The influence of polyunsaturated fatty acids on probiotic growth and adhesion

The establishment of the intestinal microflora, and probiotic bacteria, may control the inflammatory conditions in the gut. As polyunsaturated fatty acids (PUFA) possess antimicrobial activities, they may deter the action of probiotics. We assessed whether free linoleic, gamma-linolenic, arachidonic, alpha-linolenic and docosahexaenoic acids at physiological concentrations in the growth media would influence the growth and adhesion of Lactobacillus GG (probiotic), Lactobacillus casei Shirota (probiotic) and Lactobacillus bulgaricus (dairy strain). Higher concentrations of PUFA (10-40 microg PUFA ml(-1)) inhibited growth and mucus adhesion of all tested bacterial strains, whilst growth and mucus adhesion of L. casei Shirota was promoted by low concentrations of gamma-linolenic acid and arachidonic acid (at 5 microg ml(-1)), respectively. PUFA also altered bacterial adhesion sites on Caco-2 cells. Caco-2 cells grown in the presence of arachidonic acid were less adhered to by all three bacterial strains. Yet, L. casei Shirota adhered better on Caco-2 cells grown in the presence of alpha-linolenic acid. As the adhesion to mucosal surfaces is pivotal in health promoting effects by probiotics, our results indicate that the action of probiotics in the gut may be modulated by dietary PUFA.     

细菌对肉鸡肠粘液的粘附作用

研究两歧双歧杆菌、嗜酸乳杆菌、禽大肠杆菌O78、大肠杆菌 ATCC 25922、鸡白痢沙门氏菌和鼠伤寒沙门氏菌与肉鸡不同部位肠粘液糖蛋白的粘附性能,探讨两歧双歧杆菌和嗜酸乳杆菌对所试病原菌的抗粘附作用。结果表明：在不同的肠道部位,两歧双歧杆菌、嗜酸乳杆菌、鸡白痢沙门氏菌和鼠伤寒沙门氏菌与肠粘液糖蛋白均有不同的粘附作用,而禽大肠杆菌O78、大肠杆菌 ATCC 25922在各肠段粘液上的粘附性能则相近;在相同的肠道部位,所试益生菌的粘附能力大于病原菌;两歧双歧杆菌和嗜酸乳杆菌对所试病原菌的粘附有不同的阻断作用,同时二者有时还存在互补抗粘附作用。     

Wheat or rye supplemented diets do not affect faecal mucus concentration or the adhesion of probiotic micro-organisms to faecal mucus

In a crossover study, volunteers consumed a diet supplemented with either wheat or rye bread. Levels of faecal mucus were determined and the adhesion of five selected probiotics to immobilized intestinal mucus, was assessed. No significant difference in the concentration of faecal mucus or adhesion of the probiotics to the mucus was observed during the two dietary periods. However, male subjects were observed to excrete significantly more mucus during the rye bread supplemented diet. From these observations it was concluded that these two diets, with a low or high fibre content, did not change the potential binding sites for the tested probiotics or affect the level of mucus excreted in the faeces, however, on a high fibre diet men excrete more faecal mucus.     

Diversity of probiotic adhesion genes in the gastrointestinal tract of goats

Gastrointestinal (GI) microflora is an important system in the host, as it has both pathogenic and probiotic bacteria. Most of the studies were focused on the human gut microflora and the available information on the intestinal microflora of goats was limited. This urged the need to inspect the impacts of the goat's gut microflora. Metagenomic investigation of probiotic bacteria in the GI tract of goat is one of the challenging streams because of the less available data of the uncultivable bacteria. In our report, comparative analysis of metagenomic and enrichment samples of goat intestinal content was done and this approach will be helpful in analyzing the identification of uncultivable and cultivable probiotic bacteria. This study mainly focused on three key probiotic adhesion genes, such as EF-Tu, mapA, and mub. The GI of four different goats were investigated for these genes. The data from this study showed that there is a wide diversity of these genes among goat intestinal samples.     

Correlation between in vitro mucus adhesion and the in vivo colonization ability of lactic acid bacteria: screening of new candidate carp probiotics

We measured the adhesion of candidate probiotic lactic acid bacteria (LAB) to carp intestinal mucus. The percentage of adherent bacteria varied among strains. Four strains, two with high adhesion and two with low adhesion in vitro, were tested for in vivo colonization ability. Carp were fed LAB-containing feed for 12 d, and then unsupplemented feed until day 33, and the numbers and compositions of intestinal LAB were analyzed during the entire period. LAB with lower in vitro adhesion disappeared quickly from the intestine after LAB feeding stopped. LAB with higher in vitro adhesion remained in the intestine 3 weeks after LAB feeding stopped, indicating a strong correlation between mucus adhesion in vitro and colonization ability in vivo. Next we isolated nine candidate probiotic LAB with high in vitro mucus-binding ability. Three of them were fed to carp, and all three were stably maintained in the intestine.     

Comparison of faecal Lactobacillus populations in experimental animals from different breeding facilities and possible consequences for probiotic studies

AIMS: The effect of probiotic lactobacilli is likely dependent on the indigenous Lactobacillus strains in the intestinal tract. Since a substantial number of probiotic studies is performed in rodents, we compared the Lactobacillus strains of different rat and mouse populations in three animal facilities. METHODS AND RESULTS: SDS-PAGE and 16S rDNA analysis of cultured faecal lactobacilli revealed that different Lactobacillus strains were detected in genetically similar Wistar rats bred at different locations. Further, within the same animal facility host genetics did not affect the types of the predominant lactobacilli strains. CONCLUSIONS: Our results show that the environmental background of laboratory animals rather than host genetics determines the indigenous Lactobacillus strains that are found. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings underline the importance of microflora analysis in probiotic studies.     

Measurement of bacterial adhesion-in vitro evaluation of different methods

The adhesion of bacteria to host tissue is the first step in pathogenesis. Similarly, bacterial adhesion to inanimate surfaces is the first step in formation of biofilms-a real problem in industrial processes and medical devices. Various agents capable of blocking the adhesion of bacteria to surfaces have been identified, such as probiotics, which are supposed to prevent the adhesion of pathogenic bacteria to the intestinal mucosa. Although measurement of bacterial adhesion is important itself, especially when agents used to prevent adhesion are developed, a relative small number of techniques can be used in the measurement of adhesion. These techniques are not well validated and there is lack of studies where those methods are compared to each other. Here we have compared different commonly used methods to measure adhesion of bacteria; radioactive labelling, fluorescence tagging, and staining of bacteria. The methods were used to measure the adhesion of Escherichia coli and Salmonella enterica serovar Typhimurium to intestinal mucus. Moreover, selected probiotic strains were used to study whether probiotics or the adhesion method used affected the results. As a result, we show that the best reproducibility and sensitivity were obtained using radioactive labelling. With other methods, the sensitivity was too low due to poorly adhering bacteria and low signal-to-background ratio.     

Influence of carp intestinal mucus molecular size and glycosylation on bacterial adhesion

The first step of the pathogenesis of many infectious diseases is the colonisation of the mucosal surface by the pathogen. Bacterial colonisation of the mucosal surface is promoted by adherence to high molecular weight mucus glycoproteins. We examined the effect of carp intestinal mucus glycoproteins on the adhesion of different bacteria. The bacteria used were 3 strains of Aeromonas hydrophila, and A. salmonicida, Edwardsiella tarda and Yersinia ruckeri. All bacteria adhered to mucus, but at varying intensities. All tested bacteria adhered best to molecules of 670 to 2000 kDa in size, less to molecules larger than 2000 kDa and weakest to molecules of 30 to 670 kDa. In general, bacteria that showed a stronger adhesion to intestinal mucus were cytotoxic to cells in vitro, and bacteria that showed a weaker adhesion to intestinal mucus did not lead to alterations of monolayers of EPC-cells. Furthermore, the involvement of glycan side chains of the glycoproteins for bacterial adhesion was analysed for one A. hydrophila strain. After cleavage of terminal sugar residues by treatment of mucus glycoproteins with different glycosidases, binding of bacteria was modulated. When mannose was cleaved off, adhesion significantly increased. Blocking of glycan receptors by incubation of bacteria with different oligosaccharides had no clear effect on bacterial binding to mucus glycoproteins. Our results suggest that bacteria interact with carbohydrate side chains of mucus glycoproteins, and that the carbohydrates of the core region are involved in bacterial binding.     

Some factors affecting the adherence of probiotic Propionibacterium acidipropionici CRL 1198 to intestinal epithelial cells

Adhesion to the intestinal mucosa is generally considered an important property of probiotic microorganisms and has been related to many of their health benefits. This study investigated some factors that could affect or be involved in the adherence of Propionibacterium acidipropionici CRL 1198, a dairy strain with suggested probiotic effects and high adherence in vitro and in vivo to intestinal epithelial cells. In vitro adhesion of propionibacteria was decreased by gastric digestion but not affected by bile and pancreatic enzymes. Adherence was also decreased by pretreatment of bacterial cells with protease, sodium metaperiodate, and trichloroacetic acid, revealing that different features of the cell surface, like protein factors, carbohydrates, and teichoic acids, are involved in the process. Adherence to intestinal epithelial cells was enhanced by calcium and was dependent on other divalent cations. Adhesion to intestinal mucus was also demonstrated. The results should explain the metabolic effects in the host previously obtained with this strain and support the potential of Propionibacterium for development of new probiotics.     

Adhesion of probiotic lactobacilli to chick intestinal mucus

In the present work, interactions between three Lactobacillus strains (Lactobacillus fermentum CRL1015, Lactobacillus animalis CRL1014, and Lactobacillus fermentum CRL1016) and chicken small intestinal mucus were determined. Three lactobacilli isolated from chicken and selected by their potentially probiotic properties were able to grow in mucus preparations. Three peaks from gel filtration chromatography of intestinal mucus were obtained. The adhesion to three mucus fractions (I, II, and III), especially fraction III, was higher (P < 0.01) in L. fermentum CRL1015 than L. animalis CRL1014. Pretreatment of this fraction with proteases and metaperiodate showed lower (P < 0.01) adhesion values than that of the control, suggesting that a glycoprotein from the mucus acts as a receptor for L. fermentum CRL1015. Highest adhesion values were obtained at pH 7 and 42 degrees C, and neither the removal of divalent cations with ethylenediaminetetraacetic acid (EDTA) nor the addition of calcium produced significant variation from the adhesion values of the control (P > 0.01). This adhesion was only inhibited by N-acetyl-glucosamine. Salmonella pullorum and Salmonella gallinarum showed high (P < 0.01) values of adhesion to chick intestinal mucus. The results obtained from assays of the inhibition of adherence of Salmonella spp. to mucus, immobilized in polystyrene tissue culture wells, indicated that the pathogen adhesion was not reduced by lactobacilli (P > 0.05) or their spent culture supernatants (P > 0.05), suggesting that these strains did not interfere with the binding sites for Salmonella spp. adhesion to the small intestinal mucus.     

Role of commercial probiotic strains against human pathogen adhesion to intestinal mucus

AIMS: The aims of this study present were to assess and to evaluate in vitro the abilities of commercial probiotic strains derived from fermented milk products and related sources currently marketed in European countries, to inhibit, compete and displace the adhesion of selected potential pathogens to immobilized human mucus. METHODS AND RESULTS: The adhesion was assessed by measuring the radioactivity of bacteria adhered to the human mucus. We tested 12 probiotic strains against eight selected pathogens. All strains tested were able to adhere to mucus. All probiotic strains tested were able to inhibit and displace (P<0.05) the adhesion of Bacteroides, Clostridium, Staphylococcus and Enterobacter. In addition, the abilities to inhibit and to displace adhered pathogens depended on both the probiotic and the pathogen strains tested suggesting that several complementary mechanisms are implied in the processes. CONCLUSIONS: Our results indicate the need for a case-by-case assessment in order to select strains with the ability to inhibit or displace a specific pathogen. Probiotics could be useful to correct deviations observed in intestinal microbiota associated with specific diseases and also, to prevent pathogen infections. SIGNIFICANCE AND IMPACT OF THE STUDY: The competitive exclusion properties of probiotics as well as their ability to displace and inhibit pathogens are the most importance for therapeutic manipulation of the enteric microbiota. The application of such strategies could contribute to expand the beneficial properties on human health against pathogen infection.     

Regulation of immune response at intestinal and peripheral sites by probiotics

The gut associated lymphoid tissue (GALT) should protect intestinal mucosa against pathogens, but also avoid hypersensitivity reactions to food proteins, normal bacterial flora and other environmental macromolecules. The interaction between epithelial cells and microflora is fundamental to establish gut mucosal barrier and GALT development. The normal colonization of intestine by commensal bacteria is thus crucial for a correct development of mucosal immune system. Probiotic bacteria are normal inhabitants of microflora and may confer health benefits to the host. The modification of the intestinal microflora towards a healthier probiotics enriched microflora may generate beneficial mucosal immunomodulatory effects and may represent a new strategy to cure intestinal and allergic diseases. The health benefits may be specific for different probiotic strains. Ongoing research is providing new insights into the probiotic beneficial effects and related mechanisms. This review represents an update of immunomodulatory activity of different probiotics and of the more accredited mechanisms underlying such activities. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.     

Probiotic Strains and Their Combination Inhibit <Emphasis Type="Italic">In Vitro</Emphasis> Adhesion of Pathogens to Pig Intestinal Mucosa

The aim of this study was to investigate in vitro the protective effect of commercial probiotic strains (Bifidobacterium lactis Bb12 and Lactobacillus rhamnosus LGG) alone and in combination on the adhesion of pathogenic strains as Salmonella, Clostridium, and Escherichia coli to pig intestinal mucus obtained from different intestinal regions. In combination, probiotic strains enhanced each other’s adhesion, mainly in large intestinal mucus. Treatment of intestinal mucus with Bb12 and LGG, alone or in combination, significantly reduced (P < 0.05) the adhesion of the tested pathogens. The ability to inhibit pathogen adhesion appears to depend on the specific probiotics and pathogens and on the mucosal site. B. lactis Bb12 and L. rhamnosus LGG in combination revealed a better ability to inhibit adhesion of all pathogens tested to pig intestinal mucus than probiotic strains. Probiotic combinations could be useful for counteracting disease-associated aberrations in intestinal microbiota. Specific protective probiotics could be selected for particular pig pathogens. Probiotic strains from human origin and intended for human use also adhere to pig intestinal mucus and are able to displace and inhibit pathogens.     

Evaluation of the cytoprotective effects of bovine lactoferrin against intestinal toxins using cellular model systems

This study investigated the effects of bovine lactoferrin (BLf) on the growth of different groups of bacteria in vitro. BLf showed a significant inhibitory effect on the growth of selected pathogens but not probiotics. BLf, in combination with probiotics, has the potential to influence the composition of the gut microflora via inhibition of intestinal pathogens with no significant effect on probiotic bacteria.     

The effect of probiotic bacteria on the adhesion of pathogens to human intestinal mucus

Human intestinal glycoproteins extracted from faeces were used as a model for intestinal mucus to investigate adhesion of pathogenic Escherichia coli and Salmonella strains, and the effect of probiotics on this adhesion. S-fimbriated E. coli expressed relatively high adhesion in the mucus model, but the other tested pathogens adhered less effectively. Probiotic strains Lactobacillus GG and L. rhamnosus LC-705 as well as a L. rhamnosus isolated from human faeces were able to slightly reduce S-fimbria-mediated adhesion. Adhesion of S. typhimurium was significantly inhibited by probiotic L. johnsonii LJ1 and L. casei Shirota. Lactobacillus GG and L. rhamnosus (human isolate) increased the adhesion of S. typhimurium suggesting that the pathogen interacts with the probiotic.     

Characterization of the properties of human- and dairy-derived probiotics for prevention of infectious diseases in fish

The present study aimed to investigate the potential probiotic properties of six lactic acid bacteria (LAB) intended for human use, Lactobacillus rhamnosus ATCC 53103, Lactobacillus casei Shirota, Lactobacillus bulgaricus, L. rhamnosus LC 705, Bifidobacterium lactis Bb12, and Lactobacillus johnsonii La1, and one for animal use, Enterococcus faecium Tehobak, for use as a fish probiotic. The strains for human use were specifically chosen since they are known to be safe for human use, which is of major importance because the fish are meant for human consumption. The selection was carried out by five different methods: mucosal adhesion, mucosal penetration, inhibition of pathogen growth and adhesion, and resistance to fish bile. The adhesion abilities of the seven LAB and three fish pathogens, Vibrio anguillarum, Aeromonas salmonicida, and Flavobacterium psychrophilum, were determined to mucus from five different sites on the surface or in the gut of rainbow trout. Five of the tested LAB strains showed considerable adhesion to different fish mucus types (14 to 26% of the added bacteria). Despite their adhesive character, the LAB strains were not able to inhibit the mucus binding of A. salmonicida. Coculture experiments showed significant inhibition of growth of A. salmonicida, which was mediated by competition for nutrients rather than secretion of inhibitory substances by the probiotic bacteria as measured in spent culture liquid. All LAB except L. casei Shirota showed tolerance against fish bile. L. rhamnosus ATCC 53103 and L. bulgaricus were found to penetrate fish mucus better than other probiotic bacteria. Based on bile resistance, mucus adhesion, mucus penetration, and suppression of fish pathogen growth, L. rhamnosus ATCC 53103 and L. bulgaricus can be considered for future in vivo challenge studies in fish as a novel and safe treatment in aquaculture.     

Clinical uses of probiotics for stabilizing the gut mucosal barrier: successful strains and future challenges

Probiotic bacteria are used to treat disturbed intestinal microflora and increased gut permeability which are characteristic to many intestinal disorders. Examples include children with acute rotavirus diarrhoea, subjects with food allergy, subjects with colonic disorders and patients undergoing pelvic radiotherapy and sometimes changes associated with colon cancer development. In all such disease states altered intestinal microflora, impaired gut barrier and different types of intestinal inflammation are present. Successful probiotic bacteria are able to survive gastric conditions and colonize the intestine, at least temporarily, by adhering to the intestinal epithelium. Such probiotic microorganisms appear to be promising candidates for the treatment of clinical conditions with abnormal gut microflora and altered gut mucosal barrier functions. They are also promising ingredients to future functional foods and clinical foods for specific disease states provided that basic requirements for strains and clinical studies are carefully followed.     

Veterinary probiotic preparation influence on the biochemical blood indices of young farm animals

The veterinary probiotic preparation was developed on the basis of aerobic part of bacterial strains of normal human intestinal microflora: 3 strains of Escherichia coli--G35No1-413, G35No2-412, G35No3-411, and one Enterococcus faecalis G35No4-410 strain. The influence of a pilot lot of the prepapration on the biochemical blood indices and the state of intestinal microflora of different age groups of young farm animal (calves and piglets) was studied during the process of the commission tests. It has been shown, that the developed probiotic promotes the improvement of protein and mineral metabolism, normalization of level of enzyme activities, which show state of the native immunity of the young farm animals' organism. It was the result of normalization of the intestinal microflora composition. The optimal doses and periods of intake have been determined. On the basis of executed researches the veterinary probiotic preparation has been presented for registration to the State Department of Veterinary Medicine of Ukraine.     

Effect of Lactobacillus isolates on the adhesion of pathogens to chicken intestinal mucus in vitro

AIMS: The aims of this study were to investigate in vitro the effects of Lactobacillus isolates from a chicken on adhesion of pathogenic Salmonella and Escherichia coli to chicken intestinal mucus obtained from different intestinal regions. METHODS AND RESULTS: Bacteria were labelled by using methyl-1,2-[(3)H]-thymidine. The bacterial adhesion was assessed by measuring the radioactivity of bacteria adhered to the mucus. The results showed that the abilities of Lactobacillus spp. to bind to the same intestinal mucus were higher than those of pathogenic Salmonella and E. coli. Pretreatment of intestinal mucus with Lactobacillus fermentum and Lactobacillus acidophilus, alone or in combination, reduced the adhesion of the tested pathogens, but the reductive extent of pathogenic adhesion by Lactobacillus spp. in combination was relatively high. CONCLUSIONS: The tested bacteria had different adhesions to mucus glycoproteins isolated from different intestinal regions of chicken. Lactobacillus acidophilus and Lact. fermentum in combination revealed a better ability to inhibit attachments of Salmonella and E. coli to chicken intestinal mucus than Lactobacillus sp. alone. SIGNIFICANCE AND IMPACT OF THE STUDY: A mixture of intestinal Lactobacillus spp. from a chicken may play a protective role in excluding pathogenic Salmonella and E. coli from the intestine of chicken.     

Human ileostomy glycoproteins as a model for small intestinal mucus to investigate adhesion of probiotics

Human ileostomy glycoproteins were used as a model for small intestinal mucus to investigate the adhesion of 12 Lactobacillus strains, one Lactococcus strain and one Propionibacterium strain, Both probiotic and dairy strains were tested. Adhesive and non-adhesive Escherichia coli strains were used as controls. All the strains were also tested for their adhesion to polystyrene. Adhesion to ileostomy glycoproteins and to polystyrene varied significantly among the strains tested. Lactobacillus rhamnosus (human isolate), Lactobacillus GG, Lact. acidophilus 1 and P. freudenreichii adhered to ileostomy glycoproteins. Adhesion was concentration-dependent and the most adhesive strains were able to saturate the substratum. These results indicate that human ileostomy glycoproteins can be used as a model system to select potential probiotic strains to complement the adhesion test with intestinal cell lines.