17A and Uropathogenic E. coli among Patients in Holy Karbala, iraq

The current study amid to investigate association of Interleukin-17A with uropathogenic Escherichia coli among patients with urinary tract infection in karbala province, Iraq. Bacterial infections are widespread in urinary tract infections with a global extention. Uropathogenic E.coli (UPeC) is the most common causes of these infections. Out of 110 patients were examined by urologists for urinary tract infection, 25 patients showed positive result for UPEC and other 25 showed positive result for other bacterial pathogens. UPEC were diagnosed depended on the cultural, microscopical, biochemical examinations and confirm the identification by using Vitek2 system. Polymerase chain reaction was used to detection of four genes (pap C, cnfA, fim H, and fyu A). Interleukin-17A concentration in urine was measured by using ELISA kit. out of 110 urine samples, 56 (44.90%) with significant bacteriuria, 44(40%) with non-significant bacteriuria and 10 (9.09 %) with negative culture. The presence of UPEC among significant bacteriuria was 25/56 (44.64 %). The distribution of pap C, cnfA, fim H, and fyu A genes among UPEC were 17(68%), 17(68%), 16(64%) and 15(60%) respectively. Through UTI patients, 50 gave positive (121.70) pg/ml results compared to 30 of control (13. 94) pg/ml. Among uropathogenic Escherichia coli patients, 25 gave positive (92.80) pg/ml results, while 25 of other bacterial pathogens gave positive (15.40) pg/ml results.


iNtRODUCtiON
Urinary tract infections (UTIs) are the most common type of infection in the human 1 . Escherichia coli (E. coli) are responsible for about 80% of these infections that have not passed the urinary bladder 2 . Uropathogenic E. coli has been seen to employ multiple virulence factors that facilitate the colonization and infected the urinary tract. These virulence factors involve adherence molecules (FimH and PapC adhesion molecules) 3 , iron chelating molecule (like FyuA molecule) that bind ferric iron, and finally secreted toxin like alpha-hemolysin and cytonecrotizing factor 1 (CNF 1) 4 . One of the most immune defenses against uropathogenic E.coli is the IL-17A. It is upregulated from six hours to one week after inoculation, and remained over the baseline through the two-week of experimental duration 5 . Interleukin-17A plays role in the innate immune response of infection by enhancing neutrophil migration to infected tissue 6 . The aim of the study was to evaluate the role of IL-17A as the immunological marker and fimberial adhesin of UPEC in patients with urinary tract infections.

Patients
One hundred and ten patients were clinically diagnosed that have UTIs in the outpatient urology clinic in Imam AL-Hussain Teaching Hospital in Karbala City/Iraq. The study was performed between September 2017 to the February 2018.

Diagnosis of bacteria
All urine samples were cultured on MacConkey agar and incubated overnight at 37°C. E. coli identification was done depending on morphological features biochemical tests (methyl red, indole, Voges-Proskauer, Simmons citrate and urease production) 7 and confirmation by using Vitek-2 system (bioMerieux) .

Molecular diagnosis of uropathogenic E.coli genes Extraction of the DNA
Bacterial DNA was extracted by using commercial kit from Geneaid manifecture's (Korea). The primers for (pap C, cnfA, fim H, and fyu A) genes were according to 8,9,10 . The sequence of the primer and amplification condition of PCR as in Table 1.

Detection of PCR products
PCR products were separated on a 1.5% gel electrophoresis agarose and visualized by ultraviolet light after addition of ethidium bromide.

Statistical analysis
Data were analyzed using SPSS statistical package version 21. Chi square (x 2 ), were used to determine the incidence of UPEC among significant bacteriuria. t-test was used to estimate the significant level of the IL-17A 28 .
UPEC is the main causative agent of community-acquired UTIs which colonize the urinary bladder using a different virulence agents that play important roles in the pathogenesis of UTI 4 these involve surface antigenic structures, such as lipopolysaccharide (LPS), polysaccharide capsule, flagella, outer-membrane vesicles, pili, curli, non-pilus adhesins, as well as secreted toxins, secretion systems, and TonB-dependent iron-uptake receptors 11 .
The results show significant difference (P < 0.05) between the incidence of UPEC and other pathogens among significant bacteriuria. The significant bacteriuria among results was (50.90%) of specimens. These results were higher than results of 12 and 13 , whose found that the significant bacteriuria were (22.7%) and (20.8%) respectively, while, lower than result of 14 , who reported that the significant bacteruria was (54.80%). As expected E. coli was the most frequently encountered species in the present study (44.64%) which was in the same line with the study conducted in Najaf city by 13 , who found that E.coli (41.3%) was the predominant causative agent of UTI. On the other hand, these results were lower than the results of 15,16,17 , whose found that the presence of UPEC were (58.57%), (57.7%) and (67.6%) respectively, while higher than the result of 18 , who revealed that the presence of E.coli was (33.8%). The differences of these results may be due to different environmental condition and sample size. The highest isolation rate of Escherichia coli infection in the urinary tract may be attributed to the contaminated with fecal flora such as virulence E. coli that facilitate the ascent of bacteria from fecal flora, up the urethra in to the bladder. The urine negative culture may be attributed to other infections such as viral or fungal infections or may be due to recent use of antibiotic 19 .
A genotypic assay was used to detection of different virulence factors of uropathogenic Escherichia coli such as adhesion-encoding genes and other virulence factors that can also associated to virulence in the urinary tract infection. In the present study, these adhesive agents, P fimbriae represents the most common adhesin in UPEC followed by type I fimbriae (fimH) 20 . The FimH adhesin mediates the bacterial adherence and    invasion of host cells and associated with the development of intracellular biofilms formation by UPEC 21 . The current study were differed from the study of 22 , who found that fimH (97%) and papC (43%) in UPEC isolates respectively. The second highest prevalence belongs to cytotoxic necrotizing factor A (68%), which encoded by cnf1 gene. The current study result in the same line with study conducted by 23 , which revealed that the prevalence of cnf gene with the rate (67%). But the present results were lower than the result of 24 , who found that the prevalence rate for cnf was (79.67%).
In this study, the percent of ferric Yersiniabactin uptake (fyuA) gene was (60%). The results of this study were lower than a study conducted by 22 , who reported that the percentage of fyuA gene was (83%) in UPEC. Also 25 , revealed that fyuA gene was (94%) which is also higher than result of current study. The differences in UPEC  Journal of Pure and Applied Microbiology virulence genes prevalence may be attributed to the climate of different geographic region 23,24 . serum level of interleukin -17A In present study among 50 patients of urinary tract infection compared to 30 of control, The IL-17A mean urine level of patients with UTI were (121.70) pg/ml, but it was (13.94) pg/ml for control and E.coli patients were (92.80) pg/ml, but it was (15.40) pg/ml for other bacterial pathogens patients. There was highly significant difference (p <0.001), (Table 3 and Table 4).
The role of cytokine IL-17A was characterized during urinary tract infection caused by UPEC. The result showed that the men of IL-17A were higher in UTI caused by uropathogenic E.coli than UTI caused by other pathogens and in control groups. This results was agreed with result of studies on mice, and because many of the genes influenced by IL-17A have the same function during UTI in mice and humans 26 , we expect that all IL-17A results in mice also have the same causes during UTI in humans. IL-17A which is an immune-modulatory cytokines, considers as an innate immune response important factor in the UTI associated with UPEC 27 . IL-17A contributed to innate clearance of UPEC through a mechanism involving secretion of cytokines and chemokine and cells influx such as neutrophils and macrophages to the bladder 5 . The γδ-positive cells is the key source of IL-17A production 27 .
In the study of murine model, the mice that lacking of IL-17A display deficient cytokine transcript up regulation and cellular responses during acute urinary tract infection, resulting in suboptimal clearance of uropathogenic E. coli, This clearance defect is likely a result of deficient cytokine and chemokine transcription and impaired of macrophage and neutrophil influx during infection, this result detect IL-17A seems to be unnecessary for the generation of protective immunity, and also IL-17A seems to play a role in regulating the response of innate immunity to UTI 27. IL-17A transcript is up regulated in response to acute bladder infection by uropathogenic E. coli time points during a 28-day period, and remains elevated throughout two weeks. A study on infected mice with uropathogenic E. colireported increased in the IL-17A, with median values peaking at 48 hours post infection, while other cytokines will be retained to near base line level one week's post infections, refers to a role for IL-17A in the response of innate immunity to UTI 5,27 .

CONClUsiON
The current study is the first study that revealed the association between human urine level of Interleukin -17A and Escherichia coli in Iraq and the world. The results of current study confirmed that the urine level of Interleukin 17A for E.coli patients were more than that in patients with other bacterial pathogens and control groups.