Microbial Biofilm – II: The 6th Lecture for 2nd-year students

1. Institute for Microbiology, Medical Faculty of Masaryk Universityand St. Anna Faculty Hospital in Brno Miroslav Votava MICROBIAL BIOFILM – II The 6th lecture for 2nd-year students March 24, 2014

2. Definition of biofilm – revision Microbial biofilm is a community of microorganisms that • forms at the boundary of phases (usually of the solid and fluid phase) • sticks to inert as well as to live surfaces • is surrounded by an extracellular matter, in which a complex system of channels may form

3. Development of the biofilm – revision Development of biofilm = cyclic process 1. Attraction of planktonic cells to a surface 2. Adhesion of planktonic cells to the surface 3. Aggregation of cells and the development of colonies –quorum-sensing phenomenon 4. Accumulation of exopolysaccharide matrix (slime) – development of typical architecture 5. Dispersal of cells from the surface of biofilm

4. Quorum sensing – revision During division individual cells emit chemical signals After reaching a particular number of cells (quorum) the elevatedconcentration of signals causes the change of cellular properties: - switching off some so far functioninggenes (e.g. a gene for the production of flagellin) - expression of other genes,and from this ensuing - production of new molecules (in particular exopolysaccharides)

5. Architecture of biofilm I – revision It depends above all on theconcentration of nutrients • <10 mg/L(mountain streams, lakes, open sea): heterogeneous mosaic(a thin layer + columns of microcolonies) • 10-1000 mg/L(majority of our rivers and ponds): complex system with channels(created by mushroom-like, partially merging microcolonies) • 1000 mg/L(in the environment of macroorganism): compact biofilm(almost without traces of channels)

6. Architecture of biofilm II– revision Low concentrations of nutrients (0.1 – 10 mg/L – mountain streams, lakes, open sea) Heterogeneous mosaic = thin layer of individual cells above which columned microcolonies rise here and there

7. Architecture of biofilm III – revision Medium concentration of nutrients (10 – 1000 mg/L – eutrophic water environment) System with channels = mushroom-shaped microcolonies partially merging together, interwoven with water channels

8. Architecture of biofilm IV – revision

9. Architecture of biofilm V – revision • High concentrations of nutrients (>1000 mg/L – in the macroorganism) • compact biofilm = closely interconnected numerous microcolonies almost without traces of possible channels • polymicrobial = e.g. dental plaque, normal microflora of mucous membranes

10. Architecture of biofilm VI – revision High concentrations of nutrients (>1000 mg/L – in the macroorganism) compact biofilm = closely interconnected numerous microcolonies almost without traces of possible channels b) monomicrobial = e.g. chronic osteomyelitis biofilm on inert surfaces of medical devices

11. Properties of microbes in biofilm – revision The properties of microbes growing in the biofilm form are fundamentally differentfrom the properties of microbes growing in the planktonic form; the microbes in biofilm • express different genes • produce different products (extracellular matrix  flagella) • enjoy a higher degree of protection

12. Properties of biofilm – summary &revision • Biofilm is a higher and morecomplex 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 effectivehomeostasis • It shows features of a primitive circulation system • It provides a high protection against antimicrobial factors • It plays an important part in many significantoccasions including medically relevant conditions

13. Importance of biofilm for the life of microorganisms I – revision More favourable environment for the life of microorganisms Possibility of effective cooperation and specialization of cells Considerably easier transfer of genes Effective homeostasis Primitive circulation system

14. Importance of biofilm for the life of microorganisms II – revision Protection against harmful influences in environment: against amoebae, phages, dessication, washing away, toxic substances in macroorganism: against phagocytes, washing away, complement, antibodies, antibiotics

15. Resistance of biofilm towards toxic substances– revision MICROBES IN THEBIOFILM FORMAREALWAYS MORE RESISTANTTHAN IN THE PLANKTONIC FORM • Higher resistance applies also to disinfectants and antibiotics • Differences in sensitivity sometimes amount up to 3 orders • General mechanism of the higher resistance is not known • In each microbe-antimicrobial combination the mechanism can be different

16. Possible causes of higher resistance of biofilm – revision • More difficultpenetration of toxic matter through the biofilm • Character of environment in the biofilmisaltered • Also the microbial population in the biofilmisaltered …

17. 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)

18. Biofilm and disease – 2 Chronic infections of natural bodily surfaces dental caries(oral streptococci, mainlyStreptococcus 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)

19. 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(Staphylococcusaureus, Staph. epidermidis) surgical sutures(Staphylococcusaureus, 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)

20. 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

21. Possibilities of affecting the biofilm – I Prevention of the biofilm development Now:modifying thesurface of biomaterials (change of charge) impregnation of biomaterials with antimicrobials (antibiotics, antiseptics) In future:interferencewith 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

22. 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

23. 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

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

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

26. Detection of biofilm – 2 • Genotypic methods • e.g. proof of a gene set called ica- operon responsible for the production of intercellular adhesin inStaphylococcus epidermidis

27. Slime and ica-operon in staphylococciisolated from blood and skin

28. 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?

29. 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

30. Determination ofMBEC – 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 survivingcells are searched for

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

32. Biofilm and health In the body the biofilm plays even a beneficial role: • Our mucosae are coated with the biofilm of normal microbial flora • This provides them with relatively efficient protection againstpathogens gaining the • foothold

33. 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 bothmetabolically 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

34. 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 Hans Zinsser: Rats, Lice, and History mvotava@med.muni.cz Thank you for your attention