*2.2. Antibiograms*

All *Salmonella* spp. strains were tested for their sensitivity against antibiotics commonly used in human medicine. As indicated in Table 2, all *S.* Typhimurium strains, with the exception of the monophasic variant, were sensitive to most antibiotics except amoxicillin/clavulanic acid and ciprofloxacin, to which about 31% (S24, S31, S32 S34) and 54% (S3, S7, S12, S17, S18, S21 S32) of strains were resistant, respectively. Only the S17 and S32 strains were also resistant to gentamicin, while S24 and S31 were also resistant to trimethoprim/sulfamethoxazole. All monophasic *S.* Typhimurium strains were resistant to amoxicillin/clavulanic acid; S19 and S28 strains were resistant to ciprofloxacin, and only S29 to trimethoprim/sulfamethoxazole. Whereas, S19 and S29 strains were susceptible by increased exposure sensitivity to piperacillin/tazobactam and ceftazidime, respectively. The S13 strain was sensitive to all antibiotics tested. *S.* Infantis strains showed various multi-resistances. Specifically, no strain was resistant to piperacillin/tazobactam, ertapenem, imipenem, meropenem, amikacin, and gentamicin, while all strains were resistant to ciprofloxacin. In total, 75% of the tested strains (i.e., all excluding S10, S26, and S42) were resistant to trimethoprim/sulfamethoxazole, 67% (all except S4, S10, S40, and S42) to amoxicillin/clavulanic acid, 58% (all minus S4, S25, S38, S39, and S42) to cefotaxime, and 50% (all apart S4, S10, S25, S38, S39 and S42) to cefepime. Increased exposure sensitivities were shown by the S10 strain vs. cefepime, and by 58% of the strains vs. ceftazidime (i.e, all strains except S4, S25, S38, S39, and S42).

#### *2.3. Broth Microdilution Susceptibility Testing*

The broth microdilution susceptibility test was performed to study the in vitro sensitivity of *Salmonella* spp. vs. *O. vulgare* EO and GR-OLI. As shown in Table 1, the two natural products have the same activity against both *S.* Typhimurium and *S.* Infantis. In particular, the *O. vulgare* EO and the GR-OLI have a respective minimal inhibitory concentration of the 90% of strains (MIC90) equal to 2% *v*/*v* (20 μL/mL) and 16% *v*/*v* (equal to 40 μL of EOs content /mL), for both *S.* Typhimurium and *S.* Infantis.

#### *2.4. Biofilm Formation Assay*

The ability of low concentrations of *O. vulgare* EO and GR-OLI to inhibit bacterial biofilm formation or disaggregate a preformed biofilm was evaluated. As shown in Figure 1, the two concentrations tested of GR-OLI were capable of inhibiting biofilm formation or disaggregating any preformed biofilm for *S.* Typhimurium. Both concentrations showed activity in the inhibition of the biofilm formation of *S.* Infantis, but only the higher concentration was active in disaggregating the preformed biofilm. *O. vulgare* EO was active only in the disaggregation of mature biofilm of both serotypes, but no inhibiting activity was exerted on biofilm formation.

**Figure 1.** Control-related ratios of the OD values measured for *S.* Typhimurium and *S.* Infantis biofilm inhibition and biofilm disaggregation by GR-OLI and *O. vulgare* EO. Vertical bars indicate ± standard errors.



sensitivity, R = Resistance, S.T. = *S.* Typhimurium, m.S.T. = monophasic *S.* Typhimurium, S.I. = *S.* Infantis.

#### *2.5. Cell Adhesion Assay*

Since GR-OLI was e ffective not only in the disaggregation of the preformed biofilm but also on the inhibition of biofilm formation, we decided to investigate the interference of this natural mixture on bacterial adhesion to target cells. An adhesion assay was performed using eight *Salmonella* spp. strains. In particular, two *Salmonella* spp. strains sensitive to almost all antibiotics (one sensible *S.* Typhimurium and one *S.* Infantis resistant only to ciprofloxacin), and six multi-resistant strains (two *S.* Typhimurium strains, two monophasic *S.* Typhimurium, and two *S.* Infantis strains) were tested. Figure 2 shows the control-related ratios of the colony-forming unit (CFU) count obtained after the adhesion of the bacteria strains, pre-treated with GR-OLI and untreated, to the Caco-2 monolayer. The ratios refer to bacteria that are still able to adhere to Caco-2 cells after treatment with GR-OLI. Data show that *S.* Typhimurium strains were inhibited in a proportion to the GR-OLI concentration. Particularly, the inhibition was significant at the lower concentration for both monophasic and non-monophasic *S.* Typhimurium (bars of standard error not attaining the 100% level of the control), while the higher concentration was e ffective only on non-monophasic *S.* Typhimurium. In reverse, the only significant inhibition of *S.* Infantis adhesion was obtained at the minimal concentration tested.

**Figure 2.** Control-related ratios of the CFU count recovered from GR-OLI-treated bacteria strains (S.T. = *S.* Typhimurium, S. I. = *S.* Infantis, mono S. T. = monophasic *S.* Typhimurium) after the adhesion to the Caco-2 monolayer. Vertical bars indicate ± standard errors.

#### *2.6. Growth Curves*

The same strains selected for the cell adhesion assay were treated with GR-OLI and *O. vulgare* EO to evaluate the impact of the two compounds on the bacterial growth curve. Growth curves showed that both products, when tested at the first sub-MIC concentrations, were able to inhibit the growth of most of the tested *Salmonella* strains. Furthermore, the inhibition determined by *O. vulgare* EO on *S.* Typhimurium strains was significantly stronger than that of the GR-OLI at 15 and 20 h (Table 3). Whereas, if compared to the control (Ctrl), both products showed an equal and significant inhibition of *S.* Infantis growth at all the time points tested.


**Table 3.** Relative growth of *S.* Typhimurium and *S.* Infantis at 10, 15, and 20 h after treatment with *O. vulgare* EO and GR-OLI vs. untreated control (=100).

Note. In each *Salmonella* species and time after treatment, different letters indicate significantly different means (LSD test at *p* < 0.05). Ctrl = Control.

#### *2.7. Checkerboard Titration Method*

The four strains (S26, S35, S36, S37) equally resistant to amoxicillin/clavulanic acid, cefotaxime, and ciprofloxacin were chosen to test the capability of GR-OLI to synergically reactivate the sensitivity to one or more of these drugs. Table 4 shows the values of the minimal bactericidal concentration (MBC) of each single compound, those of the GR-OLI/antibiotics interaction, and the relative values of the fractional bactericidal concentration index (FBCI). Due to the turbidity of the wells containing both the dilutions of the GR-OLI and those of the antibiotic, it was not possible to perform the OD450 to obtain the MIC values and the relative fractional bactericidal concentration (FIC) index. Ciprofloxacin is the only antibiotic showing synergy with the GR-OLI for all the samples analyzed, while amoxicillin/clavulanic and cefotaxime had a variable interaction depending on the strain analyzed.

**Table 4.** Average MBC values of amoxicillin/clavulanic acid, cefotaxime, and ciprofloxacin alone and combined with GR-OLI, and relative FBCI values.


Note. AMC = Amoxicillin/clavulanic acid, CTX = cefotaxime, CIP = ciprofloxacin, (s) = synergy, (a) = additivity, (i) = indifference.
