Nina Cramer

Pseudomonas aeruginosa microevolution during cystic fibrosis lung infection establishes clones with adapted virulence

RATIONALE During long-term lung infection in patients with cystic fibrosis (CF), Pseudomonas aeruginosa strains develop mutations leading to clonal expansion. This microevolution is believed to be correlated with a reduced virulence. OBJECTIVES We tested this hypothesis in models of lung infection, using mice with different genetic backgrounds. METHODS From infected airways of six patients with CF, 25 P. aeruginosa clones were isolated during a period of up to 16.3 years and genotypically and phenotypically characterized. Virulence of the 8 early, 6 intermediate, and 11 late CF isolates and 5 environmental strains was assessed by monitoring acute mortality versus survival and P. aeruginosa chronic persistence versus lung clearance in mice of different genetic backgrounds, including CF mice. MEASUREMENTS AND MAIN RESULTS Different patients harbored clonally unrelated strains, but early, intermediate, and late P. aeruginosa isolates from single patients were clonally related, allowing comparative in vivo analysis. Although late isolates were attenuated in causing acute mortality in the mouse models, compared with early and intermediate clonal isolates and environmental strains, they did not differ from early and intermediate clonal isolates in their capacity to establish chronic infection and cause extensive inflammation in the murine respiratory tract. CONCLUSIONS Our findings indicate that clonal expansion of P. aeruginosa strains during microevolution within CF lungs leads to populations with altered but not reduced virulence. These P. aeruginosa clones with adapted virulence play a critical role in the pathogenesis of chronic infections and may serve to define virulence determinants as targets for novel therapies.

Pseudomonas aeruginosa Genomic Structure and Diversity.

The Pseudomonas aeruginosa genome (G + C content 65–67%, size 5.5–7 Mbp) is made up of a single circular chromosome and a variable number of plasmids. Sequencing of complete genomes or blocks of the accessory genome has revealed that the genome encodes a large repertoire of transporters, transcriptional regulators, and two-component regulatory systems which reflects its metabolic diversity to utilize a broad range of nutrients. The conserved core component of the genome is largely collinear among P. aeruginosa strains and exhibits an interclonal sequence diversity of 0.5–0.7%. Only a few loci of the core genome are subject to diversifying selection. Genome diversity is mainly caused by accessory DNA elements located in 79 regions of genome plasticity that are scattered around the genome and show an anomalous usage of mono- to tetradecanucleotides. Genomic islands of the pKLC102/PAGI-2 family that integrate into tRNALys or tRNAGly genes represent hotspots of inter- and intraclonal genomic diversity. The individual islands differ in their repertoire of metabolic genes that make a large contribution to the pangenome. In order to unravel intraclonal diversity of P. aeruginosa, the genomes of two members of the PA14 clonal complex from diverse habitats and geographic origin were compared. The genome sequences differed by less than 0.01% from each other. One hundred ninety-eight of the 231 single nucleotide substitutions (SNPs) were non-randomly distributed in the genome. Non-synonymous SNPs were mainly found in an integrated Pf1-like phage and in genes involved in transcriptional regulation, membrane and extracellular constituents, transport, and secretion. In summary, P. aeruginosa is endowed with a highly conserved core genome of low sequence diversity and a highly variable accessory genome that communicates with other pseudomonads and genera via horizontal gene transfer.

Interclonal gradient of virulence in the Pseudomonas aeruginosa pangenome from disease and environment

The population genomics of Pseudomonas aeruginosa was analysed by genome sequencing of representative strains of the 15 most frequent clonal complexes in the P. aeruginosa population and of the five most common clones from the environment of which so far no isolate from a human infection has been detected. Gene annotation identified 5892-7187 open reading frame (ORFs; median 6381 ORFs) in the 20 6.4-7.4 Mbp large genomes. The P. aeruginosa pangenome consists of a conserved core of at least 4000 genes, a combinatorial accessory genome of a further 10 000 genes and 30 000 or more rare genes that are present in only a few strains or clonal complexes. Whole genome comparisons of single nucleotide polymorphism synteny indicated unrestricted gene flow between clonal complexes by recombination. Using standardized acute lettuce, Galleria mellonella and murine airway infection models the full spectrum of possible host responses to P. aeruginosa was observed with the 20 strains ranging from unimpaired health following infection to 100% lethality. Genome comparisons indicate that the differential genetic repertoire of clones maintains a habitat-independent gradient of virulence in the P. aeruginosa population.

Molecular Epidemiology of Mutations in Antimicrobial Resistance Loci of Pseudomonas aeruginosa Isolates from Airways of Cystic Fibrosis Patients

ABSTRACT The chronic airway infections with Pseudomonas aeruginosa in people with cystic fibrosis (CF) are treated with aerosolized antibiotics, oral fluoroquinolones, and/or intravenous combination therapy with aminoglycosides and β-lactam antibiotics. An international strain collection of 361 P. aeruginosa isolates from 258 CF patients seen at 30 CF clinics was examined for mutations in 17 antimicrobial susceptibility and resistance loci that had been identified as hot spots of mutation by genome sequencing of serial isolates from a single CF clinic. Combinatorial amplicon sequencing of pooled PCR products identified 1,112 sequence variants that were not present in the genomes of representative strains of the 20 most common clones of the global P. aeruginosa population. A high frequency of singular coding variants was seen in spuE, mexA, gyrA, rpoB, fusA1, mexZ, mexY, oprD, ampD, parR, parS, and envZ (amgS), reflecting the pressure upon P. aeruginosa in lungs of CF patients to generate novel protein variants. The proportion of nonneutral amino acid exchanges was high. Of the 17 loci, mexA, mexZ, and pagL were most frequently affected by independent stop mutations. Private and de novo mutations seem to play a pivotal role in the response of P. aeruginosa populations to the antimicrobial load and the individual CF host.

Habitat‐associated skew of clone abundance in the Pseudomonas aeruginosa population

The population structure of the cosmopolitan Pseudomonas aeruginosa was investigated by genotyping 2921 isolates from 1448 independent habitats with a custom-made 58 binary marker microarray. Of 323 identified clone types, 109 clones made up 82% of the population. The 20 most frequent clones had an absolute share of 44% indicating that the P. aeruginosa population is dominated by few epidemic clonal complexes. The frequency distribution of common clones was different between inanimate habitats and human niches. The three most abundant clones in the environment were rare among isolates from human infection. Conversely, disease-associated isolates either belonged to ubiquitous clones such as C and PA14 or to clones that were uncommon in the environment. The P. aeruginosa population consists of major clones that are just as versatile in their habitat and geographic origin as the whole species and of minor clones with preference for a peculiar niche.

Intraclonal genome diversity of the major Pseudomonas aeruginosa clones C and PA14

Summary Bacterial populations differentiate at the subspecies level into clonal complexes. Intraclonal genome diversity was studied in 100 isolates of the two dominant P seudomonas aeruginosa clones C and PA14 collected from the inanimate environment, acute and chronic infections. The core genome was highly conserved among clone members with a median pairwise within‐clone single nucleotide sequence diversity of 8 × 10−6 for clone C and 2 × 10−5 for clone PA14. The composition of the accessory genome was, on the other hand, as variable within the clone as between unrelated clones. Each strain carried a large cargo of unique genes. The two dominant worldwide distributed P. aeruginosa clones combine an almost invariant core with the flexible gain and loss of genetic elements that spread by horizontal transfer.

Comparative genomic analysis of bovine, environmental, and human strains of Pseudomonas aeruginosa

Genomic analyses on versatility of the ubiquitous opportunistic pathogen Pseudomonas aeruginosa have been focusing on clinical strains from humans but much less on animal and environmental strains. Here, we aimed to compare genomic patterns of bovine, environmental, and human strains of P. aeruginosa. A collection of 71 strains, equally representing bovine (non-clinical), environmental (aquatic), and human (clinical) isolates from all main subregions of Hungary was genotyped by PCR microarray. Results were interpreted in comparison with internationally established human clinical and environmental clones, based on single nucleotide polymorphisms, on di- and multiallelic loci (fliC and fpvA) of the conserved core genome, and on genetic markers for the flexible accessory genome. As a result, a total of 33 clones were identified, with one bovine, 10 environmental, and five human clones regarded as new ones. In spite of general clonal diversity, bovine and human clones seemed to be habitat related. Bovine strains were characterized by significant overrepresentation of type III FpvA pyoverdine receptor, while the environmental and human strains showed the dominance of type I FpvA. Genotypes of non-clinical bovine strains of P. aeruginosa differed from those of human clinical strains, supporting the hypothesis about specific groups of strains colonizing specific habitats.

Multilocus amplicon sequencing of Pseudomonas aeruginosa cystic fibrosis airways isolates collected prior to and after early antipseudomonal chemotherapy

BACKGROUND Early antimicrobial chemotherapy can prevent or at least delay chronic cystic fibrosis (CF) airways infections with Pseudomonas aeruginosa. METHODS During a 10-year study period P. aeruginosa was detected for the first time in 54 CF patients regularly seen at the CF centre Hannover. Amplicon sequencing of 34 loci of the P. aeruginosa core genome was performed in baseline and post-treatment isolates of the 15 CF patients who had remained P. aeruginosa - positive after the first round of antipseudomonal chemotherapy. RESULTS Deep sequencing uncovered coexisting alternative nucleotides at in total 33 of 55,284 examined genome positions including six non-synonymous polymorphisms in the lasR gene, a key regulator of quorum sensing. After early treatment 42 of 50 novel nucleotide substitutions had emerged in exopolysaccharide biosynthesis, efflux pump and porin genes. CONCLUSIONS Early treatment selects pathoadaptive mutations in P. aeruginosa that are typical for chronic infections of CF lungs.

WS13.1 Microevolution of Pseudomonas aeruginosa in cystic fibrosis lungs

Objectives Serial P. aeruginosa isolates in half year intervals from 35 CF patients at our local CF clinic who became colonized with P. aeruginosa in the 1980s were selected. The microevolution of P. aeruginosa in CF lungs was investigated in the six patients with the mildest and the six patients with the most severe course of their chronic P. aeruginosa infection. Methods Serial isolates were genotyped with a customized microarray. Sequential isolates of the initially colonizing clone were then subjected to whole genome sequencing by SOLiD5500 technology. Nucleotide variations compared to the PA14 genome were extracted, filtered, annotated and used for the reconstruction of clades. The 250 sequenced bacterial isolates were characterized in mutation rates, morphology, motility and secretion of virulence effectors. Results Exopolysaccharide biosynthesis, antimicrobial resistance and global regulators of lifestyle and metabolism are the most common functional categories whose genes were hit by mutations in the CF lungs. Microevolution was not uniform. The P. aeruginosa clone inhabiting severely affected lungs repetitively generated descendants with stop mutations or drastic amino acid changes in key genes of lifestyle, but these loss-of-function mutants were not recovered later. In contrast, P. aeruginosa clones predominantly acquired benign amino acid substitutions in patients who maintained a normal function of their chronically colonized lungs for up to 30 years. Conclusion Modes of microevolution of P. aeruginosa in CF lungs are associated with the severity of the chronic lung infection. Supported by DFG (SFB900) and Christiane Herzog Stiftung.