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 March 24, 2014

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

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

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)

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) mg/L (majority of our rivers and ponds): 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)

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

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

Architecture of biofilm IV – revision

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

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

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

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

Importance of biofilm for the life of microorganisms I – revision More favourable environment for the life of microorganisms 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

Importance of biofilm for the life of microorganisms II – revision 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

Resistance of biofilm towards toxic substances – revision 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

Possible causes of higher resistance of biofilm – revision 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 …

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)

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)

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)

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

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

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

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

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

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

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

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

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?

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

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

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

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 This provides them with relatively efficient protection against pathogens gaining the This provides them with relatively efficient protection against pathogens gaining the foothold foothold

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

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 Thank you for your attention