1. Institute for Microbiology, Medical Faculty of Masaryk University and St. Anna Faculty Hospital in Brno Miroslav Votava MICROBIAL BIOFILM – II MICROBIAL BIOFILM – II The 6th lecture for 2nd-year students of Dentistry March 28, 2012

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)forms at the boundary of phases (usually of the solid and fluid phase) sticks to inert as well as to live surfacessticks to inert as well as to live surfaces is surrounded by an extracellular matter, in which a complex system of channels may formis 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 elevated concentration of signals causes the change of cellular properties: - switching off some so far functioning genes (e.g. a gene for the production of flagellin) - expression of other genes, and from this ensuing ensuing - production of new molecules (in particular exopolysaccharides) exopolysaccharides)

5. Architecture of biofilm I – revision It depends above all on the concentration of nutrients <10 mg/L (mountain streams, lakes, open sea):<10 mg/L (mountain streams, lakes, open sea): heterogeneous mosaic (a thin layer + columns of microcolonies) microcolonies) 10-1000 mg/L (majority of our rivers and ponds):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):  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) lakes, open sea) Heterogeneous mosaic = thin layer of individual cells above which columned microcolonies rise here and there 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 a)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 biofilm on inert surfaces of medical devices

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

12. Properties of biofilm – summary & revision Properties of biofilm – summary & revision Biofilm is a higher and more complex form of microbial growthBiofilm 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 relevant conditionsIt plays an important part in many significant occasions including medically relevant conditions

13. Importance of biofilm for the life of microorganisms – I More favourable environment for the life of microorganisms More favourable environment for the life of microorganisms Possibility of effective cooperation and specialization of cells because of stable mutual position of cells of different species in the intercellular matrix and corresponding presence of various enzymes Considerably easier transfer of genes Effective homeostasis Inside the aerobically established biofilm anaerobic places may occur Primitive circulation system brings in and carries away nutrients, waste products as well as signal molecules

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

15. Resistance of biofilm towards toxic substances MICROBES IN THE BIOFILM FORM 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

16. Possible causes of higher resistance of biofilm 1.More difficult penetration of toxic matter through the biofilm 2.Character of environment in the biofilm is altered 3.Also the microbial population in the biofilm is altered

17. Ad 1. Difficult penetration EXTRACELLULAR MATRIX SERVES AS THE PENETRATION BARRIER difficult diffusion of the antimicrobial:difficult diffusion of the antimicrobial: logical explanation not always demonstrable influence of surface charge seems more importantinfluence of surface charge seems more important e.g. aminoglycosides (+) combine with e.g. aminoglycosides (+) combine with alginate (-) of P. aeruginosa alginate (-) of P. aeruginosa

18. Ad 2. Altered environment Ad 2. 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 of microbes beta-lactams, glycopeptides beta-lactams, glycopeptides

19. Ad 3. 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

20. Dental plaque as a biofilm Dental plaque = adhesive layer at the tooth surface composed of microbes + their products + components from the saliva Localization: 1. supragingival plaque 2. subgingival, a) adherent 2. subgingival, a) adherent b) nonadherent b) nonadherent Composition: in all places Actinomyces spp. Ad 1. yellow, green & purple complex Ad 2. red & orange complex

21. Microbial composition of supragingival dental plaque Higher No. of G+ rods & fibers and G+ cocci (actinomycetes and streptococci) Higher No. of G+ rods & fibers and G+ cocci (actinomycetes and streptococci) Overall bacteria of yellow, green & purple complex Overall bacteria of yellow, green & purple complex Yellow: Streptococcus mutans, S. oralis, S. sanguis Green: Eikenella corrodens, Capnocytophaga sputigena, C. gingivalis, C. ochracea, Aggregatibacter actinomycetemcomitans Green: Eikenella corrodens, Capnocytophaga sputigena, C. gingivalis, C. ochracea, Aggregatibacter actinomycetemcomitans Purple: Veillonela parvula, Actinomyces odontolyticus

22. Microbial composition of subgingival dental plaque Subgingival, a) adherent narrow layer at the tooth root analogy of the supragingival plaque (mainly the green & purple complex) Subgingival, b) nonadherent between the adherent plaque and the gum above all motile G– anaerobes: red & orange complex Red: Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola Orange: Prevotella intermedia, P. nigrescens, Peptostreptococcus micros, Fusobacterium nucleatum ssp. nucl., ssp. vincentii, ssp. polymoprphum, F. periodontium

23. 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)

24. 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)

25. 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)

26. 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

27. 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) 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

28. 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 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

29. 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

30. 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

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

32. 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

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

34. 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?

35. 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

36. 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

37. 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 red ultrasound

38. 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

39. 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

40. 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