Onur KARAALİOĞLU, Sıne OZMEN TOGAY, Mustafa AY, Gözde SOYSAL, Mine ÇARDAK, Ufuk BAGCI, Özlem EROL TINAZTEPE

Çiğ balık örneklerinden izole edilen Enterococcus faecium ve Enterococcus faecalis suşlarının gıda güvenliği yönünden bazı özelliklerinin değerlendirilmesi

Amaç: Bu çalışmada çiğ balık örneklerinden izole edilen Enterococcus faecium ve E. faecalis carrying the antibiotic resistance and virulence genes of suşlarının antibiyotik direnç ve virülans genleri taşıma Enterococcus faecium and E. faecalis strains isolated yönünden değerlendirilmesi ve antibakteriyel aktivite potansiyellerinin araştırılması amaçlanmıştır.Yöntem: Sardalye, istavrit, barbun ve hamsi örneklerinden Kanamisin Azid Eskülin agar besiyeri kullanılarak izole edilip Gram boyama, katalaz testi, eskulin hidrolizi, pH 9,6 ve %40’lık safra tuzu ortamında üreme, 10ºC ve 45ºC’de üreme testleri ile cins düzeyinde, API 20 Strep biyokimyasal test kiti ile de tür düzeyinde tanımlanan 33 adet izolatın antibiyotik streptomisin, kloramfenikol, eritromisin, tetrasiklin, gentamisin ve vankomisin direnç özellikleri disk difüzyon yöntemiyle ve virülans gen agg2, gelE, of 33 enterococcal strains were evaluated by using disk cylA, cylB, cylM taşıma durumları ise polimeraz zincir diffusion method. PCR were performed for evaluate reaksiyonu ile araştırılmıştır. İzolatların referans test bakterilerine Staphylococcus aureus ATCC 6538, E. coli ATCC 25922, Listeria monocytogenes ATCC 7644, L. innocua ATCC 33090, E. faecalis NCIMB 700584, E. faecium M74 karşı antibakteriyel aktivite potansiyelleri faecium M74 of the isolates were evaluated by using the agar drop methode.Results: All E. faecalis and E. faecium isolates were resistant to streptomycin, whereas 30 90.9% isolates to gentamycin and 14 42.4% isolates to vancomycin were found high level resistant. For erythromycin 32 96.7% of isolates showed intermediate level of resistancy. Other tested antibiotics, chloramphenicol and tetracycline, were found mostly susceptible, 26 78.8% and 30 90.9% isolates respectively. Multiple antibiotic resistance was also observed in isolates. In addition, gelE and agg2 genes related to pathogenic function were found in some isolates and ß-hemolytic activity was detected in 4 of isolates. However, there is a potential for significant antibacterial activity against test bacteria including S. aureus and L. monocytogenes in some of the isolates.Conclusion: It is thought that E. faecium and E. faecalis isolates may carry a risk for food safety and public health due to antibiotic resistance and virulence gene transmission, but they also have potential for antimicrobial activity in isolates and all these properties should be evaluated on strain specific

Anahtar Kelimeler:

Enterococcus, su ürünü, antibakteriyel aktivite, virülans faktör, antibiyotik direnci

Evaluation of some food safety-related characteristics of Enterococcus faecium and Enterococcus faecalis strains isolated from raw fish samples

Objective: The aim of this study was to evaluate faecium M74 of the isolates were evaluated by using the agar drop methode.Results: All E. faecalis and E. faecium isolates were resistant to streptomycin, whereas 30 90.9% isolates to gentamycin and 14 42.4% isolates to vancomycin were found high level resistant. For erythromycin 32 96.7% of isolates showed intermediate level of resistancy. Other tested antibiotics, chloramphenicol and tetracycline, were found mostly susceptible, 26 78.8% and 30 90.9% isolates respectively. Multiple antibiotic resistance was also observed in isolates. In addition, gelE and agg2 genes related to pathogenic function were found in some isolates and ß-hemolytic activity was detected in 4 of isolates. However, there is a potential for significant antibacterial activity against test bacteria including S. aureus and L. monocytogenes in some of the isolates.Conclusion: It is thought that E. faecium and E. faecalis isolates may carry a risk for food safety and public health due to antibiotic resistance and virulence gene transmission, but they also have potential for antimicrobial activity in isolates and all these properties should be evaluated on strain specific

Keywords:

Enterococcus, seafood, antibacterial activity, virulence factor, antibiotic resistance,

PDF

___

1. Franz CMAP, Holzapfel WH, Stiles ME. Enterococci at the crossroads of food safety? Int J Food Microbiol, 1999; 47: 1-24. DOI: 10.1016/S0168- 1605(99)00007-0.
2. Franz CMAP, Stiles ME, Schleifer KH, Holzapfel WH. Enterococci in foods-a conundrum for food safety. Int J Food Microbiol, 2003; 88: 105-22. DOI: 10.1016/S0168-1605(03)00174-0.
3. Foulquie Moreno MR, Sarantinopoulos P, Tsakalidou E, De Vuyst L. The role and application of enterococci in food and health. Int J Food Microbiol, 2006; 106: 1-24. DOI: 10.1016/j. ijfoodmicro.2005.06.026.
4. Harwood VJ, Whitlock J, Withington V. Classification of antibiotic resistance patterns of ındicator bacteria by discriminant analysis: use in predicting the source of fecal contaminaton in subtropical waters. Appl Environ Microbiol, 2000; 66 (9): 3698-704. DOI: 10.1128/AEM.66.9.3698- 3704.2000.
5. Valenzuela AS, Benomar N, Abriouel H, Canamero MM, Galvez A. Isolation and identification of Enterococcus faecium from seafoods: Antimicrobial resistance and production of bacteriocin-like substances. Food Microbiol, 2010; 27: 955-961. DOI: 10.1016/j.fm.2010.05.033.
6. Hammad AM, Shimamoto T, Shimamoto T. Genetic characterization of antibiotic resistance and virulence factors in Enterococcus spp. from Japanese retail ready-to-eat raw fish. Food Microbiol, 2014; 38: 62-6. DOI: 10.1016/j. fm.2013.08.010.
7. Pinto AL, Fernandes M, Pinto C, Albano H, Castilho F, Teixeira P, Gibbs PA. Characterization of antilisteria bacteriocins isolated from shellfish: Potential antimicrobials to control non- fermented seafood. Int J food Microbiol, 2009; 129: 50-8. DOI: 10.1016/j.ijfoodmicro.2008.11.005.
8. Francoise L. Occurence and role of lactic acid bacteria in seafood products. Food Microbiol, 2010; 27: 698-709. DOI: 10.1016/j.fm.2010.05.016.
9. Sarantinopoulos P, Andrighetto C, Georgalaki M, Rea MC, Lombardi A, Cogan TM, Kalantzopoulos G, Tsakalidou E. Biochemical properties of enterococci relevant to their technological performance. Int Dairy J, 2001; 11: 621-47. DOI: 10.1016/S0958-6946(01)00087-5.
10. Hugas M, Garriga M, Aymerich MT. Functionality of enterococci in meat products. Int J Food Microbiol, 2003; 88: 223-33. DOI: 10.1016/S0168- 1605(03)00184-3.
11. Klein G. Taxonomy, ecology and antibiotic resistance of enterococci from food and the gastro-intestinal tract. Int J Food Microbiol, 2003; 88: 123-31. DOI: 10.1016/S0168-1605(03)00175-2.
12. Peters J, Mac K, Schauer HW, Klein G, Ellerbroek L. Species distribution and antibiotic resistance patterns of enterococci isolated from food of animal origin in Germany. Int J Food Microbiol, 2003; 88: 311-314. DOI: 10.1016/S0168- 1605(03)00193-4.
13. Eaton TJ, Gasson MJ. Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates. Appl Environ Microbiol, 2001; 67(4): 1628-1635. DOI: 10.1128/AEM.67.4.1628- 35.2001.
14. Reviriego C, Eaton T, Martin R, Jimenez E, Fernandez L, Gasson MJ, et al. Screening of virulence determinants in Enterococcus faecium strains isolated from breast milk. J Hum Lact, 2005; 21 (2): 131-7. DOI: 10.1177/0890334405275394.
15. Aral M, Paköz NİE, Aral İ, Doğan S. Çeşitli klinik örneklerden izole edilen Enterococcus faecalis ve Enterococcus faecium suşlarının antibiyotik direnci. Turk Hij Den Biyol Derg, 2011; 68 (2): 85- 92. DOI: 10.5505/TurkHijyen.2011.53315.
16. Oladipo IC, Sanni AI, Swarnakar S. Virulence potential of Enterococcus gallinarum strains isolated from selected Nigerian traditional fermented foods. J BioSci Biotech, 2014; 3(2): 97-104. ISSN: 1314-6246.
17. Hammad AM, Hassan HA, Shimamoto T. Prevalence, antibiotic resistance and virulence of Enterococcus spp. in Egyptian fresh raw milk cheese. Food Control, 2015; 50: 815-20. DOI: 10.1016/j.foodcont.2014.10.020.
18. Pieniz S, De Moura TM, Cassenego APV, Andreazza R, Frazzon APG, Camargo FAO Brandelli A. Evaluation of resistance genes and virulence factors in a food isolated Enterococcus durans with potential probiotic effect. Food Control, 2015; 51: 49-54. DOI: 10.1016/j.foodcont.2014.11.012.
19. Migaw S, Ghrairi T, Belguesmia Y, Choiset Y, Berjeaud JM, Chobert JM et al. Diversity of bacteriocinogenic lactic acid bacteria isolated from Mediterranean fish viscera. World J Microbiol Biotechnol, 2014; 30: 1207-17. DOI: 10.1007/ s11274-013-1535-6.
20. Baixas-Nogueras S, Bover-Cid S, Veciana-Nogues MT, Vidal-Carou MC. Amino acid-decarboxylase activity in bacteria associated with Mediterranean hake spoilage. Eur Food Res Technol, 2003; 217: 164-167. DOI: 10.1007/s00217-003-0730-3.
21. Sinton LW, Donnison AM, Hastie CM. Faecal streptococci as faecal pollution indicators: A review. Part II: Sanitary significance, survival, and use. New Zeal J Mar Fresh, 1993; 27(1): 117-37. DOI: 10.1080/00288330.1993.9516549
22. Harwood VJ, Delahoya NC, Ulrich RM, Kramer MF, Whitlock JE, Garey JR, Lim DV. Molecular confirmation of Enterococcus faecalis and E. faecium from clinical, faecal and environmental sources. Lett Appl Microbiol, 2004; 38: 476-82. DOI: doi:10.1111/j.1472-765X.2004.01518.x
23. Aslım B, Beyatlı Y. Antibiotic resistance and plasmid DNA contents of Streptoccoccus thermophilus strains isolated from Turkish yogurts. Turk J Vet Anim Sci, 2004; 28: 257-63.
24. Çıtak S, Yucel N, Orhan S. Antibiotic resistance and incidence of Enterococcus species in Turkish white cheese. Int J Dairy Technol, 2004; 57(1): 27- 31. DOI: 10.1111/j.1471-0307.2004.00122.x
25. Martin B, Garriga M, Hugas M, Aymerich T. Genetic diversity and safety aspects of enterococci from slightly fermented sausages. J Appl Microbiol, 2005; 98: 1177-90. DOI: 10.1111/j.1365- 2672.2005.02555.x
26. Charteris WP, Kelly PM, Morelli L, Collins JK. Antibiotic susceptibility of potentially probiotic Lactobacillus species. J Food Prot, 1998; 61 (12): 1636-1643.
27. Savaşan S, Kaya O, Kırkan Ş, Çiftci A. Balık kökenli Enterococcus faecalis suşlarının antibiyotik dirençlilikleri. Ankara Üniv Vet Fak Derg, 2008; 55: 107-10.
28. Durlu-Ozkaya F, Xanthopoulos V, Tunain N, Tzanetaki EL. Technologically important properties of lactic acid bacteria isolates from Beyaz cheese made from raw ewes’ milk. J Appl Microbiol, 2001; 91: 861-70. DOI: 10.1046/j.1365- 2672.2001.01448.x
29. Aslım B, Yüksekdağ ZN, Sarıkaya E, Beyatlı Y. Determination of the bacteriocin-like substances produced by some lactic acid bacteria isolated from Turkish dairy products. LWT- Food Sci Technol, 2005; 38: 691-4. DOI: 10.1016/j.lwt.2004.08.001
30. Tükel Ç, Avşaroğlu MD, Şimşek Ö, Akçelik M. Isolation and partial characterization of a novel bacteriocin produced by Lactococcus lactis spp. lactis MC38. J Food Saf, 2007; 27: 17-29.
31. Diop MB, Dauphin RD, Tine E, Ngom A, Destain J, Thonart P. Bacteriocin producers from traditional food products. Biotechnol Agron Soc Environ, 2007; 11(4): 275-81.
32. Ghrairi T, Frere J, Berjeaud JM, Manai M. Purification and characterisation of bacteriocins produced by Enterococcus faecium from Tunisian rigouta cheese. Food Control, 2008; 19: 162-9. DOI: 10.1016/j.foodcont.2007.03.003
33. Høıer E, Janzen T, Rattray F, Sørensen K, Børsting MW, Brockmann E, Johansen E. The Production, Application and Action of Lactic Cheese Starter Cultures. In: Law BA, Tamime AY eds. Technology of Cheesemaking, 2nd edition, Wiley- Blackwell publication, 2010: 166-89.
34. Ben Said L, Hamdaoui M, Klibi A, Ben Slama K, Torres C, Klibi N. Diversity of species and antibiotic resistance in enterococci isolated from seafood in Tunisia. Ann Microbiol, 2017; 67: 135-41. DOI: 10.1016/j.scitotenv.2015.05.091
35. Çıtak S, Yucel N, Mendi A. Antibiotic resistance of enterococcal isolates in raw milk. J Food Process Preserv, 2005; 29: 183-95. DOI: 10.1111/j.1745- 4549.2005.00022.x
36. Hajikhani R, Beyatlı R, Aslım B. Antimicrobial activity of enterococci strains isolated from white cheese. Int J Dairy Technol, 2007; 60(2): 105-8. DOI: 10.1111/j.1471-0307.2007.00304.x
37. Gülhan T, Aksakal A, Ekin İH, Savaşan S, Boynukara B. Virulence factors of Enterococcus faecium and Enterococcus faecalis strains isolated from humans and pets. Turk J Vet Anim Sci, 2006; 30: 477-82.
38. Toğay SO, Keskin AÇ, Açık L, Temiz A. Virulence genes, antibiotic resistance and plasmid profiles of Enterococcus faecalis and Enterococcus faecium from naturally fermented Turkish foods. J Appl Microbiol, 2010; 109: 1084-92. DOI: 10.1111/j.1365-2672.2010.04763.x
39. Özden Tuncer B, Ay Z, Tuncer Y. Occurrence of enterocin genes, virulence factors, and antibiotic resistance in 3 bacteriocin-producer Enterococcus faecium strains isolated from Turkish Tulum cheese. Turk J Biol, 2013; 37: 443-9. DOI:10.3906/ biy-1209-26
40. Özmen Toğay S, Temiz A, Çelebi A, Açık L, Yalçın SS. Investigation of potential virulence genes and antibiotic resistance characteristics of Enterococcus faecalis isolates from human milk and colostrum samples. Turk J Biol, 2014; 38: 357- 64. DOI: 10.3906/biy-1311-34
41. Harris LJ, Daeschel MA, Stiles ME, Klaenhammer TR. Antimicrobial activity of lactic acid bacteria against Listeria monocytogenes. J Food Prot, 1989; 52(6): 384-7.
42. Chadad OB, El Bour M, Calo-Mata P, Boudabous A, Velazquez JB. Discovery of novel biopreservation agents with inhibitory effects on growth of foodborne pathogens and their application to seafood products. Res Microbiol, 2012; 163: 44-54. DOI: 10.1016/j.resmic.2011.08.005
43. Gomez Sala B, Munoz Atienza E, Sanchez J, Basanta A, Herranz C, Hernandez PE, et al. Bacteriocin production by lactic acid bacteria isolated from fish, seafood and fish products. Eur Food Res Technol, 2015; 241: 341-56. DOI 10.1007/s00217- 015-2465-3
44. Katırcıoğlu H, Beyatlı Y. Gökkuşağı alabalığı (O. Mykisss Richardson, 1846) ve aynalı sazandan (C.Carpio Linnaeus, 1758) izole edilen laktik asit bakterilerinin genel inhibisyon ve bakteriyosin ve/veya bakteriyosin madde üretimi açısından incelenmesi. Gıda, 2003; 28(6): 589-94.