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دراسة جزيئية وبكتريولوجية لبكتريا Porphyromonas gingivalis المعزوله من المرضى المصابين بامراض اللثه == Molecular and Bacteriological Study of Porphyromonas gingivalis Isolated from Patients with Periodontitis

Author name: فاطمة مالك عبود

Supervisor name: محمد صبري عبد الرزاق السعيد | الهام عباس بنيان

General topic: Medicine

Specific topic: Microbiology

Degree: Doctorate

University: University of Babylon - Faculty Of Medicine

Language: English

University location: Babylon

First pages:

Abstract: تم جمع 103عينة من الصفيحة الجرثومية العميقة (subgingival dental plaque) من المرضى الذين يعانون من التهاب اللثة المزمن والمتقدم (72, 31) على التوالي لمراجي العيادة الاستشارية لامراض اللثة في كلية طب الاسنان/ جامعة بابل والمركز التخصصي لطب الاسنان في مدينة الحلة. للفترة من كانون الاول 2013 الى حزيران 2014. تم تشخيص وعزل بكتريا P. gingivalis بطرق مختلفة منها استخدام الطرق البكتيرية التقليدية واظهرت النتائج ان 69.5%) 16) منها تم عزلها من التهاب اللثة المزمن وتم عزل 7 (30.5) من التهاب اللثة المتقدم باستخدام التنمية على الوسط الانتقائي الخاص لهذه البكتريا. وكذلك تم استخدام طريقة التشخيص الجزيئي باستعمال المعلم الوراثي المستند على دور جين 16s rRNA حيث اظهر ان12 عزلة من اصل 23 عزلة تم تشخيصها بشكل نهائي انها تعود لبكتريا P. gingivalis. منها حوالي 8 تعود الى التهاب اللثة المزمن و4 تعود الى التهاب اللثة المتقدم. بالاضافة الى ذلك, تم التحري عن قابلية هذه البكتريا للالتصاق(adherence ability) بالخلايا الطلائية المبطنة للفم حيث وجد ان جميع العزلات لها القابلية على الالتصاق بهذه الخلايا وتم ايضا اختبار قابليتها على انتاج انزيم (Gingpain) ووجد ان جميع العزلات منتجة الى هذا الانزيم.في هذه الدراسة تم التحري المظهري عن عملية تكوين الاغشية الحيوية(Biofilm formation) للعزلات المدروسة باستخدام فحص tissue culture plate test (TCP) اذ كانت مكونة للاغشية الحيوية بنسبة 100% وكذلك تم فحص تاثير بعض المواد المستخدمة في طب الاسنان على انتاج biofilm ووجد ان اعلى تثبيط سجل للكلورهكسدين (غسول الفم) والشب (Alum)ويليه تاثير Clove مقارنة باقل تاثير لمادة السعد ((Cyprus rotundus. وكذلك تم الكشف عن حساسية العزلات لبعض المضادات الحيوية ووجد بان كل العزلات كانت حساسة الى Co - amoxiclave وعلى العكس اغلب العزلات تظهر مقاومة عالية لل Amoxicillin بينما سجلت مقاومة قليلة جدا لل Ampicillinو Metronidazole(16.3%, 20.3%, ) على التوالي. واخيرا تم فحص تاثير بعض المواد المستخدمة في طب الاسنان على تثبيط نمو هذه البكتريا وتوضح النتائج ان اعلى تاثير سجل للكلورهكسدين (غسول الفم) والشب (Alum)ويليه تاثير القرنفل(Clove) mm) 25,30,40) على التوالي مقارنة باقل تاثير لمادة السعدC. rotundus (10mm) .في هذه الدراسة ايضا تم التحري عن التغاير الجيني ((TLR - 4 Polymorphisms باستخدام تقنية البلمرة والقطع بالانزيم ((PCR - RFLP. وتبين من النتائج وجود ثلاث انماط جينية في موقع طفرة Thr399Ile : CC وCT وTT ، اذ بلغت نسبة انتشارها ((12.5%,10%,77.5% في مجموعة المرضى على التوالي بينما هناك فقط نمطين في مجموعة السيطرة TT,CCوبنسبة انتشار(36.6% ,63.4%) على التوالي ، فيما بلغت نسبة انتشار الاليلC السائد في المرضى 82.5 % بالمقارنة للسيطرة 63.3% مع وجود اختلاف معنوي. وفي المقابل سجل وجود ثلاث انماط جينية في موقع طفرة Asp299Gly : AA وAG وGG وبنسبة انتشار % 67.5 و12.5% و20% في مجموعة المرضى على التوالي بينما هناك فقط نمطين في مجموعة السيطرة GG, AA وبنسبة انتشار34.4%) 66.6%,) على التوالي. حيث سجلت نسبة انتشار الاليل A السائد (73.75%) في مجموعة المرضى بالمقارنة بمجموعة السيطرة (66.6%) وبفرق معنوي. | In this study, 103 clinical samples were collected from subgingival dental plaque of patients with chronic (CP) and aggressive periodontitis (AP) (72 and 31) respectively, and (n=30) a healthy group admitted to teaching Hospital in college of Dentistry / Babylon University and specialized center of Dentistry in Hilla city, From February 2013 to June 2014). Out of which (n=40) with a chronic periodontitis group from them blood sample were collected for detection of gene polymorphisms in Toll - like receptor - 4 (TLR - 4). These dental plaque samples were subjected to different methods for identification of P. gingivalis mainly traditional bacteriological method. It was found that 23 P. gingivalis isolates were recovered by using selective media where 19 isolates (26.3%) obtained from (CP) and 4 isolates (12.5%) from (AP). Furthermore, molecular detection method was applied by using 16s rRNA gene as a genetic marker for confirmation of detection of P. gingivalis isolates, 12 isolates of P.gingivalis out of 23were detected by molecular method focusing on the role of 16s rRNA gene of P. gingivalis. 8 isolates were isolated from CP, and 4 isolates were isolated from AP subgingival plaques. In addition, these 12 isolates were investigated to detect Adherence of P.gingivalis to oral epithelial cells. The result showed that all isolates have ability to adhere to oral epithelial cells. The study also detected the ability of this bacteria for production of Gingipain, it was found that all isolates were positive for this enzyme at a rate 100%.Furthermore, biofilm formation was tested in the semi quantitative microtiter plate test. The results revealed that all isolates were biofilm former, high and moderate biofilm formation mode were accounted for (50%) while there are no isolates that express non biofilm formation.Regarding to effect of Chlorhexidine (CHX) gluconate (0.12%) on biofilm formation, the results of this study demonstrated (CHX) effectively and reduced the viability of biofilm - forming bacteria from moderate to 25%, weak to 75% and no strong. The study was also evaluating the effect of some plant extracts on biofilm formation. The results showed the highest effect for Alum potassium phosphate followed by Clove, in contrast to lowest effect of C. rotundus. And after screening the effect of Flagyl solution on production of bacterial pigment. The results showed that the metronidazole produced the highest inhibition activity range (30 - 10mm). Furthermore, the susceptibility of isolates to a variety of antibiotics had been investigated and it had been found that all isolates were sensitive to co - amoxiclave and, in contrast, the isolates showed high resistance to other type of beta lactam such as amoxicillin (54.3%). In addition, the result showed that the resistance of p. gingivalis was very low for ampicillin and metronidazole (16.3% and 20.3%), respectively.The screening of antimicrobial activity of (CHX) gluconate (0.12%) was carried out and the results showed that it produced the highest inhibition activity whether against p. gingivalis with inhibition zone range 40 - 15mm. Finally in vitro antibacterial activity of Alum, Clove and Cyperus rotundus plant extracts was studied and the results revealed that all tested isolates were inhibited by aqueous extracts at 50% concentration. The maximum inhibition zone was observed in Alum and Clove extracts respectively (30mm, 25mm), compared to the minimum inhibition by Cyperus rotundus (10mm). The antibacterial actions of 20% concentration of plant extract gave lower inhibition zone than 50% concentration of that extract which represented by (19mm, 16mm, and 10 mm) of the previous three mentioned extracts. This study also aimed to investigate the association of Thr399Ile and Asp299Gly single nucleotide polymorphisms (SNPs) in the (TLR - 4) with the incidence of (CP).The results revealed at the site of SNPThr399Ile, there were three genotypes for this SNP among CP patients; CC, CT and TT with frequency of 77.5%, 10% and 12.5 % respectively, whereas, there were only two genotypes among control group; CC and TT with frequency of 63.4% and 36.6% respectively with no significance differences between patients and control. However, the frequency of allele C (dominant) was higher among CP group (82.5%) than control (63.3) with significant difference. The site of SNP of Asp299Gly also showed three genotypes among CP patients; AA, AG and GG with frequency of 67.5%, 12.5% and 20% respectively, whereas, there were only two genotypes among control group; AA and GG respectively with frequency of 66.6 and 34.4% respectively with no significance differences between patients and control. However, the frequency of allele A (dominant) was higher among CP group 73.75 % than control 66.6%.

Author name: ميسم محمد حسين الكعبي

Supervisor name: سامي رحيم الكاتب

General topic: Medicine

Specific topic: Physiology

Degree: Doctorate

University: University of Kufa - Faculty Of Medicine - Department Of Physiology And Medical Physics

Language: English

University location: Najaf

First pages:

Abstract: انخفاض الخصوبة مشكلة شائعة ولكن علاجها في بعض الاحيان غير كافي لعدم تشخيص الاسباب بالكامل. الحديد عنصر ناقل للاوكسجين ولذلك فهو محفز لحيوية كل الخلايا والانسجة في الجسم. نقص الحديد من اكثر مشاكل نقص الغذاء الشائعة في العالم. لذلك ربط الحديد مع الخصوبة يح | Subfertility is a common problem, but the treatment is sometimes inadequate because the causes are not fully diagnosed. Iron is essential for oxygen transport and therefore promotes health of all cells and tissues. Iron deficiency is the most common nutritional deficiency in the world. Therefore, the connection between iron and fertility need more explanation. Some researchers have suggested that iron may be important for ovulation as the egg has an increased demand for iron while it matures and women who do not get sufficient amounts of iron may suffer anovulation and possibly poor egg health, which can inhibit pregnancy at a rate 60% higher than those with sufficient iron stores in their blood. However women planning to become pregnant should test for serum ferritin level and accordingly if low they can use iron supplements because they may help them to prevent iron deficiency and also improve fertility. Vitamin C is essential for the uptake and absorption of iron. The objective of this study was to assess the iron status, vitamin C and some hormone levels in subfertile women. The sample of study consists of 106 women subdivided into two groups : first consist of 76 subfertile women were taken from the outpatient to fertility center in the gynecological and obstetrical teaching hospital in Kerbala city and from multiple gynecology/obstetrics privet clinics. Second consist of 30 fertile women (control and comparative) who obtained from outpatients and some relatives. The subfertile women subdivided into two subgroups according to the cause of subfertility in presented study : first group : ovarian dysfunction (OD) either due to polycystic ovary syndrom (PCOS) or due to other cause (OC) like anovulation, hyperprolactenemia, premature ovarian failure. Second group : unexplained (idiopathic). Also the subfertile women subdivided into two subgroups according to the type of subfertility : primary and secondary. All of them were at reproductive age (16 - 45) years and there BMI ranged between (20 - 45 kg/m2). The study was achieved throughout a period which extends from July 2012 - October 2013. The protocol of study summarized as following : At day 3±1of menstrual cycle (menstrual phase), all women underwent the measurement of concentrations for multiple hormones in the blood like luteinizing hormone, follicle stimulating hormone, estradiole, prolactine and Testosterone. In addition to iron status (serum iron, serum ferrtin and total iron binding capacity) and vitamin C also assessed. And at day 13 of menstrual cycle (near time of ovulation), all women underwent two investigations : first : the measurement of concentrations of luteinizing hormone, serum Leptin and antioxidants as vitamin C and glutathione. In addition to Iron status (serum iron, serum ferrtin and total iron binding capacity) and hemoglobin concentration also estimated. Second : estimation the size and the number of dominant follicles in both ovaries by ultrasonograghy. While at day 23±1 of menstrual cycle (secretary phase), all women underwent two investigations : first : estimation the concentration of serum progesterone to detect ovulation. Second : estimation the endometrial thickness by ultrasonograghy. The results of study shows : • A significant increase (P<0.05) in number of subfertile women with age group (16 - 25) years and there were highly significant decreases (P<0.001) in number of women with age 36 - 45 years for both primary and secondary subfertility. While no significant difference (p>0.05) in age group (26 - 35) years.• A significant increases (P<0.05) in body mass index for subfertile women due to polycystic ovary syndrome. Also a significant increase (P<0.05) in serum leptin for women with overweight and obese as compared with normal weight.• A significant increase (P<0.05) in serum ferritin level for polycystic ovary syndrome at day 13 of menstrual cycle. Also, a significant decreases (P<0.05) in number of polycystic ovary syndrome in those having serum ferritin lower than normal at day 13 of menstrual cycle.• A significant increase (P<0.05) in the number of subfertile women due to ovarian dysfunction other than polycystic ovary syndrome who having a lower serum level of vitamin C at day 3 of menstrual cycle. • A significant increase (P<0.05) in serum testosterone level in polycystic ovary syndrome group at day 3 of menstrual cycle. • A significant increase (P<0.05) in serum prolactine level in group of ovarian dysfunction due to other cause at day 3 of menstrual cycle. when serum ferritin level lower than or within normal value. • A significant decrease (P<0.05) in serum level of luteinizing hormone for group of ovarian dysfunction due to other cause at day 13 of menstrual cycle when serum ferritin less than normal but no significant differences (P>0.05) in serum luteinizing hormone when serum ferritin within normal value. • A significant decrease (P<0.05) in serum luteinizing hormone for group of ovarian dysfunction due to O.C when vitamin C within normal value at day 13 of menstrual cycle.• A highly significant decrease (p<0.001) in endometrial thickness for all subfertile group at day 23 of menstrual cycle.• No significant difference (P>0.05) in serum levels of follicle stimulating hormone, estradiole and glutathione as compared with other parameters in fertile and subfertile women.It was concluded that the decrease in serum ferritin and vitamin C levels in the blood may be associated directly or indirectly with subfertility caused by ovarian dysfunction.