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05 microbial biofilm_ii_2008


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  • 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
  • 2. Properties of biofilm – review
    • Biofilm is a higher and more complex form of microbial growth
    • It utilizes the opportunity of mutual cooperation of cells
    • It enables the easier transfer of genes
    • It is characterized by an effective homeostasis
    • It shows features of a primitive circulation system
    • It provides a high protection against antimicrobial factors
    • It plays an important part in many significant occasions including medically important conditions
  • 3. Difficult penetration
    • difficult diffusion of the antimicrobial
    • logical explanation
    • not always demonstrable
    • influence of surface charge
    • seems more important
    • e.g. aminoglycosides (+) combine with
    • alginate (-) of P. aeruginosa
  • 4. Altered environment
    • consumption of O 2 in certain areas
    • increase in osmotic pressure
    • accumulation of acidic products of metabolism
    • directly – suppression of the action of
    • antibiotic s
      • chinolones, aminoglycosides
      • indirectly – reducing the growth rate
      • beta-lactams, glycopeptides
  • 5. Altered microbial population
    • common dosage of a toxic substance kills 99 % microbes in the biofilm
    • a subpopulation of cells remains that is highly resistant to the particular substance
    • it is not obvious if the subpopulation existed from the
    • beginning,
    • or if it has evolved by the action of the toxic
    • substance
    • the subpopulation is responsible for the increased resistance of biofilm
  • 6. Biofilm and disease – 1
    • Biofilm takes part in the pathogenesis of
    • chronic infections in general
    • infections of implanted devices
    • the progress of these infections is slow
    • they are without distinctive symptoms
    • acute exacerbations occur occasionally
    • the effect of antibiotic therapy is transitory only
    • after stopping antibiotics infections recur (even if
    • bacteria grown from them appear sensitive in vitro )
  • 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)
  • 9. Problems with biofilm outside the medicine
    • Soiling of surfaces
    • Increase in turbulence of flowing fluid
    • Narrowing the lumen up to blocking the tube
    • Corrosion of pipelines, fuel tanks in aircrafts
    • Blackening of fluids by reduced metals
    • Insulating layer in heat exchangers
    • Resistance of ship hull during passage
    • and many others
  • 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
  • 12. Detection of biofilm – 1 Phenotypic methods
    • staining of biofilm on the inner wall of a vessel (test tube, well in microplate)
    • = Christensen method
    • universal for most microbes
    • character of colonies on agar with Congo red
    • for staphylococci only
    • negative – colonies red , glossy
    • positive – colonies black , rough
  • 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
  • 15. Detection of biofilm – 2
    • Genotypic methods
    • e.g. proof of a gene set called ica - operon responsible for the production of intercellular adhesin in Staphylococcus epidermidis
  • 16. Slime and ica-operon in staphylococci isolated from blood and skin
  • 17. Clinical importance of biofilm detection
    • Biofilm = marker of clinical importance of the strain
    • Is the strain isolated from blood culture clinically relevant?
    • Is it not a contaminant?
    • Detection of biofilm can bring valuable clinical information
    • How to proceed in further treatment?
    • Which antibiotics should be used for destroying the
    • biofilm?
    • Will the common dosage suffice?
  • 18. MIC, MBC and MBEC
    • MIC = minimal inhibition concentration
    • the lowest concentration of an antimicrobial capable of stopping the growth of the tested microbial strain
    • MBC = minimal bactericidal concentration
    • the lowest concentration of an antimicrobial capable of killing the examined strain
    • MBEC = minimal biofilm eradicating concentration
    • the lowest concentration of an antimicrobial capable of killing the strain growing as a biofilm
  • 19. Determination of MBEC – I
    • On U-type microtitration plates with a 96-pin lid (so-called „hedgehog“)
    • Biofilm of the examined strain is grown on the pins
    • The accumulated biofilm is treated with antimicrobials in different concentrations
    • The treated biofilm is broken up with ultrasound
    • The subsequent cell suspensions are cultured and surviving cells are searched for
  • 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
  • 21. Biofilm and health In the body the biofilm plays even a beneficial role :
    • O ur mucosae are coated with the biofilm of normal microbial flora
    • which provides them with relatively efficient protection against pathogens gaining the
    • foothold
  • 22. Summary
    • Biofilm is the natural way of microbial growth
    • It is a microbial community placed in a structured intercellular matter
    • It sticks firmly to solid surfaces
    • Its structure depends on the amount of nutrients in the environment
    • It is more advantageous for microbes both metabolically as well as a protection against adverse conditions
    • Microbes in the biofilm have different properties
    • Biofilm brings problems in many fields
    • Getting rid of biofilm is very difficult
  • 23. Recommended reading material
    • Paul de Kruif: Microbe Hunters
    • Paul de Kruif: Men against Death
    • Axel Munthe: The Story of San Michele
    • Sinclair Lewis: Arrowsmith
    • André Maurois: La vie de Sir Alexander Fleming
    • Could you kindly supply me with another work in
    • connection with microbes or at least medicine?
    • Please mail me your suggestions at:
    • [email_address]
    • Thank you for your attention