DETEKSI Staphylococcus aureus KOAGULASE POSITIF DAN Staphylococcus aureus KOAGULASE NEGATIF DARI SUSU SERTA ANALISIS GEN KOAGULASE (coa)

Irene Linda M. S., Dr. drh. A.E.T.H Wahyuni, M.Si

2014 | Tesis | S2 Sain Veteriner

Staphylococcus aureus merupakan bakteri penyebab mastitis dengan tingkat kejadian tinggi dan merugikan peternak. Patogenesitas S. aureus dipengaruhi oleh kemampuanya menghasilkan koagulase, yaitu protein menyerupai enzim yang dapat menggumpalkan plasma oksalat atau sitrat. Staphylococcus aureus koagulase negatif dianggap kurang patogen dibandingkan S. aureus koagulase positif karena hilangnya kemampuan menghindari fagositosis. Penelitian sebelumnya menemukan S. aureus dengan koagulase negatif pada uji tabung, namun belum diketahui apakah S. aureus koagulase negatif tersebut memiliki gen koagulase (coa) atau tidak. Oleh karena itu perlu dilakukan deteksi S. aureus koagulase positif dan S. aureus koagulase negatif dari susu serta analisis gen coa. Sebanyak 32 isolat S. aureus asal susu dari Boyolali, Pacitan dan Ponorogo serta isolat Cowan 1 dan MRSA diidentifikasi ulang dengan mengkultur pada plat agar darah (PAD), pengecatan Gram, uji katalase, penanaman pada Mannitol Salt Agar (MSA) dan uji voges proskauer (VP). Identifikasi S. aureus dengan Polymerase Chain Reaction (PCR) dilakukan dengan deteksi gen 23S rRNA menggunakan primer spesifik 5’-ACGGAGTTACAAAGGACGAC-3’ (forward) dan 5’-AGCTCAGCCTTAACGAGTAC-3’ (reverse) dengan produk amplifikasi sebesar 1250 bp. Uji koagulase dilakukan dengan uji koagulase tabung. Desain primer gen coa menggunakan software online Primer3 menghasilkan urutan primer 5’–GCT GGT ACA GGT ATC CGT GAA T-3’ (forward) dan 5’-CCA TAT GTC GCA GTA CCA TCT G-3’ (reverse). Staphylococcus aureus yang memiliki gen coa disekuensing, kemudian dianalisis dengan software MEGA 5.1. Jumlah S. aureus koagulase tabung positif adalah 22 sampel (64,7%) sedangkan S. aureus koagulase tabung negatif adalah 12 sampel (35,3%). Hasil amplifikasi dari S. aureus koagulase positif terdapat 18 sampel (81,8%) memiliki gen coa dan 4 sampel (18,2%) tidak memiliki gen coa, sedangkan dari S. aureus koagulase negatif terdapat 9 sampel (75%) memiliki gen coa dan 3 sampel (25%) tidak memiliki gen coa. Total S. aureus yang teramplifikasi gen coa adalah 27 sampel (79,4%) dan yang tidak teramplifikasi adalah 7 sampel (20,6%). Variasi amplikon sampel positif memiliki gen coa antara 371-695 bp, terdiri dari 26 sampel (96,3%) dengan pita tunggal dan 1 sampel (3,7%) dengan pita ganda. Terdapat enam kelompok gen coa S. aureus berdasarkan analisis sekuen nukleotida dan asam amino.

Staphylococcus aureus is a bacteria causing mastitis in dairy cows, it has high incidence and often detrimental to farmers. The pathogenicity of Staphylococcus aureus mastitis was influenced by its ability to produce coagulase, a protein enzyme which is able to agglutinate oxalate or citrate plasma. Coagulase-negative S. aureus were considered less pathogenic than coagulasepositive S. aureus because they were not able to avoid phagocytosis. Some previous studies found S. aureus with coagulase negative properties based on tube coagulase test, but it is not known whether they have coagulase gene (coa) or not. Therefore, it become necessary to study the phenotypic differences between coagulase-positive S. aureus and coagulase-negative S. aureus as well as the analysis of the presence of coa gene. A total of 32 isolates of S. aureus from dairy cow’s milk in Boyolali, Pacitan and Ponorogo, and also Cowan 1 and MRSA were reidentified by culturing in blood agar, Gram staining, catalase test, mannitol fermentation ability in Mannitol Salt Agar (MSA), and Voges Proskauer (VP) test. Identification of S. aureus with Polymerase Chain Reaction (PCR) based on detection of 23S rRNA gene using specific primers 5’-ACGGAGTTACAAAGGACGAC-3’(forward) and 5’-AGCTCAGCCTTAACGAGTAC-3’ (reverse) with 1250 bp amplification product. Coagulation test were performed with tube coagulation test. Coa gene detection were also conducted by PCR using primer 5’–GCT GGT ACA GGT ATC CGT GAA T-3’ (forward) and 5’-CCA TAT GTC GCA GTA CCA TCT G- 3’ (reverse) which were determined by Primer3 online software. Staphylococcus aureus positive coa gene were sequenced and analyzed by MEGA 5.1 software. The number of S. aureus with positive tube coagulase test were 22 samples (64.7%) while S. aureus with negative tube coagulase test were 12 samples (35.3%). From the amplification of 22 coagulase-positive S. aureus, 18 samples (81,8%) had coa gene while four samples (18.2%) did not have the coa gene, while from the amplification of 12 coagulase-negative S. aureus, nine samples (75%) had coa gene while three samples (25%) did not have the coa gene. The number of sample with positive coa gene were 27 (79.4%) and samples with negative coa gene were seven (20.6%). The amplicon variations among the samples with coa gene positive were between 371-695 bp, consisting of 26 samples (96.3%) with single band and one sample (3.7%) with double band. There were six groups of S. aureus coa gene based on nucleotides and amino acids sequence.

Kata Kunci : Staphylococcus aureus , koagulase positif, koagulase negatif, gen coa