Effect of the probiotic Enterococcus faecium NCIMB10415 on cell numbers of total Enterococcus spp., E. faecium and E. faecalis in the intestine of piglets

Sows and their piglets were fed a diet supplemented with or without the probiotic E. faecium NCIMB10415 (also known as SF68). Piglets were sacrificed 14, 28, 35 and 56 days after birth and DNA from intestinal segments was extracted and purified. A real time PCR assay was used to distinguish Enterococcus spp. (16s rDNA based), E. faecium (Efaafm gene), E. faecalis (Efaafs gene) as well as the probiotic strain (unique plasmid sequence). Extracts of autoclaved sow feces inoculated with E. faecium and E. faecalis cultures were used to calibrate real time PCR results. The probiotic strain was detected in 14 day old suckling piglets before the piglets had access to the starter diet. In piglets of the probiotic group, probiotic E. faecium cell counts were always a significant proportion of total E. faecium cells in stomach digesta (4-20%), however only a small fraction of the total Enterococcus spp. cell number on day 14 and 28 in all intestinal segments (0.1-0.7%). Compared to control samples, the probiotic E. faecium strain significantly (p < or = 0.05) decreased the amount of total Enterococcus spp. and E. faecalis cells in the colon of 14 day old suckling piglets as well as in jejunum and colon samples one week after weaning. E. faecium cell counts were not modified on any sampling day or intestinal segment. This study showed that the presence of probiotic E. faecium NCIMB10415 coincided with reduced total E. faecalis, but not total E. faecium cell numbers in the intestine of piglets. In view of unchanged cell numbers and ratios in sow feces, modifications must have taken place within the intestine of suckling piglets.     

Specific enumeration of the probiotic strain Enterococcus faecium NCIMB 10415 in the intestinal tract and in faeces of piglets and sows

The intestinal bacterium Enterococcus faecium NCIMB 10415 (E. faecium SF68) has been used for more than a decade as a probiotic strain in animal nutrition as well as in the prevention and treatment of diarrhoea in humans. Beneficial effects have been shown in feeding and clinical trials. However, the strain has no selective growth markers and monitoring in the intestinal tract is impossible by cultivation. Using specific nucleotide sequences, in this study a probe for colony hybridization was constructed in order to quantify this probiotic strain in feed and intestinal and faecal samples from piglets and sows. The probiotic strain showed almost constant amounts in sow faeces (1.8 x 10(5) cfu/g wet weight), while contents in digesta and piglet faeces varied on a lower level depending on gut section and piglet age. The ratio of specific probiotic counts and total enterococci was much lower than in sow faeces however the strain could be detected reliably in faeces already on the 14th day of life. The application of the colony hybridization method enables for the first time the selective detection of the widely used probiotic E. faecium NCIMB 10415 strain among total Enterococcus spp. counts of digesta, faeces and feed. It is now possible to monitor the presence of the probiotic in the intestinal tract and faeces. Results of this study have implications for the proposed modes of action of probiotics in animal nutrition.     

Effects of the Dietary Probiotic,Enterococcus faecium NCIMB11181, on the Intestinal Barrier and System Immune Status in Escherichia coli O78-Challenged Broiler Chickens

Probiotics and Antimicrobial Proteins - The effects of Enterococcus faecium on growth, intestinal barrier function, and immune response in Escherichia coli O78-challenged broiler chickens were...     

Impact of the probiotic bacteria Enterococcus faecium NCIMB 10415 (SF68) and Bacillus cereus var. toyoi NCIMB 40112 on the development of serum IgG and faecal IgA of sows and their piglets

Abstract

To examine the influence of two different probiotic bacteria on the humoral immune system of swine, two animal studies were carried out with sows and their litters. The sows' feed was supplemented with either Enterococcus faecium NCIMB 10415 (SF68) or Bacillus cereus var. toyoi NCIMB 40112 beginning early in pregnancy. The total IgA content in the faeces as well as the total IgG concentration in the blood of the sows was recorded before and after weaning. The same parameters were determined in the blood and faeces of the piglets. In sows, only feed supplementation with B. cereus led to a clear increase in faecal IgA. Serum IgG levels were not significantly affected by any probiotic feeding in sows. In piglets, the group that was fed B. cereus showed significantly higher faecal IgA levels shortly before weaning, whereas in the E. faecium group, a significant decrease in IgA levels was observed one week after weaning. In both probiotic fed groups the post-weaning IgG levels were significantly decreased compared to the respective control groups. We conclude that B. cereus var. toyoi feed supplementation led to an increased intestinal IgA secretion both in sows and piglets. This effect could be related to a more successful mucosal defence which in turn led to a lower level in systemic IgG production in piglets after weaning.     

Impact of the probiotic bacteria Enterococcus faecium NCIMB 10415 (SF68) and Bacillus cereus var. toyoi NCIMB 40112 on the development of serum IgG and faecal IgA of sows and their piglets

To examine the influence of two different probiotic bacteria on the humoral immune system of swine, two animal studies were carried out with sows and their litters. The sows' feed was supplemented with either Enterococcusfaecium NCIMB 10415 (SF68) or Bacillus cereus var. toyoi NCIMB 40112 beginning early in pregnancy. The total IgA content in the faeces as well as the total IgG concentration in the blood of the sows was recorded before and after weaning. The same parameters were determined in the blood and faeces of the piglets. In sows, only feed supplementation with B. cereus led to a clear increase in faecal IgA. Serum IgG levels were not significantly affected by any probiotic feeding in sows. In piglets, the group that was fed B. cereus showed significantly higher faecal IgA levels shortly before weaning, whereas in the E. faecium group, a significant decrease in IgA levels was observed one week after weaning. In both probiotic fed groups the post-weaning IgG levels were significantly decreased compared to the respective control groups. We conclude that B. cereus var. toyoi feed supplementation led to an increased intestinal IgA secretion both in sows and piglets. This effect could be related to a more successful mucosal defence which in turn led to a lower level in systemic IgG production in piglets after weaning.     

A Strain of Enterococcus faecium (18C23) Inhibits Adhesion of Enterotoxigenic Escherichia coli K88 to Porcine Small Intestine Mucus

Few studies, if any, have addressed the adhesion of enterococci to the intestinal mucosa and their interference with the adhesion of pathogens, although more than 60% of probiotic preparations in the market contain strains of enterococci. The objective of this study was to investigate if Enterococcus faecium 18C23 has the ability to inhibit the adhesion of Escherichia coli K88ac and K88MB to the small intestine mucus of piglets. Approximately 9% of E. faecium 18C23 organisms adhered to the small intestine mucus, and the adhesion was found to be specific. Living E. faecium 18C23 culture efficiently inhibited the adhesion of E. coli K88ac and K88MB to the piglet intestine mucus. Inhibition of the adhesion of E. coli K88ac to the small intestine mucus was found to be dose dependent. Inhibition of >90% was observed when 10⁹ CFU or more of living E. faecium 18C23 culture per ml was added simultaneously with E. coli to immobilized mucus. The substances from both the 18C23 cells and the spent culture supernatant contributed to the inhibition of adhesion of E. coli K88 to the small intestine mucus receptors. The inhibiting effect was not solely a pH effect since considerable inhibitory action was demonstrated after neutralizing the mixture or spent culture supernatant to pH 7.0. Part of the inhibition of adhesion of E. coli K88ac by E. faecium 18C23 or its supernatant might occur through steric hindrance.     

Attempts to Displace the Indigenous Antibiotic Resistant Gut Flora of Chicken by Feeding Sensitive Strains of Escherichia coli Prior to Slaughter

Attempts to limit the use of antibiotics have not, in general, resulted in the gut flora in farm animals becoming predominantly sensitive. Partial success has been demonstrated, however, by feeding chickens with antibiotic sensitive Escherichia coli known to be good colonizers of the chicken gut. Where feeding was done prior to slaughter a corresponding reduction in carcass contamination by resistant E. coli was observed.     

Experimental Administration of the Probiotic Escherichia coli Strain Nissle 1917 Results in Decreased Diversity of E. coli Strains in Pigs

The strain Escherichia coli Nissle 1917 (EcN) is widely used as an efficient probiotic in therapy and prevention of human infectious diseases, especially of the intestinal system. Concurrently, small adult pigs are being used as experimental omnivore models to study human gastrointestinal functions. EcN bacteria were applied to 6 adult healthy female pigs in a 2-week trial. 6 Control animals remained untreated. Altogether, 164 and 149 bacterial strains were isolated from smear samples taken from gastrointestinal mucosa in the experimental and control group, respectively. Each individual E. coli strain was then tested for the presence of 29 bacteriocin-encoding determinants as well as for DNA markers of A, B1, B2 and D phylogenetic groups. A profound reduction of E. coli genetic variance (from 32 variants to 13 ones, P?=?0.0006) was found in the experimental group, accompanied by a lower incidence of bacteriocin producers in the experimental group when compared to control (21.3 and 34.9%, respectively; P?=?0.007) and by changes in the incidence of individual bacteriocin types. The experimental administration of EcN strain was not sufficient for stable colonization of porcine gut, but induced significant changes in the enterobacterial microbiota.     

A strain of Enterococcus faecium (18C23) inhibits adhesion of enterotoxigenic Escherichia coli K88 to porcine small intestine mucus

Few studies, if any, have addressed the adhesion of enterococci to the intestinal mucosa and their interference with the adhesion of pathogens, although more than 60% of probiotic preparations in the market contain strains of enterococci. The objective of this study was to investigate if Enterococcus faecium 18C23 has the ability to inhibit the adhesion of Escherichia coli K88ac and K88MB to the small intestine mucus of piglets. Approximately 9% of E. faecium 18C23 organisms adhered to the small intestine mucus, and the adhesion was found to be specific. Living E. faecium 18C23 culture efficiently inhibited the adhesion of E. coli K88ac and K88MB to the piglet intestine mucus. Inhibition of the adhesion of E. coli K88ac to the small intestine mucus was found to be dose dependent. Inhibition of >90% was observed when 10(9) CFU or more of living E. faecium 18C23 culture per ml was added simultaneously with E. coli to immobilized mucus. The substances from both the 18C23 cells and the spent culture supernatant contributed to the inhibition of adhesion of E. coli K88 to the small intestine mucus receptors. The inhibiting effect was not solely a pH effect since considerable inhibitory action was demonstrated after neutralizing the mixture or spent culture supernatant to pH 7.0. Part of the inhibition of adhesion of E. coli K88ac by E. faecium 18C23 or its supernatant might occur through steric hindrance.     

The First Steps of Adaptation of Escherichia coli to the Gut Are Dominated by Soft Sweeps

The accumulation of adaptive mutations is essential for survival in novel environments. However, in clonal populations with a high mutational supply, the power of natural selection is expected to be limited. This is due to clonal interference - the competition of clones carrying different beneficial mutations - which leads to the loss of many small effect mutations and fixation of large effect ones. If interference is abundant, then mechanisms for horizontal transfer of genes, which allow the immediate combination of beneficial alleles in a single background, are expected to evolve. However, the relevance of interference in natural complex environments, such as the gut, is poorly known. To address this issue, we have developed an experimental system which allows to uncover the nature of the adaptive process as Escherichia coli adapts to the mouse gut. This system shows the invasion of beneficial mutations in the bacterial populations and demonstrates the pervasiveness of clonal interference. The observed dynamics of change in frequency of beneficial mutations are consistent with soft sweeps, where different adaptive mutations with similar phenotypes, arise repeatedly on different haplotypes without reaching fixation. Despite the complexity of this ecosystem, the genetic basis of the adaptive mutations revealed a striking parallelism in independently evolving populations. This was mainly characterized by the insertion of transposable elements in both coding and regulatory regions of a few genes. Interestingly, in most populations we observed a complete phenotypic sweep without loss of genetic variation. The intense clonal interference during adaptation to the gut environment, here demonstrated, may be important for our understanding of the levels of strain diversity of E. coli inhabiting the human gut microbiota and of its recombination rate.     

Properties of a Cell Fraction That Repairs Damage to the Cell Division Mechanism of Escherichia coli

A factor in bacterial cell extracts which induces cell division in filaments of Escherichia coli produced by irradiation was found to be associated with a heat-labile particulate fraction which sediments at about 100S. Frozen or lyophilized samples of the cell extracts were stable for considerable periods of time. Fractions purified by centrifugation contained 2% of the total protein of the extract but no measurable ribonucleic acid or deoxyribonucleic acid. The extracts were inactivated by incubation with phospholipase A, lipases, and detergents, but not by incubation with selected nucleases and proteases.     

Administration of the Probiotic Escherichia coli Strain A0 34/86 Resulted in a Stable Colonization of the Human Intestine During the First Year of Life

Probiotics and Antimicrobial Proteins - Colinfant New Born (CNB) is an orally administered probiotic preparation containing the Escherichia coli strain A0 34/86, which is specially marketed for use...     

Strain improvement to enhance the production of recombinant penicillin acylase in high-cell-density Escherichia coli cultures

Using fed-batch operation for high-cell-density cultivation, efforts are frequently made for optimization of culture parameters, particularly feeding strategy. The current study also emphasized the importance of selecting strains for the production of recombinant proteins in high-cell-density cultures. With Escherichia coli penicillin acylase (PAC) as a target protein, the host/vector system of MDdeltaP7 harboring pTrcKnPAC2902 and pKS12 was designed for optimization of fed-batch cultivation for recombinant protein production. The host, MDdeltaP7, potentially had a high translational and periplasmic processing efficiency for pac expression. On the other hand, the vector, pTrcKnPAC2902, was genetically constructed for pac overexpression. Coexistence of the other vector, pKS12, significantly enhanced PAC production by improving cell physiology and reducing the amount of inclusion body formation upon pac overexpression. An extremely high volumetric PAC activity at 37,500 U/L was obtained with the use of the developed host/vector system under optimum fed-batch culture conditions.     

Heterologous expression of enterocin A, a bacteriocin from Enterococcus faecium, fused to a cellulose-binding domain in Escherichia coli results in a functional protein with inhibitory activity against Listeria

The genes for the bacteriocins enterocin A and B were isolated from Enterococcus faecium ATB 197a. Using the pET37b(+) vector, the enterocin genes were fused to an Escherichia coli specific export signal sequence, a cellulose-binding domain (CBD_cenA) and a S-tag under the control of a T7lac promotor. The constructs were subsequently cloned into E. coli host cells. The expression of the recombinant enterocins had different effects on both the host cells and other Gram-positive bacteria. The expression of entA in Esc. coli led to the synthesis and secretion of functional active enterocin A fusion proteins, which were active against some Gram-positive indicator bacteria, but did not influence the viability of the host cells. In contrast, the expression of enterocin B fusion proteins led to a reduced viability of the host cells, indicating a misfolding of the protein or interference with the cellular metabolism of Esc. coli. Indicator strains of Gram-positive bacteria were not inhibited by purified enterocin B fusion proteins. However, recombinant enterocin B displayed inhibitory activity after the proteolytic cleavage of the fused peptides.     

A Mutational Hotspot and Strong Selection Contribute to the Order of Mutations Selected for during Escherichia coli Adaptation to the Gut

The relative role of drift versus selection underlying the evolution of bacterial species within the gut microbiota remains poorly understood. The large sizes of bacterial populations in this environment suggest that even adaptive mutations with weak effects, thought to be the most frequently occurring, could substantially contribute to a rapid pace of evolutionary change in the gut. We followed the emergence of intra-species diversity in a commensal Escherichia coli strain that previously acquired an adaptive mutation with strong effect during one week of colonization of the mouse gut. Following this first step, which consisted of inactivating a metabolic operon, one third of the subsequent adaptive mutations were found to have a selective effect as high as the first. Nevertheless, the order of the adaptive steps was strongly affected by a mutational hotspot with an exceptionally high mutation rate of 10⁻⁵. The pattern of polymorphism emerging in the populations evolving within different hosts was characterized by periodic selection, which reduced diversity, but also frequency-dependent selection, actively maintaining genetic diversity. Furthermore, the continuous emergence of similar phenotypes due to distinct mutations, known as clonal interference, was pervasive. Evolutionary change within the gut is therefore highly repeatable within and across hosts, with adaptive mutations of selection coefficients as strong as 12% accumulating without strong constraints on genetic background. In vivo competitive assays showed that one of the second steps (focA) exhibited positive epistasis with the first, while another (dcuB) exhibited negative epistasis. The data shows that strong effect adaptive mutations continuously recur in gut commensal bacterial species.     

Selection and Characterization of a Candidate Therapeutic Bacteriophage That Lyses the Escherichia coli O104:H4 Strain from the 2011 Outbreak in Germany

In 2011, a novel strain of O104:H4 Escherichia coli caused a serious outbreak of foodborne hemolytic uremic syndrome and bloody diarrhea in Germany. Antibiotics were of questionable use and 54 deaths occurred. Candidate therapeutic bacteriophages that efficiently lyse the E. coli O104:H4 outbreak strain could be selected rather easily from a phage bank or isolated from the environment. It is argued that phage therapy should be more considered as a potential armament against the growing threat of (resistant) bacterial infections.     

Production and Evaluation of Antibody to the Heat-Stable Enterotoxin from a Human Strain of Enterotoxigenic Escherichia coli

Escherichia coli heat-stable enterotoxin was coupled to bovine serum albumin by a carbodiimide reagent. Antibody to the conjugate was produced by immunization of rabbits. Data from radioimmunoassay and infant mouse tests indicate the presence of antibody to the enterotoxin. The antisera can be used in a radioimmunoassay to measure enterotoxin in various fluids.