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      Plasmid-Borne and Chromosomal ESBL/AmpC Genes in Escherichia coli and Klebsiella pneumoniae in Global Food Products

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          Abstract

          Plasmid-mediated extended-spectrum beta-lactamase (ESBL), AmpC, and carbapenemase producing Enterobacteriaceae, in particular Escherichia coli and Klebsiella pneumoniae, with potential zoonotic transmission routes, are one of the greatest threats to global health. The aim of this study was to investigate global food products as potential vehicles for ESBL/AmpC-producing bacteria and identify plasmids harboring resistance genes. We sampled 200 food products purchased from Finland capital region during fall 2018. Products originated from 35 countries from six continents and represented four food categories: vegetables ( n = 60), fruits and berries ( n = 50), meat ( n = 60), and seafood ( n = 30). Additionally, subsamples ( n = 40) were taken from broiler meat. Samples were screened for ESBL/AmpC-producing Enterobacteriaceae and whole genome sequenced to identify resistance and virulence genes and sequence types (STs). To accurately identify plasmids harboring resistance and virulence genes, a hybrid sequence analysis combining long- and short-read sequencing was employed. Sequences were compared to previously published plasmids to identify potential epidemic plasmid types. Altogether, 14 out of 200 samples were positive for ESBL/AmpC-producing E. coli and/or K. pneumoniae. Positive samples were recovered from meat (18%; 11/60) and vegetables (5%; 3/60) but were not found from seafood or fruit. ESBL/AmpC-producing E. coli and/or K. pneumoniae was found in 90% (36/40) of broiler meat subsamples. Whole genome sequencing of selected isolates ( n = 21) revealed a wide collection of STs, plasmid replicons, and genes conferring multidrug resistance. bla CTX–M–15-producing K. pneumoniae ST307 was identified in vegetable ( n = 1) and meat ( n = 1) samples. Successful IncFII plasmid type was recovered from vegetable and both IncFII and IncI1-Iγ types from meat samples. Hybrid sequence analysis also revealed chromosomally located beta-lactamase genes in two of the isolates and indicated similarity of food-derived plasmids to other livestock-associated sources and also to plasmids obtained from human clinical samples from various countries, such as IncI type plasmid harboring bla TEM–52C from a human urine sample obtained in the Netherlands which was highly similar to a plasmid obtained from broiler meat in this study. Results indicate certain foods contain bacteria with multidrug resistance and pose a possible risk to public health, emphasizing the importance of surveillance and the need for further studies on epidemiology of epidemic plasmids.

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          Prokka: rapid prokaryotic genome annotation.

          T Seemann (2014)
          The multiplex capability and high yield of current day DNA-sequencing instruments has made bacterial whole genome sequencing a routine affair. The subsequent de novo assembly of reads into contigs has been well addressed. The final step of annotating all relevant genomic features on those contigs can be achieved slowly using existing web- and email-based systems, but these are not applicable for sensitive data or integrating into computational pipelines. Here we introduce Prokka, a command line software tool to fully annotate a draft bacterial genome in about 10 min on a typical desktop computer. It produces standards-compliant output files for further analysis or viewing in genome browsers. Prokka is implemented in Perl and is freely available under an open source GPLv2 license from http://vicbioinformatics.com/. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
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            Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads

            The Illumina DNA sequencing platform generates accurate but short reads, which can be used to produce accurate but fragmented genome assemblies. Pacific Biosciences and Oxford Nanopore Technologies DNA sequencing platforms generate long reads that can produce complete genome assemblies, but the sequencing is more expensive and error-prone. There is significant interest in combining data from these complementary sequencing technologies to generate more accurate “hybrid” assemblies. However, few tools exist that truly leverage the benefits of both types of data, namely the accuracy of short reads and the structural resolving power of long reads. Here we present Unicycler, a new tool for assembling bacterial genomes from a combination of short and long reads, which produces assemblies that are accurate, complete and cost-effective. Unicycler builds an initial assembly graph from short reads using the de novo assembler SPAdes and then simplifies the graph using information from short and long reads. Unicycler uses a novel semi-global aligner to align long reads to the assembly graph. Tests on both synthetic and real reads show Unicycler can assemble larger contigs with fewer misassemblies than other hybrid assemblers, even when long-read depth and accuracy are low. Unicycler is open source (GPLv3) and available at github.com/rrwick/Unicycler.
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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
                03 February 2021
                2021
                : 12
                : 592291
                Affiliations
                [1] 1Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, University of Helsinki , Helsinki, Finland
                [2] 2Finnish Food Authority , Seinäjoki, Finland
                [3] 3Wageningen Bioveterinary Research , Lelystad, Netherlands
                [4] 4Customs Laboratory , Espoo, Finland
                Author notes

                Edited by: Vincent Burrus, Université de Sherbrooke, Canada

                Reviewed by: Jian-Hua Liu, South China Agricultural University, China; Rosa Del Campo, Ramón y Cajal Institute for Health Research, Spain

                *Correspondence: Paula Kurittu, paula.kurittu@ 123456helsinki.fi

                This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2021.592291
                7886708
                33613476
                0bed52d4-3f96-40aa-8fb5-bc453aaad867
                Copyright © 2021 Kurittu, Khakipoor, Aarnio, Nykäsenoja, Brouwer, Myllyniemi, Vatunen and Heikinheimo.

                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
                : 06 August 2020
                : 14 January 2021
                Page count
                Figures: 4, Tables: 4, Equations: 0, References: 93, Pages: 19, Words: 0
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                antimicrobial resistance,whole genome sequencing,extended-spectrum beta-lactamases,multidrug resistance,imported food,hybrid sequencing,one health

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