Project description:RationaleStaphylococcus aureus is commonly cultured from the sputum of patients with bronchiectasis; however, little is known about the prevalence of the organism in these patients, the characteristics of patients who have grown the organism, or its implications.ObjectivesDetermine the relationship between S. aureus and pulmonary function, frequency of exacerbations, and frequency of hospitalization in patients with bronchiectasis Methods: The Bronchiectasis Research Registry is a database of adults with non-cystic fibrosis bronchiectasis identified from 13 sites within the United States. Baseline and follow-up demographic, spirometric, microbiologic, and therapeutic data were entered into a central web-based database. Patients were grouped into three cohorts based on their previous respiratory cultures at the time of entry into the Registry: 1) no prior S. aureus or glucose-nonfermenting gram-negative bacilli (NF-GNB) (Pseudomonas, Stenotrophomonas, or Burkholderia spp.); 2) prior S. aureus at least once; or 3) no prior S. aureus but prior NF-GNB at least once. The association between S. aureus isolation and pulmonary function and frequency of exacerbations and hospital admissions was assessed, both at baseline and after 1 year of follow-up.ResultsS. aureus was cultured from 94 of 830 patients (11.3%) included in the analysis. Patients who had grown S. aureus before entry into the Registry had a frequency of prior exacerbations and baseline pulmonary function that was between that of patients who had grown NF-GNB and those who had grown neither NF-GNB or S. aureus. Similarly, at the first follow-up visit after study entry, patients who had grown S. aureus had a frequency of exacerbations and hospitalizations that was between those of patients who had grown NF-GNB and those who had grown neither NF-GNB nor S. aureus. However, in multivariate analysis, S. aureus was not associated with pulmonary function, frequency of exacerbation, or hospital admissions. There were no significant differences in patient characteristics or outcomes between patients who had methicillin-sensitive and methicillin-resistant S. aureus.ConclusionsStaphylococcus aureus does not appear to be an independent risk factor for severe disease in patients with bronchiectasis enrolled in the Bronchiectasis Research Registry.

Project description:Staphylococcus aureus is one of the first and most prevalent pathogens cultured from the airways of cystic fibrosis (CF) patients, which can persist there for extended periods. Airway infections in CF patients are characterized by a strong inflammatory response of highly recruited neutrophils. One killing mechanism of neutrophils is the formation of neutrophil extracellular traps (NETs), which capture and eradicate bacteria by extracellular fibers of neutrophil chromatin decorated with antimicrobial granule proteins. S. aureus secretes nuclease, which can degrade NETs. We hypothesized, that S. aureus adapts to the airways of CF patients during persistent infection by escaping from NET-mediated killing via an increase of nuclease activity. Sputum samples of CF patients with chronic S. aureus infection were visualized by confocal microscopy after immuno-fluorescence staining for NET-specific markers, S. aureus bacteria and overall DNA structures. Nuclease activity was analyzed in sequential isogenic long persisting S. aureus isolates, as confirmed by whole genome sequencing, from an individual CF patient using a FRET-based nuclease activity assay. Additionally, some of these isolates were selected and analyzed by qRT-PCR to determine the expression of nuc1 and regulators of interest. NET-killing assays were performed with clinical S. aureus isolates to evaluate killing and bacterial survival depending on nuclease activity. To confirm the role of nuclease during NET-mediated killing, a clinical isolate with low nuclease activity was transformed with a nuclease expression vector (pCM28nuc). Furthermore, two sputa from an individual CF patient were subjected to RNA-sequence analysis to evaluate the activity of nuclease in vivo. In sputa, S. aureus was associated to extracellular DNA structures. Nuclease activity in clinical S. aureus isolates increased in a time-and phenotype-dependent manner. In the clinical isolates, the expression of nuc1 was inversely correlated to the activity of agr and was independent of saeS. NET-mediated killing was significantly higher in S. aureus isolates with low compared to isolates with high nuclease activity. Importantly, transformation of the clinical isolate with low nuclease activity with pCM28nuc conferred protection against NET-mediated killing confirming the beneficial role of nuclease for protection against NETs. Also, nuclease expression in in vivo sputa was high, which underlines the important role of nuclease within the highly inflamed CF airways. In conclusion, our data show that S. aureus adapts to the neutrophil-rich environment of CF airways with increasing nuclease expression most likely to avoid NET-killing during long-term persistence.

Project description:Laboratory models have been invaluable for the field of microbiology for over 100?years and have provided key insights into core aspects of bacterial physiology such as regulation and metabolism. However, it is important to identify the extent to which these models recapitulate bacterial physiology within a human infection environment. Here, we performed transcriptomics (RNA-seq), focusing on the physiology of the prominent pathogen Staphylococcus aureus in situ in human cystic fibrosis (CF) infection. Through principal-component and hierarchal clustering analyses, we found remarkable conservation in S. aureus gene expression in the CF lung despite differences in the patient clinic, clinical status, age, and therapeutic regimen. We used a machine learning approach to identify an S. aureus transcriptomic signature of 32 genes that can reliably distinguish between S. aureus transcriptomes in the CF lung and in vitro The majority of these genes were involved in virulence and metabolism and were used to improve a common CF infection model. Collectively, these results advance our knowledge of S. aureus physiology during human CF lung infection and demonstrate how in vitro models can be improved to better capture bacterial physiology in infection.IMPORTANCE Although bacteria have been studied in infection for over 100?years, the majority of these studies have utilized laboratory and animal models that often have unknown relevance to the human infections they are meant to represent. A primary challenge has been to assess bacterial physiology in the human host. To address this challenge, we performed transcriptomics of S. aureus during human cystic fibrosis (CF) lung infection. Using a machine learning framework, we defined a "human CF lung transcriptome signature" that primarily included genes involved in metabolism and virulence. In addition, we were able to apply our findings to improve an in vitro model of CF infection. Understanding bacterial gene expression within human infection is a critical step toward the development of improved laboratory models and new therapeutics.

Dataset Information

High Prevalence of Staphylococcus aureus Enterotoxin Gene Cluster Superantigens in Cystic Fibrosis Clinical Isolates.

Publications

High Prevalence of <i>Staphylococcus aureus</i> Enterotoxin Gene Cluster Superantigens in Cystic Fibrosis Clinical Isolates.

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