1. Institute for Microbiology, Medical Faculty of Masaryk University and St. Anna Faculty Hospital in Brno Miroslav Votava MICROBIAL BIOFILM – II MICROBIAL BIOFILM – II Lecture for 2nd-year students March 17, 2008

2. Properties of biofilm – review Properties of biofilm – review Biofilm is a higher and more complex form of microbial growth It utilizes the opportunity of mutual cooperation of cellsIt utilizes the opportunity of mutual cooperation of cells It enables the easier transfer of genesIt enables the easier transfer of genes It is characterized by an effective homeostasisIt is characterized by an effective homeostasis It shows features of a primitive circulation systemIt shows features of a primitive circulation system It provides a high protection against antimicrobial factorsIt provides a high protection against antimicrobial factors It plays an important part in many significant occasions including medically important conditionsIt plays an important part in many significant occasions including medically important conditions

3. Protection given by biofilm to microorganisms in the environment: against amoebae, phages, phages, desiccation, desiccation, washing away, washing away, toxic substances toxic substances in the body: against phagocytes, washing away, washing away, complement, complement, antibodies, antibodies, antibiotics antibiotics

4. Resistance of biofilm towards toxic substances – review MICROBES IN THE BIOFILM ARE ALWAYS MORE RESISTANT THAN IN THE PLANKTONIC FORM Higher resistance applies also to disinfectants and antibioticsHigher resistance applies also to disinfectants and antibiotics Differences in sensitivity sometimes amount up to 3 ordersDifferences in sensitivity sometimes amount up to 3 orders General mechanism of the higher resistance is not knownGeneral mechanism of the higher resistance is not known In each microbe-antimicrobial combination the mechanism can be differentIn each microbe-antimicrobial combination the mechanism can be different

5. Possible causes of higher resistance of biofilm – review 1.More difficult penetration of toxic matter through the biofilm 2.Altered character of environment in the biofilm 3.Altered microbial population in the biofilm

6. Difficult penetration EXTRACELLULAR MATRIX AS THE PENETRATION BARRIER difficult diffusion of the antimicrobialdifficult diffusion of the antimicrobial logical explanation not always demonstrable influence of surface chargeinfluence of surface charge seems more important e.g. aminoglycosides (+) combine with e.g. aminoglycosides (+) combine with alginate (-) of P. aeruginosa alginate (-) of P. aeruginosa

7. Altered environment Altered environment LOCAL ALTERATION IN THE BIOFILM ENVIRONMENT consumption of O 2 in certain areas increase in osmotic pressure accumulation of acidic products of metabolism AFFECTS THE ACTION OF ANTIBIOTICS directly – suppression of the action of directly – suppression of the action of antibiotics antibiotics chinolones, aminoglycosides chinolones, aminoglycosides indirectly – reducing the growth rate beta-lactams, glycopeptides beta-lactams, glycopeptides

8. Altered microbial population common dosage of a toxic substance kills 99 % microbes in the biofilmcommon dosage of a toxic substance kills 99 % microbes in the biofilm a subpopulation of cells remains that is highly resistant to the particular substancea subpopulation of cells remains that is highly resistant to the particular substance it is not obvious if the subpopulation existed from the it is not obvious if the subpopulation existed from the beginning, beginning, or if it has evolved by the action of the toxic or if it has evolved by the action of the toxic substance substance the subpopulation is responsible for the increased resistance of biofilmthe subpopulation is responsible for the increased resistance of biofilm

9. Biofilm and disease – 1 Biofilm takes part in the pathogenesis of 1. chronic infections in general 2. infections of implanted devices the progress of these infections is slow the progress of these infections is slow they are without distinctive symptoms they are without distinctive symptoms acute exacerbations occur occasionally acute exacerbations occur occasionally the effect of antibiotic therapy is transitory only the effect of antibiotic therapy is transitory only after stopping antibiotics infections recur (even if after stopping antibiotics infections recur (even if bacteria grown from them appear sensitive in vitro) bacteria grown from them appear sensitive in vitro)

10. 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)

11. 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)

12. Problems with biofilm outside the medicine Soiling of surfacesSoiling of surfaces Increase in turbulence of flowing fluidIncrease in turbulence of flowing fluid Narrowing the lumen up to blocking the tubeNarrowing the lumen up to blocking the tube Corrosion of pipelines, fuel tanks in aircraftsCorrosion of pipelines, fuel tanks in aircrafts Blackening of fluids by reduced metalsBlackening of fluids by reduced metals Insulating layer in heat exchangersInsulating layer in heat exchangers Resistance of ship hull during passageResistance of ship hull during passage and many others and many others

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

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

15. Detection of biofilm – 1 Phenotypic methods staining of biofilm on the inner wall of a vessel (test tube, well in microplate) 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 character of colonies on agar with Congo red for staphylococci only negative – colonies red, glossy positive – colonies black, rough

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

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

18. 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 e.g. proof of a gene set called ica- operon responsible for the production of intercellular adhesin in Staphylococcus epidermidis

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

20. Clinical importance of biofilm detection Biofilm = marker of clinical importance of the strainBiofilm = 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 informationDetection of biofilm can bring valuable clinical information How to proceed in further treatment? Which antibiotics should be used for destroying the Which antibiotics should be used for destroying the biofilm? biofilm? Will the common dosage suffice?

21. MIC, MBC and MBEC MIC, MBC and MBEC MIC = minimal inhibition concentrationMIC = minimal inhibition concentration the lowest concentration of an antimicrobial capable of stopping the growth of the tested microbial strain MBC = minimal bactericidal concentrationMBC = minimal bactericidal concentration the lowest concentration of an antimicrobial capable of killing the examined strain MBEC = minimal biofilm eradicating concentrationMBEC = minimal biofilm eradicating concentration the lowest concentration of an antimicrobial capable of killing the strain growing as a biofilm

22. Determination of MBEC – I On U-type microtitration plates with a 96-pin lid (so-called „hedgehog“)On U-type microtitration plates with a 96-pin lid (so-called „hedgehog“) Biofilm of the examined strain is grown on the pinsBiofilm of the examined strain is grown on the pins The accumulated biofilm is treated with antimicrobials in different concentrationsThe accumulated biofilm is treated with antimicrobials in different concentrations The treated biofilm is broken up with ultrasoundThe treated biofilm is broken up with ultrasound The subsequent cell suspensions are cultured and surviving cells are searched forThe subsequent cell suspensions are cultured and surviving cells are searched for

23. Determination of MBEC – II BIOFILM EXPOSED TO ANTIBIOTICS Biofilm grown on pins of the „hedgehog“ Detection of viable bacteria after the influence of ATB PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN 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

24. Biofilm and health In the body the biofilm plays even a beneficial role: Our mucosae are coated with the biofilm of normal microbial flora Our mucosae are coated with the biofilm of normal microbial flora which provides them with relatively efficient protection against pathogens gaining the which provides them with relatively efficient protection against pathogens gaining the foothold foothold

25. Summary Biofilm is the natural way of microbial growthBiofilm is the natural way of microbial growth It is a microbial community placed in a structured intercellular matterIt is a microbial community placed in a structured intercellular matter It sticks firmly to solid surfacesIt sticks firmly to solid surfaces Its structure depends on the amount of nutrients in the environmentIts 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 conditionsIt is more advantageous for microbes both metabolically as well as a protection against adverse conditions Microbes in the biofilm have different propertiesMicrobes in the biofilm have different properties Biofilm brings problems in many fieldsBiofilm brings problems in many fields Getting rid of biofilm is very difficultGetting rid of biofilm is very difficult

26. 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: mvotava@med.muni.cz Thank you for your attention