Mesorhizobium comanense sp. nov., isolated from groundwater.

Strain 3P27G6T was isolated from an artesian well connected to the thermal water basin of Comano Terme, Province of Trento, Italy. In phylogenetic analyses based on multilocus sequence analysis, strain 3P27G6T clustered together with Mesorhizobium australicum WSM2073T. Genome sequencing produced a 99.51 % complete genome, with a length of 7 363 057 bp and G+C content of 63.53 mol%, containing 6897 coding sequences, 55 tRNA and three rRNA. Average nucleotide identity analysis revealed that all distances calculated between strain 3P27G6T and the other Mesorhizobium genomes were below 0.9, indicating that strain 3P27G6T represents a new species. Therefore, we propose the name Mesorhizobium comanense sp. nov. with the type strain 3P27G6T (=DSM 110654T=CECT 30067T). Strain 3P27G6T is a Gram-negative, rod-shaped, aerobic bacterium. Growth condition, antibiotic susceptibility, metabolic and fatty acid-methyl esters profiles of the strain were determined. Only few nodulation and nitrogen fixation genes were found in the genome, suggesting that this strain may not be specialized in nodulation or in nitrogen fixation.

Para-cresol production by Clostridium difficile affects microbial diversity and membrane integrity of Gram-negative bacteria.

Clostridium difficile is a Gram-positive spore-forming anaerobe and a major cause of antibiotic-associated diarrhoea. Disruption of the commensal microbiota, such as through treatment with broad-spectrum antibiotics, is a critical precursor for colonisation by C. difficile and subsequent disease. Furthermore, failure of the gut microbiota to recover colonisation resistance can result in recurrence of infection. An unusual characteristic of C. difficile among gut bacteria is its ability to produce the bacteriostatic compound para-cresol (p-cresol) through fermentation of tyrosine. Here, we demonstrate that the ability of C. difficile to produce p-cresol in vitro provides a competitive advantage over gut bacteria including Escherichia coli, Klebsiella oxytoca and Bacteroides thetaiotaomicron. Metabolic profiling of competitive co-cultures revealed that acetate, alanine, butyrate, isobutyrate, p-cresol and p-hydroxyphenylacetate were the main metabolites responsible for differentiating the parent strain C. difficile (630Δerm) from a defined mutant deficient in p-cresol production. Moreover, we show that the p-cresol mutant displays a fitness defect in a mouse relapse model of C. difficile infection (CDI). Analysis of the microbiome from this mouse model of CDI demonstrates that colonisation by the p-cresol mutant results in a distinctly altered intestinal microbiota, and metabolic profile, with a greater representation of Gammaproteobacteria, including the Pseudomonales and Enterobacteriales. We demonstrate that Gammaproteobacteria are susceptible to exogenous p-cresol in vitro and that there is a clear divide between bacterial Phyla and their susceptibility to p-cresol. In general, Gram-negative species were relatively sensitive to p-cresol, whereas Gram-positive species were more tolerant. This study demonstrates that production of p-cresol by C. difficile has an effect on the viability of intestinal bacteria as well as the major metabolites produced in vitro. These observations are upheld in a mouse model of CDI, in which p-cresol production affects the biodiversity of gut microbiota and faecal metabolite profiles, suggesting that p-cresol production contributes to C. difficile survival and pathogenesis.

Staphylococcus aureus Impacts Pseudomonas aeruginosa Chronic Respiratory Disease in Murine Models.

Background: Staphylococcus aureus and Pseudomonas aeruginosa are key bacterial pathogens of the respiratory tract in patients with cystic fibrosis (CF). Although P. aeruginosa chronic bronchial infection is associated with a poorer prognosis, the consequences of S. aureus colonization on CF outcomes are controversial. Methods: In this paper, murine models of infection resembling traits of the CF human airways disease have been revisited using an infection schedule that mimics the sequence of events of pulmonary disease in CF patients. First, mice were infected with S. aureus, embedded in agar beads; this was followed by P. aeruginosa infection and analysis of bacterial load, leukocyte infiltration, and lung tissue damage. Results: We reveal that (1) S. aureus promotes severe lesions including abscess formation, (2) S. aureus increases the risk of subsequent chronic P. aeruginosa respiratory infection, and (3) once the chronic infection has been established, P. aeruginosa influences most of the inflammatory responses independent of S. aureus. Conclusions: Our findings established the significance of S. aureus colonization per se and the impact on the subsequent P. aeruginosa infection. This would point towards a thorough assessment for the need of treatment against S. aureus.

Comparative genomics and biological characterization of sequential Pseudomonas aeruginosa isolates from persistent airways infection.

BACKGROUND: Pseudomonas aeruginosa establishes life-long chronic airway infections in cystic fibrosis (CF) patients. As the disease progresses, P. aeruginosa pathoadaptive variants are distinguished from the initially acquired strain. However, the genetic basis and the biology of host-bacteria interactions leading to a persistent lifestyle of P. aeruginosa are not understood. As a model system to study long term and persistent CF infections, the P. aeruginosa RP73, isolated 16.9 years after the onset of airways colonization from a CF patient, was investigated. Comparisons with strains RP1, isolated at the onset of the colonization, and clonal RP45, isolated 7 years before RP73 were carried out to better characterize genomic evolution of P. aeruginosa in the context of CF pathogenicity. RESULTS: Virulence assessments in disease animal model, genome sequencing and comparative genomics analysis were performed for clinical RP73, RP45, RP1 and prototype strains. In murine model, RP73 showed lower lethality and a remarkable capability of long-term persistence in chronic airways infection when compared to other strains. Pathological analysis of murine lungs confirmed advanced chronic pulmonary disease, inflammation and mucus secretory cells hyperplasia. Genomic analysis predicted twelve genomic islands in the RP73 genome, some of which distinguished RP73 from other prototype strains and corresponded to regions of genome plasticity. Further, comparative genomic analyses with sequential RP isolates showed signatures of pathoadaptive mutations in virulence factors potentially linked to the development of chronic infections in CF. CONCLUSIONS: The genome plasticity of P. aeruginosa particularly in the RP73 strain strongly indicated that these alterations may form the genetic basis defining host-bacteria interactions leading to a persistent lifestyle in human lungs.

The spore-associated protein BclA1 affects the susceptibility of animals to colonization and infection by Clostridium difficile.

The BclA protein is a major component of the outermost layer of spores of a number of bacterial species and Clostridium difficile carries three bclA genes. Using insertional mutagenesis each gene was characterized and spores devoid of these proteins had surface aberrations, reduced hydrophobicity and germinated faster than wild-type spores. Therefore the BclA proteins were likely major components of the spore surface and when absent impaired the protective shield effect of this outermost layer. Analysis of infection and colonization in mice and hamsters revealed that the 50% infectious dose (ID50 ) of spores was significantly higher (2-logs) in the bclA1(-) mutant compared to the isogenic wild-type control, but that levels of toxins (A and B) were indistinguishable from animals dosed with wild-type spores. bclA1(-) spores germinated faster than wild-type spores yet mice were less susceptible to infection suggesting that BclA1 must play a key role in the initial (i.e. pre-spore germination) stages of infection. We also show that the ID50 was higher in mice infected with R20291, a 'hypervirulent' 027 strain, that carries a truncated BclA1 protein.

Integrated whole-genome screening for Pseudomonas aeruginosa virulence genes using multiple disease models reveals that pathogenicity is host specific.

Pseudomonas aeruginosa is a multi-host opportunistic pathogen causing a wide range of diseases because of the armoury of virulence factors it produces, and it is difficult to eradicate because of its intrinsic resistance to antibiotics. Using an integrated whole-genome approach, we searched for P. aeruginosa virulence genes with multi-host relevance. We constructed a random library of 57 360 Tn5 mutants in P. aeruginosa PAO1-L and screened it in vitro for those showing pleiotropic effects in virulence phenotypes (reduced swarming, exo-protease and pyocyanin production). A set of these pleiotropic mutants were assayed for reduced toxicity in Drosophila melanogaster, Caenorhabditis elegans, human cell lines and mice. Surprisingly, the screening revealed that the virulence of the majority of P. aeruginosa mutants varied between disease models, suggesting that virulence is dependent on the disease model used and hence the host environment. Genomic analysis revealed that these virulence-related genes encoded proteins from almost all functional classes, which were conserved among P. aeruginosa strains. Thus, we provide strong evidence that although P. aeruginosa is capable of infecting a wide range of hosts, many of its virulence determinants are host specific. These findings have important implication when searching for novel anti-virulence targets to develop new treatments against P. aeruginosa.

Characterization of Verona Integron-Encoded Metallo-ß-Lactamase-Type Carbapenemase-Producing Escherichia coli Isolates Collected over a 16-Year Period in Bolzano (Northern Italy).

Multidrug-resistant Escherichia coli, particularly carbapenemase producers, are a major source of concern. This study aims to investigate the long-term epidemiology of Verona integron-encoded metallo-ß-lactamase (VIM)-producing E. coli in the health district of Bolzano, Northern Italy, by examining the phenotypic and genotypic characteristics of 26 isolates obtained during 2005-2020. Isolates were identified with matrix-assisted laser desorption/ionization time-of-flight, susceptibility testing was by Vitek 2, Sensititre, and Etest; carbapenemase activity was confirmed by Etest and Carbapenemase Inactivation Method (CIM) test; and the VIM-antigen was identified by the NG-Test CARBA 5. Genome sequencing was performed on an Illumina MiSeq platform. Carbapenem minimum inhibitory concentrations varied across methodologies, and overall category agreement between phenotypic methods was low. All 23 sequenced isolates contained blaVIM-1. Eleven (47.8%) isolates belonged to the clonal lineage ST131, with fimH30 being the most common subclone. In Bolzano ST131-fimH30 was present as early as 2005. While the ST131 clonal lineage predominated for the first 10 years, various clonal lineages were present, especially in subsequent years, indicating the concurrent circulation of multiple clonal lineages. Future efforts should focus on the implementation of surveillance methods, including genomic analysis, as well as the use of updated infection control strategies and antibiotic stewardship programs to prevent the spread of these carbapenem-resistant strains.

Positive signature-tagged mutagenesis in Pseudomonas aeruginosa: tracking patho-adaptive mutations promoting airways chronic infection.

The opportunistic pathogen Pseudomonas aeruginosa can establish life-long chronic infections in the airways of cystic fibrosis (CF) patients. Persistent lifestyle is established with P. aeruginosa patho-adaptive variants, which are clonal with the initially-acquired strains. Several reports indicated that P. aeruginosa adapts by loss-of-function mutations which enhance fitness in CF airways and sustain its clonal expansion during chronic infection. To validate this model of P. aeruginosa adaptation to CF airways and to identify novel genes involved in this microevolution, we designed a novel approach of positive-selection screening by PCR-based signature-tagged mutagenesis (Pos-STM) in a murine model of chronic airways infection. A systematic positive-selection scheme using sequential rounds of in vivo screenings for bacterial maintenance, as opposed to elimination, generated a list of genes whose inactivation increased the colonization and persistence in chronic airways infection. The phenotypes associated to these Pos-STM mutations reflect alterations in diverse aspects of P. aeruginosa biology which include lack of swimming and twitching motility, lack of production of the virulence factors such as pyocyanin, biofilm formation, and metabolic functions. In addition, Pos-STM mutants showed altered invasion and stimulation of immune response when tested in human respiratory epithelial cells, indicating that P. aeruginosa is prone to revise the interaction with its host during persistent lifestyle. Finally, sequence analysis of Pos-STM genes in longitudinally P. aeruginosa isolates from CF patients identified signs of patho-adaptive mutations within the genome. This novel Pos-STM approach identified bacterial functions that can have important clinical implications for the persistent lifestyle and disease progression of the airway chronic infection.

Extended Staphylococcus aureus persistence in cystic fibrosis is associated with bacterial adaptation.

Staphylococcus aureus often persists in the airways of cystic fibrosis (CF) patients. There is only limited knowledge about bacterial persistence in and adaptation to this new ecological environment. Therefore, we used S. aureus isolates from a unique strain collection, in which all S. aureus isolates recovered from CF patients from two CF centers were stored from more than 150 CF patients for more than a decade. S. aureus early and late isolates from 71 CF patients with long-term staphylococcal colonization of the airways (≥ 5 years) were preselected by genotyping of agr and cap. Identical pairs were subjected to spa-typing and MLST. S. aureus strain pairs of individual patients with the same or closely related spa-type and identical MLST were compared for adaptive changes in important phenotypic and virulence traits. The virulence of three S. aureus strain pairs (early and late isolates) was analyzed in a murine chronic pneumonia model. Strain pairs of 29 individual patients belonged to the same MLST and same or closely related spa-types. The mean persistence of the same clone of S. aureus in 29 CF patients was 8.25 years. Late compared to early isolates were altered in production of capsule (48%), hemolysis (45%), biofilm formation (41%), as well as antibiotic susceptibility (41%), cytotoxicity (34%), colony size (28%), and spa-type (17%). Adaptive changes positively correlated with the length of S. aureus persistence. For seven patients from whom the initial colonizing isolate was recovered, staphylococcal adaptation was most apparent, with capsule production being reduced in five of seven late isolates. In a mouse chronic pneumonia model, all tested isolates strongly induced chronic pneumonia with severe lesions in bronchi and pulmonary parenchyma. Adaptive changes in S. aureus accumulated with the length of persistence in the CF airways, but differed in patients infected with the same S. aureus clonal lineage indicating that individual host factors have an impact on adaptation.

Current Uses and Future Perspectives of Genomic Technologies in Clinical Microbiology.

Recent advancements in sequencing technology and data analytics have led to a transformative era in pathogen detection and typing. These developments not only expedite the process, but also render it more cost-effective. Genomic analyses of infectious diseases are swiftly becoming the standard for pathogen analysis and control. Additionally, national surveillance systems can derive substantial benefits from genomic data, as they offer profound insights into pathogen epidemiology and the emergence of antimicrobial-resistant strains. Antimicrobial resistance (AMR) is a pressing global public health issue. While clinical laboratories have traditionally relied on culture-based antimicrobial susceptibility testing, the integration of genomic data into AMR analysis holds immense promise. Genomic-based AMR data can furnish swift, consistent, and highly accurate predictions of resistance phenotypes for specific strains or populations, all while contributing invaluable insights for surveillance. Moreover, genome sequencing assumes a pivotal role in the investigation of hospital outbreaks. It aids in the identification of infection sources, unveils genetic connections among isolates, and informs strategies for infection control. The One Health initiative, with its focus on the intricate interconnectedness of humans, animals, and the environment, seeks to develop comprehensive approaches for disease surveillance, control, and prevention. When integrated with epidemiological data from surveillance systems, genomic data can forecast the expansion of bacterial populations and species transmissions. Consequently, this provides profound insights into the evolution and genetic relationships of AMR in pathogens, hosts, and the environment.

Identification and Characterization of an RRM-Containing, RNA Binding Protein in Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative pathogen, known to acquire resistance to antibiotics used in the clinic. The RNA-binding proteome of this bacterium is poorly characterized, in particular for what concerns the proteins containing RNA Recognition Motif (RRM). Here, we browsed the A. baumannii proteome for homologous proteins to the human HuR(ELAVL1), an RNA binding protein containing three RRMs. We identified a unique locus that we called AB-Elavl, coding for a protein with a single RRM with an average of 34% identity to the first HuR RRM. We also widen the research to the genomes of all the bacteria, finding 227 entries in 12 bacterial phyla. Notably we observed a partial evolutionary divergence between the RNP1 and RNP2 conserved regions present in the prokaryotes in comparison to the metazoan consensus sequence. We checked the expression at the transcript and protein level, cloned the gene and expressed the recombinant protein. The X-ray and NMR structural characterization of the recombinant AB-Elavl revealed that the protein maintained the typical ß1α1ß2ß3α2ß4 and three-dimensional organization of eukaryotic RRMs. The biochemical analyses showed that, although the RNP1 and RNP2 show differences, it can bind to AU-rich regions like the human HuR, but with less specificity and lower affinity. Therefore, we identified an RRM-containing RNA-binding protein actually expressed in A. baumannii.

NirA Is an Alternative Nitrite Reductase from Pseudomonas aeruginosa with Potential as an Antivirulence Target.

The opportunistic pathogen Pseudomonas aeruginosa produces an arsenal of virulence factors causing a wide range of diseases in multiple hosts and is difficult to eradicate due to its intrinsic resistance to antibiotics. With the antibacterial pipeline drying up, antivirulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treat P. aeruginosa infections. To identify P. aeruginosa genes required for virulence in multiple hosts, a random library of Tn5 mutants in strain PAO1-L was previously screened in vitro for those showing pleiotropic effects in the production of virulence phenotypes. Using this strategy, we identified a Tn5 mutant with an insertion in PA4130 showing reduced levels of a number of virulence traits in vitro Construction of an isogenic mutant in this gene presented results similar to those for the Tn5 mutant. Furthermore, the PA4130 isogenic mutant showed substantial attenuation in disease models of Drosophila melanogaster and Caenorhabditis elegans as well as reduced toxicity in human cell lines. Mice infected with this mutant demonstrated an 80% increased survival rate in acute and agar bead lung infection models. PA4130 codes for a protein with homology to nitrite and sulfite reductases. Overexpression of PA4130 in the presence of the siroheme synthase CysG enabled its purification as a soluble protein. Methyl viologen oxidation assays with purified PA4130 showed that this enzyme is a nitrite reductase operating in a ferredoxin-dependent manner. The preference for nitrite and production of ammonium revealed that PA4130 is an ammonia:ferredoxin nitrite reductase and hence was named NirA.IMPORTANCE The emergence of widespread antimicrobial resistance has led to the need for development of novel therapeutic interventions. Antivirulence strategies are an attractive alternative to classic antimicrobial therapy; however, they require identification of new specific targets which can be exploited in drug discovery programs. The host-specific nature of P. aeruginosa virulence adds complexity to the discovery of these types of targets. Using a sequence of in vitro assays and phylogenetically diverse in vivo disease models, we have identified a PA4130 mutant with reduced production in a number of virulence traits and severe attenuation across all infection models tested. Characterization of PA4130 revealed that it is a ferredoxin-nitrite reductase and hence was named NirA. These results, together with attenuation of nirA mutants in different clinical isolates, high level conservation of its gene product in P. aeruginosa genomes, and the lack of orthologues in human genomes, make NirA an attractive antivirulence target.

Expression, purification, and characterization of the recombinant putative periplasmic hemin-binding protein (HutB) of Photobacterium damselae subsp. piscicida.

HutB, the periplasmic hemin binding protein of Photobacterium damselae subsp. piscicida, was produced as a recombinant protein. UV-Vis spectrophotometrical analysis showed absorption spectral changes in hemin upon mixing it with the recombinant protein, indicating complex formation. Spectrophotometric titration of HutB with hemin showed saturation at a heme/HutB ratio of 1:1 and a binding affinity (K(d)) of 10 microM.

Genomic and metagenomic insights into the microbial community of a thermal spring.

BACKGROUND: Water springs provide important ecosystem services including drinking water supply, recreation, and balneotherapy, but their microbial communities remain largely unknown. In this study, we characterized the spring water microbiome of Comano Terme (Italy) at four sampling points of the thermal spa, including natural (spring and well) and human-built (storage tank, bathtubs) environments. We integrated large-scale culturing and metagenomic approaches, with the aim of comprehensively determining the spring water taxonomic composition and functional potential. RESULTS: The groundwater feeding the spring hosted the most atypical microbiome, including many taxa known to be recalcitrant to cultivation. The core microbiome included the orders Sphingomonadales, Rhizobiales, and Caulobacterales, and the families Bradyrhizobiaceae and Moraxellaceae. A comparative genomic analysis of 72 isolates and 30 metagenome-assembled genomes (MAGs) revealed that most isolates and MAGs belonged to new species or higher taxonomic ranks widely distributed in the microbial tree of life. Average nucleotide identity (ANI) values calculated for each isolated or assembled genome showed that 10 genomes belonged to known bacterial species (> 95% ANI), 36 genomes (including 1 MAG) had ANI values ranging 85-92.5% and could be assigned as undescribed species belonging to known genera, while the remaining 55 genomes had lower ANI values (< 85%). A number of functional features were significantly over- or underrepresented in genomes derived from the four sampling sites. Functional specialization was found between sites, with for example methanogenesis being unique to groundwater whereas methanotrophy was found in all samples. CONCLUSIONS: Current knowledge on aquatic microbiomes is essentially based on surface or human-associated environments. We started uncovering the spring water microbiome, highlighting an unexpected diversity that should be further investigated. This study confirms that groundwater environments host highly adapted, stable microbial communities composed of many unknown taxa, even among the culturable fraction.

Genome-Based Approach Delivers Vaccine Candidates Against Pseudomonas aeruginosa.

High incidence, severity and increasing antibiotic resistance characterize Pseudomonas aeruginosa infections, highlighting the need for new therapeutic options. Vaccination strategies to prevent or limit P. aeruginosa infections represent a rational approach to positively impact the clinical outcome of risk patients; nevertheless this bacterium remains a challenging vaccine target. To identify novel vaccine candidates, we started from the genome sequence analysis of the P. aeruginosa reference strain PAO1 exploring the reverse vaccinology approach integrated with additional bioinformatic tools. The bioinformatic approaches resulted in the selection of 52 potential antigens. These vaccine candidates were conserved in P. aeruginosa genomes from different origin and among strains isolated longitudinally from cystic fibrosis patients. To assess the immune-protection of single or antigens combination against P. aeruginosa infection, a vaccination protocol was established in murine model of acute respiratory infection. Combinations of selected candidates, rather than single antigens, effectively controlled P. aeruginosa infection in the in vivo model of murine pneumonia. Five combinations were capable of significantly increase survival rate among challenged mice and all included PA5340, a hypothetical protein exclusively present in P. aeruginosa. PA5340 combined with PA3526-MotY gave the maximum protection. Both proteins were surface exposed by immunofluorescence and triggered a specific immune response. Combination of these two protein antigens could represent a potential vaccine to prevent P. aeruginosa infection.

Pseudomonas aeruginosa mutants defective in glucose uptake have pleiotropic phenotype and altered virulence in non-mammal infection models.

Pseudomonas spp. are endowed with a complex pathway for glucose uptake that relies on multiple transporters. In this work we report the construction and characterization of Pseudomonas aeruginosa single and multiple mutants with unmarked deletions of genes encoding outer membrane (OM) and inner membrane (IM) proteins involved in glucose uptake. We found that a triple ΔgltKGF ΔgntP ΔkguT mutant lacking all known IM transporters (named GUN for Glucose Uptake Null) is unable to grow on glucose as unique carbon source. More than 500 genes controlling both metabolic functions and virulence traits show differential expression in GUN relative to the parental strain. Consistent with transcriptomic data, the GUN mutant displays a pleiotropic phenotype. Notably, the genome-wide transcriptional profile and most phenotypic traits differ between the GUN mutant and the wild type strain irrespective of the presence of glucose, suggesting that the investigated genes may have additional roles besides glucose transport. Finally, mutants carrying single or multiple deletions in the glucose uptake genes showed attenuated virulence relative to the wild type strain in Galleria mellonella, but not in Caenorhabditis elegans infection model, supporting the notion that metabolic functions may deeply impact P. aeruginosa adaptation to specific environments found inside the host.

Persistence and Microevolution of Pseudomonas aeruginosa in the Cystic Fibrosis Lung: A Single-Patient Longitudinal Genomic Study.

Background: During its persistence in cystic fibrosis (CF) airways, P. aeruginosa develops a series of phenotypic changes by the accumulation of pathoadaptive mutations. A better understanding of the role of these mutations in the adaptive process, with particular reference to the development of multidrug resistance (MDR), is essential for future development of novel therapeutic approaches, including the identification of new drug targets and the implementation of more efficient antibiotic therapy. Although several whole-genome sequencing studies on P. aeruginosa CF lineages have been published, the evolutionary trajectories in relation to the development of antimicrobial resistance remain mostly unexplored to date. In this study, we monitored the adaptive changes of P. aeruginosa during its microevolution in the CF airways to provide an innovative, genome-wide picture of mutations and persistent phenotypes and to point out potential novel mechanisms allowing survival in CF patients under antibiotic therapy. Results: We obtained whole genome sequences of 40 P. aeruginosa clinical CF strains isolated at Trentino Regional Support CF Centre (Rovereto, Italy) from a single CF patient over an 8-year period (2007-2014). Genotypic analysis of the P. aeruginosa isolates revealed a clonal population dominated by the Sequence Type 390 and three closely related variants, indicating that all members of the population likely belong to the same clonal lineage and evolved from a common ancestor. While the majority of early isolates were susceptible to most antibiotics tested, over time resistant phenotypes increased in the persistent population. Genomic analyses of the population indicated a correlation between the evolution of antibiotic resistance profiles and phylogenetic relationships, and a number of putative pathoadaptive variations were identified. Conclusion: This study provides valuable insights into the within-host adaptation and microevolution of P. aeruginosa in the CF lung and revealed the emergence of an MDR phenotype over time, which could not be comprehensively explained by the variations found in known resistance genes. Further investigations on uncharacterized variations disclosed in this study should help to increase our understanding of the development of MDR phenotype and the poor outcome of antibiotic therapies in many CF patients.

Draft Genome Sequences of 40 Pseudomonas aeruginosa Clinical Strains Isolated from the Sputum of a Single Cystic Fibrosis Patient Over an 8-Year Period.

We report draft genome sequences of 40 Pseudomonas aeruginosa strains, isolated from the sputum of a single cystic fibrosis patient over eight years. Analyses indicated a correlation between multidrug-resistant phenotypes and population structure. Our data provide new insights into the mechanisms leading to acquisition of antibiotic resistance in P. aeruginosa.

Persistent cystic fibrosis isolate Pseudomonas aeruginosa strain RP73 exhibits an under-acylated LPS structure responsible of its low inflammatory activity.

Pseudomonas aeruginosa, the major pathogen involved in lethal infections in cystic fibrosis (CF) population, is able to cause permanent chronic infections that can persist over the years. This ability to chronic colonize CF airways is related to a series of adaptive bacterial changes involving the immunostimulant lipopolysaccharide (LPS) molecule. The structure of LPSs isolated from several P. aeruginosa strains showed conserved features that can undergo chemical changes during the establishment of the chronic infection. In the present paper, we report the elucidation of the structure and the biological activity of the R-LPS (lipooligosaccharide, LOS) isolated from the persistent CF isolate P. aeruginosa strain RP73, in order to give further insights in the adaptation mechanism of the pathogen in the CF environment. The complete structural analysis of P. aeruginosa RP73 LOS was achieved by chemical analyses, NMR spectroscopy and MALDI MS spectrometry, while the assessment of the biological activity was attained testing the in vivo pro-inflammatory capacity of the isolated LOS molecule. While a typical CF LPS is able to trigger a high immune response and production of pro-inflammatory molecules, this P. aeruginosa RP73 LOS showed to possess a low pro-inflammatory capacity. This was possible due to a singular chemical structure possessing an under-acylated lipid A very similar to the LPS of P. aeruginosa found in chronic lung diseases such as bronchiectstasis.

Adaptation of Pseudomonas aeruginosa in Cystic Fibrosis airways influences virulence of Staphylococcus aureus in vitro and murine models of co-infection.

Cystic fibrosis (CF) airways disease represents an example of polymicrobial infection whereby different bacterial species can interact and influence each other. In CF patients Staphylococcus aureus is often the initial pathogen colonizing the lungs during childhood, while Pseudomonas aeruginosa is the predominant pathogen isolated in adolescents and adults. During chronic infection, P. aeruginosa undergoes adaptation to cope with antimicrobial therapy, host response and co-infecting pathogens. However, S. aureus and P. aeruginosa often co-exist in the same niche influencing the CF pathogenesis. The goal of this study was to investigate the reciprocal interaction of P. aeruginosa and S. aureus and understand the influence of P. aeruginosa adaptation to the CF lung in order to gain important insight on the interplay occurring between the two main pathogens of CF airways, which is still largely unknown. P. aeruginosa reference strains and eight lineages of clinical strains, including early and late clonal isolates from different patients with CF, were tested for growth inhibition of S. aureus. Next, P. aeruginosa/S. aureus competition was investigated in planktonic co-culture, biofilm, and mouse pneumonia model. P. aeruginosa reference and early strains, isolated at the onset of chronic infection, outcompeted S. aureus in vitro and in vivo models of co-infection. On the contrary, our results indicated a reduced capacity to outcompete S. aureus of P. aeruginosa patho-adaptive strains, isolated after several years of chronic infection and carrying several phenotypic changes temporally associated with CF lung adaptation. Our findings provide relevant information with respect to interspecies interaction and disease progression in CF.