Influence of fecal sample storage on bacterial community diversity

Abstract

Previous studies have identified a correlation, either positive or negative, between specific stool bacteria strains and certain autoimmune diseases. These conflicting data may relate to sample collection. The aim of this work was to evaluate the influence of the collection parameters of time and temperature on bacterial community composition. Samples were taken from healthy children and immediately divided in 5 sub-samples. One sample was frozen immediately at -80 ° C, while the other aliquots were frozen 12, 24, 48, and 72h later DNA extracted from each sample was used to amplify the 16S rRNA with barcoded primers. The amplified products were pooled and partial 16S rRNA sequences were obtained by pyrosequencing. Person-to-person variability in community diversity was high. A list of those taxa that comprise at least 1% of the community was made for each individual. None of these were present in high numbers in all individuals. The Bacteroides were present in the highest abundance in three of four subjects. A total of 23,701 16S rRNA sequences were obtained with an average of 1,185 reads per sample with an average length of 200 bases. Although pyrosequencing of amplified 16S rRNA identified changes in community composition over time (~10%), little diversity change was observed at 12 hours (3.06%) with gradual changes occurring after 24 (8.61%), 48 (9.72%), and 72 h (10.14%), post collection.

Fig. (1)

The % change in bacterial community composition in the stool samples compared to samples frozen immediately. Each point is the mean of samples from four individuals with the bars representing the standard error about the mean.

Fig. (2)

Heat map showing the proportion of bacteria found at different times of storage (immediately (IM), after 12h, 24h, 48h and 72h) and the significance of the variation observed. Maps A, B, C and D represent the subjects analyzed. The scale on the right represents the contribution of a particular OTU and it is expressed in percentage of the total. The closest bacterial relative is shown in the left side of the map. Note that OTUs were classified to the highest specificity level, ranging from phylum to genus.