Koyunlardan İzole Edilen Shiga Toksin Üreten Escherichia coli Suşlarının Virülens Faktörlerinin Araştırılması

Yıl 2023, , 36 - 45, 20.06.2023

Nuray Şahin , Murat Yıldırım , Sibel Kızıl

https://doi.org/10.35864/evmd.1264065

Öz

Tüm dünyada, Shiga toksin üreten Escherichia coli (STEC), önemli zoonotik patojen olarak kabul edilmektedir. Başta sığırlar olmak üzere geviş getirenler ana rezervuarlarıdır, ancak küçükbaş hayvanların insan enfeksiyonlarının epidemiyolojisindeki rolü pek çok ülkede kapsamlı bir şekilde değerlendirilmemiştir. Bu çalışmada koyunlarda Escherichia coli suşları içerisinde Shiga toksin üreten izolatların Stx1, Stx2, eae ve ehxa genlerinin shiga toksin alt tiplerini belirlenmesi ve bunlara ırk, cinsiyeti gibi değişkenlerin karşılaştırmalı etkisini belirlemek amaçlanmıştır. Bu çalışmada, Ankara ili ve çevresindeki 12 farklı koyun sürüsünden alından 272 rektal sıvaptan izole edilen 215 E. coli izolatlarında STEC suşlarının virulens genleri ile shiga toksin alt tipleri tanımlanmıştır. 215 adet izolattan sadece 19 (%8,84) adet Stx1, 3 (%1,4) adet Stx2, 5 (%2,33) adet eae ve 9 (%4,19) adet ehxa geni, 13 (%6,05) adet Stx1-Stx2-ehxa, 5 (%2,33) adet Stx1-eae -ehxa, 3 (%1,4) adet Stx2-eae-ehxa birlikte ve 1(%0,47) adet Stx1- eae, 30 (%13,95) adet Stx1- ehxa, 1(%0,47) adet Stx2- eae, 15 (%6,98) adet Stx2-ehxa ve 10 (%4,65) adet eae – ehxa birlikte tespit edilmiştir. 101 (%46,98) adet izolattan ise Stx1, Stx2, eae ve ehxa genlerinden herhangi biri tespit edilmemiştir. Koyunlarda stx1 alt tiplendirmesinde ise yüksek oranda (%82,35) stx1c izole edilmiştir. Stx2 alt tiplendirmesi de ise sırasıyla stx2f (%62,86), stx2c (%54,29), stx2b (%45,71), stx2d (%22,86), stx2h (%22,86) alt tipleri farklı kombinasyonlarda daha yüksek oranda tespit edilmiştir. Bu veriler koyunlarda STEC varlığını ve insanlarda ciddi enfeksiyona neden olma potansiyellerinin olduğunu göstermektedir.

Anahtar Kelimeler

Shiga Toksin Üreten E. coli (STEC), Stx1, Stx2, Alt Tip, Koyunlar

Investigation of virulence factors of shiga toxin-producing Escherichia coli strains isolated from sheep

Öz

All over the world, Escherichia coli (STEC), which produces Shiga toxin, is recognized as an important zoonotic pathogen. Ruminants, particularly cattle, are their main reservoirs, but the role of small ruminants in the epidemiology of human infections has not been extensively evaluated in many countries. In this study, it was aimed to determine the Shiga toxin subtypes of Stx1, Stx2, eae and ehxa genes of Shiga toxin producing isolates within Escherichia coli strains in sheep and to determine the comparative effect of variables such as race and sex on them. In this study, virulence genes of STEC strains and Shiga toxin subtypes were identified in 215 E. coli isolates isolated from 272 rectal swab from 12 different sheep herds in and around Ankara. Of the 215 isolates, only 19 (8.84%) Stx1, 3 (1.4%) Stx2, 5 (2.33%) eae and 9 (4.19%) ehxa genes, 13 (6%, 05) Stx1-Stx2-ehxa, 5 (2.33%) Stx1-eae -ehxa, 3 (1.4%) Stx2-eae-ehxa together and 1 (0.47%) Stx1- eae, 30 (13.95%) Stx1- ehxa, 1 (0.47%) Stx2- eae, 15 (6.98%) Stx2-ehxa and 10 (4.65%) eae – ehxa were detected together. Of the 101 (46.98%) isolates, none of the Stx1, Stx2, eae and ehxa genes were detected. In stx1 subtyping in sheep, a high rate (82.35%) of stx1c was isolated. In stx2 subtyping stx2f (%62,86), stx2c (%54,29), stx2b (%45,71), stx2d (%22,86), stx2h (%22,86) subtypes are more common in different combinations. Detected at a high rate. These data indicate the presence of STEC in sheep and their potential to cause serious infection in humans

Anahtar Kelimeler

Shiga Toxin Producing E. coli, Stx1, Stx2, Subtype, Sheep.

Kaynakça

• Amézquita-López BA, Quiñones B, Cooley MB, León-Félix J, Castro-del Campo N, Mandrell RE, Jiménez M, Chaidez C. (2012). Genotypic analyses of shiga toxin-producing Escherichia coli O157 and non-O157 recovered from feces of domestic animals on rural farms in Mexico. PLoS One.7(12): e51565. doi:10.1371/journal.pone.0051565
• Bai X, Fu S, Zhang J, Fan R, Xu Y, Sun H, He X, Xu J, Xiong Y. (2018). Identification and pathogenomic analysis of an Escherichia coli strain producing a novel Shiga toxin 2 subtype. Sci Rep. 8(1):6756. Published 2018 Apr 30. doi:10.1038/s41598-018-25233-x
• Bélanger L, Garenaux A, Harel J, Boulianne M, Nadeau E, Dozois CM (2011). Escherichia coli from animal reservoirs as a potential source of human extraintestinal pathogenic E. coli. FEMS Immunol Med Microbiol. 62(1):1-10. doi:10.1111/j.1574-695X.2011.00797.x
• Beutin L, Geier D, Steinrück H, Zimmermann S, Scheutz F (1993). Prevalence and some properties of verotoxin (Shiga-like toxin)-producing Escherichia coli in seven different species of healthy domestic animals. J Clin Microbiol.31(9):2483-2488. doi:10.1128/jcm.31.9.2483-2488.1993
• Beutin L, Krüger U, Krause G, Miko A, Martin A, Strauch E (2008). Evaluation of major types of Shiga toxin 2E-producing Escherichia coli bacteria present in food, pigs, and the environment as potential pathogens for humans. Appl Environ Microbiol. 74(15):4806-4816. doi:10.1128/AEM.00623-08
• Blanco J, Blanco M, Blanco JE, Mora A, González EA, Bernárdez MI, Alonso MP, Coira A, Rodriguez A, Rey J, Alonso JM, Usera MA (2003). Verotoxin-producing Escherichia coli in Spain: prevalence, serotypes, and virulence genes of O157:H7 and non-O157 VTEC in ruminants, raw beef products, and humans. Exp Biol Med (Maywood).228(4):345-351. doi:10.1177/153537020322800403
• Brandal LT, Sekse C, Lindstedt BA, Sunde M, Løbersli I, Urdahl AM, Kapperud G. (2012). Norwegian sheep are an important reservoir for human-pathogenic Escherichia coli O26:H11. Appl Environ Microbiol.78(12):4083-4091. doi:10.1128/AEM.00186-12
• Brett KN, Ramachandran V, Hornitzky MA, Bettelheim KA, Walker MJ, Djordjevic SP (2003). stx1c Is the most common Shiga toxin 1 subtype among Shiga toxin-producing Escherichia coli isolates from sheep but not among isolates from cattle. J Clin Microbiol. 41(3):926-936. doi:10.1128/JCM.41.3.926-936.2003
• Caprioli A, Morabito S, Brugère H, Oswald E (2005). Enterohaemorrhagic Escherichia coli: emerging issues on virulence and modes of transmission. Vet Res.36(3):289-311. doi:10.1051/vetres:2005002
• Caprioli A, Scavia G, Morabito S (2014). Public Health Microbiology of Shiga Toxin-Producing Escherichia coli. Microbiol Spectr. 2(6):10.1128/microbiolspec.EHEC-0014-2013. doi:10.1128/microbiolspec.EHEC-0014-2013
• Chapman PA, Siddons CA, Gerdan Malo AT, Harkin MA (1997). A 1-year study of Escherichia coli O157 in cattle, sheep, pigs and poultry. Epidemiol Infect.119(2):245-250. doi:10.1017/s0950268897007826 Cody EM, Dixon BP (2019). Hemolytic Uremic Syndrome. Pediatr Clin North Am. 66(1):235-246. doi:10.1016/j.pcl.2018.09.011
• Dallenne C, Da Costa A, Decré D, Favier C, Arlet G (2010). Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in Enterobacteriaceae. J Antimicrob Chemother. 65(3):490-495. doi:10.1093/jac/dkp498
• de Boer RF, Ferdous M, Ott A, Scheper HR, Wisselink GJ, Heck ME, Rossen JW, Kooistra-Smid AM. (2015). Assessing the public health risk of Shiga toxin-producing Escherichia coli by use of a rapid diagnostic screening algorithm. J Clin Microbiol. 53(5):1588-1598. doi:10.1128/JCM.03590-14
• Djordjevic SP, Hornitzky MA, Bailey G, Gill P, Vanselow B, Walker K, Bettelheim KA (2001). Virulence properties and serotypes of Shiga toxin-producing Escherichia coli from healthy Australian slaughter-age sheep. J Clin Microbiol. May;39(5):2017-21. doi: 10.1128/JCM.39.5.2017-2021.2001. PMID: 11326039; PMCID: PMC88074.
• Ferhat L, Chahed A, Hamrouche S, Korichi-Ouar M, Hamdi TM (2019). Research and molecular characteristic of Shiga toxin-producing Escherichia coli isolated from sheep carcasses. Lett Appl Microbiol. 68(6):546-552. doi: 10.1111/lam.13142. Epub 2019 Mar 19. PMID: 30801745.
• Friedrich AW, Bielaszewska M, Zhang WL, Pulz M, Kuczius T, Ammon A, Karch H (2002). Escherichia coli harboring Shiga toxin 2 gene variants: frequency and association with clinical symptoms. J Infect Dis.185(1):74-84. doi:10.1086/338115 Gill A, Dussault F, McMahon T, Petronella N, Wang X, Cebelinski E, Scheutz F, Weedmark K, Blais B, Carrillo C (2022). Characterization of Atypical Shiga Toxin Gene Sequences and Description of Stx2j, a New Subtype. J Clin Microbiol. 2022;60(3):e0222921. doi:10.1128/jcm.02229-21
• Gonzalez AG, Cerqueira AM, Guth BE, Coutinho CA, Liberal MH, Souza RM, Andrade JR, (2016). Serotypes, virulence markers and cell invasion ability of Shiga toxin-producing Escherichia coli strains isolated from healthy dairy cattle. J Appl Microbiol. 121(4):1130-1143. doi:10.1111/jam.13230
• Gökçe E, Ünver A, Erdoğan, HM (2010). İshalli neonatal kuzularda enterik patojenlerin belirlenmesi. Kafkas Univ Vet Fak Derg, 16(5), 717-722. Göncüoglu M, Bilir Ormanci FS, Ayaz ND, Erol, I (2010). Antibiotic resistance of Escherichia coli O157: H7 isolated from cattle and sheep. Annals of microbiology, 60(3), 489-494.
• Gülhan T (2003). Sağlıklı görünen hayvanların dışkılarından izole edilen Escherichia coli suşlarının biyokimyasal, enterotoksijenik ve verotoksijenik özelliklerinin belirlenmesi. Yüzüncü Yıl Üniversitesi Veteriner Fakültesi Dergisi, 14(1), 102-109.
• Gyles CL (2007). Shiga toxin-producing Escherichia coli: an overview. J Anim Sci. 85(13 Suppl): E45-E62. doi:10.2527/jas.2006-508
• Han Y, Liu M, Han Y, Shi N, Wang Q, Cui T, Yang L, Zhang X, Zhu L, Qian H, Jin H, Dong C (2022). Genetic and phylogenetic characterization of Shiga toxin-producing Escherichia coli and enteropathogenic E. coli from livestock in Jiangsu by using whole-genome sequencing. J Appl Microbiol.132(5):3925-3936. doi:10.1111/jam.15494
• Hu B, Yang X, Liu Q, Zhang Y, Jiang D, Jiao H, Yang Y, Xiong Y, Bai X, Hou P, (2022). High prevalence and pathogenic potential of Shiga toxin-producing Escherichia coli strains in raw mutton and beef in Shandong, China. Curr Res Food Sci. 5:1596-1602. Published 2022 Sep 15. doi:10.1016/j.crfs.2022.08.021
• Hua Y, Bai X, Zhang J, Jernberg C, Chromek M, Hansson S, Frykman A, Yang X, Xiong Y, Wan C, Matussek A,(2020). Molecular characteristics of eae-positive clinical Shiga toxin-producing Escherichia coli in Sweden. Emerg Microbes Infect. 9(1):2562-2570. doi:10.1080/22221751.2020.1850182
• Jajarmi M, Imani Fooladi AA, Badouei MA, Ahmadi A (2017). Virulence genes, Shiga toxin subtypes, major O-serogroups, and phylogenetic background of Shiga toxin-producing Escherichia coli strains isolated from cattle in Iran. Microb Pathog.109:274-279. doi:10.1016/j.micpath.2017.05.041
• Kaper JB, O'Brien AD (2014). Overview and Historical Perspectives. Microbiol Spectr. 2(6):10.1128/microbiolspec.EHEC-0028-2014.doi:10.1128/microbiolspec.EHEC-0028-2014 Kudva IT, Hatfield PG, Hovde CJ (1996). Escherichia coli O157:H7 in microbial flora of sheep. J Clin Microbiol.34(2):431-433. doi:10.1128/jcm.34.2.431-433.1996
• Kudva IT, Hatfield PG, Hovde CJ(1997). Characterization of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli serotypes isolated from sheep. J Clin Microbiol. 35(4):892-899. doi:10.1128/jcm.35.4.892-899.1997 Liu Y, Li H, Chen X, Tong P, Zhang Y, Zhu M, Su Z, Yao G, Li G, Cai W (2022). Characterization of Shiga toxin-producing Escherichia coli isolated from Cattle and Sheep in Xinjiang province, China, using whole-genome sequencing. Transbound Emerg Dis.69(2):413-422. doi:10.1111/tbed.13999
• Martin A, Beutin L, (2011). Characteristics of Shiga toxin-producing Escherichia coli from meat and milk products of different origins and association with food producing animals as main contamination sources. Int J Food Microbiol. 146(1):99-104. doi:10.1016/j.ijfoodmicro.2011.01.041
• Martins FH, Guth BE, Piazza RM, Leão SC, Ludovico A, Ludovico MS, Dahbi G, Marzoa J, Mora A, Blanco J, Pelayo JS, (2015). Diversity of Shiga toxin-producing Escherichia coli in sheep flocks of Paraná State, southern Brazil. Vet Microbiol.175(1):150-156. doi:10.1016/j.vetmic.2014.11.003
• McCarthy SC, Macori G, Duggan G, Burgess CM, Fanning S, Duffy G (2021). Prevalence and Whole-Genome Sequence-Based Analysis of Shiga Toxin-Producing Escherichia coli Isolates from the Recto-Anal Junction of Slaughter-Age Irish Sheep. Appl Environ Microbiol.87(24):e0138421. doi:10.1128/AEM.01384-21
• Momtaz H, Safarpoor Dehkordi F, Rahimi E, Ezadi H, Arab R (2013). Incidence of Shiga toxin-producing Escherichia coli serogroups in ruminant's meat. Meat Sci.95(2):381-388. doi:10.1016/j.meatsci.2013.04.051
• Mora A, Herrrera A, López C, Dahbi G, Mamani R, Pita JM, Alonso MP, Llovo J, Bernárdez MI, Blanco JE, Blanco M, Blanco J, (2011). Characteristics of the Shiga-toxin-producing enteroaggregative Escherichia coli O104:H4 German outbreak strain and of STEC strains isolated in Spain. Int Microbiol.14(3):121-141. doi:10.2436/20.1501.01.142
• Mühlen S, Dersch P, (2020) Treatment Strategies for Infections With Shiga Toxin-Producing Escherichia coli. Front Cell Infect Microbiol.10:169. Published 2020 May 6. doi:10.3389/fcimb.2020.00169
• Ogden ID, MacRae M, Strachan NJ, (2005). Concentration and prevalence of Escherichia coli O157 in sheep faeces at pasture in Scotland. J Appl Microbiol.98(3):646-651. doi:10.1111/j.1365-2672.2004.02493.x
• Orth D, Grif K, Khan AB, Naim A, Dierich MP, Würzner R, (2007) The Shiga toxin genotype rather than the amount of Shiga toxin or the cytotoxicity of Shiga toxin in vitro correlates with the appearance of the hemolytic uremic syndrome. Diagn Microbiol Infect Dis.59(3):235-242. doi:10.1016/j.diagmicrobio.2007.04.013
• Paton AW, Paton JC, (2002). Direct detection and characterization of Shiga toxigenic Escherichia coli by multiplex PCR for stx1, stx2, eae, ehxA, and saa. J Clin Microbiol. 40(1):271-274. doi:10.1128/JCM.40.1.271-274.2002
• Paton AW, Paton JC (2005). Multiplex PCR for direct detection of Shiga toxigenic Escherichia coli strains producing the novel subtilase cytotoxin. J Clin Microbiol.43(6):2944-2947. doi:10.1128/JCM.43.6.2944-2947.2005
• Probert WS, McQuaid C, Schrader K, (2014). Isolation and identification of an Enterobacter cloacae strain producing a novel subtype of Shiga toxin type 1. J Clin Microbiol. 52(7):2346-2351. doi:10.1128/JCM.00338-14
• Scheutz F, Teel LD, Beutin L, Piérard D, Buvens G, Karch H, Mellmann A, Caprioli A, Tozzoli R, Morabito S, Strockbine NA, Melton-Celsa AR, Sanchez M, Persson S, O'Brien AD, (2012),Multicenter evaluation of a sequence-based protocol for subtyping Shiga toxins and standardizing Stx nomenclature. J Clin Microbiol. 50(9):2951-2963. doi:10.1128/JCM.00860-12
• Shen J, Zhi S, Guo D, Jiang Y, Xu X, Zhao L, Lv J, (2022). Prevalence, Antimicrobial Resistance, and Whole Genome Sequencing Analysis of Shiga Toxin-Producing Escherichia coli (STEC) and Enteropathogenic Escherichia coli (EPEC) from Imported Foods in China during 2015-2021. Toxins (Basel). 14(2):68. Published 2022 Jan 19. doi:10.3390/toxins14020068
• Skinner C, McMahon S, Rasooly R, Carter JM, He X, (2013). Purification and characterization of Shiga toxin 2f, an immunologically unrelated subtype of Shiga toxin 2. PLoS One. 8(3):e59760. doi:10.1371/journal.pone.0059760
• Türütoğlu H, Öztürk D, Güler L, Pehlivanoğlu F (2007). Sağlıklı koyun dışkısında sorbitol negatif Escherichia coli O157'nin varlığı ve özellikleri.
• Url 1: https://arastirma.tarimorman.gov.tr/tepge/Belgeler/E.T: 8 Mayıs 2021.
• Url 2: https://gida.erciyes.edu.tr/upload/IVKSWT4mIkro-6.pdf/ E.T. 10 Mayıs 2021
• Vettorato MP, Leomil L, Guth BE, Irino K, Pestana de Castro AF, (2003). Properties of Shiga toxin-producing Escherichia coli (STEC) isolates from sheep in the State of São Paulo, Brazil. Vet Microbiol. 2003;95(1-2):103-109. doi:10.1016/s0378-1135(03)00153-6
• Yang X, Liu Q, Bai X, Hu B, Jiang D, Jiao H, Lu L, Fan R, Hou P, Matussek A, Xiong Y, (2022). High Prevalence and Persistence of Escherichia coli Strains Producing Shiga Toxin Subtype 2k in Goat Herds. Microbiol Spectr. 10(4):e0157122. doi:10.1128/spectrum.01571-22
• Yang X, Liu Q, Sun H, Xiong Y, Matussek A, Bai X, (2022). Genomic Characterization of Escherichia coli O8 Strains Producing Shiga Toxin 2l Subtype. Microorganisms. 2022;10(6):1245. Published 2022 Jun 17. doi:10.3390/microorganisms10061245
• Zweifel C, Blanco JE, Blanco M, Blanco J, Stephan R, (2004). Serotypes and virulence genes of ovine non-O157 Shiga toxin-producing Escherichia coli in Switzerland. Int J Food Microbiol. 2004;95(1):19-27. doi:10.1016/j.ijfoodmicro.2004.01.015