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All Journal Medicina Buletin Veteriner Udayana E-Jurnal Medika Udayana Jurnal Veteriner Indonesia Medicus Veterinus ISM (Intisari Sains Medis) : Jurnal Kedokteran Jurnal Kedokteran: Media Informasi Ilmu Kedokteran dan Kesehatan Journal of Health Sciences and Medicine Journal of Global Pharma Technology
Komang Januartha Putra Pinatih
Departemen Mikrobiologi, Fakultas Kedokteran, Universitas Udayana, Denpasar, Bali, Indonesia
Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Chemistry Health Professions Immunology & microbiology Medicine & Pharmacology Public Health Veterinary

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Journal : Jurnal Veteriner

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Penentuan Marka Genetik Escherichia coli O157:H7 Asal Hewan dan Manusia dengan Metode Random Amplified Polymorphic DNA (GENETIC MARKERS IDENTIFICATION OF ESCHERICHIA COLI O157:H7 ORIGINATED FROM ANIMALS AND HUMAN BY USING RANDOM AMPLIFIED POLYMORPHIC DNA I Wayan Suardana; Dyah Ayu Widiasih; Komang Januartha Putra Pinatih
Jurnal Veteriner Vol 15 No 3 (2014)
Publisher : Faculty of Veterinary Medicine, Udayana University and Published in collaboration with the Indonesia Veterinarian Association

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (153.57 KB)

Abstract

The use of random amplified polymorphic DNA (RAPD) as a method to identify a genetic markerof bacteria is widely used by researcher. This method is known as a simple, faster, and reliabletechnicque. This study is to find out the aplication of RAPD method in order to identify specific markersof E. coli O157:H7 as a zoonotic agent. The study began by cultivating of 20 isolates of E. coli O157:H7colected by previous study that consist of 2 isolates originated from cattle feces, 2 isolates originatedfrom beef, 2 isolates originated from chicken feces, 2 isolates originated from healthy human and 11isolates originated from unhealthy human (human with kidney failure). All isolates were confirmed byculturing on selective medium sorbitol MacConkey agar (SMAC). Confirmation were followed by testingon O157 latex aglutination, and finally by testing on H7 antiserum. RAPD method as molecularanalysis was performed using decamer primers mixture OPA-01, OPA-02, OPA-03, and OPA-04.Results of study showed both bands 1721 and 700 bp are specifically to differentiate of isolatesoriginated from cases of healthy and unhealthy human. On the other hand, bands with position 1721 bp,300 bp, and 250 bp indicate the isolates originated from unhealthy human, healthy human and chicken,respectively. Isolates from beef are characterized by both bands 1400 and 429 bp, and the isolates fromcattle feces are identified by band with position 342 bp. The specific bands are considered as markers inorder to know the source of E. coli O157:H7 fastly.
Sekuen Nukleotida Gene Shiga like toxin-2 dari Isolat Lokal Escherichia coli O157:H7 asal Hewan dan Manusia (NUCLEOTIDES SQUENCES OF SHIGA-LIKE TOXIN 2 GENES OF ESCHERICHIA COLI O157:H7 LOCAL ISOLATES ORIGINATED FROM ANIMALS AND HUMAN) I Wayan Suardana; Dyah Ayu Widiasih; Komang Januartha Putra Pinatih
Jurnal Veteriner Vol 18 No 1 (2017)
Publisher : Faculty of Veterinary Medicine, Udayana University and Published in collaboration with the Indonesia Veterinarian Association

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (364.77 KB) | DOI: 10.19087/jveteriner.2017.18.1.83

Abstract

Animals/livestock, especially cattle, are known as the main reservoir of Escherichia coli O157: H7. As the only one of zoonotic E. coli, the pathogenicity of these bacteria is determined by its ability to produce one or more very potent cytotoxin known as Shiga-like toxin (Stx) or verocytotoxin, particularly of the Stx2 type that is closely related to the incidence of hemolytic uremic syndrome (HUS) in humans. This study analyzed the nucleotide sequences of stx2 gene between isolates from animals and humans in an effort to assess the potential zoonoses of the agent. The research activity was initiated by cultivating 20 isolates of E. coli O157:H7 collection based on result in the previous study i.e. 2 isolates originated from cattle feces, 2 isolates originated from beef, 2 isolates originated from chicken feces, 2 isolates originated from human feces, and 12 non-clinical isolates originated from human fecal who were suffering with renal failure. All isolates were confirmed on selective medium Sorbitol MacConkey Agar (SMAC) followed by testing on aglutination O157 latex test, and H7 antisera. Molecular analysis of stx2 gene covering open reading frame (ORF) of the stx2 gene was performed using the primer which was designed by researcher i.e. Stx2 (F)/Stx2 (R). The results showed, there were 2 isolates i.e. KL-48 (2) originated from human feces and SM-25 (1) originated from cattle feces were positive for carrying a stx2 gene, which was marked by the 1587 bp PCR product. Analysis of sequencing showed both isolates had identical to stx2 nucleotide squences with E. phaga 933 as well as E. coli ATCC 933. These results indicate the both local isolates are potential as zoonotic agents with clinical effects similar to E. phaga 933 and E. coli ATCC 43894. ABSTRAK Hewan ternak khususnya sapi, dikenal sebagai reservoir utama Escherichia coli O157:H7. Sebagai satu-satunya serotipe E. coli yang bersifat zoonosis, patogenitas bakteri ini ditentukan oleh kemampuannya untuk menghasilkan satu atau lebih cytotoxin yang sangat potensial yang dikenal dengan nama Shiga-like toxin (Stx) atau verocytotoxin, khususnya dari jenis Stx2 yang terkait erat dengan kejadian hemolytic uremic syndrome (HUS) pada manusia. Studi ini bertujuan menganalisis susunan nukleotida dari gen stx2 antara isolat asal hewan dan manusia dalam upaya mengkaji potensi zoonosis yang ditimbulkannya. Kegiatan penelitian diawali dengan kultivasi 20 isolat E. coli O157:H7 koleksi hasil penelitian sebelumnya dengan rincian dua isolat asal tinja sapi, dua isolat asal daging sapi, dua isolat asal tinja ayam, dua isolat asal tinja manusia non-klinis, dan 12 isolat asal tinja manusia klinis (asal penderita gagal ginjal). Isolat sebanyak 20 tersebut dikonfirmasi pada media selektif sorbitol MacConkey agar (SMAC) yang dilanjutkan dengan uji latex O157 aglutination test serta uji antiserum H7. Analisis molekuler komplit gen stx2 yang meliputi open reading frame (ORF) dari gen stx2 dilakukan menggunakan primer rancangan peneliti yaitu Stx2(F)/Stx2(R). Hasil penelitian menunjukkan bahwa ada dua isolat yaitu KL-48 (2) asal tinja manusia dan SM-25 (1) asal tinja sapi positif membawa gene stx2 yang ditandai dengan produk PCR 1587 bp. Analisis hasil sekuensing menunjukkan kedua isolat memiliki susunan gene stx2 yang identik dengan E. phaga 933 dan E. coli ATCC 43894. Hasil ini mengindikasikan kedua isolat lokal berpotensi sebagai agen zoonosis dengan efek klinis yang serupa dengan E. phaga 933 dan E. coli ATCC 43894.
Co-Authors Agus Eka Darwinata Agus Roy Rusly Hariantana Hamid Amy Yelly D. A. Widiasih Dwi Lestari Dyah Ayu Widiasih Gede Wara Samsarga Samsarga I Dewa Ayu Rayna Nareswari Wikananda I Dewa Made Sukrama I G A Aristi Dhika Pranani I Gede Sathya Agastya I Gede Wikania Wira Wiguna I Gusti Agung Istri Gladys Elsyaningrat I Gusti Agung Utama Surya Putra I Gusti Putu Hendra Sanjaya I Kadek Jaya Santika I Made Gustama Heryawan I Nyoman Arnatha I Putu Gede Septiawan Saputra I Wayan Suardana Ida Ayu Putu Putri Andari Ida Ayu Santhi Pertiwi Manuaba Ida Bagus Aditya Bhaskara Ida Sri Iswari Kadek Adit Wiryadana Luh Putu Dea Sasmita Pralambari Made Agus Hendrayana Mita Ekamelinda Ni Luh Ranthi Kurniawathi Ni Made Ratih Purnama Dewi Ni Nengah Dwi Fatmawati Ni Nyoman Sri Budayanti Ni Putu Eka Umarista Apriliani Oktivia Chandra Mustika, Oktivia Chandra Putu Indri Widiani Rachmy Hamdiyati Rian Ka Praja Yosi Kusuma