1. Institute for Microbiology, Medical Faculty of Masaryk University and St. Anna Faculty Hospital in Brno Miroslav Votava, Vladana Woznicová MICROBIAL BIOFILM – II Lecture for 2nd-year students March 1 6 , 200 9

7. Biofilm and disease – 2 Chronic infections of natural bodily surfaces dental caries (oral streptococci , mainly Streptococcus mutans ) periodontitis (Gram-negative oral anaerobes) otitis media ( Haemophilus influenzae ) osteomyelitis ( Staphylococcus aureus ) cholecystitis and cholangoitis (enterobacteria) prostatitis ( Escherichia coli ) subacute bacterial endocarditis (oral streptococci) pneumonia in cystic fibrosis ( Pseudomonas aeruginosa )

8. Biofilm and disease – 3 Chronic infections of artificial surfaces central venous catheters (coagul. neg. staphylococci, candidae) prosthetic heart valves ( Staph . aureus , Staph . epidermidis ) joint prostheses ( Staphylococcus aureus , Staph . epidermidis ) surgical sutures ( Staphylococcus aureus , Staph . epidermidis ) vascular grafts (Gram-positive cocci) endotracheal tubes (various bacteria and yeasts) intrauterine contraceptive devices ( Actinomyces israelii ) urinary catheters ( E. coli or others, mainly Gram-negative rods) contact lenses ( Pseudomonas aeruginosa , Gram-positive cocci)

10. Possibilities of affecting the biofilm – I Prevention of the biofilm development Now: modifying the surface of biomaterials (change of charge) impregnation of biomaterials with antimicrobials (antibiotics, antiseptics) In future: interference with quorum-sensing signals inhibition of extracellular matrix production inhibition of highly resistant persistors development ce se signály typu quorum-sensing inhibice tvorby mimobuněčné hmoty inhibice vzniku vysoce odolných perzistorů

11. Possibilities of affecting the biofilm – II Disrupting the already present biofilm Now: high concentration of an antimicrobial – so-called antibiotic plug in a venous catheter combination of antimicrobials with different mechanisms of action disruption of extracellular matrix – e.g. with enzymes (polysaccharide lyases) In future: use of molecules causing the autodestruction of biofilm

13. Inoculum: 0.5 McFarland scale; culture: Sabouraud broth with 8 % glucose, 48 hrs, 37 °C PS = polystyrene, S = glass PS PS S S Biofilm + Biofilm ─ Biofilm production on glass and on hardened polystyrene

14. Positive production of slime on agar with Congo red Black colonies of a biofilm-positive staphylococcus strain

16. Slime and ica-operon in staphylococci isolated from blood and skin

20. Determination of MBEC – II BIOFILM EXPOSED TO ANTIBIOTICS Biofilm grown on pins of the „hedgehog“ Detection of viable bacteria after the influence of ATB The concentration of ATB decreases from above down Live microbes betray themselves by yellowing the medium in the detection microplate Microbes killed: the medium remains carmine ultrasound PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN