**1. Introduction**

The serotypes of *Salmonella* spp, a pathogenic genus of Enterobacteriaceae, are responsible for animal infections from a sub-clinical to severe level, as well as typhoid fever and severe diarrhea in humans [1]. These Gram-negative bacteria are one of the major causes of concern for the veterinary industry. In particular, chickens and pigs are known as *Salmonella*'s major vehicles. Great efforts are aimed at controlling the *Salmonella* spp. colonization in pig and chicken reservoirs since these animals, if infected, generally host the bacterium asymptomatically in the tonsils, intestine, and lymphoid tissue associated with the intestine [2]. According to the community summary report on trends and sources of zoonoses and zoonotic agents and foodborne outbreaks in the European Union [3], the three most commonly reported zoonoses in Europe are the foodborne enteric diseases campylobacteriosis, salmonellosis, and yersiniosis. Recent studies have identified *Salmonella* spp. multi-resistance to the most common antibiotics used in livestock [4,5]. For this reason, the European Food Safety Authority (EFSA) considers *Salmonella* serotypes isolated from livestock as a danger to public health [6], and the European Union banned the use of antibiotics in animal food production as growth promoters [7]. Subsequently, control measures are aimed at reducing the prevalence of *Salmonella* spp. in livestock, especially in chickens and pigs, and the search for valid alternatives to the use of antibiotics has been stimulated. Probiotics, prebiotics, acidifiers, plant extracts, and nutraceuticals are new alternatives to antibiotics that are widely investigated by researchers [8,9]. Among them, essential oils (EOs) have a prominent place [9].

According to the International Organization for Standardization, essential oils are volatile products obtained from "natural raw material of plant origin by steam distillation, by mechanical processes from the epicarp of citrus fruits, or by dry distillation, after separation of the aqueous phase—if any—by physical processes" [10].

To date, many studies have evaluated the in vitro and in vivo efficacy of EOs on livestock microbial strains, including Salmonella spp. Several studies have analyzed the effects of EOs or their preparations on live animals from pig and poultry farms [11–13], on strains isolated from farms, on reference strains [14], and even on meat after slaughtering [15–18], in order to reduce the infection of Salmonella spp. or increase the shelf-life of the products intended for human consumption. However, not all EOs have been shown to be suitable for in vivo use, as the minimum inhibitory concentration (MIC) of most EOs is much higher than the acceptable dose levels in animal industry in terms of cost-effectiveness and feed palatability [9].

In November 2019, the EFSA published a report expressing the Agency's Endorsement of the safety and efficacy of *Origanum vulgare* L. EO use in feed of all animal species [19]. For each animal species, this report indicates the allowed dosages of the EO expressed in mg/kg live weight. The doses of 22 mg/kg for fattening chickens, 33 mg/kg for laying hens, 30 mg/kg for fattening turkeys, 40 mg/kg for piglets, and 48 mg/kg for fattening pigs are established as safe for both humans and animals, and they are not expected to pose a risk for the environment. According to European Commission Regulation No 1334/20083, *O. vulgare* EO can be used as a flavouring additive in all animal feed, without additional evaluation and approval [4]. The antimicrobial action of *O. vulgare* EO on both bacteria and fungi has been documented in several studies. Generally, the antimicrobial action of this EO is more effective in fungi than it is in bacteria. More specifically, it is more effective against Gram-positive bacteria than Gram-negative bacteria [20]. The EO of *O. vulgare* L. acts on fungal cells by thinning the morphology of the hyphae, and inducing an oxidative stress until cell lysis [21,22]. In bacteria, the main target of the EO active chemicals is the cellular phospholipid bilayer [23]. In particular, the two major chemicals of the EO (thymol and carvacrol) disrupt the outer membrane, alter the proton gradient, and inhibit the production of ATP of Gram-negative cells (including *Salmonella* spp cells) [24,25]. Carvacrol is the principal active compound of the *O. vulgare* phytocomplex, and it belongs to phenols that could exert toxic effects.

The aim of this study was to compare the in vitro antimicrobial effectiveness of the *O. vulgare* EO vs. a mixture of feed additives, namely GR-OLI, characterised by a lower concentration of carvacrol and already approved for use in animal feed, on 29 strains of *Salmonella* spp. isolated from poultry and pig farms.
