Effect of DNA extraction and sample preservation method on rumen bacterial population

Abstract

The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isolated from cow rumen content stored under different conditions was conducted. The influence of rumen fluid treatment (cheesecloth squeezed, centrifuged, filtered), storage temperature (RT, -80 °C) and cryoprotectants (PBS-glycerol, ethanol) on quality and quantity parameters of extracted DNA was evaluated by bacterial DGGE analysis, real-time PCR quantification and metabarcoding approach using high-throughput sequencing. Samples clustered according to the type of extracted DNA due to considerable differences between iDNA and eDNA bacterial profiles, while storage temperature and cryoprotectants additives had little effect on sample clustering. The numbers of Firmicutes and Bacteroidetes were lower (P < 0.01) in eDNA samples. The qPCR indicated significantly higher amount of Firmicutes in iDNA sample frozen with glycerol (P < 0.01). Deep sequencing analysis of iDNA samples revealed the prevalence of Bacteroidetes and similarity of samples frozen with and without cryoprotectants, which differed from sample stored with ethanol at room temperature. Centrifugation and consequent filtration of rumen fluid subjected to the eDNA isolation procedure considerably changed the ratio of molecular operational taxonomic units (MOTUs) of Bacteroidetes and Firmicutes. Intracellular DNA extraction using bead-beating method from cheesecloth sieved rumen content mixed with PBS-glycerol and stored at -80 °C was found as the optimal method to study ruminal bacterial profile.

Extracellular DNA in natural environments: features, relevance and applications

“…Zhang et al (2013) found that several antimicrobial resistance genes were present in the exDNA and iDNA pool of such environments and that HGT is a potential mechanism for the spread of antimicrobial resistance. Investigating rumen-borne microbial communities, considerable differences between exDNA and iDNA bacterial profiles have been found (Fliegerová et al 2014), suggesting differing lysis and/or DNA secretion of the microorganisms.…”

“…Extracellular DNA has also been investigated within dead wood (Gómez-Brandón et al 2017a), cattle rumen and manure (Chroňáková et al 2013; Fliegerová et al 2014; Nagler et al 2018), aerobic and anammox granules (Cheng et al 2011; Xiong and Liu 2012; Dominiak et al 2011) and human epithelial cells used in forensics (Wang et al 2017). In addition, exDNA was found to act as a trap for infectious organisms in mammalians (reviewed by Ciesluk et al 2017) and during root tip growth of plants (Hawes et al 2012; Pietramellara et al 2013).…”

Studies on bacterial community composition are affected by the time and storage method of the rumen content

“…It can also be affected by the sampling technique of rumen contents and the fractionation of the rumen sample; in addition, sample handling and collection times can affect the results of bacterial composition studies [6, 8–12]. …”

Effects of Oregano, Attapulgite, Benzoic Acid and their Blend on Chicken Performance, Intestinal Microbiology and Intestinal Morphology

“…Lactobacillus acidophilus was grown on MRS medium agar anaerobically at 37℃. The total number of cfu of each culture was calculated by plating 100 μl of the appropriate 10-fold dilution series on LB and MRS plates for aerobic and anaerobic bacteria respectively (Rawsthorne and Phister, 2006;Skånseng et al, 2006;Fliegerova et al, 2014) .…”