اسم المؤلف:
ميس عماد احمد
اسم المشرف:
منى تركي موسى الموسوي
المستخلص:
One hundred fifty clinical samples (burns, wounds, urine, nasal and ear swabs) were collected from patients of Al - Yarmouk Hospital and Teaching Baghdad Hospital during the period from November / 2015 to January / 2016.Multi drug resistant strains are serious problems in public health worldwide. So, the continueous development of new classes of antimicrobial agents has become of increasing importance for medicine. Therefore was aimed to produce of Crude and purified bacteriocins from antibiotic resistant bacteria (G - ve MRSA and G+ve P. aeruginosa) isolated from clinical samples and determine the optimization conditions for production of bacteriocin and their purification and characterization. In addition to characterize their inhibitory activity in - vitro and in - vivo .Cultural and morphological characteristice examination ,biochemical tests were conducted and confirmed the diagnosis by API20E and Staph API and antibiotics sensitivity test confirmed by Vitek - 2 system.The results showed 102(68%) isolates of S.aureus, all of which were Methicillin Resistace S.aureus (MRSA), constituted 50% from the ratio in the burns and wounds samples, while the lowest 13(12.7%) isolates were from ear samples. Highly significant differences (P˂0.01) recorded in both isolates among clinical samples. The isolates showed multi - resistant to Methicillin, Penicillin G , Cefoxitin, Erythromycin, Oxacillin, Chloramphenicol and Tetracyclin, while sensitive to vancomycin.Also 77(51.3%) isolates were P.aeruginosa, 62(80.5%) from which were multi - resistant to antibiotics that constituted more than half (59.8% and 51.6%) from the ratio in burn and wound samples respectively, while the lowest 8(12.9%) isolates were from ear samples too. Highly significant differences (P˂0.01) recorded in both isolates among clinical samples. There were about completely resist to Cefoxitin 75(97.40 %) and also show a higher resist to Ceftazidime, Tetracycline and Cefepime, While less resistance to Azithromycin.Vitek 2 system gave confirmation of positive results for MRSA and resistant P.aeruginosa and as a selected organism with a probability 98 - 99%."II "The MRSA isolate S12 and P.aeruginosa isolate (P26) were chosen among five bacterial isolates as a good producer for crude MRSAcin and MAR - pyocin according to their widest inhibition zone .Five methods were used to detect of MRSAcin and MAR - pyocin production by (well diffusion assay WDA , flip - streak , spot on the lawn , Cup disc and paper disc method) against indicator pathogens (G+ve , G - ve bacteria and yeast) which included, Staphylococcus aureus, Escherichia coli and Candida albicans .Well diffusion assay was the best method for identify of bacteriocin production and antibacterial activity against indicator pathogens.The crude and purified MRSAcin and MAR - pyocin recorded non - significant differences in activity whether as alone or synergistic against pathogenic bacteria (G - ve and G+ve) and yeast compared with higher sensitive antibiotics.The optimum conditions for bacteriocins production were in trypticase soya broth and brain heart infusion broth supplemented with )Carbon ,nitrogen, Salts, surfactants and Vitamins) with pH 7, at temperature 37°C for 48 hrs. and inoculum size of 2% from bacterial culture 6×108 cell/ml."Purification of bacteriocin was made by two steps method including extraction by ammonium sulphate 70% followed by ion - exchange chromatography DEAE - cellulose that the specific activity for these fractions was 1.375and1.000 (AU/mg) with 6 and 5 purification folds and 78 and 29 % yield and gel filtration chromatography on using Sephadex G - 50 column. The resulted fractions were 1.646 and 1.160 (AU/mg) protein with 8 and 6 purification folds and 38 and 28% yield respectively."Physical characteristics were also studied and the results showed that the molecular weight of MRSAcin and MAR - pyocin was )15and 35) KDa respectively by Sodium Dodecyl Sulfate - polyacrylamide gel electrophoresis (SDS - PAGE). Purified bacteriocins were stable at wide range of pH values 1 - 8, and remained active (50%) at pH 9 .The whole activity was lost at the pH value 10 and thermostabile at 100°C for 30 min.A strong stability of bacteriocins were recorded against organic solvents 10% and surfactants (0.5% EDTA), mineral salts 0.5% of each (CuSo4 and FeSo4), (10%) proteolytic enzymes lipase recorded significantly different (P≤0.05) compared with other enzyme (trypsin, pepsin and papain) which show complete inactivation of antibacterial activity.Also crud bacteriocins are still active for at least 3 months of freezing storage, while in purified after 2 months of freezing storage. Showed that the minimum inhibition zone against E. coli was 62.5 μg/ml for both MRSAcin and R - pyocin in vitro. The effect of MRSAcin and MAR - pyocin on the cell permeability was studied and the results showed that the absorbance at 260nm increased, indicating that DNA leaked from cells due to the cell lysis.Experimental infection induced by therapeutic effectiveness of the bacteriocin invivo skin injury bacteria MRSA and resistance P. aeruginosa which was marked at the beginning of her appearance inflammation of skin, swelling and redness of the affected area with the appearance of festering spots and abscesses in the surface layer of the skin and with the progress of the abscess spotted Purple happens .The antibacterial action of synergistic affected of (MRSAcin and MAR - pyocin) extracts .The results confirmed that the treated invivo skin injury bacteria MRSA and resistance P. aeruginosa was damaged . MRSAcin , MAR - pyocin and synergistic were giving good healing process with limited tissue damage in vivo (mice) against superficial skin infection by MRSA and resistance P. aeruginosa isolates at 1,2,3 day after infection.
