1. Skapandet av "oslemmiga" ytor Olena Rzhepishevska, Umeå Universitet

2. Vilka är vi? Madeleine Ramstedt, kemist, docent Shoghik Hakobyan, kemist, doktorand & Olena Rzhepishevska, mikrobiolog, forskare

3. 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 Kaneko, Y., J Clin Invest, 2007 Rzhepishevska & Ramstedt, unpublished Deffinition: complex communities of microorganisms attached to a surface or interface enclosed in an exsopolysacchraride matrix of microbial and host origin to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)

4. 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

5. Importance of biofilms Biofilms Industry Public health Clinical Pharmathutical Processing Shipping Water Food Distribution pipes Process surfaces Drinking water Home Prosthesis & Biomaterials Orthopaedics Catheters Contact lenses Shunts Pins/staples Infusion lines Tissue Gut Urinary tract Lungs Bone Heart Dental Teeth Gums Tongue Implants 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.

6. 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.

7. Steps in biofilm formation Planctonic cells ReversibleIrreversibleMicrocolonies Macrocolonies (mashroom bodies ) attachment monolayer Pseudomonas aeruginosa Staphylococcus aureus & Staphylococcus epidermidis Primary attachment Accumulative growth Red tails are single protein molecules Modified from Caiazza & O’Toole, 2004 Mack et al. 2004

8. Steps in biofilm formation Vibrio cholerea Van Dellen et al., JBact., 2008 Environment/host (VPS) Host (TCP) Sea water (Ca 2+ ) Reversible (transient) attachment Irreversible (permanent) attachment Monolayer Planctonic cells TCP - toxin-coregulated pilus VPS - Vibrio polysaccharide

9. Biofilm, CLSM, 1:1 mixture of yellow fluorescent P. aeruginosa PAO1 wt & cyan fluorescent P. aeruginosa pilA, Klausen et al, 2003 Flagella and pili are needed to build biofilm FlagellaPili

10. Matrix - the slime Exopolysaccharides (EPS) - physical & chemical protection Alginate – P.aeruginosa; colanic acid – E.coli Rhamnolipids - support mashroom structures Proteins -attachment, protection from host DNA -support structure, antibiotic resistance DNAse treatment of S.aureus biofilm, Mann et al, 2009 Day 2 pretreatment Day 3 post-treatment Mono-rhamnolipidDi-rhamnolipid

11. Communication is everywhere – quorum sensing homoserine lactone

12. N-(3-oxohexanoyl) homoserine lactone, Vibrio fischeri & a shining squid

13. 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 Ga 3+ 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

14. or P OLIMER BRUSH WITH POSITIVE CHARGE P OLIMER BRUSH WITH NEGATIVE CHARGE Polymer brushes vs bacteria

15. Surface charge: METAC +27mV SPM -35mV MEDSAH -16mV POEGMA -2mV PMMA neutral MEDSAHPMMAPOEGMA SPM

16. PUM P Flow breakers Bubble traps Waste Flow chamber Fresh medium Polymer brushes vs bacteria- confocal microscopy

17. Biofilm, name of the polymer brush, and its charge in mV Structure of polymer METAC +27mV POEGMA -2mV PMMA neutral GLASS SPM -35mV MEDSAH -16mV NO MODIFICATION Fluorescence intensity, 515nm B IOFILM AFTER 3 DAYS Polymer brushes vs bacteria- confocal microscopy

18. Polymer brushes vs bacteria - motility ?

19. GaNO 3 Torbamycin Y. Kaneko, 2007 Red–dead; green-alive Antibiotics & gallium Penicillin – blocks the key enzyme in bacterial cell wall synthesis Bacteria produce beta-lactamase to break penicillin Streptomycine – sabotages bacterial protein synthesis Bacteria mutate in a ribosomal proteins

20. Fe 3+ Fe 2+ Iron Hemoproteins- respiration Enzyme active center- metabolic reactions Signaling-regulation of gene expression Iron has many functions in a bacterial cell Ga 3+ Gallium Hemoproteins- respiration Enzyme active center- metabolic reactions Signaling-regulation of gene expression Y. Kaneko, 2007 Bernstein, L. R. 1998 Gallium

21. Bacteria +Ga citrate Bacteria, no Ga Bacteria+Ga DFO No bacteria Gallium protects cells from bacteria

22. Gallium against bacterial toxins

23. Ongoing work

24. 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

25. Tack!!