Research Open Access
Like 0



In 2017, a food-borne Agona outbreak caused by infant milk products from a French supplier occurred in Europe. Simultaneously, Agona was detected in animal feed samples in Bavaria.


Using next generation sequencing (NGS) and three data analysis methods, this study’s objectives were to verify clonality of the Bavarian feed strains, rule out their connection to the outbreak, explore the genetic diversity of Bavarian Agona isolates from 1993 to 2018 and compare the analysis approaches employed, for practicality and ability to delineate outbreaks caused by the genetically monomorphic Agona serovar.


In this observational retrospective study, three 2017 Bavarian feed isolates were compared to a French outbreak isolate and 48 Agona isolates from our strain collections. The later included human, food, feed, veterinary and environmental isolates, of which 28 were epidemiologically outbreak related. All isolates were subjected to NGS and analysed by: (i) a publicly available species-specific core genome multilocus sequence typing (cgMLST) scheme, (ii) single nucleotide polymorphism phylogeny and (iii) an in-house serovar-specific cgMLST scheme. Using additional international Agona outbreak NGS data, the cluster resolution capacity of the two cgMLST schemes was assessed.


We could prove clonality of the feed isolates and exclude their relation to the French outbreak. All approaches confirmed former Bavarian epidemiological clusters.


Even for Agona, species-level cgMLST can produce reasonable resolution, being standardisable by public health laboratories. For single samples or homogeneous sample sets, higher resolution by serovar-specific cgMLST or SNP genotyping can facilitate outbreak investigations.


Article metrics loading...

Loading full text...

Full text loading...



  1. Rabsch W, Fruth A, Simon S, Szabo I, Malorny B. The zoonotic agent Salmonella. In: Sing A (eds), Zoonoses - Infections Affecting Humans and Animals. Dordrecht: Springer; 2015. p. 179-211. 10/1007978-94-017-9457-2.
  2. Koch J, Schrauder A, Alpers K, Werber D, Frank C, Prager R, et al. Salmonella agona outbreak from contaminated aniseed, Germany. Emerg Infect Dis. 2005;11(7):1124-7.  https://doi.org/10.3201/eid1107.041022  PMID: 16022796 
  3. Rabsch W, Prager R, Koch J, Stark K, Roggentin P, Bockemühl J, et al. Molecular epidemiology of Salmonella enterica serovar Agona: characterization of a diffuse outbreak caused by aniseed-fennel-caraway infusion. Epidemiol Infect. 2005;133(5):837-44.  https://doi.org/10.1017/S0950268805004152  PMID: 16181503 
  4. Nicolay N, Thornton L, Cotter S, Garvey P, Bannon O, McKeown P, et al. Salmonella enterica serovar Agona European outbreak associated with a food company. Epidemiol Infect. 2011;139(8):1272-80.  https://doi.org/10.1017/S0950268810002360  PMID: 20950515 
  5. Mba-Jonas A, Culpepper W, Hill T, Cantu V, Loera J, Borders J, et al. A Multistate Outbreak of Human Salmonella Agona Infections Associated With Consumption of Fresh, Whole Papayas Imported From Mexico-United States, 2011. Clin Infect Dis. 2018;66(11):1756-61.  https://doi.org/10.1093/cid/cix1094  PMID: 29471372 
  6. Thompson CK, Wang Q, Bag SK, Franklin N, Shadbolt CT, Howard P, et al. Epidemiology and whole genome sequencing of an ongoing point-source Salmonella Agona outbreak associated with sushi consumption in western Sydney, Australia 2015. Epidemiol Infect. 2017;145(10):2062-71.  https://doi.org/10.1017/S0950268817000693  PMID: 28462733 
  7. Hörmansdorfer S, Messelhäußer U, Rampp A, Schönberger K, Dallman T, Allerberger F, et al. Re-evaluation of a 2014 multi-country European outbreak of Salmonella Enteritidis phage type 14b using recent epidemiological and molecular data. Euro Surveill. 2017;22(50).  https://doi.org/10.2807/1560-7917.ES.2017.22.50.17-00196  PMID: 29258650 
  8. Achtman M, Wain J, Weill FX, Nair S, Zhou Z, Sangal V, et al. S. Enterica MLST Study Group. Multilocus sequence typing as a replacement for serotyping in Salmonella enterica. PLoS Pathog. 2012;8(6):e1002776.  https://doi.org/10.1371/journal.ppat.1002776  PMID: 22737074 
  9. Sandt CH, Krouse DA, Cook CR, Hackman AL, Chmielecki WA, Warren NG. The key role of pulsed-field gel electrophoresis in investigation of a large multiserotype and multistate food-borne outbreak of Salmonella infections centered in Pennsylvania. J Clin Microbiol. 2006;44(9):3208-12.  https://doi.org/10.1128/JCM.01404-06  PMID: 16954249 
  10. Long SG, DuPont HL, Gaul L, Arafat RR, Selwyn BJ, Rogers J, et al. Pulsed-field gel electrophoresis for Salmonella infection surveillance, Texas, USA, 2007. Emerg Infect Dis. 2010;16(6):983-5.  https://doi.org/10.3201/edi1606.091276  PMID: 20507752 
  11. Piras F, Spanu C, Mocci AM, Demontis M, Santis EPL, Scarano C. Occurrence and traceability of Salmonella spp. in five Sardinian fermented sausage facilities. Ital J Food Saf. 2019;8(1):8011.  https://doi.org/10.4081/ijfs.2019.8011  PMID: 31008088 
  12. van Belkum A. The role of short sequence repeats in epidemiologic typing. Curr Opin Microbiol. 1999;2(3):306-11.  https://doi.org/10.1016/S1369-5274(99)80053-8  PMID: 10383858 
  13. Lindstedt B-A. Multiple-locus variable number tandem repeats analysis for genetic fingerprinting of pathogenic bacteria. Electrophoresis. 2005;26(13):2567-82.  https://doi.org/10.1002/elps.200500096  PMID: 15937984 
  14. Wuyts V, Mattheus W, De Laminne de Bex G, Wildemauwe C, Roosens NH, Marchal K, et al. MLVA as a tool for public health surveillance of human Salmonella Typhimurium: prospective study in Belgium and evaluation of MLVA loci stability. PLoS One. 2013;8(12):e84055.  https://doi.org/10.1371/journal.pone.0084055  PMID: 24391880 
  15. Vignaud ML, Cherchame E, Marault M, Chaing E, Le Hello S, Michel V, et al. MLVA for Salmonella enterica subsp. enterica Serovar Dublin: Development of a Method Suitable for Inter-Laboratory Surveillance and Application in the Context of a Raw Milk Cheese Outbreak in France in 2012. Front Microbiol. 2017;8:295.  https://doi.org/10.3389/fmicb.2017.00295  PMID: 28289408 
  16. Peters T, Bertrand S, Björkman JT, Brandal LT, Brown DJ, Erdõsi T, et al. Multi-laboratory validation study of multilocus variable-number tandem repeat analysis (MLVA) for Salmonella enterica serovar Enteritidis, 2015. Euro Surveill. 2017;22(9):30477.  https://doi.org/10.2807/1560-7917.ES.2017.22.9.30477  PMID: 28277220 
  17. Malorny B, Junker E, Helmuth R. Multi-locus variable-number tandem repeat analysis for outbreak studies of Salmonella enterica serotype Enteritidis. BMC Microbiol. 2008;8(1):84.  https://doi.org/10.1186/1471-2180-8-84  PMID: 18513386 
  18. European Centre for Disease Prevention and Control (ECDC). Laboratory standard operating procedure for multiple-locus variable-number tandem repeat analysis of Salmonella enterica serotype Enteritidis. Stockholm: ECDC; 2016.  https://doi.org/http://dx.doi.org/10.2900/973540 
  19. Dallman T, Inns T, Jombart T, Ashton P, Loman N, Chatt C, et al. Phylogenetic structure of European Salmonella Enteritidis outbreak correlates with national and international egg distribution network. Microb Genom. 2016;2(8):e000070.  https://doi.org/10.1099/mgen.0.000070  PMID: 28348865 
  20. Kanagarajah S, Waldram A, Dolan G, Jenkins C, Ashton PM, Carrion Martin AI, et al. Whole genome sequencing reveals an outbreak of Salmonella Enteritidis associated with reptile feeder mice in the United Kingdom, 2012-2015. Food Microbiol. 2018;71:32-8.  https://doi.org/10.1016/j.fm.2017.04.005  PMID: 29366466 
  21. Zhou Z, McCann A, Litrup E, Murphy R, Cormican M, Fanning S, et al. Neutral genomic microevolution of a recently emerged pathogen, Salmonella enterica serovar Agona. PLoS Genet. 2013;9(4):e1003471.  https://doi.org/10.1371/journal.pgen.1003471  PMID: 23637636 
  22. Yoshida CE, Kruczkiewicz P, Laing CR, Lingohr EJ, Gannon VP, Nash JH, et al. The Salmonella In Silico Typing Resource (SISTR): An Open Web-Accessible Tool for Rapidly Typing and Subtyping Draft Salmonella Genome Assemblies. PLoS One. 2016;11(1):e0147101.  https://doi.org/10.1371/journal.pone.0147101  PMID: 26800248 
  23. Jourdan-da Silva N, Fabre L, Robinson E, Fournet N, Nisavanh A, Bruyand M, et al. Ongoing nationwide outbreak of Salmonella Agona associated with internationally distributed infant milk products, France, December 2017. Euro Surveill. 2018;23(2).  https://doi.org/10.2807/1560-7917.ES.2018.23.2.17-00852  PMID: 29338811 
  24. European Food Safety Authority (EFSA) and European Centre for Disease Prevention and Control (ECDC). Multi‐country outbreak of Salmonella Agona infections linked to infant formula. EFSA Supporting Publications.2018;15(1): EN-1365E.  https://doi.org/10.2903/sp.efsa.2018.EN-1365 
  25. Grimont PA, Weill FX. Antigenic Formulae of the Salmonella Serovars, 9th Edition; 2007. Paris: WHO Collaborating Center for Reference and Research on Salmonella.
  26. National Center for Biotechnology Information (NCBI). National Center for Biotechnology Information (NCBI) - Sequence Read Archive (SRA). [Accessed Apr 2018]. Available from: https://www.ncbi.nlm.nih.gov/sra
  27. Jünemann S, Sedlazeck FJ, Prior K, Albersmeier A, John U, Kalinowski J, et al. Updating benchtop sequencing performance comparison. Nat Biotechnol. 2013;31(4):294-6.  https://doi.org/10.1038/nbt.2522  PMID: 23563421 
  28. Warwick-Medical-School. Enterobase website - Salmonella database; v1.1.2:Available from: http://enterobase.warwick.ac.uk/species/index/senterica
  29. Alikhan NF, Zhou Z, Sergeant MJ, Achtman M. A genomic overview of the population structure of Salmonella. PLoS Genet. 2018;14(4):e1007261.  https://doi.org/10.1371/journal.pgen.1007261  PMID: 29621240 
  30. Ridom_GmbH. Ridom SeqSphere+ Documentation - Core Genome MLST Cluster Type. Münster: Ridom GmbH. [Accessed May 2018]. Available from: https://www.ridom.de/seqsphere/ug/v40/Core_Genome_MLST_Cluster_Type.html
  31. Ridom SeqSphere+ version 4.0 User Guide: Available from: https://www.ridom.de/seqsphere/ug/v40/User_Guide.html
  32. Kidgell C, Reichard U, Wain J, Linz B, Torpdahl M, Dougan G, et al. Salmonella typhi, the causative agent of typhoid fever, is approximately 50,000 years old. Infect Genet Evol. 2002;2(1):39-45.  https://doi.org/10.1016/S1567-1348(02)00089-8  PMID: 12797999 
  33. Public Health England (PHE). PHEnix SNP calling pipeline. Copyright 2016. London: PHE. [Accessed Mar 2018]. Available from: https://github.com/phe-bioinformatics/PHEnix
  34. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114-20.  https://doi.org/10.1093/bioinformatics/btu170  PMID: 24695404 
  35. Li H, Durbin R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics. 2010;26(5):589-95.  https://doi.org/10.1093/bioinformatics/btp698  PMID: 20080505 
  36. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010;20(9):1297-303.  https://doi.org/10.1101/gr.107524.110  PMID: 20644199 
  37. Stamatakis A. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics. 2006;22(21):2688-90.  https://doi.org/10.1093/bioinformatics/btl446  PMID: 16928733 
  38. Pearce ME, Alikhan NF, Dallman TJ, Zhou Z, Grant K, Maiden MCJ. Comparative analysis of core genome MLST and SNP typing within a European Salmonella serovar Enteritidis outbreak. Int J Food Microbiol. 2018;274:1-11.  https://doi.org/10.1016/j.ijfoodmicro.2018.02.023  PMID: 29574242 
  39. Petzold M, Prior K, Moran-Gilad J, Harmsen D, Lück C. Epidemiological information is key when interpreting whole genome sequence data - lessons learned from a large Legionella pneumophila outbreak in Warstein, Germany, 2013. Euro Surveill. 2017;22(45).  https://doi.org/10.2807/1560-7917.ES.2017.22.45.17-00137  PMID: 29162202 

Data & Media loading...

Submit comment
Comment moderation successfully completed
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error