Bacillus spp. as potential probiotics: promoting piglet growth by improving intestinal health

The application of Bacillus spp. as probiotics in the swine industry, particularly for piglet production, has garnered significant attention in recent years. This review aimed to summarized the role and mechanisms of Bacillus spp. in promoting growth and maintaining gut health in piglets. Bacillus spp. can enhance intestinal barrier function by promoting the proliferation and repair of intestinal epithelial cells and increasing mucosal barrier integrity, thereby reducing the risk of pathogenic microbial invasion. Additionally, Bacillus spp. can activate the intestinal immune system of piglets, thereby enhancing the body’s resistance to diseases. Moreover, Bacillus spp. can optimize the gut microbial community structure, enhance the activity of beneficial bacteria such as Lactobacillus, and inhibit the growth of harmful bacteria such as Escherichia coli, ultimately promoting piglet growth performance and improving feed efficiency. Bacillus spp. has advantages as well as challenges as an animal probiotic, and safety evaluation should be conducted when using the newly isolated Bacillus spp. This review provides a scientific basis for the application of Bacillus spp. in modern piglet production, highlighting their potential in improving the efficiency of livestock production and animal welfare.


Introduction
Weaning is one of the key events in the life cycle of pigs.Changes in diet and environment during weaning usually result in reduced feed intake, intestinal inflammation, and imbalances in intestinal microbial composition, leading to diarrhea in weaned piglets (1)(2)(3).In practical production, the addition of antibiotics (4,5) or high doses of zinc oxide (ZnO) (2,(6)(7)(8) to the diets has been found to effectively alleviate diarrhea in piglets after weaning and promote their growth and development.However, while acknowledging the positive effects of antibiotics and high doses of ZnO on weaned piglets, it is also necessary to recognize their negative effects.Long-term or excessive of antibiotics in swine will lead to the imbalance of pig intestinal microbiota, the increase of antibiotic-resistant pathogens, and the residue of antibiotics in pork, which will directly or indirectly harm human health (9,10).Excessive or long-term use of high doses of ZnO may lead to dull skin and coarse hair, inhibit the growth and development of pigs, reduce the bioavailability of ZnO, and may also lead to an increase in the percentage of intestinal bacteria resistant to multiple drugs, causing waste of zinc resources and environmental pollution (11)(12)(13).The prohibition or restriction of dietary antibiotics, and the reduction of ZnO in feed have formed a broad consensus in the world.Therefore, the search for green, safe and efficient non-antibiotic additives and ZnO substitutes, such as essential oils (14), organic acids (15), prebiotics (16), and probiotics (17), and so on to improve intestinal health and reduce diarrhea of weaned piglets has become a hot field of animal nutrition.
Probiotics are live bacteria that can improve the beneficial flora of the host with single or multiple bacteria (18).Previous studies have demonstrated that probiotics can improve the immune function, enhance the ability to resist bacterial infection, regulate the balance of intestinal flora, and thus improve production performance of piglets (19)(20)(21)(22).There are many kinds of probiotics, including Bifidobacteria, Lactobacillus, yeast, Bacillus spp., etc.Among these probiotics, Bacillus spp.has gained increasing attention as an alternative to antibiotics or ZnO due to its advantages such as heat resistance during feed granulation, resistance to low pH in the stomach, and stability at ambient temperatures (23,24).
Bacillus spp.are Gram-positive aerobic or facultative anaerobic bacteria capable of producing resistant endospores (25).Bacillus spp.can adapt to a variety of environments and are widely distributed around the world because their spores are highly resistant to ultraviolet radiation, high temperatures, strong acids, ionizing radiation, and many toxic chemicals (26,27), making it widely used in industrial, agricultural, medical and other fields (25,(28)(29)(30).This is also the advantage of Bacillus spp. as a probiotic for animals.In the field of animal nutrition and feed science, compared with other types of probiotics, Bacillus spp.has more advantages.First of all, in the process of feed processing, Bacillus spp.can resist high temperature because of the presence of spores, and therefore, Bacillus spp.can maintain biological activity after processing, and its spore-forming ability is beneficial for long-term storage compared to that of nonpore-forming bacteria (31,32).For example, Amerah et al. (33) showed that Bacillus spores are resistant to pelleting temperatures of up to 90 ° C, and more than 90% of spores are still alive in feed samples.Second, because of the strong stress resistance of Bacillus spores, they can tolerate cholate and stomach acid after entering the gastrointestinal tract, and successfully colonize and play their role in the intestine (23,24).For example, Dong et al. (34) showed that Bacillus licheniformis (B.licheniformis) WBL009 had strong acid and bile salt resistance.The survival rate of B. licheniformis WBL009 was 35.8% after 2 h treatment in artificial gastric juice with pH value of 2.5, and 51.6% after 12 h treatment with bile salt concentration of 0.3%.
Numerous studies have shown that Bacillus spp.can produce extracellular enzymes such as glycoenzyme, protease and lipase, and also has antibacterial and immune regulation effects, thereby improving the nutrient digestibility, reducing the colonization of harmful bacteria such as, Escherichia coli and Clostridium perfringens of piglets, and thus improving the growth performance of piglets (35)(36)(37)(38)(39)(40).There are many types of Bacillus spp., including Bacillus subtilis (B.subtilis), B. licheniformis, Bacillus coagulans (B.coagulans), Bacillus megaliformis (B.megaliformis), Bacillus cereus (B.cereus), and so on.However, not all Bacillus spp.are effective probiotics, some strains cannot be used because of limitations such as poor colonization effect or virulence factors, and some Bacillus spp.strains have been identified as opportunistic pathogens that causing food spoilage and toxin release in the hosts (41).Therefore, although most of the Bacillus spp.are generally recognized as safe for animal consumption, it is necessary to summarize the application effect of different Bacillus spp. on piglets.In this paper, the effects of Bacillus spp. in piglet production were summarized from the perspective of growth promotion and health benefits, so as to provide a reference for the rational utilization of Bacillus spp. in swine production.

Growth promoting effect of Bacillus spp. on piglets
Early weaning technology is the production method commonly used in most intensive pig farms at present.The weaning process of piglets is accompanied by a series of changes in diet, society and environment, which can easily cause weaning stress (3).The transition from high digestibility liquid milk to non-digestible solid feed is a great challenge for the immature gastrointestinal tract of the piglets, which usually causes diarrhea due to indigestion (42).Due to the combination of sudden separation from the sow, social factors, and other influences, piglets experience anxiety, leading to a reduction in feed intake or even refusal to eat (3,42,43).It is generally believed that the production performance of the whole pig farm is determined by the production performance of the piglets, and diarrhea is the culprit leading to the low production performance of the piglets.Therefore, the control of post-weaning diarrhea of piglets is of great significance to improve the economic benefits of pig production.
In addition to the addition of single strains of Bacillus spp., the combination of Bacillus spp. with other probiotics or functional substances has shown a synergistic effect in promoting the growth performance of piglets.For example, Liu et al. (35) showed that the combination of B. subtilis QST713 (100 mg/kg) with β-mannanase (150 mg/kg) effectively decreased the feed conversion ratio (FCR) of piglets throughout the trial period.Jiao et al. (37) demonstrated that compared to the control group, the combination of Bacillus spp.
(1.3 × 10 9 CFU/kg; B. licheniformis and B. subtilis with the ratio of 1: 1) with medium-chain fatty acid (0.588 g/kg) significantly increased the average daily gain (ADG) and dry matter intake of piglets in phase 1 (days 1 to 9), and significantly increased the ADG of piglets in the whole period (days 1 to 36).Liu et al. (80) showed that the combination of B. subtilis with Lactobacillus plantarum (L.plantarum) jointly increased average daily feed intake (ADFI) and ADG of weaned piglets in d 14 ~ 28 and d 28 ~ 42.Pu et al. (81) showed that piglets fed a diet added with benzoic acid (3,000 g/t) and B. coagulans (400 g/t)  85) showed that dietary supplementation with Bacillus spp.alone has no effect on the growth performance of piglets.The different effects of Bacillus spp. on alleviating diarrhea and improving growth performance of weaned piglets may be closely related to the different types of Bacillus spp., dosage, adding stage, use mode, diet composition and physiological health status of piglets.Although the effect of Bacillus spp. on the growth performance of weaned piglets is inconsistent, it is certain that Bacillus spp.has some other benefits, such as promoting intestinal health.

Intestinal health benefits of Bacillus spp. on piglets
Weaning is the transitional period when piglets shift from relying on maternal milk nutrition to solid feed.During this time, the digestive system of the piglets faces significant challenges, making them susceptible to intestinal dysfunction and diseases (86,87).Promoting intestinal development and intestinal health of weaned piglets is the key to cope with weaning stress.In previous studies, there are many ways to promote the intestinal health of piglets, among which probiotics, especially Bacillus spp., are prominent in promoting the intestinal health of piglets.

Bacillus spp. promotes intestinal development
The gut is the main site for the digestion and absorption of nutrients in animals and serves as a selective barrier for the body to prevent exogenous harmful substances from entering the circulatory system (3, 87).The gastrointestinal digestive system of piglets is still immature, and external stress will further damage the intestinal structure of piglets, such as intestinal villi shedding, crypt hyperplasia, intestinal mucosal atrophy, etc., and then destroy the intestinal mucosal barrier function and digestive absorption capacity (3, 88, 89).Previous studies have shown that Bacillus spp.has good nutritional physiological effects on intestinal development of piglets, including the maintenance of intestinal morphology and structure (50, 71, 73, 75, 85, 90), promoting the secretion of intestinal digestive enzymes (49, 75, 91, 92), and reducing intestinal permeability (59,90,91).
Bacillus spp.have a positive effect on the intestinal morphological structure and function of piglets, and can improve the intestinal damage caused by various stresses, so as to maintain the integrity of intestinal mucosa (65,66).Bacillus spp.can increase the intestinal villus height (VH), decrease the intestinal crypt depth (CD), increase the intestinal villus height to crypt depth ratio (VCR) and reduce the apoptosis of intestinal mucosal epithelial cells of piglets (50, 67,68,92).For instance, Du et al. (67) showed that dietary supplementation of B. amyloliquefaciens (5.0× 10 9 CFU/kg) significantly increased intestinal VH and VCR, while decreased intestinal CD.Hu et al. (92) reported that dietary supplementation with B. subtilis PB6 can improve intestinal damage of suckling piglets caused by intrauterine growth retardation (IUGR), as shown by higher VH and VCR, and lower CD than that of IUGR piglets without B. subtilis supplementation.Du et al. (93) showed that dietary supplementation with B. amyloliquefaciens SC06 (2.0 × 10 8 CFU/kg) significantly increased intestinal villus length, and intestinal villi morphology was improved.Wang et al. (36) showed that the combination of B. subtilis with B. licheniformis significantly increased the ileum VH and the jejunum and ileum VCR, and decreased the jejunum CD of piglets.The increase in VH and the decrease in CD on the intestinal mucosa effectively increase the surface area of the intestinal tract in contact with the digested food, thus improving the absorption efficiency of nutrients.
Most Bacillus spp.have a strong enzyme production capacity and can produce a variety of extracellular enzymes, such as cellulase, xylanase, amylase, protease, lipase (94)(95)(96)(97)(98), which can assist animals to digest feed and improve nutrient absorption.In addition to the exogenous enzymes secreted by themselves, Bacillus spp.can also stimulate the secretion of various digestive enzymes in the intestines of piglets (49, 75, 91, 92).For example, Deng et al. (49) showed that B. subtilis supplementation significantly increased ileum lipase, amylase, lactase, and maltase activities compared to control group, and significantly increased ileum lactase, and maltase activities compared to antibiotic group.Hu et al. (91) showed that replacement of aureomycin with B. amyloliquefaciens (2 × 10 8 cfu/Kg) significantly

Bacillus spp. promotes intestinal mucosal barrier
The intestinal epithelium serves as the primary barrier, facilitating nutrient breakdown and absorption via brush border enzymes and various transport proteins at the apical and basolateral membranes, while also protecting against antigenic invasion (3).Tight junctions are vital components of the intestinal mucosal barrier against harmful pathogens, predominantly made up of membrane protein complexes, including zonula occludens proteins (ZOs), occludin, and claudins (109).Alterations in the structure and function of tight junctions may directly impair the integrity of the intestinal mucosal barrier, leading to the infiltration of pathogens and other undesirable substances through the epithelial layer (110).Previous studies have confirmed the regulatory role of Bacillus spp. on intestinal tight junction proteins in piglets (35,75,105,(111)(112)(113).For instance, in a study using an IPEC-J2 cells model infected with E. coli, Sudan et 105) demonstrated that piglets supplemented with B. licheniformis, either alone or in combination with Clostridium butyricum significantly up-regulated the protein expression of ZO-1 and Occludin in the jejunum, as well as ZO-1, Claudin-1 and Occludin in the ileum of piglets.In addition, Yang et al. (114) showed that dietary supplementation with a low dose (3.9 × 10 8 CFU/day) of a mixture of B. licheniformis and B. subtilis significantly up-regulated the protein expression of ZO-1 in the jejunum of piglets infected with E. coli, thereby preventing the loss of intestinal epithelial barrier integrity.
Another important molecule in the intestinal mucosal barrier is mucins (MUCs), which are secreted by intestinal goblet cells (87, 115).As principal constituents of the mucus layer, MUCs facilitate gut lubrication and form the initial line of defense within the mucosal barrier, which can promote the colonization of symbiotic bacteria, inhibit the attachment of pathogens, and maintain the homeostasis of the intestinal environment (116,117).Therefore, the differentiation and proliferation of intestinal goblet cells, along with the normal secretion of MUCs, are crucial for maintaining intestinal health.Stress commonly disrupts these processes in piglets, leading to compromised cellular functions.For example, studies by Li et al. (77), Zhang et al. (118), and Xu et al. (119) showed that the number of intestinal goblet cells decreased significantly when piglets were infected with E. coli.Conversely, Bacillus spp.have been shown to enhance the maturation and differentiation of intestinal goblet cells and the secretion of MUCs in piglets (60,68,80,93,107,118).Specifically, research by He et al. (60) revealed that dietary inclusion of B. pumilus significantly increased the number of goblet cells in duodenal villi, while supplementation of B. subtilis significantly up-regulated the expression of MUC2 gene in the jejunum mucosa of piglets.Liu et al. (80) showed that the combination of B. subtilis with L. plantarum at dosage of 1 kg/t significantly increased the number of colonic goblet cells of weaned piglets.Zhang et al. (118) demonstrated that dietary supplementation a dosage of 7.8 × 10 8 CFU/kg Bacillus spp.probiotics mixture significantly increased the number of ileal goblet cells, and up-regulated the expression of MUC2 gene in the ileum of piglets infected with E. coli.In conclusion, the protective effect of Bacillus spp. on intestinal mucosal barrier of piglets is achieved by promoting the expression of intestinal tight junction proteins and the secretion of MUCs.

Bacillus spp. promotes intestinal immune function and inhibits inflammatory response
The intestine is not only the most important digestive organ in animals but also the largest immune organ.The gut immune system recognizes and combats pathogens, such as bacteria, viruses, and parasites, that traverse the gut surface to prevent them from entering the bloodstream and causing systemic infections, thus playing a pivotal role in maintaining overall health (3).The intestinal Initially, Bacillus spp.enhances the intestinal immune capacity of piglets by stimulating the activity and proliferation of immune cells.The intestinal immune system of piglets is not fully matured in their early stages, and Bacillus spp.can stimulate immune cells in the intestinal submucosa, particularly T cells and B cells in the gastrointestinal-associated lymphoid tissue (GALT), to promote their proliferation and differentiation (114,122,123).This proliferation helps to establish a stronger and more active immune defense network, thereby enhancing the ability to resist pathogenic invasions.For instance, Xie et al. (107) showed that dietary supplementation with L. acidophilus and B. subtilis mixture increased CD4+ T cells and sIgA+ cells in intestinal mucosa of piglets, and Zhang et al. (121) showed that oral administration of B. subtilis significantly increased the number of IgA secreting cells and CD3+ T cells in intestinal tract of piglets, which suggests a direct influence on the mucosal immune response.Yang et al. (114) demonstrated that dietary supplementation with high-dose (7.8 × 10 8 CFU/day) of B. licheniformis and B. subtilis mixture significantly increased the percentage of CD4 − CD8 − T cells in the inflamed intestine of piglets challenged with E. coli.Similarly, Zhou et al. (122) showed that adding a combination of B. licheniformis and B. subtilis to the diet notably increased the percentage of CD4 + Foxp3+ T regulatory cells among the intraepithelial lymphocytes.It also increased the presence of CD4 + IL-10+ T cells in the Peyer's patches and the lamina propria of small intestines in E. coliinfected piglets.
Secondly, Bacillus spp.enhances the intestinal immune function of piglets by producing a variety of antimicrobial substances, such as antimicrobial peptides (AMPs) and short-chain fatty acids (SCFAs), which directly inhibit the growth of pathogens, thereby protecting piglets from infections (77,105,107,124).For example, Xie et al. (107) observed that dietary inclusion of a combination of L. acidophilus and B. subtilis upregulated the gene expression of AMPs, including porcine beta defensin-2 (PBD-2), PBD-3 and regenerating islet-derived IIIγ (RegIIIγ), and short-chain fatty acid receptors including GPR43, GPR41and GPR109A in the ileum mucosa of piglets.Fu et al. (105) showed that the use of B. licheniformis alone had no effect on the expression of AMPs genes, while the combination of B. licheniformis and Clostridium butyricum could significantly increase the expression of intestinal AMPs genes, including PBD-1, PBD-2, PBD-3 and PR-39 in piglets.At the same time, the use of B. licheniformis alone or in combination with Clostridium butyricum significantly increased the content of acetic, propionic acid, butyric acid, and total acid in the ileal contents of piglets.
Finally, Bacillus spp.also plays an important role in inhibiting inflammation by regulating the production of inflammatory cytokines, such as reducing the release of pro-inflammatory factors (such as TNF-α, IL-1β, and IL-6), while increasing the production of antiinflammatory cytokines (such as IL-10, and IL-22), thereby preventing intestinal damage caused by excessive immune activity (47, 59,72,80,105,107).For example, Yu et al. (72) showed that dietary B. licheniformis supplementation significantly increased antiinflammatory factors (IL-10), and reduced pro-inflammatory factors (TNF-α and IL-6) levels in the jejunal mucosa of piglets challenged with LPS.Li et al. (77) showed that B. halotolerans can inhibit the expression of various inflammatory factors in intestines of piglets by suppressed the activation of the toll-like receptor (TLR)2/TLR4myeloid differentiation factor 88 (MyD88)-nuclear transcription factor-κB (NF-κB) pathway.Xie et al. (107) demonstrated that dietary inclusion of a combination of L. acidophilus and B. subtilis upregulated the expression of IL-22 in the ileum mucosa of piglets.In an E. coliinfected IPEC-1 model, Ji et al. (69) showed that B. amyloliquefaciens can inhibit the mRNA expression of IL-1α, IL-6, IL-8, and TNF-α by suppression of mitogen-activated protein kinase (MAPK) signaling pathways.In addition, Bacillus spp.can promote intestinal immune function by promoting the secretion of intestinal secretory immunoglobulin A (sIgA), which is also an important mechanism for suppressing excessive immune responses and inflammation (44, 77,80,107).

Bacillus spp. inhibits pathogenic bacteria and regulates intestinal flora homeostasis
The gastrointestinal tract of animals is inhabited by a large number of microorganisms, and these microorganisms and their metabolites play an important role in host health in terms of nutrition, intestinal barrier and immunity through interaction with intestinal mucosa (125,126).Changes in diet, environment, and other factors impact (3, 87).The optimization of intestinal microbial structure of piglets by nutritional strategies is one of the current research hotspots (127)(128)(129).Among which, the use of probiotics including Bacillus spp.have a significant effect on the regulation of intestinal microbial homeostasis in piglets (76,(130)(131)(132)(133).For example, Wang et al. (36) showed that dietary supplementation with B. subtilis and B. licheniformis mixture decreased the abundance of Blautia and Clostridium, while increased the abundances of Bacteroidetes and Ruminococcaceae.Hu et al. (46), Wang et al. (52), and Li et al. (77) showed that piglets fed Bacillus spp.significantly increased the number of intestinal Lactobacillus and decreased the number of E. coli.In which, Bacteroidetes is benefits for the degradation of proteins and carbohydrates (134), and the activation of host's immune system (135).Ruminococcaceae are associated with energy production and can ferment cellulose and hemicellulose to produce SCFAs (136).Clostridium is associated with diarrhea, and high abundance of intestinal Clostridium increases the risk of diarrhea in piglets (137).The abnormal increase of E. coli abundance can cause intestinal oxidative damage, reduce immune function and destroy intestinal integrity, making it a primary pathogen causing diarrhea of piglets (77,138).Lactobacillus plays an important role in intestinal health, such as preventing diarrhea and intestinal infections, so it is considered to be a beneficial bacterium to maintain the balance of intestinal flora (46).Previous studies shown that an important sign of piglet diarrhea is primarily distinguished by the increased number of E. coli and the decreased number of Lactobacillus in intestines (46,77,138).
Therefore, the potential mechanisms by which Bacillus spp.modulate the gut microbial composition of piglets involve the promotion of beneficial bacterial proliferation and the suppression of pathogenic bacterial growth.On one hand, Bacillus spp.competes with opportunistic pathogens for adhesion sites and nutrients, thereby inhibiting the attachment and colonization of harmful microbes in the intestinal tract (139).On the other hand, Bacillus spp.ferments carbohydrates to produce a large amount of L-lactic acid, which reduces intestinal pH value, forms an anaerobic acidic environment conducive to the growth of beneficial bacteria such as Lactobacillus and bifidobacterium, and prevents the invasion of aerobic and eosinophilic pathogens (140,141).In addition, Bacillus spp.secretes a variety of antibacterial substances, such as MUCs, AMPs, sIgA, and SCFAs, which exhibit marked antagonistic activity against various pathogens, thereby regulating the balance of intestinal microbiota (80,107,118).The beneficial effects of Bacillus spp. on the intestinal health of piglets are summarized in Table 2.A review of these literatures revealed that Bacillus spp.exert a positive influence on intestinal health of piglets.Firstly, Bacillus spp.facilitates intestinal development and reduces gut permeability in piglets.Secondly, Bacillus spp.enhances the function of the intestinal mucosal barrier by upregulating tight junction proteins and stimulating MUCs secretion.Thirdly, Bacillus spp.enhances intestinal immunity by activating immune cells within the gut, modulating the secretion of both pro-inflammatory and anti-inflammatory cytokines, and increasing the production of secretory immunoglobulins.Lastly, Bacillus spp.helps maintain a dynamic balance of the intestinal microbiota by encouraging the growth of beneficial microorganisms and suppressing the proliferation of pathogenic bacteria.Numerous studies have shown that a healthy gut can promote the digestion and absorption of nutrients, thus promoting animal growth (3).Analysis of the existing literatures found that Bacillus spp.had good regulatory effects on the intestinal tract of piglets which can promote intestinal development, promote intestinal mucosal barrier, promote intestinal immune function, inhibit inflammatory response, inhibit pathogenic bacteria and regulate intestinal flora homeostasis.Moreover, most of the Bacillus can promote the growth of piglets.Given the close connection between intestinal health and animal growth, we can speculate that Bacillus spp.promotes growth by promoting intestinal health of piglets.

Potential risks and concerns of Bacillus spp. as probiotics
Over the past decade, concerns about the overuse of antibiotics have shifted attention to probiotics in the animal feed industry as they can improve growth performance and reduce disease risk (32,144).The spore-forming Bacillus spp.has received extensive scientific and commercial attention, and their beneficial effects have been widely reported and acknowledged (32,41).However, with a better understanding of their positive role, many questions have been raised about their safety and the relevance of spore formation in the practical application of this class of microorganisms.The first is the safety concerns of the Bacillus spp.Some strains of Bacillus can cause infections such as bacteremia and endocarditis when they enter the bloodstream (27, 41).For example, A study by Deng et al. (145) who had collected 50 commercial probiotic products and isolated bacillus from the products, which showed that 34 probiotic products (68%) exhibited hemolysis, including 19 human probiotics, 9 animal probiotics, and 6 plant probiotics.19 of 28 B. cereus isolates maintained to exhibit hemolysis after heat treatment.Secondly, pathogenic potential.Some species within the Bacillus genus are known to be pathogenic.For example, Bacillus cereus is notorious for causing food poisoning, which causes great harm to food safety and animal health by producing enterotoxin and vomitoxin (146, 147).For example, Li et al. (146) showed that piglets received Bacillus cereus caused diarrhea, weight loss, and reduced IgG titers of swine fever virus (CSFV) and porcine epidemic diarrhea (PED).The potential for probiotic strains to switch from a beneficial to a pathogenic state is a concern.The third risk factor is antimicrobial and antibiotic resistance issues.There is a worry that the use of Bacillus spp. as probiotics could contribute to the spread of antibiotic resistance genes, particularly in the context of their extensive use in the food industry and as a biological control agent in agriculture (41, 148).For example, Deng et al. (145) showed that all 48 Bacillus spp.isolates exhibited resistance to lincomycin, and 5 were resistant to tetracycline.Zhu et al. (149) evaluated the safety of 15 strains of Bacillus cereus and found that nearly half of the strains carried the antimicrobial resistance gene tet (45).In one strain, tet (45) is located on a mobile genetic element that encodes site-specific recombination mechanisms and is transferred to Staphylococcus aureus and B. subtilis by electrical transformation.Zhai et al. (150) tested the antimicrobial resistance and antibiotic resistance of 114 isolates of Bacillus spp., and the antibiotic susceptibility tests showed that the intrinsic resistance rates of Bacillus to ampicillin and penicillin were 80 and 86%, respectively.Bacillus strains with acquired antibiotic resistance may spread antibiotic resistance between Bacillus and other clinical pathogens through horizontal gene transfer.Fourth, strain-specificity concerns.As probiotics, the action of Bacillus spp.appears to be strain-specific, which means that not all strains will have the same benefits or risks.This adds a layer of complexity to their use and regulation (41).Fifth, lack of standardization.There is a lack of standardization in the production and use of Bacillus spp.probiotics, which can lead to variability in efficacy and safety (27).Lastly, regulatory challenges.The regulatory framework for probiotics, including Bacillus spp., varies by country and is not always clear, leading to challenges in ensuring the safety and quality of probiotic products (27).In conclusion, Bacillus spp.has advantages as well as challenges as an animal probiotic, and safety evaluation should be conducted when using the newly isolated Bacillus spp.

Conclusion
The use of Bacillus spp. as probiotics in piglets offers a promising approach to promote growth and enhance intestinal health.The mechanisms by which Bacillus spp.exert their beneficial effects include improving growth performance, enhancing intestinal mucosal barrier function, improving intestinal immune function and producing antimicrobial compounds, as well as modulating gut microbiota.Further research is needed to identify the most effective strains and optimal application strategies to maximize the benefits of Bacillus spp. as probiotics in piglets.

TABLE 1
Growth promoting effect of Bacillus spp. on piglets.Bacillus spp.alone or in combination with other additives on weaned piglets, some studies have shown that Bacillus spp.has no effect on the growth performance of weaned piglets.For example, Jiao et al. (37), Huting et al. (38), Kritas and Morrison (83), Luise et al. (84), and Ding et al. (

TABLE 1 (
Continued) (108)5,(99)(100)(101)a + /K + -ATPase activities of piglets, and half replacement of aureomycin with B. amyloliquefaciens (1 × 10 8 cfu/Kg), the activity of intestinal chymotrypsin was significantly improved.Many studies have shown that dietary addition of Bacillus spp.can significantly improve the nutrient digestibility of piglets(38,45,(99)(100)(101), which may be related to the secretion of intestinal digestive enzymes.For instance, Huting et al.(38)showed that dietary addition of multi-strains of Bacillus spp.significantly improved the apparent total tract digestibility (ATTD) of dry matter (DM) and organic matter (OM) of piglets.Hu and Kim(45)showed that dietary B. subtilis C-3102 supplementation significantly improved the apparent the ATTD of DM, crude protein (CP), and energy of piglets.Cai et al.(99)and Lewton et al.(100)showed that piglets fed with Bacillus spp.based direct-fed microbial had a higher protein utilization, as indicated by increased the ATTD of nitrogen.Cui et al.(101)showed that the ether extract (EE) and phosphate digestibility was increased when piglets fed with B. subtilis.When exposed to stress conditions such as weaning stress, Escherichia coli (E.coli) infection, IUGR and lipopolysaccharide (LPS) stimulation, piglets are susceptible to intestinal mucosal injury, potentially leading to increased intestinal permeability (3, 87, 102-104).In general, intestinal permeability is assessed by quantifying the passage of small molecules like horseradish peroxidase(59), monitoring plasma levels of D-lactic acid, endotoxins, and diamine oxidase (DAO) (37, 44, 105), as well as by measuring trans-epithelial electrical resistance (TEER) in intestinal tissues(106).Studies have shown that Bacillus spp.can reduce intestinal permeability of piglets.Km et al.(59)demonstrated that a high dosage of B. subtilis (2.56 × 10 9 CFU/kg) supplementation lowered both intestinal intercellular and intracellular permeability in E. coli infected pigs.Hu et al.(91)showed that substituting aureomycin with B. amyloliquefaciens (2 × 10 8 cfu/Kg) preserved intestinal integrity, as evidenced by significantly decreased DAO activity.Xie et al.(107)showed that a combination of Lactobacillus acidophilus and B. subtilis reduced serum DAO level in piglets.In an IPEC-J2 cell model of deoxynivalenol (DON)-induced injury, Gu et al.(108)reported that TEER was higher in cells treated with B. subtilis compared to non-B.subtilis treated cells.
al. (37) demonstrated that B. subtilis significantly upregulated the expression of ZO-1, claudin-1, and occludin genes.Similarly, Li et al. (77) found that B. halotolerans markedly increased both the gene and protein expression levels of ZO-1, claudin-1, and Occludin in weaned piglets suffering from E. coli-induced diarrhea.Sun et al. (75) showed that dietary inclusion of B. licheniformis significantly promoted the expression of Occludin and ZO-1 in jejunum mucosa of piglets.Additionally, Fu et al. (

TABLE 2
Beneficial effects of Bacillus spp. on the intestinal health of piglets.