Abstract
The widespread presence of antibiotic-resistance genes (ARGs) and opportunistic pathogens/antibiotic-resistant bacteria (ARB) in wastewater has emerged as a serious threat to human health and the environment. This study aimed to evaluate the efficiency of selected Ultrafiltration (UF) and Nanofiltration (NF) membranes with a molecular weight cut-off between 10,000 Da to 300 Da for removing ARGs and opportunistic pathogens/ARBs from wastewater in a lab-scale setup, and shotgun metagenomic analysis of wastewater from various sources. ARG plasmid spiked WWTP effluents were filtered and ARG plasmid and Enterobacteriaceae were detected and quantified using qPCR in influent, concentrate, and permeate. In addition, shotgun metagenomic sequencing of wastewater samples from various sources was performed using Oxford Nanopore MinION Mk1B device with Rapid Barcoding Kit 24 with V14 chemistry. Bioinformatic tools; kraken2 in Omicsbox for taxonomic and microbial diversity analysis, and BLAST and EPI2ME for ARG detection in hospital wastewater and WWTP effluents, were used.
Our study shows that all tested UF and NF membranes remove Enterobacteriaceae with 100% efficiency and ARG plasmid with efficiency between 99.97% and 99.99%, achieving LRV of ~6 or higher for both. The MinION yielded 4.56 million reads, with 99.89% of reads basecalled in 68 hours run. Hospital wastewater contained more classifiable reads (~33%) and comparable microbial diversity with WWTP effluent. More than 85% of the classified data were assigned to bacteria and Moraxellaceae, Streptomycetaceae, Arcobacteraceae, Bacteroidaceae, and Vibrionaceae, the most common families. Aliarcobacter butzleri, Acinetobacter cumulans, Acidovorax temperans, Aliarcobacter cryaerophilus, and Acinetobacter johnsonii were the top common species. Enterococcus faecalis and Streptomyces sp were present in UF-1 influent (LF2 and LQ1) which were reduced or absent in UF-1 permeate (LPF1 and LQ1). ARGs like AAC(6')-Ib, OXA, tet, ANT(2'')-Ia, sul1, TEM, and ermB were detected in higher abundance in hospital wastewater than WWTP effluent. Some ARGs were detected in the permeate of the UF membrane and no ARGs in the permeate of the NF membrane. In conclusion, UF and NF membranes can effectively eliminate ARGs, ARBs and pathogens from wastewater. NF shows optimal performance and metagenomic sequencing can be used to track these contaminants in the wastewater.