Characterization and antibacterial activity of purified microcin produced by Klebsiella pneumoniae K15
Paper Details
Characterization and antibacterial activity of purified microcin produced by Klebsiella pneumoniae K15
Abstract
This study has interested with the characterization and antibacterial activity of purified microcin of Klebsiella pneumoniae. Isolates were collected and identified by morphological characteristics and biochemical tests of API 20E system. The diagnosis was confirmed by detection of 16S r RNA gene. Fifty-six (56%) of K. pneumoniae isolates were microcin producing, The majority of producer isolates are urine origin. Optimum conditions for production were in brain heart infusion broth with 5% Glycerol, pH 7, 37 °C / 24 hrs. The efficient producer isolate of K. pneumoniae (K15) was chosen for microcin extraction and purification, inhibition zone ≥16mm. Antimicrobial susceptibility test was done for microcin producing isolates. No correlation was noticed between antibiotics resistance and microcin production. Extraction and Purification of microcin by method based on retention of the protein lying on hydrophobic medium silica gel C60, eluted by methanol/water gradient from 20% – 80% concentration. Protein concentration 80 µg/ml and specific activity 1U/µg. Final purification was done by HPLC. Molecular weight was determined by SDS-PAGE; 8-9 KDa. Amino acid analysis by PITC, amino acids as following : (Asp), (Asn), (Glu), (Gln), (Ser), (Gly), (His), (Arg), (Thr), (Ala), (Pro), (Tyr), (Val), (Met), (Cys), (Trp), (Ile), (Leu), (Phe) at concentration of: 23.81, 53.44, 19.78, 17.39, 16.82, 17.73, 15.92, 15.16, 45.67, 31.17, 29.78, 28.90, 18.39, 28.29, 27.85, 22.31, 17.81, 30.47, 24.53 µg/ml respectively. Antibacterial activity of microcin( 40 &80 µg/ml ) against 12 local isolates were examined. In conclusion, local clinical K15 produce microcin exhibit inhibitory action on other pathogenic bacteria.
Acuña L, Picariello G, Sesma F, Morero RD, Bellomio A. 2012. A new hybrid bacteriocin, Ent35-MccV, displays antimicrobial activity against pathogenic Gram-positive and Gram-negative bacteria. FEBS Open Biology 2, 12–19.
Al-Charrakh AH, Yousif SY, Al-Janabi HS. 2011. Antimicrobial spectrum of the action of bacteriocins from Klebsiella isolates from Hilla/Iraq. Iranian Journal of Microbiology 2(5), 76-78.
Al-Qassab AO, Al-Khafaji ZM. 1992. Effect of different conditions on inhibition activity of enteric lactobacilli against diarrhea-causing enteric bacteria. Journal of Agricultural Science 3(1), 18-26.
Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 7(72), 248-54.
CLSI. Clinical and Laboratory Standards Institute. 2016. Performance standard for antimicrobial susceptibility testing; Twenty-six informational supplement.
De Lorenzo V. 1984. Isolation and characterization of microcin E492 from Klebsiella pneumoniae. Archives of Microbiology 139, 72-75.
Duquesne S, Destoumieux-Garzo´n D, Peduzzi J, Rebuffat S. 2007. Microcins, gene-encoded antibacterial peptides from enterobacteria. Natural Product Report 24, 708–734.
Etayash H, Azmi S, Dangeti R. 2015. Peptide bacteriocins – structure activity relationships. Current Topics in Medicinal Chemistry 16, 220-241.
Gaillard-Gendron S, Vignon D, Cottenceau G, Graber M, Zorn N, Van Dorsselaer A, Pons AM. 2000. Isolation, purification and partial amino acid sequence of a highly hydrophobic new microcin named microcin L produced by Escherichia coli. FEMS Microbiology Letters 193, 95-98.
Garcia-Gutierrez E, Mayer MJ, Cotter PD, Narbad A. 2018. Gut microbiota as a source of novel antimicrobials. Gut Microbes 10(10), 1-21.
Gupta U, Radramma A, Rati ER, Joseph R. 1998. Nutritional quality of lactic acid fermented bitter. International Journal of Food Science and Nutrition 49(2), 101-108.
Hancock WS, Sparraw JT. 1981. Use of mixed- mode, high performance liquid chromatography for the separation of peptide and protein mixtures; Journal of Chromatography 206, 71-82.
Kolter R, Moreno F. 1992.Genetics of ribosomally synthesized peptide antibiotics. Annuls Reviews in Microbiology 46, 141–163.
Lafta A. 2010. Klebocin effect partially purified from bacteria Klebsiella pneumonia isolated locally on some intestinal bacteria and some immune cells. M.Sc. thesis. College of Science/ Al-Mustansiriyah University.
Lagos R, Wilkens M, Vergara C, Cecchi X, Monasterio O. 1993. Microcin E492 forms ion channels in phospholipid bilayer membranes. FEBS Lett. 321, 145-148.
Mayr-Harting A, Hedges AJ, Berkeley RCW. 1972. Methods for studying bacteriocins. In Methods in Microbiology ed. Norris, J.R. and Ribbons, D.W., p 315–342. New York: Academic Press.
McIntosh J, Donia MS, Schmidt EW. 2009. Ribosomal peptide natural products: bridging the ribosomal and nonribosomal worlds. Natural Product Report 26, 537–559.
Ozdemir GB, Oryasin E, Biyik HH, Ozteber M, Bozdogan B. 2011. Phenotypic and genotypic characterization of bacteriocins in enterococcal isolates of different sources. Indian Journal of Microbiology 51(2), 182–187.
Rebuffat S. 2013. Handbook of Biologically Active Peptides: Bacterial/Antibiotic Peptides. Elsevier Inc 129-137.
Sassone-Corsi M, Nuccio SP, Liu H, Hernandez D, Vu CT, Takahashi AA, Edwards RA, Raffatellu M. 2016. Microcins mediate competition among Enterobacteriaceae in the inflamed gut Nature. International Journal of Science 540, 280–283.
Severinov K, Semenova E, Kazakov A, Kazakov T, Gelfand MS. 2007. Low-molecular-weight post-translationally modified microcins. Molecular Microbiology 65, 1380–1394.
Aqeela Abdul-Zahraa, Ikbal Khudhur Al-joofy, Intesar Nadhum Khelkal, 2019. Characterization and antibacterial activity of purified microcin produced by Klebsiella pneumoniae K15. Int. J. Biosci., 14(1), 146-155.
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