Aktivitas Antibakteri dan Antibiofilm Metabolit Actinomycetes Isolat amo.128 dari Pulau Seribu Jakarta

Heru Agus Cahyanto, Prof. Dr. Mustofa, Apt, M.Kes; Dr. Rofiq Sunaryanto, S.Si, M.Si

2024 | Tesis | S2 Ilmu Kedokteran Dasar dan Biomedis

INTISARI

Latar Belakang : Prevalensi bakteri resisten semakin meningkat setiap tahunnya di Indonesia. Resistensi ini terjadi pada beberapa kelompok agen antimikroba sekaligus. Salah satu penyebab terjadinya resistensi mikroba adalah kemampuan mikroba dalam membentuk suatu biofilm. Biofilm merupakan kumpulan mikroba, biasanya polimikroba, didalam matriks ekstraseluler. Mekanisme pembentukan biofilm melalui proses yang melibatkan aktivitas yang disebut quorum-sensing. Actinomycetes merupakan mikroba yang dikenal sebagai produsen antimikroba. Amo.128 merupakan actinomycete koleksi Laboratorium Bioteknologi, BRIN Serpong, diharapkan memiliki aktivitas antimikroba dan antibiofilm. Isolat amo.128 merupakan aktinomisete yang diisolasi dari hewan laut di Pulau Seribu, Jakarta. Identifikasi lebih lengkap perlu dilakukan untuk memastikan identitas dan karakteristik isolat yang akan digunakan untuk menghasilkan senyawa antibakteri dan antibiofilm.  Selain itu, mekanisme penghambatan quorum-sensing juga dilakukan secara in silico.

Tujuan Penelitian : Mengidentifikasi actinomycete amo.128 secara makroskopis, mikroskopis, dan molekuler, mengetahui aktivitasnya metabolitnya sebagai  antibakteri dan antibiofilm, mengidentifikasi kandungan metabolit sekunder, dan untuk memahami mekanisme penghambatan quorum sensing secara in silico.

Metode : Actinomycetes amo.128 diidentifikasi secara makroskopis, mikroskopis, dan molekuler menggunakan mikroskop dan PCR. Identifikasi kandungan senyawa dilakukan dengan LC-HRMS dan FTIR. Penentuan aktivitas antibakteri dan antibiofilm dilakukan dengan mikrodilusi dan uji kristal violet. Mekanisme penghambatan quorum sensing secara in silico dengan target protein adalah SdiA, AgrA dan CviR.

Hasil Penelitian : Isolat amo.128 memiliki kemiripan 100?ngan Streptomyces parvus strain NBRC 14599. Metabolit amo.128 mengandung senyawa antara lain N-asetyltyramin (16,66%), cyclophenylalanilprolyl (7,97%), dan golongan senyawa pyrrol-pyrazine (6,45%). MIC/MBC/MIC50 ekstrak amo.128 terhadap S. aureus adalah 25/50/28,48 ppm sementara pada E. coli adalah 100/200/49,38 ppm. Metabolit isolat amo.128 menghambat dan mereduksi biofilm masing-masing terhadap S. aureus dan E. coli dengan nilai BIC50 3.425 ppm; 57.07 ppm dan BRC50 62,07 ppm; 60,44 ppm. Senyawa metabolit amo.128 memiliki potensi sebagai quorum sensing. inhibitor.

Kesimpulan : Isolat amo.128 memiliki kemiripan 100?ngan Streptomyces parvus strain NBRC 14599, memiliki kandungan metabolit sekunder  N-asetyltyramin, cyclophenylalanylprolyl, dan golongan senyawa pyrrol-pyrazine. Metabolit amo.128 memiliki potensi sebagai antibakteri terutama bakteri Gram + dan potensi untuk dikembangkan sebagai antibakteri Gram +. Senyawa metabolit amo.128 memiliki potensi sebagai  quorum sensing inhibitor.

ABSTRACT

Background: The prevalence of resistant bacteria is increasing every year in Indonesia. This resistance occurs in several antimicrobial groups. One of the causes of microbial resistance is the ability of microbes to form biofilms. Biofilms are collections of microbes, usually polymicrobial, in an extracellular matrix. The mechanism for biofilm formation is through a process that involves an activity called quorum sensing. Actinomycetes are microbes known as antimicrobial producers. Amo.128 is an actinomycete from the collection of the Laboratory Biotechnology, BRIN Serpong, which is expected to have both antimicrobial and antibiofilm activity. The amo.128 isolate is an actinomycete isolated from marine animals at Seribu Island, Jakarta. More complete identification needs to be carried out to ensure the identity and characteristics of the isolate that will be used to produce antibacterial and antibiofilm compounds. Besides, the quorum sensing inhibition mechanism was also carried out using in silico.

Objective: to identify amo.128 macroscopically, microscopically, and molecularly; to determine the antibacterial and antibiofilm activity; to identify secondary metabolites; and to understand the mechanism of quorum sensing inhibition by in silico

Method: Actinomycete amo.128 was identified macroscopically, microscopically, and molecularly using a microscope and PCR. Identification of components was performed by LC-HRMS and FTIR. Determining antibacterial and antibiofilm activity was performed by microdilution and crystal violet assay. The mechanism of quorum sensing inhibition by in silico with proteins target was SdiA, AgrA, and CviR.

Results: Isolate amo.128 is a genus of Streptomyces based on microscopic, macroscopic, and molecular identification. Phylogenetic analysis of the amo.128 isolate has a 100% similarity to the Streptomyces parvus strain NBRC 14599. Metabolites amo.128 contains several compounds, including N-acetyltyramine (16.66%), cyclophenylalanylprolyl/cFP (7.97%), and pyrrol-pyrazine groups (6.45%). The MIC/MBC/MIC50 of amo.128 metabolites against S. aureus is 25/50/28.48 ppm while in E. coli is 100/200/49.38 ppm. Amo.128 isolate metabolites inhibited and reduced biofilms against S. aureus and E. coli, respectively, with BIC50 values of 3,425 ppm and 57.07 ppm and BRC50 62.07 ppm and 60.44 ppm. Amo.128 metabolite compound has the potential activity as a quorum-sensing inhibitor.

Conclusion: Isolate amo.128 has 100% similarity to Streptomyces parvus strain NBRC 14599 and contains secondary metabolites N-acetyltyramin, cyclophenylalanylprolyl, and pyrrol-pyrrazine compounds. Isolate amo.128 metabolites have the potential to be developed as antibacterials especially againts Gram (+) bacteria. Amo.128 metabolite compounds have the potential activity as quorum-sensing inhibitors. 

Kata Kunci : isolate amo.128, antibacterial, antibiofilm, quorum sensing inhibitor