Urinary Tract Infection (UTIs) are common diseases and spread widely among members of society and different age groups, but their presence in females is more compared to males due to several reasons, including the proximity of the urinary opening to the genital and anal areas, urethral shortening and pregnancy, during which physiological changes occur that cause this disease [1]. The microorganisms that cause urinary tract infections vary, but most of them are caused by bacteria and there may be more than one pathogen at the same time[2]. Bacterial infection is caused by both Gram-positive and Gram-negative bacteria [3]. However, Gram-negative bacteria recorded the highest percentage of infection events compared to Gram-positive bacteria, the most common of which is E.coli. Gram-negative bacteria are often the cause of complex urinary tract infections [4]. The use of antibiotics has contributed to the treatment of many diseases, including the treatment of urinary tract infection and for a long time occupied the center stage in the resistance to pathogens.

The majority of urinary infections that affect men are complex [5], while uncomplicated infections are very low in men, and the majority of men with bacterial infections have urinary tract abnormalities [6]. Women UTIs have a distinct pattern in that women show an increased susceptibility to infection, while men have an increased severity of infection and it is more consistent. The prevalence of bacterial infection in women is estimated to be about 10% compared to 0.1% in men. [7]. The infection occurs in women of all age groups [8]. Women exposed to infection have weak IgA in most cases compared to the virulence of pathogenic bacteria [9]. Urinary tract infections are the third most common bacterial infection 

among children, especially in developing countries, after gastrointestinal and respiratory infections. The infection is often associated with urinary tract abnormalities that have resulted in long-term complications including high blood pressure and chronic renal failure [10]. In newborns, urinary tract infections can cause septicemia and general ill health with growth failure [11]. Breastfeeding is important for children because breast milk contains defensive factors represented by immunoglobulin A (IgA), which can Binding to pathogenic bacteria and destroying its outer wall by Lysozyme enzyme in synergy with Lactoferrin, thus preventing infection [12].

The detection of serological UTI by measuring the cellular motility in the serum of the infected, which is a vital indicator of the level of inflammation, including Interloukin 22 (IL-22), which is associated with UTI and its severity [13].             

Antibiotics used: Antibiotics

In the current study, antibiotic tablets were used for the purpose of testing the sensitivity and resistance of isolated bacterial species to antibiotics, including (AK) Amikacin, Cefixime (CFM), Gentamicin (CN), Meropenem (ME), and Nitrofurantoin (F).

Preparation of Culture Media

The following culture media was prepared Blood agar, Nutrient broth, Nutrient Agar, MacConkey Agar, Mannitol Salt Aga, Eosin- methylene blue agar, Brain-heart infusion agar, Muller-Hinton agar.

Sample Collection and Culture

The current study included the collection of urine and blood samples from patients aged between years from the auditors of Al-Shifa Laboratory and Pharma Laboratory in the city of Tikrit / Salah Al-Din / Iraq during the period from 20/9/2020 to 10/5/2021. The samples included urine samples blood samples from people suffering from symptoms of urinary tract infections, in addition to urine and blood samples from healthy people as a control sample. A drop was taken from each urine sample by means of the bacterial vector Loop and planted on three culture media: Blood agar, Maconkey agar and saline mannitol. Pure colonies were preserved using tilted nutrient agar at 4°C until subsequent diagnostic tests were performed [14].

Identification of Isolated Under Study

The isolates under study were diagnosed according to the following tests: 

• Morphological Diagnosis and Microscopical Diagnosis

Antibiotic Sensitivity Test

Enzyme-Linked Immunosorbent Assay (ELISA) Technology: 

This technique was used to estimate the levels of immunoglobulin proteins (IL22) in the sera of the samples used in the study.

Work Steps

 All samples, materials and reagents were left at room temperature for 30 minutes before use, then the standard concentrations were prepared by conducting a series of dilutions in five sterile tubes to obtain the concentrations of interleukin-22 (50-100-200-400-800) as in Figure 1.

Figure 1: Series Dilutions of the Standard Solution Substrate for IL-22

Then, by pipette, 50 μl of diluted standard solution tubes were transferred to the first five holes on the special plate (for each tube a hole used). The rest of the pits were mixed gently, the plate was covered with an adhesive cover and incubated for half an hour at a temperature of 37 0 C. The concentrated washing solution was diluted with distilled water (30 times), the cap was gently removed from the plate, then the plate pits were washed with the washing solution, and the process was repeated five times, turning the plate over Blotting paper to get rid of the remaining solution add 50 μl of HRP-Conjugate enzyme solution to all pits of the plate except for the control plank pits, return the adhesive cap and incubate as in step 4, wash the plate as in step 6, add 50 μl of staining solution A and 50 μl of staining solution B in the dark to all the pits and mix gently, then the plate was incubated for 15 minutes at 37°C, 50 μl of stop solution was added to all pits to finish the reaction, absorbance reading O.D. The samples were etched into a calibration plate using an ELISA device at a wavelength of 450 nm.

Results of Culture Samples

200 samples of them gave bacterial growth when grown on the culture media as shown in the sample collection paragraph in the separation of materials and methods of work, at a rate of 80.0% of the total of 250 samples, median urine samples, while the remaining 50 samples, at a rate of 20.0%, did not give any bacterial growth as shown in the Table 1, which is a growth result completely identical to what was reached by the researcher[15], who obtained 160 positive growth samples with a percentage of 80.0% and 20% negative growth, which is a high growth result compared to what was reached by the researcher [13] who obtained On 86 samples positive for bacterial growth at a rate of 66.15% and 44 samples did not show bacterial growth at a rate of 33.85%, the lack of growth of some samples may be due to a viral or fungal cause or from anaerobic bacteria that we cannot isolate by the usual culture methods that were used in this study, which needs to Culture media and special conditions for development or as a result of the patient's use of random antibiotics led to the disappearance of pathogenic bacteria [16].

Table 1: Results of Transplantation of Samples Taken from Patients with Urinary Tract Infections

Samples grown on culture media showed two types of growth: Gram positive, 37.5%, and Gram negative, 62.5%, as shown in Table 2. This result is close to what was found by [17] 43% of bacteria Positive bacteria and 57% negative bacteria and [18] in Tikrit, which recorded 39.5% of gram-positive bacteria and 57.5% of gram-negative bacteria, and as a result, the gram-negative bacteria of those infected had the highest percentage of gram-positive bacteria and this agrees with the study current.

Table 2: Number and Percentages of Gram-Positive and Gram-Negative Bacterial Isolates

Phenotypic and Microscopic Identification of Bacterial Isolates Causing Urinary Tract Infections

The results of phenotypic and microscopic tests that were performed on the growing bacteria (using Gram stain) showed that all Gram-negative bacteria grow on Maconkey medium, which does not allow Gram-positive bacteria to grow on it and are bacillary in shape. Escherichia coli colonies appeared in pink on MacConkey medium as a result of fermentation of lactose sugar (Figure 2), and its medium-sized colonies are regular, dry, and convex. 1) This bacteria is one of the natural flora found in the intestines of humans and animals, and one of the most common types of urinary tract infections [19].

Figure 2: Shows (A) E.Coli Colonies on Maconkey Medium And (B) Shows on EMB Mediu

Resistance of Bacterial iIolates to Antibiotics

The results of the bacterial isolates showed different types of resistance to the antibiotics used as shown in (Table 3) and (Figure 3), and the results were interpreted according to.

Figure 3: Shows Colonies of Klebsiella Bacteria on Maconkey Medium

 Table 3: Sensitivity of Bacterial Species Isolated from Urinary Tract Infections to Antibiotics 

S: sensitive, R:resistance Me: Meropenen,| CFM: Cefixime, CN: Gentamicin, F: Nitrofurantion, AK: Amikacin

E.coli isolates showed absolute resistance to CFM, and low resistance to both F and CN antigens, with different percentages (30,36.6) respectively, and very weak resistance to Me, AK antigens with ratios (11.6 and 8.3). As for E.coli isolates, they showed resistance with ratios (11.6 and 8.3). % (i.e. sensitive towards isolates with a percentage of 91.7%), and this agrees with all patel and its group 2019 with percentages (91.89%). As for the anti-F, the results of our study agreed with [20]by 34.1%, and our study did not agree with the researcher [13], where the proportion of her study was 89.4%, and the anti-CN of E.coli was resistant to this antibody by 30%, and this converges with the study of [15]where it was (and 37.5%), but it does not agree with (95.8%), but it does not agree with[17] with a percentage of (95.8%). % and 2.8%) respectively, while the anti-CFM matched the results of the researcher [13], where it was 89.74%. One of the important reasons for the resistance of E.coli bacteria to antibiotics may be due to changes in the permeability barrier, which leads to difficulty in reaching the target site and thus obstructing its work, which is a special feature in Gram-negative bacteria [21]. The resistance of these bacteria may be the result of a change in the composition of certain enzymes or the loss of their function and thus lead to negative changes in the site on which the antibiotic works, or the resistance results from enzymes that remove the toxicity of the antibiotic [22], as for the resistance of bacteria to lactam-antibiotics. β comes from bacterial production of β-lactamase enzymes that degrade the β-lactam ring [23].

S.aureus bacteria where the absolute resistance to the anti-CFM is 100%, and the resistance to anti-Me in our study is 36%, and this result is similar to the study of the researcher [24]where the result of her study was 28.1% and our study does not agree with the researcher[25] where This antagonist is sensitive to 100%, and anti-F is 10% resistant, and this does not agree with [26], where the resistance ratios in their studies were (72.2%). As for the anti-gentamicin, it was moderately resistant by 49%, and this agrees with the study [15]. The result of the resistance to the antigen was 51.4%, and our study does not agree with [25] with rates (14.7%). As for the anti-Amikacin, it is 25.5% resistant, and this agrees with [25] where it is 23.3% resistant, and we do not agree with [26], where it is 11.1%. As for Cefixime, it is absolutely resistant, and this corresponds to the study [26] by 100%, while K.pnumonia bacteria is isolated as it has absolute resistance to CFM. As for meropenen, it was 17% resistant (i.e. 83% sensitive) and this The result is similar to the study[24], where it was 100% sensitive.

Anti-Nitrofurantion showed a medium resistance of 57% and this is in agreement with the study of [15] and [26], where the resistance they had was (65% and 64.7%), respectively. We also used the antigen in our study, gentamicin, and it had a resistance of 22.8% and this agrees with the study [27]by 33.3%, but it does not agree with [15] by 89.7%, and Amikacin has a high sensitivity of 94.3% and this agrees with the study of [26] and a study [28] with a percentage of 100% for each of them, and an approach to the study [15], where it was sensitive with a percentage of 75.9%. Cefixime has absolute resistance, and this is similar to [26] and[15] with a ratio of 82.3 and 79.3, respectively. The most important of these mechanisms is its production of β-lactamase enzymes, as studies have proven that it possesses many types of these enzymes that destroy a wide range of β-lactamases . And Proteus.mirabilis bacteria have absolute resistance to the anti-CFM, and they are resistant to anti-Me by 20%, anti-F and CN are resistance by 33.3% each, while anti-Me is sensitive by 20% and here we agree with the study [24] where it is resistant by 25%, and the result of anti-F is completely identical to the study[26] with a percentage of 33.3%, anti-CN and in our study it agrees with the study of [13] where it is 25%, but anti-AK is sensitive in our study by 80% and this is close to a result [26] 100%. The reason for the resistance of these bacteria isolates to the antibiotic is due to several reasons, including the presence of mutations or plasmids that show the character of resistance towards the antibiotic, and the bacteria may have the ability to form a biofilm that has a role in this resistance [29] (Figure 4).

Figure 4: Susceptibility of Proteus.Mirabilis, S.Aureus and K.Pnumonia Isolated To Antibiotics

The results of the current study showed a high concentration of interleukin in the serum of patients with urinary tract infection compared to healthy controls, as its level in interleukin 22 reached its level in the infected (445.0±16.4 mg/dl) and in the healthy (135.3±18.4) and there are highly significant differences as the probability value reached p<0.001 as shown in (Table 4).

Table 4: Measurement of Interleukin IL22

Our study matched with the study of [30] where the levels of IL-22 were significantly elevated in their study in patients with Candida compared to control samples, and showed that the presence of IL-22 in urine is an important means in diagnosing Candida infection in the urinary tract.

• The results showed that the prevalence of urinary tract infections in patients was of different ages ranging from (15-60) years, and females recorded higher infections than males, and it was found that the highest infection appeared in ages ranging from (31-45) years for both genders, and E.coli bacteria were the predominant bacteria over the rest of the bacterial species while the least bacterial types causing the disease were K. kristinae and E.aerogenes

• The results showed a high percentage of IL-22 used in the study compared to control samples

Recommendation

• Expanding the study of other types of cellular compounds, including chemical attraction and interleukins and determining their relationship with the various causes of urinary tract infections

• Assessment of IL-22 levels in viral infections

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