Transcript Document
Skapandet av "oslemmiga" ytor Olena Rzhepishevska, Umeå Universitet Vilka är vi? Madeleine Ramstedt, kemist, docent Shoghik Hakobyan, kemist, doktorand & Olena Rzhepishevska, mikrobiolog, forskare What’s biofilm? Kaneko, Y., J Clin Invest , 2007 Rzhepishevska & Ramstedt, unpublished Deffinition: complex communities of microorganisms attached to a surface or interface enclosed in an exsopolysacchraride matrix of microbial and host origin to produce a spatially organized three dimentional structure (Costerton et al. 1995) Biofilm is a natural way to exist for microorganisms The biofilm on pyritic sediments, Richmond mine, California Edwards et al., Science, 2000 Bacteria appear red around the vessel wall. Red blood cells within the lumen appear pink, and DAPI stained host cell nuclei appear blue. Schaber et al. Infect Immun. 2007 Importance of biofilms Biofilms Industry Pharmathutical Processing Shipping Clinical Prosthesis & Biomaterials Orthopaedics Catheters Contact lenses Shunts Pins/staples Infusion lines Tissue Dental Gut Urinary tract Lungs Bone Heart Teeth Gums Tongue Implants Public health Water Distribution pipes Drinking water Food Process surfaces Home Modified from Jass, Surman, Walker, 2003 Biofilms are responsible for approximately 80% of all microbial infections, and cause 100,000 deaths annually in the USA alone. Surfaces and bacteria - catheters Most common infections Pseudomonas aeruginosa Staphylococcus aureus Biofilms are responsible for approximately 80% of all microbial infections and cause 100,000 deaths annually in the USA alone. Pseudomonas aeruginosa Planctonic cells Steps in biofilm formation monolayer Reversible Irreversible attachment Microcolonies Macrocolonies (mashroom bodies) Modified from Caiazza & O’Toole, 2004 Primary attachment Accumulative growth Red tails are single protein molecules Mack et al. 2004 Staphylococcus aureus & Staphylococcus epidermidis Steps in biofilm formation Vibrio cholerea Van Dellen et al., JBact., 2008 Planctonic cells Monolayer Reversible (transient) attachment Irreversible (permanent) attachment Host (TCP) Environment/host (VPS) Sea water (Ca2+) TCP - toxin-coregulated pilus VPS - Vibrio polysaccharide Flagella and pili are needed to build biofilm Flagella Pili Biofilm, CLSM, 1:1 mixture of yellow fluorescent P. aeruginosa PAO1 wt & cyan fluorescent P. aeruginosa pilA, Klausen et al, 2003 Matrix - the slime Exopolysaccharides (EPS) - physical & chemical protection Alginate – P.aeruginosa; colanic acid – E.coli Mono-rhamnolipid Rhamnolipids - support mashroom structures Proteins -attachment, protection from host DNA -support structure , antibiotic resistance Day 2 pretreatment Day 3 post-treatment DNAse treatment of S.aureus biofilm, Mann et al, 2009 Di-rhamnolipid Communication is everywhere – quorum sensing homoserine lactone N-(3-oxohexanoyl) homoserine lactone, Vibrio fischeri & a shining squid Our contribution to the field 1. The surface charge of anti-bacterial coatings alters motility and biofilm architecture Rzhepishevska O, Hakobyan S, Ruhal R, Gautrot J, Barbero D, Ramstedt M RSC Biomaterials Science 2013 March 2. The antibacterial activity of Ga3+ is influenced by ligand complexation as well as the bacterial carbon source Rzhepishevska O, Ekstrand-Hammarström B, Popp M, Björn E, Bucht A, Sjöstedt A, Antti H, Ramstedt M Antimicrobial Agents & Chemotherapy 2011 Dec 3. The Gallium-saliciliden acylhydrazide complex shows synergistic anti-biofilm effect and inhibits toxin Production by Pseudomonas aeruginosa Journal of Inorganic Biochemistry 2014 Rzhepishevska O, Hakobyan S, Ekstrand-Hammarström B, Nygren Y, Karlsson T, Bucht A, Elofsson M, Boily JF, Ramstedt M Polymer brushes vs bacteria or POLIMER BRUSH WITH POLIMER BRUSH WITH NEGATIVE CHARGE POSITIVE CHARGE Polymer brushes vs bacteria Surface charge: PMMA neutral POEGMA -2mV MEDSAH -16mV PMMA POEGMA SPM -35mV MEDSAH METAC +27mV SPM Polymer brushes vs bacteria- confocal microscopy Flow breakers Bubble traps Flow chamber Waste Fresh medium Polymer brushes vs bacteria- confocal microscopy Biofilm, name of the polymer brush, and its charge in mV Structure of polymer n O METAC +27mV + O n O m POEGMA -2mV N Cl OH O O PMMA neutral NO MODIFICATION n SPM -35mV S O-3 K + O O MEDSAH -16mV n O O + N S O3- Fluorescence intensity, 515nm GLASS O n 1200 1000 800 600 400 200 0 BIOFILM AFTER 3 DAYS Polymer brushes vs bacteria - motility ? Antibiotics & gallium Penicillin – blocks the key enzyme in bacterial cell wall synthesis Red–dead; green-alive Bacteria produce beta-lactamase to break penicillin Streptomycine – sabotages bacterial protein synthesis Bacteria mutate in a ribosomal proteins GaNO3 Torbamycin Y. Kaneko, 2007 Gallium Hemoproteinsrespiration Enzyme active centermetabolic reactions Iron Fe3+ Fe2+ Signaling-regulation of gene expression Iron has many functions in a bacterial cell Hemoproteinsrespiration Enzyme active centermetabolic reactions Gallium Ga3+ Signaling-regulation of gene expression Y. Kaneko, 2007 Bernstein, L. R. 1998 Gallium protects cells from bacteria Bacteria, no Ga No bacteria Bacteria +Ga citrate Bacteria+Ga DFO Gallium against bacterial toxins O H N H2N OH N O Ga H2O H2O O H2O OH Ongoing work O O H N H2N H2N OH H2O O H2O OH H2O H N H2N N Ga H2O Ga O OH O N O H2O H N O H2O OH OH N O Ga H2O H2O O H2O OH Conclusions Biofilm is a natural way of living for bacteria Biofilms are both good and bad On certain surfaces biofilms grow better that on other There are special methods to fight biofilms and these methods are improving Tack!!