Metabolic Network Analysis of Pseudomonas aeruginosa during Chronic Cystic Fibrosis Lung Infection

System-level modeling is beginning to be used to decipher high throughput data in the context of disease. In this study, we present an integration of expression microarray data with a genome-scale metabolic reconstruction of Pseudomonas aeruginosa in the context of a chronic cystic fibrosis (CF) lung infection. A genome-scale reconstruction of P. aeruginosa metabolism was tailored to represent the metabolic states of two clonally related lineages of P. aeruginosa isolated from the lungs of a CF patient at different points over a 44-month time course, giving a mechanistic glimpse into how the bacterial metabolism adapts over time in the CF lung. Metabolic capacities were analyzed to determine how tradeoffs between growth and other important cellular processes shift during disease progression. Genes whose knockouts were either significantly growth reducing or lethal in silico were also identified for each time point and serve as hypotheses for future drug targeting efforts specific to the stages of disease progression.The last decade has witnessed an explosion in both the quantity and the pace of biological discovery. High throughput methods have been developed and leveraged at an expanding rate, with the accumulation of high throughput data outstripping the capacity for analysis using conventional methods (16, 21). To face these new challenges, systems-focused methods have come to the forefront of biological discovery, enabling a synergistic merging of network analysis with the existing reductionist paradigms that have fueled biology for the past half-century (25, 40).One of the most pressing applications of systems analysis is unraveling the myriad factors that combine to form human disease. This ambitious goal has motivated a surge of interest in the collection and analysis of microarray data, which has emerged as a dominant technology for gathering genome-scale data due to its relatively low cost, ubiquity, ease, and increasingly high resolution and reproducibility (42). In particular, microarrays for gene expression profiling have been used in longitudinal studies of disease, as it enables a glimpse at the internal changes cells undergo as a disease progresses. While many such studies have been published, very little model-driven analysis has been leveraged toward interpreting these data at the network level. There is a tremendous need for this next level of analysis, as a network approach promises a deeper mechanistic understanding of whole-cell phenotypes that will be crucial for determining better therapies in the future.With the increase in life span of cystic fibrosis (CF) patients over the last several decades, bacterial infections of the thickened mucus of the lung have become the primary disease burden that must be managed in these patients today (23). The peculiarities of the CF lung mucosal environment render it a ripe environment for growth of Pseudomonas aeruginosa in particular, a notorious opportunistic pathogen that chronically infects the lungs of nearly every CF patient by an early age (32). Due to the ability of P. aeruginosa to thrive in many varied environments and its possession of a large number of regulators, it has been hypothesized that an important determinant of the virulence of this pathogen is its exceptional metabolic versatility and adaptability (37).CF lung infections involve many adaptive stages as the bacteria respond to the host lung environment and as the lungs contemporaneously remodel based on the stresses of infection (18, 20, 35). Long-term bacterial adaptations have been studied in part through gene expression profiling, and it has been noted that a significant percentage of genes differentially expressed during chronic infection encode physiological or metabolic functions (12, 36). This finding reinforces the hypothesis that the metabolic versatility of P. aeruginosa is a large factor in its pathogenicity. As a tool in studying the metabolism of this opportunistic human pathogen, we have previously published a genome-scale reconstruction of the P. aeruginosa PAO1 strain (26). This reconstruction accounts for the functions of 1,056 genes, 883 reactions, and 760 metabolites, incorporating the functions of approximately 20% of the genes in the genome into a functional computational model that is amenable to metabolic flux-level analysis (9, 17, 31). Methods for integrating high-throughput data, including gene expression array data, with genome-scale models of metabolism in order to study tissue- or condition-dependent metabolic phenotypes are developing (1, 4-6, 22, 34). By integrating gene expression data from a longitudinal study of P. aeruginosa growth (12) with our model of P. aeruginosa metabolism (26), we are providing the first network-driven analysis of metabolic changes in P. aeruginosa growing in the CF lung. By evaluating the metabolic changes that occur in this environment, we offer a deeper understanding of how the metabolism of this pathogen adapts during a chronic CF infection and present a new way to view its evolving metabolic lifestyle.     

Nitrous Oxide Production in Sputum from Cystic Fibrosis Patients with Chronic Pseudomonas aeruginosa Lung Infection

Chronic lung infection by Pseudomonas aeruginosa is the major severe complication in cystic fibrosis (CF) patients, where P. aeruginosa persists and grows in biofilms in the endobronchial mucus under hypoxic conditions. Numerous polymorphonuclear leukocytes (PMNs) surround the biofilms and create local anoxia by consuming the majority of O₂ for production of reactive oxygen species (ROS). We hypothesized that P. aeruginosa acquires energy for growth in anaerobic endobronchial mucus by denitrification, which can be demonstrated by production of nitrous oxide (N₂O), an intermediate in the denitrification pathway. We measured N₂O and O₂ with electrochemical microsensors in 8 freshly expectorated sputum samples from 7 CF patients with chronic P. aeruginosa infection. The concentrations of NO₃ ⁻ and NO₂ ⁻ in sputum were estimated by the Griess reagent. We found a maximum median concentration of 41.8 µM N₂O (range 1.4–157.9 µM N₂O). The concentration of N₂O in the sputum was higher below the oxygenated layers. In 4 samples the N₂O concentration increased during the initial 6 h of measurements before decreasing for approximately 6 h. Concomitantly, the concentration of NO₃ ⁻ decreased in sputum during 24 hours of incubation. We demonstrate for the first time production of N₂O in clinical material from infected human airways indicating pathogenic metabolism based on denitrification. Therefore, P. aeruginosa may acquire energy for growth by denitrification in anoxic endobronchial mucus in CF patients. Such ability for anaerobic growth may be a hitherto ignored key aspect of chronic P. aeruginosa infections that can inform new strategies for treatment and prevention.     

Mucoidy,Quorum Sensing,Mismatch Repair and Antibiotic Resistance in Pseudomonas aeruginosa from Cystic Fibrosis Chronic Airways Infections

Survival of Pseudomonas aeruginosa in cystic fibrosis (CF) chronic infections is based on a genetic adaptation process consisting of mutations in specific genes, which can produce advantageous phenotypic switches and ensure its persistence in the lung. Among these, mutations inactivating the regulators MucA (alginate biosynthesis), LasR (quorum sensing) and MexZ (multidrug-efflux pump MexXY) are the most frequently observed, with those inactivating the DNA mismatch repair system (MRS) being also highly prevalent in P. aeruginosa CF isolates, leading to hypermutator phenotypes that could contribute to this adaptive mutagenesis by virtue of an increased mutation rate. Here, we characterized the mutations found in the mucA, lasR, mexZ and MRS genes in P. aeruginosa isolates obtained from Argentinean CF patients, and analyzed the potential association of mucA, lasR and mexZ mutagenesis with MRS-deficiency and antibiotic resistance. Thus, 38 isolates from 26 chronically infected CF patients were characterized for their phenotypic traits, PFGE genotypic patterns, mutations in the mucA, lasR, mexZ, mutS and mutL gene coding sequences and antibiotic resistance profiles. The most frequently mutated gene was mexZ (79%), followed by mucA (63%) and lasR (39%) as well as a high prevalence (42%) of hypermutators being observed due to loss-of-function mutations in mutL (60%) followed by mutS (40%). Interestingly, mutational spectra were particular to each gene, suggesting that several mechanisms are responsible for mutations during chronic infection. However, no link could be established between hypermutability and mutagenesis in mucA, lasR and mexZ, indicating that MRS-deficiency was not involved in the acquisition of these mutations. Finally, although inactivation of mucA, lasR and mexZ has been previously shown to confer resistance/tolerance to antibiotics, only mutations in MRS genes could be related to an antibiotic resistance increase. These results help to unravel the mutational dynamics that lead to the adaptation of P. aeruginosa to the CF lung.     

Affecting Pseudomonas aeruginosa Phenotypic Plasticity by Quorum Sensing Dysregulation Hampers Pathogenicity in Murine Chronic Lung Infection

In Pseudomonas aeruginosa quorum sensing (QS) activates the production of virulence factors, playing a critical role in pathogenesis. Multiple negative regulators modulate the timing and the extent of the QS response either in the pre-quorum or post-quorum phases of growth. This regulation likely increases P. aeruginosa phenotypic plasticity and population fitness, facilitating colonization of challenging environments such as higher organisms. Accordingly, in addition to the factors required for QS signals synthesis and response, also QS regulators have been proposed as targets for anti-virulence therapies. However, while it is known that P. aeruginosa mutants impaired in QS are attenuated in their pathogenic potential, the effect of mutations causing a dysregulated timing and/or magnitude of the QS response has been poorly investigated so far in animal models of infection. In order to investigate the impact of QS dysregulation on P. aeruginosa pathogenesis in a murine model of lung infection, the QteE and RsaL proteins have been selected as representatives of negative regulators controlling P. aeruginosa QS in the pre- and post-quorum periods, respectively. Results showed that the qteE mutation does not affect P. aeruginosa lethality and ability to establish chronic infection in mice, despite causing a premature QS response and enhanced virulence factors production in test tube cultures compared to the wild type. Conversely, the post-quorum dysregulation caused by the rsaL mutation hampers the establishment of P. aeruginosa chronic lung infection in mice without affecting the mortality rate. On the whole, this study contributes to a better understanding of the impact of QS regulation on P. aeruginosa phenotypic plasticity during the infection process. Possible fallouts of these findings in the anti-virulence therapy field are also discussed.     

A gacS Deletion in Pseudomonas aeruginosa Cystic Fibrosis Isolate CHA Shapes Its Virulence

Pseudomonas aeruginosa, a human opportunistic pathogen, is capable of provoking acute and chronic infections that are associated with defined sets of virulence factors. During chronic infections, the bacterium accumulates mutations that silence some and activate other genes. Here we show that the cystic fibrosis isolate CHA exhibits a unique virulence phenotype featuring a mucoid morphology, an active Type III Secretion System (T3SS, hallmark of acute infections), and no Type VI Secretion System (H1-T6SS). This virulence profile is due to a 426 bp deletion in the 3′ end of the gacS gene encoding an essential regulatory protein. The absence of GacS disturbs the Gac/Rsm pathway leading to depletion of the small regulatory RNAs RsmY/RsmZ and, in consequence, to expression of T3SS, while switching off the expression of H1-T6SS and Pel polysaccharides. The CHA isolate also exhibits full ability to swim and twitch, due to active flagellum and Type IVa pili. Thus, unlike the classical scheme of balance between virulence factors, clinical strains may adapt to a local niche by expressing both alginate exopolysaccharide, a hallmark of membrane stress that protects from antibiotic action, host defences and phagocytosis, and efficient T3S machinery that is considered as an aggressive virulence factor.     

Clonal Dissemination,Emergence of Mutator Lineages and Antibiotic Resistance Evolution in Pseudomonas aeruginosa Cystic Fibrosis Chronic Lung Infection

Chronic respiratory infection by Pseudomonas aeruginosa is a major cause of mortality in cystic fibrosis (CF). We investigated the interplay between three key microbiological aspects of these infections: the occurrence of transmissible and persistent strains, the emergence of variants with enhanced mutation rates (mutators) and the evolution of antibiotic resistance. For this purpose, 10 sequential isolates, covering up to an 8-year period, from each of 10 CF patients were studied. As anticipated, resistance significantly accumulated overtime, and occurred more frequently among mutator variants detected in 6 of the patients. Nevertheless, highest resistance was documented for the nonmutator CF epidemic strain LES-1 (ST-146) detected for the first time in Spain. A correlation between resistance profiles and resistance mechanisms evaluated [efflux pump (mexB, mexD, mexF, and mexY) and ampC overexpression and OprD production] was not always obvious and hypersusceptibility to certain antibiotics (such as aztreonam or meropenem) was frequently observed. The analysis of whole genome macrorestriction fragments through Pulsed-Field Gel Electrophoresis (PFGE) revealed that a single genotype (clone FQSE-A) produced persistent infections in 4 of the patients. Multilocus Sequence typing (MLST) identified clone FQSE-A as the CF epidemic clone ST-274, but striking discrepancies between PFGE and MLST profiles were evidenced. While PFGE macrorestriction patterns remained stable, a new sequence type (ST-1089) was detected in two of the patients, differing from ST-274 by only two point mutations in two of the genes, each leading to a nonpreviously described allele. Moreover, detailed genetic analyses revealed that the new ST-1089 is a mutS deficient mutator lineage that evolved from the epidemic strain ST-274, acquired specific resistance mechanisms, and underwent further interpatient spread. Thus, presented results provide the first evidence of interpatient dissemination of mutator lineages and denote their potential for unexpected short-term sequence type evolution, illustrating the complexity of P. aeruginosa population biology in CF.     

The Susceptibility of Pseudomonas aeruginosa Strains from Cystic Fibrosis Patients to Bacteriophages

Phage therapy may become a complement to antibiotics in the treatment of chronic Pseudomonas aeruginosa infection. To design efficient therapeutic cocktails, the genetic diversity of the species and the spectrum of susceptibility to bacteriophages must be investigated. Bacterial strains showing high levels of phage resistance need to be identified in order to decipher the underlying mechanisms. Here we have selected genetically diverse P. aeruginosa strains from cystic fibrosis patients and tested their susceptibility to a large collection of phages. Based on plaque morphology and restriction profiles, six different phages were purified from “pyophage”, a commercial cocktail directed against five different bacterial species, including P. aeruginosa. Characterization of these phages by electron microscopy and sequencing of genome fragments showed that they belong to 4 different genera. Among 47 P. aeruginosa strains, 13 were not lysed by any of the isolated phages individually or by pyophage. We isolated two new phages that could lyse some of these strains, and their genomes were sequenced. The presence/absence of a CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and Crisper associated genes) was investigated to evaluate the role of the system in phage resistance. Altogether, the results show that some P. aeruginosa strains cannot support the growth of any of the tested phages belonging to 5 different genera, and suggest that the CRISPR-Cas system is not a major defence mechanism against these lytic phages.     

Pseudomonas aeruginosa Exploits Lipid A and Muropeptides Modification as a Strategy to Lower Innate Immunity during Cystic Fibrosis Lung Infection

Pseudomonas aeruginosa can establish life-long airways chronic infection in patients with cystic fibrosis (CF) with pathogenic variants distinguished from initially acquired strain. Here, we analysed chemical and biological activity of P. aeruginosa Pathogen-Associated Molecular Patterns (PAMPs) in clonal strains, including mucoid and non-mucoid phenotypes, isolated during a period of up to 7.5 years from a CF patient. Chemical structure by MS spectrometry defined lipopolysaccharide (LPS) lipid A and peptidoglycan (PGN) muropeptides with specific structural modifications temporally associated with CF lung infection. Gene sequence analysis revealed novel mutation in pagL, which supported lipid A changes. Both LPS and PGN had different potencies when activating host innate immunity via binding TLR4 and Nod1. Significantly higher NF-kB activation, IL-8 expression and production were detected in HEK293hTLR4/MD2-CD14 and HEK293hNod1 after stimulation with LPS and PGN respectively, purified from early P. aeruginosa strain as compared to late strains. Similar results were obtained in macrophages-like cells THP-1, epithelial cells of CF origin IB3-1 and their isogenic cells C38, corrected by insertion of cystic fibrosis transmembrane conductance regulator (CFTR). In murine model, altered LPS structure of P. aeruginosa late strains induces lower leukocyte recruitment in bronchoalveolar lavage and MIP-2, KC and IL-1β cytokine levels in lung homogenates when compared with early strain. Histopathological analysis of lung tissue sections confirmed differences between LPS from early and late P. aeruginosa. Finally, in this study for the first time we unveil how P. aeruginosa has evolved the capacity to evade immune system detection, thus promoting survival and establishing favourable conditions for chronic persistence. Our findings provide relevant information with respect to chronic infections in CF.     

Effect of Quorum Sensing Inhibitor Agents against Pseudomonas aeruginosa

Russian Journal of Bioorganic Chemistry - The abuse of antibiotics in therapy has lead to the development of resistance in the target organisms. The failure of presented antibiotics to control...     

铜绿假单胞菌中群体感应系统研究进展

群体感应系统(Quorum-sensing system,QS)是一个依赖于细胞数量的基因调控系统。系统中的自诱导物(Autoinducer或AI)随细胞的数量增加而变化,当细胞数达到一定数量时,系统中的自诱导物达到一定的域值时可以与一类转录调节蛋白结合,开始诱导或抑制数量众多的基因表达,使细菌表现多细胞特性的群体行为。同时,群体感应系统受到许多外界环境因素的影响,其调节途径是一个极其复杂的级联过程。此外,以群体感应系统为药物靶点来筛选新型抗菌药物越来越受到人们的重视。结合作者本人的工作及铜绿假单胞菌中群体感应系统的最新研究进展,对该系统在铜绿假单胞菌中的作用及其调控途径进行分析、探讨和总结。     

Genomic Variation among Contemporary Pseudomonas aeruginosa Isolates from Chronically Infected Cystic Fibrosis Patients

The airways of individuals with cystic fibrosis (CF) often become chronically infected with unique strains of the opportunistic pathogen Pseudomonas aeruginosa. Several lines of evidence suggest that the infecting P. aeruginosa lineage diversifies in the CF lung niche, yet so far this contemporary diversity has not been investigated at a genomic level. In this work, we sequenced the genomes of pairs of randomly selected contemporary isolates sampled from the expectorated sputum of three chronically infected adult CF patients. Each patient was infected by a distinct strain of P. aeruginosa. Single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were identified in the DNA common to the paired isolates from different patients. The paired isolates from one patient differed due to just 1 SNP and 8 indels. The paired isolates from a second patient differed due to 54 SNPs and 38 indels. The pair of isolates from the third patient both contained a mutS mutation, which conferred a hypermutator phenotype; these isolates cumulatively differed due to 344 SNPs and 93 indels. In two of the pairs of isolates, a different accessory genome composition, specifically integrated prophage, was identified in one but not the other isolate of each pair. We conclude that contemporary isolates from a single sputum sample can differ at the SNP, indel, and accessory genome levels and that the cross-sectional genomic variation among coeval pairs of P. aeruginosa CF isolates can be comparable to the variation previously reported to differentiate between paired longitudinally sampled isolates.     

Complementation of Methionine Auxotrophs of Pseudomonas aeruginosa from Cystic Fibrosis

Auxotrophic Pseudomonas aeruginosa are exclusive to respiratory infections in cystic fibrosis (CF) and bronchiectatic patients, and isolates require specific amino acids for growth on minimal media, particularly methionine. Since auxotrophic and prototrophic P. aeruginosa from CF are identical by genotyping, we investigated the genetic events leading to methionine auxotrophy (Met⁻). Most (10/13) Met⁻ strains had the same pattern of growth on methionine precursors and required methionine exclusively for growth. Back mutation to prototrophy was very low (frequencies 10⁻⁸ to <10⁻¹⁰). Complementation of the mutations leading to auxotrophy was achieved for five strains with a genomic library of P. aeruginosa PAO1. Strains with different patterns of growth on methionine precursors were complemented by clones with different restriction patterns, while identical clones complemented strains with the same pattern of growth on methionine precursors. Methionine auxotrophy in P. aeruginosa from CF results from stable chromosomal mutations, and the commonest defect is probably in gene(s) encoding enzymes that convert homocysteine to methionine. Received: 2 August 1997 / Accepted: 23 September 1997     

Adaptation of Iron Homeostasis Pathways by a Pseudomonas aeruginosa Pyoverdine Mutant in the Cystic Fibrosis Lung

Cystic fibrosis (CF) patients suffer from chronic bacterial lung infections, most notably by Pseudomonas aeruginosa, which persists for decades in the lungs and undergoes extensive evolution. P. aeruginosa requires iron for virulence and uses the fluorescent siderophore pyoverdine to scavenge and solubilize ferric iron during acute infections. Pyoverdine mutants accumulate in the lungs of some CF patients, however, suggesting that the heme and ferrous iron acquisition pathways of P. aeruginosa are more important in this environment. Here, we sought to determine how evolution of P. aeruginosa in the CF lung affects iron acquisition and regulatory pathways through the use of longitudinal CF isolates. These analyses demonstrated a significant reduction of siderophore production during the course of CF lung infection in nearly all strains tested. Mass spectrometry analysis of one of these strains showed that the later CF isolate has streamlined the metabolic flux of extracellular heme through the HemO heme oxygenase, resulting in more-efficient heme utilization. Moreover, gene expression analysis shows that iron regulation via the PrrF small RNAs (sRNAs) is enhanced in the later CF isolate. Finally, analysis of P. aeruginosa gene expression in the lungs of various CF patients demonstrates that both PrrF and HemO are consistently expressed in the CF lung environment. Combined, these results suggest that heme is a critical source of iron during prolonged infection of the CF lung and that changes in iron and heme regulatory pathways play a crucial role in adaptation of P. aeruginosa to this ever-changing host environment.     

Proteomic Profiling of Burkholderia cenocepacia Clonal Isolates with Different Virulence Potential Retrieved from a Cystic Fibrosis Patient during Chronic Lung Infection

Respiratory infections with Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) are associated with a worse prognosis and increased risk of death. In this work, we assessed the virulence potential of three B. cenocepacia clonal isolates obtained from a CF patient between the onset of infection (isolate IST439) and before death with cepacia syndrome 3.5 years later (isolate IST4113 followed by IST4134), based on their ability to invade epithelial cells and compromise epithelial monolayer integrity. The two clonal isolates retrieved during late-stage disease were significantly more virulent than IST439. Proteomic profiling by 2-D DIGE of the last isolate recovered before the patient’s death, IST4134, and clonal isolate IST439, was performed and compared with a prior analysis of IST4113 vs. IST439. The cytoplasmic and membrane-associated enriched fractions were examined and 52 proteins were found to be similarly altered in the two last isolates compared with IST439. These proteins are involved in metabolic functions, nucleotide synthesis, translation and protein folding, cell envelope biogenesis and iron homeostasis. Results are suggestive of the important role played by metabolic reprogramming in the virulence potential and persistence of B. cenocepacia, in particular regarding bacterial adaptation to microaerophilic conditions. Also, the content of the virulence determinant AidA was higher in the last 2 isolates. Significant levels of siderophores were found to be secreted by the three clonal isolates in an iron-depleted environment, but the two late isolates were more tolerant to low iron concentrations than IST439, consistent with the relative abundance of proteins involved in iron uptake.     

Inhaled Lactonase Reduces Pseudomonas aeruginosa Quorum Sensing and Mortality in Rat Pneumonia

Rationale

The effectiveness of antibiotic molecules in treating Pseudomonas aeruginosa pneumonia is reduced as a result of the dissemination of bacterial resistance. The existence of bacterial communication systems, such as quorum sensing, has provided new opportunities of treatment. Lactonases efficiently quench acyl-homoserine lactone-based bacterial quorum sensing, implicating these enzymes as potential new anti-Pseudomonas drugs that might be evaluated in pneumonia.

Objectives

The aim of the present study was to evaluate the ability of a lactonase called SsoPox-I to reduce the mortality of a rat P. aeruginosa pneumonia.

Methods

To assess SsoPox-I-mediated quorum quenching, we first measured the activity of the virulence gene lasB, the synthesis of pyocianin, the proteolytic activity of a bacterial suspension and the formation of biofilm of a PAO1 strain grown in the presence of lactonase. In an acute lethal model of P. aeruginosa pneumonia in rats, we evaluated the effects of an early or deferred intra-tracheal treatment with SsoPox-I on the mortality, lung bacterial count and lung damage.

Measurements and Primary Results

SsoPox-I decreased PAO1 lasB virulence gene activity, pyocianin synthesis, proteolytic activity and biofilm formation. The early use of SsoPox-I reduced the mortality of rats with acute pneumonia from 75% to 20%. Histological lung damage was significantly reduced but the lung bacterial count was not modified by the treatment. A delayed treatment was associated with a non-significant reduction of mortality.

Conclusion

These results demonstrate the protective effects of lactonase SsoPox-I in P. aeruginosa pneumonia and open the way for a future therapeutic use.     

Molecular Epidemiology of Chronic Pseudomonas aeruginosa Airway Infections in Cystic Fibrosis

Background/Methods

The molecular epidemiology of the chronic airway infections with Pseudomonas aeruginosa in individuals with cystic fibrosis (CF) was investigated by cross-sectional analysis of bacterial isolates from 51 CF centers and by longitudinal analysis of serial isolates which had been collected at the CF centers Hanover and Copenhagen since the onset of airway colonization over 30 years.

Results

Genotyping revealed that the P. aeruginosa population in CF is dominated by a few ubiquitous clones. The five most common clones retrieved from the CF host also belonged to the twenty most frequent clones in the environment and in other human disease habitats. Turnover of clones in CF airways was rare. At the Hanover clinic more than half of the patient cohort was still harbouring the initially acquired clone after twenty years of airway colonization. At the Copenhagen clinic, however, two rare clones replaced the initially acquired individual clones in all but one patient.

Conclusion

The divergent epidemiology at the two sites is explained by their differential management of hygiene and antipseudomonal chemotherapy. Hygienic measures to prohibit patient-to-patient transmission and the modalities of antipseudomonal chemotherapy modify the epidemiology of the chronic P. aeruginosa infections in CF.