Antistaphylococcal nanocomposite films based on enzyme-nanotube conjugates

Abstract

Infection with antibiotic-resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) is one of the primary causes of hospitalizations and deaths. To address this issue, we have designed antimicrobial coatings incorporating carbon nanotube-enzyme conjugates that are highly effective against antibiotic-resistant pathogens. Specifically, we incorporated conjugates of carbon nanotubes with lysostaphin, a cell wall degrading enzyme, into films to impart bactericidal properties against Staphylococcus aureus and Staphylococcus epidermidis. We fabricated and characterized nanocomposites containing different conjugate formulations and enzyme loadings. These enzyme-based composites were highly efficient in killing MRSA (>99% within 2 h) without release of the enzyme into solution. Additionally, these films were reusable and stable under dry storage conditions for a month. Such enzyme-based film formulations may be used to prevent growth of pathogenic and antibiotic-resistant microorganisms on various common surfaces in hospital settings. Polymer and paint films containing such antimicrobial conjugates, in particular, could be advantageous to prevent risk of staphylococcal-specific infection and biofouling.

Figure 1

Kinetics of Lst activity against N-acetylhexaglycine and microbes (S. aureus). (a) Michaelis-Menten kinetics of Lst activity based on the colorimetric assay. Rates in nM·s⁻¹ for native Lst (◆), Lst-MWNT (■) and Lst-PEG-MWNT (▲) are plotted as a function of substrate concentrations. (b) Comparison of bactericidal effect of Lst-MWNT (■) and Lst-PEG-MWNT (▲) with the native enzyme (◆), determined by using a cell-based assay.

Figure 2

Effect of Lst loading on the bactericidal activity shown by composite films containing Lst-PEG-MWNT conjugates (□). Controls for these experiments consisted of paints with heat-inactivated conjugates (■), paints with MWNT (◇) and BSA-MWNT (△). Data from leaching tests (○) suggest minimal bactericidal effect due to release of the enzyme or conjugates from films. Inset pictures show confocal images of S. aureus cells exposed to films containing 0% w/w and 4% w/w Lst, confirming the bactericidal effect of Lst. The scale bar corresponds to 10 μm.

Figure 3

Percent viable cells obtained after exposing the microbial suspension containing 10⁶ CFU/mL MRSA to control and Lst-containing films (4% w/w Lst in the form of Lst-PEG-MWNT) for 2 h. The results are normalized with respect to the colony counts obtained for control samples.

Figure 4

Operational (●) and storage stability (■) of films containing 4% w/w Lst in the form of Lst-PEG-MWNT. The films were stored in dry conditions, and at room temperature in between the two use cycles.

Scheme 1

Antimicrobial nanocomposite films containing Lst-nanotube conjugates (not drawn to scale): (a) Preparation of Lst-nanotube conjugates via two (no PEG linker) and four (with PEG linker) step EDC-NHS coupling reactions. Nanocomposite films were prepared by filtering a mixture of diluted paint and conjugates onto a polycarbonate membrane. (b) Cell-based assay to determine bactericidal activity of Lst-containing nanocomposite films.