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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
production of hemolysin enzyme from local isolated of vibrio cholerae bacteria isolated from diarrhea patients
Vibrio من العزلة المحلیة لبكتریا Hemolysin إنتاج إنزیم الھیمولایسین المعزولة من مرضى مصابین بالإسھال cholerae

Author: *محمد ابراھیم نادر
Journal: Diyala Journal For Pure Science مجلة ديالى للعلوم الصرفة ISSN: 83732222 25189255 Year: 2012 Volume: 8 Issue: 2 Pages: 18-31
Publisher: Diyala University جامعة ديالى

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Abstract

Diagnosed 26 isolate of vibrio cholera was isolated from clinical sources responsibleof causing diarrhea. All isolates showed high productivity of hemolysin by differentefficiency. Semi quantitative screening on a selective solid medium (blood ager additive 7%blood) showed that all isolates have the ability to produce hemolysin as indicated by theformation of clear zone around the colonies.The selected isolate characterized as Vibrio cholera AMK6 based on its highproduction of enzyme and used in the present study. The optimum conditions for hemolysinproduction by submerged cultures (brain heart infusion containing 3%Glycerol) with aninoculums size of 3x108 CFU at an initial pH of 8 with shaking incubation at 35oC, 150cycle/min for 24 hours

تم عزل تشخيص 26 عزل من ضمة الكوليرا من مصادر مسؤولة السريرية من تسبب الاسهال. وأظهرت جميع العزلات إنتاجية عالية من حالة دموية من قبل مختلف كفاءة. الكمية فحص نصف في المتوسط ​​صلبة انتقائي (الدم المضافة اجير 7٪ وأظهرت الدم) أن جميع العزلات لها القدرة على إنتاج حالة دموية كما يدل على ذلك تشكيل منطقة واضحة حول المستعمرات. العزلة تميزت مختارة مثل الكوليرا الضمة AMK6 على أساس ارتفاع لها انتاج الانزيم، واستخدمت في هذه الدراسة. الظروف المثلى لحالة دموية الإنتاج من الثقافات المغمورة (الدماغ ضخ القلب التي تحتوي على الجلسرين 3٪) مع اللقاح حجم 3x108 CFU في الأس الهيدروجيني الأولي لل8 مع اهتزاز عند 150 حضانة، 35oC دورة / دقيقة لمدة 24 ساعة


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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