Study the Antibacterial Mechanism of Diclofenac and its Activity Alone or Combined with Ciprofloxacin in Treating Urinary Tract Infection

: This study aimed to compare the effects of diclofenac, ciprofloxacin and their combination groups on urine tract infection (U.T.I.s) caused by resistance E coli 0157 H7 in vitro and in vivo . Two hundred urine samples were collected from adult women patients suffering from U.T.I.s from February 2022 to July 2022. The urine specimen was cultured on the blood agar and then incubated at 37°C for (1-2) days; MacConkey agar is the media used for determining the biochemistry results, and identification by Rapidchek test which was performed on typical colonies from the selective plate. The M.I.C effect of Diclofenac began at (25600µg/ml) against resistance E coli 0157H7 while M.I.C of ciprofloxacin it was at (0.96mg/ml), but M.I.C of a combination between (diclofenacn+ciprofloxacin) it was at (400+0.48) mg/ml. Resistance induction assay,with the ciprofloxacin has increased four times, while with the diclofenac has doubled, whilst the combination between (diclofenacn+ciprofloxacin) has no changed after 21 days, according to M.I.C.in the time-kill assay, it was proved that the ciprofloxacin and the combination between (diclofenacn+ciprofloxacin) works after 6 hours, compared to the diclofenac, which started to reduce bacterial counting after 40. After that, the examination was carried out by scanning electronic microscope (S.E.M.) to exam the mechanics of diclofenac,work which was found that it was works as anti-biofilm In this study, forty-eighth (48) rabbits were used and divided into six groups as follow positive control (P.C.), negative control (N.C.), diclofenac (D.C.) in 1mg/kg group, ciprofloxacin (C.I.P.)in7mg/kg group, combination (ciprofloxacin 3.5 mg/kg+diclofenac1mg/kg)COM1 group and combination (ciprofloxacin 1.75 mg/kg+diclofenac1mg/kg) com2 All animals (injected with 0.1 ml in 1 × 10 8 CFU E coli 0157 H7 by urinary catheterization route excepting negative control all group excepting negative control resulted in a significant increase in pus cell, epithelium cell concentration in the urine after three days E coli 0157 H7 infection comparison with negative control while in all experimental period after (14 days) showed no significant (P < 0.05) between positive control (P.C.) and diclofenac treated groups but combination COM1 group and com2 decrease significantly (P< 0.05) after (seven and fourteen) days comparison with positive control. diclofenac can be combined with antibiotics as an anti-virulence agent, enhancing the immune system's ability to eradicate infection.

Pathogens that are regarded the focus of infection, including outpatients and hospitalized patients, are becoming more resistant to antibiotics on a global scale.According to (Al-Zubaidy et al., 2018) these resistances might develop via mutation or by the appropriation of resistance genes from different species.Antimicrobial usage in animals and humans and the transfer of resistant strains among them are the major causes of rising antibiotic resistance.Antibiotic resistance is connected to the release of pharmaceutical industry effluents that have not been properly handled, particularly in nations where bulk medicines are produced (Najim et al., 2017).The overall result fraction of resistant bacteria that continue to proliferate rises as a result of antibiotics' increased selection pressure on bacterial populations, which kills more susceptible germs.Even at relatively low antibiotic concentrations, resistant bacteria may proliferate more quickly than susceptible germs.Alternative therapies are more necessary as antibiotic resistance spreads.Urinary tract infections (U.T.I.s) are among the most common bacterial infections seen in primary healthcare, and they are among the diseases with increasing antibiotic resistance (Al-Iraqi, 2017).Calls for new antibiotic therapies have been made, but new drug development is becoming rarer (Kapoor et al., 2017).With up to 35% of nosocomial diseases occurring in hospitals and being the second leading cause of bacteremia in patients in hospitals, these diseases have also emerged as the most prevalent hospitalacquired illnesses.Therefore they affecting patients of all ages and sexes but in Females account for 87.5% in compared to males (71.3%), affecting patients of all ages and sexes (AL-Dujaily and Mahmood, 2022).This is related to women's small urethral tubes and the anus's near closeness to the urethral entrance, which makes it easier for germs to enter the urethra.According to estimates by( Al-Taii and Yousif ,2022), 50% of women repeated cases of acute cystitis throughout their adult lives.Although it is disputed and controversial, combination antibiotic treatment is routinely used to treat severe infections by gram negative.In comparison to monotherapy, combinations may be more effective because they have a wider antibacterial range, synergistic effects, and a lower likelihood of resistance developing during treatment.Combinations are being used more often to boost the antibacterial activity of existing medications against multidrug-resistant pathogens in the lack of evidence-based therapeutic choices.However, excessive usage of combinations must be avoided as it may lead to greater expenses, superinfections, resistant strain selection, and increased toxicity risks (Malema et al., 2018),.In addition to infecting adults,as well as these organisms are also responsible for various outbreaks in animals, poultry, and humans around the world.These organisms cause food-borne diarrhea, urinary tract infection, bloody diarrhea, and hemolytic uremic syndrome, which occur with an average of 4% of infections in women and children (Khudhir et al., 2022).One of the most serious illnesses in babies and children which induce renal failure or result in patient death is urinary tract infection brought on by E. coli O157:H7 (Al-Rudha, 2016).

Identification of E.coli O157:H7
The culture of bacteria on Sorbitol MacConkey showed as amber or colorless, negative to sorbitol fermentation, while positive sorbitol of E. coli showed pink color Fig (1).
Figer(1) Culturing E. coli O157:H7on Chromogenic agar base medium with cefixime tellurite the culture of E. coli O157:H7 on Chromogenic medium with cefixime tellurite isolates belong to serotype resistance E. coli O157:H7; colonies showed pink to red in the color.

Specific biochemical tests for resistance E. coli O157:H7
The biochemical examination of the resistance E. coli O157:H7 cannot ferment cellobiose sugar (cellobiose fermentation -ve), cannot grow in the presence of potassium cyanide (K.C.N. -ve), and it can produce enterohemolysin on blood agar (enterohemolysin production +ve),table (1),fig( 3and4).

A:Tube with turbid solution (negative result). A: tube with yellow color (cellobiose fermentation) B: tube with clear solution (positive result). B: Tube with red color (no cellobiose fermentation)
Resistance E. coli O157:H7 isolates lack the ability to grow in the presence of potassium cyanide Culturing of resist.E. coli O157:H7 in potassium cyanide broth showed a clear solution after a period of incubation because it was unable to grow in the presence of potassium cyanide while E. hermanii is able to grow in potassium cyanide broth (B.A.M., 2001).E. hermanii is biochemically similar to resistance E. coli O157:H7.Therefore the main biochemical tests required to distinguish between them was their ability to ferment Cellobiose and grow in the presence of potassium cyanide (K.C.N.) (Al-Dawmy and Yousif, 2013).
The third confirmatory test involved the production of enterohemolysin.when E. coli O157:H7 isolates grew on washing sheep blood agar, they demonstrated their capacity to produce enterohemolysin; -hemolysis manifested as small, turbid, unclear areas around the colonies.these findings were compatible with (Gould et al., 2009).One of the serum risk indicators for HUS. is IL-1, which is produced as a result of the pore-forming toxin enterohemolysin (Taneike, 2002).

Identification of E.coli O157:H7 by Rapidchek test:
The two lines of the positive test of the sample broth is transferred to a test tube after the appropriate enrichment, and a test strip was then placed inside the tube.No heat treatment is necessary.Ten minutes will pass before the outcome.whentwo lines had appeared indicate a positive sample, whereas one line indicates a negative sample.The lateral flow strip includes a built-in control line, letting you know if the test was successful (Guerini et al., 2006).fig.The M.I.C. of selected antibiotics was obtained for resist.E. coli O157: H7 isolate used.Serial dilutions of ciprofloxacin, Diclofenac, and a combination of (ciprofloxacin + Diclofenac) were made in broth, and a standard inoculum of E. coli O157: H7 of the tested bacteria was added and incubated for 24 hours.Then determination of the highest dilution showed no turbidity.A control tube was prepared by adding culture to broth without antibiotic in addition to blank (no bacteria and no antibiotic).After overnight incubation at 37°C, tubes were inspected for growth which was indicated by turbidity.Tubes which the antibiotic was present in sufficient concentrations to inhibit bacterial growth remained clear.The last "clear" tube in the M.I.C. results showed that bacterial isolates were more affected by combination at low concentrations (0.48 µg/ml ciprofloxacin + 400 µg/ml Diclofenac) against resistance E.coli o157 H7, whilst ciprofloxacin showed a mild activity against resistance E.coli 0157 H7 (0.96 µg/ml ), the result is considered resistant compared to CLSL (2020) which it was ≤ 0.25.The clinical E. coli detected in the hospitals showed that Diclofenac has an antibacterial effect against different bacteria at (5-50) μg/ml and could treat U.T.I.s.(Mazumdar at el., 2009;Salem-Milani et al., 2013).The results have depicted significant antibacterial activity of Diclofenac and Ibuprofen( I.B.P. ) at 50μg/ml diclofenac decreased the M.I.C. for ciprofloxacin against resistance E coli 0157 H7.One or more of the clinical strains utilized in the experiment showed a high ciprofloxacin M.I.C. without diclofenac (M.I.C. = 0.96 μg/ml) but a considerable decrease in M.I.C. with diclofenac at two different doses (M.I.C. = 0.48 μg/ml).This outcome is consistent with the conclusions achieved by (Abbas et al., 2020)..In addition to its antibacterial properties when combined with other medicines, diclofenac also makes bacteria more susceptible to antibiotic agents (Chan et al., 2017).Consequently, a method for treating microbial diseases and reducing inflammation is to combine antibiotic drugs with Diclofenac.Ibuprofen, diclofenac, aspirin, and celecoxib have been demonstrated to have antibacterial properties against a variety of bacteria, including K. pneumoniae, E. coli, P. aeruginosa, and S. aureus in many investigations (Chan et al., 2017; Paes Leme et al., 2021).NSAIDs, diclofenac (D.C.), as previously reported by (Abbas et al., 2020),which have inhibitory actions against the formation of S.T.X. and were able to reduce the virulence of E. coli.diclofenac may prevent the growth of a number of microorganisms, including S. aureus, E. coli, C. albicans, M. tuberculosis, and L. monocytogenes, according to investigations by (Mazumdar et al., 2009).whereas, in disagreement with (Leo et al., 2020), who said that diclofenac showed in vitro activity against E coli isolate with a high M.I.C. (2,000 mg/mL) and no measurable minimum bactericidal levels >2,000 mg/m.According to several researches, NSAIDs may decrease bacterial pathogenicity and antibiotic sensitivity through a variety of methods.According to different research,Ibuprofen ( I.B.P. )and diclofenac may bind to D.N.A. gyrase and prevent the development of bacteria by acting as antibiotics.As stated by a recent research, diclofenac has structural similarities with the quinolones and fluoroquinolone families of antimicrobials (Kahlous et al., 2017).The anti-virulence effects of NSAIDs, such as the reduction of hemolysis and staphyloxanthin formation in these bacteria, provide indications that they are active against the pathogenicity of S. aureus in addition to their anti-biofilm action (Abbas et al., 2020)

Time Kill Assay
In the time-kill assay, the results presented in terms of the changes in the log10 cfu/mL of viable colonies indicated that the diclofenac exhibited little bactericidal activity.

Scanning Electron Microscope (S.E.M.).
Electron microscopy results showed Regarding the mechanics of diclofenac work, destroyed the formed biological membranes, which increased its resistance E coli 0157 H7 ciprofloxacin.The results were consistent with (Leão et al., 2020) where mentioned published an interesting study evaluating the in vitro activity of NSAIDs against resistance E. coli 0157 H7and S. aureus biofilms.In vitro, experiments showed that aspirin (A.S.A. )diclofenac and ibuprofen have anti-biofilm activity in concentrations similar to those found in human pharmacokinetic studies (plasma concentrations).S.E.M. images of Diclofenac (1× M.I.C.) treated and untreated resistance E coli 0157 H7 cells.Normal morphology of untreated diclofenac resistance Smooth cell surfaces with no apparent ultrastructural alterations are shown in E. coli 0157 H7 fig ( 7,a); diclofenac-treated cells exhibit deformities and core holes during the exposure period fig ( 8)(b, c, and d); A large amount of cell surface damage is seen in the S.E.M. picture as shown by spurting of cellular contents, loss of form (bending), and the central hollowing of numerous biconcave cells with a central depression and cavity indicating concurrent leaking of cytoplasmic contents.A few deformed cells may be seen at the field's perimeter, and colonies with scratches, surface blebs, and plicated surfaces have also been noted.Numerous cells had the appearance of being pierced and spewing cytoplasmic material.Black solid arrows have denoted major structural modifications.Urine Culture All urine samples were collected from infected rabbits with urinary tract infection (in sterile containers, midstream urine, after cleaning the genitals and centrifuged 2000rpm for 2min) from infected rabbits with urinary tract infections.Immediately, the sediment was incubated with brain heart infusion broth at 37oC overnight and streaked (by sterile swab) on blood and MacConkey agar surface and incubated aerobically at 37oC for 24h (Collee et al., 1996).
Forty-eight female rabbits were divided randomly into six groups (8 rabbits in each group).

Table (5) pus cell score at high power field (HPF) in urine-infected groups by resist.
E coli 0157 treated by ciprofloxacin, diclofenac alone, and a combination between (ciprofloxacin +diclofenac) in two doses (mg/kg) and control groups.

Three days Seven days Fourteen days
Control -ve(NC)

2.50±0.20 Cb
The small letter compares the values in the columns, and the capital letter compares the values in the row.Common causes of pus cell concentration in urine include urinary tract infections (U.T.I.s), such as bladder or kidney infections.U.T.I.s can occur when bacteria enter the urinary tract and multiply, leading to symptoms such as frequent urination, pain or burning during urination, cloudy or foul-smelling urine, and a feeling of urgency.In addition to U.T.I.s, other conditions that can cause pus cells in urine include kidney stones, interstitial cystitis (a chronic bladder condition), prostatitis (inflammation of the prostate gland in men), and sexually transmitted infections (S.T.I.s) such as gonorrhea or chlamydia.(Muvunyi et al .,2011)In this study, increased pus cell concentration in the urine of all groups after three days of infected by resistance E colli 0157 H7 which indication by urinary catheter kidney dysfunction excepted negative control.This is evidence of infection after the injection resistance E coli 0157 H7 in this study after 14 days no ciprofloxacin (C.I.P.) group showed no significant difference (P< 0.05) compared with negative control (N.C.).However, two groups treated by combination (ciprofloxacin + Diclofenac) and in two doses 3.5+1(mg/kg) and 1.75+1(mg/kg), after 7,14 days from the beginning resulted showed a significant decrease (P< 0.05) compared with (C.I.P., DC, P.C.) and this is evidence of the additive effect of diclofenac with ciprofloxacin, while diclofenac did not give an effect on its own, even after 14 days of starting treatment compared with negative control (N.C.).

Epithelium Cells score in urine
The values of epithelium cells score in urine were normal at the starting point of the experiment in negative control group range during different times(3,7and14) days of the experiment (8.70±0.83,8.80±0.66,8.70±0.70)respectively; but However the infected group (P.C., DC, C.I.P., COM1, COM2) with resistance E.coli O157: H7 showed a significant increase epithelium cells concentration in urine (P< 0.05) (18.00±0.63,17.80±0.37,17.80±0.37,17.20±0.25,17.40±0.18)respectivelyas after three days compared with the N.C. group it was (8.70±0.83) in the same time the values after seven days demonstrated decrease the epithelium cells numbers in the urine (P< 0.05) in (COM1, COM2) groups (9.20±1.11,9.20±1.11)respectively compared (P.C., D.I.C., C.I.P.) was (20.30±0.80,18.80±0.80,17.40±0.18),incontrast the values of epithelium cells showed a significant increase (P< 0.05) in (COM1andCOM2) groups which was (9.20±1.11and 9.20±1.11)respectively compared with (N.C.) group,was (8.80±0.66)after seven days, and the values of epithelium cells after 14 days showed a significant decrease (P< 0.05) in (C.I.P., COM1, COM2) groups which was, (4.50±1.00,4.40±0.84,4.60±1.00)respectively as compared with(P.C and DC) groups which was (37.90±2.21,37.60±2.48)respectively moreover.no significance difference (P< 0.05) in (C.I.P., COM1, COM2) groups after 14 days in the values of epithelium cells was(4.50±1.00,4.40±0.84,4.60±1.00)respectively, as compared with (N.C.) groups, was(8.70±0.70) the values of epithelium cells increased significantly epithelium cells concentration in urine (P< 0.05) in(P.Cand DC)groups (37.90±2.21,37.60±2.48)respectively compared with (N.C.) group (8.70±0.70)after 14 days table (5).The small letter compares the values in the columns, and the capital letter compares the values in the row.Transitional epithelial cells: also known as urothelial cells are found in the lining of the urinary tract.These cells have a rounded or irregular shape and may be indicative of inflammation or infection in the urinary system, such as a urinary tract infection (U.T.I.).these cells: these cells are derived from the renal tubules in the kidneys The presence of renal tubular epithelial cells in urine may suggest kidney damage or diseases, such as acute tubular necrosis or other kidney disorders Columnar epithelial cells: These cells are typically found in the lining of the urinary tract and can be seen in cases of inflammation or infection.(De la Salle et al .,2017)In this study, increased epithelial cells concentration in urine all groups after three days infected by resistance E coli 0157 H7 inducation by urinary catheter kidney dysfunction excepted negative control this is evidence of infection in this study ciprofloxacin (C.I.P.) group showed no significant (P< 0.05) changed in the epithelial cells concentration in urine after 14 days no a compared with negative control (N.C.) whilst the two groups treated by combination (ciprofloxacin+ diclofenac) and in two doses 3.5+1(mg/kg),1.75+1(mg/kg),after 7and 14 days presented asignficant decreased t (P< 0.05) compared with (C.I.P., DCand P.C.) and this is evidence of the additive effect of diclofenac with ciprofloxacin, while diclofenac did not give an effect on its own, even after 14 days of starting treatment compared with negative control (N.C.).
The results of current study agreement with (Bartlett ET AL, 2010), ciprofloxacin (C.I.P.) group pus cell, epithelium cell in urine after 14 days in compared with negative control (N.C.).
The additive effect between diclofenac and ciprofloxacin against resistance Escherichia coli 0157 was found to decrease pus cell and epithelium cell significantly (p<0.05)compared diclofenac, ciprofloxacin groups alone in addition the current study agreement with many reports which demonstrated that ibuprofen, diclofenac, celecoxib, and aspirin could use against K. pneumoniae, E. coli, S. aureus, and P. aeruginosa (Chan et al.,2017;Paes Leme et al.,2021).NSAIDs (Diclofenac), the antimicrobial activity of the antibiotics combination, increase the bacteria's susceptibility to the antibiotics (Chan et al., 2017).administration of antibiotics with NSAIDs can treat bacterial and inflammatory diseases the presented study, the effect of both groups combination (diclofenac+ciprofloxacin) in dosage (3.5+1and1+75) mg/kg decreased significantly (P< 0.05) the pus, epithelium cell concentration after 7, 14 days of the duration of the experiment compared with diclofenac and ciprofloxacin alone.diclofenac is used together with antibiotics to treat diseases, decrease inflammation, fever, and pain.The mechanism of action of diclofenac depends on cyclooxygenase enzyme inhibition (Rainsford, 2007).Concentration after 14 days was no significant changed (P< 0.05) compared with negative control (N.C.).

Conclusions:
From the results of study, the following observations are deduced: 1. E. coli O157:H7 cultured from females' urine at the hospital, identified by chromogenic agar medium, biochemical and molecular assays.2.An antibiotics susceptibility test was done for E. coli O157:H7 isolates that showed multiple antibiotic resistance, including ciprofloxacin. 3. Shiga-like toxin 2 is the most important virulence marker of E. coli O157:H7.4. Used Diclofenac with ciprofloxacin in urinary tract infection (U.T.I.)caused by.for reduce the bacterial effect and inflammation

NOVELTY STATEMENT:
Due to the absence of an approved vaccination for any resistance E coli 0157 H7 caused (U.T.I.) disease and the lack of easily accessible, secure, and effective medicines for some diseases resistant to synthetic treatments, it is imperative to seek alternate antibacterial sources.The study's novelty is that it focuses on antimicrobials for Diclofenac and ciprofloxacin) that could be used as antibacterial therapy.

AUTHORS CONTRIBUTION:
All the mentioned authors contributed to the current work.

References
Figer(1) Culturing E. coli O157:H7on Chromogenic agar base medium with cefixime tellurite the culture of E. coli O157:H7 on Chromogenic medium with cefixime tellurite isolates belong to serotype resistance E. coli O157:H7; colonies showed pink to red in the color.Specific biochemical tests for resistance E. coli O157:H7The biochemical examination of the resistance E. coli O157:H7 cannot ferment cellobiose sugar (cellobiose fermentation -ve), cannot grow in the presence of potassium cyanide (K.C.N. -ve), and it can produce enterohemolysin on blood agar (enterohemolysin production +ve),table(1),fig(3and4).Table(1): biochemical examination of E. coli O157:H7 Biochemical tests Results fermentation of the Cellobiose -ve K.C.N.-ve production of the Enterohemolysin +ve

Figure ( 9 )
Figure (9) pus cell score in urine at high power field (HPF) in infected groups by resistance E coli 0157 treated by ciprofloxacin, Diclofenac alone, and combination between (ciprofloxacin +diclofenac) in two doses(mg/kg) and control groups DC=diclofenac dosage 1mg/kg CIP=ciprofloxacin dosage 7 mg/kg COM1= (ciprofloxacin3.5mg/kg+diclofenac1mg/kg) COM2= (ciprofloxacin1.75mg/kg+diclofenac1mg/kg) Pus cells, also known as leukocytes, are white blood cells that are present in the urine.The presence of pus cells in urine may indicate an infection or inflammation in the urinary tract (Akoachere et al.,2012).Common causes of pus cell concentration in urine include urinary tract infections (U.T.I.s), such as bladder or kidney infections.U.T.I.s can occur when bacteria enter the urinary tract and multiply, leading to symptoms such as frequent urination, pain or burning during urination, cloudy or foul-smelling urine, and a feeling of urgency.

Figure ( 10 )
Figure (10)epithelium cells score in urine in infected groups by resistance E coli 0157 treated by ciprofloxacin, Diclofenac alone, and a combination between (ciprofloxacin +diclofenac) in two doses(mg/kg) and control groups

Table ( 4) Killing time(log) in infected groups by resist E coli 0157 treated by ciprofloxacin, diclofenac alone, and combination between (ciprofloxacin +diclofenac) and control groups.
compares the values in the columns, and the capital letter compares the values in the row.

Table ( 5) epithelium cells score in-infected groups by resistance E coli 0157 treated by ciprofloxacin, Diclofenac alone, and a combination between (ciprofloxacin +diclofenac) in two doses(mg/kg) and control groups
(Mundt et al., 2011)urine are originate from various parts of the urinary tract, including the kidneys, bladder, ureters, and urethra.The presence of epithelial cells in urine can be an indication of certain conditions or diseases affecting the urinary system.(Mundtetal., 2011)types of epithelial cells commonly found in urine include squamous epithelial cells, which have a flat and scale-like appearance They are typically derived from the urethra and can be considered normal if present in small numbers.However, increased squamous epithelial cells in urine may indicate contamination during sample collection.