The Susceptibility of Pseudomonas aeruginosa Strains from Cystic Fibrosis Patients to Bacteriophages Christiane Essoh 1,2 , Yann Blouin 1,2 , Guillaume Loukou 3 , Arsher Cablanmian 3 , Serge Lathro 3 , Elizabeth Kutter 4 , Hoang Vu Thien 5 , Gilles Vergnaud 1,2,6 , Christine Pourcel 1,2 * 1 Univ Paris-Sud, Institut de Ge ´ne ´tique et Microbiologie, UMR 8621, Orsay, France, 2 CNRS, Orsay, France, 3 Laboratoire National de Sante ´ Publique, Abidjan, Co ˆ te d’Ivoire, 4 The Evergreen State College, Olympia, Washington, United States of America, 5 Ho ˆ pital Armand Trousseau, Assistance Publique-Ho ˆ pitaux de Paris (APHP), Bacte ´ riologie, Paris, France, 6 DGA/MRIS- Mission pour la Recherche et l’Innovation Scientifique, Bagneux, France Abstract Phage therapy may become a complement to antibiotics in the treatment of chronic Pseudomonas aeruginosa infection. To design efficient therapeutic cocktails, the genetic diversity of the species and the spectrum of susceptibility to bacteriophages must be investigated. Bacterial strains showing high levels of phage resistance need to be identified in order to decipher the underlying mechanisms. Here we have selected genetically diverse P. aeruginosa strains from cystic fibrosis patients and tested their susceptibility to a large collection of phages. Based on plaque morphology and restriction profiles, six different phages were purified from ‘‘pyophage’’, a commercial cocktail directed against five different bacterial species, including P. aeruginosa. Characterization of these phages by electron microscopy and sequencing of genome fragments showed that they belong to 4 different genera. Among 47 P. aeruginosa strains, 13 were not lysed by any of the isolated phages individually or by pyophage. We isolated two new phages that could lyse some of these strains, and their genomes were sequenced. The presence/absence of a CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and Crisper associated genes) was investigated to evaluate the role of the system in phage resistance. Altogether, the results show that some P. aeruginosa strains cannot support the growth of any of the tested phages belonging to 5 different genera, and suggest that the CRISPR-Cas system is not a major defence mechanism against these lytic phages. Citation: Essoh C, Blouin Y, Loukou G, Cablanmian A, Lathro S, et al. (2013) The Susceptibility of Pseudomonas aeruginosa Strains from Cystic Fibrosis Patients to Bacteriophages. PLoS ONE 8(4): e60575. doi:10.1371/journal.pone.0060575 Editor: Gunnar F Kaufmann, The Scripps Research Institute and Sorrento Therapeutics, Inc., United States of America Received November 22, 2012; Accepted February 28, 2013; Published April 24, 2013 Copyright: ß 2013 Essoh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was performed with the financial support of the association Vaincre La Mucoviscidose (Grant Nu 2010/IC1020). The development of tools for the surveillance of bacterial pathogens is supported by the French Direction Ge ´ne ´rale de l’Armement (DGA). CE holds a fellowship of Agence Universitaire de la Francophonie. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: christine.pourcel@u-psud.fr Introduction Pseudomonas aeruginosa is a highly heterogeneous species whose members show various levels of pathogenicity towards plants and animals [1,2]. It is widely distributed in the environment and is a normal human commensal. P. aeruginosa is naturally resistant to many drugs and its capacity to form biofilms makes it very difficult to eradicate, particularly in chronically infected cystic fibrosis (CF) patients [3,4]. Due to the recent increase in multidrug resistant bacteria, phage therapy is being considered as a therapeutic alternative to antibiotics [5,6]. It has been used since soon after the 1917 discovery of bacteriophages by Felix d’Herelle at the Pasteur Institute in Paris, mostly in countries of Eastern Europe. Although controlled studies were seldom available, its efficacy has been clearly demonstrated in numerous cases [7,8]. Phages have the capacity to reach bacteria trapped inside biofilms such as those that form in the lung of cystic fibrosis patients [9,10]. Therapeutic cocktails such as the ‘‘pyophage’’ formulation widely used in Georgia to treat purulent skin, wound and lung infections, contain many different phages against each of their target pathogens (which are staphylococcus, streptococcus, proteus, Escherichia coli and P. aeruginosa, for pyophage). Each set must be modified twice a year to retain its ability to lyse a large proportion of the target species [5,11]. P. aeruginosa bacteriophages are numerous, and current knowledge of their diversity shows that they are distributed in at least 7 genera of purely lytic phages (T7-like, WKMV-like, LUZ24- like, N4-like, PB1-like, WKZ-like, JG004-like) in addition to a similar number of temperate genera [12,13]. Within each genus, phages with a variety of different host spectra are observed, in part at least reflecting differences in their tail-associated adhesins [12,14,15]. Yet the combination of phages used to treat different types of infections remain empirical and usually target not more than 85% of the isolates necessitating the development of a ‘‘Sur- mesure’’ strategy [13]. For this strategy, at least one virulent phage for a patient’s given otherwise recalcitrant bacterial strain is identified from a collection, or even de novo isolated when none is available, and a therapeutical preparation is freshly made. A major challenge is the regular emergence of bacteria resistant to any given phage, which necessitates the ongoing isolation of new phages or variants of phages targeting different hosts or host receptors. The mechanisms of bacterial resistance to phages are diverse, driving co-evolution of bacteria and bacteriophages [16,17,18,19]. They involve not only the inhibition of the adsorption of phages on PLOS ONE | www.plosone.org 1 April 2013 | Volume 8 | Issue 4 | e60575