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تثبيط تعبير جينات fimH ,fimD ,fimC في بكتريا E. coli الممرضة للمجاري البولية باستعمال جسيمات ثنائي اوكسيد التيتانيوم النانوية المصنعة حيويا من المعززات الحيوية البكتيرية == Inhibition the Expression of fimC, fimD, fimH Genes in Uropathogenic E. coli Using TiO2 Nanoparticles Biosynthesized by Probiotics Bacteria
Author name:
سوزان عبد الرحيم حسن
Supervisor name:
سوسن ساجد الجبوري | جيهان عبد الستار سلمان
General topic:
Biology
Specific topic:
Microbiology
Degree:
Doctorate
University:
Mustansiriyah University - College Of Science
Language:
English
University location:
Baghdad
Key words:
- fimC
- fimD
- fimH Genes
- Uropathogenic E. coli
- TiO2 Nanoparticles
- Probiotics Bacteria
- biosynthesis
First pages:
T107044 - p.pdf
Abstract:
Summary ►Urinary tract infections (UTIs) are the commonest human bacterial infections and are responsible for substantial morbidity and mortality, resulting in increased healthcare costs. Specialized Escherichia coli strains referred to as Uropathogenic E. coli (UPEC) causes most UTIs. ►Total 120 isolates primarily identified as E.coli; were collected from (December 2018 to February 2019). Out of total collected isolates, 94/120 were included in this study and the contaminated bacteria or weak growth were excluded. The isolated pure colonies were re-identified using some standard morphological tests followed by genetically identification using housekeeping gene (HKG) 16S rDNA. The results confirm that all the isolates were definite as E. coli. ►The UPEC isolates showed their ability of adherence to uroepithelial cells. The aggregation ability of bacterial cells were appeared as dotes on the surface of epithelial cells. ►The antimicrobial susceptibility test (AST) was performed for 50 isolates under study. The highest resistance rate was concerning to Aztreonam; Cefazolin; Ceftazidime and Ceftriaxone (84%), Cefepime (82%), while the minimum resistant was for Imipenem; Ertapenem; Meropenem; Amikacin when it was (2%) beside that none of the isolates was able to resist Tigecycline. According to AST for fifty UPEC isolates; (88%) were multidrug resistance (MDR), while (2%) was extensively drug resistance (XDR) and (10%) were sensitive to all antibiotics thus they were non-drug resistant (NDR). ►All isolates were subjected to detect type1 fimbriae genes (fimA operon) by polymerase chain reaction using specific primers (designed in this study) followed by sequencing the amplified fragment and analyzed using Geneious prime software. The results showed that all the isolates (100%) were positive for fimA, fimI, fimC, fimD, fimG, fimH, while (96%) for fimB, fimF and (82%) for fimE. ►Raw milk samples were collected for probiotics bacteria isolation. All isolates were subjected to the cultural, microscopically, biochemical tests and Vitek2 system. Results indicated that six isolates of Lactococcus spp. Identified as: Lactococcus lactis ssp. lactis, in addition, 14 isolates of Leuconostoc spp. distributed among species as (8) isolates belonged to Leuconostoc mesenteroides ssp. cremoris (3) isolates belonged to Leuconostoc mesenteroides ssp. mesenteroides, (2) isolates identified as Leuconostoc pseudomesenteroides and only one as Leuconostoc citreum. ►All isolated probiotics bacteria were tested for Titanium dioxide (TiO2) nanoparticles production. The results showed that they had the ability to biosynthesize TiO2 NPs, biosynthesized TiO2 NPs were characterized using UVVis Spectroscopy; Atomic Force Microscope (AFM); X-ray Diffraction (XRD); Field Emission Scanning Electron Microscopy (FE-SEM) Energy dispersive Xray spectroscopy (EDX); and Fourier Transform-Infrared Spectroscopy (FTIR). The detected absorption spectrum with a wide-ranging absorption band in UV range and its cut off wavelength was (344-348) nm for all 20 probiotic isolates. ►The smoothness and roughness of all samples of biosynthesized TiO2 NPs was detected by AFM with smoothest sample for L. mesenteroides ssp. cremoris (Leu.4) as 1.137nm. XRD results indicated the formation of nano anatase crystals. ►The Scanning electron microscope images of biosynthesized TiO2 NPs biosynthesized by probiotics bacteria; that were consists of a uniform distribution of spherical shaped nanostructure crystals with a smallest size of (37.48) nm for L. citreum (Leu. 13). ►The EDX analysis of TiO2 NPs existing in medium which confirm that all the samples contained the elements (Ti, O), which again established that the biosynthesized NPs were pure TiO2. ►The determination of functional group of biosynthesized TiO2 NPs studied using FTIR. The main IR absorption peaks of TiO2 biosynthesized by probiotics bacteria were in range at (408- 445) cm-1; these peaks belonged to anatase Titania. ►The biosynthesized TiO2 NPs at concentrations (1000- 0.001) mg/mL were tested for its antibacterial activity against UPEC isolates, thus there was neither bactericidal effect against pathogenic bacteria nor been as minimum inhibitory concentration (MIC). ►There was no effect of temperature, pH and photocatalytic activation on antibacterial activity of biosynthesized TiO2 NPs against clinical UPEC isolates. ►Regardless of no growth inhibition activity showed on culture media after subjecting bacterial UPEC isolates to TiO2 NPs. There was a noticeable inhibitory effect of TiO2 NPs on the attachment ability of bacterial cells without killing them. The highest inhibition was recorded at the higher concentration of biosynthesized TiO2 NPs (1000 g/mL) with inhibition reached 92% and 90.3%, while the lowest inhibition were 52% and 54.8% at 250g/mL against E.coli74 and E.coli23, respectively. ►The effect of biosynthesized TiO2 NPs by probiotics bacteria and commercial TiO2 NPs was studied on the expression of fim genes. There was a subsequent change in the gene expression in term of folding and CT values when the expression of fimH, C, D genes start to decrease by increasing concentration of TiO2 NPs as compared with control and the maximum inhibitions was noticed by using 1000 g/mL concentration of the NPs specially against fimD.
Summary:
ce50ce6d09.pdf
References:
bad49367f4.pdf
