Lutz Wiehlmann

Population structure of Pseudomonas aeruginosa

The metabolically versatile Gram-negative bacterium Pseudomonas aeruginosa inhabits terrestrial, aquatic, animal-, human-, and plant-host-associated environments and is an important causative agent of nosocomial infections, particularly in intensive-care units. The population genetics of P. aeruginosa was investigated by an approach that is generally applicable to the rapid, robust, and informative genotyping of bacteria. DNA, amplified from the bacterial colony by circles of multiplex primer extension, is hybridized onto a microarray to yield an electronically portable binary multimarker genotype that represents the core genome by single nucleotide polymorphisms and the accessory genome by markers of genomic islets and islands. The 240 typed P. aeruginosa strains of diverse habitats and geographic origin segregated into two large nonoverlapping clusters and 45 isolated clonal complexes with few or no partners. The majority of strains belonged to few dominant clones widespread in disease and environmental habitats. The most frequent genotype was represented by the sequenced strain PA14. Core and accessory genome were found to be nonrandomly assembled in P. aeruginosa. Individual clones preferred a specific repertoire of accessory segments. Even the most promiscuous genomic island, pKLC102, had integrated preferentially into a subset of clones. Moreover, some physically distant loci of the core genome, including oriC, showed nonrandom associations of genotypes, whereas other segments in between were freely recombining. Thus, the P. aeruginosa genome is made up of clone-typical segments in core and accessory genome and of blocks in the core with unrestricted gene flow in the population.

The Neglected Intrinsic Resistome of Bacterial Pathogens

Bacteria with intrinsic resistance to antibiotics are a worrisome health problem. It is widely believed that intrinsic antibiotic resistance of bacterial pathogens is mainly the consequence of cellular impermeability and activity of efflux pumps. However, the analysis of transposon-tagged Pseudomonas aeruginosa mutants presented in this article shows that this phenotype emerges from the action of numerous proteins from all functional categories. Mutations in some genes make P. aeruginosa more susceptible to antibiotics and thereby represent new targets. Mutations in other genes make P. aeruginosa more resistant and therefore define novel mechanisms for mutation-driven acquisition of antibiotic resistance, opening a new research field based in the prediction of resistance before it emerges in clinical environments. Antibiotics are not just weapons against bacterial competitors, but also natural signalling molecules. Our results demonstrate that antibiotic resistance genes are not merely protective shields and offer a more comprehensive view of the role of antibiotic resistance genes in the clinic and in nature.

Concordant genotype of upper and lower airways P aeruginosa and S aureus isolates in cystic fibrosis

Rationale: Lower airway (LAW) infection with Pseudomonas aeruginosa and Staphylococcus aureus is the leading cause of morbidity in cystic fibrosis (CF). The upper airways (UAW) were shown to be a gateway for acquisition of opportunistic bacteria and to act as a reservoir for them. Therefore, tools for UAW assessment within CF routine care require evaluation. Objectives: The aims of the study were non-invasive assessment of UAW and LAW microbial colonisation, and genotyping of P aeruginosa and S aureus strains from both segments. Methods: 182 patients with CF were evaluated (age 0.4–68 years, median 17 years). LAW specimens were preferably sampled as expectorated sputum and UAW specimens by nasal lavage. P aeruginosa and S aureus isolates were typed by informative single nucleotide polymorphisms (SNPs) or by spa typing, respectively. Results: Of the typable S aureus and P aeruginosa isolates from concomitant UAW- and LAW-positive specimens, 31 of 36 patients were carrying identical S aureus spa types and 23 of 24 patients identical P aeruginosa SNP genotypes in both compartments. Detection of S aureus or P aeruginosa in LAW specimens was associated with a 15- or 88-fold higher likelihood also to identify S aureus or P aeruginosa in a UAW specimen from the same patient. Conclusions: The presence of identical genotypes in UAW and LAW suggests that the UAW play a role as a reservoir of S aureus and P aeruginosa in CF. Nasal lavage appears to be suitable for non-invasive UAW sampling, but further longitudinal analyses and comparison with invasive methods are required. While UAW bacterial colonisation is typically not assessed in regular CF care, the data challenge the need to discuss diagnostic and therapeutic standards for this airway compartment. Trial registration number: NCT00266474.

Genome Diversity of Pseudomonas aeruginosa PAO1 Laboratory Strains

Pseudomonas aeruginosa PAO1 is the most commonly used strain for research on this ubiquitous and metabolically versatile opportunistic pathogen. Strain PAO1, a derivative of the original Australian PAO isolate, has been distributed worldwide to laboratories and strain collections. Over decades discordant phenotypes of PAO1 sublines have emerged. Taking the existing PAO1-UW genome sequence (named after the University of Washington, which led the sequencing project) as a blueprint, the genome sequences of reference strains MPAO1 and PAO1-DSM (stored at the German Collection for Microorganisms and Cell Cultures [DSMZ]) were resolved by physical mapping and deep short read sequencing-by-synthesis. MPAO1 has been the source of near-saturation libraries of transposon insertion mutants, and PAO1-DSM is identical in its SpeI-DpnI restriction map with the original isolate. The major genomic differences of MPAO1 and PAO1-DSM in comparison to PAO1-UW are the lack of a large inversion, a duplication of a mobile 12-kb prophage region carrying a distinct integrase and protein phosphatases or kinases, deletions of 3 to 1,006 bp in size, and at least 39 single-nucleotide substitutions, 17 of which affect protein sequences. The PAO1 sublines differed in their ability to cope with nutrient limitation and their virulence in an acute murine airway infection model. Subline PAO1-DSM outnumbered the two other sublines in late stationary growth phase. In conclusion, P. aeruginosa PAO1 shows an ongoing microevolution of genotype and phenotype that jeopardizes the reproducibility of research. High-throughput genome resequencing will resolve more cases and could become a proper quality control for strain collections.

Diversity of the Abundant pKLC102/PAGI-2 Family of Genomic Islands in Pseudomonas aeruginosa

The known genomic islands of Pseudomonas aeruginosa clone C strains are integrated into tRNA(Lys) (pKLC102) or tRNA(Gly) (PAGI-2 and PAGI-3) genes and differ from their core genomes by distinctive tetranucleotide usage patterns. pKLC102 and the related island PAPI-1 from P. aeruginosa PA14 were spontaneously mobilized from their host chromosomes at frequencies of 10% and 0.3%, making pKLC102 the most mobile genomic island known with a copy number of 30 episomal circular pKLC102 molecules per cell. The incidence of islands of the pKLC102/PAGI-2 type was investigated in 71 unrelated P. aeruginosa strains from diverse habitats and geographic origins. pKLC102- and PAGI-2-like islands were identified in 50 and 31 strains, respectively, and 15 and 10 subtypes were differentiated by hybridization on pKLC102 and PAGI-2 macroarrays. The diversity of PAGI-2-type islands was mainly caused by one large block of strain-specific genes, whereas the diversity of pKLC102-type islands was primarily generated by subtype-specific combination of gene cassettes. Chromosomal loss of PAGI-2 could be documented in sequential P. aeruginosa isolates from individuals with cystic fibrosis. PAGI-2 was present in most tested Cupriavidus metallidurans and Cupriavidus campinensis isolates from polluted environments, demonstrating the spread of PAGI-2 across habitats and species barriers. The pKLC102/PAGI-2 family is prevalent in numerous beta- and gammaproteobacteria and is characterized by high asymmetry of the cDNA strands. This evolutionarily ancient family of genomic islands retained its oligonucleotide signature during horizontal spread within and among taxa.

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.

The crystal structure of SdsA1, an alkylsulfatase from Pseudomonas aeruginosa, defines a third class of sulfatases

Pseudomonas aeruginosa is both a ubiquitous environmental bacterium and an opportunistic human pathogen. A remarkable metabolic versatility allows it to occupy a multitude of ecological niches, including wastewater treatment plants and such hostile environments as the human respiratory tract. P. aeruginosa is able to degrade and metabolize biocidic SDS, the detergent of most commercial personal hygiene products. We identify SdsA1 of P. aeruginosa as a secreted SDS hydrolase that allows the bacterium to use primary sulfates such as SDS as a sole carbon or sulfur source. Homologues of SdsA1 are found in many pathogenic and some nonpathogenic bacteria. The crystal structure of SdsA1 reveals three distinct domains. The N-terminal catalytic domain with a binuclear Zn2+ cluster is a distinct member of the metallo-β-lactamase fold family, the central dimerization domain ensures resistance to high concentrations of SDS, whereas the C-terminal domain provides a hydrophobic groove, presumably to recruit long aliphatic substrates. Crystal structures of apo-SdsA1 and complexes with substrate analog and products indicate an enzymatic mechanism involving a water molecule indirectly activated by the Zn2+ cluster. The enzyme SdsA1 thus represents a previously undescribed class of sulfatases that allows P. aeruginosa to survive and thrive under otherwise bacteriocidal conditions.

Mutations in the cohesin complex in acute myeloid leukemia: clinical and prognostic implications

Mutations in the cohesin complex are novel, genetic lesions in acute myeloid leukemia (AML) that are not well characterized. In this study, we analyzed the frequency, clinical, and prognostic implications of mutations in STAG1, STAG2, SMC1A, SMC3, and RAD21, all members of the cohesin complex, in a cohort of 389 uniformly treated AML patients by next generation sequencing. We identified a total of 23 patients (5.9%) with somatic mutations in 1 of the cohesin genes. All gene mutations were mutually exclusive, and STAG1 (1.8%), STAG2 (1.3%), and SMC3 (1.3%) were most frequently mutated. Patients with any cohesin complex mutation had lower BAALC expression levels. We found a strong association between mutations affecting the cohesin complex and NPM1. Mutated allele frequencies were similar between NPM1 and cohesin gene mutations. Overall survival (OS), relapse-free survival (RFS), and complete remission rates (CR) were not influenced by the presence of cohesin mutations (OS: hazard ratio [HR] 0.98; 95% confidence interval [CI], 0.56-1.72 [P = .94]; RFS: HR 0.7; 95% CI, 0.36-1.38 [P = .3]; CR: mutated 83% vs wild-type 76% [P = .45]). The cohesin complex presents a novel pathway affected by recurrent mutations in AML. This study is registered at www.clinicaltrials.gov as #NCT00209833.

Structure of Pseudomonas aeruginosa Populations Analyzed by Single Nucleotide Polymorphism and Pulsed-Field Gel Electrophoresis Genotyping

Pseudomonas aeruginosa has a wide ecological distribution that includes natural habitats and clinical settings. To analyze the population structure and distribution of P. aeruginosa, a collection of 111 isolates of diverse habitats and geographical origin, most of which contained a genome with a different SpeI macrorestriction profile, was typed by restriction fragment length polymorphism based on 14 single nucleotide polymorphisms (SNPs) located at seven conserved loci of the core genome (oriC, oprL, fliC, alkB2, citS, oprI, and ampC). The combination of these SNPs plus the type of fliC present (a or b) allowed the assignment of a genetic fingerprint to each strain, thus providing a simple tool for the discrimination of P. aeruginosa strains. Thirteen of the 91 identified SNP genotypes were found in two or more strains. In several cases, strains sharing their SNP genotype had different SpeI macrorestriction profiles. The highly virulent CHA strain shared its SNP genotype with other strains that had different SpeI genotypes and which had been isolated from nonclinical habitats. The reference strain PAO1 also shared its SNP genotype with other strains that had different SpeI genotypes. The P. aeruginosa chromosome contains a conserved core genome and variable amounts of accessory DNA segments (genomic islands and islets) that can be horizontally transferred among strains. The fact that some SNP genotypes were overrepresented in the P. aeruginosa population studied and that several strains sharing an SNP genotype had different SpeI macrorestriction profiles supports the idea that changes occur at a higher rate in the accessory DNA segments than in the conserved core genome.

How Pseudomonas aeruginosa adapts to various environments: a metabolomic approach

In addition to transcriptome and proteome studies, metabolome analysis represents a third complementary approach to identify metabolic pathways and adaptation processes. In order to elucidate basic principles of metabolic versatility of Pseudomonas aeruginosa, we investigated the metabolome profiles of two genetically and morphologically divergent strains, the reference strain PAO1 and the mucoid clinical isolate TBCF10839 in exponential growth and stationary phase in six different carbon sources (cadaverine, casamino acids, citrate, glucose, succinate and tryptone). Both strains exhibited strong similarities in mode of growth; the metabolite patterns were mainly defined by the growth condition. Besides this adaptive response, a basic core metabolism shapes the P. aeruginosa metabolome, independent of growth phase, carbon source and genetic background. This core metabolism includes pathways related to the central energy and amino acid metabolism. These consistently utilized metabolic pathways are closely related to glutamate which represents a dominant metabolite in all conditions analysed. In nutrient-depleted media of stationary phase cultures, P. aeruginosa maintains a specific repertoire of metabolic pathways that are related to the carbon source formerly available. This specified adaptation strategy combined with the invariant basic core metabolism may represent a fundamental requirement for the metabolic versatility of this organism.