Faecium NCIMB Research Articles

Scientific Opinion on the safety and efficacy of Cylactin® (Enterococcus faecium NCIMB 10415) as a feed additive for pigs for fattening, piglets and sows

Cylactin® is the trade name for a preparation of Enterococcus faecium. It is currently authorised for use several animal species. The product is intended for use with sows at a minimum dose of 7 × 108 and a maximum dose of 1.25 × 109 colony-forming unit (CFU)/kg feed and with weaned and suckling piglets and pigs for fattening at a minimum dose of 3.5 × 108 and a maximum dose of 1.0 × 109 CFU/kg feed. The characterisation of the additive was fully described and its safety for consumers, users and the environment was assessed in previous opinions and it was found not to give rise to any concerns. The Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) is unaware of any new data which would lead it to revise its conclusions. Therefore, the focus of the present opinion is on the safety and efficacy of Cylactin® when used with the target animals. Results on the safety and efficacy apply to all forms of the additive when delivering the same dose. When used at 100 times the maximum recommended dose, Cylactin® did not show any adverse effect on the performance or mortality of piglets and sows. Therefore, the additive is considered safe for suckling and weaned piglets, pigs for fattening and sows at the maximum proposed dose. Cylactin® has the potential to be efficacious in sows and suckling piglets when supplemented to both sows and suckling piglets at 7.0 × 108 and 1 × 109 CFU/kg feed, respectively. Cylactin® also has the potential to be efficacious in weaned piglets when supplemented at the minimum recommended dose of 3.5 × 108 CFU/kg feed. Insufficient data were provided to support the efficacy of Cylactin® when used in pigs for fattening.

Probiotic Treatment Decreases the Number of CD14-Expressing Cells in Porcine Milk Which Correlates with Several Intestinal Immune Parameters in the Piglets.

Modulating the mucosal immune system of neonates by probiotic treatment of their mothers is a promising approach which can only be investigated through the use of animal models. Here, we used sows and their piglets to investigate the impact of a bacterial treatment on the sow’s milk and on the neonate piglet intestinal immune system. In previous experiments, feed supplementation of sows with the probiotic Enterococcus faecium NCIMB 10415 during pregnancy and lactation had been shown to affect intestinal microbiota and cytokine expression of the offspring during the suckling and weaning periods. We therefore investigated the composition of the milk from treated sows in comparison to samples from a control group. In treated sows, the amount of lactose increased, and the somatic cell numbers were reduced. In all milk samples, the percentage of cells expressing membranous CD14 (mCD14) was greater than the fractions of immune cells, indicating expression of mCD14 on mammary epithelial cells. However, in the milk of E. faecium-treated sows, mCD14+ cells were reduced. Furthermore, the number of CD14+ milk cells was positively correlated with the percentages of B cells and activated T cells in the ileal MLN of the piglets. This study provides evidence for the expression of mCD14 by the porcine mammary epithelium, and suggests an immunological effect of mCD14+ milk cells on the piglets’ intestinal immune system. Our study further suggests that mCD14+ mammary epithelial cell populations can be modulated by probiotic feed supplementation of the sow.

A prospective, randomized, blinded, placebo-controlled pilot study on the effect of Enterococcus faecium on clinical activity and intestinal gene expression in canine food-responsive chronic enteropathy.

BackgroundCanine chronic enteropathies (CE) are believed to be caused by an aberrant immune response towards the intestinal microbiome. Administration of probiotics can alleviate colitis in people. In vitro effects of the probiotic Enterococcus faecium NCIMB 10415 E1707 (EF) previously have been evaluated using canine cells (e.g., whole blood, intestinal biopsies), but data on in vivo efficacy are lacking.Hypothesis/ObjectivesAdministration of EF to dogs with food‐responsive CE will improve clinical outcome and decrease the intestinal inflammatory profile.AnimalsDogs diagnosed with CE were prospectively recruited to receive a hydrolyzed elimination diet plus either a synbiotic product containing EF or placebo for 6 weeks. Both veterinary staff and owners were blinded to the treatment.MethodsClinical severity index (CCECAI), clinicopathological data and gene expression using intestinal biopsies (TLR2/4/5/9, IL‐17A, IL‐22, IL‐23p19, RORC, IL‐2, IL‐12p35, TNFα, IL‐4, IFNy, IL‐10, TGFβ, IL‐1β, IL‐18, NLRP3, casp‐1, TFF1, TFF3 and PPARy) before and after 6 weeks of treatment were analyzed using linear mixed modeling.ResultsOf the 45 cases recruited, 12 finished the clinical trial. Seven received the synbiotic and 5 the placebo product. There was no difference between groups or treatments regarding clinical efficacy, histology scores or expression of any of the investigated genes.Conclusions and clinical importanceStandard dietary treatment induced rapid clinical response in all cases. Because the study was underpowered, it was not possible to determine whether or not EF had an additional effect within the time period of 6 weeks.

Analysis of in vitro and in vivo effects of probiotics against Campylobacter spp.

Campylobacter (C.) spp. are well recognised as the leading cause of bacterial food-borne diarrheal disease worldwide, with C. jejuni and C. coli as the most important species: C. coli is highly abundant in pigs and pork meat has often been implicated as a source for human infection. Intestinal colonisation of C. coli in pigs plays a role in carcass contamination during slaughter. Different pre-harvest intervention measures are proposed to reduce the C. coli burden in the porcine intestine. Among others, the use of probiotics to prevent or reduce the colonisation of intestinal pathogens is discussed. One aim of this study was to screen a variety of probiotics to evaluate their inhibitory activity against Campylobacter spp. in vitro. Therefore, cell-free culture supernatants of Lactobacillus spp., Bifidobacterium spp., Enterococcus (E.) faecium NCIMB 10415, and Escherichia coli Nissle 1917 were tested against C. jejuni and C. coli by a well-diffusion agar assay. Seven out of eleven Lactobacillus strains showed an inhibitory activity against at least one of the three tested Campylobacter strains. This antagonistic activity against Campylobacter spp. was caused by the production of organic acids that lowered the pH. Application with pH neutralised cell-free culture supernatants abolished this inhibitory effect. Other tested strains with probiotic properties showed no inhibitory activity against any Campylobacter spp. strain. The strain E. faecium NCIMB 10415 was chosen to test its inhibitory activity against C. coli in vivo. Twenty weaned piglets were allocated into two groups, a probiotic group and a control group.The diet of the probiotic group was supplemented with E. faecium NCIMB 10415 (10(9) cfu/kg feed, Cylactin) since weaning, whereas the control group received no probiotic treatment. All piglets were naturally colonised with C. coli. The excretion load of C. coli was monitored for 28 days. The results indicate that dietary supplementation of E. faecium NCIMB 10415 did not significantly affect C. coli excretion levels in pigs. In this study, E. faecium NCIMB 10415 showed no antagonistic activity against C. coli in vitro and in vivo and had no impact on the growth performance of weaned piglets.

Effects of probiotic Enterococcus faecium NCIMB 11181 administration on swine fecal microbiota diversity and composition using barcoded pyrosequencing

Although our understanding of probiotics is increasing, basic information regarding their effect on the composition of the gut microbiome is scarce. The purpose of this study was to characterize the fecal microbiota of weaned piglets before and after probiotic intervention. The probiotic bacterium Enterococcus faecium NCIMB 11181 was administered to pigs for 2 weeks and fecal bacterial diversity using 16S rRNA gene pyrosequencing was analyzed. A more stable bacterial community, as indicated by increased bacterial diversity and richness, was observed after administration of the probiotic, compared with the control group. Regardless of the diet, Firmicutes and Bacteroidetes were found to be the dominant bacterial phyla in the pigs; however, inhibition of Proteobacteria was clearly evident in the probiotic group. In addition, the probiotic-fed group featured elevated levels of Lactobacillus, Enterococcus, Oscillibacter, Succinivibro, and Clostridium genera and decreased levels of Escherichia and Dialister genera, compared with the control group. This study demonstrated that the probiotic E. faecium NCIMB 11181 modulates the fecal microbiota of weaned piglets. This modulation is characterized by an increase in other bacterial species including L. johnsonii, L. fermentum, and L. kimchii and several Clostridium spp., which might be beneficial to the health of weaned piglets.

Intraepithelial lymphocyte numbers and histomorphological parameters in the porcine gut after Enterococcus faecium NCIMB 10415 feeding in a Salmonella Typhimurium challenge

Salmonellae are among the most widespread sources of foodborne infections and Salmonella Typhimurium, in particular, is correlated with human disease caused by the consumption of contaminated pork. Intraepithelial lymphocytes (IEL) have early contact with intestinal antigens and play an important role in the detection of pathogenic bacteria. The objective of this study was to determine whether a presumed probiotic Enterococcus faecium strain could improve histomorphological and immune system-related parameters of gut function after a Salmonella challenge in weaned pigs. In particular the morphological parameters villus length and width, crypt depth and width as well as the actual enlargement of the intestinal epithelial surface were calculated and the number of IEL was evaluated in sections of the porcine gut.Weaned piglets were challenged with Salmonella enterica serovar Typhimurium DT 104, and half of them also received Enterococcus faecium NCIMB 10415 in the diet. Animals were sacrificed at days post infection (DPI) 2 and 28. The effect of the factors “time post-infection/age” and “probiotic treatment” on jejunal morphology and IEL numbers and distribution was evaluated by light microscopy. The time post-infection had significant effects in both feeding groups. Animals sacrificed at DPI 28 had longer and wider villi, deeper and wider crypts, a higher villus enlargement factor, a higher ratio between villus and crypt enlargement factors as well as more IEL. Probiotic treatment resulted in longer villi, a higher ratio of villus surface/crypt circumference enlargement factors and significantly more IEL. The larger total number of IEL displayed by the probiotic group resulted from significantly higher numbers of IEL at the nuclear and apical levels of the intraepithelial compartment but not from the number of IEL situated at the basement membrane. The probiotic effects were only measurable 28 DPI. It is proposed that Enterococcus faecium NCIMB 10415 exerts an immune modulatory effect by increasing the numbers of intraepithelial lymphocytes.

Effects of the probiotic Enterococcus faecium NCIMB 10415 on selected lactic acid bacteria and enterobacteria in co-culture.

Enterococcus faecium NCIMB 10415 is used as a probiotic for piglets and has been shown to modify the porcine intestinal microbiota. However, the mode of action of this probiotic modification is still unclear. One possible explanation is the direct growth inhibiting or stimulating effect of the probiotic on other indigenous bacteria. Therefore, the aim of the present study was to examine the growth interactions of the probiotic with different indigenous porcine bacteria in vitro. Reference strains were cultivated with the probiotic E. faecium strain NCIMB10415 (SF68) in a checkerboard assay with 102 to 105 cells/ml inoculum per strain. Growth kinetics were recorded for 8 h and used to determine specific growth of the co-cultures. Additionally, total DNA was extracted from the co-cultures at the end of the incubation to verify which strain in the co-culture was affected. Co-cultivation with eight Enterococcus spp. tester strains showed strain-specific growth differences. Three of four E. faecium strains were not influenced by the probiotic strain. PCR results showed reduced growth of the probiotic strain in co-culture with E. faecium DSM 6177. Three of four Enterococcus faecalis strains showed reduced specific growth in co-culture with the probiotic strain. However, E. faecalis DSM 20478 impaired growth of the probiotic E. faecium strain. The growth of Lactobacillus johnsonii DSM 10533 and Lactobacillus reuteri DSM 20016 was enhanced in co-culture with the probiotic strain, but co-cultivations with Lactobacillus mucosae DSM13345 or Lactobacillus amylovorus DSM10533 showed no differences. Co-cultures with the probiotic E. faecium showed no impact on the growth rate of four different enterobacterial reference strains (2 strains of Salmonella enterica and 2 strains of Escherichia coli), but PCR results showed reduced cell numbers for a pathogenic E. coli isolate at higher concentration of the probiotic strain. As the in vitro effect of the probiotic E. faecium on enterococci was strain specific and the growth of certain Lactobacillus spp. was enhanced by the probiotic, these results indicate a direct effect of the probiotic on certain members of the porcine gastro intestinal microbiota.

Enterococcus faecium NCIMB 10415 modulates epithelial integrity, heat shock protein, and proinflammatory cytokine response in intestinal cells.

Probiotics have shown positive effects on gastrointestinal diseases; they have barrier-modulating effects and change the inflammatory response towards pathogens in studies in vitro. The aim of this investigation has been to examine the response of intestinal epithelial cells to Enterococcus faecium NCIMB 10415 (E. faecium), a probiotic positively affecting diarrhea incidence in piglets, and two pathogenic Escherichia coli (E. coli) strains, with specific focus on the probiotic modulation of the response to the pathogenic challenge. Porcine (IPEC-J2) and human (Caco-2) intestinal cells were incubated without bacteria (control), with E. faecium, with enteropathogenic (EPEC) or enterotoxigenic E. coli (ETEC) each alone or in combination with E. faecium. The ETEC strain decreased transepithelial resistance (TER) and increased IL-8 mRNA and protein expression in both cell lines compared with control cells, an effect that could be prevented by pre- and coincubation with E. faecium. Similar effects were observed for the increased expression of heat shock protein 70 in Caco-2 cells. When the cells were challenged by the EPEC strain, no such pattern of changes could be observed. The reduced decrease in TER and the reduction of the proinflammatory and stress response of enterocytes following pathogenic challenge indicate the protective effect of the probiotic.

Scientific Opinion on the safety and efficacy of Cylactin® (Enterococcus faecium NCIMB 10415) as a feed additive for chickens for fattening, chickens reared for laying, minor poultry species for fattening and minor poultry species reared for laying

Scientific Opinion on the safety and efficacy of Cylactin® (Enterococcus faecium NCIMB 10415) as a feed additive for chickens for fattening, chickens reared for laying, minor poultry species for fattening and minor poultry species reared for laying

Efficacy of a Feed Probiotic Bacteria (Enterococcus faecium NCIMB 10415), Spore (Bacillus subtilis ATCC PTA-6737) and Yeast (Saccharomyces cerevisiae) in Japanese quails

Efficacy of a bacteria ( enterococcus faecium NCIMB 10415), spore ( bacillus subtilis ATCC PTA-6737 ) and yeast ( saccharomyces cerevisiae ) probiotic was tested in quails from day 2 to day 35. A control diet (CON) was supplemented at the recommended dosages to obtain three diets containing a bacteria (D-bacteria), a spore (D-spore) and a yeast (D-yeast). The results indicated that the birds fed on D-yeast significantly (P<0.05) consumed more feed at the age of 16, 30 and 37 days than the birds fed on CON, D-bacteria and D-spore, whose food intake (FI) were almost similar throughout the fattening period. Body weights (BW) and weight gains (WG) of birds fed on probiotic diets were higher than the birds fed on CON diet, especially BW and GW of the birds fed on D-yeast were significantly (P<0.05) greater than birds on CON at the age of 9, 16 and 30 days, and than birds on D-spore at the age of 30 days. Feed conversion ratio (FCR) was (P<0.05) improved in the birds of all probiotic diets, compared to the control bird group at the age of 9 and 16 days. As the birds get older the differences in FCR between control group and probiotic groups were not significant; all FCR were almost similar. Carcass yield was significantly (P<0.05) high in the birds of D-bacteria. A significant increase in the weight and length of digestive tract was seen with the birds of D-spore group. The results indicated that the use of selected probiotics enhanced bird performance, and the effect of D-yeast probiotic was better.

The general composition of the faecal virome of pigs depends on age, but not on feeding with a probiotic bacterium.

BackgroundThe pig faecal virome, which comprises the community of viruses present in pig faeces, is complex and consists of pig viruses, bacteriophages, transiently passaged plant viruses and other minor virus species. Only little is known about factors influencing its general composition. Here, the effect of the probiotic bacterium Enterococcus faecium (E. faecium) NCIMB 10415 on the pig faecal virome composition was analysed in a pig feeding trial with sows and their piglets, which received either the probiotic bacterium or not.ResultsFrom 8 pooled faecal samples derived from the feeding trial, DNA and RNA virus particles were prepared and subjected to process-controlled Next Generation Sequencing resulting in 390,650 sequence reads. In average, 14% of the reads showed significant sequence identities to known viruses. The percentage of detected mammalian virus sequences was highest (55–77%) in the samples of the youngest piglets and lowest (8–10%) in the samples of the sows. In contrast, the percentage of bacteriophage sequences increased from 22–44% in the youngest piglets to approximately 90% in the sows. The dominating mammalian viruses differed remarkably among 12 day-old piglets (kobuvirus), 54 day-old piglets (boca-, dependo- and pig stool-associated small circular DNA virus [PigSCV]) and the sows (PigSCV, circovirus and “circovirus-like” viruses CB-A and RW-A). In addition, the Shannon index, which reflects the diversity of sequences present in a sample, was generally higher for the sows as compared to the piglets. No consistent differences in the virome composition could be identified between the viromes of the probiotic bacterium-treated group and the control group.ConclusionThe analysis indicates that the pig faecal virome shows a high variability and that its general composition is mainly dependent on the age of the pigs. Changes caused by feeding with the probiotic bacterium E. faecium could not be demonstrated using the applied metagenomics method.

Dietary Enterococcus faecium NCIMB 10415 and Zinc Oxide Stimulate Immune Reactions to Trivalent Influenza Vaccination in Pigs but Do Not Affect Virological Response upon Challenge Infection

Swine influenza viruses (SIV) regularly cause significant disease in pigs worldwide. Since there is no causative treatment of SIV, we tested if probiotic Enterococcus (E.) faecium NCIMB 10415 or zinc (Zn) oxide as feed supplements provide beneficial effects upon SIV infection in piglets. Seventy-two weaned piglets were fed three different diets containing either E. faecium or different levels of Zn (2500 ppm, Znhigh; 50 ppm, Znlow). Half of the piglets were vaccinated intramuscularly (VAC) twice with an inactivated trivalent SIV vaccine, while all piglets were then infected intranasally with H3N2 SIV. Significantly higher weekly weight gains were observed in the E. faecium group before virus infection, and piglets in Znhigh and E. faecium groups gained weight after infection while those in the control group (Znlow) lost weight. Using ELISA, we found significantly higher H3N2-specific antibody levels in the E. faecium+VAC group 2 days before and at the day of challenge infection as well as at 4 and 6 days after challenge infection. Higher hemagglutination inhibition (HI) titers were also observed in the Znhigh+VAC and E. faecium+VAC groups at 0, 1 and 4 days after infection. However, there were no significant differences in virus shedding and lung lesions between the dietary groups. Using flow cytometry analysis significantly higher activated T helper cells and cytotoxic T lymphocyte percentages in the PBMCs were detected in the Znhigh and E. faecium groups at single time points after infection compared to the Znlow control group, but no prolonged effect was found. In the BAL cells no influence of dietary supplementation on immune cell percentages could be detected. Our results suggest that feeding high doses of zinc oxide and particularly E. faecium could beneficially influence humoral immune responses after vaccination and recovery from SIV infection, but not affect virus shedding and lung pathology.

Characterization of CD4+ subpopulations and CD25+ cells in ileal lymphatic tissue of weaned piglets infected with Salmonella Typhimurium with or without Enterococus faecium feeding

The aim of the present study was to test the effect of Enterococcus faecium NCIMB 10415 (E. faecium) on CD4+ T helper immune cell subpopulations and CD25+ cells in ileal lymphatic tissue after challenge with Salmonella (S.) Typhimurium DT 104. German Landrace piglets treated with E. faecium (n=16) as a feed additive and untreated controls (n=16) were challenged with S. Typhimurium 10 days after weaning. The expression of lineage specific T helper cell subtype master transcription factors on mRNA level was measured in the whole tissue of the gut associated lymphoid tissues (ileocecal mesenteric lymph node, ileum with Peyer's patches and papilla ilealis) and in magnetically sorted T helper cells from blood and ileocecal mesenteric lymph nodes at two and 28 days post infection. CD25 protein expression of T helper cells was studied by flow cytometry in ileal Peyer's patches, lymph nodes and blood. Distribution and morphology of CD25+ cells was demonstrated in situ by immunohistochemistry in paraffin embedded specimens of the ileum and the ileocecal mesenteric lymph nodes.The data provide evidence for a higher T helper 2 cell driven immune response in the control group compared to the E. faecium treated group (P<0.05) in CD4+ magnetically sorted lymphocytes from the ileocecal mesenteric lymph nodes at two and 28 days post infection. We did not observe differences for CD25+ cells in immunohistochemistry and flow cytometry between E. faecium fed pigs and the control group, but provided a detailed description of the occurrence and morphology of these cells in the gut associate lymphoid tissues of piglets. In conclusion we suggest that (i) prolonged feeding with E. faecium can result in changes of the T helper cell response leading to a stronger infection with S. Typhimurium and (ii) that it is important to examine purified immune cells to be able to detect effects on T helper cell subpopulations.

Effects of age and controlled oral dosing of Enterococcus faecium on epithelial properties in the piglet small intestine.

Enterococcus faecium NCIMB 10415 is a licensed probiotic for piglets that has been shown to positively affect diarrhoea incidence and to act on transport properties and immunological parameters in the porcine intestine. The aim of the present study was to examine its effects on jejunal absorptive and secretory capacities around weaning. Furthermore, the possible involvement of heat shock proteins in the effects of probiotics on epithelial functions was investigated. A significant part of the probiotic was dosed orally to reduce the variability of intake of the probiotic. The piglets were randomly assigned to a control and a probiotic feeding group, the latter receiving 4.5×109 cfu/day of E. faecium directly into the mouth for 34 days starting after birth. Additionally, their feed was supplemented with the probiotic strain. Piglets were weaned at day 29 after birth. Ussing chamber studies were conducted with the mid-jejunum of piglets aged 14, 28, 31, 35 and 56 days. Changes in short-circuit current (ΔIsc) were measured after stimulation of Na+-coupled absorption with L-glutamine or glucose or with the secretagogue prostaglandin E2 (PGE2). The mRNA expression for SGLT1, CFTR and various heat shock proteins was determined. The transport properties changed significantly with age. The glucose-, L-glutamine- and PGE2-induced changes in Isc were highest at day 31 after birth. No significant differences between the feeding groups were observed. The mRNA of HSP60, HSC70, HSP70 and HSP90 was expressed in the jejunal tissues. The mRNA expression of HSC70 was higher and that of HSP60 was lower in the probiotic group. HSC70 expression increased with age. In conclusion, whereas age effects were observed on absorptive and secretory functions, controlled E. faecium dosing had no measurable effects on these functional parameters in this experimental setup. The possible role of heat shock proteins should be further evaluated.

Enterococcus faecium NCIMB 10415 supplementation affects intestinal immune-associated gene expression in post-weaning piglets

In a Salmonella challenge study of weaned piglets supplemented with the probiotic Enterococcus faecium NCIMB 10415 (SF68), we observed a delayed, post-infection proliferative response of purified blood mononuclear cell fractions towards Salmonella antigens. In order to clarify this observation, we examined the patterns of immune-associated gene expression in long-term feeding trials of both pre- and post-weaning piglets. Piglets supplemented with E. faecium NCIMB 10415 showed a post-weaning dysregulation in the expression patterns of both pro- and anti-inflammatory cytokine expression in intestinal tissues and spleen. Piglets of the supplemented group showed significantly reduced levels of IL-8, IL-10 and the co-stimulatory molecule CD86 mRNA expression in ileal Peyer's patches. The expression of CTLA4, an inhibitor of T-cell activation/proliferation, showed similar levels of expression in all tissues examined, particularly in ileal Peyer's patches post-weaning where IL-8, IL-10 and CD86 transcript levels were significantly reduced relative to control animals. Blood serum cytokine protein levels showed elevated TGFβ in pre-weaning piglets which, together with IL-6, may have suppressed IFNγ production in the probiotic-fed animals. In a second Salmonella challenge study, post-weaning, E. faecium-fed animals showed significantly elevated levels of IL-8 gene expression in mesenteric lymph nodes, but reduced levels in the spleen. At early times post-infection, the probiotic-fed group showed similar levels of IL-10, CD86 and CTLA4 mRNA expression as the control animals in intestinal Peyer's Patches, despite high relative levels of IL-8 expression in mesenteric lymph nodes. The sum of the observations suggests that supplementation of pre-weaning piglets with E. faecium affects intestinal immune-associated gene expression, which is aggravated post-weaning when the animals receive increased levels of the probiotic in feed. We suggest the post-weaning reductions in gene expression may delay the host response to infections, and provide pathogenic bacteria such as Salmonella with a “window of opportunity”, leading to the increased bacterial loads and shedding observed in challenge trials. Possible mechanisms explaining these effects of E. faecium NCIMB 10415 are discussed.

Characterization of the effects of Enterococcus faecium on intestinal epithelial transport properties in piglets1

Probiotics have been shown to have positive effects on growth performance traits and the health of farm animals. The objective of the study was to examine whether the probiotic strain Enterococcus faecium NCIMB 10415 (E. faecium) changes the absorptive and secretory transport and barrier properties of piglet jejunum in vitro and thereby to verify tendencies observed in a former feeding trial with E. faecium. Further aims were to assess a potential mechanism of probiotics by testing effects of IL-α, which is upregulated in the peripheral blood mononuclear cells of E. faecium-supplemented piglets, and to test the hypothesis that IL-1α induces a change in ion transport. Sows and their piglets were randomly assigned to a control group and a probiotic group supplemented with E. faecium. The sows received the probiotic supplemented feed from d 28 before parturition and the piglets from d 12 after birth. Piglets were killed at the age of 12 ± 1, 26 ± 1, 34 ± 1, and 54 ± 1 d. Ussing chamber studies were conducted with isolated mucosae from the mid jejunum. Samples were taken for mRNA expression analysis of sodium-glucose-linked transporter 1 (SGLT1) and cystic fibrosis transmembrane conductance regulator (CFTR). The Na(+)/glucose cotransport was increased in the probiotic group compared with the control group at 26 (P = 0.04) and 54 d of age (P = 0.01). The PGE2-induced short circuit current (Isc) was greater at 54 d of age in the probiotic group compared with the control group (P = 0.03). In addition, effects of age on the absorptive (P < 0.01) and secretory (P < 0.01) capacities were observed. Neither SGLT1 nor CFTR mRNA expression was changed by probiotic supplementation. Mannitol flux rates as a marker of paracellular permeability decreased in both groups with increasing age and were less in the probiotic group at the 26 d of age (P = 0.04), indicating a tighter intestinal barrier. The ΔIsc induced by IL-1α was inhibited by bumetanide (P < 0.01), indicating an induction of Cl(-) secretion. Thus, in this experimental setup, E. faecium increased the absorptive and secretory capacity of jejunal mucosae and enhanced the intestinal barrier. Furthermore, the results indicated that IL-1α induces bumetanide-sensitive chloride secretion. The effects of cytokines as potential mediators of probiotic effects should, therefore, be the subject of further studies.

Inhibitory Influence of Enterococcus faecium on the Propagation of Swine Influenza A Virus In Vitro

The control of infectious diseases such as swine influenza viruses (SwIV) plays an important role in food production both from the animal health and from the public health point of view. Probiotic microorganisms and other health improving food supplements have been given increasing attention in recent years, but, no information on the effects of probiotics on swine influenza virus is available. Here we address this question by assessing the inhibitory potential of the probiotic Enterococcus faecium NCIMB 10415 (E. faecium) on the replication of two porcine strains of influenza virus (H1N1 and H3N2 strain) in a continuous porcine macrophage cell line (3D4/21) and in MDBK cells. Cell cultures were treated with E. faecium at the non-toxic concentration of 1×106 CFU/ml in growth medium for 60 to 90 min before, during and after SwIV infection. After further incubation of cultures in probiotic-free growth medium, cell viability and virus propagation were determined at 48 h or 96 h post infection. The results obtained reveal an almost complete recovery of viability of SwIV infected cells and an inhibition of virus multiplication by up to four log units in the E. faecium treated cells. In both 3D4/21- and MDBK-cells a 60 min treatment with E. faecium stimulated nitric oxide (NO) release which is in line with published evidence for an antiviral function of NO. Furthermore, E. faecium caused a modified cellular expression of selected mediators of defence in 3D4-cells: while the expression of TNF-α, TLR-3 and IL-6 were decreased in the SwIV-infected and probiotic treated cells, IL-10 was found to be increased. Since we obtained experimental evidence for the direct adsorptive trapping of SwIV through E. faecium, this probiotic microorganism inhibits influenza viruses by at least two mechanisms, direct physical interaction and strengthening of innate defence at the cellular level.

Antiviral effects of a probiotic Enterococcus faecium strain against transmissible gastroenteritis coronavirus

The enteropathogenic coronavirus transmissible gastroenteritis virus (TGEV) causes severe disease in young piglets. We have studied the protective effects of the probiotic Enterococcus faecium NCIMB 10415 (E. faecium), which is approved as a feed additive in the European Union, against TGEV infection. E. faecium was added to swine testicle (ST) cells before, concomitantly with, or after TGEV infection. Viability assays revealed that E. faecium led to a dose-dependent rescue of viability of TGEV-infected cells reaching nearly to complete protection. Virus yields of the E. faecium–treated cultures were reduced by up to three log10 units. Western blot analysis of purified TGEV revealed that the levels of all viral structural proteins were reduced after E. faecium treatment. Using transmission electron microscopy, we observed attachment of TGEV particles to the surface of E. faecium which might be a means to trap virus and to prevent infection. Increased production of nitric oxide in the cells treated with E. faecium and elevated expression of interleukin 6 and 8 pointed to stimulated cellular defense as a mechanism to fight TGEV infection.

Influence of age and Enterococcus faecium NCIMB 10415 on development of small intestinal digestive physiology in piglets

This study aimed at studying the effect of age and Enterococcus faecium NCIMB 10415 on jejunal physiological measures in suckling and weaned piglets. A total of 112 piglets from 16 landrace sows were used. Sows received either no probiotic (n=8, control) or were fed a diet with 4.2 to 4.3×106cfu/g E. faecium NCIMB 10415 (n=8, EF) from 28days ante partum onwards. Piglets were offered creep feed beginning at 12 d of age and piglets were weaned to a pre-starter diet at 26 d of age. For EF piglets, the creep feed (pre-starter) and starter diets contained 5.1×106cfu/g and 3.6×106cfu EF/g, respectively. Twenty-seven randomly selected piglets from each group were euthanized at 12 (n=6), 26 (n=6), 34 (n=7), and 54d (n=8) of age and a 45cm mid-jejunal segment was taken. Histomorphometry was performed and relative transcript abundance of caspase-3, proliferating cell nuclear antigen, intestinal alkaline phosphatase (IAP), lactase-phlorizin hydrolase (LPH), sucrase (SUC), aminopeptidase-N (APN) and sodium/glucose cotransporter 1 (SGLT1) was determined. Lactase (LAC), maltase (MAL), SUC, APN and IAP activities were measured in brush border membranes. The EF treatment did not affect (P>0.05) performance variables like the pre- or post-weaning daily weight gain, feed intake, incidence of diarrhoea and ileal and total tract nutrient digestibility, except the feed conversion ratio which was reduced (P=0.033). Jejunal villus length and crypt depth did not differ between groups (P>0.05) but increased age-dependently (P=0.003 and P<0.001, respectively). Feeding EF did not markedly influence brush border enzyme expression or activity. Enzyme activity increased (SUC, MAL) or decreased (LAC, IAP) with age reflecting maturation and adaptation to creep feed (P<0.001). The gene expression of SUC, SGLT1 and IAP changed over time (P=0.012, P=0.003 and P=0.013). Furthermore, gene expression was not affected by treatment. The current study suggests that EF did not markedly affect physiological variables associated with digestive function. Age of piglets seems to be the main factor determining function and morphology of the jejunal tissue, reflecting both maturation and adaptation to creep feed.

Enterococcus faecium NCIMB 10415 does not protect interleukin-10 knock-out mice from chronic gut inflammation

Enterococcus faecium NCIMB 10415 reduces diarrhoea incidence and duration in animals and human study subjects. We tested whether the strain is also capable of reducing chronic gut inflammation and aimed to identify mechanisms that are involved in possible probiotic effects. To identify health-promoting mechanisms of the strain, we used interleukin-10-deficient mice that spontaneously develop gut inflammation and fed these mice a diet containing NCIMB 10415 for 3, 8 and 24 weeks, respectively. Control mice were fed a diet which was identically composed but did not contain the strain. After 3 weeks of intervention the experimental animals were less inflamed in the caecum than the control animals. This effect was not observed in the colon and there were no differences between experimental and control mice at any other time point. The application of the strain was associated with higher expression levels of interferon gamma and interferon gamma-induced protein 10 after 3 and 24 but not after 8 weeks of feeding. No differences between the animals were observed in intestinal barrier function or intestinal microbiota composition. However, we observed a low abundance of the mucin-degrading bacterium Akkermansia muciniphila in the mice that were fed NCIMB 10415 for 8 weeks. These low cell numbers were associated with a significantly lower caecal inflammation score and improved paracellular permeability as compared to the NCIMB-treated mice that were killed after 3 and 24 weeks of intervention. In conclusion, NCIMB 10415 is not capable of reducing gut inflammation in the IL-10-/- mouse model. The exact role of A. muciniphila and of a possible interaction between this bacterium, NCIMB 10415 and the host in gut inflammation requires further investigation.