Metagenomics Reveals the Impact of Wastewater Treatment Plants on the Dispersal of Microorganisms and Genes in Aquatic Sediments

Abstract

Wastewater treatment plants (WWTPs) release treated effluent containing mobile genetic elements (MGEs), antibiotic resistance genes (ARGs), and microorganisms into the environment, yet little is known about their influence on nearby microbial communities and the retention of these factors in receiving water bodies. Our research aimed to characterize the genes and organisms from two different WWTPs that discharge into Lake Michigan, as well as from surrounding lake sediments to determine the dispersal and fate of these factors with respect to distance from the effluent outfall. Shotgun metagenomics coupled to distance-decay analyses showed a higher abundance of genes identical to those in WWTP effluent genes in sediments closer to outfall sites than in sediments farther away, indicating their possible WWTP origin. We also found genes attributed to organisms, such as those belonging to Helicobacteraceae, Legionellaceae, Moraxellaceae, and Neisseriaceae, in effluent from both WWTPs and decreasing in abundance in lake sediments with increased distance from WWTPs. Moreover, our results showed that the WWTPs likely influence the ARG composition in lake sediments close to the effluent discharge. Many of these ARGs were located on MGEs in both the effluent and sediment samples, indicating a relatively broad propensity for horizontal gene transfer (HGT). Our approach allowed us to specifically link genes to organisms and their genetic context, providing insight into WWTP impacts on natural microbial communities. Overall, our results suggest a substantial influence of wastewater effluent on gene content and microbial community structure in the sediments of receiving water bodies.IMPORTANCE Wastewater treatment plants (WWTPs) release their effluent into aquatic environments. Although treated, effluent retains many genes and microorganisms that have the potential to influence the receiving water in ways that are poorly understood. Here, we tracked the genetic footprint, including genes specific to antibiotic resistance and mobile genetic elements and their associated organisms, from WWTPs to lake sediments. Our work is novel in that we used metagenomic data sets to comprehensively evaluate total gene content and the genetic and taxonomic context of specific genes in environmental samples putatively impacted by WWTP inputs. Based on two different WWTPs with different treatment processes, our findings point to an influence of WWTPs on the presence, abundance, and composition of these factors in the environment.

Keywords: antibiotic resistance; freshwater; lakes; metagenomics; wastewater treatment.

FIG 1

Normalized abundance of genes found in Lake Michigan that are identical to those from WWTP effluent from Manitowoc (A) and Sheboygan (C), and the correlation between identical gene abundance with the distance from WWTPs (B and D). (E and F) Mean abundances of those genes from Manitowoc sites E) (all sites from panel A are combined as “M sites”) and from Sheboygan sites (F) (all sites from panel C are combined as “S sites”) are shown along with abundances from reference samples (LMS11, LMS18, and LMS41) for comparison. ND, not detected. Filled circles show the scale of gene abundance. Red stars mark the positions of the Manitowoc WWTP (MTP) and Sheboygan WWTP (STP). (Source: Esri.)

FIG 2

Normalized relative abundance (to mean coverage of 108 single-copy genes) of ARG categories in WWTP effluent and sediments (A) and of shared specific ARGs that appear in at least one sediment sample and effluent (B). ME and SE, effluent from Manitowoc and Sheboygan WWTPs, respectively; M1 to M14, Lake Michigan sites around Manitowoc WWTP; S2 to S6, sites around Sheboygan WWTP. The order of the sites from left to right corresponds to increasing distance from WWTPs. Locations of the sampling sites can be found in Fig. 1A and C.

FIG 3

Correlation between geographic distance and Jaccard dissimilarity index for shared ARG composition in sediments and Manitowoc WWTP effluent.

FIG 4

Percentage of mobile ARGs that are located on plasmids in total ARGs detected from sediments near the WWTP effluent outfall (sites M1 to M14 and S2 to S6) and reference sediment samples (LMS11, LMS18, and LMS41). The order of the sites from left to right corresponds to increasing distance from WWTPs.