Secondary messenger signalling influences Pseudomonas aeruginosa adaptation to sinus and lung environments.

Pseudomonas aeruginosa is a cause of chronic respiratory tract infections in people with cystic fibrosis (CF), non-CF bronchiectasis, and chronic obstructive pulmonary disease. Prolonged infection allows the accumulation of mutations and horizontal gene transfer, increasing the likelihood of adaptive phenotypic traits. Adaptation is proposed to arise first in bacterial populations colonizing upper airway environments. Here, we model this process using an experimental evolution approach. Pseudomonas aeruginosa PAO1, which is not airway adapted, was serially passaged, separately, in media chemically reflective of upper or lower airway environments. To explore whether the CF environment selects for unique traits, we separately passaged PAO1 in airway-mimicking media with or without CF-specific factors. Our findings demonstrated that all airway environments-sinus and lungs, under CF and non-CF conditions-selected for loss of twitching motility, increased resistance to multiple antibiotic classes, and a hyper-biofilm phenotype. These traits conferred increased airway colonization potential in an in vivo model. CF-like conditions exerted stronger selective pressures, leading to emergence of more pronounced phenotypes. Loss of twitching was associated with mutations in type IV pili genes. Type IV pili mediate surface attachment, twitching, and induction of cAMP signalling. We additionally identified multiple evolutionary routes to increased biofilm formation involving regulation of cyclic-di-GMP signalling. These included the loss of function mutations in bifA and dipA phosphodiesterase genes and activating mutations in the siaA phosphatase. These data highlight that airway environments select for traits associated with sessile lifestyles and suggest upper airway niches support emergence of phenotypes that promote establishment of lung infection.

Identification of the chaA and fwA Spore Color Genes of Aspergillus nidulans.

Wild-type Aspergillus nidulans asexual spores (conidia) are green due to a pigment that protects the spores against ultraviolet light. The pigment is produced by a biosynthetic pathway, the genes of which are dispersed in the genome. The backbone molecule of the pigment is a polyketide synthesized by a polyketide synthase encoded by the wA gene. If wA is not functional, the conidia are white. The polyketide is modified by a laccase encoded by the yA gene and inactivation of yA in an otherwise wild-type background results in yellow spores. Additional spore color mutations have been isolated and mapped to a locus genetically, but the genes that correspond to these loci have not been determined. Spore color markers have been useful historically, and they remain valuable in the molecular genetics era. One can determine if a transforming fragment has been successfully integrated at the wA or yA locus by simply looking at the color of transformant conidia. The genes of the potentially useful color loci chaA (chartreuse conidia) and fwA (fawn conidia) have not been identified previously. We chose a set of candidate genes for each locus by comparing the assembled genome with the genetic map. By systematically deleting these candidate genes, we identified a cytochrome P450 gene (AN10028) corresponding to chaA. Deletions of this gene result in chartreuse conidia and chartreuse mutations can be complemented in trans by a functional copy of this gene. With fwA, we found that the existing fawn mutation, fwA1, is a deletion of 2241 base pairs that inactivates three genes. By deleting each of these genes, we determined that fwA is AN1088, an EthD domain protein. Deletion of AN1088 results in fawn conidia as expected. Neither deletion of chaA nor fwA restricts growth and both should be valuable target loci for transformations. Combinations of deletions have allowed us to investigate the epistasis relationships of wA, yA, chaA and fwA.

Airway metabolic profiling during Streptococcus pneumoniae infection identifies branched chain amino acids as signatures of upper airway colonisation.

Streptococcus pneumoniae is a leading cause of community-acquired pneumonia and bacteraemia and is capable of remarkable phenotypic plasticity, responding rapidly to environmental change. Pneumococcus is a nasopharyngeal commensal, but is responsible for severe, acute infections following dissemination within-host. Pneumococcus is adept at utilising host resources, but the airways are compartmentalised and those resources are not evenly distributed. Challenges and opportunities in metabolite acquisition within different airway niches may contribute to the commensal-pathogen switch when pneumococcus moves from nasopharynx into lungs. We used NMR to characterise the metabolic landscape of the mouse airways, in health and during infection. Using paired nasopharynx and lung samples from naïve animals, we identified fundamental differences in metabolite bioavailability between airway niches. Pneumococcal pneumonia was associated with rapid and dramatic shifts in the lung metabolic environment, whilst nasopharyngeal carriage led to only modest change in upper airway metabolite profiles. NMR spectra derived from the nasopharynx of mice infected with closely-related pneumococcal strains that differ in their colonisation potential could be distinguished from one another using multivariate dimensionality reduction methods. The resulting models highlighted that increased branched-chain amino acid (BCAA) bioavailability in nasopharynx is a feature of infection with the high colonisation potential strain. Subsequent analysis revealed increased expression of BCAA transport genes and increased intracellular concentrations of BCAA in that same strain. Movement from upper to lower airway environments is associated with shifting challenges in metabolic resource allocation for pneumococci. Efficient biosynthesis, liberation or acquisition of BCAA is a feature of adaptation to nasopharyngeal colonisation.

Preclinical testing of antimicrobials for cystic fibrosis lung infections: current needs and future priorities.

A workshop was held by the PIPE-CF strategic research centre to consider preclinical testing of antimicrobials for cystic fibrosis (CF). The workshop brought together groups of people from the CF community to discuss current challenges and identify priorities when developing CF therapeutics. This paper summarizes the key points from the workshop from the different sessions, including talks given by presenters on the day and round table discussions. Currently, it is felt that there is a large disconnect throughout the community, with communication between patients, clinicians and researchers being the main issue. This leads to little consideration being given to factors such as treatment regimes, routes of administration and side effects when developing new therapies, that could alter the day-to-day lifestyles of people living with CF. Translation of numerical data that are obtained in the laboratory to successful outcomes of clinical trials is also a key challenge facing researchers today. Laboratory assays in preclinical testing involve basing results on bacterial clearance and decrease in viable cells, when these are not factors that are considered when determining the success of a treatment in the clinic. However, there are several models currently in development that seek to tackle some of these issues, such as the organ-on-a-chip technology and adaptation of a hollow-fibre model, as well as the development of media that aim to mimic the niche environments of a CF respiratory tract. It is hoped that by summarizing these opinions and discussing current research, the communication gap between groups can begin to close.

An innate pathogen sensing strategy involving ubiquitination of bacterial surface proteins.

Sensing of pathogens by ubiquitination is a critical arm of cellular immunity. However, universal ubiquitination targets on microbes remain unidentified. Here, using in vitro, ex vivo, and in vivo studies, we identify the first protein-based ubiquitination substrates on phylogenetically diverse bacteria by unveiling a strategy that uses recognition of degron-like motifs. Such motifs form a new class of intra-cytosolic pathogen-associated molecular patterns (PAMPs). Their incorporation enabled recognition of nonubiquitin targets by host ubiquitin ligases. We find that SCFFBW7 E3 ligase, supported by the regulatory kinase, glycogen synthase kinase 3ß, is crucial for effective pathogen detection and clearance. This provides a mechanistic explanation for enhanced risk of infections in patients with chronic lymphocytic leukemia bearing mutations in F-box and WD repeat domain containing 7 protein. We conclude that exploitation of this generic pathogen sensing strategy allows conservation of host resources and boosts antimicrobial immunity.

Influence of Streptococcus pneumoniae Within-Strain Population Diversity on Virulence and Pathogenesis.

The short generation time of many bacterial pathogens allows the accumulation of de novo mutations during routine culture procedures used for the preparation and propagation of bacterial stocks. Taking the major human pathogen Streptococcus pneumoniae as an example, we sought to determine the influence of standard laboratory handling of microbes on within-strain genetic diversity and explore how these changes influence virulence characteristics and experimental outcomes. A single culture of S. pneumoniae D39 grown overnight resulted in the enrichment of previously rare genotypes present in bacterial freezer stocks and the introduction of new variation to the bacterial population through the acquisition of mutations. A comparison of D39 stocks from different laboratories demonstrated how changes in bacterial population structure taking place during individual culture events can cumulatively lead to fixed, divergent change that profoundly alters virulence characteristics. The passage of D39 through mouse models of infection, a process used to standardize virulence, resulted in the enrichment of high-fitness genotypes that were originally rare (<2% frequency) in D39 culture collection stocks and the loss of previously dominant genotypes. In the most striking example, the selection of a <2%-frequency genotype carrying a mutation in sdhB, a gene thought to be essential for the establishment of lung infection, was associated with enhanced systemic virulence. Three separately passaged D39 cultures originating from the same frozen stocks showed considerable genetic divergence despite comparable virulence. IMPORTANCE Laboratory bacteriology involves the use of high-density cultures that we often assume to be clonal but that in reality are populations consisting of multiple genotypes at various abundances. We have demonstrated that the genetic structure of a single population of a widely used Streptococcus pneumoniae strain can be substantially altered by even short-term laboratory handling and culture and that, over time, this can lead to changes in virulence characteristics. Our findings suggest that caution should be applied when comparing data generated in different laboratories using the same strain but also when comparing data within laboratories over time. Given the dramatic reductions in the cost of next-generation sequencing technology in recent years, we advocate for the frequent sampling and sequencing of bacterial isolate collections.

Twelve-Week Daily Gluteal and Hamstring Electrical Stimulation Improves Vascular Structure and Function, Limb Volume, and Sitting Pressure in Spinal Cord Injury: A Pilot Feasibility Study.

OBJECTIVE: We examined the long-term effects of low-intensity electrical stimulation on (micro)vasculature and sitting pressure of a home-based, wearable electrical stimulation device in a pilot feasibility study. DESIGN: In a cohort observation before-after trial, nine middle-aged male (n = 8) and female (n = 1) individuals (48 ± 15 yrs) with American Spinal Injury Association A-C classified chronic (1-24 yrs) spinal cord injury underwent 12 wks of self-administered daily, low-intensity gluteal and hamstring electrical stimulation (50 Hz, 6 hrs [30-min electrical stimulation, 15-min rest]). Common femoral artery diameter and blood blow were determined with ultrasound, skin vascular function during local heating was assessed using Laser-Doppler flowmetry, thigh volume was estimated using leg circumferences and skinfolds, and interface sitting pressure was measured using pressure mapping. RESULTS: Resting common femoral artery diameter increased (0.73 ± 0.20 to 0.79 ± 0.22 cm, P < 0.001) and baseline common femoral artery blood flow increased (0.28 ± 0.12 to 0.40 ± 0.15 l/min, P < 0.002). Gluteal cutaneous vascular conductance showed a time*temperature interaction (P = 0.01) with higher conductance at 42°C after 12 wks. Ischial peak pressure decreased (P = 0.003) by 32 ± 23 mm Hg and pressure gradient decreased (23 ± 7 to 16 ± 6 mm Hg, P = 0.007). Thigh volume increased (+19%, P = 0.01). CONCLUSIONS: Twelve-week daily home-based gluteal and hamstring electrical stimulation is feasible and effective to improve (micro)vasculature and sitting pressure, and electrical stimulation may have clinical implications for ameliorating pressure ulcers and (micro)vascular complications in spinal cord injury.

Challenges and opportunities in the development of novel antimicrobial therapeutics for cystic fibrosis.

Chronic respiratory infection is the primary driver of mortality in individuals with cystic fibrosis (CF). Existing drug screening models utilised in preclinical antimicrobial development are unable to mimic the complex CF respiratory environment. Consequently, antimicrobials showing promising activity in preclinical models often fail to translate through to clinical efficacy in people with CF. Model systems used in CF anti-infective drug discovery and development range from antimicrobial susceptibility testing in nutrient broth, through to 2D and 3D in vitro tissue culture systems and in vivo models. No single model fully recapitulates every key aspect of the CF lung. To improve the outcomes of people with CF (PwCF) it is necessary to develop a set of preclinical models that collectively recapitulate the CF respiratory environment to a high degree of accuracy. Models must be validated for their ability to mimic aspects of the CF lung and associated lung infection, through evaluation of biomarkers that can also be assessed following treatment in the clinic. This will give preclinical models greater predictive power for identification of antimicrobials with clinical efficacy. The landscape of CF is changing, with the advent of modulator therapies that correct the function of the CFTR protein, while antivirulence drugs and phage therapy are emerging alternative treatments to chronic infection. This review discusses the challenges faced in current antimicrobial development pipelines, including the advantages and disadvantages of current preclinical models and the impact of emerging treatments.

Pneumococcal Colonization and Virulence Factors Identified Via Experimental Evolution in Infection Models.

Streptococcus pneumoniae is a commensal of the human nasopharynx and a major cause of respiratory and invasive disease. We examined adaptation and evolution of pneumococcus, within nasopharynx and lungs, in an experimental system where the selective pressures associated with transmission were removed. This was achieved by serial passage of pneumococci, separately, in mouse models of nasopharyngeal carriage or pneumonia. Passaged pneumococci became more effective colonizers of the respiratory tract and we observed several examples of potential parallel evolution. The cell wall-modifying glycosyltransferase LafA was under strong selection during lung passage, whereas the surface expressed pneumococcal vaccine antigen gene pvaA and the glycerol-3-phosphate dehydrogenase gene gpsA were frequent targets of mutation in nasopharynx-passaged pneumococci. These mutations were not identified in pneumococci that were separately evolved by serial passage on laboratory agar. We focused on gpsA, in which the same single nucleotide polymorphism arose in two independently evolved nasopharynx-passaged lineages. We describe a new role for this gene in nasopharyngeal carriage and show that the identified single nucleotide change confers resistance to oxidative stress and enhanced nasopharyngeal colonization potential. We demonstrate that polymorphisms in gpsA arise and are retained during human colonization. These findings highlight how within-host environmental conditions can determine trajectories of bacterial evolution. Relative invasiveness or attack rate of pneumococcal lineages may be defined by genes that make niche-specific contributions to bacterial fitness. Experimental evolution in animal infection models is a powerful tool to investigate the relative roles played by pathogen virulence and colonization factors within different host niches.

Traditional Cardiovascular Risk Factors Strongly Underestimate the 5-Year Occurrence of Cardiovascular Morbidity and Mortality in Spinal Cord Injured Individuals.

OBJECTIVES: To explore whether traditional models of cardiovascular disease (CVD) risk prediction correctly predict CVD events across a median 5.7-year follow-up period in individuals with spinal cord injury (SCI) and whether adding SCI-related characteristics (ie, lesion level) to the prediction model can improve the prognostic value. DESIGN: Retrospective analysis of patient records. SETTING: Observation at the start of active rehabilitation of participants in a multicenter cohort study, "Restoration of (Wheelchair) Mobility in SCI Rehabilitation," in the Netherlands. PARTICIPANTS: Patients with SCI (N=200) The patients were 74% men, aged 40±14 years, and with an American Spinal Injury Association (ASIA) impairment score of A through D. Forty percent had tetraplegia, and 69% were motor complete. INTERVENTIONS: Risk profiling/not applicable. MAIN OUTCOME MEASURES: Survival status and cardiovascular morbidity and mortality qwere obtained from medical records. Five-year Framingham Risk Scores (FRS) and the FRS ability to predict events assessed using receiver operating characteristic (ROC) curves with corresponding areas under the curve (AUC) and 95% confidence intervals (CI). Kaplan-Meier curves and the log-rank test were used to assess the difference in clinical outcome between participants with an FRS score lower or higher than the median FRS score for the cohort. SCI-related factors associated with CVD events, ASIA impairment, motor completeness, level of injury, and sports participation before injury were explored using univariate and multivariate Cox proportional hazard regression. RESULTS: The median 5-year FRS was 1.36%. Across a median follow-up period of 5.7 years, 39 developed a CVD event, including 10 fatalities. Although the FRS markedly underestimated the true occurrence of CVD events, the Kaplan-Meier curves and the log-rank test showed that the risk ratio for individuals with an FRS score less than the median FRS (eg, low risk) versus a score greater than the median FRS (high risk) was 3.2 (95% CI, 1.6-6.5; P=.001). Moreover, ROC with corresponding AUCs suggests acceptable accuracy of the FRS to identify individuals with increased risk for future CVD events (ROC AUC of 0.71; 95% CI, 0.62-0.82). Adding ASIA impairment (0.74; 95% CI, 0.66-0.82), motor impairment (0.74; 95% CI, 0.66-0.83), level of injury (0.72; 95% CI, 0.63-0.81), or active engagement in sport before injury (0.72; 95% CI, 0.63-0.88) to the FRS did not improve the level of discrimination. CONCLUSIONS: Our 5.7-year retrospective study reveals that cardiovascular risk factors and risk models markedly underestimate the true risk for CVD events in individuals with SCI. Nonetheless, these markers successfully distinguish between SCI individuals at high versus low risk for future CVD events. Our data may have future clinical implications, both related to (cutoff values of) CVD risk factors, but also for (earlier) prescription of (non)pharmacologic strategies against CVD in SCI individuals.

Femoral Artery Blood Flow and Microcirculatory Perfusion During Acute, Low-Level Functional Electrical Stimulation in Spinal Cord Injury.

OBJECTIVE: Functional electrical stimulation (FES) may help to reduce the risk of developing macrovascular and microvascular complications in people with spinal cord injury. Low-intensity FES has significant clinical potential because this can be applied continuously throughout the day. This study examines the acute effects of low-intensity FES using wearable clothing garment on vascular blood flow and oxygen consumption in people with spinal cord injury. DESIGN: This was a cross-sectional observation study. METHODS: Eight participants with a motor complete spinal cord injury received four 3-min unilateral FES to the gluteal and hamstring muscles. Skin and deep femoral artery blood flow and oxygen consumption were measured at baseline and during each bout of stimulation. RESULTS: Femoral artery blood flow increased by 18.1% with the application of FES (P = 0.02). Moreover, femoral artery blood flow increased further during each subsequent block of FES (P = 0.004). Skin perfusion did not change during an individual block of stimulation (P = 0.66). Skin perfusion progressively increased with each subsequent bout (P < 0.001). There was no change in femoral or skin perfusion across time in the nonstimulated leg (all P > 0.05). CONCLUSION: Low-intensity FES acutely increased blood flow during stimulation, with a progressive increase across subsequent FES bouts. These observations suggest that continuous, low-intensity FES may represent a practical and effective strategy to improve perfusion and reduce the risk of vascular complications.

What does the future hold for advanced nursing?

This third in a three-part series on advanced nursing explores the future demand for a flexible but regulated nursing career framework. Part 1 explored the historical evolution of advanced nursing, while part 2 discussed the development of a governance framework.

A governance framework for advanced nursing.

This second in a three-part series outlines the introduction of a governance framework for advanced nursing practice. Part 1 explored the history of the evolution of advanced practice, while part 3, to be published next week, will discuss the future of advanced practice and how it may shape the career structure of nursing.

Advanced nursing. Part 1: The development of advanced nursing roles.

This first in a three-part series on advanced nursing practice reviews its historical evolution. Part 2, to be published next week, reviews the introduction of a governance framework, while part 3 explores the future of advanced practice and how it may shape nursing career structures.

Hydroxyl-directed cyclizations of 1,6-enynes.

The palladium-catalyzed, hydroxyl-directed cyclization reactions of 1,6-enynes provide a highly diastereoselective process for the syntheses of stereochemically defined cyclopentanes. Consistently high levels of cis-selectivity are possible using homopropargyl alcohols in contrast to the corresponding propargyl alcohols. Hydroborylative enyne cyclizations coupled with this directing group effect provide a useful method for the syntheses of multifaceted compounds bearing all carbon quaternary centers.

Understanding practitioner ethnography.

Thomas David Barton provides an overview of practitioner ethnography, a research approach that provides practitioners with a way of exploring the culture of their workplace. He compares practitioner and traditional ethnography and looks at the pros and cons of the method.

Clinical mentoring of nurse practitioners: the doctors' experience.

The clinical development of nurse practitioners (NPs) has historically been dependent on mentorship from medical practitioners, yet their experience of this mentorship is generally unexplored. NPs have an ambiguous relationship with medicine as they have been dependent on medical mentorship to develop clinical skills, and they substitute into roles traditionally associated with medical practice. Consequently, NPs challenge professional boundaries and present particular concerns to their medical mentors. Practitioner ethnography examined the experiences of medical mentors, nurse practitioner students and academic staff during a clinical degree programme. This paper reports specifically on the medical mentors, focusing primarily on their professional authority relationship with their students and on their experience of imparting and sharing clinical knowledge. These experiences fell into three perspective stages, the provisional perspective, transitional perspective, and final perspective. Medical mentors were instrumental to the advanced clinical role of the student NP. This resulted in a conflicting experience of promoting a clinical role that challenged traditional medical authority. The effect of this was a cautious re-negotiation of professional boundaries. In future NP students (and their academic teachers) need to acknowledge this if they are to mutually gain the most from their relationship with their medical mentors.

Nurse practitioners - or advanced clinical nurses?

This article reviews a specific finding from a research project that examined the experiences of students, teachers and clinicians involved in a nurse practitioner degree programme. The development of advanced clinical nursing roles has presented challenges to the professional structure of nursing, particularly in the area of the unregulated and confusing array of titles adopted by nurses that infer advanced clinical practice. Over a 2-year period, practitioner ethnography was used to examine a sample of 10 student nurse practitioners who were undertaking a clinical degree programme (BSc (Hons) Nurse Practitioner). Data were also collected from 11 other individuals involved in the degree programme: teachers, medical mentors and senior academics. The data were systematically analysed and structured, leading to the inductive identification of themes and frameworks. The sample's experience of the development of advanced clinical nursing roles led to consideration of the evolution of new career structures and identities associated with advanced nursing practice. This evidence enabled a view of advanced clinical nursing roles within a framework of advanced nursing practice. The development of advanced clinical nursing is dependent on the cooperation of clinical nurses, educators, managers, doctors and politicians. Nevertheless, that development can only succeed if strategic policy is backed by the development of a professional clinical nursing career framework that enables the process.