Farklı Kaynaklardan İzole Edilen Listeria Türlerinin Moleküler Tiplendirilmesi

Abstract

Bu çalışmada farklı kaynaklardan (sığır-koyun kıyma ve dışkısı, süt, silaj) Listeria türlerinin varlığının saptanması ve bu kaynaklardan elde edilen izolatlar ile Erciyes Üniversitesi Tıp Fakültesi Mikrobiyoloji Anabilim Dalından temin edilen insan kaynaklı L. monocytogenes izolatlarının Rep-PCR tekniği ile genotiplendirilmesi amaçlandı. Çalışmada Aralık 2008-Mayıs 2009 tarihleri arasında toplam 350 örnek konvansiyonel yöntemler ve immunomanyetik separasyon metodu ile Listeria spp. yönünden incelendi. İzolatların identifikasyonu Microbact 12L Listeria identifikasyon kiti (Oxoid, MB1128A) ile yapıldı ve L. monocytogenes izolatlarını doğrulamak için PCR yöntemi kullanıldı. Listeria türleri, 50 sığır dışkı, 50 koyun dışkı, 25 sığır kıyma ve 25 koyun kıyma örneğinden sırasıyla % 22, % 10, % 48, % 16 oranında izole edildi. İncelenen koyun ve sığır sütleri, tank sütü örnekleri ile silaj örneklerinden Listeria türleri izole edilemedi. Toplamda 32 Listeria spp. izolatı elde edildi. Bu izolatların 12’si L. monocytogenes, 12’si L. innocua, 6’sı L. grayi, 2’si L. welshimeri olarak identifiye edildi. Rep-PCR profillerine göre, Listeria türleri A, B, C olarak dizayn edilen 3 farklı genotipik gruba ayrıldı. Gruplar içerisindeki aynı türlerin % 90-99 oranında genetik benzerlik gösterdiği tespit edildi. İnsan, kıyma, sığır-koyun dışkı örneklerinden izole edilen L. monocytogenes suşları arasında yüksek düzeyde genotipik yakınlık saptandı. Bu sonuçlar gıda kaynaklı zoonoz bir patojen olan Listeria türlerinin önemini ortaya koymaktadır

Keywords

• İmmunomanyetik separasyon
• koyun
• Listeria spp.
• Rep-PCR
• sığır

Molecular typing of Listeria spp. isolated from different sources

Abstract

The aim of this study was to determine the presence of Listeria spp. from different sources (beef and sheep minced meat, feces, milk and silage), and perform the genotyping of Listeria spp. strains isolated from these sources and L. monocytogenes isolates obtained from Erciyes University, Faculty of Medicine, Department of Microbiology by Rep-PCR using the DiversiLab system. A total of 350 samples were examined for the presence of Listeria spp. by conventional culture method and immunomagnetic separation method, between December 2008 and May 2009. Identification of obtained isolates was made with Microbact 12L Listeria identification test kit (Oxoid, MB1128A) and PCR was used to confirm the L. monocytogenes isolates. Listeria spp. were isolated from 22%, 10%, 48% and 16% of 50 cattle feces, 50 sheep feces, 25 beef minced meat and 25 sheep minced meat samples respectively. No Listeria spp. were obtained from the sheep and cow’s milks, bulk tank milks and silage samples examined. In total, 32 Listeria spp. isolates were obtained. Of these isolates, 12 were identified as L. monocytogenes, 12 as L. innocua, 6 as L. grayi and 2 as L. welshimeri. Listeria species were divided into three different genotypic groups designated as A, B, C by Rep-PCR profiles. The same species in the groups were detected as genetically similar at a ratio of 90-99 %. High level genotypic relations were determined among the L. monocytogenes strains isolated from cattle-sheep feces, minced meat and human samples. The results reveal the importance of Listeria spp. as a foodborne pathogen, possessing zoonotic potential

Keywords

• Cattle
• immunomagnetic separation
• Listeria spp.
• Rep-PCR
• sheep

References

1. Adesiyun AA, Webb LA, Romain H, Kaminjolo JS (1996): Prevalence of Salmonella, Listeria monocytogenes, Campylobacter spp., Yersinia enterocolitica and Cryptosporidium spp. in bulk milk, cows’ faeces and effluents of dairy farms in Trinidad. Rev Elev Med Vet Pays Trop, 49, 303–309.
2. Atıl E, Ertas HB, Ozbey G (2011): Isolation and molecular characterization of Listeria spp. from animals, foood and environmental samples. Veterinarni Medicina, 56, 386-394.
3. Chou CH, Wang C (2006): Genetic relatedness between Listeria monocytogenes isolates from seafood and humans PFGE and Rep-PCR. Int J Food Microbiol, 110, 135-148.
4. Dhanashree B, Otta SK, Karunasagar I, Goebel W (2003): Incidence of Listeria spp. in clinical and food samples in Mangalore, India. Food Microbiol, 20, 447- 453.
5. Dumen E, Issa G, Ikiz S, Bagcıgil F, Ozgur Y, Kahraman T, Ergin S, Yesil O (2011): Determining existance and antibiotic susceptibility status of Listeria monocytogenes isolated from dairy products, serological and moleculer typing of the isolates. Kafkas Univ Vet Fak Derg, 17, 111-119
6. Ertas HS, Seker E (2005): Isolation of Listeria monocytogenes from fish intestines and RAPD analysis. Turk J Vet Anim Sci, 29, 1007-1011.
7. Fedio WM, Jackson H (1990): Incidence of Listeria monocytogenes in raw bulk milk in Alberta. Food Res Int, 23, 236–238.
8. Fugett EB, Schoonmaker-Boop D, Dumas NB., Corby J, Wiedmann M (2007): Pulsed Field Gel Electrophoresis (PFGE) analysis of temporally matched Listeria monocytogenes isolates from human clinical cases, foods, ruminant farms and urban and natural environments reveals source-associated as well as widely distributed PFGE types. J Clin Microbiol, 45, 865-873.

9. Garrido V, Torroba L, Garcia-Jalon I, Vitas AI (2008): Surveillance of Listeriosis in Navarre, Spain, 1995-2005- Epidemiological patterns and characterization of clinical and food isolates. Eurosurveillance, 13, 1-6.
10. Gaya P, Sanchez J, Medina M, Nunez M (1998): Incidence of Listeria monocytogenes and other Listeria species in raw milk produced in Spain. Food Microbiol, 15, 551–555.
11. Gouws AP, Liedemann I (2005): Evaluation of Diagnostic PCR for the detection of L. monocytogenes in food products. Food Technol Biotechnol, 43, 201-205.
12. Graves LM, Helsel LO, Steigerwalt AG, Morey RE, Daneshvar MI, Roof SE, Orsi RH, Fortes ED, Milillo SR, den Bakker HC, Wiedmann M, Swaminathan B, Sauders BD (2010): Listeria marthii sp. nov., isolated from the natural environment, Finger Lakes National Forest. Int J Syst Evol Microbiol, 60, 1280–1288.
13. Hamdi TM, Naim M, Martin P, Jacquet C (2007): Identification and molecular characterization of Listeria monocytogenes isolated in raw milk in the region of Algiers. Int J Food Microbiol, 116, 190-193.
14. Healy M, Huong J, Bittner T, Lising M, Frye S, Raza S, Schrock R, Manry J, Renwick A, Nieto R, Woods C, Versalovic J, James R, Lupski JR (2005): Microbial DNA typing by automated repetitive-sequence-based PCR. J Clin Microbiol, 43, 199-207.
15. Husu JR (1990): Epidemiological studies on the occurrence of Listeria monocytogenes in the feces of dairy cattle. Zentralbl Veterinarmed B, 37, 276-282.
16. Jalali M, Abedi D (2008): Prevalence of Listeria species in food products in Isfahan, Iran. Int J Food Microbiol, 122, 336-340.
17. Jersek B, Gilot P, Gubina N, Klun J, Mehle E, Tcherneva N, Rijpens N, Herman L (1999): Typing of Listeria monocytogenes strains by repetitive element sequence-based PCR. J Clin Microbiol, 37, 103-109.
18. Kalorey DR, Warke SR, Kurkure NV, Rawool DB, Barbuddhe SB (2008): Listeria species in bovine raw milk: A large survey of Central India. Food Control, 19, 109-112.
19. Leclercq A, Clermont D, Bizet C, Grimont PA, Le Flèche-Matéos A, Roche SM, Buchrieser C, Cadet- Daniel V, Le Monnier A, Lecuit M, Allerberger F (2010): Listeria rocourtiae sp. nov. Int J Syst Evol Microbiol, 60, 2210-2214.
20. Liu D (2006): Identification, subtyping and virulence determination of Listeria monocytogenes, an important foodborne pathogen. J Med Microbiol, 55, 645-659.
21. Massa SM, Cesaroni D, Poda G (1990): The incidence of Listeria spp. in soft cheeses, butter and raw milk in the province of Bologna. J Appl Microbiol, 68, 153-156.
22. Molla B, Yilma R, Alemayehu D (2004): Listeria monocytogenes and other Listeria species in retail meat and milk products in Addis Ababa, Ethiopia. Ethiop J Health Dev, 18, 208-211.
23. Neves E, Lourenco A, Silva AC, Coutinho R, Brito L (2008): Pulsed Field Gel Electrophoresis (PFGE) analysis of Listeria monocytogenes isolates from different sources and geographical origins and representative of the twelve serovars. Syst Appl Microbiol, 31, 387-392.
24. Ozbey G, Ertas HB, Kok F (2006): Prevalence of Listeria species in camel sausages from retail markets in Aydin province in Turkey and RAPD analysis of Listeria monocytogenes isolates. Irish Vet J, 59, 342-344.
25. Perrin M, Bemer M, Delemare C (2003): Fatal Case of Listeria innocua Bacteremia. J Clin Microbiol, 41, 5308– 5309.
26. Rocourt, J, Hof H, Schrettenbrunner A, Malinverni R, Brille J (1986): Méningite purulente aigüe à Listeria seeligeri chez un adulte immunocompétent. Schweiz Med Wochenschr, 116, 248-251.
27. Roussel S, Félix B, Colanéri C, Vignaud ML, Dao TT, Marault M, Brisabois A (2010): Semi-Automated Repetitive-Sequence-Based Polymerase Chain Reaction Compared to Pulsed-Field Gel Electrophoresis for Listeria monocytogenes subtyping. Foodborne Pathog Dis, 7, 1005- 1012.
28. Seeliger HPR, Jones D (1986): Listeria. 1235-1245. In: Sneath PHA, Mair N, Sharpe ME, Holt JG (Eds), Bergeys Manual of Systematic Bacteriology. Williams and Wilkins, Baltimore.
29. Skovgaard N, Morgen CA (1988): Detection of Listeria spp. in faeces from animals, in feeds, and in raw foods of animal origin. Int J Food Microbiol, 6, 229-242.
30. Unnerstad H, Romell A, Ericsson H, Danielsson-Tham ML, Tham W (2000): Listeria monocytogenes in faeces from clinically healthy dairy cows in Sweden. Acta Vet Scand, 41, 167-171.
31. Van Coillie E, Werbrouck H, Heyndrickx M, Herman L, Rijpens NJ (2004): Prevalence and typing of Listeria monocytogenes in ready-to-eat food products on the Belgian market. J Food Prot, 67, 2480-2487.
32. Van Kessel JS, Karns JS, Gorski L, McCluskey BJ, Perdue ML (2004): Prevalence of Salmonella, Listeria monocytogenes, and fecal coliforms in bulk tank milk on US dairies. J Dairy Sci, 87, 2822–2830.
33. Van Kessel JS, Karns JS, Gorski L, Perdue ML (2005): Subtyping Listeria monocytogenes from Bulk Tank Milk Using Automated Repetitive Element–Based PCR. J Food Prot, 68, 2707–2712.
34. Versalovic J, Koeuth T, Lupski JR (1991): Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res, 19, 6823–6831.
35. Vilar MJ, Yus E, Sanjuan ML, Dieguez FJ, Rodriguez- Otero JL (2007): Prevalence of and risk factors for Listeria species on dairy farms. J Dairy Sci, 90, 5083- 5088.
36. Waak E, Tham W, Danielsson-Tham ML (2002): Prevalence and fingerprinting of Listeria monocytogenes strains isolated from raw whole milk in farm bulk tanks and dairy plant receiving tanks. Appl Environ Microbiol, 68, 3366-3370.
37. Weber A, Potel J, Schafer-Schmidt R, Prell A, Datzmann C (1995): Studies on the occurrence of Listeria monocytogenes in fecal samples of domestic and companion animals. Zentralbl Hyg Umweltmed, 198, 117- 123.
38. Wojciech L, Kowalczyk K, Staroniewicz Z, Kosek K, Molenda J, Ugorski M (2004): Genotypic characterization of Listeria monocytogenes isolated from foodstuffs and farm animals in Poland. Bull Vet Inst Pulawy, 48, 427-435.