Abstract

Background. Infections caused by Pseudomonas aeruginosa are the main cause of morbidity and mortality in Cystic Fibrosis (CF) patients and occur via primary colonisation of the airway followed by the accumulation of pathoadaptive mutations in the bacterial genome which increase fitness in the lung environment and result in chronicization. A better understanding of i) the evolutionary dynamics occurring during chronic airway infections in CF patients and ii) the genetic adaptation of strains to the CF lung environment, might give further clues for preventive measures or novel therapies to control CF infections in the future. In this work, we obtained genomic sequences of 40 P. aeruginosa isolates from a single CF patient collected over an eight-year period (2007-2014) and analysed the population in terms of clonality of the isolates, phylogenetic relationships, and presence of polymorphisms and variants between the strains. Population structure and microevolution. In silico Multilocus Sequence Typing (MLST) analysis revealed a characteristic single clonal population dominated by a previously characterized sequence type (ST390) and a small number of new, closely related ST variants (ST1863, ST1864, ST1923). EBURST analysis of the sequence types revealed that all members of this population belong to the same clonal lineage and likely evolved from a single ancestral colonizing strain. Furthermore, the phylogenetic analysis based on SNPs also divided the population into two subpopulations derived from the evolution of the first infecting strain. The annotation of SNPs allowed us to identify mutations with moderate or high impact. Genes with high impact variants encoded respiratory nitrate reductase subunit gamma nail, polyprotein signal peptidase lspA, the ABC transporter-binding protein aaltP, the copper resistance protein A precursor pcoAin, and four hypothetical proteins. The evolution of strains in the CF airway is characterized by the loss of many virulence traits, including motility and protease secretion, along with the acquisition of multidrug resistance. Functional phenotypic assays of the collection, including motility and secretion of proteases, showed a decrease over time in the persistent isolates. We also determined the antibiotic susceptibility profile of the collection; while early isolates were found to be susceptible to almost all these antibiotics, resistant phenotypes dramatically increased over time in the population. Functional studies on specific strains. To identify additional functional variations related to pathoadaptive mutations occurring in the course of chronic infection in CF, we then selected three isolates for further characterization: one early CF isolate (TNCF_23 isolated in 2007); one clonal late CF isolate (TNCF_175 isolated in 2014); one clinical isolate (VrPa97) from a non-CF patient belonging to the same sequence type (ST390) as the former isolates. With this approach, we aimed to identify additional phenotypic and functional variations between isolates with a very homogeneous genomic background, in an attempt to find out new pathoadaptive mutations occurring in the course of chronic infection in CF. Specifically, the following traits were investigated: killing of C. elegans and G. mellonella (in vivo virulence); immunomodulatory properties (IL-8 ELISA assay); competitive growth in Artificial Sputum Medium (ASM); functionality of Type Six Secretion System (T6SS). Despite their close genetic relatedness, considerable variations were observed between the three isolates, among which the late isolate TNCF_175 showed several alterations 7 putatively resulting from the adaptation process to the CF lung. TNCF_175 presented a mutation in tssK3, part of H3-T6SS; this mutation (C958T) was therefore introduced in the reference strains PAO1 and PA14, and mutated strains were subsequently complemented; killing rate on C. elegans and growth rate in ASM in mutant and complemented strains were evaluated. Conclusions. A rare feature of this strain collection is the consistent number of clonal isolates obtained from a single patient over a rather long period of 8 eight years, thus providing a model to look at microevolutionary trends within a highly homogenous bacterial population, and avoiding potential biases due to the host genetic background and clinical history. In spite of the close genomic relatedness of all isolates, a surprisingly high diversity was observed for the majority of tested phenotypes. Investigating the competitive ability of early versus late strains we propose a role for T6SS in the adaptation process to the CF lung environment. Our data suggest that once persistence has been established, a strain no longer requires its T6SS, allowing loss of function mutations to occur. Conversely, acute and early CF strains still carry a number of virulence factors, including T6SS that potentially provide an advantage in outcompeting other microorganisms in the initial stage of CF infection.