title: Single-molecule long-read 16S sequencing to characterize the lung microbiome from mechanically ventilated patients with suspected pneumonia.
abstract: In critically ill patients, the development of pneumonia results in significant morbidity and mortality and additional health care costs. The accurate and rapid identification of the microbial pathogens in patients with pulmonary infections might lead to targeted antimicrobial therapy with potentially fewer adverse effects and lower costs. Major advances in next-generation sequencing (NGS) allow culture-independent identification of pathogens. The present study used NGS of essentially full-length PCR-amplified 16S ribosomal DNA from the bronchial aspirates of intubated patients with suspected pneumonia. The results from 61 patients demonstrated that sufficient DNA was obtained from 72% of samples, 44% of which (27 samples) yielded PCR amplimers suitable for NGS. Out of the 27 sequenced samples, only 20 had bacterial culture growth, while the microbiological and NGS identification of bacteria coincided in 17 (85%) of these samples. Despite the lack of bacterial growth in 7 samples that yielded amplimers and were sequenced, the NGS identified a number of bacterial species in these samples. Overall, a significant diversity of bacterial species was identified from the same genus as the predominant cultured pathogens. The numbers of NGS-identifiable bacterial genera were consistently higher than identified by standard microbiological methods. As technical advances reduce the processing and sequencing times, NGS-based methods will ultimately be able to provide clinicians with rapid, precise, culture-independent identification of bacterial, fungal, and viral pathogens and their antimicrobial sensitivity profiles.
authors.affiliation: Department of Medicine, Division of Genomic Medicine, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA Department of Physical Therapy and Health Care Sciences, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA itoma@gwu.edu msiegel@mfa.gwu.edu.
authors.forename: Ian
authors.identifier: http://orcid.org/0000-0002-9637-5899
authors.initials: I
authors.lastname: Toma
authors.affiliation: Division of Infectious Diseases, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA itoma@gwu.edu msiegel@mfa.gwu.edu.
authors.forename: Marc O
authors.initials: MO
authors.lastname: Siegel
authors.affiliation: Department of Pathology, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA.
authors.forename: John
authors.initials: J
authors.lastname: Keiser
authors.affiliation: Department of Medicine, Division of Genomic Medicine, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA.
authors.forename: Anna
authors.initials: A
authors.lastname: Yakovleva
authors.affiliation: Department of Medicine, Division of Genomic Medicine, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA.
authors.forename: Alvin
authors.initials: A
authors.lastname: Kim
authors.affiliation: Children's National Medical Research Center, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA.
authors.forename: Lionel
authors.initials: L
authors.lastname: Davenport
authors.affiliation: Children's National Medical Research Center, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Devaney
authors.affiliation: Children's National Medical Research Center, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA.
authors.forename: Eric P
authors.initials: EP
authors.lastname: Hoffman
authors.affiliation: Department of Medicine, Division of Genomic Medicine, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA.
authors.forename: Rami
authors.initials: R
authors.lastname: Alsubail
authors.affiliation: Computational Biology Institute, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA.
authors.forename: Keith A
authors.initials: KA
authors.lastname: Crandall
authors.affiliation: Computational Biology Institute, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Castro-Nallar
authors.affiliation: Computational Biology Institute, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA Centro de Investigação em Biodiversidade e Recursos Genéticos (CIBIO), Vairão, Portugal.
authors.forename: Marcos
authors.initials: M
authors.lastname: Pérez-Losada
authors.affiliation: Computational Biology Institute, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA.
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Hilton
authors.affiliation: Department of Anesthesiology, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA.
authors.forename: Lakhmir S
authors.initials: LS
authors.lastname: Chawla
authors.affiliation: Department of Medicine, Division of Genomic Medicine, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA Department of Microbiology, Immunology, and Tropical Medicine, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA.
authors.forename: Timothy A
authors.initials: TA
authors.lastname: McCaffrey
authors.affiliation: Division of Infectious Diseases, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA.
authors.forename: Gary L
authors.initials: GL
authors.lastname: Simon
mesh_terms: D000368:Aged; D001419:Bacteria / Q000145:classification* / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D005260:Female; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008168:Lung / Q000382:microbiology*; D008297:Male; D064307:Microbiota; D008875:Middle Aged; D008969:Molecular Sequence Data; D053717:Pneumonia, Ventilator-Associated / Q000382:microbiology*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S