2.8.1. Bacterial Strains

Five *Enterobacteriaceae* isolates, four *Escherichia coli*, and one *Klebsiella pneumonia* were selected to carry out the study. The strains were isolated from poultry in the Regional Veterinary Laboratory of Mostaganem, Algeria, and identified using matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI-TOF-MS), as previously reported [23]. *E. coli* ATCC 25922 and *Staphylococcus aureus* ATCC 6538 (American Type Culture Collection, Rockville, MD, USA) were also tested.

#### 2.8.2. Antimicrobial Susceptibility Testing

Disk diffusion method was used to test and confirm the antimicrobial susceptibility of the Enterobacteriaceae isolates using Muller–Hinton agar (MHA, Oxoid, Milan, Italy) and an incubation time of 16–18 h at 37 ◦C, following the Clinical and Laboratory Standards Institute Guidelines (CLSI) [24]. The antimicrobial used were: ciprofloxacin (CIP, 5 μg), nalidixic acid (NA, 30 μg), amoxicillin/clavulanic acid (AMC, 20/10 μg), amoxicillin (AML, 25 μg), levofloxacin (LEV, 5 μg), cefotaxime (CTX, 30 μg), sulphonamides (SSS, 300 μg), tetracycline (TE, 30 μg), trimethoprim/sulphamethoxazole (SXT, 1,25/23,75 μg), trimethoprim (TMP, 5 μg), chloramphenicol (C, 30 μg), and neomycin (N, 30 μg) (Bio-Rad, Marnes la Coquette, France). The results were assessed following the CLSI guidelines [24].

2.8.3. Determination of the Antibacterial Activity of all Extracts of *Rhus Coriaria* by Disk Diffusion Assays

The antibacterial activity of the different extracts (SE, SM, SA, SEW, SMW, SAW, and SW) against the selected Enterobacteriaceae isolates was assessed by the disk diffusion method, as previously described [25]. Briefly, the bacterial colonies were suspended in 10 mL of saline water, and the turbidity of the bacterial suspension was adjusted to 0.5 McFarland standard. MHA plates were inoculated with bacteria by spreading overnight cultures on MHA using sterile cotton swabs. Filter paper disks (6 mm diameter; Thermo Fisher, Milan, Italy) containing 10 μL of each extract at a concentration of 10 mg/mL were then applied on the agar plates. Cefotaxime served as a positive control, and a disk impregnated with sterile distilled water was used as a negative control. The plates were incubated for 24 h at 37 ◦C, and the antibacterial activity was evaluated by measuring the diameters of the inhibition zones. Each assay was performed in triplicate.

#### 2.8.4. Determination of the Minimum Inhibitory Concentration (MIC) of the SM Extract

In order to determine the minimum inhibitory concentration (MIC) of SM, the serial double dilution method was performed according to CLSI guidelines [26]. Briefly, overnight bacterial cultures in log phase were used to prepare the suspension of cells adjusted to 10<sup>6</sup> CFU in Muller-Hinton Broth (MHB). Serial dilutions were performed in the growth medium in a concentration range between 2000 and 2 μg/mL for the SM extract. Wells containing compound-free MHB with bacteria were used as the positive control. Plates were incubated at 37 ◦C for 24 h. The MIC value was defined as the lowest concentration of the tested compound that inhibits the growth of bacteria at the end of the 24 h incubation. MICs were determined in triplicate. The MIC was defined as the lowest concentration inhibiting the visible growth of the tested strains after incubation.

#### *2.9. Embryo Acute Toxicity Test*

The embryo acute toxicity test was carried out according to the Organisation for Economic Cooperation and Development (OECD) guidelines for the testing of chemicals [27]. The ZFET was conducted on fertilized eggs from the Centre for Experimental Fish Pathology of Sicily (CISS, Sicily, Italy). Adult zebrafish (*Danio rerio*) were kept in a standalone facility (ZebTec, Tecniplast, West Chester, PA, United States) in water-controlled conditions: temperature 28 ◦C, conductivity 600 μS/cm, pH 7.5, and 14/10 h dark/light regimen. Twice a day, fish were fed with *Artemia salina* at 3% of body weight and Gemma micro 300 (Skretting, Varese, Italy). Following mating, the eggs were placed in steel grids inside tanks to avoid predation by adults and to guarantee their collection. The fertilized eggs were collected using a stereomicroscope (Leica M205 C) and exposed to *R. coriaria* extract, which was previously prepared at a concentration of 9.37 μg/mL, in a sterilized embryo medium (15 mM NaCl, 0.5 mM KCl, 1 mM CaCl2, 1 mM MgSO4, 0.15 mM KH2PO4, 0.05 mM Na2HPO4, 0.7 mM NaHCO3; pH 7.3). The control group was held in an embryo medium. The Fish Embryo Acute Toxicity (FET) was performed, as described by Pecoraro et al. [28]. Right after the fertilization, embryos were collected, bleached as reported by Westerfield [29], and distributed as one embryo per well into 24-well plates (LABSOLUTE, Th. Geyer GmbH & Co.KG, Berlin, Germany). Embryos were incubated with a 10/14 h dark/light regimen at 26 ◦C for 96-hours post-fertilization (hpf). The test solutions and controls were replaced daily [27]. The exposure period started from 180 min post-fertilization and ended at 96 h. The following endpoints were used to evaluate the toxicity: embryo coagulation, tail non-detachment, somite formation lack, heartbeat nondetection, and the hatched embryos number. Acute toxicity was determined at the end of the exposure period.
