Newly introduced genomic prophage islands are critical determinants of in vivo competitiveness in the Liverpool Epidemic Strain of Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa isolates have a highly conserved core genome representing up to 90% of the total genomic sequence with additional variable accessory genes, many of which are found in genomic islands or islets. The identification of the Liverpool Epidemic Strain (LES) in a children's cystic fibrosis (CF) unit in 1996 and its subsequent observation in several centers in the United Kingdom challenged the previous widespread assumption that CF patients acquire only unique strains of P. aeruginosa from the environment. To learn about the forces that shaped the development of this important epidemic strain, the genome of the earliest archived LES isolate, LESB58, was sequenced. The sequence revealed the presence of many large genomic islands, including five prophage clusters, one defective (pyocin) prophage cluster, and five non-phage islands. To determine the role of these clusters, an unbiased signature tagged mutagenesis study was performed, followed by selection in the chronic rat lung infection model. Forty-seven mutants were identified by sequencing, including mutants in several genes known to be involved in Pseudomonas infection. Furthermore, genes from four prophage clusters and one genomic island were identified and in direct competition studies with the parent isolate; four were demonstrated to strongly impact on competitiveness in the chronic rat lung infection model. This strongly indicates that enhanced in vivo competitiveness is a major driver for maintenance and diversifying selection of these genomic prophage genes.

Strain-dependent diversity in the Pseudomonas aeruginosa quorum-sensing regulon

“…3). As previously documented for LESB58 (18), the plt gene cluster was found at the same chromosomal location adjacent to PA2593 in both PaE2 and BE171.…”

“…The plt operon has been identified in a few P. aeruginosa isolates, namely, PACS171b, PACS88, and LESB58 (18,28). LESB58, the earliest archived P. aeruginosa isolate from the Liverpool CF epidemic, carries the plt gene cluster on a genomic island, suggesting that it was acquired through horizontal transfer (18). Curiously, in LESB58 there is a frameshift mutation caused by a deletion in the pltB gene, and the operon is nonfunctional.…”

“…The plt gene cluster is found in plant-associated pseudomonads (e.g., P. fluorescens Pf-5 and CHAO) and contributes to the ecological fitness of these pseudomonads in the rhizosphere (27). The plt operon has been identified in a few P. aeruginosa isolates, namely, PACS171b, PACS88, and LESB58 (18,28). LESB58, the earliest archived P. aeruginosa isolate from the Liverpool CF epidemic, carries the plt gene cluster on a genomic island, suggesting that it was acquired through horizontal transfer (18).…”

Temperate phages both mediate and drive adaptive evolution in pathogen biofilms

“…Specifically, we propagated six replicate populations of P. aeruginosa PAO1 in the presence vs. absence of an assemblage of three temperate phages (LESɸ2, ɸ3, and ɸ4) for ∼240 bacterial generations. These temperate phages naturally coexist as prophages in the genome of the P. aeruginosa Liverpool epidemic strain (LESB58) (27), the dominant clone infecting the UK CF population (28), and contribute to its competitiveness in vivo (27,(29)(30)(31). Whereas ɸ2 and ɸ3 are insertion site specific, ɸ4 is closely related to D3112, which is known to insert randomly throughout the P. aeruginosa chromosome (9, 10) and may therefore play an important role in facilitating the evolutionary adaptation of P. aeruginosa by increasing mutational supply.…”

Transmissible strains ofPseudomonas aeruginosain cystic fibrosis lung infections

“…LESB58 out competes both PAO1 and PA14 in a rat model of chronic lung infection by using the unusual tactic of limited dissemination [59,60]. Signature-tagged mutagenesis (STM) was used to demonstrate that novel prophage and genomic island regions play critical roles in the competitiveness of P. aeruginosa LESB58 in the rat model [48]. STM mutations in specific genes within either prophage 2, 3 or 5 were found to cause a 7-to 58-fold decrease in competitiveness, thus suggesting that these prophages could play an important role in the establishment of infection in this model [48].…”