A central metabolic circuit controlled by QseC in pathogenic Escherichia coli

The QseC sensor kinase regulates virulence in multiple Gram-negative pathogens, by controlling the activity of the QseB response regulator. We have previously shown that qseC deletion interferes with dephosphorylation of QseB thus unleashing what appears to be an uncontrolled positive feedback loop stimulating increased QseB levels. Deletion of QseC downregulates virulence gene expression and attenuates enterohaemorrhagic and uropathogenic Escherichia coli (EHEC and UPEC), Salmonella typhimurium, and Francisella tularensis. Given that these pathogens employ different infection strategies and virulence factors, we used genome-wide approaches to better understand the role of the QseBC interplay in pathogenesis. We found that deletion of qseC results in misregulation of nucleotide, amino acid, and carbon metabolism. Comparable metabolic changes are seen in EHEC ΔqseC, suggesting that deletion of qseC confers similar pleiotropic effects in these two different pathogens. Disruption of representative metabolic enzymes phenocopied UPEC ΔqseC in vivo and resulted in virulence factor downregulation. We thus propose that in the absence of QseC, the constitutively active QseB leads to pleiotropic effects, impairing bacterial metabolism, and thereby attenuating virulence. These findings provide a basis for the development of antimicrobials targeting the phosphatase activity of QseC, as a means to attenuate a wide range of QseC-bearing pathogens.     

AiiA Quorum-Sensing Quenching Controls Proteolytic Activity and Biofilm Formation by Enterobacter cloacae

The aim of this work was to evaluate a quorum-quenching approach to identify functions regulated by quorum sensing in Enterobacter cloacae. We employed an aiiA transconjugant strain of E. cloacae that synthesizes a lactonase enzyme that hydrolyzes N-acyl homoserine lactone signaling molecules to compare bacterial phenotypes in the presence and absence of quorum signals. The aiiA-expressing strain displayed increased proteolytic activity and intensity of a milk-clotting reaction when compared to the wild-type strain. Although both strains growing on polystyrene plates in rich media and a minimal medium of salts formed biofilms, the wild-type strain exhibited a higher number of adhered cells. On the surface of stainless steel coupons that were submerged in culture media, the number of adhered cells of the wild type contained up to one log more cells compared with the aiiA transconjugant. However, after 48?h of incubation, there was no significant difference between the strains. The results demonstrated that the quorum-sensing system negatively regulates proteolytic activity and is likely involved in the early steps of biofilm formation by E. cloacae 067.     

Inhibition of Candida albicans Biofilm Formation by Farnesol, a Quorum-Sensing Molecule

Farnesol is a quorum-sensing molecule that inhibits filamentation in Candida albicans. Both filamentation and quorum sensing are deemed to be important factors in C. albicans biofilm development. Here we examined the effect of farnesol on C. albicans biofilm formation. C. albicans adherent cell populations (after 0, 1, 2, and 4 h of adherence) and preformed biofilms (24 h) were treated with various concentrations of farnesol (0, 3, 30, and 300 μM) and incubated at 37°C for 24 h. The extent and characteristics of biofilm formation were then assessed microscopically and with a semiquantitative colorimetric technique based on the use of 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide. The results indicated that the effect of farnesol was dependent on the concentration of this compound and the initial adherence time, and preincubation with 300 μM farnesol completely inhibited biofilm formation. Supernatant media recovered from mature biofilms inhibited the ability of planktonic C. albicans to form filaments, indicating that a morphogenetic autoregulatory compound is produced in situ in biofilms. Northern blot analysis of RNA extracted from cells in biofilms indicated that the levels of expression of HWP1, encoding a hypha-specific wall protein, were decreased in farnesol-treated biofilms compared to the levels in controls. Our results indicate that farnesol acts as a naturally occurring quorum-sensing molecule which inhibits biofilm formation, and we discuss its potential for further development and use as a novel therapeutic agent.     

Biofilm Formation Protects Escherichia coli against Killing by Caenorhabditis elegans and Myxococcus xanthus

Enteric bacteria, such as Escherichia coli, are exposed to a variety of stresses in the nonhost environment. The development of biofilms provides E. coli with resistance to environmental insults, such as desiccation and bleach. We found that biofilm formation, specifically production of the matrix components curli and cellulose, protected E. coli against killing by the soil-dwelling nematode Caenorhabditis elegans and the predatory bacterium Myxococcus xanthus. Additionally, matrix-encased bacteria at the air-biofilm interface exhibited ∼40-fold-increased survival after C. elegans and M. xanthus killing compared to the non-matrix-encased cells that populate the interior of the biofilm. To determine if nonhost Enterobacteriaceae reservoirs supported biofilm formation, we grew E. coli on media composed of pig dung or commonly contaminated foods, such as beef, chicken, and spinach. Each of these medium types provided a nutritional environment that supported matrix production and biofilm formation. Altogether, we showed that common, nonhost reservoirs of E. coli supported the formation of biofilms that subsequently protected E. coli against predation.     

Alkaloids Modulate Motility,Biofilm Formation and Antibiotic Susceptibility of Uropathogenic Escherichia coli

Alkaloid-containing natural compounds have shown promise in the treatment of microbial infections. However, practical application of many of these compounds is pending a mechanistic understanding of their mode of action. We investigated the effect of two alkaloids, piperine (found in black pepper) and reserpine (found in Indian snakeroot), on the ability of the uropathogenic bacterium Escherichia coli CFT073 to colonize abiotic surfaces. Sub-inhibitory concentrations of both compounds (0.5 to 10 µg/mL) decreased bacterial swarming and swimming motilities and increased biofilm formation. qRT-PCR revealed a decrease in the expression of the flagellar gene (fliC) and motility genes (motA and motB) along with an increased expression of adhesin genes (fimA, papA, uvrY). Interestingly, piperine increased penetration of the antibiotics ciprofloxacin and azithromycin into E. coli CFT073 biofilms and consequently enhanced the ability of these antibiotics to disperse pre-established biofilms. The findings suggest that these alkaloids can potentially affect bacterial colonization by hampering bacterial motility and may aid in the treatment of infection by increasing antibiotic penetration in biofilms.     

F1C Fimbriae Play an Important Role in Biofilm Formation and Intestinal Colonization by the Escherichia coli Commensal Strain Nissle 1917

Bacterial biofilm formation is thought to enhance survival in natural environments and during interaction with hosts. A robust colonizer of the human gastrointestinal tract, Escherichia coli Nissle 1917, is widely employed in probiotic therapy. In this study, we performed a genetic screen to identify genes that are involved in Nissle biofilm formation. We found that F1C fimbriae are required for biofilm formation on an inert surface. In addition, these structures are also important for adherence to epithelial cells and persistence in infant mouse colonization. The data suggest a possible connection between Nissle biofilm formation and the survival of this commensal within the host. Further study of the requirements for robust biofilm formation may improve the therapeutic efficacy of Nissle 1917.     

IscR Controls Iron-Dependent Biofilm Formation in Escherichia coli by Regulating Type I Fimbria Expression

Biofilm formation is a complex developmental process regulated by multiple environmental signals. In addition to other nutrients, the transition metal iron can also regulate biofilm formation. Iron-dependent regulation of biofilm formation varies by bacterial species, and the exact regulatory pathways that control iron-dependent biofilm formation are often unknown or only partially characterized. To address this gap in our knowledge, we examined the role of iron availability in regulating biofilm formation in Escherichia coli. The results indicate that biofilm formation is repressed under low-iron conditions in E. coli. Furthermore, a key iron regulator, IscR, controls biofilm formation in response to changes in cellular Fe-S homeostasis. IscR regulates the FimE recombinase to control expression of type I fimbriae in E. coli. We propose that iron-dependent regulation of FimE via IscR leads to decreased surface attachment and biofilm dispersal under iron-limiting conditions.     

Genetic Analysis of the RcsC Sensor Kinase from Escherichia coli K-12

The Rcs two-component pathway is involved in the regulation of capsule production in Escherichia coli. RcsC is predicted to be the sensor component of this two-component pathway, and in this study we present the first genetic data that support the role of RcsC as a hybrid sensor kinase.     

Effect of Brominated Furanones on the Formation of Biofilm by Escherichia coli on Polyvinyl Chloride Materials

To study the influence of brominated furanones on the biofilm (BF) formation by Escherichia coli (E. coli) on polyvinyl chloride (PVC) material, and to provide new ways of surface modification of materials to clinically prevent biomaterial centered infection. Three brominated furanones, dissolved in ethanol, furanone-1(3,4-dibromo-5-hydroxyl-furanone), furanone-2(4-bromo-5-(4-methoxypheny)-3-(methylamino)-furanone), and furanone-3(3,4-dibromo-5,5-dimethoxypheny-2(5H)-furanone) with representative chemical structure, were coated on the surfaces of separate PVC materials (1 × 1 cm), respectively. The surface-modified PVC materials were incubated with E. coli and for controls, 75 % ethanol-treated PVC materials were used. This treatment played as control group. The cultivation incubations were for 6, 12, 18, and 24 h. The thickness of bacterial BF and bacterial community quantity unit area on the PVC materials was determined by confocal laser scanning microscopy (CLSM), and the surface structure of bacterial BF formation was examined by scanning electron microscopy (SEM). The results of CLSM indicated the thickness of bacterial BF and bacterial community quantity unit area on PVC materials treated with furanone-3 were significantly lower than that of control at all time points (P < 0.05), whereas, the differences between furanone-1 and furanone-2 groups and control group were not significantly different (P > 0.05). The results of SEM indicated that after 6 h incubation, the quantity of bacterial attachment to the surface of PVC material treated with furanone-3 was lower than the control group. By 18 h incubation there was completely formed BF structure on the surface of control PVC material. However, there was no significant BF formation on the surface of PVC material treated with furanone-3. The impact of different brominated furanones on SA biofilm formation on the surface of PVC materials are different, furanone-3 can inhibit E. coli biofilm formation on the surface of PVC material.     

Biofilm Formation and Dispersal under the Influence of the Global Regulator CsrA of Escherichia coli

The predominant mode of growth of bacteria in the environment is within sessile, matrix-enclosed communities known as biofilms. Biofilms often complicate chronic and difficult-to-treat infections by protecting bacteria from the immune system, decreasing antibiotic efficacy, and dispersing planktonic cells to distant body sites. While the biology of bacterial biofilms has become a major focus of microbial research, the regulatory mechanisms of biofilm development remain poorly defined and those of dispersal are unknown. Here we establish that the RNA binding global regulatory protein CsrA (carbon storage regulator) of Escherichia coli K-12 serves as both a repressor of biofilm formation and an activator of biofilm dispersal under a variety of culture conditions. Ectopic expression of the E. coli K-12 csrA gene repressed biofilm formation by related bacterial pathogens. A csrA knockout mutation enhanced biofilm formation in E. coli strains that were defective for extracellular, surface, or regulatory factors previously implicated in biofilm formation. In contrast, this csrA mutation did not affect biofilm formation by a glgA (glycogen synthase) knockout mutant. Complementation studies with glg genes provided further genetic evidence that the effects of CsrA on biofilm formation are mediated largely through the regulation of intracellular glycogen biosynthesis and catabolism. Finally, the expression of a chromosomally encoded csrA'-'lacZ translational fusion was dynamically regulated during biofilm formation in a pattern consistent with its role as a repressor. We propose that global regulation of central carbon flux by CsrA is an extremely important feature of E. coli biofilm development.     

High-Throughput Microfluidic Method To Study Biofilm Formation and Host-Pathogen Interactions in Pathogenic Escherichia coli

Biofilm formation and host-pathogen interactions are frequently studied using multiwell plates; however, these closed systems lack shear force, which is present at several sites in the host, such as the intestinal and urinary tracts. Recently, microfluidic systems that incorporate shear force and very small volumes have been developed to provide cell biology models that resemble in vivo conditions. Therefore, the objective of this study was to determine if the BioFlux 200 microfluidic system could be used to study host-pathogen interactions and biofilm formation by pathogenic Escherichia coli. Strains of various pathotypes were selected to establish the growth conditions for the formation of biofilms in the BioFlux 200 system on abiotic (glass) or biotic (eukaryotic-cell) surfaces. Biofilm formation on glass was observed for the majority of strains when they were grown in M9 medium at 30°C but not in RPMI medium at 37°C. In contrast, HRT-18 cell monolayers enhanced binding and, in most cases, biofilm formation by pathogenic E. coli in RPMI medium at 37°C. As a proof of principle, the biofilm-forming ability of a diffusely adherent E. coli mutant strain lacking AIDA-I, a known mediator of attachment, was assessed in our models. In contrast to the parental strain, which formed a strong biofilm, the mutant formed a thin biofilm on glass or isolated clusters on HRT-18 monolayers. In conclusion, we describe a microfluidic method for high-throughput screening that could be used to identify novel factors involved in E. coli biofilm formation and host-pathogen interactions under shear force.     

Pseudomonas aeruginosa Promotes Escherichia coli Biofilm Formation in Nutrient-Limited Medium

Biofilms have been implicated as an important reservoir for pathogens and commensal enteric bacteria such as Escherichia coli in natural and engineered water systems. However, the processes that regulate the survival of E. coli in aquatic biofilms have not been thoroughly studied. We examined the effects of hydrodynamic shear and nutrient concentrations on E. coli colonization of pre-established Pseudomonas aeruginosa biofilms, co-inoculation of E. coli and P. aeruginosa biofilms, and P. aeruginosa colonization of pre-established E. coli biofilms. In nutritionally-limited R2A medium, E. coli dominated biofilms when co-inoculated with P. aeruginosa, and successfully colonized and overgrew pre-established P. aeruginosa biofilms. In more enriched media, P. aeruginosa formed larger clusters, but E. coli still extensively overgrew and colonized the interior of P. aeruginosa clusters. In mono-culture, E. coli formed sparse and discontinuous biofilms. After P. aeruginosa was introduced to these biofilms, E. coli growth increased substantially, resulting in patterns of biofilm colonization similar to those observed under other sequences of organism introduction, i.e., E. coli overgrew P. aeruginosa and colonized the interior of P. aeruginosa clusters. These results demonstrate that E. coli not only persists in aquatic biofilms under depleted nutritional conditions, but interactions with P. aeruginosa can greatly increase E. coli growth in biofilms under these experimental conditions.     

Glycopeptidolipid Acetylation Affects Sliding Motility and Biofilm Formation in Mycobacterium smegmatis

The absence of glycopeptidolipids (GPLs) abolishes the ability of mycobacteria both to slide over the surface of motility plates and to form biofilms on polyvinyl chloride. In a screen for biofilm-defective mutants of Mycobacterium smegmatis mc(2)155, a new mutant was obtained that resulted in partial inhibition of both processes and also showed an intermediate rough colony morphology. The mariner transposon insertion mapped to a GPL biosynthesis gene (atf1) which encodes a putative acetyltranferase involved in the transfer of acetyl groups to the glycopeptide core. Physical characterization of the GPLs from the atf1 mutant demonstrated that they were not acetylated.     

Motility influences biofilm architecture in Escherichia coli

Eight Escherichia coli strains were studied in minimal medium with a continuous flow system using confocal microscopy. K12 wild-type strains ATCC 25404 and MG1655 formed the best biofilms (∼43 μm thick, 21 to 34% surface coverage). JM109, DH5α, and MG1655 motA formed intermediate biofilms (∼13 μm thick, 41 to 58% surface coverage). BW25113, MG1655 qseB, and MG1655 fliA had poor biofilms (surface coverage less than 5%). The best biofilm-formers, ATCC 25404 and MG1655, displayed the highest motility, whereas the worst biofilm former, BW25113, was motility-impaired. The differences in motility were due to differences in expression of the motility loci qseB, flhD, fliA, fliC, and motA (e.g., qseB expression in MG1655 was 139-fold higher than BW25113 and 209-fold higher than JM109). Motility affected the biofilm architecture as those strains which had poor motility (E. coli JM109, E. coli MG1655 motA, and DH5α) formed flatter microcolonies compared with MG1655 and ATCC 25404, which had more dramatic vertical structures as a result of their enhanced motility. The presence of flagella was also found to be important as qseB and fliA mutants (which lack flagella) had less biofilm than the isogenic paralyzed motA strain (threefold less thickness and 15-fold less surface coverage).