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: Tn5706-like transposon Tn6298, IS200/605-related transposon Tn6303, Tn3 family transposon Tn6299, and three ISCR2-related transposons, Tn62300, Tn62301, and Tn62302.
IMPORTANCEFertilization 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. U pon their discovery in the late 1940s, the broad-spectrum efficiency, ease of use, and relatively low price of tetracycline derivatives placed them among the antibiotics of choice for the treatment of infections inside and outside clinics (1, 2). Their extensive use soon also brought the development of high-level resistance in most human pathogens, and tetracycline use progressively decayed in hospitals, especially after new broad-spectrum antibiotics became available (1). More than 40 distinct tetracycline resistance genes (TRGs) have been identified, classified according to the mechanism of action of their encoded proteins: energy-dependent efflux of the drug out of the cell, ribosomal protection (since tetracyclines bind to the 30S ribosomal subunit to inhibit protein translation), and drug inactivation through enzymatic activity (3). The classical nomenclature has also been challenged by the discovery of mosaic resistance genes resulting from fragment exchange between different genes encoding ribosomal protection (4, 5). Like many other antibiotic resistance genes, TRGs are widely and efficiently disseminated among bacteria through mobile genetic elements (MGEs). Most of them can be found on small nonconjugative transposons that are themselves embedded in conjugative plasmids or transposons (6-10). However, despite the fai...