Lea Mette Madsen Sommer

Convergent evolution and adaptation of Pseudomonas aeruginosa within patients with cystic fibrosis

Little is known about how within-host evolution compares between genotypically different strains of the same pathogenic species. We sequenced the whole genomes of 474 longitudinally collected clinical isolates of Pseudomonas aeruginosa sampled from 34 children and young individuals with cystic fibrosis. Our analysis of 36 P. aeruginosa lineages identified convergent molecular evolution in 52 genes. This list of genes suggests a role in host adaptation for remodeling of regulatory networks and central metabolism, acquisition of antibiotic resistance and loss of extracellular virulence factors. Furthermore, we find an ordered succession of mutations in key regulatory networks. Accordingly, mutations in downstream transcriptional regulators were contingent upon mutations in upstream regulators, suggesting that remodeling of regulatory networks might be important in adaptation. The characterization of genes involved in host adaptation may help in predicting bacterial evolution in patients with cystic fibrosis and in the design of future intervention strategies.

Deciphering the resistome of the widespread P. aeruginosa ST175 international high-risk clone through whole genome sequencing

ABSTRACT Whole-genome sequencing (WGS) was used for the characterization of the frequently extensively drug resistant (XDR) Pseudomonas aeruginosa sequence type 175 (ST175) high-risk clone. A total of 18 ST175 isolates recovered from 8 different Spanish hospitals were analyzed; 4 isolates from 4 different French hospitals were included for comparison. The typical resistance profile of ST175 included penicillins, cephalosporins, monobactams, carbapenems, aminoglycosides, and fluoroquinolones. In the phylogenetic analysis, the four French isolates clustered together with two isolates from one of the Spanish regions. Sequence variation was analyzed for 146 chromosomal genes related to antimicrobial resistance, and horizontally acquired genes were explored using online databases. The resistome of ST175 was determined mainly by mutational events; resistance traits common to all or nearly all of the strains included specific ampR mutations leading to ampC overexpression, specific mutations in oprD conferring carbapenem resistance, or a mexZ mutation leading to MexXY overexpression. All isolates additionally harbored an aadB gene conferring gentamicin and tobramycin resistance. Several other resistance traits were specific to certain geographic areas, such as a streptomycin resistance gene, aadA13, detected in all four isolates from France and in the two isolates from the Cantabria region and a glpT mutation conferring fosfomycin resistance, detected in all but these six isolates. Finally, several unique resistance mutations were detected in single isolates; particularly interesting were those in genes encoding penicillin-binding proteins (PBP1A, PBP3, and PBP4). Thus, these results provide information valuable for understanding the genetic basis of resistance and the dynamics of the dissemination and evolution of high-risk clones.

Evolutionary insight from whole-genome sequencing of Pseudomonas aeruginosa from cystic fibrosis patients

The opportunistic pathogen Pseudomonas aeruginosa causes chronic airway infections in patients with cystic fibrosis (CF), and it is directly associated with the morbidity and mortality connected with this disease. The ability of P. aeruginosa to establish chronic infections in CF patients is suggested to be due to the large genetic repertoire of P. aeruginosa and its ability to genetically adapt to the host environment. Here, we review the recent work that has applied whole-genome sequencing to understand P. aeruginosa population genomics, within-host microevolution and diversity, mutational mechanisms, genetic adaptation and transmission events. Finally, we summarize the advances in relation to medical applications and laboratory evolution experiments.

Evolution of the Pseudomonas aeruginosa mutational resistome in an international Cystic Fibrosis clone

Emergence of epidemic clones and antibiotic resistance development compromises the management of Pseudomonas aeruginosa cystic fibrosis (CF) chronic respiratory infections. Whole genome sequencing (WGS) was used to decipher the phylogeny, interpatient dissemination, WGS mutator genotypes (mutome) and resistome of a widespread clone (CC274), in isolates from two highly-distant countries, Australia and Spain, covering an 18-year period. The coexistence of two divergent CC274 clonal lineages was revealed, but without evident geographical barrier; phylogenetic reconstructions and mutational resistome demonstrated the interpatient transmission of mutators. The extraordinary capacity of P. aeruginosa to develop resistance was evidenced by the emergence of mutations in >100 genes related to antibiotic resistance during the evolution of CC274, catalyzed by mutator phenotypes. While the presence of classical mutational resistance mechanisms was confirmed and correlated with resistance phenotypes, results also showed a major role of unexpected mutations. Among them, PBP3 mutations, shaping up β-lactam resistance, were noteworthy. A high selective pressure for mexZ mutations was evidenced, but we showed for the first time that high-level aminoglycoside resistance in CF is likely driven by mutations in fusA1/fusA2, coding for elongation factor G. Altogether, our results provide valuable information for understanding the evolution of the mutational resistome of CF P. aeruginosa.

Bacterial evolution in PCD and CF patients follows the same mutational steps.

Infections with Pseudomonas aeruginosa increase morbidity in primary ciliary dyskinesia (PCD) and cystic fibrosis (CF) patients. Both diseases are associated with a defect of the mucociliary clearance; in PCD caused by non-functional cilia, in CF by changed mucus. Whole genome sequencing of P. aeruginosa isolates from CF patients has shown that persistence of clonal lineages in the airways is facilitated by genetic adaptation. It is unknown whether this also applies to P. aeruginosa airway infections in PCD. We compared within-host evolution of P. aeruginosa in PCD and CF patients. P. aeruginosa isolates from 12 PCD patients were whole genome sequenced and phenotypically characterised. Ten out of 12 PCD patients were infected with persisting clone types. We identified convergent evolution in eight genes, which are also important for persistent infections in CF airways: genes related to antibiotic resistance, quorum sensing, motility, type III secretion and mucoidity. We document phenotypic and genotypic parallelism in the evolution of P. aeruginosa across infected patients with different genetic disorders. The parallel changes and convergent adaptation and evolution may be caused by similar selective forces such as the intensive antibiotic treatment and the inflammatory response, which drive the evolutionary processes.

Is genotyping of single isolates sufficient for population structure analysis of Pseudomonas aeruginosa in cystic fibrosis airways

BackgroundThe primary cause of morbidity and mortality in cystic fibrosis (CF) patients is lung infection by Pseudomonas aeruginosa. Therefore much work has been done to understand the adaptation and evolution of P. aeruginosa in the CF lung. However, many of these studies have focused on longitudinally collected single isolates, and only few have included cross-sectional analyses of entire P. aeruginosa populations in sputum samples. To date only few studies have used the approach of metagenomic analysis for the purpose of investigating P. aeruginosa populations in CF airways.ResultsWe analysed five metagenomes together with longitudinally collected single isolates from four recently chronically infected CF patients. With this approach we were able to link the clone type and the majority of SNP profiles of the single isolates to that of the metagenome(s) for each individual patient.ConclusionBased on our analysis we find that when having access to comprehensive collections of longitudinal single isolates it is possible to rediscover the genotypes of the single isolates in the metagenomic samples. This suggests that information gained from genome sequencing of comprehensive collections of single isolates is satisfactory for many investigations of adaptation and evolution of P. aeruginosa to the CF airways.

SERS detection of the biomarker hydrogen cyanide from Pseudomonas aeruginosa cultures isolated from cystic fibrosis patients

Pseudomonas aeruginosa is the primary cause of chronic airway infections in cystic fibrosis (CF) patients. Persistent infections are seen from the first P. aeruginosa culture in about 75% of young CF patients, and it is important to discover new ways to detect P. aeruginosa at an earlier stage. The P. aeruginosa biomarker hydrogen cyanide (HCN) contains a triple bond, which is utilized in this study because of the resulting characteristic C≡N peak at 2135 cm−1 in a Raman spectrum. The Raman signal was enhanced by surface-enhanced Raman spectroscopy (SERS) on a Au-coated SERS substrate. After long-term infection, a mutation in the patho-adaptive lasR gene can alter the expression of HCN, which is why it is sometimes not possible to detect HCN in the breath of chronically infected patients. Four P. aeruginosa reference strains and 12 clinical P. aeruginosa strains isolated from CF children were evaluated, and HCN was clearly detected from overnight cultures of all wild type-like isolates and half of the later isolates from the same patients. The clinical impact could be that P. aeruginosa infections could be detected at an earlier stage, because daily breath sampling with an immediate output could be possible with a point-of-care SERS device.

Evolutionary highways to persistent infection by Pseudomonas aeruginosa

Persistent infections require bacteria to evolve from their navïe colonization state by optimizing fitness in the host. Bacteria may follow the same adaptive path, but many distinct paths could enable equally successful persistence. Here, we map the development of persistent infection over 10 years by screening 8 infection-relevant phenotypes of 443 longitudinal Pseudomonas aeruginosa isolates from 39 young cystic fibrosis patients. Using Archetype Analysis to map the multi-trait evolutionary continuum and Generalized Additive Mixed Models to identify trait correlations accounting for patient-specific influences, we find: 1) a 2-3 year timeline of rapid substantial adaptation after colonization, 2) variant “navïe” and “adapted” states reflecting discordance between phenotypic and molecular adaptation and linked by distinct evolutionary trajectories, and 3) new phenotypes associated with pathoadaptive mutations. Our results underline the environmental influences affecting evolution of complex natural populations, while providing a clinically accessible approach for tracking patient-specific pathogen adaptation to guide treatment.

127 Collateral sensitivity cycling of antibiotics for cystic fibrosis airway infections

Methods: Epidemiological ambispective multicenter study. Health status was measured by EQ-5D-3L questionnaire [Descriptive part (DS) (5 dimensions) + EQ-VAS]. PEx were defined as the European Consensus Group proposal. Patients were included consecutively. Informed consent was collected and the study was approved by the EC of the Ramon y Cajal Hospital. The communication corresponds to an interim analysis. Results: Data from 145 patients were collected (49% from the theoretical number). 54% were female. 66 patients were included without PEx, 47 had mild PEx and 32 had severe PEx. 63, 47 and 35 patients had a mild, moderate or severe disease respectively. DS score in patients with severe PEx were worse than patients with mild PEx (p-values from 0.039 to <0.001 among different dimensions), the same was seen with 5Q-VAS score (52.58±14.25 vs 70.98±18.22; p < 0.001). DS score by state of lung disease was different between moderate and severe patients in two of five dimensions whereas 5Q-VAS score was not different (63.50±19.28 vs 56.91±21.52; p=N.S.). Conclusion: Health status in patients with more severe status (either lung disease or PEx at inclusion) is worse than in the remaining patients as expected. Patients suffering a severe PEx have health status measurements (dimensions and VAS) lower than patients with a severe lung status which seems to show the important role of PEx in patient’s quality of life.

WS19.6 Diversity of Pseudomonas aeruginosa in cystic fibrosis airways

Objectives Previous investigations of the population dynamics of P. aeruginosa in the CF airways have been based on longitudinally collected single isolates, but recently this approach has been questioned. The aim of this study is to determine whether genotypic characterization of single isolates can represent the adaptation of P. aeruginosa populations in CF airways. Methods Five metagenomes from sputum samples from four CF patients were sequenced without pre-culturing. The metagenomes were analysed and compared with single isolates from the same patients. Results For all the investigated patients the most recent clone type identified among the single isolates was compared with the metagenome of the specific patient. In all cases the metagenomes were linked to a significantly higher degree of the mutations found in the single isolates sampled from the same patient, than to single isolates sampled from other patients(p Conclusion Our results document that single isolates represent the dominant clone type in the population of P. aeruginosa in CF lungs, as observed by the comparison between single isolate genomes and corresponding metagenomes. Furthermore, single isolates also show the same mutational patterns as metagenomes sampled from the same patient, and is different from metagenomes and single isolates sampled from other patients. The evolutionary dynamics of the population represented by single isolates is supported by the dominant SNP alleles from metagenomes. According to these results, the genomic information from longitudinally collected single isolates is sufficient for investigations of the dynamics of P. aeruginosa populations in the CF airways.