Journal
CANADIAN JOURNAL OF MICROBIOLOGY
Volume 55, Issue 2, Pages 163-178Publisher
CANADIAN SCIENCE PUBLISHING, NRC RESEARCH PRESS
DOI: 10.1139/W08-109
Keywords
NEXAFS; STXM; biofilm; antimicrobial; CLSM
Categories
Funding
- Advanced Food and Materials Network (AFMNet)
- Environment Canada
- Natural Sciences and Engineering Research Council of Canada (NSERC)
- Canada Research Chair program (APH)
- National Science Foundation [DMR-9975694]
- Department of Energy [DOE DE-FG02-98ER45737]
- Dow Chemical
- NSERC
- Canadian Foundation for Innovation
- Director, Office of Energy Research, Office of Basic Energy Sciences, Materials Sciences Division of the United States Department of Energy [DE-AC03-76SF00098]
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Confocal laser scanning microscopy (CLSM) and scanning transmission X-ray microscopy (STXM) were used to examine the morphological and biochemical changes in Pseudomonas fluorescens biofilms grown in the presence of subinhibitory concentrations of 4 antimicrobial agents: triclosan, benzalkonium chloride, chlorhexidine dihydrochloride, and trisodium phosphate. CLSM analyses using the stains SYTO9 and propidium iodide indicated that the antimicrobial agents affected cell membrane integrity and cellular density to differing degrees. However, fluorescein diacetate assays and plate counts demonstrated that the cells remained metabolically active. Fluorescent lectin binding assays showed that changes in the arrangement and composition of the exopolymer matrix of the biofilms also occurred and that these changes depended on the antimicrobial agent. Detailed single cell analyses using STXM provided evidence that the cell morphology, and the spatial distribution and relative amounts of protein, lipids and polysaccharides in the biofilms and within the cells were different for each antimicrobial. The distribution of chlorhexidine in the biofilm, determined from its distinct spectral signature, was localized mainly inside the bacterial cells. Each antimicrobial agent elicited a unique response; P. fluorescens cells and biofilms changed their morphology and architecture, as well as the distribution and abundance of bio-macromolecules, in particular the exopolymer matrix. Pseudomonas fluorescens also exhibited adaptation to benzalkonium chloride at 10 mu g/mL. Our observations point to the importance of changes in the quantity and chemistry of the exopolymeric matrix in the response to antimicrobial agents and suggest their importance as targets for control.
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