Biomolecular Mechanisms of Pseudomonas aeruginosa and Escherichia coli Biofilm Formation

Pseudomonas aeruginosa and Escherichia coli are the most prevalent Gram-negative biofilm forming medical device associated pathogens, particularly with respect to catheter associated urinary tract infections. In a similar manner to Gram-positive bacteria, Gram-negative biofilm formation is fundamentally determined by a series of steps outlined more fully in this review, namely adhesion, cellular aggregation, and the production of an extracellular polymeric matrix. More specifically this review will explore the biosynthesis and role of pili and flagella in Gram-negative adhesion and accumulation on surfaces in Pseudomonas aeruginosa and Escherichia coli. The process of biofilm maturation is compared and contrasted in both species, namely the production of the exopolysaccharides via the polysaccharide synthesis locus (Psl), pellicle Formation (Pel) and alginic acid synthesis in Pseudomonas aeruginosa, and UDP-4-amino-4-deoxy-l-arabinose and colonic acid synthesis in Escherichia coli. An emphasis is placed on the importance of the LuxR homologue sdiA; the luxS/autoinducer-II; an autoinducer-III/epinephrine/norepinephrine and indole mediated Quorum sensing systems in enabling Gram-negative bacteria to adapt to their environments. The majority of Gram-negative biofilms consist of polysaccharides of a simple sugar structure (either homo- or heteropolysaccharides) that provide an optimum environment for the survival and maturation of bacteria, allowing them to display increased resistance to antibiotics and predation.

Biofilm formation by Bacillus subtilis: new insights into regulatory strategies and assembly mechanisms

Biofilm formation is a social behaviour that generates favourable conditions for sustained survival in the natural environment. For the Gram-positive bacterium Bacillus subtilis the process involves the differentiation of cell fate within an isogenic population and the production of communal goods that form the biofilm matrix. Here we review recent progress in understanding the regulatory pathways that control biofilm formation and highlight developments in understanding the composition, function and structure of the biofilm matrix.

Tyrosine phosphorylation and dephosphorylation in Burkholderia cenocepacia affect biofilm formation, growth under nutritional deprivation, and pathogenicity

Burkholderia cenocepacia, a member of the B. cepacia complex (Bcc), is an opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis. Tyrosine phosphorylation has emerged as an important post-translational modification modulating the physiology and pathogenicity of Bcc bacteria. Here, we investigated the predicted bacterial tyrosine kinases BCAM1331 and BceF, and the low molecular weight protein tyrosine phosphatases BCAM0208, BceD and BCAL2200 of B. cenocepacia K56-2. We show that BCAM1331, BceF, BCAM0208 and BceD contributed to biofilm formation, while BCAL2200 was required for growth in nutrient-limited conditions. Multiple deletions of either tyrosine kinase or low molecular weight protein tyrosine phosphatases genes resulted in attenuation of B. cenocepacia intramacrophage survival and reduced pathogenicity in the Galleria mellonella larvae infection model. Experimental evidence indicates that BCAM1331 displays a reduced
tyrosine autophosphorylation activity compared to BceF. Using the artificial substrate p-nitrophenyl phosphate, the phosphatase activity of the three low molecular weight protein tyrosine phosphatases demonstrated similar kinetic parameters. However, only BCAM0208 and BceD could dephosphorylate BceF. Further, BCAL2200 becomes tyrosine phosphorylated in vivo and catalyzes its auto-dephosphorylation. Together, our data suggest that despite having similar biochemical activities low molecular weight protein tyrosine phosphatases and tyrosine kinases have both overlapping and specific roles in the physiology of B. cenocepacia.

Zinc as an agent for the prevention of biofilm formation by pathogenic bacteria

Les biofilms sont des communautés structurées de micro-organismes enrobées dans une matrice extracellulaire. Les biofilms sont impliqués dans la persistance de plusieurs maladies infectieuses et la matrice extracellulaire du biofilm protège les bactéries contre les cellules du système immunitaire de l'hôte, les antibiotiques et les désinfectants. Récemment notre laboratoire a démontré que le zinc inhibe la formation de biofilm chez Actinobacillus pleuropneumoniae, une bactérie pathogène du porc. Le but de cette étude est d'évaluer l'effet du zinc sur la croissance et la formation du biofilm chez différentes bactéries pathogènes du porc, telles que Bordetella bronchiseptica, Escherichia coli, Haemophilus parasuis, Salmonella, Staphylococcus aureus et Streptococcus suis. Les bactéries ont été cultivées dans des plaques de 96 puits sous condition optimale de formation de biofilm et les biofilms ont été colorés au cristal violet. La présence du biofilm a été confirmée par microscopie confocale à balayage laser à l’aide du marqueur fluorescent FilmTracerTM FM ® 1-43. À des concentrations micromolaires, le zinc inhibe faiblement la croissance bactérienne et bloque d'une manière dose-dépendante la formation de biofilm d’A. pleuropneumoniae, Salmonella Typhimurium et H. parasuis. De plus, la formation de biofilm de E. coli, S. aureus et S. suis a été faiblement inhibée par le zinc. Nos résultats indiquent que le zinc a un effet inhibiteur sur la formation de biofilm de la plupart des pathogènes bactériens d'origine porcine. Cependant, le mécanisme sous-jacent de l'activité anti-biofilm du zinc reste à être caractérisé.

Biofilm formation as a novel phenotypic feature of adherent-invasive Escherichia coli (AIEC)

Crohn's disease (CD) is a high morbidity chronic inflammatory disorder of unknown aetiology. Adherent-invasive Escherichia coli (AIEC) has been recently implicated in the origin and perpetuation of CD. Because bacterial biofilms in the gut mucosa are suspected to play a role in CD and biofilm formation is a feature of certain pathogenic E. coli strains, we compared the biofilmformation capacity of 27 AIEC and 38 non-AIEC strains isolated from the intestinal mucosa. Biofilmformation capacity was then contrasted with the AIEC phenotype, the serotype, the phylotype, andthe presence of virulence genes. Results: Specific biofilm formation (SBF) indices were higher amongst AIEC than non-AIEC strains(P = 0.012). In addition, 65.4% of moderate to strong biofilms producers were AIEC, whereas74.4% of weak biofilm producers were non-AIEC (P = 0.002). These data indicate that AIEC strainswere more efficient biofilm producers than non-AIEC strains. Moreover, adhesion (P = 0.009) andinvasion (P = 0.003) indices correlated positively with higher SBF indices. Additionally, motility(100%, P < 0.001), H1 type flagellin (53.8%, P < 0.001), serogroups O83 (19.2%, P = 0.008) and O22(26.9%, P = 0.001), the presence of virulence genes such as sfa/focDE (38.5%, P = 0.003) and ibeA(26.9%, P = 0.017), and B2 phylotype (80.8%, P < 0.001) were frequent characteristics amongstbiofilm producers.Conclusion: The principal contribution of the present work is the finding that biofilm formationcapacity is a novel, complementary pathogenic feature of the recently described AIEC pathovar. Characterization of AIEC specific genetic determinants, and the regulatory pathways, involved in biofilm formation will likely bring new insights into AIEC pathogenesis

Influence of the Photorhabdus luminescens phosphomannose isomerase gene, manA, on mannose utilization, exopolysaccharide structure, and biofilm formation

Extracellular polysaccharide (EPS) is produced by diverse bacterial pathogens and fulfills assorted roles, including providing a structural matrix for biofilm formation and more specific functions in virulence, such as protection against immune defenses. We report here the first investigation of some of the genes important for biofilm formation in Photorhabdus luminescens and demonstrate the key role of the phosphomannose isomerase gene, manA, in the structure of functional EPS. Phenotypic analyses of a manA-deficient mutant showed the importance of EPS in motility, insect virulence, and biofilm formation on abiotic surfaces as well as the requirement of this gene for the use of mannose as the sole carbon source. Conversely, this defect had no apparent impact on symbiosis with the heterorhabditid nematode vector. A more detailed analysis of biofilm formation revealed that the manA mutant was able to attach to surfaces with the same efficiency as that of the wild-type strain but could not develop the more extended biofilm matrix structures. A compositional analysis of P. luminescens EPS reveals how the manA mutation has a major effect on the formation of a complete, branched EPS.

Effects of single- and multi-strain probiotics on biofilm formation and in vitro adhesion to bladder cells by urinary tract pathogens

Inhibition of biofilm seems to be a major mechanism of urinary tract pathogen exclusion, related to, and possibly dependent upon, the probiotic ability to reduce environmental pH. Exclusion via competition of binding sites is a possible in vivo mechanism for these probiotics. If an additive or synergistic effect exists between strains within a mixture, it does not manifest itself in a greater effect through these two inhibitory mechanisms.

Effects of the antimicrobial peptide gomesin on the global gene expression profile, virulence and biofilm formation of Xylella fastidiosa

In the xylem vessels of susceptible hosts, such as citrus trees, Xylella fastidiosa forms biofilm-like colonies that can block water transport, which appears to correlate to disease symptoms. Besides aiding host colonization, bacterial biofilms play an important role in resistance against antimicrobial agents, for instance antimicrobial peptides (AMPs). Here, we show that gomesin, a potent AMP from a tarantula spider, modulates X. fastidiosa gene expression profile upon 60 min of treatment with a sublethal concentration. DNA microarray hybridizations revealed that among the upregulated coding sequences, some are related to biofilm production. In addition, we show that the biofilm formed by gomesin-treated bacteria is thicker than that formed by nontreated cells or cells exposed to streptomycin. We have also observed that the treatment of X. fastidiosa with a sublethal concentration of gomesin before inoculation in tobacco plants correlates with a reduction in foliar symptoms, an effect possibly due to the trapping of bacterial cells to fewer xylem vessels, given the enhancement in biofilm production. These results warrant further investigation of how X. fastidiosa would respond to the AMPs produced by citrus endophytes and by the insect vector, leading to a better understanding of the mechanism of action of these molecules on bacterial virulence.

The ΔF508-CFTR mutation results in increased biofilm formation by Pseodomonas aeruginosa by increasing iron availability

Enhanced antibiotic resistance of Pseudomonas aeruginosa in the cystic fibrosis (CF) lung is thought to be due to the formation of biofilms. However, there is no information on the antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells or on the effects of airway cells on biofilm formation by P. aeruginosa. Thus we developed a coculture model and report that airway cells increase the resistance of P. aeruginosa to tobramycin (Tb) by >25-fold compared with P. aeruginosa grown on abiotic surfaces. Therefore, the concentration of Tb required to kill P. aeruginosa biofilms on airway cells is 10-fold higher than the concentration achievable in the lungs of CF patients. In addition, CF airway cells expressing ΔF508-CFTR significantly enhanced P. aeruginosa biofilm formation, and ΔF508 rescue with wild-type CFTR reduced biofilm formation. Iron (Fe) content of the airway in CF is elevated, and Fe is known to enhance P. aeruginosa growth. Thus we investigated whether enhanced biofilm formation on ΔF508-CFTR cells was due to increased Fe release by airway cells. We found that airway cells expressing ΔF508-CFTR released more Fe than cells rescued with WT-CFTR. Moreover, Fe chelation reduced biofilm formation on airway cells, whereas Fe supplementation enhanced biofilm formation on airway cells expressing WT-CFTR. These data demonstrate that human airway epithelial cells promote the formation of P. aeruginosa biofilms with a dramatically increased antibiotic resistance. The ΔF508-CFTR mutation enhances biofilm formation, in part, by increasing Fe release into the apical medium.

The type II secretion system and its ubiquitous lipoprotein substrate, SsIE are required for biofilm formation and virulence of enteropathogenic escherichia coli

Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrhea in infants in developing countries. We have identified a functional type II secretion system (T2SS) in EPEC that is homologous to the pathway responsible for the secretion of heat-labile enterotoxin by enterotoxigenic E. coli. The wild-type EPEC T2SS was able to secrete a heat-labile enterotoxin reporter, but an isogenic T2SS mutant could not. We showed that the major substrate of the T2SS in EPEC is SslE, an outer membrane lipoprotein (formerly known as YghJ), and that a functional T2SS is essential for biofilm formation by EPEC. T2SS and SslE mutants were arrested at the microcolony stage of biofilm formation, suggesting that the T2SS is involved in the development of mature biofilms and that SslE is a dominant effector of biofilm development. Moreover, the T2SS was required for virulence, as infection of rabbits with a rabbit-specific EPEC strain carrying a mutation in either the T2SS or SslE resulted in significantly reduced intestinal colonization and milder disease.

A kinetic model for Xylella fastidiosa adhesion, biofilm formation, and virulence

Xylella fastidiosa is the causal agent of citrus variegated chlorosis and Pierce's disease which are the major threat to the citrus and wine industries. The most accepted hypothesis for Xf diseases affirms that it is a vascular occlusion caused by bacterial biofilm, embedded in an extracellular translucent matrix that was deduced to be the exopolysaccharide fastidian. Fourier transform infrared spectroscopy analysis demonstrated that virulent cells which form biofilm on glass have low fastidian content similar to the weak virulent ones. This indicates that high amounts of fastidian are not necessary for adhesion. In this paper we propose a kinetic model for X fastidiosa adhesion, biofilm formation, and virulence based on electrostatic attraction between bacterial surface proteins and xylem walls. Fastidian is involved in final biofilm formation and cation sequestration in dilute sap. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.