Widespread alterations in systemic immune profile are linked to lung function heterogeneity and airway microbes in cystic fibrosis.

BACKGROUND: Excessive inflammation and recurrent airway infections characterize people with cystic fibrosis (pwCF), a disease with highly heterogeneous clinical outcomes. How the overall immune response is affected in pwCF, its relationships with the lung microbiome, and the source of clinical heterogeneity have not been fully elucidated. METHODS: Peripheral blood and sputum samples were collected from 28 pwCF and an age-matched control group. Systemic immune cell subsets and surface markers were quantified using multiparameter flow cytometry. Lung microbiome composition was reconstructed using metatranscriptomics on sputum samples, and microbial taxa were correlated to circulating immune cells and surface markers expression. RESULTS: In pwCF, we found a specific systemic immune profile characterized by widespread hyperactivation and altered frequencies of several subsets. These included substantial changes in B-cell subsets, enrichment of CD35+/CD49d+ neutrophils, and reduction in dendritic cells. Activation markers and checkpoint molecule expression levels differed from healthy subjects. CTLA-4 expression was increased in Tregs and, together with impaired B-cell subsets, correlated with patients' lung function. Concentrations and frequencies of key immune cells and marker expression correlated with the relative abundance of commensal and pathogenic bacteria in the lungs. CONCLUSION: The CF-specific immune signature, involving hyperactivation, immune dysregulation with alteration in Treg homeostasis, and impaired B-cell function, is a potential source of lung function heterogeneity. The activity of specific microbes contributes to disrupting the balance of the immune response. Our data provide a unique foundation for identifying novel markers and immunomodulatory targets to develop the future of cystic fibrosis treatment and management.

Adaptive Evolution of Pseudomonas aeruginosa in Human Airways Shows Phenotypic Convergence Despite Diverse Patterns of Genomic Changes.

Selective forces in the environment drive bacterial adaptation to novel niches, choosing the fitter variants in the population. However, in dynamic and changing environments, the evolutionary processes controlling bacterial adaptation are difficult to monitor. Here, we follow 9 people with cystic fibrosis chronically infected with Pseudomonas aeruginosa, as a proxy for bacterial adaptation. We identify and describe the bacterial changes and evolution occurring between 15 and 35 yr of within-host evolution. We combine whole-genome sequencing, RNA sequencing, and metabolomics and compare the evolutionary trajectories directed by the adaptation of 4 different P. aeruginosa lineages to the lung. Our data suggest divergent evolution at the genomic level for most of the genes, with signs of convergent evolution with respect to the acquisition of mutations in regulatory genes, which drive the transcriptional and metabolomic program at late time of evolution. Metabolomics further confirmed convergent adaptive phenotypic evolution as documented by the reduction of the quorum-sensing molecules acyl-homoserine lactone, phenazines, and rhamnolipids (except for quinolones). The modulation of the quorum-sensing repertoire suggests that similar selective forces characterize at late times of evolution independent of the patient. Collectively, our data suggest that similar environments and similar P. aeruginosa populations in the patients at prolonged time of infection are associated with an overall reduction of virulence-associated features and phenotypic convergence.

Long-term evolution of antibiotic tolerance in Pseudomonas aeruginosa lung infections.

Pathogenic bacteria respond to antibiotic pressure with the evolution of resistance but survival can also depend on their ability to tolerate antibiotic treatment, known as tolerance. While a variety of resistance mechanisms and underlying genetics are well characterized in vitro and in vivo, an understanding of the evolution of tolerance, and how it interacts with resistance in situ is lacking. We assayed for tolerance and resistance in isolates of Pseudomonas aeruginosa from chronic cystic fibrosis lung infections spanning up to 40 years of evolution, with 3 clinically relevant antibiotics: meropenem, ciprofloxacin, and tobramycin. We present evidence that tolerance is under positive selection in the lung and that it can act as an evolutionary stepping stone to resistance. However, by examining evolutionary patterns across multiple patients in different clone types, a key result is that the potential for an association between the evolution of resistance and tolerance is not inevitable, and difficult to predict.

Longitudinal Evolution of the Pseudomonas-Derived Cephalosporinase (PDC) Structure and Activity in a Cystic Fibrosis Patient Treated with ß-Lactams.

Traditional studies on the evolution of antibiotic resistance development use approaches that can range from laboratory-based experimental studies, to epidemiological surveillance, to sequencing of clinical isolates. However, evolutionary trajectories also depend on the environment in which selection takes place, compelling the need to more deeply investigate the impact of environmental complexities and their dynamics over time. Herein, we explored the within-patient adaptive long-term evolution of a Pseudomonas aeruginosa hypermutator lineage in the airways of a cystic fibrosis (CF) patient by performing a chronological tracking of mutations that occurred in different subpopulations; our results demonstrated parallel evolution events in the chromosomally encoded class C ß-lactamase (blaPDC). These multiple mutations within blaPDC shaped diverse coexisting alleles, whose frequency dynamics responded to the changing antibiotic selective pressures for more than 26 years of chronic infection. Importantly, the combination of the cumulative mutations in blaPDC provided structural and functional protein changes that resulted in a continuous enhancement of its catalytic efficiency and high level of cephalosporin resistance. This evolution was linked to the persistent treatment with ceftazidime, which we demonstrated selected for variants with robust catalytic activity against this expanded-spectrum cephalosporin. A "gain of function" of collateral resistance toward ceftolozane, a more recently introduced cephalosporin that was not prescribed to this patient, was also observed, and the biochemical basis of this cross-resistance phenomenon was elucidated. This work unveils the evolutionary trajectories paved by bacteria toward a multidrug-resistant phenotype, driven by decades of antibiotic treatment in the natural CF environmental setting. IMPORTANCE Antibiotics are becoming increasingly ineffective to treat bacterial infections. It has been consequently predicted that infectious diseases will become the biggest challenge to human health in the near future. Pseudomonas aeruginosa is considered a paradigm in antimicrobial resistance as it exploits intrinsic and acquired resistance mechanisms to resist virtually all antibiotics known. AmpC ß-lactamase is the main mechanism driving resistance in this notorious pathogen to ß-lactams, one of the most widely used classes of antibiotics for cystic fibrosis infections. Here, we focus on the ß-lactamase gene as a model resistance determinant and unveil the trajectory P. aeruginosa undertakes on the path toward a multidrug-resistant phenotype during the course of two and a half decades of chronic infection in the airways of a cystic fibrosis patient. Integrating genetic and biochemical studies in the natural environment where evolution occurs, we provide a unique perspective on this challenging landscape, addressing fundamental molecular mechanisms of resistance.

Macrolide therapy in Pseudomonas aeruginosa infections causes uL4 ribosomal protein mutations leading to high-level resistance.

OBJECTIVES: Pseudomonas aeruginosa colonizes the cystic fibrosis (CF) airways causing chronic bacterial lung infections. CF patients are routinely treated with macrolides, however, P. aeruginosa is considered insusceptible as consequence of inadequate susceptibility testing leaving resistance mechanism completely overlooked. Here, we investigated a new mechanism of macrolide resistance caused by ribosomal protein mutations. METHODS: Investigating a longitudinal collection of 529 isolates from CF patients and analysing 5758 protein sequences from different sources, mutations in P. aeruginosa's ribosomal proteins connected to macrolide resistance were identified. Using a modified susceptibility testing protocol, isolates harbouring a mutated uL4 ribosomal protein were tested for resistance against macrolide antibiotics and macrolide-induced quorum sensing modulation. Proteome and ribosome profiling were applied to assess the impact of the mutations on the bacterial physiology. RESULTS: Five uL4 mutations were identified in isolates from different CF patients. Most mapped to the conserved loop region of uL4 and resulted in increased macrolide tolerance (>10-fold relative to wt strains). Greater concentrations (>10-fold) of macrolide antibiotic were needed to inhibit the growth, reduce swimming motility, and induce redox sensitivity of the uL4 mutants. 16 proteins involved in ribosome adaptation displayed altered expression possibly to compensate for the uL4 mutations, which changed the ribosome stoichiometry without negatively affecting bacterial physiology. CONCLUSIONS: Macrolide antibiotics should, therefore, be considered as active antimicrobial agents against P. aeruginosa and resistance development should be contemplated when patients are treated with prolonged courses of macrolides. Importantly, improved macrolide susceptibility testing is necessary for the detection of resistant bacteria.

Polymicrobial infections can select against Pseudomonas aeruginosa mutators because of quorum-sensing trade-offs.

Bacteria with increased mutation rates (mutators) are common in chronic infections and are associated with poorer clinical outcomes, especially in the case of Pseudomonas aeruginosa infecting cystic fibrosis (CF) patients. There is, however, considerable between-patient variation in both P. aeruginosa mutator frequency and the composition of co-infecting pathogen communities. We investigated whether community context might affect selection of mutators. Using an in vitro CF model community, we show that P. aeruginosa mutators were favoured in the absence of other species but not in their presence. This was because there were trade-offs between adaptation to the biotic and abiotic environments (for example, loss of quorum sensing and associated toxin production was beneficial in the latter but not the former in our in vitro model community) limiting the evolvability advantage of an elevated mutation rate. Consistent with a role of co-infecting pathogens selecting against P. aeruginosa mutators in vivo, we show that the mutation frequency of P. aeruginosa population was negatively correlated with the frequency and diversity of co-infecting bacteria in CF infections. Our results suggest that co-infecting taxa can select against P. aeruginosa mutators, which may have potentially beneficial clinical consequences.

Adaptive Interactions of Achromobacter spp. with Pseudomonas aeruginosa in Cystic Fibrosis Chronic Lung Co-Infection.

In the lungs of patients with cystic fibrosis (CF), the main pathogen Pseudomonas aeruginosa is often co-isolated with other microbes, likely engaging in inter-species interactions. In the case of chronic co-infections, this cohabitation can last for a long time and evolve over time, potentially contributing to the clinical outcome. Interactions involving the emerging pathogens Achromobacter spp. have only rarely been studied, reporting inhibition of P. aeruginosa biofilm formation. To evaluate the possible evolution of such interplay, we assessed the ability of Achromobacter spp. isolates to affect the biofilm formation of co-isolated P. aeruginosa strains during long-term chronic co-infections. We observed both competition and cohabitation. An Achromobacter sp. isolate secreted exoproducts interfering with the adhesion ability of a co-isolated P. aeruginosa strain and affected its biofilm formation. Conversely, a clonal Achromobacter sp. strain later isolated from the same patient, as well as two longitudinal strains from another patient, did not show similar competitive behavior against its P. aeruginosa co-isolates. Genetic variants supporting the higher virulence of the competitive Achromobacter sp. isolate were found in its genome. Our results confirm that both inter-species competition and cohabitation are represented during chronic co-infections in CF airways, and evolution of these interplays can happen even at the late stages of chronic infection.

Compensatory evolution of Pseudomonas aeruginosa's slow growth phenotype suggests mechanisms of adaptation in cystic fibrosis.

Long-term infection of the airways of cystic fibrosis patients with Pseudomonas aeruginosa is often accompanied by a reduction in bacterial growth rate. This reduction has been hypothesised to increase within-patient fitness and overall persistence of the pathogen. Here, we apply adaptive laboratory evolution to revert the slow growth phenotype of P. aeruginosa clinical strains back to a high growth rate. We identify several evolutionary trajectories and mechanisms leading to fast growth caused by transcriptional and mutational changes, which depend on the stage of adaptation of the strain. Return to high growth rate increases antibiotic susceptibility, which is only partially dependent on reversion of mutations or changes in the transcriptional profile of genes known to be linked to antibiotic resistance. We propose that similar mechanisms and evolutionary trajectories, in reverse direction, may be involved in pathogen adaptation and the establishment of chronic infections in the antibiotic-treated airways of cystic fibrosis patients.

Omics-based tracking of Pseudomonas aeruginosa persistence in "eradicated" cystic fibrosis patients.

Whenever Pseudomonas aeruginosa is cultured from cystic fibrosis (CF) patient airways, the primary goal is eradication by antibiotic therapy. Success is defined by ≥6 months of negative bacterial airway cultures. However, we suspect that P. aeruginosa persists in airways without clinical detection for long periods.Out of 298 P. aeruginosa-infected Copenhagen CF patients, we identified 80 with complete P. aeruginosa monitoring records and measured their maximum P. aeruginosa-free eradication periods (MEP). Isolates from 72 patients were whole-genome sequenced (n=567) and clone typed. Select isolate relatedness was examined through phylogenetic analysis and phenotypic multivariate modelling.69 (86%) patients exhibited eradication in the monitoring period (2002-2018). Sequenced isolates bridged the MEP of 42 patients, and the same clone type persisted over the MEP in 18 (43%) patients. Patients with failed eradication were on average treated more intensively with antibiotics, but this may be linked to their more severe pre-MEP infection trajectories. Of the 42 patients, 26 also had sinus surgery; the majority (n=15) showed MEPs adjacent to surgery, and only five had persisting clone types. Importantly, combined phylogenetic-phenomic evaluation suggests that persisting clone types are a result of re-emergence of the same strain rather than re-infection from the environment, and similar relatedness is exhibited by paired lower and upper airway samples and in transmission cases.In conclusion, nearly half of CF patients with supposed eradication may not truly be cleared of their original bacteria according to omics-based monitoring. This distinct cohort that is persistently infected would probably benefit from tailored antibiotic therapy.

Pseudomonas aeruginosa adaptation and evolution in patients with cystic fibrosis.

Intense genome sequencing of Pseudomonas aeruginosa isolates from cystic fibrosis (CF) airways has shown inefficient eradication of the infecting bacteria, as well as previously undocumented patient-to-patient transmission of adapted clones. However, genome sequencing has limited potential as a predictor of chronic infection and of the adaptive state during infection, and thus there is increasing interest in linking phenotypic traits to the genome sequences. Phenotypic information ranges from genome-wide transcriptomic analysis of patient samples to determination of more specific traits associated with metabolic changes, stress responses, antibiotic resistance and tolerance, biofilm formation and slow growth. Environmental conditions in the CF lung shape both genetic and phenotypic changes of P. aeruginosa during infection. In this Review, we discuss the adaptive and evolutionary trajectories that lead to early diversification and late convergence, which enable P. aeruginosa to succeed in this niche, and we point out how knowledge of these biological features may be used to guide diagnosis and therapy.

Bacterial persisters in long-term infection: Emergence and fitness in a complex host environment.

Despite intensive antibiotic treatment, Pseudomonas aeruginosa often persists in the airways of cystic fibrosis (CF) patients for decades, and can do so without antibiotic resistance development. Using high-throughput screening assays of bacterial survival after treatment with high concentrations of ciprofloxacin, we have determined the prevalence of persisters in a large patient cohort using 460 longitudinal isolates of P. aeruginosa from 39 CF patients. Isolates were classed as high persister variants (Hip) if they regrew following antibiotic treatment in at least 75% of the experimental replicates. Strain genomic data, isolate phenotyping, and patient treatment records were integrated in a lineage-based analysis of persister formation and clinical impact. In total, 19% of the isolates were classified as Hip and Hip emergence increased over lineage colonization time within 22 Hip+ patients. Most Hip+ lineages produced multiple Hip isolates, but few Hip+ lineages were dominated by Hip. While we observed no strong signal of adaptive genetic convergence within Hip isolates, they generally emerged in parallel or following the development of ciprofloxacin resistance and slowed growth. Transient lineages were majority Hip-, while strains that persisted over a clinically diagnosed 'eradication' period were majority Hip+. Patients received indistinguishable treatment regimens before Hip emergence, but Hip+ patients overall were treated significantly more than Hip- patients, signaling repeated treatment failure. When subjected to in vivo-similar antibiotic dosing, a Hip isolate survived better than a non-Hip in a structured biofilm environment. In sum, the Hip phenotype appears to substantially contribute to long-term establishment of a lineage in the CF lung environment. Our results argue against the existence of a single dominant molecular mechanism underlying bacterial antibiotic persistence. We instead show that many routes, both phenotypic and genetic, are available for persister formation and consequent increases in strain fitness and treatment failure in CF airways.

Gene Loss and Acquisition in Lineages of Pseudomonas aeruginosa Evolving in Cystic Fibrosis Patient Airways.

Genome analyses have documented that there are differences in gene repertoire between evolutionary distant lineages of the same bacterial species; however, less is known about microevolutionary dynamics of gene loss and acquisition within bacterial lineages as they evolve over years. Here, we analyzed the genomes of 45 Pseudomonas aeruginosa lineages evolving in the lungs of cystic fibrosis (CF) patients to identify genes that are lost or acquired during the first years of infection. On average, lineage genome content changed by 88 genes (range, 0 to 473). Genes were more often lost than acquired, and prophage genes were more variable than bacterial genes. We identified convergent loss or acquisition of the same genes across lineages, suggesting selection for loss and acquisition of certain genes in the host environment. We found that a notable proportion of such genes are associated with virulence; a trait previously shown to be important for adaptation. Furthermore, we also compared the genomes across lineages to show that the within-lineage variable genes (i.e., genes that had been lost or acquired during the infection) often belonged to genomic content not shared across all lineages. In sum, our analysis adds to the knowledge on the pace and drivers of gene loss and acquisition in bacteria evolving over years in a human host environment and provides a basis to further understand how gene loss and acquisition play roles in lineage differentiation and host adaptation.IMPORTANCE Bacterial airway infections, predominantly caused by P. aeruginosa, are a major cause of mortality and morbidity of CF patients. While short insertions and deletions as well as point mutations occurring during infection are well studied, there is a lack of understanding of how gene loss and acquisition play roles in bacterial adaptation to the human airways. Here, we investigated P. aeruginosa within-host evolution with regard to gene loss and acquisition. We show that during long-term infection P. aeruginosa genomes tend to lose genes, in particular, genes related to virulence. This adaptive strategy allows reduction of the genome size and evasion of the host's immune response. This knowledge is crucial to understand the basic mutational steps that, on the timescale of years, diversify lineages and adds to the identification of bacterial genetic determinants that have implications for CF disease.

Electrochemical Detection of Pyocyanin as a Biomarker for Pseudomonas aeruginosa: A Focused Review.

Pseudomonas aeruginosa (PA) is a pathogen that is recognized for its advanced antibiotic resistance and its association with serious diseases such as ventilator-associated pneumonia and cystic fibrosis. The ability to rapidly detect the presence of pathogenic bacteria in patient samples is crucial for the immediate eradication of the infection. Pyocyanin is one of PA's virulence factors used to establish infections. Pyocyanin promotes virulence by interfering in numerous cellular functions in host cells due to its redox-activity. Fortunately, the redox-active nature of pyocyanin makes it ideal for detection with simple electrochemical techniques without sample pretreatment or sensor functionalization. The previous decade has seen an increased interest in the electrochemical detection of pyocyanin either as an indicator of the presence of PA in samples or as a tool for quantifying PA virulence. This review provides the first overview of the advances in electrochemical detection of pyocyanin and offers an input regarding the future directions in the field.

Visualizing the invisible: class excursions to ignite children's enthusiasm for microbes.

We have recently argued that, because microbes have pervasive - often vital - influences on our lives, and that therefore their roles must be taken into account in many of the decisions we face, society must become microbiology-literate, through the introduction of relevant microbiology topics in school curricula (Timmis et al. 2019. Environ Microbiol 21: 1513-1528). The current coronavirus pandemic is a stark example of why microbiology literacy is such a crucial enabler of informed policy decisions, particularly those involving preparedness of public-health systems for disease outbreaks and pandemics. However, a significant barrier to attaining widespread appreciation of microbial contributions to our well-being and that of the planet is the fact that microbes are seldom visible: most people are only peripherally aware of them, except when they fall ill with an infection. And it is disease, rather than all of the positive activities mediated by microbes, that colours public perception of 'germs' and endows them with their poor image. It is imperative to render microbes visible, to give them life and form for children (and adults), and to counter prevalent misconceptions, through exposure to imagination-capturing images of microbes and examples of their beneficial outputs, accompanied by a balanced narrative. This will engender automatic mental associations between everyday information inputs, as well as visual, olfactory and tactile experiences, on the one hand, and the responsible microbes/microbial communities, on the other hand. Such associations, in turn, will promote awareness of microbes and of the many positive and vital consequences of their actions, and facilitate and encourage incorporation of such consequences into relevant decision-making processes. While teaching microbiology topics in primary and secondary school is key to this objective, a strategic programme to expose children directly and personally to natural and managed microbial processes, and the results of their actions, through carefully planned class excursions to local venues, can be instrumental in bringing microbes to life for children and, collaterally, their families. In order to encourage the embedding of microbiology-centric class excursions in current curricula, we suggest and illustrate here some possibilities relating to the topics of food (a favourite pre-occupation of most children), agriculture (together with horticulture and aquaculture), health and medicine, the environment and biotechnology. And, although not all of the microbially relevant infrastructure will be within reach of schools, there is usually access to a market, local food store, wastewater treatment plant, farm, surface water body, etc., all of which can provide opportunities to explore microbiology in action. If children sometimes consider the present to be mundane, even boring, they are usually excited with both the past and the future so, where possible, visits to local museums (the past) and research institutions advancing knowledge frontiers (the future) are strongly recommended, as is a tapping into the natural enthusiasm of local researchers to leverage the educational value of excursions and virtual excursions. Children are also fascinated by the unknown, so, paradoxically, the invisibility of microbes makes them especially fascinating objects for visualization and exploration. In outlining some of the options for microbiology excursions, providing suggestions for discussion topics and considering their educational value, we strive to extend the vistas of current class excursions and to: (i) inspire teachers and school managers to incorporate more microbiology excursions into curricula; (ii) encourage microbiologists to support school excursions and generally get involved in bringing microbes to life for children; (iii) urge leaders of organizations (biopharma, food industries, universities, etc.) to give school outreach activities a more prominent place in their mission portfolios, and (iv) convey to policymakers the benefits of providing schools with funds, materials and flexibility for educational endeavours beyond the classroom.

Antibiotic resistance in Pseudomonas aeruginosa and adaptation to complex dynamic environments.

Antibiotic resistance has become a serious threat to human health (WHO Antibacterial Agents in Clinical Development: an Analysis of the Antibacterial Clinical Development Pipeline, Including Tuberculosis. Geneva: World Health Organization; 2017), and the ability to predict antibiotic resistance from genome sequencing has become a focal point for the medical community. With this genocentric prediction in mind, we were intrigued about two particular findings for a collection of clinical Pseudomonas aeruginosa isolates (Marvig et al. Nature Genetics 2015;47:57-64; Frimodt-Møller et al. Scientific Reports 2018;8:12512; Bartell et al. Nature Communications 2019;10:629): (i) 15 out of 52 genes found to be frequently targeted by adaptive mutations during the initial infection stage of cystic fibrosis airways ('candidate pathoadaptive genes') (Marvig et al. Nature Genetics 2015;47:57-64) were associated with antibiotic resistance (López-Causapé et al. Fronters in Microbiology 2018;9:685; López-Causapé et al. Antimicrobal Agents and Chemotherapy 2018;62:e02583-17); (ii) there was a parallel lack of resistance development and linkage to the genetic changes in these antibiotic-resistance-associated genes (Frimodt-Møller et al. Scientific Reports 2018;8:12512; Bartell et al. Nature Communications 2019;10:629). In this review, we highlight alternative selective forces that potentially enhance the infection success of P. aeruginosa and focus on the linkage to the 15 pathoadaptive antibiotic-resistance-associated genes, thereby showing the problems we may face when using only genomic information to predict and inform about relevant antibiotic treatment.

Nanograss sensor for selective detection of Pseudomonas aeruginosa by pyocyanin identification in airway samples.

Pyocyanin is a virulence factor solely produced by the pathogen Pseudomonas aeruginosa. Pyocyanin is also a redox active molecule that can be directly detected by electrochemical sensing. A nanograss (NG) based sensor for sensitive quantification of pyocyanin in sputum samples from cystic fibrosis (CF) patients is presented here. The NG sensors were custom made in a cleanroom environment by etching nanograss topography on the electrode surface followed by depositing 200 nm gold. The NG sensors were utilized for amperometric quantification of pyocyanin in spiked hypertonic saline samples, resulting in a linear calibration curve with a R2 value of 0.9901 and a limit of detection of 172 nM. The NG sensors were applied in a small pilot test on five airway samples from five CF patients. The NG sensor was capable of identifying P. aeruginosa in the airway samples in 60 s without any sample pretreatment.

Structure of Pseudomonas aeruginosa ribosomes from an aminoglycoside-resistant clinical isolate.

Resistance to antibiotics has become a major threat to modern medicine. The ribosome plays a fundamental role in cell vitality by the translation of the genetic code into proteins; hence, it is a major target for clinically useful antibiotics. We report here the cryo-electron microscopy structures of the ribosome of a pathogenic aminoglycoside (AG)-resistant Pseudomonas aeruginosa strain, as well as of a nonresistance strain isolated from a cystic fibrosis patient. The structural studies disclosed defective ribosome complex formation due to a conformational change of rRNA helix H69, an essential intersubunit bridge, and a secondary binding site of the AGs. In addition, a stable conformation of nucleotides A1486 and A1487, pointing into helix h44, is created compared to a non-AG-bound ribosome. We suggest that altering the conformations of ribosomal protein uL6 and rRNA helix H69, which interact with initiation-factor IF2, interferes with proper protein synthesis initiation.

Adapting to the Airways: Metabolic Requirements of Pseudomonas aeruginosa during the Infection of Cystic Fibrosis Patients.

Pseudomonas aeruginosa is one of the major causes of morbidity and mortality of cystic fibrosis patients. During the infection, the bacteria colonize the nutritional rich lung mucus, which is present in the airway secretions in the patients, and they adapt their phenotype accordingly to the lung environment. In the airways, P. aeruginosa undergoes a broad metabolic rewiring as a consequence of the nutritional and stressful complexity of the lungs. However, the role of such metabolic rewiring on the infection outcome is poorly understood. Here, we review the metabolic evolution of clinical strains of P. aeruginosa during a cystic fibrosis lung infection and the metabolic functions operating in vivo under patho-physiological conditions. Finally, we discuss the perspective of modeling the cystic fibrosis environment using genome scale metabolic models of P. aeruginosa. Understanding the physiological changes occurring during the infection may pave the way to a more effective treatment for P. aeruginosa lung infections.

Filamentous bacteriophages are associated with chronic Pseudomonas lung infections and antibiotic resistance in cystic fibrosis.

Filamentous bacteriophage (Pf phage) contribute to the virulence of Pseudomonas aeruginosa infections in animal models, but their relevance to human disease is unclear. We sought to interrogate the prevalence and clinical relevance of Pf phage in patients with cystic fibrosis (CF) using sputum samples from two well-characterized patient cohorts. Bacterial genomic analysis in a Danish longitudinal cohort of 34 patients with CF revealed that 26.5% (n = 9) were consistently Pf phage positive. In the second cohort, a prospective cross-sectional cohort of 58 patients with CF at Stanford, sputum qPCR analysis showed that 36.2% (n = 21) of patients were Pf phage positive. In both cohorts, patients positive for Pf phage were older, and in the Stanford CF cohort, patients positive for Pf phage were more likely to have chronic P. aeruginosa infection and had greater declines in pulmonary function during exacerbations than patients negative for Pf phage presence in the sputum. Last, P. aeruginosa strains carrying Pf phage exhibited increased resistance to antipseudomonal antibiotics. Mechanistically, in vitro analysis showed that Pf phage sequesters these same antibiotics, suggesting that this mechanism may thereby contribute to the selection of antibiotic resistance over time. These data provide evidence that Pf phage may contribute to clinical outcomes in P. aeruginosa infection in CF.