EpicPCR approach uncovers the diverse hosts of newly-emerging tigecycline resistance gene tet(X4) in anaerobic digesters

The hosts of newly-emerging and clinically important antibiotic resistance genes (e.g., tet(X4)) are largely uninvestigated due to lacking appropriate approaches. Herein, an culture-independent and high-throughput epicPCR (emulsion, paired isolation, and concatenation polymerase chain reaction) method was developed, optimized, and demonstrated for identifying tet(X4) bacterial hosts from environmental bacterial community. Considering the high sequence similarity between tet(X4) and other tet(X)-variant genes, specific primers were screened and verified for identifying tet(X4) accurately, linking tet(X4) with 16S rRNA, which were validated within an artificially constructed bacterial community. The epicPCR targeting tet(X4) was further applied for identifying the bacterial hosts of tet(X4) in anaerobic digesters treating swine manure. Totally 19 genera which distributed among Proteobacteria, Bacteroidota, Firmicutes and Caldatribacteriota were identified as tet(X4) hosts. The latter two phyla were previously unknown to possess tet(X4). The results indicated that a far more diverse range of bacteria is involved in tet(X4) than previously realized. Compared with the tet(X4) hosts determination results of network analysis and metagenomic binning, the epicPCR revealed the high diversity of tet(X4) hosts. The epicPCR method developed in this study will be effective to accurately determine tet(X4) bacterial hosts from different microbiota.