A case of food-borne botulism occurred in Slovakia in 2015. Clostridium botulinum type A was isolated from three nearly empty commercial hummus tubes. The product, which was sold in Slovakia and the Czech Republic, was withdrawn from the market and a warning was issued immediately through the European Commission's Rapid Alert System for Food and Feed (RASFF). Further investigation revealed the presence of botulinum neurotoxin (BoNT) subtype BoNT/A3, a very rare subtype implicated in only one previous outbreak (Loch Maree in Scotland, 1922). It is the most divergent subtype of BoNT/A with 15.4% difference at the amino acid level compared with the prototype BoNT/A1. This makes it more prone to evading immunological and PCR-based detection. It is recommended that testing laboratories are advised that this subtype has been associated with food-borne botulism for the second time since the first outbreak almost 100 years ago, and to validate their immunological or PCR-based methods against this divergent subtype.

F D Roosevelt Teaching Hospital Department of Neurology Slovak Medical University Banská Bystrica Slovakia

Regional Authority of Public Health Banská Bystrica Banská Bystrica Slovakia

Robert Koch Institute Centre for Biological Threats and Special Pathogens Berlin Germany

Robert Koch Institute Consultant laboratory for neurotoxin producing clostridia Berlin Germany

Slovak Medical University Faculty of Public Health Bratislava Slovakia

See more in PubMed

Johnson EA, Montecucco C. Botulism. Handb Clin Neurol. 2008;91:333-68. 10.1016/S0072-9752(07)01511-4 PubMed DOI

Peck MW. Biology and genomic analysis of Clostridium botulinum. Adv Microb Physiol. 2009;55:183-265, 320. 10.1016/S0065-2911(09)05503-9 PubMed DOI

Williamson CH, Sahl JW, Smith TJ, Xie G, Foley BT, Smith LA, et al. Comparative genomic analyses reveal broad diversity in botulinum-toxin-producing Clostridia. BMC Genomics. 2016;17(1):180. 10.1186/s12864-016-2502-z PubMed DOI PMC

Kull S, Schulz KM, Weisemann J, Kirchner S, Schreiber T, Bollenbach A, et al. Isolation and functional characterization of the novel Clostridium botulinum neurotoxin A8 subtype. PLoS One. 2015;10(2):e0116381. 10.1371/journal.pone.0116381 PubMed DOI PMC

Peck MW, Smith TJ, Anniballi F, Austin JW, Bano L, Bradshaw M, et al. Historical perspectives and guidelines for botulinum neurotoxin subtype nomenclature. Toxins (Basel). 2017;9(1):38. 10.3390/toxins9010038 PubMed DOI PMC

Montecucco C, Rasotto MB. On botulinum neurotoxin variability. MBio. 2015;6(1):e02131-14. 10.1128/mBio.02131-14 PubMed DOI PMC

Rummel A. The long journey of botulinum neurotoxins into the synapse. Toxicon. 2015;107(Pt A):9-24. 10.1016/j.toxicon.2015.09.009 PubMed DOI

Dorner MB, Schulz KM, Kull S, Dorner BG. Complexity of botulinum neurotoxins: challenges for detection technology. Curr Top Microbiol Immunol. 2013;364:219-55.10.1007/978-3-662-45790-0_11 PubMed DOI

Siegel L. Destruction of Botulinum Toxins in Food and Water. In: Hauschild A, Dodds K, editors. Clostridium botulinum: Ecology and Control in Foods. New York: Marcel Dekker Inc; 1992. p. 323-41.

Weingart OG, Schreiber T, Mascher C, Pauly D, Dorner MB, Berger TFH, et al. The case of botulinum toxin in milk: experimental data. Appl Environ Microbiol. 2010;76(10):3293-300. 10.1128/AEM.02937-09 PubMed DOI PMC

Cowden J. Food-borne Clostridium botulinum intoxication from mass produced foodstuffs in Europe. Euro Surveill. 2011;16(49):20033. PubMed

Peck MW, Stringer SC, Carter AT. Clostridium botulinum in the post-genomic era. Food Microbiol. 2011;28(2):183-91. 10.1016/j.fm.2010.03.005 PubMed DOI

Lindström M, Vuorela M, Hinderink K, Korkeala H, Dahlsten E, Raahenmaa M, et al. Botulism associated with vacuum-packed smoked whitefish in Finland, June-July 2006. Euro Surveill. 2006;11(7):E060720.3. PubMed

European Centre for Disease Prevention and Control (ECDC). Table 1. Reported confirmed botulism cases: number and rate per 100 000 population, EU/EEA, 2010–2014. Stockholm: ECDC. [Accessed 13 Jul 2017]. Available from: https://ecdc.europa.eu/en/publications-data/table-1-reported-confirmed-botulism-cases-number-and-rate-100-000-population

Public Health Authority of the Slovak Republic (UVZSR). Analýza epidemiologickej situácie a činnosti odborov epidemiológie v Slovenskej republike za rok 2012. Z poverenia hlavného hygienika SR vypracovali pracovníci RÚVZ so sídlom v Banskej Bystrici. [Analysis of epidemiological situation and the activities of the departments of epidemiology in Slovak Republic in 2012. Drafted by the epidemiologists on behalf of Chief Public Health Officer of Slovak Republic]. Bratislava: UVZSR. [Accessed 20 Jul 2017]. Slovak. Available from: www.epis.sk/InformacnaCast/Publikacie/VyrocneSpravy/Files/VS_SR_2012.aspx

De Medici D, Anniballi F, Wyatt GM, Lindström M, Messelhäußer U, Aldus CF, et al. Multiplex PCR for detection of botulinum neurotoxin-producing clostridia in clinical, food, and environmental samples. Appl Environ Microbiol. 2009;75(20):6457-61. 10.1128/AEM.00805-09 PubMed DOI PMC

Kirchner S, Krämer KM, Schulze M, Pauly D, Jacob D, Gessler F, et al. Pentaplexed quantitative real-time PCR assay for the simultaneous detection and quantification of botulinum neurotoxin-producing clostridia in food and clinical samples. Appl Environ Microbiol. 2010;76(13):4387-95. 10.1128/AEM.02490-09 PubMed DOI PMC

Raphael BH, Andreadis JD. Real-time PCR detection of the nontoxic nonhemagglutinin gene as a rapid screening method for bacterial isolates harboring the botulinum neurotoxin (A-G) gene complex. J Microbiol Methods. 2007;71(3):343-6. 10.1016/j.mimet.2007.09.016 PubMed DOI

Jacobson MJ, Lin G, Raphael B, Andreadis J, Johnson EA. Analysis of neurotoxin cluster genes in Clostridium botulinum strains producing botulinum neurotoxin serotype A subtypes. Appl Environ Microbiol. 2008;74(9):2778-86. 10.1128/AEM.02828-07 PubMed DOI PMC

Pauly D, Kirchner S, Stoermann B, Schreiber T, Kaulfuss S, Schade R, et al. Simultaneous quantification of five bacterial and plant toxins from complex matrices using a multiplexed fluorescent magnetic suspension assay. Analyst (Lond). 2009;134(10):2028-39. 10.1039/b911525k PubMed DOI

Simon S, Fiebig U, Liu Y, Tierney R, Dano J, Worbs S, et al. Recommended immunological strategies to screen for botulinum neurotoxin-containing samples. Toxins (Basel). 2015;7(12):5011-34. 10.3390/toxins7124860 PubMed DOI PMC

Hill KK, Smith TJ, Helma CH, Ticknor LO, Foley BT, Svensson RT, et al. Genetic diversity among botulinum neurotoxin-producing clostridial strains. J Bacteriol. 2007;189(3):818-32. 10.1128/JB.01180-06 PubMed DOI PMC

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215(3):403-10. 10.1016/S0022-2836(05)80360-2 PubMed DOI

Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30(4):772-80. 10.1093/molbev/mst010 PubMed DOI PMC

Weigand MR, Pena-Gonzalez A, Shirey TB, Broeker RG, Ishaq MK, Konstantinidis KT, et al. Implications of genome-based discrimination between Clostridium botulinum Group I and Clostridium sporogenes strains for bacterial taxonomy. Appl Environ Microbiol. 2015;81(16):5420-9. 10.1128/AEM.01159-15 PubMed DOI PMC

Scottish Board of Health Botulism at Loch Maree. Analyst (Lond). 1923;48(564):118-20. 10.1039/an9234800118 DOI

Lúquez C, Raphael BH, Joseph LA, Meno SR, Fernández RA, Maslanka SE. Genetic diversity among Clostridium botulinum strains harboring bont/A2 and bont/A3 genes. Appl Environ Microbiol. 2012;78(24):8712-8. 10.1128/AEM.02428-12 PubMed DOI PMC

Dowell VR, Jr, McCroskey LM, Hatheway CL, Lombard GL, Hughes JM, Merson MH. Coproexamination for botulinal toxin and Clostridium botulinum. A new procedure for laboratory diagnosis of botulism. JAMA. 1977;238(17):1829-32. 10.1001/jama.1977.03280180033021 PubMed DOI

Caya JG, Agni R, Miller JE. Clostridium botulinum and the clinical laboratorian: a detailed review of botulism, including biological warfare ramifications of botulinum toxin. Arch Pathol Lab Med. 2004;128(6):653-62. PubMed

Lindström M, Korkeala H. Laboratory diagnostics of botulism. Clin Microbiol Rev. 2006;19(2):298-314. 10.1128/CMR.19.2.298-314.2006 PubMed DOI PMC

European Commission. Commission Decision 2002/253/EC of 19 March 2002 laying down case definitions for reporting communicable diseases to the Community network under Decision No 2119/98/EC of the European Parliament and of the Council. Official Journal of the European Union. Luxembourg: Publications Office of the European Union.03.04.2002: L 086. Available from: http://data.europa.eu/eli/dec/2002/253/oj

Browning LM, Prempeh H, Little C, Houston C, Grant K, Cowden JM, United Kingdom Botulism Incident Management Team An outbreak of food-borne botulism in Scotland, United Kingdom, November 2011. Euro Surveill. 2011;16(49):20036. PubMed

Jalava K, Selby K, Pihlajasaari A, Kolho E, Dahlsten E, Forss N, et al. Two cases of food-borne botulism in Finland caused by conserved olives, October 2011. Euro Surveill. 2011;16(49):20034. PubMed

Pingeon JM, Vanbockstael C, Popoff MR, King LA, Deschamps B, Pradel G, et al. Two outbreaks of botulism associated with consumption of green olive paste, France, September 2011. Euro Surveill. 2011;16(49):20035. PubMed

Peck MW, Goodburn KE, Betts RP, Stringer SC. Assessment of the potential for growth and neurotoxin formation by non-proteolytic Clostridium botulinum in short shelf-life commercial foods designed to be stored chilled. Trends Food Sci Technol. 2008;19(4):207-16. 10.1016/j.tifs.2007.12.006 DOI

Lindström M, Korkeala H. Food-borne Pathogenic Clostridia. In: Tham W, Danielsson-Tham ML, editors. Food Associated Pathogens. Boca Raton, Florida: CRC Press; 2013. p. 67-87

Tepp WH, Lin G, Johnson EA. Purification and characterization of a novel subtype A3 botulinum neurotoxin. Appl Environ Microbiol. 2012;78(9):3108-13. 10.1128/AEM.07967-11 PubMed DOI PMC

Gibson AM, Modi NK, Roberts TA, Shone CC, Hambleton P, Melling J. Evaluation of a monoclonal antibody-based immunoassay for detecting type A Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system. J Appl Bacteriol. 1987;63(3):217-26. 10.1111/j.1365-2672.1987.tb04939.x PubMed DOI

Fach P, Gibert M, Griffais R, Guillou JP, Popoff MR. PCR and gene probe identification of botulinum neurotoxin A-, B-, E-, F-, and G-producing Clostridium spp. and evaluation in food samples. Appl Environ Microbiol. 1995;61(1):389-92. PubMed PMC

Boyle B, Dallaire N, MacKay J. Evaluation of the impact of single nucleotide polymorphisms and primer mismatches on quantitative PCR. BMC Biotechnol. 2009;9(1):75. 10.1186/1472-6750-9-75 PubMed DOI PMC

Bru D, Martin-Laurent F, Philippot L. Quantification of the detrimental effect of a single primer-template mismatch by real-time PCR using the 16S rRNA gene as an example. Appl Environ Microbiol. 2008;74(5):1660-3. 10.1128/AEM.02403-07 PubMed DOI PMC

Bhattacharjee Y. Biosecurity. Panel selects most dangerous select agents. Science. 2011;332(6037):1491-2. 10.1126/science.332.6037.1491 PubMed DOI

Hooper DC, Hirsch MS. Novel Clostridium botulinum toxin and dual use research of concern issues. J Infect Dis. 2014;209(2):167. . Available from: 10.1093/infdis/jit52810.1093/infdis/jit528 PubMed DOI PMC