Uncovering the diverse hosts of tigecycline resistance gene tet(X4) from environmental microbiota (using epicPCR)

The environmental hosts of newly-emerging and clinically important antibiotic resistance genes are largely uninvestigated due to lacking appropriate approaches. This study developed, optimized, and demonstrated an epicPCR (emulsion, paired isolation, and concatenation polymerase chain reaction) method for identifying the tet(X4) bacterial hosts in the environment. Because the sequence of tet(X4) is highly similar to the other tet(X)-variants gene, a specificity validation step was needed to ensure accurate identification and linkage of tet(X4) with 16S rRNA gene. The epicPCR targeting tet(X4) was successfully demonstrated on anaerobic digesters samples. Totally 19 genera which belonging to 14 classified families, four classified phyla were identified as tet(X4) bacterial hosts. The phyla of Firmicutes and Caldatribacteriota were previously unknown to possess tet(X4). There were 16 genera have not been reported as tet(X4) hosts in literatures. 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 more diverse tet(X4) hosts. The results substantiated that the epicPCR was an effective approach to explore the diversity and dynamics of tet(X4) hosts from environmental microbiota. This culture-independent epicPCR method established in this study provides high specificity, accuracy and throughput for exploring the distribution, diversity, and dynamic of tet(X4) bacterial hosts from environmental samples.