Tetracycline antibiotics are widely used in livestock, and tetracycline resistance genes (TRG) are frequently reported in the manure of farmed animals. However, the diversity of TRG-carrying transposons in manure has still been rarely investigated. Using a culture-free functional metagenomic procedure, combined with large-insert library construction and sequencing, bioinformatic analyses, and functional experiments, we identified 17 distinct TRGs in a single pig manure sample, including two new tet genes: tet (59), encoding a tetracycline efflux pump, and tet (W/N/W), encoding mosaic ribosomal protection. Our study also revealed six new TRG-carrying putative nonconjugative transposons: Tn 5706 -like transposon Tn 6298 , IS 200 / 605 -related transposon Tn 6303 , Tn 3 family transposon Tn 6299 , and three IS CR2 -related transposons, Tn 62300 , Tn 62301 , and Tn 62302 .IMPORTANCE Fertilization of agricultural fields with animal manure is believed to play a major role in antibiotic resistance dissemination in the environment. There is growing concern for the possible spread of antibiotic resistance from the environment to humans since genetic resistance determinants may be located in transposons and other mobile genetic elements potentially transferable to pathogens. Among the various antibiotic resistance genes found in manure, tetracycline resistance genes (TRGs) are some of the most common. The present study provides a detailed snapshot of the tetracycline mobilome in a single pig manure sample, revealing an unappreciated diversity of TRGs and potential TRG mobility vectors. Our precise identification of the TRG-carrying units will enable us to investigate in more details their mobility effectiveness.
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