is the second human bacterial zoonosis delivered especially by chicken and pork meats, but also milk, eggs, and seafood. The WHO estimates 550 million people (including 220 million children under the age of 5 years) fall ill each year due to diarrhoeal diseases due to unsafe food [26
]. In 2018, 91,857 confirmed cases of salmonellosis in humans were reported with an EU notification (EFSA, 2019), while the U.S. Center for Disease Control and Prevention (CDC) estimates that Salmonella
bacteria cause about 1.35 million infections per year in the United States, of which only 41,930 in 2011 were laboratory confirmed [27
]. Along with the world population increase, the consumption of meat is also increasing. Foley et al. [19
] reported that since the early 1900s, the consumption of chicken in the U.S. has increased about sixfold, while pork consumption by about 20%. Whereas, the European Union data show that in 2018, Europe increased its chicken meat production by a quarter, and 70% of this production was in six member states: Poland (16.8%), the United Kingdom (12.9%), France (11.4%), Spain (10.7%), Germany (10.4%), and Italy (8.5%) [18
]. An upward trend, although less steep than in the case of poultry meat, was recorded for pork meat whose consumption, in Europe, has increased by about 3.5% per person in 10 years [19
]. In order to meet consumer demands, unavoidable changes in animal production were necessary. The introduction of intensive animal husbandry practices has on the one hand increased the exposure of consumers to zoonosis, and, on the other hand, has probably modified the characteristics of Salmonella
spp. colonization in farms by selecting strains resistant to antibiotics. In animals, Salmonella
infection can cause fever, diarrhea, prostration, and mortality. Most of the animals that survive this infection remain asymptomatic carriers, posing a threat to human health as, during slaughtering, their carcasses can contaminate others [20
]. Within Salmonella
Montevideo, and S.
Infantis are among the major pig and poultry serotypes most frequently associated with human infections [1
]. Strains of Salmonella
spp. with antimicrobial drug resistance acquired in the animal host are now widespread in all countries [22
]. Resistance to ciprofloxacin, which belongs to the group of fluoroquinolones and was, until the last decade, the treatment of choice, and to cephalosporins is increasingly being documented [22
]. Therefore, the WHO listed resistant Salmonella
spp. among priority pathogens for which new antibiotics were urgently needed, and several countries have established Salmonella
surveillance and control programmes. Our data agree with the above-reported concern, because the presence of widespread resistance to ciprofloxacin is confirmed by the circumstance that 21 of the 29 analyzed strains (72.4%) were resistant to fluoroquinolone, and highlight the resistance or a reduced sensitivity to cephalosporins (cefotaxime, ceftazidime, and cefepime), especially in the S.
Infantis serotype. Amoxicillin/clavulanic acid is another drug showing decreased efficacy especially against S.
Infantis and monophasic S.
Typhimurium strains. Although the combination of amoxicillin with clavulanic acid overcomes the intrinsic resistance of beta-lactamase-producing strains, and therefore makes it one of the main antimicrobial substances in swine medicine for the treatment and control of infections, the fair percentage of resistance (55.2% of strains) supports the choice of the European Medicine Agency [26
] to classify this association in category C. This category includes antibiotics that are approved for use in livestock and pet animals, but which must be used with caution, only when there are few or no alternatives belonging to category D [26
]. Natural substances represent a valid resource in the search for alternatives to current antibiotics. Thanks to their high antimicrobial potential, EOs are widely studied to counteract the development of antibiotic resistance and respond to the growing demand of consumers for antibiotic-free foods [12
]. As noted in the introduction, the O. vulgare
EO was found to be active against a broad spectrum of microorganisms. The antimicrobial activity is essentially mediated by the main chemicals carvacrol and thymol, which, because of their amphipathic nature, interact with the bacterial and fungal cell membrane. In particular, carvacrol is able to accumulate in the cell membrane of Salmonella
spp and other bacteria strains, where it can bind to hydrogen by altering the cell membrane potential and inducing a conformational and metabolic modification (decrease of ATP production) up to the time of cell death [20
]. This antimicrobial activity of the O. vulgare
EO on bacterial and fungal membranes is common to many EOs caracterised by the same amphypathic chemical compounds. Despite their strong antimicrobial action, the use of EOs in farms is limited by their poor water solubility. This characteristic makes it necessary to convey them with suitable surfactants or through biotechnological processes. The Italian product GR-OLI is a water-soluble mixture of EOs emulsified in an inert carrier additive, which is regularly authorized as additive for use in animal feed. This mixture has been compared with the activity of the O. vulgare
OE that recently received a positive opinion from the EFSA for use in animal production. The chemical analysis of both products shows that the O. vulgare
OE and GR-OLI have respectively three (carvacrol, p-cymene, and γ-terpinene) and eight (limonene, carvacrol, 1-8 cineol, p-cimene, linalool, terpinen-4-ol, and thymol) chemicals with a concentration >5%. Furthermore, if compared to the O. vulgare
EO, the GR-OLI has a lower concentration of carvacrol and a higher concentration of the other terpenic molecules with known antimicrobial action. If, on the one hand, the antimicrobial action of carvacrol is well known [29
], on the other hand, this phenolic compound is acknowledged to be potentially toxic, depending on the concentration of use [31
]. For this reason, a preliminary in vitro comparison between the antimicrobial properties of O. vulgare
EO and this commercial aromatic mixture was needed. Data show that the MIC90 of the O. vulgare
EO is slightly lower than that of GR-OLI against the different Salmonella
strains tested, and that the sub-MIC of O. vulgare
EO inhibits over time the S.
Typhimurium growth more effectively than GR-OLI. However, while the O. vulgare
EO is only capable of disaggregating a formed biofilm, GR-OLI is simultaneously capable of inhibiting the formation of the biofilm and disaggregating the formed one at minimal concentrations potentially compatible with animal palatability. The ability to prevent the early stages of bacterial adhesion to intestinal cells is critical for the establishment of chronic colonization in animals, which are the reservoir for acute events. In this regard, data obtained from the cell adhesion assay confirmed that GR-OLI, at very low concentrations, is actually able to inhibit bacterial adhesion to the intestinal cell line Caco-2. Inhibition occurs in different ways depending on the serotype. Specifically, the monophasic S.
Typhimurium and S.
Infantis strains showing the greatest resistance to antibiotics were sensitive only to the higher concentration tested, while the other strains tested were sensitive to both concentrations. These activities could be useful also with animals carrying Salmonella
spp. asymptomatically. In these animals, it is important to inhibit both the adhesion and the formation of the biofilm to prevent contamination of the carcasses at the time of slaughtering. Furthermore, data obtained from the checkerboard test indicate that GR-OLI has synergistic action with ciprofloxacin at concentrations much lower than MIC. This data identifies a possible new resource in the fight against antibiotic resistances, as it indicates the possibility of reactivating the sensitivity to ciprofloxacin with low doses of natural compounds mixed with commercial antibiotics. Moreover, given the heterogeneity of the phytocomplex of each EO, the use of concentrations lower than MIC is not currently correlated with the development of resistance [32
]. This makes the use of sub-MIC of the EOs mixtures safer against the development of potential resistances.