Antibiotic resistance genes associated with coliforms and plasmids in municipal WWTPs revealed by metagenomic and metatranscriptomic methods

Abstract

Antibiotic resistance genes (ARGs) have been considered as global emerging environmental pollutants. Wastewater treatment plants (WWTPs), which connect the human society and nature environments, are the important hubs for ARGs dissemination. However, many aspects of the ARGs exploration in WWTPs were not fully-studied yet. Thus, this Ph.D. study was conducted to (1) evaluate the methods for WWTP sample pretreatment and DNA extraction about ARGs exploration; (2) explore the ARGs expression level during the antibiotic degradation in WWTP systems; (3) correlate the phenotype and genotype of the antibiotic resistance; and (4) investigate the plasmid-mediated ARGs in the WWTP systems. Sample pretreatment and DNA extraction were evaluated for ARGs exploration of influent, activated sludge (AS) and effluent in WWTPs. Considering DNA yield, purity, the abundance and diversity of ARGs and microbial taxa, plus the reproducibility of the extraction, sample pretreatment by fixation in 50% ethanol is suggested and the FastDNA® SPIN Kit for Soil is recommended for DNA extraction. ARGs expression level during ampicillin degradation was investigated using metagenomic, metatranscriptomic approaches and UPLC-MS/MS. In the simulated AS systems, ampicillin degradation fitted the first-order biodegradation kinetics model well (R2: 0.983 - 0.991), and noticeable up-regulation of a class A beta-lactamase gene (P14171 gene) was observed. Based on the community structure analysis, this up-regulated ARG was carried by Alphaproteobacteria, which could contribute significantly for the degradation of ampicillin. Functional metagenomics and cultivation based metagenomics were conducted to correlate the genotype and phenotype of antibiotic resistance. The obtained results showed that ARGs co-expression and antibiotic cross-resistance were prevalent in WWTP influent. The following ARGs: beta-lactamase genes, aminoglycoside 3-phosphotransferase genes, phosphotransferase type 2 genes, tetA genes, etc. contributed significantly to the related resistance of the isolated coliforms. A novel portable sequencing approach – Nanopore sequencing was conducted to review the resistome of the isolated coliforms. Meanwhile, a hybrid assembly of combined Nanopore and Illumina sequences was used to reconstruct the genome structure of a multidrug resistant E. coli strain, indicating that horizontal gene transfer contributed to the obtaining of its chromosomal beta-lactamase genes. Plasmid capture and plasmid metagenome extraction were combined with high-throughput sequencing to exhibit the profile of plasmid-mediated functional genes in WWTP systems. Eighteen types of ARGs were detected in the plasmid metagenomes. Compared to the corresponding total DNA metagenomes, more ARGs were encoded by plasmid metagenomes.