Assessment of sample handling practices on microbial activity in sputum samples from patients with cystic fibrosis.
OBJECTIVE
The aim of this study was to quantitatively and qualitatively assess the effect of sample storage on the metabolically active microbial community found in sputum samples from patients with cystic fibrosis (CF).


METHODS
Sputum samples were collected and split in two equal aliquots one of which was immersed in RNAlater and refrigerated immediately, the second stored at room temperature for 24 h and RNAlater was subsequently added. mRNA was extracted, and RT-PCR-DGGE and qPCR analysis of the bacterial and fungal communities was carried out.


RESULTS
Significant differences in the bacterial communities between the two protocols were observed but there were no significant difference seen in the fungal community analyses. Analysis by qPCR demonstrated that room temperature storage gave statistically significant increases in eubacteria and Pseudomonas spp. and a statistically significant decrease in those of Haemophilus influenzae.


CONCLUSIONS
The analysis of metabolically active microbial communities from CF sputum using molecular techniques indicated that samples should be stored at 4 degrees C upon addition of RNAlater to obtain an accurate depiction of the CF lung microbiota. Also, storing respiratory samples at room temperature may cause an over representation of Pseudomonas aeruginosa and mask the presence of other clinically significant organisms.
Nelson
A
A
School of Applied Sciences, Ellison Building, University of Northumbria, Newcastle upon Tyne, UK.
De Soyza
A
A
Bourke
S J
SJ
Perry
J D
JD
Cummings
S P
SP
D000328:Adult; D001419:Bacteria / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D001424:Bacterial Infections / Q000175:diagnosis / Q000382:microbiology; D044822:Biodiversity; D003550:Cystic Fibrosis / Q000150:complications*; D004591:Electrophoresis, Polyacrylamide Gel; D005658:Fungi / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D054892:Metagenome; D008969:Molecular Sequence Data; D009181:Mycoses / Q000175:diagnosis / Q000382:microbiology; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013048:Specimen Handling / Q000379:methods*; D013183:Sputum / Q000382:microbiology*; D013696:Temperature; D013997:Time Factors