AVS2012 Session MB+BI-ThM: Biofilms and Biofouling in Medicine

The control of biomolecules adsorption (such as proteins) and other microorganisms is of high interest for various fields of biotechnology, such as bioanalytics, cell biology, tissue engineering and biomaterials. An efficient method to control adsorption includes the use of well-defined oligo(ethylene glycol)-terminated self-assembled monolayer (SAM). However, multiple processing steps are often required to prepare SAM onto substrate. To address these problems of surface modification, we have developed a simple method using the powerful thiolene reaction to prepare self-assembled monolayer on different metal oxides to control bioadhesion on surfaces starting from commercially available building blocks, and taking the advantage of the photoreaction to easily create adhesive and anti-adhesive patterns.

Such well-controlled grafting strategy has been applied to different metal oxides: from silicon substrates for the methodology set-up to model metal oxides formed on biocompatible metals and alloys. The obtained films are robust; the process is low-cost, simple, and efficient.

Essential oils such as tea tree oil are known for their antibacterial properties. They have been used extensively as effective topical antimicrobial agents and are active against a wide range of micro-organisms. Traditionally, the antimicrobial properties of tea tree oil has been linked to the constituents of the oil including terpinen-4-ol and 1,8-cineole. To translate these antimicrobial properties into medical devices, methods for incorporating or coating materials are required. Recently, thin polymer films from terpinen-4-ol have been fabricated using plasma polymerisation. Initial data suggests that some antimicrobial activity was maintained. While plasma polymer films of 1,8-cineole have been fabricated previously, little focus has been placed on the antibacterial activity of this constituent.

The activity of 1,8-cineole against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) was evaluated based on the time-kill approach. Several concentrations of the oil (0.05 %, 0.2 %, 0.3 % and 0.5 % v/v) were added to broth media containing 3x10⁸ CFU/mL of each bacterium. The optical density of the resulting solutions was measured at intervals over 24-hrs to monitor bacterial growth rates. The results demonstrated that concentrations above 0.35% v/v gave a 90% kill rate in the gram-negative E. coli after 8-hrs. Interestingly, the oil was less effective against the gram-positive S. aureus, producing a lower kill rate and rapid growth recovery in a 24-hour period.

Plasma polymers thin films of 1,8-cineole and 1,7-octadiene (hydrophobic control) were prepared using a stainless steel plasma reactor as described previously, while uncoated glass slides were used as a hydrophilic, positive control. Samples were then exposed to broth media containing 3x10⁸ CFU/mL of each bacteriumto determine the effects of the coatings on bacterial attachment and growth. In this work, bacterial studies of the coating behaviour have focused on the attachment of S.aureus and E. coli, and biofilm formation of E. coli. Comparison of the plasma polymerised cineole (ppCineole) coating with the two controls demonstrated a reduction in bacterial attachments over 18-hrs. The reduction in bacterial attachment was more obvious in the case of E.coli in comparison with S. aureus. There were not significant differences between plasma polymerised cineole and octadiene in the inhibition of E.coli attachment and growth over a period of 18-hours. However, the biofilm studies indicated that only the 1,8-cineole film demonstrated antimicrobial behaviour over a period of time of 5 days.

Keywords: plasma polymerisation, antibacterial activity, cineole

Pseudomonas aeruginosa is extremely harmful to immune-compromised individuals. An atomic force microscope (AFM) can be used to measure the forces between the AFM tip and the bacterial exopolymers, with which the bacteria attach themselves to surfaces. These forces are characterized with a model that is a function of brush (exopolymer) layer thickness, probe radius, temperature, separation distance, and a molecular volume. Initial experiments with limited data sets are consistent with expected brush thicknesses of a few hundred nanometers. In order to progress – now with rigor – we have just developed a high throughput method for the analysis of force curves on the exopolymers of P. aeruginosa [1]. The above-described model is only valid for the region where the tip is in contact with the exopolymers, yet is not perturbing the bacterial membrane. MatLab code was written to determine the location of this region in each force curve, crop the curve to that region, and apply the force model in order to obtain parameters of the exopolymers. The standard deviation of the mean and Chauvenet’s Criterion are then applied to the results of sets of one-hundred force curves to increase measurement precision and objectively remove outliers. This procedure removes user subjectivity in cropping, fitting, and outlier removal, decreases analysis time by two orders of magnitude, and increases the precision of fitted results by a factor of ten (for one-hundred curves), which is necessary for demonstrating the statistical significance of our data.

[1] Anderson et al., to be submitted May 2012.

Quaternary ammonium compounds (QAC) are potent cationic antimicrobials used in everyday consumer products like contact lens solutions and mouthrinses as well as in numerous industrial processes, like water purification. Unlike the case for many antibiotics, the development of bacterial resistance against QACs is considered unlikely, although Pseudomonas aeruginosa strains isolated from contact lens cases have been shown to possess resistance against QACs. The first step in the antimicrobial action of QACs is the approach of the QAC molecule towards the bacterial cell surface. This is mediated by hydrophobic and electrostatic attractions between positively charged QAC molecules and the negatively charged bacterial cell surface. Upon their subsequent adsorption, QAC molecules replace Ca²⁺ or Mg²⁺ ions from the cytoplasmic membrane in order to maintain charge neutrality in the membrane. The replacement of Ca²⁺ and Mg²⁺ ions by QACs destabilizes the intracellular matrix of a bacterium, as the hydrophobic tail interdigitates into the hydrophobic bacterial membrane causing leakage of intracellular fluid and loss of turgor pressure. Antimicrobial efficacy of QACs remains preserved when QAC molecules are immobilized on a substratum surface. It is difficult to envisage how immobilized QAC molecules can exert the same mechanism of antimicrobial activity as do QACs in solution. Immobilized QACs, especially after adsorption of a protein film as developing rapidly in the human body, are strongly hindered in their search for heterogeneously distributed negative charges on bacterial cell surfaces which is crucial for their efficacy in solution. Hence, it has been often hypothesized that QAC molecules immobilized to a substratum surface possess other generic mechanisms of action than QACs in solution, but these have never been elucidated. Immobilized QACs do not cause directly visual membrane damage. Instead, the strong adhesion forces arising from immobilized QACs enter bacterial adhesion forces into the lethal regime, i.e. where the stress exerted on the bacterial cell membrane is causing killing.