarRS two-component regulatory system. Through epistasis analysis we determined that CarR acts in parallel with HapR, the negative regulator of vps gene expression.

Observing the Biofilm Matrix of Staphylococcus epidermidis ATCC 35984 Grown Using the CDC Biofilm Reactor

Abstract: Bacteria flourish in nearly every environment on earth. Contributing to their ability to grow in many esoteric locations is their development into a biofilm structure. In an effort to more accurately model the growth environment of biofilms in nature, a Center for Disease Control and Prevention (CDC) biofilm reactor has been developed that mimics nature-like shear forces and renewable nutrient sources. To date, there has been no confirmation by scanning electron microscopy (SEM) that mature biofilms develop on a surface when grown using the CDC biofilm reactor. Three different SEM methods were used to collect images of Staphylococcus epidermidis ATCC 35984 that was to be grown using the CDC biofilm reactor. In addition, two different fixative techniques were used in each of the imaging methods. Results indicated that after 48 hours of growth in the reactor, S. epidermidis ATCC 35984 does produce a significant network of matrix components and 3D mushroom- or pillar-like structures with signs of water channel development. In conclusion, S. epidermidis ATCC 35984 grown using the CDC biofilm reactor does appear to display signs of mature biofilm development. These results could be important for studies wherein mature biofilms are needed for in vitro and/or in vivo applications.

Three-dimensional macromolecular organization of cryofixed Myxococcus xanthus biofilms as revealed by electron microscopic tomography.

Abstract: Despite the fact that most bacteria grow in biofilms in natural and pathogenic ecosystems, very little is known about the ultrastructure of their component cells or about the details of their community architecture. We used high-pressure freezing and freeze-substitution to minimize the artifacts of chemical fixation, sample aggregation, and sample extraction. As a further innovation we have, for the first time in biofilm research, used electron tomography and three-dimensional (3D) visualization to better resolve the macromolecular 3D ultrastructure of a biofilm. This combination of superb specimen preparation and greatly improved resolution in the z axis has opened a window in studies of Myxococcus xanthus cell ultrastructure and biofilm community architecture. New structural information on the chromatin body, cytoplasmic organization, membrane apposition between adjacent cells, and structure and distribution of pili and vesicles in the biofilm matrix is presented.

BcsQ is an essential component of the Escherichia coli cellulose biosynthesis apparatus that localizes at the bacterial cell pole.

Abstract: Biofilms are microbial communities characterized by three-dimensional growth resulting from the ability of individual cells to adhere to each other as well as to produce an extracellular matrix that ensures biofilm physical cohesion. Numerous bacteria produce cellulose as a biofilm matrix polymer, a property relying on the expression of bacterial cellulose synthesis (Bcs) proteins and their post-translational activation upon binding of cyclic di-guanosine mono-phosphate second messenger (c-di-GMP) produced by diguanylate cyclases. In Escherichia coli and other Enterobacteriaceae, two genes of unknown function, yhjR and yhjQ, are located upstream of the bcs genes. Here, we show that yhjQ, but not yhjR, is essential for cellulose biosynthesis; it has therefore been renamed bcsQ. Using a green fluorescent protein (GFP) fusion approach, we demonstrate that BcsQ, a MinD homologue, displays a polar localization and that cell-to-cell adhesion is initiated through production of cellulose at the BcsQ-labelled pole. Although we did not detect a similar localization for other Bcs proteins, immunogold labelling of cellulose itself at the pole of individual bacteria indicates the localized activity of the cellulose biosynthesis apparatus. These results therefore suggest that BcsQ could participate in spatial restriction of cellulose biosynthesis activity in Enterobacteriaceae.

Survival potential of wild type cellulose deficient Salmonella from the feed industry

Abstract: Biofilm has been shown to be one way for Salmonella to persist in the feed factory environment. Matrix components, such as fimbriae and cellulose, have been suggested to play an important role in the survival of Salmonella in the environment. Multicellular behaviour by Salmonella is often categorized according to colony morphology into rdar (red, dry and rough) expressing curli fimbriae and cellulose, bdar (brown, dry and rough) expressing curli fimbriae and pdar (pink, dry and rough) expressing cellulose. The aim of the study was to look into the distribution of morphotypes among feed and fish meal factory strains of Salmonella, with emphasis on potential differences between morphotypes with regards to survival in the feed factory environment. When screening a total of 148 Salmonella ser. Agona, Salmonella ser. Montevideo, Salmonella ser. Senftenberg and Salmonella ser. Typhimurium strains of feed factory, human clinical and reference collection origin, as many as 99% were able to express rough morphology (rdar or bdar). The dominant morphotype was rdar (74%), however as many as 55% of Salmonella ser. Agona and 19% of Salmonella ser. Senftenberg displayed the bdar morphology. Inconsistency in Calcofluor binding, indicating expression of cellulose, was found among 25% of all the strains tested, however Salmonella ser. Agona showed to be highly consistent in Calcofluor binding (98%). In biofilm, Salmonella ser. Agona strains with bdar mophology was found to be equally tolerant to disinfection treatment as strains with rdar morphotype. However, rdar morphology appeared to be favourable in long term survival in biofilm in a very dry environment. Chemical analysis showed no major differences in polysaccharide content between bdar and rdar strains. Our results indicate that cellulose is not a major component of the Salmonella biofilm matrix. The bdar morphotype is common among Salmonella ser. Agona strains isolated from the factory environment. The rdar and the bdar strains were found to be equally tolerant to disinfectants, while the rdar strain was found to be more tolerant to long-term desiccation and nutrient depletion in biofilm than the bdar strain. Cellulose does not appear to be a major component of the Salmonella biofilm matrix.

3D Finite Element Model of Biofilm Detachment Using Real Biofilm Structures From CLSM Data

Abstract: In this work, a three-dimensional model of fluid-structure interactions (FSI) in biofilm systems is developed in order to simulate biofilm detachment as a result of mechanical processes. Therein, fluid flow past the biofilm surface results in a mechanical load on the structure which in turn causes internal stresses in the biofilm matrix. When the strength of the matrix is exceeded parts of the structure are detached. The model is used to investigate the influence of several parameters related to the mechanical strength of the biofilm matrix, Young's modulus, Reynolds number, and biofilm structure on biofilm detachment. Variations in biofilm strength and flow conditions significantly influence the simulation outcome. With respect to structural properties the model is widely independent from a change of Young's modulus. A further result of this work indicates that the change of biofilm structure due to growth or other processes will significantly change the stress distribution in the biofilm and thereby the detachment rate. An increase of the mechanical load by increasing fluid flow results in a flat surface of the remaining biofilm structure. It is concluded that the change of structure during biofilm development is the key determinant in terms of the detachment behavior.

Phagocytosis of staphylococci biofilms by polymorphonuclear neutrophils: S. aureus and S. epidermidis differ with regard to their susceptibility towards the host defense.

Abstract: Bacteria organized in biofilms are a common cause of relapsing or persistent infections. In patients receiving orthopedic implants, such as endoprostheses or osteosynthesis materials, Staphylococcus aureus and S. epidermidis are prevalent and it is widely assumed that bacteria in biofilms are not only relatively resistant towards antibiotics and biocides, but also towards host defense mechanisms. In that context, we addressed the question how polymorphonuclear neutrophils (PMN), the "first line defense" against bacterial infection, interact with biofilms generated in vitro. By time-lapse video microscopy, we observed migration of PMN towards the biofilms. In the case of S. aureus, the PMN moved across the biofilm and took up bacteria as they moved along. On S. epidermidis, in contrast, the PMN were rather immobile, and phagocytosis was limited to bacteria in the immediate vicinity. By labeling the bacteria within the biofilm with H-thymidine we found that S. aureus biofilms were more sensitive towards the PMN attack than S. epidermidis. Following phagocytosis of either bacteria strain, the PMN underwent apoptosis, in line with the dogma, that phagocytosis induces programmed cell-death in order to prevent spilling of the bactericidal and cytotoxic entities. In conclusion, biofilms are not inherently protected against the attack by phagocytic cells; their sensitivity, however, varies among bacterial strains, presumably due to properties of the extracellular biofilm matrix affecting the motility of PMN on the film.

Candida albicans biofilm formation and its clinical consequences.

Abstract: In the last several years there has been increasing recognition in the Microbiology field that biofilms constitute the predominant mode of growth for most microorganisms in their natural habitats [1, 2]. Different from planktonic (free floating) organisms, biofilms can be defined as structured microbial communities, attached to a biotic or abiotic surface, and most frequently encapsulated within a matrix of self-produced exopolymeric material. Most importantly, the same is also true for the relative minority of microorganisms that are pathogenic to humans, and according to the CDC today it is estimated that about 65% of all treated infections are associated with microbial biofilm formation on the surface of tissues, organs or medical devices. Biofilm formation carries important clinical implications, as sessile cells typically display increased levels of resistance to most antibiotics and also to host defence mechanisms. In addition, the protective structure of a biofilm provides cells with a safe sanctuary in which they are able to withstand adverse environmental conditions. In essence, biofilms act as reservoirs for persistent sources of infection. Thus, the net effect is that microbial biofilms negatively impact the health of a growing number of patients, that ultimately translates to a soaring financial burden to our health care system [3]. Candida albicans is no exception to this rule, and this often benign commensal of humans is now the fungal species most frequently associated with formation of biofilms affecting different types of immunosuppressed patients [4]. There is little doubt that different manifestations of candidiasis, including oropharyngeal candidiasis, denture stomatitis, endocarditis and catheter-related candidemia and candiduria, among others, are intimately associated with the formation of biofilms on host surfaces and/or implantable medical devices [5, 6]. A variety of biomaterials used in clinical practice are able to support biofilm formation by Candida and, ironically, the increase in candidiasis in recent years (now the third to fourth most common nosocomial infection in US hospitals and abroad) has been virtually concomitant with the increase in use of a broad range of medical implant devices, mostly in immunocompromised patients [7, 8]. The increasing awareness of the importance of Candida biofilms is reflected by the number of publications on this topic: a simple PubMed search using the terms “Candida” and “biofilm” returned 549 articles, with over 90% of these articles having been published in the last ten years. But, how does C. albicans form a biofilm and what are the most important characteristics linked to biofilm formation? In order to answer these fundamental questions multiple groups of investigators have developed different models of C. albicans biofilm formation, with varying degrees of sophistication, both in vitro and most recently in vivo. Using these models and now armed with state of the art analytical techniques (including advanced microscopy techniques, genomics and proteomics, etc.) researchers have been able to shed some light on the structural characteristics and molecular mechanisms governing biofilm formation by this opportunistic pathogenic fungus [4]. From these studies it is now clear that these biofilms are not a simple accumulation of cells, but rather highly structured microbial communities, postulated to represent an optimal spatial arrangement to facilitate the influx of nutrients and disposal of waste products. In general, most investigators in the field agree that C. albicans biofilm development encompasses different phases, including initial adherence, colonization, proliferation, maturation and ultimately dispersion so that the “biofilm life-cycle” can be repeated all over again [4, 9, 10]. Mature C. albicans biofilms typically consist of an intricate network of yeasts, hyphae and pseudohyphae within ramifying water channels and are encased within exopolymeric material. They exhibit a rather complex three-dimensional architecture, likely indicative of a high degree of specialization reminiscent of what is found in primitive tissue. Our current understanding at the molecular level of the mechanisms controlling C. albicans biofilm formation is still somewhat limited; however, in recent years studies by multiple groups of investigators have begun to unravel some of the major driving forces behind the transition to the biofilm life style [4, 11]. Besides some insights into biofilm metabolism, these studies have revealed a pivotal role for morphogenetic conversions (the ability to reversibly switch between yeast and filamentous forms in response to different environmental stimuli), adhesive interactions and quorum sensing mechanisms in C. albicans biofilm development, with also some very important implications in mating. Two very interesting reviews published in this very issue of this Journal provide readers with up to date information on the Ras/cAMP/PKA signaling pathway that regulates filamentation and virulence in C. albicans and on the role of farnesol as a quorum sensing molecule: both of these phenomena are critical for the C. albicans biofilm mode of growth. For example, the C. albicansΔefg1 mutant strain is locked in the yeast form and forms only a rudimentary (monolayer) biofilm, thus pointing to the Efg1p regulator protein (a main component of the Ras/cAMP/PKA signaling pathway) as a key factor in C. albicans biofilm formation [12]. Moreover, HWP1 and ALS3, whose expression is under the control of Efg1p, encode hypha-specific cell wall proteins that play complementary adhesive functions critical in C. albicans biofilm development [13]. Likewise, farnesol, and other autoregulatory molecules also control C. albicans biofilm formation via quorum sensing mechanisms [14, 15]. As mentioned before, C. albicans biofilm formation carries important negative clinical implications, mostly due to the fact that cells in biofilms are recalcitrant to antifungal therapy. As such, biofilm formation is now widely considered one of the major virulence attributes of C. albicans and a key contributing factor to the unacceptably high mortality rates associated with candidiasis. A plethora of articles have now been published reporting that fungal biofilms show intrinsic resistance to azole derivatives and display high levels of resistance against polyenes, two of the most common classes of antifungal agents. Contrary to these observations echinocandins, a new class of antifungal agents targeting cell wall glucan, seem to display excellent anti-biofilm activity at therapeutic concentrations [4, 16]. Antifungal drug resistance of C. albicans cells within biofilms is likely multifactorial and, among other mechanisms, may be due to i) metabolic and physiological state of sessile fungal cells, ii) elevated cellular density within the biofilm; iii) the protective effect of the biofilm matrix, including presence of glucans which may bind to molecules of certain antifungal agents (azoles) ; iv) differential expression of genes linked to resistance, including those encoding efflux pumps; v) differences in sterol composition of the cell wall membrane; and vi) presence of a subpopulation of “persister” cells [4]. In conclusion, sessile C. albicans cells within biofilms possess distinct developmental properties and phenotypic characteristics that are in stark contrast to planktonic cells. Because of this, infections associated with C. albicans biofilm formation represent an escalating problem in health care and negatively impact the health of an increasing number of individuals as progress in modern medicine prolong the lives of severely ill patients. The increased recognition by the research and medical community of the role that biofilms play during infection should, without any doubt, lead to major advances in the diagnosis, prevention and treatment of biofilm-associated candidiasis in the near future.