Identifikasi gen enteroktoksin dan accessory gene regulator (agr) staphylococcus aureus dari berbagai pangan asal hewan dan infeksi kulit manusia

SUGIYONO, Prof Dr. drh. Siti Isrina Oktavia Salasia

2008 | Tesis | S2 Sain Veteriner

Accessory gen regulator (agr) merupakan gen regulator ekspresi berbagai faktor virulen yang salah satunya adalah enterotoksin. Dalam penelitian ini telah dilakukan identifikasi gen enterotoksin dan agr yang berperan dalam meregulasi gen penyandi enterotoksin Staphylococcus aureus yang diisolasi dari berbagai produk pangan asal ternak dan infeksi kulit manusia. Penelitian ini bertujuan untuk identifikasi berbagai macam gen penyandi enterotoksin yang dihasilkan oleh S. aureus yang berasal dari berbagai bahan pangan asal ternak dan infeksi kulit manusia dan identifikasi agr S. aureus sebagai regulator enterotoksin S. aureus dengan teknik polymerase chain reaction (PCR). Sampel yang digunakan sebanyak 47 isolat S. aureus yang terdiri dari 21 isolat berasal dari bahan pangan asal ternak, 5 isolat berasal dari bumble foot ayam, 11 isolat berasal dari produk pangan asal ternak (susu kemasan, roti, keju, rolade, bakso dan sosis) serta 10 isolat berasal dari infeksi kulit manusia dari RS. Dr. Sardjito, Yogyakarta. Identifikasi S. aureus dilakukan dengan mengamplifikasi gen 23S rRNA dengan menggunakan primer spesies spesifik. Gen penyandi enterotoksin dideteksi dengan teknik PCR menggunakan primer spesifik sea, seb, sec, sed, see, seg, seh, sei dan sej . Accessory gen regulator diidentifikasi dengan melakukan amplifikasi menggunakan primer agrI dan agrII. Hasil amplifikasi gen 23S rRNA, enterotoksin dan agr dari 47 isolat dianalisis secara deskriptif dengan membandingkannya terhadap beberapa S. aureus strain referen (kontrol positif) dan Staphylococcus epidermidis (kontrol negatif). Hasil penelitian memperlihatkan bahwa semua isolat (100%) dapat diamplifikasi terhadap gen 23S rRNA, yang menunjukkan bahwa semua isolat yang digunakan dalam penelitian adalah spesifik S. aureus. Sebanyak 9 isolat (19,15%) tidak terdeteksi adanya gen stafilokokal enterotoksin. Sebanyak 38 isolat (80,85%) mengandung berbagai gen stafilokokal enterotoksin dengan perincian sebagai berikut : 2,63% (sea), 15,79% (sec), 10,53% (see), 5,26% (seh), kombinasi gen se(a,c), se(a,e), se(c,e), se(g,i), se(a,c,e) masing-masing 5,26%, kombinasi gen se(b,i), se(c,g), se(e,h), se(b,c,i), se(c,e,i), se(c,g,i), se(b,g,h,i) masing-masing 2,63%, kombinasi gen se(a,h) 7,89% dan kombinasi gen se(b,c) 13,56%. Distribusi berbagai gen stafilokokal enterotoksin dapat dikelompokkan berdasar accessory gene regulator (agr) menjadi 3 klaster yaitu klaster I (agrI), klaster II (agrII) dan klaster III (agrIII).

Accessory gene regulator (agr) is a gene regulator of expression of various virulence genes including enterotoxin genes. The aims of this research were to identifiy various enterotoxin genes of S. aureus isolated from various foods of animal origin and human skin infection and to indentifiy agr of S. aureus as enterotoxin gene regulators. The total number of isolates used in this research were 47 S. aureus isolates including 21 isolates from foods of animal origin, 5 isolates from bumble foot diseases of chickens, 11 isolates from food products of animal origins (packaging milks, breads, cheeses, rolade, meat balls and sausages) and 10 isolates from human skin infection that were obtained from Dr. Sarjito Hospital Yogyakarta. Polymerase chain reaction based procedures were used to detect for spesific 23S rRNA, staphylococcal enterotoxin genes and agr genes. Molecular identification was conducted for detection of S.aureus 23S rRNA gene by using oligonucleotides spesies specific primers. The enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei and sej) of S. aureus were determined by using spesific primers for staphylococcal enterotoxin sequences. The accessory gene regulator were detected by using agrI and agrII spesific primers. The amplicons of 23S rRNA, enterotoxin genes and agr of S aureus were analysed descriptively by comparing to S. aureus referen control (positive control) with S. epidermidis (negative control) The results of the studies showed that 47 isolates (100%) could be amplified by using oligonucleotide spesies spesific primer for 23S rRNA indicated that all isolates were S. aureus. Based on the detection of enterotoxin genes there were 9 isolates (19,15%) negative for staphylococcal enterotoxin genes. Thirty-eight isolates (80,85%) harboured for one or more staphylococcal enterotoxin genes such as follows : 2,63% (sea), 15,79% (sec), 10,53% (see), 5,26% (seh), combination of genes se(a,c), se(a,e), se(c,e), se(g,i), se(a,c,e) of 5,26% each, combination of genes se(b,i), se(c,g), se(e,h), se(b,c,i), se(c,e,i), se(c,g,i), se(b,g,h,i) of 2,63% each, combination of genes se(a,h) 7,89% and 13,56% was combination of gene se(b,c). All isolates could be grouped into 3 clusters such as cluster I (agrI), cluster II (agrI and agrII) and cluster III (agrII). The clustering of S aureus isolates were based on accessory gene regulator and staphylococcal enterotoxin genes distributions.

Kata Kunci : Staphylococcus aureus,Enterotoksin,Accessory gene regulator,Polymerase Chain Reaction,Produk pangan asal hewan,Infeksi kulit manusia, Staphylococcus aureus, enterotoxin, accessory gene regulator, polymerase chain reaction (PCR), foods of animal origin, hu