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      Highly sensitive detection of antimicrobial resistance genes in hospital wastewater using the multiplex hybrid capture target enrichment

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          ABSTRACT

          Wastewater can be useful in monitoring the spread of antimicrobial resistance (AMR) within a hospital. The abundance of antibiotic resistance genes (ARGs) in hospital effluent was assessed using metagenomic sequencing (mDNA-seq) and hybrid capture (xHYB). mDNA-seq analysis and subsequent xHYB targeted enrichment were conducted on two effluent samples per month from November 2018 to May 2021. Reads per kilobase per million (RPKM) values were calculated for all 1,272 ARGs in the constructed database. The monthly numbers of patients with presumed extended-spectrum β-lactamase (ESBL)-producing and metallo-β-lactamase (MBL)-producing bacteria, methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant enterococci (VRE) were compared with the monthly RPKM values of bla CTX-M, bla IMP, mecA, vanA, and vanB by xHYB. The average RPKM value for all ARGs detected by xHYB was significantly higher than that of mDNA-seq (665, 225, and 328, respectively, and P < 0.05). The average number of patients with ESBL producers and RPKM values of bla CTX-M-1 genes in 2020 were significantly higher than that in 2019 (17 and 13 patients per month and 921 vs 232 per month, respectively, both P < 0.05). The average numbers of patients with MBL-producers, MRSA, and VRE were 1, 28, and 0 per month, respectively, while the average RPKM values of bla IMP, mecA, vanA, and vanB were 6,163, 6, 0, and 126 per month, respectively. Monitoring ARGs in hospital effluent using xHYB was found to be more useful than conventional mDNA-seq in detecting ARGs including bla CTX-M, bla IMP, and vanB, which are important for infection control.

          IMPORTANCE

          Environmental ARGs play a crucial role in the emergence and spread of AMR that constitutes a significant global health threat. One major source of ARGs is effluent from healthcare facilities, where patients are frequently administered antimicrobials. Culture-independent methods, including metagenomics, can detect environmental ARGs carried by non-culturable bacteria and extracellular ARGs. mDNA-seq is one of the most comprehensive methods for environmental ARG surveillance; however, its sensitivity is insufficient for wastewater surveillance. This study demonstrates that xHYB appropriately monitors ARGs in hospital effluent for sensitive identification of nosocomial AMR dissemination. Correlations were observed between the numbers of inpatients with antibiotic-resistant bacteria and the ARG RPKM values in hospital effluent over time. ARG surveillance in hospital effluent using the highly sensitive and specific xHYB method could improve our understanding of the emergence and spread of AMR within a hospital.

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          Most cited references29

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          VSEARCH: a versatile open source tool for metagenomics

          Background VSEARCH is an open source and free of charge multithreaded 64-bit tool for processing and preparing metagenomics, genomics and population genomics nucleotide sequence data. It is designed as an alternative to the widely used USEARCH tool (Edgar, 2010) for which the source code is not publicly available, algorithm details are only rudimentarily described, and only a memory-confined 32-bit version is freely available for academic use. Methods When searching nucleotide sequences, VSEARCH uses a fast heuristic based on words shared by the query and target sequences in order to quickly identify similar sequences, a similar strategy is probably used in USEARCH. VSEARCH then performs optimal global sequence alignment of the query against potential target sequences, using full dynamic programming instead of the seed-and-extend heuristic used by USEARCH. Pairwise alignments are computed in parallel using vectorisation and multiple threads. Results VSEARCH includes most commands for analysing nucleotide sequences available in USEARCH version 7 and several of those available in USEARCH version 8, including searching (exact or based on global alignment), clustering by similarity (using length pre-sorting, abundance pre-sorting or a user-defined order), chimera detection (reference-based or de novo), dereplication (full length or prefix), pairwise alignment, reverse complementation, sorting, and subsampling. VSEARCH also includes commands for FASTQ file processing, i.e., format detection, filtering, read quality statistics, and merging of paired reads. Furthermore, VSEARCH extends functionality with several new commands and improvements, including shuffling, rereplication, masking of low-complexity sequences with the well-known DUST algorithm, a choice among different similarity definitions, and FASTQ file format conversion. VSEARCH is here shown to be more accurate than USEARCH when performing searching, clustering, chimera detection and subsampling, while on a par with USEARCH for paired-ends read merging. VSEARCH is slower than USEARCH when performing clustering and chimera detection, but significantly faster when performing paired-end reads merging and dereplication. VSEARCH is available at https://github.com/torognes/vsearch under either the BSD 2-clause license or the GNU General Public License version 3.0. Discussion VSEARCH has been shown to be a fast, accurate and full-fledged alternative to USEARCH. A free and open-source versatile tool for sequence analysis is now available to the metagenomics community.
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            Tackling antibiotic resistance: the environmental framework.

            Antibiotic resistance is a threat to human and animal health worldwide, and key measures are required to reduce the risks posed by antibiotic resistance genes that occur in the environment. These measures include the identification of critical points of control, the development of reliable surveillance and risk assessment procedures, and the implementation of technological solutions that can prevent environmental contamination with antibiotic resistant bacteria and genes. In this Opinion article, we discuss the main knowledge gaps, the future research needs and the policy and management options that should be prioritized to tackle antibiotic resistance in the environment.
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              Global epidemiology of CTX-M β-lactamases: temporal and geographical shifts in genotype.

              Globally, rates of ESBL-producing Enterobacteriaceae are rising. We undertook a literature review, and present the temporal trends in blaCTX-M epidemiology, showing that blaCTX-M-15 and blaCTX-M-14 have displaced other genotypes in many parts of the world. Explanations for these changes can be attributed to: (i) horizontal gene transfer (HGT) of plasmids; (ii) successful Escherichia coli clones; (iii) ESBLs in food animals; (iv) the natural environment; and (v) human migration and access to basic sanitation. We also provide explanations for the changing epidemiology of blaCTX-M-2 and blaCTX-M-27. Modifiable anthropogenic factors, such as poor access to basic sanitary facilities, encourage the spread of blaCTX-M and other antimicrobial resistance (AMR) genes, such as blaNDM, blaKPC and mcr-1. We provide further justification for novel preventative and interventional strategies to reduce transmission of these AMR genes.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: InvestigationRole: MethodologyRole: Writing – original draft
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – review and editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: Methodology
                Role: ConceptualizationRole: Funding acquisitionRole: MethodologyRole: SupervisionRole: Writing – review and editing
                Role: Editor
                Journal
                mSphere
                mSphere
                mSphere
                mSphere
                American Society for Microbiology (1752 N St., N.W., Washington, DC )
                2379-5042
                24 May 2023
                Jul-Aug 2023
                24 May 2023
                : 8
                : 4
                : e00100-23
                Affiliations
                [1 ]Department of Infectious Diseases, Internal Medicine, Tohoku University Graduate School of Medicine; , Sendai, Miyagi, Japan
                [2 ]Pathogen Genomics Center, National Institute of Infectious Diseases; , Tokyo, Japan
                JMI Laboratories, North Liberty; , Iowa, USA
                Author notes
                Address correspondence to Hiroaki Baba, hbaba48@ 123456med.tohoku.ac.jp
                Address correspondence to Hajime Kanamori, kanamori@ 123456med.tohoku.ac.jp

                The authors declare no conflict of interest.

                Author information
                https://orcid.org/0000-0001-5487-6698
                Article
                00100-23 msphere.00100-23
                10.1128/msphere.00100-23
                10449491
                37222510
                99783b06-ef8d-4726-919a-5a417ba54ad8
                Copyright © 2023 Baba et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

                History
                : 27 February 2023
                : 18 April 2023
                Page count
                supplementary-material: 2, authors: 4, Figures: 3, Tables: 1, Equations: 1, References: 31, Pages: 12, Words: 6229
                Funding
                Funded by: The Ministry of Health, Labour and Welfare, Japan;
                Award ID: H30 Shinkogyosei-Ippan-002 and 21HA1002
                Award Recipient :
                Categories
                Research Article
                clinical-microbiology, Clinical Microbiology
                Custom metadata
                July/August 2023

                antimicrobial resistance,antibiotic resistant genes,metagenomics,water environment,hospital wastewater,one health

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