Temperate Bacteriophages (Prophages) in Pseudomonas aeruginosa Isolates Belonging to the International Cystic Fibrosis Clone (CC274)

Abstract

Bacteriophages are important in bacterial ecology and evolution. Pseudomonas aeruginosa is the most prevalent bacterial pathogen in chronic bronchopulmonary infection in cystic fibrosis (CF). In this study, we used bioinformatics, microbiological and microscopy techniques to analyze the bacteriophages present in 24 P. aeruginosa isolates belonging to the international CF clone (ST274-CC274). Interestingly, we detected the presence of five members of the Inoviridae family of prophages (Pf1, Pf4, Pf5, Pf6, Pf7), which have previously been observed in P. aeruginosa. In addition, we identified a new filamentous prophage, designated Pf8, in the P. aeruginosa AUS411.500 isolate belonging to the international CF clone. We detected only one prophage, never previously described, from the family Siphoviridiae (with 66 proteins and displaying homology with PHAGE_Pseudo_phi297_NC_016762). This prophage was isolated from the P. aeruginosa AUS531 isolate carrying a new gene which is implicated in the phage infection ability, named Bacteriophage Control Infection (bci). We characterized the role of the Bci protein in bacteriophage infection and in regulating the host Quorum Sensing (QS) system, motility and biofilm and pyocyanin production in the P. aeruginosa isogenic mutant AUS531Δbci isolate. The findings may be relevant for the identification of targets in the development of new strategies to control P. aeruginosa infections, particularly in CF patients.

Keywords: CC274 clone; Pseudomonas; cystic fibrosis; inovirus; prophages; siphovirus.

FIGURE 1

(A) Schematic representation and comparison of filamentous phages Pf1, Pf4, Pf5, and Pf8. Genes are classified by function into assembly and secretion, structural, and replication/integration genes. Dark gray regions represent >90% of nucleotide sequence identity between Pf genome regions. (B) Schematic representation of the genome of siphovirus phage AUS531phi and position of the Bci protein (GenBank: MN585195.1).

FIGURE 2

(A) TEM of the Inoviridae Pf8 bacteriophage of P. aeruginosa clinical isolate AUS411 (Pf8_ST274-AUS411). (B) TEM of Siphoviridae bacteriophage of P. aeruginosa clinical isolates AUS531 (AUS531phi*) and AUS531Δbci (AUS531Δbciphi**).

FIGURE 3

Relative expression of the bci gene in AUS531 isolate under the quorum sensing inducers C4-HSL and 3OXO-C12-HSL. Standard deviations are indicated. (^∗) Statistically significant differences (p < 0.05) were determined by Student’s t-test (GraphPad Prism v.6).

FIGURE 4

Relative expression of quorum sensing-related genes of the AUS531Δbci isolate infected with AUS531phi and AUS531phiΔbci bacteriophages. Standard deviations are indicated. (^∗) Statistically significant differences (p < 0.05) were determined by Student’s t-test (GraphPad Prism v.6).

FIGURE 5

Infection curves for the lysogenic phages AUS531phi (A) and AUS531phiΔbci at MOIs 0.1 (A), 1 (B), and 10 (C) during 6 h. Standard deviations are indicated. Statistically significant differences were determined by Student’s t-test for each point on the curve (GraphPad Prism v.6). (*) indicates a strongly significant difference (p < 0.0001).

FIGURE 6

(A) Motility assay in AUS531Δbci under normal conditions (1), adding AUS531phi phage (2), and adding AUS531phiΔbci phage (3). (B) Biofilm production after 24 h of AUS531 wild type isolate, AUS531Δbci under normal conditions, AUS531Δbci in response to addition of the wild type AUS531phi phage and the mutant AUS531Δbci phage. (^∗) Statistically significant differences were determined by Student’s t-test (GraphPad Prism v.6). (C) Pyocyanin production of controls AUS531 and AUS531Δbci and AUS531Δbci in response to infection with the wild-type strain.