Characterization of tetracycline resistant bacterial community in saline activated sludge using batch stress incubation with high-throughput sequencing analysis

•An innovative and cost-effective method was developed to identify TRB in AS.•TRB consists of 13 genera with Haliea, Microbacterium and Paracoccus as major genera.•Six new TRB genera were first found in AS treating saline sewage.•The increase of tetG and tetA abundance might be related to the Pseudomonas augment.•tetS abundance showed significant increase after 20 mg/L tetracycline treatment.

An innovative and cost-effective method, i.e., batch stress incubation with tetracycline in combination with Cloning-Sanger sequencing and Illumina high-throughput sequencing was developed to identify tetracycline resistant bacteria (TRB) in activated sludge (AS) treating saline sewage. This method overcomes the drawbacks of culture-based approach (unrepresentative and biased results) and utilizes both the advantages of Cloning-Sanger sequencing and Illumina high-throughput sequencing, that is, long length read for correct taxonomic assignment at lower ranks and enough sequencing depth for accurate quantification of TRB communities with medium to low abundances, respectively. High precision (relative deviation ≤16.1%) was obtained for all taxon ranks with relative abundances over 0.01%. In the AS sample, TRB consisted of 13 genera with Haliea, Microbacterium and Paracoccus as dominate genera and 6 new TRB genera, i.e., Haliea, Rheinheimera, Alishewanella, Idiomarina, Pseudorhodobacter and Algoriphagus. The increase of tetG and tetA abundance might be associated with the significant increase of Pseudomonas (tetG and tetA host) in the AS after tetracycline stress incubation. tetS abundance also showed an obvious increase after 20 mg/L tetracycline treatment. This method provided a new tool to screen other antibiotic resistant bacteria, bacteria resistant to heavy metals or disinfectants in AS samples.

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