The uronic acids assay: a method for the determination of chemical activity on biofilm EPS

Abstract

In this work, the uronic acids assay was evaluated for its potential to function as a bioassay to screen for antagonistic activity against the production of microbial biofilm exopolysaccharide (EPS). The assay was first applied to biofilms produced in the presence of two universal disinfectants (sodium hypochlorite and sodium dodecyl sulfate) known to inhibit microbial growth and biofilm formation. The performance of the assay was then characterized through statistical assessment of threshold concentrations for disinfection efficiency and consistency relative to values reported in the literature. The assay was then evaluated for its utility in screening for enzymatic or chemical inhibitors of biofilm formation (eg glycosidases, halogenated furanones, and semi-crude fractions extracted from minimally fouled marine plants) and its ability to distinguish between true anti-biofilm activity and simple disinfection. Activity was characterized as (i) no effect, (ii) a true positive effect (ie increased biofilm EPS), (iii) anti-bacterial activity (ie decreased biofilm EPS and analogous decrease in planktonic growth), and (iv) anti-biofilm EPS activity (ie decreased biofilm EPS, without analogous decrease in planktonic growth). Results demonstrate that the uronic acids assay can augment existing biofilm characterization methods by providing a quantitative measure of biofilm EPS.

Keywords:

• uronic acid
• biofilm
• exopolysaccharide
• assay
• C. lytica

Acknowledgments

This research is funded in part by a grant/cooperative agreement from the National Oceanic and Atmospheric Administration, Project # R/MP-19, which is sponsored by the University of Hawaii Sea Grant College Program, SOEST, under Institutional Grant No. NA05OAR4171048 from NOAA Office of Sea Grant, Department of Commerce. The views expressed herein are those of the authors and do not necessarily reflect the views of NOAA or any of its subagencies. UNIHI-SEAGRANT-JC-06-25. This research was also supported in part by the National Institutes of Health (NIH/NCRR)-funded grant entitled ‘Integrated Technology Resource for Biomedical Glycomics’ (grant no. 1 P41 RR018502-01) to the Complex Carbohydrate Research Center. The authors gratefully acknowledge Bill Costerton for his many helpful discussions and extended support. The authors also thank the PBRC Biological Electron Microscope Facility and Tina Weatherby for use of their microscope facility and guidance. Finally, the authors thank Philip Williams for the donations of several extracts and guidance with microbial screening protocols.