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Genetic approaches to the study of oral microflora: a review.

As the study of oral microorganisms intensified almost 2 decades ago, the application of genetic techniques resulted in important contributions to the understanding of this clinically and ecologically important group of bacteria. The isolation and characterization of mutants of cariogenic streptococci helped to focus attention on traits that were important in colonization and virulence. Such classic genetic approaches gave way to molecular genetic techniques, including recombinant DNA methodology in the late 1970s. Gene cloning systems and methods to move DNA into cells have been developed for oral streptococci. Many streptococcal genes thought to be important in colonization and virulence have since been cloned and their nucleotide sequence determined. Mutant strains have been constructed using defective copies of cloned genes in order to create specific genetic lesions on the bacterial chromosome. By testing such mutants in animal models, a picture of the cellular and molecular basis of dental caries is beginning to emerge. These modern genetic methodologies also are being employed to develop novel and efficacious cell-free or whole cell vaccines against this infection. Genetic approaches and analyses are now being used to dissect microorganisms important in periodontal disease as well. Such systems should be able to exploit advances made in genetically manipulating related anaerobes, such as the intestinal Bacteroides. Gene cloning techniques in oral anaerobes, Actinomyces and Actinobacillus, are already beginning to pay dividends in helping understand gene structure and expression. Additional effort is needed to develop facile systems for genetic manipulation of these important groups of microorganisms.

Bacteria↗

Monoassociation with Lactobacillus acidophilus UFV-H2b20 stimulates the immune defense mechanisms of germfree mice.

Probiotics are formulations containing live microorganisms or microbial stimulants that have some beneficial influence on the maintenance of a balanced intestinal microbiota and on the resistance to infections. The search for probiotics to be used in prevention or treatment of enteric infections, as an alternative to antibiotic therapy, has gained significant impulse in the last few years. Several studies have demonstrated the beneficial effects of lactic acid bacteria in controlling infection by intestinal pathogens and in boosting the host's nonspecific immune response. Here, we studied the use of Lactobacillus acidophilus UFV-H2b20, a lactic acid bacterium isolated from a human newborn from Viçosa, Minas Gerais, Brazil, as a probiotic. A suspension containing 10(8) cells of Lactobacillus acidophilus UFV-H2b20 was inoculated into groups of at least five conventional and germfree Swiss mice to determine its capacity to stimulate the host mononuclear phagocytic activity. We demonstrate that this strain can survive the stressing conditions of the intestinal tract in vivo. Moreover, the monoassociation of germfree mice with this strain for seven days improved the host's macrophage phagocytic capacity, as demonstrated by the clearance of a Gram-negative bacterium inoculated intravenously. Monoassociated mice showed an undetectable number of circulating E. coli, while 0.1% of the original inoculum was still present in germfree animals. Mice treated with viable or heat-killed Lactobacillus acidophilus UFV-H2b20 presented similarly improved clearance capacity when compared with germfree controls. In addition, monoassociated mice had twice the amount of Kupffer cells, which are responsible for the clearance of circulating bacteria, compared to germfree controls. These results suggest that the L. acidophilus strain used here stimulates a nonspecific immune response and is a strong candidate to be used as a probiotic.

Animals↗

[Pharmacological properties in vitro of various extracts of Mimosa tenuiflora (tepescohuite)].

The in vitro biological effects of polar extracts of Mimosa tenuiflora (Willd.) Poir. trunk bark (tepescohuite) were studied. Ethyl acetate extract, with a high tannin content, inhibited the growth of different microorganisms. Alkaloids were particularly abundant in the butanol extract and this product strongly inhibited the intestinal peristalsis and produced contraction of uterine and gastric strips of rat and guinea pig. Saponins were detected in butanol and methanol extracts producing hemolysis. The screenings performed showed the diversity of bioactive compounds present in this plant product.

Alkaloids↗

Restricted IgA repertoire in both B-1 and B-2 cell-derived gut plasmablasts.

Mucosal IgA is the most abundantly produced Ig upon colonization of the intestinal tract with commensal organisms in the majority of mammals. The repertoire of these IgA molecules is still largely unknown; a large amount of the mucosal IgA cannot be shown to react with the inducing microorganisms. Analysis of the repertoire of used H chain Ig (V(H)) genes by H-CDR3 spectrotyping, cloning, and sequencing of V(H) genes from murine intestinal IgA-producing plasma cells reveals a very restricted usage of V(H) genes and multiple clonally related sequences. The restricted usage of V(H) genes is a very consistent observation, and is observed for IgA plasma cells derived from B-1 or conventional B-2 cells from different mouse strains. Clonal patterns from all analyzed V(H) gene sequences show mainly independently acquired somatic mutations in contrast to the clonal evolution patterns often observed as a consequence of affinity maturation in germinal center reactions in peripheral lymphoid organs and Peyer's patches. Our data suggest a model of clonal expansion in which many mucosal IgA-producing B cells develop in the absence of affinity maturation. The affinity of most produced IgA might not be the most critical factor for its possible function to control the commensal organisms, but simply the abundance of large amounts of IgA that can bind with relatively unselected affinity to redundant epitopes on such organisms.

Animals↗

Hansenula anomala killer toxin induces secretion and severe acute injury in the rat intestine.

BACKGROUND & AIMS: The yeast Hansenula anomala has been associated with gastrointestinal symptomatology and damage to the intestinal wall in humans. In vitro and in vivo, H. anomala secretes a toxin, killer toxin, which is lethal to other microorganisms. In view of the very high rate of killer phenotype expression recorded for H. anomala strains in nature, this study aimed to investigate the hypothesis that H. anomala killer toxin plays a role in the pathogenesis of H. anomala-induced enteritis. METHODS: Effects of active and heat-inactivated H. anomala killer toxin on intestinal fluid homeostasis and electrolyte balance were investigated in rat small intestine using a standard intestinal perfusion technique. Sections of the perfused jejunum tracts were examined histologically. RESULTS: H. anomala killer toxin induced a significant secretion of water and electrolytes. No significant change was observed when either heat-inactivated H. anomala killer toxin or control growth medium were tested. Histological analysis showed ischemic degeneration of villi and sloughing of surface epithelium in 50% of active H. anomala killer toxin-perfused jejuna. CONCLUSIONS: This paper presents original observations compatible with the hypothesis that H. anomala killer toxin plays a role in the pathogenesis of H. anomala-induced enteritis.

Animals↗

Shigella's ways of manipulating the host intestinal innate and adaptive immune system: a tool box for survival?

Shigella, a Gram-negative invasive enteropathogenic bacterium, causes the rupture, invasion and inflammatory destruction of the human colonic epithelium. This complex and aggressive process accounts for the symptoms of bacillary dysentery. The so-called invasive phenotype of Shigella is linked to expression of a type III secretory system (TTSS) injecting effector proteins into the epithelial cell membrane and cytoplasm, thereby inducing local but massive changes in the cell cytoskeleton that lead to bacterial internalization into non-phagocytic intestinal epithelial cells. The invasive phenotype also accounts for the potent pro-inflammatory capacity of the microorganism. Recent evidence indicates that a large part of the mucosal inflammation is initiated by intracellular sensing of bacterial peptidoglycan by cytosolic leucine-rich receptors of the NOD family, particularly NOD1, in epithelial cells. This causes activation of the nuclear factor kappa B and c-JunNH(2)-terminal-kinase pathways, with interleukin-8 appearing as a major chemokine mediating the inflammatory burst that is dominated by massive infiltration of the mucosa by polymorphonuclear leukocytes. Not unexpectedly, this inflammatory response, which is likely to be very harmful for the invading microbe, is regulated by the bacterium itself. A group of proteins encoded by Shigella, which are injected into target cells by the TTSS, has been recently recognized as a family of potent regulators of the innate immune response. These enzymes target key cellular functions that are essential in triggering the inflammatory response, and more generally defense responses of the intestinal mucosa. This review focuses on the mechanisms employed by Shigella to manipulate the host innate response in order to escape early bacterial killing, thus ensuring establishment of its infectious process. The escape strategies, the possible direct effect of Shigella on B and T lymphocytes, their impact on the development of adaptive immunity, and how they may help explain the limited protection induced by natural infection are discussed.

Animals↗

Identification by in situ hybridization of segmented filamentous bacteria in the intestine of diarrheic rainbow trout (Oncorhynchus mykiss).

Nonculturable segmented filamentous bacteria (SFB) have been described in the gut of rats, mice and chickens, and 16S rRNA sequences for these organisms are available. These organisms, peripherically related to Clostridium phylogenetic group I, have been provisionally named 'Candidatus Arthromitus'. This work reports the observation of similar bacteria in the intestinal content of the distal intestine, preferentially, in the adult rainbow trout (Oncorhynchus mykiss) that exhibited episodic acute diarrhea, usually during the summer. Abdominal distension, intestinal fluid-mucus content and epithelium detachment were observed in trout. The demonstration that the observed microorganisms are bacteria and belong in the 'Candidatus Arthromitus' group was achieved by in situ hybridization with, respectively, a eubacterial probe and an oligonucleotide probe designed to react specifically with SFB 16S rRNA (encoded by the rrs gene) sequences. The sequenced rrs gene was compared with published sequences and found to be closely related to (although distinct from) other SFB sequences. Implication of these bacteria in trout diarrheic illness remains hypothetical.

Animals↗

[Pathogenic intestinal bacteria: adhesion and invasion].

Enteric bacterial pathogens cause considerable morbidity and mortality in developing countries. However, also industrialised countries are burdened with these microorganisms, and there is an increasing public awareness of the problems associated with foodborne diarrhoeal diseases. The ability to interact with the gut epithelium is essential for these bacteria. During the last 10-15 years our insight into the mechanisms for bacterial adhesion and invasion has increased dramatically, and, based upon this, new strategies for prevention and treatment of diarrhoeal diseases have been suggested. This review summarises the present general knowledge about bacterial adhesion and invasion in the gut and gives details for bacteria, that are recognised enteric pathogens. Examples are given about possible future use of this knowledge in the prevention or treatment of diarrhoeal disease.

Bacterial Adhesion↗

Intestinal macrophages: unique effector cells of the innate immune system.

The gastrointestinal mucosa is the largest reservoir of macrophages in the body. These important effector cells are derived from blood monocytes that are recruited to the lamina propria by endogenous chemoattractants in the non-inflamed mucosa and by inflammatory chemokines and bacterial products during inflammation. In the non-inflamed mucosa, newly recruited pro-inflammatory monocytes are exposed to lamina propria stromal (extracellular matrix) factors that induce phenotypic and functional differentiation into non-inflammatory macrophages. As a consequence of this differentiation, resident lamina propria macrophages are strikingly downregulated for the expression of innate response receptors, such as the receptors for lipopolysaccharide, immunoglobulin G (IgG), and IgA, and the production of pro-inflammatory cytokines, including interleukin-1 (IL-1), IL-6, IL-8, and tumor necrosis factor-alpha. Despite downregulated pro-inflammatory function, strong phagocytic and bactericidal activities remain intact. Thus, in the non-inflamed intestinal mucosa, lamina propria macrophages are non-inflammatory but retain avid scavenger and host defense functions, a unique but ideal phenotype and functional profile for effector cells in close proximity to immunostimulatory microorganisms and products.

Animals↗

The beneficial effects of microflora, especially obligate anaerobes, and their products on the colonic environment in inflammatory bowel disease.

Because intestinal microflora play a pivotal role in the development of inflammatory bowel disease (IBD), there is currently some interest in alternating the composition of the microflora toward a potentially more remedial community. This paper summarizes the clinical and experimental efficacy of the manipulation of microflora by the use of antibiotics, probiotics, and prebiotics in IBD. Germinated barley foodstuff (GBF) is a prebiotic whose unique characteristics make it highly suitable for applications in IBD. It also helps prolong remission in remissive ulcerative colitis (UC) patients and also attenuates clinical activity in non-remissive UC patients. GBF has shown to be converted into a preferential nutrient, butyrate, for colonocytes through the action of Eubacterium and Bifidobacterium, and this bacterial butyrate can provide anti-inflammatory effects. The probiotic approaches for IBD include VSL#3, Nissle1917, Clostridium butyricum, and Bifidobacterium-fermented milk. In this paper, we summarize the distinctive role of another probiotic, Eubacterium limosum (E. limosum), which is a commensal microorganism that is promoted by GBF administration. The metabolites of E. limosum included butyrate, which can accelerate intestinal epithelial growth and inhibit IL-6 production. This new probiotic approach may be useful as an adjunctive IBD treatment in the future. Although these strategies hold great promise and appear to be useful in some settings, more experimental and clinical studies are needed to firmly establish their relevance.

Animals↗

Prevention of febrile neutropenia in cancer patients by probiotic strain Enterococcus faecium M-74. Pilot study phase I.

Febrile neutropenia (FN) remains a potentially life-threatening complication of anticancer chemotherapy. Bacterial translocation via intestinal mucosa is a significant mechanism of FN development. Competitive inhibition of bowel colonization by pathogenic microorganisms by lactic acid bacteria could be a useful prevention of FN. The aim of the study was the evaluation of dose and safety of probiotic strain Enterococcus faecium M-74 enriched with organic selenium in patients with solid and hematological malignancies. Eleven (9 M/2F) patients were included in the study. In the first phase six patients with germ cell tumors treated by chemotherapy were included. They received prophylaxis by nonpathogenic strain E. faecium M-74 during 2 cycles of chemotherapy. The planned daily dose was 6 x 10(9) bacteria. Regarding the insufficient colonization of the gut, the dose was further increased up to 18 x 10(9) tid. After safety evaluation, five patients were included with relapse of acute leukemia. In patients with germ cell cancer, severe neutropenia G3/4 was noted in 10 of 12 cycles of chemotherapy. The febrile episode was not observed in any of the patients. The gut colonization by enterococci reaches 10(6) CFU/g stool. In 5 patients with acute leukemia during 127 days of severe neutropenia 12 febrile episodes occurred. There was not noted any febrile episode or infection provoked by the tested strain. Tolerance of therapy was excellent without significant undesirable effects. Optimal dose was assessed and safety of probiotic strain was evaluated in neutropenic patients with solid, or hematological malignancies. Based on these results we plan phase II study to evaluate the effectiveness of this strain in FN prophylaxis.

Administration, Oral↗

Electron microscopic study of Tetratrichomonas didelphidis and its interaction with a prokaryotic cell.

Tetratrichomonas didelphidis is a flagellate protozoan found in the intestine of the opossum. The parasite lives in a hostile and stressed environment, where it interacts with microorganisms and can survive under extreme conditions for growth, involving strict anaerobiosis or equilibration with air and abundance or absence of nutrients. The in vitro cultivation of this protozoan depends upon Escherichia coli as a growth-promoting partner. In this study, we used scanning and transmission electron microscopy to observe the phagocytosis of bacteria by the protozoan, confirming the strong association between both cells and the growth dependence of T. didelphidis upon E. coli. After adherence to the protozoan surface, the bacteria induced the appearance of crater-like depressions and the ingested bacteria were intracellularly degraded.

Animals↗

[Role of normal microflora in the treatment of vibrio carrier state in rat gnotobionts].

Materials on the study of interactions between Vibrio cholera El Tor and some indigenous bacteria of human intestinal tract, which proved to be active antagonists in vitro, are presented in this work. Experiments were conducted on gnotobiotic rats (Fisher), using various combinations and variants of the order of the microorganism administration; there were revealed no antagonistic relations between Virbrio cholerae El Tor and Escherichia coli M-17, Lactobacillus fermenti, Lactobacillus plantarum. Vibrio cholera El Tor disappeared completely from the intestinal tract of the rats after its contact with the fecal microflora of healthy rats.

Animals↗

Introduction. Membrane transport of peptides.

Carrier-mediated membrane transport of small peptides is now realized to be a process of wide biological distribution, occurring not only in the small intestine and elsewhere in the animal body but also in bacteria, yeast, the mould Neurospora crassa, and probably in higher plants during the germination of seeds. The important features of peptide transport are outlined, and possible relationships between peptide transport and hydrolysis are discussed. Peptide transport is a stereochemically specific active process, and is independent of the transport of free amino acids. It is frequently, though not always, more rapid than the transport of amino acids. In the intestine, it is probably limited to dipeptides and tripeptides, but certain other animal cells and bacteria can take up larger peptides of seven or more amino acid residues. The ability to take up small peptides on a large scale is nutritionally important in some microorganisms, and might be of nutritional importance to the intact animal and to animal cells in culture. In the absorptive cells of the small intestine, and in Escherichia coli, peptide transport into the cells is followed by intracellular hydrolysis; transport and hydrolysis are quite distinct processes. Whether hydrolysis and amino acid transport can be coupled processes, or whether peptide transport and hydrolysis are different aspects of the same process, remains to be seen. This question is one of those where a close integration of studies of peptide transport with those of peptide hydrolysis should be particularly helpful.

Bacteria↗

[Enterotoxin production as a diagnostic test in intestinal dysbacterioses ].

The new approach to advancing diagnostic of dysbacterioses grounded on determination of the pathogenic factors of potential-pathogenic microflora is proposed. The method of reacting of an indirect hemagglutination with usage of erythrocital diagnosticums ostend availability of production of staphylococcal enterotoxines of types A and B in 40% of the strains of staphylococcuses, isolated at dysbacterioses. The intensity of production of staphylococcal enterotoxines for strains of microorganisms, isolated at dysbacterioses and carrying of S. aureus, and at food borne diseases, is various. The availability of clinical developments of an intestinal dysfunction in faeces of the patients contacts with persistence of enterotoxigenics S. aureus, and their elimination--with successful police of the disturbed microbiocenosis.

Bacterial Infections↗

[Clinical observation on acupuncture combined with microorganism pharmaceutical preparations for treatment of irritable bowel syndrome of constipation type].

OBJECTIVE: To explore the best program for treatment of irritable bowel syndrome (IBS) of constipation type. METHODS: Ninety-five cases of IBS were randomly divided into 3 groups. Group A (n = 30) were treated by acupuncture combined with microorganism pharmaceutical preparations, group B (n = 35) by oral administration of medicine for loosening the bowel to relieve constipation plus microorganism pharmaceutical preparations, and group C (n = 30) by simple acupuncture. RESULTS: The total effective rates were 90.0%, 77.2% and 66.7%, in the group A, B and C, respectively, with a very significant differences as the group A compared with those in the groups B, C (P < 0.01), and with no significant difference as the group B compared with that of the group C (P > 0. 05). The intestinal available bacteria, bilidobacteria and lactobacillus, increased and enteric bacilli decreased in varying degrees in the 3 groups. CONCLUSION: Acupuncture combined with microorganism pharmaceutical preparations has a better therapeutic effect on irritable bowel syndrome of constipation type.

Acupuncture Therapy↗