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      Genomic Investigation of Salmonella Isolates Recovered From a Pig Slaughtering Process in Hangzhou, China

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          Abstract

          The pig industry is the principal source of meat products in China, and the presence of pathogens in pig-borne meat is a crucial threat to public health. Salmonella is the major pathogen associated with pig-borne diseases. However, route surveillance by genomic platforms along the food chain is still limited in China. Here, we conducted a study to evaluate the dynamic prevalence of Salmonella in a pig slaughtering process in Hangzhou, Zhejiang Province, China. Fifty-five of 226 (24.37%) samples were positive for Salmonella; from them, 78 different isolates were selected and subjected to whole genome sequencing followed by bioinformatics analyses to determine serovar distribution, MLST patterns, antimicrobial resistance genes, plasmid replicons, and virulence factors. Moreover, phenotypic antimicrobial resistance was performed using the broth dilution method against 14 antimicrobial agents belonging to 10 antimicrobial classes. Our results showed that samples collected from the dehairing area (66.66%) and the splitting area (57.14%) were the most contaminated. Phenotypic antimicrobial resistance classified 67 of 78 isolates (85.90%) as having multidrug resistance (MDR), while the highest resistance was observed in tetracycline (85.90%; 67/78) followed by ampicillin (84.62%; 66/78), chloramphenicol (71.80%; 56/78), and nalidixic acid (61.54%; 48/78). Additionally, serovar prediction showed the dominance of Salmonella Typhimurium ST19 (51.28%; 40/78) among the 78 studied isolates, while plasmid prediction reported the dominance of IncHI2A_1 (20.51%; 16/78), followed by IncX1_1 (17.95%; 14/78) and IncHI2_1 (11.54%; 9/78). Virulence factor prediction showed the detection of cdtB gene encoding typhoid toxins in two Salmonella Goldcoast ST358 and one Salmonella Typhimurium ST19, while one isolate of Salmonella London ST155 was positive for genes encoding for the siderophore “yersiniabactin” and the gene senB encoding for enterotoxin production. From this study, we conclude that pig slaughterhouses are critical points for the dissemination of virulent and multidrug-resistant Salmonella isolates along the food chain which require the implementation of management systems to control the critical points. Moreover, there is an urgent need for the implementation of the whole genome sequencing platform to monitor the emergence of virulent and multidrug-resistant clones along the food chain.

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          SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing.

          The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online ( http://bioinf.spbau.ru/spades ). It is distributed as open source software.
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            In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing.

            In the work presented here, we designed and developed two easy-to-use Web tools for in silico detection and characterization of whole-genome sequence (WGS) and whole-plasmid sequence data from members of the family Enterobacteriaceae. These tools will facilitate bacterial typing based on draft genomes of multidrug-resistant Enterobacteriaceae species by the rapid detection of known plasmid types. Replicon sequences from 559 fully sequenced plasmids associated with the family Enterobacteriaceae in the NCBI nucleotide database were collected to build a consensus database for integration into a Web tool called PlasmidFinder that can be used for replicon sequence analysis of raw, contig group, or completely assembled and closed plasmid sequencing data. The PlasmidFinder database currently consists of 116 replicon sequences that match with at least at 80% nucleotide identity all replicon sequences identified in the 559 fully sequenced plasmids. For plasmid multilocus sequence typing (pMLST) analysis, a database that is updated weekly was generated from www.pubmlst.org and integrated into a Web tool called pMLST. Both databases were evaluated using draft genomes from a collection of Salmonella enterica serovar Typhimurium isolates. PlasmidFinder identified a total of 103 replicons and between zero and five different plasmid replicons within each of 49 S. Typhimurium draft genomes tested. The pMLST Web tool was able to subtype genomic sequencing data of plasmids, revealing both known plasmid sequence types (STs) and new alleles and ST variants. In conclusion, testing of the two Web tools using both fully assembled plasmid sequences and WGS-generated draft genomes showed them to be able to detect a broad variety of plasmids that are often associated with antimicrobial resistance in clinically relevant bacterial pathogens. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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              Identification of acquired antimicrobial resistance genes

              Objectives Identification of antimicrobial resistance genes is important for understanding the underlying mechanisms and the epidemiology of antimicrobial resistance. As the costs of whole-genome sequencing (WGS) continue to decline, it becomes increasingly available in routine diagnostic laboratories and is anticipated to substitute traditional methods for resistance gene identification. Thus, the current challenge is to extract the relevant information from the large amount of generated data. Methods We developed a web-based method, ResFinder that uses BLAST for identification of acquired antimicrobial resistance genes in whole-genome data. As input, the method can use both pre-assembled, complete or partial genomes, and short sequence reads from four different sequencing platforms. The method was evaluated on 1862 GenBank files containing 1411 different resistance genes, as well as on 23 de- novo-sequenced isolates. Results When testing the 1862 GenBank files, the method identified the resistance genes with an ID = 100% (100% identity) to the genes in ResFinder. Agreement between in silico predictions and phenotypic testing was found when the method was further tested on 23 isolates of five different bacterial species, with available phenotypes. Furthermore, ResFinder was evaluated on WGS chromosomes and plasmids of 30 isolates. Seven of these isolates were annotated to have antimicrobial resistance, and in all cases, annotations were compatible with the ResFinder results. Conclusions A web server providing a convenient way of identifying acquired antimicrobial resistance genes in completely sequenced isolates was created. ResFinder can be accessed at www.genomicepidemiology.org. ResFinder will continuously be updated as new resistance genes are identified.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                08 July 2021
                2021
                : 12
                : 704636
                Affiliations
                [1] 1Zhejiang Provincial Center for Disease Control and Prevention , Hangzhou, China
                [2] 2Hainan Institute of Zhejiang University , Sanya, China
                [3] 3Department of Veterinary Medicine, Institute of Preventive Veterinary Sciences, Zhejiang University College of Animal Sciences , Hangzhou, China
                [4] 4State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, National Medical Center for Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University , Hangzhou, China
                [5] 5Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine , Hangzhou, China
                Author notes

                Edited by: Bojana Bogovic Matijasic, University of Ljubljana, Slovenia

                Reviewed by: Baowei Yang, Northwest A&F University, China; Jing Han, National Center for Toxicological Research (FDA), United States

                *Correspondence: Min Yue, myue@ 123456zju.edu.cn

                These authors have contributed equally to this work

                This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2021.704636
                8298193
                34305874
                a6529ad9-85fe-470a-9741-66461b2e69c9
                Copyright © 2021 Wu, Ed-Dra, Pan, Dong, Jia and Yue.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 03 May 2021
                : 11 June 2021
                Page count
                Figures: 5, Tables: 5, Equations: 0, References: 65, Pages: 12, Words: 0
                Funding
                Funded by: National Key Research and Development Program of China 10.13039/501100012166
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                salmonella,antimicrobial resistance,plasmid replicons,virulence factors,pig slaughterhouse,whole genome sequencing

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