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

Abstract

BackgroundStaphylococcus 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.MethodsWe 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.Results87 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.ConclusionSequencing 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.

Caracterización molecular de bacterias patógenas de las vías respiratorias de pacientes peruanos con fibrosis quística

“…En términos de diversidad, la familia Enterobacteriacae presentó 10 especies, seguida por Bacillaceae (6) y Streptococcaceae (4). A nivel de cepas, las bacterias cultivables más aisladas fueron P. aeruginosa (40), S. aureus (16), Pseudomonas sp (15), Klebsiella oxytoca (4). Otros microorganismos que incluían a familias como Enterobacteriaceae y Bacillaceae se presentaron en menores cantidades (Tabla 1).…”

“…aureus fue el segundo patógeno más frecuente (12,6% de los aislados) en la población estudiada; también es reportado como uno de los patógenos más virulentos y predominante en pacientes con FQ (16) . Dentro de los S. aureus, existen varios fenotipos como los MRSA (Methicillin-resistant S. aureus) antiguamente conocidos como SCV (Small colony variant) que representan una amenaza para los pacientes infectados.…”

Impact of enzymatic digestion on bacterial community composition in CF airway samples

“…Further explanation of selection of samples to be used for this study are included in a previous publication (Johnson et al, 2016). Excess specimen was stored frozen at −80 °C for molecular assessment of infection.…”

Molecular Identification of Gram-Negative Bacteria in Respiratory Samples of Cystic Fibrosis Patients from a Children Referral Hospital in Tehran

“…Culturebased methods are time-consuming in the detection of bacteria from the airways of CF patients and they usually give false positive and false negative results due to colonization and previous antibiotic use, respectively. This is while molecular techniques can detect causal bacteria rapidly (10,11). Polymerase chain reaction (PCR) assays targeting the 16srRNA gene can be used for the detection of lung infection bacteria in CF patients (10,12).…”

“…This is while molecular techniques can detect causal bacteria rapidly (10,11). Polymerase chain reaction (PCR) assays targeting the 16srRNA gene can be used for the detection of lung infection bacteria in CF patients (10,12).…”