Variation in the response to antibiotics and life-history across the major Pseudomonas aeruginosa clone type (mPact) panel.

Pseudomonas aeruginosa is a ubiquitous, opportunistic human pathogen. Since it often expresses multidrug resistance, new treatment options are urgently required. Such new treatments are usually assessed with one of the canonical laboratory strains, PAO1 or PA14. However, these two strains are unlikely representative of the strains infecting patients, because they have adapted to laboratory conditions and do not capture the enormous genomic diversity of the species. Here, we characterized the major P. aeruginosa clone type (mPact) panel. This panel consists of 20 strains, which reflect the species' genomic diversity, cover all major clone types, and have both patient and environmental origins. We found significant strain variation in distinct responses toward antibiotics and general growth characteristics. Only few of the measured traits are related, suggesting independent trait optimization across strains. High resistance levels were only identified for clinical mPact isolates and could be linked to known antimicrobial resistance (AMR) genes. One strain, H01, produced highly unstable AMR combined with reduced growth under drug-free conditions, indicating an evolutionary cost to resistance. The expression of microcolonies was common among strains, especially for strain H15, which also showed reduced growth, possibly indicating another type of evolutionary trade-off. By linking isolation source, growth, and virulence to life history traits, we further identified specific adaptive strategies for individual mPact strains toward either host processes or degradation pathways. Overall, the mPact panel provides a reasonably sized set of distinct strains, enabling in-depth analysis of new treatment designs or evolutionary dynamics in consideration of the species' genomic diversity. IMPORTANCE: New treatment strategies are urgently needed for high-risk pathogens such as the opportunistic and often multidrug-resistant pathogen Pseudomonas aeruginosa. Here, we characterize the major P. aeruginosa clone type (mPact) panel. It consists of 20 strains with different origins that cover the major clone types of the species as well as its genomic diversity. This mPact panel shows significant variation in (i) resistance against distinct antibiotics, including several last resort antibiotics; (ii) related traits associated with the response to antibiotics; and (iii) general growth characteristics. We further developed a novel approach that integrates information on resistance, growth, virulence, and life-history characteristics, allowing us to demonstrate the presence of distinct adaptive strategies of the strains that focus either on host interaction or resource processing. In conclusion, the mPact panel provides a manageable number of representative strains for this important pathogen for further in-depth analyses of treatment options and evolutionary dynamics.

A VirB4 ATPase of the mobile accessory genome orchestrates core genome-encoded features of physiology, metabolism, and virulence of Pseudomonas aeruginosa TBCF10839.

Pseudomonas aeruginosa TBCF10839 is a highly virulent strain that can persist and replicate in human neutrophils. Screening of a signature-tagged mutagenesis (STM) TBCF10839 transposon library in phagocytosis tests identified a mutant that carried the transposon in the VirB4 homolog 5PG21 of an integrative and conjugative element (ICE)-associated type IV secretion system of the pKLC102 subtype. 5P21 TBCF10839 insertion mutants were deficient in metabolic versatility, secretion, quorum sensing, and virulence. The mutants were efficiently killed in phagocytosis tests in vitro and were avirulent in an acute murine airway infection model in vivo. The inactivation of 5PG21 silenced the rhl, las, and pqs operons and the gene expression for the synthesis of hydrogen cyanide, the antimetabolite l-2-amino-4-methoxy-trans-3-butenoic acid, and the H2- and H3-type VI secretion systems and their associated effectors. The mutants were impaired in the utilization of carbon sources and stored compounds that are not funneled into intermediary metabolism. This showcase demonstrates that a single gene of the mobile accessory genome can become an essential element to operate the core genome-encoded features of metabolism and virulence.

Microevolution of Pseudomonas aeruginosa in the airways of people with cystic fibrosis.

The chronic infections of cystic fibrosis (CF) airways with Pseudomonas aeruginosa are a paradigm of how environmental bacteria can conquer, adapt, and persist in an atypical habitat and successfully evade defense mechanisms and chemotherapy in a susceptible host. The within-host evolution of intraclonal diversity has been examined by whole-genome sequencing, phenotyping, and competitive fitness experiments of serial P. aeruginosa isolates collected from CF airways since onset of colonization for a period of up to 40 years. The spectrum of de novo mutations and the adaptation of phenotype and fitness of the bacterial progeny were more influenced by the living conditions in the CF lung than by the clone type of their ancestor and its genetic repertoire.

Structural genome variants of Pseudomonas aeruginosa clone C and PA14 strains.

Plasticity of Pseudomonas aeruginosa chromosomes is mainly driven by an extended accessory genome that is shaped by insertion and deletion events. Further modification of the genome composition can be induced by chromosomal inversion events which lead to relocation of genes in the affected genomic DNA segments, modify the otherwise highly conserved core genome synteny and could even alter the location of the replication terminus. Although the genome of the first sequenced strain, PAO1, displayed such a large genomic inversion, knowledge on such recombination events in the P. aeruginosa population is limited. Several large inversions had been discovered in the late 1990s in cystic fibrosis isolates of the major clonal lineage C by physical genome mapping, and subsequent work on these examples led to the characterization of the DNA at the recombination breakpoints and a presumed recombination mechanism. Since then, the topic was barely addressed in spite of the compilation of thousands of P. aeruginosa genome sequences that are deposited in databases. Due to the use of second-generation sequencing, genome contig assembly had usually followed synteny blueprints provided by the existing reference genome sequences. Inversion detection was not feasible by these approaches, as the respective read lengths did not allow reliable resolution of sequence repeats that are typically found at the borders of inverted segments. In this study, we applied PacBio and MinION long-read sequencing to isolates of the mentioned clone C collection. Confirmation of inversions predicted from the physical mapping data demonstrated that unbiased sequence assembly of such read datasets allows the detection of genomic inversions and the resolution of the recombination breakpoint regions. Additional long-read sequencing of representatives of the other major clonal lineage, PA14, revealed large inversions in several isolates, from cystic fibrosis origin as well as from other sources. These findings indicated that inversion events are not restricted to strains from chronic infection background, but could be widespread in the P. aeruginosa population and contribute to genome plasticity. Moreover, the monitored examples emphasized the role of small mobile DNA units, such as IS elements or transposons, and accessory DNA elements in the inversion-related recombination processes.

Phylogroup-specific variation shapes the clustering of antimicrobial resistance genes and defence systems across regions of genome plasticity in Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen consisting of three phylogroups (hereafter named A, B, and C). Here, we assessed phylogroup-specific evolutionary dynamics across available and also new P. aeruginosa genomes. METHODS: In this genomic analysis, we first generated new genome assemblies for 18 strains of the major P. aeruginosa clone type (mPact) panel, comprising a phylogenetically diverse collection of clinical and environmental isolates for this species. Thereafter, we combined these new genomes with 1991 publicly available P. aeruginosa genomes for a phylogenomic and comparative analysis. We specifically explored to what extent antimicrobial resistance (AMR) genes, defence systems, and virulence genes vary in their distribution across regions of genome plasticity (RGPs) and "masked" (RGP-free) genomes, and to what extent this variation differs among the phylogroups. FINDINGS: We found that members of phylogroup B possess larger genomes, contribute a comparatively larger number of pangenome families, and show lower abundance of CRISPR-Cas systems. Furthermore, AMR and defence systems are pervasive in RGPs and integrative and conjugative/mobilizable elements (ICEs/IMEs) from phylogroups A and B, and the abundance of these cargo genes is often significantly correlated. Moreover, inter- and intra-phylogroup interactions occur at the accessory genome level, suggesting frequent recombination events. Finally, we provide here the mPact panel of diverse P. aeruginosa strains that may serve as a valuable reference for functional analyses. INTERPRETATION: Altogether, our results highlight distinct pangenome characteristics of the P. aeruginosa phylogroups, which are possibly influenced by variation in the abundance of CRISPR-Cas systems and are shaped by the differential distribution of other defence systems and AMR genes. FUNDING: German Science Foundation, Max-Planck Society, Leibniz ScienceCampus Evolutionary Medicine of the Lung, BMBF program Medical Infection Genomics, Kiel Life Science Postdoc Award.

The Cystic Fibrosis Upper and Lower Airway Metagenome.

The microbial metagenome in cystic fibrosis (CF) airways was investigated by whole-genome shotgun sequencing of total DNA isolated from nasal lavage samples, oropharyngeal swabs, and induced sputum samples collected from 65 individuals with CF aged 7 to 50 years. Each patient harbored a personalized microbial metagenome unique in microbial load and composition, the exception being monocultures of the most common CF pathogens Staphylococcus aureus and Pseudomonas aeruginosa from patients with advanced lung disease. The sampling of the upper airways by nasal lavage uncovered the fungus Malassezia restricta and the bacterium Staphylococcus epidermidis as prominent species. Healthy and CF donors harbored qualitatively and quantitatively different spectra of commensal bacteria in their sputa, even in the absence of any typical CF pathogen. If P. aeruginosa, S. aureus, or Stenotrophomonas maltophilia belonged to the trio of the most abundant species in the CF sputum metagenome, common inhabitants of the respiratory tract of healthy subjects, i.e., Eubacterium sulci, Fusobacterium periodonticum, and Neisseria subflava, were present only in low numbers or not detectable. Random forest analysis identified the numerical ecological parameters of the bacterial community, such as Shannon and Simpson diversity, as the key parameters that globally distinguish sputum samples from CF and healthy donors. IMPORTANCE Cystic fibrosis (CF) is the most common life-limiting monogenetic disease in European populations and is caused by mutations in the CFTR gene. Chronic airway infections with opportunistic pathogens are the major morbidity that determines prognosis and quality of life in most people with CF. We examined the composition of the microbial communities of the oral cavity and upper and lower airways in CF patients across all age groups. From early on, the spectrum of commensals is different in health and CF. Later on, when the common CF pathogens take up residence in the lungs, we observed differential modes of depletion of the commensal microbiota in the presence of S. aureus, P. aeruginosa, S. maltophilia, or combinations thereof. It remains to be seen whether the implementation of lifelong CFTR (cystic fibrosis transmembrane conductance regulator) modulation will change the temporal evolution of the CF airway metagenome.

The Pseudomonas aeruginosa whole genome sequence: A 20th anniversary celebration.

Toward the end of August 2000, the 6.3 Mbp whole genome sequence of Pseudomonas aeruginosa strain PAO1 was published. With 5570 open reading frames (ORFs), PAO1 had the largest microbial genome sequenced up to that point in time-including a large proportion of metabolic, transport and antimicrobial resistance genes supporting its ability to colonize diverse environments. A remarkable 9% of its ORFs were predicted to encode proteins with regulatory functions, providing new insight into bacterial network complexity as a function of network size. In this celebratory article, we fast forward 20 years, and examine how access to this resource has transformed our understanding of P. aeruginosa. What follows is more than a simple review or commentary; we have specifically asked some of the leaders in the field to provide personal reflections on how the PAO1 genome sequence, along with the Pseudomonas Community Annotation Project (PseudoCAP) and Pseudomonas Genome Database (pseudomonas.com), have contributed to the many exciting discoveries in this field. In addition to bringing us all up to date with the latest developments, we also ask our contributors to speculate on how the next 20 years of Pseudomonas research might pan out.

Sequence diversity of the Pseudomonas aeruginosa population in loci that undergo microevolution in cystic fibrosis airways.

Five hundred and thirty-four unrelated Pseudomonas aeruginosa isolates from inanimate habitats, patients with cystic fibrosis (CF) and other human infections were sequenced in 19 genes that had been identified previously as the hot spots of genomic within-host evolution in serial isolates from 12 CF lungs. Amplicon sequencing confirmed a significantly higher sequence diversity of the 19 loci in P. aeruginosa isolates from CF patients compared to those from other habitats, but this overrepresentation was mainly due to the larger share of synonymous substitutions. Correspondingly, non-synonymous substitutions were either rare (gltT, lepA, ptsP) or benign (nuoL, fleR, pelF) in some loci. Other loci, however, showed an accumulation of non-neutral coding variants. Strains from the CF habitat were often mutated at evolutionarily conserved positions in the elements of stringent response (RelA, SpoT), LPS (PagL), polyamine transport (SpuE, SpuF) and alginate biosynthesis (AlgG, AlgU). The strongest skew towards the CF lung habitat was seen for amino acid sequence variants in AlgG that clustered in the carbohydrate-binding/sugar hydrolysis domain. The master regulators of quorum sensing lasR and rhlR were frequent targets for coding variants in isolates from chronic and acute human infections. Unique variants in lasR showed strong evidence of positive selection indicated by d N/d S values of ~4. The pelA gene that encodes a multidomain enzyme involved in both the formation and dispersion of Pel biofilms carried the highest number of single-nucleotide variants among the 19 genes and was the only gene with a higher frequency of missense mutations in P. aeruginosa strains from non-CF habitats than in isolates from CF airways. PelA protein variants are widely distributed in the P. aeruginosa population. In conclusion, coding variants in a subset of the examined loci are indeed characteristic for the adaptation of P. aeruginosa to the CF airways, but for other loci the elevated mutation rate is more indicative of infections in human habitats (lasR, rhlR) or global diversifying selection (pelA).

Phenotypic and Genomic Comparison of the Two Most Common ExoU-Positive Pseudomonas aeruginosa Clones, PA14 and ST235.

Genotyping of 2,882 Pseudomonas aeruginosa isolates that had been collected during the last 40 years identified the ExoU-positive lineages PA14 (ST253) and ST235 as the second and third most frequent clones in the P. aeruginosa population. Both clones were approximately 2-fold more frequently detected in animate habitats than in soil or aquatic habitats. While ST253 clone isolates were causing mainly acute and chronic infections in humans, ST235 isolates had been preferentially collected from hospitalized patients with severe acute infections, particularly, keratitis, urinary tract infections, burn wounds, and ventilator-associated pneumonia. The two major exoU clones differed substantially in the composition and flexibility of the accessory genome and by more than 8,000 amino acid sequences. Pronounced sequence variation between orthologs was noted in genes encoding elements of secretion systems and secreted effector molecules, including the type III secretion system, indicating the modes of action of the different clones. When comparing representatives of the two clones in batch culture, the PA14 strain orchestrated the quorum sensing circuitry for the expression of pathogenic traits and stopped growing in batch culture when it entered the stationary phase, but the quorum sensing-deficient ST235 strain expressed high type III secretion activity and continued to grow and to divide. In summary, unrestricted growth, high constitutive type III secretion activity, and facilitated uptake of foreign DNA could be major features that have made ST235 a global high-risk clone associated with poor outcomes of acute nosocomial infections.IMPORTANCE The ubiquitous and metabolically versatile environmental bacterium Pseudomonas aeruginosa can cause infections in a wide variety of hosts, including insects, plants, animals, and humans. P. aeruginosa is one of the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens that are the major cause of nosocomial infections in the United States and are a threat all over the world because of their capacity to become increasingly resistant to all available antibiotics. Most experimental work on P. aeruginosa has been performed with reference strains PAO1 and PA14, providing deep insight into key metabolic and regulatory pathways thought to be applicable to all P. aeruginosa strains. However, this comparative study on the two most common exoU-positive clones taught us that there are major lineages in the population such as the global high-risk clone ST235 that exhibit uncommon traits of lifestyle, genome mobility, and pathogenicity distinct from those in our knowledge gained from the studies with the reference strains.

Why? - Successful Pseudomonas aeruginosa clones with a focus on clone C.

The environmental species Pseudomonas aeruginosa thrives in a variety of habitats. Within the epidemic population structure of P. aeruginosa, occassionally highly successful clones that are equally capable to succeed in the environment and the human host arise. Framed by a highly conserved core genome, individual members of successful clones are characterized by a high variability in their accessory genome. The abundance of successful clones might be funded in specific features of the core genome or, although not mutually exclusive, in the variability of the accessory genome. In clone C, one of the most predominant clones, the plasmid pKLC102 and the PACGI-1 genomic island are two ubiquitous accessory genetic elements. The conserved transmissible locus of protein quality control (TLPQC) at the border of PACGI-1 is a unique horizontally transferred compository element, which codes predominantly for stress-related cargo gene products such as involved in protein homeostasis. As a hallmark, most TLPQC xenologues possess a core genome equivalent. With elevated temperature tolerance as a characteristic of clone C strains, the unique P. aeruginosa and clone C specific disaggregase ClpG is a major contributor to tolerance. As other successful clones, such as PA14, do not encode the TLPQC locus, ubiquitous denominators of success, if existing, need to be identified.

Metabolite profiling of the cold adaptation of Pseudomonas putida KT2440 and cold-sensitive mutants.

Free-living bacteria such as Pseudomonas putida are frequently exposed to temperature shifts and non-optimal growth conditions. We compared the transcriptome and metabolome of the cold adaptation of P. putida KT2440 and isogenic cold-sensitive transposon mutants carrying transposons in their cbrA, cbrB, pcnB, vacB, and bipA genes. Pseudomonas putida changes the mRNA expression of about 43% of all annotated open reading frames during this initial phase of cold adaptation, but only a small number of 6-93 genes were differentially expressed at 10°C between the wild-type strain and the individual mutants. The spectrum of metabolites underwent major changes during cold adaptation particularly in the mutants. Both the KT2440 strain and the mutants increased the levels of the most abundant sugars and amino acids which were more pronounced in the cold-sensitive mutants. All mutants depleted their pools for core metabolites of aromatic and sugar metabolism, but increased their pool of polar amino acids which should be advantageous to cope with the cold stress.

Genomic characterisation of an international Pseudomonas aeruginosa reference panel indicates that the two major groups draw upon distinct mobile gene pools.

Pseudomonas aeruginosa is an important opportunistic pathogen, especially in the context of infections of cystic fibrosis (CF). In order to facilitate coordinated study of this pathogen, an international reference panel of P. aeruginosa isolates was assembled. Here we report the genome sequencing and analysis of 33 of these isolates and 7 reference genomes to further characterise this panel. Core genome single nucleotide variant phylogeny demonstrated that the panel strains are widely distributed amongst the P. aeruginosa population. Common loss-of-function mutations reported as adaptive during CF (such as in mucA and mexA) were identified amongst isolates from chronic respiratory infections. From the 40 strains analysed, 37 unique resistomes were predicted, based on the Resistance Gene Identifier method using the Comprehensive Antibiotic Resistance Database. Notably, hierarchical clustering and phylogenetic reconstructions based on the presence/absence of genomic islands (GIs), prophages and other regions of genome plasticity (RGPs) supported the subdivision of P. aeruginosa into two main groups. This is the largest, most diverse analysis of GIs and associated RGPs to date, and the results suggest that, at least at the largest clade grouping level (group 1 vs group 2), each group may be drawing upon distinct mobile gene pools.

Long-Term Microevolution of Pseudomonas aeruginosa Differs between Mildly and Severely Affected Cystic Fibrosis Lungs.

Chronic airway infections with Pseudomonas aeruginosa determine morbidity in most individuals with cystic fibrosis (CF). P. aeruginosa may persist for decades in CF lungs, which provides a rare opportunity to study the long-term within-host evolution of a bacterial airway pathogen. In this work, we sought to resolve the genetic adaptation of P. aeruginosa in CF lungs from the onset of colonization until the patient's death or permanent replacement by another P. aeruginosa clone. We followed the microevolution of the first persisting P. aeruginosa clone by whole-genome sequencing of serial isolates from highly divergent clinical courses of airway infection, i.e., a fatal outcome because of respiratory insufficiency within less than 15 years, or a rather normal daily life 25-35 years after acquisition of P. aeruginosa. Nonneutral mutations predominantly emerged in P. aeruginosa genes relevant for protection against and communication with signals from the lung environment, i.e., antibiotic resistance, cell wall components, and two-component systems. Drastic and loss-of-function mutations preferentially happened during the severe courses of infection, and the bacterial lineages of the mild courses more proficiently incorporated extra metabolic genes into their accessory genome. P. aeruginosa followed different evolutionary paths depending on whether the bacterium had taken up residence in a patient with CF and normal or already compromised lung function.

The Pseudomonas aeruginosa ExoY phenotype of high-copy-number recombinants is not detectable in natural isolates.

The nucleotidyl cyclase ExoY is an effector protein of the type III secretion system of Pseudomonas aeruginosa We compared the cyclic nucleotide production and lung disease phenotypes caused by the ExoY-overexpressing strain PA103ΔexoUexoT::Tc pUCPexoY, its vector control strain PA103ΔexoUexoT::Tc pUCP18, its loss-of-function control PA103ΔexoUexoT::Tc pUCPexoY K81M and natural ExoY-positive and ExoY-negative isolates in a murine acute airway infection model. Only the P. aeruginosa carrier of the exoY-plasmid produced high levels of cUMP and caused the most severe course of infection. The pathology ascribed to ExoY from studies using the high-copy-number plasmid on mammalian cells in vitro and in vivo was not observed with natural P. aeruginosa isolates. This indicates that the role of ExoY during infection with real-life P. aeruginosa still needs to be resolved.

Recent advances in understanding Pseudomonas aeruginosa as a pathogen.

The versatile and ubiquitous Pseudomonas aeruginosa is an opportunistic pathogen causing acute and chronic infections in predisposed human subjects. Here we review recent progress in understanding P. aeruginosa population biology and virulence, its cyclic di-GMP-mediated switches of lifestyle, and its interaction with the mammalian host as well as the role of the type III and type VI secretion systems in P. aeruginosa infection.

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.

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

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 ß-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.

Oxygen-dependent regulation of c-di-GMP synthesis by SadC controls alginate production in Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces increased levels of alginate in response to oxygen-deprived conditions. The regulatory pathway(s) that links oxygen limitation to increased synthesis of alginate has remained elusive. In the present study, using immunofluorescence microscopy, we show that anaerobiosis-induced alginate production by planktonic PAO1 requires the diguanylate cyclase (DGC) SadC, previously identified as a regulator of surface-associated lifestyles. Furthermore, we found that the gene products of PA4330 and PA4331, located in a predicted operon with sadC, have a major impact on alginate production: deletion of PA4330 (odaA, for oxygen-dependent alginate synthesis activator) caused an alginate production defect under anaerobic conditions, whereas a PA4331 (odaI, for oxygen-dependent alginate synthesis inhibitor) deletion mutant produced alginate also in the presence of oxygen, which would normally inhibit alginate synthesis. Based on their sequence, OdaA and OdaI have predicted hydratase and dioxygenase reductase activities, respectively. Enzymatic assays using purified protein showed that unlike OdaA, which did not significantly affect DGC activity of SadC, OdaI inhibited c-di-GMP production by SadC. Our data indicate that SadC, OdaA and OdaI are components of a novel response pathway of P. aeruginosa that regulates alginate synthesis in an oxygen-dependent manner.

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

Bacterial populations differentiate at the subspecies level into clonal complexes. Intraclonal genome diversity was studied in 100 isolates of the two dominant Pseudomonas 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.

Clinical utilization of genomics data produced by the international Pseudomonas aeruginosa consortium.

The International Pseudomonas aeruginosa Consortium is sequencing over 1000 genomes and building an analysis pipeline for the study of Pseudomonas genome evolution, antibiotic resistance and virulence genes. Metadata, including genomic and phenotypic data for each isolate of the collection, are available through the International Pseudomonas Consortium Database (http://ipcd.ibis.ulaval.ca/). Here, we present our strategy and the results that emerged from the analysis of the first 389 genomes. With as yet unmatched resolution, our results confirm that P. aeruginosa strains can be divided into three major groups that are further divided into subgroups, some not previously reported in the literature. We also provide the first snapshot of P. aeruginosa strain diversity with respect to antibiotic resistance. Our approach will allow us to draw potential links between environmental strains and those implicated in human and animal infections, understand how patients become infected and how the infection evolves over time as well as identify prognostic markers for better evidence-based decisions on patient care.