I. Khan1, S. Niraula2, S. Stirewalt2, J. Jung3, M. Alagna2, P. Seed4, K. Ho2 1Southern Illinois University School Of Medicine, Springfield, IL, USA 2Northwestern University, Division Of Vascular Surgery, Chicago, IL, USA 3Penn Medicine, Department Of Surgery, Philadelphia, PA, USA 4Stanley Manne Children’s Research Institute, Chicago, IL, USA
Introduction:
The gut microbiome is known to be affected by cardiovascular diseases, including peripheral artery disease (PAD). While it is well-known that sample collection and storage methods impact microbiome community composition, the optimal storage and processing method for PAD samples is unclear. Our goal is to investigate the effect of sample collection and storage methods on the viability and community composition of fecal samples from participants with and without PAD.
Methods:
Participants with PAD and non-PAD controls performed home stool sample collection. Samples were either immediately frozen (FR) or placed in Cary-Blair (CB) storage media and returned to the study team. Glycerol stocks (20%) were prepared from each sample aerobically and anaerobically and frozen at -80°C. Glycerol stocks were thawed, serially diluted (10-3 to 10-5 for FR and 10-4 to 10-6 for CB), and anaerobically cultured on Gifu agar media. Colony-forming units (CFU) were quantified every 24 hours for 4 days. Microbial profiling was done using 16S rRNA (V4) sequencing on an Illumina MiSeq sequencer. Raw paired-end reads (150 bp) were analyzed in QIIME2 and plotted in R.
Results:
Sample preservation methods significantly influenced microbial community structure based on generalized UniFrac distances (PERMANOVA, p=.0001). In both aerobically and anaerobically processed samples, CB glycerol stocks showed significantly higher CFUs compared to FR glycerol stocks at 48 hours (aerobic, p=.035; anaerobic, p=0.0004). Among samples that were processed within 24 hours of collection, the anaerobically processed CB glycerol stocks had significantly higher CFUs than FR glycerol stocks (p=.0098). There was no difference in viability based on processing time when samples were processed aerobically. Within the CB and FR groups, there was no difference in viability based on processing modality. There was no difference in viability between revivified PAD and non-PAD samples.
Conclusion:
Glycerol stocks of fecal samples collected using CB storage media had greater viability when processed anaerobically compared to FR samples. Future 16S rRNA sequencing of revivified CFUs will assess community composition and enable the use of these samples for downstream research applications.