{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,5]],"date-time":"2026-11-05T00:52:20Z","timestamp":1793839940897,"version":"4.6.1"},"reference-count":46,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2022,5,28]],"date-time":"2022-05-28T00:00:00Z","timestamp":1653696000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ricerca di Base 2020, University of Perugia"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Antibiotics"],"abstract":"<jats:p>This work aimed to evaluate phenotypically and genotypically the colistin susceptibility of 85 Salmonella Infantis strains isolated in Italy from the broiler production chain, and to apply a whole-genome approach for the determination of genes conferring antimicrobial resistance (AMR). All isolates were tested by the broth microdilution method to evaluate the colistin minimum inhibitory concentrations (MICs). A multiplex PCR was performed in all isolates for the screening of mcr-1, mcr-2, mcr-3 mcr-4, mcr-5 genes and whole-genome sequencing (WGS) of six S. Infantis was applied. Three out of 85 (3.5%) S. Infantis strains were colistin resistant (MIC values ranged from 4 to 8 mg\/L) and mcr-1 positive. The mcr-1.1 and mcr-1.2 variants located on the IncX4 plasmid were detected in three different colistin-resistant isolates. The two allelic variants showed identical sequences. All six isolates harbored blaCTXM-1, aac(6\u2032)-Iaa and gyrA\/parC genes, mediating, respectively, beta-lactam, aminoglycoside and quinolone resistance. The pESI-megaplasmid carrying tet(A) (tetracycline resistance), dfrA1, (trimethoprim resistance) sul1, (sulfonamide resistance) and qacE (quaternary ammonium resistance) genes was found in all isolates. To our knowledge, this is the first report of the mcr-1.2 variant described in S. Infantis isolated from broilers chickens. Our results also showed a low prevalence of colistin- resistance, probably due to a reduction in colistin use in poultry. This might suggest an optimization of biosecurity control both on farms and in slaughterhouses.<\/jats:p>","DOI":"10.3390\/antibiotics11060728","type":"journal-article","created":{"date-parts":[[2022,5,29]],"date-time":"2022-05-29T08:50:51Z","timestamp":1653814251000},"page":"728","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["mcr-1-Mediated Colistin Resistance and Genomic Characterization of Antimicrobial Resistance in ESBL-Producing Salmonella Infantis Strains from a Broiler Meat Production Chain in Italy"],"prefix":"10.3390","volume":"11","author":[{"given":"Patrizia","family":"Casagrande Proietti","sequence":"first","affiliation":[{"name":"Department of Veterinary Medicine, University of Perugia, Via S. Costanzo 4, 06126 Perugia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1106-2130","authenticated-orcid":false,"given":"Laura","family":"Musa","sequence":"additional","affiliation":[{"name":"Department of Veterinary Medicine, University of Perugia, Via S. Costanzo 4, 06126 Perugia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9621-3315","authenticated-orcid":false,"given":"Valentina","family":"Stefanetti","sequence":"additional","affiliation":[{"name":"Department of Veterinary Medicine, University of Perugia, Via S. Costanzo 4, 06126 Perugia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Massimiliano","family":"Orsini","sequence":"additional","affiliation":[{"name":"Istituto Zooprofilattico Sperimentale delle Venezie, 35020 Padova, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Valeria","family":"Toppi","sequence":"additional","affiliation":[{"name":"Department of Veterinary Medicine, University of Perugia, Via S. Costanzo 4, 06126 Perugia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4428-9016","authenticated-orcid":false,"given":"Raffaella","family":"Branciari","sequence":"additional","affiliation":[{"name":"Department of Veterinary Medicine, University of Perugia, Via S. Costanzo 4, 06126 Perugia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francesca","family":"Blasi","sequence":"additional","affiliation":[{"name":"Istituto Zooprofilattico Sperimentale dell\u2019Umbria e Delle Marche \u2018Togo Rosati\u2019, 06124 Perugia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7580-4861","authenticated-orcid":false,"given":"Chiara Francesca","family":"Magistrali","sequence":"additional","affiliation":[{"name":"Istituto Zooprofilattico Sperimentale dell\u2019Umbria e Delle Marche \u2018Togo Rosati\u2019, 06124 Perugia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0004-9272","authenticated-orcid":false,"given":"Stefano","family":"Capomaccio","sequence":"additional","affiliation":[{"name":"Department of Veterinary Medicine, University of Perugia, Via S. Costanzo 4, 06126 Perugia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4676-4108","authenticated-orcid":false,"given":"Tana Shtylla","family":"Kika","sequence":"additional","affiliation":[{"name":"Faculty of Veterinary Medicine, Agricultural University of Tirana, 1029 Tirana, Albania"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria Pia","family":"Franciosini","sequence":"additional","affiliation":[{"name":"Department of Veterinary Medicine, University of Perugia, Via S. Costanzo 4, 06126 Perugia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,5,28]]},"reference":[{"key":"ref_1","unstructured":"Benedict, R.G., and Langlykke, A.F. (1947). Antibiotic Activity of Bacillus Polymyxa. J. Bacteriol., 54."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"557","DOI":"10.1128\/CMR.00064-16","article-title":"Polymyxins: Antibacterial Activity, Susceptibility Testing, and Resistance Mechanisms Encoded by Plasmids or Chromosomes","volume":"30","author":"Poirel","year":"2017","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"598","DOI":"10.1016\/j.ijantimicag.2016.09.016","article-title":"Colistin Use and Colistin Resistance in Bacteria from Animals","volume":"48","author":"Kempf","year":"2016","journal-title":"Int. J. Antimicrob. Agents"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2023","DOI":"10.3201\/eid2312.170883","article-title":"High Rate of MCR-1-Producing Escherichia coli and Klebsiella pneumoniae among Pigs, Portugal","volume":"23","author":"Kieffer","year":"2017","journal-title":"Emerg. Infect. Dis."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Rhouma, M., Beaudry, F., Th\u00e9riault, W., and Letellier, A. (2016). Colistin in Pig Production: Chemistry, Mechanism of Antibacterial Action, Microbial Resistance Emergence, and One Health Perspectives. Front. Microbiol., 7.","DOI":"10.3389\/fmicb.2016.01789"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Kumar, H., Chen, B.-H., Kuca, K., Nepovimova, E., Kaushal, A., Nagraik, R., Bhatia, S.K., Dhanjal, D.S., Kumar, V., and Kumar, A. (2020). Understanding of Colistin Usage in Food Animals and Available Detection Techniques: A Review. Animals, 10.","DOI":"10.3390\/ani10101892"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"297","DOI":"10.1016\/j.ijantimicag.2015.06.005","article-title":"Use of Colistin-Containing Products within the European Union and European Economic Area (EU\/EEA): Development of Resistance in Animals and Possible Impact on Human and Animal Health","volume":"46","author":"Catry","year":"2015","journal-title":"Int. J. Antimicrob. Agents"},{"key":"ref_8","unstructured":"World Health Organization (2021). GLASS: The Detection and Reporting of Colistin Resistance, World Health Organization."},{"key":"#cr-split#-ref_9.1","unstructured":"(2018). European Food Safety Authority"},{"key":"#cr-split#-ref_9.2","unstructured":"European Centre for Disease Prevention and Control. The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-Borne Outbreaks in 2017. EFSA J., 16."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"977","DOI":"10.1111\/1462-2920.12351","article-title":"A Unique Megaplasmid Contributes to Stress Tolerance and Pathogenicity of an Emergent Salmonella Enterica Serovar Infantis Strain","volume":"16","author":"Aviv","year":"2014","journal-title":"Environ. Microbiol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Franco, A., Leekitcharoenphon, P., Feltrin, F., Alba, P., Cordaro, G., Iurescia, M., Tolli, R., D\u2019Incau, M., Staffolani, M., and Di Giannatale, E. (2015). Emergence of a Clonal Lineage of Multidrug-Resistant ESBL-Producing Salmonella Infantis Transmitted from Broilers and Broiler Meat to Humans in Italy between 2011 and 2014. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0144802"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1016\/j.fm.2017.03.011","article-title":"Molecular Epidemiology of the Endemic Multiresistance Plasmid PSI54\/04 of Salmonella Infantis in Broiler and Human Population in Hungary","volume":"71","author":"Szmolka","year":"2018","journal-title":"Food Microbiol."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Alba, P., Leekitcharoenphon, P., Carfora, V., Amoruso, R., Cordaro, G., Di Matteo, P., Ianzano, A., Iurescia, M., Diaconu, E.L., and Study Group, E.-E.-A.N. (2020). Molecular Epidemiology of Salmonella Infantis in Europe: Insights into the Success of the Bacterial Host and Its Parasitic PESI-like Megaplasmid. Microb. Genom., 6.","DOI":"10.1099\/mgen.0.000365"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1016\/S1473-3099(15)00424-7","article-title":"Emergence of Plasmid-Mediated Colistin Resistance Mechanism MCR-1 in Animals and Human Beings in China: A Microbiological and Molecular Biological Study","volume":"16","author":"Liu","year":"2016","journal-title":"Lancet Infect. Dis."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"424","DOI":"10.1038\/s41598-017-00095-x","article-title":"Genetic Analysis of the IncX4 Plasmids: Implications for a Unique Pattern in the Mcr-1 Acquisition","volume":"7","author":"Sun","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_16","first-page":"113","article-title":"Plasmid-Mediated Antimicrobial Resistance in Salmonella Enterica","volume":"5","author":"Carattoli","year":"2003","journal-title":"Curr. Issues Mol. Biol."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Yang, Q.-E., Sun, J., Li, L., Deng, H., Liu, B.-T., Fang, L.-X., Liao, X.-P., and Liu, Y.-H. (2015). IncF Plasmid Diversity in Multi-Drug Resistant Escherichia coli Strains from Animals in China. Front. Microbiol., 6.","DOI":"10.3389\/fmicb.2015.00964"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Casagrande Proietti, P., Stefanetti, V., Musa, L., Zicavo, A., Dionisi, A.M., Bellucci, S., Mensa, A.L., Menchetti, L., Branciari, R., and Ortenzi, R. (2020). Genetic Profiles and Antimicrobial Resistance Patterns of Salmonella Infantis Strains Isolated in Italy in the Food Chain of Broiler Meat Production. Antibiotics, 9.","DOI":"10.3390\/antibiotics9110814"},{"key":"ref_19","unstructured":"(2022, March 19). ISO 6579-1:2017\/AMD 1:2020 ISO 6579-1:2017\/Amd 1:2020. Available online: https:\/\/www.iso.org\/cms\/render\/live\/en\/sites\/isoorg\/contents\/data\/standard\/07\/66\/76671.html."},{"key":"ref_20","unstructured":"Grimont, P.A.D., and Weill, F.-X. (2007). Antigenic Formulae of the Salmonella Serovars, WHO."},{"key":"ref_21","unstructured":"EUCAST (2022, March 19). European Committee on Antimicrobial Susceptibility Testing. Available online: https:\/\/www.eucast.org\/fileadmin\/src\/media\/PDFs\/EUCAST_files\/Breakpoint_tables\/v_10.0_Breakpoint."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Rebelo, A.R., Bortolaia, V., Kjeldgaard, J.S., Pedersen, S.K., Leekitcharoenphon, P., Hansen, I.M., Guerra, B., Malorny, B., Borowiak, M., and Hammerl, J.A. (2018). Multiplex PCR for Detection of Plasmid-Mediated Colistin Resistance Determinants, Mcr-1, Mcr-2, Mcr-3, Mcr-4 and Mcr-5 for Surveillance Purposes. Eurosurveillance, 23.","DOI":"10.2807\/1560-7917.ES.2018.23.6.17-00672"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1089\/cmb.2012.0021","article-title":"SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing","volume":"19","author":"Bankevich","year":"2012","journal-title":"J. Comput. Biol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1072","DOI":"10.1093\/bioinformatics\/btt086","article-title":"QUAST: Quality assessment tool for genome assemblies","volume":"29","author":"Gurevich","year":"2013","journal-title":"Bioinformatics"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2068","DOI":"10.1093\/bioinformatics\/btu153","article-title":"Prokka: Rapid prokaryotic genome annotation","volume":"30","author":"Seemann","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1016\/j.bsheal.2020.05.001","article-title":"Prevalence and Transmission of Mobilized Colistin Resistance (Mcr) Gene in Bacteria Common to Animals and Humans","volume":"2","author":"Luo","year":"2020","journal-title":"Biosaf. Health"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Carfora, V., Alba, P., Leekitcharoenphon, P., Ballar\u00f2, D., Cordaro, G., Di Matteo, P., Donati, V., Ianzano, A., Iurescia, M., and Stravino, F. (2018). Corrigendum: Colistin Resistance Mediated by Mcr-1 in ESBL-Producing, Multidrug Resistant Salmonella Infantis in Broiler Chicken Industry, Italy (2016\u20132017). Front. Microbiol., 9.","DOI":"10.3389\/fmicb.2018.02395"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"V\u00e1zquez, X., Garc\u00eda, V., Fern\u00e1ndez, J., Bances, M., de Toro, M., Ladero, V., Rodicio, R., and Rodicio, M.R. (2021). Colistin Resistance in Monophasic Isolates of Salmonella Enterica ST34 Collected from Meat-Derived Products in Spain, with or without CMY-2 Co-Production. Front. Microbiol., 12.","DOI":"10.3389\/fmicb.2021.735364"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Maron, D.F., Smith, T.J.S., and Nachman, K.E. (2013). Restrictions on Antimicrobial Use in Food Animal Production: An International Regulatory and Economic Survey. Glob. Health, 9.","DOI":"10.1186\/1744-8603-9-48"},{"key":"ref_30","unstructured":"Moulin, G., Catry, B., Baptiste, K., Cavaco, L., Jukes, H., and Kluytmans, J. (2016). Updated Advice on the Use of Colistin Products in Animals within the European Union: Development of Resistance and Possible Impact on Human and Animal Health. Eur. Med. Agency, 44."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1102","DOI":"10.1016\/S1473-3099(16)30329-2","article-title":"China Bans Colistin as a Feed Additive for Animals","volume":"16","author":"Walsh","year":"2016","journal-title":"Lancet Infect. Dis."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Uddin, M.B., Hossain, S.M.B., Hasan, M., Alam, M.N., Debnath, M., Begum, R., Roy, S., Harun-Al-Rashid, A., Chowdhury, M.S.R., and Rahman, M.M. (2021). Multidrug Antimicrobial Resistance and Molecular Detection of Mcr-1 Gene in Salmonella Species Isolated from Chicken. Animals, 11.","DOI":"10.3390\/ani11010206"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1089\/fpd.2011.1015","article-title":"Tentative Colistin Epidemiological Cut-off Value for Salmonella spp","volume":"9","author":"Torpdahl","year":"2012","journal-title":"Foodborne Pathog. Dis."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"66","DOI":"10.1016\/j.diagmicrobio.2017.12.015","article-title":"Spread of Colistin Resistance Gene Mcr-1 in Italy: Characterization of the Mcr-1.2 Allelic Variant in a Colistin-Resistant Blood Isolate of Escherichia Coli","volume":"91","author":"Simoni","year":"2018","journal-title":"Diagn. Microbiol. Infect. Dis."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"5612","DOI":"10.1128\/AAC.01075-16","article-title":"Mcr-1.2, a New Mcr Variant Carried on a Transferable Plasmid from a Colistin-Resistant KPC Carbapenemase-Producing Klebsiella Pneumoniae Strain of Sequence Type 512","volume":"60","author":"Arena","year":"2016","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Alba, P., Leekitcharoenphon, P., Franco, A., Feltrin, F., Ianzano, A., Caprioli, A., Stravino, F., Hendriksen, R.S., Bortolaia, V., and Battisti, A. (2018). Molecular Epidemiology of Mcr-Encoded Colistin Resistance in Enterobacteriaceae from Food-Producing Animals in Italy Revealed through the EU Harmonized Antimicrobial Resistance Monitoring. Front. Microbiol., 9.","DOI":"10.3389\/fmicb.2018.01217"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Vi\u00f1es, J., Cusc\u00f3, A., Napp, S., Alvarez, J., Saez-Llorente, J.L., Ros\u00e0s-Rodoreda, M., Francino, O., and Migura-Garcia, L. (2021). Transmission of Similar Mcr-1 Carrying Plasmids among Different Escherichia coli Lineages Isolated from Livestock and the Farmer. Antibiotics, 10.","DOI":"10.3390\/antibiotics10030313"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"EFSA Panel on Biological Hazards (EFSA BIOHAZ Panel), Koutsoumanis, K., Allende, A., Alvarez-Ord\u00f3\u00f1ez, A., Bolton, D., Bover-Cid, S., Chemaly, M., De Cesare, A., Herman, L., and Hilbert, F. (2019). Salmonella Control in Poultry Flocks and Its Public Health Impact. EFSA J., 17.","DOI":"10.2903\/j.efsa.2019.5596"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"358","DOI":"10.1016\/j.ijantimicag.2005.02.006","article-title":"Mechanisms of Quinolone Resistance in Escherichia coli and Salmonella: Recent Developments","volume":"25","author":"Hopkins","year":"2005","journal-title":"Int. J. Antimicrob. Agents"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"4012","DOI":"10.1128\/AAC.48.10.4012-4015.2004","article-title":"Prevalence of Mutations within the Quinolone Resistance-Determining Region of GyrA, GyrB, ParC, and ParE and Association with Antibiotic Resistance in Quinolone-Resistant Salmonella Enterica","volume":"48","author":"Eaves","year":"2004","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"14242","DOI":"10.1021\/acsomega.9b01660","article-title":"Characterization and Solution Properties of Quaternary-Ammonium-Salt-Type Amphiphilic Gemini Ionic Liquids","volume":"4","author":"Kawai","year":"2019","journal-title":"ACS Omega"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"275","DOI":"10.17221\/6013-VETMED","article-title":"Genetic Basis of Resistance to Quaternary Ammonium Compounds\u2014The Qac Genes and Their Role: A Review","volume":"57","author":"Jaglic","year":"2012","journal-title":"Vet. Med."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1211\/0022357001773742","article-title":"Do Biocides Select for Antibiotic Resistance?","volume":"52","author":"Russell","year":"2000","journal-title":"J. Pharm. Pharmacol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1016\/j.ijfoodmicro.2017.07.009","article-title":"Characterization and Horizontal Transfer of QacH-Associated Class 1 Integrons in Escherichia coli Isolated from Retail Meats","volume":"258","author":"Jiang","year":"2017","journal-title":"Int. J. Food Microbiol."},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Aconiti Mandolini, N., Perugini, G., Filippini, G., Pierucci, P., Baiguini, A., Vaccaro, A., Pelagalli, G., Marinelli, F., and Capuccella, M. (2020). Evaluation of Colistin Consumption in Swine and Poultry of Marche Region during the 2017\u20132019 Period. Eur. J. Public Health, 30.","DOI":"10.1093\/eurpub\/ckaa166.1305"}],"container-title":["Antibiotics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-6382\/11\/6\/728\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T23:20:43Z","timestamp":1760138443000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-6382\/11\/6\/728"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,5,28]]},"references-count":46,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2022,6]]}},"alternative-id":["antibiotics11060728"],"URL":"https:\/\/doi.org\/10.3390\/antibiotics11060728","relation":{},"ISSN":["2079-6382"],"issn-type":[{"value":"2079-6382","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,5,28]]}}}