ABSTRACT: With the issue of the antimicrobial additive ban in feed in Chinese animal husbandry, it is important to determine the potential drivers of the spread of the newly discovered tigecycline-resistant tet(X)-variant genes. Here, we investigated the correlations between residues of heavy metals, antimicrobials, and pesticides and the relative abundance of tet(X)-variant genes in 94 commercial organic-fertilizer samples collected from 9 Chinese provinces. A total of 5 heavy metals (mercury, lead, arsenic, chromium, and cadmium), 10 antimicrobials, and 18 pesticides were detected. The tet(X)-variant genes, including tet(X)/(X2), tet(X3), tet(X4), tet(X5), and tet(X6) were detected in 39 (41.5%) samples. Although tet(X)-variant-carrying bacteria were not isolated from these samples, the tet(X4)-carrying plasmids could be captured by exogenous Escherichia coli. Correlation analysis revealed that heavy metals, other than antimicrobials, showed a significant positive association with the relative abundance of the tet(X)-variant genes, especially tet(X3) and tet(X4) (R = 0.346 to 0.389, P < 0.001). The correlation was attributed to the coselection of the tet(X3)/tet(X4) gene on the same plasmid and the conjugation-promoting effect of tet(X3)/tet(X4)-carrying plasmids by subinhibitory concentrations of heavy metals. The heavy metals increased the permeability of the bacterial outer membrane and upregulated the transcription of type IV secretion system (T4SS)-encoding genes on tet(X)-variant-carrying plasmids, therefore enhancing the bacterial conjugation rates. Taken together, our findings have indicated that heavy metals may play an important role in spreading tet(X)-variant genes within the animal manure-related environment. IMPORTANCE An antimicrobial resistance gene (ARG) is considered a novel contaminant for the environment. Most animal feces are usually made into commercial organic fertilizers in China and will pose a threat to the farmland soil and agricultural product if fertilizers harboring clinically significant antimicrobial-resistant (AMR) genes are applied on farmland. This study has indicated that heavy metals may play an important role in the transmission of transferable tigecycline resistance genes [tet(X3) and tet(X4)]. The mechanism was that heavy metals posed a coselection effect of the tet(X3)/tet(X4) gene on the same plasmid and could increase the conjugation ability of tet(X3)/tet(X4)-carrying plasmids. The conjugation-promoting concentrations of heavy metals are lower than the maximal limits defined in the national standard for fertilizers, indicating a high transmission risk of tet(X3)/tet(X4) genes within the animal manure-related environment. The findings in this study will provide scientific evidence for the future development of effective measures to reduce AMR dissemination.
