Abstract
Animals/livestock, especially cattle, are known as the main reservoir of Escherichia coli O157: H7. As theonly one of zoonotic E. coli, the pathogenicity of these bacteria is determined by its ability to produce oneor more very potent cytotoxin known as Shiga-like toxin (Stx) or verocytotoxin, particularly of the Stx2 typethat is closely related to the incidence of hemolytic uremic syndrome (HUS) in humans. This study analyzedthe nucleotide sequences of stx2 gene between isolates from animals and humans in an effort to assess thepotential zoonoses of the agent. The research activity was initiated by cultivating 20 isolates of E. coliO157:H7 collection based on result in the previous study i.e. 2 isolates originated from cattle feces, 2isolates originated from beef, 2 isolates originated from chicken feces, 2 isolates originated from humanfaeces and 12 non-clinical isolates originated from human fecal who were suffering with renal failure. Allisolates were confirmed on selective medium Sorbitol MacConkey Agar (SMAC) followed by testing onaglutination 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 bpPCR product. Analysis of sequencing showed both isolates had identical to stx2 nucleotide squences withE. phaga 933 as well as E. coli ATCC 933. These results indicate the both local isolates are potential aszoonotic agents with clinical effects similar to E. phaga 933 and E. coli ATCC 43894
Highlights
Kultivasi Stok Isolat Kultivasi terhadap 20 isolat stok yang terdiri dari satu isolat kontrol positif E. coli ATCC 43894 serta dua isolat asal tinja sapi, dua isolat asal daging sapi, dua isolat asal tinja ayam, dua isolat asal tinja manusia nonklinis, dan 12 isolat asal tinja manusia klinis
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, of the Shiga toxin type 2 (Stx2) type that is closely related to the incidence of hemolytic uremic syndrome (HUS) in humans
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 faeces and 12 non-clinical isolates originated from human fecal who were suffering with renal failure
Summary
Kultivasi Stok Isolat Kultivasi terhadap 20 isolat stok (tersimpan dalam gliserol 30% dengan suhu penyimpanan -20OC) yang terdiri dari satu isolat kontrol positif E. coli ATCC 43894 serta dua isolat asal tinja sapi, dua isolat asal daging sapi, dua isolat asal tinja ayam, dua isolat asal tinja manusia nonklinis, dan 12 isolat asal tinja manusia klinis (asal penderita gagal ginjal). Lysate dimasukan ke dalam penyaring yang terletak dalam tabung kecil 2 mL (QIAamp Mini spin column) dan dipusing dengan kecepatan 6000xg (8000 rpm) selama satu menit. Dan filter yang telah mengandung DNA dimasukan ke dalam tabung kecil 2 mL yang baru untuk selanjutnya dilakukan tahapan washing DNA. Maret 2017 Vol 18 No 1 : 83-93 ditambah 500 μL wash buffer (buffer AW1) dan didiamkan selama lima menit, dipusing dengan kecepatan 6000xg atau 8000 rpm selama satu menit. Tabung kecil dipusing dengan kecepatan penuh 14.000 rpm selama tiga menit, sisa cairan yang tertampung dibuang dan dilanjutkan untuk eluting DNA. QIAamp Mini spin column yang mengandung DNA dimasukan ke dalam tabung kecil (Nunc, Inc) steril baru, ditambahkan 50 μL elution buffer (buffer AE), didiamkan pada suhu kamar selama lima menit, dan dipusing dengan kecepatan 6000xg atau 8000 rpm selama satu menit untuk selanjutnya disimpan pada 4OC. Nilai persentase replikasi pohon dari taksa/isolat yang membentuk clade diolah dengan bootstrap test 1000 dan diperlihatkan dekat cabang, serta analisis struktur proteinnya dilakukan dengan program DDBJ GTOP
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