Antibiofilm Metabolit Proteus myxofaciens JB 20B untuk Pencegahan dan Pengobatan Udang Vaname yang Diinfeksi Bakteri Vibrio harveyi

Abstract

Udang merupakan salah satu komoditas unggulan perikanan Indonesia, yang memiliki nilai ekspor cukup tinggi. Budidaya udang masih mengalami kendala penyakit, terutama vibriosis yang disebabkan oleh infeksi dari bakteri genus vibrio diantaranya Vibrio harveyi. Kematian udang yang disebabkan oleh infeksi V. harveyi dapat merugikan baik bagi petambak udang dalam skala besar maupun skala kecil yang ada di Indonesia. Bakteri V. harveyi mampu membentuk biofilm yang dapat meningkatkan sifat virulensi, resistansi serta mampu melindungi bakteri dari lingkungan eksternal. Biofilm juga dapat menyediakan nutrisi bagi sel bakteri sehingga menyebabkan bakteri menjadi resisten terhadap berbagai kondisi lingkungan. Struktur biofilm mempersulit dalam pengobatan maupun pencegahan dengan menggunakan antibiotik, karena biofilm sulit untuk ditembus oleh antibiotik sehingga memerlukan dosis antiobiotik yang lebih tinggi. Metabolit bakteri Proteus myxofaciens JB 20B merupakan salah satu alternatif yang diharapkan dapat digunakan untuk pencegahan dan pengobatan penyakit vibriosis pada budidaya udang vaname. Penelitian ini bertujuan untuk menguji efektivitas metabolit bakteri P. myxifaciens JB 20B, sebagai agen antibiofilm untuk pencegahan dan pengobatan udang vaname (L. vannamei) yang diinfeksi bakteri V. harveyi. Hewan uji yang digunakan pada penelitian merupakan udang vaname dengan berat 3±0,5 g ekor -1. Perlakuan menggunakan rancangan acak lengkap dengan tujuh perlakuan dan tiga ulangan yang terdiri dari perlakuan K- (kontrol negatif, tanpa pemberian perlakuan serta tidak uji tantang), K+ (kontrol positif, tanpa pemberian perlakuan namun dilakukan uji tantang), PCA (pencegahan antibiotik, udang diberi pakan dengan coating antibiotik oksitetrasiklin 50 g ml-1 sebelum uji tantang), PGA (pengobatan antibiotik, udang diberi pakan dengan coating antibiotik oksitetrasiklin 50 g ml-1 setelah diuji tantang), PCE (pencegahan ekstrak, udang diberi pakan dengan coating metabolit bakteri P. myxofaciens JB 20B dosis 0,1 ml Kg-1 pakan sebelum diuji tantang), PCS (pencegahan supernatan, udang diberi pakan dengan coating metabolit bakteri P. myxofaciens JB 20B dosis 20 ml Kg-1 pakan sebelum diuji tantang), PGE (pengobatan ekstrak, udang diberi pakan dengan coating metabolit bakteri P. myxofaciens JB 20B dosis 0,1 ml Kg-1 pakan setelah diuji tantang) dan PGS (pengobatan supernatan, udang diberi pakan dengan coating metabolit bakteri P. myxofaciens JB 20B dosis 20 ml Kg-1 pakan setelah diuji tantang). Parameter yang diamati yakni kinerja pertumbuhan (tingkat kelangsungan hidup (TKH), laju pertumbuhan spesifik (LPS), rasio konversi pakan (RKP)), gejala klinis, total haemocyte count (THC), differential haemocyte count (DHC), aktivitas fagositik (AF), aktivitas phenoloxidase (PO), respiratory burst (RB), aktivitas lisozim (AL), histopatologi hepatopankreas dan total plate count diamati pada hari ke-0, 14, 16, 19 dan 28. Hasil pengamatan kinerja pertumbuhan selama 30 hari pemeliharaan menunjukkan bahwa pemberian metabolit bakteri P. myxofaciens JB 20B mampu meningkatkan nilai TKH, LPS dan RKP walaupun tidak berbeda nyata iii (P>0,05) terhadap kontrol negatif. Kepadatan bakteri pada organ internal menghasilkan kepadatan bakteri 106-108 CFU g-1 yang tidak berbeda nyata (P>0,05) antar perlakuan. Selanjutnya gejala klinis pasca uji tantang terjadi penurunan nafsu makan pada udang, berenang tidak normal, karapas lunak, usus kosong, hepatopankreas pucat. Hasil histopatologi menunjukkan nekrosis pada perlakuan PCE (12,23-38,23%), PCS (12,25-35,14%), PGE (12,25-37,62%), PGS (12,25-36,42%) berbeda nyata (P<0,05) dibandingkan perlakuan K+ (12,23-50,52%). Kecilnya persentase nekrosis pada PGS disebabkan adanya penekanan pertumbuhan bakteri V. harveyi yang disebabkan aktivitas dari metabolit bakteri P. myxofaciens JB 20B selama pemberian pakan dan pemeliharaan berlangsung. Pengukuran respons imun dilakukan dengan pengamatan DHC, THC, AF, PO, RB dan AL. Udang vaname yang diberi perlakuan metabolit bakteri P. myxofaciens JB 20B mampu meningkatkan respons imun pada udang. Hasil pengamatan pada penelitian ini menyatakan bahwa pemberian metabolit bakteri P. myxofaciens JB 20B dapat memberikan efek yang lebih baik dari kontrol antibiotik, baik sebagai pencegahan dan pengobatan udang vaname dari serangan bakteri V. harveyi. Kesimpulan dari penelitian ini adalah perlakuan metabolit bakteri P. myxofaciens JB 20B (PCE, PCS, PGE dan PGS) lebih unggul dibandingkan dengan perlakuan kontrol positif (K+) dalam hal mengurangi nekrosis hepatopankreas udang, meningkatkan nilai DHC, THC, AF, PO, RB, AL dan performa pertumbuhan (TKH, JKP, RKP) udang vaname melalui pemberian pakan baik pada perlakuan pencegahan maupun pengobatan. Penggunaan metabolit bakteri P. myxofaciens JB 20B untuk mengendalikan infeksi V. harveyi pada udang vaname memberikan hasil terbaik pada perlakuan pengobatan supernatan (PGS).

Shrimp is one of Indonesia’s leading fisheries commodity, which has a high export value is quite high. Shrimp farming still experiences disease constrainsts, especially vibriosis caused by infection from vibrio genus bacteria including vibrio harveyi. Shrimp mortality caused by Vibrio harveyi. Shrimp mortality caused by V. harveyi infection can be detrimental for both shrimp farmers on a large scale and small-scale shrimp farmers in indonesia. V. harveyi are able to form biofilms that can increase virulence, resistance and protect the bacteri from the ecternal environment. Protect the bacteria from the external environment. Biofilm can also provide nutrients for cells bacteria, causing the bacteria to become resistant to various environmental conditions. The biofilm structure makes it difficult in treatment or prevention using antibiotic, because biofilm is difficult to penetrate by antibiotics, thus requiring higher doses of antibiotics. Metabolites Proteus myxifaciens JB 20B is one of the alternatives that is expected to be used for the prevention and treatment of biofilms. That is expected to be used for the prevention and treatment of vibriosis disease in vaname shrimp farming. This research to test the effectiveness of the bacterial metabolite P. myxifaciens JB 20B, as an antibiofilm agent for the prevention and treatment of vaname shrimp (L. vannamei) infected with V. harveyi bacteria. The test animals used in the research were vaname shrimp weighing 3 ± 0.5 g tail -1. The treatment used a completely randomized design with seven treatments and three replications consisting of treatment K- (negative control, without treatment and no challenge test), K+ (positive control without no treatment but challenge test), PCA (prevention of shrimp fed with antibiotic coated feed oxytetracycline 50 g ml-1 before challenge test), PGA (treatment of shrimp fed with antibiotic coated feed oxytetracycline 50 g ml-1 after challenge test), PCE (prevention of shrimp extract fed with feed dipcoated metabolite of P. myxofaciens JB 20B bacteria dose 0.1 ml Kg-1 feed before challenge test), PCS (prevention of supernatant of shrimp fed coated feed metabolite of P. myxofaciens JB bacteria 20B dose of 20 ml Kg-1 feed before challenge test), PGE (treatment of shrimp extract given food coated with metabolites of P. myxofaciens JB 20B bacteria dose 0.1 ml Kg-1 feed after challenge test) and PGS (treatment of shrimp supernatant fed coated feed with metabolites of P. myxofaciens JB 20B bacteria dose 20 ml Kg-1 feed after challenge test). The parameters observed were growth performance (survival rate (TKH), specific growth rate (LPS), feed conversion ratio (RKP)), clinical symptoms, total haemocyte count (THC), differential haemocyte count (DHC), phagocytic activity (AF), phenoloxidase activity (PO), respiratory burst (RB), lysozyme activity (AL), hepatopancreatic histopathology and total plate count were observed on days 0,14, 16, 19 and 28. Observation results of growth performance during 30 days of maintenance showed that administration of the bacterial metabolite P. myxofaciens JB 20B was able to increase good TKH, LPS and RKP values, although it had no significant effect (P>0,05) on the negative control. This density of bacteria in the v internal organs resulted in a bacterial density of 108 CFU mL-1 which was not significantly different (P>0,05) between treatments. Furthermore, clinical symptoms after the challenge test were decreased appetite in shrimp, abnormal swimming, soft carapace, no intestines, pale hepatopancreas. Histopathology results showed necrosis in the treatment of PCE PCE (12,23-38,23%), PCS (12,25-35,14%), PGE (12,25-37,62%), PGS (12,25-36,42%) berbeda nyata (P<0,05) dibandingkan perlakuan K+ (12,23-50,52%). The small percentage of necrosis in PGS is due to the suppression of the growth of V. harveyi bacteria due to the activity of the bacterial metabolite P. myxofaciens JB 20B during feeding and maintenance. Measurement response of the immune response was carried out by observing DHC, THC, AF, PO, RB dan AL. Vannamei shrimp treated with the bacterial metabolite P. myxofaciens JB 20B were improve response the immune response in shrimp. The results of observations in this study state that administration of metabolites of the phyllosphere bacteria P. myxofaciens JB 20B can provide a better effect than antibiotic control, both as a prevention and treatment for vaname shrimp from being attacked by the bacteria V. harveyi. conclusion of this research, the treatment of bacterial metabolites P. myxofaciens JB 20B (PCE, PCS, PGE, and PGS) is superior to the positive control (K+) treatment in terms of reducing shrimp hepatopancreas necrosis, increasing the value of DHC, THC, AF, PO, RB, AL, and growth performance (TKH, JKP, RKP), of vaname shrimp through post-infection feeding in both prevention and treatment. The use of P. myxofaciens JB 20B bacterial metabolites to control V. harveyi infection in vaname shrimp yielded the best results in the supernatant treatment (PGS).