Miroslav Votava MICROBIAL BIOFILM – I

1. Institute for Microbiology, Medical Faculty of Masaryk Universityand St. Anna Faculty Hospital in Brno Miroslav Votava MICROBIAL BIOFILM – I The 5th lecture for 2nd-year students of General Medicine March 18, 2013

2. Resistance of microbes to drying up – revision Very sensitive: agents of STD – gonococci, treponemes Less sensitive:all Gram-negative bacteria A bit more resistant: skin flora – staphylococci, corynebacteria acidoresistant rods– mycobacteria Rather resistant: xerophiles – actinomycetes, nocardiae, moulds parasite cysts, helminth eggs Highly resistant: bacterial spores

3. Practicalapplication of water shortage – revision Lowering water activity stops action of most microbes → we use it forfoodpreservation Examples: • drying – meat, mushroom, fruit (prunes) • concentration – plum jam • salting – meat, fish, butter • sugaring – sirups, jams, candied fruit

4. The influence of heat – revision The temperature higher than optimum → heatshock and gradual dying of cells The number of killed cells depends on the duration of the exposure to higher temperature The relation between the number of surviving cells and the duration of heating is inversely logarithmic one The time needed for exterminating the whole microbial population depends on its size (on the initial number of microbes)

5. Decimal reduction time – revision The relation between the duration of heating and the number of surviving microbes: Log10 number of survivors 6 D =decimal reduction time= 5 = the time required to reduce 4 the No of microbes to 1/10 = 3 = the time required to kill 90 % of 2 Dmicrobes present (at the 1 specific temperature) 1 2 3 4 5 6 (min)

6. Toxic substances – revision Their influence depends on the concentrationand durationof exposure Various microbes markedly differ in relativeresistance to different types of toxic substances In general (and contrary to drying):G– bacteria are more resistant to toxic substances than G+ bacteria(because of different structure of bacterial cell wall → presence of enzymes in periplasmatic space of G– bacteria) For application it is vital to know the effects of the particular substances used for disinfection

7. Bacterial cell wall – revision G+G– lipoteichoic acidO-antigenlipopoly- inner polysaccharidesaccharide lipidA(endotoxin) murein porin outer membrane lipoprotein ENZYMESperiplasmatic space innermembrane (G–) cytoplasmatic membrane (G+)

8. Sterilization versus disinfection – revision Sterilization = removal of all microorganisms from objects or environment Disinfection = removal of infectious agents from objects and environment or from the body surface Disinfection aims at breaking the chain of infection transmission Biocides = a new general term including also disinfectants

9. Types of disinfectants – revision • Oxidizing agents (peracetic acid, H2O2, O3) • Halogens(hypochlorite, sol. iodi) • Alkylating agents (aldehydes) • Cyclic compounds (cresol, chlorophenols) • Biguanides (chlorhexidine) • Strong acids and alkali(e.g. slaked lime) • Heavy metal compounds (Hg, Ag, Cu, Sn) • Alcohols(ethanol, propanols) • Surface active agents (QAS; e.g. cetrimid) • Others(e.g. crystal violet& other dyes)

10. Relative resistance of different agents to biocides – revision Enveloped viruses herpesviruses Some protozoa very susceptible Trichomonas Gram-positive bacteria Streptococcus Gram-negative bacteria Salmonella Yeasts susceptible Candida Moulds Trichophyton Naked virusesenteroviruses Protozoal cystsrelatively resistant Giardia Acidoresistant rodsMycobacterium Helminth eggsAscaris Bacterial sporesvery resistant Clostridium Coccidia Cryptosporidium Prionsextremely resistant agent of CJD - - -

11. Two forms of microbial growth • Growth in planktonic form Isolated microbial cells float freely in a fluid environment • Growth in biofilm form Result of the natural tendency of microbial cells to stick to one another and to a solid surface and to form a community connected by an extracellular matter

12. Which form is more frequent? • Planktonic form • fairly common in the laboratory (e.g. in nutrient broth) • relatively scarce in a natural environment • Biofilm form • standard and crucial in the natural environment • more advantageous for microbes

13. Definition of biofilm 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

14. Three examples of biofilm • Have you ever slipped on a wet stone in a creek? Certainly – and in was biofilm that you slipped on • Have you an aquarium and do you clean its walls? If you do, what you wipe from them is the biofilm formed by algae • Do you clean your teeth regularly? I hope so and by doing this you remove the biofilm called dental plaque

15. History of biofilm • 1676 Antony van Leeuwehoek bacteria in dental plaque • 1935 C. E. Zobel the first description of biofilm in marine bacteria • 1950 – 1960 first information about problems with the biofilm • 1978 J. W. Costerton drawing attention to the ubiquity of biofilm • 1999 Costerton, Stewart  Greenberg biofilm involvement in persistent infections

16. Microbiology lead astray – 1 • For 100 years since Pasteur and Koch times it never occurred to anybody that in nature bacteria grow in other ways than as a freely floating plankton in seas or as colonies on agar • From the half of the 19th to the half of the 20th century, throughout the whole „golden age of bacteriology“, the only subject of study were planktonic forms If signs of the biofilm growth appeared the experiment was quickly „sewered“

17. Microbiology lead astray – 2 The whole microbiology has been mislead by efforts to examine and investigate pure cultures of planktonically growing cells only, whereas the natural microbial growth is in the form of biofilm The last area of microbiology that started to be concerned with thebiofilm is regrettably the medical microbiology, proud of its achievements with planktonic forms

18. How does the biofilm develop? 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

19. Development of biofilm – attraction Attraction does not concern solid surfaces only but in general the boundaries of phases Prominent in mobile bacteria with flagella How does the bacterium know the proximity of a surface? It sends out chemical signals that diffuse more quickly into free areas while they concentrate in the vicinity of boundaries of phases

20. Development of biofilm – adhesion Bacterialadhesins fimbriae (pilli) colonization factors of enteropathogenic E. coli proteins and lipopolysaccharides of outer membrane generally in most of Gram-negative bacteria slime bothcoagulase-negative and golden staphylococci curli E. coli

21. Development of biofilm – aggregation I 1. Movement by means of flagella (E. coli, Vibrio cholerae) by means of fimbriae (type IV pilli of Pseudomonas aeruginosa) divergent – continuous layer of cells forms convergent – aggregates develop, even of different species (e.g. coaggregation of Streptococcus gordonii + Fusobacterium nucleatum in dental plaque) 2. Multiplication both aggregation and cell division in aggregates lead to the development of microcolonies

22. Development of biofilm – aggregation II 3. Quorum sensing During division individual cells emit chemical signals (homoserinlactones in P. aeruginosa) 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)

23. Development of biofilm – accumulation Production of exopolysaccharides colanic acid (E. coli) alginate (P. aeruginosa) polysaccharide intercellular adhesin (Staph. epidermidis) leads to the development oftypical biofilm architecture Its appearance depends mainly on thenature of the environment

24. Development ofbiofilm – dispersal After reaching the critical amount of biomass or after the reduction of the amount of nutrients in the environment the character of cells at the surface of biofilm changes • e.g. in P. aeruginosa the superficial cells - cease producing alginate - begin producing lyase and flagellin • superficial layer of biofilm starts to disintegrate • cells grow flagella and get loose of biofilm The cells as a planktonicpopulation drift away to look for more suitable environment and to colonize new surfaces The cycle closes…

25. Architecture of biofilm – I 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)

26. Architecture of biofilm – II 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

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

28. Architecture of biofilm – IV

29. Architecture of biofilm – V • 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

30. Architecture of biofilm – VI 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

31. Architecture of biofilm – VII Candida albicans biofilm. Alcian blue has coloured extracellular polysaccharides. Photo: Veronika Holá

32. Architecture of biofilm – VIII Candida albicans biofilm. Toluidin blue. At the photo mushroom-likestructure of the biofilm is obvious. Photo: Veronika Holá

33. Importance of biofilm for the life of microorganisms – I 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

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

35. Resistance of biofilm towards toxic substances 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

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

37. Ad 1. Difficult penetration EXTRACELLULAR MATRIX SERVES AS THE PENETRATION BARRIER • difficultdiffusion of the antimicrobial: logical explanation not always demonstrable • influence of surfacechargeseems more important e.g. aminoglycosides (+) combine with alginate (-) of P. aeruginosa

38. Ad 2. Altered environment LOCAL ALTERATION IN THE BIOFILM ENVIRONMENT (consumption of O2in certain areas, increase in osmotic pressure, accumulation of acidic products of metabolism) AFFECTS THE ACTION OF ANTIBIOTICS directly – suppression of the action of antibiotics chinolones, aminoglycosides indirectly – reducing the growth rateof microbes beta-lactams, glycopeptides

39. Ad 3. 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 • thesubpopulation is responsible for the increased resistance of biofilm

40. Properties of biofilm • 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 important conditions

41. Properties of microbes in biofilm Summary: The properties of microbes growing in the biofilm form arefundamentally different from the properties of microbes growing in the planktonic form; the microbes • express different genes • produce different products (extracellular matrix  flagella) • enjoy a higher degree of protection

42. 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