التمنيع باضداد الخمل النوع الاول المنتجة من بكتريا الاشريكية القولونية المسببة لالتهاب المجاري البولية ضد استيطان واصابة المثانة == Immunization With Type 1 Fimbriae of Uropathogenic Escherichia Coli Against Colonization And Infection of Bladder
Author name:
اروى علي شكر
Supervisor name:
رسمية عبد ابو ريشة
General topic:
Biology
Specific topic:
Microbiology
Degree:
Master
University:
University of Baghdad
Language:
English
University location:
Baghdad
First pages:
24T2953 - p.pdf
Abstract:
Between September and November 2013 a total of 140 urine specimens obtained from patients in Al - Kadhmiah Teaching Hospital, Ibn - Al Naffees Teaching Hospital, and Educational Laboratories in Medical city. These midstream urine specimens were obtained from patients clinically suspected to have urinary tract infection (UTI) in sterilized containers. All specimens were processed immediately after collection.A total number of 140 specimens of mid - stream urine were collected from patients suffering urinary tract infection symptoms. These isolates were obtained from 41 male and 99 female patients. They were distributed in the age group of 5 - 60 years old. All specimens were identified using biochemical tests and API 20 E system. 60 (42.85%) of urine specimens was Escherichia coli and 15(10.71%) of specimens growth was Klebsiella spp. and 25(17.85%) growth was Proteus spp. and 40 (28.57%) was G+ve bacteria.The adherence ability of E.coli isolates were evaluated by using Congo red agar and detected their ability to produce biofilm by using methylene blue staining technique in polystyrene microtiter plates and then Optical density was determined at 580 nm.All sixty isolates of E.coli were grown on Congo red agar medium to detect their adherence ability. Twenty (33.3%) isolates were given negative result by forming pink colonies on congo red agar, were as, forty (66.6%) isolates were given positive result by forming black colonies with a dry crystalline. Three isolates give strong results E.coli (13, 8, and 40). Consistency indicated biofilm production by microtiter plate. E. coli 40 which isolated produced the thickest biofilm (O.D. : =3.17).Type 1 fimbriae expression by E.coli 40 isolate was detected by mannose - resistant hemagglutination (MRHA) of human blood group (O) IRBCs in the presence of 2% D - mannose. The isolate showed strong MRHA characteristics of type 1 fimbriae under microscope (40x). The E.coli E.40isolate which produced the thickest biofilm and gave strong MRHA was elected to be the source of type 1 fimbriae.Type 1 fimbriae was extracted by heating and mechanical shearing and partially purified by Ultracentrifugation (48, 000xg for 3hrs). SDS - PAGE with a molecular weight 20, 000 Dalton.Anti - type1 fimbriae antisera were prepared in rabbits. The specificity and titration of anti - type1fimbriae antibodies were determined using IgG protein by radial immunodiffusion plate and bacterial agglutination. IgG antibodies to fimbriae type 1 were also detected in rabbit sera from immunized rabbit and non - immunized. Immunized rabbit test 1 had increases in Fim1 - specific IgG antibodies (9.5) mg/dl. Immunized rabbit test 2 had increases in Fim1 - specific IgG antibodies (8.6) mg/dl. The non - immunized rabbit had no increase in absolute IgG antibodies to Fim1.The antisera agglutinated bacterial cells up to 1/80 dilution confirming the presence and titer of specific antibodies against type1 fimbriae.The microtiter plate method was applied to estimate the role of the prepared anti - type1fimbriae antibodies in reducing the biofilim formation by E.coli. Prevention of bacterial adherence and subsequent biofilm formation to polystyrene microtiter plate was studied by employing different dilutions (1/10, 1/20, 1/40, 1/80, 1/160, 1/320, 1/640) of rabbit sera containing anti - type 1 fimbriae antibodies. The maximum inhibition of biofilm formation in terms of optical density (580 nm) was found in lowest dilution (1/10) (highest concentration of Abs). However, the minimum inhibition was observed in highest dilution of rabbit antisera (1/640) (lowest concentration of Abs). Here the inhibition occurs in a dose dependent manner as the biofilm formation increased dramatically with increasing in antisera dilution. The results also showed that there is a significant difference(P<0.05) among data treated with sera and data without sera and among serial dilutions.General urine examination and culture for urine that absorbed from test 2 and control was done (GUE) show that pus cell in urine was (1 - 2/HPF). There was no growth of bacteria in urine culture.The Histological section show that the Control and test 1 rabbit (have been immunized with fimbriae and adjuvant and injected with E.coli intra muscular) and Test 2rabbit (that has been immunized with fimbriae and adjuvant and injected with E.coli directly in bladder has normal epithelial cells and mucosa.
