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- Inter-laboratory calibration of quantitative analyses of antibiotic resistance genesPublication . Rocha, Jaqueline; Cacace, Damiano; Kampouris, Ioannis; Guilloteau, Hélène; Jäger, Thomas; Marano, Roberto B.M.; Karaolia, Popi; Manaia, Célia M.; Merlin, Christophe; Fatta-Kassinos, Despo; Cytryn, Eddie; Berendonk, Thomas U.; Schwartz, ThomasAntibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are widely distributed in the environment where they represent potential public health threats. Quantitative PCR (qPCR) is a suitable approach to detect and quantify ARGs in environmental samples. However, the comparison of gene quantification data between different laboratories is challenging since the data are predominantly obtained under non-harmonized conditions, using different qPCR protocols. This study aimed at carrying out an inter-laboratory calibration in order to assess the variability inherent to the qPCR procedures for quantification of ARGs. With this aim, samples of treated wastewater collected in three different countries were analysed based on common DNA extract pools and identical protocols as well as distinct equipment, reagents batches, and operators. The genes analysed were the 16S rRNA, vanA, blaTEM, qnrS, sul1, blaCTXM-32 and intI1 and the artificial pNORM1 plasmid containing fragments from the seven targeted genes was used as a reference. The 16S rRNA gene was the most abundant, in all the analysed samples, followed by intI1, sul1, qnrS, and blaTEM, while blaCTXM-32 and vanA were below the limit of quantification in most or all the samples. For the genes 16S rRNA, sul1, intI1, blaTEM and qnrS the inter-laboratory variation was below 28% (3–8%, 6–18%, 8–21%, 10–24%, 15–28%, respectively). While it may be difficult to fully harmonize qPCR protocols due to equipment, reagents and operator variations, the inter-laboratory calibration is an adequate and necessary step to increase the reliability of comparative data on ARGs abundance in different environmental compartments and/or geographic regions.
- Performance of secondary wastewater treatment methods for the removal of contaminants of emerging concern implicated in crop uptake and antibiotic resistance spread: a reviewPublication . Krzeminski, Pawel; Tomei, Maria Concetta; Karaolia, Popi; Langenhoff, Alette; Almeida, C. Marisa R.; Felis, Ewa; Gritten, Fanny; Andersen, Henrik Rasmus; Fernandes, Telma; Manaia, Célia M.; Rizzo, Luigi; Fatta-Kassinos, DespoContaminants of emerging concern (CEC) discharged in effluents of wastewater treatment plants (WWTPs), not specifically designed for their removal, pose serious hazards to human health and ecosystems. Their impact is of particular relevance to wastewater disposal and re-use in agricultural settings due to CEC uptake and accumulation in food crops and consequent diffusion into the food-chain. This is the reason why the chemical CEC discussed in this review have been selected considering, besides recalcitrance, frequency of detection and entity of potential hazards, their relevance for crop uptake. Antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) have been included as microbial CEC because of the potential of secondary wastewater treatment to offer conditions favourable to the survival and proliferation of ARB, and dissemination of ARGs. Given the adverse effects of chemical and microbial CEC, their removal is being considered as an additional design criterion, which highlights the necessity of upgrading conventional WWTPs with more effective technologies. In this review, the performance of currently applied biological treatment methods for secondary treatment is analysed. To this end, technological solutions including conventional activated sludge (CAS), membrane bioreactors (MBRs), moving bed biofilm reactors (MBBRs), and nature-based solutions such as constructed wetlands (CWs) are compared for the achievable removal efficiencies of the selected CEC and their potential of acting as reservoirs of ARB&ARGs. With the aim of giving a picture of real systems, this review focuses on data from full-scale and pilot-scale plants treating real urban wastewater. To achieve an integrated assessment, technologies are compared considering also other relevant evaluation parameters such as investment and management costs, complexity of layout and management, present scale of application and need of a post-treatment. Comparison results allow the definition of design and operation strategies for the implementation of CEC removal in WWTPs, when agricultural reuse of effluents is planned.
- Antibiotic resistance genes in treated wastewater and in the receiving water bodies: a pan-European survey of urban settingsPublication . Cacace, Damiano; Fatta-Kassinos, Despo; Manaia, Célia M.; Cytryn, Eddie; Kreuzinger, Norbert; Rizzo, Luigi; Karaolia, Popi; Schwartz, Thomas; Alexander, Johannes; Merlin, Christophe; Garelick, Hemda; Schmitt, Heike; Vries, Daisy de; Schwermer, Carsten U.; Meric, Sureyya; Ozkal, Can Burak; Pons, Marie-Noelle; Kneis, David; Berendonk, Thomas U.There is increasing public concern regarding the fate of antibiotic resistance genes (ARGs) during wastewater treatment, their persistence during the treatment process and their potential impacts on the receiving water bodies. In this study, we used quantitative PCR (qPCR) to determine the abundance of nine ARGs and a class 1 integron associated integrase gene in 16 wastewater treatment plant (WWTP) effluents from ten different European countries. In order to assess the impact on the receiving water bodies, gene abundances in the latter were also analysed. Six out of the nine ARGs analysed were detected in all effluent and river water samples. Among the quantified genes, intI1 and sul1 were the most abundant. Our results demonstrate that European WWTP contribute to the enrichment of the resistome in the receiving water bodies with the particular impact being dependent on the effluent load and local hydrological conditions. The ARGs concentrations in WWTP effluents were found to be inversely correlated to the number of implemented biological treatment steps, indicating a possible option for WWTP management. Furthermore, this study has identified blaOXA-58 as a possible resistance gene for future studies investigating the impact of WWTPs on their receiving water.