[en] [en] BACKGROUND: Antimicrobial resistance has been identified as a major threat to global health. The pig food chain is considered an important source of antimicrobial resistance genes (ARGs). However, there is still a lack of knowledge on the dispersion of ARGs in pig production system, including the external environment.
RESULTS: In the present study, we longitudinally followed one swine farm located in Italy from the weaning phase to the slaughterhouse to comprehensively assess the diversity of ARGs, their diffusion, and the bacteria associated with them. We obtained shotgun metagenomic sequences from 294 samples, including pig feces, farm environment, soil around the farm, wastewater, and slaughterhouse environment. We identified a total of 530 species-level genome bins (SGBs), which allowed us to assess the dispersion of microorganisms and their associated ARGs in the farm system. We identified 309 SGBs being shared between the animals gut microbiome, the internal and external farm environments. Specifically, these SGBs were characterized by a diverse and complex resistome, with ARGs active against 18 different classes of antibiotic compounds, well matching antibiotic use in the pig food chain in Europe.
CONCLUSIONS: Collectively, our results highlight the urgency to implement more effective countermeasures to limit the dispersion of ARGs in the pig food systems and the relevance of metagenomics-based approaches to monitor the spread of ARGs for the safety of the farm working environment and the surrounding ecosystems.
Disciplines :
Microbiology
Author, co-author :
Scicchitano, Daniel; Fano Marine Center, Fano, Italy ; Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy
Leuzzi, Daniela; Fano Marine Center, Fano, Italy ; Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy
Babbi, Giulia; Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy
Palladino, Giorgia; Fano Marine Center, Fano, Italy ; Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy
Turroni, Silvia; Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy
LACZNY, Cedric Christian ; University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB) > Systems Ecology
WILMES, Paul ; University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB) > Systems Ecology
Correa, Federico; Department of Agricultural and Food Sciences, University of Bologna, Bologna, Italy
Leekitcharoenphon, Pimlapas; Tecnhical University of Denmark, Kongens Lyngby, Denmark
Savojardo, Castrense; Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy
Luise, Diana; Department of Agricultural and Food Sciences, University of Bologna, Bologna, Italy
Martelli, Pierluigi; Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy
Trevisi, Paolo; Department of Agricultural and Food Sciences, University of Bologna, Bologna, Italy
Aarestrup, Frank Møller; Tecnhical University of Denmark, Kongens Lyngby, Denmark
Candela, Marco; Fano Marine Center, Fano, Italy. marco.candela@unibo.it ; Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy. marco.candela@unibo.it
Rampelli, Simone; Fano Marine Center, Fano, Italy ; Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy
This work was carried out in the context of the “Controlling Microbiomes Circulations for Better Food Systems” (CIRCLES) project, which was funded by the European Union’s Horizon 2020 research and innovation program under grant agreement no. 818290.
B.P. Alcock W. Huynh R. Chalil K.W. Smith A.R. Raphenya M.A. Wlodarski et al. CARD 2023: expanded curation, support for machine learning, and resistome prediction at the comprehensive antibiotic resistance database Nucleic Acids Res 2023 51 D1 D690 D699 1:CAS:528:DC%2BB3sXht1yhsLbP 10.1093/nar/gkac920 36263822
F. Beghini L.J. McIver A. Blanco-Míguez L. Dubois F. Asnicar S. Maharjan A. Mailyan P. Manghi M. Scholz A.M. Thomas M. Valles-Colomer G. Weingart Y. Zhang M. Zolfo C. Huttenhower E.A. Franzosa N. Segata Integrating taxonomic, functional, and strain-level profiling of diverse microbial communities with bioBakery 3 Elife 2021 10 e65088 1:CAS:528:DC%2BB3MXislantrzF 10.7554/eLife.65088 33944776 8096432
M. Bergamaschi F. Tiezzi J. Howard Y.J. Huang K.A. Gray C. Schillebeeckx N.P. McNulty C. Maltecca Gut microbiome composition differences among breeds impact feed efficiency in swine Microbiome 2020 8 1 110 1:CAS:528:DC%2BB38XjsVeht7w%3D 10.1186/s40168-020-00888-9 32698902 7376719
J.P. Brooks M.R. McLaughlin Antibiotic resistant bacterial profiles of anaerobic swine lagoon effluent J Environ Qual 2009 38 6 2431 2437 1:CAS:528:DC%2BD1MXhsVCjsLnJ 10.2134/jeq2008.0471 19875799
A. Checcucci P. Trevisi D. Luise M. Modesto S. Blasioli I. Braschi P. Mattarelli Exploring the animal waste resistome: the spread of antimicrobial resistance genes through the use of livestock manure Front Microbiol 2020 11 1416 10.3389/fmicb.2020.01416 32793126 7387501
C. Chen Y. Zhou H. Fu X. Xiong S. Fang H. Jiang J. Wu H. Yang J. Gao L. Huang Expanded catalog of microbial genes and metagenome-assembled genomes from the pig gut microbiome Nat Commun 2021 12 1 1106 1:CAS:528:DC%2BB3MXksFSms70%3D 10.1038/s41467-021-21295-0 33597514 7889623
A. Dawangpa P. Lertwatcharasarakul A. Boonsoongnern N. Ratanavanichrojn A. Sanguankiat N. Pinniam S. Jala S. Laopiem P. Tulayakul Multidrug resistance problems targeting piglets and environmental health by Escherichia coli in intensive swine farms Emerg Contam 2022 8 123 133 1:CAS:528:DC%2BB38XitFCqtL7M 10.1016/j.emcon.2022.03.002
G. De Angelis B. Fiori G. Menchinelli T. D'Inzeo F.M. Liotti G.A. Morandotti M. Sanguinetti B. Posteraro T. Spanu Incidence and antimicrobial resistance trends in bloodstream infections caused by ESKAPE and Escherichia coli at a large teaching hospital in Rome, a 9-year analysis (2007–2015) Eur J Clin Microbiol Infect Dis Off Publ Eur Soc Clin Microbiol 2018 37 9 1627 1636 1:CAS:528:DC%2BC1cXhtFeqtrbE 10.1007/s10096-018-3292-9
L. de Nies S. Lopes S.B. Busi et al. PathoFact: a pipeline for the prediction of virulence factors and antimicrobial resistance genes in metagenomic data Microbiome 2021 9 49 1:CAS:528:DC%2BB38XjsVemt7o%3D 10.1186/s40168-020-00993-9 33597026 7890817
G.C. da Silva O.S. Gonçalves J.N. Rosa K.C. França J.T. Bossé M.F. Santana P.R. Langford D.M.S. Bazzolli Mobile genetic elements drive antimicrobial resistance gene spread in pasteurellaceae species Front Microbiol 2022 12 10.3389/fmicb.2021.773284 35069478 8777487
G.V. De Socio P. Rubbioni D. Botta E. Cenci A. Belati R. Paggi M.B. Pasticci A. Mencacci Measurement and prediction of antimicrobial resistance in bloodstream infections by ESKAPE pathogens and Escherichia coli J Global Antimicrob Res 2019 19 154 160 10.1016/j.jgar.2019.05.013
J. Dewulf P. Joosten I. Chantziaras E. Bernaerdt W. Vanderhaeghen M. Postma D. Maes Antibiotic use in European pig production: less is more Antibiotics 2022 11 11 1493 10.3390/antibiotics11111493 36358148 9686698
Dragulescu A, Arendt C (2020) xlsx: Read, Write, Format Excel 2007 and Excel 97/2000/XP/2003 Files_. R package version 0.6.5, https://CRAN.R-project.org/package=xlsx
ECDC 2023 https://www.ecdc.europa.eu/en/publications-data/antimicrobial-resistance-surveillance-europe-2023-2021-data. Accessed June 2023
M. Exner S. Bhattacharya B. Christiansen J. Gebel P. Goroncy-Bermes P. Hartemann P. Heeg C. Ilschner A. Kramer E. Larson W. Merkens M. Mielke P. Oltmanns B. Ross M. Rotter R.M. Schmithausen H.G. Sonntag M. Trautmann Antibiotic resistance: what is so special about multidrug-resistant Gram- negative bacteria? GMS Hyg Infect Control 2017 12 Doc05 10.3205/dgkh000290 28451516 5388835
T.N. Gaire H.M. Scott N.R. Noyes A.C. Ericsson M.D. Tokach M.B. Menegat J. Vinasco B. Roenne T. Ray T.G. Nagaraja V.V. Volkova Age influences the temporal dynamics of microbiome and antimicrobial resistance genes among fecal bacteria in a cohort of production pigs Animal microbiome 2023 5 1 2 1:CAS:528:DC%2BB3sXntFakuw%3D%3D 10.1186/s42523-022-00222-8 36624546 9830919
T.N. Gaire H.M. Scott L. Sellers T.G. Nagaraja V.V. Volkova Age dependence of antimicrobial resistance among fecal bacteria in animals: a scoping review Front Vet Sci. 2021 7 622495 10.3389/fvets.2020.622495 33575279 7870994
Gao C (2022) ggVennDiagram: A 'ggplot2' Implement of Venn Diagram_. R package version 1.2.2, https://CRAN.R-project.org/package=ggVennDiagram
Q. Gao S. Gao C. Bates Y. Zeng J. Lei H. Su Q. Dong Z. Qin J. Zhao Q. Zhang D. Ning Y. Huang J. Zhou Y. Yang The microbial network property as a bio-indicator of antibiotic transmission in the environment Sci Total Environ 2021 758 1:CAS:528:DC%2BB3cXisVOnt77N 10.1016/j.scitotenv.2020.143712 33277004
D.B. Holman A. Kommadath J.P. Tingley D.W. Abbott Novel insights into the pig gut microbiome using metagenome-assembled genomes Microbiol Spectr 2022 10 4 e02380 e2422 10.1128/spectrum.02380-22 35880887 9431278
K. He J. Xiong W. Yang L. Zhao T. Wang W. Qian S. Hu Q. Wang M.T. Aleem W. Miao W. Yan Metagenome of gut microbiota provides a novel insight into the pathogenicity of Balantioides coli in weaned piglets Int J Mol Sci 2023 24 13 10791 1:CAS:528:DC%2BB3sXhsFWms7%2FK 10.3390/ijms241310791.PMID:37445967;PMCID:PMC10342044 37445967 10342044
A. Herrero-Fresno C. Zachariasen M.H. Hansen A. Nielsen R.S. Hendriksen S.S. Nielsen J.E. Olsen Apramycin treatment affects selection and spread of a multidrug-resistant Escherichia coli strain able to colonize the human gut in the intestinal microbiota of pigs Vet Res 2016 47 12 1:CAS:528:DC%2BC2sXlslGqs7o%3D 10.1186/s13567-015-0291-z 26739225 4704421
D.B. Holman A. Kommadath J.P. Tingley D.W. Abbott Novel insights into the pig gut microbiome using metagenome-assembled genomes Microbiol Spectr 2022 10 4 1:CAS:528:DC%2BB38XisVyqtbrK 10.1128/spectrum.02380-22 35880887
P.S. Krawczyk L. Lipinski A. Dziembowski PlasFlow: predicting plasmid sequences in metagenomic data using genome signatures Nucleic Acids Res 2018 46 6 e35 e35 1:CAS:528:DC%2BC1cXitlGisrvM 10.1093/nar/gkx1321 29346586 5887522
D.G.J. Larsson C.F. Flach Antibiotic resistance in the environment Nat Rev Microbiol 2022 20 5 257 269 1:CAS:528:DC%2BB3MXisVahsb%2FL 10.1038/s41579-021-00649-x 34737424
X. Liao X. Lu C. Rensing V.P. Friman S. Geisen Z. Chen Z. Yu Z. Wei S. Zhou Y. Zhu Hyperthermophilic composting accelerates the removal of antibiotic resistance genes and mobile genetic elements in sewage sludge Environ Sci Technol 2018 52 1 266 276 1:CAS:528:DC%2BC2sXhvFSlu7jM 10.1021/acs.est.7b04483 29199822
F. Lorenzo-Díaz C. Fernández-López R. Lurz A. Bravo M. Espinosa Crosstalk between vertical and horizontal gene transfer: plasmid replication control by a conjugative relaxase Nucleic Acids Res 2017 45 13 7774 7785 1:CAS:528:DC%2BC1cXhs1aju7k%3D 10.1093/nar/gkx450 28525572 5737340
F. Ma S. Xu Z. Tang Z. Li L. Zhang Use of antimicrobials in food animals and impact of transmission of antimicrobial resistance on humans Biosafety Health 2021 3 1 32 38 10.1016/j.bsheal.2020.09.004
A. Mann K. Nehra J.S. Rana T. Dahiya Antibiotic resistance in agriculture: perspectives on upcoming strategies to overcome upsurge in resistance Curr Research Microbial Sci 2021 2 1:CAS:528:DC%2BB38XhvFCjt7nF 10.1016/j.crmicr.2021.100030
D.F. Maron T.J. Smith K.E. Nachman Restrictions on antimicrobial use in food animal production: an international regulatory and economic survey Global Health 2013 9 48 10.1186/1744-8603-9-48 24131666 3853314
M. Krassowski krassowski/complex-upset 2020 10.5281/zenodo.3700590 Zenodo
X.C. Monger A.A. Gilbert L. Saucier A.T. Vincent Antibiotic resistance: from pig to meat Antibiotics (Basel, Switzerland) 2021 10 10 1209 1:CAS:528:DC%2BB3MXisleisbvI 10.3390/antibiotics10101209 34680790
D. Mun H. Kim M. Shin S. Ryu M. Song S. Oh Y. Kim Decoding the intestinal microbiota repertoire of sow and weaned pigs using culturomic and metagenomic approaches J Anim Sci Technol 2021 63 6 1423 1432 1:CAS:528:DC%2BB38XovFCrt7w%3D 10.5187/jast.2021.e124 34957455 8672247
E. Nanetti G. Palladino D. Scicchitano G. Trapella N. Cinti M. Fabbrini et al. Composition and biodiversity of soil and root-associated microbiome in Vitis vinifera cultivar Lambrusco distinguish the microbial terroir of the Lambrusco DOC protected designation of origin area on a local scale Front Microbiol 2023 14 1108036 10.3389/fmicb.2023.1108036 36910169 9992870
Neuwirth E _RColorBrewer: ColorBrewer Palettes_. R package version 1.1-3, https://CRAN.R-project.org/package=RColorBrewer. (2022)
Oksanen J, Simpson G, Blanchet FG, Kindt R, Legendre P, Minchin P et al. (2022). Vegan Community Ecology Package Version 2.6-2 April 2022. The Comprehensive R Archive Network. Available online: http://cran.r-project.Org
D.H. Parks M. Imelfort C.T. Skennerton P. Hugenholtz G.W. Tyson Assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes Genome Res 2014 25 1043 1055 10.1101/gr.186072.114
E. Pasolli F. Asnicar S. Manara M. Zolfo N. Karcher F. Armanini F. Beghini P. Manghi A. Tett P. Ghensi M.C. Collado B.L. Rice C. DuLong X.C. Morgan C.D. Golden C. Quince C. Huttenhower N. Segata Extensive unexplored human microbiome diversity revealed by over 150,000 genomes from metagenomes spanning age, geography, and lifestyle Cell. 2019 176 3 649 662.e20 1:CAS:528:DC%2BC1MXhsVars7w%3D 10.1016/j.cell.2019.01.001 30661755 6349461
S. Petrin I. Patuzzi A. Di Cesare A. Tiengo G. Sette G. Biancotto G. Corno M. Drigo C. Losasso V. Cibin Evaluation and quantification of antimicrobial residues and antimicrobial resistance genes in two Italian swine farms Environ Pollut 2019 255 Pt 1 113183 1:CAS:528:DC%2BC1MXhvVSrtLvO 10.1016/j.envpol.2019.113183 31541814
J. Pollock A. Muwonge M.R. Hutchings G. Mainda B.M. Bronsvoort D.L. Gally A. Corbishley Resistance to change: AMR gene dynamics on a commercial pig farm with high antimicrobial usage Sci Rep 2020 10 1 1708 1:CAS:528:DC%2BB3cXjs1Ojt7g%3D 10.1038/s41598-020-58659-3 32015392 6997390
D. Poulin-Laprade J.S. Brouard N. Gagnon A. Turcotte A. Langlois J.J. Matte C.D. Carrillo R. Zaheer T. McAllister E. Topp G. Talbot Resistance determinants and their genetic context in enterobacteria from a longitudinal study of pigs reared under various husbandry conditions Appl Environ Microbiol 2021 87 8 e02612 e2620 1:CAS:528:DC%2BB3MXhtF2qur3L 10.1128/AEM.02612-20 33514521 8091121
L.B. Rice Progress and challenges in implementing the research on ESKAPE pathogens Infect Control Hosp Epidemiol 2010 31 Suppl 1 S7 S10 10.1086/655995 20929376
Rudis B (2020) hrbrthemes: Additional Themes, Theme Components and Utilities for 'ggplot2'_. R package version 0.8.0, https://CRAN.R-project.org/package=hrbrthemes
S. Santajit N. Indrawattana Mechanisms of antimicrobial resistance in ESKAPE pathogens Biomed Res Int 2016 2016 2475067 1:CAS:528:DC%2BC2sXnsFKlsL8%3D 10.1155/2016/2475067 27274985 4871955
S. Sarrazin P. Joosten L. Van Gompel R.E. Luiken D.J. Mevius J.A. Wagenaar D.J. Heederik J. Dewulf Quantitative and qualitative analysis of antimicrobial usage patterns in 180 selected farrow-to-finish pig farms from nine European countries based on single batch and purchase data J Antimicrob Chemother 2019 74 3 807 816 1:CAS:528:DC%2BC1MXitVejurfI 10.1093/jac/dky503 30544242
M. Savin G. Bierbaum J.A. Hammerl C. Heinemann M. Parcina E. Sib A. Voigt J. Kreyenschmidt Antibiotic-resistant bacteria and antimicrobial residues in wastewater and process water from German pig slaughterhouses and their receiving municipal wastewater treatment plants Sci Total Environ 2020 727 1:CAS:528:DC%2BB3cXotVGgtb4%3D 10.1016/j.scitotenv.2020.138788 32498197
H.M. Scott G. Acuff G. Bergeron M.W. Bourassa J. Gill D.W. Graham L.H. Kahn P.S. Morley M.J. Salois S. Simjee R.S. Singer T.C. Smith C. Storrs T.E. Wittum Critically important antibiotics: criteria and approaches for measuring and reducing their use in food animal agriculture Ann N Y Acad Sci 2019 1441 1 8 16 10.1111/nyas.14058 30924540 6850619
Simon G, Noam R, Robert R, Antônio PC, Marco Sciaini, and Cédric Scherer (2021). Rvision–Colorblind-Friendly Color Maps for R. R package version 0.6.2
Stier A, Ter Braak C, Weedon J (2022) vegan: Community Ecology Package_. R package version 2.6-2, https://CRAN.R-project.org/package=vegan
P.J. Stogios A. Savchenko Molecular mechanisms of vancomycin resistance Protein Sci Publ Protein Soc 2020 29 3 654 669 1:CAS:528:DC%2BB3cXhsVSntL0%3D 10.1002/pro.3819
C.H. Teng P.C. Wu S.L. Tang Y.C. Chen M.F. Cheng P.C. Huang W.C. Ko J.L. Wang A large spatial survey of colistin-resistant gene mcr-1-Carrying E. coli in rivers across Taiwan Microorganisms 2021 9 4 722 1:CAS:528:DC%2BB3MXhvFygt7nN 10.3390/microorganisms9040722 33807253 8066897
TrimBWAstyle.usingBam.pl, 2010. https://github.com/genome/genome/blob/master/lib/perl/Genome/Site/TGI/Hmp/HmpSraProcess/trimBWAstyle.usingBam.pl. Accessed June 2023
P.J. Turnbaugh R.E. Ley M. Hamady C.M. Fraser-Liggett R. Knight J.I. Gordon The human microbiome project Nature 2007 449 7164 804 810 1:CAS:528:DC%2BD2sXhtFOjt7nP 10.1038/nature06244 17943116 3709439
D.T. Truong A. Tett E. Pasolli C. Huttenhower N. Segata Microbial strain-level population structure and genetic diversity from metagenomes Genome Res 2017 27 4 626 638 1:CAS:528:DC%2BC2sXmtlSqsbs%3D 10.1101/gr.216242.116 28167665 5378180
M.E. Velazquez-Meza M. Galarde-López B. Carrillo-Quiróz C.M. Alpuche-Aranda Antimicrobial resistance: one Health approach Veterinary world 2022 15 3 743 749 1:CAS:528:DC%2BB38XhvVOkurbL 10.14202/vetworld.2022.743-749 35497962 9047147
M. Valles-Colomer A. Blanco-Míguez P. Manghi F. Asnicar L. Dubois D. Golzato N. Segata The person-to-person transmission landscape of the gut and oral microbiomes Nature 2023 614 125 135 1:CAS:528:DC%2BB3sXhslKisbY%3D 10.1038/s41586-022-05620-1 36653448 9892008
X. Wang S. Howe X. Wei F. Deng T. Tsai J. Chai Y. Xiao H. Yang C.V. Maxwell Y. Li J. Zhao Comprehensive cultivation of the swine gut microbiome reveals high bacterial diversity and guides bacterial isolation in pigs mSystems. 2021 6 4 e0047721 10.1128/mSystems.00477-21 34282935
Warnes G, Bolker B, Bonebakker L, Gentleman R, Huber W, Liaw A, Lumley T, Maechler M, Magnusson A, Moeller S, Schwartz M, Venables B (2022). gplots: Various R Programming Tools for Plotting Data. R package version 3.1.3, https://CRAN.R-project.org/package=gplots
World Health Organization, WHO, https://www.who.int/. Accessed June 2023
H. Wickham Reshaping data with the reshape package J Stat Softw 2007 10.18637/jss.v021.i12
H. Wickham ggplot2: elegant graphics for data analysis 2016 New York Springer-Verlag 10.1007/978-3-319-24277-4
H. Wickham M. Averick J. Bryan W. Chang L.D. McGowan R. François G. Grolemund A. Hayes L. Henry J. Hester M. Kuhn T.L. Pedersen E. Miller S.M. Bache K. Müller J. Ooms D. Robinson D.P. Seidel V. Spinu K. Takahashi D. Vaughan C. Wilke K. Woo H. Yutani Welcome to the tidyverse J Open Source Softw 2019 4 43 1686 10.21105/joss.01686
Wood J (2022) RcppAlgos: High Performance Tools for Combinatorics and Computational Mathematics. R package version 2.6.0, https://CRAN.R-project.org/package=RcppAlgos
Y. Wu Y. Meng L. Qian B. Ding H. Han H. Chen L. Bai D. Qu Y. Wu The Vancomycin resistance-associated regulatory system VraSR modulates biofilm formation of staphylococcus epidermidis in an ica-dependent manner mSphere 2021 6 5 e0064121 10.1128/mSphere.00641-21 34550006