RESUMEN
RATIONALE: The clinical utility of culture-independent testing of pediatric BAL specimens is unknown. In addition, the variability of the pediatric pulmonary microbiome with patient characteristics is not well understood. OBJECTIVES: To compare testing with 16S rRNA gene-based sequencing to conventional cultures of BAL specimens in children Methods: Study subjects were not more than 22 years old and underwent BAL from May 2013 to August 2015 as part of clinical care. DNA extracted from BAL specimens was used for 16S rRNA gene-based analysis, and results were compared with routine cultures from the same samples. Indices of microbial diversity and relative taxon abundances were compared on the basis of subject characteristics (diagnosis and antibiotic use). RESULTS: From 81 participants (male, 51%; median age, 9 yr), 89 samples were collected. The 16S rRNA genes of 77 samples (86.5%) from 70 subjects were successfully analyzed. These 70 subjects included 23 with cystic fibrosis, 19 who were immunocompromised, and 28 who were nonimmunocompromised. Of 68 organisms identified in culture, 16S rRNA gene-based analyses detected corresponding taxa in 66 (97.1%) and also identified potentially clinically significant organisms missed by cultures (e.g., Staphylococcus, Legionella, and Pseudomonas). Taxa that varied significantly with diagnosis and antibiotic use included Veillonella, Corynebacterium, Haemophilus, and Streptococcus. The microbiota of cystic fibrosis samples was less diverse. A "core" group of 15 taxa present in all three diagnosis groups was identified. CONCLUSIONS: Culture-independent analysis was concordant with routine cultures and showed the potential to detect noncultured pathogens. Although culture-independent testing identified relative changes in organism abundance associated with clinical characteristics, distinct microbiome profiles associated with disease states were not identified.
Asunto(s)
Líquido del Lavado Bronquioalveolar/microbiología , Fibrosis Quística/microbiología , Neumonía Bacteriana/diagnóstico , ARN Bacteriano/análisis , ARN Ribosómico 16S/análisis , Adolescente , Líquido del Lavado Bronquioalveolar/química , Broncoscopía , Niño , Preescolar , Corynebacterium/genética , Corynebacterium/aislamiento & purificación , Técnicas de Cultivo , Femenino , Haemophilus/genética , Haemophilus/aislamiento & purificación , Humanos , Huésped Inmunocomprometido , Lactante , Recién Nacido , Legionella/genética , Legionella/aislamiento & purificación , Pulmón/microbiología , Masculino , Microbiota/genética , Neumonía Bacteriana/microbiología , Pseudomonas/genética , Pseudomonas/aislamiento & purificación , Staphylococcus/genética , Staphylococcus/aislamiento & purificación , Streptococcus/genética , Streptococcus/aislamiento & purificación , Veillonella/genética , Veillonella/aislamiento & purificación , Adulto JovenRESUMEN
We postulated that the activation of proinflammatory signaling by methicillin-resistant Staphylococcus aureus (MRSA) strain USA300 is a major factor in the pathogenesis of severe pneumonia and a target for immunomodulation. Local activation of T cells in the lung was a conserved feature of multiple strains of S. aureus, in addition to USA300. The pattern of Vß chain activation was consistent with known superantigens, but deletion of SelX or SEK and SEQ was not sufficient to prevent T-cell activation, indicating the participation of multiple genes. Using Rag2(-/-), Cd4(-/-), and Cd28(-/-) mice, we observed significantly improved clearance of MRSA from the airways and decreased lung pathology, compared with findings for wild-type controls. The improved outcome correlated with decreased production of proinflammatory cytokines (tumor necrosis factor, KC, interleukin 6, and interleukin 1ß). Our data suggest that T-cell-mediated hypercytokinemia induced by infection with MRSA strain USA300 contributes to pathogenesis and may be a therapeutic target for improving outcomes of this common infection in a clinical setting.