LAPORAN AKHIR HASIL
PENELITIAN UNGGULAN PERGURUAN TINGGI
HIBAH BERSAING (TAHUN KE-1)
TAHUN ANGGARAN 2012
Judul
:
Kemampuan Quorum Quenching Bacillus Sebagai
Biokontrol Untuk Mencegah Vibriosis Pada
Budidaya Tambak Udang
Ketua
: Ating Yuniarti, S.Pi, M.Aqua
Anggota : 1. Dr. Ir. Maftuch, M.Si
2.Dr. Ir. Anik M. Hariati, M.Sc
Dibiayai oleh Direktorat Jenderal Pendidikan Tinggi, Kementerian
Pendidikan dan Kebudayaan, Melalui DIPA Universitas Brawijaya
nomor : 0636/023-04.2.16/15/2012, tanggal 9 Desember 2011, dan
berdasarkan SK Rektor Universitas Brawijaya Nomor : 058/SK/2012
tanggal 8 Pebruari 2012
LembagaPenelitiandanPengabdiankepadaMasyarakat
UniversitasBrawijaya
2012
ABSTRAK
Infeksi yang diakibatkan oleh bakteri pathogen telah menyebabkan kerugian yang cukup besar pada industri akuakultur. Beberapa bakteri pathogen pada industri akuakultur seperti Vibriosp dan Aeromonass p diketahui mengatur ekspresi gen virulensinya berdasarkan sistem Quorum Sensing (QS). QS merupakan sistem komunikasi antar sel dengan menggunakan molekul signal sedangkan QQ (Quorum Quenching) adalah mekanisme yang dapat menganggu proses kunci dari sistem QS. Penghambatan N-acyl homoserine lactone (AHL) sebagai salah satu molekul signal QS diharapkan akan menghambat ekspresi virulensi dari pathogen. Tujuan dari penelitian ini adalah untuk mengetahui kekerabatan 5 isolatBacillus (UB1-UB5) yang diisolasi dari tambak udang di JawaTimur Indonesia dan kemampuannya untuk mendegradasi signal molekul AHL. Dengan menggunakan sekuensing 16SrRNA maka didapatkan hasil bahwa isolat Bacillus yang ada berkerabat dekat dengan B. cereus, B. thuringiensis, B.
subtilisdan Bacillus anthracis. Skreening dengan bioassay. Semua isolat Bacillus
yang ada juga mampu mengurai molekul signal AHL melalui uji bioassay dengan bioreporter Agrobacterium tumefaciens NTL4 (traR; tra::lacZ749). Laju degradasi AHL yang diobservasi pada media Luria-Bertani yang ditambah dengan 0.5 nM of N-Octanoyl-L-homoserine lactone adalah berkisar antara1.18 E-04 – 2.99 E-04nM min -1l-1. Kemampuan mengurai molekul signal AHL ini juga dikonfirmasi
dengan adanya gen aiiA, gen pengkodeenzim pengurai AHL, pada semua isolat. Keywords: Bacillussp, N-acylhomoserine lactone, quorum quenching, quorum sensing, tambakudang
ABSTRACT
Infections caused by antibiotic-resistant pathogenic bacteria can cause considerable losses in aquaculture. Many aquaculture pathogens sich as Vibrio
sp and Aeromonassp regulate the expression of virulence genes through Quorum
Sensing (QS). QS is a bacterial cell-to-cell communication with small signal molecules, while QQ (Quorum Quenching) is any mechanism that can effectively interfere the key processes in quorum sensing. Therefore, the degradation of N-acyl homoserine lactone (AHL) as a signal molecule of QS offers an alternative to control the virulence of pathogen. The objectives of this research were to evaluate the five strain of Bacilli (UB1-UB5) isolated from the shrimp pond at East Java Indonesia and their ability to degrade the AHL signal molecules. By using 16SrRNA sequencing, all Bacillus isolates were identified closely related to B.
cereus, B. thuringiensis, B. subtilisdan B. anthracis. All isolates showed a various
ability to degrade N-acylhomoserine lactone (AHL) revealed by the bioassay using bioreporter Agrobacterium tumefaciens NTL4 (tra dan traG::lacZ). The degradation rates observed in Luria–Bertani medium supplemented with 0.5 nM of N-Octanoyl-L-homoserine lactone were ranged from 1.18 E-04 – 2.99 E-04nM min -1l-1. The aiiA homologue gen, encoding an autoinducer inactivation enzyme
catalyzing the degradation of AHL’s, were also detected in all Bacillus isolates.
Keywords: Bacillussp, N-acylhomoserine lactone, quorum quenching, quorum sensing, shrimp pond
RINGKASAN
Vibriosis ialah penyakit yang merugikan usaha budidaya tambak udang. Secara berantai, vibriosis menjadi pemicu prevalensi virus, seperti white-spot disease. Kurasi melalui aplikasi antibiotik menyebabkan timbulnya species vibrio yang resistan. Antibiotik yang terakumulasi menyebabkan udang ditolak oleh pasar internasional. Quorum Quenching (QQ) ialah proses enzimatik untuk memutus komunikasi antar bakteri, termasuk kemampuannya dalam menimbulkan penyakit. QQ dapat dihasilkan oleh bakteri Bacillus. Strategi biokontrol, melalui mekanisme self-internal remediation sangat menjanjikan karena kemungkinan terjadinya resistansi sangat minimal dan selanjutnya diharapkan dapat meningkatkan produksi tambak. Tujuan penelitian ialah mengidentifikasi Bacillus (diisolasi dari tambak) dengan kemampuan QQ maksimal, dan menelusuri faktor lingkungan optimal pendukung kinerja QQ secara maksimal. Hasil penelitian pendahuluan (morphologi, staining dan uji bio-kimia) menunjukkan keberadaan beberapa jenis Bacillus. Penelitian tahun pertama ditujukan untuk menelusuri kekerabatan dan kemampuan QQ dari masing-masing isolat Bacillus. Melalui sekuensing 16Sr RNA di tahun pertama menunjukkan bahwa isolat Bacillus UB1-UB5 diidentifikasi dekat dengan B.
cereus, B. thuringiensis, B. subtilis dan B.anthracis.Dengan menggunakan
bio-reporter, Agrobacterium tumefaciensNTL4(traR; tra::lacZ749) dan skreening homologi gen Aii didapatkan hasil bahwa kelima isolat Bacillus memiliki kemampuan QQ untuk mengurai signal AHL. Kemampuan QQ dari kelima isolat tersebut bervariasi dari 1,18 E-04 – 2,99 E-04 nM menit-1 µl-1. Isolat UB2 yang
mempunyai kemampuan QQ terbesar selanjutnya akan digunakan pada penelitian tahun kedua. Penelitian tahun kedua ditujukan untuk mencari media optimal (suhu dan pH ) serta kemampuannya dalam mencegah vibriosis. Uji in-vitro dilakukan pada target V. harveyi, sedangkan secara in-vivo melalui uji prevalensi vibriosis pada hewan udang.
SUMMARY
Vibriosisisa detrimental disease toshrimpfarming and it also can be a trigger of viral diseases such as white-spot. A control strategy with applicationof antibioticsled tothe emergence ofdrug-resistantspecies. Furthermore, acumulation of antibiotics often causethe rejection of shrimp productby the international market. Quorum Quenching (QQ) isan enzymatic processto cut off the communication between bacteria which then theability tocause disease. QQ can beproduced by the bacterium Bacillus. A biocontrol strategy through the mechanism of internal self-remediation is quite promising as the occurrence of resistanceis minimal. That then expected to increasethe shrimp farm production. The research objectiveis to identify Bacilliwith QQ maximum capability, and track the optimal environmental factors supporting QQ maximum performance. Preliminary results (morphology, staining and bio-chemical test) showed the presence of several types of Bacilli. The first year of researchis aimed to trace the kinship and QQ activity of each Bacillus. The results of 16Sr RNA sequencing study in the first year showed that the bacilli isolates identified as B. cereus, B.
thuringiensis, B. subtilis and Bacillus sp.By usingbio-reporter, Agro bacteri umtumefaciens (traR; tra::lacZ749) and screening of the aiiA homolog gen, it was
confirmed that all Bacilli isolates had QQ ability to degrade AHL signal. The QQ activities of the Bacilli isolates varied from 1.18 E-04 to 2.99 E-04 nM min-1 µl-1.
As isolate of UB 2 Bacilli was found to have the biggest degradation activity toward AHL, then this isolate will be used for the second year study. The second year study aimed to find the optimal medium (temperature and pH) to prevent
vibriosis for Bacilli UB 2. In-vitro testwill be performed on the target Vibrio harveyi,
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