Keragaman Aeromonas spp. pada Lele (Clarias sp.) yang Dibudidayakan di Jawa, Indonesia

DINI SISWANI MULIA, Dr. Ir. Alim Isnansetyo, M.Sc.; Dr. Rarastoeti Pratiwi, M.Sc.; Prof. drh. Widya Asmara, S.U., Ph.D.

2021 | Disertasi | DOKTOR BIOLOGI

Aeromonas spp. merupakan bakteri Gram negatif yang hidup di perairan, dapat menyerang ikan dan biota air lainnya, tidak terkecuali ikan lele. Bakteri ini memiliki tingkat patogenisitas yang tinggi dan beragam. Infeksi Aeromonas spp. dapat menyebabkan penyakit aeromoniasis atau Motile Aeromonas Septicemia (MAS) dan merupakan salah satu kendala dalam budidaya lele di Indonesia. Selain menyebabkan ikan sakit, serangan bakteri ini dapat menyebabkan kematian massal dalam waktu yang singkat. Tujuan penelitian ini adalah mengetahui keragaman Aeromonas spp. pada lele (Clarias sp.) yang dibudidayakan di Jawa, Indonesia. Penelitian menggunakan metode survey dan teknik purposive sampling untuk menentukan wilayah kolam budidaya lele serta teknik random purposive sampling untuk pengambilan sampel lele sakit. Pengambilan sampel dilakukan di kolam budidaya di empat provinsi di Pulau Jawa, yaitu DIY Yogyakarta, Jawa Tengah, Jawa Barat, dan Jawa Timur. Penelitian diawali dengan mengambil sampel lele sakit selanjutnya dilakukan inokulasi bakteri dari organ ginjal pada medium glutamate starch phenyl (GSP) secara aseptis. Isolasi dan pemurnian bakteri dilakukan pada medium yang sama. Tujuh puluh empat isolat bakteri berhasil dikumpulkan. Selanjutnya dilakukan uji postulat Koch dan diperoleh 16 isolat patogen. Gejala klinis yang muncul antara lain depigmentasi kulit, erosi, hiperemia, dan hemoragik; abdominal dropsi, asites, abses, dan inflamasi pada bagian perut; lesi, hemoragik, dan pucat pada ginjal; lesi dan pucat pada hati dan insang. Dampak uji postulat Koch juga terjadi pada organ histologis ikan, yaitu nekrosis pada ginjal, hati, dan insang, hemoragik pada ginjal, kongesti pada hati, serta fusi pada lamella insang. Selanjutnya, 16 isolat patogen hasil penelitian ini dan dua isolat koleksi laboratorium dikarakterisasi secara fenotipe (morfologis dan karakteristik biokimiawi) dan genotipe (16S rDNA), diperoleh 10 isolat Aeromonas. Sepuluh isolat Aeromonas tersebut terdiri dari empat spesies, yaitu A. dhakensis (empat isolat, 40%), A. veronii bv veronii (empat isolat, 40%), A. caviae (satu isolat, 10%), dan A. hydrophila (satu isolat, 10%). Berdasarkan indeks keragaman Shannon-Wiener yang mencapai 1,1936, Aeromonas spp. patogen di Jawa memiliki keragaman spesies sedang. Aeromonas spp. patogen menyebar di tiga provinsi, yaitu DIY, Jawa Tengah, dan Jawa Barat. A. dhakensis menyebar di DIY (Sleman dan Kulonprogo) dan Jawa Tengah (Magelang dan Kedung Ombo), masing-masing provinsi terdiri dari dua isolat. A. veronii bv veronii menyebar di DIY (Sleman), Jawa Tengah (Demak), dan Jawa Barat (Bogor), masing-masing satu isolat, kecuali Jawa Barat terdiri dari dua isolat. A. caviae berasal dari Jawa Tengah (Magelang), sedangkan A. hydrophila dari Jawa Barat (Bogor). A. dhakensis merupakan spesies baru yang belum pernah dipublikasikan di Indonesia, bahkan di dunia pun sebagian besar dilaporkan dari manusia dan sebagian kecil spesies ikan, tetapi belum pernah diisolasi dari lele. Selain itu, A. veronii bv veronii ditemukan sebagai biovar baru di Indonesia. Deteksi gen virulen, yaitu aerolysin/haemolysin (aerA/haem), heat-labile cytotonic enterotoxin (alt), heat-stable cytotonic enterotoxin (ast), polar flagella (flaA), lateral flagella (lafA), dan ferric siderophore receptor (fstA) dan uji LD50 dilakukan untuk analisis virulensi Aeromonas spp., sedangkan analisis resistensi Aeromonas spp. meliputi deteksi gen resisten tetracycline (tetA), aminoglicoside (strA-strB), dan quinolone (qnrA) dan uji resistensi Aeromonas spp. berdasarkan minimum inhibitor concentration (MIC) dan minimum bactericidal concentration (MBC) antibiotik. Aeromonas spp. memiliki keragaman gen virulen sedang, berdasarkan indeks keragaman Shannon-Wiener mencapai 1,7490. Setiap isolat minimal memiliki empat jenis gen virulen. Berdasarkan prevalensinya, gen aerA/haem dan flaA mencapai 100%, ast mencapai 90%, alt mencapai 80%, fstA mencapai 60%, dan lafA mencapai 40%. Gen virulen pada Aeromonas spp. sebagian besar menyebar mengikuti pola sebaran spesiesnya. Secara umum, lima gen virulen, yaitu aer/haem, alt, ast, flaA, dan lafA menyebar di tiga provinsi, yaitu DIY, Jawa Tengah, dan Jawa Barat, sedangkan gen fstA hanya menyebar di dua provinsi, yaitu DIY dan Jawa Tengah. Aeromonas spp. pada lele memiliki virulensi tinggi dengan nilai LD50, yaitu 7,61 x 103 CFU/mL sampai 1,4907 x 105 CFU/mL. Sebagian Aeromonas spp. memiliki satu gen resisten, dari tiga jenis gen yang dideteksi, yaitu gen tetA dan strA-strB, tetapi tidak memiliki gen qnrA. Aeromonas spp. resisten terhadap oksitetrasiklin, ampisilin, dan eritromisin dan sensitif terhadap kanamisin, enrofloksasin, dan kloramfenikol. Antibiotik yang masih efektif digunakan untuk mengobati ikan yang terserang Aeromonas spp. adalah kanamisin, enrofloksasin, dan kloramfenikol.

Aeromonas spp. is Gram negative bacteria living in waters and able to attack fish as well as other aquatic biota, including catfish. It has high degree of pathogenicity and variety. It can cause aeromoniasis or Motile Aeromonas Septicemia (MAS), which is one of the obstacles in catfish cultivation in Indonesia. Beside causing disease to the fish, it can also cause mass mortality within a short time. The objective of this study was to determine the genotype biodiversity of Aeromonas spp. pathogens in catfish cultivated in Java, Indonesia. This study used the survey methods and purposive sampling techniques to determine the area of catfish cultivation ponds, as well as random purposive sampling techniques for diseased catfish sampling. Sampling was carried out in aquaculture ponds of four provinces in Java: Yogyakarta, Central Java, West Java and East Java. The study was started by taking sample of diseased catfish and aseptically inoculating the kidney on glutamate starch phenyl (GSP) medium. Isolation and purification of bacteria were carried out on the same medium. Seventy-four bacterial isolates were collected. Furthermore, the Koch Postulate test was carried out and 16 pathogenic isolates were obtained. Subsequent to the Koch postulate test, emerging clinical symptoms included skin depigmentation; erosion, hyperaemia and haemorrhagic; abdominal dropsy, acites, abscesses, and inflammation of the abdomen; lesions, haemorrhagic and pallor of the kidneys; lesions and pallor on the liver on the gills. The impacts of the Koch postulate test is also happened on the observation of the histological organs: necrosis of the kidneys, the liver, and the gill, haemorrhagic of the kidneys, congestion of the liver, and fusion of the gill lamellae. Furthermore, 16 isolates from this study and two isolates from the laboratory collection were characterized phenotypically (morphological and biochemical characteristics) and genotypically (16S rDNA) obtained 10 Aeromonas isolates. The ten Aeromonas isolates consisted of four species, namely A. dhakensis (four isolates, 40%), A. veronii bv veronii (four isolates, 40%), A. caviae (one isolate, 10%), and A. hydrophila (one isolate, 10%). Based on the Shannon-Wiener diversity index, which reaches 1.1936, Aeromonas spp. pathogens in Java indicate moderate species diversity. Aeromonas spp. pathogens spread in three provinces, namely DIY, Central Java, and West Java. Meanwhile, A. dhakensis spreads in DIY (Sleman and Kulonprogo) and Central Java (Magelang and Kedung Ombo), with each province consisting of two isolates. A. veronii bv veronii spreads in DIY (Sleman), Central Java (Demak), and West Java (Bogor), with each province consisting of one isolate, except two isolates in West Java. A. caviae comes from Central Java (Magelang), and A. hydrophila comes from West Java (Bogor). A. dhakensis is a new species and there has not been any publication about it in Indonesia and even in the world. Most of it has been reported to be found in humans and a small number of fish species, but never in isolated catfish. In addition, A. veronii bv veronii is found to be a new biovar identified in Indonesia. Detection of virulent genes i.e. aerolysin/haemolysin (aerA/haem), heat-labile cytotonic enterotoxin (alt), heat-stable cytotonic enterotoxin (ast), polar flagella (flaA), lateral flagella (lafA), and ferric siderophore receptor (fstA) and LD50 test were carried out in the purpose of virulence analysis for Aeromonas spp., meanwhile, the resistance analysis included the detection of resistance genes i.e. tetracyclin (tetA), aminoglycoside (strA-strB), and quinolone (qnrA) and resistance test based on the minimum inhibitor concentration (MIC) and the minimum bactericidal concentration (MBC) of antibiotics. Based on the Shannon-Wiener diversity index, with the value of reaching 1.7490, Aeromonas spp. indicates virulent gene diversity. Each isolate has at least four types of virulent genes. Based on the prevalence, the aerA/haem and flaA genes reached 100%, the ast genes reached 90%, the alt reached 80%, the fstA reached 60%, and the lafA reached 40%. Virulent genes in Aeromonas spp. are mostly spread in accordace to the distribution pattern of its species. In general, five virulent genes, i.e. aerA/haem, alt, ast, flaA, and lafA, spread in three provinces, namely DIY, Central Java and West Java. Meanwhile the fstA gene only spreads in two provinces, which are Yogyakarta and Central Java. Aeromonas spp. in catfish has high virulence with an LD50 value of 7.61 x 103 CFU/mL to 1.4907 x 105 CFU/mL. Some of Aeromonas spp. had one resistance gene out of the three types of detected genes, which were tetA and strA-strB genes, but did not have the qnrA gene. Aeromonas spp. was resistant to oxytetracycline, ampicillin and erythromycin and sensitive to kanamycin, enrofloxacin and chloramphenicol. Effective antibiotics to treat fish attacked by Aeromonas spp. include kanamycin, enrofloxacin and chloramphenicol.

Kata Kunci : Aeromonas spp., 16S rDNA, fenotipe, gen resisten, gen virulen, genotipe, lele