## 2 Materials and Methods
### Antimicrobial Susceptibility Testing  
#### 2.7.1 Determination of Minimum Inhibitory Concentrations (MICs) for _E. coli_ Isolates  
A single colony from each _E. coli_ isolate was initially inoculated into 5 mL of Luria-Bertani medium (LB broth) and then incubated overnight (-20 h) at 37 \({}^{\circ}\)C. Fifteen different concentrations of each antibiotic were prepared in Mueller-Hinton Broth using a stock solution via serial dilution: 0.03, 0.06, 0.12, 0.25, 0.50, 1, 2, 4, 8, 16, 32, 64, 128, 256, and 512 \(\upmu\)g/mL. The isolates were tested for susceptibility to Ampicillin (AMP), Amikacin (AM), Tetracycline (TET), Chloramphenicol (CHL), Gentamicin (GEN), Streptomycin (STR), Trimethoprim/Sulfamethoxazole (TRI/SU), Nalidixic acid (NAL), Cephalothin (CEP), Cefotaxime (CEF), Sulfathiazole (SUF), and Ciprofloxacin (CIP).  
Subsequently, 50 \(\upmu\)L of each antibiotic solution was added to individual wells of a 96-well microplate. Then, 50 \(\upmu\)L of the bacterial suspension (10\({}^{6}\)-10\({}^{7}\) CFU/mL) was dispensed into each well, where the various dilutions of antibiotics had been placed. The microplate was incubated aerobically at 37 \({}^{\circ}\)C for 24-32 h. The Minimum Inhibitory Concentration (MIC) was recorded as the lowest concentration of the antibiotic that halted the growth of the tested isolates. The results were categorized as sensitive, intermediate, or resistant based on the standardized table provided by NCCLS 2000, CLSI 2018, and EUCAST 2013 [51; 52; 53]. _E. coli_ ATCC 25922 and _Staphylococcus aureus_ ATCC 6538 were used as quality controls. The experiment was repeated three times. Multidrug-resistant (MDR) _E. coli_ was defined as bacteria that were resistant to at least three different classes of antimicrobials.  
2.7.2 Detection of Extended-Spectrum Beta-Lactamase (ESBL) Producers Phenotype in _E. coli_ Isolates  
All isolates were assessed for extended-spectrum beta-lactamase (ESBL) production according to the criteria established by the CA-SFM/EUCAST [54; 55], using the Combined Disc Synergy Method with MAST IDTM ESBL Detection Discs (Mast Group, Bootle, UK). The MAST Extended IDTM ESBL Detection Discs consist of discs containing cefpodoxime 30 \(\upmu\)g, cefpodoxime 30 \(\upmu\)g with clavulanic acid 10 \(\upmu\)g, ceftazidine 30 \(\upmu\)g, ceftazidine 30 \(\upmu\)g with clavulanic acid 10 \(\upmu\)g, cefotaxime 30 \(\upmu\)g, and cefotaxime 30 \(\upmu\)g with clavulanic acid 10 \(\upmu\)g. A suspension of the test isolates in distilled water, equivalent in density to a McFarland 0.5 opacity standard, was prepared. This suspension was evenly spread across the surface of Mueller-Hinton agar plates (Merck KGaA, Darmstadt, Germany), using a sterile swab. One of each MAST IDTM ESBL Detection Disc was then placed onto the inoculated medium using sterile forceps, ensuring even spacing. The plates were aerobically incubated at 35-37 \({}^{\circ}\)C for 18-20 h. Any observed zones of inhibition were measured and recorded. The diameter of the inhibition zones for cefpodoxime, ceftazidine, and cefotaxime was compared to those of the respective combination discs containing clavulanic acid. An increase in zone diameter of \(\geq\)5 mm in the presence of clavulanic acid from any or all sets of MAST IDTM ESBL Detection Discs indicates the presence of ESBL in the tested _E. coli_ isolates [56; 57; 58].