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.
Genera Lactobacillus and Bifidobacterium include a large number of species and strains exhibiting important properties in an applied context, especially in the area of food and probiotics. An updated list of species belonging to those two genera, their phylogenetic relationships and other relevant taxonomic information are reviewed in this paper. The conventional nature of taxonomy is explained and some basic concepts and terms will be presented for readers not familiar with this important and fast-evolving area, which importance is often underestimated. The analysis of biodiversity and its cataloguing, i.e. taxonomy, constitute the basis for applications and scientific communication: reliable identification and correct naming of bacterial strains are not only primary aims of taxonomic studies, but also fundamental elements in an applied context, for the tracking of probiotic strains and a non fraudulent labelling of fermented milks and pharmaceutical products containing probiotic microorganisms. A number of resources freely available have been listed and their use is suggested for people concerned with different aspects of taxonomy. Some perspectives in taxonomy have been outlined, in particular considering the role of culture independent analyses to reveal the still unknown and uncultured microorganisms. Finally, the impact of the availability of whole-genome sequences in taxonomy is briefly explained: they have already begun to give insights on bacterial evolution, which will surely have implications on taxonomy, even if the analysis of data for lactic acid bacteria is still limited to few species.
Introduction Antibiotics have been used as therapeutic and prophylactic treatments to control a variety of bacterial infections in livestock for more than 50 years. Different types of antibiotics have also been fed at sub-therapeutic levels to cattle, poultry and swine to increase productivity and feed efficiency (McDermott et al., 2002). The emergence of antibiotic-resistance in many bacteria relevant for animal and public health stresses the importance of decreasing the use of antibiotics in animal production. The reduction of antibiotic application in livestock can only be achieved if alternative antimicrobial strategies are available. Among those interventions that have been investigated and applied are: immunization, diet modification, sanitation, feed additives, and probiotic bacteria (Callaway et al., 2004; Gillor et al., 2004). A variety of probiotic bacteria have been tested to control animal and foodborne pathogenic bacteria in livestock, but in many of them the beneficial effects have not been fully elucidated (Fuller, 1999). The mechanism of inhibition of pathogenic bacteria for several of those probiotic microorganisms is mediated by the production of bacteriocins. The application of bacteriocins such as colicins in livestock has been largely achieved by feeding bacteriocin-producing strains. Feeding purified bacteriocins to humans for control of diarrhea was reported in a few publications during the 1900’s (Papavassiliou, 1961), but there is very little evidence that administering bacteriocins alone to livestock has ever been done. The absence of that type of studies could be due to the likely rapid degradation of these proteinaceous compounds in the digestive tract of mammals. Because of this lack of evidence, our discussion on the use of bacteriocins in livestock will be largely based on those studies that reported feeding or applying bacteriocin-producing bacteria (BPB).
In the last few years, advances in immunology, metabolomics and microbial ecology have shown that the contribution of the intestinal microbiota to the overall health status of the host has been so far underestimated. In this context, intestinal epithelial cells play a crucial role in the maintenance of intestinal homoeostasis. Indeed, at the interface between the luminal content and host tissues, the intestinal epithelium must integrate pro- and anti-inflammatory signals to regulate innate and adaptative immune responses, i.e. to control inflammation. However, under the influence of environmental factors, disturbance of the dialog between enteric bacteria and epithelial cells contributes to the development of chronic inflammation in genetically susceptible hosts. The present review covers the state of knowledge of the host response, especially in intestinal epithelial cells, to enteric bacteria, including colitogenic and probiotic bacteria. It also seeks to give an overview of potential regulatory mechanisms involved in the maintenance of intestinal homeostasis, and discusses the clinical implications for inflammatory bowel diseases.
Coeliac disease (CD) is an immune-mediated enteropathy with a multifactorial aetiology, characterized by chronic inflammation of the small intestinal mucosa. Although evidence suggests that the gut microbiota contributes to other chronic inflammatory disorders, its possible role in CD has not been determined. In this study, the composition of the fecal microbiota of coeliac children and age-matched controls was investigated by culture-dependent and -independent methodologies, using fluorescent in situ hybridization (FISH). The levels of Bacteroides, Clostridium and Staphylococcus were significantly higher (p < 0.05) in fecal samples from coeliac patients than in healthy subjects when analysed by culture methods. The numbers of Bacteroides-Prevotella, Clostridium histolyticum, Eubacterium rectale-C. coccoides, Atopobium, and sulfate reducing bacterial groups were also significantly higher (p < 0.05) in fecal samples from coeliac infants when analysed by FISH. The counts of Bifidobacterium tended to be higher in healthy controls by the two type of analysis but the differences were not significant. This is the first report on the identification of the specific bacterial groups responsible for alterations in the intestinal microecology of children with active CD. The bacterial pattern detected in coeliac patients, correlates with the epidemiological data and metabolic deviations associated with CD, and involve bacterial groups link to other chronic inflammatory disorders.
Gluco-oligosaccharides produced by Gluconobacter oxydans NCIMB 4943 from maltodextrin as the source, were evaluated for their fermentability by the human colonic microflora. The selectivity of growth of desirable bacteria in the human colon was studied in a three-stage continuous model of the human large intestine. Populations of bacteria, and their fluctuations as a response to the fermentation, were enumerated using fluorescent in situ hybridization (FISH). The gluco-oligosaccharides resulted in increases in numbers of bifidobacteria and the Lactobacillus/Enterococcus group in all 3 vessels of the system, representing the proximal, transverse and distal colonic areas. The prebiotic indices of the gluco-oligosaccharides were 2.29, 4.23 and 2.74 in V1, V2 and V3 respectively.
Terminal-restriction fragment length polymorphism (T-RFLP) was used to evaluate how to store intestinal specimens for bacterial community analysis. Bacterial communities are increasingly often described by means of DNA-based methods and it is common practice to store intestinal or faecal specimens either at -20 degrees C or -80 degrees C. In this study, samples of intestines from five different pigs were stored at -80 degrees C and -20 degrees C, respectively and a thawing and freezing procedure was carried out three times for each intestinal per pig per temperature. The cumulative sum of the T-RFLP peak heights (T-RF intensities) decreased as the temperature decreased. The composition of the bacterial community changed when stored at -80 degrees C compared to the samples stored at -20 degrees C. Thus it is recommended from this study that samples of intestinal content are stored at -20 degrees C before use for bacterial community analysis, instead of the current practice at -80 degrees C.
Total parenteral nutrition (TPN) has been associated with mucosal atrophy, impaired gut barrier function, and translocation of luminal bacteria with resultant sepsis in preterm human infants. Currently, we examined the effects of enteral (ENT) or TPN treatments on translocation events in neonatal pigs and on colonization and composition of microbiota in the neonatal gut. Newborn, colostrum-deprived pigs (<24 hours old) were fitted with intravenous catheters and were fed either ENT (n = 13) or TPN (n = 13) for 7 days. After 7 days of treatment, pigs were euthanized and samples were collected for bacterial culture from the blood, intestinal tract and organs. ENT pigs had increased numbers of bacterial genera isolated, higher concentrations of bacteria (CFU/g), and increased colonization of all segments of the intestinal tract compared to the TPN pigs. Translocation of bacteria from the intestinal tract to tissues or blood was similar (8 of 13) for both groups. The ENT group had 1/13 positive for Clostridium difficile toxin A whereas the TPN group had 5/13. We concluded that ENT favored increased bacterial concentrations comprised of more speciation in the gastrointestinal tract compared to TPN, and that TPN-treated piglets were at higher risk of colonization by toxin-expressing strains of C. difficile.
While prebiotic substances have attracted considerable attention in terms of their stimulatory effect on intestinal calcium absorption, the potential influence of probiotic bacteria on calcium absorption has received little research emphasis. Therefore, the objective of this study was to investigate the effect of well-characterized probiotics (Lactobacillus salivarius (UCC 118) and Bifidobacterium infantis (UCC 35624)) on calcium uptake and transepithelial calcium transport in human intestinal-like, Caco-2, cells in culture. Cells were seeded onto permeable transport membranes and allowed to differentiate, over 16 d, into intestinal-like cell monolayers. Monolayers (n=12-20/ treatment) were then exposed to E. coli UCC 118, UCC 35624 (10(7) cfu/ml) or no bacteria (control) for 6 or 24 h prior to calcium transport studies. Calcium transport was unaffected by exposure of Caco-2 cells to E. coli, UCC 118 or UCC 35624 for 6 or 24 h. Calcium uptake into Caco-2 cell monolayers after 24 h was unaffected by UCC 35624, but was significantly (P<0.05) or tended (P=0.079) to be increased by UCC 118 and E. coli, respectively, relative to the control. In conclusion, the findings of this study which suggest that bacteria can enhance intestinal calcium uptake, if not calcium transport, highlights the need to undertake further studies in this, to date, vastly underinvestigated area.
Acidified feeding formulas have been proposed as a method of controlling gastrointestinal colonization and nosocomial infection in critically ill patients. We examined possible mechanisms by which chronic acid water feeding might protect the host against lethal gut derived sepsis by assessing its effect on both local intestinal epithelial barrier function to bacteria as well as on local and systemic heat shock protein expression. Heat shock protein expression measured by immunoblot demonstrated that HSP25 was increased in the stomach, aorta and kidney of mice chronically fed acid water (8 weeks) compared to tap water fed controls. HSP72 expression was also increased in the aorta of mice drinking acid water. The protein content of cecum and its barrier function were enhanced in mice ingesting acidified water. The direct effect of an acid environment on intestinal epithelial barrier function was tested in cultured human intestinal epithelial cells. An acidified environment protected against bacterial mediated disruption of the intestinal epithelial barrier. Finally, the protective effect of chronic acid water feeding on gut-derived sepsis due to P. aeruginosa was tested in mice. Chronic acid water feeding protected mice from the lethal gut derived sepsis due to P. aeruginosa.
The aim of this study was to develop selectively fermented (prebiotic) carbohydrate molecules which would also result in the generation of butyric acid. Gluco-oligosaccharides produced by Gluconobacter oxydans NCIMB 4943 from various types of maltodextrins were evaluated for their fermentation by mixed cultures of human colonic microflora. The selectivity of growth of desirable bacteria (bifidobacteria, lactobacilli) was studied in stirred pH-controlled (6.8) batch cultures. Bacterial populations were enumerated using fluorescent in situ hybridization (FISH). Gluco-oligosaccharides resulted in significantly (P<0.05) increased numbers of bifidobacteria and lactobacilli within 24 hours. Bacteroides, clostridial and eubacterial populations were slightly decreased at 48 h. There was very little difference in selectivity between the maltodextrin substrates and the products, although maltodextrin displayed a slightly less selective fermentation than the gluco-oligosaccharide products, also stimulating the growth of bacteroides, clostridia and eubacteria. Gluco-oligosaccharides, produced from G19 maltodextrin, resulted in the best prebiotic effect with the highest prebiotic index (PI) of 5.90 at 48 hours. Acetate, propionate and butyrate were all produced from gluco-oligosaccharides, derived from G19 maltodextrin, at 48 hours but no lactate or formate were detected.
A number of Lactobacillus species, Bifidobacterium sp, Saccharomyces boulardii, and some other microbes have been proposed as and are used as probiotic strains, i.e. live microorganisms as food supplement in order to benefit health. The health claims range from rather vague as regulation of bowel activity and increasing of well-being to more specific, such as exerting antagonistic effect on the gastroenteric pathogens Clostridium difficile, Campylobacter jejuni, Helicobacter pylori and rotavirus, neutralising food mutagens produced in colon, shifting the immune response towards a Th2 response, and thereby alleviating allergic reactions, and lowering serum cholesterol (Tannock, 2002). Unfortunately, most publications are case reports, uncontrolled studies in humans, or reports of animal or in vitro studies. Whether or not the probiotic strains employed shall be of human origin is a matter of debate but this is not a matter of concern, as long as the strains can be shown to survive the transport in the human gastrointestinal (GI) tract and to colonise the human large intestine. This includes survival in the stressful environment of the stomach - acidic pH and bile - with induction of new genes encoding a number of stress proteins. Since the availability of antioxidants decreases rostrally in the GI tract production of antioxidants by colonic bacteria provides a beneficial effect in scavenging free radicals. LAB strains commonly produce antimicrobial substance(s) with activity against the homologous strain, but LAB strains also often produce microbicidal substances with effect against gastric and intestinal pathogens and other microbes, or compete for cell surface and mucin binding sites. This could be the mechanism behind reports that some probiotic strains inhibit or decrease translocation of bacteria from the gut to the liver. A protective effect against cancer development can be ascribed to binding of mutagens by intestinal bacteria, reduction of the enzymes beta-glucuronidase and beta-glucosidase, and deconjugation of bile acids, or merely by enhancing the immune system of the host. The latter has attracted considerable interest, and LAB have been tested in several clinical trials in allergic diseases. Characteristics ascribed to a probiotic strain are in general strain specific, and individual strains have to be tested for each property. Survival of strains during production, packing and storage of a viable cell mass has to be tested and declared.
Isoflavones are recognized to be estrogenic compounds that are often associated with a reduced risk of cancers. The estrogenic activity can be enhanced after metabolization to more active compounds such as genistein and daidzein by gut microorganisms. The direct use of these metabolites has been investigated in laboratory rats and farm animals over the last decade. This paper reviews the research progress on the effect of isoflavonic compounds including metabolites on the physiology, gut microbiology and performance of farm animals in China.
"Segregated early weaning" (SEW) of pigs reduces exposure to pathogenic bacteria, but upon arrival at grower facilities pigs may be co-mingled regardless of farm of origin. The present study was designed to examine the effect of mixing (social) stress on populations of Salmonella enterica Typhimurium in SEW pigs. Piglets (7 days old; n = 28 in each of 2 replicates) were separated into 2 treatments (control and mixed groups) of 2 pens per treatment (7 piglets/pen). One (n = 1) "seeder" pig/pen was inoculated with 10(9) CFU of S. Typhimurium. Each seeder was placed with non-inoculated "contact" piglets (n = 6). A"contact" piglet was swapped each day between the "mixed" pens for 5 days; pigs in control pens were not exchanged. On day 5, the incidence of fecal Salmonella shedding was higher in the mixed contact pigs (P < 0.05). Rectal Salmonella and cecal coliform populations in mixed pigs were significantly (P < 0.05) greater than in control pigs but cecal Salmonella populations were not different. Mixed pigs were more susceptible to tissue invasiveness (i.e., Salmonella-positive tonsils and lymph nodes) than control pigs. These results indicate that social stress of weaned pigs may increase susceptibility to and/or fecal shedding of Salmonella. Food-borne Salmonella infections in the United States are estimated to cost the economy dollar 2.4 billion annually (ERS/USDA, 2001). Approximately 6-9% of human salmonellosis is associated with the consumption of pork products (Frenzen et al., 1999). Salmonella is relatively common on swine farms and has been isolated from all stages of the pork production chain (Davies et al., 1999; Fedorka-Cray et al., 1997b; Rostagno et al., 2003). Salmonella is a threat to the pork industry not only from a food-safety perspective as a public health concern, but some Salmonella serotypes can cause clinical illnesses in swine, negatively impacting production efficiency and profitability (Schwartz, 1991).
The gastrointestinal tract (GIT) of adult mammals is colonized by a complex and dynamic community of microorganisms. Most protection against potential pathogens occurs via a mucosal immune system involving mechanisms of innate immunity as well as a secondary lymphoid organ, the gut-associated lymphoid tissue (GALT). However, the bacterial community also supports its host against invasion by potential pathogens, by a mechanism called 'colonization resistance'. Young animals need time to develop both a complex bacterial community and their immature GIT immune system, and until such developments have taken place, they are vulnerable to the presence of potential pathogens in their GIT. Initial protection against invading pathogens is provided by milk and colostrum, which contain antibodies and other bioactive components. At weaning, with the introduction of solid food and deprivation of the mother's milk, the young must also cope with a rapidly changing microbiota. The colonizing microbiota not only provides colonization resistance to potentially pathogenic bacteria. It also has a major role in the development of the intestinal immune system, both in terms of GALT development and mucosal immunity, and the induction of oral tolerance. Studies using gnotobiotic animal models have revealed that the presence of even limited numbers of the indigenous microbiota may influence the GIT immune system. Regulation of the composition of the GIT microbiota, e.g. by the use of pre- and probiotics, offers the possibility to influence the development of mucosal, and also systemic immunity.
In a young evolving science, there are always more questions than answers. That is also the situation in the emerging field of Probiotics, and this was made very clear at the International Probiotics Workshop in Amsterdam. In the report of this workshop, we present a selection of the most urgent questions in the field of probiotics. In addition, we propose a few strategies for the future of probiotics research. During the workshop, 120 experts--from disciplines including Human Nutrition, Gastroenterology, Nutritional Therapy, Cell Biology, Microbiology and Immunology--discussed new views on microbe-host interactions and the role of probiotics in prevention and alleviation of gastro-intestinal, atopic and auto-immune diseases. There is a general consensus among the experts that administering defined strains can help in preventing and curing gut flora related diseases: the first clinical trials show a promising role for probiotics. But the system is very complex, and most underlying mechanisms are still unclear. Rapid progress in this field will depend largely on the collaboration between fundamental researchers from different disciplines and medical specialists. Besides, more clinical studies are required to convince authorities and the public of the value of microbial therapies.
Genetically-engineered animals are known to be useful in clarifying the functions of many genes and as animal models for human diseases. However, it has been widely reported that pathophysiology is not expressed in these animals when they become germfree or SPF animals, i.e., the pathophysiology is not the result of genes alone and a combination of gene function and intestinal flora as an environmental factor are necessary. It is important to determine the roles of each of these two factors by pathophysiological analysis. Gnotobiotic mice were produced by establishment of specified bacterial species in germfree animals to form the intestinal flora of SPF animals and they were placed in barrier facilities. Measures have been taken against infections by bacteria such as Pseudomonas aeruginosa and Enterobacter cloacae. In addition, gnotobiotic mice with a highly normal physiology are required. Analysis of the effects of each bacterial species and combinations of bacteria on in vivo functions, i.e., the cross-talk between the host and intestinal flora, is essential in the creation of better laboratory animals. Monitoring of the intestinal flora, a key factor in the colonies produced, is a topic for future research.
The recent increased interest in probiotics among clinicians has many causes, primarily the concern about the limitations of the current armamentarium of pharmaceutical agents. Although probiotics have been used mostly in dietary supplements and foods to maintain health, scientific and clinical studies are recognizing the potential of some probiotics to be therapeutic in function. Scientific breakthroughs in understanding the source and composition of the human microbiota, the key nutritional factors that influence these microbes, and their immunomodulatory effects; the creation of disease-targeted recombinant strains; the isolation and characterization of signaling molecules that can modulate microbial biofilms and infectious processes; and advances in biomedical engineering that will provide new delivery systems for probiotics will shape the future of clinical applications of probiotics. In time and with rigorous documentation, some probiotics will likely find an important place in medical practice.
On August 29-31, 2004, 84 academic and industry scientists from 16 countries gathered in Copper Mountain, Colorado USA to discuss certain issues at the forefront of the science of probiotics and prebiotics. The format for this invitation only meeting included six featured lectures: engineering human vaginal lactobacilli to express HIV-inhibitory molecules (Peter Lee, Stanford University), programming the gut for health (Thaddeus Stappenbeck, Washington University School of Medicine), immune modulation by intestinal helminthes (Joel Weinstock, University of Iowa Hospitals and Clinics), hygiene as a cause of autoimmune disorders (G. A. Rook, University College London), prebiotics and bone health (Connie Weaver, Purdue University) and prebiotics and colorectal cancer risk (Ian Rowland, Northern Ireland Centre for Food and Health). In addition, all participants were included in one of eight discussion groups on the topics of engineered probiotics, host-commensal bacteria communication, 'omics' technologies, hygiene and immune regulation, biomarkers for healthy people, prebiotic and probiotic applications to companion animals, development of a probiotic dossier, and physiological relevance of prebiotic activity. Brief conclusions from these discussion groups are summarized in this paper.
Protozoan programmed cell death or apoptosis is an important factor in the survival of the parasite and its pathogenicity. The most amazing aspect of protozoan cell death is in its molecular architecture. To date, protozoa lack most of the components of the highly complex cell death machinery studied in multicellular organisms. Hence the unique apoptotic machinery in protozoa can be exploited for the development of therapeutic drugs and diagnostic markers. This review focuses on human intestinal protozoa undergoing cell death and inducing or inhibiting host cell apoptosis. The first part of this review focuses on intestinal protozoa that undergo PCD under various stress conditions. The second part focuses on protozoa that induce or inhibit PCD in their host cell. Although these intestinal parasites differ in their mechanism of infection and intracellular localization, they may activate conserved cell death pathways within themselves and in the host cell. Understanding conserved cell death pathways in the intestinal protozoa and their host-parasite PCD relationship may lead to drug targets which can be used for a broad range of parasitic diseases.
