Iraqi Journal of Biotechnology

The present study aimed to Detection of exoU,exoS genes Pseudomonas aeruginosa isolates
in Baghdad and Wasit,Iraq . 150 samples of P. aeruginosa were collected and identified by biochemical
tests, to identify the isolates of P. aeruginosa at the genus and species levels by means of primers
targeting oprI and oprL genes. and were characterized for antibiotic resistance. and the presence of exo
genes was evaluated by allele-specific PCR (polymerase chain reaction). The exoU (60.31%), exoS
(90.47%) genes were detected P. aeruginosa in different clinical specimens. Among 63 isolates of
P.aeruginosa.

This study was aimed to detect STRA6 gene polymorphism in Iraqi type 2 diabetes
mellitus patients (T2DM) to found the correlation between the SNP (rs 736118) polymorphism in
STRA6 gene and lipid metabolism and impact on the incidence of type 2 diabetes mellitus (T2DM) .
Genomic DNA was extracted from the blood samples of T2DM patients and apparently healthy as a
control group by using genomic DNA purification kits (Geneaid), the purity of the DNA was between
1.7 -1.9 and the concentration between 30 -100 ng / μl,. total cholesterol TC , high density lipids HDL,
low density lipids LDL and Triglycride and fasting blood sugar FBS were measured by kits supplied by
(Biolabo, France), while glycosylated hemoglobin HbA1C measured by kit supplied by (Nycocard,
Norway). Mean value of triglycerides in diabetic patients was significantly (p<0.001). The result show
high Significant difference was observed in FBS level (p<0.001) in patient group (183.47 ± 6.32) and in
control (88.61 ± 3.70), in other hand high Significant difference was observed in HBA1C level was
(p<0.001) in patient group ( 8.87 ± 0.21 ), while in control group was (4.86 ± 0.10). Cholesterol mean
level value in diabetic patients was significantly higher than those of control group (p<0.001) , LDL
Cholesterol mean value in diabetic patients was statistically significant (p<0.005) higher than the mean
value of control group, (p<0.001), Serum HDL-Cholesterol mean value was significantly (p<0.001),
VLDL-Cholesterol mean value in diabetic patients was significantly (p<0.001) increased compared to the
mean of control group. Real time PCR (Taq man ) RT-PCR were used to detect SNP (rs 736118) C>T
in STRA6 gene by using specific probes and primers, As a related with SNP (rs 736118) C>T in
STRA6 gene , the percentage of those CT polymorphism genotype in the patients group was significant
(p<0.001) compare with control group (58% versus 0% respectively) and the individuals with the CT
genotype in the patients group is significantly higher than those with CC genotype, the percentage of
those with TT polymorphism genotype in the patients group is significantly higher as compared with
control group (6% versus 0% respectively . The percentage of those with CC polymorphism genotype in
the patients group show highly significant differences as a compared with control healthy group (28%,
0% respectively), as a conclusion of this study there was a relationship between polymorphism of STRA
6 gene SNP (rs736118) and the incidence of T2DM in sample of Iraqi patients.

This study aimed to isolation and characterization of E.coli from Children urine , the phenotypic detection of biofilm formation , bacteria ability to adhesion, and the detection of fimH gene using PCR-based molecular diagnostic methods. In order to isolate E.coli, 250 urine samples were collected from children in camp saad of displaced / Diyala.Fifteen isolates(30%) of Escherichia coli were recovered according to the bactereiological , biochemical tests , used Api20E system and VITEK 2 system for confirmation. The study found that bacterial growth in females higher than in males and the incidence of infection in children under the age of 3 years is more common than in older age groups. The sensitivity test was performed for Ten antibiotics (Ampicillin, Piperacillin/tazobactam, Cefoxitin, Cefixime, Ceftazidime, Imipenem, Amikacin, Gentamicin, Ciprofloxacin, Trimethoprim /sulfamethoxacole) Where the percentage of resistance were (93.3% , 20.0% , 26.7% , 80.0% , 80.0% , 0.0% , 0.0% , 46.7% , 20.0% , 73.3%) respectively.Minimal inhibitory concentrations (MIC) were determined for Ceftazidime,Piperacillin /tazobactam, Gentamicin and Trimethoprim/ sulfamethoxacole. The MIC values were (4-32), (4-128), (<16), (20-320)μg / mL.Some of the virulence factors were detected, that included detection of biofilm formation and isolates ability to adhesion, all isolates showed ability to produce a biofilm and adhesion .Gene fimH was detected by Polymerase Chain Reaction(PCR), the percentage of isolates containing the gene was 100%.

Uropathogenic Escherichia coli produce cytotoxic necrotizing factor-1 (CNF-1). Which is mediates its effects on epithelial cells, and acts as one of uropathogenic E. coli virulence factors . Therefore this study was aimed to know the behavior of peripheral blood lymphocytes during the intravenous injection of CNF-1toxin , which has been poorly investigated in vivo .By using flow cytometery and light microscope, the effect of CNF1 was explored in rabbits, after certified the chromosomal cnf1 gene is found in the bacterial isolate. PCR analysis confirmed the foundation of the chromosomal cnf1 gene in the selected uropathogenic isolate. Whereas the assays of lymphocytes apoptosis and necrosis, revealed different effects, which were correlated with the investigated doses (1.82,3.65,5.47, and 7.3 µg/kg).The last two doses shown severe effects on some vital organs (liver, kidneys, and lungs )of rabbits. Histological evaluation of rabbits treated with the dose 3.65 µg/kg revealed different changes occurred grossly due to the cytotoxic effect of CNF1. They ranged between severe congestion in liver and lung tissues to paleness of kidney with changing in their textures. Microscopic examination showed, infiltration of inflammatory cells with fibrin network and cellular necrosis were observed. A conclusion of study revealed that,CNF1of uropathogenic E. coli , coded by chromosomal cnf1 gene with 498bp ,has cytotoxic effects on immune system represented by lymphocytes apoptosis and necrosis. As well as, different histopathological effects in some vital organs have been observed.

A Total of 150 clinical specimens from different clinical sources including burns, wounds, and urinary tract infections in Baquba General Teaching Hospital for the period from September 2016 to May 2017. 14 isolate were identified as A.baumannii. Sensitivity of 14 clinical isolates were tested against 14 antibiotics. and the result showed all isolate was MDR. All Acinetobacter isolates were investigated for the production of virulence factors which include the ability for the biofilms formation by microtiter plate method, and detection of efflux pumps. 92% of the isolates were biofilms former, and 85.7% of the isolates possess the efflux pumps. also investigate for bap gene responsible for biofilm formation.

The objective of this study was to investigate the association of polymorphisms in IL1B gene with the keratoconus disease in samples of Iraqi patients. Sample collection was carried out in the duration of two months from November 2016 to January 2017. The genomic DNA was extracted using Promega kits, with subsequent amplification of the fragments via PCR with specific primers followed by molecular detection of single nucleotide polymorphisms (SNPs) in specific regions along IL1B gene by DNA sequence analysis. The -511 (C>T) rs16944 is considered to be a risk factor for KC disease as the C allele frequency being higher in patients than controls (76% versus 40% respectively, OR 4.7500, 95% CI 2.1741-10.377, P 0.0001) also CC genotype (64% in patients versus 30% in controls, OR 4.1481, 95% CI 1.3573-12.677, P 0.0126). In addition -31 TATA box (T>C) rs1143627 was also considered as a risk factor to KC disease due to the results of allele and genotype frequency (T allele frequency 72% in patients to 33% in controls, OR 5.3407, 95% CI 2.4173-11.799, P 0.0001) and (TT genotype frequency 60% in patients to 25% in controls, OR 4.5000. 95% CI 1.4113-14.348, P 0.0110). Furthermore the two promoter SNPs (-31 and -511) are proved to be in complete linkage disequilibrium (LD) and the frequency of (T>C) haplotype (84% in KC patients versus 35% in controls, OR 9.7500, 95% CI 2.9663-32.047 and P 0.0002) which make it clear that the presence of those two SNPs together considered as a risk factor of KC disease for Iraqi patients. It was expected that the expression of IL1B may be enhanced due to the presence of T>C haplotype in the promoter region. Thus promote apoptosis consideres as a fundamental cause for keratoconus disease.

Polymerase chain reaction assay (PCR) used for the detection of Mycobacterium tuberculosis strain and fingerprint study. Ten positive cultures on Lowenstein Jensen (LJ) media were collected from specialized center of chest and respiratory disease\Baghdad .genomic DNA was extracted from all isolates and subjected to genetic characterization by (PCR) methods. two specific primers were used in pcr analysis for detection 16S rRNA and insertion sequence IS6110 where commonly used as a target of Mycobacterium. The result of gel electrophoresis showed that all isolates belong to Mycobacterium, and a sequence showed that 99% identity in sequence of 16S rRNA of M. tuberculosis and there is one nucleotide changes, the isolate submission in gen bank NCBI with accession number (MG03060) and 100 identity in sequence of IS6110 of M. tuberculosis. The isolate showed there is Ten polymorphism diversity according to copy number of IS6110 that have, where 80% of M.tuberculosis has large copy number (6_10) and 20% have low copy number (1-5) of isolate. This study showed that M.tuberculosis isolated in Iraq belong to two groups. Polymorphism groups depended on copy number of IS6110.

Current study we aimed to study the correlation of anti TSH receptor autoantibody with the thyroid hormones in the Graves ' disease patients . This cross –sectional study includes 60 appearantly healthy control and 60 patients who were early diagnosed with thyrotoxicosis diagnosed as Graves ' disease by the physicians. Those patients were attended Hormonal Unite at Specialized Center for Endocrinology and Diabetes in Baghdad / Rusafa – Iraq for the period between May- 2016 to January - 2017. Hormonal estimation including T3, T4 and TSH and immunological study including anti-TSHR antibody. According to the results, parameters (T4, TSH, anti TSHR) showed no significant difference between male and female (P value were 42.8, 0.26 and 4.17 respectively), thus , sex had no effect on development of disease, no significant differences between (T3 ,T4,TSH) regarding to smoking (p value = 2.066 ,45.78, 0.235 respectively) , but there is significant difference in Anti TSHR between smokers and non-smokers. there is differences between levels of (T3,T4,TSH and Anti TSHR Ab ) regarding to age groups , in patients age less than 40 years , there is a highly significant difference with T3 ,T4 and Anti TSHR Ab. There is correlation between Anti-TSHR with T3 and T4 showed highly Significant positive (r= +0.96, +0.75 respectively) , in contrast there is a highly significant negative between Anti-TSHR Ab and TSH (r= -0.66) . Measurement of Anti TSHR Ab in patients with thyrotoxicosis in a blood can help to establish the diagnosis and sometimes to predict the clinical course and response to treatment.

Atrazine (ATZ)is remaining as one of the most widely broad spectrum pesticide used to control on annual grasses and broadleaf weeds. While Chromium Cr (III) it has been used currently as food supplemented. The current study was designed to determine impact of Cr at 7,30 and 300ppm on liver was in absence or in the present of (25 and 50mg/kg) of ATZ for 6 weeks in rats. Results indicated that deficiency or supplementation with Cr did not cause measurable toxicity and has no obvious effect on oxidative stress or anti-oxidant enzymes and lipid metabolism. Furthermore, the results showed that the mixture increased level of Aryl Hydrocarbon Receptor (AhR), Reactive Oxygen Species (ROS). Moreover, the mixture had effects on antioxidants such as super oxide dismutase (SOD), Catalase (CAT), Reduced glutathione (GSH), activity where demonstrated more sever in liver tissue compared to Cr alone, especially as dose of ATZ increased. Moreover, as dose of Cr in the mixture was increased, the ROS, AhR, CAT, GSH activity was increased compared to low Cr values. However, SOD activity showed reduction in elevation of mixture concentrations. The results of this study suggest that there is synergic impact of co-exposure to ATZ and Cr on the parameters used as markers for liver toxicity in the current study. Further studies are warranted to investigate the interact of Cr and ATZ toxicity in more detail and to avoid having the Cr supplement where ATZ exposure is identified.

Escherichia coli frequently causing urinary tract, wound and blood infection resulting in significant morbidity and mortality due to had plasmid encoded ESBLs lead to failure treatment .Tow hundred samples were collected from (urine, wound, sputum), Escherichia coli. isolated and identified by using microscopical, morphological, biochemical tests and Vitek 2 compact system. Antibiotic susceptibility testing was screening according to the CLSI guideline and Vitek 2 compact system. Phenotypic screening of ESBLs was undertaken using (Double disk diffusion and Standard disk diffusion) Methods, also PCR technique was used for genotypic detection of ESBL genes (blaCTX-M) according to the standard protocol. We obtained in this study 60 (29.33%) total positive results of Escherichia coli.40 (20%) isolates for Escherichia coli isolated from urine, 15 (7.5%) wound,5 (2.5%) sputum,from patient attending e Rizgary hospital fromSemptemper 2015 to March 2016.Susceptibility profile has been done for all Escherichia coli isolated by using 13 antimicrobial agent,Our multifinding pointed out that highest resistance be Ceftriaxone 45(75%) Ceftazidime 44(73.3) Tobromycin 39(65%) Piperacillin 39(65%),most of Escherichia coli isolates were resistance to more than three antibiotics belonging to different classes used and these were considered to be multidrug resistant (MDR) isolates. The incidence rate of ESBL-producing Escherichia coli was 48 (80%) by Standard disk diffusion Method,46 (76.7%) by Double disk diffusion and 38(66.7%) of ESBL producer in urine samples. Remarkably, dissemination of blaCTX-M 44 (73.3%) genes among ESBLs-positive isolates and the length of amplified genes (550) bp for blaCTX-M genes. It can be said that the incidence rate of Escherichia coli carrying genes encoding for ESBL enzyme representing their commonness in our institute and multi resistance to many classes of antibiotic, resulting in limited treatment options.

Polycystic ovary syndrome (PCOS) is among the most common disorders of premenopausal women , cause infertility. This study was conducted to investigate whether the vitamin D receptor (VDR) gene polymorphisms are associated with the enhancement of sister chromatid exchanges(SCE) . This study was carried out in the Institute of Genetic Engineering and Biotechnology for Postgraduate Studies - University of Baghdad through the period from 1 November 2016 until the end of August 2017. The PCOS samples were taken from the Kamal Al-Samarraee infertility treatment Hospital in Bag hdad. The genomic instability were evaluated by measured the frequency of sister chromatid exchange in the presence of 5-bromodeoxy- uridine (BrdU) and using the Hoechst stain in 32 PCOS patients who have homozygous mutant result in genotyping assay , and from 34 PCOS patients who have heterozygous result assay. as well as 14 apparently healthy women of 50 women as control group. PCOS are classified depending on the results of the genotyping assay. The result showed significant differences in the level of sister chromatid exchange in patients with PCOS for each of VDR gene polymorphisms compared with normal women. This study concluded that the SCE as affected by polycystic ovary syndrome and the genotypes of VDR gene.

The study include 50 subject in period (20/12/2016- 28/3/2017) of iraqi women in Diyala governorate (18-25 years), 40 subject with Polycystic ovary syndrome (PCOs) 20 with physical activity who practice sport for 2 hours in day and 20 with no physical activity) and 10 subject control. The study showed no significant difference of the age(˃20, ≤20) and body mass index of PCOs and control, but there were highly significant of sex hormones (LH, Prolactin, Testesteron) in PCOs compared with control, results showed no significant difference in the age, body mass index with physical activity and PCOs, and a highly significant effect of the physical activity on sex hormones (LH, Prolactin, Testestosterone) in the physical activity PCOs women compared with no physical activity PCOs women. This study showed that physical activity improves levels of hormones (LH, Prolactine, and Testosterone) in PCOs Diyala women.