The gastrointestinal tract is a complex ecosystem that associates a resident microbiota and cells of various phenotypes lining the epithelial wall expressing complex metabolic activities. The resident microbiota in the digestive tract is a heterogeneous microbial ecosystem containing up to 1×1014 colony-forming units (CFUs) of bacteria. The intestinal microbiota plays an important role in normal gut function and maintaining host health. The host is protected from attack by potentially harmful microbial microorganisms by the physical and chemical barriers created by the gastrointestinal epithelium. The cells lining the gastrointestinal epithelium and the resident microbiota are two partners that properly and/or synergistically function to promote an efficient host system of defence. The gastrointestinal cells that make up the epithelium, provide a physical barrier that protects the host against the unwanted intrusion of microorganisms into the gastrointestinal microbiota, and against the penetration of harmful microorganisms which usurp the cellular molecules and signalling pathways of the host to become pathogenic. One of the basic physiological functions of the resident microbiota is that it functions as a microbial barrier against microbial pathogens. The mechanisms by which the species of the microbiota exert this barrier effect remain largely to be determined. There is increasing evidence that lactobacilli and bifidobacteria, which inhabit the gastrointestinal microbiota, develop antimicrobial activities that participate in the host's gastrointestinal system of defence. The objective of this review is to analyze the in vitro and in vivo experimental and clinical studies in which the antimicrobial activities of selected lactobacilli and bifidobacteria strains have been documented.

Antagonistic activities of lactobacilli and bifidobacteria against microbial pathogens

Bifidobacteria are Gram-positive prokaryotes that naturally colonize the human gastrointestinal tract (GIT) and vagina. Although not numerically dominant in the complex intestinal microflora, they are considered as key commensals that promote a healthy GIT. We determined the 2.26-Mb genome sequence of an infant-derived strain of Bifidobacterium longum, and identified 1,730 possible coding sequences organized in a 60%–GC circular chromosome. Bioinformatic analysis revealed several physiological traits that could partially explain the successful adaptation of this bacteria to the colon. An unexpectedly large number of the predicted proteins appeared to be specialized for catabolism of a variety of oligosaccharides, some possibly released by rare or novel glycosyl hydrolases acting on “nondigestible” plant polymers or host-derived glycoproteins and glycoconjugates. This ability to scavenge from a large variety of nutrients likely contributes to the competitiveness and persistence of bifidobacteria in the colon. Many genes for oligosaccharide metabolism were found in self-regulated modules that appear to have arisen in part from gene duplication or horizontal acquisition. Complete pathways for all amino acids, nucleotides, and some key vitamins were identified; however, routes for Asp and Cys were atypical. More importantly, genome analysis provided insights into the reciprocal interactions of bifidobacteria with their hosts. We identified polypeptides that showed homology to most major proteins needed for production of glycoprotein-binding fimbriae, structures that could possibly be important for adhesion and persistence in the GIT. We also found a eukaryotic-type serine protease inhibitor (serpin) possibly involved in the reported immunomodulatory activity of bifidobacteria.

https://www.pnas.org/content/pnas/99/22/14422.full.pdf

The genome sequence of Bifidobacterium longum reflects its adaptation to the human gastrointestinal tract

B. K OS, J. SUSKOVIC ´ ,S. V U K O V I C´ ,M. SIMPRAGA, J. F RECE A ND S. M ATOSIC ´ . 2003. Aims: To investigate aggregation and adhesiveness of Lactobacillus acidophilus M92 to porcine ileal epithelial cells in vitro, and the influence of cell surface proteins on autoaggregation and adhesiveness of this strain. Methods and Results: Lactobacillus acidophilus M92 exhibits a strong autoaggregating phenotype and manifests a high degree of hydrophobicity determined by microbial adhesion to xylene. Aggregation and hydrophobicity were abolished upon exposure of the cells to pronase and pepsin. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of cell surface proteins revealed the presence of potential surface layer (S-layer) proteins, approximated at 45 kDa, in L. acidophilus M92. The relationship between autoaggregation and adhesiveness to intestinal tissue was investigated by observing the adhesiveness of L. acidophilus M92 to porcine ileal epithelial cells. Removal of the S-layer proteins by extraction with 5 mol l )1 LiCl reduced autoaggregation and in vitro adhesion of this strain. Conclusions: These results demonstrate that there is relationship between autoaggregation and adhesiveness ability of L. acidophilus M92, mediated by proteinaceous components on the cell surface. Significance and Impact of the Study: This investigation has shown that L. acidophilus M92 has the ability to establish in the human gastrointestinal tract, which is an important determinant in the choice of probiotic strains.

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Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92

The ability of probiotic bacteria to adhere to the intestinal epithelium play an important role in colonization of the gastrointestinal tract, preventing their elimination by peristalsis and providing a competitive advantage in this ecosystem. To identify bacterial traits related to adhesion the probiotic strain L. acidophilus M92 was examined for autoaggregation ability and cell surface hydrophobicity L. acidophilus M92 exhibits a strong autoaggregation phenotype and also coaggregation with some pathogen microorganisms that may form a barrier that prevents their colonization. The examined probiotic strain manifests a good degree of hydrophobicity determined by microbial adhesion to hydrocarbons. Aggregation and hydrophobicity were abolished upon exposure of the cells to pronase, which suggests the possible role of cell surface layer (S-layer) proteins, approximated at 45 kDa, in a L. acidophilus M92. The relationship between autoaggregation and adhesion ability to intestinal tissue was investigated by observing the adhesivity of L. acidophilus M92 to porcine ileal epithelial cells. Removal of the S-layer proteins by extraction with 5 M LiCl reduced autaggregation and in vitro adhesion of this strain.

To identify bacterial traits related to adhesion ability in human bifidobacteria, 13 strains of Bifidobacterium longum isolated from human gastric juice and intestine were studied. Strains were tested for their capability to adhere to Caco-2 cells and classified as adhesive (Adh+) or non-adhesive (Adh-). Adh+ and Adh- strains were then investigated for their autoaggregation ability and surface hydrophobicity. Comparing the properties of Adh+ and Adh-, we observed that strains were able to adhere to cell monolayers if they autoaggregate and manifest a good degree of hydrophobicity as determined by microbial adhesion to hydrocarbons. These two traits could be used for preliminary screening to identify potentially adherent isolates.

Adhesion, autoaggregation and hydrophobicity of 13 strains of Bifidobacterium longum

On the basis of autoaggregation ability, two different phenotypes (Agg+ and Agg-) were selected from a strain (BSu895) of Bifidobacterium suis. The relationship between autoaggregation and adhesion of bacteria to intestinal tissue was investigated by observing the adhesivity of the two phenotypic variants to ileum and colon tissue pieces collected from six new-born piglets. The results suggest that there is a good relationship between autoaggregation and adhesion as variant Agg+ (autoaggregating) has a stronger adhesion ability than Agg- (non-autoaggregating).

Autoaggregation and adhesion ability in a Bifidobacterium suis strain

Previous assays with weak sinusoidal magnetic fields (SMF) have shown that bacteria that had been exposed to a 50 Hz magnetic field (0.1-1 mT) gave colonies with significantly lower transposition activity as compared to sham-exposed bacteria. These experiments have now been extended by using a pulsed-square wave magnetic field (PMF) and, unexpectedly, it was found that bacteria exposed to PMF showed a higher transposition activity compared to the controls. The increase of the transposition activity was positively correlated with the intensity of the magnetic fields (linear dose-effect relation). This phenomenon was not affected by any bacterial cell proliferation, since no significant difference was observed in number and size of PMF-exposed and sham-exposed colonies. In addition, the cell viability of E. coli was significantly higher than that of the controls when exposed to SMF, and lower than that of the controls when exposed to PMF. Under our experimental conditions it was shown that exposure to PMF stimulates the transposition activity and reduces cell viability of bacteria, whereas exposure to SMF reduces the transposition mobility and enhances cell viability. These results suggest that the biological effects of magnetic fields may critically depend on the physical characteristics of the magnetic signal, in particular the wave shape.

Various effects on transposition activity and survival of Escherichia coli cells due to different ELF-MF signals

The effect of various antineoplastic drugs (1-β-d-arabinofuranosylcytosine, 5-azacytidine, cisplatin, dactinomycin, epirubicin, vincristine, and the activated metabolite of cyclophosphamide, mafosfamide) on cell differentiation in vitro was investigated using a human alveolar rhabdomyosarcoma cell clone, RMZ-RC2. These cells are able to differentiate spontaneously from small mononuclear proliferating elements to terminal, extremely elongated multinuclear structures resembling myotubes; morphological differentiation is accompanied by the expression of myosins, in particular the embryonic isoform, which was used in this study as a specific marker of myogenic differentiation. The proportion of differentiated myosin-positive cells, which was around 10–15% in control cultures 10–15 days after seeding, was increased by some drug treatments up to 30–40%; the proportion of multinuclear elements was also increased. 1-β-d-arabinofuranosylcytosine and 5-azacytidine were the most effective drugs, while dactinomycin had no effect; other molecules ranked in between. Since significant increments were usually observed after treatment with drug doses inhibiting cell growth, the kinetic behavior of the absolute number of myosin-positive cells or nuclei was analyzed to assess whether some effects could be due to a negative selection of proliferating, undifferentiated cells. This appeared to be the case for vincristine and epirubicin, while 1-β-d-arabinofuranosylcytosine, 5-azacytidine, and, to a lesser degree, mafosfamide and cisplatin actually seemed to increase differentiation ability.

https://cancerres.aacrjournals.org/content/canres/49/13/3631.full.pdf

Myogenic differentiation of human rhabdomyosarcoma cells induced in vitro by antineoplastic drugs.

The aim of this study was to verify whether extremely low frequency (ELF) magnetic fields (MF) could affect transposition activity like some environmental stress factors such as heat shock or UV irradiation. Using an Escherichia coli Lac Z(-) strain transformed with a plasmid containing a Tn 10 derivative element expressing beta-galactosidase only after transposition, it was possible to determine the events of transposition evaluating the rate at which the colonies developed dark coloured papillae (Lac Z(+)). We found that those bacteria that had been exposed for a long time (58 h) to a 50 Hz low intensity MF (0.1-1 mT) gave colonies with significantly lower transposition activity compared to sham-exposed bacteria. Such reduction in transposition activity was positively correlated to the intensity of the MF, in a dose-effect manner. This phenomenon was not affected by bacterial cell proliferation, since no significant differences were observed in number, diameter and perimeter between sham-exposed and MF-exposed colonies.

B. Del Re

Papers

Adhesion, autoaggregation and hydrophobicity of 13 strains of Bifidobacterium longum

Autoaggregation and adhesion ability in a Bifidobacterium suis strain

Various effects on transposition activity and survival of Escherichia coli cells due to different ELF-MF signals

Myogenic differentiation of human rhabdomyosarcoma cells induced in vitro by antineoplastic drugs.

Extremely low frequency magnetic fields affect transposition activity in Escherichia coli