*3.1. Antibacterial Activity of the Extracts*

The antibacterial capability of the polyphenol (PF) extracts of *Ogliarola*, *Ravece*, and *Ruvea antica* EVOOs was assayed against different Gram-positive and Gram-negative bacteria, through the inhibition zone test and the determination of the Minimal Inhibitory Concentration (MIC). Results are shown in Tables 1 and 2 respectively.

**Table 1.** Antibacterial activity evaluated through the inhibition zone test of the three polyphenol (PF) extracts of *Ogliariola*, *Ravece* and *Ruvea antica* EVOOs, against different pathogens. The test was performed using 2.5 and 4.9 μg of extract. Data are expressed in mm. Results are shown as mean (± SD) (*n* = 3). For details, see Materials and Methods.


**Table 2.** Minimal Inhibitory Concentration (MIC, μg/mL) of the PF extracts of 'Ogliarola', 'Ravece' and 'Ruvea antica' EVOOs, evaluated through the resazurin test, as reported in the Materials and Methods section.


The minimum concentration necessary to inhibit the growth of the pathogenic tester strains was low for all the PF extracts, usually equal to 1–2 μg, except when PF of *Ravece* were tested against *S. aureus* (MIC > 15 μg), and when those of *Ruvea antica* were assayed against *E. faecalis* (MIC > 10 μg). This confirms that polyphenols present in the EVOO have a general capacity to inhibit the growth of pathogenic or unwanted microorganisms [3]. Therefore, different in vitro studies demonstrated that

some polyphenols from olive oil are able to inhibit the growth of different bacteria, including those responsible for some respiratory infection and intestinal diseases, as well as against bacteria, such as *Helicobacter pylori*, one of the agents of peptic ulcers and some types of cancer [4,20].

In general, 4.9 μg of the PF extract from *Ogliarola* were very effective in inhibiting the microbial growth of all the strains considered, with inhibition zone not lesser than 10.67 (against *L. innocua*) up to 18.33 mm (against *B. cereus* 4313). Overall, 4.9 μg of the polyphenol extract from *Ravece* produced inhibition zones also superior to 17 mm (17.33 mm, against *B. cereus* 4313 and *E. faecalis*). 4.9 μg of PF extract from *Ruvea antica* resulted less effective, producing zones not greater than 12.67 mm. All three EVOO PF extracts were effective in inhibiting the growth of *E. coli*, producing (with 4.9 μg of PF extracts from *Ravece* and *Ogliarola*) inhibition zones up to 13 mm. This result, in our opinion, could find an interesting practical application. *E. coli* is the most frequent cause of urinary tract infections. Like other *E. coli* pathotypes, the strain used in our experiments differs from the commensal *E. coli*, due to the presence of some virulence factors, which can concur, with other microbial systems, to increase its resistance against conventional antibiotics, to form biofilm, as well as to contaminate food or medical support (e.g., catheters), with difficulty to eradicate the infection and serious damage to health [21]. Thus, the capability of EVOO polyphenols to avoid the growth of this pathogen strain could be exploited not only for the EVOO per se, or for the great bioavailability of EVOO PFs, but also taking into account that the EVOO by-products are rich in polyphenols, which can convert them from a problem for the environment to a resource of biomolecules of high added value, potentially useful for food and pharmaceutical purposes. Therefore, other olive by-products, such as leaves demonstrated activity against different species of pathogens, including those used in our experiments [22]. The three PF extracts were also capable of inhibiting the growth of *Ps. aeruginosa*. Such microorganism, similar to *E. coli*, not only is a well-known pathogen, but it is also capable to form biofilm, increasing its resistance to the conventional drugs [23]. The effect was well visible, so that we measured inhibition halos until 8.67 mm just using 2.5 μg. In both cases, the extracts *Ogliarola* and *Ravece* were more effective than those of *Ruvea antica* in inhibiting the growth of the strain; in particular, 2.5 μg of PF extract of *Ravece* were twice as effective as that of *Ruvea antica* against *Ps. aeruginosa*; 4.9 μg of *Ravece* PF extracts were even three times more effective than the *Ruvea antica* ones. The different effectiveness exhibited by the extracts against the two strains of *B. cereus* (DSM 4313 and DSM 4384) proved once again that the resistance/sensitivity of a microorganism to a natural extract or to a singular compound might be not only linked to the genera or species but, in some cases, it might even be strain-specific [24,25].
