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Article
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
Biofilm Shows Independency form Hemolysin Genes Arsenal in Methicillin Resistant Staphylococcus Aureus

Authors: Nihad Taha Mohammed Jaddoa --- Harith Jabbar Fahad Al-Mathkhury
Journal: Iraqi Journal of Science المجلة العراقية للعلوم ISSN: 00672904/23121637 Year: 2018 Volume: 59 Issue: 4C Pages: 2184-2194
Publisher: Baghdad University جامعة بغداد

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Abstract

Normally, bacteria exposed to antibiotics at sub minimal inhibitory concentrations (MIC) inside the host. Therefore, the current study aimed to comprehend the association among hemolysins, biofilm, as well as gentamicin resistance in local MRSA isolates. Around 35 Staphylococcus aureus locally isolated from different clinical specimens were employed in this study. Methicillin resistance was detected via cefoxitin disk diffusion and mecA amplification methods. MIC of gentamicin was estimated by broth microdilution method. Hemolysin genes involving hla, hlb, hld, and hlg were determined using multiplex polymerase chain reaction (PCR) technique. Microtiter plate method was employed for biofilm assessment in the presence and absence of gentamicin at sub MIC. Moreover, atomic force microscopy technique was employed for confirming the effect of gentamicin on biofilm. The present findings revealed that methicillin resistant S. aureus (MRSA) constituted, nearly, 94.29% (33 isolates) of all S. aureus isolates. Around 12 (36.36%), four (12.12%), and 17 (51.51%) isolates were gentamicin-sensitive, intermediate, and resistant to gentamicin, respectively. hla and hld were located in 32 out 33 MRSA isolates. All MRSA isolates succeeded in forming biofilm; however, three (0.09%), 23 (69.69%), and seven (21.21%) isolates formed weak,moderate, andstrong biofilm, respectively. Gentamicin at sub MIC reduced the intensity of biofilm and the AFM confirmed this finding. In conclusion, very weak correlation linked the biofilm formation capacity and isolate MIC. On the other hand, possession of hemolysin genes seems has no correlation with biofilm formation. Nevertheless, gentamicin at sub MIC reduced the intensity of MRSA biofilm.

Keywords

MRSA --- hemolysin --- biofilm --- gentamicin


Article
Molecular Study of Aeromonasspp. Isolated from Clinical and Environmental Samples

Author: Safaa M. Salman
Journal: journal of al-qadisiyah for pure science(quarterly) مجلة القادسية للعلوم الصرفة (فصلية). ISSN: 19972490 Year: 2015 Volume: 2 Issue: 20 Pages: 45-55
Publisher: Al-Qadisiyah University جامعة القادسية

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Abstract

Receved :7/4/2015 Accepted : 9/6/2015 and Azhar N. HussienAL-Qadisiya University / Collegeof Education / Biology DepartmentE-Mails: AbstractA total of 259 specimen from different clinical sources from patients of AL-Diwanyia'shospitals and environmental sources ( water) were collected. Biochemical and morphologicalcharacterization tests besides the use of API 20 E and Vitek System and Polymerase ChainReaction (PCR)Technique which used 16S rDNA geneshowed that seventeen isolates wereidentified as Aeromonas spp. These Isolates distributed as : 8 isolates belong to A. hydrophila, 4 isolates belong to A. sobria , 4 isolates belong to A. caviae and one isolate belong to A.salmonicida . PCR was used for detecting virulence factors such as exotoxin genes :hemolysin (hly A) , aerolysin(aer A) and cytolytic enterotoxin (aer) , results showed that77.7% and 62.5% of environmental and clinical isolates had hly A while 44.4 % and 50 %542015 ﺔﻨﺳ 2 ﺩﺪﻌﻟﺍ 20 ﺪﻠﺠﻤﻟﺍ ﺔﻓﺮﺼﻟﺍ ﻡﻮﻠﻌﻠﻟ ﺔﻴﺳﺩﺎﻘﻟﺍ ﺔﻠﺠﻣhad aer A , aer gene just found in clinical isolates in rate 25% while didn't found inenvironmental isolates .The same technique (PCR)was also used for detecting resistancegenes of beta - lactam antibiotic : blaCMY , blaTEM , blaCTX-M , and blaSHV genes which used inthis study . The results showed that 11.76% had blaCMYgene , 5.88% had blaTEM gene and17.64% had blaCTX-M gene of all isolates , while blaSHV gene didn't found in all the types ofisolates which isolated from clinical and environmental sources .


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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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
GROWTH OF STAPHYLOCOCCUS AUREUS AND PRODUCTION OF HEMOLYSIN IN DIFFERENT PLANT AND ANIMAL WASTES MEDIA
نمو بكتريا Staphylococcus aureusوانتاج حال الدم (hemolysin) في أوساط من مخلفات نباتية وحيوانية مختلفة

Author: شذى سلمان حسن
Journal: Iraqi Journal of Biotechnology المجلة العراقية للتقانات الحياتية ISSN: 18154794 Year: 2008 Volume: 7 Issue: 2 Pages: 157-167
Publisher: Baghdad University جامعة بغداد

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Abstract

Four different media prepared from plant and animal wastes included rice bran, sunflower meal, potato peels and chicken bones, further more Brain Heart Infusion was used as commercial medium. The media was prepared as solid media (with agar and blood) and liquid media in two concentrations 5% and 10%.The media were cultured with S. aureus, growth and hemolysin were detected in solid media and quantitatively estimated in liquid media. S. aureus grew and produced hemolysin in all these media. The highest growth was observed in 5% and 10% rice bran. Hemolysin activity in 5% rice bran was more than in other media, followed by sunflower meal, ckicken bones and potato peels respectively. The bacterial growth in 10% concentration was more than in 5% in most media, while hemolysin activity was nearly equal in the two concentrations, except in rice bran in which hemolysin in 10% was more than in 5%. These results indicated that the plant and animal wastes used are suitable for bacterial growth and hemolysin production, rice bran is the best one for this purpose.

استعملت أربعة أوساط محضرة من مخلفات نباتية وحيوانية وهي نخالة الرز وكسبة زهرة الشمس وقشور البطاطا وعظام الدجاج فضلاً عن الوسط التجاري نقيع القلب والدماغ Brain Heart Infusion لتنمية Staphylococcus aureus وإنتاج حال الدم ( الهيمولايسن ). حضرت الأوساط بشكلين صلب (مضافاً له الاكار والدم) وسائل بتركيزين 5% و10%. زرعت البكتريا في هذه الأوساط ولوحظت كثافة النمو ومناطق تحلل الدم في الوسط الصلب، وتم قياس النمو وفعالية حال الدم في الأوساط السائلة. نمت البكتريا في هذه الأوساط وأنتجت حال الدم، وقد أعطى الوسط المحضر من نخالة الرز أعلى نمو للبكتريا بتركيزيه 5% و10% كما أعطى أعلى فعالية لحال الدم بتركيز 10% تلاه وسط كسبة زهرة الشمس ثم مستخلص عظام الدجاج وأخيراً الوسط المحضر من قشور البطاطا، وكان النمو في الأوساط المحضرة بتركيز 10% أعلى مما عليه بتركيز 5% في غالبيتها، أما حال الدم فكانت فعاليته متقاربة في التركيزين في معظم الأوساط إلاّ أن فعاليته كانت أعلى في تركيز 10% نخالة الرز مما عليه في تركيز 5% . يتضح من هذه النتائج أن المخلفات النباتية والحيوانية المستعملة في هذه الدراسة ملائمة لتنمية البكتريا وإنتاج حال الدم عموماً، وأن وسط نخالة الرز هو الأفضل لهذا الغرض ويمكن أن تستعمل مثل هذه الأوساط بديلاً عن الأوساط التجارية المكلفة مما يحقق فائدة عملية وإقتصادية.


Article
Molecular detection of Hemolycin in Escherichia coli and attempt to inhibition by using the Probiotics
الكشف الجزيئي عن الهيمولايسين في بكترياE.coli ومحاولة التثبيط بأستخدام المعززات الحيوية

Author: Halah A. A. Al-Hadithi هالة عبد الخالق عوض الحديثي
Journal: Tikrit Journal of Pure Science مجلة تكريت للعلوم الصرفة ISSN: 18131662 Year: 2018 Volume: 23 Issue: 6 Pages: 79-90
Publisher: Tikrit University جامعة تكريت

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The current study included of (60) urine samples were collected from patients suffering from urinary tract infection in Tikrit Hospital during the period from April 2017 to June 2017 The results of identification showed that among (49) at percentage (81.6 %) positive samples were found (24) of samples with percentage (48.97 %) were Escherichia coli isolates . The susceptibility of isolates to avariety of antibiotics has been investigated, the results revealed that the isolates were (100%) resistant to (Erythromycin and Nalidixic acid), also these isolates have the highest percentage of resistant to Ceftriaxone (91.6%), Cephalothin (95.8%), Ampicillin sulbactam (91.6%), Nitrofurantoin (87.5%), where as isolates have shown lowest percentage of resistant to Chloramphenicol (12.5), Trimethoprime (20.83%). antagonistic activity of four types of probiotics bacteria against (24) isolates Escheriachia coli were performed. Results showed that all probiotics types have inhibition activity against E.coli isolates and the diameter of inhibition zone was highest (20.8 mm) when probiotic L.casei used ,while Bifido.bifidum showed less inhibitory activity against E.coli isolates with (14.5mm)of inhibition zoneIn this study the Polymerase chain reaction technique (PCR) were used for detection of hemolysin gene (hly A). Two primers ( hlyA) with 561 bp and (hlyA) with 1177 bp were used for this purpose. The results were shown that only (62.5%) and (50%) of 24 E.coli isolates were carried the genes hlyA (561bp), hly A (1177bp) respectively. Furthermore the investigate of molecular influence of probiotics on hemolysin genes were done by using PCR. The results showed some of them lost hly A gene after treating with probiotics. In conclusion, hemolysin toxin gene is important virulence factor for uropathogenic E.coli and using PCR technique was appeared highly specific,very sensitive method, more than it serves as asuitable molecular diagnostic tool for detection UPEC producing hemolysin toxin .

تضمنت الدراسة الحالية جمع (60) عينة ادرار من المرضى المصابين بألتهاب المجاري البولية في مستشفى تكريت وللفترة من شهر نيسان 2017م ولغاية شهر حزيران 2017 م، وقد أظهرت نتائج التشخيص أن من بين (49) عينة بنسبة (81.6%) وجدت عزلات بكتريا Escherichia coli في (24) عينة بنسبة (48.97%). وقد تم التحري عن حساسية العزلات تجاه أنواع مختلفة من المضادات الحيوية، وقد كشفت النتائج مقاومة العزلات بنسبة (100%) تجاه المضادين (Erythromycin , Nalidixic acid )، وقد امتلكت العزلات نسبة مقاومة عالية تجاه المضادات Ceftriaxone (91.6%), Cephalothin (95.8%), Ampicillin sulbactam (91.6%), Nitrofurantoin (87.5%). بينما أظهرت العزلات نسبة مقاومة واطئة تجاه Chloramphenicol (%12.5) ,Trimethoprime (20.83%).، كما تم أختبار الفعالية التضادية لأربعة أنواع من المعززات الحيوية تجاه (24) عزلة من بكتريا E.coli ، وقد أظهرت النتائج أمتلاك جميع المعززات الحيوية للفعالية التضادية تجاه عزلات E.coli ، وقد أعطى أعلى قطر تثبيط ((20.8 ملم عند أستخدام بكتريا Lactobacillus casei ، بينما أظهرت بكتريا Bifidobacterium . bifidum أنها أقل فعالية تثبيطية تجاه عزلات E.coli مع قطر تثبيط ( (14.5ملم. أستخدمت في هذه الدراسة تقنية الPCR للكشف عن جين الهيمولايسين hly A))، وقد أستخدم أثنين من هذه البادئات النوعية (hly A (561 bp، hly A ( 1177bp)، وقد أظهرت النتائج أن (62.5%) و ((50% من مجموع (24) عزلة من بكترياE.coli تمتلك جين hly A(561bp)، hly A (1177bp)بالتتابع. أضافة الى ذلك، تم التحري عن التأثير الجزيئي للمعززات الحيوية على جين الهيمولايسين بأستعمالPCR . وقد أظهرت النتائج أن بعض من هذه العزلات قد فقدت الجين hly A بعد المعاملة بالمعززات الحيوية، يستنتج من ذلك أن جين ذيفان الهيمولايسين هو عامل ضراوة مهم للمرض البولي E.coli وأن أستعمال تقنية الPCR تعد طريقة جدا حساسة وذات خصوصية عالية، وأكثر من ذلك أنها تعد أداة تشخيصية جزيئية مناسبة للكشف عن ذيفان الهيمولايسين الذي ينتجه الممرض البولي.


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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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
The Location of Aerobactin Determinants of Uropathogenic E. coli in Association with Hemolysin Production Antibiotic Resistance and Patient Characteristics.

Author: Ibtesam Ghadban Auda
Journal: Iraqi Academic Scientific Journal المجلة العراقية للاختصاصات الطبية ISSN: 16088360 Year: 2006 Volume: 5 Issue: 2 Pages: 223-229
Publisher: The Iraqi Borad for Medical Specialization المجلس العراقي للاختصاصات الطبية

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ABSTRACT:BACKGROUND:Aerobactin, Hemolysin and the resistance to some antibiotics axe important Features ofUropathogenic E. coli (UPEC). The characteristics of patients from which we isolate theUPEC have associate with these Features and their determinants location.AIM:We determined the phenotypic expression and gene location of Aerobactin and phenotypicexpression of Hemolysin among 60 UPEC isolates. We correlated the presence of Aerobactin systemwith antibacterial agents resistance. The expression of Hemolysin and the characteristics of patients.METHODS:Two methods of plasmid curin, sodium dodecyl sulfate and elevated temperature plasmid curing, areused then plasmid extraction and agarose gel electrophoresis are performed to determine thelocation of Aerobactin determinants and the size of Aerobactin plasmids, as well as the location ofthe determinants of some antibiotics resistance which were ampicillin (Am), Tetracycline (Tc),gentamicin (GM), Chloramphenicol (C) and co- trimazole (Co).RESULTS:Aerobactin and hemolysin expression among UPEC isolates were 93.3% and 35% respectively. Theisolates of non compromised patients produce statistically higher rate of expressed hemolysin (90.5%, p<0.01). Plasmid- borne Aerobactin was absent in UPEC isolates of non compromised patients(89.5%, p<0.01). On the contrary compromised patient isolates express plasmid Aerobactin of 59.5%(p<0.02). We also found that Aerobactin determinants are located on a plasmid in compromisedpatient isolates and associated with the absence of chromosomal Hemolysin production (82.9%,p<0.01). Yet, The chromosomal aerobactin is associated with hemolysin production (61.9%,p<0.02). Furthermore, UPEC isolates of compromised patients carry relatively large plasmid ofAerobactin (85.7%. p<0.05) and these large plasmids carry either chloramphenicol (83.3%.p<0.02) orgentamicin determinants (100%. P<0.01) but not co- trimazole, tetracycline or ampicillin.CONCLUSION:The isolates of non compromised patients carry chromosomal Aerobactin and hemolysin. While theisolates of compromised patients express plasmid Aerobactin and do not express chromosomalhemolysin. Aeobactin plasmids are relatively large plasmids and carry either chloramphenicol orgentamicin resistance determinants.


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.


Article
DETECTION OF ENTEROCIN PRODUCTION FROM LOCAL ISOLATES OF ENTEROCOCCUS FAECALIS AND RELATIONSHIP WITH SOME VIRULENCE FACTORS AND RESISTANCE TO ANTIBIOTICS
التحري عن إنتاج الانتروسين من العزلات المحلية لبكتريا Enterococcus faecalis وعلاقتـه ببعض عوامـل الضراوة والمقاومة للمضادات الحيوية

Author: نهى جوزيف قندلا1
Journal: Iraqi Journal of Biotechnology المجلة العراقية للتقانات الحياتية ISSN: 18154794 Year: 2010 Volume: 9 Issue: 3 Pages: 642-656
Publisher: Baghdad University جامعة بغداد

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Abstract

A total of 290 clinical specimens were collected from various hospitals in Baghdad from patient suffering from Urinary tract infection ,wound ,vaginal inflammation and (80) stool samples collected from healthy individuals. The results showed that 50 isolates belonged to Enterococcus faecalis according to the cultural, microscopically, biochemical examination and monovalent test (agglutination test) of which, 23 isolates (46%) from stool samples,27 (54%) isolates from clinical cases distributed between 16(32%) urine samples, 6(12%) wound swabs and 5(10%) vaginal swabs. The cup agar method considered as most efficiency methods that use in this study to detection the ability of E.faecalis to produce enterocin. The results showed that 56% of the isolates produced enterocin while 44% of isolates did not show any inhibitory action. The isolates showed multi resistance to many antibiotics, but Augmentin and Nitrofurantion were found to be the most effective agents against the isolates, while others were variable in their sensitivity, and only 6 isolates (12%) from clinical sources showed ability to produce B- lactamase Detection of some virulence factors in local isolates of E. faecalis such as Hemolysin , Gelatinase, cytolysin (production hemolysin and enterocin), showed that E.faecalis isolates possessed 14% hemolysin, 8% from clinical sources, and 6% of this isolates showed ability to produce enterocin (cytolysin), 6% of normal flora isolates produced hemolycin was distributed between 4% isolates produce cytolycin( hemolycin and enterocin), and all isolates of E. faecalis did not show ability to produce Gelatinase

جمعت 290 عينة من مستشفيات مختلفة في بغداد من مرضى يعانون من التهابات السبيل البولي والجروح والتهابات المهبل و80 عينة من براز أشخاص أصحاء (نبيت طبيعي ). شخصت 50 عزلة عائدة الى بكتريا المكورات المعوية البرازيــة Enterococcus faecalis إعتماداً على الفحوصات المجهرية والزرعية والكيموحيوية والمصلية، تضمنت 23 عزلة(46%) من عينات البراز و27 عزلة(54%)من حالات سريرية توزعت بين 16 عزلة(32%) من عينات الادرار و6 عزلات(12%) من مسحات الجروح و5 عزلات (10%) من عينات المهبل. عدت طريقة أقراص آلاكار Cup agar من أكفا ألطرائق المستعملة في التحري عن قابلية العزلات المحلية من بكتريا E. faecalis على إنتاج الانتروسين، وقد أظهرت النتائج إن 56 % من العزلات لها القابلية على الانتاج، بينما كانت نسبة العزلات غير المنتجة 44%. أظهرت العزلات مقاومة متعددة تجاه المضادات الحيوية, وتأثرت أغلب العزلات بمضادي Augmentin و Nitrofurantion في حين تباينت في حساسيتها لبقية المضادات المستعملة، وكانت 12% (6عزلات) من هذه العزلات منتجة لانزيمات البيتالاكتاميز جميعها عائدة أخذت من المصادر السريرية. تبين عند التحري عن قابلية العزلات المحلية من بكتريا E.faecalis على إنتاج بعض عوامل الضراوة مثل الهيمولايسين والجيلاتينيز والسايتولايسين ( الهيمولايسين والانتروسين) إن 14% من العزلات كانت منتجة لانزيم الهيمولايسين (الأنزيم الحال للدم ) الشكلت 8% من عزلات المصادر السريرية، وأبدت 6% من هذه العزلات قدرة على إنتاج الانتروسين (السايتولايسين). وقد بلغت نسبة عـزلات النبيت الطبيعي المنتجة للهيمولايسين 6% توزعت بين 4% منتجـة للسايتولايسيـن( الهيمولايسين والانتروسين ) و2%غير منتجة له. ولم تظهر أي عزلة من العزلات المعزولة قدرة على إنتاج أنزيم الجيلاتينيز.

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