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Study the effect of the toxin extracted from the E.coli bacteria in the pathogenesis and virulence of E.coli
دراسة تأثير الذيفان المستخلص من بكتريا E.coli في إمراضية وضراوة بكتريا E.coli

Authors: ذكرى عدنان جواد --- وقار عدنان حمدان
Journal: journal of kerbala university مجلة جامعة كربلاء ISSN: 18130410 Year: 2016 Volume: 14 Issue: 2 Pages: 217-227
Publisher: Kerbala University جامعة كربلاء

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Abstract

A local isolation of E.coli bacteria used in these study, and The toxin (Hemolysin) were extracted from it after their culture in the Chemical Defined Media (CDM). Hemolysin extraction was done and partialy purification applied, by using ammonium sulfate. It was found that filtrate contained hemolytic activity at a level higher than that before purification and was the highest dilution gave the hemolytic activity on blood cells 1/32 (320 units / ml)before the partial purification, but after partial purification found that dilution (1/64 ) caused degradation of 50% of the blood cells compared to the standard curve of sodium chloride, LD50 of the mouse for the toxin was calculated and the effect of LD50 of the toxin on the value of LD50 of the bacteria E.coli (non hemolytic) isolated from the stool, The results of the research confirmed role of the toxin on the pathogenesis and virulence of E.coli bacteria through lowering LD50 of this bacteria when injected in the mice with LD50 of the toxin (1.56 micrograms / mL) from (3.16 × 107 cells / mL) to (2.3 × 106 cells / mL).The injection of different concentrations of the toxin in mice effect on the viability of the mice where the concentration of the toxin that gave hemolytic activity (100, 90%) gave the percentage loss of 100%, while the concentration of the toxin that gave hemolytic activity 50% cause death of of 50% of the mice. Also injection of concentrations different from the toxin, the concentrations that gave hemolytic activity (100,90,70,50,25%), with the lethal dose of the E.coli bacteria is not producing toxin led to increase the proportion of the loss of the mice.

إستخدمت في هذه الدراسة عزلة محلية من بكتريا E.coli ، و تم إستخلاص الذيفان حال الدم (Hemolysin) منها بعد تنميتها في الوسط المعرف كيميائيا CDM) ) Chemical Defined Media وتم تنقيته جزئيا بالترسيب بكبريتات الأمونيوم وكان أعلى تخفيف أعطى تحلل لكريات الدم (للراشح) 1/32 ( 320 وحدة/ مليلتر) ، أما بعد التنقية الجزئية وجد أن التخفيف (1/ 64( سبب تحلل 50% من كريات الدم مقارنة مع المنحنى القياسي لكلوريد الصوديوم ، وتم حساب الجرعة المهلكة لنصف الفئران من الذيفان حال الدم وكذلك تأثير الجرعة المهلكة للنصف من الذيفان على قيمة الجرعة المهلكة للنصف من بكتريا E.coli غير منتجة للذيفان حال الدم معزولة من البراز وعلى ضراوتها ، أكدت نتائج البحث الدور الذي يلعبه الذيفان حال الدم في إمراضية وضراوة بكتريا E.coli من خلال إنخفاض الجرعة القاتلة للنصف (LD50) لهذه البكتريا عند حقنهـــــــا مع التركيز القاتــــــــــل لنصف الفئـــران من الذيفان (1.56 مايكروغرام / مليلتر) من (3.16× 107 خلية/ مليلتر) إلى ( 2.3 × 106 خلية / مليلتر) . كما إن لحقن تراكيز مختلفة من الذيفان حال الدم تأثير على عيوشة الفئران حيث إن تركيز الذيفان الذي أعطى فعالية تحـلل ( 100 و 90 ) % أعطى نسبة هلاك 100% بينما التركيز الذي أعطى فعالية تحلل 50% سبب هلاك 50% من الفئران كما إن حقن الفئران بتراكيز مختلفة من الذيفان ، التراكيز التي أعطت فعاليات تحلل ) 100،90،70،50،25) % ، مع الجرعة القاتلة للنصف لبكتريا E.coli غير منتجة للذيفان أدت إلى زيادة نسبة هلاك الفئران حيث إن الجرعة القاتلة للنصف مع التركيز الـ(100)% تسبب هلاك 100% من الفئران


Article
Extraction and studying the effect of pH and temperature on hemolysin production by a local isolates of Staphylococcus aureus

Authors: Mohammed I. Nadir --- Hussain S. Al-Hasani --- Amer H. Al-Shammary
Journal: Karbala Journal of Medicine مجلة كربلاء الطبية ISSN: 19905483 Year: 2012 Volume: 5 no 1 Issue: 11 Pages: 1283-1294
Publisher: Kerbala University جامعة كربلاء

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Abstract

background: Staphylococcus aureus is a ubiquitous bacterium that is generating increasingly bad press coverage due to its propensity to adopt a pathogenic lifestyle in hospital and community settings. S. aureus colonies are found in approximately 30% of the general population. It colonizes the skin readily and can lead to a wide range of pathological conditions from skin lesions to osteomyelitis, endocarditis, and septicemia. Many bacteria produce substances that are cytolysins i.e they dissolve red blood cells (hemolysins) or kill tissue cells or leukocytes (leukocidins). The β-toxin degrades sphingomyelin and therefore is toxic for many kinds of cells, including human red blood cells. Hemolysins were extracted and the optimum conditions for their production were extensively studied including optimum pH, temperature, incubation period, and various manipulations of the culture media.Objectives1-Extraction of hemolysin from a local isolate of S. aureus.2-Studying the effects of pH and temperature on hemolysin production.Methods : Bacterial samples were identified by subjecting them to the standard laboratory procedures while semi quantitative screening on blood agar (containing 5% human blood) revealed that all isolates were hemolysin producer but in different efficiencies. Determination of the optimal conditions for hemolysin production including the optimum pH and temperature were also performed.Results : Bacterial samples were identified by subjecting them to the standard laboratory procedures and the results showed that forty isolates out of the total of 100 were identified as Staphylococcus aureus. Semi quantitative screening on blood agar (containing 5% human blood) revealed that all isolates were hemolysin producer but in different efficiencies. Depending on the semi-quantitative screening and hemolytic assays isolate SW-14 ofStaphylococcus aureus was the higher hemolysin producing isolate. Determination of the optimal conditions for hemolysin production including the optimum pH and temperature were performed, the results demonstrated that the best hemolysin production was in the pH near neutrality (pH 7-7.5) and in temperature of 35-40oC.Conclusions1.Conventional methods can be performed to extract hemolysins.2.Hemolysin was maximally produced when the pH was near neutrality and incubation temperature was 37oC and this conclusion indicates that hemolysin was produced when the conditions were similar to that of the host.


Article
Purification and characterization of hemolysin produced by a local isolates of Staphylococcus aureus

Authors: Mohammed I. Nadir --- Hussain S. Al-Hassani --- Amer H. Al-Shammary
Journal: Karbala Journal of Medicine مجلة كربلاء الطبية ISSN: 19905483 Year: 2012 Volume: 5 no 2 Issue: 12 Pages: 1455-1463
Publisher: Kerbala University جامعة كربلاء

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Abstract

background: Staphylococcus aureus is a ubiquitous bacterium that is generating increasingly bad press coverage due to its propensity to adopt a pathogenic lifestyle in hospital and community settings. S. aureus colonies are found in approximately 30% of the general population. It colonizes the skin readily and can lead to a wide range of pathological conditions from skin lesions to osteomyelitis, endocarditis, and septicemia. Hemolysins are extracellular toxic proteins which are produced by many gram negative (e.g. Escherichia coli, Serratia spp., Proteus spp., Vibrio spp., Pasteurella spp., Pseudomonas aeruginosa) and gram positive bacteria (e.g. Streptococcus spp., Staphylococcus aureus, Listeria spp., Bacillus cerius, Clostridium tetani), all of which possess a certain pathogenic potential. Hemolysins have been therefore always considered as virulence factors. Most hemolysins cause lysis of erythrocytes by forming pores of varying diameters in the membrane and are designated as such because they have the ability to lyse red blood cells (RBCs). Objectives1-Purification of hemolysin from a local isolate of S. aureus.2-Characterization of hemolysin produced by a local isolate of S. aureus.Methods: Bacterial samples were identified by subjecting them to the standard laboratory procedures while semi quantitative screening on blood agar (containing 5% human blood) revealed that all isolates were hemolysin producer but in different efficiencies. Hemolysin was extracted by cooling centrifugation and purified by many steps including: precipitation by ammonium sulphate, dialysis, ionic exchange chromatography by using DEAE-Cellulose, and gel filtration chromatography by using Sephadex G-100. The molecular weight of hemolysin was determined by gel filtration chromatography on Sephadex G-100 while the optimum pH and temperature for hemolysin stability were also determined.Results: The results showed that forty isolates out of 100 were identified as Staphylococcus aureus. Hemolysin was extracted by cooling centrifugation and purified by many steps including: precipitation by ammonium sulphate with 50-75% saturation percentage, dialysis, ionic exchange chromatography by using DEAE-Cellulose, and gel filtration chromatography by using Sephadex G-100. The results showed that hemolysin was purified 135 fold with a yield of 1.16%.The molecular weight of hemolysin determined by gel filtration chromatography on Sephadex G-100 column was about 35000 daltons, while the optimum pH for enzyme stability was 7 and the optimum temperature for enzyme stability was between 25-35oC.Conclusions1.Conventional methods can be performed to extract hemolysins.2.Hemolysin was maximally produced when the pH was near neutrality and incubation temperature was 37oC and this conclusion indicates that hemolysin was produced when the conditions were similar to that of the host.

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