<i>intI</i>1 primer selection for class 1 integron integrase gene and transcript quantification – validation and application for monitoring<i>intl</i>1 gene abundance within septic tanks in Thailand-Reference-Cited by-同舟云学术

intI1 primer selection for class 1 integron integrase gene and transcript quantification – validation and application for monitoringintl1 gene abundance within septic tanks in Thailand

Published:2023-06-19 Issue: Volume: Page:
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Okonkwo Valentine^ORCID,Cholet Fabien^ORCID,Ijaz Umer Z.^ORCID,Koottatep Thammarat,Pussayanavin Tatchai,Polpraset Chongrak,Sloan William T.^ORCID,Connelly Stephanie^ORCID,Smith Cindy J.^ORCID

Abstract

AbstractAntimicrobial resistance (AMR) poses serious global public health threat and wastewater treatment (WWT), including septic tanks, are a significant source of AMR genes to the environment. Environmental monitoring of broad-range AMR genes remains a challenge. The class-1 integron-integrase (intI1) gene has been proposed as a proxy for overall AMR abundance, but there is no consensus on the qPCR primer set to use. A systematic review of the literature found 65 primer sets. The coverage and specificity of each, including newly designed MGB-TaqMan primer-probe, was evaluated against a database of clinical and environmentalintl1, intl1-like and non-intl1 sequences. Three primer sets were selected, laboratory validated for DNA and mRNA quantification and used to quantityintl1 gene abundance from household and healthcare conventional septic tanks (CST) and novel household Solar Septic Tanks (SST) in Thailand. Specificity of theintl1septic tank amplicons showed that no primer set could distinguish betweenintl1andintl-1like sequences. Each primer set showed the same trends across septic tanks, with highest gene abundance in influent>sludge>effluent. There was no statistical difference between the same sample quantified by the three primer sets. However, when comparing gene abundances from the same primer set across septic tanks, statistical differences between different sample types were observed for one primer set but not the others. This may lead to different interpretation of risk associated with each reactor in spreadingintl1 to the environment. Comparing reactor types, intI1 abundance in the effluent was lowest in the SST-household<CST-household<CST-healthcare. Depending on primer set used, 31 to 42% ofintI1 was removed from effluent of the CST-household tank with accessible influent. Our study provided insight into the importance ofintl1primer choice. We propose the use of the validated set (F3-R3) for optimalintI1 quantification and towards the goal of achieving standardisation across environmental studies.

Publisher

Cold Spring Harbor Laboratory

Reference55 articles.

1. Amos, G. C. A. , Ploumakis, S. , Zhang, L. , Hawkey, P. M. , Gaze, W. H. and Wellington, E. M. H . (2018) ‘The widespread dissemination of integrons throughout bacterial communities in a riverine system.’ ISME Journal, 12(3).

2. Barraud, O. , Baclet, M. C. , Denis, F. and Ploy, M. C . (2010) ‘Quantitative multiplex real-time PCR for detecting class 1, 2 and 3 integrons.’ Journal of Antimicrobial Chemotherapy, 65(8).

3. Berglund, B. , Fick, J. and Lindgren, P. E . (2015) ‘Urban wastewater effluent increases antibiotic resistance gene concentrations in a receiving northern European river.’ Environmental Toxicology and Chemistry, 34(1).

4. Bolyen, E. , Rideout, J. R. , Dillon, M. R. , Bokulich, N. A. , Abnet, C. C. , Al-Ghalith, G. A. , Alexander, H. , Alm, E. J. , Arumugam, M. , Asnicar, F. , Bai, Y. , Bisanz, J. E. , Bittinger, K. , Brejnrod, A. , Brislawn, C. J. , Brown, C. T. , Callahan, B. J. , Caraballo-Rodríguez, A. M. , Chase, J. , Cope, E. K. , Da Silva, R. , Diener, C. , Dorrestein, P. C. , Douglas, G. M. , Durall, D. M. , Duvallet, C. , Edwardson, C. F. , Ernst, M. , Estaki, M. , Fouquier, J. , Gauglitz, J. M. , Gibbons, S. M. , Gibson, D. L. , Gonzalez, A. , Gorlick, K. , Guo, J. , Hillmann, B. , Holmes, S. , Holste, H. , Huttenhower, C. , Huttley, G. A. , Janssen, S. , Jarmusch, A. K. , Jiang, L. , Kaehler, B. D. , Kang, K. Bin , Keefe, C. R. , Keim, P. , Kelley, S. T. , Knights, D. , Koester, I. , Kosciolek, T. , Kreps, J. , Langille, M. G. I. , Lee, J. , Ley, R. , Liu, Y. X. , Loftfield, E. , Lozupone, C. , Maher, M. , Marotz, C. , Martin, B. D. , McDonald, D ., McIver, L. J. , Melnik, A. V. , Metcalf, J. L. , Morgan, S. C. , Morton, J. T. , Naimey, A. T. , Navas-Molina, J. A. , Nothias, L. F. , Orchanian, S. B. , Pearson, T. , Peoples, S. L. , Petras, D. , Preuss, M. L. , Pruesse, E. , Rasmussen, L. B. , Rivers, A. , Robeson, M. S. , Rosenthal, P. , Segata, N. , Shaffer, M. , Shiffer, A. , Sinha, R. , Song, S. J. , Spear, J. R. , Swafford, A. D. , Thompson, L. R. , Torres, P. J. , Trinh, P. , Tripathi, A. , Turnbaugh, P. J. , Ul-Hasan, S. , van der Hooft, J. J. J. , Vargas, F. , Vázquez-Baeza, Y. , Vogtmann, E. , von Hippel, M. , Walters, W. , Wan, Y. , Wang, M. , Warren, J. , Weber, K. C. , Williamson, C. H. D. , Willis, A. D. , Xu, Z. Z. , Zaneveld, J. R. , Zhang, Y. , Zhu, Q. , Knight, R. and Caporaso, J. G. (2019) ‘Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2.’ Nature Biotechnology.

5. Bourlat, S. J. , Haenel, Q. , Finnman, J. and Leray, M. (2016) ‘Preparation of amplicon libraries for metabarcoding of marine eukaryotes using illumina MiSeq: The dual-PCR method.’ In Methods in Molecular Biology.