Bacterial hosts and horizontal transfer characteristics of clinically important tet(X)-variant genes in municipal wastewater treatment plants using epicPCR-directed cultivation strategy

Mobile tet(X)-variant genes confer resistance to a wide range of tetracyclines, including the antibiotic of last-resort, tigecycline, raising significant concerns regarding their potential spread cross-environmental dissemination. However, the bacterial hosts and environmental spread of these genes remain poorly understood. Herein, a retrospective study unveiled the prevalence of tet(X)-variant genes (ranging from tet(X3) to tet(X6)) in activated sludge samples from five municipal wastewater treatment plants (WWTPs) from 2013 to 2021. Among these variants, tet(X4) exhibited the highest detection frequency (100%) and abundance [(2.48 ± 3.07) × 10⁷ copies/g dry weight] with an increasing trend. An epicPCR-directed cultivation strategy was proposed to facilitate the targeted isolation of tet(X4)-carrying bacterial hosts in activated sludge. This strategy involves the identification of bacterial host profiles using epicPCR and subsequent selective isolating target bacteria. Enterobacteriaceae emerged as the primary bacterial host for tet(X4), alongside previously unreported genera like Providencia, Advenella, and Moheibacter. Subsequent selective isolation of the most abundant Enterobacteriaceae based on the epicPCR-informed host spectrum yielded 39 tet(X4)-carrying Escherichia coli strains from the WWTP. Whole genome sequencing of tet(X4)-positive strains revealed that plasmid-mediated horizontal gene transfer is the primary mechanism driving tet(X4) dissemination. Plasmids including IncFIA(HI1)/IncHI1A/IncHI1B(R27) and IncX1, commonly reported in various clinical and animal settings, were identified as the predominant carriers of tet(X4). E. coli strains harbouring tet(X4) in the WWTP showed substantial genetic similarity to strains from hospital and animal sources, underscoring concerns about the potential risk of across diverse sectors. This study provided the first glimpse of the presence of mobile tet(X)-variants in WWTPs, and highlighted the promise of the epicPCR-directed cultivation strategy for exploring bacterial hosts of clinically important ARGs in different habitats from a One Health perspective.