Tavuk Dışkıları ve Çevresel Örneklerden Salmonella Infantis Fajlarının İzolasyonu ve Karakterizasyonu

Bu çalışmada, Türkiye’de en çok izole edilen kanatlı Salmonella serotipi olan S. Infantis bakteriyofajlarının izolasyonu ve bu fajların konak spektrumunun belirlenmesi ayrıca bu fajların su, yem ve altlık materyallerindeki etki ve yaşam süreleri ile saklama sürelerinin belirlenmesi amaçlandı. Çalışmada, 50 adet dışkı-altlık ve 50 adet atık su örneğinden izole edilen 38 adet S. Infantis fajının, rutin test dilüsyonları, litik spektrumları ve litik profilleri belirlenerek seçilen fajlar RAPD-PCR ile genotiplendirildi. Litik profilleri ve RAPD homoloji düzeyleri birbirinden farklı olanlar arasından seçilen en yüksek litik spektruma sahip fajların (SF-In7, SF-In20) faj-bakteri dinamikleri incelendi. SF-In7, SF-In20 fajlarının adsorbsiyon oranı 20 dk’da %95 ve latent dönemleri ise sırasıyla 57 dk ve 65 dk olarak belirlendi. Deneysel çalışmalarda SF-In7 ve SF-In20 fajlarının 24 saatte canlı S. Infantis sayısını su materyalinde 4 log10 cfu/ml (p<0.001), altlık ve yem materyalinde 2-3 log10 cfu/ml (p<0.001) azalttığı, konak hücre bulundurmayan su materyalinde 4 hafta, altlık ve yem materyallerinde ise 3 hafta yaşadığı tespit edildi. Ayrıca çalışmada, SF-In7 ve SF-In20 fajlarının oda ısında (20-22°C) 6 hafta, +4°C’de 9 ay, -20°C ve -80°C’de ise 4 yıldan fazla canlılıklarını korudukları belirlendi. Çalışma sonucunda, SF-In7 ve SF-In20 fajlarının S. Infantis kontaminasyonunu azaltmada biyokontrol ajanı olarak kullanılabileceği, geniş saklama ısısı ve uzun yaşam süresi sebebiyle saha, kümes, kesimhane gibi ortamlarda uygulanmadan önce uzun süre kolaylıkla saklanabileceği sonucuna varıldı.

Anahtar Kelimeler:

Bakteriyofaj, biyokontrol, tavuk, Salmonella Infantis

Isolation and Characterization of Salmonella Infantis Phages from Poultry Faces and Environmental Samples

In this study, it was aimed to isolate the bacteriophages of S. Infantis, the most isolated Salmonella serotype of poultry in Turkey, to determine the effect and lifespan in the water, litter and feed and to detect the host spectrum and the storage time of these phages. In this study, the routine test dilutions, lytic spectra and lytic profiles of 38 S. Infantis phages isolated from 50 stool-litter and 50 wastewater samples were determined and the selected phages were genotyped by RAPD-PCR. Phage-bacterial dynamics of phages with the highest litic spectrum (SF-In7, SF-In20) selected among the different litic profiles and RAPD homology levels were investigated. The adsorption rate of SF-In7, SF-In20 phages was determined as 95% in 20 min and latency periods were determined as 57 min and 65 min, respectively. Inexperimental studies, it was determined that the SF-In7 and SF-In20 phages decreased the number of alive S. Infantis in 24 h at 4 log10 cfu/ml (p<0.001) in water and at 2-3 log10 cfu/ml (p<0.001) in feed and litter and found to be alive for 4 weeks in the water and 3 weeks in the feed and litter without host cells. In addition, it was also determined that the SF-In7 and SF-In20 phages survived for 6 weeks at room temperature (20-22°C), 9 months at 4°C, and more than 4 years at -20°C and -80°C. As a result of the study, SF-In7 and SF-In20 phages can be used as biocontrol agents to reduce the S. Infantis contamination, and can be stored easily for a longtime period before application in environments such as field, poultry house, slaughterhouse due to its large storage temperature and long life span.

PDF

___

Ackermann HW, Tremblay D, Moineau S, (2004). Long-term bacteriophage preservation. World Federation for Culture Collections Newsletter. 38, 35-40.
Akhtar M, Viazis S, Diez-Gonzalez S, (2014). Isolation, identification and characterization of lytic, wide host range bacteriophages from waste effluents against Salmonella enterica serovars. Food Control, 38, 67-74.
Aksakal A, (2003). Bazı Kanatlıların Dışkılarında Salmonella Türlerinin Varlığı ve Yaygınlığı ile Antibiyotiklere Duyarlılıkları. YYÜ Vet Fak Derg. 14 (1), 95-101.
Anonim (2018) Ulusal Salmonella Kontrol Programı. Erişim adresi: https://tuyekad.org.tr/wpcontent/uploads/2018/09/ULUSAL_SALMONELLA_KONTROL_PROGRAMI__.pdf Erişim tarihi: 18.10.2019
Atterbury RJ, Van Bergen MAP, Ortiz F, Lovell MA, Harris JA, De Boer A, Wagenaar JA, Allen VM, Barrow PA, (2007). Bacteriophage Therapy To Reduce Salmonella Colonization Broiler Chickens. Appl Environ Microbio. 73, 4543-4549.
Bao H, Zhang H, Wang R, (2011). Isolation and characterization of bacteriophages of Salmonella enterica serovar Pullorum. Poultry Sci. 90, 2370-2377.
Bardina C, Spricigo DA, Cortes P, Lagosteraa M, (2012). Significance of the Bacteriophage Treatment Schedule in Reducing Salmonella Colonization of Poultry. Appl Environ Microb. 78(18), 6600-6607.
Borie C, Albala I, Sanchez P, Sanchez ML, Ramirez S, Navarro C, Morales MA, Retamales J, Robeson J, (2008). Bacteriophage Treatment Reduces Salmonella Colonization of Infected Chickens. Avian Dis. 52, 64-7.
Bourdin G, Schmitt B, Guy LM, Germond JE, Zuber S, Michot L, Reuteler G, Brussow H, (2014). Amplification and Purification of T4-Like Escherichia coli Phages for Phage Therapy: from Laboratory to Pilot Scale. Appl Environ Microbiol. 80(4), 1469–1476.
Carey-Smith GV, Billington C, Cornelius AJ, Hudson JA, Heinemann JA, (2006). Isolation and characterization of bacteriophages infecting Salmonella spp. FEMS Microbiol Lett. 258, 182-186.
Cortes P, Spricigo DA, Bardina C, Llagostera M, (2015). Remarkable diversity of Salmonella bacteriophages in swine and poultry. FEMS Microbiol Lett. 362, 1-7.Duc HM, Minh SH, Ken-Ichi H, Miyamoto T, (2018). Isolation and application of bacteriophages to reduce Salmonella contamination in raw chicken meat. Food Scı Tech-Brazıl. 91, 353-360.
EFSA, (2017). The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2016. Efsa Journal, 15(12), 5077
EFSA, (2016). The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2015. Efsa Journal, 14(12),4634
Field A, (2009). DISCOVERING STATISTICS USING SPSS. Third Edition. Dubai: Oriental Press. Chapter 6 p. 166
Fiorentin LI, Vieira NDI, Barioni Junior WI, Barros SII, (2004). In vitro characterization and in vivo properties of Salmonellae lytic bacteriophages isolated from free-range layers. Rev Bras Cienc Avic. 6(2), 121-128.
Gutierrez D, Martin-Platero AM, Rodriguez A, Martinez-Bueno M, Garcia P, Martinez B, (2011). Typing of bacteriophages by randomly amplified polymorphic DNA (RAPD) PCR to assess genetic diversity. FEMS Microbiol Lett. 322, 90-97
Huang C, Virk SM, Shı J, Zhou Y, Willias SP, Morsy MK, Abdelnabby HE, Liu J, Wang X, Li J, (2018). Isolation, Characterization, and Application of Bacteriophage LPSE1 Against Salmonella enterica in Ready to Eat (RTE) Foods. Front Microbiol, 9: 1046
Hyman P, Abedon ST, (2009). Practical Methods for Determining Phage Growth Parameters. Clokie MRJ, Kropinski AM. eds. Bacteriophages Methods and Protocols, Volume 1: Isolation, Characterization, and Interactions. Humana Press, New York. p. 175-202.
Kropinski AM, Mazzocco A, Waddell TE, Lingohr E, Johnson PR, (2009). Enumeration of Bacteriophages by Double Agar Overlay Plaque Assay. Clokie MRJ, Kropinski AM. eds. Bacteriophages Methods and Protocols, Volume 1: Isolation, Characterization, and Interactions. Humana Press, New York. p. 69-77.Kutter E, (2009). Phage Host Range and Efficiency of Plating. Clokie MRJ, Kropinski AM. eds. Bacteriophages Methods and Protocols, Volume 1: Isolation, Characterization, and Interactions. Humana Press, New York. p. 141-151.
Madsen L, Bertelsen SK, Dalsgaard I, Mıddelboeb M, (2013). Dispersal and Survival of Flavobacterium psychrophilum Phages In Vivo in Rainbow Trout and In Vitro under Laboratory Conditions: Implications for Their Use in Phage Therapy. Appl Envirol Microbiol. 79(16), 4853-4861.
Mahmoud M, Askorab A, Barakata AB, Rabiea OEF, Hassanc ES, (2018). Isolation and characterization of polyvalent bacteriophages infecting multi drug resistant Salmonella serovars isolated from broilers in Egypt. Int J Food Microbiol. 266, 8-13
Mirzaei MK, Nilsson AS, (2015). Isolation of Phages for Phage Therapy: A Comparison of Spot Tests and Efficiency of Plating Analyses for Determination of Host Range and Efficacy. PLoS One, 10(3), e0118557.
Nakai T, Sugimoto R, Park KH, Matsuoka S, Mori K, Nishioka T, Maruyama K, (1999). Protective effects of bacteriophage on experimental Lactococcus garvieae infection in yellowtail. Dis Aquat Org. 37(1), 33-41.
Ngangbam AK, Devi NB, (2012). Molecular Characterization of Salmonella Bacteriophages Isolated from Natural Environment and its Potential Role in Phage Therapy. Banglad J Microbiol. 29(1), 33-36.
Pereira C, Silva YJ, Santos AL, Cunha A, Gomes NC, Almeida A, (2011). Bacteriophages with potential for inactivation of fish pathogenic bacteria: survival, host specificity and effect on bacterial community structure. Mar Drugs. 9(11), 2236-55.
Rahaman MT, Rahman M, Rahman MB, Khan MFR, Hossen ML, Parvej MS, Ahmed S, (2014). Poultry Salmonella Specıfıc Bacterıophage Isolatıon And Characterızatıon. Banglad. J Vet Med. 12(2), 107-114.
Sanders ME, Klaenhammer TR, (1980). Restriction and modification in group N streptococci: effect of heat on development of modified lytic bacteriophage. Appl Environ Microbiol. 40(3), 500-6.
Sengül SS, Türkyılmaz S, (2007). Broylerlerde Salmonella Enteritidis ve Salmonella Typhimurium Infeksiyonlarının ELISA ve Drag Sıvap Yöntemleri ile Incelenmesi. J Fac Vet Med Univ Erciyes. 4(2), 85-90.
Soykut EA, Tunail N (2009). Süt Endüstrisinde Sorun Yaratan Termofilik Fajlar. J Food. 34 (2), 107-113.
Spricigo DA, Bardina C, Cortes P, Llagostera M, (2013). Use of a bacteriophage cocktail to control Salmonella in food and the food industry. Int J Food Microbiol. 165, 169-174.
Thung TY, Premarathne JMKJK, Chang WS, Loo YY, Chin YZ, Kuan CH, Tan CW, Basri DF, Radzi CWJWM, Radu S, (2017). Use of a lytic bacteriophage to control Salmonella Enteritidis in retail food. Food Scı Tech-Brazıl. 78, 222-225.
Warren JC, Hatch MT, (1969). Survival of T3 coliphage in varied extracellular environments. I. Viability of the coliphage during storage and in aerosols. Appl Microbiol. 17(2), 256-61.
Wiggins AB, Alexander M, (1985). Minimum Bacterial Density for Bacteriophage Replication: Implications for Significance of Bacteriophages in Natural Ecosystems. Appl Environ Microbiol. 49(1), 19-23.
Woolston J, Parks AR, Abuladze T, Anderson B, Li M, Carter C, Hanna LF, Heyse S, Charbonneau D, Sulakvelidze A, (2013). Bacteriophages lytic for Salmonella rapidly reduce Salmonella contamination on glass and stainless steel surfaces. Bacteriophage. 3(3), e25697.
Zierdt CH (1988). Stabilities of lyophilized Staphylococcus aureus typing bacteriophages. Appl Environ Microbiol. 54(10), 2590.