Genome diversity of Pseudomonas aeruginosa isolates from cystic fibrosis patients and the hospital environment

Abstract

Pseudomonas aeruginosa is a gram-negative rod that is ubiquitous in nature. P. aeruginosa is also the quintessential opportunistic pathogen, causing a wide variety of infections in compromised hosts. In cystic fibrosis patients, P. aeruginosa is the leading cause of death. In this study, the evolutionary genetic relationships among 17 P. aeruginosa isolates were examined by comparative sequence analysis of the housekeeping gene encoding malate dehydrogenase and the chaperone groEL. The P. aeruginosa isolates examined included the sequenced strain PAO1, 11 strains recovered from cystic fibrosis patients in Ireland, 4 environmental isolates recovered from a hospital environment, and 1 isolate recovered from a plant rhizosphere. Phylogenetically, clinical and environmental isolates clustered together with one another on the mdh gene tree. At the groEL locus, among the 17 isolates examined, only two polymorphic sites were observed, highlighting the close genetic relationship between isolates from these different environments. Phenotypic analysis of 12 traits among our isolates, however, found that only clinical isolates produced phenazines and elastase. Furthermore, molecular analysis of the distribution of 15 regions associated with virulence showed that two of the environmental isolates examined lacked the majority of regions. Among the clinical isolates examined, the 15 virulence regions were variably present. The distribution of two prophages (Bacto1, Pf1) was also determined, with most isolates encoding both these regions. Of the four genomic islands (the flagellum island and PAGI-1, -2, and -3) examined, only two isolates contained the flagellum island, and PAGI-1, -2, and -3 were absent from all isolates tested. Our data demonstrate the significant role horizontal gene transfer and recombination, together with gene loss, play in the evolution of this important human pathogen.

FIG. 1.

Neighbor-joining tree constructed by the Jukes-Cantor method with the nucleotide sequences of mdh gene fragments of P. aeruginosa strains. Construction and bootstrapping of the tree were carried out with the MEGA suite of programs. One thousand bootstrap replicates were performed for each analysis. Bootstrap values are given at the nodes.

FIG. 2.

PCR and Southern blot analysis of the pilB genes from our P. aeruginosa isolates. (A) PCR analysis. Lane 1, 1-kb marker; lane 2, negative control; lanes 3 to 19, PAO1 (positive control), CF93, CF95, CF139, CF144, CF175, CF177, CF194, CF195, CF198, CF208, CF242, BR220, BR227, BR257, BR642, and BR764, respectively. (B) Southern blot analysis. Lane 1, negative control; lane 2, PAO1 (positive control); lanes 3 to 19, P. aeruginosa isolates CF93, CF95, CF139, CF144, CF175, CF177, CF194, CF195, CF198, CF208, CF242, BR220, BR227, BR257, BR642, and BR764.

FIG. 3.

PCR and Southern blot analysis of the exoT genes of our P. aeruginosa isolates. (A) PCR analysis. Lane 1, 1-kb marker; lane 2, negative control; lanes 3 to 19, PAO1 (positive control), CF93, CF95, CF139, CF144, CF175, CF177, CF194, CF195, CF198, CF208, CF242, BR220, BR227, BR257, BR642, and BR764, respectively. (B) Southern blot analysis. Lanes 1 to 17, P. aeruginosa isolates CF93, CF95, CF139, CF144, CF175, CF177, CF194, CF195, CF198, CF208, CF242, BR220, BR227, BR257, BR642, and BR764; lane 18, negative control; lane 19, PAO1 (positive control).

FIG. 4.

PCR and Southern blot analysis of the pilA genes of our P. aeruginosa isolates. (A) PCR analysis. Lane 1, 1-kb marker; lane 2, PAO1 (positive control); lanes 3 to 18, P. aeruginosa isolates CF93, CF95, CF139, CF144, CF175, CF177, CF194, CF195, CF198, CF208, CF242, BR220, BR227, BR257, BR642, and BR764, respectively; lane 19, negative control. (B) Southern blot analysis. Lanes 1 to 17, P. aeruginosa PAO1 (positive control) and isolates CF93, CF95, CF139, CF144, CF175, CF177, CF194, CF195, CF198, CF208, CF242, BR220, BR227, BR257, BR642, and BR764, respectively; lane 19, negative control.