ISOLASI, EFEKTIVITAS, DAN KARAKTERISASI BAKTERIOFAG LITIK Salmonella enterica SEBAGAI BIOKONTROL PENYAKIT GASTROENTERITIS
ISOLATION, EFFECTIVENESS, AND CHARACTERIZATION OF Salmonella enterica LYTIC BACTERIOPHAGE FOR BIOCONTROL OF GASTROENTHERITIS
Abstract
Salmonella enterica merupakan salah satu bakteri patogen penyebab gastroenteritis yang ditransmisikan melalui air dan makanan terkontaminasi yang sering terjadi pada negara berkembang. Beberapa strain Salmonella enterica multi-resisten terhadap berbagai antibiotika. Bakteriofag litik pada famili Siphoviridae dapat menjadi solusi alternatif dalam mengurangi kejadian gastroenteritis oleh Salmonella enterica. Tujuan penelitian ini adalah untuk mengisolasi, mengetahui kemampuan bakteriofag litik Lytic Bacteriophage 1 (LB1) dalam melisis inangnya yaitu Salmonella enterica penyebab gastroenteritis, serta mengetahui karakterisasi bakteriofag litik LB 1 sebagai biokontrol penyakit gastroenteritis. Bakteriofag litik diisolasi dari pembuangan limbah domestik menggunakan teknik double layer plaque. Bakteriofag litik diidentifikasi berdasarkan morfologi plak, struktur litik, inang, aktivitas lisis sel bakteri Salmonella enterica, stabilitas dalam kondisi buffer yang berbeda dan karakterisasi protein. Bakteriofag litik LB1 hanya menginfeksi sel Salmonella enterica. Hasil pemeriksaan dengan menggunakan Transmission Electron Microscope (TEM), bakteriofag litik LB1 termasuk ke dalam famili Siphoviridae. Morfologi kepala hexagonal-icosahedral berdiameter 72.7 nm, dengan ekor non-kontraktil berdiameter 17.3 nm dan panjang 100 nm. Bakteriofag litik LB1 memiliki stabilitas terbaik dalam buffer Ringers suhu 4 oC yang ditunjukkan dengan penurunan plak sebesar 28% setelah 3 minggu penyimpanan. Hasil pengujian efektivitas menunjukkan bahwa bakteriofag litik LB1 dapat mengurangi populasi sel Salmonella sebanyak 67,12% setelah 8 jam inkubasi. Bakteriofag litik LB1 memiliki 8 protein yang berbeda dengan berat molekul yang beragam 11.4 kDa, 19.6 kDa, 23 kDa, 33 kDa, 58.3 kDa, 77 kDa, 94.5 kDa, 133 kDa. Studi ini menunjukkan bahwa bakteriofag litik LB1 yang diisolasi pembuangan limbah domestik dapat secara efektif mengurangi Salmonella enterica dengan cara melisis sel bakteri. Bakteriofag litik LB1 berpeluang dapat digunakan sebagai biokontrol penyakit gastroenteritis yang disebabkan oleh Salmonella enterica. Stabilitas terbaik bakteriofag litik LB1 pada penyimpanan dalam buffer Ringer di suhu dingin (4oC), memiliki karakterisasi famili Siphoviridae, dapat mengurangi Salmonella enterica sebanyak 67.12% setelah 8 jam inkubasi, dan memiliki berat molekul 11.4-133 kDa.
Salmonella enterica is one of pathogenic bacteria causing gastroenteritis transmitted by water and food contamination which commonly occur in developing country. Some study reported that Salmonella serovar enterica strains were multi-resistant to various of antibiotics. Lytic bacteriophage in Siphoviridae family offered a good solution to reduce gastroenterytis disease caused by Salmonella enterica. This reseach aim was to isolate, effectivity test of LB 1 and to characterize lytic bacteriophage as biocontrol of gastroenterytis. Methodology and results were LB1 lytic bacteriophage was isolated from domestic waste using double layer plaque technique, was determined by the plaque morphology, the structure, the host range, the activity to lyse bacterial host cells, the stability of phage on different buffer conditions, and the protein characterization. The results showed that LB1 only infects Salmonella enterica. Based on Electron Microscope Observation showed that LB1 is grouped into Siphoviridae. It has hexagonal-icosahedral head with 72.7 nm in diameter and long-non contractile tail with 100 nm in diameter. LB1 had a good storage stability in Ringers buffer at low temperature (40C), with viability of bacteriophage decreased by 28% after 3 weeks of storage. The effectiveness showed that LB1 could reduce Salmonella enterica by 67.12% after 8 hours of incubation. LB 1 has different proteins with molecular weights: 11.4 kDa, 19.6 kDa, 23 kDa, 33 kDa, 58.3 kDa, 77 kDa, 94.5 kDa, and 133 kDa. The conclusion was LB1 was isolated from sewage water were identified to reduces Salmonella enterica effectively with concentration of 8.2x108 CFU/mL. LB 1 can be used as a biocontrol of gastroenterytis caused by Salmonella enterica, LB 1 has the best stability in buffer ringers in cold temperatures (4oC) and proven as Siphoviridae family, reduced Salmonella enterica by 67.12% after 8 hours of incubation, and has protein molecule with molecular weight 11.4 to 133 kDa.
References
Bhardwaj, N., Bhardwaj, S.K., Deep, A., Dahiya, S., and Kapoor, S. 2015. Lytic Bacteriophages as biocontrol agents of foodborne pathogens. Asian journal of animal and veterinary advances 10(11): 708-723.
Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry, 72(1-2), 248-254.
Carey-Smith, G. V., Billington, C., Cornelius, A. J., Hudson, J. A., & Heinemann, J. A. 2006. Isolation and characterization of bacteriophages infecting Salmonella spp. FEMS microbiology letters 258(2): 182-186.
Cele, N. 2009. Strategies to control bacteriophage infection in a threonine bioprocess. Dissertation of Master of Technology: Biotechnology. Durban University of Technology. Durban.
Centers for Disease Control and Prevention [CDC]. 2015. Salmonella enteritidis infection linked to raw, frozen, stuffed chicken entrees. http://www.cdc.gov/salmonella/frozen-chicken-entrees-part2-07-15/index.html.
Cheng, J.H., Yang, H., Liu, M.L., Su, W., Feng, P.M., Ding, H., et al. 2018. Prediction of bacteriophage proteins located in the host cell using hybrid features. Chemometrics and intelligent laboratory systems 180: 64-69.
Deshanda R.P., Lingga R., Hidayati N.A., Sari E., Hertati R. 2018. Fag Salmonella asal limbah pasar ikan dan air sungai di sekitar Kampus Universitas Bangka Belitung. Ekotonia: Jurnal penelitian biologi, botani, zoologi dan mikrobiologi 3(2): 45-49.
Eng, S.K., Pusparajah, P., Ab Mutalib, N.S., Ser, H.L., Chan, K.G. and Lee, L.H. 2015. Salmonella: a review on pathogenesis, epidemiology and antibiotic resistance. Frontiers in life science 8(3): 284-293.
Hagens, S., and Loessner, M.J. 2010. Bacteriophage for biocontrol of foodborne pathogens: calculations and considerations. Current pharmaceutical biotechnology 11(1): 58-68.
Harada, L.K., Silva, E.C., Campos, W.F., Del Fiol, F.S., Vila, M., Dabrowska, K., et al. 2018. Biotechnological applications of bacteriophages: state of the art. Microbiological research 212-213: 38-58.
Hardanti S., Wardani A.K., Putri W.D.R. 2018. Isolasi dan karakterisasi bakteriofag spesifik Salmonella typhi dari kulit ayam. Jurnal teknologi pertanian 19(2).
Jamal, M., Hussain, T., Das, C.R., and Andleeb, S. 2015. Characterization of Siphoviridae phage z and studying it efficacy against multidrug-resistant planktonic cells and biofilm. Journal of medical microbiology 64: 454-462.
Jatmiko Y. D., Purwanto A.P., Ardyati T. 2018. Uji Aktivitas Bakteriofage Litik Dari Limbah Rumah Tangga Terhadap Salmonella Typhi. Jurnal biodjati 3(2).
Jończyk, E., Kłak, M., Międzybrodzki, R., and Górski, A. 2011. The influence of external factors on bacteriophages. Folia microbiologica 56(3): 191-200.
Kementerian Kesehatan Republik Indonesia. 2014. Situasi diare di Indonesia. Buletin Jendela Data dan Informasi Kesehatan. ISSN 2088-270x.
Kittler, S., Wittmann, J., Mengden, R.A.L.P., Klein, G., Rohde, C., and Lehnherr, H. 2017. The use of bacteriophages as one health approach to reduce multidrug resistant bacteria. Sustainable chemistry and pharmacy 5: 80-83.
Kropinski, A.M., and Lavigne, R. 2009. Bacteriophages. M.R. Clokie (Ed.). Vol 1. Humana Press. India.
Mahamuni, P.P., Patil, A.R., and Ghosh, J.S. 2017. Proteolytic and lipolytic properties of endotoxins (enterotoxins) produced by Salmonella typhi NCIM 5255, Salmonella typhimurium NCIM 2501 and Shigella exneri NCIM 5265. Internatonal food research journal 24(6): 2685-2688.
Merabishvili, M., Pirnay, J.P., Verbeken, G., Chanishvili, N., Tediashvili, M., Lashkhi, N., et al. 2009. Quality-controlled small-scale production of a well-defined bacteriophage cocktail for use in human clinical trials. PloS one 4(3): e4944.
Odonkor, S.T., and Addo, K.K. 2011. Bacteria resistance to antibiotics: recent trends and challenges. Int j biol med res 2(4): 1204-10.
Pelzek, A.J. 2013. Isolation of Bacteriophages from Environmental Sources, and Creation and Functional Screening of Phage DNA Libraries. Current Protocols in Essential Laboratory Techniques 7(1). 13.3.1-13.3.35.
Phothaworn, P., Dunne, M., Supokaivanich, R., Ong, C., Lim, J., Taharnklaew, R., et al. 2019. Characterization of flagellotropic, Chi-like Salmonella phage isolated from Thai Poultry Farm. Viruses 11 (520): 10-17.
Phumkhachorn, P., and Rattanachaikunsopon, P. 2010. Isolation and partial characterization of a bacteriophage infecting the shrimp pathogen Vibrio harveyi. Afr. J. microbiol. res 4(16): 1794-1800.
Rakhuba, D.V., Kolomiets, E.I., Dey, E.S., and Novik, G.I. 2010. Bacteriophage receptors, mechanisms of phage adsorption and penetration into host cell. Pol. j. microbiol 59(3): 145-155.
Saefunida D.M., Wjanarka, Rukmi M.D.I., Hidayat N.N. 2016. Isolasi bakteriofag Escherichia coli dari sistem distribusi air minum isi ulang sebagai antibiofilm. Jurnal biologi 5(2).68-75.
Santos, S.B., Costa, A.R., Carvalho, C., Nóbrega, F.L., Azeredo, J. 2018. Exploiting bacteriophage proteomes: the hidden biotechnological potential. Trends in biotechnology 36(9): 966-984.
Seockmo, K., Eduardo, X., Thomas, K., and Michael, R.L. 2016. Salmonella in shell eggs: mechanisms, prevention and detection. J nutr food sci 6(1): 1-7.
Shende, R.K., Hirpurkar, S.D., Sannat, C., Rawat, N., Pandey, V. 2017. Isolation and characterization of bacteriophages with lytic activity against common bacterial pathogens. Veterinary world 10(8): 973-978.
Silva, J.B., Storms, Z., and Sauvageau, D. 2016. Host receptors for bacteriophage adsorption. FEMS microbiology letters 363(4): 1-11.
Strydom, A., and Witthuhn, C.R. 2015. Listeria monocytogenes: a target for bacteriophage biocontrol. Comprehensive reviews in food science and food safety 14(6): 694-704.
