Salmonella enterica subspecies enterica serotype Choleraesuis is a swine adapted serovar. S. Choleraesuis variant Kunzendorf is responsible for the majority of outbreaks among pigs. S. Choleraesuis is rare in Europe, although there have been serious outbreaks in pigs including two outbreaks in Denmark in 1999-2000 and 2012-2013. Here, we elucidate the epidemiology, possible transmission routes and sources, and clonality of European S. Choleraesuis isolates including the Danish outbreak isolates. A total of 102 S. Choleraesuis isolates from different European countries and the United States, covering available isolates from the last two decades were selected for whole genome sequencing. We applied a temporally structured sequence analysis within a Bayesian framework to reconstruct a temporal and spatial phylogenetic tree. MLST type, resistance genes, plasmid replicons, and accessory genes were identified using bioinformatics tools. Fifty-eight isolates including 11 out of 12 strains from wild boars were pan-susceptible. The remaining isolates carried multiple resistance genes. Eleven different plasmid replicons in eight plasmids were determined among the isolates. Accessory genes were associated to the identified resistance genes and plasmids. The European S. Choleraesuis was estimated to have emerged in ∼1837 (95% credible interval, 1733-1983) with the mutation rate of 1.02 SNPs/genome/year. The isolates were clustered according to countries and neighbor countries. There were transmission events between strains from the United States and European countries. Wild boar and pig isolates were genetically linked suggesting cross-border transmission and transmission due to a wildlife reservoir. The phylogenetic tree shows that multiple introductions were responsible for the outbreak of 2012-2013 in Denmark, and suggests that poorly disinfected vehicles crossing the border into Denmark were potentially the source of the outbreak. Low levels of single nucleotide polymorphisms (SNPs) differences (0-4 SNPs) can be observed between clonal strains isolated from different organs of the same animal. Proper disinfection of livestock vehicles and improved quality control of livestock feed could help to prevent future spread of S. Choleraesuis or other more serious infectious diseases such as African swine fever (ASF) in the European pig production system.

Bakteriológiai Laboratórium Állategészségügyi Diagnosztikai Igazgatóság Nemzeti Élelmiszerlánc biztonsági Hivatal Budapest Hungary

Department of Agriculture Food and the Marine Laboratories Celbridge Ireland

Division of Animal and Food Microbiology Center for Veterinary Medicine United States Food and Drug Administration Laurel MD United States

European Union Reference Laboratory for Antimicrobial Resistance WHO Collaborating Center for Antimicrobial Resistance in Food Borne Pathogens and Genomics Research Group for Genomic Epidemiology National Food Institute Kongens Lyngby Denmark

French Agency for Food Environmental and Occupational Health and Safety Maisons Alfort France

Istituto Zooprofilattico Sperimentale del Lazio e della Toscana National Reference Laboratory for Antimicrobial Resistance Rome Italy

National Salmonella Reference Laboratory Unit Molecular Microbiology and Genome Analysis Federal Institute for Risk Assessment Berlin Germany

National Veterinary Institute Technical University of Denmark Kongens Lyngby Denmark

National Veterinary Research Institute Department of Microbiology National Reference Laboratory for Salmonellosis and Antimicrobial Resistance Puławy Poland

NRC Salmonella Austrian Agency for Health and Food Safety Graz Austria

RISE Research Institutes of Sweden Lund Sweden

Státní Veterinární Ústav Praha Prague Czechia

Veterinaar ja Toidulaboratoorium Tartu Estonia

Zobrazit více v PubMed

Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. (1990). Basic local alignment search tool. J. Mol. Biol. 215 403–410. 10.1016/S0022-2836(05)80360-2 PubMed DOI

Baggesen D., Christensen J., Jensen T., Skov M., Sørensen G., Sørensen V. (2000). Udbrud af Salmonella enterica subsp. enterica serovar Choleraesuis var. Kunzendorf (S. Choleraesuia) i en dansk svinebesætning. Dansk Veterinærtidsskrift 83 6–12.

Barnes D., Sorensen D. (1975). “Salmonellosis,” in Diseases of Swine, eds Dunne H. W., Leman A. (Ames: Iowa State University Press; ), 554–564.

Bielejec F., Baele G., Vrancken B., Suchard M. A., Rambaut A., Lemey P. (2016). SpreaD3: interactive visualization of spatiotemporal history and trait evolutionary processes. Mol. Biol. Evol. 33 2167–2169. 10.1093/molbev/msw082 PubMed DOI PMC

Biester H. (1958). Diseases of Swine. Ames: The Iowa State College Press, 317–333.

Carattoli A., Zankari E., Garciá-Fernández A., Larsen M. V., Lund O., Villa L., et al. (2014). In Silico detection and typing of plasmids using plasmidfinder and plasmid multilocus sequence typing. Antimicrob. Agents Chemother. 58 3895–3903. 10.1128/AAC.02412-14 PubMed DOI PMC

Chiu C.-H., Su L.-H., Chu C. (2004). Salmonella enterica serotype Choleraesuis: epidemiology, pathogenesis, clinical disease, and treatment. Clin. Microbiol. Rev. 17 311–322. 10.1128/CMR.17.2.311-322.2004 PubMed DOI PMC

Chiu C.-H., Tang P., Chu C., Hu S., Bao Q., Yu J., et al. (2005). The genome sequence of Salmonella enterica serovar Choleraesuis, a highly invasive and resistant Zoonotic pathogen. Nucleic Acids Res. 33 1690–1698. 10.1093/nar/gki297 PubMed DOI PMC

Chiu C.-H., Wu T.-L., Su L.-H., Chu C., Chia J.-H., Kuo A.-J., et al. (2002). The emergence in Taiwan of fluoroquinolone resistance in Salmonella enterica serotype choleraesuis. N. Engl. J. Med. 346 413–419. 10.1056/NEJMoa012261 PubMed DOI

Coyle E. F., Palmer S. R., Ribeiro C. D., Jones H. I., Howard A. J., Ward L., et al. (1988). Salmonella enteritidis phage type 4 infection: association with hen’s eggs. Lancet 2 1295–1297. 10.1016/S0140-6736(88)92902-9 PubMed DOI

Drummond A. J., Ho S. Y. W., Phillips M. J., Rambaut A. (2006). Relaxed phylogenetics and dating with confidence. PLoS Biol. 4:e88. 10.1371/journal.pbio.0040088 PubMed DOI PMC

Drummond A. J., Rambaut A. (2007). BEAST: bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 7:214. 10.1186/1471-2148-7-214 PubMed DOI PMC

Drummond A. J., Suchard M. A., Xie D., Rambaut A. (2012). Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol. Biol. Evol. 29 1969–1973. 10.1093/molbev/mss075 PubMed DOI PMC

Elder J. R., Chiok K. L., Paul N. C., Haldorson G., Guard J., Shah D. H. (2016). The Salmonella pathogenicity island 13 contributes to pathogenesis in streptomycin pre-treated mice but not in day-old chickens. Gut Pathog. 8:16. 10.1186/s13099-016-0098-0 PubMed DOI PMC

Fedorka-Cray P. J., Gray J. T., Wray C. (2000). “Salmonella infections in pigs,” in Salmonella in Domestic Animals, (Wallingford: CABI; ), 191–207. 10.1079/9780851992617.0191 DOI

Frantz L. A. F., Schraiber J. G., Madsen O., Megens H.-J., Cagan A., Bosse M., et al. (2015). Evidence of long-term gene flow and selection during domestication from analyses of Eurasian wild and domestic pig genomes. Nat. Genet. 47 1141–1148. 10.1038/ng.3394 PubMed DOI

Galán J. E. (2001). Salmonella interactions with host cells: type III secretion at work. Annu. Rev. Cell Dev. Biol. 17 53–86. 10.1146/annurev.cellbio.17.1.53 PubMed DOI

Gilmour M. W., Thomson N. R., Sanders M., Parkhill J., Taylor D. E. (2004). The complete nucleotide sequence of the resistance plasmid R478: defining the backbone components of incompatibility group H conjugative plasmids through comparative genomics. Plasmid 52 182–202. 10.1016/j.plasmid.2004.06.006 PubMed DOI

Griffith R., Schwartz K., Meyerholz D. (2006). Diseases in Swine, Oxford: Blackwell Publishing, 739–754.

Guindon S., Dufayard J. F., Lefort V., Anisimova M., Hordijk W., Gascuel O. (2010). New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59 307–321. 10.1093/sysbio/syq010 PubMed DOI

Hawkey J., Edwards D. J., Dimovski K., Hiley L., Billman-Jacobe H., Hogg G., et al. (2013). Evidence of microevolution of Salmonella Typhimurium during a series of egg-associated outbreaks linked to a single chicken farm. BMC Genomics 14:800. 10.1186/1471-2164-14-800 PubMed DOI PMC

Helms M., Ethelberg S., Mølbak K., Lightfoot D., Powling J., Berghold C., et al. (2005). International Salmonella Typhimurium DT104 infections, 1992-2001. Emerg. Infect. Dis. 11 859–867. 10.3201/eid1106.041017 PubMed DOI PMC

Hendriksen R. S., Vieira A. R., Karlsmose S., Lo Fo Wong D. M. A., Jensen A. B., Wegener H. C., et al. (2011). Global monitoring of Salmonella serovar distribution from the World Health Organization global foodborne infections network country data bank: results of quality assured laboratories from 2001 to 2007. Foodborne Pathog. Dis. 8 887–900. 10.1089/fpd.2010.0787 PubMed DOI

Humphrey T. J., Hart R. J. (1988). Campylobacter and Salmonella contamination of unpasteurized cows’ milk on sale to the public. J. Appl. Bacteriol. 65 463–467. 10.1111/j.1365-2672.1988.tb01918.x PubMed DOI

Hyatt D., Chen G.-L., Locascio P. F., Land M. L., Larimer F. W., Hauser L. J. (2010). Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11:119. 10.1186/1471-2105-11-119 PubMed DOI PMC

Jori F., Laval M., Maestrini O., Casabianca F., Charrier F., Pavio N. (2016). Assessment of domestic pigs, wild boars and feral hybrid pigs as reservoirs of Hepatitis E Virus in Corsica, France. Viruses 8:E236. 10.3390/v8080236 PubMed DOI PMC

Kaas R. S., Leekitcharoenphon P., Aarestrup F. M., Lund O. (2014). Solving the problem of comparing whole bacterial genomes across different sequencing platforms. PLoS One 9:e104984. 10.1371/journal.pone.0104984 PubMed DOI PMC

Kingsley R. A., Humphries A. D., Weening E. H., De Zoete M. R., Winter S., Papaconstantinopoulou A., et al. (2003). Molecular and phenotypic analysis of the CS54 island of Salmonella enterica serotype typhimurium: identification of intestinal colonization and persistence determinants. Infect. Immun. 71 629–640. 10.1128/IAI.71.2.629-640.2003 PubMed DOI PMC

Kingsley R. A., Msefula C. L., Thomson N. R., Kariuki S., Holt K. E., Gordon M. A., et al. (2009). Epidemic multiple drug resistant Salmonella Typhimurium causing invasive disease in sub-Saharan Africa have a distinct genotype. Genome Res. 19 2279–2287. 10.1101/gr.091017.109 PubMed DOI PMC

Komano T., Kubo A., Nisioka T. (1987). Shufflon: multi-inversion of four contiguous DNA segments of plasmid R64 creates seven different open reading frames. Nucleic Acids Res. 15 1165–1172. 10.1093/nar/15.3.1165 PubMed DOI PMC

Kröger C., Dillon S. C., Cameron A. D. S., Papenfort K., Sivasankaran S. K., Hokamp K., et al. (2012). The transcriptional landscape and small RNAs of Salmonella enterica serovar Typhimurium. Proc. Natl. Acad. Sci. U.S.A. 109 E1277–E1286. 10.1073/pnas.1201061109 PubMed DOI PMC

Larsen M. V., Cosentino S., Rasmussen S., Friis C., Hasman H., Marvig R. L., et al. (2012). Multilocus sequence typing of total-genome-sequenced bacteria. J. Clin. Microbiol. 50 1355–1361. 10.1128/JCM.06094-11 PubMed DOI PMC

Leekitcharoenphon P., Hendriksen R. S., Le Hello S., Weill F.-X., Baggesen D. L., Jun S.-R., et al. (2016). Global genomic epidemiology of Salmonella enterica serovar typhimurium DT104. Appl. Environ. Microbiol. 82 2516–2526. 10.1128/AEM.03821-15 PubMed DOI PMC

Leekitcharoenphon P., Kaas R. S., Thomsen M. C. F., Friis C., Rasmussen S., Aarestrup F. M. (2012a). snpTree–a web-server to identify and construct SNP trees from whole genome sequence data. BMC Genomics 13(Suppl. 7):S6. 10.1186/1471-2164-13-S7-S6 PubMed DOI PMC

Leekitcharoenphon P., Lukjancenko O., Friis C., Aarestrup F. M., Ussery D. W. (2012b). Genomic variation in Salmonella enterica core genes for epidemiological typing. BMC Genomics 13:88. 10.1186/1471-2164-13-88 PubMed DOI PMC

Lemey P., Rambaut A., Drummond A. J., Suchard M. A. (2009). Bayesian phylogeography finds its roots. PLoS Comput. Biol. 5:e1000520. 10.1371/journal.pcbi.1000520 PubMed DOI PMC

Lewiński T., Rozvany G. I. N., Sokół T., Bołbotowski K. (2013). Exact analytical solutions for some popular benchmark problems in topology optimization III: L-shaped domains revisited. Struct. Multidiscip. Optim. 47 937–942. 10.1007/s00158-012-0865-6 DOI

Li H., Durbin R. (2009). Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25 1754–1760. 10.1093/bioinformatics/btp324 PubMed DOI PMC

Li H., Handsaker B., Wysoker A., Fennell T., Ruan J., Homer N., et al. (2009). The sequence alignment/map format and SAMtools. Bioinformatics 25 2078–2079. 10.1093/bioinformatics/btp352 PubMed DOI PMC

Liu W.-Q., Feng Y., Wang Y., Zou Q.-H., Chen F., Guo J.-T., et al. (2009). Salmonella paratyphi C: genetic divergence from Salmonella choleraesuis and pathogenic convergence with Salmonella typhi. PLoS One 4:e4510. 10.1371/journal.pone.0004510 PubMed DOI PMC

Okoro C. K., Kingsley R. A., Connor T. R., Harris S. R., Parry C. M., Al-Mashhadani M. N., et al. (2012). Intracontinental spread of human invasive Salmonella Typhimurium pathovariants in sub-Saharan Africa. Nat. Genet. 44 1215–1221. 10.1038/ng.2423 PubMed DOI PMC

Parkhill J., Dougan G., James K. D., Thomson N. R., Pickard D., Wain J., et al. (2001). Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18. Nature 413 848–852. 10.1038/35101607 PubMed DOI

Pedersen K., Sørensen G., Löfström C., Leekitcharoenphon P., Nielsen B., Wingstrand A., et al. (2015). Reappearance of Salmonella serovar Choleraesuis var. Kunzendorf in Danish pig herds. Vet. Microbiol. 176 282–291. 10.1016/j.vetmic.2015.01.004 PubMed DOI

Sherburne C. K., Lawley T. D., Gilmour M. W., Blattner F. R., Burland V., Grotbeck E., et al. (2000). The complete DNA sequence and analysis of R27 a large IncHI plasmid from Salmonella typhi that is temperature sensitive for transfer. Nucleic Acids Res. 28 2177–2186. 10.1093/nar/28.10.2177 PubMed DOI PMC

Sirichote P., Hasman H., Pulsrikarn C., Schønheyder H. C., Samulioniené J., Pornruangmong S., et al. (2010). Molecular characterization of extended-spectrum cephalosporinase-producing Salmonella enterica serovar Choleraesuis isolates from patients in Thailand and Denmark. J. Clin. Microbiol. 48 883–888. 10.1128/JCM.01792-09 PubMed DOI PMC

Suchard M. A., Lemey P., Baele G., Ayres D. L., Drummond A. J., Rambaut A. (2018). Bayesian phylogenetic and phylodynamic data integration using BEAST 1.10. Virus Evol. 4:vey016. 10.1093/ve/vey016 PubMed DOI PMC

Tettelin H., Masignani V., Cieslewicz M. J., Donati C., Medini D., Ward N. L., et al. (2005). Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: implications for the microbial “pan-genome”. Proc. Natl. Acad. Sci. U.S.A. 102 13950–13955. 10.1073/pnas.0506758102 PubMed DOI PMC

Yu H., Wang J., Ye J., Tang P., Chu C., Hu S., et al. (2006). Complete nucleotide sequence of pSCV50 the virulence plasmid of Salmonella enterica serovar Choleraesuis SC-B67. Plasmid 55 145–151. 10.1016/j.plasmid.2005.09.001 PubMed DOI

Zankari E., Hasman H., Cosentino S., Vestergaard M., Rasmussen S., Lund O., et al. (2012). Identification of acquired antimicrobial resistance genes. J. Antimicrob. Chemother. 67 2640–2644. 10.1093/jac/dks261 PubMed DOI PMC

Zerbino D. R., Birney E. (2008). Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 18 821–829. 10.1101/gr.074492.107 PubMed DOI PMC

Zhang S., Yin Y., Jones M. B., Zhang Z., Deatherage Kaiser B. L., Dinsmore B. A., et al. (2015). Salmonella serotype determination utilizing high-throughput genome sequencing data. J. Clin. Microbiol. 53 1685–1692. 10.1128/JCM.00323-15 PubMed DOI PMC

Zhou Z., McCann A., Weill F.-X., Blin C., Nair S., Wain J., et al. (2014). Transient darwinian selection in Salmonella enterica serovar Paratyphi A during 450 years of global spread of enteric fever. Proc. Natl. Acad. Sci. U.S.A. 111 12199–12204. 10.1073/pnas.1411012111 PubMed DOI PMC