Evolution and diversification of Pseudomonas aeruginosa in the paranasal sinuses of cystic fibrosis children have implications for chronic lung infection

Abstract

The opportunistic pathogen Pseudomonas aeruginosa is a frequent colonizer of the airways of patients suffering from cystic fibrosis (CF). Depending on early treatment regimens, the colonization will, with high probability, develop into chronic infections sooner or later, and it is important to establish under which conditions the switch to chronic infection takes place. In association with a recently established sinus surgery treatment program for CF patients at the Copenhagen CF Center, colonization of the paranasal sinuses with P. aeruginosa has been investigated, paralleled by sampling of sputum from the same patients. On the basis of genotyping and phenotypic characterization including transcription profiling, the diversity of the P. aeruginosa populations in the sinuses and the lower airways was investigated and compared. The observations made from several children show that the paranasal sinuses constitute an important niche for the colonizing bacteria in many patients. The paranasal sinuses often harbor distinct bacterial subpopulations, and in the early colonization phases there seems to be a migration from the sinuses to the lower airways, suggesting that independent adaptation and evolution take place in the sinuses. Importantly, before the onset of chronic lung infection, lineages with mutations conferring a large fitness benefit in CF airways such as mucA and lasR as well as small colony variants and antibiotic-resistant clones are part of the sinus populations. Thus, the paranasal sinuses potentially constitute a protected niche of adapted clones of P. aeruginosa, which can intermittently seed the lungs and pave the way for subsequent chronic lung infections.

Figures

Figure 1

Colony morphotypes of sinus and lower airway isolates from patients B22 and B34. Diversity in colony morphotypes is seen in and often shared between the paranasal sinuses and lower airways. (a) All morphotypes from patient B22. (*) A morphotype and genotype identical to B22-9_H was isolated again in June 2009 (B22-10_H). (b) All morphotypes from patient B34. A representative of each morphotype is displayed with sample date and isolate name.

Figure 2

Ciprofloxacin resistance profile of sinus and lower airways isolates of patient B34. The MIC of ciprofloxacin for patient B34 isolates was determined by E-test. Isolates are grouped into morphotype lineages with B34-1_A as a model ancestor. An increase in resistance was observed for the mucoid isolates, of which B34-sin_M has the highest level of resistance. The B morphotype and SCV found in the left side of the sinuses had increased resistance to the same level as the previously isolated identical morphotypes from lower airways. Patient isolates obtained after sinus surgery are not included. At least two replicate experiments were performed for each strain.

Figure 3

SCVs from the paranasal sinuses display similar characteristics as SCVs from chronic lung infection. (a) Colony morphology of first isolate and SCV from patients B34, B28, B42 and B11. SCVs were routinely grown on LB and not PIA amp plates owing to a higher instability on the latter. On PIA amp plates, the morphology of SCVs resembled isolate B42-sin_SCV. (b) Motility was generally reduced or lost in SCVs when compared with the first isolate. Swimming motility was also reduced in all SCVs; however, some activity still remained (except for B34-sin_SCV). Motility zone diameters are presented as mean±s.d. for at least three replicates. White bars, twitching motility; gray bars, swimming motility; black bars, swarming motility. (c) Biofilm formation abilities in microtiter plates were significantly increased for all SCVs. Data are presented as mean±s.d. for seven replicates. (d) The antibiotic resistance profiles of the SCVs were strain specific. Resistances to ciprofloxacin (black bars) and tobramycin (white bars) were determined by E-test. At least two replicate experiments were performed for each strain.

Figure 4

Reconstruction model of the evolution of sinus populations in patient B22. On the basis of the phenotypic and genetic profile, relatedness of isolated variants from patient B22 could be estimated. All isolates are of identical SNP genotype. The profiles of lung isolates reveal that they likely represent previous stages in the evolution of variants from the sinus populations. The observed evolutionary events of a very common left sinus morphotypes (B22-sin_F) could, for example, be followed in a step-like manner through the longitudinal lower airway isolates. This was also the case in the right sinus population; however, only one major change was observed. Swarming motility was already lost in the first lower airway isolate (B22-1_A), but not in all successive lower airway isolates, suggesting that the first isolate from this patient does not fully represent the first colonizing strain (termed ‘WT') and that segregation into each side of the sinuses happened before the collection of first lower airway isolate. As the WT phenotype is unknown, first isolate B22-1_A was chosen as a reference strain for phenotypic comparison, except for swarming motility where B22-6_E was used as reference strain. Each vertical step represents an evolutionary event that occurred at some point before the sample was collected (see timeline) and all major phenotypic changes observed (relative to first isolate) at that point are shown. ↓, considerably reduced (>70%) or lost phenotype.