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Title:	Bakteriosin daripada pencilan Pseudomonas putida FStm2 dan potensinya sebagai bahan anti-biofilem
Authors:	Mohamad Rahimi Hamid @ Abdul Hamid (P45128)
Supervisor:	Asmat Ahmad, Prof. Madya Dr.
Keywords:	Bakteriosin Pseudomonas Anti-biofilem Marine microbiology
Issue Date:	31-Aug-2014
Description:	Kajian telah dijalankan untuk memencil dan mengenal pasti bakteria marin yang berpotensi menghasilkan peptida antimikrob. Selain itu, kajian ini juga bertujuan untuk menciri serta mengenal pasti aktiviti peptida antimikrob bakteriosin terhadap bakteria penghasil biofilem. Satu pencilan bakteria daripada ikan yu yang diberi kod FStm2 telah dikenal pasti sebagai Pseudomonas putida melalui ujian biokimia, hemolisin, API 20NE, Biolog, penjujukan nukleotida gen ribosom 16S dan gyrB, mikroskop elektron imbasan (SEM) dan analisis asid lemak. Perlakuan dengan beberapa enzim menunjukkan hanya proteinase K menyebabkan penurunan dalam aktiviti bakteriosin terhadap bakteria penunjuk Serratia marcescens ATCC13880. Bakteriosin ini tidak aktif pada suhu >80oC dan aktif dalam julat pH 3-9. Kaldu soya triptik (TSB) didapati paling sesuai untuk penghasilan bakteriosin ini. Hasil ujian kepekatan perencatan minimum (MIC), kepekatan bakterisidal minimum (MBC), lengkuk pertumbuhan dan pemerhatian menggunakan mikroskop elektron transmisi (TEM) menunjukkan aktiviti bakteriosin terhadap S. marcescens ATCC13880 adalah bakterisidal. Berat molekul bakteriosin adalah ~32 kDa. Nilai CC50 bakteriosin adalah 16 μg/mL dan bakteriosin pada kepekatan MBC iaitu 1.25 μg/mL tidak toksik terhadap sel darah merah melalui ujian hemolisin. Analisis profil bakteriosin menggunakan matrix-assisted laser desorption ionization time of flight (MALDI-TOF MS/MS) menunjukkan tiada padanan dengan bakteriosin yang sudah diketahui dan ini mencadangkan ia adalah bakteriosin novel. Melalui analisis penjujukan peptida de novo didapati sebahagian kandungan asid amino bagi bakteriosin ini adalah CHWRHLNLSTGK, yang juga tiada persamaan dengan kandungan asid amino bakteriosin Pseudomonas atau bakteria lain yang pernah dikaji. Bakteriosin ini mempunyai aktiviti terhadap bakteria yang mampu membentuk biofilem iaitu Burkholderia cepacia strain UC(A)a3, UC(A)a5 dan UC(A)b8 dan Staphylococcus hominis strain UC(A)b1 yang dipencilkan daripada kateter urinari. Pemerhatian melalui SEM pada pasak kit asai kepekatan minimum membasmi biofilem (MBEC) menunjukkan lapisan biofilem berkurangan selari dengan pengurangan bakteria pembentuk biofilem selepas perlakuan dengan bakteriosin. Pemerhatian menggunakan mikroskop imbasan konfokal laser (CLSM) menunjukkan kematian sel bakteria pembentuk biofilem lebih ketara pada bakteriosin berkepekatan 20 μg/mL berbanding dengan bakteriosin berkepekatan 10 μg/mL. Lapisan biofilem B. cepacia, S. hominis dan S. marcescens pada kateter foley juga berkurangan apabila ditindakkan dengan bakteriosin ini. Bakteriosin ini berpotensi dimajukan sebagai agen antibakteria terutamanya terhadap bakteria yang telah rintang terhadap antibiotik lazim.,A study was conducted to isolate and identify marine bacteria with potentials to produce antimicrobial peptides. In addition, the study also aimed to characterize the activity of the antimicrobial peptide bacteriocin against biofilm forming bacteria. One isolate obtained from shark skin and given the code FStm2, was identified as Pseudomonas putida based on biochemical tests, hemolysin assay, API 20NE test, Biolog test, 16S ribosomal RNA and gyrB gene sequences, scanning electron microscopy (SEM) and fatty acid analysis. Treatment with some enzymes showed that only proteinase K reduced the activity of the bacteriocin against the indicator bacterium Serratia marcescens ATCC13880. The bacteriocin was inactive at temperatures >80o C and was active over the range of pH 3-9. The tryptic soy broth (TSB) medium was the best for bacteriocin production. Minimum inhibitory concentration (MIC) assay, minimum bactericidal concentration (MBC) assay, growth curve and observation by transmission electron microscopy (TEM) all indicated that the action of the bacteriocin against S. marcescens ATCC13880 was bactericidal. The molecular weight of the bacteriocin was ~32 kDa. CC50 value of bacteriocin was determined at 16 μg/mL and at the MBC concentration of 1.25 μg/mL did not cause hemolysis of red blood cells. Profile analysis of this bacteriocin by matrix-assisted laser desorption ionization time of flight (MALDI-TOF MS/MS) showed no matches against any of the known bacteriocins which suggested that it is a novel bacteriocin. De novo peptide sequencing produced a partial amino acid sequence containing CHWRHLNLSTGK which again did not match with any of the known Pseudomonas or other bacteria bacteriocins. The bacteriocin was active against biofilm forming bacteria Burkholderia cepacia strain UC(A)a3, UC(A)a5 and UC(A)b8, and Staphylococcus hominis strain UC(A)b1 isolated from urinary catheter. Observation of minimum biofilm eradication concentration (MBEC) assay kit pegs by SEM showed that the biofilm layer decreased together with the decrease in biofilm bacteria density after treatment with bacteriocin. Confocal laser scanning microscopy (CLSM) showed that bactericidal effect on biofilm forming bacteria was markedly higher at bacteriocin concentration of 20 μg/mL as compared to that at 10 μg/mL. Biofilm thickness of B. cepacia, S. hominis and S. marcescens in foley catheter was reduced after treatment with the bacteriocin. This bacteriocin has very good potential to be developed as an antibacterial agent especially against bacteria that are resistant toward commonly-used antibiotics.,PhD
Pages:	295
Call Number:	QR82.P78M837 2014 tesis
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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