The green tea polyphenol EGCG inhibits E. coli biofilm formation by impairing amyloid curli fibre assembly and downregulating the biofilm regulator CsgD via the σE‐dependent sRNA RybB

In bacterial biofilms, which are often involved in chronic infections, cells are surrounded by a self‐produced extracellular matrix that contains amyloid fibres, exopolysaccharides and other biopolymers. The matrix contributes to the pronounced resistance of biofilms against antibiotics and host immune systems. Being highly inflammatory, matrix amyloids such as curli fibres of Escherichia coli can also play a role in pathogenicity. Using macrocolony biofilms of commensal and pathogenic E. coli as a model system, we demonstrate here that the green tea polyphenol epigallocatachin gallate (EGCG) is a potent antibiofilm agent. EGCG virtually eliminates the biofilm matrix by directly interfering with the assembly of curli subunits into amyloid fibres, and by triggering the σ^E cell envelope stress response and thereby reducing the expression of CsgD – a crucial activator of curli and cellulose biosynthesis – due to csgD mRNA targeting by the σ^E‐dependent sRNA RybB. These findings highlight EGCG as a potential adjuvant for antibiotic therapy of biofilm‐associated infections. Moreover, EGCG may support therapies against pathogenic E. coli that produce inflammatory curli fibres along with Shigatoxin.     

Sum of the Parts: Composition and Architecture of the Bacterial Extracellular Matrix

Bacterial biofilms are complex multicellular assemblies that exhibit resistance to antibiotics and contribute to the pathogenesis of serious and chronic infectious diseases. New approaches and quantitative data are needed to define the molecular composition of bacterial biofilms. Escherichia coli biofilms are known to contain polysaccharides and functional amyloid fibers termed curli, yet accurate determinations of biofilm composition at the molecular level have been elusive. The ability to define the composition of the extracellular matrix (ECM) is crucial for the elucidation of structure–function relationships that will aid the development of chemical strategies to disrupt biofilms. We have developed an approach that integrates non-perturbative preparation of the ECM with electron microscopy, biochemistry, and solid-state NMR spectroscopy to define the chemical composition of the intact and insoluble ECM of a clinically important pathogenic bacterium—uropathogenic E. coli. Our data permitted a sum-of-all-the-parts analysis. Electron microscopy revealed supramolecular shell-like structures that encapsulated single cells and enmeshed the bacterial community. Biochemical and solid-state NMR measurements of the matrix and constitutive parts established that the matrix is composed of two major components, curli and cellulose, each in a quantifiable amount. This approach to quantifying the matrix composition is widely applicable to other organisms and to examining the influence of biofilm inhibitors. Collectively, our NMR spectra and the electron micrographs of the purified ECM inspire us to consider the biofilm matrix not as an undefined slime, but as an assembly of polymers with a defined composition and architecture.     

Analyses of the red-dry-rough phenotype of an Escherichia coli O157:H7 strain and its role in biofilm formation and resistance to antibacterial agents

In a previous study, we identified Congo red-binding and -nonbinding phase variants of Escherichia coli serotype O157:H7 strain ATCC 43895. The Congo red-binding variant, strain 43895OR, produced a dry, aggregative colony that was similar to the red, dry, and rough (rdar) phenotype characteristic of certain strains of Salmonella. In contrast, variant 43895OW produced a smooth and white colony morphology. In this study, we show that, similar to rdar strains of Salmonella enterica serovar Typhimurium, strain 43895OR forms large aggregates in broth cultures, firm pellicles at the air-medium interface on glass, and dense biofilms on glass and polystyrene. However, unlike S. enterica serovar Typhimurium, strain 43895OR does not stain positive for cellulose production. When strain 43895OR was fixed on agar, scanning electron microscopy showed cells expressing extracellular matrix (ECM) containing curli fibers. Strain 43895OW was devoid of any ECM or curli fibers on agar but showed expression of curli fibers during attachment to glass. Strain 43895OR produced >4-fold-larger amounts of biofilm than strain 43895OW on polystyrene, glass, stainless steel, and Teflon; formation was >3-fold higher in rich medium than in nutrient-limited medium. Biofilm-associated cells of both strains showed statistically greater resistance (P < 0.05) to hydrogen peroxide and quaternary ammonium sanitizer than their respective planktonic cells. This study shows that the rdar phenotype of E. coli O157:H7 strain 43895OR is important in multicellular growth, biofilm formation, and resistance to sanitizers. However, the lack of cellulose production by strain 43895OR indicates important differences in the ECM composition compared to that of Salmonella.     

Analyses of the Red-Dry-Rough Phenotype of an Escherichia coli O157:H7 Strain and Its Role in Biofilm Formation and Resistance to Antibacterial Agents

In a previous study, we identified Congo red-binding and -nonbinding phase variants of Escherichia coli serotype O157:H7 strain ATCC 43895. The Congo red-binding variant, strain 43895OR, produced a dry, aggregative colony that was similar to the red, dry, and rough (rdar) phenotype characteristic of certain strains of Salmonella. In contrast, variant 43895OW produced a smooth and white colony morphology. In this study, we show that, similar to rdar strains of Salmonella enterica serovar Typhimurium, strain 43895OR forms large aggregates in broth cultures, firm pellicles at the air-medium interface on glass, and dense biofilms on glass and polystyrene. However, unlike S. enterica serovar Typhimurium, strain 43895OR does not stain positive for cellulose production. When strain 43895OR was fixed on agar, scanning electron microscopy showed cells expressing extracellular matrix (ECM) containing curli fibers. Strain 43895OW was devoid of any ECM or curli fibers on agar but showed expression of curli fibers during attachment to glass. Strain 43895OR produced >4-fold-larger amounts of biofilm than strain 43895OW on polystyrene, glass, stainless steel, and Teflon; formation was >3-fold higher in rich medium than in nutrient-limited medium. Biofilm-associated cells of both strains showed statistically greater resistance (P < 0.05) to hydrogen peroxide and quaternary ammonium sanitizer than their respective planktonic cells. This study shows that the rdar phenotype of E. coli O157:H7 strain 43895OR is important in multicellular growth, biofilm formation, and resistance to sanitizers. However, the lack of cellulose production by strain 43895OR indicates important differences in the ECM composition compared to that of Salmonella.     

Cellulose as an Architectural Element in Spatially Structured Escherichia coli Biofilms

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.     

Quantitative Analysis of Amyloid-Integrated Biofilms Formed by Uropathogenic Escherichia coli at the Air-Liquid Interface

Bacterial biofilms are complex multicellular assemblies, characterized by a heterogeneous extracellular polymeric matrix, that have emerged as hallmarks of persistent infectious diseases. New approaches and quantitative data are needed to elucidate the composition and architecture of biofilms, and such data need to be correlated with mechanical and physicochemical properties that relate to function. We performed a panel of interfacial rheological measurements during biofilm formation at the air-liquid interface by the Escherichia coli strain UTI89, which is noted for its importance in studies of urinary tract infection and for its assembly of functional amyloid fibers termed curli. Brewster-angle microscopy and measurements of the surface elasticity (G_s′) and stress-strain response provided sensitive and quantitative parameters that revealed distinct stages during bacterial colonization, aggregation, and eventual formation of a pellicle at the air-liquid interface. Pellicles that formed under conditions that upregulate curli production exhibited an increase in strength and viscoelastic properties as well as a greater ability to recover from stress-strain perturbation. The results suggest that curli, as hydrophobic extracellular amyloid fibers, enhance the strength, viscoelasticity, and resistance to strain of E. coli biofilms formed at the air-liquid interface.     

Community behavior and amyloid-associated phenotypes among a panel of uropathogenic E. coli

Uropathogenic Escherichia coli (UPEC) are the major causative agents of urinary tract infection and engage in a coordinated genetic and molecular cascade to colonize the urinary tract. Disrupting the assembly and/or function of virulence factors and bacterial biofilms has emerged as an attractive target for the development of new therapeutic strategies to prevent and treat urinary tract infection, particularly in the era of increasing antibiotic resistance among human pathogens. UPEC vary widely in their genetic and molecular phenotypes and more data are needed to understand the features that distinguish isolates as more or less virulent and as more robust biofilm formers or poor biofilm formers. Curli are extracellular functional amyloid fibers produced by E. coli that contribute to pathogenesis and influence the host response during urinary tract infection (UTI). We have examined the production of curli and curli-associated phenotypes including biofilm formation among a specific panel of human clinical UPEC that has been studied extensively in the mouse model of UTI. Motility, curli production, and curli-associated biofilm formation attached to plastic were the most prevalent behaviors, shared by most clinical isolates. We discuss these results in the context on the previously reported behavior and phenotypes of these isolates in the murine cystitis model in vivo.     

Common plant flavonoids prevent the assembly of amyloid curli fibres and can interfere with bacterial biofilm formation

Like all macroorganisms, plants have to control bacterial biofilm formation on their surfaces. On the other hand, biofilms are highly tolerant against antimicrobial agents and other stresses. Consequently, biofilms are also involved in human chronic infectious diseases, which generates a strong demand for anti-biofilm agents. Therefore, we systematically explored major plant flavonoids as putative anti-biofilm agents using different types of biofilms produced by Gram-negative and Gram-positive bacteria. In Escherichia coli macrocolony biofilms, the flavone luteolin and the flavonols myricetin, morin and quercetin were found to strongly reduce the extracellular matrix. These agents directly inhibit the assembly of amyloid curli fibres by driving CsgA subunits into an off-pathway leading to SDS-insoluble oligomers. In addition, they can interfere with cellulose production by still unknown mechanisms. Submerged biofilm formation, however, is hardly affected. Moreover, the same flavonoids tend to stimulate macrocolony and submerged biofilm formation by Pseudomonas aeruginosa. For Bacillus subtilis, the flavonone naringenin and the chalcone phloretin were found to inhibit growth. Thus, plant flavonoids are not general anti-biofilm compounds but show species-specific effects. However, based on their strong and direct anti-amyloidogenic activities, distinct plant flavonoids may provide an attractive strategy to specifically combat amyloid-based biofilms of some relevant pathogens.     

Toll‐like receptors 1 and 2 cooperatively mediate immune responses to curli,a common amyloid from enterobacterial biofilms

Responses to host amyloids and curli amyloid fibrils of Escherichia coli and Salmonella enterica serotype Typhimurium are mediated through Toll‐like receptor (TLR) 2. Here we show that TLR2 alone was not sufficient for mediating responses to curli. Instead, transfection experiments with human cervical cancer (HeLa) cells and antibody‐mediated inhibition of TLR signalling in human macrophage‐like (THP‐1) cells suggested that TLR2 interacts with TLR1 to recognize curli amyloid fibrils. TLR1/TLR2 also serves as a receptor for tri‐acylated lipoproteins, which are produced by E. coli and other Gram‐negative bacteria. Despite the presence of multiple TLR1/TLR2 ligands on intact bacterial cells, an inability to produce curli amyloid fibrils markedly reduced the ability of E. coli to induce TLR2‐dependent responses in vitro and in vivo. Collectively, our data suggest that curli amyloid fibrils from enterobacterial biofilms significantly contribute to TLR1/TLR2‐mediated host responses against intact bacterial cells.     

Functional amyloid in Pseudomonas

Amyloids are highly abundant in many microbial biofilms and may play an important role in their architecture. Nevertheless, little is known of the amyloid proteins. We report the discovery of a novel functional amyloid expressed by a Pseudomonas strain of the P. fluorescens group. The amyloid protein was purified and the amyloid‐like structure verified. Partial sequencing by MS/MS combined with full genomic sequencing of the Pseudomonas strain identified the gene coding for the major subunit of the amyloid fibril, termed fapC. FapC contains a thrice repeated motif that differs from those previously found in curli fimbrins and prion proteins. The lack of aromatic residues in the repeat shows that aromatic side chains are not needed for efficient amyloid formation. In contrast, glutamine and asparagine residues seem to play a major role in amyloid formation as these are highly conserved in curli, prion proteins and FapC. fapC is conserved in many Pseudomonas strains including the opportunistic pathogen P. aeruginosa and is situated in a conserved operon containing six genes, of which one encodes a fapC homologue. Heterologous expression of the fapA–F operon in Escherichia coli BL21(DE3) resulted in a highly aggregative phenotype, showing that the operon is involved in biofilm formation.     

Involvement of curli fimbriae in the biofilm formation of Enterobacter cloacae

In this study, we examined the biofilm forming ability, the mRNA expression of curli genes and the morphologies of curli fimbriae and biofilms in clinical isolates of Enterobacter cloacae. The csgBA operon was found in 11 (78.6%) of the 14 isolates. The ability of E. cloacae isolates to form biofilms was significantly correlated with the mRNA expression level of the csgA and csgD genes. The curli protein fimbriae appeared as tangled fibers and the curli-proficient strain formed mature biofilms. Our data suggest that the expression of the curli fimbriae play an important role in biofilm formation in E. cloacae.     

Diversity in biofilm formation and production of curli fimbriae and cellulose of Salmonella Typhimurium strains of different origin in high and low nutrient medium

The biofilm forming behavior of 51 Salmonella Typhimurium strains was determined in Tryptone Soya Broth (TSB) and 20 times diluted TSB (1/20TSB) at 25°C and 37°C. The results indicated that biofilm forming behavior is influenced by environmental conditions and associated with the origin of the strains. Clinical, outbreak-associated and retail product isolates showed dense biofilm formation in both media at 25°C, and in TSB also at 37°C. However, industrial isolates only showed dense biofilm formation in 1/20TSB at 25°C. By enumeration of biofilm cells, LIVE/DEAD staining and SEM analysis of biofilms it was found that the ratio of cells and extracellular matrix is affected by environmental conditions. Indeed, the genes involved in curli fimbriae and cellulose production are highly induced during biofilm formation at 25°C in 1/20TSB. This indicates that these are important matrix components during biofilm formation in 1/20TSB at 25°C and that other factors contribute to biofilm formation of clinical, outbreak-associated and retail product isolates at 37°C and/or nutrient-rich conditions.     

Amyloid peptides derived from CsgA and FapC modify the viscoelastic properties of biofilm model matrices

The bacterial biofilm is a complex environment of cells, which secrete a matrix made of various components, mainly polysaccharides and proteins. An understanding of the precise role of these components in the stability and dynamics of biofilm architecture would be a great advantage for the improvement of anti-biofilm strategies. Here, artificial biofilm matrices made of polysaccharides and auto-assembled peptides were designed, and the influence of bacterial amyloid proteins on the mechanical properties of the biofilm matrix was studied. The model polysaccharides methylcellulose and alginate and peptides derived from the amyloid proteins curli and FapC found in biofilms of Enterobacteriaceae and Pseudomonas, respectively, were used. Rheological measurements showed that the amyloid peptides do not prevent the gelation of the polysaccharides but influence deformation of the matrices under shear stress and modify the gel elastic response. Hence the secretion of amyloids could be for the biofilm a way of adapting to environmental changes.     

Transposon Mutagenesis Identifies Uropathogenic Escherichia coli Biofilm Factors

Uropathogenic Escherichia coli (UPEC), which accounts for 85% of urinary tract infections (UTI), assembles biofilms in diverse environments, including the host. Besides forming biofilms on biotic surfaces and catheters, UPEC has evolved an intracellular pathogenic cascade that culminates in the formation of biofilm-like intracellular bacterial communities (IBCs) within bladder epithelial cells. Rapid bacterial replication during IBC formation augments a build-up in bacterial numbers and persistence within the host. Relatively little is known about factors mediating UPEC biofilm formation and how these overlap with IBC formation. To address this gap, we screened a UPEC transposon mutant library in three in vitro biofilm conditions: Luria broth (LB)-polyvinyl chloride (PVC), YESCA (yeast extract-Casamino Acids)-PVC, and YESCA-pellicle that are dependent on type 1 pili (LB) and curli (YESCA), respectively. Flagella are important in all three conditions. Mutants were identified that had biofilm defects in all three conditions but had no significant effects on the expression of type 1 pili, curli, or flagella. Thus, this approach uncovered a comprehensive inventory of novel effectors and regulators that are involved in UPEC biofilm formation under multiple conditions. A subset of these mutants was found to be dramatically attenuated and unable to form IBCs in a murine model of UTI. Collectively, this study expands our insights into UPEC multicellular behavior that may provide insights into IBC formation and virulence.     

Anti-biofilm activities of essential oils rich in carvacrol and thymol against Salmonella Enteritidis

The aim of the present study was to determine the bioactive compounds in four essential oils (EO’s) from Origanum heracleoticum, Origanum vulgare, Thymus vulgaris and Thymus serpyllum and to assess their antimicrobial and anti-biofilm activity against Salmonella Enteritidis. Strains were previously characterized depending on the expression of the extracellular matrix components cellulose and curli fimbriae as rdar (red, dry and rough) and bdar morphotype (brown, dry and rough). This study revealed that the EO’s and EOC’s (carvacrol and thymol) investigated showed inhibition of biofilm formation at sub-minimum inhibitory concentration. Comparing the efficacy of EO’s and EOC’s in the inhibition of biofilm formation between the strains with different morphotype (rdar and bdar) did not show a statistically significant difference. Results related to the effectiveness of EO’s and EOC’s (the essential oil components, carvacrol and thymol) on eradication of preformed 48?h old biofilms indicated that biofilm reduction occurred in a dose-dependent manner over time.     

大肠杆菌卷曲菌毛的生物合成和致病性

大肠杆菌卷曲菌毛是其菌体表面的一种含纤维素样蛋白质附着器官,出现在大肠杆菌生理和病理过程中。卷曲菌毛可以通过黏附等作用介导大肠杆菌侵袭宿主;作为一种细菌淀粉样蛋白,卷曲菌毛有可能引起淀粉样蛋白相关疾病;卷曲菌毛可以诱导宿主炎症因子水平升高,引起脓毒血症;卷曲菌毛可以和纤维素等一起构成菌外基质,参与生物膜的形成。我们简要综述了大肠杆菌卷曲菌毛的生物合成、生物学功能和致病性。