The susceptibility of Pseudomonas aeruginosa strains from cystic fibrosis patients to bacteriophages - PubMed (original) (raw)

The susceptibility of Pseudomonas aeruginosa strains from cystic fibrosis patients to bacteriophages

Christiane Essoh et al. PLoS One. 2013.

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Morphology of plaques produced by three different phages of the pyophage A) P1-15pyo on PAO1, B) P1-14pyo on C1-14, C) PTr60pyo on Tr60.

Figure 2

Figure 2. Restriction analysis of phage DNA.

The DNA of each of six phages from pyophage was digested by _Hind_III and electrophorezed on a 0.8% agarose gel. From 1 to 6: P1-15pyo, P8-13pyo, P2-10pyo, P3-20pyo, PTr60pyo, P1-14pyo. MW is a size marker. On the side are indicated the sizes of three DNA fragments. The DNA of P1-14pyo is not totally digested.

Figure 3

Figure 3. Electron microscopy observation of four phages from pyophage: P8-13pyo, P1-14pyo, P1-15pyo, P2-10pyo.

Figure 4

Figure 4. Electron microscopy observation of the new phages P1-14Or01 and P2-10Ab01.

Figure 5

Figure 5. Alignment of P1-14Or01, MR199-2 and LUZ24 genomes.

Putative open reading frames (ORF) are shown with arrows. In red are shown the tentative cluster of genes encoding structural proteins and in yellow the genes involved in DNA replication. tRNA genes are shown with triangles. Boxes with different shades of grey represent degrees of similarities between genomes.

Figure 6

Figure 6. Alignment of P2-10Ab01, JG004 and PAKP1 genomes.

The genomes are separated into two sections (A and B) of approximately 46 kb. The legend is that of Figure 4.

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Grants and funding

This study was performed with the financial support of the association Vaincre La Mucoviscidose (Grant N° 2010/IC1020). The development of tools for the surveillance of bacterial pathogens is supported by the French Direction Générale 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.

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