Study and use of the probiotic Lactobacillus reuteri in pigs: a review

Probiotics are living microorganisms that provide a wide variety of health benefits to the host when ingested in adequate amounts. The bacterial strains most frequently used as probiotic agents are lactic acid bacteria, such as Lactobacillus reuteri, which is one of the few endogenous Lactobacillus species found in the gastrointestinal tract of vertebrates, including humans, rats, pigs and chickens. L. reuteri is one of the most well documented probiotic species and has been widely utilized as a probiotic in humans and animals for many years. Initially, L. reuteri was used in humans to reduce the incidence and the severity of diarrhea, prevent colic and necrotic enterocolitis, and maintain a functional mucosal barrier. As interest in alternatives to in-feed antibiotics has grown in recent years, some evidence has emerged that probiotics may promote growth, improve the efficiency of feed utilization, prevent diarrhea, and regulate the immune system in pigs. In this review, the characteristics of L. reuteri are described, in order to update the evidence on the efficacy of using L. reuteri in pigs.


Introduction
Antibiotics are a common additive in livestock feed which have been widely used for growth promotion and prophylaxis purposes in farm animals during the past several decades [1]. However, antibiotic resistance is a looming public health crisis. The use of antibiotics as growth promoters has been forbidden in the European Union, Korea, and Japan. Other countries including the United States and China may ban the feeding of antibiotics within the next few years. As a result, there is increasing interest concerning alternatives to in-feed antibiotics, such as probiotics, prebiotics, plant products and organic acids in the livestock industry [2].
Probiotics are living microorganisms, which, when consumed in adequate amounts, can confer a health benefit to the host [3]. In farm animals, probiotics have been shown to promote growth, improve the efficiency of feed utilization, modulate the gastrointestinal ecosystem, stimulate the immune system and protect the host from gastrointestinal tract (GIT) diseases [4]. Therefore, probiotics provide a potential alternative strategy to infeed antibiotics [5].
The properties of probiotics are strain-specific, and suitable probiotic strains for pigs are usually selected based on some criteria including pig origin, acid and bile tolerance, their ability to adhere to intestinal cells and to colonise the intestinal tract, production of antimicrobial substances, antibiotic resistance patterns, demonstrable efficacy and safety, and stability to the conditions used in industrial processes [6][7][8]. The organisms most frequently used as probiotic agents are lactic acid bacteria (LAB) [9], such as Lactobacillus, which are a normal inhabitant of the GIT [10]. Lactobacillus reuteri is one of the dominant species in the GIT of vertebrates such as humans, rats, pigs and chickens [11]. It is one of the most well documented probiotic species and has been widely utilized as a probiotic in humans and animals [12][13][14][15].
In recent years, numerous probiotic strains have been used in pig production. The application of probiotics provide a potential alternative strategy to the use of antibiotics. The aim of this review is to systematically review and update the evidence on the efficacy of using L. reuteri in pigs.

Characteristics of Lactobacillus reuteri
L. reuteri is a heterofermentative bacterium, and is considered to be one of the few true autochthonous Lactobacillus species in humans and animals. Many researchers have already selected some specific L. reuteri strains isolated from human feces, breast milk, the human vagina, the human oral cavity, guinea pigs, rats, pigs, broilers and sourdough. There is now mounting evidence to show that selected L. reuteri strains have probiotic characteristics, and can provide health benefits to their hosts. We have constructed a summary table (Table 1), in order to provide an overview of the reported L. reuteri strains used as probiotics.

Probiotic properties
Probiotic bacteria encounter various stresses after ingestion by the host, including exposure to a low pH in the stomach and contact with bile in the small intestine. L. reuteri I5007, initially known as Lactobacillus fermentum I5007, was selected from over 7,000 native Lactobacilli colonies according to criteria including resistance to heat, low pH, copper, and bile salts, as well as storage stability and antagonism to pathogenic agents [16]. Other L. reuteri strains also show resistance to low pH and bile salts [12,[17][18][19].
Adhesion of a probiotic strain to the host GIT is important for bacterial colonization, pathogen exclusion, L. reuteri ATCC 53608 pig recognize immunoglobulins [24,41] L. reuteri BSA131 pig resistance to pH, oxgall and antibiotics, and antimicrobial activities against enteric pathogenic, improved pig performance [12] L. reuteri Pg4 broilers tolerate acid and bile salts; inhibit pathogenic bacteria; adhere to intestinal epithelial cells; improved broiler performance [17] L. reuteri ATCC 55730 breast milk colonize the intestinal tract; resistance to tetracycline and lincomycin; maintain intestinal health; prevent diarrhea; modulate the immune system; used in treatment of Helicobacter pylori [13,14,64,65] L. reuteri DSM 17938 ATCC 55730 a daughter strain derived from ATCC 55730 and has the same properties as ATCC 55730 [42] L. reuteri ATCC PTA 4659 breast milk partly prevent diet-induced obesity possibly via the mechanism of inducing liver expression of Cpt1a.
L. reuteri has been reported to produce a variety of antimicrobial substances such as lactic acid, hydrogen peroxide [28], reuterin [29][30][31], and reutericyclin [32], which have beneficial effects for the host organism. L. reuteri strains have been demonstrated to inhibit the in vitro growth of many enteric pathogens, including Escherichia coli, Salmonella Typhimurium, Staphylococcus epidermidis, Staphylococcus aureus, Helicobacter pylori, and rotavirus [12,19,33]. In addition, L. reuteri can produce vitamin B 12 [31,34], and has the capacity to de novo synthesize L-lysine and folic acid based on a computer simulation model [15].
L. reuteri exhibited free radical-scavenging capacity in vitro [35], and encoded various antioxidant enzymes [15]. Studies in animals and humans have shown that oral administration of L. reuteri reduced the incidence and the severity of diarrhea, decreased visceral pain, prevented colic and necrotic enterocolitis, maintained a functional mucosal barrier, and induced colonization and immunomodulation [36][37][38][39].

Safety and stability aspects
L. reuteri has the most extensive safety assessment record of any probiotic strain. A number of studies conducted both in vivo and in vitro indicate that L. reutei is safe for human consumption, even in large amounts [38,40]. However, as is the case for all other species of LAB, plasmids can be found in some strains of L. reuteri [15,41,42], and some of these plasmids have been shown to encode for antibiotic resistance genes [42]. According to the European Food Safety Authority, probiotics should not contain known antibiotic resistance traits. L. reuteri ATCC 55730 is a commercially available probiotic strain which has been found to carry potentially transferable resistance traits for tetracycline and lincomycin. Therefore, it has been replaced by L. reuteri DSM 17938, a strain where the two resistance plasmids have been removed without losing any probiotic characteristics [42].
Probiotic strains must be able to resist any adverse conditions encountered during industrial production in order to survive [43]. L. reuteri is sensitive to heat, and therefore, freeze-drying is commonly used for maintaining the stability of L. reuteri. Subjecting L. reuteri to a higher fermentation temperature (47°C) or a neutral pH (pH 6.7) has been shown to increase the survival of L. reuteri during subsequent freeze-drying [44].

Strains
Not all L. reuteri strains are the same or provide a beneficial response, and the evolution of L. reuteri with vertebrates resulted in the emergence of host specialization [45]. Probiotic strains need to be carefully chosen, and evaluated for their safety and effectiveness using in vitro assays, animal models, and clinical trials. There are numerous strains of L. reuteri, which have some minor differences that make them unique (Table 1).
L. reuteri DSM 17938, L. reuteri NCIMB 30242, L. reuteri ATCC PTA 6475 which are of human origin are the most commonly used in dietary supplements and have been researched the most. In pigs, L. reuteri I5007, was isolated from the colonic mucosa of healthy weaning piglets, and has been demonstrated in several studies to have probiotic properties [15,21,35,[46][47][48][49].
Applications of probiotic L. reuteri for pigs In pigs, the administration of L. reuteri has been shown to have beneficial effects on performance, prevention of diarrhea, stress relief, altered gut microbiota, and immunomodulation. The applications of L. reuteri for pigs are listed in Table 2. Noticeably, L. reuteri is mainly used in neonatal piglets and during the post-weaning period.

Improved performance
In the pig industry, the use of probiotics improves intestinal health which can improve pig performance. Supplementation of L. reuteri has resulted in improved growth and feed efficiency in neonatal and growing pigs. Liu et al. [46] reported that L. reuteri I5007 (6 × 10 9 CFU/d) supplementation increased average daily gain (ADG) in formula-fed piglets. Wang et al. [47] found that administration of L. reuteri I5007 significantly increased weight gain and feed conversion compared with weaned pigs fed without L. reuteri I5007. Also, Wang et al. [50] reported that weaned piglets supplemented with L. reuteri had faster growth and higher feed intakes than unsupplemented piglets. However, feed conversion was unaffected by L. reuteri supplementation. In addition, Wang et al. [48] showed that dietary supplementation with L. reuteri or aureomycin significantly improved the performance of weanling piglets, and there was no difference between the two feed additives. Other studies using L. reuteri BSA131 tended to show improved ADG and feed conversion in weaned pigs [12]. Wang et al. [51] also reported that supplementation with L. reuteri X-1 increased ADG and feed conversion. Yu et al. [52] determined the influence of different levels of L. reuteri I5007 on performance, nutrient digestibility and immunity of weaned pigs. The results demonstrated that the ideal supplemental concentration of L. reuteri was 5.8 × 10 7 CFU/g feed.

Prevention of diarrhea
Diarrhea is one of the most frequent causes of heavy economic losses in swine operations [53]. The effect of L. reuteri against diarrhea in pigs was confirmed in several reports [16,46,47,54,55]. Diarrhea incidence was lower in piglets fed L. reuteri I5007 compared with a control [46]. Enterotoxigenic E. coli (ETEC) are a major cause of diarrhea in neonatal and weaned pigs [55]. Huang et al. [16] showed that a native Lactobacilli complex preparation (including L. gasseri, L. reuteri, L. acidophilus and L. fermentum) could effectively prevent weaning piglet diarrhea when administered before challenge with an E. coli solution (serovars K99, K88 and 987P at a ratio of 1:1:1). Wang et al. [47] reported that 12, 24, and 48 h after challenge, pigs challenged with E. coli had mild diarrhea and mild fecal scores. Supplementation of L. reuteri I5007 did not alleviate these effects. Only on day 10, did feeding L. reuteri I5007 decrease the occurrence of diarrhea. Chen et al. [54] demonstrated that reuteran produced by L. reuteri may prevent piglet diarrhea by reducing adhesion of ETEC K88.

Alleviate stress
Pigs in industrial farming systems are frequently exposed to oxidative stress, which results in decreased performance and reduced immune function. L. reuteri has been shown to be effective in scavenging free radicals in vitro, and could be used to alleviate oxidative stress [35,37]. Wang et al. [35] reported that supplementation of L. reuteri I5007 improved the antioxidant status of growing-finishing pigs (from 50 to 90 kg) as evidenced by increased levels of antioxidant enzymes such as superoxide dismutase and glutathione peroxidase, and decreased levels of malondialdehyde. Wang et al. [50] determined the anti-oxidative effect of L. reuteri I5007 in weaning piglets using an oxidative stress model induced by diquat. Their results showed that diquatinjection decreased the performance of weaning pigs and increased plasma levels of cortisol, adrenaline, carbonyl and malondialdehyde. L. reuteri supplementation alleviated oxidative stress and enhanced the performance of weanling pigs.
Weaning is one of the most stressful periods that results in gastrointestinal, immunological, and behavioral changes [56]. Wang et al. [48] demonstrated that L. reuteri I5007 Table 2 The application of probiotic L. reuteri in pigs Strain Dose Animal Significant results Reference L. reuteri I5007 6 × 10 9 CFU/d newborn piglets increased average daily gain; reduced diarrhea incidence; affected the colonic microbial communities , in particular, reduced numbers of Clostridium sp; reduced mRNA expression of IL-1β in the ileum [46] L. reuteri I5007 2× 10 9 CFU/d weaned pigs increased weight gain and feed conversion; decreased the occurrence of diarrhea; enhanced T-cell differentiation and induced cytokine expression in the ileum [47] L. reuteri I5007 2× 10 9 CFU/d weaned pigs had faster growth and higher feed intakes; improved the anti-oxidative defence system and alleviated damage caused by diquat [50] L. reuteri I5007 5.8 × 10 7 CFU/g weaned pigs increased weight gain, feed intake and apparent crude protein digestibility; increased serum specific anti-OVA IgG level [52] Lactobacilli complex 10 5 CFU/g weaned pigs increased weight gain and feed intake compared with carbadox; prevented diarrhea; decreased E. coli and aerobe counts, increased Lactobacilli and anaerobe counts in digesta and mucosa [16] L. reuteri BSA131 2× 10 6 , 2× 10 8 CFU/g weaned pigs improved weight gain and feed conversion; decreased the number of enterobacteria in the feces [12] L. reuteri X-1 10 8 CFU/g weaned pigs improved weight gain and feed conversion; decreased serum IgG and IgM concentrations, incresed serum DAO and D-lactate concentrations [51] L. reuteri I5007 1.02 × 10 8 CFU/g growing pigs increased total antioxidant capacity [35] L. reuteri I5007 10 8 CFU/g weaned pigs increaed weight gain, neither body weights nor weight gains differed between the L. reuteri and aureomycin groups; alleviated weaning stress syndrome [48] L. reuteri and L. plantarum complex 10 6 CFU/g weaned pigs increased apparent total tract digestibility of nitrogen, gross energy, and fecal Lactobacillus concentration; decreased fecal gas emission, diarrhea score, and E. coli concentration [78] L. reuteri 3S7 and L. plantarum 4.1 10 10 CFU/d sows and piglets were found in the faeces; decreased the population of Enterobacteriaceae; decreased β-glucuronidase activity of all pigs [79] Note: L. reuteri I5007 was initially known as L. fermentum I5007. Lactobacilli complex including L. gasseri, L. reuteri, L. acidophilus and L. fermentum (renamed L. reuteri I5007). alleviated weaning stress syndrome by enhancing the levels of proteins involved in energy metabolism, lipid metabolism, cell structure and mobility, protein synthesis, and immune response, thereby facilitating cellular proliferation and depressing apoptosis.

Modulation of gut microbiota
L. reuteri in neonatal piglets can be used to support the development of a stable microbiota, to stimulate the immune system and to prevent diarrheal diseases. During the weaning and post-weaning periods, L. reuteri is used in pigs to modulate the gastrointestinal microbiota as it aims to prevent post-weaning diarrhea and stimulate growth. Liu et al. [46] reported that L. reuteri I5007 plays a positive role in gut development in neonatal piglets by modulating the microbial composition and intestinal development, Denaturing gradient gel electrophoresis (DGGE) revealed that L. reuteri I5007 affected the colonic microbial communities on day 14 and, in particular, reduced numbers of Clostridium spp. In weaning pigs, administration of L. reuteri BSA131 decreased the number of enterobacteria in the feces [12]. Huang et al. [16] showed that a Lactobacilli compound (including L. gasseri, L. reuteri, L. acidophilus and L. fermentum) significantly decreased E. coli and aerobe counts, and increased Lactobacilli and anaerobe counts in the digesta and mucosa of most sections of the GIT compared with a control group.
In addition, oral administration of L. reuteri I5007 not only increased the concentration of butyrate and other branched chain fatty acids but also decreased Clostridium strains accompanied by a lowered pH in the colonic digesta [46]. This indicates that administration of L. reuteri modulates gut microbiota, and thereby affected the microbial metabolites.

Immunomodulation
Probiotics such as L. reuteri may stimulate or suppress innate immune responses via several mechanisms including modulation of pro-inflammatory cytokines. L. reuteri strains can be divided into two subsets, immunosuppressive (ATCC PTA 6475 and ATCC PTA 5289) and immunostimulatory strains (ATCC 55730 and CF48-3A), and each subset has potential therapeutic value [57]. The effects of L. reuteri on immunomodulation were documented in pigs. Wang et al. [47] reported that oral administration of L. reuteri I5007 could enhance T-cell differentiation and induce ileal cytokine expression, which suggests that this probiotic strain could modulate immune function in weaned piglets. Yu et al. [52] showed that L. reuteri I5007 supplementation increased serum specific anti-OVA IgG levels. In neonatal piglets, L. reuteri has been found to decrease the mRNA expression of IL-1β in the ileum [46]. Azevedo et al. [58] found that L. reuteri combined with L. acidophilus could help to maintain Figure 1 Mechanisms of L. reuteri modulating in the gut. ① L. reuteri can produce a variety of antimicrobial substances (AMS) such as lactic acid, and reuterin [28][29][30]. ② L. reuteri has the capacity to colonize, and can adhere to mucin and intestinal epithelial cells [17,21,22] . ③ L. reuteri has been shown to stimulate or suppress innate immune responses by affected the production of cytokines in macrophages (M), monocytes, and dendritic cells (DCs). The modulation of dendritic cells by L. reuteri has been shown to be mediated through dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) and promote development of regulatory T cells producing high amounts of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) [59,60]. ④ L. reuteri has been reported affected the colonic microbial communities and short chain fatty acid (SCFA) concentration [46]. Please see text for details and references.
immunological homeostasis in neonatal gnotobiotic pigs infected with human rotavirus by regulating TGF-β production.

Conclusions and perspectives
In conclusion, L. reuteri is a probiotic bacteria that is one of the few true autochthonous Lactobacillus species. Numerous studies have demonstrated that they can positively improve performance, prevent diarrhea, alleviate stress, alter gastrointestinal microbiota, regulate the immune system, and thereby improve pig performance and health. The beneficial effects of L. reuteri in pigs have been related to different modes of action. The improvements in pig performance of supplemental L. reuteri are mostly due to the fact that L. reuteri has the ability to colonize the GIT, produce antimicrobial substances and stimulate the intestinal immune system (Figure 1), thereby promoting nutrient metabolism and improve health. However, a clear mode of action has yet to be described. It appears from the data presented that the benefical effects of L. reuteri are strain specific. It will be important to select more powerful or targeted strains. Unfortunately, the viability of L. reuteri is a key criteria for developing L. reuteri products. To expand the probiotic L. reuteri application in pigs, care must be taken during processing techniques such as microencapsulation to maintain bacterial stability. Pig husbandry has entered an era when the use of antibiotics is increasingly unwelcomed. Probiotics, which are a potential alternative to in feed antibiotics, can expect a promising future. Besides selection of excellent strains and improved processing techniques, more research, especially in the form of well-designed animal trials, is needed to evaluate the efficacy of L. reuteri. More studies are also needed to explore the mechanisms of action of L. reuteri in pigs. An important fact is that L. reuteri added to pig diets may potentially help improve performance. With evolving knowledge, effective use of L. reuteri will be possible in the future.