*2.6. Antimicrobial Activity*

## 2.6.1. Bacterial Media

Nutrient Broth (NB), Nutrient Agar (NA), Mueller Hinton Broth (MHB), Mueller Hinton Agar (MHA), Mc Farland standard 0.5, Tripticase Soy Broth (TSB), were purchased from Oxoid (Basingstone, UK).

#### 2.6.2. Bacterial Strains and Growth Conditions

The pathogenic bacterial strains were obtained from the American Type Culture Collection (ATCC). Gram-negative bacteria [*Escherichia coli* (ATCC 25922), *Salmonella enterica* ser. *Typhimurium* (ATCC 14028), and *Enterobacter aerogenes* (ATCC 13048)], and Gram-positive bacteria [*Enterococcus faecalis* (ATCC 29212) and *Staphylococcus aureus* (ATCC 25923)] were employed to test the antimicrobial activities of cladode extracts. All bacteria strains were grown on NB and MHB and incubated overnight at 37 ◦C under aerobic conditions.

#### 2.6.3. Inhibition Assay–MINIMUM Inhibitory Concentration (MIC)

The minimal inhibitory concentration (MIC) against selected bacteria was determined according to [7] with some modifications. Cladode extracts were diluted in sterile water to the concentration of 2000 μg/mL; then, further dilutions were made up to the concentration of 50 μg/mL.

Tested pathogenic microorganisms were cultured in MHB at 37 ◦C for 16 h. Initially, the cultures were diluted to match the turbidity of 0.5 McFarland standard; thereafter, further dilutions with sterile MHB made it possible to obtain a suspension of about 1–5 × 10<sup>5</sup> CFU/mL. Aliquot of bacterial suspensions (50 μL) were added to a sterile 96-well plate containing 100 μL of MHB and 100 μL of cladode extract dilutions. A positive control (without cladode extract) was included on each microplate.

The plates were incubated in aerobic conditions at 37 ◦C for 24 h. A microplate reader (Eti-System Fast Reader Sorin Biomedica, Modena, Italy) was used to record the optical density (OD) at 600 nm. The MIC was defined as the lowest concentration of cladode extract able to inhibit the microorganism's growth.

#### *2.7. Biofilm Production and Inhibition Assay*

The biofilm production was determined using the method described by [2] with some modifications. The assay was performed using two *Staphylococcus* strains: the biofilm producer *Staphylococcus aureus* (ATCC 35556) and *Staphylococcus epidermidis* (ATCC 12228), used as a negative control, since it does not produce biofilm.

The strains were activated by culturing in 5 mL of TSB at 37 ◦C for 24 h. After 24 h, the optical density (OD) was measured at 600 nm and appropriate dilutions were made in TSB + 1% sucrose, to obtain an optical density of 0.1 corresponding to about 10<sup>6</sup> cells/mL. The assay was performed in sterile 96-well polystyrene plate (Greiner Bio-One Gmbh, Austria). Briefly, 100 μL *Staphylococcus aureus* cells were inoculated and cultured with or without 100 μL of cladodes extract (at concentrations ranging from 50 to 1500 μg/mL), without shaking at 37 ◦C. After 24 h incubation, non-adherent cells were removed by dipping each sample three times in sterile PBS. Samples were fixed at 60 ◦C for 1 h and the biofilms were stained with 0.1% solution of crystal violet in water, according to [17]. After staining, samples were washed thrice with distilled water. The quantitative analysis of biofilm production was performed by adding 125 μL of 30% acetic acid to de-stain the samples. Afterwards, the OD at 492 nm was detected using the microplate reader. The percentage of biofilm inhibition was determined by the formula:

$$Biofillm\text{ }reduction\text{ }\%= \frac{OD\text{ }control-OD\text{ }sample}{OD\text{ }control} \times 100\%$$
