Microbial Adhesion to Surfaces

Transcription

1 Microbial Adhesion to Surfaces René P. Schneider Laboratório de Microbiologia Ambiental Departamento de Microbiologia Instituto de Ciências Biomédicas Universidade de São Paulo São Paulo, Brasil

2 First phase: macroscopic approach to surface cell Cells are transported from the medium to the vicinity of the surface, where the further approach becomes controlled by surface forces. cell Conditioning film substratum

3 Macroscopic approach to surface Stagnant fluids Bacteria approach surfaces either by: Active displacement: motile microbes (speeds of up to 500mm/s) Passive displacement: non-motile organisms, through settling and diffusion (approx. 1 cell length/minute). cell diffusion settling

4 Macroscopic approach to surface Dynamic fluids: laminar flow Velocity profile Diffusive boundary layer

5 Macroscopic approach to surface Dynamic fluids: turbulent flow Diffusive boundary layer Velocity distribution Transition zone Viscous sublayer (laminar flow) Hydrodynamic boundary layer

6 Second phase: further approach to surface, controlled by surface forces Adhesion in the secondary minimum cell cell Conditioning film substratum

7 Forces between surfaces: The electric double layer

8 Forces between surfaces: The electric double layer Fonte: Myers: Surfaces, Interfaces & Colloids

9 Forces between surfaces: The electric double layer Fonte: Shaw, Introdução à Química dos Colóides e de Superfícies, Edgar Blücher, 1975

10 Electrostatic repulsive forces between surfaces: thickness of the double layer Double layer thickness: effect of concentration and valency of ions of electrolyte Concentration thickness (nm) (M) 1:1 1:2 2:2 1:3 2: ,4 17,6 15,2 15,2 13, ,6 5,6 4,8 4,8 4, ,0 1,8 1,5 1,5 1, ,0 0,6 0,5 0,5 0,4 1 0,3 0,2 0,2 0,2 0,1

11 Ionic strengths typical of biofilm-supporting media Rijnaarts et al. Coll. Surf. B: Biointerfaces 14: 179 (1999)

12 Intermolecular and interionic forces Act between molecules and ions. Ensure structural stability of substrata Mediate the interaction of substrates with other components: - particles (bacteria) - dissolved molecules - dissolved ions - other materials - solvents

13 Intermolecular and interionic forces Tipe of force Energy Equilibrium Distance (kj/mol) (Å) Primary forces: Ionic Covalent Metalic Secondary forces: molecules particles London nm Debye 3 gas phase Keesom 25 gas phase Hidrogen bonds ,4-3,1 Ionic (attraction/repulsion) nm Ion-dipole

14 Microbial adhesion: standardisation of interaction force data The standard unit for comparison of forces of interaction between molecules or surfaces is the product of the Boltzmann constant (1,38 x ) and the absolute temperature (K). The value of this product at 300K (aproximately room temperature) is: 1 kt = 4,1 x J If the attraction energy remains below this value, thermal motion (Brownian motion) will predominate and the adhesive interactions will be unstable.

15 Long range interaction forces between surfaces in liquids Two classes: 1. Electrostatic (or Coulombic) depend on the availability of charged entities (ions, molecules, particles). 2. Van der Waals: - London universal forces which act between all molecules.

16 Interaction energy (kt) Forces emanating from hydrophilic surface 0,0001M -40 vdw 0,001M -60 AB 0,1M 1M Distance from the substratum surface (nm) electrostatic forces 30

17 Net forces acting between negatively charged substratum and bacterium: classical DLVO approach Interaction energy (kt) ,0001M 0,001M 0,1M 1M Distance from the substratum surface (nm)

18 Net forces acting between negatively charged substratum and bacterium: extended DLVO approach Interaction energy (kt) ,0001M 0,001M 0,1M 1M Distance from substratum surface (nm)

19 Third phase: irreversible adhesion Irreversible adhesion through the establishment of strong bonds between microbial adhesive polimers and substratum cell cell Conditioning film substratum

20 Substratum : Surface roughness, texture Corrosion products on glass surface Linsmeier SW 90, Dez. 2000

21 Substratum : Surface roughness, texture % of total attached Citrobacter freundii located at grain boundaries Stainless steel surface Geesey et al., Corr. Sci. 38: 73 (1996) % of total surface allocated to grain boundaries

22 Substratum : Surface roughness, texture Corrosion products on metal surface Pannoni e Wolynec CH 57,p 58, 1989

23 Substrata : microscopic heterogeneities Local ceramic Imported ceramic Cristalline structure of ceramics from Saby Abyad, Syria, 8000 anos Mommsen & Schneiderl SW 82, Nov 2000

24 Substrata: cell surfaces Cell surface of Gram-negative microbe Tortora et al., Artmed

25 Cell surface: morphological heterogeneity Atomic force microscopic image of the surface of a Xenopus levis cell Anczykowski SW 97, Dez 1999

26 Substrata: cell surfaces Glicocalix of Psychrobacter Immobilis sp. SW8. Bar: 0,1 mm Leslie et al., Coll. Surf. A Physicochem. Eng. Asp. 73, 165 (1993). S-layer of Thermoanaerobacter thermohydrosulfuricus Sára & Sleytr., SW 95, nov. 1999

27 Comparison of force profiles near substratum surface of bacterium and a polimer (protein) Y P = Y B = -30mV Electrolyte 1:1, 0,1M Temperature: 25 C R B : 0,5 mm A 123B : 1x10-21 J R P : 5nm A 123P : 5 x J Fonte: Garbassi et al., Polymer Surfaces

28 Measurement of forces between interfaces Surface force apparatus

29 Interactions between Streptavidin and Biotin surfaces: parameters Leckband et al., Biochemistry, 33: 4611 (1994)

30 Leckband et al., Biochemistry, 33: 4611 (1994) Steric repulsion between two Streptavidin surfaces

31 Effect of melting temperature of phospholipid bilayer on the interaction between Streptavidin and Biotin Leckband et al., Biochemistry, 33: 4611 (1994)

32 Interaction of Streptavidin and biotin surfaces: force profiles at different separation distances Interaction Force Leckband et al., Biochemistry, 33: 4611 (1994)

33 LPS and adhesion of E. coli Ong et al., Langmuir 15: (1999)

34 LPS and adhesion of E. coli D21f2: D21 Ong et al., Langmuir 15: (1999)

35 LPS and adhesion of E. coli D21f2: D21 Ong et al., Langmuir 15: (1999)

36 LPS and adhesion of Pseudomonas aeruginosa Makin & Beveridge. 142: (1996). A-band: B-band

37 Steric hindrance in microbial adhesion Pseudomonas putida Burkholderia cepacia Camesano & Logan Env. Sci. Tecnol. 34: (2000)

38 Mechanisms that control adhesion of different bacteria to surfaces at different ionic strengths C1: Arthrobacter sp.; C3: Rhodococcus sp. C5: Corybnebacterium sp. C6: Corynebacterium sp. P1: Pseudomonas oleovorans; P3: Pseudomonas sp. P4: Pseudomonas putida -log [ionic strength (M)] Rijnaarts et al. Coll. Surf. B: Biointerfaces 14: 179 (1999)

39 Adsorption : Termodynamic analysis D ads G = (D ads H TD ads S) < 0 D ads G Gibbs free energy of adsorption D ads H energy released as heat (enthalpy) TD ads S energy released as entropy (a measure of the degree of freedom of movement of molecules)

40 Protein adsorption: thermodynamic analysis D ads G = (D ads H TD ads S) < 0 Fonte: Norde: Pure & Applied Chemistry, 66, 491 (1994)

41 Protein adsorption: thermodynamic analysis Processes involved in protein adsoprtion to surfaces: Dehydration of substratum surface Dehydration of protein surface Protonation/deprotonation generating or cancelling electric surface charges Overlap of electrical fields of protein and substratum Incorporation or expulsion of ions from the protein-substratum interface Alteration of protein structure

42 Adhesion What is the role of the conditioning film? cell cell substratum

43 Microbial footprints Different types of microbial footprints

44 Removal of cells in washing steps of adhesion assays Most papers report Retention and not Adhesion! Fonte: Busscher et al. In Microbial Cell Surface Hydrophobicity (Doyle, Rosenberg, eds.)

45 Removal of cells in washing steps of adhesion assays Fonte:Bos et al. FEMS Microbiol. Rev. 23, 179 (1999).

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