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Appl. Environ. Microbiol. doi:10.1128/AEM.02304-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

DIFFUSION-REACTION OF BACTERIOPHAGES INSIDE BIOFILMS: A DYNAMIC APPROACH USING FLUORESCENCE CORRELATION SPECTROSCOPY

INRA, UMR Bioadhésion et Hygiène des Matériaux, F- 91300 Massy, France; Laboratoire de Photophysique Moléculaire, CNRS UPR 3361, Université Paris-Sud, F-91405 Orsay Cedex, France; Centre de Photonique Biomédicale, Centre Laser de l'Université Paris-Sud, Univ Paris-Sud, F-91405 Orsay Cedex, France; INRA, Unité Génétique Microbienne, Domaine de Vilvert, F- 78352 Jouy-en-Josas, France; AgroParisTech, UMR Bioadhésion et Hygiène des Matériaux, F- 91300 Massy, France

^* To whom correspondence should be addressed. Email: romain.briandet{at}jouy.inra.fr.

	Abstract

In the natural environment, most of the phages that target bacteria are thought to exist in biofilm ecosystems. The purpose of this paper was to gain a clearer understanding of reactivity of these viral particles when they come into contact with bacteria embedded in biofilms. Experimentally, we quantified the lactococcal c2 phage diffusion-reaction through model biofilms using in situ Fluorescence Correlation Spectroscopy under Two Photon Excitation. Correlation curves of fluorescently-labeled c2 phages in non-reacting Stenotrophomonas maltophilia biofilms indicated that exopolymeric substances (EPS) did not confer significant resistance on phage penetration and diffusion, even though this was sometimes restricted because of the non-contractile tail of the viral particle.

Fluctuations in the fluorescence intensity of the labeled phages were detected through the thickness of c2-sensitive and c2-resistant strains of Lactococcus lactis biofilms, but could never be correlated with time, revealing their immobilization. This finding confirm the presence of recognition binding receptors of the viral particle also on the resistant bacterial cell wall. Taken together, our results suggest that biofilms may act as an "active" phage reservoir that can entrap, amplify and protect these particles from harsh environments.