TY - JOUR
T1 - Inter-laboratory calibration of quantitative analyses of antibiotic resistance genes
AU - Rocha, Jaqueline
AU - Cacace, Damiano
AU - Kampouris, Ioannis
AU - Guilloteau, Hélène
AU - Jäger, Thomas
AU - Marano, Roberto B. M.
AU - Karaolia, Popi
AU - Manaia, Célia M.
AU - Merlin, Christophe
AU - Fatta-Kassinos, Despo
AU - Cytryn, Eddie
AU - Berendonk, Thomas U.
AU - Schwartz, Thomas
N1 - Funding Information:
This work was supported by Working Group 5: Wastewater Reuse and Contaminants of Emerging Concern from Norman activities ( http://www.norman-network.net/?q=node/106 ). UCP was supported by the Portuguese National Funds from FCT – Fundação para a Ciência e a Tecnologia STARE – “Stopping Antibiotic Resistance Evolution” (WaterJPI/0001/2013). Jaqueline Rocha was supported by the International PhD Programme in Biotechnology – BIOTECH. DOC (NORTE-08-5369-FSE-000007). LCPME was supported by the QualiOrne project funded by the French “Agence de l’Eau Rhin-Meuse”. ARO was partially supported by the Israel Ministry of Agriculture Chief Scientist (grant # 821-0000-13). Roberto Marano is part of the ANSWER project that has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Grant Agreement No 675530.
Funding Information:
The authors wish to warmly acknowledge the support of NORMAN network, in particular, Valeria Dulio and Jaroslav Slobodnik. The authors would like to acknowledge the COST Action ES1403 NEREUS “New and emerging challenges and opportunities in wastewater reuse”, supported by COST (European Cooperation in Science and Technology) for enabling the collaboration among the authors of the paper.
Publisher Copyright:
© 2018 Elsevier Ltd.
PY - 2020/2
Y1 - 2020/2
N2 - Antibiotic 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.
AB - Antibiotic 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.
KW - Antibiotic resistance gene
KW - Inter-laboratory calibration
KW - Quantitative PCR
KW - Wastewater
UR - http://www.scopus.com/inward/record.url?scp=85042650412&partnerID=8YFLogxK
U2 - 10.1016/j.jece.2018.02.022
DO - 10.1016/j.jece.2018.02.022
M3 - Article
AN - SCOPUS:85042650412
SN - 2213-3437
VL - 8
SP - 1
EP - 6
JO - Journal of Environmental Chemical Engineering
JF - Journal of Environmental Chemical Engineering
IS - 1
M1 - 102214
ER -