Dissemination and persistence of antimicrobial resistance (AMR) along the wastewater-river continuum

Abstract

Antimicrobial resistance (AMR) is a global health hazard. Although clinical and agricultural environments are well-established contributors to the evolution and dissemination of AMR, research on wastewater treatment works (WwTWs) has highlighted their potential role as disseminators of AMR in freshwater environments. Using metagenomic sequencing and analysis, we investigated the changes in resistomes and associated mobile genetic elements within untreated wastewater influents and treated effluents of five WwTWs, and sediments collected from corresponding river environments in Oxfordshire, UK, across three seasonal periods within a year. Our analysis demonstrated a high diversity and abundance of antimicrobial resistance genes (ARGs) in untreated wastewater influents, reflecting the varied anthropogenic and environmental origins of wastewater. WwTWs effectively reduced AMR in the final effluent, with an average 87 % reduction in normalised ARG abundance and an average 63 % reduction in richness. However, wastewater effluents significantly impacted the antimicrobial resistome of the receiving rivers, with an average 543 % increase in ARG abundance and a 164 % increase in richness from upstream sediments to downstream sediments. The normalised abundance of the human gut-associated bacteriophage crAssphage was highly associated with both ARG abundance and richness. We observed seasonal variation in the resistome of raw influent which was not found in the effluent-receiving sediments. We illustrate the potential of WwTWs as focal points for disseminating ARGs and resistance-selecting chemicals, contributing to the elevation of environmental AMR. Our study emphasises the need for a comprehensive understanding of the anthropogenic impacts on AMR evolution and dissemination in wastewater and river environments, informing efforts to mitigate this growing public health crisis.

Keywords: Antimicrobial resistance; Resistome; River; Sediment; Wastewater.

Fig. 1

(A) Locations of the five wastewater treatment works, Oxford, Didcot, Witney, Wantage, and Watlington, within the River Thames, UK catchment. (B) Schematic of the sediment sampling sites at each location, showing representative locations of samples taken from the wastewater influent, wastewater effluent, and river sites upstream and downstream of the wastewater effluent point source.

Fig. 2. The relative abundance of AMR gene families was grouped according to the resistance categories.

Abbreviations: FCA = fluoroquinolone, quinolone, florfenicol, chloramphenicol, and amphenicol. MLSB = macrolide-lincosamide-streptogramin B. PhLOPSA = Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A, Other = grouped low abundance categories.

Fig. 3

Box plots showing the normalised abundance (genes per cell) of; (A) ARGs, (B) insertion sequences, (C) Enterobacterales plasmids, and the richness of; (D) ARGs, (E) insertion sequences, and (F) Enterobacterales plasmids, from upstream sediment, influent, effluent, and downstream sediment samples aggregated across sites. Non-metric multidimensional scaling plots showing the relationship between samples based on (G) composition of ARGs, (H) insertion sequences (ISs), and (I) Enterobacterales plasmids.

Fig. 4

Volcano plots showing differentially abundant: Antimicrobial resistance genes (ARGs) in; (A) untreated influent and treated effluent, and in (B) upstream and downstream sediments. Insertion Sequences (ISs) in; (C) untreated influent and treated effluent, and in (D) upstream and downstream sediments. Enterobacterales plasmids in; (E) untreated influent and treated effluent, and in (F) upstream and downstream sediments. The vertical dotted line represents a P-value of 0.001.

Fig. 5

(A) Source estimates of antimicrobial resistance genes (ARGs) from untreated wastewater assigned to each sediment sample from all river sites. For each site, data from three sampling time points are represented. The relationship between the normalised abundance of crAssphage against; (B) normalised ARG abundance and (C) normalised ARG richness, where the lines represent fitted generalised additive models (GAMs) across all sample types (influent, effluent, upstream and downstream sediments).

Fig. 6

Euler diagram showing the overlap of antimicrobial resistance genes (ARGs) between untreated wastewater (influent), treated wastewater effluent (effluent), and sediments downstream of the effluent entry point to the river, pooled across all sampling locations and time points. Circle packing plots show unique ARGs associated with each environmental compartment. FCA = fluoroquinolone, quinolone, florfenicol, chloramphenicol, and amphenicol. MLSB = macrolide-lincosamide-streptogramin B.

Fig. 7

Non-metric multidimensional scaling (NMDS) plots show seasonal differences in the resistome of (A) untreated wastewater influent, (B) treated effluent, and (C) river sediment. UpSet plots show the number of shared ARGs among (D) untreated wastewater influent, (E) treated effluent, and (F) river sediment samples.