Resistensi Staphylococcus aureus terhadap Metisilin serta Hubungan Pola Genetik antara Sapi Perah dan Manusia

TATO, Syarifuddin , Siti Isrina Oktavia Salasia

2012 | Disertasi |

Staphylococcus aureus merupakan penyebab utama mastitis klinis maupun subklinis pada sapi perah Staphylococcus aureus juga menyebabkan berbagai penyakit patogen pada manusia. Kasus infeksi methicillin resistant Staphylococcus aureus (MRSA) semakin meningkat, dan strain ini sering bersifat resisten terhadap berbagai antibiotik yang lain. Penelitian ini bertujuan untuk mengidentifikasi resistensi S. aureus terhadap metisilin dan mengetahui hubungan klonal antar isolat S. aureus dari manusia maupun sapi perah. Dalam penelitian ini digunakan S. Aureus yang diisolasi dari kulit manusia di RSUP Dr. Sardjito (10 isolat) dan dari susu sapi perah (11 isolat) yang berasal dari wilayah Yogyakarta, Solo dan Boyolali. Identifikasi bakteri dilakukan berdasar pengamatan pertumbuhan pada plat agar darah (PAD), pewarnaan Gram, fermentasi pada mannitol salt agar (MSA), uji katalase, koagulase dan clumping factor, serta amplifikasi gen spesifik 23S rRNA. Uji resistensi S. Aureus terhadap metisilin digunakan metode disc diffusion dan deteksi gen mecA menggunakan polymerase chain reaction (PCR) dengan primer spesifik. Hubungan genetik antar isolat S. aureus asal manusia dan sapi ditentukan berdasar analisis single enzyme amplified fragment length polymorphism (AFLP), dan skuen gen mecA dari isolat MRSA ditentukan berdasar analisis pohon filogenetik. Hasil penelitian menunjukkan bahwa semua isola (21 isolat) adalah S. aureus berdasar identifikasi secara fenotipik dan genotipik. Hasil uji sensitivitas terhadap metisilin menunjukkan bahwa S. aureus resisten terhadap metisilin sebanyak 7 (33,3%) isolat deengan metode disc diffusion, dengan uji molekuler terdapat 9 (42,9%) isolat mampu mengekspresikan gen mecA. Analisis AFLP menunjukkan adanya 15 pola S. aureus dengan kode A sampai O dan dapat diklasterisasi menjadi 7 klaster (! Sampai VII). Staphylococcus aureus isolat sapi perah asal Boyolali dan Solo berada dalam satu klaster (kecuali 1 isolat asal Yogyakarta), isolat sapi dan manusia di Yogyakarta mengelompok menjadi beberapa klaster. Pada masing-masing klaster terdapat galur MRSA. Analisis pohon filogenetik terhadap 9 S. aureus yang positif mengandung gen mecA (6 isolat manusia dan 3 isolat sapi), dapat dikelompokkan menjadi 3 klaster MRSA. Hasil penelitian ini memperlihatkan bahwa teknik molekuler merupakan metode yang lebih sensitif dalam deteksi MRSA dibanding dengan metode disc diffusion. Analisis genotipik dengan AFLP dan pohon filogenetik terhadap MRSA dapat digunaklan untuk pengendalian infeksi MRSA. Kata kunci : Staphylococcus aureus, resisten, metisilin, gen

Staphylococcus aureus is the causative agent of clinical or subclinical mastitis in dairy cow. The bacteria cause also several diseases in human. The case of methicillin resistant Staphylococcus aureus (MRSA) increase frequently, and this strain was resistant to several antibiotics. The aim of the study were to identify the resistance of S. aureus to methicillin and the clonal association between dairy cow and human isolates. Staphylococcus aureus strains used in this study were isolated from human skin infections of RSUP Dr. Sardjito Yogyakarta (10 isolates) and from dairy cows milk (10 isolates) originated from Yogyakarta, Solo and Boyolali. Bacterial identifications were performed based on the growth of bacteria on blood agar plate (BAP) media, Gram staining, MSA fermentation, catalase, coagulase and clumping factor test, and amplification of specific section of the 23S rRNA gene. The resistancy assay of S.aureus to methicillin were performed by the disc diffusion method and the detection of mecA genes by using polymerase chain reaction (PCR) with specific primers. Determination of the genetic relationships between S. aureus isolated from dairy cow and human used single enzyme amplified fragment length polymorphism (AFLP) technique and sequencing of mecA genes of MRSA strains were analysed by phylogenetic tree. The results showed that all isolates from human and dairy cow (21 isolates) were S. aureus based on the phenotypic and genotypic identification. The bacteria were resistant to methicillin for 7 (33.3%) isolates by disc diffusion test, but there were 9 (42.9%) isolates expressed mecA gene. Seven S. aureus isolates methicillin resistant, there were 5 isolates expressed mecA gene and 2 isolates without expression of mecA gene. On the other hand, there were 4 S. aureus isolates sensitive to methicillin, expressed mecA gene. A single enzyme AFLP analysis revealed 15 patterns named A to O and could be clustered into 7 clusters (I to VII). Staphylococcus aureus isolated from dairy cow wich location is close to each other in Boyolali and Solo, grouped in one cluster (exept 1 dairy cow isolate from Yogyakarta), dairy cow and human isolates in Yogyakarta grouped into several clusters. Each cluster could be detected MRSA strains. Based on the phylogenetic tree analysis of the sequence of 9 S. aureus were positive mecA gene (6 human isolates and 3 dairy cow isolates), there were grouped into 3 types of MRSA. In conclusion, the molecular technique was more sensitive method in detecting MRSA compared to the classical disc diffusion method. The genotypic analysis of the present stydy might help to understand the distribution of S. aureus clones between human and dairy cow isolates and might help to control MRSA infection. Key words : Staphylococcus aureus, resistant, methicillin,mecA gene, AFLP, phylogenetic tree

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