27
sitoskeletal di antaranya aktin juga berperan dalam penempelan EPEC. Akhirnya terjadi perubahan struktur sitoskeletal setelah penempelan Tir-intimin,
menghasilkan bentuk pedestal Lu Walker 2001. Hal inilah yang menyebabkan EPEC mampu menempel lebih kuat daripada bakteri patogen lainnya Gambar
2.8.
Gambar 2.8 Mekanisme penempelan EPEC di sel epitel usus Lu Walker 2001
Identifikasi BAL
Identifikasi BAL sangat diperlukan untuk mengetahui genus, spesies bahkan galurnya. Menurut FAOWHO 2002, untuk dapat diketahui dan didekati
sifat fungsionalnya sebagai probiotik, maka BAL harus diketahui spesies bahkan sampai galur untuk klaim sifat fungsional tertentu. Dalam perkembangannya,
metode identifikasi BAL dapat dibagi menjadi dua yaitu :
1. Metode klasik
Metode klasik meliputi metode untuk menguji morfologi dan sifat biokimiawi BAL. Uji morfologi yang dimaksud adalah reaksi Gram, produksi
katalase dan morfologi sel. Uji biokimiawi meliputi produksi CO
2
dari glukosa, produksi NH
3
dari arginin, produksi dekstran dari sukrosa, pertumbuhan pada suhu 15 dan 45°C, pertumbuhan pada NaCl 6.5 dan reaksi pada litmus milk.
Semuanya akan dapat menentukan genus dari BAL. Untuk mengetahui sampai tingkat spesies BAL, maka untuk metode klasik digunakan pengujian pola
28
fermentasi pada berbagai gula 49 jenis gula, yang umumnya digunakan adalah API CHL dan API CHS Counter et al. 2005; Reque et al. 2000; Vassu et al.
2001. Namun demikian, metode klasik ini mempunyai beberapa kelemahan antara lain : a identifikasi hanya terbatas sampai tingkat spesies, b identifikasi
hanya tergantung pada pola hasil fermentasi beberapa gula sehingga terkadang bias untuk beberapa spesies tertentu atau bahkan tidak dapat teridentifikasi.
Pengujian dengan sistem identifikasi API 50 CH yang dilakukan oleh Counter et al. 2005 membuktikan bahwa dari 90 isolat BAL yang diisolasi dari sosis
fermentasi, terdapat 10 dari keseluruhan isolat tidak dapat diidentifikasi unidentified dan ada kesalahan software dimana Lactobacillus sakei subsp. sakei
diidentifikasi sebagai Lactococcus lactis subsp. lactis. Namun demikian, identifikasi dengan metode klasik tetap diperlukan
untuk mengetahui sifat fisiologis dan biokimiawi dari BAL. Pada seleksi BAL, metode untuk mengetahui sampai tingkat genus umumnya menggunakan metode
klasik, penentuan dugaan awal sampai tingkat spesies juga digunakan metode klasik. Namun, untuk mengkonfirmasi spesies BAL tersebut perlu dilakukan
identifikasi secara molekuler Vamanu et al. 2006.
2. Metode modern molekuler
Metode molekuler dikembangkan untuk mengatasi kelemahan metode identifikasi klasik. Di samping itu, perkembangan beberapa metode molekuler
ditujukan untuk mempertimbangkan kedekatan beberapa galur bakteri BAL.
Teknik PFGE Pulse Field Gel Electrophoresis dan RFLP Restricted
Fragment Length Polymorphism
Dasar teknik PFGE adalah penggunaan sistem getaran elektrik untuk migrasi fragmen DNA yang besar melewati gel agarosa. Fragmen DNA diperoleh
dari digesting genom dengan enzim restriksi yang mempunyai recognition site 8 bp dan 6 bp. Fragmen DNA yang direstriksi di gel agarosa dimasukkan ke setiap
sumur agarosa dan dipisahkan dengan PFGE berdasarkan ukuran fragmennya Tannock 1999. Teknik RFLP mirip dengan teknik PFGE. Perbedaannya adalah
jika sebelum dielektroforesis restriksi DNA dilakukan pada gel untuk PFGE, namun tidak untuk RFLP. Teknik RFLP dan PFGE ini dapat mendeteksi BAL
sampai karakteristik spesifik bakteri pada tingkat galur Beasley 2004.
29
Ribotyping
Tahapan utama teknik ini adalah mengisolasi DNA dari isolat kultur, dilakukan restriksi dan pemisahan ukuran di dalam gel agarosa. Selanjutnya gel
dihibidrisasi dengan menggunakan penanda rRNA yang dilabel, yang mengikat fragmen berisi salinan dari operon rRNA. Untuk deteksi penanda, fragmen yang
terikat pada penanda selanjutnya divisualisasikan dan membentuk karakteristik
RFLP Tannock 1999. Analisis urutan basa
sequencing gen 16S rRNA
Teknik ini merupakan teknik cepat yang mempunyai tahapan amplifikasi gen 16S rRNA, menggunakan PCR dengan primer target yang berlaku secara
umum dan disepakati secara internasional untuk gen tersebut. Analisis urutan basa filogenetik 16S rRNA merupakan metode molekuler yang akurat untuk
identifikasi mikroba Walter et al. 2000; Anukam et al. 2005; Ennahar et al. 2003. Sekuen nukleotida-nukleotida pada gen 16S rRNA dapat diidentifikasi
lebih lanjut dengan menggunakan PCR-DGGE Denaturating Gradient Gel Electrophoresis Ennahar et al. 2003 dan TGGE Temperature Gradient Gel
Electrophoresis Cocolin et al. 2000. Tahapan yang dilakukan pada teknik analisis PCR-TGGE didahului
dengan ekstraksi DNA dan reaksi PCR dengan prinsip yang sama seperti dijelaskan pada Anukam et al. 2005 kemudian dilanjutkan dengan analisis
TGGE. Tahapan prosedur analisis TGGE seperti yang dilakukan oleh Walter et al. 2000 adalah sebagai berikut : elektroforesis dilakukan dengan menggunakan
gel poliakrilamid dan bufer TAE tris asetat pada tegangan 110 V selama 5 jam. Gradien dari temperatur 65.5 – 68°C digunakan secara paralel pada saat
elektroforesis berjalan running. Setelah elektroforesis selesai komplet maka dilakukan pewarnaan pada TAE, selanjutnya gel divisualisasikan dengan
menggunakan sistem kamera polaroid. Hibridisasi DNA
Hibridisasi DNA dilakukan untuk mengetahui kandungan G+C mol dari BAL. Metode yang digunakan adalah isolasi DNA lalu penentuan
kandungan G+C dilakukan dengan menggunakan thermal melting temperature
30
DNA menggunakan
spektrofotometer contohnya
: Gilford
Response Spectrophotometer Krockel et al 2003.
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3. METODOLOGI UMUM
