İnsan ve Sığır Dışkı Örneklerinde Shiga Toksin Üreten Escherichia coli Varlığının Kültür, ELİSA ve Moleküler Yöntemlerle Araştırılması

Year 2024, , 81 - 88, 30.06.2024

Selahattin Ünlü , Hamdi Murat Tuğrul

https://doi.org/10.30607/kvj.1423778

Abstract

Shiga Toksin üreten Escherichia coli (STEC) 2021 EFSA verilerine göre insanlarda Campylobacter, Salmonella ve Yersinia türlerinden sonra en sıklıkla örülen dördüncü zoonotik etkendir. Hemolitik üremik sendrom (HÜS), hemorojik kolit (HK) gibi çok ciddi komplikasyonlara neden olabilmektedir. 2011 yılında Almanya’da 54 kişinin ölümüne ve birçok kişide HÜS oluşumuna sebep olan salgın gibi büyük salgınlara neden olabilir. Tanıda STEC bakterisinin tespiti ve toksin oluşumunu göstermek önemli bir yol göstericidir. Bu çalışmada, dışkı kültürü istemi yapılan hastalarda STEC sıklığının belirlenmesi ve bir entegre et tesisinde bulunan sığırlarda bu mikroorganizmanın kolonizasyon oranlarının saptanması amaçlanmıştır. İnsanlardan toplam 250 dışkı örneği ve bir entegre et tesisinden toplanan 180 sığıra ait dışkı örnekleri STEC varlığı yönünden incelenmiştir. Örnekler, CHROMagar STEC besiyeri ve MacConkey sıvı besiyerlerinde ekimi yapılmış, daha sonra enzyme linked immunosorbent assay (ELISA) ve polimeraz zincir reaksiyonu (PZR) yöntemleri kullanılarak STEC varlığı araştırılmıştır, PZR’de pozitif çıkan patojen gen bölgeleri için dizileme, kültürde üreyen STEC’ler için serotiplendirme yapılmıştır. İnsanlarda araştırılan örneklerin hiçbirinde STEC tespit edilmemiştir. Hayvan öreklerinin 11’i ELISA ile pozitif bulunmuştur. ELISA yöntemiyle pozitif bulunan örneklerin 8’i aynı zamanda PZR yöntemiyle de pozitif saptanmıştır. PZR yöntemiyle pozitif bulunan örneklerin 3’ü CROMagar STEC besiyerinde üretilebilmiştir. İzole edilen 3 bakteriden 1’i O103:NM olarak serotiplendirilmiş diğerleri serotiplendirilememiştir. Ülkemizde STEC varlığının tespiti için yapılan çalışmaların büyük bir kısmı O 157 serotipi hakkında bilgi vermektedir, ancak O 157 dışı suşlarında büyük salgınlara sebep olabilmesi nedeniyle multipleks PZR yöntemiyle, Stx üreten tüm suşların tespit edilmesi gerekmektedir.

Keywords

Shiga Toksin üreten Escherichia coli, Dışkı, ELİSA, PCR

Ethical Statement

Bu araştırma Trakya Üniversitesi Tıp Fakültesi Girişimsel Olmayan Klinik Araştırmalar Etik Kurulu, Trakya Üniversitesi Etik Kurulu tarafından onaylandı (Karar Numarası: 27/06, Protokol Numarası:TÜTF-GOKAEK 2013/183, Onay Tarihi: 25.12.2013) )

Supporting Institution

Bu makale Selahattin Ünlü'nün "Dışkı örneklerinde Shiga toksini üreten Escherichia coli suşlarının görülme sıklığı" başlıklı tıpta uzmanlık tezindeki verilerin güncel literatür verileriyle sentezlenmesiyle oluşturulmuştur. Tez Trakya Üniversitesi Bilimsel Araştırma Projeleri tarafından desteklenmiştir, Proje numarası: 2014-30

Project Number

2014-30

Research into the Presence of Shiga Toxin-producing Escherichia coli in Human and Cattle Feces with Culture, ELISA and Molecular Methods

Abstract

According to 2021 European Food Safety Authority (EFSA) data, Shiga-toxin (stx) producing Escherichia coli (STEC) is the fourth most frequently observed zoonotic agent in humans after Campylobacter, Salmonella and Yersinia. It may cause very serious infections like hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC). In 2011 it caused a large epidemic, leading to the death of 54 people and the development of HUS in several people in Germany. For diagnosis, the detection of STEC bacteria is an important marker to indicate the formation of the toxin. In this study, the aim was to determine the frequency of STEC in patients referred for fecal cultures and to identify the colonization rates of this microorganism among cattle in an integrated meat facility. A total of 250 human fecal samples and fecal samples from 180 cattle collected from an integrated meat facility were investigated for the presence of STEC. None of the samples from humans had STEC identified. Of the animal samples, 11 were positive with enzyme linked immunosorbent assay (ELISA). Of the samples positive with ELISA, 8 were also positive with polymerase chain reaction (PCR). Of the samples positive with PCR, 3 could proliferate on CROMagar STEC medium. Of the 3 isolated bacteria, 1 was serotyped as O103:NM and the other two could not serotyped. The majority of studies performed for the detection of STEC in our country provide information about the O157 serotype; however, it is necessary to identify all strains producing stx with the multiplex PCR method as non-O157 strains may be responsible for large epidemics.

Keywords

ELISA, Feces, PCR, Shiga toxin-producing Escherichia coli

Ethical Statement

This research was approved by The Ethics Committee of the Trakya University Faculty of Medicine Non-Invasive Clinical Research Ethics Committee, Trakya University (Decision Number: 27/06, Protocol Number:TÜTF-GOKAEK 2013/183, Date of Approvel: 25.12.2013)

Supporting Institution

This article was created by synthesizing data from a specialization in medicine thesis entitled “Incidence of Shiga toxin-producing Escherichia coli strains in fecal samples” by Selahattin Ünlü with current literature data. The thesis was supported by Trakya University Scientific Research Projects, Project number is: 2014-30

Project Number

2014-30

References

• Ahmed, M. K. A. A. (2017). Hayvansal kaynaklı Esherichia coli izolatlarında bazı virulens genlerinin PCR ile saptanması. (Doctoral dissertation). Retrieved from https://www.proquest.com/openview/2eb8d3073cba6733ce0f30effc0ca24f/1?pq-origsite=gscholar&cbl=2026366&diss=y
• Aslan, S., Altındiş, M., Yaman, H. (2016). Sığır ve İshalli İnsan Dışkıları ile Bazı Gıdalarda Escherıchıa coli O157: H7 ve stx1/stx2 Gen Varlığının Araştırılması. Nobel Medicus Journal, 12(3).
• Aslantaş, Ö., Erdoğan, S., Cantekin, Z., Gülaçtı, İ., Evrendilek, G. A. (2006). Isolation and characterization of verocytotoxin-producing Escherichia coli O157 from Turkish cattle. International journal of food microbiology, 106(3), 338-342.
• Ball, H. J., Finlay, D., Zafar, A., Wilson, T. (1996). The detection of verocytotoxins in bacterial cultures from human diarrhoeal samples with monoclonal antibody-based ELISAs. Journal of medical microbiology, 44(4), 273-276.https://doi.org/10.1099/00222615-44-4-273
• Bergan, J., Lingelem, A. B. D., Simm, R., Skotland, T., Sandvig, K. (2012). Shiga toxins. Toxicon, 60(6), 1085-1107.https://doi.org/10.1016/j.toxicon.2012.07.016
• Blanco, M., Blanco, J. E., Mora, A., Rey, J., Alonso, J. M., Hermoso, M., ... Blanco, J. (2003). Serotypes, virulence genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from healthy sheep in Spain. Journal of clinical microbiology, 41(4), 1351-1356.https://doi.org/10.1128/jcm.41.4.1351-1356.2003
• Centers for Disease Control and Prevention (CDC). (2021) National Shiga toxin-producing Escherichia coli (STEC) Surveillance Annual Report, 2017. Atlanta, Georgia: US Department of Health and Human Services.Retrieved August 1, 2023, from https://www.cdc.gov/ecoli/surv2017/index.html
• Dastmalchi, S. H., & Ayremlou, N. (2012). Characterization of Shiga toxin-producing Escherichia coli (STEC) in feces of healthy and diarrheic calves in Urmia region, Iran. Iranian journal of microbiology, 4(2), 63.
• Ekinci, Z., Candan, C., Alpay, H., Canpolat, N., Akyüz, S. G., Gündüz, Z., ... Söylemezoğlu, O. (2013). Hemolytic uremic syndrome outbreak in Turkey in 2011. Turkish Journal of Pediatrics, 55(3),246-52.
• Eklund, M. (2005). Enterohemorrhagic Escherichia coli (EHEC) findings from humans in Finland. Retrieved August 1, 2023, from https://helda.helsinki.fi/server/api/core/bitstreams/db4b85b2-5d82-4172-872c-f86c02693aff/content
• Erol, İ. (2016). Yeni ve yeniden önem kazanan gıda kaynaklı bakteriyel zoonozların epidemiyolojisi. Veteriner Hekimler Derneği Dergisi, 87(2), 63-76.
• European Food Safety Authority, & European Centre for Disease Prevention and Control. (2022). The European Union One Health 2021 Zoonoses Report. EFSA Journal, 20(12). Retrieved August 1, 2023, from https://efsa.onlinelibrary.wiley.com/doi/full/10.2903/j.efsa.2022.7666 https://doi.org/10.2903/j.efsa.2022.7666
• Frank, C., Werber, D., Cramer, J. P., Askar, M., Faber, M., an der Heiden, M., … Krause, G. (2011). Epidemic profile of Shiga-toxin–producing Escherichia coli O104: H4 outbreak in Germany. New England Journal of Medicine, 365(19), 1771-1780.
• Garmendia, J., Frankel, G., Crepin, V. F. (2005). Enteropathogenic and enterohemorrhagic Escherichia coli infections: translocation, translocation, translocation. Infection and immunity, 73(5), 2573 2585.https://doi.org/10.1128/iai.73.5.2573-2585.2005
• Gulesen, R., Levent, B., Demir, T., Akgeyik, M., & Kuran, S. (2016). Characterization of shiga toxin-producing Escherichia coli isolated from humans between 2011 and 2014. Japanese Journal of Infectious Diseases, 69(5), 390-394.https://doi.org/10.7883/yoken.JJID.2015.346
• Hala, O., Ehab, E. (2010). Detection of enterohemorrhagic escherichia coli by conventional methods versus multiplex polymerase chain reaction.Egypt. J. Med. Lab. Sci.. 19 (1), 1-7.
• Harada, T., Wakabayashi, Y., Seto, K., Lee, K., Iyoda, S., Kawatsu, K. (2023). Real-time PCR assays to detect 10 Shiga toxin subtype (Stx1a, Stx1c, Stx1d, Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g) genes. Diagnostic Microbiology and Infectious Disease, 105(3). https://doi.org/10.1016/j.diagmicrobio.2022.115874
• Nuhay, Ç., Gülhan, T. (2017). Samsun ili ve ilçelerinde yetiştirilen Anadolu mandalarının dışkı örneklerinde Escherichia coli O157: H7’nin tespiti. Etlik Veteriner Mikrobiyoloji Dergisi, 28(1), 39-45.https://doi.org/10.35864/evmd.530097
• Okumuş E.(2021) Hemolitik Üremik Sendrom Tanılı Hastalardan İzole Edilen Shiga Toksin Üreten Escherichia coli İzolatlarının O Serotiplerinin ve Toksin Varyantlarının Klinik Durumla İlişkisinin Araştırılması.(Specialization in Medicine thesis). Retrieved from the National Thesis Center of the Council of Higher Education. (Thesis No: 666023)
• Paton, J. C.,Paton, A. W. (1998). Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli infections. Clinical microbiology reviews, 11(3), 450-479.https://doi.org/10.1128/cmr.11.3.450
• Pinaka, O., Pournaras, S., Mouchtouri, V., Plakokefalos, E., Katsiaflaka, A., Kolokythopoulou, F., ... Hadjichristodoulou, C. (2013). Shiga toxin-producing Escherichia coli in Central Greece: prevalence and virulence genes of O157: H7 and non-O157 in animal feces, vegetables, and humans. European journal of clinical microbiology & infectious diseases, 32, 1401-1408.
• Prager, R., Fruth, A., Busch, U., Tietze, E. (2011). Comparative analysis of virulence genes, genetic diversity, and phylogeny of Shiga toxin 2g and heat-stable enterotoxin STIa encoding Escherichia coli isolates from humans, animals, and environmental sources. International Journal of Medical Microbiology, 301(3), 181-191.https://doi.org/10.1016/j.ijmm.2010.06.003
• Pulz, M., Matussek, A., Monazahian, M., Tittel, A., Nikolic, E., Hartmann, M., ... Gunzer, F. (2003). Comparison of a shiga toxin enzyme-linked immunosorbent assay and two types of PCR for detection of shiga toxin-producing Escherichia coli in human stool specimens. Journal of Clinical Microbiology, 41(10), 4671-4675.https://doi.org/10.1128/jcm.41.10.4671-4675.
• Rosensweig, J. N., & Gourley, G. R. (1991). Verotoxic Escherichia coli in human disease. Journal of pediatric gastroenterology and nutrition, 12(3), 295-304
• Sánchez, S., Martínez, R., García, A., Benítez, J. M., Blanco, J., Blanco, J. E., ... & Rey, J. (2010). Variation in the prevalence of non-O157 Shiga toxin-producing Escherichia coli in four sheep flocks during a 12-month longitudinal study. Small Ruminant Research, 93(2-3), 144-148.
• Schmidt, H., Beutin, L., Karch, H. (1995). Molecular analysis of the plasmid-encoded hemolysin of Escherichia coli O157: H7 strain EDL 933. Infection and immunity, 63(3), 1055-1061.https://doi.org/10.1128/iai.63.3.1055-1061.1995
• Torres, A. G. (2010). Pathogenic Escherichia coli in Latin America. Retrieved August 1, 2023, from https://books.google.com.tr/books?id=651HFhtY748C&pg=PA1&hl=tr&source=gbs_toc_r&cad=3#v=onepage&q&f=false
• Ünlü S. (2015). Dışkı Örneklerinde Shiga Toksin Üreten Escherıchia coli Suşlarının Görülme Sıklığı.(Specialization in Medicine thesis). Retrieved from the National Thesis Center of the Council of Higher Education. (Thesis No: 392898)
• Wang, G., Clark, C. G., Rodgers, F. G. (2002). Detection in Escherichia coli of the genes encoding the major virulence factors, the genes defining the O157: H7 serotype, and components of the type 2 Shiga toxin family by multiplex PCR. Journal of clinical microbiology, 40(10), 3613-3619.
• Welch, R. A. (1991). Pore‐forming cytolysins of Gram‐negative bacteria. Molecular microbiology, 5(3), 521-528.https://doi.org/10.1111/j.1365-2958.1991.tb00723.x
• Welch, R. A., Pellett, S.(1988). Transcriptional organization of the Escherichia coli hemolysin genes. Journal of bacteriology, 170(4), 1622-1630.https://doi.org/10.1128/jb.170.4.1622-1630.1988
• World Health Organization. (1998). Zoonotic Non-0157 Shiga Toxin-Producing Escherichia Coli (STEC): report of a WHO scientific working group meeting, Berlin, Germany 23-26 June 1998 (No. WHO/CSR/APH/98.8). World Health Organization. Retrieved August 1, 2023, from https://iris.who.int/bitstream/handle/10665/68880/WHO_CSR_APH_98.8.pdf.