Characterization of broad-spectrum antibiotic resistance genes in wastewater treatment reactors through metagenomic approaches

Abstract

The spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) have attracted great concerns worldwide. Wastewater treatment plants (WWTPs) are reservoirs of ARGs while wastewater/sludge treatment processes are considered as important means to control these emerging biological pollutants. However, the full profiles of ARGs in WWTPs or the removal efficiency of ARGs by wastewater/sludge treatment process was not well characterized yet. Thus, the major tasks in this study were (1) to reveal the broad-spectrum profiles of ARGs in WWTPs; (2) to investigate the removal efficiencies of ARGs using wastewater/sludge treatment process; (3) to examine the differences and similarities of ARGs in samples from WWTPs and other environments; (4) to develop an efficient platform for examination on the broad-spectrum profiles of environmental ARGs through metagenomic analysis.

Activated sludge is an important segment for harboring ARGs and horizontal gene transfer of ARGs among different microbial communities. Existence of six-lactam resistance genes were found through PCR and qPCR approaches in activated sludge collected from 15 WWTPs in East Asia and North America. Abundances of these resistance genes ranged from 5.34×101 copies/ng DNA to 5.49×104 copies/ng DNA. Broad-spectrum profiles of ARGs in activated sludge from a local WWTP in a period of four years were investigated through metagenomic analysis. A total of 16 ARGs types were detected. During the 4-year period, a descending trend of total ARGs abundance was found but the abundances of _-lactam and quinolone resistance genes increased. Moreover, resistance genes of tetracycline, sulfonamide and vancomycin showed seasonal variation.

Metagenomic analysis was also applied to study the fate of ARGs in wastewater/sludge treatment processes in a full-scale municipal WWTP. Results suggested that 99.82% of ARGs in influent could be removed by wastewater treatment process while only 20.70% of ARGs could be removed in sludge anaerobic digestion. The distribution of ARGs in WWTP was significantly correlated with six genera and five of which included potential pathogens.

Effect of temperature on ARGs removal from activated sludge by anaerobic digestion was examined through metagenomic analysis. No significant change was found for the removal efficiency of total ARGs using bench-scale thermophilic or mesophilic anaerobic digestion reactor. But results suggested that more ARGs subtypes can be effectively removed by anaerobic digestion under mesophilic condition.

ARGs profiles in 10 typical environments, including different sections in WWTPs, river water, drinking water, soils, sediments and faeces from livestock and human, were revealed through metagenomic analysis. Generally the total ARGs abundances in different environments increased with the impacts of anthropogenic activities. Co-occurrence of ARGs subtypes in different environments was studied using network analysis, tetM and aminoglycoside resistance genes were recognized as the indicators to estimate the abundances of other co-occurred ARGs subtypes.

The processes in metagenomic analysis were improved to shorten the analysis time and simplify the classification of ARGs-like sequences. Error and redundant sequences were removed from the widely used reference database of ARGs. A structured database was then constructed to realize the automatic classification of ARGs in metagenomic data. A hybrid alignment process was optimized for rapid ARGs or other specific functional genes annotation as well.