Dataset Information

Molecular Identification of Staphylococcus aureus in Airway Samples from Children with Cystic Fibrosis.

ABSTRACT: BACKGROUND:Staphylococcus aureus is a common and significant pathogen in cystic fibrosis. We sought to determine if quantitative PCR (qPCR) and 16S rRNA gene sequencing could provide a rapid, culture-independent approach to the identification of S. aureus airway infections. METHODS:We examined the sensitivity and specificity of two qPCR assays, targeting the femA and 16S rRNA gene, using culture as the gold standard. In addition, 16S rRNA gene sequencing to identify S. aureus directly from airway samples was evaluated. DNA extraction was performed with and without prior enzymatic digestion. RESULTS:87 samples [42 oropharyngeal (OP) and 45 expectorated sputum (ES)] were analyzed. 59 samples (68%) cultured positive for S. aureus. Using standard extraction techniques, sequencing had the highest sensitivity for S. aureus detection (85%), followed by FemA qPCR (52%) and 16SrRNA qPCR (34%). For all assays, sensitivity was higher from ES samples compared to OP swabs. Specificity of the qPCR assays was 100%, but 21.4% for sequencing due to detection of S. aureus in low relative abundance from culture negative samples. Enzymatic digestion increased the sensitivity of qPCR assays, particularly for OP swabs. CONCLUSION:Sequencing had a high sensitivity for S. aureus, but low specificity. While femA qPCR had higher sensitivity than 16S qPCR for detection of S. aureus, neither assay was as sensitive as sequencing. The significance of S. aureus detection with low relative abundance by sequencing in culture-negative specimens is not clear.

SUBMITTER: Johnson EJ

PROVIDER: S-EPMC4726467 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Molecular Identification of Staphylococcus aureus in Airway Samples from Children with Cystic Fibrosis.

Johnson Emily J EJ Zemanick Edith T ET Accurso Frank J FJ Wagner Brandie D BD Robertson Charles E CE Harris J Kirk JK

PloS one 20160125 1

<h4>Background</h4>Staphylococcus aureus is a common and significant pathogen in cystic fibrosis. We sought to determine if quantitative PCR (qPCR) and 16S rRNA gene sequencing could provide a rapid, culture-independent approach to the identification of S. aureus airway infections.<h4>Methods</h4>We examined the sensitivity and specificity of two qPCR assays, targeting the femA and 16S rRNA gene, using culture as the gold standard. In addition, 16S rRNA gene sequencing to identify S. aureus dire ...[more]

PMID: 26808658

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Project description:Staphylococcus aureus is a major cause of chronic respiratory infection in patients with cystic fibrosis (CF). We recently showed that Pseudomonas aeruginosa exhibits enhanced biofilm formation during respiratory syncytial virus (RSV) coinfection on human CF airway epithelial cells (AECs). The impact of respiratory viruses on other bacterial pathogens during polymicrobial infections in CF remains largely unknown. To investigate if S. aureus biofilm growth in the CF airways is impacted by virus coinfection, we evaluated S. aureus growth on CF AECs. Initial studies showed an increase in S. aureus growth over 24 h, and microscopy revealed biofilm-like clusters of bacteria on CF AECs. Biofilm growth was enhanced when CF AECs were coinfected with RSV, and this observation was confirmed with S. aureus CF clinical isolates. Apical conditioned medium from RSV-infected cells promoted S. aureus biofilms in the absence of the host epithelium, suggesting that a secreted factor produced during virus infection benefits S. aureus biofilms. Exogenous iron addition did not significantly alter biofilm formation, suggesting that it is not likely the secreted factor. We further characterized S. aureus-RSV coinfection in our model using dual host-pathogen RNA sequencing, allowing us to observe specific contributions of S. aureus and RSV to the host response during coinfection. Using the dual host-pathogen RNA sequencing approach, we observed increased availability of nutrients from the host and upregulation of S. aureus genes involved in growth, protein translation and export, and amino acid metabolism during RSV coinfection.IMPORTANCE The airways of individuals with cystic fibrosis (CF) are commonly chronically infected, and Staphylococcus aureus is the dominant bacterial respiratory pathogen in CF children. CF patients also experience frequent respiratory virus infections, and it has been hypothesized that virus coinfection increases the severity of S. aureus lung infections in CF. We investigated the relationship between S. aureus and the CF airway epithelium and observed that coinfection with respiratory syncytial virus (RSV) enhances S. aureus biofilm growth. However, iron, which was previously found to be a significant factor influencing Pseudomonas aeruginosa biofilms during virus coinfection, plays a minor role in S. aureus coinfections. Transcriptomic analyses provided new insight into how bacterial and viral pathogens alter host defense and suggest potential pathways by which dampening of host responses to one pathogen may favor persistence of another in the CF airways, highlighting complex interactions occurring between bacteria, viruses, and the host during polymicrobial infections.

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.

Project description:Early-life viral infections are responsible for pulmonary exacerbations that can contribute to disease progression in young children with cystic fibrosis (CF). The most common respiratory viruses detected in the CF airway are human rhinoviruses (RV), and augmented airway inflammation in CF has been attributed to dysregulated airway epithelial responses although evidence has been conflicting. Here, we exposed airway epithelial cells from children with and without CF to RV in vitro. Using RNA-Seq, we profiled the transcriptomic differences of CF and non-CF airway epithelial cells at baseline and in response to RV. There were only modest differences between CF and non-CF cells at baseline. In response to RV, there were 1,442 and 896 differentially expressed genes in CF and non-CF airway epithelial cells, respectively. The core antiviral responses in CF and non-CF airway epithelial cells were mediated through interferon signaling although type 1 and 3 interferon signaling, when measured, were reduced in CF airway epithelial cells following viral challenge consistent with previous reports. The transcriptional responses in CF airway epithelial cells were more complex than in non-CF airway epithelial cells with diverse over-represented biological pathways, such as cytokine signaling and metabolic and biosynthetic pathways. Network analysis highlighted that the differentially expressed genes of CF airway epithelial cells' transcriptional responses were highly interconnected and formed a more complex network than observed in non-CF airway epithelial cells. We corroborate observations in fully differentiated air-liquid interface (ALI) cultures, identifying genes involved in IL-1 signaling and mucin glycosylation that are only dysregulated in the CF airway epithelial response to RV infection. These data provide novel insights into the CF airway epithelial cells' responses to RV infection and highlight potential pathways that could be targeted to improve antiviral and anti-inflammatory responses in CF.

Dataset Information

Molecular Identification of Staphylococcus aureus in Airway Samples from Children with Cystic Fibrosis.

Publications

Molecular Identification of Staphylococcus aureus in Airway Samples from Children with Cystic Fibrosis.

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