Effects of preservation method on canine (Canis lupus familiaris) fecal microbiota

Abstract

Studies involving gut microbiome analysis play an increasing role in the evaluation of health and disease in humans and animals alike. Fecal sampling methods for DNA preservation in laboratory, clinical, and field settings can greatly influence inferences of microbial composition and diversity, but are often inconsistent and under-investigated between studies. Many laboratories have utilized either temperature control or preservation buffers for optimization of DNA preservation, but few studies have evaluated the effects of combining both methods to preserve fecal microbiota. To determine the optimal method for fecal DNA preservation, we collected fecal samples from one canine donor and stored aliquots in RNAlater, 70% ethanol, 50:50 glycerol:PBS, or without buffer at 25 °C, 4 °C, and −80 °C. Fecal DNA was extracted, quantified, and 16S rRNA gene analysis performed on Days 0, 7, 14, and 56 to evaluate changes in DNA concentration, purity, and bacterial diversity and composition over time. We detected overall effects on bacterial community of storage buffer (F-value = 6.87, DF = 3, P < 0.001), storage temperature (F-value=1.77, DF = 3, P = 0.037), and duration of sample storage (F-value = 3.68, DF = 3, P < 0.001). Changes in bacterial composition were observed in samples stored in −80 °C without buffer, a commonly used method for fecal DNA storage, suggesting that simply freezing samples may be suboptimal for bacterial analysis. Fecal preservation with 70% ethanol and RNAlater closely resembled that of fresh samples, though RNAlater yielded significantly lower DNA concentrations (DF = 8.57, P < 0.001). Although bacterial composition varied with temperature and buffer storage, 70% ethanol was the best method for preserving bacterial DNA in canine feces, yielding the highest DNA concentration and minimal changes in bacterial diversity and composition. The differences observed between samples highlight the need to consider optimized post-collection methods in microbiome research.

Interpersonal Variations in Gut Microbiota Profiles Supersedes the Effects of Differing Fecal Storage Conditions

“…During DNA extraction, we found that it was difficult to fully separate RNAlater and feces using centrifugation, an observation that has also previously been reported 27 , 33 . Since RNAlater can influence subsequent cell lysis during DNA extraction 33 , this could affect the microbiota profile.…”

“…During DNA extraction, we found that it was difficult to fully separate RNAlater and feces using centrifugation, an observation that has also previously been reported 27 , 33 . Since RNAlater can influence subsequent cell lysis during DNA extraction 33 , this could affect the microbiota profile. In two out of three participants, we observed a slightly reduced composition of bacteria belonging to the Bacteroidetes phylum and especially the genus Prevotella in samples stored in RNAlater, similar to observations by Hale et al .…”

How should we store avian faecal samples for microbiota analyses? Comparing efficacy and cost-effectiveness

“…Faecal extracts are characterized by low DNA concentration and high degradation (Dai et al, 2015;Demay et al, 2013), and sparrow samples analysed here are no exception. Avian DNA concentrations and purity are consistently lower compared to those reported for mammal faeces DNA (Bubb et al, 2011;Costa et al, 2017;Horng et al, 2018).…”

“…Results are variable and often contradictory. RNAlater is frequently used to store faecal samples for microbiota analysis (Al et al, 2018;Broquet et al, 2007;Horng et al, 2018;Vlčková et al, 2012;Vogtmann et al, 2017), yet there is evidence that its performance decreases after a period of time at room temperature (Flores et al, 2015), and that the bacterial community profiles differ to those of frozen samples (Choo et al, 2015). Ethanol is also regularly used and has been shown to produce microbial profiles comparable to those obtained with RNAlater (Vogtmann et al, 2017).…”

“…Ethanol is also regularly used and has been shown to produce microbial profiles comparable to those obtained with RNAlater (Vogtmann et al, 2017). However, some results when stored at 70% ethanol have shown higher species diversity compared to fresh samples (Horng et al, 2018) with particular disparity in bacterial counts of Enterobacteriaceae (Vlčková et al, 2012) and overall poor performance, showing an increase in relative abundance of certain taxa (Song et al 2016).…”