{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,16]],"date-time":"2026-10-16T16:16:43Z","timestamp":1792167403103,"version":"4.4.0"},"reference-count":39,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2025,7,16]],"date-time":"2025-07-16T00:00:00Z","timestamp":1752624000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"\u201cFondo de Investigaci\u00f3n Sanitaria, Instituto de Salud Carlos III, Ministerio de Econom\u00eda y Competitividad\u201d","award":["FIS PI17\/00474"],"award-info":[{"award-number":["FIS PI17\/00474"]}]},{"name":"\u201cFondo de Investigaci\u00f3n Sanitaria, Instituto de Salud Carlos III, Ministerio de Econom\u00eda y Competitividad\u201d","award":["BP17-08"],"award-info":[{"award-number":["BP17-08"]}]},{"name":"European Regional Development Fund of the European Union","award":["FIS PI17\/00474"],"award-info":[{"award-number":["FIS PI17\/00474"]}]},{"name":"European Regional Development Fund of the European Union","award":["BP17-08"],"award-info":[{"award-number":["BP17-08"]}]},{"name":"Program \u201cSevero Ochoa\u201d","award":["FIS PI17\/00474"],"award-info":[{"award-number":["FIS PI17\/00474"]}]},{"name":"Program \u201cSevero Ochoa\u201d","award":["BP17-08"],"award-info":[{"award-number":["BP17-08"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Antibiotics"],"abstract":"<jats:p>Background\/Objective: The present study focused on the analysis of the Spanish clone belonging to the successful 4,[5],12:i:- monophasic variant of Salmonella enterica serovar Typhimurium. Methods: All isolates of the clone recovered in a Spanish region from human clinical samples between 2008 and 2018 (N = 14) were investigated using microbiological approaches and genome sequence analysis. In addition, they were compared with isolates from the years 2000 to 2003 (N = 21), which were previously characterized but had not yet been sequenced. Results: Phylogenetic analyses indicate that all isolates are closely related (differing by 1 to 103 SNPs) but belong to two clades termed A and B. With few exceptions, clade A comprised isolates of the first period, also including two \u201colder\u201d control strains, LSP 389\/97 and LSP 272\/98. Clade B only contained isolates from the second period. Isolates from both periods were resistant to antibiotics and biocides, with almost all resistance genes located on large IncC plasmids, additionally carrying pSLT-derived virulence genes. The number of resistance genes was highly variable, resulting in a total of 22 ABR (antibiotic biocide resistance) profiles. The number of antibiotic resistance genes, but not that of biocide resistance genes, was considerably lower in isolates from the second than from the first period (with averages of 5.5 versus 9.6 genes). Importantly, IS26, which resides in multiple copies within these plasmids, appears to be playing a crucial role in the evolution of resistance, and it was also responsible for the monophasic phenotype, which was associated with four different deletions eliminating the fljAB region. Conclusions: the observed reduction in the number of antibiotic resistance genes could correlate with the loss of adaptive advantage originating from the ban on the use of antibiotics as feed additives implemented in the European Union since 2006, facilitated by the intrinsic instability of the IncC plasmids. Two consecutive IS26 transposition events, which can explain both the clonal relationship of the isolates and their variability, may account for the observed fljAB deletions.<\/jats:p>","DOI":"10.3390\/antibiotics14070711","type":"journal-article","created":{"date-parts":[[2025,7,16]],"date-time":"2025-07-16T10:46:05Z","timestamp":1752662765000},"page":"711","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Epidemiological Surveillance, Variability, and Evolution of Isolates Belonging to the Spanish Clone of the 4,[5],12:i:- Monophasic Variant of Salmonella enterica Serovar Typhimurium"],"prefix":"10.3390","volume":"14","author":[{"given":"Xenia","family":"V\u00e1zquez","sequence":"first","affiliation":[{"name":"Departamento de Biolog\u00eda Funcional, \u00c1rea de Microbiolog\u00eda, Universidad de Oviedo (UO), 33006 Oviedo, Spain"},{"name":"Grupo de Microbiolog\u00eda Traslacional, Instituto de Investigaci\u00f3n Sanitaria del Principado de Asturias (ISPA), 33011 Oviedo, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0418-296X","authenticated-orcid":false,"given":"Patricia","family":"Garc\u00eda","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Biomedical Research Institute A Coru\u00f1a (INIBIC), University Hospital A Coru\u00f1a (CHUAC), 15006 A Coru\u00f1a, Spain"},{"name":"Centro de Investigaci\u00f3n Biom\u00e9dica en Red de Enfermedades Infecciosas (CIBERINFECT), Instituto de Salud Carlos III, 28029 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7081-0936","authenticated-orcid":false,"given":"Javier","family":"Fern\u00e1ndez","sequence":"additional","affiliation":[{"name":"Departamento de Biolog\u00eda Funcional, \u00c1rea de Microbiolog\u00eda, Universidad de Oviedo (UO), 33006 Oviedo, Spain"},{"name":"Grupo de Microbiolog\u00eda Traslacional, Instituto de Investigaci\u00f3n Sanitaria del Principado de Asturias (ISPA), 33011 Oviedo, Spain"},{"name":"Servicio de Microbiolog\u00eda, Hospital Universitario Central de Asturias (HUCA), 33011 Oviedo, Spain"},{"name":"Centro de Investigaci\u00f3n Biom\u00e9dica en Red-Enfermedades Respiratorias, 30627 Madrid, Spain"},{"name":"Research & Innovation, Artificial Intelligence and Statistical Department, Pragmatech AI Solutions, 33001 Oviedo, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7613-3745","authenticated-orcid":false,"given":"V\u00edctor","family":"Ladero","sequence":"additional","affiliation":[{"name":"Department of Technology and Biotechnology, Instituto de Productos L\u00e1cteos de Asturias, Consejo Superior de Investigaciones Cient\u00edficas (IPLA-CSIC), 33011 Oviedo, Spain"},{"name":"Grupo de Microbiolog\u00eda Molecular, Instituto de Investigaci\u00f3n Sanitaria del Principado de Asturias (ISPA), 33011 Oviedo, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0495-4712","authenticated-orcid":false,"given":"Carlos","family":"Rodr\u00edguez-Lucas","sequence":"additional","affiliation":[{"name":"Grupo de Microbiolog\u00eda Traslacional, Instituto de Investigaci\u00f3n Sanitaria del Principado de Asturias (ISPA), 33011 Oviedo, Spain"},{"name":"Servicio de Microbiolog\u00eda, Hospital Universitario Central de Asturias (HUCA), 33011 Oviedo, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4197-4285","authenticated-orcid":false,"given":"J\u00fcrgen J.","family":"Heinisch","sequence":"additional","affiliation":[{"name":"Department of Genetics, Faculty of Biology and Chemistry, University of Osnabr\u00fcck, Barbarastrasse 11, D-49076 Osnabr\u00fcck, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rosaura","family":"Rodicio","sequence":"additional","affiliation":[{"name":"Grupo de Microbiolog\u00eda Traslacional, Instituto de Investigaci\u00f3n Sanitaria del Principado de Asturias (ISPA), 33011 Oviedo, Spain"},{"name":"Departamento de Bioqu\u00edmica y Biolog\u00eda Molecular, Universidad de Oviedo (UO), 33006 Oviedo, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3402-2908","authenticated-orcid":false,"given":"M. Rosario","family":"Rodicio","sequence":"additional","affiliation":[{"name":"Departamento de Biolog\u00eda Funcional, \u00c1rea de Microbiolog\u00eda, Universidad de Oviedo (UO), 33006 Oviedo, Spain"},{"name":"Grupo de Microbiolog\u00eda Traslacional, Instituto de Investigaci\u00f3n Sanitaria del Principado de Asturias (ISPA), 33011 Oviedo, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,7,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"882","DOI":"10.1086\/650733","article-title":"The global burden of nontyphoidal Salmonella gastroenteritis","volume":"50","author":"Majowicz","year":"2010","journal-title":"Clin. Infect. Dis."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"526","DOI":"10.1016\/j.resmic.2014.07.004","article-title":"Supplement 2008-2010 (no. 48) to the White-Kauffmann-Le Minor scheme","volume":"165","author":"Roggentin","year":"2014","journal-title":"Res. Microbiol."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"ECDC (European Food Safety Authority and European Centre for Disease, Prevention and Control) (2023). The European Union One Health 2022 Zoonoses Report. EFSA J., 21, e8442.","DOI":"10.2903\/j.efsa.2023.8442"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"EFSA (European Food Safety Authority) Panel on Biological Hazards (BIOHAZ) (2010). Scientific Opinion on monitoring and assessment of the public health risk of \u201cSalmonella typhimurium-like\u201d strains. EFSA J., 8, 1826.","DOI":"10.2903\/j.efsa.2010.1826"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"ECDC (European Food Safety Authority and European Centre for Disease, Prevention and Control) (2022). The European Union One Health 2021 Zoonoses Report. EFSA J., 20, e07666.","DOI":"10.2903\/j.efsa.2022.7666"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1089\/fpd.2019.2676","article-title":"The epidemiology of monophasic Salmonella Typhimurium","volume":"17","author":"Sun","year":"2020","journal-title":"Foodborne Pathog. Dis."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"407","DOI":"10.1089\/fpd.2008.0213","article-title":"Emergence, distribution, and molecular and phenotypic characteristics of Salmonella enterica serotype 4,5,12:i","volume":"6","author":"Switt","year":"2009","journal-title":"Foodborne Pathog. Dis."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"845","DOI":"10.1016\/0092-8674(80)90075-6","article-title":"Phase variation: Genetic analysis of switching mutants","volume":"19","author":"Silverman","year":"1980","journal-title":"Cell"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"3546","DOI":"10.1128\/JCM.00546-09","article-title":"Salmonella enterica serotype 4,5,12:i:-, an emerging Salmonella serotype that represents multiple distinct clones","volume":"47","author":"Soyer","year":"2009","journal-title":"J. Clin. Microbiol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2028","DOI":"10.1093\/jac\/dkr228","article-title":"Leakage of emerging clinically relevant multidrug-resistant Salmonella clones from pig farms","volume":"66","author":"Antunes","year":"2011","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2395","DOI":"10.1128\/JCM.41.6.2395-2400.2003","article-title":"Several Salmonella enterica subsp. enterica serotype 4,5,12:i:- phage types isolated from swine samples originate from serotype Typhimurium DT U302","volume":"41","author":"Zapata","year":"2003","journal-title":"J. Clin. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Echeita, M.A., Aladuena, A., Cruchaga, S., and Usera, M.A. (1999). Emergence and spread of an atypical Salmonella enterica subsp. enterica serotype 4,5,12:i:- strain in Spain. J. Clin. Microbiol., 37.","DOI":"10.1128\/JCM.37.10.3425-3425.1999"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2981","DOI":"10.1128\/JCM.39.8.2981-2983.2001","article-title":"Atypical, fljB-negative Salmonella enterica subsp. enterica strain of serovar 4,5,12:i:- appears to be a monophasic variant of serovar Typhimurium","volume":"39","author":"Echeita","year":"2001","journal-title":"J. Clin. Microbiol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"543","DOI":"10.1093\/jac\/dkq481","article-title":"IncA\/C plasmids mediate antimicrobial resistance linked to virulence genes in the Spanish clone of the emerging Salmonella enterica serotype 4,[5],12:i","volume":"66","author":"Garcia","year":"2011","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"973","DOI":"10.1128\/JCM.02777-12","article-title":"Genetic types, gene repertoire, and evolution of isolates of the Salmonella enterica serovar 4,5,12:i:- Spanish clone assigned to different phage types","volume":"51","author":"Garcia","year":"2013","journal-title":"J. Clin. Microbiol."},{"key":"ref_16","first-page":"341","article-title":"Molecular characterisation of emergent multiresistant Salmonella enterica serotype [4,5,12:i:-] organisms causing human salmonellosis","volume":"190","author":"Guerra","year":"2000","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1305","DOI":"10.1128\/AAC.45.4.1305-1308.2001","article-title":"Multidrug resistance is mediated by large plasmids carrying a class 1 integron in the emergent Salmonella enterica serotype [4,5,12:i:-]","volume":"45","author":"Guerra","year":"2001","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Arrieta-Gisasola, A., Atxaerandio-Landa, A., Garrido, V., Grillo, M.J., Martinez-Ballesteros, I., Laorden, L., Garaizar, J., and Bikandi, J. (2020). Genotyping study of Salmonella 4,[5],12:i:- monophasic variant of serovar Typhimurium and characterization of the second-phase flagellar deletion by whole genome sequencing. Microorganisms, 8.","DOI":"10.3390\/microorganisms8122049"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2074","DOI":"10.1128\/JCM.40.6.2074-2078.2002","article-title":"DNA microarray-based typing of an atypical monophasic Salmonella enterica serovar","volume":"40","author":"Garaizar","year":"2002","journal-title":"J. Clin. Microbiol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"4563","DOI":"10.1128\/JCM.00337-10","article-title":"Genetic evolution of the Spanish multidrug-resistant Salmonella enterica 4,5,12:i:- monophasic variant","volume":"48","author":"Laorden","year":"2010","journal-title":"J. Clin. Microbiol."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"V\u00e1zquez, X., Garc\u00eda, P., Garc\u00eda, V., de Toro, M., Ladero, V., Heinisch, J.J., Fern\u00e1ndez, J., Rodicio, R., and Rodicio, M.R. (2021). Genomic analysis and phylogenetic position of the complex IncC plasmid found in the Spanish monophasic clone of Salmonella enterica serovar Typhimurium. Sci. Rep., 11.","DOI":"10.1038\/s41598-021-90299-z"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Hancock, S.J., Phan, M.D., Peters, K.M., Forde, B.M., Chong, T.M., Yin, W.F., Chan, K.G., Paterson, D.L., Walsh, T.R., and Beatson, S.A. (2017). Identification of IncA\/C plasmid replication and maintenance genes and development of a plasmid multilocus sequence typing scheme. Antimicrob. Agents Chemother., 61.","DOI":"10.1128\/AAC.01740-16"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2175","DOI":"10.1093\/jac\/dkr230","article-title":"GIsul2, a genomic island carrying the sul2 sulphonamide resistance gene and the small mobile element CR2 found in the Enterobacter cloacae subspecies cloacae type strain ATCC 13047 from 1890, Shigella flexneri ATCC 700930 from 1954 and Acinetobacter baumannii ATCC 17978 from 1951","volume":"66","author":"Nigro","year":"2011","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"852","DOI":"10.1038\/35101614","article-title":"Complete genome sequence of Salmonella enterica serovar Typhimurium LT2","volume":"413","author":"McClelland","year":"2001","journal-title":"Nature"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Branchu, P., Charity, O.J., Bawn, M., Thilliez, G., Dallman, T.J., Petrovska, L., and Kingsley, R.A. (2019). SGI-4 in monophasic Salmonella typhimurium ST34 is a novel ICE that enhances resistance to copper. Front. Microbiol., 10.","DOI":"10.3389\/fmicb.2019.01118"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"237","DOI":"10.1016\/S1286-4579(01)01375-2","article-title":"Non-typhoidal salmonellosis: Emerging problems","volume":"3","author":"Rabsch","year":"2001","journal-title":"Microbes Infect."},{"key":"ref_27","first-page":"mgen000456","article-title":"Whole-genome epidemiology links phage-mediated acquisition of a virulence gene to the clonal expansion of a pandemic Salmonella enterica serovar Typhimurium clone","volume":"6","author":"Tassinari","year":"2020","journal-title":"Microb. Genom."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Arrieta-Gisasola, A., Martinez-Ballesteros, I., Martinez-Malaxetxebarria, I., Bikandi, J., and Laorden, L. (2025). Detection of mobile genetic elements conferring resistance to heavy metals in Salmonella 4,[5],12:i:- and Salmonella Typhimurium serovars and their association with antibiotic resistance. Int. J. Food Microbiol., 426.","DOI":"10.1016\/j.ijfoodmicro.2024.110890"},{"key":"#cr-split#-ref_29.1","unstructured":"European Parliament and the Council of the European Union (2003). Regulation"},{"key":"#cr-split#-ref_29.2","unstructured":"(EC) No 1831\/2003 of the European Parliament and of the Council of 22 September 2003. Off. J. Eur. Union, 4."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Cadel-Six, S., Cherchame, E., Douarre, P.E., Tang, Y., Felten, A., Barbet, P., Litrup, E., Banerji, S., Simon, S., and Pasquali, F. (2021). The spatiotemporal dynamics and microevolution events that favored the success of the highly clonal multidrug-resistant monophasic Salmonella Typhimurium circulating in Europe. Front. Microbiol., 12.","DOI":"10.3389\/fmicb.2021.651124"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Mastrorilli, E., Pietrucci, D., Barco, L., Ammendola, S., Petrin, S., Longo, A., Mantovani, C., Battistoni, A., Ricci, A., and Desideri, A. (2018). A comparative genomic analysis provides novel insights Into the ecological success of the monophasic Salmonella serovar 4,[5],12:i. Front. Microbiol., 9.","DOI":"10.3389\/fmicb.2018.00715"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"610","DOI":"10.1016\/j.ijantimicag.2015.01.013","article-title":"Metal tolerance in emerging clinically relevant multidrug-resistant Salmonella enterica serotype 4,[5],12:i:- clones circulating in Europe","volume":"45","author":"Mourao","year":"2015","journal-title":"Int. J. Antimicrob. Agents"},{"key":"ref_33","unstructured":"Council of the European Union (1999). Council directive 1999\/29\/EC of 22 April 1999 on the undesirable substances and products in animal nutrition. Counc. Dir., 9, 32\u201346."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Harmer, C.J., and Hall, R.M. (2024). IS26 and the IS26 family: Versatile resistance gene movers and genome reorganizers. Microbiol. Mol. Biol. Rev., 88.","DOI":"10.1128\/mmbr.00119-22"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"He, S., Hickman, A.B., Varani, A.M., Siguier, P., Chandler, M., Dekker, J.P., and Dyda, F. (2015). Insertion sequence IS26 reorganizes plasmids in clinically isolated multidrug-resistant bacteria by replicative transposition. mBio, 6.","DOI":"10.1128\/mBio.00762-15"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"V\u00e1zquez, X., Fernandez, J., Heinisch, J.J., Rodicio, R., and Rodicio, M.R. (2024). Insights into the evolution of IncR plasmids found in the Southern European clone of the monophasic variant of Salmonella enterica serovar Typhimurium. Antibiotics, 13.","DOI":"10.3390\/antibiotics13040314"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1016\/j.mimet.2005.03.018","article-title":"Identification of plasmids by PCR-based replicon typing","volume":"63","author":"Carattoli","year":"2005","journal-title":"J. Microbiol. Methods"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"783","DOI":"10.2307\/2408678","article-title":"Confidence limits on phylogenies: An approach using the bootstrap","volume":"39","author":"Felsenstein","year":"1985","journal-title":"Evolution"}],"container-title":["Antibiotics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-6382\/14\/7\/711\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T18:10:30Z","timestamp":1760033430000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-6382\/14\/7\/711"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,7,16]]},"references-count":39,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2025,7]]}},"alternative-id":["antibiotics14070711"],"URL":"https:\/\/doi.org\/10.3390\/antibiotics14070711","relation":{},"ISSN":["2079-6382"],"issn-type":[{"value":"2079-6382","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,7,16]]}}}