Showing papers on "Biofilm matrix published in 2013"

The extracellular matrix protects Pseudomonas aeruginosa biofilms by limiting the penetration of tobramycin.

Abstract: Biofilm cells are less susceptible to antimicrobials than their planktonic counterparts. While this phenomenon is multifactorial, the ability of the matrix to reduce antibiotic penetration into the biofilm is thought to be of limited importance studies suggest that antibiotics move fairly rapidly through biofilms. In this study, we monitored the transport of two clinically relevant antibiotics, tobramycin and ciprofloxacin, into non-mucoid Pseudomonas aeruginosa biofilms. To our surprise, we found that the positively charged antibiotic tobramycin is sequestered to the biofilm periphery, while the neutral antibiotic ciprofloxacin readily penetrated. We provide evidence that tobramycin in the biofilm periphery both stimulated a localized stress response and killed bacteria in these regions but not in the underlying biofilm. Although it is unclear which matrix component binds tobramycin, its penetration was increased by the addition of cations in a dose-dependent manner, which led to increased biofilm death. These data suggest that ionic interactions of tobramycin with the biofilm matrix limit its penetration. We propose that tobramycin sequestration at the biofilm periphery is an important mechanism in protecting metabolically active cells that lie just below the zone of sequestration.

Cellulose as an Architectural Element in Spatially Structured Escherichia coli Biofilms

Abstract: Morphological form in multicellular aggregates emerges from the interplay of genetic constitution and environmental signals. Bacterial macrocolony biofilms, which form intricate three-dimensional structures, such as large and often radially oriented ridges, concentric rings, and elaborate wrinkles, provide a unique opportunity to understand this interplay of "nature and nurture" in morphogenesis at the molecular level. Macrocolony morphology depends on self-produced extracellular matrix components. In Escherichia coli, these are stationary phase-induced amyloid curli fibers and cellulose. While the widely used "domesticated" E. coli K-12 laboratory strains are unable to generate cellulose, we could restore cellulose production and macrocolony morphology of E. coli K-12 strain W3110 by "repairing" a single chromosomal SNP in the bcs operon. Using scanning electron and fluorescence microscopy, cellulose filaments, sheets and nanocomposites with curli fibers were localized in situ at cellular resolution within the physiologically two-layered macrocolony biofilms of this "de-domesticated" strain. As an architectural element, cellulose confers cohesion and elasticity, i.e., tissue-like properties that-together with the cell-encasing curli fiber network and geometrical constraints in a growing colony-explain the formation of long and high ridges and elaborate wrinkles of wild-type macrocolonies. In contrast, a biofilm matrix consisting of the curli fiber network only is brittle and breaks into a pattern of concentric dome-shaped rings separated by deep crevices. These studies now set the stage for clarifying how regulatory networks and in particular c-di-GMP signaling operate in the three-dimensional space of highly structured and "tissue-like" bacterial biofilms.

Biofilm matrix and its regulation in Pseudomonas aeruginosa.

Abstract: Biofilms are communities of microorganisms embedded in extracellular polymeric substances (EPS) matrix. Bacteria in biofilms demonstrate distinct features from their free-living planktonic counterparts, such as different physiology and high resistance to immune system and antibiotics that render biofilm a source of chronic and persistent infections. A deeper understanding of biofilms will ultimately provide insights into the development of alternative treatment for biofilm infections. The opportunistic pathogen Pseudomonas aeruginosa, a model bacterium for biofilm research, is notorious for its ability to cause chronic infections by its high level of drug resistance involving the formation of biofilms. In this review, we summarize recent advances in biofilm formation, focusing on the biofilm matrix and its regulation in P. aeruginosa, aiming to provide resources for the understanding and control of bacterial biofilms.

BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm

Abstract: Biofilms represent the predominant mode of microbial growth in the natural environment. Bacillus subtilis is a ubiquitous Gram-positive soil bacterium that functions as an effective plant growth-promoting agent. The biofilm matrix is composed of an exopolysaccharide and an amyloid fiber-forming protein, TasA, and assembles with the aid of a small secreted protein, BslA. Here we show that natively synthesized and secreted BslA forms surface layers around the biofilm. Biophysical analysis demonstrates that BslA can self-assemble at interfaces, forming an elastic film. Molecular function is revealed from analysis of the crystal structure of BslA, which consists of an Ig-type fold with the addition of an unusual, extremely hydrophobic "cap" region. A combination of in vivo biofilm formation and in vitro biophysical analysis demonstrates that the central hydrophobic residues of the cap are essential to allow a hydrophobic, nonwetting biofilm to form as they control the surface activity of the BslA protein. The hydrophobic cap exhibits physiochemical properties remarkably similar to the hydrophobic surface found in fungal hydrophobins; thus, BslA is a structurally defined bacterial hydrophobin. We suggest that biofilms formed by other species of bacteria may have evolved similar mechanisms to provide protection to the resident bacterial community.

Multiple factors modulate biofilm formation by the anaerobic pathogen Clostridium difficile

Abstract: Bacteria within biofilms are protected from multiple stresses, including immune responses and antimicrobial agents. The biofilm-forming ability of bacterial pathogens has been associated with increased antibiotic resistance and chronic recurrent infections. Although biofilms have been well studied for several gut pathogens, little is known about biofilm formation by anaerobic gut species. The obligate anaerobe Clostridium difficile causes C. difficile infection (CDI), a major health care-associated problem primarily due to the high incidence of recurring infections. C. difficile colonizes the gut when the normal intestinal microflora is disrupted by antimicrobial agents; however, the factors or processes involved in gut colonization during infection remain unclear. We demonstrate that clinical C. difficile strains, i.e., strain 630 and the hypervirulent strain R20291, form structured biofilms in vitro, with R20291 accumulating substantially more biofilm. Microscopic and biochemical analyses show multiple layers of bacteria encased in a biofilm matrix containing proteins, DNA, and polysaccharide. Employing isogenic mutants, we show that virulence-associated proteins, Cwp84, flagella, and a putative quorum-sensing regulator, LuxS, are all required for maximal biofilm formation by C. difficile. Interestingly, a mutant in Spo0A, a transcription factor that controls spore formation, was defective for biofilm formation, indicating a possible link between sporulation and biofilm formation. Furthermore, we demonstrate that bacteria in clostridial biofilms are more resistant to high concentrations of vancomycin, a drug commonly used for treatment of CDI. Our data suggest that biofilm formation by C. difficile is a complex multifactorial process and may be a crucial mechanism for clostridial persistence in the host.

The extracellular matrix Component Psl provides fast-acting antibiotic defense in Pseudomonas aeruginosa biofilms.

Abstract: Bacteria within biofilms secrete and surround themselves with an extracellular matrix, which serves as a first line of defense against antibiotic attack. Polysaccharides constitute major elements of the biofilm matrix and are implied in surface adhesion and biofilm organization, but their contributions to the resistance properties of biofilms remain largely elusive. Using a combination of static and continuous-flow biofilm experiments we show that Psl, one major polysaccharide in the Pseudomonas aeruginosa biofilm matrix, provides a generic first line of defense toward antibiotics with diverse biochemical properties during the initial stages of biofilm development. Furthermore, we show with mixed-strain experiments that antibiotic-sensitive “non-producing” cells lacking Psl can gain tolerance by integrating into Psl-containing biofilms. However, non-producers dilute the protective capacity of the matrix and hence, excessive incorporation can result in the collapse of resistance of the entire community. Our data also reveal that Psl mediated protection is extendible to E. coli and S. aureus in co-culture biofilms. Together, our study shows that Psl represents a critical first bottleneck to the antibiotic attack of a biofilm community early in biofilm development.

Staphylococcus epidermidis Esp Degrades Specific Proteins Associated with Staphylococcus aureus Biofilm Formation and Host-Pathogen Interaction

Abstract: Staphylococcus aureus exhibits a strong capacity to attach to abiotic or biotic surfaces and form biofilms, which lead to chronic infections. We have recently shown that Esp, a serine protease secreted by commensal Staphylococcus epidermidis, disassembles preformed biofilms of S. aureus and inhibits its colonization. Esp was expected to degrade protein determinants of the adhesive and cohesive strength of S. aureus biofilms. The aim of this study was to elucidate the substrate specificity and target proteins of Esp and thereby determine the mechanism by which Esp disassembles S. aureus biofilms. We used a mutant Esp protein (Esp(S235A)) with defective proteolytic activity; this protein did not disassemble the biofilm formed by a clinically isolated methicillin-resistant S. aureus (MRSA) strain, thereby indicating that the proteolytic activity of Esp is essential for biofilm disassembly. Esp degraded specific proteins in the biofilm matrix and cell wall fractions, in contrast to proteinase K, which is frequently used for testing biofilm robustness and showed no preference for proteolysis. Proteomic and immunological analyses showed that Esp degrades at least 75 proteins, including 11 biofilm formation- and colonization-associated proteins, such as the extracellular adherence protein, the extracellular matrix protein-binding protein, fibronectin-binding protein A, and protein A. In addition, Esp selectively degraded several human receptor proteins of S. aureus (e.g., fibronectin, fibrinogen, and vitronectin) that are involved in its colonization or infection. These results suggest that Esp inhibits S. aureus colonization and biofilm formation by degrading specific proteins that are crucial for biofilm construction and host-pathogen interaction.

d-Amino Acids Indirectly Inhibit Biofilm Formation in Bacillus subtilis by Interfering with Protein Synthesis

Abstract: The soil bacterium Bacillus subtilis forms biofilms on surfaces and at air-liquid interfaces. It was previously reported that these biofilms disassemble late in their life cycle and that conditioned medium from late-stage biofilms inhibits biofilm formation. Such medium contained a mixture of D-leucine, D-methionine, D-tryptophan, and D-tyrosine and was reported to inhibit biofilm formation via the incorporation of these D-amino acids into the cell wall. Here, we show that L-amino acids were able to specifically reverse the inhibitory effects of their cognate D-amino acids. We also show that D-amino acids inhibited growth and the expression of biofilm matrix genes at concentrations that inhibit biofilm formation. Finally, we report that the strain routinely used to study biofilm formation has a mutation in the gene (dtd) encoding D-tyrosyl-tRNA deacylase, an enzyme that prevents the misincorporation of D-amino acids into protein in B. subtilis. When we repaired the dtd gene, B. subtilis became resistant to the biofilm-inhibitory effects of D-amino acids without losing the ability to incorporate at least one noncanonical D-amino acid, D-tryptophan, into the peptidoglycan peptide side chain. We conclude that the susceptibility of B. subtilis to the biofilm-inhibitory effects of D-amino acids is largely, if not entirely, due to their toxic effects on protein synthesis.

Life after death: the critical role of extracellular DNA in microbial biofilms.

Abstract: The death and lysis of microbial cells leads to the release of cytoplasmic contents, many of which are rapidly degraded by enzymes. However, some macromolecules survive intact and find new functions in the extracellular environment. There is now strong evidence that DNA released from cells during lysis, or sometimes by active secretion, becomes a key component of the macromolecular scaffold in many different biofilms. Enzymatic degradation of extracellular DNA can weaken the biofilm structure and release microbial cells from the surface. Many bacteria produce extracellular deoxyribonuclease (DNase) enzymes that are apparently tightly regulated to avoid excessive degradation of the biofilm matrix. Interfering with these control mechanisms, or adding exogenous DNases, could prove a potent strategy for controlling biofilm growth.

Small Regulatory RNAs in the Control of Motility and Biofilm Formation in E. coli and Salmonella

Abstract: Biofilm formation in Escherichia coli and other enteric bacteria involves the inverse regulation of the synthesis of flagella and biofilm matrix components such as amyloid curli fibres, cellulose, colanic acid and poly-N-acetylglucosamine (PGA). Physiologically, these processes reflect the transition from growth to stationary phase. At the molecular level, they are tightly controlled by various sigma factors competing for RNA polymerase, a series of transcription factors acting in hierarchical regulatory cascades and several nucleotide messengers, including cyclic-di-GMP. In addition, a surprisingly large number of small regulatory RNAs (sRNAs) have been shown to directly or indirectly modulate motility and/or biofilm formation. This review aims at giving an overview of these sRNA regulators and their impact in biofilm formation in E. coli and Salmonella. Special emphasis will be put on sRNAs, that have known targets such as the mRNAs of the flagellar master regulator FlhDC, the stationary phase sigma factor σS (RpoS) and the key biofilm regulator CsgD that have recently been shown to act as major hubs for regulation by multiple sRNAs.

The EAL domain protein YciR acts as a trigger enzyme in a c-di-GMP signalling cascade in E. coli biofilm control

Abstract: C-di-GMP—which is produced by diguanylate cyclases (DGC) and degraded by specific phosphodiesterases (PDEs)—is a ubiquitous second messenger in bacterial biofilm formation. In Escherichia coli, several DGCs (YegE, YdaM) and PDEs (YhjH, YciR) and the MerR-like transcription factor MlrA regulate the transcription of csgD, which encodes a biofilm regulator essential for producing amyloid curli fibres of the biofilm matrix. Here, we demonstrate that this system operates as a signalling cascade, in which c-di-GMP controlled by the DGC/PDE pair YegE/YhjH (module I) regulates the activity of the YdaM/YciR pair (module II). Via multiple direct interactions, the two module II proteins form a signalling complex with MlrA. YciR acts as a connector between modules I and II and functions as a trigger enzyme: its direct inhibition of the DGC YdaM is relieved when it binds and degrades c-di-GMP generated by module I. As a consequence, YdaM then generates c-di-GMP and—by direct and specific interaction—activates MlrA to stimulate csgD transcription. Trigger enzymes may represent a general principle in local c-di-GMP signalling.

Analytical techniques to study microbial biofilm on abiotic surfaces: pros and cons of the main techniques currently in use.

Abstract: Biofilm is a bacterial lifestyle widespread in microbial world and represents a concern in health care. Despite the great life expectancy related to advanced health care, the increasing numbers of biofilm-mediated infections remain a significant public health challenge. Moreover, the problem of biofilm-mediated infections becomes much more severe when biofilm colonizes medical devices and biomaterials. The public health risk due to microbial biofilm-related infections is a concern that requires full attention. However, the complexity of biofilm makes difficult its exhaustive analysis. Although biofilm represents a major challenge in both microbiological and hygiene areas, at now methods aimed to analyse biofilm formation and development are not standardized yet. Different methods have been employed to qualitatively and quantitatively evaluate biofilm each of which is useful to estimate a peculiar aspect of biofilm lifestyle. In the present review, fifteen assays for the qualitative and quantitative evaluation of bacterial biofilm colonizing abiotic substrates, such as medical devices, prosthesis or surfaces for food production together with advantages and limitations of each method were described and compared. Some methods are suited to quantify biofilm matrix while others are capable to evaluate both living and dead cells or quantify exclusively viable cells in biofilm. In particular, colorimetric methods to evaluate biofilm matrix (crystal violet; 1,9-dimethyl methylen blue and fluorescein-di-acetate methods) or viable cells (LIVE/DEAD BacLight, BioTimer Assay, resazurin, tetrazolium hydroxide salt methods) and genetic methods to estimate the bacterial population (PCR and FISH) are reported. Moreover, a section is dedicated to examine the performances of advanced microscopic techniques employed to study microbial biofilms (mass spectrometry; confocal laser scanning microscopy; Raman spectroscopy and electron microscopy). Because of its complexity, an exhaustive study of biofilm requires a combination of different experimental approaches as biochemical, genetic or physical ones.

Coordinated Cyclic-Di-GMP Repression of Salmonella Motility through YcgR and Cellulose

Abstract: Cyclic di-GMP (c-di-GMP) is a secondary messenger that controls a variety of cellular processes, including the switch between a biofilm and a planktonic bacterial lifestyle. This nucleotide binds to cellular effectors in order to exert its regulatory functions. In Salmonella, two proteins, BcsA and YcgR, both of them containing a c-di-GMP binding PilZ domain, are the only known c-di-GMP receptors. BcsA, upon c-di-GMP binding, synthesizes cellulose, the main exopolysaccharide of the biofilm matrix. YcgR is dedicated to c-di-GMP-dependent inhibition of motility through its interaction with flagellar motor proteins. However, previous evidences indicate that in the absence of YcgR, there is still an additional element that mediates motility impairment under high c-di-GMP levels. Here we have uncovered that cellulose per se is the factor that further promotes inhibition of bacterial motility once high c-di-GMP contents drive the activation of a sessile lifestyle. Inactivation of different genes of the bcsABZC operon, mutation of the conserved residues in the RxxxR motif of the BcsA PilZ domain, or degradation of the cellulose produced by BcsA rescued the motility defect of ΔycgR strains in which high c-di-GMP levels were reached through the overexpression of diguanylate cyclases. High c-di-GMP levels provoked cellulose accumulation around cells that impeded flagellar rotation, probably by means of steric hindrance, without affecting flagellum gene expression, exportation, or assembly. Our results highlight the relevance of cellulose in Salmonella lifestyle switching as an architectural element that is both essential for biofilm development and required, in collaboration with YcgR, for complete motility inhibition.

Bacteriophage therapy for Staphylococcus aureus biofilm-infected wounds: a new approach to chronic wound care.

Abstract: : Background: Bacterial biofilms, which are critical mediators of chronic wounds, remain difficult to treat with traditional methods. Bacteriophage therapy against biofilm has not been rigorously studied in vivo. The authors evaluate the efficacy of a species-specific bacteriophage against Staphylococcus aureus biofilm infected wounds using a validated, quantitative, rabbit ear model. Methods: Six-millimeter dermal punch wounds in New Zealand rabbit ears were inoculated with wild-type or mutant, biofilm-deficient S. aureus. In vivo biofilm was established and maintained using procedures from our previously published wound biofilm model. Wounds were left untreated, or treated every other day with topical S. aureus specific bacteriophage, sharp de bridement, or both. Histologic wound healing and viable bacterial count measurements, and scanning electron microscopy were performed following harvest. Results: Wild-type S. aureus biofilm wounds demonstrated no differences in healing or viable bacteria following bacteriophage application or sharp debridement alone. However, the combination of both treatments significantly improved all measured wound healing parameters (p 0.05) and reduced bacteria counts (p = 0.03), which was confirmed by scanning electron microscopy. Bacteriophage treatment of biofilm-deficient S. aureus mutant wounds alone also resulted in similar trends for both endpoints (p 0.05). Conclusions: Bacteriophages can be an effective topical therapy against S. aureus biofilm infected wounds in the setting of a deficient (mutant) or disrupted (de bridement) biofilm structure. Combination treatment aimed at disturbing the extracellular biofilm matrix, allowing for increased penetration of species-specific bacteriophages, represents a new and potentially effective approach to chronic wound care. These results establish principles for biofilm therapy that may be applied to several different clinical and surgical problems.

Novel Role for the Streptococcus pneumoniae Toxin Pneumolysin in the Assembly of Biofilms

Abstract: Streptococcus pneumoniae is an important commensal and pathogen responsible for almost a million deaths annually in children under five. The formation of biofilms by S. pneumoniae is important in nasopharyngeal colonization, pneumonia, and otitis media. Pneumolysin (Ply) is a toxin that contributes significantly to the virulence of S. pneumoniae and is an important candidate as a serotype-independent vaccine target. Having previously demonstrated that a luxS knockout mutant was unable to form early biofilms and expressed less ply mRNA than the wild type, we conducted a study to investigate the role of Ply in biofilm formation. We found that Ply was expressed in early phases of biofilm development and localized to cellular aggregates as early as 4 h postinoculation. S. pneumoniae ply knockout mutants in D39 and TIGR4 backgrounds produced significantly less biofilm biomass than wild-type strains at early time points, both on polystyrene and on human respiratory epithelial cells, cultured under static or continuous-flow conditions. Ply’s role in biofilm formation appears to be independent of its hemolytic activity, as S. pneumoniae serotype 1 strains, which produce a nonhemolytic variant of Ply, were still able to form biofilms. Transmission electron microscopy of biofilms grown on A549 lung cells using immunogold demonstrated that Ply was located both on the surfaces of pneumococcal cells and in the extracellular biofilm matrix. Altogether, our studies demonstrate a novel role for pneumolysin in the assembly of S. pneumoniae biofilms that is likely important during both carriage and disease and therefore significant for pneumolysin-targeting vaccines under development. IMPORTANCE The bacterium Streptococcus pneumoniae (commonly known as the pneumococcus) is commonly carried in the human nasopharynx and can spread to other body sites to cause disease. In the nasopharynx, middle ear, and lungs, the pneumococcus forms multicellular surface-associated structures called biofilms. Pneumolysin is an important toxin produced by almost all S. pneumoniae strains, extensively studied for its ability to cause damage to human tissue. In this paper, we demonstrate that pneumolysin has a previously unrecognized role in biofilm formation by showing that strains without pneumolysin are unable to form the same amount of biofilm on plastic and human cell substrates. Furthermore, we show that the role of pneumolysin in biofilm formation is separate from the hemolytic activity responsible for tissue damage during pneumococcal diseases. This novel role for pneumolysin suggests that pneumococcal vaccines directed against this protein should be investigated for their potential impact on biofilms formed during carriage and disease.

Base pairing interaction between 5'- and 3'-UTRs controls icaR mRNA translation in Staphylococcus aureus.

Abstract: The presence of regulatory sequences in the 3′ untranslated region (3′-UTR) of eukaryotic mRNAs controlling RNA stability and translation efficiency is widely recognized. In contrast, the relevance of 3′-UTRs in bacterial mRNA functionality has been disregarded. Here, we report evidences showing that around one-third of the mapped mRNAs of the major human pathogen Staphylococcus aureus carry 3′-UTRs longer than 100-nt and thus, potential regulatory functions. We selected the long 3′-UTR of icaR, which codes for the repressor of the main exopolysaccharidic compound of the S. aureus biofilm matrix, to evaluate the role that 3′-UTRs may play in controlling mRNA expression. We showed that base pairing between the 3′-UTR and the Shine-Dalgarno (SD) region of icaR mRNA interferes with the translation initiation complex and generates a double-stranded substrate for RNase III. Deletion or substitution of the motif (UCCCCUG) within icaR 3′-UTR was sufficient to abolish this interaction and resulted in the accumulation of IcaR repressor and inhibition of biofilm development. Our findings provide a singular example of a new potential post-transcriptional regulatory mechanism to modulate bacterial gene expression through the interaction of a 3′-UTR with the 5′-UTR of the same mRNA.

Alginate Lyase Exhibits Catalysis-Independent Biofilm Dispersion and Antibiotic Synergy

Abstract: More than 2 decades of study support the hypothesis that alginate lyases are promising therapeutic candidates for treating mucoid Pseudomonas aeruginosa infections. In particular, the enzymes' ability to degrade alginate, a key component of mucoid biofilm matrix, has been the presumed mechanism by which they disrupt biofilms and enhance antibiotic efficacy. The systematic studies reported here show that, in an in vitro model, alginate lyase dispersion of P. aeruginosa biofilms and enzyme synergy with tobramycin are completely decoupled from catalytic activity. In fact, equivalent antibiofilm effects can be achieved with bovine serum albumin or simple amino acids. These results provide new insights into potential mechanisms of alginate lyase therapeutic activity, and they should motivate a careful reexamination of the fundamental assumptions underlying interest in enzymatic biofilm dispersion.

Iron induces bimodal population development by Escherichia coli

Abstract: Bacterial biofilm formation is a complex developmental process involving cellular differentiation and the formation of intricate 3D structures. Here we demonstrate that exposure to ferric chloride triggers rugose biofilm formation by the uropathogenic Escherichia coli strain UTI89 and by enteric bacteria Citrobacter koseri and Salmonella enterica serovar typhimurium. Two unique and separable cellular populations emerge in iron-triggered, rugose biofilms. Bacteria at the air–biofilm interface express high levels of the biofilm regulator csgD, the cellulose activator adrA, and the curli subunit operon csgBAC. Bacteria in the interior of rugose biofilms express low levels of csgD and undetectable levels of matrix components curli and cellulose. Iron activation of rugose biofilms is linked to oxidative stress. Superoxide generation, either through addition of phenazine methosulfate or by deletion of sodA and sodB, stimulates rugose biofilm formation in the absence of high iron. Additionally, overexpression of Mn-superoxide dismutase, which can mitigate iron-derived reactive oxygen stress, decreases biofilm formation in a WT strain upon iron exposure. Not only does reactive oxygen stress promote rugose biofilm formation, but bacteria in the rugose biofilms display increased resistance to H2O2 toxicity. Altogether, we demonstrate that iron and superoxide stress trigger rugose biofilm formation in UTI89. Rugose biofilm development involves the elaboration of two distinct bacterial populations and increased resistance to oxidative stress.

Role of matrix β-1,3 glucan in antifungal resistance of non-albicans Candida biofilms.

Abstract: Candida biofilm infections pose an increasing threat in the health care setting due to the drug resistance associated with this lifestyle. Several mechanisms underlie the resistance phenomenon. In Candida albicans, one mechanism involves drug impedance by the biofilm matrix linked to β-1,3 glucan. Here, we show this is important for other Candida spp. We identified β-1,3 glucan in the matrix, found that the matrix sequesters antifungal drug, and enhanced antifungal susceptibility with matrix β-1,3 glucan hydrolysis.

Rapid degradation of Streptococcus pyogenes biofilms by PlyC, a bacteriophage-encoded endolysin

Abstract: Objectives Streptococcus pyogenes, or Group A streptococcus (GAS), has a propensity to colonize human tissues and form biofilms. Significantly, these biofilms are a contributing mechanism of antibiotic treatment failure in streptococcal disease. In this study, we evaluate a streptococcal-specific bacteriophage-encoded endolysin (PlyC), which is known to lyse planktonic streptococci, on both static and dynamic streptococcal biofilms. Methods PlyC was benchmarked against antibiotics for MIC, MBC and minimum biofilm eradication concentration (MBEC). A biomass eradication assay based on crystal violet staining of the biofilm matrix was also used to quantify the anti-biofilm properties of PlyC. Finally, conventional fluorescence microscopy and laser scanning confocal microscopy were used to study the effects of PlyC on static and dynamic biofilms of GAS. Results PlyC and antibiotics had similar MIC (range 0.02-0.08 mg/L) and MBC (range 0.02-1.25 mg/L) values on planktonic GAS. However, when GAS grew in biofilms, the MBEC values for antibiotics rose to clinically resistant values (≥400 mg/L) whereas PlyC had MBEC values two orders of magnitude lower by mass and four orders of magnitude lower by molarity than the conventional antibiotics. Laser scanning confocal microscopy revealed that PlyC destroys the biofilm as it diffuses through the matrix in a time-dependent fashion. Conclusions Our findings indicate that while streptococcal cells within a biofilm rapidly become refractory to traditional antibiotics, the biofilm matrix is readily destroyed by the lytic actions of PlyC.

CovR and VicRK regulate cell surface biogenesis genes required for biofilm formation in Streptococcus mutans.

Abstract: The two-component system VicRK and the orphan regulator CovR of Streptococcus mutans co-regulate a group of virulence genes associated with the synthesis of and interaction with extracellular polysaccharides of the biofilm matrix. Knockout mutants of vicK and covR display abnormal cell division and morphology phenotypes, although the gene function defects involved are as yet unknown. Using transcriptomic comparisons between parent strain UA159 with vicK (UAvic) or covR (UAcov) deletion mutants together with electrophoretic motility shift assays (EMSA), we identified genes directly regulated by both VicR and CovR with putative functions in cell wall/surface biogenesis, including gbpB, wapE, smaA, SMU.2146c, and lysM. Deletion mutants of genes regulated by VicR and CovR (wapE, lysM, smaA), or regulated only by VicR (SMU.2146c) or CovR (epsC) promoted significant alterations in biofilm initiation, including increased fragility, defects in microcolony formation, and atypical cell morphology and/or chaining. Significant reductions in mureinolytic activity and/or increases in DNA release during growth were observed in knockout mutants of smaA, wapE, lysM, SMU.2146c and epsC, implying roles in cell wall biogenesis. WapE and lysM mutations also affected cell hydrophobicity and sensitivity to osmotic or oxidative stress. Finally, vicR, covR and VicRK/CovR-targets (gbpB, wapE, smaA, SMU.2146c, lysM, epsC) are up-regulated in UA159 during biofilm initiation, in a sucrose-dependent manner. These data support a model in which VicRK and CovR coordinate cell division and surface biogenesis with the extracellular synthesis of polysaccharides, a process apparently required for formation of structurally stable biofilms in the presence of sucrose.

A spider web strategy of type IV pili-mediated migration to build a fibre-like Psl polysaccharide matrix in Pseudomonas aeruginosa biofilms.

Abstract: Bacterial motilities participate in biofilm development. However, it is unknown how/if bacterial motility affects formation of the biofilm matrix. Psl polysaccharide is a key biofilm matrix component of Pseudomonas aeruginosa. Here we report that type IV pili (T4P)-mediated bacterial migration leads to the formation of a fibre-like Psl matrix. Deletion of T4P in wild type and flagella-deficient strains results in loss of the Psl-fibres and reduction of biofilm biomass in flow cell biofilms as well as pellicles at air-liquid interface. Bacteria lacking T4P-driven twitching motility including those that still express surface T4P are unable to form the Psl-fibres. Formation of a Psl-fibre matrix is critical for efficient biofilm formation, yet does not require flagella and polysaccharide Pel or alginate. The Psl-fibres are likely formed by Psl released from bacteria during T4P-mediated migration, a strategy similar to spider web formation. Starvation can couple Psl release and T4P-driven twitching motility. Furthermore, a radial-pattern Psl-fibre matrix is present in the middle of biofilms, a nutrient-deprived region. These imply a plausible model for how bacteria respond to nutrient-limited local environment to build a polysaccharide-fibre matrix by T4P-dependent bacterial migration strategy. This strategy may have general significance for bacterial survival in natural and clinical settings.

Extracellular DNA within a Nontypeable Haemophilus influenzae-Induced Biofilm Binds Human Beta Defensin-3 and Reduces Its Antimicrobial Activity

Abstract: Biofilms formed by nontypeable Haemophilus influenzae (NTHI) are associated with multiple chronic infections of the airway, including otitis media. Extracellular DNA (eDNA) is part of the biofilm matrix and serves as a structural component. Human β-defensin-3 (hBD-3) is a cationic antimicrobial host defense protein (AMP) critical to the protection of the middle ear. We hypothesized that anionic eDNA could interact with and bind hBD-3 and thus shield NTHI in biofilms from its antimicrobial activity. We demonstrated that recombinant hBD-3 [(r)hBD-3] bound eDNA in vitro and that eDNA in biofilms produced by NTHI in the chinchilla middle ear co-localized with the orthologue of this AMP. Incubation of physiological concentrations of (r)hBD-3 with NTHI genomic DNA abrogated the ability of this innate immune effector to prevent NTHI from forming robust biofilms in vitro. Establishment of NTHI biofilms in the presence of both DNase I and (r)hBD-3 resulted in a marked reduction in the overall height and thickness of the biofilms and rescued the antimicrobial activity of the AMP. Our results demonstrated that eDNA in NTHI biofilms sequestered hBD-3 and thus diminished the biological activity of an important effector of innate immunity. Our observations have important implications for chronicity of NTHI-induced diseases.

Interaction between extracellular lipase LipA and the polysaccharide alginate of Pseudomonas aeruginosa

Abstract: As an opportunistic human pathogen Pseudomonas aeruginosa is able to cause acute and chronic infections. The biofilm mode of life significantly contributes to the growth and persistence of P. aeruginosa during an infection process and mediates the pathogenicity of the bacterium. Within a biofilm mucoid strains of P. aeruginosa simultaneously produce and secrete several hydrolytic enzymes and the extracellular polysaccharide alginate. The focus of the current study was the interaction between extracellular lipase LipA and alginate, which may be physiologically relevant in biofilms of mucoid P. aeruginosa. Fluorescence microscopy of mucoid P. aeruginosa biofilms were performed using fluorogenic lipase substrates. It showed a localization of the extracellular enzyme near the cells. A microtiter plate-based binding assay revealed that the polyanion alginate is able to bind LipA. A molecular modeling approach showed that this binding is structurally based on electrostatic interactions between negatively charged residues of alginate and positively charged amino acids of the protein localized opposite of the catalytic centre. Moreover, we showed that the presence of alginate protected the lipase activity by protection from heat inactivation and from degradation by the endogenous, extracellular protease elastase LasB. This effect was influenced by the chemical properties of the alginate molecules and was enhanced by the presence of O-acetyl groups in the alginate chain. We demonstrate that the extracellular lipase LipA from P. aeruginosa interacts with the polysaccharide alginate in the self-produced extracellular biofilm matrix of P. aeruginosa via electrostatic interactions suggesting a role of this interaction for enzyme immobilization and accumulation within biofilms. This represents a physiological advantage for the cells. Especially in the biofilm lifestyle, the enzyme is retained near the cell surface, with the catalytic centre exposed towards the substrate and is protected from denaturation and proteolytic degradation.

Metabolic spatial variability in electrode-respiring Geobacter sulfurreducens biofilms

Abstract: In this study, we quantified electron transfer rates, depth profiles of electron donor, and biofilm structure of Geobacter sulfurreducens biofilms using an electrochemical-nuclear magnetic resonance microimaging biofilm reactor. Our goal was to determine whether electron donor limitations existed in electron transfer processes of electrode-respiring G. sulfurreducens biofilms. Cells near the top of the biofilms consumed acetate and were metabolically active; however, acetate concentration decreased to below detection within the top 100 microns of the biofilms. Additionally, porosity in the biofilms fell below 10% near the electrode surface, exacerbating exclusion of acetate from the lower regions. The dense biofilm matrix in the acetate-depleted zone acted as an electrical conduit passing electrons generated at the top of the biofilm to the electrode. To verify the distribution of cell metabolic activity, we used uranium as a redox-active probe for localizing electron transfer activity and X-ray absorption spectroscopy to determine the uranium oxidation state. Cells near the top reduced UVI more actively than the cells near the base. High-resolution transmission electron microscopy images showed intact, healthy cells near the top and plasmolyzed cells near the base. Contrary to models proposed in the literature, which hypothesize that cells nearest the electrode surface are the most metabolically active because of a lower electron transfer resistance, our results suggest that electrical resistance through the biofilm does not restrict long-range electron transfer. Cells far from the electrode can respire across metabolically inactive cells, taking advantage of their extracellular infrastructure produced during the initial biofilm formation.

Efficacy of a Marine Bacterial Nuclease against Biofilm Forming Microorganisms Isolated from Chronic Rhinosinusitis

Abstract: Background The persistent colonization of paranasal sinus mucosa by microbial biofilms is a major factor in the pathogenesis of chronic rhinosinusitis (CRS). Control of microorganisms within biofilms is hampered by the presence of viscous extracellular polymers of host or microbial origin, including nucleic acids. The aim of this study was to investigate the role of extracellular DNA in biofilm formation by bacteria associated with CRS. Methods/Principal Findings Obstructive mucin was collected from patients during functional endoscopic sinus surgery. Examination of the mucous by transmission electron microscopy revealed an acellular matrix punctuated occasionally with host cells in varying states of degradation. Bacteria were observed in biofilms on mucosal biopsies, and between two and six different species were isolated from each of 20 different patient samples. In total, 16 different bacterial genera were isolated, of which the most commonly identified organisms were coagulase-negative staphylococci, Staphylococcus aureus and α-haemolytic streptococci. Twenty-four fresh clinical isolates were selected for investigation of biofilm formation in vitro using a microplate model system. Biofilms formed by 14 strains, including all 9 extracellular nuclease-producing bacteria, were significantly disrupted by treatment with a novel bacterial deoxyribonuclease, NucB, isolated from a marine strain of Bacillus licheniformis. Extracellular biofilm matrix was observed in untreated samples but not in those treated with NucB and extracellular DNA was purified from in vitro biofilms. Conclusion/Significance Our data demonstrate that bacteria associated with CRS form robust biofilms which can be reduced by treatment with matrix-degrading enzymes such as NucB. The dispersal of bacterial biofilms with NucB may offer an additional therapeutic target for CRS sufferers.