Broiler Karkaslarından İzole Edilen Campylobacter jejuni İzolatlarının Makrolid, Kinolon ve Tetrasiklin Grubu Antibiyotiklere Karşı Direnç Durumu*

Öz

Çalışmada Haziran 2012 ve Ocak 2013 dönemlerinde, Kayseri şehir merkezinde bulunan farklı satış noktalarında tü- ketime sunulan toplam 200 broiler karkasından Campylobacter jejuni izolasyonu ve izolatların makrolid, kinolon ve tetrasiklin antibiyotiklerine karşı direnç durumlarının fenotipik ve genotipik yöntemlerle belirlenmesi amaçlanmıştır. Çalışmada but, göğüs, kanat ve bütün tavuk olmak üzere toplam 200 örnek incelendi. Örneklerden elde edilen izolatların identifikasyonu, fenotipik testler ve multipleks Polimeraz Zincir Reaksiyonu (mPZR) ile gerçekleştirildi. C. jejuni’lerin antibiyotiklere direnç durumları disk difüzyon testi ile; MİK dağılımları ise E test ile saptandı. Aynı zamanda antibiyotiklere dirençlilikleri; PZR ve sekans analizi gibi moleküler yöntemlerle test edildi. Haziran döneminde toplanan 100 örneğin 81’i, Ocak döneminde ise 100 örneğin 77’si Campylobacter spp. yönünden pozitif bulundu. mPZR testi sonucunda Haziran dönemindeki 124 izolatın 88’i; Ocak dönemindeki 109 izolatın 68’i C. jejuni olarak tespit edildi. Disk difüzyon testinde en yüksek direnç oranları, Haziran ve Ocak dönemleri için sırasıyla nalidiksik asit (%78.4- %92.6), siprofloksasin (%67.1-%86.7) ve tetrasikline (%55.6-%64.7) karşı, en düşük oranlar ise azitromisin (%1.1-%4.4), doksisiklin (%2.2-%1.4) ve eritromisine (%11.3-%4.4) karşı bulundu. E test sonuçları ile disk difüzyon testi sonuçlarının uyumlu olduğu belirlendi. Tetrasiklin direnci yönünden genomik tetO geni; Haziran ve Ocak döneminde sırasıyla %88 ve %85.7’inde gösterilirken; plazmid aracılı tetO geni izolatların %93.7 ve %85.7’sinde tespit edildi. Makrolid direncinde A2074C mutasyonuna izolatların hiçbirinde rastlanmazken; A2075G mutasyonuna Haziran ve Ocak dönemlerinde %18.4 ve %14.8 oranında rastlandı. Genomik kinolon direncinde gyrA geni üzerindeki mutasyonlar, Haziran ve Ocak dönemlerinde, izolatların %83.5 ve %81.8’inde tespit edilirken; plazmid aracılı kinolon direnci amacıyla incelenen qnr geni, izolatların hiçbirinde bulunamadı. PZR ile saptanan kinolon ve makrolid direnci, sekans analizi ile doğrulandı. Sekans analizinde kinolon dirençli izolatlarda Tre-86-İle mutasyonuna rastlanırken; duyarlı izolatlarda bu mutasyona rastlanmadı. Ayrıca, makrolid dirençli izolatlarda A2075G mutasyonu tespit edildi. Ancak, hiçbir izolatta A2074C mutasyonuna rastlanmadı. Duyarlı izolatların hiçbirinde de bu iki mutasyon görülmedi. Sonuç olarak; tavuk etindeki antibiyotiklere dirençli C. jejuni varlığı nedeniyle koruyucu tedbirlerin alınması, dar spektrumlu antibiyotik kullanımının tercih edilmesi ve antibiyotiklerin organizmadan atılma süreleri tamamlanmadan hayvanların kesilip tüketime sunulmaması, ayrıca tedavide ilk olarak tercih edilen eritromisine ilaveten azitromisinin ve doksisiklinin de alternatif olarak kullanılabileceği kanısına varıldı.

Anahtar Kelimeler

• Broiler,Campylobacter jejuni,kinolon,makrolid,tetrasiklin

The Resistance State of Campylobacter jejuni Isolated from Broiler Carcasses Against to Macrolide, Quinolone and Tetracycline Groups of Antibiotics

Öz

: In the study, in June 2012 and in January 2013, the isolation of Campylobacter jejuni from a total of 200 broiler carcasses in different sales points in the city center of Kayseri and the determination of resistance states to macrolides, quinolone and tetracycline groups of antibiotics by using phenotypical and genotypical methods were aimed. Two hundered samples including chicken thigh, breast, wings and carcasses were examined in the study. The identification of the isolates obtained from samples were performed by phenotypical tests and multiplex Polymerase Chain Reaction (mPCR) method. The antibiotic resistance and MIC values of C. jejuni isolates were detected with disk diffusion and E test method, respectively. In addition, the antibiotic resistances were tested by using of PCR and sequencing. Eighty-one of 100 samples collected in June and 77 of 100 samples in January were found to be positive in terms of Campylobacter spp. As a result of mPCR, 88 of 124 and 68 of 109 Campylobacter spp. isolates were identified as C. jejuni from during June and January terms, respectively. In disk diffusion test, while, the highest resistance rates were observed as nalidixic acid (78.4% - 92.6%), ciprofloxacin (67.1% - 86.7%) and tetracycline (55.6% - 64.7%), the lowest resistance rates as azithromycin (1.1% - 4.4%), doxycycline (2.2% - 1.4%) and erythromycin (11.3% - 4.4% ), for the period June and January, were respectively found. It was determined that E test results were compatible with disk diffusion test. Whilst, chromosomal tetO gene was shown in June and January isolates at 88 % and 85.7 % rates, plasmid mediated tetO gene was found at 93.7% and 85.7% rates, respectively. In macrolide resistance, while A2075G mutation was detected in 23SrRNA at rates 18.4% and 14.8% of June and January isolates, A2074C mutation was not observed at any of June or January isolates, respectively. When, in chromosomal quinolone resistance, the mutations in gyrA gene were detected at rates of 83.5% and 81.8% in June and January isolates, respectively, qnr gene examined for plasmid mediated quinolone resistance was found in none of C. jejuni isolates. Quinolone and macrolide resistances detected by PCR were confirmed by DNA sequencing. While, Tre-86-Ile mutation was found in quinolone resistant isolates with sequencing, but these mutations were not detected in susceptible isolates. Additionally, A2075G mutation was detected in macrolide resistant isolates. However, A2074C mutation was observed in any isolates by using sequencing. It was not observed that none of the macrolide susceptible isolates had any of these two mutations. In conclusion, due to the presence of antibiotic resistant C. jejuni detected in chicken meat, protective measures should be taken for, the using of narrow-spectrum antibiotics may be preferred, animals treated with antibiotics, before the completion of withdrawal period of antibiotics from organisms, animals may not be slaughtered or supplied for consumption. Besides, it is considered that in the treatment, azythromycin and doxycycline may be used as alternate to erythromycin in the drug of choice.

Anahtar Kelimeler

• Broiler carcass,Campylobacter jejuni,macrolide,quinolone,tetracycline

Kaynakça

1. Abay S, Kayman T, Otlu B, Hizlisoy H, Aydin F, Ertas N. Genetic diversity and antibiotic resistance profiles of Campylobacter jejuni isolates from poultry and humans in Turkey. Int J Food Microbiol 2014; 178: 29-38.
2. 2. Akan ÖA, Hasçelik G, Akyön Y, Yurdakök K. Campylobacter türlerinin çeşitli antibiyotiklere in vitro duyarlılığı. Mikrobiyol Bul 1994; 28(2): 122-6.
3. 3. Alfredson DA, Korolik V. Antibiotic resistance and resistance mechanisms in Campylobacter jejuni and Campylobacter coli. Fems Microbiol Lett 2007; 277: 123-32.
4. 4. Alonso R, Mateo E, Churruca E, Martinez I, Girbau C, Fernandez-Astorga A. MAMA-PCR assay for the detection of point mutations associated with high-level erythromycin resistance in Campylobacter jejuni and Campylobacter coli strains. J Micro Methods 2005; 63: 99-103.
5. 5. Atabay HI, Corry JEL. Evaluation of a new arcobacter enrichment medium and comparison with two media developed for enrichment of Campylobacter spp. Int J Food Microbiol 1998; 41: 53-8.
6. 6. Bahrani Mougeot FK, Donnenberg, MS. Enteropathogenic bacteria. Schaechter M. Lederberg J. eds. In: The Desk Encyclopedia of Microbiology. USA, Elsevier Ltd, 2004; pp. 403-14.
7. 7. Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1966; 45(4):493–6.
8. 8. Carter GR, Wise DJ. Essentials of Veterinary Bacteriology and Mycology. Sixth Edition. Iowa: Iowa State Press, 2004; p.155-9.

9. 9. Clinical and Laboratory Standards Institute. Methods for antimicrobial dilution and disk susceptibility test of infrequently isolated or fastidious bacteria. Approved Guideline. Second Edition. CLSI Document M45-A2, Vol. 30 No.18 Replaces M-45-A Vol. 26 No.19 2010. CLSI, Wayne, PA.
10. 10. Cokal Y, Caner V, Sen A, Cetin C, Karagenc N. Campylobacter spp. and their antimicrobial resistance patterns in poultry: an epidemiological survey study in Turkey. Zoonoses Public Hlth 2009; 56: 105–10.
11. Coker AO, Isokpehi RD, Thomas BN, Amisu KO, Obi CL. Human campylobacteriosis in developing countries. Emerg Infect Dis 2002; 8(3): 237-44.
12. 12. Collins CH, Lyne PM, Grange JM, Falkinham JO. Collins&Lyne’s Microbiological Methods. Eighth Edition. UK: Arnold Publisher, 2004; 314-6.
13. 13. Diker KS. Gram negatif mikroaerofilik hareketli bakteriler, Campylobacteriaceae familyası. Arda M, Mimbay A, Leloğlu N, Aydın N, Kahraman M, Akay Ö, Ilgaz A, İzgür M, Diker K.S. eds. In: Özel Mikrobiyoloji. Ankara: Medisan Yayınevi, Beşinci Baskı, 1999; pp. 134.
14. 14. Gibreel A, Tracz DM, Nonaka L, Ngo TM, Connell SR, Taylor DE. Incidence of antibiotic resistance in Campylobacter jejuni isolated in Alberta, Canada, from 1999 to 2002, with special reference to tet(O)-mediated tetracycline resistance. Antimicrob Agents Chemother 2004; 48(9): 3442-50.
15. 15. Gür D, Hasçelik G, Akyön Y, Akalın HE, Diker S. Campylobacter jejuni ve Campylobacter coli’nin quinolone grubu antibiyotiklere in vitro duyarlılıkları. Mikrobiyol Bul 1989; 23(3): 185-9.
16. 16. Habib I, Sampers I, Uyttendaele M, Berkvens D, De Zutter L. Baseline data from a Belgium-wide survey of Campylobacter species contaminationin chicken meat preparations and considerations for a reliable monitoring program. Appl Environ Microbiol 2008;74(17): 5483–9.
17. 17. Haruna M, Sasaki Y, Murakami M, Ikeda A, Kusukawa M, Tsujiyama Y, Ito K, Asai T, Yamada Y. Prevalence and antimicrobial susceptibility of Campylobacter in broiler flocks in Japan. Zoonoses Public Hlth 2012; 59: 241–5.
18. 18. Iovine NM. Resistance mechanisms in Campylobacter jejuni. Virulence 2013; 4(3): 230-40.
19. 19. ISO 10272. Microbiology of food and animal feeding stuffs-horizontal method for detection of thermotolerant Campylobacter. The International Organization for Standardization 1995.
20. 20. Jacoby GA, Chow N, Waites KB. Prevalence of plasmid-mediated quinolone. Antimicrob Agents Chemother 2003; 47(2): 559-62.
21. Kayman T, Abay S, Hızlısoy H. Campylobacter türlerinin fenotipik yöntemler ve multipleks polimeraz zincir reaksiyonu ile tanımlanması ve antibiyotik duyarlılıkları. Mikrobiyol Bul 2013; 47(2): 230-9.
22. 22. Kayman T, Abay S, Hızlısoy H, Gödekmerdan A, Aydın F. Campylobacter jejuni izolatlarında siprofloksasin direncinin fenotipik ve moleküler yöntemlerle analizi. İkinci Ulusal Klinik Mikrobiyoloji Kongresi. Kasım, 10-13, 2013; ANTALYA.
23. 23. Luber P, Wagner J, Hahn H, Bartelt E. Antimicrobial resistance in Campylobacter jejuni and Campylobacter coli strains isolated in 1991 and 2001-2002 from poultry and humans in Berlin, Germany. Antimicrob Agents Chemother 2003; 47(12): 3825-30.
24. 24. Lucey B, O’Halloran F, Fanning S, Molecular-based identification and typing of Campylobacter jejuni and C. coli. Methods Mol Biol 2004; 268: 33-47.
25. 25. Mansouri-najand L, Salehe AA, Wai SS. Prevalence of multidrug resistance Campylobacter jejuni and Campylobacter coli in chickens slaughtered in selected markets, Malaysia. Trop Biomed 2012; 29(2): 231–8.
26. 26. Mazi W, Senok A, Al-Mahmeed A, Arzese A, Bindayna K, Botta G. Trends in antibiotic sensitivity pattern and molecular detection of tet(O)-mediated tetracycline resistance in Campylobacter jejuni isolates from human and poultry sources. Jpn J Infect Dis 2008; 61: 82-4.
27. 27. Mead GC, Hudson WR, Hinton MH. Effect of changes in processing to improve hygiene control on contamination of poultry carcasses with Campylobacter. Epidemiol Infect 1995;115: 495- 500.
28. 28. Miflin JK, Templeton JM, Blackall PJ. Antibiotic resistance in Campylobacter jejuni and Campylobacter coli isolated from poultry in the South-East Queensland region. J Antimicrob Chemother 2007; 59: 775-8.
29. 29. Oyarzabal OA, Backert S, Williams LL, Lastovica AJ, Miller RS, Pierce SJ, Vieira SL, Rebollo-Carrato F. Molecular typing, serotyping and cytotoxicity testing of Campylobacter jejuni strains isolated from commercial broilers in Puerto Rico. J Appl Microbiol 2008; 105: 800-12.
30. 30. Öztürk R, Midilli K, Okyay K Eroğlu C, Aygün G, Kenani Y, Sarsan A. Çocuk ve erişkin yaş grubu sürgün olgularında Campylobacter jejuni ve Campylobacter coli sıklığının araştırılması. Türk Mikrobiyol Cem Derg 1994; 24(1): 42-5.
31. Rahimi E, Ameri M. Antimicrobial resistance patterns of Campylobacter spp. isolated from raw chicken, turkey, quail, partridge, and ostrich meat in Iran. Food Control 2011; 22: 1165-70.
32. 32. Rodrigo S, Adesiyun A, Asgarali Z, Swanston W. Antimicrobial resistance of Campylobacter spp. isolated from broilers in small poultry processing operations in Trinidad. Food Control 2007; 18: 321-5.
33. 33. Rosenquist H, Boysen L, Krogh AL, Jensen AN, Nauta M. Campylobacter contamination and the relative risk of illness from organic broiler meat in comparison with conventional broiler meat. Int J Food Microbiol 2013; 162(3): 226-30.
34. 34. SPSS, 2001. SPSS Statistical Package for Windows, Version 14.0.1, (Serial: 9869264). SPSS Inc. Chicago, USA.
35. 35. Taylor DE, Tracz DM. Mechanisms of Antimicrobial Resistance in Campylobacter. Ketley JM, Konkel ME. eds. In: Campylobacter Molecular and Cellular Biology. Norfolk, UK: Horizon Bioscience, 2005; pp.193-204.
36. 36. Uzunovic-Kamberovic S, Zorman T, Berce I, Herman L, Mozine SS. Comparison of the frequency and the occurrence of antimicrobial resistance among C. jejuni and C. coli isolated from human infections retail poultry meat and poultry in Zenica-Doboj Canton, Bosnia and Herzegovina. Med Glas 2009; 6(2): 173-80.
37. 37. Vandamme P, De Ley J. Proposal for a new family, Campylobacteraceae. Int J Syst Bacteriol 1991; 41: 451-5.
38. 38. Wang G, Clifford GC, Tracy MT, Pucknell C, Barton C, Price L, Woodward DL, Rodgers FG. Colony multiplex PCR assay for identification and differentiation of Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus subsp. fetus. J Clinical Microbiol 2002; 40: 4744–7.
39. 39. Wieczorek K, Osek J. Antimicrobial resistance mechanisms among Campylobacter. Biomed Res Int 2013; 2013: 340605. doi: 10.1155/2013/340605.
40. 40. Williams A, Oyarzabal OA. Prevalence of Campylobacter spp. in skinless, boneless retail broiler meat from 2005 through 2011 in Alabama, USA. BMC Microbiology 2012; 184: 1-7.
41. Wirz SE, Overesch G, Kuhnert P, Korczak BM. Genotype and antibiotic resistance analyses of Campylobacter isolates from ceca and carcasses of slaughtered broiler flocks. Appl Environ Microbiol 2010; 76(19): 6377-86.
42. 42. Yildirim M, Istanbulluoglu E, Ayvali B. Prevalence and antibiotic susceptibility of thermophilic Campylobacter species in broiler chickens. Turk J Vet Anim Sci 2005; 29: 655-60.
43. 43. Zirnstein G, Li Y, Swaminathan B, Angulo F. Ciprofloxacin resistance in Campylobacter jejuni isolates: detection of gyrA resistance mutations by mismatch amplification mutation assay PCR and DNA sequence analysis. J Clin Microbiol 1999; 37(10): 3276-80.