DNA extraction, clone library construction and DNA sequencing

DNA was extracted from 300 mg of wet colonic digesta using the bead-beating method followed by the Soil DNA kit (Omega, USA). The mcrA gene was amplified using primer pairs, and the amplification protocols were utilized according to previously report [33]. PCR products were purified using the EasyPure Quick Gel Extraction Kit (Trans, Beijing, China), ligated into pEASY-T3 (Trans, Beijing, Chain) and transformed into Trans1-T1 Phage resistant chemically competent cell. Plasmid DNA was recovered from recombinant cell colonies and the DNA library was screened by PCR analysis using previously described primer pairs [33]. A total of 219 positive insert-containing clones were randomly selected, and the nucleotide sequences of the clones’ inserts were determined by Beijing AuGCT DNA-SYN Biotechnology Co., Ltd.