Pyocyanin Asal Bakteri Laut Pseudomonas aeruginosa yang Berpotensi sebagai Antimikrob

Abstract

Pyocyanin merupakan pigmen biru kehijauan yang diproduksi oleh 90-95% strain bakteri Pseudomonas aeruginosa. Selain warnanya yang menarik, pyocyanin juga memiliki kemampuan bioaktivitas yang menjanjikan seperti antimikrob. Pencarian senyawa antimikrob masih menjadi urgensi dalam dunia kesehatan. Urgensi tersebut berkaitan dengan angka kematian akibat kasus resistansi mikrob patogen terhadap antibiotik yang terus meningkat. Oleh karena itu, penelitian ini bertujuan menapis bakteri potensial sebagai produsen pyocyanin, mengarakterisasi pyocyanin yang diperoleh, dan menguji aktivitas pyocyanin tersebut sebagai antimikrob. Penelitian diawali dengan peremajaan tujuh isolat bakteri pada media Sea Water Complete (SWC). Seluruh isolat dapat ditumbuhkan, namun menunjukkan perbedaan pada aspek pigmentasi. Pada media padat, satu isolat memiliki koloni berwarna biru kehijauan (P1.S9), empat isolat memiliki koloni berwarna hijau (P1.Z39, D2.Z12, D2.Z13, dan D2.Z15), dan dua isolat memiliki koloni berwarna putih (P1.S21 dan D2.Z14). Pigmentasi yang sama juga ditunjukkan pada kultur bakteri di media cair, perbedaannya hanya pada isolat P1.S21 dan D2.Z14 yang tidak menunjukkan ciri pigmentasi sehingga warna kultur tetap kuning seperti warna media awal. Setelah mengidentifikasi ciri koloni dan kultur, uji hemolisis pada Sheep Blood Agar (SBA) digunakan untuk mengetahui ciri patogenisitas isolat. Hasilnya, lima isolat teridentifikasi mempunyai kemampuan ß-hemolisis (P1.S9, P1.Z39, D2.Z12, D2.Z13, dan D2.Z15) dan dua isolat teridentifikasi mempunyai kemampuan a-hemolisis (P1.S21 dan D2.Z14). Kedua aspek yang telah disampaikan sebelumnya, kemudian dilengkapi oleh analisis gen 16S rRNA dalam upaya penapisan lebih lanjut. Semua isolat teridentifikasi sebagai P. aeruginosa, kecuali D2.Z14 yang teridentifikasi sebagai Alcaligenes faecalis. Merujuk hasil pengamatan koloni dan kultur, uji hemolisis, dan analisis gen 16S rRNA, isolat P1.S21 dan D2.Z14 dieliminasi. P1.S21 dinilai tidak mampu menghasilkan pyocyanin berdasarkan ciri pigmentasi, sama dengan D2.Z14 yang selain tidak menunjukkan ciri pigmentasi juga tidak teridentifikasi sebagai P. aeruginosa. Analisis gen 16S rRNA hanya terbatas hingga pengelompokan pada tingkat spesies. Analisis gen ini tidak dapat membedakan strain bakteri dalam spesies yang sama. Oleh karena itu, lima isolat tersisa dinilai karakter intraspesiesnya menggunakan metode Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR). Analisis pola ERIC-PCR yang diperoleh membagi kelima isolat menjadi lima strain yang berbeda. Pengelompokan ini menjadi dasar penggunaan kelima isolat pada tahap optimasi produksi pyocyanin. Optimasi dibagi menjadi dua tahap. Tahap pertama fokus pada faktor isolat, jenis media, dan temperatur. Konsentrasi pyocyanin tertinggi yang diperoleh adalah 16,179 ± 0,517 µg/mL, dengan kombinasi perlakuan berupa isolat P. aeruginosa P1.S9 yang ditumbuhkan pada media King's A dan diinkubasi pada 27°C. Sementara itu, tahap kedua fokus pada komposisi media seperti sumber karbon, sumber nitrogen, dan sumber ion logam-garam anorganik. Pada tahap ini, isolat P. aeruginosa P1.S9 dijadikan isolat tunggal, suhu inkubasi dipertahankan pada 27°C, dan media King’s A digunakan sebagai acuan untuk menyusun perlakuan. Hasilnya, konsentrasi pyocyanin tertinggi yang diperoleh adalah 29,057 ± 0,691 µg/mL, dengan perlakuan berupa media King’s A yang menggunakan pati sebagai sumber karbon. Berdasarkan hasil tahap optimasi, P. aeruginosa P1.S9 dipilih untuk didalami pada tahap berikutnya. P. aeruginosa P1.S9 dijadikan isolat tunggal pada tahap analisis gen penyandi (phzM dan phzS) dan produksi ekstrak pyocyanin. Sekuens protein PhzM yang diperoleh dari hasil translasi gen phzM teridentifikasi sebagai phenazine-1-carboxylate N-methyltransferase, sedangkan protein PhzS yang diperoleh dari hasil translasi gen phzS teridentifikasi sebagai flavin-dependent oxidoreductase. Kedua protein tersebut adalah enzim yang bertanggung jawab di dua tahap terakhir pada jalur sintesis pyocyanin. Selain pensejajaran (alignment) sekuens dengan protein pada database, analisis lebih lanjut mengenai kedua protein tersebut juga didukung oleh pemodelan tiga dimensi, analisis superposisi, identifikasi domain, dan deteksi fingerprint motif. Berdasarkan hasil tersebut, secara molekuler P. aeruginosa P1.S9 dinilai mampu menghasilkan pyocyanin. Tahapan berikutnya adalah karakterisasi secara kimiawi ekstrak pyocyanin yang dihasilkan oleh P. aeruginosa P1.S9. Ekstrak pyocyanin P. aeruginosa P1.S9 berwarna biru pekat dengan tekstur pasta. Karakterisasi ekstrak dilakukan dengan bantuan spektrofotometer UV-Visible (UV-Vis), Fourier Transform Infrared (FTIR), dan Nuclear Magnetic Resonance (1HNMR). Hasil analisis UV-Vis menunjukkan tiga puncak spektrum pada posisi 279, 389, dan 522 nm; hasil analisis FTIR menunjukkan keberadaan beberapa gugus fungsi seperti O-H, C-H, C=N, C=C, C-N, dan C-H bend; sedangkan hasil analisis 1HNMR menunjukkan keberadaan ikatan metil-nitrogen dan cincin aromatik sebagai penciri pyocyanin. Karakteristik tersebut sesuai dengan keterangan beberapa literatur yang digunakan sebagai rujukan untuk karakterisasi pyocyanin. Terakhir, kemampuan antimikrob pyocyanin diuji dengan metode difusi cakram dan penentuan nilai Minimum Inhibitory Concentration (MIC). Zona bening pada uji difusi cakram terbentuk melawan Bacillus subtilis ATCC 19659, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 15442, dan Staphylococcus aureus ATCC 6538. Sementara itu, nilai MIC terkuat adalah sebesar 62,5 µg/mL melawan B. subtilis ATCC 19659.

Pyocyanin is a blue-green pigment produced by 90-95% of Pseudomonas aeruginosa strains. Besides its attractive colour, pyocyanin also possesses promising biological activities such as antimicrobial. Efforts to find antimicrobial compounds are still a matter of urgency in the world of health. The urgency is related to the mortality rate due to antibiotic resistance of pathogens that continue to increase. Therefore, this study aims to screen a potential bacterium as pyocyanin producer, to characterize the pyocyanin obtained, and to test the antimicrobial activity of the pyocyanin. The study began with the rejuvenation of seven bacterial isolates on Sea Water Complete (SWC). On SWC agar, all isolates could be grown but showed differences in the pigmentation aspect. One isolate had a blue-green colour (P1.S9), four isolates were green (P1.Z39, D2.Z12, D2.Z13, and D2.Z15), and two isolates were white (P1.S21 and D2.Z14). The same results were also obtained for the SWC broth, the difference was only shown by P1.S21 and D2.Z14 which did not have any pigmentation and still maintained the colour of the SWC broth. After identifying the morphological characteristics of the colonies and cultures, the haemolysis test using Sheep Blood Agar (SBA) was used to determine the isolates pathogenicity. As a result, five isolates were identified for having ß-haemolysis ability (P1.S9, P1.Z39, D2.Z12, D2.Z13, and D2.Z15) and two isolates had a-haemolysis ability (P1.S21 and D2.Z14). Both aspects mentioned previously were then complemented by 16S rRNA gene analysis. All isolates were identified as P. aeruginosa, except D2.Z14 which was identified as Alcaligenes faecalis. According to isolates colonies and cultures characteristics, haemolysis test, and 16s rRNA gene analysis, P1.S21 and D2.Z14 were eliminated. P1.S21 was considered incapable of producing pyocyanin based on the pigmentation features on the media, similar to D2.Z14 which neither showed pigmentation characteristics nor identified as P. aeruginosa. The 16S rRNA gene analysis is limited to the species level classification. The analysis is unable to distinguish bacterial strains within the same species. Therefore, the remaining five isolates were assessed for intraspecies characters using the Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR). The ERIC-PCR patterns thus divided the five isolates into five separated strains. This separation was the reason for using all five isolates in the optimization step of pyocyanin production. Optimization was divided in two stages. The first stage focused on isolates, media, and temperature as parameters. The highest pyocyanin concentration obtained was 16,179 ± 0,517 µg/mL, with the treatment combination of isolate P. aeruginosa P1.S9 cultured in King's A medium and incubated at 27°C. Meanwhile, the second stage focused on the composition of media such as carbon sources, nitrogen sources, and metal ion-inorganic salt ion sources. In this stage, P. aeruginosa P1.S9 was used as a single isolate, the incubation temperature was kept at 27°C, and King's A medium was set as the baseline for the treatments. As a result, the highest pyocyanin concentration obtained was 29,057 ± 0,691 µg/mL, with the treatment of King's A media that used starch as a carbon source. Based on the outcome of the optimization stage, P. aeruginosa P1.S9 was selected to be further investigated in the next stages. P. aeruginosa P1.S9 was used as a single subject for the analysis of the encoding genes (phzM and phzS) and the production of pyocyanin extract. The PhzM protein sequence obtained from the translation of the phzM gene was identified as phenazine-1-carboxylate N-methyltransferase, while the PhzS protein obtained from the translation of the phzS gene was identified as flavin-dependent oxidoreductase. Both proteins are enzymes responsible in the last two stages of pyocyanin synthesis pathway. In addition to sequence alignment with the database, further analysis of both proteins was also supported by three-dimensional modelling, superposition analysis, domain identification, and fingerprint motif detection. Thus, P. aeruginosa P1.S9 was considered capable of producing pyocyanin molecularly. The subsequent step was to chemically characterize the pyocyanin extract produced by P. aeruginosa P1.S9. The pyocyanin extract of P. aeruginosa P1.S9 was blue in colour with a paste texture. Characterization of the extract was carried out using UV-Visible spectrophotometer (UV-Vis), Fourier Transform Infrared (FTIR), and Nuclear Magnetic Resonance (1HNMR). The UV-Vis analysis showed three peaks at 279, 389, and 522 nm; FTIR analysis showed the presence of several functional groups such as O-H, C-H, C=N, C=C, C-N, and C-H bend; while 1HNMR analysis showed the presence of methyl-nitrogen bonds and aromatic rings as the pyocyanin markers. These characteristics are consistent with some literatures used as references for pyocyanin characterization. Ultimately, the antimicrobial activity of pyocyanin was examined by the disc diffusion method and determination of the Minimum Inhibitory Concentration (MIC). The zone of inhibition in the disc diffusion test was formed against Bacillus subtilis ATCC 19659, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 15442, and Staphylococcus aureus ATCC 6538. Meanwhile, the strongest MIC value was 62,5 µg/mL against B. subtilis ATCC 19659.