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Fluorescence Correlation Spectroscopy To Study Diffusion and Reaction of Bacteriophages inside Biofilms

INRA, UMR Bioadhésion et Hygiène des Matériaux, F-91300 Massy, France,¹ Laboratoire de Photophysique Moléculaire, CNRS UPR 3361, Université Paris-Sud, F-91405 Orsay Cedex, France,² Centre de Photonique Biomédicale, Centre Laser de l'Université Paris-Sud, Université Paris-Sud, F-91405 Orsay Cedex, France,³ INRA, Unité Génétique Microbienne, Domaine de Vilvert, F-78352 Jouy-en-Josas, France,⁴ AgroParisTech, UMR Bioadhésion et Hygiène des Matériaux, F-91300 Massy, France⁵

Received 11 October 2007/ Accepted 25 January 2008

In the natural environment, most of the phages that target bacteria are thought to exist in biofilm ecosystems. The purpose of this study was to gain a clearer understanding of the reactivity of these viral particles when they come into contact with bacteria embedded in biofilms. Experimentally, we quantified lactococcal c2 phage diffusion and reaction through model biofilms using in situ fluorescence correlation spectroscopy with two-photon excitation. Correlation curves for fluorescently labeled c2 phage in nonreacting Stenotrophomonas maltophilia biofilms indicated that extracellular polymeric substances did not provide significant resistance to phage penetration and diffusion, even though penetration and diffusion were sometimes restricted because of the noncontractile tail of the viral particle. Fluctuations in the fluorescence intensity of the labeled phage were detected throughout the thickness of biofilms formed by c2-sensitive and c2-resistant strains of Lactococcus lactis but could never be correlated with time, revealing that the phage was immobile. This finding confirmed that recognition binding receptors for the viral particles were present on the resistant bacterial cell wall. Taken together, our results suggest that biofilms may act as "active" phage reservoirs that can entrap and amplify viral particles and protect them from harsh environments.

Published ahead of print on 1 February 2008.