Staphylococcus aureus harbouring egc cluster coding for non-classical enterotoxins, involved in a food poisoning outbreak, Romania, 2012 / Staphylococcus aureus purtător de gene codante pentru enterotoxine non-clasice (cluster egc), implicat într-un focar de toxiinfecţie alimentară, România, 2012
In March 2012, a food poisoning outbreak was reported in a Romanian county, with a total number of 30 children affected. The symptoms (vomiting, diarrhea and abdominal pain), with onset within 1-2 hours after the ingestion of a particular food (milk), suggested a possible staphylococcal aetiology. An outbreak investigation was carried out, in accordance with the national surveillance methodology and 25 samples: stool (n=9), vomit (n=5), nasal swabs (n=9), and milk (n=2) were collected from the affected children, food handlers and suspected food. All isolated strains were sent to the Reference Centre for Staphylococci within the “Cantacuzino” National Institute of Research-Development for Microbiology and Immunology, Bucharest, Romania, for confirmation and further analysis. The aim of this study was to increase the reference laboratory capacity to confirm staphylococcal food poisoning (SFP) outbreak by defining the molecular basis of toxicity of Staphylococcus aureus (S. aureus) isolates and assessing their genetic relatedness. PCR methods have been used to detect 14 enterotoxin genes and the expression of some of these genes was proved by using a reverse transcription real-time PCR. Pulsed-field gel electrophoresis (PFGE) and Staphylococcus protein A coding gene sequence typing (spa typing) have been used to track the origin of the S. aureus contamination and to confirm the food poisoning outbreak.
Two enterotoxigenic S. aureus strains isolated from milk, twelve isolated from patients and two from food handlers were of the same spa- type (t902) and revealed an indistinguishable SmaI macrorestriction pattern after a PFGE analysis. All these strains harboured the same toxin genes profile, namely the enterotoxin gene cluster (egc), which strongly supports the evidence that the milk was the incriminated food vehicle of the outbreak and a food-handler was the most likely source of the staphylococcal food poisoning (SFP) incident.
1. Bianchi DM, Gallina S, Bellio A, Chiesa F, Civera T, Decastelli L. Enterotoxin gene profiles of Staphylococcus aureus isolated from milk and dairy products in Italy. Lett Applied Microbiol. 2013;58(2):190-6. DOI: 10.1111/lam.12182
2. Hennekinne JA, De Buyser ML, Dragacci S. Staphylococcus aureus and its food poisoning toxins: characterization and outbreak investigation. FEMS Microbiol Rev. 2012;36(4):815-36. DOI: 10.1111/j.1574-6976.2011.00311.x
3. Argudin MA, Mendoza MC, Rodicio MR. Food Poisoning and Staphylococcus aureus enterotoxins. Tox ins. 2010;2(7):1751-73. DOI: 10.3390/toxins2071751
4. Tang J, Tang C, Chen J, Du Y, Yang XN, Wang C, et al. Phenotypic characterization and prevalence of enterotoxin genes in Staphylococcus aureus isolates from outbreaks of illness in Chengdu City. Foodborne Pathog Dis. 2011;8(12):1317-20. DOI: 10.1089/fpd.2011.0924
5. Dinges MM, Orwin PM, Schlievert PM. Exotoxins of Staphylococcus aureus. Clin Microbiol 2000;13(1):16-34. DOI: 10.1128/CMR.13.1.16-34.2000
6. Murray RJ. Recognition and Management of Staphylococcus aureus Toxin-Mediated Disease. Intern Med J. 2005;35(2): 106-19. DOI: 10.1111/j.1444-0903.2005.00984.x
7. Balaban N, Rasooly A. Staphylococcal enterotoxins. Int J Food Microbiol. 2000;61(1):1-10. DOI: 10.1016/ S0168-1605(00)00377-9
8. Omoe K, Hu DL, Takahashi-Omoe H, Nakane A, Shinagawa K. Identification and characterization of a new staphylococcal enterotoxin-related putative toxin encoded by two kinds of plasmids. Infect Immun. 2003;71(10):6088-94. DOI: 10.1128/IAI.71.10.6088-6094.2003
9. Ono HK, Omoe K, Imanishi K, Iwakabe Y, Hu DL, Kato H, et al. Identification and characterization of two novel staphylococcal enterotoxins, types S and T. Infect Immun. 2008;76(11):4999-5005. DOI: 10.1128/ IAI.00045-08
10. Thomas DY, Jarraud S, Lemercier B, Cozon G, Echasserieau K, Etienne J, et al. Staphylocccal enterotoxin- like toxins U2 and V, two new staphylococcal superantigens arising from recombination within the enterotoxin gene cluster. Infect Immun. 2006;74(8):4724-34. DOI: 10.1128/IAI.00132-06
11. Wilson GJ, Seo KS, Cartwright RA, Connelley T, Chuang- Smith ON, Merriman JA, et al. A novel core genome- encoded superantigen contributes to lethality of community-associated MRSA necrotizing pneumonia. PLoS Pathog. 2011;7(10):e1002271. DOI: 10.1371/ journal.ppat.1002271
12. Lina G, Bohach GA, Nair SP, Hiramatsu K, Jouvin- Marche E, Mariuzza R. Standard nomenclature for the superantigens expressed by Staphylococcus. J Infect Dis. 2004;189(12):2334-6. DOI: 10.1086/420852
13. Omoe K, Hu DL, Ono HK, Shimizu S, Takahashi-Omoe H, Nakane A, et al. Emetic potentials of newly identified staphylococcal enterotoxin-like toxins. Infect Immun. 2013;81(10):3627-31. DOI: 10.1128/IAI.00550-13
14. Ikeda T, Tamate N, Yamaguchi K, Makino S. Mass Outbreak of Food Poisoning Disease Caused by Small Amounts of Staphylococcal Enterotoxins A and H. Appl Environ Microbiol. 2005;71(5):2793-5. DOI: 10.1128/ AEM.71.5.2793-2795.2005
15. Jorgensen HJ, Mathisen T, Lovseth A, Omoe K, Qvale KS, Loncarevic S. An outbreak of staphylococcal food poisoning caused by enterotoxin H in mashed potato made with raw milk. FEMS Microbiol Lett. 2005;252(2):267-72. DOI: 10.1016/j. femsle.2005.09.005
16. Blaiotta G, Ercolini D, Pennacchia C, Fusco V, Casaburi A, Pepe O, et al. PCR detection of staphylococcal enterotoxin genes in Staphylococcus spp. strains isolated from meat and dairy products. Evidence for new variants of seG and seI in S. aureus AB-8802. J Appl Microbiol. 2004;97(4):719-30. DOI: 10.1111/j.1365-2672.2004.02349.x
17. Bania J, Dabrowska A, Bystron J, Korzekwa K, Chrzanowska J, Molenda J. Distribution of newly described enterotoxin-like genes in Staphylococcus aureus from food. Int J Food Microbiol. 2006;108(1):36-41. DOI: 10.1016/j.ijfoodmicro.2005.10.013
18. Bennett RW, Monday SR. Staphylococcus aureus. eds Miliotis MD and Bier JW. International Handbook of Foodborne Pathogens. Marcel Dekker Inc: New York. 2003;p41-59.
19. Hu DL, Nakane A. Mechanism of staphylococcal enterotoxin- induced emesis. Eur J Pharmacol. 2014;722:95-107. DOI: 10.1016/j.ejphar.2013.08.050
20. Bergdoll MS. Enterotoxins. Eds Easman CSF and Adlam C. Staphylococci and Staphylococcal Infections. Academic Press Inc: London, UK. 1983;559-98.
21. Jarraud S, Peyrat MA, Lim A, Tristan A, Bes M, Mougel C, et al. egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus. J Immunol. 2001;166(1):669-77. DOI: 10.4049/jimmunol.166.1.669
22. Vicosa GN, Le Loir A, Le Loir Y, de Carvalho AF, Nero LA. egc characterization of enterotoxigenic Staphylococcus aureus isolates obtained from raw milk and cheese. Int J Food Microbiol. 2013;165(3):227-30. DOI: 10.1016/j.ijfoodmicro.2013.05.023
23. Blaiotta G, Fusco V, von Eiff C, Villani F, Becker K. Biotyping of enteterotoxigenic Staphylococcus aureus by enterotoxin gene cluster (egc) polymorphism and spa typing analyses. App Environ Microbiol. 2006;72(9):6117-23. DOI: 10.1128/AEM.00773-06
24. Hennekinne JA, Ostyn A, Guillier F, Herbin S, Prufer AL, Dragacci S. How Should Staphylococcal Food Poisoning Outbreaks Be Characterized?. Toxins. 2010;2(8):2106-16. DOI: 10.3390/toxins2082106
25. Lee YD, Moon BY, Park JH, Chang HI, Kim WJ. Expression of enterotoxin genes in Staphylococcus aureus isolates based on mRNA analysis. J Microbiol Biotechnol. 2007;17(3):461-7.
26. Codita I, Dumitrescu V, Grigore L. Thermal stable nuclease test used to detect S. aureus multiplication in food. Bacteriol Virusol Parazitol Epidemiol. 1995;40(3-4):227-9.
27. Strommenger B, Kettlitz C, Werner G, Witte W. Multiplex PCR assay for simultaneous detection of nine clinically relevant antibiotic resistance genes in Staphylococcus aureus. J Clin Microbiol. 2003;41(9):4089-94. DOI: 10.1128/JCM.41.9.4089-4094.2003
28. Murchan S, Kaufmann ME, Deplano A, de Ryck R, Struelens M, Zinn CE, et al. Harmonization of pulsed field gel electrophoresis protocols for epidemiological typing of strains of methicillin-resistant Staphylococcus aureus: a single approach developed by consensus in 10 European laboratories and its application for tracing the spread of related strains. J Clin Microbiol. 2003; 41(4):1574-85. DOI: 10.1128/JCM.41.4.1574-1585.2003
29. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al. Iterpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol. 1995;33(9):2233-39.
30. Harmsen D, Claus H, Witte W, Rothgänger J, Claus H, Turnwald D, et al. Typing of methicillin- resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management. J Clin Microbiol. 2003;41(12):5442-8. DOI: 10.1128/JCM.41.12.5442-5448.2003
31. Mellmann A, Weniger T, Berssenbrügge C, Keckevoet U, Friedrich AW, Harmsen D, et al. Characterization of clonal relatedness among the natural population of Staphylococcus aureus strains by using spa sequence typing and the BURP (based upon repeat patterns) algorithm. J Clin Microbiol. 2008;46(8):2805-8. DOI: 10.1128/JCM.00071-08
32. Johnson W, Tyler MS, Ewan SD, Ashton EP, Polland FE, Rozee KR. Detection of genes for enterotoxins, exofoliative toxins and toxic shock syndrome toxin 1 in Staphylococcus aureus by polymerase chain reaction. J Clin Microbiol. 1991;29:426-30.
33. McLauchlin J, Narayanan GL, Mithani V, O’Neill G. The detection of enterotoxins and toxic shock syndrome toxin genes in Staphylococcus aureus by polymerase chain reaction. J Food Protect. 2000;63:479-88.
34. Rosec JP, Gigaud O. Staphylococcal enterotoxin genes of classical and new types detected by PCR in France. Int J Food Microbiol. 2002;77:61-70. DOI: 10.1016/ S0168-1605(02)00044-2
35. Xie YP, He YP, Gehring A, Hu Y, Li QQ, Tu SI, et al. Genotypes and toxin gene profiles of Staphylococcus aureus clinical isolates from China. PLoS One. 2011;6(12):28276-87. DOI: 10.1371/journal. pone.0028276
36. Coldea IL, Dragulescu EC, Lixandru BE, Dragomirescu CC, Codita I. Phenotypic and genotypic characterization of Staphylococcus aureus strains isolated from a familial foodborne outbreak. Roum Arch Microbiol Immunol. 2013;72(3):210-17.
37. Martin MC, Fueyo JM, González-Hevia MA, Mendoza MC. Genetic procedures for identification of enterotoxigenic strains of Staphylococcus aureus from three food poisoning outbreaks. Int J Food Microbiol. 2004;94(3):279-86. DOI: 10.1016/j.ijfoodmicro. 2004.01.011
38. Jorgensen HJ, Mørk T, Caugant DA, Kearns A, Rorvik LM. Genetic variation among Staphylococcus aureus strains from Norwegian bulk milk. Appl Environ Microbiol. 2005b;71(12):8352-61. DOI: 10.1128/ AEM.71.12.8352-8361.2005
39. Zhang C, Shen Y, Dong M. Distribution, polymorphism and temporal expression of egc in Staphylococcus aureus isolates from various foods in China. Food Control. 2013;29(1):279-85. DOI: 10.1016/j.foodcont. 2012.06.024
40. Hwang SY, Kim SH, Jang EJ, Kwon NH, Park YK, Koo HC, et al. Novel multiplex PCR for the detection of the Staphylococcus aureus superantigen and its application to raw meat isolates in Korea. Int J Food Microbiol. 2007;117(1):99-105. DOI: 10.1016/j.ijfoodmicro. 2007.02.013
41. Naik S, Smith F, Ho J, Croft NM, Domizio P, Price E, et al. Staphylococcal enterotoxins G and I, a cause of severe but reversible neonatal enteropathy. Clin Gastroenterol Hepatol. 2008; 6(2):251-4. DOI: 10.1016/j. cgh.2007.09.004