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Antibiotic-resistant bifidobacteria have the ability to actively interact with mucosal tissue from the intestine of mice and rats in vitro and to adapt well in the intestinal tract of normal and germ-free rats, when introduced both with and without antibiotics; in some cases antibiotics enhance the ability of bifidobacteria to adapt in the intestine. The interaction between antibiotic-resistant bifidobacteria and mucosal tissue from the intestine remains unaffected by the prolonged action of 0.1M SDS and Tween-80 solutions, 0.1% trypsin and hyaluronidase. Antibiotic-resistant bifidobacteria introduced into the intestinal tract of rats in combination with gentamycin effectively inhibit the multiplication of exogenous enterobacteria infecting the digestive tract after the cessation of the antibiotic administration.

Volunteers were vaccinated subcutaneously and orally against typhoid fever; a determination was made of immunological reactions in the digestive tract secretions. Oral vaccination caused a much greater production of the secretory IgA than the subcutaneous one. Immunoglobulins of classes G and M were revealed in the intestinal contents only in a few volunteers. In studying the coprofiltrates obtained before the vaccination there was shown for the first time the presence of normal coproantibodies to typhoid fever, dysentery, tetanus, diphteria, botulism and gas gangrene; in the majority of the cases there was found a correlation between the presence of normal serum and secretory antibodies. Subcutaneous and oral vaccination caused an increase of coproantibodies specific for the vaccines used. Besides, the same vaccines stimulated the nonspecific response expressed in a rise of the level of other species of antibodies. Specificity of coproantibodies in the inhibitary passive hemagglutination reaction was demonstrated in the majority of the coprofiltrates examined.

Gnotobiological studies were conducted on germ-free guinea pigs. Association of the animals with the citrobacteria 5396/38 strain led to the reproduction of these bacteria in numbers exceeding the sum total number of all the representatives of the normal microbial flora in the conventional animals. Association lasting for a period of 46 days caused no antibody formation in the blood serum of these animals in difference to conventional guinea pigs immunized parenterally. Conventionalization of the monoflora guinea pigs served as the beginning of population of the intestinal tract by other microbial representatives which replaced the first culture. Biochemical properties of the citrobacteria strains isolated from the monoflora strains were identical to the properties of the initial culture. After the monoflora animals were let out of the isolation cage the citrobacteria strains displayed changes of some differential-diagnostic signs. Passage through the macroorganism of the citrobacteria stain proved to lead to the changes in the antigenic structure.

An unusual ninhydrin-positive compound has been isolated from feces of accidentally contaminated Sprague-Dawley autbred rats and identified by mass spectrometry and 1H-NMR spectroscopy techniques as 5-aminovaleric acid. The described procedure of isolation, purification and structure determination can be recommended as a general method of identification of unusual ninhydrin-positive compounds in complex mixtures of biological origin. The 5-aminovaleric acid was found to be produced by an anaerobic bacterium Clostridium bifermentans. It is shown that not all Clostridium spp. excrete his amino acid and that the majority of microorganisms, normally inhabiting intestine of rats, simians and man, do not possess this ability. On the basis of data obtained, the test for 5-aminovaleric acid is proposed to be included into the taxonomy of bacteria.

The ability of S. flexneri 2a virulent and avirulent (vaccine) strains No. 516 and No. 516 m to displace one another during long-term carrier state in germ-free rats has been studied. The long-term persistence of the vaccine strain in the intestine of the rats has been shown to produce colonization resistance to subsequent infection with the virulent culture of these bacteria. During carrier state in rats progressive S--R dissociation of the bacteria occurs, type II antigen is lost with simultaneous retention of group 3, 4 antigens and the resulting transformation of S. flexneri, serotype 2a, into variant y; the virulence of Shigella S-forms is also lost.

When cephalexin and erythromycin were administered intragastrically with intervals, adhesion of facultative pathogenic bacteria such as Klebsiella, Staphylococcus and E. coli to enterocytes of the small intestine in vitro was more pronounced than with their continuous administration with drinking water. The adhesive capacity of the staphylococcal strains resistant to the antibiotics was higher than that of the sensitive ones.

When lactobacilli are administered to gnotobiotic rats (Fischer strain), a gradual increase of lymphocytic, eosinophilic and mast cell infiltration of the intestinal connective tissue stroma, dilatation of vessels and their filling with lymphocytes is observed. Radioautographically 7 months later, migration and differentiation of enterocytes in the crypta--villus system of the iliac mucous tunic increases. In the mesenteric lymph nodes the postcapillary venules increase in number, lymphocytes migrate out of them, focal plasmocytic reaction with presence of some blastic forms takes place. Specific antibodies for lactobacilli in the blood serum either are absent or occur in low titers (1:2-1:8). Owing to the data obtained, a conclusion can be made on a weak immunogenecity of the lactobacilli. Their administration to the germ-free rats produces a certain cellular reaction, probably T-dependent, though any definite signs of hormonal response are absent. One--14 days after lactobacilli have been injected, in the rat intestine mucous tunic no microorganisms are revealed to fix to enterocytes. In cytoplasm of the absorbtive cell villi, the Golgi complex is subjected to hyperplasia and occupies an essential area over the nucleus. Within its cysterns, as well as in the mitochondrial and nuclear membrane areas, myelin-like bodies are revealed; evidently, they demonstrate certain changes in peroxidic oxidation of lipids..

The dynamics of the accumulation of phase II S. sonnei in the infectious process in germ-free rats, during cultivation and in subcultures was studied. The in vivo variability of S. sonnei showed a sharply defined phasic character of the process with the periods of the apparent absence of phase II, the increase of its occurrence and stabilization with the preservation of S-forms in minor amounts. Kanamycin-resistant phase II accumulated in vivo more rapidly than in vitro. Cultivation with sodium desoxycholate was found to accelerate the process of disassociation. The characteristic feature of all dissociants detected in all systems was their plasmid profile: in phase I, plasmids of 120 and 60 Md, as well as small plasmids, were detected; in phase II disassociants, plasmid with a molecular weight of 120 Md was absent. The restriction analysis of total plasmid DNA by means of restrictase EcoR1 showed the absence of 3 fragments in phase II while other 13 fragments were retained. These results open prospects for the molecular cloning of the gene antigen of phase II.

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.

Ultrastructure of the small intestinal mucosa of gnotobiotic rats, carriers of Vibrio eltor or V. Cholerae was studied. The cytoplasm of enterocytes manifested an increased number of lysosome-like structures and hypertrophy of the Golgi complex. Unlike V. eltor, V. cholerae induced more intensive extrusion of enterocytes and appreciable infiltration of the epithelial layer with lymphocytes. It is concluded that chronic vibrio-carrier state in gnotobiotic rats is not associated with pathological alterations.

In experiments on Fischer rats (F-344), both with common microflora and germ-free, the influence of the systemic destination of different antibiotics (tetracycline + ampicillin, gentamicin + kefzol, gentamicin, fradizine) on the intestinal microflora, the content of beta-ospartylglycine in feces and the colonization resistance of the intestinal tract to Staphylococcus aureus B-243 and Pseudomonas aeruginosa No. 93 has been studied. The early appearance of beta-aspartyl-glycine in the supernatant of fecal samples has been shown to be the first sign of dysbacteriosis and to indicate the decrease of the colonization resistance of the intestine to opportunistic bacteria.

Gnotobiological studies showed that under natural conditions normal microflora (microsymbiocenosis) is necessary for normal vital activity. In addition to autochthonous flora microsymbiocenosis includes an insignificant number of facultative pathogenic bacteria. In etiotropic antibiotic therapy it is important to preserve the autochthonous portion of the microsymbiocenonosis: bifidobacteria, lactobacilli, propionic bacteria and others. However, with respect to certain indications (diseases associated with suppression of resistance to infections) total decontamination with obligatory isolation of the patient under gnotobiological conditions is possible. In selective decontamination the facultative pathogenic portion of the intestinal microflora is the sole target of antibiotics. Selective decontamination is one of the methods for control of nosocomial infections, dysbacteriosis. It is indicated that establishment of departments in charge of investigation of intestinal microflora of patients and medical staff in hospitals is required for detecting risk groups which can be both the source of infection and the population most susceptible to hospital strains of antibiotic resistant facultative pathogenic bacteria.

The last two decades indicate that one of the main features of modern gnotobiology are comparative experiments on germfree and conventional counterparts to study the significance of normal (or dysbiotic) microflora (or individual species) in various physiological and pathological processes of the host organism. There are some immunological consequences of absence of normal microflora. Interactions between some indigenous and pathogenic bacteria in the intestine of gnotobiotic and conventional rats are discussed.

A total of 39 germ-free and 28 conventional germ-free Fisher rats were used to study cyclophosphamide-induced specific (hemagglutinins) and nonspecific (IgG2) immunosuppression. Most pronounced effect of the drug was observed in germ-free animals. 180 mg/kg of cyclophosphamide completely suppressed hemagglutinin formation for at least 8 weeks.

In the course of chronic carrier state of El-Tor vibrio cholerae gnotobiotic rats develop an immunomorphological reaction in the mucous membranes of the small and large intestines, Peter's patches, and mesenterial lymph nodes consisting in activation of reactive centers of follicles, hyperplasia of reticular cells, and an increase in the number of pyroninophilic plasma cells. The vibrio cholerae caused no pathological changes in enterocytes and other cells but its inoculation was accompanied by a "minor inflammation" in the intestinal wall and mesenterial lymph nodes. Eosinophilic infiltration in the intestinal tract, intensive mast cell reaction with degranulation in the small and large intestines and regional lymph nodes was observed.

The effect of long-term use of cephalexin and erythromycin (for 17 days) on large intestine microflora was studied on rats. It was found that intragastric administration of cephalexin in a dose of 800 mg/kg and erythromycin in a dose of 400 mg/kg was followed by changes in the quantitative and qualitative composition of the large intestine aerobic and anaerobic microflora. However, it did not induce production of beta-aspartyl glycine which is a biochemical indicator of deep changes in intestinal microflora. The long-term use of the above antibiotics resulted in increased levels and persistence of intestine colonization with facultative pathogenic enterobacteria and staphylococci resistant to the chemotherapeutic agents.

Histological and electron microscopic investigations of mucosal strucutre of the small intestine in gnotobiotic and conventional rats have demonstrated that mucosal index (lenear dimentions of villi/crypt dimentions ratio) is equal to 3--4 and 1.4--2.5, respectively. In gnotobiotic rats the number of mitotic figures in crypts is less, migratory-desquamative parameters of enterocytes are 5--6 days, cranio-caudal gradient of linear dimentions of villi, crypts and the number of goblet-shaped cells is not expressed. The goblet-shaped cells and cells with acidophilic granules have secretion of moderate intensity. The number of cells infiltrating epithelium of villi and crypts is greater when microbes are present in the gut lumen. A considerable difference in the structure of the connective tissue basis of the gnotobiotic and conventional rats mucosa are also connected with presence or absence of microflora in the gut lumen.

The dissociation variants of S. sonnei in phase I, isolated from germ-free rats and obtained in vitro, have been studied. Such dissociation variants have been found to form colonies with classical and atypical morphology. The electronmicroscopic study has revealed that different dissociation variants include small dense cells with the markedly thickened cell wall and pronounced microcapsule and spheroplasts with the damaged cell wall and less pronounced microcapsule. The formation of these cells is supposed to be the way of the adaptation of S. sonnei in the course of the infectious process and linked with changes in their virulence in the population cycle.

The enteral administration of antibiotics has been shown to induce the occult carriership (persistence) of sporulating Gram-positive bacilli inhabiting the intestine before the use of antibiotics and V. eltor introduced orally after the administration of antibiotics is stopped. After the prolonged use of some antibiotics the intestinal tract has been found to become an inadequate environment for the life of nonpathogenic E. coli easily colonizing the intestine of intact germ-free animals. Such form of resistance of colonization is an unfavorable consequence of antibiotic therapy.

Macro- and microscopic changes were studied in case of reproduction of the local Schwartzmann phenomenon in animals with various extent of bioisolation. It was revealed that the Schwartzmann phenomenon was positive in the usual animals and negative in the germ-free guinea pigs and in the animals with reduced enteric microbial flora given sterile diet. In the absence of microscopic changes in the germ-free animals there was revealed in their skin a neutrophilic-mononuclear infiltration of the derma, dilatation of the vessels, thrombosis of individual vessels. Apart from the same changes in guinea pigs with a reduced microbial flora, there were found focal extravasation and thrombosis of a somewhat greater number of vessels. There was no marked thromboses of small branches of the vessels, extensive hemorrhages or necroses which usually accompanied positive Schwartzmann phenomenon.

The development of allergy in germ-free rats in chronic vibriocarriership was studied. The formation of specific delayed type hypersensitivity was found to occur to a limited extent in the animals devoid of normal microflora. The picture of hyperergic inflammation was revealed in the lymph nodes of vibriocarrier rats, the test for specific allergen being positive.

Repeated immune response to diphtheria toxoid was investigated in monocontaminated rats. It was shown that repeated immunization of these animals led to a sufficiently intensive immune response by the secondary type, but it ended much earlier than in the usual animals (by the 12th day). A supposition is put forward that earlier termination of the secondary response in gnotobionts is caused by the absence in their lymph nodes of an additional differentiation of cell-precursors at the late periods of secondary immune response.

Eleven germfree and two monoassociated with the Citrobacter guinea pigs, and 25 conventional animals were injected with the heat-inactivated Citrobacter or E. coli for the induction of the local Schwartzman phenomenon. All the gnotobiotic pigs gave a positive reaction. Infiltration at the site of intracutaneous injection was found in all the conventionals, but in none of the gnotobiotics. The data are discussed from the aspect of primary and secondary sequelae of the absence of host microflora.

Many authors have demonstrated simplification of the species composition of the normal microflora in animals long kept in a closed environment. The purpose of the present work is to reveal the effect of such simplification (without reduction of the total number of bacteria) on the immunocompetent system of the animals. Three months old germ-free, monocontaminated (Staph. albus) and conventional guinea pigs with reduced fecal microflora were used in this study. Guinea pigs of the second group were fed a sterile diet but were kept in an ordinary vivarium. Two groups of control animals were: 1) conventionals fed regular diets; 2) conventionals fed the diet contaminated with fecal microflora of guinea pigs after sterilization. The fecal microflora was examined. The total number of bacteria in faeces of monocontaminated and microflora-reduced conventional animals was approximately the same as in conventional controls. Various lymphoid organs were examined. Their weight, size, number and activity of germinal centres, amount of globulin-producing cells, their differentiation and amount of globulins in single cells were determined. The lymphoid tissue in microflora-reduced animals and especially in monocontaminated guinea pigs was found to be underdeveloped. These changes were, however, less pronounced than in germ-free animals. It is suggested that simplification of the microflora in a macro-organism during long-term enclosure reduces the lymphoid tissue; such a macro-organism may develop severe reactions of the microbial shock, when occurring in a normal microbial environment.