## 2 Materials and Methods
### Methodology: Isolation and Phenotypic Characterization of Escherichia coli  
Each sample underwent thorough mixing in its sampling vial via 20-25 inversion movements. Subsequently, within 3 min of agitation, 10 mL of the sample was drawn using a sterile pipette and transferred to a dilution bottle containing 90 mL of homogenization liquid (Buffered Peptone Water, BPW, BioMerieux, Marcy l'Etoile, France), resulting in a 1:10 dilution. Decimal dilutions were subsequently carried out up to a dilution factor of \(10^{-8}\), using Ringer's solution. A variety of selective and differential plating media has been used for the recovery of _E coli_ isolates. The rationale for adopting this approach was to support the growth of both starved and unstarved _E. coli_ cells. The following commercial media were used: Violet Red Bile Glucose Agar (VRBGA, Merck KGaA, Darmstadt, Germany), Endo Agar (Merck KGaA, Darmstadt, Germany), Sorbitol McConkey agar containing 2.5 mg/L potassium tellurite and 0.05 mg/L cefixime (CT-SMAC, Merck KGaA, Darmstadt, Germany), CT-SMAC additionally supplemented with 50 mg/L 5-Brom-4-chlor-indolyl-beta-D-glucuronoid cyclohexylammonium-salt (Merck KGaA, Darmstadt, Germany), CHROMagarTM_E.coli_, EC O157:H7 ChromoSelect Agar, modified and supplemented with 0.25 mL of 1% potassium tellurite solution/L (Merck KGaA, Darmstadt, Germany), CHROMagarTM O157 (CHROMagar Microbiology, Paris, France), supplemented with 2.5 mg/L tellurite and 2.5 mg/L cefusolodin, CHROMagarTM STEC (with 50 mg/L STEC supplement), and CHROMagar STEC supplemented with 1 mg/mL cefotaxime. The inoculated different solid media were incubated aerobically at 37 \({}^{\circ}\)C for 24-32 h.  
Additionally, apart from employing standard procedures with chromogenic agar media, as described above, an enrichment protocol was implemented that consisted of two steps. Initially, 25 mL of homogenized sample is transferred to a sterile container containing 225 mL of modified Trypticase Soy Broth with the addition of 20 mg/L novobiocin (mTSB, Merck KGaA, Darmstadt, Germany). After 24 h of incubation at 37 \({}^{\circ}\)C, the second step involves the after-enrichment process, where a fraction of 100 mL of the 24 h TCS culture is inoculated onto sorbitol MacConkey agar (CT-SMAC) and supplemented with enrichment agents cefixime (0.05 mg/L) and potassium tellurite (2.5 mg/L), CHROMagarTM STEC (with 50 mg/L STEC supplement), and CHROMagar STEC supplemented with 1 mg/mL cefotaxime, followed by incubation at 37 \({}^{\circ}\)C for 24 h [37; 38; 39].  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Experimental Group** & **Substance** & **Therapeutic Protocol** \\ \hline \multirow{4}{*}{**Group 1**} & Penicillin/streptomycin & \multirow{2}{*}{1 mL/25 kg; BW a qd b IM c} \\  & (Commercial preparation & \\  & containing per mL 200,000 IU of & \\  & proclaim penicillin G and 250 mg & \\  & dihydrostreptomycinycine & \\ \hline \multirow{2}{*}{**Group 2**} & Oxytetracycline & 8 mg/kg BW; qd SC d or IM \\  & & for 5 days \\ \hline \multirow{2}{*}{**Group 3**} & Enrofloxacin & 2.5–5 mg/kg qd SC for 5 days \\  & & \\ \hline \hline \end{tabular}
\end{table}
Table 1: Antimicrobial substances and therapeutic protocols in the different experimental groups.  
Bio-Typing: By Conventional Biochemical Tests and Platform VITEK 2 System  
After incubation, presumptive _E. coli_ isolates were enumerated and subjected to phenotypic bio-typing procedures. These procedures involved a series of tests aimed at detecting specific metabolic characteristics, such as Gram staining, motility testing, cytochrome oxidase and catalase production, Triple Sugar Iron (TSI) Agar, and subsequently, the APIE 20E system (Biomerieux SA, Marcy I'Etoile, France) and the VITEK 2 automated microbiology system (bioMerieux, Marcy I'Etoile, France), all conducted according to the manufacturer's instructions. The latter system, employing the Vitek 2\({}^{\text{\text{\text  
(scored as negative), and as resistant if more than 90% of organisms survived after 180 min (scored as positive) [44].