Mastitisli Sığırlardan İzole Edilen Enterococcus faecium İzolatlarında gelE, esp ve efaAfm Genlerinin Varlığının İncelenmesi

Öz

Bu çalışmada, 600 mastitisli sığır sütünden izole edilen Enterococcus faecium izolatlarının önemli virülens genlerinin [jelatinaz (gelE), enterokokal yüzey proteini (esp), adezyonla ilgili protein (efaAfm)] polimeraz zincir reaksiyonu (PZR) ile incelenmesi amaçlandı. Enterokok izolasyonu selektif besiyerleri kullanılarak gerçekleştirildikten sonra cins ve tür düzeyinde identifikasyonlar PZR ile doğrulandı. Çalışmada 96 Enterococcus spp. izole edilirken; bunların %13,5’i E. faecium olduğu belirlendi. Bu izolatların virulens genlerinin incelenmesi sonucunda %69,2’sinin efaAfm, %30,7’sinin gelE ve %30,7’sinin esp genlerini taşıdıkları saptandı. İzolatların %23,1’inin herhangi bir virulens geni taşımıyordu. Beş farklı virulens genotipi saptandı. Sonuç olarak bu çalışma, mastitisli sığır sütlerinden izole edilen E. faecium izolatlarının yüksek patojenite potansiyeline sahip olduklarını gösterdi. E. faecium ile ilişkili diğer virulens faktörlerin tanımlanması, bu virulens faktörlerinin antibiyotiklerle ilişkilerinin ortaya konması konusunda daha geniş kapsamlı araştırmaların yapılması gerekmektedir.

Anahtar Kelimeler

• E. Faecium,Efaafm,Esp,Gele,Mastitis

Investigation of the Presence of gelE, esp and efaAfm Genes in Enterococcus faecium Isolates Isolated from Mastitic Bovine

Öz

In this study, it was aimed to investigate the important virulence genes [gelatinase (gelE), adhesion-associated protein (efaAfm) and enterococcal surface protein (esp)] of Enterococcus faecium isolates isolated from 600 mastitic bovine milk samples with polymerase chain reaction (PCR). Enterococci isolation was performed with using selective agars. Identifications based on genus and species were also performed with PCR. Only 13.5% isolates were determined as E. faecium in 96 Enterococcus spp. It was determined that 69.2%, 30.7% and 30.7% isolate were carrying efaAfm, gelE, and esp genes, respectively. 23.1% of the isolates did not carry any virulence genes. Five different genotypes were detected. Finally, it can be said that E. faecium isolates isolated from mastitic bovine milk have high pathogenity. It is thought that further studies should be conducted on the definition of virulence factors related to severity of the infection and expending traits of E. faecium and their relationships with some kind of antibiotics should also be revealed.

Anahtar Kelimeler

• E. Faecium,Efaafm,Esp,Gele,Mastitis

Kaynakça

1. Abriouel H, Omar NB, Molinos AC. Comparative analysis of genetic diversity and incidence of virulence factors and antibiotic resistance among enterococcal populations from raw fruit and vegetable foods, water and soil, and clinical samples. Int J Food Microbiol 2008; 123: 38-49.
2. Baylan O, Nazik H, Bektore B, Citil BE, Turan D, Ongen B, Özyurt M, Açıkel CH, Haznedaroğlu T. The relationship between antibiotic resistance and virulence factors in urinary enterococcus isolates. Mikrobiyol Bul 2011; 45: 430-445.
3. Belgacem ZB, Abriouel H, Omar NB, Lucas R, Martinez-Canamero M, Galvez A, Manai M. Antimicrobial activity, safety aspects, and some technological properties of bacteriocinogenic Enterococcus faecium from artisanal Tunisian fermented meat. Food Control 2010; 21: 462–470.
4. Bensalah F, Flores MJ, Mouats A. A rapid PCR based method to distinguish between Enterococcus species by using degenerate and species-specific sodA gene primers. African J Biotechnol 2006; 5: 607-702.
5. Cengiz S, Tekin O, Akan M. Mastitislerden izole edilen enterokokların moleküler tiplendirilmesi. Ankara Üniv Vet Fak Derg 2011; 58: 17-20.
6. Charles MAPF, Albrecth BMS, Nuham KY, Marc V, Jean S, Wilhelm HH. Incidence of virulence factors and antibiotic resistance among nterococci isolated from food. Appl Environ Microbiol 2001; 67: 4385-4389.
7. Creti R, Imperi M, Bertuccini L. Survey for virulence determinants among Enterococcus faecalis isolated from different sources. J Med Microbiol 2004; 53: 13-20.
8. Devriese LA, Hommez J, Laevens H, Pot B, Vandamme P, Haesebrouck F. Identification of aesculinhydrolyzing Streptococci, Lactococci, Aerococci and Enterococci from subclinical intramammary infections in dairy cows. Vet Microbiol 1999; 70: 87-94.

9. Eaton TJ, Gasson MJ. Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates. Appl Environ Microb 2001; 67: 1628-1635.
10. Ke D, Picard FJ, Martineau F, Menard C, Roy PH, Ouellette M, Bergeron M. Development of a PCR assay for rapid detection of Enterococci. J Clin Microbiol 1999; 37: 3497-3503.
11. Kuyucuoğlu Y. Antibiotic resistances of enterococci isolated from bovine subclinical mastitis. Eurasian J Vet Sci 2011; 27: 231- 234.
12. Lanthier M, Scott A, Lapen D, Zhang Y, Topp E. Frequency of virulence genes and antibiotic resistances in Enterococcus sp. isolates from wastewater and feces of domesticated mammals and birds, and wildlife. Canadian J Microbiol 2010b; 56: 715–29.
13. Lanthier M, Scott A, Zhang Y, Cloutier M, Durie D, Henderson VC, Wilkes G, Lapen DR, Topp E. Distribution of selected virulence genes and antibiotic resistance in Enterococcus species isolated from the South Nation River drainage basin, Ontario, Canada. J Appl Microbiol 2010a; 110: 407–421.
14. Lund B, Adamsson I, Edlund C. Gastrointestinal Transit Survival of an Enterococcus faecium probiotic strain administered with or without vancomycin. Intl J Food Microbiol 2002; 77: 109-115.
15. Nam HM, Lim SK, Moon JS, Kang HM, Kim JM, Jang KC, Kang MI, Joo YS, Jung SC. Antimicrobial resistance of Enterococci isolated from mastitic bovine milk samples in Korea. Zoonoses Public Health 2009; 57: e59-64.
16. Petersson-Wolfe CS, Adams S, Wolf SL, Hogan JS. Genomic typing of Enterococci isolated from bovine mammary glands and environmental sources. J Dairy Sci 2007b; 91: 615–619
17. Petersson-Wolfe CS, Wolf SL, Hogan JS. In vitro growth of Enterococci of bovine origin in bovine mammary secretions from various stages of lactation. J Dairy Sci 2007a; 90, 4246-4231.
18. Semedo T, Santos MA, Lopes MF, Figueiredo Marques JJ, Barreto Crespo MT, Tenreiro R. Virulence factors in food, clinical and reference Enterococci: a common trait in the genus? Syst Appl Microbiol 2003; 26: 13–22.
19. Shankar V, Baghdayan AS, Huycke MM, Lindahl G, Gilmore MS. Infection-derived Enterococcus faecalis strains are enriched in esp, a gene encoding a novel surface protein. Infect Immun 1999; 67: 193–200.
20. Smith KL, Hogan JS. Environmental mastitis caused by species of Streptococcus and Enterococcus: risk factors and control. Ohio Agricultural Research and Development Center The Ohio State University Wooster, USA, 2003.
21. Tenhagen BA, Köster G, Wallmann J, Heuwieser W. Prevalence of mastitis pathogens and their resistance against antimicrobial agents in dairy cows in Brandenburg, Germany. J Dairy Sci 2006; 89: 2542–2551.
22. Tuncer Y, Inoğlu ZN. Safety assessment of Enterococcus faecium and Enterococcus faecalis strains isolated from Turkish tulum cheese. J Safety 2013; 33: 369-377.
23. Vankerckhoven V, Van Autgaerden T, Vael C. Development of a multiplex PCR for the detection of asa1, gelE, cylA, esp, and hyl genes in enterococci and survey for virulence determinants among European hospital isolates of Enterococcus faecium. J Clin Microbiol 2004; 42: 4473-4479.
24. Vilela, MA, Souz, SL, Palazzo ICV, Ferreira JC, Morais Jr MA, Darini ALC, Morais MMC. Identification and molecular characterization of Van A-type vancomycin-resistant Enterococcus faecalis in Northeast of Brazil. Mem. Inst. The Mem I Oswaldo Cruz 2006; 101: 716-719.
25. Waar K, Muscholl-Silberhorn AB, Willems RJ, Slooff MJ, Harmsen HJ, Degener JE. Genogrouping and incidence of virulence factors of Enterococcus faecalis in liver transplant patients differ from blood culture and fecal isolates. J Infect Dis 2002; 185: 1121-1127.
26. Yıldız Ö, Turkyilmaz S. Investigation of virulence genes of Enterococcus faecalis strains isolated from mastitic bovine milk. Isr J Vet Med 2015; 70: 16-22.