title: Time between collection and storage significantly influences bacterial sequence composition in sputum samples from cystic fibrosis respiratory infections.
abstract: Spontaneously expectorated sputum is traditionally used as the sampling method for the investigation of lower airway infections. While guidelines exist for the handling of these samples for culture-based diagnostic microbiology, there is no comparable consensus on their handling prior to culture-independent analysis. The increasing incorporation of culture-independent approaches in diagnostic microbiology means that it is of critical importance to assess potential biases. The aim of this study was to assess the impact of delayed freezing on culture-independent microbiological analyses and to identify acceptable parameters for sample handling. Sputum samples from eight adult cystic fibrosis (CF) patients were collected and aliquoted into sterile Bijou bottles. Aliquots were stored at room temperature before being frozen at -80 °C for increasing intervals, up to a 72-h period. Samples were treated with propidium monoazide to distinguish live from dead cells prior to DNA extraction, and 16S rRNA gene pyrosequencing was used to characterize their bacterial compositions. Substantial variation was observed in samples with high-diversity bacterial communities over time, whereas little variation was observed in low-diversity communities dominated by recognized CF pathogens, regardless of time to freezing. Partitioning into common and rare species demonstrated that the rare species drove changes in similarity. The percentage abundance of anaerobes over the study significantly decreased after 12 h at room temperature (P = 0.008). Failure to stabilize samples at -80 °C within 12 h of collection results in significant changes in the detected community composition.
authors.affiliation: NERC Centre for Ecology & Hydrology, Wallingford, United Kingdom Institute of Pharmaceutical Science, Molecular Microbiology Research Laboratory, King's College London, London, United Kingdom.
authors.forename: Leah
authors.initials: L
authors.lastname: Cuthbertson
authors.affiliation: Institute of Pharmaceutical Science, Molecular Microbiology Research Laboratory, King's College London, London, United Kingdom SAHMRI Infection and Immunity Theme, School of Medicine, Flinders University, Bedford Park, Adelaide, Australia.
authors.forename: Geraint B
authors.initials: GB
authors.lastname: Rogers
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, United Kingdom.
authors.forename: Alan W
authors.initials: AW
authors.lastname: Walker
authors.affiliation: NERC Centre for Ecology & Hydrology, Wallingford, United Kingdom.
authors.forename: Anna
authors.initials: A
authors.lastname: Oliver
authors.affiliation: Cystic Fibrosis Unit, Southampton University Hospitals, NHS Trust, Southampton, United Kingdom.
authors.forename: Tarana
authors.initials: T
authors.lastname: Hafiz
authors.affiliation: Department of Pediatrics, University of Washington, Seattle, Washington, USA Department of Microbiology, University of Washington, Seattle, Washington, USA.
authors.forename: Lucas R
authors.initials: LR
authors.lastname: Hoffman
authors.affiliation: Cystic Fibrosis Unit, Southampton University Hospitals, NHS Trust, Southampton, United Kingdom.
authors.forename: Mary P
authors.initials: MP
authors.lastname: Carroll
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, United Kingdom.
authors.forename: Julian
authors.identifier: http://orcid.org/0000-0002-7069-5958
authors.initials: J
authors.lastname: Parkhill
authors.affiliation: Institute of Pharmaceutical Science, Molecular Microbiology Research Laboratory, King's College London, London, United Kingdom.
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Bruce
authors.affiliation: NERC Centre for Ecology & Hydrology, Wallingford, United Kingdom cjvdg@ceh.ac.uk.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: van der Gast
mesh_terms: D000328:Adult; D001419:Bacteria / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D001424:Bacterial Infections / Q000382:microbiology*; D016000:Cluster Analysis; D003550:Cystic Fibrosis / Q000150:complications*; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012141:Respiratory Tract Infections / Q000382:microbiology*; D017422:Sequence Analysis, DNA; D013048:Specimen Handling / Q000379:methods*; D013183:Sputum / Q000382:microbiology*; D013696:Temperature; D013997:Time Factors
chemical_list: D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S