Microbiocenosis of parietal mucin in gastrointestinal tract of rats with induced disbiosis

Comparative study of qualitative and quantitative characteristics of parietal mucin microbiocenosis in rats with alimentary induced disbiosis was performed. It has been shown that disbiosis was associated with changes in parietal microflora which were observed mainly in colon mucosa. Disbiosis in parietal area, in contrast to central area manifested by changes in quantitative characteristics of microbiocenosis. Results of the study allowed to suppose that parietal mucosal microbiocenosis was more stable than gut one and that this stability was determined by endogenous homeostatic factors of the host.     

Microbiocenosis of parietal mucin in the gastrointestinal tract of rats

The qualitative and quantitative composition of the microbial community in parietal mucin at different areas of the gastrointestinal tract (GIT) of rats was revealed. The pronounced variability in the quantitative and qualitative characteristics of microbiocenosis in parietal mucin of rats at different sections was revealed. The differences were most pronounced in the passage from upper to lower GIT sections, the large intestine found to be the richest biocenosis. The microbial composition of rat feces was faintly associated with the GIT parietal microbiocenosis. The individual areas of GIT mucosa were unique of their microbial characteristics and organization. This makes it possible to regard them as relatively independent biotopes and indicates that it is impossible to evaluate the microbial community by one of the colonic mucosal sifes.     

Study of the parietal microflora in the rat intestine

The article deals with the adaptation of the existing method of the isolation of microorganisms from parietal mucin of the intestine of experimental animals (rats) with a view to ensure the methodological uniformity of investigations. The effectiveness of the method of sparing disintegration of parietal mucin taken from the mucous membrane of the gastrointestinal tract, specially developed for human biomaterial, and its adequacy for microbiological investigations of the digestive organs of rats have been confirmed. A certain similarity between the microbiocenosis of the parietal mucin in the intestine of humans and rats has been established.     

Comparative study of parietal and lumen microflor in the large intestine in experiments on mice

The methodological aspects of the study of the microflora of the parietal mucous layer (parietal mucin) of the large intestine is discussed; the study is difficult because the mucin layer is thin closely associated with epithelium and the impossibility of the mechanical separation of this layer from the epithelium without damaging the latter is possible. The method of homogenization fails to determine of the composition of parietal mucin proper. Such possibility becomes real after the dissolution of mucin and obtaining the suspension of microbial cells. As experimentally shown in vitro, urea solution, reducing disulfide bonds, effectively depolymerized mucin and exhibited no antibacterial and cytolytic activity. With the use of urea treatment, microflora of parietal mucin of the large intestine was studied on 44 non-inbred mice. This newly developed method was shown to have higher resolution in comparison with the traditional one (homogenization). Some qualitative and quantitative characteristics of the microflora of parietal mucin were established. In the same group of mice the study of fecal microflora was made and compared with microflora of parietal mucin.     

The autologous intestinal microflora in the postresuscitation period after acute blood loss in rats

In this work the data obtained in the study of intestinal microbiocenosis in rats on the third day of the postresuscitation period after acute blood loss are presented. The quantitative and qualitative shifts of microflora at different biotopes, such as the wall of the small intestine, the parietal microflora of Peyer's patches, the contents and wall of the large intestine, have been characterized. Some specific features of dysbiotic changes have been revealed in comparison with the shifts of intestinal microflora in cases of dysbacteriosis caused by other reasons. The translocation of different microorganisms, including Bacterium bifidum and lactobacilli, into mesenteric lymph nodes, the liver, the spleen and the blood has been observed.     

Variation of mucin distribution in the rat intestine, caecum and colon: effect of the bacterial flora.

The influence of the intestinal microflora on mucin types was studied in the small intestine, caecum and colon of conventional (CV) rats as compared to germ-free (GF) rats. A colorimetric method was used on purified water-soluble mucin extracted from mucosal scrapings and contents. Variations occurred between the three anatomical sites both in the mucosas and intestinal contents of GF rats. In CV rats, the presence of the bacterial flora led to different effects depending on the intestinal site: in the small intestinal mucosa, neutral and sulphomucins values were higher whereas sialomucin was much lower. Conversely, sialomucin was higher in the caecal and colonic mucosas and contents whereas sulphated mucins were decreased significantly in caecal contents and caecal and colonic mucosas. These variations in the contents may reflect the bacterial mucolytic activity and the effect of bacterial metabolites on the mucosa.     

Parital microflora of human intestine

The modified method of the microbiological study of parietal mucin has been developed. The proposed method makes it possible to evaluate the parietal microflora of the intestine. The advantages of using physiological saline and Hanks' solution as medium ensuring the storage and self-thinning of mucin have been proved. The optimum time of making the microbiological study of intestinal mucin has also been determined.     

New experimental models in microbial ecology

Peculiar features of dysbiosis development in persons under extreme conditions were studied. It was shown that a number of extreme factors participated in formation of dysbiotic disorders in intestinal microflora. Of paramount importance was the neuro-emotional stress. Lability of bifido- and lactoflora was considered as the starting mechanism in dysbacteriosis under the extreme conditions. In the experimental models with rats SPF and Primates during flights of biosatellites of the Kosmos series the role of indigenous++ microflora in maintaining the microecological homeostasis, as well as the need for development of artificial and controlled intestinal microflora promising in prophylaxis of dysbacteriosis under extreme conditions was shown. The theoretical and experimentally grounded necessity of maintaining constant intestine microbiocenosis was confirmed by the practice of using the system of measures for recovery, stabilization and optimization of microflora in persons under extreme conditions.     

Influence of enterococci on the recovery of intestinal microflora changed by antibiotic therapy and carbohydrate nutrition

The process of intestinal microflora normalization after a course of antibiotic therapy was studied on mice and in persons with using S. faecium UDS-86. It was shown that oral inoculation of strain UDS-86 influenced correction of the intestinal microflora in the mice and persons after the antibiotic therapy and carbohydrate nutrition. Oral inoculation of S. faecium UDS-86 resulted in lower quantities of potentially pathogenic organisms and higher levels of lactobacteria in the intestine at the background of dysbacteriosis induced by the antibiotic therapy and carbohydrate nutrition. Possible development of a preparation, eubiotic based on S. faecium UDS-86 is discussed.     

Fate and effect of ingested Bacillus cereus spores and vegetative cells in the intestinal tract of human-flora-associated rats

The fate and effect of Bacillus cereus F4433/73R in the intestine of human-flora-associated rats was studied using bacteriological culturing techniques and PCR-denaturing gradient gel electrophoresis in combination with cell assays and immunoassays for detection of enterotoxins. In faecal samples from animals receiving vegetative cells, only few B. cereus cells were detected. Spores survived the gastric barrier well, and were in some cases detected up to 2 weeks after ingestion. Selective growing revealed no major changes in the intestinal flora during passage of B. cereus. However, denaturing gradient gel electrophoresis analysis with universal 16S rRNA gene primers revealed significant changes in the intestinal microbiota of animals dosed with spores. Vero cell assays and a commercial kit (BCET-RPLA) did not reveal any enterotoxin production from B. cereus F4433/73R in the intestinal tract.     

Screening of plasmids in the bacteria of the genus Bacillus with antilysozyme activity

In the study of the microbiocenosis of the distal section of the patients' large intestine Bacillus strains with antilysozyme activity (ALA) were isolated. In B. cereus strain 26 with pronounced expression of antilysozyme factor the plasmid sized approximately 100 kb was detected. The transformation of the isolated plasmid in cells of B. cereus non-plasmid strain IP5832 the localization of genes encoding ALA and resistance to kanamycin was determined. The production of ALA factor in the recombinant clone of B. cereus strain IP5832 corresponded to the clinical isolate of B. cereus 26. The replicon of the detected plasmid could be used for the determination of the coding sequence of the antilysozyme sign of bacilli. Genetic determinants of antilysozyme factors and kanamycin resistance may be used for the construction of vector systems of cloning in bacilli.     

Dysbacteriosis in patients with colon polyposis

The study revealed the most profound changes in the composition of intestinal microflora in patients with polyposis of the large intestine. In these patients anaerobic microflora (bifidobacteria, lactic acid bacteria) was more often suppressed than in other examined groups, in particular, patients with cholelithic disease. The associations of hemolytic Escherichia with Klebsiella pneumoniae, Pseudomonas aeruginosa, Citrobacter freundii were often observed as well as the increased content of enterococci and fungi of the genus Candida. The determination of frequency and degree of manifestations showed that dysbacteriosis was registered in the absolute majority of patients (97.4%) with polyposis of the large intestine. Among patients with cholelithic disease disturbances in microbiocenosis were detected in 60.0% of cases, the profundity and character of the microflora composition changes being less pronounced.     

Formation of intestinal microflora in children during the first year of their life

In 525 young children the state of intestinal microbiocenosis was studied every month of the first year their life. The study revealed that the process of the microflora formation lasted throughout the first year of their life and was characterized by dysbiotic disturbances. During this period the aggravation of dysbiotic changes in the intestine of these children on months 3, 6-7 and 11-12 was of particular importance. The formation of stable dysbacteriosis led to a decrease in the immunological status of the child, which was manifested by the increased content of such microorganisms as hemolytic cocci, Proteus and a decrease in the quantitative level of bifidobacteria in the total intestinal microbiocenosis by the end of the first year of child's life.     

Microecological aspects of small intestinal bacterial overgrowth syndrome

Small intestinal bacterial overgrowth syndrome (SIBOS) means chronic recurrent diarrhea with malabsorption, intoxication and increased risk of endogenous infection. This syndrome are accompanied by increase of overall bacterial burden in biotope >10(5) CFU/ml in adults and >10(4) CFU/ml in children, emergence of different species of enterobacteria, bacteroides, clostridia and fusobacteria et al. in small intestine. Such characteristics of the syndrome allow to consider it as syndrome of disturbances of intestinal microflora (dysbacteriosis). Microecological changes are accompanied by B12 vitamin deficiency anemia, hypovitaminosis, protein deficiency, translocation of bacteria and their toxins from intestine in blood, emergence of endotoxinemia and possible generalization of infection. SIBOS is diagnosed by concentration of hydrogen in expiratory flow (lactulosa load test) or by bacteriological study of aspirate from proximal part of small intestine. Complex treatment includes containing lacto- and bifidobacteria probiotics and, in more severe cases, antimicrobial agents (vancomycine, metronidazole, aminoglycosides, amoxicillin clavulanate, tetracycline, and cephalosporines of 2nd generation) with following correction of disturbed microbiocenosis by different probiotics.     

In vitro evaluation of influence of medicinal plant decoctions and antibacterial antibodies to bifidobacteria on bifidobacterium adhesion

Natural antibodies to the surface antigens of bifidobacteria contained in blood sera and determined in the passive hemagglutination test blocked the adhesion of bifidobacteria to human red blood cells in vitro. The decoctions of medicinal plants, often used by pediatricians for therapeutic purposes, were found to have a pronounced inhibiting effect on the adhesion of bifidobacteria after the preliminary treatment of both microorganisms and human red blood cells by these decoctions. Suggestion was made on the possible role of natural human antibodies in changing the contents of the indigenous intestinal microflora; the prolonged use of preparations obtained from medicinal plants could probably produce negative influence on the microbiocenosis of the intestine.     

The frequency of staphylococcal colonization of the intestines in children with the manifestations of dysbacteriosis

In 2100 children of different age groups the microbiocenosis of the large intestine was studied. The study revealed that the colonization of the mucous membrane of the large intestine with staphylococci developed in 30% of children with intestinal dysbacteriosis. Young children were mainly affected (91%). The prevailing species among isolated staphylococci was S. aureus (86%), capable of persistence in the intestine (30.9%). In children non typing S. aureus strains mainly circulated (70%), and among phage-typing strains isolates of phage group III prevailed (70.2%). The colonization of the intestine with coagulase-negative staphylococci was possible (14%). Microecological intestinal disturbances in children of different age groups were characterized by different degrees of changes in normal microflora with the prevalence of opportunistic microorganisms in the microbial picture.     

Secretion of endogenous lectin by chicken intestinal goblet cells

The two lactose-binding lectins found in adult chicken intestine, chicken-lactose-lectin-1 (CLL-1) and chicken-lactose-lectin-11 (CLL- 11), were localized within the vesicles of the mucin-secreting goblet cells by indirect immunofluorescence and immunoperoxidase staining methods. Attention was concentrated on CLL-11 which is 200 time more abundant than CLL-1 in adult intestine. The localization of CLL-11 in secretory vesicles, combined with its demonstration on the intestinal epithelial surface by immune staining methods and by specific elution with lactose, suggested that at least a portion of the CLL-11 in the vesicles was secreted by the goblet cells and then became associated with the mucosal surface. In support of this, treatment of isolated intestinal strips with a cholinergic agent, bethanechol (10(-7 M) produced a small but significant increase in the amount of CLL-11 that could be eluted from their surface with lactose. Secretion of lectin may occur in conjunction with mucin because both are localized in the secretory vesicles and CLL-1 and CLL-11 apparently bind to purified chicken intestinal mucin, which is a potent inhibitor of their hemagglutination activities. The mucin is six orders of magnitude more potent than lactose as a hemagglutination inhibitor of CLL-1 or CLL-11 on a molar basis, and three orders of magnitude more potent when expressed per mole of hexose. These results suggest that CLL-11, and perhaps CLL-1, are secreted from the goblet cells along with mucin. They may function in the organization of mucin for secretion and/or in its association with the intestinal mucosal surface.     

The rheology of pig small intestinal and colonic mucus: weakening of gel structure by non-mucin components.

Mechanical spectroscopy has been used to study the structure and properties of pig small intestinal and colonic adherent mucus gel. Both mucus secretions had properties of viscoelastic gels, but that from the small intestine was substantially weaker in quality. Small intestinal mucus gel was disrupted by acid (pH 1), detergents (bile) and protein denaturants while that from the colon remained stable following these treatments. Concentration of purified colonic mucin produced a gel with the same rheological properties as the native secretion. Purified small intestinal mucin when concentrated produced a stronger gel than the native secretion and, in contrast to the latter, one which was not disrupted by acid or denaturants. The instability of native small intestinal mucus was shown not to be a function of the mucin components (which alone could account for the gel-forming properties), but to arise from the presence of insoluble material largely from sloughed mucosal cells. These studies show (1) that mucus gels from the colon and small intestine have similar mechanical behaviour and properties to those from the stomach and duodenum, and (2) emphasise the caution that should be exercised when interpreting the rheological properties of mucus preparations, particularly with respect to their content of mucosal cellular material.     

A method for assessing the microflora status of the large intestine using a computer

The qualitative and quantitative composition of the microflora of the large intestine has been studied in 31 healthy adults and in 137 patients with acute viral hepatitides A and B. A set of quantitative tests ensuring the complete characterization of the microbiocenosis under study has been proposed. The results obtained in this investigation have been processed by means of a computer with the use of the principles of numeric taxonomy, thus making it possible to obtain the objective criterion of the state of the microflora of the large intestine, expressed by the similarity index.     

Mucin degradation in the intestine

Rat intestinal mucin was labelled biologically by intraperitoneal injection of radioactive amino acids and monosaccharides 3–6 h prior to killing, followed by isolation and purification of the mucin from mucosal scrapings. The labelled product was then introduced into intestinal segments of rats under ether anesthesia for periods up to 3 h, removed by washing and assessed for evidence of degradation. In segments containing the pancreatic ducts the total mucin precipitable by cetyltrimethylammonium bromide fell from 80% to 5% in 3 h. At 3 h, chromotography on Sephadex G-100 and Sepharose 4B revealed multiple products, including very small molecular weight fragments deficient in carbohydrate label. With the introduction of neomycin sulfate into the segments to reduce bacterial growth, only two products were found, one corresponding in size to the original mucin and one somewhat smaller, although still in excess of 200 000 daltons. These products occurred independently of the presence of the pancreatic ducts in the segments, and in chronically pancreatectomized rats. The smaller product could not be produced by incubation with trypsin or elastase. Both products were altered antigenically as compared with the original mucin. Both products also retained the same ratio of carbohydrate and protein label as the original. It is concluded that mucins undergo early degradative changes in the intestine which do not involve deglycosylation but which involve partial loss of antigenicity and a fall in molecular weight. The pancreas is not responsible for these changes.