Wastewater is among the most important reservoirs of antibiotic resistance in urban environments. The abundance of carbon sources and other nutrients, a variety of possible electron acceptors such as oxygen or nitrate, the presence of particles onto which bacteria can adsorb, or a fairly stable pH and temperature are examples of conditions favouring the remarkable diversity of microorganisms in this peculiar habitat. The wastewater microbiome brings together bacteria of environmental, human and animal origins, many harbouring antibiotic resistance genes (ARGs). Although numerous factors contribute, mostly in a complex interplay, for shaping this microbiome, the effect of specific potential selective pressures such as antimicrobial residues or metals, is supposedly determinant to dictate the fate of antibiotic resistant bacteria (ARB) and ARGs during wastewater treatment. This paper aims to enrich the discussion on the ecology of ARB&ARGs in urban wastewater treatment plants (UWTPs), intending to serve as a guide for wastewater engineers or other professionals, who may be interested in studying or optimizing the wastewater treatment for the removal of ARB&ARGs. Fitting this aim, the paper overviews and discusses: i) aspects of the complexity of the wastewater system and/or treatment that may affect the fate of ARB&ARGs; ii) methods that can be used to explore the resistome, meaning the whole ARB&ARGs, in wastewater habitats; and iii) some frequently asked questions for which are proposed addressing modes. The paper aims at contributing to explore how ARB&ARGs behave in UWTPs having in mind that each plant is a unique system that will probably need a specific procedure to maximize ARB&ARGs removal.

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Wastewater (WW) reuse is expected to be increasingly indispensable in future water management to mitigate water scarcity. However, this increases the risk of antibiotic resistance (AR) dissemination via irrigation. Herein, a conventional (chlorination) and an advanced oxidation process (heterogeneous photocatalysis (HPC)) were used to disinfect urban WW to the same target of Escherichia coli <10 CFU/100 mL and used to irrigate lettuce plants ( Lactuca sativa ) set up in four groups, each receiving one of four water types, secondary WW (positive control), fresh water (negative control), chlorinated WW, and HPC WW. Four genes were monitored in water and soil, 16S rRNA as an indicator of total bacterial load, intI1 as a gene commonly associated with anthropogenic activity and AR, and two AR genes bla OXA-10 and qnrS . Irrigation with secondary WW resulted in higher dry soil levels of intI1 (from 1.4 × 10 4 copies/g before irrigation to 3.3 × 10 5 copies/g after). HPC-treated wastewater showed higher copy numbers of intI1 in the irrigated soil than chlorination, but the opposite was true for bla OXA-10 . The results indicate that the current treatment is insufficient to prevent dissemination of AR markers and that HPC does not offer a clear advantage over chlorination.

Antibiotic resistant bacteria and antibiotic resistance genes (ARGs) are major human-health threats, widely distributed in the environment. Quantitative PCR (qPCR) is a standard approach to detect and quantify ARGs in environmental compartments. However, the comparison of gene quantification reported by different laboratories is challenging since data are predominantly obtained under nonharmonized conditions, using different qPCR protocols. Objectives The aim of this study was to develop and calibrate standardized qPCR procedures for quantification of key ARGs, analyzing the same samples with common protocols and distinct equipment, reagents batches and operators. Methods Treated wastewater from three European countries were processed immediately after collection and transported to the laboratory for total DNA extraction. DNA extracts from each sample were pooled and aliquots were distributed by five partners involved in the calibration procedure. The genes 16S rRNA, vanA, blaTEM, qnrS, sul1, blaCTXM-32 and intI1 were analyzed using harmonized qPCR protocols and the constructed pNORM1 plasmid, which contains fragments of the seven targeted genes, was used for generating standard curves. Conclusions The 16S rRNA gene was the most abundant, followed by sul1, intI1, qnrS and blaTEM. Quantifications made by different partners were reproducible and inter-laboratory variation was < 20%. The notorious exception was for the qnrS gene, and therefore protocol improvement is recommended. The genes blaCTXM-32 and vanA were below the limit of quantification in most or all of the samples analyzed. The inter-laboratory calibration is an adequate approach to reliably assess ARG abundance and environmental contamination in different environments and geographic locations.

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Treated-wastewater (TW) irrigation transfers antibiotic-resistant bacteria (ARB) to soil, but persistence of these bacteria is generally low due to resilience of the soil microbiome. Nonetheless, wastewater-derived bacteria and associated antibiotic resistance genes (ARGs) may persist below detection levels and potentially proliferate under copiotrophic conditions. To test this hypothesis, we exposed soils from microcosm, lysimeter, and field experiments to short-term enrichment in copiotroph-stimulating media. In microcosms, enrichment stimulated growth of multidrug-resistant Escherichia coli up to 2 weeks after falling below detection limits. Lysimeter and orchard soils irrigated in-tandem with either freshwater or TW were subjected to culture-based, qPCR and shotgun metagenomic analyses prior, and subsequent, to enrichment. Although native TW- and freshwater-irrigated soil microbiomes and resistomes were similar to each other, enrichment resulted in higher abundances of cephalosporin- and carbapenem-resistant Enterobacteriaceae and in substantial differences in the composition of microbial communities and ARGs. Enrichment stimulated ARG-harboring Bacillaceae in the freshwater-irrigated soils, whereas in TWW-irrigated soils, ARG-harboring γ-proteobacterial families Enterobacteriaceae and Moraxellaceae were more profuse. We demonstrate that TW-derived ARB and associated ARGs can persist at below detection levels in irrigated soils and believe that similar short-term enrichment strategies can be applied for environmental antimicrobial risk assessment in the future.

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