Novel insight into diversity of active tetracycline-resistant bacteria and genes in sludge by in situ microcapsule-entrapped cultivation.

Antibiotic resistance poses a growing threat to global health and increases healthcare costs. Considering the limitations of traditional methods identifying antibiotic-resistant bacteria and genes, this study established an in situ microcapsule-entrapped cultivation (IMEC) strategy to screen the active tetracycline-resistant bacteria and genes in activated sludge from a wastewater treatment plant. Besides six tetracycline-resistant bacterial genera identified by conventional isolation and direct sequencing, IMEC uncovered extra 20 bacterial genera resistant to tetracycline, and their metabolic activities and tetracycline resistance were confirmed by Raman-Deuterium isotope probing. Among them, Romboutsia, Janthinobacterium, and Hydrogenoanaerobacterium were reported to have tetracycline resistance for the first time. The diversity of active tetracycline-resistant genes identified by IMEC was much lower than that by direct sequencing. In particular, one new tetG-like gene encoding a tetracycline outer periplasmic pump was confirmed by molecular docking. These findings documented the underestimated diversity of tetracycline-resistant bacteria but overestimated diversity of tetracycline-resistant genes in sludge. Overall, IMEC provides an effective tool for resolving active antibiotic-resistant bacteria and genes in activated sludge, offering new insights for water epidemiology.