Original Scientific Article
Detection of enterotoxigenic potential of Staphylococcus aureus isolates from cheese samples with two different methods
Marija Ratkova Manovska * ,
Mirko Prodanov ,
Dean Jankuloski ,
Pavle Sekulovski ,
Katerina Blagoevska

Mac Vet Rev 2022; 45 (1): 27 - 33

10.2478/macvetrev-2022-0010

Received: 21 September 2021

Received in revised form: 04 November 2021

Accepted: 08 November 2021

Available Online First: 12 January 2022

Published on: 15 March 2022

Correspondence: Marija Ratkova Manovska, ratkova.marija@fvm.ukim.edu.mk
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Abstract

The primary objective of our study was to detect the occurrence of enterotoxigenic Staphylococcus aureus in diverse types of cheese (cow’s milk cheese and mixed milk cheese) samples from R.N. Macedonia. Cheese samples were analyzed for enumeration and isolation of the S. aureus strains according to ISO 6888-1. We detected the toxigenic potential of the strains by the use of the Enzyme Link Fluorescent Assay VIDAS system, and we confirmed the presence of the SEs (sea, seb, sec, sed, see) genes by multiplex PCR. The results showed that out of 270 samples of cheese, coagulase-positive Staphylococcus (CPS) were detected in 27 (10%), and coagulase-negative staphylococci in five samples (1.8%). Biochemically, all 27 CPS samples were confirmed to be Staphylococcus aureus. With VIDAS SET2 test we confirmed that 11 isolates are producers of one of the toxins limited by the test. With the conventional PCR we confirmed genes in only 7 isolates. Most common detected gene was seb n=3 (42.8%), followed by sea n=2 (28.6%), and sec n=2 (28.6%). Additionally, sed and see genes were not detected in any of the S. aureus isolates. Discrepancies between the two test methods for detection of enterotoxigenic potential are not uncommon. The presence of viable Staphylococcus aureus cells that have enterotoxin potency demonstrates the importance of appropriate hygiene practices in the diary process and also the maintenance of the products in order to obtain a safe final product for the consumers.

Keywords: cheese, enterotoxigenic, Staphylococcus aureus, VIDAS SET2, PCR


References

  1. Fetsch, A., Johler, S. (2018). Staphylococcus aureus as a foodborne pathogen. Curr Clin Microbiol. Rpt. 5, 88-96. https://doi.org/10.1007/s40588-018-0094-x
  2. De Buyser, M.L., Dufour B., Maire M., Lafarge, V. (2001). Implication of milk and milk products in food-borne diseases in France and in different industrialized countries. Int J Food Microbiol. 67(1-2): 1-17. https://doi.org/10.1016/S0168-1605(01)00443-3
  3. Ikeda, T., Tamate, N., Yamaguchi, K., Makino, S. (2005). Mass outbreak of food poisoning disease caused by small amounts of Staphylococcal enterotoxins A and H. Appl Env Microbiol. 71(5): 2793- 2795. https://doi.org/10.1128/AEM.71.5.2793-2795.2005 PMid:15870376 PMCid:PMC1087516            
  4. Andrade, G.P., Zelante, F. (1989). Simultaneous occurrence of enterotoxigenic Staphylococcus aureus on the hands and in the mouth and stools of asymptomatic carriers. Rev Saude Publica. 23(4): 277-284. https://doi.org/10.1590/S0034-89101989000400002 PMid:2631181       
  5. Pereira, M.L., Carmo, L.S., Lara, M.A., Silva, S.O., Dias, R.S., Bergdoll, M.S. (1994). Enterotoxigenic staphylococci from food handlers working in an industrial kitchen in Belo Horizonte, MG (Brazil). Rev Microbiol. 25(3): 161-165.         
  6. D'Amico, D.J., Donnelly, C.W. (2011). Characterization of Staphylococcus aureus strains isolated from raw milk utilized in small-scale artisan cheese production. J Food Prot. 74 (8): 1353-1358. https://doi.org/10.4315/0362-028X.JFP-10-533 PMid:21819666 
  7. Sutherland, J.P., Bayliss, A.J., Roberts, T.A. (1994). Predictive modelling of growth of Staphylococcus aureus: the effects of temperature, pH and sodium chloride. Int J Food Microbiol. 21(3): 217-236. https://doi.org/10.1016/0168-1605(94)90029-9
  8. Smyth, D.S., Hartigan, P.J., Meaney, W.J., Fitzgerald, J.R., Deobald, C.F., Bohach, G.A., Smyth, C.J. (2005). Superantigen genes encoded by the egc cluster and SaPIbor are predominant among Staphylococcus aureus isolates from cows, sheep, rabbits and poultry. J Med Microbiol. 54(Pt 4): 401-411. https://doi.org/10.1099/jmm.0.45863-0 PMid:15770028             
  9. Mucchetti, G., Bottari, B., Gatti, M., Neviani, E., Lazzi, C. (2013). Invited review: Microbial evolution in raw milk, long ripened cheeses produced using undefined natural whey starters, J. Dairy Sci. 97(2): 573-591. https://doi.org/10.3168/jds.2013-7187 PMid:24290824 
  10. Charlier, C., Cretenet, M., Even, S., Le Loir, Y. (2009). Interactions between Staphylococcus aureus and lactic acid bacteria: an old story with new perspectives. Int J Food Microbiol.131(1): 30-39. https://doi.org/10.1016/j.ijfoodmicro.2008.06.032 PMid:18687499        
  11. Rajkovic, A. (2016). Staphylococcus: food poisoning. In B. Caballero, P.M. Finglas, F. Toldrá (Eds). Encyclopedia of food and health (pp. 133- 139). 1st ed. Oxford: Academic Press https://doi.org/10.1016/B978-0-12-384947-2.00655-3
  12. Clemente, M., das G, do Valle R.H.P. (2003). Staphylococcus in cheeses made from raw and pasteurized milk. Food Hyg Mag. 17(104-105): 38-39.            
  13. Jarraud, S., Cozon, G., Vandenesch, F., Bes, M., Etiene, J., Lina, G. (1999). Involvement of enterotoxins G and I in staphylococcal toxic shock syndrome and staphylococcal scarlet fever. J Clin Microbiol. 37(8): 2446-2449. https://doi.org/10.1128/JCM.37.8.2446-2449.1999 PMid:10405382 PMCid:PMC85251
  14. Argudín, M.A., Fetsch, A., Tenhagen, B.A., Hammerl, J.A., Hertwig, S., Kowall, J., Rodicio, M.R., Kasbohrer, A., Helmuth, R., Schroeter, A., Mendoza, M.C., Braunig, J., Appel, B., Guerra, B. (2010). High heterogeneity within methicillin-resistant Staphylococcus aureus ST398 isolates, defined by Cfr9I macrorestriction-pulsed-field gel electrophoresis profiles and spa and SCCmec types. Appl Environ Microbiol. 76(3): 652-658. https://doi.org/10.1128/AEM.01721-09 PMid:20023093 PMCid:PMC2813030
  15. Priego, R., Medina, L.M., Jordano, R. (2009). Comparison between the Vitek immunodiagnostic assay system and PCR for the detection of pathogenic microorganisms in an experimental dry sausage during its curing process. J Food Prot. 72(9): 1977-1981. https://doi.org/10.4315/0362-028X-72.9.1977 PMid:19777902   
  16. ISO 6888:1999 - Microbiology of food and feed - Horizontal method for detection of coagulase positive staphylococci (Staphylococcus aureus and other spp.).   
  17. Pinto, B., Chenolla, E., Aznar, R. (2005). Identification and typing of food-borne Staphylococcus aureus by PCR-based techniques. Syst Appl Microbiol. 28(4): 340-352. https://doi.org/10.1016/j.syapm.2005.01.002PMid:15997707
  18. Shiun-Bi, Su., Yu-Lan, W., Shiu-Ing, Ch., Jing-Lai, T., Cheng-Ing, Ch. (2005). Establishing the use of Real-time PCR for staphylococcal enterotoxin. Epidemiol Bull. 21(12): 387-395.      
  19. Mehrotra, M., Wang, G., Johnson, W.M. (2000). Multiplex PCR for detection of genes for Staphylococcus aureus enterotoxins, exfoliative toxins, toxic shock syndrome toxin 1, and methicillin resistance. J Clinical M. 38(3): 1032-1035. https://doi.org/10.1128/JCM.38.3.1032-1035.2000 PMid:10698991 PMCid:PMC86330         
  20. Sharma, K.N., Rees, D.E.C., Dood, R.E.C. (2000). Development of a single-reaction multiplex PCR toxin typing assay for Staphylococcus aureus strains. Appl Environ Microbiol. 66 (4): 1347-1353. https://doi.org/10.1128/AEM.66.4.1347-1353.2000 PMid:10742210 PMCid:PMC91991  
  21. Kateete, D., Kimani, C., Katabazi, F., Okeng, A., Okee, M., Nanteza, A., Joloba, M., Najjuka, F. (2010). Identification of Staphylococcus aureus: DNase and Mannitol salt agar improve the efficiency of the tube coagulase test. Ann Clin Microbiol Antimicrob. 9, 23. https://doi.org/10.1186/1476-0711-9-23 PMid:20707914 PMCid:PMC2927478     
  22. Official Gazette of R.M. Book of Rules from 21.02.2012, concerning Requirements for the number of somatic cells and the number of microorganisms in raw milk. Article 19, Off. G. of R.M. 26/2012, (p. 25). 
  23. Medeiros, M.I.M., Nader Filho, A., Jordano, R., Ruz, V., Medina, L.M., García, V. (2019). Occurrence of Staphylococcus aureus and its toxins in cheeses from the region of Andalusia, Spain. J Dairy Vet Anim Res. 8(1): 33-36. https://doi.org/10.15406/jdvar.2019.08.00239
  24. Sampaio, E., Nader-Filho, A. (2000). Occurrence of Staphylococcus aureus in cheese made in Brazil. Rev Saude Publica. 34(6): 578-580. https://doi.org/10.1590/S0034-89102000000600003 PMid:11175601
  25. Savić Radovanović, R., Zdravković, N., Velebit, B. (2020). Occurrence and characterization of enterotoxigenic Staphylococci isolated from soft cheeses in Serbia. Acta Veterinaria 70(2): 238-254. https://doi.org/10.2478/acve-2020-0017
  26. Vernozy-Rozand, C., Mazuy, C., Perrin, G., Haond, F., Bes, M., Brun, Y., Fleurette, J. (1996). Identification Micrococcaceae isolated from goat's milk and cheese in the Poitou-Charentes region. Int J Food Microbiol. 30(3): 373-378. https://doi.org/10.1016/0168-1605(96)00953-1
  27. Ratkova Manovska, M., Prodanov, M., Angelovski, Lj., Jankuloski, D., Sekulovski, P. (2021). Prevalence of the enterotoxigenic Staphylococcus aureus strains isolated from raw milk and cheese. Mac Vet Rev. 44(1): 71-77. https://doi.org/10.2478/macvetrev-2021-0014
  28. Holeckova, B., Holoda, E., Fotta, M., Kalinacova, V., Gondol, J., Grolmus, J. (2002). Occurrence of enterotoxigenic Staphylococcus aureus in food. Ann Agric Environ Med. 9(2): 179-182.   
  29. Aitichou, M., Henkens, R., Sultana, A.M., Ulrich, R.G., Ibrachim, M.S. (2004). Detection the Staphylococcus aureus enterotoxin A and B genes with PCR-EIA and a hand-held electrochemical sensor. Mol Cell Probes 18(6): 373-377. https://doi.org/10.1016/j.mcp.2004.06.002 PMid:15488376
  30. Hennekinne, J.A., De Buyser, M.L., Dragacci, S. (2011). Staphylococcus aureus and its food poisoning toxins: characterization and outbreak investigation. FEMS Microbiol Rev. 36(4): 815-836. https://doi.org/10.1111/j.1574-6976.2011.00311.x PMid:22091892        
  31. Arcuri, F.E, Angelo, F.F., Guimaraes, M.F.M., Talon, R., Borges, M.F.B., Leroy, S., Loiseau, G., Lange, C.C., Andrade, N.J., Montet, D. (2010). Toxigenic status of Staphylococcus aureus isolated from bovine raw milk and Minas frescal cheese in Brazil. J Food Prot. 73(12): 2225-2231. https://doi.org/10.4315/0362-028X-73.12.2225 PMid:21219740       
  32. Najera-Sanches, G., Rodriges, M.R., Olvera, P.R., de la Garza, M.L. (2003). Development of two multiplex polymerase chain reactions for the detection of enterotoxigenic strains of Staphyloccus aureus isolated from foods. J Food Prot. 66(6): 1055-1062. https://doi.org/10.4315/0362-028X-66.6.1055 PMid:12801009            
  33. Kav, K., Col, R., Ardic, M. (2011). Characterisation of Staphylococcus aureus isolates from white-brined Urfa cheese. J Food Prot. 74(11): 1788-1796. https://doi.org/10.4315/0362-028X.JFP-11-179 PMid:22054178 
  34. Mousa, W.S., Abdeen, E., Hussein, H., Hadad, G. (2017). Prevalence and multiplex PCR for enterotoxin genes of Staphylococcus aureus isolates from subclinical mastitis and Kareish cheese. J Infect Dis Preve Med. 5(4). https://doi.org/10.4172/2329-8731.1000174
  35. Ertas, N., Gonulalan, Z., Yildirim, Y., Kum, E. (2010). Detection of Staphylococcus aureus enterotoxins in sheep cheese and dairy desserts by multiplex PCR technique. Int J Food Microbiol 142(1-2): 74-77. https://doi.org/10.1016/j.ijfoodmicro.2010.06.002 PMid:20573416
  36. Jørgensen, H.J., Mathisen, T., Lovseth, A., Omoe, K., Qvale, K.S., Loncarevic, S. (2005). An outbreak of staphylococcal food poisoning caused by enterotoxin H in mashed potato made with raw milk. FEMS Microbiol Lett. 252(2): 267-272. https://doi.org/10.1016/j.femsle.2005.09.005 PMid:16213675   
  37. Kérouanton, A., Hannekinne, A.J., Leterte, C., Petit, L., Chesneau, O., Brisabois, A., De Buyser, L.M. (2007). Characterization of Staphylococcus aureus strains associated with food poisoning outbreaks in France. Int J Food Microbiol. 115(3): 369-375. https://doi.org/10.1016/j.ijfoodmicro.2006.10.050 PMid:17306397    


Copyright

© 2022 Ratkova Manovska M. This is an open-access article published under the terms of the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Conflict of Interest Statement

The authors have declared that no competing interests exist.

Citation Information

Macedonian Veterinary Review. Volume 45, Issue 1, Pages 27-33, e-ISSN 1857-7415, p-ISSN 1409-7621, DOI: 10.2478/macvetrev-2022-0010, 2022