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Assessing bacterial populations in the lung by replicate analysis of samples from the upper and lower respiratory tracts.

ABSTRACT: Microbes of the human respiratory tract are important in health and disease, but accurate sampling of the lung presents challenges. Lung microbes are commonly sampled by bronchoscopy, but to acquire samples the bronchoscope must pass through the upper respiratory tract, which is rich in microbes. Here we present methods to identify authentic lung microbiota in bronchoalveolar lavage (BAL) fluid that contains substantial oropharyngeal admixture. We studied clinical BAL samples from six selected subjects with potential heavy lung colonization. A single sample of BAL fluid was obtained from each subject along with contemporaneous oral wash (OW) to sample the oropharynx, and then DNA was extracted from three separate aliquots of each. Bacterial 16S rDNA sequences were amplified and products analyzed by 454 pyrosequencing. By comparing replicates, we were able to specify the depth of sequencing needed to reach a 95% chance of identifying a bacterial lineage of a given proportion--for example, at a depth of 5,000 tags, OTUs of proportion 0.3% or greater would be called with 95% confidence. We next constructed a single-sided outlier test that allowed lung-enriched organisms to be quantified against a background of oropharyngeal admixture, and assessed improvements available with replicate sequence analysis. This allowed identification of lineages enriched in lung in some BAL specimens. Finally, using samples from healthy volunteers collected at multiple sites in the upper respiratory tract, we show that OW provides a reasonable but not perfect surrogate for bacteria carried into to the lung by a bronchoscope. These methods allow identification of microbes that can replicate in the lung despite the background due to oropharyngeal microbes derived from aspiration and bronchoscopic carry-over.

SUBMITTER: Charlson ES

PROVIDER: S-EPMC3435383 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Assessing bacterial populations in the lung by replicate analysis of samples from the upper and lower respiratory tracts.

Charlson Emily S ES Bittinger Kyle K Chen Jun J Diamond Joshua M JM Li Hongzhe H Collman Ronald G RG Bushman Frederic D FD

PloS one 20120906 9

Microbes of the human respiratory tract are important in health and disease, but accurate sampling of the lung presents challenges. Lung microbes are commonly sampled by bronchoscopy, but to acquire samples the bronchoscope must pass through the upper respiratory tract, which is rich in microbes. Here we present methods to identify authentic lung microbiota in bronchoalveolar lavage (BAL) fluid that contains substantial oropharyngeal admixture. We studied clinical BAL samples from six selected s ...[more]

PMID: 22970118

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Project description:BackgroundUnderstanding the complex structures and interactions of the bacterial communities inhabiting the upper (URT) and lower (LRT) respiratory tract of pigs is at an early stage. The objective of this study was to characterize the bacterial topography of three URT (nostrils, choana, and tonsils) and LRT (proximal trachea, left caudal lobe and secondary bronchi) sites in pigs. Thirty-six post-mortem samples from six pigs were analysed by 16S rRNA gene quantification and sequencing, and the microbiota in nostrils and trachea was additionally profiled by shotgun sequencing.ResultsThe bacterial composition obtained by the two methods was congruent, although metagenomics recovered only a fraction of the diversity (32 metagenome-assembled genomes) due to the high proportion (85-98%) of host DNA. The highest abundance of 16S rRNA copies was observed in nostrils, followed by tonsils, trachea, bronchi, choana and lung. Bacterial richness and diversity were lower in the LRT compared to the URT. Overall, Firmicutes and Proteobacteria were identified as predominant taxa in all sample types. Glasserella (15.7%), Streptococcus (14.6%) and Clostridium (10.1%) were the most abundant genera but differences in microbiota composition were observed between the two tracts as well as between sampling sites within the same tract. Clear-cut differences were observed between nasal and tonsillar microbiomes (R-values 0.85-0.93), whereas bacterial communities inhabiting trachea and lung were similar (R-values 0.10-0.17). Moraxella and Streptococcus were more common in bronchial mucosal scraping than in lavage, probably because of mucosal adherence. The bacterial microbiota of the choana was less diverse than that of the nostrils and similar to the tracheal microbiota (R-value 0.24), suggesting that the posterior nasal cavity serves as the primary source of bacteria for the LRT.ConclusionWe provide new knowledge on microbiota composition and species abundance in distinct ecological niches of the pig respiratory tract. Our results shed light on the distribution of opportunistic bacterial pathogens across the respiratory tract and support the hypothesis that bacteria present in the lungs originate from the posterior nasal cavity. Due to the high abundance of host DNA, high-resolution profiling of the pig respiratory microbiota by shotgun sequencing requires methods for host DNA depletion.

Project description:Background Respiratory tract infection (RTI) is a common disease among children of all ages that causes high hospitalization and mortality rates. Infection with more than one pathogen has been reported in RTI; however, the association of the pathogen spectrum in upper and lower respiratory tract infections remains unclear. Methods A prospective study was conducted during February to October 2016. Fifty-five nasopharyngeal swabs (NPS) and 30 bronchoalveolar lavage fluid (BALF) samples from 55 hospitalized children aged less than 14 years (mean age 40 months) and diagnosed with an RTI were collected. All samples were detected for 18 respiratory pathogens using the Filmarray assay, real-time PCR, or nested PCR methods. Detection results and clinical characteristics of all cases were analyzed using chi-square and t tests. Results Forty-one of 55 (74.5%) NPS obtained from children were positive for at least one pathogen by the Filmarray assay. Of these cases, 53.7% (22/41) were co-infected. The most commonly detected pathogen was rhinovirus (RV), followed by Mycoplasma pneumoniae (MP) and respiratory syncytial virus (RSV). Infection by both RV and MP was the most frequently observed pattern of co-infection. Similar results were observed using real-time PCR. The pathogens in the NPS from 76.6% of cases detected by Filmarray and 80.0% of cases by real-time PCR included all the pathogens detected in the BALF sample from the same individual. The Filmarray assay showed an 80% concordance rate with real-time PCR and had a turnaround time of less than 1.2 h. No significant differences were observed between the association of single-infection and co-infection with clinical characteristics, neither by Filmarray nor real-time PCR. Conclusion The spectrum of pathogens is mostly concordant in the upper and lower respiratory tract. Collecting NPS for detection can be a non-invasive and more convenient option compared with BALF. Although co-infection is common in children with an RTI, the clinical significance of co-infection remains unclear and warrants further analysis.

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Dataset Information

Assessing bacterial populations in the lung by replicate analysis of samples from the upper and lower respiratory tracts.

Publications

Assessing bacterial populations in the lung by replicate analysis of samples from the upper and lower respiratory tracts.

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