Comparative genomics and biological characterization of sequential Pseudomonas aeruginosa isolates from persistent airways infection

Abstract

Background: Pseudomonas aeruginosa establishes life-long chronic airway infections in cystic fibrosis (CF) patients. As the disease progresses, P. aeruginosa pathoadaptive variants are distinguished from the initially acquired strain. However, the genetic basis and the biology of host-bacteria interactions leading to a persistent lifestyle of P. aeruginosa are not understood. As a model system to study long term and persistent CF infections, the P. aeruginosa RP73, isolated 16.9 years after the onset of airways colonization from a CF patient, was investigated. Comparisons with strains RP1, isolated at the onset of the colonization, and clonal RP45, isolated 7 years before RP73 were carried out to better characterize genomic evolution of P. aeruginosa in the context of CF pathogenicity.

Results: Virulence assessments in disease animal model, genome sequencing and comparative genomics analysis were performed for clinical RP73, RP45, RP1 and prototype strains. In murine model, RP73 showed lower lethality and a remarkable capability of long-term persistence in chronic airways infection when compared to other strains. Pathological analysis of murine lungs confirmed advanced chronic pulmonary disease, inflammation and mucus secretory cells hyperplasia. Genomic analysis predicted twelve genomic islands in the RP73 genome, some of which distinguished RP73 from other prototype strains and corresponded to regions of genome plasticity. Further, comparative genomic analyses with sequential RP isolates showed signatures of pathoadaptive mutations in virulence factors potentially linked to the development of chronic infections in CF.

Conclusions: The genome plasticity of P. aeruginosa particularly in the RP73 strain strongly indicated that these alterations may form the genetic basis defining host-bacteria interactions leading to a persistent lifestyle in human lungs.

Fig. 1

P. aeruginosa sequential isolates from patient RP. Two clone types (OC2E and OC4A) of P. aeruginosa strains were isolated from patient RP who is heterozygous for F508del and R1162X mutations in the CFTR gene. OC2E was isolated at the onset of chronic colonization for the first eleven years. Thereafter OC4A became the dominant clone. Strain RP1 belongs to the clone type OC2E and was the first P .aeruginosa strains isolated. Strains RP45 and RP7 belong to the clone type OC4A and were isolated after 10 and 16.9 years respectively after the onset of chronic colonization of the patient’s airways with P. aeruginosa (Additional file 1 and Cramer et al. [12]). Lung function parameters at the time of P. aeruginosa isolation are indicated

Fig. 2

Virulence of P. aeruginosa RP isolates in comparison with prototype strains in murine model. C57Bl/6NCrlBR mice were infected with 1-2*10⁶ CFU/lung P. aeruginosa RP1, RP45, RP73, PAO1 and PA14 strains embedded in agar beads. Mortality induced by bacteremia (black) and survival (light gray) was evaluated on challenged mice. Clearance (white) and capacity to establish chronic infection (dark gray) were determined on surviving mice after 14 days. The data show the percentage of mice infected with single P. aeruginosa strains analyzed in two to three independent experiments. Statistical significance by Chi-square test is indicated: *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

Histological lesions after chronic P. aeruginosa infection in mice. Mice were infected with 2x10⁶ cfu/lung of P. aeruginosa RP73 strain embedded in agar beads and lung harvested after 14 days from challenge. Histopathological analysis of lungs chronically infected by RP73 strain are characterised by acute and chronic lesions; the pulmonary parenchyma is infiltrated by macrophages, lymphocytes and some neutrophils (a). Agar beads (arrow) containing bacteria macrocolonies (*) are localised in the bronchia and surrounded by a massive neutrophils inflammation (b). Alcian blue staining shows mucus secretory cells hyperplasia (circle) (c)

Fig. 4

Circular map of P. aeruginosa RP isolates and prototype strain PA14. Circular map constructed with the CGView Comparison Tool [56]. Starting from the outside: genomic islands predicted with IslandViewer (see Table 1 for details) [52], RP73, RP45, RP1, PA14 and GC content. Colored regions are shared with RP73 according to blast search. Dotted lines: known genomic islands (GIs) that distinguish RP73 because they are incomplete or absent in the 12 complete P. aeruginosa genomes available at pseudomonas.com. RP isolates also carry LESGI-4, identified in the Liverpool epidemic strain

Fig. 5

Core genome phylogeny for RP isolates and strains representative of P. aeruginosa diversity. The figure represents a partial view of the tree to show the relationships between RP1, RP45 and RP73. The position of RP1 is indicated in blue, while the position of RP45 and RP73 is indicated in red. PA14 is distantly related to all these strains