Abstract: The overuse of antibiotics in medicine, animal husbandry, and aquiculture industry increases the emergence of antibiotic resistant bacteria and antibiotic resistance genes (ARGs), and also, accelerates the dissemination of ARGs within environmental bacteria. In this study, the total DNA was directly extracted from environmental samples, and the upstream and downstream of antibiotic resistance genes were directly amplified by thermal asymmetric interlaced PCR (Tail-PCR) technique.By optimizing the Tail-PCR program, the multiple flanking sequences of tetW,including 6 upstream sequences and 9 downstream sequences, were simultaneously acquired. Through the bioinformatics analysis, the upstream of tetW presented a perfect inverted repeat (IR), a known tetW regulator peptide, and an insertional sequence (IS), whereas the downstream of tetW presented a most conservative fragment and a common open reading frame (ORF) coding methyltransferase. This study not only revealed several conserved flanking tetW gene modules, but also supplied a highly-efficient and convenient methodology for the research of tetW's dissemination within bacteria, i.e., several flanking sequences could be concisely obtained from one sample by using Tail-PCR program.

Key words: antibiotic resistance gene, real-time PCR, thermal asymmetric interlaced PCR (Tail-PCR), tetW flanking sequencing