Kanatlı Orijinli Escherichia coli suşlarının virülens özelliklerinin fenotipik ve moleküler metotlarla belirlenmesi

Yıl 2015, Cilt: 26 Sayı: 2, 71 - 76, 01.08.2015

Zafer Cantekın Müjgan İzgür

Öz

Bu çalışmada, kolibasillozisli kanatlı hayvanlardan izole edilen 200 adet Escherichia (E.) coli suşunda, özellikle ekstra-intestinal infeksiyonlar için önemli olan virülens faktörlerinden serum direnci, aerobaktin demir elde etme sistemleri, çeşitli adezinler (Tip 1 fimbrialar, P fimbrialar, S fimbrialar ve afimbrial adezinler), hemoliz ve sitotoksik nekrotizan faktör 1 gibi virülens özellikleri fenotipik yöntemlerle ve moleküler yöntemlerle araştırıldı. Çalışmada, suşlar koyun kanlı agar kullanılarak yapılan testle hemoliz üretimi ve moleküler olarak hemoliz özelliğini kodlayan gen yönünden negatif bulundu. Suşların tamamının demir kısıtlayıcı besiyerinde ürediği ve bu suşların %88’inin aerobaktin özelliğini kodlayan İucD genine sahip oldukları saptandı. Vero hücre kültürleri kullanılarak yapılan toksin analizlerinde suşların %40’ında ısıya duyarlı toksin belirlenirken suşların hiç birinde fenotipik ve moleküler olarak sitotoksik nekrotizan faktör 1 belirlenmedi. İnsan O grubu ve değişik hayvanlara ait eritrositler kullanılarak yapılan hemaglutinasyon testi ile suşların %90’ında hemaglutinasyon aktivitesi saptandı ve bunların %26’sı mannoz resistans hemaglutinasyon özelliğinde olduğu belirlendi. Tip 1 fimbriaları kodlayan FimH geni suşların %88’inde, P fimbriaları kodlayan PapEF geni %26’sında bulundu ve bu izolatların %14’ü aynı zamanda PapC genini de bulundurduğu saptandı. Ancak, suşlarda S fimbrialar ve afimbrial adezinleri kodlayan Sfa ve Afa genleri belirlenemedi. Değişik hayvanlara ait serumlarla yapılan testte suşların tamamında serum direnci fenotipik olarak saptandı ve serum direncinden sorumlu TraT geni suşların %90’ında PCR ile belirlendi. Bu çalışmada elde edilen sonuçlara göre, kanatlı orijinli E. coli suşlarında en önemli virülens özelliklerinin aerobaktin demir elde etme sistemleri, fimbrial adezinler ve serum direnci olduğu belirlenmiştir

Anahtar Kelimeler

Escherichia coli, Kanatlı hayvanlar, Virülens özellikleri

Detection of Virulence Properties Escherichia coli Originated From Poultry by Phenotypic and Molecular Methods

Öz

In this study, virulence factors important for extra-intestinal infections such as serum resistance, aerobactin iron uptake systems, some adhesins (type 1 fimbria, P fimbria, S fimbria, afimbrail adhesins), haemolysis and cytotoxic necrotizing factor 1 in 200 Escherichia coli isolates from poultry with colibacillosis, were investigated by both phenotypic methods and molecular methods. Strains were detected to be negative in haemolysin synthesis tests performed with sheep blood agar, and they were negative in molecular tests investigating the haemolysin gene. All strains grew on iron deficient medium and 88% of these were found to harbor iucD gene encoding aerobactin. Heat-labile toxin was detected in 40% of the isolates in the toxin analyses performed in Vero cells, however, cytotoxic necrotizing factor 1 could not be determined in the strains with phenotypic and molecular tests. Haemagglutination activity was determined in 90% of strains in haemagglutination tests performed with human O group erythrocytes and erythrocytes derived from different animals. Twenty six percent of these strains were found to have MRHA activity. FimH gene encoding type 1 fimbria, papEF gene encoding P fimbria, were detected respectively in 88% and 26% of the strains, and 14% of all isolates were also found to harbor the papC gene also encoding the P fimbria. However, sfa and afa genes encoding S fimbria and afimbrial adhesins, respectively, could not be detected in the study. Serum resistance was phenotypically determined in all strains in the tests performed with sera derived from different animals and traT gene encoding serum resistance were detected in 90% of strains by PCR. In the study, serum resistance, aerobactin iron uptake systems and the existence of fimbrial adhesins were determined as the most important virulence factors of avian pathogenic E. coli isolates

Anahtar Kelimeler

Escherichia coli, Poultry, Virulence Properties

Kaynakça

• Arda M, İzgür M, Akay, Ayhan H (1989). Tavuklardan izole edilen Escherichia coli suşlarının Kongo red’e bağlanma, mannoz direnci, patojenite ve antibiyotiklere duyarlılık özelliklerinin incelenmesi. Türk Mikrobiyol Cem Derg.19, 185-198.
• Barnes JH, Gross WB (1997). Colibacillosis. In: Calnek, B.W., Barnes, J.H., Beard, C.W., Mac Dougald, R.L., Saif, Y.M. (Eds.), Diseases of Poultry, Iowa State University Press, Ames, Iowa, pp. 131–141.
• Binns MM, Mayden J, Levine RP (1982). Further Characterization of Complement Resistance Conferred on Escherichia coli by the Plasmid Genes traT of R100 and iss of ColV, I-K94. Infect Immun 35, 654-65.
• Blanco JE, Blanco M, Gonzalez EA, Alonso MP, Garabal JI (1990). Comparative evaluation of three tests for the detection of Escherichia coli cytotoxic necrotizing factors (CNF1 and CNF2) using filtrates of cultures treated with mitomycin C. FEMS Microbiol Lett 69, 311–316.
• Blanco JE, Blanco M, Mora A, Blanco J (1997). Production of toxins (enterotoxins, verotoxins and necrotoxins) and colicins by Escherichia coli strains isolated from septicaemic and healthy chickens: relationship with in vivo pathogenicity. J Clin Microbiol, 35, 2953–2957.
• Da Silveira WD, Ferreira A,Brocchi ML, Hollanda MDA, Castro FPD, Yamada AT, Lancellotti M, Da Silveira WD, De Hollanda LM, De Castro, AFP (2002). Biological characteristics and pathogenicity of avian Escherichia coli strains. Vet Microbiol, 85, 47-53.
• Delicato ER, De Brito BG, Gaziri L J, Vidotto MC (2003). Virulenceassociated genes in Escherichia coli isolates from poultry with colibacillosis. Vet Microbiol, 94, 97-103.
• Dho-Moulin M, Fairbrother JM (1999). Avian pathogenic Escherichia coli (APEC). Vet Res, 30, 299–316.
• Ellis MG, Arp LH, Lamont SJ (1988). Serum resistance and virulence of Escherichia coli isolated from turkeys. Am J Vet Res, 49, 2034-2037.
• Emery DA, Nagaraja KV, Shawd P, Newman JA, White DG (1992). Virulence factors of Escherichia coli associated with colisepticemia in chickens and turkeys. Avian Dis, 36, 504-511.
• Fantinatti F, Silveira WD, Castro AF (1994). Characteristics associated with pathogenicity of avian septicaemic Escherichia coli strains. Vet Microbiol, 41, 75-86.
• İzgür M (1981). Sağlıklı koyunlardan izole edilen E. coli suşlarının çeşitli özellikleri üzerinde incelemeler. Doktora Tezi. Ankara Üniv. Vet. Fak., Ankara
• Janssen T, Schwarz C, Preıkschat P, Voss M, Philipp HC, Wieler LH (2001). Virulence-associated genes in avian pathogenic Escherichia coli (APEC) isolated from internal organs of poultry having died from colibacillosis. Int J Med Microbiol, 291, 371-378.
• Johnson JR, Stell AL (2000). Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise. J Infect Dis, 181, 261–272.
• Kaipainen T, Pohjanvirta T, Shpigel NY, Shwimmer A, Pyörala S, Pelkonen S (2002). Virulence factors of Escherichia coli isolated from bovine clinical mastitis. Vet Microbiol, 85i 37-46.
• Knöbl T, Baccaro MR, Moreno AM, Gomes TAT, Vieira MAM, Ferreira CSA, Ferreira AJP(2001). Virulence properties of Escherichia coli isolated from ostriches with respiratory disease. Vet Microbiol, 83: 71- 80.
• La Ragıone, R. M., Woodward, M. J. (2002). Virulence factors of Escherichia coli serotypes associated with avian colisepticaemia. Res Vet Science, 73, 27–35.
• Lafont JP, Dho M, D'hauteville HM, Brée A, Sansonetti PJ (1987). Presence and expression of aerobactin genes in virulent avian strains of Escherichia coli. Infect Immun, 55, 193–197.
• Linggood M, Roberts M, Ford S, Parry SH, Williams PH (1987). Incidence of aerobactin iron uptake system among Escherichia coli izolates from infections of farm animals. J Gen Microbiol, 133,835-842.
• Ngeleka M, Kwaga JK, White DG, Whittam TS, Riddell C, Goodhope R, Potter AA, Allan B (1996). Escherichia coli celulitis in broiler chickens: clonal relationship among strains and analysis of virulenceassociated factors of izolates from diseased birds. Infect Immun, 64, 3118-3126.
• Ørskov I, Ørskov F (1985). Escherichia coli in extra-intestinal infections. J Hyg Camb, 95, 551–575.
• Payne SM (1988). Iron and virulence in the family Enterobacteriacae. CRC Critical Reviews in Microbiology, 16: 81–111.
• Pfaff-Mcdonough SJ, Horne SM, Giddings CW, Ebert JO, Doetkott C, Smith MH, Nolan LK (2000). Complement resistance-related traits among Escherichia coli isolates from apparently healthy birds and birds with colibacillosis. Avian Dis, 44, 23–33.
• Quinn PJ, Carter ME, Markey BK, Carter GR (1994). Clinical Veterinary Microbiology. Mosby-Year Book Europe Limited, Lynton House, London WC1H9LB, England. s.: 209-236.
• Reingold J, Starr N, Maurer J, Lee M D (1999). Identification of a new Escherichia coli She haemolysin homolog in avian E. coli. Vet. Microbiol. 66, 125–134.
• Sambrook J, Russell W (2002). Molecular cloning: a laboratory manual (3rd ed.), Cold Spring Harbor Press, New York, NY (2002).
• Sanchez CV, Mcdonald JS, Packer RA (1984). Virulence factors of Escherichia coli isolated from cows with acute mastitis. Am J Vet Res, 45, 1775–1777.
• Smith JL, Fratomıco PM, Gunther NW (2007). Extraintestinal pathogenic Escherichia coli. Foodborne Pathog Dis, 4, 134-163.
• Stuart ST, Grenwod KT, Luke RKJ(1980). Hidroxamate-mediated transport of iron controlled by Col V plasmid. J Bacteriol, 143, 35–42.
• Tsuji T, Joya, JE, Honda T, Miwatani T (1990). A heat-labile enterotoxin (LT) purified from chicken enterotoxigenic Escherichia coli is identical to porcine LT. FEMS Microbiol Lett, 55, 329-161.
• Van Den Bogaard AE, London N, Driessen C, Stobberingh EE (2001). Antibiotic resistance of faecal Escherichia coli in poultry, poultry farmers and poultry slaughterers. J Antimicrob Chemother, 47, 763- 771.
• Van Den Bosch JF, Hendriks JHIM, Gladigau I, Willems H MC, Storm, PK, De Graff FK (1993). Identification of F11 fimbriae in chicken Escherichia coli strains. Infect Immun, 61, 800–806.
• Vidotto MC, Müller EE, De Freitas JC, Alfieri AA, Guimarães IG, Santos DS (1990). Virulence factors of avian Escherichia coli. Avian Dis, 34, 531–538.
• Winn W, Allen S, Janda W, Koneman E, Procop G, Schreckenberger P, Woods G (2006). The Enterobacteriaceae. İn: Koneman's Color Atlas and Textbook of Diagnostic Microbiology, 6th ed. Philadelphia: Lippincott Williams & Wilkins s.: 211-308.
• Yamamoto S, Terai A, Yuri K, Kurazono H, Takeda Y, Yoshida O (1995). Detection of Urovirulence Factors in Escherichia coli by Multiplex Polymerase Chain Reaction. FEMS Immunol Med Microbiol, 12, 85-90. , Yogaratnam V (1995). Analysis of the causes of high rates of carcase rejection at a poultry processing plant. Vet Rec, 137, 215-217.