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Abstract Pathogenic resistance to antibiotics, used for treating diseases, became an increased phenomenon with increasing pathogens. This study was conducted on real samples taken from cases admitted to Urology department at El-Mansoura hospital and did not normally respond to treatments by regular used antibiotics. The primarily samples investigation concluded (642) urine samples collected from patients suffered from urinary tract infections (UTI), 458 samples showed pathogenic bacterial growth on nutrient agar, while 184 samples showed no growth and considered free from UTI pathogens. The Gram reaction for picked colonies belonging to these levels revealed that 170 isolated were G-ve, resistant to 2 types of out of 12 used antibiotics. 19 cases were G+ve, resistant to 3 types. severe infection with number of pus less than 500 cells per field were represented by 98 cases. The pathogenic bacteria isolated from these cases were 91 G-ve and 7 were G-ve, resp. The biochemical characterization of the 485 UTI samples showed 373 cases were infected with Escherichia coli, 47 cases with Klebsiella pneumonia, 10 cases with Staphylococcus saprophyticus, 3 cases with Enterobacter faecalis, and 25 cases with other causes. However, E.coli bacteria is one of the most members of the enteric family that causes about 90% of UTI. Three E.coli strains were selected from the three infection levels named as: E.coli EMG03 (minor infection level), E.coli EMG04 (moderate) and E.coli EMG05, which represents the (severe level of infection). Two reference multi drug resistant strains were used: E.coli EMG01 is resistant to Ampicillin, Neomycin, and strain E.coli EMG02 is resistant to Ampicillin and Chloramphenicol. In this study, the theme oil was used as natural additives to antibiotics. The minor (GC-Mass) component enclouded the phenolic compounds extracted from the thyme essential oil; 26 compounds were detected. Thymol, P-Cymene, and the other Terpinene represented the largest percentage of component by 32.3, 21.8 and 13.9%, resp. While Carvacrol, Cineol and Linalool represented the second important percentage of component by 5.1, 4.4 and 3.4%, respectively. The other important component like Camphor, β-Caryophyllene and other Terpen did not ENGLISH SUMMARY -104- represent an detectible percentage. While the emulsification process was synthesized using highly purified thyme crude oil or thymol crystal substance. The nano-emulsion forms were characterized, the main thyme nanoform was lie in sized 53,99 nm, the thymol nanoform was lie in 51,09 nm with total zeta average of 67.64 and 66.05 nm, resp. The resistance to specific 12 antibiotics was measured using antibiotics resistance scale according to CLSI,(2017), which assigned three levels of resistance (resistant, intermediate, and susceptible). The used antibiotics were Ampicillin and Amoxicillin at a concentration of 10 µg that showed inhibition zones of 6.41 and 6.73 mm, respectively., and showed 100% resistance. Cefoxitin at concentration of 30 µg showed inhibition zones of 15.96 mm and 98% resistance. Ten µg concentration of Imipenem showed inhibition zones of 15.92 mm and the strain was 98% resistant. Vancomycin at a concentration of 30 µg, showed average inhibition zones of 11.05 mm and the strain was 95% resistant, Erythromycin, Gentamycin, Streptomycin, and Neomycin with a concentration of 10 µg showed inhibition zones of 15.03, 13.31 14.64 and 16.81 mm, resp., and showed 85.4, 86.3, 78.7 mm and 82.6% resistance, resp. the Chloramphenicol of 30 µg revealed inhibition zone of 16.36 mm and the strains were 78.5% resistant. Ciprofloxacin 5 µg showed inhibition zone of 19.85 mm and strains were 56.5% resistant. Tetracycline at a concentration of 30 µg showed area of inhibition of 20.54 mm and the strains were 75.9% resistant. Also, the antibiotic susceptibility tests were conducted against the selected three E.coli strains under study compared with the reference strains. the three E.coli isolates were resistant to Ampicillin and amoxicillin as same as reference strains EMG01 and EMG02. The isolates EMG04 and EMG05 were resistant to Cefoxitin, but EMG03 showed inhibition zone diameter (around 14.23 mm) similar to reference strains. All isolates as well as reference strains showed similar inhibition zone diameter (around 15.00 mm) to Imipenem. The isolate EMG05 was resistant to Vancomycin, but EMG03 and EMG04 was similar to EMG01 and EMG02 in their sensitivity to Vancomycin as showed inhibition zone ranged between 9.17-10.07 mm. The isolates EMG05 was resistant to Gentamycin, but EMG03 and EMG04 showed ENGLISH SUMMARY -105- average inhibition zone of 12,5 mm similar to those of EMG01 and EMG02. All isolates as well as reference strains showed similar inhibition zone diameter (around 14 and 14.7 mm) Erythromycin and Streptomycin, respectively. On other side, the effect of (1.5, 2 and 2.5%) concentrations of thyme and thymol either as emulsion or nano-emulsion on the growth of isolated UTI E.coli were estimated and showed slight inhibition zones around isolated and references strains. The differences in diameters of inhibition zones between 1.5, 2 and 2.5% concentrations were insignificant compared with reference strains. While the increasing on the nano-emulsion of thyme oil concentrations caused significant increased inhibition zones when used against the three E.coli isolates, also they were significantly different from those of references strains. It was noticeable the significant differences among the used concentrations of nanocrystals against either isolated or reference E.coli strains. Moreover, uses of 2.5% concentrations of nan-form of crystal oil was the most effective among other concentrations as showed a significant inhibition zone against all isolated and reference strains. Besides, the effect of 10 µg of Ampicillin, Amoxicillin, Erythromycin, 30 µg of Neomycin, Chloramphenicol and 5 µg of Ciprofloxacin were achieved when mixed with emulsion and nano-emulsion forms of both thyme oil and thymol crystal at three different concentrations 1.5%, 2.0%, and 2.5% on isolated E.coli UTI and reference strains. The use of emulsions forms did not show any significant differences in inhibition zone diameters between isolated and reference strains, while the use of nano-emulsion forms caused significant differences in inhibition zone diameters, the nano-emulsion of thyme crystals was the most effective. For Ampicillin, the inhibition zones diameters were 6.45, 6.32, 6.25 for EMG03; 6.07, 6.32 and 6.25 for EMG04 and 6.04, 6.25, and 6.15 for EMG05, resp., compared with 6.27, 6.64 and 6.92 mm for EMG01 and 6.05, 6.17 and 6.25 for EMG02. The effect of nanocrystal form on isolated or reference strains was the effective, which were 8.35, 11.20 and 16.43 for EMG03; 8.23, 10.88 and 16.25 mm for EMG04 and 8.11, 10.52 and 16.08, compared with those of reference strains 7.82, 10.64 and 16.70 for EMG01 7.98, 10.72 and 16.54 mm for EMG02, resp. ENGLISH SUMMARY -106- The effect of Amoxicillin was 6.22, 6.55 and 7.63 mm for EMG03; 6.19, 6.41 and 7.23 mm for EMG04 and 6.15, 6.24 and 7.05 for EMG05, compared with those of references strains that recorded 6.89, 6.98 and 7.85 mm for EMG01 and 6.92, 6.95 and 7.91 for EMG02, respectively. The use of thymol nano-emulsion inhibited the growth of tested bacteria. The inhibition zones were 8.81, 12.82 and 19.87 for EMG03; 8.25, 12.33 and 19.22 for EMG04 and 8.10, 12.13 and 19.14 for EMG05, compared with reference strain that showed 8.92, 12.67 and 19.90 for EMG01 and 8.89, 12.72 and 19.76 for EMG02, resp. For the Erythromycin, the inhibition zones were 15.45, 15.66 and 17.46 for EMG03; 15.04, 15.32 and 17.18 for EMG04 and 14.79, 15.03 and 16.78 mm for EMG05, compared with 15.68, 15.75 and17.63 for EMG01 and 15.26, 15.53 and 17.27 mm for EMG02, resp. In case of crystal nano-emulsion were 20.88, 24.87 and 29.86 for EMG03; 20.76, 24.66 and 29.32 for EMG04 and 20.35, 24.57 and 29.13 for EMG05, compared with 20.53 mm, 24.68 and 29.89 mm for EMG01 and 20.19, 24.26 mm and 29.73 mm for EMG02, resp. Also, the effect of Neomycin showed inhibition zones of 16.93,17.31 and 18.02 for EMG03; 16.65,17.19 and 17.91 for EMG04 and 16.22, 17.98 and 17.86 for EMG05, Resp., compared with 11.83, 11.96 and 12.06 for EMG01 and 16.89, 1723 and 17.93 for EMG02, resp. Although the reference strain EMG01 was already resistant to Neomycin, it became slightly more sensitive with significant difference after the addition of emulsions or nano-emulsions, as significant differences in inhibition zone diameters were found between the rest of tested E.coli and reference strain EMG02. The inhibition zones diameters recorded for nano-emulsion of thymol crystals were 22.96, 27.04 and 30.25 for EMG03; 22.72, 26.81 and 29.94 for EMG04 and 22.53, 26.65 and 29.86 for EMG05 compared with 15.09, 17.01 and 19.53 for EMG01 and 22.83, 26.95 and 30.16 for EMG02, resp. However, the effect of Chloramphenicol showed inhibition zones of 16.58, 17.13 and 17.80 for EMG03; 16.46, 16.98 and 17.72 for EMG04 and 16.01, 16.73 and 17.64 for EMG05, compared with 16.60, 16.71 and 16.44 for EMG01 and 11.65, 11.79 and 11.86 for EMG02, resp. ENGLISH SUMMARY -107- Although the reference strain EMG02 was already resistant to Chloramphenicol, it turned slightly sensitive to used treatments under used various concentrations, The differences in inhibition zone diameters were significant between all isolated strains treated with nanocrystal. The inhibition zones were 22.49, 26.78 and 30.02 for EMG03, 22.57, 26.58 and 29.79 for EMG04 and22.35 mm, 26.46 mm, and 29.65 mm for EMG05, compared with 22.31, 26.45 and 29.66 for EMG01 and 14.67 mm, 16.91 mm and 19.35 for EMG02, under used treatments, resp. Also, the effect of Ciprofloxacin under the nanocrystal treatments, the inhibition zone diameters were 21.09, 23.14 and 25.62 for EMG03, 21.73, 23.02 and 25.47 for EMG04 and 21.56 mm, 22.86 mm, and 25.35 mm, compared with 21.24, 23.21 and 25.79 for EMG01 and 20.93 mm, 23.06 mm, and 25.53 mm for EMG02, resp. Forth more, the increased inhibition zones after adding the nanoforms of oil or crystals are consistent with some evidence that components of essential oils interact synergistically with antibiotics by interfering with antibiotic resistance mechanisms. On other side, the G-ve pathogenic K. pneumonia which isolated from UTI patient was resistant to Ampicillin and Erythromycin 10 µg, however the addition of 2.5% concentration of thyme or thymol emulsion significantly affected differences between all oil forms (emulsion and nano-emulsion) in inhibition zone diameters. The antibiotics of Neomycin and Chloramphenicol 30 µg showed similar effects when used with emulsion of thyme oil or thymol crystals. Also, nano-emulsions forms were similar in their effect, and it was noticeable the significant of nanoforms. Also, G+ve S. saprophyticus isolated from UTI patients showed resistance to Ampicillin (10 µg) and Ciprofloxacin (5 µg). However it became sensitive to these antibiotics after addition of emulsion or nano-emulsion of thyme oil or thymol crystals. But the inhibition zones were increased when nano-emulsion forms of were used, The same effect with the two antibiotics, Neomycin and Chloramphenicol 30 µg, while the use of Erythromycin with nano-emulsion always showed significant differences in inhibition zone diameters. ENGLISH SUMMARY -108- Moreover, the determination of the inhibition’s zones was dramatically increased by increasing the natural additives concentrations, and it is normal to record the highest concentrations levels of minimal inhibitory concentrations and minimal inhibitory bacterial concentrations (MICs and MBC) which reached to 220 and 440 mg/ml, resp. with bactericidal efficiency (MBC/MIC) by 200% from the bacteriostatic effects. It’s clear to noted that, the nano-emulsion forms can sharply decrease the used amounts of thyme oil or thymol crystal to reach the MIC and MBC by 50%. The using of 180 to 220 mg/ml of nanoforms sufficient to reach MBCs for the G-ve bacteria from the G+ve UTI E.coli and K. penumenae genera, while 145 to 180 mg/ml of nanoforms sufficient to reach MBCs for the G+ve UTI S. saprophyticus. The Transmission electron microscopy was used to study the effect of oil on the shape and walls of UTI pathogenic E. coli and treated thymol nano-emulsion 2.5%. The images of the cells confirmed the change in the external shape of the E. coli cells, also showed the presence of damage to the cell walls resulting from the addition of the natural form of nano thymol, which allows the entry of the antibiotic into resistant bacteria and enhances its effect when used together as a step to break the ability of microbes to resist antibiotics. Conclusion: The study concluded that adding a nanoemulsion of thyme oil at a concentration of 2.5% to antibiotic treatment for bacteria causing urinary tract infections increases the effectiveness and effect of antibiotics, strengthens their action, and reduces bacterial resistance to antibiotics, and thus reduces the doses used and the duration of treatment and economic cost . . Recommendations: 1- Using the active compounds (thymol and thyme oil )in nanometers form at a concentration of 2.5% to support antibiotics. 2- Including thyme flowers an leaves in therapeutic nutrition programs due to its important components of thymol, cavacrol, phenols, and carbohydrates, and its ability to resist pathogenic microorganisms. |