Date of Award

May 2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical and Chemical Engineering

Advisor(s)

Dacheng Ren

Second Advisor

Anthony Garza

Keywords

bacterial adhesion, bacterial mechanosensing, biofilm, cell tracking, material stiffness, phagocytosis

Subject Categories

Engineering

Abstract

Biofilms are communities of microbial cells attached on surfaces and embedded in a self-produced extracellular matrix comprised of polysaccharides, DNA, and proteins. Biofilms of pathogenic bacteria cause serious chronic infections due to high tolerance to antibiotics and host immune systems compared to their planktonic compartments. Biofilm formation is known to be influenced by many properties of substrate materials, such as surface chemistry, hydrophobicity, roughness, topography, and charge. However, few studies have been conducted to investigate the effects of substrate stiffness. In this study, Escherichia coli RP437 and Pseudomonas aeruginosa PAO1 were used as model strains to investigate the early stage biofilm formation on poly(dimethylsil-oxane) (PDMS) with varying stiffness of 0.1 MPa to 2.6 MPa, which were prepared by controlling the degree of crosslinking.

An inverse correlation between cell adhesion and substrate stiffness was observed for both E. coli and P. aeruginosa. Interestingly, it was found that the cells attached on relatively stiff substrates were significantly shorter than those on relatively soft substrates, and the distribution of cell length was narrower on stiff substrates. In addition to the difference in size, the cells on stiff substrates were also found to be less susceptible to antimicrobials, such as ofloxacin, ampicillin, tobramycin and lysozyme, than the cells attached on soft substrates. The cell tracking results revealed that the E. coli cells on stiff surfaces were more mobile than those on soft surfaces, suggesting that the cells attached on soft surfaces may enter biofilm stage faster. Consistently, the intracellular level of c-di-GMP (an important signal for biofilm formation) in the cells on soft surfaces was higher than that of cells on stiff surfaces.

Comparison of the wild-type strains and isogenic mutants revealed that the motB mutant of E. coli RP437 has defects in response to the stiffness of PDMS, which was rescued by complementation of the motB gene. Additionally, the cell tracking results indicate that the mutation of motB rendered the cells much less mobile compared to wild type E. coli RP437 strains, and the decrease in the velocity of motility is higher on stiff surfaces than on soft surfaces. Those results suggest that motB may play a role in mechanosensing of material stiffness by E. coli. Similarly, mutation of oprF in P. aeruginosa also caused major defects in response to PDMS stiffness and abolished the difference in adhesion, growth, morphology and antibiotic susceptibility of attached cells between soft and stiff PDMS surfaces. These defects were rescued by genetic complementation of oprF, suggesting that oprF is involved in mechnosensing of P. aeruginosa.

In summary, the findings from this study indicate that material stiffness has potent effects on bacterial adhesion and the physiology of attached cells. To our best knowledge, this is the first study on the effects of material stiffness of silicon-based polymers on biofilm formation, and the first report of the effects of material stiffness on the physiology of attached cells. These results are helpful for designing better anti-fouling materials.

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