Farnesol induces cell detachment from established S. epidermidis biofilms

Antibiotic resistance is a serious problem in Staphylococcus epidermidis infections as many clinical isolates of this organism are resistant to up to eight different antibiotics. The increased resistance to conventional antibiotic therapy has lead to the search for new antimicrobial therapeutic agents. Farnesol, an essential oil found in many plants, has been shown to be active against S. epidermidis. Using a type control strain we recently described that although farnesol was not efficient at killing biofilm bacteria, a strong reduction on biofilm biomass was detected, and we hypothesize that farnesol could, somehow, induce biofilm detachment. In this report, to test our hypothesis we used 36 representative clinical strains of S. epidermidis from different geographic locations and characterized them in terms of genetic variability by multilocus sequence typing and staphylococcal chromosome cassette mec. Strains were tested for biofilm formation, and the presence of ica, bhp and aap genes was determined. Stronger biofilms had always the presence of ica operon but often co-harbored bhp and aap genes. Farnesol was then used in biofilm-forming strains, and biofilm detachment was detected in half of the strains tested. Furthermore, we also showed that farnesol inability to kill biofilm bacteria was not the result of the biofilm structure but was related to high cell density. Our results demonstrate, for the first time, that the biomass reduction previously found by us, and many other groups, is the result not of cell killing but instead is the result of biofilm detachment.We thank Herminia de Lencastre for reviewing the manuscript. Support for this work was provided by project P-99911 from Fundacao Calouste Gulbenkian and CONCORD-HEALTH-F3-2008/Project Number 222718/European Commission. This work was also supported by Fundacao para a Ciencia e a Tecnologia through grant #PEst-OE/EQB/LA0004/2011 awarded to ITQB

Genetically Engineered Alginate Lyase-PEG Conjugates Exhibit Enhanced Catalytic Function and Reduced Immunoreactivity

Alginate lyase enzymes represent prospective biotherapeutic agents for treating bacterial infections, particularly in the cystic fibrosis airway. To effectively deimmunize one therapeutic candidate while maintaining high level catalytic proficiency, a combined genetic engineering-PEGylation strategy was implemented. Rationally designed, site-specific PEGylation variants were constructed by orthogonal maleimide-thiol coupling chemistry. In contrast to random PEGylation of the enzyme by NHS-ester mediated chemistry, controlled mono-PEGylation of A1-III alginate lyase produced a conjugate that maintained wild type levels of activity towards a model substrate. Significantly, the PEGylated variant exhibited enhanced solution phase kinetics with bacterial alginate, the ultimate therapeutic target. The immunoreactivity of the PEGylated enzyme was compared to a wild type control using in vitro binding studies with both enzyme-specific antibodies, from immunized New Zealand white rabbits, and a single chain antibody library, derived from a human volunteer. In both cases, the PEGylated enzyme was found to be substantially less immunoreactive. Underscoring the enzyme's potential for practical utility, >90% of adherent, mucoid, Pseudomonas aeruginosa biofilms were removed from abiotic surfaces following a one hour treatment with the PEGylated variant, whereas the wild type enzyme removed only 75% of biofilms in parallel studies. In aggregate, these results demonstrate that site-specific mono-PEGylation of genetically engineered A1-III alginate lyase yielded an enzyme with enhanced performance relative to therapeutically relevant metrics.Cystic Fibrosis Foundation (Research Development Program)National Center for Research Resources (U.S.) (P20RR018787-06

The Extracellular Matrix Component Psl Provides Fast-Acting Antibiotic Defense in Pseudomonas aeruginosa Biofilms

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.National Institutes of Health (U.S.). National Institute of Environmental Health Sciences (Training Grant in Toxicology 5 T32 ES7020-37

Investigating the Responses of Human Epithelial Cells to Predatory Bacteria

One beguiling alternative to antibiotics for treating multi-drug resistant infections are Bdellovibrio-and-like-organisms (BALOs), predatory bacteria known to attack human pathogens. Consequently, in this study, the responses from four cell lines (three human and one mouse) were characterized during an exposure to different predatory bacteria, Bdellovibrio bacteriovorus HD100, Bacteriovorus BY1 and Bacteriovorax stolpii EB1. TNF-alpha levels were induced in Raw 264.7 mouse macrophage cultures with each predator, but paled in comparison to those obtained with E. coli. This was true even though the latter strain was added at an 11.1-fold lower concentration (p < 0.01). Likewise, E. coli led to a significant (54%) loss in the Raw 264.7 murine macrophage viability while the predatory strains had no impact. Tests with various epithelial cells, including NuLi-1 airway, Caco2, HT29 and T84 colorectal cells, gave similar results, with E. coli inducing IL-8 production. The viabilities of the NuLi-1 and Caco-2 cells were slightly reduced (8%) when exposed to the predators, while T84 viability remained steady. In no cases did the predatory bacteria induce actin rearrangement. These results clearly demonstrate the gentle natures of predatory bacteria and their impacts on human cells.ope

Elevated expression of both mRNA and protein levels of IL-17A in sputum of stable Cystic Fibrosis patients

<p>Abstract</p> <p>Background</p> <p>T helper 17 (Th17) cells can recruit neutrophils to inflammatory sites through production of IL-17, which induces chemokine release. IL-23 is an important inducer of IL-17 and IL-22 production. Our aim was to study the role of Th17 cells in cystic fibrosis (CF) lung disease by measuring IL-17 protein and mRNA levels and IL-22 and IL-23 mRNA in sputum of clinically stable CF patients and by comparing these levels with healthy controls.</p> <p>Methods</p> <p>Sputum induction was performed in adult CF patients outside of an exacerbation and healthy control subjects. IL-17A protein levels were measured in supernatants with cytometric bead array (CBA) and RNA was isolated and quantitative RT-PCR was performed for IL-17A, IL-22 and IL-23.</p> <p>Results</p> <p>We found significantly higher levels of IL-17A protein and mRNA levels (both: p < 0.0001) and IL-23 mRNA levels (p < 0.0001) in the sputum of CF group as compared to controls. We found very low levels of IL-22 mRNA in the CF group. The levels of IL-17 and IL-23 mRNA were higher in patients chronically infected with <it>Pseudomonas aeruginosa </it>(<it>P. aeruginosa</it>) as compared to those who were not chronically infected with <it>P. aeruginosa</it>. The presence of <it>Staphylococcus aureus </it>(<it>S. aureus</it>) on sputum did not affect the IL-17 or IL-23 levels. There was no correlation between IL-17 or IL-23 levels and FEV₁nor sputum neutrophilia.</p> <p>Conclusion</p> <p>The elevated levels of IL-17 and IL-23 might indicate that Th17 cells are implicated in the persistent neutrophil infiltration in CF lung disease and chronic infection with <it>P. aeruginosa</it>.</p

Fructooligosacharides Reduce Pseudomonas aeruginosa PAO1 Pathogenicity through Distinct Mechanisms

Pseudomonas aeruginosa is ubiquitously present in the environment and acts as an opportunistic pathogen on humans, animals and plants. We report here the effects of the prebiotic polysaccharide inulin and its hydrolysed form FOS on this bacterium. FOS was found to inhibit bacterial growth of strain PAO1, while inulin did not affect growth rate or yield in a significant manner. Inulin stimulated biofilm formation, whereas a dramatic reduction of the biofilm formation was observed in the presence of FOS. Similar opposing effects were observed for bacterial motility, where FOS inhibited the swarming and twitching behaviour whereas inulin caused its stimulation. In co-cultures with eukaryotic cells (macrophages) FOS and, to a lesser extent, inulin reduced the secretion of the inflammatory cytokines IL-6, IL-10 and TNF- a . Western blot experiments indicated that the effects mediated by FOS in macrophages are associated with a decreased activation of the NF- k B pathway. Since FOS and inulin stimulate pathway activation in the absence of bacteria, the FOS mediated effect is likely to be of indirect nature, such as via a reduction of bacterial virulence. Further, this modulatory effect is observed also with the highly virulent ptxS mutated strain. Co-culture experiments of P. aeruginosa with IEC18 eukaryotic cells showed that FOS reduces the concentration of the major virulence factor, exotoxin A, suggesting that this is a possible mechanism for the reduction of pathogenicity. The potential of these compounds as components of antibacterial and anti-inflammatory cocktails is discussed.The authors acknowledge financial support from FEDER funds and Fondo Social Europeo through grants from the Spanish Ministry of Economy and Competitiveness (grants SAF2011-22922, SAF2011-22812) the Andalusian regional government Junta de Andalucía (grant CVI-7335) and the Centre of Networked Biomedical Research on Hepatic and Digestive Diseases (CIBERehd) which is funded by the Carlos III Health Institute and the Ramón Areces Foundation, Spain

Utilization and control of ecological interactions in polymicrobial infections and community-based microbial cell factories

Microbial activities are most often shaped by interactions between co-existing microbes within mixed-species communities. Dissection of the molecular mechanisms of species interactions within communities is a central issue in microbial ecology, and our ability to engineer and control microbial communities depends, to a large extent, on our knowledge of these interactions. This review highlights the recent advances regarding molecular characterization of microbe-microbe interactions that modulate community structure, activity, and stability, and aims to illustrate how these findings have helped us reach an engineering-level understanding of microbial communities in relation to both human health and industrial biotechnology

Pseudomonas aeruginosa PilY1 Binds Integrin in an RGD- and Calcium-Dependent Manner

PilY1 is a type IV pilus (tfp)-associated protein from the opportunistic pathogen Pseudomonas aeruginosa that shares functional similarity with related proteins in infectious Neisseria and Kingella species. Previous data have shown that PilY1 acts as a calcium-dependent pilus biogenesis factor necessary for twitching motility with a specific calcium binding site located at amino acids 850–859 in the 1,163 residue protein. In addition to motility, PilY1 is also thought to play an important role in the adhesion of P. aeruginosa tfp to host epithelial cells. Here, we show that PilY1 contains an integrin binding arginine-glycine-aspartic acid (RGD) motif located at residues 619–621 in the PilY1 from the PAK strain of P. aeruginosa; this motif is conserved in the PilY1s from the other P. aeruginosa strains of known sequence. We demonstrate that purified PilY1 binds integrin in vitro in an RGD-dependent manner. Furthermore, we identify a second calcium binding site (amino acids 600–608) located ten residues upstream of the RGD. Eliminating calcium binding from this site using a D608A mutation abolished integrin binding; in contrast, a calcium binding mimic (D608K) preserved integrin binding. Finally, we show that the previously established PilY1 calcium binding site at 851–859 also impacts the protein's association with integrin. Taken together, these data indicate that PilY1 binds to integrin in an RGD- and calcium-dependent manner in vitro. As such, P. aeruginosa may employ these interactions to mediate host epithelial cell binding in vivo