Hadi, Wiyono (2019) Ekstrak Etanol Propolis Trigona Sp. Malang Indonesia Sebagai Quorum Sensing Inhibitor Isolat Biofilm Staphylococcus Aureus Dari Rinosinusitis Kronis. Doctor thesis, Universitas Brawijaya.
Abstract
Rinosinusitis kronis (RSK) merupakan peradangan mukosa hidung dan sinus paranasal yang ditandai dengan dua gejala atau lebih salah satunya pilek dan buntu hidung disertai dengan nyeri/ rasa tertekan pada wajah dan gangguan penciuman, yang berlangsung lebih dari tiga bulan. Etiologi RSK multifaktorial, salah satunya adalah biofilm bakteri. Keberadaan biofilm bakteri menyebabkan RSK sulit diatasi. Kekambuhan sering terjadi yang menyebabkan morbiditas berulang, penurunan kualitas hidup, dapat menyebabkan komplikasi dan memerlukan biaya tinggi. Keberadaan biofilm menyebabkan bakteri lebih resisten. Staphylococcus aureus merupakan salah satu bakteri yang dapat membentuk biofilm dan merupakan salah satu penyebab RSK. Pembentukan biofilm Staphylococcus aureus melalui 4 tahap yaitu tahap perlekatan, quorum sensing (QS), maturasi dan pelepasan, QS merupakan tahap paling penting pada proses pembentukan biofilm. Quorum sensing Inhibitor (QSI) merupakan salah satu pendekatan strategis untuk pengembangan terapi baru pengobatan biofilm bakteri. Penghambatan proses ini diharapkan dapat menghentikan proses pembentukan biofilm. QS merupakan proses komunikasi antar bakteri baik intra maupun inter spesies. Pada Staphylococcus aureus komunikasi diperantarai oleh sinyal mikromolekul autoinducer peptide (AIP). Pada kepadatan tertentu (quorum) AIP akan dikenali oleh reseptor transmembran yang disebut AgrC-histidine kinase. Phosporilasi histidine kinase akan mengaktifkan AgrA sebagai regulator promotor P2 dan P3. Promotor P2 menyebabkan peningkatan transkripsi operon agrB, agrD, agrC, agrA dan autoamplification dari Agr system. Aktivasi promotor P3 menginduksi transkripsi RNAIII, yang berfungsi sebagai molekul efektor utama untuk mengatur faktor virulensi. Propolis merupakan produk alami yang dihasilkan oleh lebah madu. Propolis dikenal sebagai bahan penyembuhan yang berkualitas sejak peradaban Mesir dan Yunani. Secara klinis propolis diketahui efektif sebagai antimikroba. Propolis menghambat pertumbuhan Staphylococcus aureus, Staphylococcus epidermidis, Entero-coccus Sp., Corynebacterium Sp., B. catarrahlis, dan B. cereus. Pengenceran tersebut sebagian menghambat pertumbuhan Pseudomonas aeruginosa dan Esterechia coli. Propolis mampu menghambat pembentukan biofilm dengan cara mendegradasi extracellular polymeric substance (EPS), mengurangi motilitas bakteri Pseudomonas aeruginosa dan mengacaukan fungsi autoinducer AHL. Tujuan penelitian ini adalah membuktikan Ekstrak Etanol Propolis (EEP) Trigona Sp. Malang Indonesia sebagai QSI pada isolat biofilm Staphylococcus aureus dari RSK berdasarkan penurunan AIP, histidine kinase, ekspresi gen agr dan morfologi biofilm. Metode penelitian. Jenis penelitian adalah True experimental yang xiii dilakukan di laboratorium (invitro) dengan rancangan Pre and post test control group design. Sampel penelitian adalah penderita RSK yang menjalani operasi bedah sinus endoskopi fungsional di RS PHC Surabaya. Jumlah sampel (ulangan) dihitung menggunakan rumus Frederer. Jumlah perlakuan EEP Tigona Sp. Malang Indonesia sebanyak 7 dan jumlah ulangan sebanyak 3. Penelitian dibagi menjadi 2 tahap. Pertama, Isolasi dan karakterisasi Staphylococcus aureus dan Eksplorasi dosis EEP Trigona Sp. Malang Indonesia sebagai quorum sensing inhibitor isolat biofilm Staphylococcus aureus dari RSK. Kedua, EEP Trigona Sp. Malang Indonesia sebagai QSI pada isolat biofilm Staphylococcus aureus dari RSK. Penelitian tahap pertama telah dilakukan isolasi dan karakterisasi Staphylococcus aureus. Isolasi menggunakan media Manitol Salt Agar tampak pertumbuhan bakteri berwarna kuning. Pewarnaan gram tampak koloni tersusun berjajar seperti rantai memenjang membentuk seperti buah anggur berwarna ungu. Uji katalase positif dengan terbentuknya gelembung-gelembung udara. Uji koagulase positif dengan terbentuknya gumpalan. Kultur Congo Red didapatkan biofilm Staphylococcus aureus berwarna hitam. Tes kepekaan antibiotika Staphylococcus aureus multiresisten terhadap antibiotika. Pada tes ini Staphylococcus aureus resisten terhadap Cefoxitin (FOX), Vancomycin (VA), Oxacillin (OX), Penicillin (P), Erythromycin (E) dan Lincomycin (MY). Eksplorasi dosis menggunakan tehnik mikrotiter pada larutan EEP Trigona Sp Malang Indonesia dosis 0%, 0,2%, 0,4%, 0,6%, 0,8%, 1%, 2%, 4%, 6%, 8%, 10% dan kontrol negatif (tanpa biofilm Staphylococcus aureus), ulangan (n) sebanyak 3 (tiga) kali. Inkubasi mikrotiter 108 Cfu/ml selama 48 jam. Pemeriksaan hasil mikrotiter menggunakan Confocal Laser Scanning Microscopy (CLSM) Olympus type FV1000 dengan pewarna Syto9 green fluorescent nucleic acid marker 1:500 pembesaran 400x. Kuantifikasi menggunakan Software Olympus Fluoview version 1.7a. Pengukuran dinilai dari intensitas ekspresi Syto9 dengan satuan arbitrary unit (au) dan ketebalan biofilm dengan satuan mikro meter (μm). Pada penelitian tahap pertama ini didapatkan penurunan morfologi biofilm secra bermakna dengan semakin meningkatnya dosis EEP Malang Indonesia. Berdasarkan hasil uji Kruskal Wallis, didapatkan hasil bahwa variabel Intensitas ekspresi Syto9 dan variabel ketebalan biofilm memiliki nilai signifikansi sebesar 0.001 < (α = 0,05). Penelitian tahap kedua isolat biofilm Staphylococcus aureus diberikan perlakuan menggunakan EEP Trigona Sp. Malang Indonesia dosis 0%; 0,4%, 0,8%, 2%, 6% dan 10%. Kontrol (-) adalah Staphylococcus epdermidis non biofilm. Kadar AIP diukur menggunakan Western blot dan ekspresi gen agr diperiksa menggunakan PCR, kuantifikasi menggunakan free software image-J. Histidine kinase dan intensitas ekspresi Syto9 diukur menggunakan doubel staining CLSM Olympus type FV1000 dengan pewarna rhodamin dan Syto9 green fluorescent nucleic acid marker 1:500 pembesaran 400x. Kuantifikasi menggunakan Software Olympus Fluoview version 1.7a. Hasil pengukuran ekspresi gen agr dilakukan uji Anova didapatkan hasil signifikansi (p) sebesar 0,000 (sig < α = 0,05). Hasil uji Anova pada variabel ketebalan biofilm didapatkan hasil signifikansi (p) sebesar 0,001 (sig < α = 0.05). Uji t-test variabel AIP kelompok Kontrol (-) terhadap kelompok propolis dosis 0,4%; 0,8%; 2%; 6% dan 10% menunjukkan signifikansi (p) < α (alfa) = 0.05. Uji t-test untuk variabel Histidine kinase kelompok Kontrol (-) terhadap kelompok propolis dosis 2%, 6% dan 10% didapatkan nilai signifikansi (p) < α (alfa) = 0.05. Uji t-test untuk variabel Intensitas ekspresi Syto9 kelompok Kontrol (-) terhadap kelompok propolis dosis 2%, 6% dan 10% didapatkan nilai signifikansi (p) < α (alfa) = 0,05, sehingga terdapat perbedaan bermakna antara kedua kelompok tersebut. Dari data di atas dapat disimpulkan bahwa EEP Trigona Sp. Malang Indonesia dapat digunakan sebagai QSI terhadap isolat biofilm Staphylococcus aureus dari RSK.
English Abstract
Chronic rhinosinusitis (CRS) is an inflammation of the nasal mucosa and paranasal sinuses that characterized by two or more symptoms, one of those symptoms is a runny nose and a nasal congestion accompanied by facial pain / pressure and olfactory disorders, which last for more than three months. The Chronic rhinosinusitis (CRS) becomes difficult to overcome because the existence of bacteria biofilms. Recurrence of CRS take place and give uch as in recurrent morbidity, decrease quality of life, makes complications and require much money. The existence of biofilms causes bacteria to be more resistant. Staphylococcus aureus is one of the bacteria that can form biofilms and one of the causes of CRS. Staphylococcus aureus biofilm formation through 4 stages, namely the attachment stage, quorum sensing (QS), maturation and release. QS is the most important stage in the process of biofilm formation. The Quorum sensing Inhibitor (QSI) is one of the strategic approaches in developing new therapies for bacterial biofilm. The inhibition of this process is expected to stop the biofilm formation process. QS is a communication system between intra and inter-species bacteria. In Staphylococcus aureus communication is mediated by micromolecule autoinducer peptide (AIP) signals. At a certain density (quorum) AIP will be recognized by a transmembrane receptor called AgrC-histidine kinase. Phosphorylation of histidine kinase will activate AgrA as a P2 and P3 promoter regulator. P2 promoter increase transcription of agrB, agrD, agrC, agrA and autoamplification operons from Agr system. Activation of the P3 promoter induces transcription of RNAIII, which has functions the main effector molecule to regulate virulence factors. Propolis is a natural product produced by honey bees. Propolis is known as a quality healing ingredient since Egyptian and Greek civilizations. Clinically propolis is known as an effective antimicrobial. Propolis inhibits growth of Staphylococcus aureus, Staphylococcus epidermidis, Entero-coccus Sp., Corynebacterium Sp., B. catarrahlis, and B. cereus. This dilution partially inhibits the growth of Pseudomonas aeruginosa and Esterechia coli. Propolis is able to inhibit biofilm formation by degrading extracellular polymeric substance (EPS), reducing the motility of the bacterium Pseudomonas aeruginosa and disrupting the AHL autoinducer function. The purpose of this study was to prove the Ethanol Extract of Propolis (EEP) Trigona Sp. Malang Indonesia as QSI in Staphylococcus aureus biofilm isolates from CRS based on AIP reduction, histidine kinase, agr gene expression and biofilm morphology. This type of research is True experimental conducted in the laboratory (invitro) with the design of Pre and post test control group design. The study sample were CRS patients who have functional sinus endoscopic surgery at Surabaya PHC Hospital. The number of samples (replications) were calculated using the Frederer formula. Amount of treatment of propolis Tigona Sp. as many as 7 and the number of replications were 3. The study was divided into 2 stages. First, Isolation and characterization of Staphylococcus aureus and exploration of Propolis dose Trigona Sp. as quorum sensing inhibitors of Staphylococcus aureus biofilm isolates from CRS. Secondly, Propolis Trigona Sp. Malang Indonesia as QSI in Staphylococcus aureus biofilm isolates from CRS. The first stage of the study was isolation and characterization of Staphylococcus aureus. The Isolation were used Manitol Salt media To make the growth of yellow bacteria. Gram staining appears the colony which arranged in a row like a tiered chain forming like purple grapes. Positive catalase tested with air bubbles formation. Positive coagulase test formed clots. Congo Red culture found black Staphylococcus aureus biofilms. The antibiotic sensitivity test of Staphylococcus aureus is multiresistant to antibiotics. In this test Staphylococcus aureus resistant to Cefoxitin (FOX), Vancomycin (VA), Oxacillin (OX), Penicillin (P), Erythromycin (E) and Lincomycin (MY). The doses exploration used microtiter techniques in Trigona Sp propolis solution doses of 0%, 0.2%, 0.4%, 0.6%, 0.8%, 1%, 2%, 4%, 6%, 8%, 10 % and negative control (without Staphylococcus aureus biofilm), repeat (n) 3 (three) times. Microtiter incubation 108 cfu / ml for 48 hours. The result of microtiter using Confocal Laser Scanning Microscopy (CLSM) Olympus type FV1000 with dyes Syto9 green fluorescent nucleic acid marker 1: 500 400x magnification. Quantification used the Olympus Fluoview Software version 1.7a. Measurements were assessed from the intensity of Syto9 expression with arbitrary unit (au) units and biofilm thickness with units of micro meters (μm). Based on the results of the Kruskal Wallis test, the results showed that the intensity of the Syto9 expression and the biofilm thickness variable had a significance value of 0.001 <(α = 0.05). The second phase of the study, at this stage Staphylococcus aureus biofilm isolates were given treatment using different doses of propolis, namely 0%; 0.4%, 0.8%, 2%, 6% and 10%. Control (-) is non-biofilm Staphylococcus epdermidis. The second stage of the research, At this stage isolate biofilm Staphylococcus aureus given treatment using propolis different dose, that is 0%; 0.4%, 0.8%, 2%, 6% and 10%. Control (-) is non-biofilm Staphylococcus epdermidis. The AIP levels were measured using Western blot, expression of the aged gene examined using PCR was measured using free software image-J. Histidine kinase levels and the intensity of Syto9 expression was measured using doubling staining Confocal Laser Scaning Microscope (CLSM) Olympus type FV1000 with red Rhodamine dye and Syto9 green fluorescent nucleic acid marker 1:500 magnification 400 times. Result of measurement of gene expression of gene was done by ANOVA test got result of significance (p) equal to 0.000 (sig <α = 0.05). This suggests that there are at least a pair of different dosing treatments that affect the expression of the agr gene. Anova test results on variable thickness of biofilm obtained results of significance (p) of 0.001 (sig <α = 0.05). This indicates that there are at least a pair of different dose treatments that affect the thickness of the biofilm. T-test of control group AIP variables (-) against 0.4% propolis group; 0.8%; 2%; 6% and 10% showed significance (p) <α (alfa) = 0.05. The t-test for the control group Histidine kinase variables (-) against the 2%, 6% and 10% propolis dose group obtained significant values (p) <α (alfa) = 0.05. The t-test for the variable of expression of Syto9 control group (-) on the 2%, 6% and 10% propolis dose group obtained the significance value (p) <α (alfa) = 0.05. so there is a significant difference between the two groups. Conclusion, EEP Trigona Sp. Malang Indonesia can be used as QSI against the isolated biofilm of Staphylococcus aureus from CRS.
Other obstract
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| Item Type: | Thesis (Doctor) |
|---|---|
| Identification Number: | DIS/615.36/HAD/e/2019/061906874 |
| Uncontrolled Keywords: | DRUGS O ANIMAL ORIGINS |
| Subjects: | 600 Technology (Applied sciences) > 615 Pharmacology and therapeutics > 615.3 Organics drugs > 615.36 Drugs of animal origin |
| Divisions: | S2/S3 > Doktor Ilmu Kedokteran, Fakultas Kedokteran |
| Depositing User: | Endang Susworini |
| Date Deposited: | 09 Feb 2022 07:39 |
| Last Modified: | 09 Feb 2022 07:39 |
| URI: | http://repository.ub.ac.id/id/eprint/189623 |
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