Hu, Yifan

Bakterielle biofilm er aggregater, hvor mikroorganismer danner sammenhængende kolonier fastgjort til faste overflader eller indesluttet i menneskelige og animalske værtsorganismer. Bakterielle biofilm er af stor betydning og kan være årsag til alvorlige patologiske og økonomiske skader. Bakterielle biofilms komplekse sammensætning indeholder protein, polysaccharid, ekstracellulært DNA (e-DNA), peptidoglycaner, lipider og phospholipider ”Extra-cellular polymeric substances”, EPS). Disse komponenter er vigtige både i biofilmenes indledende dannelsesfaser og i opretholdelse af selve biofilmstrukturerne. I mit ph.d.-projekt er atomar kraftspektroskopi, AFM og elektrokemi anvendt til at undersøge tre pathogene og medicinsk vigtige bakterielle biofilm, Pseudomonas aeruginosa (cystisk fibrose lungebetændelse), Staphylococcus epidermidis (kontaminering fra katedre og andet medicinsk udstyr) samt Steroptococcus mutans (dental caries).  AFM blev anvendt til en undersøgelse af levende individuelle cellers overefladeadhæsion repræsenteret ved fire forskellige Staphylococcus epidermidis stammer i vandigt medium. Stammerne var udvalgt ud fra deres specielle overfladeproteiner knyttet til indledende overfladebinding. Højopløst AFM viste ingen påviselige forskelle mellem de fire  stammer. Adhæsionskortlægning baseret på hydrofobt modificerede AFM tippe sammenlignet med rene hydrofile siliciumnitrid tippe viste heller ikke nævneværdige forskelle. Dette indikerer, at hydrofobe kræfter ikke er afgørende i bakteriel adhæsion. To centrale inhibitorer fandtes at have stor effekt på adhæsionen mellem de rene tippe og  bakterierne.  AFM og elektrokemi blev dernæst kombineret i en undersøgelse af biofilmdannelse på Au(111)-overflader for at bestemme elektrostatisk overfladeladning og vækstmønstre for Streptococcus mutans biofilm. Fem positivt [Ru(NH3)6]3+/2+, [Co(phen)3]3+/2+ og [Co(terpy)2]3+/2+ (phen = 1,10-phenanthrolin, terpy, 2,2’,2”-terpyridin) og negativt ladede, [Fe(CN)6]3-/4- og [IrCl6]3-/4- redoxprober blev udvalgt til cyklisk voltammetri. Inhibering og voltammetriske mønstre viste, at S. mutans biofilm er negativt ladede. Tilsætning af 10 DNAase peger på, at de negative ladninger i vid udstrækning kommer fra DNA udskilt af biofilmene (e-DNA).  AFM og elektrokemi blev kombineret i en videre undersøgelse af Streptococcus mutans biofilmdannelse på rene Au(111)-overflader og Au(111)-overflader modificeret af selvsamlende molekylære monolag (SAMs) af thiolbaserede molekyler. Fire SAM molekyler blev udvalgt som prober for reduktiv desorption (RD). To er lange ligekædede  thioler med enten en hydrofob (hexadecanthiol) eller en hydrofil terminal gruppe (mercaptohexadecansyre) terminal funktionel gruppe. To andre var de korte, strukturelt stive linkermolekyler L-cystein og cysteamin. Alle fire molekyler udviste stærke RD signaler både i PBS buffer ved pH 7,4 og i 0,1 M NaOH opløsning. Både AFM og de  elektrokemiske data viser videre, at biofilmene er bundet stærkere til de hydrofobe end til de hydrofile overflader.  AFM blev endelig anvendt på to andre bakterietyper, Pseudomonas aeruginosa og Pseudomonas putida og deres sammenhæng med EPS. Forskellige mutantstammer blev anvendt til at undersøge Pel og Psl polysacchariders og type IV pili under P. aeruginosa biofilm udvikling. Denne undersøgelse indikerer, at polysaccharider og e-DNA bidrager  til P. aeruginosa biofilm udviklingen. Proteinklynger kunne ses under P. putida biofilmdannelse, men flere undersøgelser er påkrævet for at kunne identificere eller skelne overfladeproteinerne Lap A og Lap F.  Kombinationen af AFM og elektrokemi er en ny indgangsvinkel til at forstå interfaseområdet mellem bakterielle biofilm, det vandige reaktionsmedium og en ren eller modificeret metaloverflade. Kommende indsatser vil være rettet mod højopløst kortlægning af levende bakterier og kontrol af biofilmdannelsen på SAM-modificerede  Au(111)-overflader ved kombination af AFM og elektrokemisk karakterisering.Bacterial biofilms are aggregates of microorganisms in which cells adhere to each other and adhere to a solid surface or an animal host cavity. Bacterial biofilms play important roles in human life, and cause serious harm for human society and huge economic losses. The complex composition of bacterial biofilms with EPS (Extracellular polymeric substances) includes proteins, polysaccharides, extracellular DNA (e-DNA), peptidoglycans, lipids and phospholipids. These substances play an important role in the initial adhesion of bacteria to the surface and maintenance of the biofilm structure. In my thesis, Atomic Force Microscopy (AFM) and electrochemistry have been applied to investigate three pathogenic medically important bacterial biofilms, i.e. Pseudomonas aeruginosa (cystic fibrosis pneumonia), Staphylococcus epidermidis (contamination of surgical catheters and indwelling equipment) and Streptococcus mutans (dental caries).  AFM was used to investigate the adhesion force on single live cell surfaces. Four different strains of Staphylococcus epidermidis in liquid aqueous environments were adressed. These strains were selected because of their special surface proteins related with the initial attachment on the surface. High-resolution AFM imaging showed no detectable differences among the four strains. Adhesion maps using hydrophobically modified tips compared with bare hydrophilic silicon nitride tips also showed small differences only. This indicates that hydrophobic effects are not the primary driving forces towards adhesion. Two chemical inhibitor compounds were found to have strong effects on the adhesion between the bare tips and the bacteria.  Secondly, AFM and electrochemistry were combined to study bacterial biofilm formation on Au(111)-surfaces, to determine the surface charge and growth pattern of Streptococcus mutans biofilms. Five redox probes were chosen for cyclic voltammetry, i.e. positively, [Ru(NH3)6]3+/2+, [Co(phen)3]3+/2+ and [Co(terpy)2]3+/2+ (phen = 1,10-phenanthroline; terpy = 2,2’,2”-terpyridine) and negatively charged, [Fe(CN)6]3-/4-. [IrCl6]3-/4-. The inhibition and voltammetric patterns showed that S. mutans biofilm are negatively charged. Addition of DNAase suggests that the negative charges to a large extent originate from DNA excreted by the biofilm. Thirdly, AFM and electrochemistry were combined to study Streptococcus mutans biofilm formation on bare Au(111) and Au(111) modified by self assembled molecular monolayers (SAMs) of thiol-based molecules. Four SAM molecules were chosen for reductive desorption (RD). Two are long straight-chain thiols with either a hydrophobic or a hydrophilic terminal group, i.e. hexadecanethiol, HS(CH2)15CH3 and mercaptohexadecanoic acid, HS(CH2)15COOH. Two others were the short rigid linker molecules L-cysteine and cysteamine. Strong RD peaks were obtained for all the four molecules both in PBS buffer at pH 7.4 and in 0.1M NaOH solution. Both AFM images and the electrochemical data show further, that the biofilms are bound more strongly to the hydrophobic surface than to the hydrophilic surfaces.  Finally, AFM was used to study two other kinds of bacteria, Pseudomonas aeruginosa and Pseudomonas putida, and their relationship with EPS. Different mutant strains were applied to investigate the roles of Pel and Psl polysaccharides and type IV pili during P. aeruginosa biofilm development. This study suggests that polysaccharides and e-DNA  contribute to the biofilm development. Protein clusters were observed during P. pudita biofilm formation, but we need further investigation to identify or distinguish the surface protein Lap A and Lap F. The combination of AFM and electrochemistry is a new approach to understand the bacterial biofilm/ medium / bare or modified Au(111) interface. Future efforts aim at observing the live bacterial biofilms with high resolution and controlling the biofilm  formation on SAM-modified Au(111)-surfaces by the combination of AFM and electrochemical characterization.</p

Online Research Database In Technology

Bacterial biofilms investigated by atomic force microscopy and electrochemistry

http://orbit.dtu.dk/ws/files/9595719/Yifan%20Hu%20Thesis.pdf

Bacterial biofilms investigated by atomic force microscopy and electrochemistry

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Online Research Database In Technology