The Cystic Fibrosis Upper and Lower Airway Metagenome.

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

Metagenome - Inferred bacterial replication rates in cystic fibrosis airways.

Bacterial replication rates were determined from metagenome sequencing of nasal lavage, throat swabs and induced sputa collected from healthy subjects and individuals with COPD or cystic fibrosis. More than 90% of peak-to-trough coverage ratios of major clones were above 1.4 indicating that the most abundant bacterial species in the microbial communities were replicating in the airways including common inhabitants such as Prevotella and Streptococcus species as well as the cystic fibrosis pathogens Staphylococcus aureus and Pseudomonas aeruginosa. The populations of P. aeruginosa and S. aureus were replicating their pool of chromosomes more slowly than the populations of the common inhabitants of a healthy airway microbial flora. The assessment of growth dynamics in microbial metagenomes could become a decision-making tool for the diagnosis and management of bacterial infections in cystic fibrosis.

Airway microbial metagenomics.

High-throughput untargeted metagenome sequencing provides information about the composition of the microbial communities of viruses, bacteria, archaea and unicellular eukaryotes in the habitat of interest. This review outlines the sampling, processing, sequencing and bioinformatic analysis of secretions of the respiratory tract and summarizes our current knowledge of the upper and lower human airways metagenome in health and disease.

Synaptic UNC13A protein variant causes increased neurotransmission and dyskinetic movement disorder.

Munc13 proteins are essential regulators of neurotransmitter release at nerve cell synapses. They mediate the priming step that renders synaptic vesicles fusion-competent, and their genetic elimination causes a complete block of synaptic transmission. Here we have described a patient displaying a disorder characterized by a dyskinetic movement disorder, developmental delay, and autism. Using whole-exome sequencing, we have shown that this condition is associated with a rare, de novo Pro814Leu variant in the major human Munc13 paralog UNC13A (also known as Munc13-1). Electrophysiological studies in murine neuronal cultures and functional analyses in Caenorhabditis elegans revealed that the UNC13A variant causes a distinct dominant gain of function that is characterized by increased fusion propensity of synaptic vesicles, which leads to increased initial synaptic vesicle release probability and abnormal short-term synaptic plasticity. Our study underscores the critical importance of fine-tuned presynaptic control in normal brain function. Further, it adds the neuronal Munc13 proteins and the synaptic vesicle priming process that they control to the known etiological mechanisms of psychiatric and neurological synaptopathies.

Impact of sample processing on human airways microbial metagenomes.

Whole metagenome shotgun sequencing provides information about the gene content and the composition of microbial communities provided that the processing of the samples does not introduce a methodology-driven bias. We tested the impact of DNA isolation and storage period on the metagenome profile. Deep throat swabs were collected from healthy adults and an infected infant. DNA was isolated by sonification or enzymatic lysis either immediately or after 24h storage in agar gel Amies transport medium at room temperature. Disruption of cells and subsequent fragmentation of DNA by sonification was as suitable as the common enzymatic lysis to generate high-quality metagenomes particularly for low total DNA input of less than ten nanograms. Conversely, storage of samples for 24h produced severely distorted metagenomes. The majority of species became less abundant or even extinct, whereas a few Streptococcus, Neisseria and Haemophilus spp. proliferated so that the total number of bacterial reads increased at the expense of human reads. We recommend that samples for metagenome analysis should be immediately processed or frozen at -80°C.

Functional and bioinformatics analysis of two Campylobacter jejuni homologs of the thiol-disulfide oxidoreductase, DsbA.

BACKGROUND: Bacterial Dsb enzymes are involved in the oxidative folding of many proteins, through the formation of disulfide bonds between their cysteine residues. The Dsb protein network has been well characterized in cells of the model microorganism Escherichia coli. To gain insight into the functioning of the Dsb system in epsilon-Proteobacteria, where it plays an important role in the colonization process, we studied two homologs of the main Escherichia coli Dsb oxidase (EcDsbA) that are present in the cells of the enteric pathogen Campylobacter jejuni, the most frequently reported bacterial cause of human enteritis in the world. METHODS AND RESULTS: Phylogenetic analysis suggests the horizontal transfer of the epsilon-Proteobacterial DsbAs from a common ancestor to gamma-Proteobacteria, which then gave rise to the DsbL lineage. Phenotype and enzymatic assays suggest that the two C. jejuni DsbAs play different roles in bacterial cells and have divergent substrate spectra. CjDsbA1 is essential for the motility and autoagglutination phenotypes, while CjDsbA2 has no impact on those processes. CjDsbA1 plays a critical role in the oxidative folding that ensures the activity of alkaline phosphatase CjPhoX, whereas CjDsbA2 is crucial for the activity of arylsulfotransferase CjAstA, encoded within the dsbA2-dsbB-astA operon. CONCLUSIONS: Our results show that CjDsbA1 is the primary thiol-oxidoreductase affecting life processes associated with bacterial spread and host colonization, as well as ensuring the oxidative folding of particular protein substrates. In contrast, CjDsbA2 activity does not affect the same processes and so far its oxidative folding activity has been demonstrated for one substrate, arylsulfotransferase CjAstA. The results suggest the cooperation between CjDsbA2 and CjDsbB. In the case of the CjDsbA1, this cooperation is not exclusive and there is probably another protein to be identified in C. jejuni cells that acts to re-oxidize CjDsbA1. Altogether the data presented here constitute the considerable insight to the Epsilonproteobacterial Dsb systems, which have been poorly understood so far.

Pre-hospital delay of treatment in patients with ST segment elevation myocardial infarction undergoing primary percutaneous coronary intervention: experience of cardiac centre located in the vicinity of the centre of Warsaw.

BACKGROUND: Early reperfusion therapy with primary percutaneous coronary intervention (PCI) in patients with ST-segment elevation myocardial infarction (STEMI) improves left ventricular function and reduces mortality. AIM: To assess the time delay in treatment of patients with STEMI referred to a twenty-four-hour interventional centre located in the vicinity of the centre of Warsaw. METHODS: We analysed 350 consecutive STEMI patients admitted to our Department between October 2005 and September 2006. The majority of the patients - 244 (69.7%), were admitted via hospitals without an interventional department. Sixty-two (17.7%) patients were transported directly by ambulance from home, 34 (9.7%) from a community health centre and 10 patients (2.9%) came by themselves from home or work. A detailed interview concerning the time of symptom onset was conducted in 342 patients (97.7%). RESULTS: Sixty-two (18%) patients arrived at the interventional centre within the first 2 hours from symptom onset: 6 women (5.5% of all women in the study population) and 56 (24.1%) men (p <0.0001). Within the first 2 hours, 32 (13.1%) patients were admitted via another hospital and 20 (32.2%) directly by ambulance (p <0.001). During the first 7 days of hospitalisation the following patients died: 2 (3.2%) patients admitted within the first 2 hours via another hospital, 6 (3.4%) patients among 178 admitted between 2 and 6 hours after pain onset, 4 (8.3%) among 48 admitted between 6 and 12 hours and 8 (14.8%) among 54 patients with the pain duration over 12 hours (p <0.02). During the first 7 days of hospitalisation 8 (3.3%) patients admitted within the first 6 hours after pain onset died compared with 12 (11.8%) admitted later (p <0.003). CONCLUSIONS: In the interventional centre located near the centre of Warsaw symptom-onset-to-door time was 120 minutes only in 18% of patients with STEMI. Almost 70% of patients underwent interhospital transfer for primary PCI. Prolongation of the time from onset of symptoms to successful PCI worsened prognosis. When transporting patients with acute coronary syndrome, efforts should be made to avoid district hospitals without a catheterisation laboratory. Direct transportation by ambulance or helicopter with educated staff equipped with ECG teletransmission data, which may substantially shorten time to treatment, should be preferred.