Deteksi Biomarker Koagulase Staphylococcus aureus dan Korelasinya dengan Enterotoksin

Handito Kurniyadi, Prof. Dr. drh. Siti Isrina Oktavia Salasia

2023 | Tesis | S2 Sain Veteriner

Staphylococcus aureus yang diisolasi dari kasus klinis hewan maupun manusia memiliki faktor virulensi koagulase. Koagulase dapat digunakan sebagai biomarker infeksi S. aureus yang terjadi pada kaskade koagulasi. Selain itu, S. aureus mampu menghasilkan Staphylococcal Enterotoxin (SE), yang merupakan superantigen dan eksotoksin gastrointestinal kuat. Toksin ini  menjadi salah satu penyebab utama keracunan makanan. Dalam penelitian dilakukan deteksi koagulase secara fenotipik maupun genotipik sebagai biomarker mekanisme infeksi S. aureus serta hubungan pola keterkaitan gen penyandi koagulase (gen coa) dan gen penyandi Staphylococcal Enterotoxin (gen se). Sebanyak 49 sampel asal susu mastitis pada sapi dan kambing dan 49 sampel isolat yang berasal dari kasus medis manusia di daerah Yogyakarta telah diidentifikasi berdasarkan gen 23S rRNA dan gen thermonuclease nuc sebagai S. aureus. Deteksi koagulase dilakukan secara fenotipik dengan uji koaguse tabung dengan menggunakan plasma kelinci dan secara genotipik dengan teknik polymerase chain reaction (PCR). Tipe polimorfisme koagulase S. aureus dan identifikasi gen se dilakukan dengan cara multipleks PCR menggunakan primer spesifik. Pengujian koagulase secara fenotipik pada 98 isolat, 67 isolat (68%) teridentifikasi sebagai koagulase positif. Staphylococcus aureus koagulase postif ditemukan paling banyak pada isolat asal sapi (88%;28/32) diikuti isolat asal kambing (76%;13/17) dan isolat asal manusia (53%;26/49). Deteksi koagulase secara molekuler menunjukkan seluruh 98 isolat memiliki gen coa (100%) menunjukkan akurasi pengujian secara molekuler. Berdasarkan deteksi polimorfisme gen coa pada 98 isolat S. aureus terdeteksi 7 jenis tipe gen coa (I, II, III, V, VI, VII, VIII). Tipe koagulase isolat S. aureus asal manusia paling tinggi adalah tipe II (61%;30/49), sedangkan isolat asal sapi adalah tipe VII (44%;14/32) dan tipe V (35%;6/17) pada isolat asal kambing. Strain S. aureus tipe gen coa II asal manusia memiliki satu atau lebih gen se (sea, seb, sec, seh) dengan presentase tertinggi pada gen sec tunggal (33,4%;10/30). Pada isolat S. aureus tipe gen coa VII asal sapi ditemukan gen sea, sec dan seh dalam bentuk tunggal maupun kombinasi dengan presentase tertingi kombinasi gen sea, sec, seh dan gen seh tunggal (masing-masing 36 %;5/14). Dari 17 isolat asal kambing terdapat 6 isolat (35%) memiliki gen se, diantaranya terdapat 2 isolat dengan tipe gen coa VII mempunyai kombinasi gen sea, sec, dan seh mirip pada isolat sapi. Hasil korelasi antara tipe coagulase dengan enterotoksin, mengindikasikan bahwa genotipe coa II dan coa VII mempunyai kemungkinan sebagai strain S. aureus yang berpotensi menyebabkan enterotoksikosis.

Staphylococcus aureus isolated from clinical cases of animals and humans can secrete coagulase. Coagulase can be used as a biomarker of S. aureus infection that occurs in the coagulation cascade. In addition, S. aureus can produce Staphylococcal enterotoxin (SE), a potent gastrointestinal superantigen, and exotoxin. This toxin is one of the leading causes of food poisoning. The detection of coagulase, both phenotypic and genotypic, as a biomarker of the mechanism of S. aureus infection and the relationship between the pattern of association of the coagulase coding gene (coa gene) and the gene encoding staphylococcal enterotoxin (se gene) will be carried out in this study. Forty-nine samples from mastitis milk in cows and goats and 49 isolates from human medical cases in Yogyakarta were identified based on the 23S rRNA gene and the nuc gene as S. aureus. Coagulase detection was carried out phenotypically by coaguse test tube using rabbit plasma and molecular detection of coagulase by amplifying the coa gene using the polymerase chain reaction (PCR) technique. The type of S. aureus coagulase polymorphism and identification of the se genes was performed by multiplex PCR using specific primers. Among 98 isolates, 67 (68%) were identified as coagulase-positive S. aureus by tube test. The most common coagulase-positive S. aureus strains were found in cattle isolates (88%;28/32), followed by goat isolates (76%;13/17) and human isolates (53%;26/49). Molecular detection of coagulase showed that all 98 isolates had the coa gene. Based on the coa gene polymorphism detection in 98 isolates of S. aureus, seven types of coa genes were detected (I, II, III, V, VI, VII, VIII). The highest coagulase type in S. aureus isolates from humans was type II (61%), while isolates from cattle were type VII (44%;14/32) and type V (35%;6/17) in isolates from goats. S. aureus strain coa II gene type of human origin has one or more se genes (sea, seb, sec, seh), with the highest percentage being a single sec gene (33,4%;10/30). In isolates of S. aureus gene type coa VII from cattle, sea, sec, and seh genes were found singly or in combination with the highest percentage of combinations of sea, sec, seh, and single seh genes (36%; 5/14 respectively). Six (35%) of the 17 goat isolates possessed the se gene, and two coa VII gene isolates exhibited a mix of sea, sec, and seh genes resembling those found in bovine isolates.. The results of the correlation between coagulase and enterotoxin types indicate that coa II and coa VII genotypes have the possibility of being S. aureus strains that have the potential to cause enterotoxicosis.

Kata Kunci : Biomarker, Koagulase, Enterotoksin, Staphylococus aureus