Zn from dietary intake is mostly absorbed in the small intestine, with the main absorption taking place in the duodenum followed by the proximal jejunum. Absorption occurs primarily through apical transport with the participation of Zn transporters in the apical membrane (25). There are two major protein families which include mammalian Zn transporters. The first group of transporters are ZIP (Zrt/Irt-like proteins), which are responsible for transporting Zn into the cytosol from either the extracellular space or from intracellular compartments. The second group of Zn transporters (SLC30 A1 - A10) transport Zn from the cytosol to the extracellular space or to intracellular organelles such as znosomes. Znosomes are vesicles able to bind large amounts of Zn (16). Metallothionein, one of the Zn binding proteins responds very rapidly to Zn status in the body (26). Up to 20% of intracellular Zn is bound to metallothionen and can be rapidly released. It has been shown that the high dietary levels of Zn lead to an increase of intestinal metallothionein synthesis, Zn sequestration, and altered binding capacity. Therefore, it appears that metallothionein may have a role in both Zn storage and regulation (7). Monitoring of metallothionein and metal transporters gene expression appears to be a promising additional marker for mineral status, mineral excretion, or antioxidant capacity in the field of pig breeding. A recently published study has shown that the inadequate dose of Zn and the antagonistic effect of other minerals can influence digestive enzyme activity and metal transporter gene expression (27).

The bioavailability of Zn depends on the composition of the food or feed. Indigestible plant ligands such as phytate, dietary fibers and lignin, can chelate Zn and inhibit its absorption. Other factors that affect the absorption of Zn are calcium and iron (16). Case et al. reported that the bioavailability of Zn from ZnO is lower than from other sources, such as ZnSO₄, Zn-methionine, and Zn-lysine. The unused ZnO in pigs' GIT might affect the overall mineral absorption (due to an antagonistic effect) and it is then excreted in the feces (12). The high amount of Zn in the slurry leads to a significant accumulation of Zn in the environment, which appears to be the biggest concern facing its application. Despite this, excess of inorganic Zn added to the diet still meets the Zn requirement for growth performance of animals (14). The deficiency of Zn may lead to severe disorders such as eye and skin lesions, hair loss, delayed sexual maturity. Weaned piglets develop first signs of clinical Zn deficiency (feed refusal) after ~10 days of low Zn intake (13). Dietary recommendations for pigs range ~80–150 mg/kg of diet (18).

In pig breeding practice, high doses of Zn have been used as an effective antibiotic replacement to prevent diarrhea in weaned piglets. The most frequently administered dose is Zn 2,000–3,000 mg/kg of diet for such purposes. Numerous studies have been conducted to clarify the antimicrobial effect on ZnO. However, the exact mechanism of Zn is not completely known, and individual results are still inconsistent. Due to the fact that a high dose of Zn is not fully absorbed into the body, a large part passes to the GIT, where the following mechanisms take place: (i) a mucosal layer formed on the intestinal epithelium, which prevents adhesion of pathogenic microorganisms; (ii) ratio of villi and crypts increases; (iii) high doses of Zn in an alkaline environment lyse the cytoplasmic membranes of microorganisms; (iv) Zn reduces ion secretion into the intestinal lumens thereby increasing water resorption and diarrhea prevention; (v) paracellular permeability reduces and thus prevents translocation of pathogenic microorganisms; (vi) free Zn ions can promote oxidation-reduction interactions (18, 28–30). Nevertheless, these mechanisms are likely to complement each other (28). Zn medication doses are given to weaned piglets for a maximum of 2 weeks after weaning. If the administration time were longer, the microbiome could be severely disrupted in terms of reducing lactobacilli as well as cellulolytic and proteolytic bacteria. Moreover, the disorders of fiber and protein digestion may occur (31). As a result, the animals' growth capacity would be reduced and fattening would be prolonged (32).

Inorganic nanoparticles are not digested in the GIT, but some of them may be fully or partially dissolved as a result of alterations in pH or dilution (48). ZnNPs can be decomposed into Zn ions in acidic solutions or biological fluids (49). It has been shown that nanoparticle chemical composition plays a major role in determining their fate in the GIT. Thus, oral application of ZnNPs can be challenging due to poor drug solubility and stability in the GIT, the decreased bioavailability due to variable pH of the biological environment and protective mucus layer, or the presence of digestive enzymes (50). After oral ingestion, mechanical forces, biological fluids, and the changes of pH along the GIT can influence the biological action of ZnNPs in GIT (51). Furthermore, these actions can be affected by any binding with several macromolecules present in feed. It is believed that lipids, proteins, and polysaccharides which could interact with NPs are digested by proteases, lipases, and amylases in GIT (48). The proposed ZnNPs' mechanism of action in piglets' GIT is illustrated on Figure 1.

The biokinetics of ZnNPs was comprehensively reviewed by Choi and Choy (52). From this summarizing article clearly accrues, that the bioavailability of ZnNPs depends on their chemical form, size, and surface charge. ZnNPs smaller than 100 nm can pass through the stomach wall, more rapidly diffuse from the intestinal mucus and enter the cells of the intestinal lining into the blood system more quickly than ordinary minerals with larger particle size (50). It has been reported that the surface-charge effect plays a crucial role in ZnNPs bioavailability. Negatively charged ZnNPs are absorbed in larger amounts than positively charged ZnNPs (52). However, the mechanism of this effect is still unclear. It is known that intestinal epithelial cells possess a negatively charged cell surface which provides electrostatic interaction with positively charged compounds (53). Penetration of NPs under normal healthy conditions cannot cross the blood vessel endothelium into the target tissue. But in certain pathological conditions, e.g., inflammation, endothelial cells lose cellular integrity due to the activation of pro-inflammatory cytokines and the distance between endothelial cells is increased. As a result, ZnNPs can extravasate from the vasculature to the diseased site through the abnormal endothelial gap (50). ZnNPs tend to accumulate in size-dependent manner in the liver, kidneys, lungs, and other internal organs (54). Additionally, the excretion of ZnNPs is controlled by nanoparticle size. The smallest ZnNPs could be eliminated via renal clearance, whereas when the NPs size increases, they are eliminated via fecal excretion (55).

ZnNPs belongs to the most studied group of inorganic nanoparticles in the field of microbiology, as many researchers want to know more about their antimicrobial effect, with a rapidly increasing trend in this research since 2012. During this time, the antimicrobial mechanisms of action on ZnNPs against various microorganisms in vitro and in vivo have been more and more scrutinized and spotlighted. Current knowledge proposes three mechanisms of the ZnNPs antimicrobial effect: (i) production of ROS; (ii) disruption of bacterial cell wall integrity; (iii) release of Zn²⁺ ions which leads to interaction with biomolecules and vital functions of bacteria (56, 57). By any of these means, it has been found that G+ bacteria are more susceptible to ZnNPs compared to G- bacteria either by the disruption of bacterial integrity, damage by ROS or downregulation the transcription of oxidative stress-resistance genes.

Due to the semiconductor and electric properties of ZnNPs, they can produce superoxide anions (O²⁻, HO⁻ and H₂O₂) which are able to interact with anionic cell walls. The mechanisms of cellular toxicity that elevate ROS production, exceeding the capacity of antioxidant defense systems, cause cells to enter a state of oxidative stress. The result of this is the damage of cellular components such as lipids, proteins, and DNA (58). Fatty acid oxidation leads to the generation of lipid peroxides that initiate a chain reaction resulting in disruption of plasma and organelle membranes, leading to cell death (59). The study in vitro (60) had indicated that various environmental conditions can cause differences in ZnNPs antimicrobial activity against E. coli and S. aureus. Environmental temperature also influences antibacterial activity due to its effect on the generation rate of ROS. When ZnNPs are stimulated by temperature, electrons are captured at the active sites. Afterward, the electrons interact with oxygen to produce ROS, thereby enhancing the antimicrobial effectiveness of ZnNPs.

Another mechanism comes from their ability to pass through the cell wall via the bacterial peptidoglycan fence due to their small size (5). The various effect on ZnNPs was observed in G+ and G- bacteria which is based on differences in bacterial cell structure. The bacterial cell wall is responsible for the osmotic pressure of the cytoplasm as well as the characteristic cell shape. This in turn it means that the construction of bacterial walls is one of the factors in the effectiveness of antibiotics. G+ bacteria have one cytoplasmic membrane with a multilayer of peptidoglycan polymer and a thicker cell wall (20–80 nm) (61). In contrast, the G- bacteria wall is composed of two cell membranes, an outer membrane and a plasma membrane with a thin layer of peptidoglycan (10) with a thickness of 7–8 nm. This points to bacteria being highly susceptible to damage because the NPs can readily pass through the peptidoglycan cell due to their high durability (62). Overall, the antimicrobial properties of ZnNPs are based on the electrostatic interactions between NPs and cell surface, in addition to aggregation and damage inside the cell (62).

In neutral or alkaline pH the ZnNPs remain intact. In acidic media, ZnNPs are able to release Zn²⁺ ions which can interact with several biomolecules such as proteins and carbohydrates (63). The partial dissolution of ZnNPs can occur in the stomach where the pH range is 2–3. Moreover, when they penetrate a bacterial cell, lysosome is formed, and the rapid decrease of pH can cause Zn²⁺ to release. Inside a bacterial cell, released Zn can affect internal metabolism and signaling pathways, which can lead to death. Al-Shabib et al. (64) reported that the released Zn²⁺ ions concentration of ZnNPs in the simulated gastric fluid was six-times higher than that of an unprotected ZnO (65) which led to higher toxicity for E. coli in vitro. Similar effects were shown in the study of Sirelkhatim et al. (10), where the antimicrobial activity of ZnNPs had been partly attributed to the ability of Zn penetration into the microbial cells and generation of ROS that damage key cellular components.

In terms of the Zn ions' release from NPs, there is less concrete knowledge about the direct effect of ZnNPs on bacterial surface components such as virulence factors (adhesins, invasins, impedins, modulins, agresins). Only a minimal number of studies were found by searching the literature. For example, it was observed the ZnNPs suppress biofilms formation in Chromobacterium violaceum, E. coli PAO1, Listeria monocytogenes, Streptococcus pneumoniae, and S. aureus in the dose-dependent manner (64, 66, 67). Green synthetized ZnO and xanthan gum nanocomposite showed inhibition effect to violacein (61%) chitinase (70%) in C. violaceum and prodigiosin (71%) and protease (72%) in S. marcescens at 128 mu g/mL concentration (68). In the term of E. coli, the ZnNPs showed efficiency as an anti-inflammatory agent and suppressed multidrug resistance producing virulence and resistance genes in E. coli isolated from chicken meat (69).

However, as with other antimicrobial compounds, concerns about bacterial resistance have been raised. Some studies reported that bacterial tolerance to NPs may be due to electrostatic repulsion, mutations, biofilm adaptation, efflux pumps, and expression of extracellular matrices in the bacterial system (70–72). At the same time, there is a possibility of deleterious effects on animal and human health due to microbiota disturbance influenced by external factors (such as diet) after exposure to toxic feed additives, antibiotics or drug use, infections, and the environment (73). In another study, it has been reported that NPs mechanisms of action did not correspond to the most of antibiotic effects and act in several ways simultaneously, thus NPs are a promising tool to bypass the bacteria's resistance mechanisms (74).

The following factors play pivotal roles in ZnNPs' mechanism of action: its morphology, concentration, size, surface charge, modification or doping. All of these factors influence not only antimicrobial properties, but also bioavailability, biodistribution, elimination, reaction of immune system, interaction with host cells or food and feed matrix. It must be remembered, however, that in vitro observations can never predict the behavior of particles in vivo and their impact on the host organism or its microbiome.

For the purpose of antimicrobial effect, there is a need to transport ZnNPs in high concentration, without any chemical changes which could influence their behavior. Simultaneously, the toxic effect and systemic damage to the organism is obviously not desirable (75). Many studies have been published which agree that the antimicrobial effect depends on their size. The smaller the NPs, the higher the antimicrobial effect. Since the most cited research has focused on clarification of size-dependent bacterial growth under ZnNPs treatment, there have been introduced several studies which shown a promising contribution in antimicrobial activity using plant extracts to synthetize ZnNPs. A promising aspect is the presence of phytoactive compounds in the NPs structure which can promote its additional properties (76). However, few studies compared these two means of synthesis, and some of them showed evidence that green synthesized ZnNPs even had lower antimicrobial effect (77, 78). Moreover, disadvantages of the green synthesis include poor control of NPs formation, difficulty in large-scale production and differences in compound composition in plant extracts (79). Similar discussion has been held regarding to efficiency of modified and doped ZnNPs. The benefits of the modification are that it protects them from the acidic environment in the stomach, prevents aggregation and/or ensures invisibility to the immune system and prevents protein corona formation (80). Also, doping of ZnNPs by metal ions such as iron shown improving effect on ZnNPs antimicrobial activity (81).

The colonization of the GIT of piglets occurs immediately after delivery and changes dynamically during the suckling, or weaning phase. Immediately after the birth, pigs' intestines are colonized by Clostridiaceae and Enterobacteriaceae families (90). The diversity of the microbial communities in the fecal samples further increase during weaning (91). In adult pigs, the number of microorganisms stabilizes at 1 × 10¹⁰-1 × 10¹¹ (91, 92). Meta-analysis of 16S rRNA amplicon sequence data showed the pig-specific species are Lactobacillus, Streptococcus, Clostridium, Desulfovibrio, Enterococcus, Fusobacterium. Also, several new genera have been described within the same study (93). The microbial diversity depends on various internal and external factors, such as are breed, sex, but also environment, feed composition, or other factors, such as stress (30, 93). Inter-farm variability of the microbial profile of piglets has shown the variability of relative abundances of Christensenellaceae and Lactobacillus in suckling piglets. However, the most abundant microbial families did not differ in weaned piglets (94). It has been shown, the microbial colonization has impact on pigs' health and performance (94). Genome-based mapping of the microbial community revealed the importance of specialized bacterial metabolites for microbe-microbe and microbe-host interactions (95). During the suckling phase, the dominating pathways are lipid metabolism pathways (such as lipid biosynthesis proteins), energy metabolism pathways (such as carbon fixation) and carbohydrate pathways (such as the citrate cycle). The nicotinate and nicotinamide metabolism which is related to the metabolism of cofactors and vitamins increased in weaned piglets compared to lactation. Moreover, weaned piglets showed a higher proportion of pathways involved in bacterial chemotaxis, motility proteins, flagellar assembly, lipopolysaccharide biosynthesis and sporulation pathway (96). Predictably, the stress response (oxidative stress and heat shock) gene families have been significantly enriched in the weaned piglets (91). The interaction network of pigs' microbiota have been recently reviewed and could be concluded, that maintaining a balance of interactions and synergistic effects of the intestinal microflora, whether diet, environment and proper welfare, has a significant effect on pig performance (97–99).

At the phylum level, the most abundant are Bacteroidetes, Firmicutes, Proteobacteria, Spirochetes in fecal microbiota of weanling piglets, whereas Fusobacteria is significantly reduced. At the genus level, the microbiome is dominated by Prevotella and Bacteroides from Bacteroidetes phyla. A significant increase of p-75-a5, Roseburia, Ruminococcus, Coprococcus, Dorea and Lachospira have been observed simultaneously with significant decrease of Bacteroides, Fusobacterium, Lactobacillus and Megashare (95). On the contrary, Guevarra et al. showed the populations of Prevotellaceae and Lactobacillaceae significantly increased in weaned piglets (91). Another study also mentions an increase of genera Veillonellaceae and Succinovibrionaceae (94). The interrelationships between microorganisms affect the metabolic and trophic functions of the microbiome and may suggest a susceptibility to certain diseases, including post-weaning diarrhea. For example, subjects with a high abundance of Prevotella usually have a lower prevalence of Bacteroides which have immunomodulatory effects (100, 101). Possible explanation could be that Prevotella produce anti-inflammatory short fatty acids which help to modulate the immune system (102). In addition, Prevotella-driven enterotype has shown a positive influence of feed intake, weight gain, and results in the decrease of diarrhea incidence (103). Comprehensive research of weaning piglets' microbiota have been conducted by Dong et al. They published a collection included 266 cultured genomes and 482 meta-genome-assembled genomes that were clustered to 428 species cross 1% phyla. They found that Limosilactobacillus reuteri was the most abundant species and represented 18% of the taxa isolated and half of these were Lactobacillus (104).

The main bacterial strains that primarily cause diarrhea in weaned pigs are considered pathogenic E. coli strains, Campylobacter spp. Clostridium perfringens and Salmonella spp. (105). The onset of post-weaning diarrhea due to E. coli is primarily caused by toxin secretion. The heat-labile (LT) and two heat-stable (STa and STb) adhesin strains K88 can secrete into target cells of the intestinal epithelium causing diarrhea. Even though they trigger different pathways, all toxins disrupt tight connections and activate the massive luminal secretion of electrolytes and water (106, 107). Despite activating various routes, all the toxins activate pro-inflammatory cytokines, affect tight junction functions, stimulate enormous luminal electrolytes, water secretions, and eventually cause diarrhea (32).

Several studies have explored the importance of the relationship between microbial composition and the occurrence of post-weaning diarrhea. It has been established that the beneficial microbiota are helpful in diarrhea prevention due to immunomodulation and interventions in defensive metabolic pathways (27). Results from recent research (88) which analyzed fecal microbiota in diarrheic piglets revealed that diarrhea was associated with the increase of Prevotella, Sutterella Campylobacter, and Fusobacterium and the decrease of Prevotellacea, Lachnospiraceae, Ruminococcaceae, Fusobacteriaceae and Lactobacillaceae (88, 91). In the case of the Fusobacterium, the higher expression of inflammatory factors that inhibits T-cell responses has been observed (108, 109). Intestinal inflammation contributes to creating such an environment, which is convenient for the growth of several diarrhea-connected pathogens. Salmonella enterica and enterotoxigenic E. coli belong to the Enterobacteriaceae family which prevail at a time when the population of Bifidobacterium, Lactobacillus is declining whereas Citrobacter, Clostridium, Rumonococcus and Diallister is increasing (110). The microbial diversity and evenness in diarrheal pigs differed over time according to their subsequent susceptibility to post-weaning diarrhea. The taxonomic composition showed the highest differences in the phyla Firmicutes and Bacteriodetes. The abundance of Lachnospiraceae, Ruminococcaceae and Prevotellaceae was increased, whereas the presence of Fusobacteriaceae and Corynebacteriaceae was decreased in healthy pigs compared to pigs with diarrhea (111). In a newly released study, the microbial colonization of healthy and pigs with diarrhea differs on the intestinal segments level. The most abundant were Lactobacillus genus (38%), Escherichia-Shigella and Enterococcus (11%), Bacteroides (9%), Fusobacterium (8%), Streptococcus and Prevotella (3%), Blautia and Clostridium (1%). The significant decrease was observed in Lactobacillus, Bacteroides in pigs with diarrhea compared to healthy pigs. Escherichia Shigella, Streptococcus, Sphaerochaeta and Enterococcus were increased in pigs with diarrhea compared to the healthy group. Surprisingly, the bacterial diversity was higher in the healthy pigs compared to pigs with diarrhea among all intestinal segments (illeum, caecum, colon and rectum) (112).

Nevertheless, it should be noted that although some studies differ in the microbial composition in the GIT of piglets, the susceptibility of piglets to diarrhea is not only based on the presence of “good” and “bad” bacteria. Some pathogenic microflora belong to the group of opportunistic pathogens and could co-exist together without any negative impact. It is necessary to take into the account the overall health as well as feed composition and welfare of animals (99).

Currently, there is a transition period between the EU ban of Zn medication doses in pig breeding and the employment of new approaches for reducing weaning pigs' diarrhea. Thus, there are some new studies emerging which are focused on the examination of the effects of both ZnO and ZnNPs on the intestinal microbiome of weaned piglets. In addition, methods such as 16S rRNA sequencing, next-generation sequencing and bioinformatics are more available among research teams. To date, there are several studies that are clarifying changes at a microbiome level in weaned piglets treated with medication doses of Zn. ZnO has been considered as effective against the abundance of opportunistic pathogens such as Campylobacterales, Enterobacteriaceae and Escherichia (5, 113, 114). Moreover, the abundance of beneficial bacteria such as Lactobacillus genera has been found in ZnO treated piglets (114, 115). Previous studies have shown the Zn is a significant factor in modulating the host microbiota and its biodiversity in dose dependent manner. Physiological doses of Zn could enhance species capable to improving gut wall integrity and immunomodulation. On the contrary, Zn overexposure could reduce bacterial diversity and lead to systemic inflammation (116). Based on analysis of growing on selective media, it was evident that 2,000 mg/L of ZnO caused the decrease of total anaerobes in stomach and small intestine, but not in caecum and colon. Lactobacilli were decreased in all parts of GIT. Coliforms were increased in caecum and colon, whereas enterococci were increased in all parts of GIT. 16S rRNA gene sequencing showed the decrease of L. amylovorus, L. reuteri and S. alactolyticus in the group treated by high doses of ZnO (2,000 mg/L) compared to the group with low dose of ZnO (100 mg/L) (117). A similar study (118), which compared porcine microbiome under 40 and 2,500 mg/L ZnO treatment, showed clear differences at genus and species level using metagenomic sequencing of colon microbiota. This study has shown the higher increases were in the genus of Prevotella, Roseburia, Bacteroides, Bacillus and the higher decreases in the genus of Lactobacillus, Megaspharea and Alisteps, whereas Clostridium, Eubacterium, Dorea, Streptococcus and Bifidobacterium remained unchanged The Prevotella/Bacteroidetes ratio in the gut microbiota has been shown to predict body weight in humans, in Prevotella pigs the rich enterotype has a beneficial effect on inflammation reduction in finish pigs due to the fact that representatives of the genus Prevotella use carbohydrates and produce anti-inflammatory short-chain fatty acids (102). The Prevotella-controlled enterotype showed a positive relationship, including feed intake and efficiency, weight gain, and prevention of diarrhea. This suggests that prevotella is important for mediating the growth performance and resistance of piglets to disease (103).

One of the first comparative studies between ZnO and ZnNPs in weaning piglets was presented by Oh et al. (119). This comprehensive study found that fecal score among various Zn forms (2,000 mg/kg ZnO, chelate-ZnO and 200 mg/kg ZnNPs) differed among phases (0–7, 7–14 days), but in the overall experimental period the control group showed lower fecal score compared to the treatment. The supplementation of various forms of Zn showed the significant increase in the genera Prevotella, Succinivibrio and Lactobacillus. Both species, Prevotella and Succinivibrio are related to the transition from milk to diet for weaning piglets, where Prevotella degrades polysaccharides in plant cell walls to short-chain fatty acids while Succinivibrio has cellulolytic activity (120–122). The specific differences between the bacterial abundance in ZnO and ZnNPs treated groups as well as the comparison among other studies can be found in Table 1. This study is in agreement with previous research where ZnNPs (600 mg/kg) increased bacterial richness and diversity in ileum and increased abundance of Streptococcus and Lactobacillus species in ileum, whereas the occurrence of Oscillospira and Prevotella decreased in colon. On the contrary, the dose of 600 mg/kg of ZnNPs was more effective in preventing pigs' diarrhea compared to the results of Oh et al. (123). Proposed mechanism of protective action of ZnNPs was explained by determination of mRNA expression of Cu-Zn SOD, GPX1, ZO-1 and Occludin in the jejunal tissue. However, an expression of mRNA was lower in ZnNPs-treated group compared to the ZnO group. Moreover, the study brings support to the evidence that in ZnO, free Zn is released more easily when compared to ZnNPs. This is probably due to the higher expression of metallothionein, which expression depends on Zn concentration in the body (127).

Recent studies (123) of ZnNPs modulation effect on pigs' microbiome is focused on the modified ZnNPs rather than crude ZnNPs. Hot melt extruded-based ZnNPs (HME-ZnO) differ from crude ZnNPs by coating ZnNPs with a polymer matrix which optimizes solubility and bioavailability of the carrier substance. It has been shown that the HME-ZnO and Zn diet decreased Clostridium spp. and coliforms in the ileum. Lactobacillus spp. were increased only in ZnO fed group. Cecal microbiota did not show differences among proposed treatments, only linear decrease of Clostridium spp. was observed with higher concentration of HME-ZnO. Dose-dependent decrease of Clostridium spp. was observed also in colon HME-ZnO-fed pigs. It should be noted, that the ZnO digestibility was lower compared to HME-ZnO which could lead to different effect on microbial composition. However, the effect on pig performance was the same for both high dose ZnO (2.5 mg/kg) and sub-pharmacological dose of HME-ZnO (500 and 1,000 mg/kg) (123).

Two types of ZnNPs based on phosphate were introduced as potential improving agents in weaning piglets' diarrhea. At the doses of 100, 500, and 2,000 mg/kg significant changes in the microbiota composition were observed compared to the control group. Significant shifts were observed in Escherichia, Streptococcus and Aerococcus genera at the doses of 500 mg/kg in ZnNPs A variant compared to the control group. Also, the abundance of Providencia and Staphylococcus were observed. Species such as Enterococcus, Yersinia, Pediococcus occurred in ZnNPs C variant compared to the control group. The higher dose of ZnNPs in ZnNPs A variant caused the increase of Escherichia compared to the ZnNPs C variant which was comparable with ZnO treated group. During the four experimental stages, there was noticeable reduction in bacterial diversity as well as the decrease of Lactobacillus occurrence. Also, the difference in the ZnNPs variant A and variant C suggested that the effect on ZnNPs strongly depends on its composition (125). Bacterial synergic effects in the intestinal microflora are well-known and could also play a role in enhancing the preventive effect against weaned piglets' diarrhea. In the study of Zheng et al. (124) the probiotic species L. plantarum have been introduced together with ZnNPs in pigs' diet after weaning. L. plantarum is also known as Zn tolerable bacteria (128) what was clearly visible from the results. The increased occurrence of Bifidobacterium and Lactobacillus and the decrease of Enterococcus and Enterobacteriaceae were predictors of improved pig's performance and reduced occurrence of diarrhea (129). In addition to L. plantarum, the microbiome of pigs comprises several bacterial strains that are tolerant to high doses of Zn. For instance, Bacillus altitudinis have been found in porcine microbiome (130). Moreover, its ability of Zn biosorption has been mentioned in the field of remediation purposes (130). Crespo-Piazuelo et al. found that supplementation of B. altitudinis spores improves porcine offspring growth performance (131). The most likely explanation is the stimulation of the immune system; however, the mechanism is not fully understood yet (93, 131).

The majority of previous studies on ZnNPs' efficiency focus mostly on the in vitro investigation of each specific microbial species. The advantage of these studies is the ability to monitor the effect of the composition and morphology of nanoparticles on the control of specific bacteria. However, in vitro studies mainly focus on important bacterial strains either as pathogens or prebiotic. Comparing the inhibitory concentrations from Table 2 (full version is available from repository: https://archive.org/details/osf-registrations-gqspd-v1), it is confirmed that ZnNPs act better on G+ bacteria, which are mainly the family Lactobacillaceae, Enterococcaceae, Streptococcaceae, although, inhibition activity is not negligible in the G- bacteria strains for family Enterobacteriaceae which are mostly pathogenic. A great deal of previous research into ZnNPs as antimicrobials has focused on the green synthesis. The purpose of green synthesis is, in addition to the ecological approach, also to use the potential of plant extracts, which have antibacterial properties, for the synergistic effect of ZnNPs. When compared to chemical route these initial observations suggest that green synthetized ZnNPs have less antimicrobial potential. However, the volume of this research annually increasing.

In vitro effectiveness of ZnNPs against pathogenic bacterial species is summarized in Table 2, but they have also been described other reviews (10). Nevertheless, it should be considered that it is not the species/strains which are potentially harmful to the host. It would be better to prevent undesirable pathological conditions by reducing the high diversity in the coliform bacteria group rather than low coliform concentrations (87). For instance, ZnNPs may inactivate the growth of diarrhea-causing E. Coli, but as it has been recorded in several studies (8, 165), it may inactivate the growth of commensal beneficial microorganisms as well in the GIT, which could lead to dysbiosis of the intestinal microbiome. It has been shown that post-weaning diarrhea control attributed to ZnO might not be directly related to effects on microbiota, like reducing Escherichia coli count in the intestine, but it may be related to the prevention of bacterial adhesion of K88 strains and internalization to the intestinal epithelium, which reduces the deleterious effects of toxins produced by these pathogens (126). In the case of ZnNPs the anti-adhesive-effect have been studied with similar results. The proposed mechanism of action relies on the internationalization of ZnNPs to the hydrophilic bacterial cell wall surface or by inhibiting exopolysaccharides which are the main component of bacterial biofilm (166).

Several concerns have been raised about ZnNPs' effects against probiotic bacteria strains which are crucial for the healthy gut in weaning piglets. In Table 2 it can be seen that the inhibitory concentration for Lactobacillus spp. is ~0.01–0.1 mg/mL, which is slightly lower concentration than, for example, for pathogenic E. Coli strains (0.06–5 mg/mL). According to Li et al. the effect could be explained by the existence of free Zn²⁺ ions which are complexed with amino acids, resulting in the decrease of ZnNPs toxicity in E. Coli (44). What has been established is that some probiotic strains are tolerant for high levels of Zn or, interestingly, they may function as a biofactory for the synthesis of ZnNPs (128). Although the effect of ZnO has some similarities to the effects of ZnNPs, in particular the release of Zn²⁺ ions may vary (for further details see Section ZnNPs Antibacterial Mechanism of Action). The proteomic study showed a dramatic change of the metabolic pathways in Bacillus subtilis exposed to ZnNPs (0.017 mg/mL, 100 nm, Sigma Aldrich). Major changes have been observed in methylglyoxal and thiol metabolisms, oxidative stress and stringent responses (167). As some researches have suggested, the effect of NPs on microorganisms is dose- and time-dependent, so the impact of ZnNPs should be carefully considered to maintain balance in the environment (167). Other research has been focused on the interaction of ZnNPs with cell membrane of probiotic bacteria strains. It has been observed the commercial ZnNPs (77 nm, Alfa Aesar, 20 mM) did not damaged the cell wall of Escherichia coli, Lactobacillus acidophilus, nor Bifidobacterium animalis. Only slight cellular morphological changes were observed (168). To contradict this study, another research team observed that commercial ZnNPs (0.1 mg/mL, 80 nm, positive surface charge, Shanghai Macklin Biochemical) significantly changed the morphology of Lactobacillus plantarum and Lactobacillus fermentum and destroyed their cell membranes in the dose-dependent manner.