High-resolution characterisation of short-term temporal variability in the taxonomic and resistome composition of wastewater influent

Abstract Wastewater-based epidemiology (WBE) for population-level surveillance of antimicrobial resistance (AMR) is gaining significant traction, but the impact of wastewater sampling methods on results is unclear. In this study we characterised taxonomic and resistome differences between single-timepoint-grab and 24H-composites of wastewater influent from a large UK-based wastewater treatment work (WWTW [population equivalent:223,435]). We autosampled hourly influent grab samples (n=72) over three consecutive weekdays, and prepared additional 24H-composites (n=3) from respective grabs. For taxonomic profiling, metagenomic DNA was extracted from all samples and 16S-rRNA gene sequenced. One composite and six grabs from day one underwent metagenomic sequencing for metagenomic dissimilarity estimation and resistome profiling. Taxonomic abundances of phyla varied significantly across hourly grab samples but followed a repeating diurnal pattern for all three days. Hierarchical clustering grouped grab samples into four time periods dissimilar in both 16S rRNA gene-based profiles and metagenomic distances. 24H-composites resembled mean daily phyla abundances and showed low variability of taxonomic profiles. Of the 122 AMR gene families (AGFs) identified across all day one samples, single grab samples identified a median of 6 (IQR:5-8) AGFs not seen in the composite. However, 36/36 of these hits were at lateral coverage <0.5 (median:0.19; IQR:0.16-0.22) and potential false positives. Conversely, the 24H-composite identified three AGFs not seen in any grab with higher lateral coverage (0.82; 0.55-0.84). Additionally, several clinically significant human AGFs (bla VIM , bla IMP , bla KPC ) were intermittently or completely missed by grab sampling but captured by the 24H-composite. Wastewater influent undergoes significant taxonomic and resistome changes on short timescales potentially affecting interpretation of results based on sampling strategy. Grab samples are more convenient and potentially capture low-prevalence/transient targets but are less comprehensive and temporally variable. Therefore, we recommend 24H-composite sampling where feasible. Further validation and optimisation of WBE methods is vital for its development into a robust AMR surveillance approach. Highlights Influent undergoes significant taxonomic/resistome changes over short timescales. Taxonomic abundances fluctuate diurnally but repeat for the 3 weekdays sampled. Detection of less prevalent AMR determinants is time-dependent for grab sampling. Single timepoint grab samples may produce temporally variable metagenomic profiles. 24H-composites reflect mean daily taxa and more reliably captured AMR determinants.