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A proteomic investigation of escherichia coli biofilm formation on a silicone coating containing a tethered quaternary ammonium compound.

Biofilms are communities of attached microorganisms that are embedded in an extracellular matrix that they themselves produce. These communities are known to be inherently resistant to a wide variety of antibiotics and disinfectants and are extremely difficult to eradicate once they are established on surfaces, such as implanted medical devices and food processing equipment. In the present study, we are investigating the differential protein expression during Escherichia coli biofilm formation on an unmodified silicone coating and a silicone coating containing a covalently tethered quaternary ammonium compound (QAC). Tethered QACs have been shown to exhibit highly effective, broad spectrum antimicrobial activity through a contact-active mechanism (1). It is anticipated that silicone coatings containing tethered QACs can be utilized to provide long term antimicrobial protection to surfaces that are susceptible to microbial biofilm formation.

E.coli biofilms were cultured in Tryptic Soy Broth on surfaces of the coatings prepared in multi-well plates (2). After incubation, the biofilms were rinsed to remove non-attached cells and analyzed with a suite of quantitative spectrophotometric techniques, including crystal violet (CV), alcian blue (AB) and ATP bioluminescence (LUM) assays. CV and AB dyes are used as total biomass indicators, staining anionic biomacromolecules and extracellular matrix carbohydrates, respectively. The ATP bioluminescence assay is used to measure cellular viability by reacting cellular ATP with a luciferase to produce luminescence. The results (Figure 1) showed a significant reduction in biofilm growth (as characterized by CV and AB) with no discernable reduction in bioluminescence, indicating that the production of biofilm extracellular matrix is being inhibited without affecting cellular viability on the surface of the QAC-containing silicone coating.

For proteomic analysis, the E.coli biofilms were recovered from the coating surfaces using nylon flocked swabs and recovered into 2mL of sterile deionized water. The recovered biofilm solutions were sonicated for six 10 second pulses, each at different power settings: 20%, 40%, 60% and 80%. The sonicated solutions were centrifuged to pellet cell debris and the supernatant was collected and concentrated for protein analysis. All samples were resolved in 10% and 12.5% Laemmli-SDS-PAGE gels to visualize band patterns. Gels were post stained with Deep Purple and images were obtained by scanning with the Ettan DIGE Imager. Based on the observed band patterns and the relative intensity of detected bands, it was determined that the 20% power setting yielded optimal protein extraction for lower molecular weight proteins (<25kD) while the 60% power setting yielded optimal protein extraction for higher molecular weight proteins. This data suggests that multiple sonication settings need to be used for detection of differentially expressed proteins on these surfaces based on the MW range of interest. Optimization of 2D SDS-PAGE gels is currently being performed, which will be followed by mass spectrometry identification of the observed differentially expressed protein spots.

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(1) Lewis K, Klibanov AM. (2005) TRENDS in Biotechnology, 23, 343.

(2) Stafslien SJ, Bahr J, Feser JM, Weisz JC, Chisholm BJ, Ready TE, and Boudjouk P. (2006) J Comb. Chem., 8, 156.

Justin W. Daniels * [1], Shane J. Stafslien [1], Bret J. Chisholm [2], Steven Meinhardt [3], Birgit M. Pruss [1]

[1] Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND 58108

[2] Center for Nanoscale Science and Engineering, North Dakota State University, Fargo, ND 58102

[3] Department of Plant Pathology, North Dakota State University, Fargo, ND 58108
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Title Annotation:COMMUNICATIONS--GRADUATE
Author:Daniels, Justin W.; Stafslien, Shane J.; Chisholm, Bret J.; Meinhardt, Steven; Pruss, Birgit M.
Publication:Proceedings of the North Dakota Academy of Science
Article Type:Report
Geographic Code:1USA
Date:Apr 1, 2009
Words:553
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