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      Contributions and Challenges of High Throughput qPCR for Determining Antimicrobial Resistance in the Environment: A Critical Review

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          Abstract

          Expansion in whole genome sequencing and subsequent increase in antibiotic resistance targets have paved the way of high throughput qPCR (HT-qPCR) for analyzing hundreds of antimicrobial resistance genes (ARGs) in a single run. A meta-analysis of 51 selected studies is performed to evaluate ARGs abundance trends over the last 7 years. WaferGen TM SmartChip is found to be the most widely used HT-qPCR platform among others for evaluating ARGs. Up till now around 1000 environmental samples (excluding biological replicates) from different parts of the world have been analyzed on HT-qPCR. Calculated detection frequency and normalized ARGs abundance (ARGs/16S rRNA gene) reported in gut microbiome studies have shown a trend of low ARGs as compared to other environmental matrices. Disparities in the HT-qPCR data analysis which are causing difficulties to researchers in precise interpretation of results have been highlighted and a possible way forward for resolving them is also suggested. The potential of other amplification technologies and point of care or field deployable devices for analyzing ARGs have also been discussed in the review. Our review has focused on updated information regarding the role, current status and future perspectives of HT-qPCR in the field of antimicrobial resistance.

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          Most cited references82

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          Antibiotic resistance genes as emerging contaminants: studies in northern Colorado.

          This study explores antibiotic resistance genes (ARGs) as emerging environmental contaminants. The purpose of this study was to investigate the occurrence of ARGs in various environmental compartments in northern Colorado, including Cache La Poudre (Poudre) River sediments, irrigation ditches, dairy lagoons, and the effluents of wastewater recycling and drinking water treatment plants. Additionally, ARG concentrations in the Poudre River sediments were analyzed at three time points at five sites with varying levels of urban/agricultural impact and compared with two previously published time points. It was expected that ARG concentrations would be significantly higher in environments directly impacted by urban/agricultural activity than in pristine and lesser-impacted environments. Polymerase chain reaction (PCR) detection assays were applied to detect the presence/absence of several tetracycline and sulfonamide ARGs. Quantitative real-time PCR was used to further quantify two tetracycline ARGs (tet(W) and tet(O)) and two sulfonamide ARGs (sul(I) and sul(II)). The following trend was observed with respect to ARG concentrations (normalized to eubacterial 16S rRNA genes): dairy lagoon water > irrigation ditch water > urban/agriculturally impacted river sediments (p < 0.0001), except for sul(II), which was absent in ditch water. It was noted that tet(W) and tet(O) were also present in treated drinking water and recycled wastewater, suggesting that these are potential pathways for the spread of ARGs to and from humans. On the basis of this study, there is a need for environmental scientists and engineers to help address the issue of the spread of ARGs in the environment.
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            Using the class 1 integron-integrase gene as a proxy for anthropogenic pollution

            Around all human activity, there are zones of pollution with pesticides, heavy metals, pharmaceuticals, personal care products and the microorganisms associated with human waste streams and agriculture. This diversity of pollutants, whose concentration varies spatially and temporally, is a major challenge for monitoring. Here, we suggest that the relative abundance of the clinical class 1 integron-integrase gene, intI1, is a good proxy for pollution because: (1) intI1 is linked to genes conferring resistance to antibiotics, disinfectants and heavy metals; (2) it is found in a wide variety of pathogenic and nonpathogenic bacteria; (3) its abundance can change rapidly because its host cells can have rapid generation times and it can move between bacteria by horizontal gene transfer; and (4) a single DNA sequence variant of intI1 is now found on a wide diversity of xenogenetic elements, these being complex mosaic DNA elements fixed through the agency of human selection. Here we review the literature examining the relationship between anthropogenic impacts and the abundance of intI1, and outline an approach by which intI1 could serve as a proxy for anthropogenic pollution.
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              rrndb: the Ribosomal RNA Operon Copy Number Database.

              The Ribosomal RNA Operon Copy Number Database (rrndb) is an Internet-accessible database containing annotated information on rRNA operon copy number among prokaryotes. Gene redundancy is uncommon in prokaryotic genomes, yet the rRNA genes can vary from one to as many as 15 copies. Despite the widespread use of 16S rRNA gene sequences for identification of prokaryotes, information on the number and sequence of individual rRNA genes in a genome is not readily accessible. In an attempt to understand the evolutionary implications of rRNA operon redundancy, we have created a phylogenetically arranged report on rRNA gene copy number for a diverse collection of prokaryotic microorganisms. Each entry (organism) in the rrndb contains detailed information linked directly to external websites including the Ribosomal Database Project, GenBank, PubMed and several culture collections. Data contained in the rrndb will be valuable to researchers investigating microbial ecology and evolution using 16S rRNA gene sequences. The rrndb web site is directly accessible on the WWW at http://rrndb.cme. msu.edu.
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                Author and article information

                Contributors
                Role: Academic Editor
                Role: Academic Editor
                Journal
                Molecules
                Molecules
                molecules
                Molecules
                MDPI
                1420-3049
                03 January 2019
                January 2019
                : 24
                : 1
                : 163
                Affiliations
                [1 ]Department of Environmental Engineering, Michigan State University, East Lansing, MI 48823, USA; waseemh1@ 123456msu.edu
                [2 ]Department of Biotechnology, University of Sialkot, Punjab 51310, Pakistan; jameels@ 123456msu.edu (S.J.); h.s.rehman15@ 123456gmail.com (H.S.U.R.)
                [3 ]Environmental Microbiology Laboratory, Department of Microbiology, Quaid-i-Azam University, Islamabad 45320, Pakistan; asifjamall@ 123456yahoo.com
                [4 ]key Laboratory of Environmental Nanotechnology and Health Effects, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, 18 Shuangqing Road, Beijing 100085, China; Jafarali_st@ 123456rcees.ac.cn
                [5 ]Department of Microbiology, University of Hazara, Mansehra 21300, Pakistan; isfahantauseef@ 123456yahoo.com
                [6 ]Department of Plant Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan; uzma.ishi@ 123456gmail.com
                Author notes
                [* ]Correspondence: ishimrl@ 123456qau.edu.pk ; Tel.: +92-51-906-43196
                Author information
                https://orcid.org/0000-0003-3246-1592
                Article
                molecules-24-00163
                10.3390/molecules24010163
                6337382
                30609875
                283c22a2-9025-4332-a1b5-c2cd85c78beb
                © 2019 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 12 December 2018
                : 29 December 2018
                Categories
                Review

                amr,high throughput qpcr,args,mges,gut microbiome
                amr, high throughput qpcr, args, mges, gut microbiome

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