**2. Natural egg defenses**

Eggshells are the primary packaging and constitute the 1st defense barrier in containing microorganisms; the priority of maintaining their integrity and quality is of great importance for producers [6, 7]. The main component of the hull is calcium carbonate in the form of calcite (94%). Apart from CaCO3, there are other inorganic components in the shell: magnesium carbonate (<1%), calcium phosphate (<1%), and silicon oxide (<1%). The approximately 4% of remaining compounds are polysaccharides, various collagens, fatty acids, and water [8]. These components make the eggshell have a unique microstructure, where the CaCO3 skeleton is characterized by a porous and rough structure with three levels of primary particles with approximately 10 nm. Calcite crystals are arranged in palisades and mammillary layers with different morphology and porosity, in addition to an absence of cell-directed assembly during calcification, compared to bone [9]. The mass of the eggshell is proportional to the egg mass and represents between 10 and 11% of the egg weight. In the eggshell, the cysteine-rich protein membrane, the mineralized layer, and the non-mineralized outer cuticle are deposited as the egg descends through the oviduct of the hens [6]. The eggshell membranes are synthesized during a period of 1.0–2.0 h, when the immature eggs travel through the proximal isthmus. Mineralized multilayers are formed in the distal isthmus and the shell gland over a period of 19–20 h. Finally, the cuticle is deposited on the eggshell in the uterus 1.5–2 h before oviposition [10]; and, at this time, the outer part of the eggshell is exposed to many contaminants that can harbor a wide range of microorganisms [11].

The cuticle covers the pores on the eggshell surface, thus forming a physical barrier against bacteria [12]; the chemical composition of the eggshell plays an important role by limiting bacterial contamination. Some antibacterial proteins (e.g., c-type lysozyme, ovotransferrin, andovocalyxin-32) have been detected in eggshell; the open pores on the eggshell surface not only serve for gas and water exchange but are also

the route of invasion [13]. Consequently, eggshell thickness is an important factor for the ingress of bacterial contamination [14, 15]. In this regard, it has been shown that good shell cuticle quality can significantly reduce the opportunity for pathogen invasion and that the amount of cuticle as a hereditary trait can be an effective strategy to reduce the transmission of microorganisms in production poultry [6].

In order to reduce Enterobacteriaceae counts on the eggshell, in some countries, such as USA, Australia, Japan, and Sweden, eggs are washed with chemicals (e.g., sodium carbonate and sodium hypochlorite) [16]. This practice may damage or partially remove the cuticle, thus increasing the risk of bacterial ingress. Class A eggs should not be washed, due to potential damage to physical barriers, such as the cuticle. Good cuticle quality is of vital importance, as the safety of table eggs depends, to a large extent, on it. The cuticle and its degree of coverage are affected by many factors, such as the age of the hen, genetic background, rearing system, and egg storage conditions [17, 18].

Eggs can be contaminated at different stages from the production stage, through processing, cooking, and consumption. Transovarial or "vertical" transmission of microorganisms occurs when eggs are infected during their formation in the hen's ovary. Horizontal transmission occurs when eggs are exposed to an environmental contaminant and microorganisms penetrate through the eggshell [13, 19].

In the past decade, Non-typhoidal *Salmonella* (NTS) caused an estimated 1.028 million cases, >19,000 hospitalizations, and 378 deaths in the USA, at a cost of \$3.3 billion [20]. Although NTS is frequently isolated in different foods of animal origin, poultry is considered an important reservoir, and contaminated poultry products are also a significant vehicle for human infection. There are >2400 recognized serotypes of NTS. However, not all are isolated from poultry; for example, *Salmonella enteritidis*, *Salmonella typhimurium*, and *Salmonella heidelberg* are historically associated with poultry. However, *Salmonella kentucky* has positioned itself as the predominant serotype associated with U.S. poultry. This change in the population dynamics of *Salmonella* in U.S. poultry has a far-reaching implication for food safety [21]. The increase in multi-drug resistance (MOR) in *Salmonella* serotypes of both animal and human origin, and, in particular, resistance to important clinical antimicrobials, is an emerging concern worldwide [22].
