Probiotics and Postbiotics as Substitutes of Antibiotics in Farm Animals: A Review

Simple Summary Breeders are searching for methods to protect farming animals against diseases caused by pathogenic bacteria. The easiest way to fight bacteria is to use antibiotics. Unfortunately, their abuse results in the presence of bacteria resistant to the most commonly used antibiotics in the environment. The restrictions on the use of antibiotics have forced the search for natural and safe ways to protect animals. It has been shown that the use of probiotics based on lactic acid bacteria may have a positive effect on the growth and use of feed by broilers, on the stabilization of the intestinal microbiota of chickens and pigs, and in the prevention of mastitis in dairy cows. The use of probiotics (live, nonpathogenic microorganisms) and postbiotics (inanimate bacteria, cell components or post-fermentation by-products) reduces the occurrence of pathogens in large-scale farms. Abstract Since 2006, the use of growth-promoting antibiotics has been banned throughout the European Union. To meet the expectations of livestock farmers, various studies have been carried out with the use of lactic acid bacteria. Scientists are trying to obtain the antimicrobial effect against the most common pathogens in large-scale farms. Supplementing the diet of broilers with probiotics (live, nonpathogenic microorganisms) stabilized the intestinal microbiota, which improved the results of body weight gain (BWG) and feed intake (FI). The positive effect of probiotics based on lactic acid bacteria has been shown to prevent the occurrence of diarrhea during piglet weaning. The antagonistic activity of postbiotics (inanimate bacteria, cell components, or post-fermentation by-products) from post-culture media after lactobacilli cultures has been proven on Staphylococcus aureus—the pathogen most often responsible for causing mastitis among dairy cows. The article aims to present the latest research examining the antagonistic effect of lactic acid bacteria on the most common pathogens in broilers, piglets, pigs, and cow farms.


Introduction
The invention of antibiotics is considered to be one of the greatest discoveries of the twentieth century and made it possible to control many diseases, but with their "overnormative" use, new threats appeared, including antibiotic resistanceto both human and veterinary medicinal products for animals [1]. It is obvious that the consumption of meat and meat products from animals fed with antibiotics (including their residues) is undoubtedly harmful to the health of the latter, and even more so to people, because even in a low concentration and regardless of the period time, it is not physiologically indifferent [2][3][4][5]. In other words, in animal husbandry, antibiotics should only be used for therapeutic purposes and not, inter alia, for reducing falls due to the crowding of animals in a small area, improving their condition, or stimulating growth and development. Thus, it is unacceptable to legally legalize the maximization of the fattening of farm animals through the use of

The Welfare Improving Poultry Farming
The most common pathogen in the poultry gastrointestinal tract in broiler breeding is the Salmonella species. Due to the limitation of the use of antibiotics, research is conducted with the use LAB bacteria in order to limit the colonization of the intestines by Salmonella sp. Wang et al. vaccinated hatched chicks with a strain of Lactiplantibacillus plantarum LTC-113. Studies have shown protection against Salmonella Typhimurium by reducing gut colonization and stabilizing tight junction gene expression in intestinal epithelial cells among treated chickens. In the control group, a Salmonella infection disrupted the intestinal epithelial barrier [28].
In contrast, another study reported that orally administered Lactobacillus johnsonii reduced intestinal colonization by Salmonella and Clostridium perfringens [29]. In another variant, the probiotic was given combined with two strains: Ligilactobacillus salivarius and Enterococcus faecium. The probiotic reduced intestinal colonization by Salmonella Enteritidis and did not cause weight loss or damage to the gastrointestinal mucosa [30]. In another experiment, the use of the E. faecium probiotic in feed increased egg weight, serum FSH (follicle-stimulating hormone) levels, and decreased Bacteroidetes (phylum) in low reproductive individuals [31]. Salmonella enters the bloodstream and then goes to the liver and spleen through damage to the intestinal barrier, which has been confirmed in many studies [32][33][34].
The administration of LAB bacterial cell membrane extracts to chickens reduced the Salmonella Enteritidis infection. Considerable protection of the intestinal epithelium against the effects of infection was evident [32]. The next experiment was very similar, however, a commercial probiotic based on LAB with proven efficacy against S. Enteritidis was used for treatment. The study aimed to understand the effect of a probiotic on intestinal colonization and intestinal permeability in infected chickens. The results were very promising. After infection of the control group with Salmonella pathogens, heterophilia and lymphopenia were observed as well as an increase in basophils and eosinophils compared to the chickens treated with a probiotic based on LAB bacteria. In the control group, increased intestinal permeability was also found [33]. In another experiment, the probiotic Bacillus subtilis C-3102 was used to control S. enteric serovar enteritidis LM-7. The specific pathogen-free chicks became infected by administering an appropriate dose of Salmonella in the food. Supplementation with B. subtilis feed reduced Salmonella infections and may accelerate the clearance of pathogens in the liver, cecum, and spleen of chicken farms [34]. Han et al. (2017) orally administered Pediococcus acidilactici mutants to chickens, modulating the microbiota and reducing the number of Salmonella enterica serovar Gallinarum, which is often found in the poultry industry. The study proved the antimicrobial activity of P. acidilactici. After treatment of the cultures with proteinase, the antimicrobial activity decreased, which may suggest the production of proteinaceous substances such as bacteriocin by P. acidilactici [35].
Another common pathogen on farms is Campylobacter sp. The pathogen is rapidly transmitted on poultry farms via the fecal-oral route. The study of Ščerbová et al. (2016) assessed the inhibition spectrum of various enterocins against Campylobacter sp. Enterocins are protein substances with antibacterial activity metabolized mainly by enterococci. The isolated strains from poultry farming were divided into two groups, Campylobacter jejunum, and Campylobacter coli. Interestingly, the strains showing resistance to antibiotics displayed sensitivity to at least one of the nine eneterocins tested [36]. On the other hand, Razmyar et al. (2017) showed that C. perfringens secretes bacteriocins, which may be responsible for facilitating intestinal colonization and causing intestinal inflammation by this pathogen [37]. Supernatants from Lactobacillus acidophilus NCFM, Lactobacillus crispatus JCM 5810, Lactobacillus gallinarum ATCC 33199, and Lactobacillus helveticus CNRZ32 cultures inhibited the growth of C. jejuni in vitro. After the analyses, the substance responsible for limiting the growth of C. jejuni was partly lactic acid. Subsequently, in in vivo studies, LAB was administered to broilers on the day of hatching, on the fourth day after hatching, and 14 days after hatching, challenged with C. jejuni F38011. Each of the four strains limited the colonization of the pathogen. It was most effective in limiting the colonization of L. crispatus JCM 5810 [38].
Another pathogenic bacterium on chicken farms is avian E. coli (APEC). Birds infected with APEC show macroscopic changes in air sacs and lungs [39]. In the experiment of , Lactobacillus animalis (ATCC 35046), Lactobacillus reuteri (ATCC 2837), and Lactobacillus rhamnosus (ATCC 23272) strains were injected into the eggs to inhibit APEC infection. However, no differences were noted in APEC-like strains infection in the probiotic-administered group and control group [40]. This result was completely different from that obtained in the experiment in which the probiotic mix with B. subtilis, Clostridium butyricum, and L. plantarum was used. The applied probiotic lowered the E. coli index in infected chickens. The positive effect of the probiotic on the modulation of the intestinal microbiota of broilers has also been proven [41].
The protection of poultry farms against microbes is one of the most important factors in good breeding. However, it is also an important factor to improve European indicators for weight gain and feed consumption. The study of Kierończyk et al. (2017) aimed to test the effect of nisin on growth efficiency, morphological parameters, the activity of digestive enzymes, the digestibility of nutrients, and the effect on intestinal morphology in chickens. It was noticed that supplementing the diet of chickens with nisin, which is a bacteriocin used in the preservation of food products [42], improved the body weight gain (BWG), feed conversion ratio (FCR), and feed intake (FI) indexes [43]. According to Hsu et al. (2004), nisin is a cyclic polypeptide that contains 34 amino acids. The lactic acid bacteria of the genus Lactococcus lactis, carries out the fermentation process and, in addition to lactic acid, produces the bacteriocin nisin. Nisin is a natural antibiotic against Gram-positive bacteria [44,45]. Research has indicated that nisin can be used as a growth simulator without adversely affecting the bird's metabolism or immunity levels [28]. According to Kierończyk et al. (2020), nisin can be considered as a new and natural growth promoter. It improves digestibility and feeds conversion. By limiting the multiplication of pathogenic bacteria, nisin has a positive influence on the modulation of the intestinal microbiota. In terms of its antibacterial properties, it is similar to monensin, an antibiotic of the coccidiostatic type [46].
The intestinal microbiota was modulated not only with probiotics but also with plant feed additives.  showed a positive effect of dietary purslane in the experiment. Purslane (Portulaca oleracea L.) is an edible wild vegetable with medicinal properties. The use of purslane in the feed increased the level of Lactobacillus and lowered Escherichia/Shigella in the digestive tract of broilers. The growth of beneficial bacteria in chicken intestines may promote high body weight gain [47]. Another study by Liang et al. (2021) applied traditional Chinese medicine based on medicinal plants combined with probiotics-a mix of B. subtilis and L. acidophilus. In the treatment of E. coli-infected chickens, the mix inhibited the survival level of E. coli, reduced the rates of diarrhea and mortality, improved body weight gain, and relieving pathological changes in the intestines and liver were observed [48].
In another study confirming the beneficial effect of LAB on the intestinal microbiota of fattening chickens conducted byŚliżewska et al. (2020), the effects of three variants of synbiotics were compared with two variants of a commercial probiotic on the chicken's performance. The synbiotics were three combinations containing the following strains: L. plantarum ŁOCK 0860, L. reuteri ŁOCK 1092, L. pentosus ŁOCK 1094, Saccharomyces cerevisiae ŁOCK 0119, L. rhamnosus ŁOCK 1087, L. paracasei ŁOCK 1091, and 2% inulin (prebiotic). Commercial probiotics included BioPlus YC (Bacillus licheniformis DSM 5749, B. subtilis DSM 5750) and Cylactin (Enterococcus faecium NCIMB 10415). The positive effect of synbiotics on the performance of fattening chickens and the balance of the intestinal microbiota was demonstrated. The number of beneficial microorganisms such as Bifidobacterium sp. and Lactobacillus sp. increased in the intestines, and the number of pathogenic bacteria such as Clostridium sp. and E. coli in the intestines and animal excretions decreased. The change in the gut microbiome increased the levels of lactic acid and SCFA (short chain fatty acid). This is another study confirming the beneficial effect of LAB on the intestinal microbiota of fattening chickens [27].
The Gram-negative bacteria Gallibacterium anatis is responsible for the decrease in the number of eggs laid by causing infections of the genital tract of hens and contributes to increased mortality [49,50]. A study conducted by  showed the antagonistic activity of the supernatant after the culture of Leuconostoc mesenteroides QZ1178 (a species of lactic acid bacteria) was used against G. anatis strains in vitro. The antagonistic effect was decreased upon increasing the pH. After analysis of the supernatant, L. mesenteroides QZ1178 mainly produced lactic acid (29 mg/mL) and acetic acid (7 mg/mL), which are probably responsible for its antibacterial properties [51]. The above information is summarized in Table 1.  * FSH-follicle-stimulating hormone; BWG-body weight gain; SCFA-short-chain fatty acids; FCR-feed conversion ratio.

Prevention of the Effects of Piglet Weaning Based on the Use of LAB
The critical moment in breeding is weaning the piglets from sow on day 28. Pigs are very sensitive to changes in their living environment. This is a very stressful time for piglets, causing destabilization of the intestinal microbiota. During this time, digestive disorders, diarrhea, growth retardation, and increased mortality occur [61][62][63][64]. The most common pathogens affecting intestinal disorders and damage to intestinal villi are E. coli, C. perfringens, Salmonella Choleraesuis, and Salmonella Typhimurium. In the case of infection, the permeability of fluids to the intestinal lumen increases and diarrhea develops. There is also an increase in pH, which prevents the multiplication of LAB [65].
Antibiotics are used to improve the intestinal microbiota and reduce the occurrence of diarrhea caused by weaning. The overuse of antibiotics has resulted in the emergence of pathogenic bacteria resistant to the basic antibiotics used in farming. Moreover, in the case of pig farms, research on the use of LAB to improve the intestinal microbiota was started [66].
Verso et al. (2018) isolated 595 pure cultures of bacteria from the small and large intestines from pre and post-weaned piglets. The selected bacteria were antagonistics to pathogens and were capable of producing antimicrobial compounds. First, the activity against pathogens E. coli MC4100, S. Choleraesuis ATCC 29628, and Listeria innocua HPB13 was tested using the Double-Agar-Layer Technique Method. At total of 51.1% of all strains showed antimicrobial activity and passed the next test to investigate the production of antimicrobial compounds. For this, the pH of the supernatant was neutralized with 1M NaOH. Activity against previous pathogens and Staphylococcus aureus ATCC 6538, Enterococcus faecalis ATCC 27275, Listeria monocytogenes LSD530, and S. Enterica ATCC 8387 was tested by the Agar-Well Diffusion Method. Loss of activity of some supernatants after protease treatment may indicate the presence of bacteriocin-like substances. These studies confirmed the ability of some LAB strains of the species L. salivarius and Lactobacillus delbrueckii subsp. lactis isolated from the digestive tract of pigs to inhibit the growth of potential pathogens by the production of organic acids in combination with bacteriocinlike proteins [67]. In another experiment, piglets were fed with an immunobiotic feed based on okara fermented soy milk with L. delbrueckii subsp. delbrueckii TUA4408L. The beneficial intestinal microbiota improved, the amount of Lactobacillus and Lactococcus increased, and the immunity also increased. The piglets showed better meat quality and growth performance [68]. In addition, the use of L. delebureckii CCTCC M 207,040 as a diet supplement by Chen et al. (2021) improved the gut structure resulting in increased gut integrity in lipolisaccharides (LPS)-challenged piglets. LPS stress induced an increase in the depth of the crypts in the jejunum and ileum. However, the use of L. delebureckii dietary supplementation reduced crypt depth compared to the non-challenged controls and LPS-challenged. Moreover, the TLRs-Btk-Nrf2 signaling pathway, which mediates oxidative stress, was mitigated [69]. Similar results were obtained in weaned piglets with C. butyricum ZJU-F1 and B. licheniformis. The intestinal permeability was reduced, and the digestibility of nutrients and the expression of antimicrobial peptides in the ileum improved [70]. In another experiment, Sobrino et al. (2021) isolated L. salivarius MP100 from sow's milk and gave inoculated feed to pregnant sows and piglets. MP100 showed antagonistic activity against the indicator bacteria: C. perfringens MP34, E. faecalis MP42, S. aureus MP83, Streptococcus suis MP205, Trueperella pyogenes MP214, E. coli MP73 (F4) and MP77 (F18), S. Typhimurium MP55, and Klebsiella pneumoniae MP87. The use of a potential probiotic resulted in a microbiological and biochemical improvement in the gut environment [71].
The Lactobacillus gasseri LA39 and Limosilactobacillus frumenti strains produce the substance gassericin A, which is a bacteriocin. A characteristic feature of this protein is that it binds to the intestinal epithelium of the host and makes it resistant to diarrhea in weaned piglets. Gassericin A bound to Keratin 19 in the plasma membrane of the intestinal epithelium increased the absorption of fluid from the intestine and reduced its secretion. The early weaning of piglets aims to shorten the slaughter cycle and improve the reproduction of the sows. In the experiment, fecal microbiota were taken from healthy Congjiang miniature pigs (a Chinese native pig breed) and administered orally to a commercial Landrace × Yorkshire (LY) pig, which shows frequent diarrhea after weaning. This treatment made the LY immune to stress-related diarrhea at weaning. The L. gasseri LA39 and L. frumenti strains can be an alternative to antibiotics in the prevention of diarrhea during increased stress in piglets [72]. In one study, the administration of L. salivarius (strains 144 and 160) to suckling piglets early in life resulted in an increase of the amount of Lactobacillus in the gastrointestinal tract and a reduction in the number of Bacteroides and Fibrobacter. The incidence of diarrhea during the most stressful time of weaning also decreased. The conclusions from the conducted research indicated the use of supplementation with L. salivarius 144 isolated from healthy pigs with a high BMI (body mass index), as it had a beneficial effect on increasing the height of intestinal villi, which influenced the pigs' growth efficiency. The same strain showed a reduction in the amount of Clostridium sp. in the feces [73].
Another way of administering LAB strains to pigs was to use of microcapsulation, i.e., administering LAB strains in sealed gelatin-alginate capsules. This method protects microorganisms against unfavorable conditions in the digestive tract. In the study of Le et al. (2019), the strains were isolated from traditional Vietnamese fermented yogurt. After the bacteria had multiplied, they were centrifuged, the supernatant was removed, and the remaining biomass was encapsulated. The L. plantarum SC01 strain showed antagonistic activity against E. coli, S. aureus, B. subtilis, Salmonella sp., and L. monocytogenes. It has been shown that the concentration of 2.5% (w/v) of alginate and 6% (w/v) of gelatin increases the production of a highly active compound that inhibits pathogens by LAB [74]. On the other hand, Pupa et al. (2021) used spray drying microencapsulation of L. plantarum 22F. The application of this method as well as alginate and chitosan usage for the production of capsules extended the viability of probiotic bacteria. The increased performance of pigs after microencapsulated probiotic supplementation was comparable to the use of live bacterial cultures. The alive bacteria: L. plantarum (strains 22F and 25F) and P. acidilactici (strain 72N) were administered as potential probiotic supplements. The best effect in reducing pathogenic intestinal strains (Enterobacteriaceae) and modulating lactobacilli in the intestinal tract was obtained with the use of P. acidilactici 72N. The administration of the probiotics to young animals resulted in the improvement of intestinal integrity, elongation of intestinal villi in the jejunum, the appearance of microorganisms positively influencing the intestinal microbiome, and improved growth of individuals in the rearing cycle [75].
The weakening of the animals also occurs during pregnancy and the lactation of sows. Wang et al. (2014) isolated L. johnsonii XS4 from the gastrointestinal mucosa of healthy laboratory pigs. In previous studies, they demonstrated a high resistance of L. johnsonii XS4 to hydrochloric acid and bile salts and an antagonistic effect against the most common pathogens in culture (S. aureus, E. coli, S. Enterica). The sows were supplemented with freeze-dried L. johnsonii XS4 from day 90 of gestation to day 25 of lactation. It was noted that the supplementation resulted in an increase in the number of piglets weaned from the sow and an increase in litter weight by 14.45% compared to the control. In the supplemented group, there was a lower loss of backfat during lactation than in the control group, but it was not statistically significant. The obtained results indicate a positive effect on the production efficiency of the sows and the obtained litter. L. johnsonii XS4 has been presented as a promising alternative to the use of antibiotics in feed [76]. In another study, 295 LAB strains were tested, and three strains (Limosilactobacillus reuteri P7, Lactobacillus amylovorus P8, and L. johnsonii P15) with high growth-inhibitory activity for enterotoxigenic E. coli K88 were selected. The strains had a positive effect on the reproductive performance of sows and the growth of weaned piglets and reduced the occurrence of diarrhea [77].
In contrast, supplementation with E. faecium DSM 7134 increased food digestibility, gross weight, and gross energy. Moreover, in this case, the supplementation affected the sow's litter. The mortality of weaned pigs decreased, and weight gain of the piglets was noticeable. In this experiment, a reduced number of E. coli in feces was demonstrated after the piglets were weaned [78].
The administration of probiotic mixes to the sow, in this case, composed of L. delbrueckii subsp. bulgaricus, L. rhamnosus, L. acidophilus, L. plantarum, Streptococcus salivarius subsp. thermophilus, Bifidobacterium bifidum, E. faecium, Candida pintolopesii, and Aspergillus oryzae also gave satisfactory results. In this study, no diarrhea occurred in either the sows or the piglets. An increased concentration of acetic, propionic, and butyric acids in the feces was shown in piglets supplemented with the mix. However, in this case, the supplementation of the sows did not affect the litter weight, but there was a visible change in the gut environment of the piglets [79]. In the case of using only the dietary supplementation L. plantarum CAM6 in sows, it had a positive effect on body weight and reduced the occurrence of diarrhea in the offspring. The nutritional value of milk in sows improved [80]. The application of a diet with L. plantarum JL01 for weaned piglets resulted in better digestion and absorption of fats in the cecum and mediated the metabolism of tryptophan [81,82].
The interest of scientists was also aroused by the influence of a probiotic diet on bacteria present in the air. The effect of administering feed supplemented with E. faecalis CICC 23,215 in a piglet house over 60 days of airborne bacterial communities in the house was investigated. Air and feces samples were tested. The enrichment of air and feces with Lactobacillus species was shown. E. faecalis reduced the abundance of Proteobacteria, Acinetobacter sp., Escherichia sp., and Shigella sp. [83].
In another study by , the effect of feeding with feed co-fermented by B. subtilis CW4 and E. faecium CWEF on lactating sows and newborn piglets was investigated. It was shown that the quality of the sows' milk was improved, which resulted in an increased weight gain in the piglets. There was a reduction in the incidence of constipation in sows and diarrhea in piglets, which was related to the modulation of the intestinal microflora. The sows showed better immunity and performance compared to the control [84]. Another experiment used the positive effects of Bacillus coagulans, oregano oil, and benzoic acid on the health, physiological, and physical condition of the piglets. An increased weight gain with a reduced feed ration was obtained in the diet supplemented with benzoic acid and B. coagulans. The addition of a third component to the diet-oregano oil-resulted in an increased number of bifidobacteria in the caecum and a decreased E. coli population in the cecum. Both the supplementation of the two-component and three-component diets positively influenced intestinal integrity, immunity, and physical condition of piglets in non-antibiotic breeding [85]. Similarly, in the study by , the use of B. coagulans and yeast hydrolysate in weaned piglets improved the intestinal barrier function, which resulted in better weight gain of the piglets. These two options can be used as alternatives to antibiotic growth promoters [86].
Not only LABs can be used as potential probiotics to prevent diarrhea. Swine commensal E. coli strains are bactericidal and compete in the environment with pathogenic porcine strains of E. coli. A reduced susceptibility of commensal E. coli to the 34 bacteriocin monoproducers was demonstrated compared to pathogenic E. coli. Finally, in vitro and in vivo, three potential probiotic E. coli were selected that could be candidates for the prophylaxis of post-weaning diarrhea [87]. The above information is briefly summarized in Table 2.  Increased concentration of acetic, propionic, and butyric acids in the feces [79] L. plantarum CAM6/probiotic per os 3 Improved nutritional value of milk [80] B. subtilis CW4 and E. faecium CWEF/probiotic per os 1,3 Improvement in the quality of sows' milk; better immunity at sows [84] L. reuteri 1/probiotic freeze-dried/per os 2 Increased carcass yield; improved meat quality and flavor [88] B. subtilis WB800 per os 1 Enhanced respiratory immunity [89] The experiment was carried out on 1-piglets, 2-pigs, 3-sows.

Prevention of Udder Infections in Cattle with Particular Emphasis on Bacterioci Like-Substance
Inflammation of a cow's udder, another name mastitis, is caused by physical trauma, chemical irritation, or bacterial infection. Mastitis can be identified in two types: clinical and subclinical. Clinical mastitis manifests itself with local and systemic symptoms. Redness, swelling, pain in the udder area, decreased appetite, increased temperature, reduced milk production, and a change in milk compounds are visible [90]. Subclinical mastitis does not alter the udder. Abnormal changes in milk are visible, such as an increased population of bacteria, a change in quality and compounds, reduced milk production, and an increased number of somatic cells [91]. Inflammation of the udder tissue has a negative impact on economical milk production and the animal's welfare due to pathologies causing edema, swelling, pain, inflammation, or udder fibrosis [92] and to a reduced reproductive efficiency [93]. Disease control is hindered by the causes of multifactorial occurrence and involving a large number of pathogens [94]. The main pathogens inducing mastitis are S. aureus, Streptococcus uberis, and Streptococcus dysgalactiae [95][96][97] and more often isolated strains of E. coli [98]. The treatment consists of identifying the pathogen and then administering an appropriate intramammary antibiotic [99]. Due to the excessive use of antibiotics, bacterial resistance increases and treatment efficacy decreases, so there is a growing interest in replacement therapies without antibiotics [100].
One experiment reviewed a large bank of engineered nisin modifications and discovered three new variants of nisin A M17Q, nisin A HTK, and nisin A T2L. These variants showed antibacterial activity against S. aureus strains associated with bovine mastitis. It has been shown to reduce the growth inhibition of commensal bacteria naturally occurring in milk, such as lactobacilli and lactococci. This is the next step in the development of substitutes for antibiotic therapies on farms [101].
Godoy-Santos et al. (2019) isolated the bovicin HC5 bacteriocin from the rumen bacteria Streptococcus equinus HC5. Bovicin HC5 is a lantibiotic with the ability to bind to lipid II in the cytoplasm such as nisin [102]. Other studies showed a bovicin HC5 bactericidal effect against L. monocytogenes, Salmonella Typhimurium, and some species of Clostridium and Bacillus [103,104]. The antibacterial effect of bovicin HC5 was tested in isolated strains from animals with mastitis. There were: S. aureus (99 strains), coagulated negative Staphylococcus sp. (CNS) (44 strains), Streptococcus agalactiae (71 strains), Streptococcus bovis (22 strains), S. uberis (20 strains), and E. coli (20 strains). Bovicin HC5 has inhibited the growth of most Streptococcus and Staphylococcus species. A total of 276 pathogenic isolates were tested. Some 18% of the isolates were not susceptible to the bacteriocin. None of the E. coli strains showed sensitivity to bovicin HC5. In the case of S. aureus, the pathogen most often responsible for the occurrence of mastitis in cows, as many as 95% of isolates showed the highest sensitivity to bovicin HC5 [102].
Due to the high costs of producing pure bacteriocins, other scientists decided to test the effect of a preparation containing live Lactococcus lactis DPC3147 cultures [105]. The effectiveness of the product was compared with the commercial antibiotic, Terrexine TM, which is used to treat cows with clinical and subclinical signs of mastitis. In cows with clinical symptoms, inflammation and/or malaise, clots, pathogens in milk, and poor milk production occurred. Cows infected with S. aureus and treated with biopreparation showed a cure rate of 45%, and cows treated with a commercial antibiotic showed a cure rate of 50%. The above data show that the biopreparation containing the bacteriocin lacticin 3147 produced by L. lactis DPC3147 shows comparable efficacy to the commercial antibiotic. Cows treated with the biopreparation showed an increased response of the immune system and a decrease in the number of somatic cells in milk. Five days after the injection into the replacement biopreparation, no DPC3147 cells were detected, which indicates that the animals quickly excrete the "live biopreparation". The above experience allows us to limit the administration of antibiotics in the treatment of mastitis in the future and shorten the withdrawal time of treated animals [105].
On the other hand, one of the in vitro studies demonstrated the bactericidal effect of the supernatant after the L. lactis ssp. lactis bacterial culture was used against the most common occurring mastitis pathogen. There were obtained uncleaned bacteriocins from supernatant after bacterial culture and used on pathogens. In this experiment, Malvisi et al. (2016) confirmed the presence of the class I bacteriocin, nisin A, in the supernatant by liquid chromatography. The highest antagonistic activity had L. lactis LL11 and SL153 supernatants after bacterial culture. Malvisi et al. (2016) applied the supernatant containing nisin A to the bovine mammary epithelial cell line BME-UV1. The stimulation of the secretion of NAGase and the LZ antibacterial enzymes by the cells was visible without causing damage or adverse inflammatory reactions, and there was no damage to cell integrity [106].
One study investigated the antimicrobial activity of 13 specific bacteria isolated in vitro from a honey bee cultivation against mastitis pathogens. Nine types of Firmicutes were isolated: Apilactobacillus kunkeei Fhon2, Apilactobacillus apinorum Fhon13, Bombilactobacillus mellis Hon2, Bombilactobacillus mellifer Bin4, Lactobacillus kullabergensis Biut2, Lactobacillus kimbladii Hma2, Lactobacillus helsingborgensis Bma5, Lactobacillus melliventris Hma8, and Lactobacillus apis Hma11 and 4 types of Actinobacteria: Bifidobacterium coryneforme Bma6, Bifidobacterium asteroides Bin2, Bifidobacterium sp. Bin7, and Bifidobacterium sp. Hma3. Three of the tested oxalicin-resistant S. uberis were inhibited by the use of a combination of a honey-based medium and 13 specific bacteria. The same was observed in ampicillin and trimethoprim-sulfamethoxazole-resistant E. coli isolates. The study demonstrated that 13 specific bacterial symbionts in combination with the heather honey matrix showed an inhibitory effect on the growth of mastitis pathogens [107].
In one of the experiments carried out by Seon-Gyu Kim et al. (2019), synergistic inhibition of the growth of S. aureus KCTC 3881 by bacteriocin and bacteriophage was used. The bacteriocin was isolated from L. lactis CJNU 3001. For comparison, bacteriocin and bacteriophage activity were also tested separately. Pure bacteriocins showed antagonistic activity against S. aureus depending on the dose. Visible effects were obtained at concentrations of 50 and 100 AU/mL. The treatment of S. aureus with a dose of 1 MOI (multiplicity of infection) of the SAP84 bacteriophage showed a viable cell count of 5.7 Log CFU/mL. The combined action of phage (0.1 MOI) and bacteriocin (100 AU/mL) showed a reduction in the number of viable S. aureus cells to 3.3 Log CFU/mL. The combination of bacteriocin and bacteriophages may be a promising strategy to combat pathogens not only associated with mastitis in cattle [108].
One of the metabolites produced by LAB is lactic acid [38,109]. Chotigarpa et al. (2018Chotigarpa et al. ( , 2019 performed the time-killing analysis of rice gel with 5% (v/v) lactic acid on E. coli ATCC 25,922 and E. coli field strains (teat skin samples from healthy dairy cows after washing udder) in the time interval of 0 to 60 min. The study showed an inhibitory effect on the growth of E. coli, which means that created gel can reduce the number of pathogenic bacteria on the cows' teats. The gel can be an alternative to the antibiotics used and in the prevention of mastitis. Additionally, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of pure lactic acid were checked for E. coli strains. The MIC and MBC were 0.5% lactic acid [110,111].
The analysis of feces in four dairy cows showed the presence of the following Lactobacillus strains: Lactobacillus gasseri, Limosilactobacillus reuteri, and Ligilactobacillus salivarius. In vitro, the supernatants of these strain cultures showed bactericidal activity against Escherichia coli O157:H7, Mycobacterium avium ssp. paratuberculosis, and the Salmonella species (Salmonella enteritidis, Salmonella typhimurium, and Salmonella Dublin). They also showed a low risk of lateral transfer of antibiotic-resistant genes despite showing resistance to streptomycin (L. gasseri) and kanamycin (L. salivarius). In an ex vivo study, they showed adherence to bovine intestinal epithelium cells. The use of such fecal isolates may be a species-specific probiotic for cattle [112]. The above information is briefly summarized in Table 3. Table 3. Overview of the application of potential probiotic bacteria in bovine farming in an in vitro study.

Antibiotics-Future Legal Regulations
In order to counteract the phenomenon of antibiotic resistance and protect broadly understood public health, comprehensive actions, including legal ones, are necessary. In reference to the data provided by OECD (Organisation for Economic Cooperation and Development), it is estimated that about 700,000 deaths may be caused globally each year by AMR (antimicrobial resistance). Compared to a world with no AMR, the economic impact associated with current rates of AMR may reach about 0.03% of GDP in 2020 in OECD countries, 0.07% in 2030, and 0.16% in 2050. This would result in a cumulative loss of about USD 2.9 trillion (NB: in the quoted report, the amount 'trillion' means 1012-'Council conclusions on the next steps under a One Health approach to combat antimicrobial resistance' (2016/C 269/05), Official Journal of the European Union 23.7.2016) by 2050 [114]. Moreover, as shown by data published by the European Medicines Agency, the use of antibiotics in Europe is more than twice as high in the treatment of animals as in humans. From 2011 to 2014, the use of antibiotics in agriculture increased by 23% [115]. The basic legal act in the European Union in the field of the use of antibiotics, which will come into force on 28 January 2022, is Regulation (EU) 2019/6 of The European Parliament and of the Council on 11 December 2018 on veterinary medicinal products, and repealing Directive 2001/82/EC [116]. The above normative act will be directly applicable in all Member States, without the obligation to implement it into the national legal order.
According to the above-mentioned legal act, in Article 4 point 12 of the Regulation (EU), 'antibiotic' means any substance with a direct action on bacteria that is used for treatment or prevention of infections or infectious diseases. The antibiotic also fits into the broader definition adopted by the EU legislator, the so-called 'antimicrobial', which means any substance with a direct action on micro-organisms used for the treatment or prevention of infections or infectious diseases, including antibiotics, antivirals, antifungals and antiprotozoals) [116]. It is also worth recalling the broadest definition in terms of the analyzed regulation (Vide Article 4 point 1) [116]: 'veterinary medicinal product' means any substance or combination of substances which fulfils at least one of the following conditions: (a) it is presented as having properties for treating or preventing disease in animals; (b) its purpose is to be used in or administered to animals with a view to restoring, correcting, or modifying physiological functions by exerting a pharmacological, immunological, or metabolic action; (c) its purpose is to be used in animals with a view to making a medical diagnosis; (d) its purpose is to be used for euthanasia of animals.
Of key importance for the issue discussed is Art. 107 of the Regulation, which expressis verbis regulates the issues of the use of antimicrobial medicinal products. It shows, inter alia, that antimicrobial medicinal products cannot be used: (a) antimicrobial medicinal products shall not be applied routinely nor used to compensate for poor hygiene, inadequate animal husbandry, or lack of care or to compensate for poor farm management; (b) in animals for the purpose of promoting growth nor to increase yield; (c) prophylaxis other than in exceptional cases for the administration to an individual animal or a restricted number of animals when the risk of an infection or of an infectious disease is very high, and the consequences are likely to be severe. In such cases, the use of antibiotic medicinal products for prophylaxis shall be limited to the administration to an individual animal only, under the conditions laid down in the first subparagraph.
A Member State may further restrict or prohibit the use of certain antimicrobials in animals on its territory if the administration of such antimicrobials to animals is contrary to the implementation of a national policy on prudent use of antimicrobials.
Antimicrobial medicinal products shall be used for metaphylaxis only when the risk of spread of an infection or of an infectious disease in the group of animals is high and where no other appropriate alternatives are available. Member States may provide guidance regarding such other appropriate alternatives and shall actively support the development and application of guidelines which promote the understanding of risk factors associated with metaphylaxis and include criteria for its initiation (Vide Article 107 Section 4) [116]. Antimicrobial medicinal products should not be used for prophylaxis other than in welldefined cases for the administration to an individual animal or restricted number of animals when the risk for infection is very high or its consequences are likely to be severe (Vide Article 107 Section 3) [116]. Member States should be able to allow the exceptional use of veterinary medicinal products without a marketing authorization where it is necessary to respond to Union-listed diseases or emerging diseases and where the health situation in a Member State so requires (Vide Section 26) [116]. It is worth emphasizing that the consequences of animal diseases, and at the same time the measures needed to combat them, can cause enormous damage to the entire population of animals, their owners, and thus the economy, and ultimately public health.
In order to fulfil the above-mentioned obligations as well as a whole range of other legal regulations regulated in the analyzed regulation Art. 107 Section 3, said Member States shall ensure that adequate financial resources are available to provide the staff and other resources necessary for the competent authorities to carry out the activities required by this Regulation [116].
The considerations carried out are only indicative, they undoubtedly prove that the widely understood processes of enactment, but above all the application of law, have a huge impact on the protection and guarantee of the highest level of public health and animal health protection as well as environmental protection. Otherwise, misuse (overuse of antibiotics) will lead to the spread of resistant pathogens, generally in animals, plants, and the environment and ultimately also in humans, representing one of the most serious global threats to public health.
The new legal regulation in the European Union does not limit the treatment of sick animals by administering them antibiotics, but its ratio legis excludes prophylactic treatment of the entire herd, with only a few diagnosed sick animals in the group. In other words, the EU legislator prohibits the massive and preventive use of antibiotics, e.g., in animal feed, water, etc. only for economic purposes. It should also be remembered that only effective supervision and control activities as well as monitoring studies of individual public administration bodies in all EU Member States will allow for a reliable assessment of the legitimacy and correctness of the use of antibiotics, and thus guarantee the protection of consumers against the consequences of their improper use.
The observation of the practice proves that in addition to the appropriate legal tools and organizational solutions, including the development of the principles of rational and safe use of antibiotics, the appropriate staffing of government and local government administration bodies to perform the above-mentioned duties is of key importance. Otherwise, staff shortages, insufficient level of financing, and high workloads will make the abovementioned tasks ineffective or perhaps more accurately simulated, due to staff fluctuations.

Conclusions
The greatest discovery of the 20th century was the control of many diseases, both animal and human with antibiotics. Unfortunately, the overuse of antibiotics has resulted in the appearance of drug-resistant pathogenic bacteria in veterinary practice and medicine. The main goal of large-scale livestock breeders is to intensify production and reduce mortality in the herd. An easy way to do this is to use antibiotics. Increased animal production keeps animals crowded, which facilitates the transmission of various diseases. As shown in the review, the use of probiotics on farms can naturally bring about a balance of gut microbes and reduce the growth of pathogens in broiler, pig, piglet, and cattle slaughter farms. Based on the review of the latest articles from 2021, an increase in interest in the effects caused by B. subtilis can be noticed ( Table 1). The results indicate that it could be a substitute for antibiotics and an effective growth promoter in broiler breeding [55][56][57][58]. The use of probiotics is of increasing importance for the alleviation of weight and immunity deficiencies as well as for lowering the mortality ratio in broiler farms [117]. The discussed studies show the positive effect of LAB and its metabolites on the welfare of farm animals. The limitations of antibiotics in farms have a positive effect on the environment and living organisms, including humans. The application of the new regulations in the law, which will come into force in 2022, is to limit completely the use of antibiotics for disease prevention before they appear in the herd. This is the next step to reduce the occurrence of resistance effects among pathogenic bacteria.