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mSphere. 2018 Aug 8;3(4). pii: e00292-18. doi: 10.1128/mSphere.00292-18.

Profiling of Bacterial and Fungal Microbial Communities in Cystic Fibrosis Sputum Using RNA.

Author information

1
Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
2
Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, Massachusetts, USA.
3
Pulmonary and Critical Care Medicine, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire, USA.
4
Department of Pathology and Laboratory Medicine, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire, USA.
5
Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA dhogan@dartmouth.edu.
#
Contributed equally

Abstract

Here, we report an approach to detect diverse bacterial and fungal taxa in complex samples by direct analysis of community RNA in one step using NanoString probe sets. We designed rRNA-targeting probe sets to detect 42 bacterial and fungal genera or species common in cystic fibrosis (CF) sputum and demonstrated the taxon specificity of these probes, as well as a linear response over more than 3 logs of input RNA. Culture-based analyses correlated qualitatively with relative abundance data on bacterial and fungal taxa obtained by NanoString, and the analysis of serial samples demonstrated the use of this method to simultaneously detect bacteria and fungi and to detect microbes at low abundance without an amplification step. Compared at the genus level, the relative abundances of bacterial taxa detected by analysis of RNA correlated with the relative abundances of the same taxa as measured by sequencing of the V4V5 region of the 16S rRNA gene amplified from community DNA from the same sample. We propose that this method may complement other methods designed to understand dynamic microbial communities, may provide information on bacteria and fungi in the same sample with a single assay, and with further development, may provide quick and easily interpreted diagnostic information on diverse bacteria and fungi at the genus or species level.IMPORTANCE Here we demonstrate the use of an RNA-based analysis of specific taxa of interest, including bacteria and fungi, within microbial communities. This multiplex method may be useful as a means to identify samples with specific combinations of taxa and to gain information on how specific populations vary over time and space or in response to perturbation. A rapid means to measure bacterial and fungal populations may aid in the study of host response to changes in microbial communities.

KEYWORDS:

NanoString; bacteria; clinical microbiology; cystic fibrosis; fungi; microbial communities

PMID:
30089648
PMCID:
PMC6083091
DOI:
10.1128/mSphere.00292-18
[Indexed for MEDLINE]
Free PMC Article

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