Comparing the Antimicrobial, Anti-Biofilm, and Cytotoxic Effects of Thymol/Ampicillin and Thymol/Cefotaxime against Escherichia coli Bacteria

Comparing the Antimicrobial, Anti-Biofilm, and Cytotoxic Effects of Thymol/Ampicillin and Thymol/Cefotaxime against Escherichia coli Bacteria | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Comparing the Antimicrobial, Anti-Biofilm, and Cytotoxic Effects of Thymol/Ampicillin and Thymol/Cefotaxime against Escherichia coli Bacteria Sajjad jafari, Mina Shirmohammadpour, Mina Tasoujlu, Bahman Mirzaei This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6177131/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction: This study aimed to compare the antimicrobial, anti-biofilm, and cytotoxic effects of thymol/ampicillin with thymol/cefotaxime on Escherichia coli ( E. coli ). Material and Methods: Antimicrobial effects of thymol/ampicillin and thymol/cefotaxime were first individually and then combined on E. coli ATCC 25922 by MIC-MBC method. Therefore, the antimicrobial effect of compounds that had synergistic effects was performed on twenty clinical strains using the MIC-MBC method. Identification of chemical bonds, functional groups, and molecular interactions of the mentioned compounds were investigated with an FT-IR device. Checkered method, time killing curve, and biofilm inhibition on E. coli ATCC 25922 bacteria, and investigation of cytotoxicity on red blood cells (RBCs) by hemolysis method and human skin fibroblast cells (Ffk) by MTT method were performed. thymol/ampicillin and thymol/cefotaxime (A1, A3 compounds) had Synergistic effects. Finally, the results of the tests were compared between the two compounds. Results: The results of this study showed that the antimicrobial effects of the thymol/ampicillin 2/8) µg/ml) were better than the thymol/cefotaxime 16/128) µg/ml(in both ATCC and clinical strains. In the examination with the FT-IR device, both compounds had bonds of OH carbohydrates proteins, polyphenols, C=O Amide I band, C-O-C polysaccharide, C-Namide III band, but one band named C=C conjugated, C≡C in both compounds showed the connection between thymol/ampicillin and thymol/cefotaxime. The biofilm inhibition effect of thymol/ampicillin (62 %) was better than thymol/cefotaxime (39.28 %) on E. coli ATCC 25922. The bacterial killing time curve of the thymol/ampicillin at a lower concentration and time was better than the combination of thymol/cefotaxime. Cytotoxicity of synergistic compounds on RBCs and human skin fibroblasts (Ffk) was not different and was lower than that of Triton X-100. Discussion: Considering the antibiotic resistance of ampicillin and cefotaxime in the treatment of diseases caused by E. coli bacteria, the thymol/ampicillin in this study showed better antimicrobial, anti-biofilm, and bacterial killing time effects than the thymol/cefotaxime. This combination can be used as a new drug in patients after further studies. Escherichia coli Thymol Cefotaxime Ampicillin anti-biofilm antimicrobial Figures Figure 2 Figure 3 Figure 4 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Escherichia coli ( E. coli ) is the most prevalent cause of infectious illnesses that extend beyond the gastrointestinal tract to extra-intestinal sites, such as urinary tract infections (UTIs), septicemia, and neonatal meningitis [ 1 ]. Gram-negative Enterobacteriaceae is a family of bacteria that includes the rod-shaped bacterium E. coli [ 2 ]. Over the past ten years, an increasing worry for public health has arisen due to the rise in antibiotic resistance in Enterobacteriaceae isolates. E. coli is a major concern when it comes to hospital and community-acquired infections, according to estimates of global antibiotic resistance from the World Health Organization (WHO) in 2014. Strains of E. coli that produce extended-spectrum beta-lactamases (ESBLs), plasmid-mediated AmpC (pAmpC), carbapenemases, resistance to the third-generation cephalosporin, and penicillin family and have spread globally as a result of the selective pressure exerted by antimicrobials [ 3 ]. A rise in resistance in E. Coli isolates has also been reported from Iran [ 4 ]. But when E. coli infections that are resistant to antibiotics proliferated, scientists and researchers searched for a strategy to combine the effects of these drugs [ 5 ]. Due to this, a great deal of research is being done on how to make antibiotics more effective and how to combat antibiotic resistance. This includes several studies on the potential benefits of using essential oils in combination with antibiotics. Research has indicated that integrating these approaches is among the most effective methods and techniques for combating bacterial infections. Thymol is one of these essential oils [ 6 ]. The main monoterpene thymol (2-isopropyl-5-methylphenol) is found in both black seed oil and thyme essential oil. Plants release it. This essential oil aids in chemical defense against plant pathogens [ 7 ]. Thymol possesses immune system modulators, antioxidant, anti-inflammatory, and anti-cancer properties. Gram-negative bacteria like E. coli are inhibited by this essential oil [ 8 ]. Using clinical strains of E. coli , we investigate the antibacterial synergism of thymol/ampicillin and thymol/cefotaxime to address widespread bacterial infections and antibiotic resistance in microorganisms. 2. Materials and methods 2.1. Antimicrobial compounds In this research, thymol, ampicillin, and cefotaxime were used for antimicrobial evaluation. Galaxir Pars Company (Mashhad, Iran) provided thymol 97%, while Dioxir Company (Tehran, Iran) supplied ampicillin and cefotaxime as antibiotics. Before their utilization for antimicrobial tests and other purposes, both were stored in a freezer at -20°C. The first 4096 µg/mL thymol concentration was diluted with distilled water to a manageable concentration of 1024 µg/mL before they were put to use. Analogously, ampicillin and cefotaxime were diluted with distilled water from 1024 µg/mL to 256 µg/mL, which was a practical quantity. 2.2. Bacterial strains In this study, E. coli ATCC 25922 was prepared from the microbiology laboratory of Zanjan University of Medical Sciences to perform antimicrobial tests. 2.3. In Vitro antimicrobial assay The antimicrobial properties of thymol, ampicillin, and cefotaxime were evaluated separately and in combination. Thymol, ampicillin, and cefotaxime were tested using the Micro Dilution Broth and Agar Dilution (MIC-MBC) method. Thymol was tested at an initial concentration of 1024 µg/ml, while ampicillin and cefotaxime were tested at an initial concentration of 256 µg/ml. In the first step, serial dilution was done in microplate wells separately for thymol, ampicillin, and cefotaxime. Then, 100 µl of E. coli ATCC 25922 suspension containing 1.5×10 6 CFU/ml was added to each well. A column containing a culture medium without bacteria was used as a negative control. The samples were incubated at 37°C for 24 h. The MBC agar dilution method was used to determine the effective concentration of thymol, ampicillin, and cefotaxime. In the second step, the same method was used to investigate the antimicrobial and synergism effects of thymol/ampicillin and thymol/cefotaxime. The combinations were tested according to Table 1 . From the wells that gave MIC, 10 µl were taken and cultured in MHB medium and placed in a 37°C incubator for 24 h. The next day, the MBC concentration of the compounds was determined [ 9 ]. Table 1 Analyzing the effects of various thymol/cefotaxime and thymol/ampicillin combinations on various strains of E. coli ATCC 25922 Strain Thymol Cefotaxime Ampicillin A1 A2 A3 A4 Thymol + Ampicillin/Cefotaxime Thymol + Ampicillin/Cefotaxime Thymol + Ampicillin/Cefotaxime Thymol + Ampicillin/Cefotaxime E. coli 1024 256 256 1024 256 1024 128 512 256 512 128 2.4. Checkerboard assay To evaluate the potential synergy between thymol/ampicillin and thymol/cefotaxime, the checkerboard method was utilized. Thymol was diluted in columns 1 through 7 of a 96-well microtiter plate, while ampicillin was diluted across rows A to G on the same plate. In the first dilution of both products, the bacterium's MIC was four times greater. Next, a bacterial slurry prepared by the McFarland standard was used to infect the plates. The plates were then incubated at the proper temperature for an entire day. The fractional inhibitory concentration index (FICI) was calculated for each chemical to determine the type of interaction between thymol/ampicillin. This calculation is based on the following formula [ 10 ]: FICI = FICA + FICB = (CA/MICA) + (CB/MICB) Thymol and cefotaxime were likewise treated using the previously mentioned methods. According to standards provided by the European Committee on Antimicrobial Sensitivity Testing [ 11 ], a FICI score of less than 0.5 indicates synergy, whereas a value between 0.5 and 1 indicates increased ability. A value of greater than 1 to 2 indicates a "lack of interaction" between the components, while values of FICI ≥ 2 demonstrate opposing effects [ 12 , 13 ]. 2.5. Fourier-Transform Infrared Spectroscopy (FT-IR) The regular deconvolution method was used to study the infrared spectroscopy of thymol, ampicillin, and cefotaxime separately and in combination [ 14 , 15 ] In short, the samples were powdered and dispersed in KBr pellets and then recorded using a Burker TENSOR 27 instrument. The FT-IR spectrometer (Thermo Scientific, USA) was used [ 14 , 15 ]. 2.6. In Vitro antimicrobial assay of clinically isolated E. coli strains Twenty different clinical strains of E. coli were obtained from laboratories and hospitals in the Tehran province. The standard and conventional biochemical microbiology tests that were used to identify these strains included indole sulfide mobility (SIM), citrate, methyl red (MR), Voges Proskauer (VP), triple sugar iron (TSI), urease (urea), gelatinases, lysine decarboxylase (LDC), and oxidases [ 16 ]. The susceptibilities of the strains to the several antibiotics listed in Table 2 were investigated, and some strains were multidrug-resistant (MDR) and some extensively drug-resistant (XDR). The method used in this test was the MIC-MBC method. Table 2 Clinical E. coli strains identified from urine samples and an inventory of antibiotic resistance Antibiotics Ciprofloxacin Cefixime Ceftriaxone Trimethoprim/sulfamethoxazole Piperacillin Ampicillin Nitrofurantoin Amikacin Gentamicin Meropenem Imipenem Amoxicillin E. coli clinical strains - - - - - - - - - - - E. coli 1 S S S R R R S S S S S R E. coli 2 I S S S R R S S S S S R E. coli 3 R R R R R R S S S I R R E. coli 4 R S S S I R I S S R I R E. coli 5 S S S R S S S S S S I S E. coli 6 I S S S R R S S S S I R E. coli 7 R R R R R R S S S S S R E. coli 8 R R R R R R S S S S S R E. coli 9 S R R R R R S S S S S R E. coli 10 S S S R R R S S S S S R E. coli 11 R R R S R R S S S R R R E. coli 12 S S S S S R R S S S S R E. coli 13 S R R R R R S S S S S R E. coli 14 R R R R R R S S R S S R E. coli 15 S S S R R R S S S S S R E. coli 16 S S S R R R S S S S S R E. coli 17 S S S R R R S S S S S R E. coli 18 S S S R R R S S S S S R E. coli 19 S S S R R R S S S S S R E. coli 20 S S S R R R S S S S S R 2.7. Antibiofilm assay The study aimed to determine the ability of thymol, ampicillin, and cefotaxime, both individually and in combination (A1 and A3 compounds, Tables 3 and 4 ), to inhibit biofilm formation in E. coli ATCC 25922. The microtiter plate method was used for this purpose. Initially, three to five colonies of the bacterial strain were suspended in 5 ml of Tryptone Soy Broth containing 1% glucose (TSB). After that, the mixture was left undisturbed for 24 h at 37°C. A 96-well plate was then filled with 100 µL of TSB medium in each well. The previously listed antimicrobial agents were added to the first well, and TSB culture medium was subsequently added to the remaining wells until the total volume reached 200 µl. The microplate was then incubated at 37°C for 24 h. After a day, the growth medium in the microplate was removed, and the wells were washed three times with phosphate-buffered saline (PBS). After that, 200 µl of 99% methanol was added to each well and let to stand at room temperature for 5 min. After the methanol was removed, 200 µl of 1% crystal violet dye was added to each well, and it was left to settle at room temperature for 5 min. PBS was used to wash the wells once more after the dye was removed. Following the injection of 200 µl of 96% ethanol, the optical density (OD) of every well was measured at a wavelength of 590 nm. The proportion of biofilm inhibition was then calculated using the following formula [ 17 , 18 ]: 2.8. Time-kill assay This test uses the MIC technique to determine how long an antimicrobial agent is effective. First, a 96-well microplate was used to dilute thymol, ampicillin, and cefotaxime to concentrations of 1024 µg/ml, 256 µg/ml, and 256 µg/ml, respectively. Next, thymol/ampicillin and thymol/cefotaxime (A1 and A3 compounds, Tables 3 and 4 ) were diluted to a concentration of 1024/256 µg/ml for this approach. Finally, the microplates were incubated at 37°C for 6, 12, 18, and 24 h. 10 µL was extracted from each well every hour and grown in MHB medium. The count and lethality kinetics were evaluated after a 24 h cultivation period. In addition, the killing activity of thymol, ampicillin, and cefotaxime alone and their combination effects was characterized as the absence of colonies in MHA after 24 h [ 19 ]. 2.9. In Vitro hemolysis assay To perform this test, first 5 ml of fresh human blood was prepared, then the fresh blood was mixed with 0/5 ml of sodium citrate dihydrate so that the blood does not clot. Then, the fresh blood was centrifuged at 10,000 rpm for 10 min, after the centrifugation, the plasma was removed and washed twice with PBS. Finally, 2 ml of fresh blood was mixed with 48 ml of PBS to prepare 2% RBCs. Next, in the 96-well microplate, thymol, ampicillin, cefotaxime, thymol/ampicillin (A1 compound), and thymol/cefotaxime (A3 compound) were serially diluted in PBS, and finally, 100 ml of fresh blood was prepared in each well, and 2% RBCs was added to the microplate for 1 and 24 h. It was placed in the incubator at 37°C, and the OD of the microplates was read by the Eliza Reader model (Agilent, USA). The toxicity percentage of the compounds was calculated with the following formula [ 20 ]: Triton X-100 (0.1%) was used as the positive control, and PBS as the negative control. 2.10. In vitro cytotoxicity assay A study was conducted to evaluate the cytotoxicity of thymol, ampicillin, and cefotaxime individually and in combination against a human fibroblast cell line (Ffk) using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) bromide assay. Initially, 3×10 4 cells were cultured in 96-well microplates using 90% DMEM (Dulbecco's modified Eagle's mix) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Subsequently, the cells were cultured at 37°C for 24 h in a humidified atmosphere containing 5% CO 2 . Serial doses of thymol, cefotaxime, and ampicillin were administered to each well, both singly and in combination. After that, the wells were incubated at 37°C for 24 h. The next day, 20 µL of MTT solution at a concentration of 5 µg/mL was added to each well, and the wells were incubated at 37°C for 24 h. After the supernatant was removed, 100 µl of dimethyl sulfoxide (DMSO) was added to each well, and the wells were shaken for 15 min to enable the cells to release the insoluble purple formazan. Finally, the samples' OD value at 570 nm was measured using a microplate spectrophotometer. The percentage of cytotoxicity was calculated using the following formula [ 20 ]: As the experiment's positive and negative controls, respectively, peptide-free cell suspension and Triton X-100 were employed. 3. Results 3.1. Investigation of antimicrobial activity (MIC-MBC) Table 3 The MIC and MBC assays were used to examine the synergistic effects of thymol/ampicillin on E. coli ATCC 25922, both separately and in combination Strain Thymol Ampicillin Thymol + Ampicillin MIC µg/ml Thymol + Ampicillin MBC µg/ml ΣFICI E. coli ATCC 25922 MIC µg/ml MBC µg/ml MIC µg/ml MBC µg/ml Thymol Ampicillin Thymol Ampicillin Single composition 256 512 4 2 - - - - - - A1 - - - - 8 2 16 4 1 Addition A2 - - - - 64 8 128 16 4.25 Antagonism A3 - - - - 2 1 4 2 0.5 Synergistic A4 - - - - 2 1 4 2 0.5 Synergistic Table 4 The MIC and MBC assays were used to examine the synergistic effects of thymol/cefotaxime on E. coli ATCC 25922, both separately and in combination. Strain Thymol Cefotaxime Thymol + Cefotaxime MIC µg/ml Thymol + Cefotaxime MBC µg/ml ΣFICI E. coli ATCC 25922 MIC µg/ml MBC µg/ml MIC µg/ml MBC µg/ml Thymol Cefotaxime Thymol Cefotaxime Single composition 256 512 32 64 - - - - --- --- A1 - - - - 128 32 256 64 0.25 Synergistic A2 - - - - 256 32 512 64 1.5 Indifferent A3 - - - - 128 16 256 32 1 Addition A4 - - - - 64 16 128 32 3 Antagonism 3.1.1. Thymol The thymol MIC and MBC measurement results are shown in Table 3 . Its MIC was reported to be 256 µg/ml. However, its MBC was shown to be 512 µg/ml. 3.1.2. Antibiotics The investigation employed ampicillin and cefotaxime as antimicrobial drugs; the MIC and MBC test findings are shown in Table 3 . The ampicillin MIC and MBC values were found to be 4 and 2 µg/ml, respectively, while the cefotaxime values were 32 and 64 µg/ml. 3.1.3. Combinations At this point, the antibacterial efficacy of combining thymol with ampicillin and cefotaxime was examined using the concentrations indicated in Table 1 . Thymol/ampicillin's MIC varied between 2–64/1–8, and its MBC ranged from 4-128/2–16 µg/ml. Likewise, thymol/cefotaxime's MIC value was found to be between 64–256/16–32 µg/ml, while its MBC was found to be between 128–512/32–64 µg/ml. As a result, the antimicrobial effect of thymol/ampicillin was better than thymol/cefotaxime on E. coli strains. 3.2. Evaluation of synergy by checkerboard assay In thymol/ampicillin, the A1 compound with FICI 1 showed an additive effect, the A2 compound with FICI 4.25 showed an antagonism effect, and compounds A3 and A4 with FICI 0.5 showed a synergistic effect. In thymol/cefotaxime, the A1 compound with FICI 0.25 showed a synergistic effect, the A2 compound with FICI 1.5 showed an indifferent effect, the A3 compound with FICI 1 showed an additive effect, and compound A4 with FICI 3 showed antagonism effect. 3.3. Fourier-Transform Infrared Spectroscopy (FT-IR) The principal peaks in the combinations of thymol, ampicillin, and cefotaxime that were subjected to FT-IR spectroscopy showed values that varied from 592.61 to 3442.64 cm-1. These peaks are associated with the following: C = O ester group, C = O Amid I band, C-N Amid II band, C-O-C-polysaccharide, CH stretching alkene group, and OH carbohydrates, proteins, and polysaccharides. Thus, thymol/ampicillin and thymol/cefotaxime combos contain all of these ingredients. The molecule C = C conjugated and C ≡ C, which denotes the connection between thymol/ampicillin and thymol/cefotaxime, is what separates the combined and individual states. The effectiveness of antibiotics may be significantly impacted by this relationship. 3.4. Antimicrobial susceptibility assay Initially, twenty clinical strains of E. coli were chosen at random within the parameters of this experiment and described using biochemical techniques and differential testing. Compounds A1 and A3 (Tables 3 and 4 ) were tested for their antibacterial activity against clinical strains of E. coli . In general, the MIC and MBC of compounds A1 (Table 5 ) are respectively (thymol 128–512 µg/ml)/(ampicillin 32–128 µg/ml) and (thymol 256–1024 µg/ml)/(ampicillin 64–256 µg/ml) respectively. Was. Also, MIC and MBC of compounds A3 (Table 5 ) were (thymol 8161024 µg/ml)/(cefotaxime 4-256 µg/ml) and (thymol 32-2024 µg/ml)/(cefotaxime 8-512 µg/ml), respectively. As a result, the antimicrobial effect of thymol/ampicillin (A1 compound) was better than thymol/cefotaxime (A3 compound) on E. coli clinical strains. Table 5 Results of A1 and A3 compounds on E. coli clinical strains E. coli clinical strains Thymol + Ampicillin Thymol + Cefotaxime - MIC (µg/ml) MBC (µg/ml) MIC (µg/ml) MBC (µg/ml) E. coli 1 R/R R/R 256/64 256/64 E. coli 2 R/R R/R 256/64 512/128 E. coli 3 R/R R/R R/R R/R E. coli 4 R/R R/R 1024/256 2024/512 E. coli 5 R/R R/R R/R R/R E. coli 6 R/R R/R 512/128 1024/256 E. coli 7 R/R R/R R/R R/R E. coli 8 R/R R/R 128/32 256/64 E. coli 9 R/R R/R R/R R/R E. coli 10 R/R R/R R/R R/R E. coli 11 R/R R/R R/R R/R E. coli 12 R/R R/R R/R R/R E. coli 13 512/128 1024/256 R/R R/R E. coli 14 R/R R/R 16/4 32/8 E. coli 15 128/32 256/64 R/R R/R E. coli 16 256/64 512/128 R/R R/R E. coli 17 R/R R/R 256/64 256/64 E. coli 18 512/128 1024/256 64/16 64/16 E. coli 19 R/R R/R 64/16 64/16 E. coli 20 R/R R/R R/R R/R 3.5. Anti-biofilm evaluation Using concentrations of 1024, 256, and 256 µg/ml, respectively, the anti-biofilm effectiveness of thymol, ampicillin, and cefotaxime combinations was evaluated at this point. When thymol/ampicillin and thymol/cefotaxime were administered, a synergistic result was observed, as shown in Tables 3 and 4 . Thymol, ampicillin, and cefotaxime individually have biofilm formation inhibition rates of 29.69%, 60%, and 16.28%, respectively. On the other hand, thymol/ampicillin (A1 compound) and thymol/cefotaxime (A3 compound) together have inhibition rates of 62% and 39.28%, respectively, indicating that the separate compounds' biofilm development is higher than that of the combined state. Therefore, the inhibition rate of biofilm formation in thymol/ampicillin (A1 compound) was higher than in thymol/cefotaxime (A3 compound). 3.6. Time-kill assay Thymol at a concentration of 1024 µg/ml in 6, 12, 18, and 24 h was able to reduce the number of bacteria to zero at a concentration of 512 µg/ml. Ampicillin at a concentration of 256 µg/ml was able to reduce the number of bacteria to zero at a concentration of 128 µg/ml in the first 6 h and at a concentration of 8 µg/ml at 18, 12 and 24 h. Cefotaxime at an initial concentration of 256 µg/ml at 6 h at a concentration of 256 µg/ml, at 12 h at a concentration of 128 µg/ml, at 18 and 24 h at a concentration of 32 µg/ml was able to reduce the number of bacteria to zero. The thymol/ampicillin at a concentration of 1024/256 µg/ml in the first 6 h at a concentration of 128/32 µg/ml was able to reduce the number of bacteria to zero, but at 12, 18, and 24 h at a concentration of 32/4 µg/ml, it was able to reduce the number of bacteria to zero. The thymol/cefotaxime at a concentration of 256/512 µg/ml in the first 6 h at a concentration of 128/512 µg/ml, at 12 h at a concentration of 64/256 µg/ml, and 18 and 24 h at a concentration of 16/128 µg/ml was able to reduce the number of bacteria to zero. Therefore, thymol/ampicillin was faster and more effective in killing bacteria than thymol/cefotaxime. 3.7. In Vitro hemolysis assay Thymol, ampicillin, and cefotaxime showed 36.12, 9.05, and 8.33 times less cytotoxic activity against human RBCs than the positive control Triton X-100, respectively. They lysed 2.63%, 10%, and 8.33% of human RBCs, respectively. Additionally, thymol/ampicillin and thymol/cefotaxime showed 9.58 and 8.38 times less cytotoxic activity against human RBCs than the positive control Triton X-100, respectively. They lysed 2.63% and 8.38% of human RBCs, respectively, while Triton X-100 lysed 95% of human RBCs (Fig. 9 ). As a result, both compounds had a lower toxicity effect against human RBCs than the positive control. 3.8. In vitro cytotoxicity assay Thymol, ampicillin, and cefotaxime showed 19.66, 8.45, and 7.08 times less cytotoxic activity than Triton X-100 against Ffk cells and lysed 4.83%, 11.23% and 13.41% of human Ffk cells. Also, thymol/ampicillin and thymol/cefotaxime showed 9.03 and 9.03 times less cytotoxic activity than Triton X-100 against Ffk cells, and lysed 10.51%, 10.51% of human Ffk cells, respectively. While Triton X-100 lysed 95% of human Ffk. (Fig. 10 ). As a result, both compounds had a lower toxicity effect against Ffk cells than the positive control. 4. Discussion The widespread use of antibiotics in the treatment of infections has led to the emergence of MDR bacteria [ 21 ]. Ampicillin and sometimes cefotaxime are among the drugs used in the treatment of infections caused by E. coli bacteria isolated from UTIs, but significant resistance to these drugs has been reported in the world, even 30–90% or more, and practically they cannot inhibit or kill E. coli bacteria [ 22 ]. The emergence of antibiotic resistance is a primary global health concern. Consequently, there is an urgent need for new strategies to combat antibiotic-resistant bacteria [ 23 ]. One of these solutions is the combination of medicinal plants and antibiotics as an alternative to treat bacterial infections [ 24 ]. One of these compounds is the active substance isolated from the extract of medicinal plants such as thymol [ 25 ]. Thymol causes death in bacteria by increasing the permeability of the cell membrane [ 25 ]. The purpose of this study was to compare the antimicrobial, anti-biofilm, and cytotoxic effects of thymol/ampicillin and thymol/cefotaxime on E. coli ATCC 25922 and clinical strains isolated from UTIs. In this study, according to the obtained results, the antimicrobial effect of thymol/ampicillin (A1 compound) on E. coli bacteria (FICI: 1) was better than thymol/cefotaxime (A3 compound) (FICI: 1). The antimicrobial effect of A1, and A3 compounds on the strains isolated from UTIs were 32–128/64–512 µg/ml and 4–16/256–1024 µg/ml, respectively, that the effect of thymol/cefotaxime was better than thymol/ampicillin. The effect of inhibiting biofilm in the A1 compound (62%) was better than in the A3 compound (39.28%). The killing time of bacteria in thymol/ampicillin was lower in concentration and time than in thymol/cefotaxime. Both compounds were examined by the FT-IR device and showed thymol/ampicillin and thymol/cefotaxime combination. A1 and A3 compounds on human RBCs and Ffk cells were not significantly different, but they were much lower than the positive control Triton X-100. In the study of Zhuocheng Yao et al. in 2022, thymol/colistin showed MIC 128/1024 µg/ml and an antimicrobial synergistic effect (FICI: 0.5) on E. coli bacteria. This compound was able to reduce the growth rate of bacteria to zero in 24 h. It also had an anti-biofilm effect on bacteria, which was consistent with our study [ 26 ]. In the study of Sumaira Noreen et al. in 2018, they investigated the antimicrobial effect of the combination of aqueous and ethanolic extracts of cloves, cinnamon, and garlic with ampicillin, imipenem, ciprofloxacin, norfloxacin and nalidixic acid on E. coli bacteria isolated from UTIs by disc diffusion method Done. Thus, the antimicrobial effect of clove extract alone was 27 mm, cinnamon 16 mm, and garlic 14 mm, and in combination with antibiotics, they showed 10 mm more, which was in line with our study [ 27 ]. In the study of Fatemeh Tavakoli et al. in 2022, the antimicrobial effect of methanolic extract of Salvia limbate with ceftazidime on Staphylococcus aureus ( S. aureus ) and Pseudomonas aeruginosa ( P. aeruginosa ) bacteria was carried out. The combination of this extract with ceftazidime could show a better antimicrobial effect with MIC 0.5 mg/ml and a slight synergistic effect against these bacteria, which was consistent with our study [ 28 ]. In our study, the MIC score was lower than in this study, which indicates that the effect of active ingredients in combination is better than raw plant extracts. In the study of Sohini Mitra et al. in 2024, the antimicrobial effect of the combination of aqueous extract of Psidium guajava (guava) plant with cefotaxime was performed. The combined effect of these two substances was able to induce 30% bactericidal inhibition in E. coli bacteria, which was consistent with the study [ 29 ]. Thymol/cefotaxime showed a better antimicrobial effect. In the study of Oumaima Amassmoud et al. in 2023, the antimicrobial effect of Thymus pallidus and T. broussonnetii plants with ciprofloxacin and streptomycin was performed. These compounds had a MIC of 128 mg/ml and a synergistic antimicrobial effect, which was consistent with our study. The active ingredient of these plants is thymol, and it has been able to induce its antimicrobial effect with the mentioned antibiotics [ 30 ]. In the study of Anwesa Bag et al. in 2011, the antimicrobial effect of Terminalia chebula fruit extract with ceftazidime was performed by MIC, checkerboard, and kinetics of killing bacteria [ 31 ]. The antimicrobial effect of these two substances caused a synergistic effect, and the bacterial killing time was better in the combined state than in the single state, which was in line with our study. Therefore, the compounds in the phenolic plant extract induce an antimicrobial effect, and the effect of these compounds in the isolated state is better than with the whole extract. Compared to previous studies, this study showed a better antimicrobial, anti-biofilm effect, kinetic killing time, and less toxicity in both thymol/ampicillin and thymol/cefotaxime. These compounds in the FT-IR device had organic compounds, including OH carbohydrates proteins and polysaccharides, CH stretching alkene group, C-O-C-polysaccharide, C = O ester group, C = O Amid I band, and C-N Amid II band. However, the antimicrobial, anti-biofilm, and other effects were due to the combination of C = C conjugated and C ≡ C, which binds these substances together and shows the inhibitory and lethal effect on bacteria. In addition to the antimicrobial and anti-biofilm effects, the fast kinetic killing time in the combined state and less toxicity against RBCs and human Ffk cells are related to this triple bond that has created the connection of these two substances. The effects of thymol are well-known in various studies. Thymol is a phenolic compound extracted from the Shirazi thyme plant. According to studies, thymol has structural isomers and a phenolic hydroxyl in the ring. The hydroxyl group increases its hydrophilicity, disrupts membrane integrity, increases membrane permeability, and finally causes proton and potassium leakage and leads to loss of membrane potential in E. coli [ 32 ]. Ampicillin and ceftazidime cause osmotic instability of bacteria by inhibiting the synthesis of the bacterial cell wall and have bactericidal effects. As a result, these compounds confirm the effect of synergism in the killing rate of E. coli bacteria compared to the individual state of these substances [ 33 ]. Conclusion This comparative study showed that the thymol/ampicillin (A1 compound) compared to the thymol/cefotaxime (A3 compound) has less antimicrobial, anti-biofilm, and toxicity effects on E. coli bacteria. According to the results obtained from different tests, thymol/ampicillin can be one of the options to reduce antibiotic resistance and treat urinary infections caused by E. coli after passing the complementary tests. Declarations Ethical Approval This study has been approved by the Vice President of the Research and Ethics Committee of Zanjan University of Medical Sciences. Consent for publication There is no limit to the publication. All authors consented to the publication of this research. Funding This work was financially supported by Zanjan University of Medical Sciences, Zanjan, Iran. Conflicts of interest The authors declare no conflict of interest. Availability of data and materials All data generated or analyzed during this work are included in this published article. Also, all data used to support the findings of this study are available from the corresponding author upon request. Acknowledgments This study was done with the financial support of the Zanjan University of Medical Sciences Research Vice-Chancellor, so the authors express their gratitude to all colleagues. Author contributions The idea of this study was designed by SJ and BM. Performing practical work and collecting clinical samples was done by SJ. Data analysis was done by MSh. The article was written by SJ, MSh, and MT. All authors read and approved the manuscript. References Pokharel P, Dhakal S, Dozois CM. The Diversity of Escherichia coli Pathotypes and Vaccination Strategies against This Versatile Bacterial Pathogen. Microorganisms. 2023;11(2):344. Jang J, et al. Environmental Escherichia coli: ecology and public health implications-a review. J Appl Microbiol. 2017;123(3):570–81. Buonsenso D, et al. Escherichia coli resistance patterns, empiric and targeted antibiotic prescriptions in children: a single center experience. Acta Biomed. 2022;93(5):e2022214. Aslam B, et al. Antibiotic resistance: a rundown of a global crisis. Infect Drug Resist. 2018;11:1645–58. Goldstein E. Rise in the prevalence of resistance to extended-spectrum cephalosporins in the USA, nursing homes and antibiotic prescribing in outpatient and inpatient settings. J Antimicrob Chemother. 2021;76(11):2745–7. Alaoui Mdarhri H et al. Alternatives Therapeutic Approaches to Conventional Antibiotics: Advantages, Limitations and Potential Application in Medicine. Antibiot (Basel), 2022. 11(12). Nagoor Meeran MF, et al. Pharmacological Properties and Molecular Mechanisms of Thymol: Prospects for Its Therapeutic Potential and Pharmaceutical Development. Front Pharmacol. 2017;8:380. Albureikan MOI, Alotaibi LMA. Antibacterial activity of chloroxylenol and thymol against pathogenic bacteria isolated from under long nails. Eur Rev Med Pharmacol Sci. 2023;27(9):3922–30. Sultan MH, et al. Bioactive Principles and Potentiality of Hot Methanolic Extract of the Leaves from Artemisia absinthium L in vitro Cytotoxicity Against Human MCF-7 Breast Cancer Cells, Antibacterial Study and Wound Healing Activity. Curr Pharm Biotechnol. 2020;21(15):1711–21. White RL, et al. Comparison of three different in vitro methods of detecting synergy: time-kill, checkerboard, and E test. Antimicrob Agents Chemother. 1996;40(8):1914–8. Definitive Document EUCAST. E.Def 1.2, May 2000: Terminology relating to methods for the determination of susceptibility of bacteria to antimicrobial agents. Clin Microbiol Infect, 2000. 6(9): pp. 503-8. Hu ZQ, et al. Epigallocatechin gallate synergistically enhances the activity of carbapenems against methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2002;46(2):558–60. Novy P, et al. Synergistic interactions of epigallocatechin gallate and oxytetracycline against various drug resistant Staphylococcus aureus strains in vitro. Phytomedicine. 2013;20(5):432–5. Buslov DK, Nikonenko NA. A Priori Estimation of the Parameters of the Method of Spectral Curve Deconvolution. Appl Spectrosc. 1998;52(4):613–20. Mirzaei B, et al. Purification and Evaluation of Polysaccharide Intercellular Adhesion (PIA) Antigen from Staphylococcus epidermidis. Curr Microbiol. 2016;73(5):611–7. Sadeghi H, et al. A retrospective cross-sectional survey on nosocomial bacterial infections and their antimicrobial susceptibility patterns in hospitalized patients in northwest of Iran. BMC Res Notes. 2021;14:1–7. Moghbel A, Hemmati A. Comparison of effects of anti-inflammatory gel prepared by glysertinic acid in licorice extract and plant rhizome with hydrocortisone ointment in rats . PhD Thesis. Ahwaz, Ahwaz Universiy of Med Sci 2008.[in Persian]. Tokam Kuaté CR, Bisso Ndezo B, Dzoyem JP. Synergistic antibiofilm effect of thymol and piperine in combination with aminoglycosides antibiotics against four Salmonella enterica serovars. Evidence-based Complementary and Alternative Medicine, 2021. 2021: pp. 1–9. Mandala SM, et al. Rustmicin, a potent antifungal agent, inhibits sphingolipid synthesis at inositol phosphoceramide synthase. J Biol Chem. 1998;273(24):14942–9. Sarfaraj HM et al. Marine natural products: A lead for Anti-cancer. 2012. Salam MA, et al. Antimicrobial Resistance: A Growing Serious Threat for Global Public Health. Volume 11. Healthcare (Basel); 2023. 13. Lee DS, Lee SJ, Choe HS. Community-Acquired Urinary Tract Infection by Escherichia coli in the Era of Antibiotic Resistance. Biomed Res Int, 2018. 2018: p. 7656752. Chinemerem Nwobodo D, et al. Antibiotic resistance: The challenges and some emerging strategies for tackling a global menace. J Clin Lab Anal. 2022;36(9):e24655. Vaou N et al. Towards Advances in Medicinal Plant Antimicrobial Activity: A Review Study on Challenges and Future Perspectives. Microorganisms, 2021. 9(10): p. 2041. Escobar A, et al. Thymol bioactivity: A review focusing on practical applications. Arab J Chem. 2020;13(12):9243–69. Yao Z, et al. Thymol Increases Sensitivity of Clinical Col-R Gram-Negative Bacteria to Colistin. Microbiol Spectr. 2022;10(4):e0018422. Naseer F. Antibacterial Activity of Medicinal Plants (Clove, Cinnamon, Garlic) Extracts and their Combined Effect with Antibiotics in Urinary Tract Infection Caused by Escherichia coli. International Journal of Pharmaceutics & Pharmacology; 2018. Tavakoli* F, et al. Synergistic Activity of Three Iranian Medicinal Plants in Combination with Ceftazidime and Neomycin against Bacterial Strains Causing Nosocomial Infections. Res J Pharmacognosy. 2022;9(3):51–9. Mitra S, Bhesania Hodiwala AV, Kar H. Susceptibility and Synergistic Effects of Guava Plant Extract and Antimicrobial Drugs on Escherichia coli. Cureus. 2024;16(1):e52345. Amassmoud O, et al. Antibacterial Activity of Essential Oils Combinations based on Thymus broussonnetii, and Their Synergism with some Antibiotics. Curr Microbiol. 2023;80(12):398. Bag A, et al. Synergistic Effect of Terminalia chebula and Antibiotics against Multidrug-resistant Uropathogenic Escherichia coli. Med aromatic plant Sci Biotechnol. 2010;5(1):70–3. Modareskia M, Fattahi M, Mirjalili MH. Thymol screening, phenolic contents, antioxidant and antibacterial activities of Iranian populations of Trachyspermum ammi (L.) Sprague (Apiaceae). Sci Rep. 2022;12(1):15645. Cho H, Uehara T, Bernhardt TG. Beta-lactam antibiotics induce a lethal malfunctioning of the bacterial cell wall synthesis machinery. Cell. 2014;159(6):1300–11. Additional Declarations No competing interests reported. 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09:53:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6177131/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6177131/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78635510,"identity":"31be96ca-f4e9-41fe-9efb-1aa3933bcd57","added_by":"auto","created_at":"2025-03-17 05:04:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":94088,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR results for ampicillin\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6177131/v1/a6af0c88f15bbdd682e817ec.png"},{"id":78636732,"identity":"8605ab58-f266-44bc-bb7c-5a134081cea9","added_by":"auto","created_at":"2025-03-17 05:28:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":73383,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR result for cefotaxime\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6177131/v1/c9111715fde0ff4751446a6c.png"},{"id":78635509,"identity":"39313f16-b40a-483d-a2e3-d010f75691c3","added_by":"auto","created_at":"2025-03-17 05:04:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":67981,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR results for thymol/ampicillin\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6177131/v1/e81c659b3128973dcdc46b1f.png"},{"id":78636735,"identity":"8a66df25-2d20-43cc-8e01-491bff55ace9","added_by":"auto","created_at":"2025-03-17 05:28:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":110466,"visible":true,"origin":"","legend":"\u003cp\u003eBiofilm results for \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6177131/v1/7cb9bb6c652515158f42880f.png"},{"id":78635521,"identity":"049612ad-904b-4fe8-8836-5074864c99f4","added_by":"auto","created_at":"2025-03-17 05:04:56","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":105125,"visible":true,"origin":"","legend":"\u003cp\u003eKinetic results of Thymol/Ampicillin on \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6177131/v1/76088cba8d1d27be6a162de1.png"},{"id":78636736,"identity":"87e6d44a-9085-4f08-94c9-3b772224c407","added_by":"auto","created_at":"2025-03-17 05:28:56","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":110565,"visible":true,"origin":"","legend":"\u003cp\u003eKinetic results of Thymol/Cefotaxime on \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-6177131/v1/ac40ee153cbdfea0eecbd0ca.png"},{"id":78635529,"identity":"c029b06e-61f2-40a2-a1d7-60a9399865dd","added_by":"auto","created_at":"2025-03-17 05:04:56","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":113233,"visible":true,"origin":"","legend":"\u003cp\u003eThymol, ampicillin, and cefotaxime’s individual and combined toxicity on human RBCs\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-6177131/v1/3cc4f890d45404aaee2a5d46.png"},{"id":78637246,"identity":"45af75b3-8a12-40c6-b210-93d1716f73c3","added_by":"auto","created_at":"2025-03-17 05:36:56","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":24642,"visible":true,"origin":"","legend":"\u003cp\u003eToxicity of thymol, ampicillin, and cefotaxime, individually and in combination, on human Ffk cells\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-6177131/v1/c0a4b1dbb7f042fa8ba567e9.png"},{"id":80738255,"identity":"33f9463d-7621-4c26-b541-7a2404af073f","added_by":"auto","created_at":"2025-04-16 13:53:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2170367,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6177131/v1/9158d95c-e8d2-48c3-a37f-72aa1407cbac.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparing the Antimicrobial, Anti-Biofilm, and Cytotoxic Effects of Thymol/Ampicillin and Thymol/Cefotaxime against Escherichia coli Bacteria","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) is the most prevalent cause of infectious illnesses that extend beyond the gastrointestinal tract to extra-intestinal sites, such as urinary tract infections (UTIs), septicemia, and neonatal meningitis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Gram-negative Enterobacteriaceae is a family of bacteria that includes the rod-shaped bacterium \u003cem\u003eE. coli\u003c/em\u003e [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOver the past ten years, an increasing worry for public health has arisen due to the rise in antibiotic resistance in Enterobacteriaceae isolates. \u003cem\u003eE. coli\u003c/em\u003e is a major concern when it comes to hospital and community-acquired infections, according to estimates of global antibiotic resistance from the World Health Organization (WHO) in 2014. Strains of \u003cem\u003eE. coli\u003c/em\u003e that produce extended-spectrum beta-lactamases (ESBLs), plasmid-mediated AmpC (pAmpC), carbapenemases, resistance to the third-generation cephalosporin, and penicillin family and have spread globally as a result of the selective pressure exerted by antimicrobials [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. A rise in resistance in E. Coli isolates has also been reported from Iran [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBut when \u003cem\u003eE. coli\u003c/em\u003e infections that are resistant to antibiotics proliferated, scientists and researchers searched for a strategy to combine the effects of these drugs [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDue to this, a great deal of research is being done on how to make antibiotics more effective and how to combat antibiotic resistance. This includes several studies on the potential benefits of using essential oils in combination with antibiotics. Research has indicated that integrating these approaches is among the most effective methods and techniques for combating bacterial infections. Thymol is one of these essential oils [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe main monoterpene thymol (2-isopropyl-5-methylphenol) is found in both black seed oil and thyme essential oil. Plants release it. This essential oil aids in chemical defense against plant pathogens [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Thymol possesses immune system modulators, antioxidant, anti-inflammatory, and anti-cancer properties. Gram-negative bacteria like \u003cem\u003eE. coli\u003c/em\u003e are inhibited by this essential oil [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUsing clinical strains of \u003cem\u003eE. coli\u003c/em\u003e, we investigate the antibacterial synergism of thymol/ampicillin and thymol/cefotaxime to address widespread bacterial infections and antibiotic resistance in microorganisms.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Antimicrobial compounds\u003c/h2\u003e\n \u003cp\u003eIn this research, thymol, ampicillin, and cefotaxime were used for antimicrobial evaluation. Galaxir Pars Company (Mashhad, Iran) provided thymol 97%, while Dioxir Company (Tehran, Iran) supplied ampicillin and cefotaxime as antibiotics. Before their utilization for antimicrobial tests and other purposes, both were stored in a freezer at -20\u0026deg;C. The first 4096 \u0026micro;g/mL thymol concentration was diluted with distilled water to a manageable concentration of 1024 \u0026micro;g/mL before they were put to use. Analogously, ampicillin and cefotaxime were diluted with distilled water from 1024 \u0026micro;g/mL to 256 \u0026micro;g/mL, which was a practical quantity.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. Bacterial strains\u003c/h2\u003e\n \u003cp\u003eIn this study, \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 was prepared from the microbiology laboratory of Zanjan University of Medical Sciences to perform antimicrobial tests.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. \u003cem\u003eIn Vitro\u003c/em\u003e antimicrobial assay\u003c/h2\u003e\n \u003cp\u003eThe antimicrobial properties of thymol, ampicillin, and cefotaxime were evaluated separately and in combination. Thymol, ampicillin, and cefotaxime were tested using the Micro Dilution Broth and Agar Dilution (MIC-MBC) method. Thymol was tested at an initial concentration of 1024 \u0026micro;g/ml, while ampicillin and cefotaxime were tested at an initial concentration of 256 \u0026micro;g/ml. In the first step, serial dilution was done in microplate wells separately for thymol, ampicillin, and cefotaxime. Then, 100 \u0026micro;l of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 suspension containing 1.5\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU/ml was added to each well. A column containing a culture medium without bacteria was used as a negative control. The samples were incubated at 37\u0026deg;C for 24 h. The MBC agar dilution method was used to determine the effective concentration of thymol, ampicillin, and cefotaxime. In the second step, the same method was used to investigate the antimicrobial and synergism effects of thymol/ampicillin and thymol/cefotaxime. The combinations were tested according to Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. From the wells that gave MIC, 10 \u0026micro;l were taken and cultured in MHB medium and placed in a 37\u0026deg;C incubator for 24 h. The next day, the MBC concentration of the compounds was determined [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAnalyzing the effects of various thymol/cefotaxime and thymol/ampicillin combinations on various strains of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStrain\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eThymol\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCefotaxime\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAmpicillin\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eA1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eA2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eA3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eA4\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eThymol\u0026thinsp;+\u0026thinsp;Ampicillin/Cefotaxime\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eThymol\u0026thinsp;+\u0026thinsp;Ampicillin/Cefotaxime\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eThymol\u0026thinsp;+\u0026thinsp;Ampicillin/Cefotaxime\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eThymol\u0026thinsp;+\u0026thinsp;Ampicillin/Cefotaxime\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e512\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e512\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Checkerboard assay\u003c/h2\u003e\n \u003cp\u003eTo evaluate the potential synergy between thymol/ampicillin and thymol/cefotaxime, the checkerboard method was utilized. Thymol was diluted in columns 1 through 7 of a 96-well microtiter plate, while ampicillin was diluted across rows A to G on the same plate. In the first dilution of both products, the bacterium\u0026apos;s MIC was four times greater. Next, a bacterial slurry prepared by the McFarland standard was used to infect the plates. The plates were then incubated at the proper temperature for an entire day. The fractional inhibitory concentration index (FICI) was calculated for each chemical to determine the type of interaction between thymol/ampicillin. This calculation is based on the following formula [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e]:\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFICI\u0026thinsp;=\u0026thinsp;FICA\u0026thinsp;+\u0026thinsp;FICB = (CA/MICA) + (CB/MICB)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThymol and cefotaxime were likewise treated using the previously mentioned methods.\u003c/p\u003e\n \u003cp\u003eAccording to standards provided by the European Committee on Antimicrobial Sensitivity Testing [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e], a FICI score of less than 0.5 indicates synergy, whereas a value between 0.5 and 1 indicates increased ability. A value of greater than 1 to 2 indicates a \u0026quot;lack of interaction\u0026quot; between the components, while values of FICI\u0026thinsp;\u0026ge;\u0026thinsp;2 demonstrate opposing effects [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5. Fourier-Transform Infrared Spectroscopy (FT-IR)\u003c/h2\u003e\n \u003cp\u003eThe regular deconvolution method was used to study the infrared spectroscopy of thymol, ampicillin, and cefotaxime separately and in combination [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e] In short, the samples were powdered and dispersed in KBr pellets and then recorded using a Burker TENSOR 27 instrument. The FT-IR spectrometer (Thermo Scientific, USA) was used [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6. \u003cem\u003eIn Vitro\u003c/em\u003e antimicrobial assay of clinically isolated \u003cem\u003eE. coli\u003c/em\u003e strains\u003c/h2\u003e\n \u003cp\u003eTwenty different clinical strains of \u003cem\u003eE. coli\u003c/em\u003e were obtained from laboratories and hospitals in the Tehran province. The standard and conventional biochemical microbiology tests that were used to identify these strains included indole sulfide mobility (SIM), citrate, methyl red (MR), Voges Proskauer (VP), triple sugar iron (TSI), urease (urea), gelatinases, lysine decarboxylase (LDC), and oxidases [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]. The susceptibilities of the strains to the several antibiotics listed in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e were investigated, and some strains were multidrug-resistant (MDR) and some extensively drug-resistant (XDR). The method used in this test was the MIC-MBC method.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eClinical \u003cem\u003eE. coli\u003c/em\u003e strains identified from urine samples and an inventory of antibiotic resistance\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAntibiotics\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCiprofloxacin\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCefixime\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCeftriaxone\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTrimethoprim/sulfamethoxazole\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePiperacillin\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAmpicillin\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNitrofurantoin\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAmikacin\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGentamicin\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMeropenem\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eImipenem\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAmoxicillin\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eclinical strains\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003e4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003e6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003e8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003e9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003e10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003e11\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003e12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003e13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003e14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003e15\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003e16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003e17\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003e18\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003e19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003e20\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7. Antibiofilm assay\u003c/h2\u003e\n \u003cp\u003eThe study aimed to determine the ability of thymol, ampicillin, and cefotaxime, both individually and in combination (A1 and A3 compounds, Tables \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e), to inhibit biofilm formation in \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922. The microtiter plate method was used for this purpose. Initially, three to five colonies of the bacterial strain were suspended in 5 ml of Tryptone Soy Broth containing 1% glucose (TSB). After that, the mixture was left undisturbed for 24 h at 37\u0026deg;C. A 96-well plate was then filled with 100 \u0026micro;L of TSB medium in each well. The previously listed antimicrobial agents were added to the first well, and TSB culture medium was subsequently added to the remaining wells until the total volume reached 200 \u0026micro;l. The microplate was then incubated at 37\u0026deg;C for 24 h. After a day, the growth medium in the microplate was removed, and the wells were washed three times with phosphate-buffered saline (PBS). After that, 200 \u0026micro;l of 99% methanol was added to each well and let to stand at room temperature for 5 min. After the methanol was removed, 200 \u0026micro;l of 1% crystal violet dye was added to each well, and it was left to settle at room temperature for 5 min. PBS was used to wash the wells once more after the dye was removed. Following the injection of 200 \u0026micro;l of 96% ethanol, the optical density (OD) of every well was measured at a wavelength of 590 nm. The proportion of biofilm inhibition was then calculated using the following formula [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]:\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\" style=\"width: 538px;\"\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8. Time-kill assay\u003c/h2\u003e\n \u003cp\u003eThis test uses the MIC technique to determine how long an antimicrobial agent is effective. First, a 96-well microplate was used to dilute thymol, ampicillin, and cefotaxime to concentrations of 1024 \u0026micro;g/ml, 256 \u0026micro;g/ml, and 256 \u0026micro;g/ml, respectively. Next, thymol/ampicillin and thymol/cefotaxime (A1 and A3 compounds, Tables \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) were diluted to a concentration of 1024/256 \u0026micro;g/ml for this approach. Finally, the microplates were incubated at 37\u0026deg;C for 6, 12, 18, and 24 h. 10 \u0026micro;L was extracted from each well every hour and grown in MHB medium. The count and lethality kinetics were evaluated after a 24 h cultivation period. In addition, the killing activity of thymol, ampicillin, and cefotaxime alone and their combination effects was characterized as the absence of colonies in MHA after 24 h [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9. \u003cem\u003eIn Vitro\u003c/em\u003e hemolysis assay\u003c/h2\u003e\n \u003cp\u003eTo perform this test, first 5 ml of fresh human blood was prepared, then the fresh blood was mixed with 0/5 ml of sodium citrate dihydrate so that the blood does not clot. Then, the fresh blood was centrifuged at 10,000 rpm for 10 min, after the centrifugation, the plasma was removed and washed twice with PBS. Finally, 2 ml of fresh blood was mixed with 48 ml of PBS to prepare 2% RBCs. Next, in the 96-well microplate, thymol, ampicillin, cefotaxime, thymol/ampicillin (A1 compound), and thymol/cefotaxime (A3 compound) were serially diluted in PBS, and finally, 100 ml of fresh blood was prepared in each well, and 2% RBCs was added to the microplate for 1 and 24 h. It was placed in the incubator at 37\u0026deg;C, and the OD of the microplates was read by the Eliza Reader model (Agilent, USA). The toxicity percentage of the compounds was calculated with the following formula [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]:\u003c/p\u003e\n \u003cp\u003e\u003cimg 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E38e7QjQF/K+oMcJaa2edfI7d/6RxIkS2+d4WlI+m1KyZ80uTZs2k/Vr18vKFSvV898bMu/VrF1TqlSrIlWrV/X5Yc2+6ytdA/0mvCGCBWHLczh+7LjTGjIoK+yPIEVpCy84h6Kw8Xwikka4WPFiunzDjP/C+QtO6z3c/qxaparUrl1HBfH8hQvURwBHUvrYu8/cf5He2BPeHZKDabvHJlc5mDJtqrzc4mVb+kaRTM9ncraGTLy48dSyERHry/1gFASDwRAqCKso9n+x48R2WoLiZsojDz6Oc6Fx/PhRHayZDbJuHhG2bN2iFgHy2vs7RurUqVVYNm/WXAUEzoUoAePHj9ftCMFp06aps2G/fv30Ptq2basCIzxhaf72vXeU4FYAf2iYp61PuIqTpzLigqDAfwPTM/cyatQo6d+/vy55YFrneSBMvUEhe9pWKLAC+PsgjFD2EOi+wBEV5YPte/bu8bufC8+YqA72Y7YcEQsCfY21CLN/QXuGHl64Z0IqEdKxYvsPrWSZqGu3rlKqdClZvWqVdOncRUqUKBFux0p/bw59ie9KsqTJ5Ny5s9K7T2/1RwkNlixUKfNSNCMboyAYDIZQiRkzYAb191++veMxjWOKvfHXDVm8ZLHT6p9t29+Tv2/+LaVLl1Ev90BsOYoiEhrMoFm7ZdBkECf3vzcIUDzuMRcvWrRIfQNKlyqt7fPmz9O8DpMnT5buPbvrTHz7e9ulU8dOqlQwkIflOiKDb7/7VqLGiBosPbEn3C/CjjwQntEWoYED5orlK2Td2nWyds1anx/W06dNnaYKlS94PpUrVlaB//EnH6tTZUgQevnF0S906SZYCuAwPGKWWogSYPbdqUMnSZY8ubPFPzwbT7A+8G5i0cmaJavTeg8UIhRWUhsTsYCz4LYt26RmzZr6nQfxPnCMu3fvSLUqVTW3A1Y3UosPGTrEpxLoCcsV/9z+x6fSF5kYBcFgMIRK/AQJdBBlbdsX+fLlk3Zt2ulAt269LXxs4e0PYsu3btkqadOklb59+jitAfB9nZHaYzEzRl+wtDDg1QFy+cpldQpr3dp3XnsEZ8tWLWXvvr06uJM7gTVmHBURFqxHY1EAohxck7xrMvZeLggJV3j4FiL28cIoW/BS37x5s0Y61K0TNJ+EWiMcQcLzRoAjPOfMnaNtLnjW7z+wP1zXH15wtMT3gqWDUSNHyR+/+84zQF9OnzFdlbFGDRups6eL9vXdgL7+y+4PX/A9+praDvgRdOrUydniH1/LM++//74+F6wrJHcC7SunXxC8RCmcPHVSFV0cEKNFDxDG7qw9rCGG3Jd2k1efByw1BQh7lL+Rr42UOLHjyJx5c+T1N1539vINljIsMSiyDxOjIBgMhlChQBADnK/1W5cBAwZoFTmcyV7p9ooWwvEeVEki07FzRzWjT5k0RfLkzuNsCYCZHgluWG8lcRCFpBAyzO5QDJYtX6ahhDiaDR82XIYMHhJstujCejcWhnHjxqkyAQhVBEPyZMn13ImTJFbFB78EwjhRGCj4QxvCd+mypYHOgpcvX5HbtuBB8bh56976PCV38YznOn759RenNYBLly7p+RBO3kSNHlU+/PBD+enHH3XwJ3KhU5dO6ucxY9qMwBk8ywncP/u41oKyZcpK0SJFVdiMe32cdO7SWXNRLFu2TGrVqaWhjpEJpn5CKdOnS68Ok63atNIcD55gOmd2zDOsXbu2jB412tkSAP4jrPXT1zhc8n2EK/d68eJFtRrQ10SADBo0SF577TXtF3+4ih0Otbt27dK/w5HDh9VJNU+evPq+uJCsCkUCXxiuhegHlj94zlTR5Hp430h+hA/C1m1bVfl1HU5dXxtvZ1COdcs+Bn3D34HfAQ6d3N9lx48AxZbiaCg09OHkyZP8KiGXfr2kCkfqNA+5UJd9wY8MWzvUPOO25qZ/94fdgZY9cOjfL/1ySYuCzJ4925o8ZbK2/b9ivxiWPehZhw9/rkU57IHJ2fL/hf0DsWwN1/nX/WP/aK3ffv3N+ZchsglLLQZ7oLOyZMtivfRyc6fFN7wHb7zxhpXp+UxW7HixrfIVylvDhg+z3hj/htW4SWMrZaqUVqXKlayDBw863wjgxo0b1qDBg7QQVIxYMayYsWNq3noKN7E/n4KFC1q58uSyXunezfrc/s2EhZ69emndB75rCxirStUqWtfAnqXr9j1792gRJM5FvQCu254da00J2qgrsXzFcmv4iOFahMoW6nptNWvWtC5cuGDt2bPHKl+xvBU7bmzdP0OmDNbUaVO1YFGv3r2sFClT6HeoR9CiRQt91tCiVQvNmx8rTiwrddrUVuGiha0sWe3n2/ylILUl5s+fZ72Q/wUt8sOH2hPzF8zXbRSxKlmqpB6Dc7Cdvzds3NCyFRPdJ7I5duyYVbduXa2BwDNr07aNNX7CeGvgoIFWocKF9NkOHTZUi3y58I7wTpQoWUKfpdvX7FuxUkXt60JFClk5c+eybMXH+vTTT51vhsy5r85Z2bJn02eQJFkS6+UWL1vNXmpmZciYQd8997ky7trKjZUjVw49L+8b793q1autiRMnWnGeiqPPkj4rVbqU1bZdW+1f3a9EMev9D963mtu/g0RJEulz58+ur3TVe7QVCCt/wfz6ffYvWaqENWbMGKtRo0ZaYCxqjKja3+7+tjKi18g76h5/y5Ytep2e9O3bV2txeBc48yQyajE8snLPnPbFF1+Uw0c+Vy2/SeOmGj/qDVoas5FGDRtKrVq1NcMZccPEXhNLTFnP/1eYbXTo0EHTuzIzaNyosSxbuszZ+mBBK8dkS7iMvxlXRGAteNTokTJp0mTJmOFehj1mfTeu31CNPGo031o/ffvbb79KFq81QWagzKRqVKuh8cGPA6z/cR+hrQFiCmRGwH2H9Jx5/7lPZibMoB4lzNILFCog/fv1V5O9P/C8JoPe/r37dV02JJjtYwE4fvy4nL9wXu8xfdr0UqpUKTWvepfu5f2kbDDvqOc2niWOdXHixlFPfZIchXZuT/hdkRSJ95T3ke/XqFEjSDbITw99Khs2bNC+wAxO1r+NmzaKLZikadOmGtJIJkV8JtiHFQPi+fF4Z6ZL6WB3ts+sMVmyZFK8WHGNiuDfrJ3ze+CDiR0v/patW8rSpUulVatWkiplKr3n8uXKS8GCBYPMkvft26eJgVzHupt/39QlkpIlS+q/mb2SYvnEyRM6Gy5durSegzH1YUEf8QyxDuGMiDWFvAd58uTRJFQZnsvg7BkA10lfM7v2fPdZbqDMM46ShJLSV+ENgcUisX//Ps0vwEw/SdIk+qzKlCkjMWMEPBP64YMPPtC8Au67xu+WpQdCFMkh8dFHH+n5mzZpqs8SiwLfa9O6jfYp18936Svunz8JnyVlMjUj3PtiX95X3mveA8YErARYlQitZZy49Msvstv+rTAmcCyiWIi88aRCxQq6nYRY/sYVoi3IyPmfKPdsPwiraLGi1sCBA7WqFjNpb7Ac1K5Ty2rarKnOKoHZ6vDhw1VLRNv8f4ZnYA8wqnWjybZq3crZ8mBhdtbSPnauPLm1nO6Dwv4haCnSGTNmOC0B2IOa1bBRQ6tchXLWi/XrWR/YGrcvevfpbdkDiM9KcPv279cZ2+bNm5yWhw9Wq9OnT1szZ820GjVuFGK5WVvIWr17B9xP6TKl7Zlgc78zn7179urx2LdM2TI6c35YMz5fhMWCAJTgTZg4oWULAqfFEFGwIDD82oLHaTEYfENJcyxvkyeHbDH/z1VzZDZGkgniWtEYvRk1epT8YGvPUyZPUe0LEiRIEJiMxA0g+n8FrZOKdPnz51ftNLJgDXOnPYs6fvSYHDt2zGm9P9CIu3btqlacLl26OK2ia8NNX2qqzkWjR46Wf22tt3HTJjrL8oR1xSVLlmhImc7KvChRvLj07d1X+vTtq7OShw1V5xo2bii2sNdQJIq+MMv1BaFRDRo2kHdWvyPt7Psh+9qRI4c1HztrzJ68++67UqdeHXvmd0WGDR2qs1ly8b/U/KUIZxR8WFSuVFnfV1tRcFoMEcW3M6PBEJw1a9fon9ToeNg8WidFW777E4yff/6ZLFy0UEaOGKmhOf9lQnK8eRAQlkSub7yBH5TJfuGiBfL9Dz+oo5gn9BlmV1LXYkomxzxOQLNnz3b2EHXwGjpsqJr+MOn6o2XLlnrto8cGdW56GGDGHTt6rDq4YT5GifFn2hv7+lhVgF4bPkIrHeIpPf6N8WpCHTp8aGB2QZ7LoMGDJF68uDJj+kwN8cOkT+w6NQE8C8I8jrA8RYrZLZs3y3fffuu0GiICpmQIq2e84cmEJSQcJdu1a6eptB82j1ZBCIE3xo+XTBkyatW3/zqWPcuObJo1ayZjx4yNcNY6T/DMnjptmjRs0CDI8Zhhf3boM80l7yauyZAhgyp4rF1fdVLXYjlgzRTlIqQ1fYQyPhqs/7Im+DDBA55QMmbNJI7x10dUI6TQD7H2VapUdVoDShoT38+6IDnZAWvCl6e/lOLFS2jxGpcG9etrMaBNmzcFeqn7gjVxnl1I7wsRADNmzgj0nn7QtHi5hWTOkkWG2ApeZFq9/qvgIU/ugC+OfaG5DvCMp+ofa+AGgzeEP5LKnGiHR8FjqSCQWIMiKy1atAzmzOQJ1ePQwHEG6v9qfw2FwWHH1c59gbMS+3Xr3k0WL14cmG9dw1CuXNESnK+//rqGJV24eEGzrPXq3UuTv7hJYpgRv7PqHTU9M8MMqawnAoLjvdKtmy6ZUNo2tIEVczZmXGLJcchcvXq1Xhv3RUYxzs02QprcJQPM08RDkyK1b7+++j13dsI9Ug6X6yCMyxME1pixY9T5kyI2YRHEm7Zs1llxk8ZBK49xfTiP4lDm9htLQ3zY/x/7ekiZ+sYbb0iXTp2DOeL4omKFipImdepHZtbGauDPcgAIfe6JmHA3XzrgpISCgECgzwAFAAi38oRyruTkp+78Z5995rQGh353E7n4grAtKtZhKQqt1GxEoR4BVQ+pXc/SiCF8EI9PyemoVhTJkzO3fPfNt/JS82ayZ8//r7O1IXLg9756zWoNeY1IeuoHAo4IjwIc9EqUKOEztG/q1KkaOoITnC82b9msIUmEBrVu09p6NtWz6mSFox/hNr7CH3FynDBhgpU1W1Z1BqxQsYIVO04sq2atmhqStH79eg0zevqZp9U5ztbcrBfyvaDH1fChuLGsbt1fsb744ohVo2YNK3mK5Fa8p+PpOXPmymmdPHnSOVMAhLD0H9BfQ39atmqpYVK58+a2non/jH2cbnpOl9lzZmuYi+ukOH78eOu5jM/p8bkfHNpsZcW6eeum9erAVzUcCGeUosWLWrYiZX3zzTcaUsQ9jR07xspfIL8VP0F868SJE9ZPP/1klSpTyoqfML6G0cyaPUvPAes3rNcwMs47ePAgvaeChQqqc6g/bOXGqlO3jobjeDsX3rp1S0OXCDHCaQ8IxSJUrHiJ4tbfN/+2unTtomFAbthqWCDMKFPmTOqgGhJcNw6ZYflwrWGBULa06dLqO3b06FGn9R62MkaKNatd+3ZOyz3Gjhur/Vq7bm3r2vVrGmrHu8S75glhX/Sdra5btpLntAbHVgKtOnXqaMgVYb6e2MqBvpex48ax5s2f57SGjbA6KXpiK5x6zfSvIezYSruGtvH+uR/+TbvB4ML4WblKZWvr1q1OS+hEhpPiY6kg1K1X18pXIJ917c97QtQTFARiVFEibG1cB26EZI+ePSx79qoC/ueff3b2DmDipIlWwkQJLXs2p/9GuBF/yqA8ZOgQFdgM6AhSPsS/vvvuu9bZs2etMWPHWgkSJtB2vPZHjx6t8b+7d++28ubLq7Gw9qxNj+uCckA7+7pwjQUKFdD2nr16Oq3BFQT4cM+HOmgnSJQgSBQAA0m1GtVU4P7xe4CQbd+hvV7biZMn9N+8XDlz5wwUFPasRePHESzz5gW0IaBz58ltFSlaJDCHwbZt21RhIKrEH3jbo6Cg5PiCqBSUpmXLlum/N27aqIrJkCFDrP0H9lvJkieztm7bat2+dduyZ98aN4wSExJcM97zREf4g4EWRQoFh+iYkD4oUOPGjXO+GTKhKQgof7xDnv3pgjJGX1eoVME6c+aMxl0T801/eILSVatOLX0HRo4a6bT6BiUBpZb3fO7cudp2+fJl/X7sOLbiMMe/guGPiCgIcOnSRc2PYDAYHizII28ZFhr/uSgGX7AGi6lV452f9h/vTCwtNb4nT56iBUvI7DWg3wBJnz69mlrPnTvn7BmwZDFp0iSNESaFKeuz1I8vVrSoUBQFsx/rPPXq1tOiHhSQWbhgodSqVUvXil/p2lVy5c4lf177Uzp37iy2EFTzeLly5eTl5i3UyZD4V3fpgPhvzP0U5cBL34Vr7P5KdzU/EwPtrk37gpzxfLjW9Rs2OK0i3377rRw7ekzrnMdPEF9N2Hj5230pX38VUK+c9fBhQ4YFpo5lPTxv7rxBll5Yq+Y5sSRAzDpUrFhR17o8Y5O9IU88yx08c1+0adNGnw1rZ2+++aaMGjVSsmXLJk2aNtH69TXtZ5o3Tx6tGtezVw+tnEfsLrHH/qBOP/d55uwZpyU49AE+AxRWKV68eIgfnCOJnrlfeJ48C5YgKATjjWbts7eRUY4lINaZuU7vZTPP73O8kODdXDB/gZQrW0769O2jS2Cdu3SRHTt2yIQJE6Rjhw7OnpFPsmTJI1yq2WAw+IfcCb6qcT5sHjsFAaH159U/NXlISCAQSWDhGSJnz/JVQUCYeJacJbSMpETHjh3VhBN8qN42feYMsWfDkip1Ks23zeDNYI2A9EzGwt+fTZFCrLuWOq95kjFDBr2O67Zi4yoI9kxcfRhYg/ZeO0I4cW8oG/hZhETDBg3t+4upuet/uXRJ23DY48UhSQtwrST4uHHjhnR5pYsMHzFcBX79+vWlerV7Ne7dvOIuRAcQWkr+8RfrvxjgY3Hzb/WqRznzByF9OBb6iyzBKXHVylVSo2YNTSZS1hZkGzds1Hsgz/zggYNk8JAh6hMxffoM2bRpk6RLm85WFnqq8uOLJE46XJKg+AM/h359+8mkiZM0giCkz+RJk6VO7TrONyMO18SaPO/iP3eCV3tDiWVbjJgxNCFKjOgx9D3zta+rvLkJd0KCRDxvvfWWvkv9BvSTLVs3y4TxE7RokcFgMDwoHlkmRYQpM3qcrlq2aOm0ijrdla9YXmeyQ4cMdVqDgtMXQo0Bcuf2nU5rgKNhg0YN1Kt8yeIlWnADKO+KkxtZsKg6h4CLZwv9BPHja1U0N2c7VoCKlSrI0/a/d+3YFSQ3Q9NmTeXtt99Wx8aXm7/stAYoA5yzWJGi9t/fU6HRsFFDzdmNx/eihYucPQMgsxa1yanC1rVLVxVWWBvIpc5z8NyfWWcF+3q4rhXLVki9evWkTNkyWmHMM+Md5XUpSkPFNBQcMpfhhNmxfUf9N7Rq3VKWr1ghM6fP1JAZIL62R88eGpXA/TMDf23Ea5rn3R9z5syWVwcNlLVr1tmzWP/7Aa8W58ciVL5CeRn46kCpUqmyFCleVK0KO97boQreyFEjNexx1sxZWk3PGxSefPnzaXYwHOQeJoQmFi5SWIX6e1vfC2Y5IRfEzFkztYgM1+8JTnwoPihqzPqpwU91QSrp1ahew9kr4L0lZwK59KdOmapFaUIDyxJ9juMjSuL0qdM1LDQihJRJkWfPNbvvkcFgeLQwrjKRIOsm47ZLZGRSfOwUBLyjK1aqKIMHDZZePXs5rUEJUUFoaCsI9sx16eKlOovm9pq91EwLfwwZNMRnOmeXiCgIhCmhEHgqCCgMFH9BCKx+Z3UQKwf3XdQWkIePHNZEQGNGj/GrIEC//v1kwqQJ0vLlltKxQ0dNd7tt6zZdrvAET3qOw9IFqW1RgiZNmKRhguBLQQASG02ZNkUryLG8Q53yje9u9FtfnyIwCPQtm7aEWI7WE66ZcMAPdn+gKU4rV62syhtlZ4FrJvUsghGFyRsUpSLFikilipVk9qx7+RQ8oe8nTZ6kAj2k0Elg3xLFS2iq79AITUEYPWa0DB46WGrXqi1r3lkj0WMEJPQClgC4ptatWquCgHK3/6P9WqQI5dCFWGeeCUWBVq5YGWpCFKJS2ndsL1u2bFGrCWluKSiDt3OLFi2cvcJOSArC/gMHZNGihaogGCXBYHj0YG0kYRkTLk+L9n8u1bIvJ8UvjhyxEidNbI0ZO8ZpCQ7FLEi1jPOXJ0Qq1KkX4OW9es1qp5WCLT3Vca5osSLq0OWPM2dOqwNe9pzZLVvIOq0BNGnaRL3VlyxZ4rQEgJcp0QYVK1YMTAeNZzve6jileRehunLlijoQ4rjnOvLhue7tpOhy4KMD6oCYNXtWq1r1apYt8J0tAVCA5K3FbwV6QZ85e8aqWr2qFT1WdIvCNG6kAc50nNPTcXHbe9v077B9x3aN8OA5jXhthNManBkzpqvDoC2QnJaQ2bhxo5X82eRa1AZ27Nhh2ULUqt+wvv4b3nnnnQDHzZ7BHf3AFtLaLxRu8QdOivUb1NfCNkQLhPTJ80IejVIJC6E5KeJoyfMgAsa7r/FC5plzfzBkyGDt59Ze/YwDI5Efz2d+3vrxxx+dVt/gXIozJs6OON4Cz4eIgqeefspasjTo+xkWQnJStBVa8zEf83kMP948EU6KceLGVSeukBK92NdNrtLA8p6euG32A9Q/AUsFa8XHjh+X10a+FpjZDpjRYiIG0gLbT16Pr+fwwE3r7N1+99+AtWN7KA3chkmZZEGsubME4cnxE8e1+AoFYSpVqqRt7rV6OhG65M2TV2fzzPIOfnxQEx55grPh0KFDtVQsZH4+s/Tp3UfrjDNTdq/JPYfWX7c5ePCgjBw5MjB9MLPz5s2b6zX4ug6XZ+In0GPh8xAazIx53o0aNNIiPUBiJZZ1Ll64GJgcBsdHjpk5S0Cddm8olHT92nUtg+sPTG0Uujqw/4Ds+XBPiJ+DHx30a53yhutyn6Ov51KoUCH1Afnxpx/V+uXy1ddfaY6JtGnSquMkVHOKs3x+5LD8dvk3bQNyU1y5fEWKFi2qviH+4DdBvor169dr6Vz3HphNYNniWrp07SLLly/X9geBazkwH/Mxn8fr8zB47BQEnPrwjMZk7gsGbPwUUARIZITgdMGJ7fsfvtcB/czpM4HCsWrlKlpp69btWzJ/wXz1oO/Tr480btxImjVvZu8f4DR24vgJHYTxJMd5z4V1WIQ91fzwHcD5DLgW1v0533n7et1oABSSDu076H6Y4w84yYcuXbykyYq4rjGjxqijH0ISMz9VzEhfe/m3y4HXDaw1VatSVa+9YIGCKkQ8SZM2jV4zSxEoC/DlqVN6XBQQBCc+BvgBcFwyGkLGTBlVMJG8iX3xjcCXgSgOlAV/kLSI+/V0AvXHzFkzdNmif//+TouoYkRUBc+R9MQoKDhekka0it1PvsChE8fT0LJAoliiGIXl49b28Ad9y3Oh1j3V6VCI+Dt/eioKKJ7dunbT/UkAxbO+feu2Rs389ONPGvXieiMjwEktzVLCjOkz9B2m5vzkKZMlWfJkofoe0FckTxk1cpQqgZ5Q6RC/m/z58utylXftC4PBYAg3ttB4JNiDrN88COXKl9NEPJiNPbEHbKtNuzZaTxuTL588efNo0pbDhw9b+fLn0/rrtLNPW3tf9xiYijt07KDbWRJgiYKlhJUrV+p2e2amOQDYzidz1szWoUOHrAMHDmi+AKppcVyS/thKhXX92jVdEnCvhe+Qu8GN1Sf5CQmbns+S2UqbPq1Vo1ZNvT7yF7gVFclXUKVqZT2me2zOZc9wdbsLeQlIsOTrWXEekvRg5uZ+qlSrqtdOQqV/7/6ruQcwu7vPhXMQM3/w44Naq5/liyLFimpVQZY+3GUPf9iKki4BUYEwJI4cOaJLC75M3uSD4HzUbqfio62sBEse5Al9RMKo8CQNuV94n1iOoO94bnzoa/Je+LoOEhelz5Deymu/j8WLF9d9Xxv5WuCykwtLTPQX/WErkppXgxwVYbm3ffv26nn47fjjm6+/sca9Ps668vsVpyV0IpoHwR8sxbi19w2GBwnvvq2k65i/du3aYL8vb+7euas5Bdif5U57cuNsiRz+uf2PLhPairx17uxZp9U/P/38k/XpId9VX8NLZCwxPHZOijB4yGB5a/FbWneesEUXLhWHNWZwOAMCs9nEiRLrDByzbfRoATNDju+G47nmGPb97vvvNEcCIWdUUXTT43JcZvyugxvnwJrBcbAouDPOfwmHjBJVj0tIpue18HeOx6zSheMyM+dPatBjjsbMDFwP7Z4mI5zhkiROEiQXAd7tpJLGcRJzsjdcNyZtlhmonZ8jR45AJ0bSQmPqd6+fZ8iHGPabN//W2uWELiZMkFDzDYSW0pOZPA51T8V7SjZt3BR4797gMEM6aJY/fOVVwLKA5QSHu4IFCkgKH9U8XUgdTMGSTw5+8kBqSYQFLDb0L/dHfwPPze1jX+GI538+rxYl+pAKpRkzZHS2BIV3itwcOMXaip/Wf39kqVRtQnJSDC/kY8Bpc87sOZLvhXxOa1CwVtlKr3xtv7NXr/4piZMk1vDYHNlzBHHodeF3Tdiwp9UH6wv9wf70BaHInh7dDwPO/7BMvY8LhCLTd4xbsePE1qJm5KOhyq4vsAQTPh3oOGxLG8ZQ/sTiR99pCLCPfvcFsoEIMSx1WbNklQ/e/8BvvzMuDhkyRDZs3KC/ZaK79u/bH2nvCWnSSb+PFfuy/c4uX7pcatWq7Wz1Dcur9kRT2rZuKy+99JLTGjGeCCdFwKGNTImkAn7SQeOtXr2a1bt3b6fl0TNixAh1GsQCEhK2IHT+FnGwkGBteLH+i06L4UHzoCwItsJnZXw+o7Vw0UKnJSjM9lasWKEpwrGckBoax9scOXOok2bFShV1VsjY4Mm0adPUuoWFjxTkWGqwlpGVlO9myZZFHU+bv9zcWrp0aYiOyA8KrCQNGzW0Ro8Z/USkSWZ2inMs/YvlsVbtWpqFNVmKZJpqnsykWMc8oR/79e+nDtaefYeVlHTrvANZsmbW7KdYdze8u8G6fv26823fnD59Wp2VcaYuU65MMCuzJ4w/WF/JNItTL++d67QdGeBAbE9krPTPpVcr9ZYtm50tIbNr9y61ou7cFWBZjiiRYUF4LBUEOp0lBlIuP+lgoic9MZ3/uEDdCZYZZsyY4bREHnv27tG6EuvWrXVaDA+aB6EgMLCXLlva6ti5o9MSFLYTgUM6aOqnfP3N16owIFzt2agqAQgbaqz06dtHzcguLC0S6UGKbKKDKlWuZJ07d06jN7759htNS07kEEKI7dRoIbokLCBwIrIcQm0V5lcIvPPng0Y8ISgOHgy6TPj/zJtvvmklTZZUnz+1X6ipQr/RRyyXFi5SWKNziKShnzyhPgrLtOnSB/RN27ZtNbU6S77nvjqnkVQIcBRE3o2q1aoGpoz3x+73d+uxmDiEpCC4IICfeuYpTbMemQoCcHyUXiLpwqogAPV3UJx4pyPKE6MgwIq3V2jxIGoePKmstYVismeTWdVqVHdaHh86dOqgPzgG78iE8EwGgsg+z5PMg1AQZs2aqValb7/91mm5B74wXV/pqjUr2nds73fdmBor/OYJDR06dIjTeo9WbVppqHHvPr6tadRNQXlgdsm1hBaK++WXX6oFIrzFrQD/pOr27xJrmqfFw/UJwhryX2DN2jXq/5Mrdy71HfIFAp8ibSgJZcuWDWbBQZHA14j+X7jQt3UJhYriePRd3hfyBlM0PEH5ixU7VpgVBCzSD0tBQHHC3yy8CoKGv+fKaQ0YGLSmT3iIDAXhsYticCGRDol4XnttRGDUwJMEURRdunSRXy79IvVCSZzzKCBBD+t6c+fOcVoePNS02LVrlwwZPMSnH4Ph8QD/gJmzZ2liME+fIZeNmzfKwkULta7JoAGD/EaQEB7crm1bXa+eNWe2ltL2JGaMkNeOOT6+D9RAIcHVsOHDQgzHJYnVyRMnw7z+7UmBAgVk86bN6mPj6YdD2DSRUp4Js/5fwX+AxHK2EJYB/QcES87mgl8QGVipE3Lg4AGNyvGE5xNa1BBj/fTp0yVF8hRy8tRJGTturLPl/nFD1B8Gtkzl/5x/hR38mlq1bClLlywNEpn3qHnkCoI+UI8/XXihyKt/8tQpmTw5eHa9/zr82EqXKSNFixSVMqXLOK2PDzj8jBs3TibZg8G+ffuc1gcHuSJ69e6l2SMpimV4fKGmCP1FHg1vcMhcsGCBChkKTKVNl9bZ4psWLVqqIy6hu2+/87bTGnYQYm3bBCgZhHqS78MbrgkFnLDkqNEDanwQFo3zHU64LjgXk6ES+YJD6YoVK+T06dO6jUkL4dY7dmxXp0vAmZm01IxdFOjCEfXIkSPq0OcJTmzUh1myZIkqwShYvuBcKEmMjTjzku2UlPFc58MAJ+PTZ05rbhUKuYUEoctkJ4X1G9bLxUsX9e/hoXix4lLHngxFiRJVtm/fHpjbxRcIfRQP+pKidyveXqHO3N7POjTox/0H9uv3cTD2Be8u74Hb97wLK1eulHc3bpRLv/yibSGBQ+WJEyfUKfujjz7STL++lIBqtoJ89+4d2eBRnO+RY798jwR3iSFjpgzqbDRq1ChnS1DsB2rZ2npgJr4nCdbvvB1/Hjdmzpyppj7vLIL3A2ZAQkm79+xu3f4nck2ChvtfYmC5CUfBX34N/g5gxk+TNo06ib311ltOq3/o7zp162gYMmGwtqLgbLHP07FDiEsMLkePHdVwXo7hq3z2/v37rdx5c2uoKWG+OJVxrkKFC6lvw/wF863S9jtNaCuOlDhLk02TcxOCPWvWLKtQkUIaxstx+I1iRseRlnvlmGxj+QLns0VvLtLz8nsmgydZN1lrJ+Mpfhc47FFa3sVWHPQ3lSpNKqtsubLWyndW6rVhto4WPZremz9H0AcF4zNlxaPGiKrOiWFh+ozp6kPCxxbwTmuAD0mlKpVCXGJweXfju/r8WBJYvmK50xoUlhg4B2X9We9PnjK5ZmLFvM46/urVQU3s+Kh4LzHw5yp7P77P8gnLWvTZzFkzdTvYipvVvUd3lU+U3R86bKg6pdIv0ezz8X4VKVZEl7ZcWD7jnfFcYuC9J0stfhbp7HeN7fhReMN3ORfPPSL8p5YYCA8iBGXYsOFahY76C74gMdCyZcuC1EV4UiBc0g3DfFzp3KmzjBs77l4Y0wMArZ5EU+PGjNNwVMPjC7NnKnNmy5pNkiYJXuGTGhyEKfJ+pEwVevla+jtL5sw6M7x2/ZrYiqezJew8m+JZrSnCMdzkZZ5QjpwKo8WKFlNLBXUxdu/aLatXrVYLBEsVsWPHUosAYWiDBg/SBF9UfeU3aQt+taAxqydkzh5HtZ1CYoMGDtJljZw5csr297bLju07tCorEKLHsajlwvLE1s1btboo5doJ9968ZbPuxzWQlI3znzh5QqZNnaY1Nt5e8bbUqVNHSFKGCd5fMrkHAdlmSa5GiC/WzLBA1teo9rhuKxchVl4NCZ4rifLu3LkjF0O5P7LCUqJ//dr1Mm/OPMmSJataPDp16Szbtm119goKVgdbYZGOnTpqsbOKFSrK3j17dYmE0Os3xr8RaP2gbykLjwWA66F8PdYbCqrNmTNXQ3M//fRTmThpou7vD4rrERZPv77z9krZsH6DWtO8wfKExRhrA0tkjwOPVEEg01/zl5prHoRCBQs5W4KTJUsW/dEaHkOiiBQuXFjTJz8oiI0uWaKk8Tv4PwABjFmX/B6+8My4SY6OsJDAVooZyBmUyeERXhhoA8ub+1gPpsANmTvJnUAJd/IwMAEhph9FpnSp0lr5lfePuP92bdtpgbT3d7+vRbdKlSyl1WaJ30cJwdzNeMYxUqRIoenXY9kKBmvzCBH2Iz02xdQKFSqklVZdvwWUhSaNm8iV36/IqFGjVEiVKFFCunbuqtdC6m2WNlDEKVHOsivXiQAmC2dkcfXPq4FLJ+61hgaCPZb9zHgWnqnEwwP+IO75QlrJ592gD1GcEKpt2rSR1e+sUlnBks2EiRMDr98Tjn3JVgA2bFiv+1K4jhwOKInPZ3pelbIff/hR96X/O3XoJOXLldelJ4q7oaRREZhlrH59+urxPLPresJzgLFjx9qv4b+ybs06HStDmkxR5fbn8z+He6kksnhkCoLBYPj/hxk+g2eSpL5nmZ71M3BqDQuugGAg9axWF1YY5JndM0AnTZbMaQ2OJuzxA0l8mAljbUBZANbiUQAgVqyYEt3HQI/CEAWt2QvWuP+yZ65FChfRJGOe1KpZS+/z7LmzWpkT4saNo9fP7N0zORrKAWm1sVqgVEQW8eLGU0WLa/DnI+EN+/LhGfhLnBQazJyZ4dP3vixSLjxjzsPHJVOmTNK+XXv97pmzZ9RXwBsUi/Tpn5O1a9fJ3NlzA5PWYTGJFTOWPtff/wiqlMaLF1fbvR1wqezKe3Lr5q0g9X2A54BuOnPWLPUzeevNxSHWWXHhuXFPEbXAPGiMgmAwGCIMdSoYdBM841sgUGOC2hcMsP6cwLzB6oBwRmhimg0vKC0oCdGiRZU8ufM4reHEHtwp3IaA9mXJYvAPaYbrCcoKxbyi2YqPr0yomLE5B4rW9999r20cP+DP4GdhOSM0z3wypOYrkE8KFi7o95M3Xx7p3LVzECHrgjWA7J4oayyBsBQTGigSCHdM82lSp3FawwdLUtdv3JDYsWJrNtLwgjWB83MdntYrb3D8pgje4cOfy+AhQ6RuvXry3Q/fqVLkjb++iB49hlo8vPtCFaWoUaRvv77y6qsD9HriPRVUKfQHVi2OGValLLIxCoLBYIgwd+/c1YETk7ovMNtmyJhB9yHahdTfIYGpliqYCK3ChQpHyAdn3bp16r/ATNG7uFm4YBbopHS+H1CguC8Eh6+BX2frTop4BEtohOV6EPBFCxeVgvlsZSC/70+hAoUlQ/qAvvEGIVW8eHH9O9EERGeExuEjh/U+WVphdh1eUAqJ7rh965amfc+eLbuzJeywPIkFAUHvmfLem48/+dhWCupKzdq15OKF8zJlymRNee5rqSC86JKT/V/lSpUk/XPp5cgXR6R3716hvvuAYsS1h6YAPiyMgmAwGCIMsyMc2fzlG8BkWrd2HRVClDrfsnWLs8U3hAYeOnRIZ1KNGzdxWsMOoYeY82NEj6nr+I/MudljfI8fP74kcWpt+ArdQ2m4/c9tNVenS5vOab0/mMHPnDlTZs2eJbNm+f7MmzdP+vTpowLVF/Xr1ddrv3bjmix6a5HT6hsE63vvbdP7qFGjZrCaKb6WXbzZum2r7Ni5w35nEkrPHj211oM/VIDah0Tp8gTrEdaDZMmSSabnMzmt9+BeWcZo1aqVmv5HvTZKFixYqJVy1Rpgv6dhudYQsS8NZadatWoyc/pMtcSsWbtGho8YHqpyx7WjULoK46PGKAgGgyHCIEBixooZokm0U6dOavplOWLMmDHq+OcLrAZTpkxRr3Ec95iBecKgy9jtvd7rQrnz3n16qRd6r5699LwhoYLA/p9fU3RYJnHs47kfssX+oDC5s1EET9ly5VSYHTr0qa0EndJ2l2PHjqpzIjNmnBhD44EIsTCAQx3RRFhR1q5ZK6veWeVsCc6qVavkww/36JJO925By5ZzvfQd9//H1T+c1qDwDAYOfFXfgbH2O1K9enVni284Fk6I3o6Im7du1ufe4uUWWsQP3GeFUsGSCb4JFy5eUMFN/gbd5lwj+HJu9AXf8Rb4XJcqLXY7+RNKliwpI4aNUOUPZW3CxAnOnr7BynDnnzthjhyJbIyCYDAYIgwzdNbPXc9vXyRMmEhmTJ8hBfIVUD+EJk2baAIjz8GVdfrefXvLshXLpEGDhjJm9BhnSwAoIKyFYzbeu2+v/f2PdDClHec+IgTKVywvR48d0+8OHTIkYKAOAXwcCCfctGmTJtvBukGlV2CWSTQCjpW+FJJLv1xSAYDA49pdqIpKxkcsBSx1fPzxx/Lhng+lfZv2tvAvLD/8+KO8Nmpk4DH57lx7Jk94J6F2WE6A8zOT/OP3P3RW6cL38K8AFKnIhgqfRJn99fdf0rVbV80S6Xk9CFWSBnXv2V2ey/CcZrL09j8g0uWnn3/S94QlBAQ0fUeEClalMWPHSNXq1eSG3TZzxkyNGgkJ12eCZ7xt2zb9O/C8ScpVu1ZtDZ13wf+D71z946o+b8INWdbh7wjszz77TEaPHi2fff6ZKhDLVyzX+3T7lb7gXSKrrSc8f5QJ9z0EFD31obHPd9Hpn/bt20unjp3kH7s/x4wZrWGRVIv1xfkL59Uq97Cq1oaK/SM1GAxPMPeTKMkeeK1SZUppAiFbYDqtviFnPzUZSPSTIGECrWVAYSYSIFGtkcQ34yeM1/r9LvZs0Oo/oL+VN19eTQLDh6RLFAsjeRBF3fiThDUDBw+yTn15yvlm6NjCxUqUNJEVPVZ0TYRTvGRxTaLU/9X+eo2ch/MVKFjAmj59mvMtyxo2bJhlC0PdTrIckiWR3AhshUKPQ00BkvlkypzRmjJlsm6zBaNWQaSdfXr26qkJl3LkyqFVLMEWvlabtm2s1GlSB56/VOlSmgSHujR8j+9z3lSpU1nDhw/X70Um9Ks9+9WKmSQA4nl37dpVE1ZRUZHraNW6lRZf8sRWZPReSELF9XIvJCRKmSql9hd9R2Ig3p83xo8PtTqsC1VDc+fOpcdLZL8HNWrV0PePYl2DBg3SYllgKzX2NfbSZ+meP1/+fNb69evt872hFRfpp3hPxdNkUNwT+3GPjZo0srbv2K6JqqgsTF+Q6Ih31VYIrGn2+8A7oMeNG0cLVvXv398qWaqk9g/781woREV9Dvq1cZPGer6Y9ncofLVr1y69Tk+aNm0amHwrvERGoqQo/J+jKxgMhicQ0r7aA7XOFnv36u20hh0SAC1eslg+PvixpEoZ+syHNMc4iTGTZJaGKThPnjyaj98NI3Rh5scMD6dDzLQutGM6xjkySeIkOuPy3B4WGPqY3ZPshhDGChUq6AyeUMO///o7sJ7Czb9v6nW5XvXu9bB0AIS5EX7o5mrh/rBGYEkg1wu5EFyYfXM+vOdZhsiRI6c6A7JmDlgNsK5gvVCPent0ZpbKucntcPSLoxIjZgyd0WJKj/9MfMmXL59+N7LBcoFFhORNLOfEjRNXQwuLFS8mWTJnCQxPdcHCwr3euXsn8FkBCY7YFx+D5MmSqxXKV/RASJAn4PPPP5czZ0/b1/W7HgNzfo7sOZw9Ap4l+/DMA/vy5k3JmCGjhixue2+bpj/GobJK5Sq6bfWa1fpMSUiF9YgcB1g+eN5YS/ANYBkIqxUWBfoC6COeB+dy21xfAt5r7u/X3361++8LiWLfO+8vS0qelgLOX7BQQe3PxW8tdlrDDvdasnRJWfzmYmnQoIHTen8YBcFgeMK5XwWB/PLVa1aX2TNnS+PGjZ1Wg8EQHvbv3y+169TWZFx1I1CgLzIUBOODYDAY7guc2Zj1LFu+LHB92GAwhI8ly5ZI6jSpH6vidEZBMBgM9wWhYwP6DVAnr499VE80GAwh4zpc9u3dVyODHheMgmAwGBR/8fBhgXLAdWvXlX4D+ocY8mgwGIKCL8nAwQOlQP4C0rBhQGGviHA/v19/GAXBYHjCcePDT546KTt37tS6+lu2bNEZTVjrJwA5DgjRGvDqAD2mwWAInXGvj9PcIFSJ5PcTFnCG3L17t/5O+b3u2LFDw39xKXyQvz3jpGgwPOGQ/750mdJy2UMZcIeFJYuXhsthCu/27j26S/fu3bUip8Fg8M/Ro0dl2IhhMnrUGMmRPeyppXEsLle+nFy4cP6e5SBKFI2cIAKiQX0TxWAwGB4AhKMRdsefnsmFGBmy24OWG4IXVkgcw7DiVsozGAy+4bdC+CS1M8IDIZFHjhwO9pvV8MkcOTR89EFgFASDwWAwGAzBMD4IBoPBYDAYgmEUBIPBYDAYDMEwCoLBYDAYDIZgGAXBYDAYDAZDMIyCYDAYDAaDIRhGQTAYDAaDweCFyP8ACmsBiN4uWAAAAAAASUVORK5CYII=\" style=\"width: 375px;\"\u003e\u003c/p\u003e\n \u003cp\u003eTriton X-100 (0.1%) was used as the positive control, and PBS as the negative control.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e2.10. \u003cem\u003eIn vitro\u003c/em\u003e cytotoxicity assay\u003c/h2\u003e\n \u003cp\u003eA study was conducted to evaluate the cytotoxicity of thymol, ampicillin, and cefotaxime individually and in combination against a human fibroblast cell line (Ffk) using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) bromide assay. Initially, 3\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells were cultured in 96-well microplates using 90% DMEM (Dulbecco\u0026apos;s modified Eagle\u0026apos;s mix) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Subsequently, the cells were cultured at 37\u0026deg;C for 24 h in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e. Serial doses of thymol, cefotaxime, and ampicillin were administered to each well, both singly and in combination. After that, the wells were incubated at 37\u0026deg;C for 24 h. The next day, 20 \u0026micro;L of MTT solution at a concentration of 5 \u0026micro;g/mL was added to each well, and the wells were incubated at 37\u0026deg;C for 24 h. After the supernatant was removed, 100 \u0026micro;l of dimethyl sulfoxide (DMSO) was added to each well, and the wells were shaken for 15 min to enable the cells to release the insoluble purple formazan. Finally, the samples\u0026apos; OD value at 570 nm was measured using a microplate spectrophotometer. The percentage of cytotoxicity was calculated using the following formula [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]:\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\" style=\"width: 305px;\"\u003e\u003c/p\u003e\n \u003cp\u003eAs the experiment\u0026apos;s positive and negative controls, respectively, peptide-free cell suspension and Triton X-100 were employed.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Investigation of antimicrobial activity (MIC-MBC)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe MIC and MBC assays were used to examine the synergistic effects of thymol/ampicillin on \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922, both separately and in combination\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eThymol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eAmpicillin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eThymol\u0026thinsp;+\u0026thinsp;Ampicillin\u003c/p\u003e \u003cp\u003eMIC \u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eThymol\u0026thinsp;+\u0026thinsp;Ampicillin\u003c/p\u003e \u003cp\u003eMBC \u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c11\" namest=\"c10\" rowspan=\"2\"\u003e \u003cp\u003eΣFICI\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e ATCC 25922\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC \u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMBC \u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIC \u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMBC \u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eThymol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAmpicillin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eThymol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAmpicillin\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSingle composition\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eA1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eAddition\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eA2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e4.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eAntagonism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eA3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eSynergistic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eA4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eSynergistic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe MIC and MBC assays were used to examine the synergistic effects of thymol/cefotaxime on \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922, both separately and in combination.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eThymol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eCefotaxime\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eThymol\u0026thinsp;+\u0026thinsp;Cefotaxime\u003c/p\u003e \u003cp\u003eMIC \u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eThymol\u0026thinsp;+\u0026thinsp;Cefotaxime\u003c/p\u003e \u003cp\u003eMBC \u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c11\" namest=\"c10\" rowspan=\"2\"\u003e \u003cp\u003eΣFICI\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e \u003cp\u003eATCC 25922\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC \u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMBC \u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIC \u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMBC \u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eThymol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCefotaxime\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eThymol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCefotaxime\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSingle composition\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eA1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eSynergistic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eA2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eIndifferent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eA3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eAddition\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eA4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eAntagonism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1. Thymol\u003c/h2\u003e \u003cp\u003eThe thymol MIC and MBC measurement results are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Its MIC was reported to be 256 \u0026micro;g/ml. However, its MBC was shown to be 512 \u0026micro;g/ml.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2. Antibiotics\u003c/h2\u003e \u003cp\u003eThe investigation employed ampicillin and cefotaxime as antimicrobial drugs; the MIC and MBC test findings are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The ampicillin MIC and MBC values were found to be 4 and 2 \u0026micro;g/ml, respectively, while the cefotaxime values were 32 and 64 \u0026micro;g/ml.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.1.3. Combinations\u003c/h2\u003e \u003cp\u003eAt this point, the antibacterial efficacy of combining thymol with ampicillin and cefotaxime was examined using the concentrations indicated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Thymol/ampicillin's MIC varied between 2\u0026ndash;64/1\u0026ndash;8, and its MBC ranged from 4-128/2\u0026ndash;16 \u0026micro;g/ml. Likewise, thymol/cefotaxime's MIC value was found to be between 64\u0026ndash;256/16\u0026ndash;32 \u0026micro;g/ml, while its MBC was found to be between 128\u0026ndash;512/32\u0026ndash;64 \u0026micro;g/ml. As a result, the antimicrobial effect of thymol/ampicillin was better than thymol/cefotaxime on \u003cem\u003eE. coli\u003c/em\u003e strains.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Evaluation of synergy by checkerboard assay\u003c/h2\u003e \u003cp\u003eIn thymol/ampicillin, the A1 compound with FICI 1 showed an additive effect, the A2 compound with FICI 4.25 showed an antagonism effect, and compounds A3 and A4 with FICI 0.5 showed a synergistic effect. In thymol/cefotaxime, the A1 compound with FICI 0.25 showed a synergistic effect, the A2 compound with FICI 1.5 showed an indifferent effect, the A3 compound with FICI 1 showed an additive effect, and compound A4 with FICI 3 showed antagonism effect.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Fourier-Transform Infrared Spectroscopy (FT-IR)\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe principal peaks in the combinations of thymol, ampicillin, and cefotaxime that were subjected to FT-IR spectroscopy showed values that varied from 592.61 to 3442.64 cm-1. These peaks are associated with the following: C\u0026thinsp;=\u0026thinsp;O ester group, C\u0026thinsp;=\u0026thinsp;O Amid I band, C-N Amid II band, C-O-C-polysaccharide, CH stretching alkene group, and OH carbohydrates, proteins, and polysaccharides. Thus, thymol/ampicillin and thymol/cefotaxime combos contain all of these ingredients. The molecule C\u0026thinsp;=\u0026thinsp;C conjugated and C\u0026thinsp;\u0026equiv;\u0026thinsp;C, which denotes the connection between thymol/ampicillin and thymol/cefotaxime, is what separates the combined and individual states. The effectiveness of antibiotics may be significantly impacted by this relationship.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Antimicrobial susceptibility assay\u003c/h2\u003e \u003cp\u003eInitially, twenty clinical strains of \u003cem\u003eE. coli\u003c/em\u003e were chosen at random within the parameters of this experiment and described using biochemical techniques and differential testing. Compounds A1 and A3 (Tables\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) were tested for their antibacterial activity against clinical strains of \u003cem\u003eE. coli\u003c/em\u003e. In general, the MIC and MBC of compounds A1 (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) are respectively (thymol 128\u0026ndash;512 \u0026micro;g/ml)/(ampicillin 32\u0026ndash;128 \u0026micro;g/ml) and (thymol 256\u0026ndash;1024 \u0026micro;g/ml)/(ampicillin 64\u0026ndash;256 \u0026micro;g/ml) respectively. Was. Also, MIC and MBC of compounds A3 (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) were (thymol 8161024 \u0026micro;g/ml)/(cefotaxime 4-256 \u0026micro;g/ml) and (thymol 32-2024 \u0026micro;g/ml)/(cefotaxime 8-512 \u0026micro;g/ml), respectively. As a result, the antimicrobial effect of thymol/ampicillin (A1 compound) was better than thymol/cefotaxime (A3 compound) on \u003cem\u003eE. coli\u003c/em\u003e clinical strains.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of A1 and A3 compounds on \u003cem\u003eE. coli\u003c/em\u003e clinical strains\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e clinical strains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eThymol\u0026thinsp;+\u0026thinsp;Ampicillin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eThymol\u0026thinsp;+\u0026thinsp;Cefotaxime\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMBC (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIC (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMBC (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e256/64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e256/64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e256/64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e512/128\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1024/256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2024/512\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e512/128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1024/256\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e128/32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e256/64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003e9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003e11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003e13\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e512/128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1024/256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003e14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16/4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e32/8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003e15\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e128/32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e256/64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003e16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e256/64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e512/128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003e17\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e256/64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e256/64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003e18\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e512/128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1024/256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64/16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e64/16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003e19\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64/16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e64/16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003e20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR/R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Anti-biofilm evaluation\u003c/h2\u003e \u003cp\u003eUsing concentrations of 1024, 256, and 256 \u0026micro;g/ml, respectively, the anti-biofilm effectiveness of thymol, ampicillin, and cefotaxime combinations was evaluated at this point. When thymol/ampicillin and thymol/cefotaxime were administered, a synergistic result was observed, as shown in Tables\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Thymol, ampicillin, and cefotaxime individually have biofilm formation inhibition rates of 29.69%, 60%, and 16.28%, respectively. On the other hand, thymol/ampicillin (A1 compound) and thymol/cefotaxime (A3 compound) together have inhibition rates of 62% and 39.28%, respectively, indicating that the separate compounds' biofilm development is higher than that of the combined state. Therefore, the inhibition rate of biofilm formation in thymol/ampicillin (A1 compound) was higher than in thymol/cefotaxime (A3 compound).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Time-kill assay\u003c/h2\u003e \u003cp\u003eThymol at a concentration of 1024 \u0026micro;g/ml in 6, 12, 18, and 24 h was able to reduce the number of bacteria to zero at a concentration of 512 \u0026micro;g/ml. Ampicillin at a concentration of 256 \u0026micro;g/ml was able to reduce the number of bacteria to zero at a concentration of 128 \u0026micro;g/ml in the first 6 h and at a concentration of 8 \u0026micro;g/ml at 18, 12 and 24 h. Cefotaxime at an initial concentration of 256 \u0026micro;g/ml at 6 h at a concentration of 256 \u0026micro;g/ml, at 12 h at a concentration of 128 \u0026micro;g/ml, at 18 and 24 h at a concentration of 32 \u0026micro;g/ml was able to reduce the number of bacteria to zero.\u003c/p\u003e \u003cp\u003eThe thymol/ampicillin at a concentration of 1024/256 \u0026micro;g/ml in the first 6 h at a concentration of 128/32 \u0026micro;g/ml was able to reduce the number of bacteria to zero, but at 12, 18, and 24 h at a concentration of 32/4 \u0026micro;g/ml, it was able to reduce the number of bacteria to zero. The thymol/cefotaxime at a concentration of 256/512 \u0026micro;g/ml in the first 6 h at a concentration of 128/512 \u0026micro;g/ml, at 12 h at a concentration of 64/256 \u0026micro;g/ml, and 18 and 24 h at a concentration of 16/128 \u0026micro;g/ml was able to reduce the number of bacteria to zero. Therefore, thymol/ampicillin was faster and more effective in killing bacteria than thymol/cefotaxime.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.7. \u003cem\u003eIn Vitro\u003c/em\u003e hemolysis assay\u003c/h2\u003e \u003cp\u003eThymol, ampicillin, and cefotaxime showed 36.12, 9.05, and 8.33 times less cytotoxic activity against human RBCs than the positive control Triton X-100, respectively. They lysed 2.63%, 10%, and 8.33% of human RBCs, respectively. Additionally, thymol/ampicillin and thymol/cefotaxime showed 9.58 and 8.38 times less cytotoxic activity against human RBCs than the positive control Triton X-100, respectively. They lysed 2.63% and 8.38% of human RBCs, respectively, while Triton X-100 lysed 95% of human RBCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). As a result, both compounds had a lower toxicity effect against human RBCs than the positive control.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.8. \u003cem\u003eIn vitro\u003c/em\u003e cytotoxicity assay\u003c/h2\u003e \u003cp\u003eThymol, ampicillin, and cefotaxime showed 19.66, 8.45, and 7.08 times less cytotoxic activity than Triton X-100 against Ffk cells and lysed 4.83%, 11.23% and 13.41% of human Ffk cells. Also, thymol/ampicillin and thymol/cefotaxime showed 9.03 and 9.03 times less cytotoxic activity than Triton X-100 against Ffk cells, and lysed 10.51%, 10.51% of human Ffk cells, respectively. While Triton X-100 lysed 95% of human Ffk. (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). As a result, both compounds had a lower toxicity effect against Ffk cells than the positive control.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe widespread use of antibiotics in the treatment of infections has led to the emergence of MDR bacteria [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Ampicillin and sometimes cefotaxime are among the drugs used in the treatment of infections caused by \u003cem\u003eE. coli\u003c/em\u003e bacteria isolated from UTIs, but significant resistance to these drugs has been reported in the world, even 30–90% or more, and practically they cannot inhibit or kill \u003cem\u003eE. coli\u003c/em\u003e bacteria [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The emergence of antibiotic resistance is a primary global health concern. Consequently, there is an urgent need for new strategies to combat antibiotic-resistant bacteria [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. One of these solutions is the combination of medicinal plants and antibiotics as an alternative to treat bacterial infections [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. One of these compounds is the active substance isolated from the extract of medicinal plants such as thymol [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Thymol causes death in bacteria by increasing the permeability of the cell membrane [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The purpose of this study was to compare the antimicrobial, anti-biofilm, and cytotoxic effects of thymol/ampicillin and thymol/cefotaxime on \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 and clinical strains isolated from UTIs.\u003c/p\u003e \u003cp\u003eIn this study, according to the obtained results, the antimicrobial effect of thymol/ampicillin (A1 compound) on \u003cem\u003eE. coli\u003c/em\u003e bacteria (FICI: 1) was better than thymol/cefotaxime (A3 compound) (FICI: 1). The antimicrobial effect of A1, and A3 compounds on the strains isolated from UTIs were 32–128/64–512 µg/ml and 4–16/256–1024 µg/ml, respectively, that the effect of thymol/cefotaxime was better than thymol/ampicillin. The effect of inhibiting biofilm in the A1 compound (62%) was better than in the A3 compound (39.28%). The killing time of bacteria in thymol/ampicillin was lower in concentration and time than in thymol/cefotaxime. Both compounds were examined by the FT-IR device and showed thymol/ampicillin and thymol/cefotaxime combination. A1 and A3 compounds on human RBCs and Ffk cells were not significantly different, but they were much lower than the positive control Triton X-100.\u003c/p\u003e \u003cp\u003eIn the study of Zhuocheng Yao \u003cem\u003eet al.\u003c/em\u003e in 2022, thymol/colistin showed MIC 128/1024 µg/ml and an antimicrobial synergistic effect (FICI: 0.5) on \u003cem\u003eE. coli\u003c/em\u003e bacteria. This compound was able to reduce the growth rate of bacteria to zero in 24 h. It also had an anti-biofilm effect on bacteria, which was consistent with our study [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In the study of Sumaira Noreen \u003cem\u003eet al.\u003c/em\u003e in 2018, they investigated the antimicrobial effect of the combination of aqueous and ethanolic extracts of cloves, cinnamon, and garlic with ampicillin, imipenem, ciprofloxacin, norfloxacin and nalidixic acid on \u003cem\u003eE. coli\u003c/em\u003e bacteria isolated from UTIs by disc diffusion method Done. Thus, the antimicrobial effect of clove extract alone was 27 mm, cinnamon 16 mm, and garlic 14 mm, and in combination with antibiotics, they showed 10 mm more, which was in line with our study [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In the study of Fatemeh Tavakoli \u003cem\u003eet al.\u003c/em\u003e in 2022, the antimicrobial effect of methanolic extract of Salvia limbate with ceftazidime on \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (\u003cem\u003eS. aureus\u003c/em\u003e) and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (\u003cem\u003eP. aeruginosa\u003c/em\u003e) bacteria was carried out. The combination of this extract with ceftazidime could show a better antimicrobial effect with MIC 0.5 mg/ml and a slight synergistic effect against these bacteria, which was consistent with our study [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In our study, the MIC score was lower than in this study, which indicates that the effect of active ingredients in combination is better than raw plant extracts. In the study of Sohini Mitra \u003cem\u003eet al.\u003c/em\u003e in 2024, the antimicrobial effect of the combination of aqueous extract of Psidium guajava (guava) plant with cefotaxime was performed. The combined effect of these two substances was able to induce 30% bactericidal inhibition in \u003cem\u003eE. coli\u003c/em\u003e bacteria, which was consistent with the study [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Thymol/cefotaxime showed a better antimicrobial effect. In the study of Oumaima Amassmoud \u003cem\u003eet al.\u003c/em\u003e in 2023, the antimicrobial effect of Thymus pallidus and T. broussonnetii plants with ciprofloxacin and streptomycin was performed. These compounds had a MIC of 128 mg/ml and a synergistic antimicrobial effect, which was consistent with our study. The active ingredient of these plants is thymol, and it has been able to induce its antimicrobial effect with the mentioned antibiotics [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In the study of Anwesa Bag \u003cem\u003eet al.\u003c/em\u003e in 2011, the antimicrobial effect of Terminalia chebula fruit extract with ceftazidime was performed by MIC, checkerboard, and kinetics of killing bacteria [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The antimicrobial effect of these two substances caused a synergistic effect, and the bacterial killing time was better in the combined state than in the single state, which was in line with our study. Therefore, the compounds in the phenolic plant extract induce an antimicrobial effect, and the effect of these compounds in the isolated state is better than with the whole extract.\u003c/p\u003e \u003cp\u003eCompared to previous studies, this study showed a better antimicrobial, anti-biofilm effect, kinetic killing time, and less toxicity in both thymol/ampicillin and thymol/cefotaxime. These compounds in the FT-IR device had organic compounds, including OH carbohydrates proteins and polysaccharides, CH stretching alkene group, C-O-C-polysaccharide, C = O ester group, C = O Amid I band, and C-N Amid II band. However, the antimicrobial, anti-biofilm, and other effects were due to the combination of C = C conjugated and C ≡ C, which binds these substances together and shows the inhibitory and lethal effect on bacteria. In addition to the antimicrobial and anti-biofilm effects, the fast kinetic killing time in the combined state and less toxicity against RBCs and human Ffk cells are related to this triple bond that has created the connection of these two substances. The effects of thymol are well-known in various studies. Thymol is a phenolic compound extracted from the Shirazi thyme plant. According to studies, thymol has structural isomers and a phenolic hydroxyl in the ring. The hydroxyl group increases its hydrophilicity, disrupts membrane integrity, increases membrane permeability, and finally causes proton and potassium leakage and leads to loss of membrane potential in \u003cem\u003eE. coli\u003c/em\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Ampicillin and ceftazidime cause osmotic instability of bacteria by inhibiting the synthesis of the bacterial cell wall and have bactericidal effects. As a result, these compounds confirm the effect of synergism in the killing rate of \u003cem\u003eE. coli\u003c/em\u003e bacteria compared to the individual state of these substances [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e "},{"header":"Conclusion","content":"\u003cp\u003eThis comparative study showed that the thymol/ampicillin (A1 compound) compared to the thymol/cefotaxime (A3 compound) has less antimicrobial, anti-biofilm, and toxicity effects on \u003cem\u003eE. coli\u003c/em\u003e bacteria. According to the results obtained from different tests, thymol/ampicillin can be one of the options to reduce antibiotic resistance and treat urinary infections caused by \u003cem\u003eE. coli\u003c/em\u003e after passing the complementary tests.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study has been approved by the Vice President of the Research and Ethics Committee of Zanjan University of Medical Sciences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is no limit to the publication. All authors consented to the publication of this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by Zanjan University of Medical Sciences, Zanjan, Iran.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this work are included in this published article. Also, all data used to support the findings of this study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was done with the financial support of the Zanjan University of Medical Sciences Research Vice-Chancellor, so the authors express their gratitude to all colleagues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe idea of this study was designed by SJ and BM. Performing practical work and collecting clinical samples was done by SJ. Data analysis was done by MSh. The article was written by SJ, MSh, and MT. All authors read and approved the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePokharel P, Dhakal S, Dozois CM. 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Eur Rev Med Pharmacol Sci. 2023;27(9):3922\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSultan MH, et al. Bioactive Principles and Potentiality of Hot Methanolic Extract of the Leaves from Artemisia absinthium L in vitro Cytotoxicity Against Human MCF-7 Breast Cancer Cells, Antibacterial Study and Wound Healing Activity. Curr Pharm Biotechnol. 2020;21(15):1711\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWhite RL, et al. Comparison of three different in vitro methods of detecting synergy: time-kill, checkerboard, and E test. Antimicrob Agents Chemother. 1996;40(8):1914\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDefinitive Document EUCAST. \u003cem\u003eE.Def 1.2, May\u003c/em\u003e 2000: Terminology relating to methods for the determination of susceptibility of bacteria to antimicrobial agents. 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Curr Microbiol. 2016;73(5):611\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSadeghi H, et al. A retrospective cross-sectional survey on nosocomial bacterial infections and their antimicrobial susceptibility patterns in hospitalized patients in northwest of Iran. BMC Res Notes. 2021;14:1\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoghbel A, Hemmati A. \u003cem\u003eComparison of effects of anti-inflammatory gel prepared by glysertinic acid in licorice extract and plant rhizome with hydrocortisone ointment in rats\u003c/em\u003e. PhD Thesis. Ahwaz, Ahwaz Universiy of Med Sci 2008.[in Persian].\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTokam Kuat\u0026eacute; CR, Bisso Ndezo B, Dzoyem JP. \u003cem\u003eSynergistic antibiofilm effect of thymol and piperine in combination with aminoglycosides antibiotics against four Salmonella enterica serovars.\u003c/em\u003e Evidence-based Complementary and Alternative Medicine, 2021. 2021: pp. 1\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMandala SM, et al. Rustmicin, a potent antifungal agent, inhibits sphingolipid synthesis at inositol phosphoceramide synthase. J Biol Chem. 1998;273(24):14942\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarfaraj HM et al. \u003cem\u003eMarine natural products: A lead for Anti-cancer.\u003c/em\u003e 2012.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalam MA, et al. Antimicrobial Resistance: A Growing Serious Threat for Global Public Health. Volume 11. Healthcare (Basel); 2023. 13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee DS, Lee SJ, Choe HS. \u003cem\u003eCommunity-Acquired Urinary Tract Infection by Escherichia coli in the Era of Antibiotic Resistance.\u003c/em\u003e Biomed Res Int, 2018. 2018: p. 7656752.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChinemerem Nwobodo D, et al. Antibiotic resistance: The challenges and some emerging strategies for tackling a global menace. J Clin Lab Anal. 2022;36(9):e24655.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVaou N et al. \u003cem\u003eTowards Advances in Medicinal Plant Antimicrobial Activity: A Review Study on Challenges and Future Perspectives.\u003c/em\u003e Microorganisms, 2021. 9(10): p. 2041.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEscobar A, et al. Thymol bioactivity: A review focusing on practical applications. 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Susceptibility and Synergistic Effects of Guava Plant Extract and Antimicrobial Drugs on Escherichia coli. Cureus. 2024;16(1):e52345.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmassmoud O, et al. Antibacterial Activity of Essential Oils Combinations based on Thymus broussonnetii, and Their Synergism with some Antibiotics. Curr Microbiol. 2023;80(12):398.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBag A, et al. Synergistic Effect of Terminalia chebula and Antibiotics against Multidrug-resistant Uropathogenic Escherichia coli. Med aromatic plant Sci Biotechnol. 2010;5(1):70\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eModareskia M, Fattahi M, Mirjalili MH. Thymol screening, phenolic contents, antioxidant and antibacterial activities of Iranian populations of Trachyspermum ammi (L.) Sprague (Apiaceae). Sci Rep. 2022;12(1):15645.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCho H, Uehara T, Bernhardt TG. Beta-lactam antibiotics induce a lethal malfunctioning of the bacterial cell wall synthesis machinery. Cell. 2014;159(6):1300\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Escherichia coli, Thymol, Cefotaxime, Ampicillin, anti-biofilm, antimicrobial","lastPublishedDoi":"10.21203/rs.3.rs-6177131/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6177131/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction:\u003c/strong\u003e This study aimed to compare the antimicrobial, anti-biofilm, and cytotoxic effects of thymol/ampicillin with thymol/cefotaxime on \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterial and Methods:\u003c/strong\u003e Antimicrobial effects of thymol/ampicillin and thymol/cefotaxime were first individually and then combined on \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 by MIC-MBC method. Therefore, the antimicrobial effect of compounds that had synergistic effects was performed on twenty clinical strains using the MIC-MBC method. Identification of chemical bonds, functional groups, and molecular interactions of the mentioned compounds were investigated with an FT-IR device. Checkered method, time killing curve, and biofilm inhibition on \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 bacteria, and investigation of cytotoxicity on red blood cells (RBCs) by hemolysis method and human skin fibroblast cells (Ffk) by MTT method were performed. thymol/ampicillin and thymol/cefotaxime (A1, A3 compounds) had Synergistic effects. Finally, the results of the tests were compared between the two compounds.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The results of this study showed that the antimicrobial effects of the thymol/ampicillin 2/8) µg/ml) were better than the thymol/cefotaxime 16/128) µg/ml(in both ATCC and clinical strains. In the examination with the FT-IR device, both compounds had bonds of OH carbohydrates proteins, polyphenols, C=O Amide I band, C-O-C polysaccharide, C-Namide III band, but one band named C=C conjugated, C≡C in both compounds showed the connection between thymol/ampicillin and thymol/cefotaxime. The biofilm inhibition effect of thymol/ampicillin (62 %) was better than thymol/cefotaxime (39.28 %) on \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922. The bacterial killing time curve of the thymol/ampicillin at a lower concentration and time was better than the combination of thymol/cefotaxime. Cytotoxicity of synergistic compounds on RBCs and human skin fibroblasts (Ffk) was not different and was lower than that of Triton X-100.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiscussion:\u003c/strong\u003e Considering the antibiotic resistance of ampicillin and cefotaxime in the treatment of diseases caused by \u003cem\u003eE. coli\u003c/em\u003e bacteria, the thymol/ampicillin in this study showed better antimicrobial, anti-biofilm, and bacterial killing time effects than the thymol/cefotaxime. This combination can be used as a new drug in patients after further studies.\u003c/p\u003e","manuscriptTitle":"Comparing the Antimicrobial, Anti-Biofilm, and Cytotoxic Effects of Thymol/Ampicillin and Thymol/Cefotaxime against Escherichia coli Bacteria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-17 05:04:51","doi":"10.21203/rs.3.rs-6177131/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8bc74685-1d3f-488c-8362-62810a8727cf","owner":[],"postedDate":"March 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-16T13:53:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-17 05:04:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6177131","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6177131","identity":"rs-6177131","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}