1. Introduction

Urinary tract infections (UTIs) exhibit an increasing point of concern for investigations. They have ranked as one of the most common hospital-acquired infection as well as occupying the second place in the list of causes of bacteremia in hospitalized patients ^{[1, 2]}. Nevertheless, UTI causes significant distress to the individual and is associated with high healthcare and social costs. Acute uncomplicated infection is the most common form of symptomatic UTI, affecting 40% of women at some point in their life. One-third of patients who develop a UTI will go on to have recurrent infections. Symptomatic infection is less common in men. Asymptomatic bacterial colonization of the urinary tract is a common finding in women and the elderly ^[3]. Studies clearly demonstrate increasing antibiotic resistance in uropathogens causing both community- and nosocomial acquired UTIs ^[4]. WHO and the European Union (EU) have recognized the importance of studying the emergence and causes of antibiotic resistance and the need for strategic development to control this public health issue ^{[5, 6]}.

The rapid and global dissemination of antibiotic-resistant bacteria has resulted in the decrease of therapeutic options for many infectious diseases, highlighting the urgent need for new therapies ^[7]. Moreover, the toxic side effects produced by antibiotics reducing their demand. Recently, remarkable antimicrobial action was observed by several compounds ^[8], belonging to various pharmacological categories, such as the antihistamines ^[9], tranquilizers ^[10], the antihypertensive ^[11], the antipsychotics ^[12] and the anti-inflammatory agents ^[13].

The anti-inflammatory, analgesic and anti-pyretic properties of non-steroidal anti-inflammatory drugs (NSAIDs) are particularly useful in treating rheumatic and other musculoskeletal disorders ^[14]. These non-steroidal anti-inflammatory drugs have demonstrated strong antimicrobial property when tested against a large number of Gram-positive and Gram-negative bacteria ^[15] and are now referred to as “non-antibiotics” ^[16]. In this study; we aimed to isolate bacteria causing UTIs, detection of antibacterial activity of some NSAIDs (diclofenac sodium, aspirin, indomethacin, and ibuprofen) against these bacteria and finally; examine the combination effect of NSAIDs with β-lactam antibiotics against standard P. aeruginosa (ATCC 10145) and K. pneumoniae (ATCC 10031) strains.

2. Patients, Materials and Methods

From May 2014 until December 2015, a total number of 100 hospitalized patients suffering from UTIs of different ages and gender were enrolled into the study. They were attending the Urology Department at Minia University Hospital (MUH). All patients were subjected to clinical examination and a sheet was filled for each patient and included: date, name, age, sex, diagnosis, and symptoms. Patients were 37 males and 63 females of different ages from 5 months to 78 years. As 19 patients were in the age group of 1 to 20 years, 29 patients were in the age group of 21 to 40 years, 33 patients were in the age group of 41 to 60 years, and 15 patients were in the age group of >60 years.

Urine samples were inoculated on cysteine lactose electrolyte deficient (CLED) media (Lab M, UK) ^[17]. All the samples were examined for presence of bacteria by streaking them onto nutrient agar (Lab M, UK), MacConkey agar (Lab M, UK), EMB agar (Himedia, India) and mannitol salt agar (Becton, USA). Further identification was done by conventional biochemical tests. All media were prepared according to the manufacturers’ instructions. Standard strains were obtained from MIRCIN center, Faculty of Agriculture, Ain Shams University, Egypt. Bacteria were maintained by storage at -70°C on tryptone soy broth (TSB) medium (Himedia, India) enriched with 20% glycerol ^{[18, 19]}.

The following antibiotics were used; Ampicillin, Amoxicillin (EIPCO, Egypt), Augmentin (Sedico, Egypt), Cephalexin (Glaxo, Egypt), Cephradin (Smithkline, Egypt), Cefotaxime (EIPCO, Egypt), Ciprofloxacin (Amriya, Egypt) and Gentamicin (Memphis, Egypt). The following NSAIDs were used: Diclofenac Sodium (Glaxo, Egypt), Ibuprofen (Kahira/Abbott, Egypt), Aspirin and Indomethacin (Kahira, Egypt). All the drugs were obtained as pure dry powder and stored at 4°C.

Stock solutions of the tested NSAIDs and antibiotics were prepared at a concentration of 2.5 mg/ml. MICs were determined using Agar dilution method according to Clinical laboratory standard institute (CLSI). Bacterial suspensions of isolated bacteria were prepared in 2ml sterile saline and turbidity was adjusted to 0.5 McFarland (1-2x10⁸ CFU/ml). Serial dilutions for the tested compounds were performed using Muller Hinton Agar plates. Then, the tested strains were inoculated on the surface of agar using multi-inoculator device.

Two drugs combined effects were determined by the Checkerboard dilution technique to determine the fractional inhibitory concentration (FIC) indices. As E. coli was the major pathogen; combination between NSAIDs and antibiotics against resistant E. coli strains was mentioned in previous publication ^[20].

Definition of FIC is as follows: MIC of substance_A tested in combination /MIC of substance_A tested alone + MIC of substance_B tested in combination /MIC of substance_B tested alone. The FIC index (FICI) was calculated using the following formula: FIC index = FIC_A + FIC_B= [A]/ MIC_A+ [B]/MIC_B. Synergism is showed as FIC index of ≤0.5, while indifference is showed as an FIC index of >0.5 ≤4 and antagonism is showed as an FIC index of>4. FIC index was an average of two independent experiments ^[21].

Statistical analysis was done using SPSS one way Anova test and paired t test. P values of <0.05 were considered with statistically significant differences.

3. Results

Patients were classified into different age groups from 5 months to 78 years. Figure 1 shows that higher prevalence of UTIs was observed in female patients (63%) than male that represented 37%. In female patients; the highest incidence of infection was in age group of 21 to 40 (39.7%) while the lowest incidence was in the age group above 60 years (11.1%). On contrary, in male patients; the highest incidence of infection was observed in the age group above 60 years (40.54%) while the lowest incidence of infection was in the age group from 21 to 40 (21.6%).

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Figure 1. Prevalence of UTIs in Relation to Age and Gender

A total of 122 isolates of 100 patients were retrieved, in which 48 isolates (39.3%) of E. coli and 32 isolates of coagulase negative Staphylococci (CoNS) (26.2%) were the most common isolates. The other identified pathogens were 12 (9.8%) S. aureus, 9 (7.4%) Klebsiella spp., 9 (7.4%) Enterococcus faecalis, 4 (3.2%) P. aeruginosa, 3 Streptococci (2.5%), 3 Proteus spp. (2.5%) and 2 Bacillus spp. (1.6%) (Figure 2).

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Figure 2. Distribution of isolated bacteria from UTIs

The different UTI isolated species were screened for their susceptibility to different antibiotics. The minimum inhibitory concentration (MIC) values of each of the tested antibiotic against the different pathogens isolated from UTI samples were determined and for better comparison, MIC₉₀ and MIC₅₀ and percentage of resistance of different UTI isolates to each antibiotic were recorded (Figure 3).

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Figure 3. Prevalence of antibiotics resistance among the isolated microorganisms

As shown in Tabe1, MIC₉₀ of diclofenac sodium against Bacilli and Streptococci spp. were 0.5μg/ml and 1μg/ml, respectively. MIC₉₀ values against Proteus spp. was 64μg/ml. MIC₉₀ values against E. coli, Klebsiella spp., E. faecalis and Pseudomonas aeruginosa were the same (256μg/ml). Diclofenac sodium showed the highest MIC₉₀ against CoNS and S. aureus (512μg/ml and 1024μg/ml, respectively). Regarding MIC₉₀ of aspirin against all isolates; the lowest MIC₉₀ value against aspirin was 4μg/ml against Bacilli. MIC₉₀ of aspirin was 512μg/ml against E. faecalis, Proteus and Pseudomonas spp., while, MIC₉₀ against E. coli, Klebsiella, S. aureus, CoNS and Streptococci was 1024μg/ml (Table 2). Regarding MIC₉₀ of indomethacin against all isolates; Bacilli exhibited the lowest MIC₉₀ value against indomethacin (1μg/ml), while; Pseudomonas spp., E. coli, and S. aureus showed the highest MIC₉₀ against indomethacin (1024μg/ml). MIC₉₀ against E. faecalis and Proteus were 128μg/ml and against Streptococci was 256μg/ml (Table 3). Data of MIC₉₀ values of ibuprofen against all isolates showed that MIC₉₀ for Bacilli was 2μg/ml represented the lowest value against ibuprofen, on contrary, S. aureus, E. coli, CoNS, Pseudomonas spp. and Streptococci revealed the highest and the same MIC₉₀ value (1024μg/ml). For E. faecalis, Proteus and Klebsiella, MIC₉₀ values were the same; 512μg/ml (Table 4).

Table 1. Distribution of minimum inhibitory concentrations of diclofenac sodium, MIC₉₀ and MIC₅₀ among the isolated bacteria

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Table 2. Distribution of minimum inhibitory concentrations of aspirin, MIC₉₀ and MIC₅₀ among the isolated bacteria

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Table 3. Distribution of minimum inhibitory concentrations of indomethacin, MIC₉₀ and MIC₅₀ among the isolated bacteria

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Table 4. Distribution of minimum inhibitory concentrations of ibuprofen, MIC₉₀ and MIC₅₀ among the isolated bacteria

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Data in Table 5 represented the MIC values of some widely used antibiotics and NSAIDS against standard strains. MIC values of nearly all antibiotics against P. aeruginosa were the highest among all species. Finally, MIC of ciprofloxacin against tested standard strains was ≤0.25μg/ml. Indomethacin showed the lowest MIC; 128μg/ml against K. pneumoniae but P. aeruginosa was more sensitive and revealed MIC of 64μg/ml. MIC of diclofenac sodium and ibuprofen were 1024μg/ml against K. pneumonia. Ibuprofen recorded MIC of 512μg/ml against P. aeruginosa, while diclofenac sodium showed MIC; 1024μg/ml. MIC of aspirin against K. pneumoniae was 256μg/ml which was doubled that against P. aeruginosa (128μg/ml).

Table 5. Distribution of minimum inhibitory concentrations of different antibiotics and NSAIDs against the standard strains

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The combined effects of NSAIDs with the beta-lactam antibiotics on standard P. aeruginosa (ATCC 10145) and K. pneumoniae (ATCC 10031) strains were shown in Table 6 and Table 7. All the tested NSAIDs significantly lowered the MICs of antibiotics against the tested bacteria and fractional inhibitory concentration indices (FICIs) for this combination ranged from 0.004 to 0.5. These results showed that NSAIDs have a synergistic effect when combined with antibiotics and this combination could effectively inhibit growth of bacteria.

Table 6. Combinations effect of NSAIDs with antibiotics against P. aeruginosa (ATCC10145)

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Table 7. Combinations effect of NSAIDs with antibiotics against K. pneumoniae (ATCC10031)

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4. Discussion

In the present study, the selected age group was comparable to two studies conducted by Gupta and Bhadelia, 2014 and Kiffer et al., 2007 who selected age group from 1 to > 60 years with mean age 35.5 ^{[7, 22]}. A study performed in Egypt made by Ghonemy et al. consisted of 62.2% males and 37.8% females and the mean age of the patients was 52.03 ± 14.67years. The highest proportion of patients (31.9%) was aged between 50 and 60 years in both males and females ^[23] and this was the same as our result, but Kiffer et al. revealed that among the positive cultures, 88.8% belonged to female and 11.2% to male patients ^[22]. Das et al. revealed that elderly (61 years or more) males had a higher incidence of UTI (49.23%) compared to the elderly females (21.75%) ^[24] and that was similar to our result regarding male patients.

Akhter et al. revealed that bacteria isolated from urine samples were E. coli 30%, CoNS 26%, S. aureus 20%, E. faecalis 10%, Proteus spp. 6%, Pseudomonas spp. 6% and Klebsiella spp.2% ^[25] and this was similar to our result regarding E. coli and CoNS. A study done in Philippine reported that E. coli was the most common organism isolated (28.8%) which was lower than ours, followed by Staphyloccocus spp. (13.5%), Klebsiella pneumoniae (9.6%) which were close to ours, Enterococcus faecalis (2.9%), and P. aeruginosa (2.9%) which were lower than ours ^[26]. Mubanga et al. revealed that the top five cultured uropathogens were E. coli (61.5%), S. aureus (14%), Pseudomonas species (6.5%), E. faecalis (5.5%) and Streptococcus agalactiae (5%) and this was higher than our result ^[27].

In the present study; diclofenac sodium showed the lowest MIC₉₀ against most strains; while indomethacin showed the lowest MIC against standard strains. A study done by Mazumdar on clinical strains of E. coli in hospitals have indicated that diclofenac sodium has shown antibacterial activity against many strains of bacteria from 5-50 μg/ml and was effective in treating UTIs and this was lower than ours ^[28]. A study on E. faecalis was done to evaluate antibacterial effect of diclofenac in comparison with ibuprofen, calcium hydroxide and amoxicillin. The results have depicted significant antibacterial activity of diclofenac and ibuprofen at 50μg/ml and above concentrations ^[29], but this was lower than our result, as in ours; MIC₉₀ of ibuprofen against E. faecalis was 512μg/ml and MIC₉₀ of diclofenac sodium was 256μg/ml.

Akhtar et al. determined the antibacterial effect of aspirin against different bacterial strains isolated from UTI. Aspirin was effective at 500 μg/ml concentrations as similar to ours. Antibacterial effect of aspirin on isolates of diabetic foot infection showed that 100μg/ml concentrations of aspirin were mostly inhibitory for S. aureus and other isolates and this was lower than our result ^[25]. Muller et al. and Polonio et al. revealed that aspirin has effect on isolates of UTI; that S. aureus and E. faecalis indicated 100% inhibition at 100, 500,1000 μg/ml concentrations ^{[30, 31]}. Al-Bakri et al. showed that aspirin possessed a broad spectrum antimicrobial activity against E. coli and P. aeruginosa ^[32]. All the previous studies were the same as ours. Another studies have reported that aspirin and other NSAIDs interfere with growth of both Gram negative and Gram positive bacteria in vitro ^[33] in contrast to ours.

Obad et al. concluded that ibuprofen may be responsible for the broad spectrum of activity, both antibacterial and antifungal activity ^[34]. Al- Janabi studied activity of ibuprofen on E. coli and showed susceptibility to tested agent at MIC of 2.5 mg/ml ^[35] which is higher than our results. NSAID is equally effective as an antibiotic, and this may lead to a reduction in the use of antibiotics and reduce antibiotic resistance ^[36].

Annadurai et al., exhibited that diclofenac sodium has shown significant antibacterial effect in synergism with aminoglycosides both in vitro and in vivo studies ^[37] and agreed with our result. The non-antibiotic drug; Diclofenac was found to protect mice from Salmonella infection more effectively when combined with streptomycin than used alone ^[38]. The synergism between diclofenac and streptomycin against S. aureus NCTC 6571 and E. coli K12 C600 was found to be statistically significant (p<0.01), when compared with their individual effects ^[39], and this agrees with our results which showed significant synergism between diclofenac sodium and β-lactam antibiotics. Ronser revealed that sodium salicylate and related compounds such as aspirin are known to have a variety of effects on microorganisms. E. coli, for example, exhibits increased resistance to chloramphenicol, ampicillin, nalidixic acid, and tetracycline after such treatment ^[40], but that study disagrees with our result, as aspirin showed FIC_index<0.5 showing synergism. On the other hand; Aumercier et al., revealed that E. coli cells grown in the presence of salicylate are more sensitive to aminoglycosides ^[33]. Del Prado et al. revealed that animals receiving amoxicillin combined with ibuprofen showed a more pronounced reduction in bacterial counts even than those receiving the antibiotic alone and this was the same as our result ^[41]. Dutta et al. used the checkerboard technique giving a FIC_index for E. coli of 0.49 for diclofenac and streptomycin, there by showing a synergistic effect ^[42] as ours and another study showed that the combination effect of diclofenac with gentamicin ⁄ampicillin which was examined by using checkerboard technique yielded FIC_index ranging from 0.4 to 0.5 for diclofenac + gentamicin and values >1 for diclofenac + ampicillin ^[43], but that FIC_index was higher than ours regarding ampicillin which was <0.5. In the present study NSAIDs alone recorded antimicrobial activity, but NSAIDs in combination with antibiotics exhibited significant synergistic effect when used together and the drugs were bactericidal in addition to the synergistic effect and prevented the bacterial regrowth.

5. Conclusion

In conclusion, diclofenac sodium, aspirin, indomethacin, and ibuprofen showed in vitro antibacterial activity against bacteria associated with UTIs. Our results indicate that a combination of these NSAIDs and antibiotics exhibited good synergism against standard strains. This new finding might provide a new way to overcome antibacterial resistance. However, in vivo and clinical studies will be required to support this finding.

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