Karakterisasi Isolat Klinik Pseudomonas aeruginosa Pembentuk Biofilm dan Faktor-faktor Pembentukan Biofilm

DIDIK WAHYUDI, Prof. Dra. A. Endang Sutariningsih Soetarto, M.Sc., Ph.D.; Dr. Niken Satuti Nur Handayani, M.Sc.; dr. Abu Tholib Aman, M.Sc., Ph.D., SpMK(K).

2020 | Disertasi | DOKTOR BIOLOGI

Pseudomonas aeruginosa merupakan bakteri patogen oportunistik, penyebab infeksi nosokomial, seperti pneumonia, infeksi saluran kemih (ISK) serta bakteremia. Bakteri tersebut sangat unik karena mampu membentuk biofilm dengan karakteristik fenotipik dan biokimia yang spesifik, dan menyebabkan infeksi berbasis biofilm yang sulit diobati. Biofilm P. aeruginosa berkembang pada infeksi kronis dan sangat terkait dengan persistensi dan resistensi terhadap antibiotik. Penelitian ini bertujuan untuk melakukan karakterisasi isolat klinis P. aeruginosa berdasarkan kemampuan membentuk biofilm dan kepekaannya terhadap berbagai antibiotik, mengkaji pengaruh faktor lingkungan (komposisi media kultur, suhu inkubasi, pH media, dan antibiotik) terhadap pembentukan biofilm P. aeruginosa, dan mendeteksi gen yang mengendalikan produksi biofilm. Isolat P. aeruginosa diperoleh dari Rumah Sakit, berasal dari berbagai jenis sampel klinis antara lain; darah, sputum, urin, cairan telinga, cairan pleura, pus, feces, dan cairan jaringan. Isolat bakteri dipurifikasi menggunakan teknik koloni sel tunggal. Karakterisasi kultur murni isolat dikerjakan berdasarkan kepekaan terhadap antibiotik dengan metode pelarutan menggunakan Vitek 2 Compact. Uji kemampuan isolat dalam membentuk biofilm dilakukan dengan menumbuhkannya pada medium cair trypticase selama 24 jam, massa biofilm yang terbentuk diukur dengan microtiter plate culture technique, secara spektrofotometri (panjang gelombang 570 nm) dengan microplate reader, isolat diseleksi berdasarkan 3 kriteria pembentukan biofilm (lemah, sedang, dan kuat), Kelompok isolat (lemah, sedang, dan kuat) terpilih (masing-masing 8 isolat yang representatif) diuji lebih lanjut terhadap faktor lingkungan yang mempengaruhi pembentukan biofilm. Uji Pengaruh faktor lingkungan dilakukan melalui percobaan kultivasi pada medium trypticase cair dengan sumber C (glukosa, mannosa) sumber N (lisin, tryptophan) dengan konsentrasi, suhu inkubasi, dan pH media berbeda. Biofilm yang terbentuk diamati dengan pewarnaan crystal violet 0,1 persen. Pengaruh antibiotik (ciprofloxacin dan amikacin) konsentrasi 1, 2, 4, 8. 16. 32. 64. dan 128 mikrogram/mililiter ditentukan dengan menggunakan 3-(4,5-dimethythiazol-2-yl)-2, 5- diphenyl tetrazolium bromide (MTT). Deteksi gen pengendali biofilm dilakukan dengan Polymerase Chain Reaction (PCR), dilanjutkan sequensing. Hasil Penelitian menunjukkan bahwa 64 isolat P. aeruginosa memiliki bentuk batang berukuran 0,5-0,8 mikron x 1.5-3.0 mikron, Gram negatif. Beberapa isolat (72 persen) sensitif terhadap piperasilin, ceftazidime, cefepime, oztreonam, meropenem, amikacin, gentamicin, dan ciprofloxacin. Berdasarkan kemampuan pembentukan biofilm, isolat P. aeruginosa meliputi tiga kriteria: kelompok pembentuk biofilm lemah (14 isolat), kelompok pembentuk biofilm sedang (32 isolat), dan kelompok pembentuk biofilm kuat (18 isolat). Penambahan glukosa pada konsentrasi 0,3mM dan manosa pada konsentrasi 0,5mM selama 24 jam telah memacu produksi biofilm, baik pada kelompok biofilm yang lemah, sedang, maupun yang kuat; sedangkan penambahan lisin tidak mempengaruhi pembentukan biofilm, dan tryptophan menghambat pembentukan biofilm. Semua isolat P. aeruginosa mampu membentuk biofilm pada suhu 28 dan 37C, sedangkan pada suhu 40C pembentukan biofilm lamban. Pembentukan biofilm terjadi dengan cepat pada pH media 7,0 dan 9,0; dan sangat lamban pada pH 5,0. Amikacin (16 mikrogram/mililiter) dan ciprofloxacin (64 mikrogram/mililiter) mampu menghambat sel P. aeruginosa dalam biofilm sebesar 50 persen (MBIC 50). Gen pslA, alg44, dan pelD terdapat pada semua kelompok isolat (kuat, sedang, dan lemah). Kesimpulan, isolat klinik P. aeruginosa memiliki kepekaan yang berbeda-beda terhadap antibiotik, dan memiliki kemampuan yang bervariasi dalam membentuk biofilm. Pembentukan biofilm dipengaruhi oleh komposisi media kultur, suhu inkubasi, pH media, dan antibiotik. Semua isolat tedeteksi adanya gen pslA, alg44, dan pelD. Pada kondisi planktonik, isolat yang lebih kuat dalam membentuk biofilm lebih resisten terhadap antibiotik, dan ketika berada di dalam biofilm isolat dengan kemampun lemah, sedang, dan kuat dalam membentuk biofilm memiliki kemampuan bertahan hidup yang sama ketika terpapar dengan antibiotik.

Pseudomonas aeruginosa is an opportunistic pathogenic bacterium, causing nosocomial infections, such as pneumonia, urinary tract infections (UTI) and bacteremia. These bacteria are very unique because they are able to form biofilms with specific phenotypic and biochemical characteristics, causing biofilm-based infections that are difficult to treat. P. aeruginosa biofilms developed in chronic infections and were strongly associated with persistence and resistance to antibiotics. The aims of the study were to characterize P. aeruginosa clinical isolates based on their ability to form biofilms and their sensitivity to various antibiotics, to assess the influence of environmental factors (culture media composition, incubation temperature, media pH, and antibiotics) on the formation of P. aeruginosa biofilms, and to detect genes that controlling biofilm production. P. aeruginosa isolates were obtained from the hospital, and originated from various types of clinical samples, among others; blood, sputum, urine, ear fluid, pleural fluid, pus, feces, and tissue fluid. Bacterial isolates were purified using a single cell colony technique. Identification of pure culture of isolates was carried out based on sensitivity to antibiotics through the dissolution method using Vitek 2 Compact. The biofilm formation ability of pure isolates culture was done by growing isolates on trypticase liquid medium for 24 hours, the mass of biofilms formed was measured by microtiter plate culture technique, spectrophotometrically (wavelength 570 nm) with a microplate reader, isolates were selected based on 3 biofilm formation criteria (weak, moderate , and strong), selected isolates (weak, medium, and strong) groups (each of 8 representative isolates) were further tested for environmental factors affecting biofilm formation. The test of the influence of environmental factors was carried out through cultivation experiments on liquid trypticase medium with a C source (glucose, mannose) N source (lysine, tryptophan) with different concentrations, incubation temperatures, and pH. Biofilms formed were observed with 0.1 percent crystal violet staining. The effect of antibiotics (ciprofloxacin and amikacin) concentrations of 1, 2, 4, 8. 16. 32. 64. and 128 microgram/milliliter was determined using 3- (4,5-dimethythiazol-2-yl) -2, 5- diphenyl tetrazolium Bromide (MTT). Detection of biofilm controlling genes is done by Polymerase Chain Reaction (PCR), followed by sequensing. The results showed that 64 P. aeruginosa isolates had rod shapes of 0.5-0.8 mikron x 1.5-3.0 mikron, Gram negative. Some isolates (72 percent) were sensitive to piperacillin, ceftazidime, cefepime, oztreonam, meropenem, amikacin, gentamicin, and ciprofloxacin. Based on the ability of biofilm formation, P. aeruginosa isolates included three criteria: weak biofilm forming groups (14 isolates), moderate biofilm forming groups (32 isolates), and strong biofilm forming groups (18 isolates). The addition of glucose at a concentration of 0.3 mM and manosa at a concentration of 0.5 mM for 24 hours had stimulated biofilm production, in the weak, moderate, or strong biofilm group; whereas the addition of lysine did not affect the formation of biofilms, and tryptophan inhibited the formation of biofilms. All of P. aeruginosa isolates were able to form biofilms at 28 and 37C, but at 40C the formation of biofilms was slow. Biofilm formation occurs rapidly at pH media 7.0 and 9.0; and very slow at pH 5.0. Amikacin (16 microgram/milliliter) and ciprofloxacin (64 microgram/milliliter) were able to inhibit P. aeruginosa cells in biofilms by 50 percent (MBIC 50). pslA, alg44, and pelD genes were present in all isolate groups (strong, moderate, and weak). Conclusion, P. aeruginosa clinical isolates had different sensitivity to antibiotics, and had varying abilities in forming biofilms. The formation of biofilms was influenced by the composition of culture media, incubation temperature, pH of the media, and antibiotics. All isolates detected the pslA, alg44, and pelD genes. In planktonic conditions, isolates that are stronger in forming biofilms are more resistant to antibiotics, and when in biofilms, isolates with weak, moderate, and strong abilities had the same survival ability when exposed to antibiotics.

Kata Kunci : P. aeruginosa, biofilm, medium, suhu, pH, antibiotik, gen.