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Study bacterial biofilms in native material (renal calculus) by electron microscopy method and developmeit of biofilm model by isolates in vitro on sterile calculi of various chemical composition.Bacterial spectra of microflora of renal calculus lavages were studied, isolated pure cultures were identified up to species. Comparisons of urine microflora obtained before operation in patients with urolithiasis with microflora of removed renal calculi were carried out.Urease activity and genes coding pathogenicity factors were detected, and the ability to form biofilms by isolates was studied. Model of formation of biofilms in vitro on sterile renal calculi was developed and candidate agents reducing the biofilm forming ability were tested.Uropathogenic microorganisms infecting renal calculi and forming biofilms on them not only support chronic infection by increased resistance to therapy but also facilitate novel lithogenesis.

By means of plasposon mutagenesis, mutants of Burkholderia cenocepacia 370 with the change in production of N-acyl-homoserine lactones (AHL), signal molecules of the Quorum Sensing system of regulation, were obtained. To localize plasposon insertions in mutant strains, fragments of chromosomal DNA containing plasposons were cloned, adjacent DNA regions sequenced, and a search for homologous nucleotide sequences in the GeneBank was initiated. It has been shown that the insertion of plasposon into gene lon encoding lon proteinase drastically decreases AHL synthesis. Upon insertion of plasposon into gene pps encoding phosphoenolpyruvate-synthase, enhancement of AHL production is observed. In mutant carrying inactivated gene lon, a strong decline of extracellular protease activity, hemolytic, and chitinolytic activities was observed in comparison with the original strain; lipase activity was not changed in this mutant. Mutation in gene pps did not affect these properties of B. cenocepacia 370. Mutations in genes lon and pps reduced the virulence of bacteria upon infection of mice.

The role of biofilms in the environment and in the hosts of pathogenic bacteria has recently attracted much attention of researchers. Microorganisms form biofilms at any biotic and abiotic surfaces and thereby cause serious problems in medical practice and other areas of human activity. Biofilms have been shown to be pathogenetic factors responsible for chronization of infectious process. The data are presented illustrating ubiquitous nature of biofilms, their structural and functional characteristics, and modern methods for the study of microbial communities. The discussion is focused on the role of biofilms in chronization of infectious process, enhanced resistance of biofilm organisms to antibiotics and its underlying mechanisms. Approaches to the search for new means for biofilm control during chronic infections are considered.

Advances in microscopic analysis and molecular genetics research methods promoted the acquisition of evidence that natural bacteria populations exist predominately as substrate attached biofilms. Bacteria in biofilms are able to exchange signals and display coordinated activity that is inherent to multicellular organisms. Formation of biofilm communities turned out to be one of the main survival strategies of bacteria in their ecological niche. Bacteria in attached condition in biofilm are protected from the environmental damaging factors and effects of antibacterial substances in the environment and host organism during infection. According to contemporary conception, biofilm is a continuous layer of bacterial cells that are attached to a surface and each other, and contained in a biopolymer matrix. Such bacterial communities may be composed of bacteria of one or several species, and composed of actively functioning cells as well as latent and uncultured forms. Particular attention has recently been paid to the role of biofilms in the environment and host organism. Microorganisms form biofilm on any biotic and abiotic surfaces which creates serious problems in medicine and various areas of economic activity. Currently, it is established that biofilms are one of the pathogenetic factors of chronic inflection process formation. The review presents data on ubiquity of bacteria existence as biofilms, contemporary methods of microbial community analysis, structural-functional features of bacterial biofilms. Particular attention is paid to the role of biofilm in chronic infection process formation, heightened resistance to antibiotics of bacteria in biofilms and possible mechanisms of resistance. Screening approaches for agents against biofilms in chronic infections are discussed.

Mice of I/St strain develop severe lung inflammation and die shortly following infection with virulent mycobacteria. The susceptibility does not depend on the Nramp1 gene, as I/St mice carry its resistant allele, but is controlled by little interacting QTL mapped to chromosomes 3, 9, 17. To find out whether the tuberculosis-susceptible I/St mice are susceptible to other intracellular bacteria taxonomically distant pathogen of Chlamydia pneumoniae was studied. Comparison of I/St and TB-resistant A/Sn mice (both Nramp1r) demonstrated that the former were more susceptible to chlamydia, displaying a significantly shortened survival time following challenge (I/St, 9.2 +/- 1.2 days; A/Sn, 22.0 +/- 0 days (p0.001)). To estimate the degree of chlamydial multiplication in the lungs, we suggested a quantitative real-time polymerase chain reaction (PCR)-based method which allows enumeration of the parasite's genome equivalents in infected tissue from 1 to 16 days after challenge. The interstrain difference of chlamydia burden in lungs was observed only after 24 hours after infection. Multiplication of chlamydia in the lungs was controlled efficiently after day 4 of infection. The numbers of genome equivalents dropped slightly by day 8 both in I/St and A/Sn mice. Lung pathology develops more rapidly in I/St compared to A/Sn mice following infection with chlamydia despite their similar ability to control bacterial multiplication. Lung tissue of susceptible I/St mice was markedly infiltrated with macrophages (p0.01), which differed significantly from the lungs of resistant A/Sn mice. In agreement with higher macrophage content in the lungs, significantly more macrophage-derived proinflammatory cytokines TNF-? and IL-6 were detected in lung tissue homogenates obtained from I/St mice (p0.05). Because the prominent difference in survival time did not correlate with permanent difference in bacterial multiplication, we suggested that both infections trigger fatal pathological processes whose dynamics depends strongly upon the host genetics.

By the methods of light microscopy and immunocytochemistry studies of interaction between S. typhimurium and corpuscular biliary components was investigated in experimental model "bile-bacteria" It was shown that the results of this interaction was bacterial-biliary sludge formation. Bacterial extracellular mucopolysaccharides matrix and flagella's play crucial role in mechanism of sludge formation.

To assess efficacy of using the method of quantitative detection of Legionella in objects of the environment by real-time polymerase chain reaction (RT-PCR).For the development of the assay, genus-specific primers from gene coding 16S rRNAas well as species-specific primers for detection of Legionella pneumophila on the basis of mip gene sequence. For quantitative detection of L. pneumophila calibration samples of pGEM plasmid containing fragment of the mip gene in known concentration were used. Samples of water and biofilms obtained from cooling stacks of production plants, systems of autonomic water supply, humidification blocks of centralized systems of air conditioning were studied.Correlation of results obtained with RT-PCR and bacteriologic methods was shown during monitoring of potentially dangerous water objects as well as during epidemic outbreak of Legionella infection. Importance of samples preparation stage, during which considerable losses of DNA and inhibition of reaction could occur, is underlined. Disinfection measures on the studied objects significantly influenced on the results of the RT-PCR and can lead to false positive results.Obtained results confirm usefulness of testing of potentially dangerous water objects on the presence of Legionella based on the preliminary screening with RT-PCR for the 24 hours followed by bacteriologic testing of samples for 8 - 12 days.

The goal of this work was to elucidate the mechanism of direct interaction of bacterial cells with tumor necrosis factor (TNF-alpha; cytokine). It was shown earlier that this interaction facilitated activation of bacterial growth and recultivation of non-cultivated forms in vitro and in vivo. It was shown in experiments with mice deficient in the genes encoding eucaryotic TNF-alpha receptors and infected with salmonella that addition of exogenous TNF-alpha to suspension of infection cells caused a one-day acceleration in the infection start (bacteria planting from spleen) in both knockouted and control mice relative to the same animals infected with the same bacteria without cytokine. Thus, bacteria are able to interact with cytokine even in the absence of eucaryotic receptors. Specificity of the bacterium-cytokine interaction and bacterial protein EF-Tu mediating direct interaction of bacteria with cytokine were identified using the method of immobilization of recombinant protein TNF-alpha-spacer-CSD on cellulose.

Ability of Legionella species to form biofilms in association with other microorganisms is the key factor of their spreading in potentially dangerous water systems. Ability of different strains of Legionella to form monospecies biofilms as well as biofilms in association with Pseudomonas aeruginosa in constant conditions was analyzed. It was shown that ability of Legionella strains to form monospecies biofilms correlates with their ability to persist in biofilms formed by P. aeruginosa.

Ability of biofilm formation was studied in 28 strains belonging to 12 species of Legionella. Optimal conditions for formation of biofilms were ascertained using reference strain Legionella pneumophila Philadelphia 1. Comparative assessment of the ability of Legionella spp. to form biofilms was performed by cultivation in proteosopepton broth (for 96 hours) and in water (for up to 2 weeks). Highest rates of biofilm formation were observed for strains of L. pneumophila and L. longbeachae. Between L. pneumophila strains the most prominent ability to form biofilms was observed in newly isolated strains BLR-05 and TOTAL 1. Opportunity to use different ability of Legionella species to biofilm formation as a epidemiologically significant marker and for modeling of biofilms of Legionella in association with other microorganisms was discussed.

Using the methods of transmission electron microscopy, the structure of the biofilms formed by the bacterium Burkholderia cepacia (clinical isolate and mutants with an increased and decreased ability to produce biofilms) were investigated. The biofilms were obtained on a liquid nutrient medium or on an abiotic surface (polystyrene). It has been demonstrated that the cultures of the studied strains differ in some morphological and functional characteristics. In biofilms, changes in the size and submicroscopic organization of all the components of bacterial cells occur. Staining biofilms with ruthenium red revealed the presence of exopolysaccharides in the intercellular space. The differences in the ultrastructure of bacterial films formed on nutrient medium and abiotic surfaces were demonstrated.

This paper is a brief review of data on bacterial biofilms that occur inside and outside of host organisms. Such biofilms are of great ecological and clinical importance. The role of interspecies communications in the development of bacterial biofilms and infectious diseases is particularly emphasized. Considerable attention is given to the electron microscopic study of biofilms formed by Salmonella typhimurium cells incubated as a broth culture in microtubes without aeration. Bacterial samples taken from the biofilm and planktonic culture grown in the same microtube were comparatively investigated by transmission electron microscopy.

Data concerning mechanisms of strengthening virulence of bacteria B.cepacia and P.aeruginosa during experimental mixed-infection are submitted. Results of in vitro studies of acylhomoserine lactones (AHL) with various length of a carbon lateral circuit have shown that butanoyl-homoser-ine lactone (C4), hexanoyl-homoserine lactone (C6) and octanoyl-homoserine lactone (C8) separately and in various combinations display different effects on growth of bacteria, formation of colonies and production of pathogenicity factors. Experiments on animals have shown strengthening virulence of bacteria B.cepacia by C4, C6 and C8 lactones when introduced simultaneously. Strengthening of virulence of the P.aeruginosa strain under the action of lactone C6 and simultaneous action of C4, C6 and C8 lactones was observed. The data obtained give grounds for assumption, that bacteria may use exogenous lactones, including those produced by closely related bacteria, during their life cycle.

A review of the literature about the bacterial systems of regulation of expression of the genes controlled by signals generated by the bacteria themselves is given. Three types of presently known Quorum sensing systems contributing to the expression of the pathogenicity factors, infection process, and formation of biofilms by pathogenic bacteria are described. Possible mechanisms of enhanced resistance of bacteria in films to antibacterial preparations and factors of immune protection of human body are discussed. The perspectives of the development of new approaches to treatment of chronic diseases caused by the pathogens producing bacterial films are considered.

The ability of 14 different strains of Salmonella typhimurium to biofilm formation depending on genotype and culture conditions was investigated in artificial systems: in 96-well plastic microtitre plates, plastic and glass tubes, plastic Petri dishes and on microscope glasses. Quantitative biofilm growth was monitored by using an assay based on crystal violet staining, while planctonic growth in the same cultures was monitored by absorbance in iEMS Reader MF, and qualitatively--by digital photo and visually. Optimal rate between growth and biofilm indications for all strains was determined at initial cell concentration 10(6-7) KOE/ml and culture incubation at t degrees 28 degrees C. The nutrient content of the medium significantly influenced the quantity of produced biofilm. The nutrient broth LB without NaCl was more effective in promoting biofilm formation, than LB itself. The least quantity of biofilm was formed in water. The genotype of the strains also critically influenced the quantity of produced biofilm. Nonmotile mutants cells had reduced ability to form biofilm. RpoS mutant cells produced significantly less biofilm as compared with cells of isogenic parent strains. The chemical content of plastic and glass also influenced biofilm formation.

The results of the study of hospital strains of the B. cepacia complex, isolated in hospitals of Moscow, with the use of phenotypical and molecular-genetic methods are presented. The phenotypical methods made it possible to differentiate Russian strains and classify them with a group of genomovars (I, III, IV). As the result the epidemic importance of the strains with epidemic markers, having specific characteristics for every clinic, was determined. The detection of the collection of genes cepI and cepR in the strains made confirmed the epidemic importance of the stains which had, due to the regulatory "quorum sensing" (QS) system, the potential capacity for inducing infection and persisting in the patient's body. The presence of gene cepR in all strains and the absence of gene cepl in 33% of strains gave evidence to suggest that in some strains the activation of the production of pathogenicity factors required the presence of other bacteria having the fully developed QS system. Thus, the new complex approach with the use of phenotypical and molecular-genetic methods permits more precise identification of the source of hospital infection induced by the bacteria of the B. cepacia complex.

Bacteria which belong to the group of facultative parasites are capable of intensive proliferation both outside the organism and inside eukaryotic cells, thus often causing severe human and animal illness. The necessity of quick adaptation to the alteration of conditions after intrusion into the macro-organism gave rise to the development of complex genetic systems, regulating the activity of various gene groups. The review, written by the example of two pathogen bacteria, the gram-positive Listeria monocytogenes and the gram-negative Legionella pneumophila, illustrates up-to-date data that concern system regulation of genes, coding pathogenicity factors, as adaptive response of a bacterium to changes in outer conditions within the infectious process.

The authors present a review containing their own and literature data on the role of cytokine TNF-alfa in infectious process on the part of the macro- and microorganism, paying special attention to the latter. The paper contains data from literature concerning the mechanism of the interaction between cytokine and eukaryotic cells and experimental data on direct interaction between cytokine and bacterial cells in vitro and in vivo.

Studies of the last decade have shown that most bacteria exist in natural ecosystems as specifically organized, attached to substrates biofilms rather than as freely floating plankton cells. The formation of these biofilms is a complex and highly regulated process. The development of biofilm communities is a primary strategy of bacterial survival not only in the external environment but also in the bodies of infected macroorganisms. In these organisms, bacteria are joined by complicated cell-cell associations, which makes them functionally similar to multicellular organisms. In the present review, we consider the structural organization of biofilms, factors affecting initiation of the biofilm formation, differential expression of bacterial genes at various stages of the biofilm development and their regulation. The significance of studies in this field for medicine, in particular, for prevention and protection against pathogenic bacteria, is discussed.

Described in the paper are characteristics of B. cepacia clinical strains isolated from patients at Moscow hospitals. The strains were investigated for the presence of proteolytic, chitinolytic, hemolytic and lipase activities as well as for presence of components of the "Quorum sensing" gene activity regulatory system by using biological test-systems and in the polymerase chain reaction with primers to genes cepI and cepR.

The review deals with the data of literature on the role of the "quorum sensing" (QS) system ensuring the social behavior of bacteria in the regulation of virulence genes. The mechanisms of the action of these systems in Gram-negative and Gram-positive bacteria, as well as the influence of acyl-homoserine lactones, one of the components of the QS system, on the immune response of the infected host are discussed. In addition, in this review the data of literature on the existence of bacteria in the form of biofilms are presented. The methods of the identification of biofilms, the methods of their experimental preparation and the role of the QS system in the process of their formation are considered.

A minireview of the data of literature on the interaction of bacteria with cytokins and the stimulation of the growth of pathogenic bacteria by some cytokins is presented. The addition of tumor necrosis factor-alpha into the culture medium has been shown to stimulate the growth of Salmonella virulent and avirulent strains 4-fold and 2-fold respectively. This cytokin has also been shown to produce a stimulating effect on the process of the in vitro and in vivo recultivation of noncultivating forms of virulent and avirulent strains.

Data on the structural organization and evolutionary role of specific bacterial DNA regions known as genomic islands are reviewed. Emphasis is placed on the most extensively studied genomic islands, pathogenicity islands (PAIs), which are present in the chromosome of Gram-negative and Gram-positive pathogenic bacteria and absent from related nonpathogenic strains. PAIs are extended DNA regions that harbor virulence genes and often differ in GC content from the remainder of the bacterial genome. Many PAI occur in the tRNA genes, which provide a convenient target for foreign gene insertion. Some PAI are highly homologous to each other and contain sequences similar to ISs, phage att sites, and plasmid ori sites, along with functional or defective integrase and transposase genes, suggesting horizontal transfer of PAI among bacteria.

The data on the interaction of bacteria of the genus Bdellovibrio with the representatives of pathogenic Salmonella typhimurium are presented. Different types of such interaction are demonstrated: in a two-component system, in fluid media, in an agar layer and on the surface of a solid carrier. As shown for the first time, Bdellovibrio cells are capable of interacting not only with actively growing bacteria, but also with their noncultivable forms. The data obtained may serve as the basis for the study of possible practical use of such bacteria for controlling Gram-negative organisms, the causative agents of sapronotic infections.

The dynamics of vegetative and dormant (noncultivable) of S. typhimurium cells of two isogenic strains in association with microalgae Scenedesmus quadricauda and under the action of the exometabolites of the algae at different stages of their growth was studied using in parallel bacteriological method and PCR. The study revealed that at the stage of active growth green algae and their metabolic products maintain the survival of salmonellae (strain TR = 1) vegetative forms in water at an optimum temperature. Low temperatures induced their gradual (3 weeks) transition to the dormant state. The exometabolites of old dying algae induced the rapid (several hours) and complete transition of the bacterial population (TR = 1) to noncultivable state. In our experiments the insertional mutation in gene pqi (strain PhoA = 8), inducing the defect of transmembrane protein and disturbances in the transition of salmonellae to dormant state, led to stable existence (lasting 7 months, i.e. the whole term of observation) of vegetative cells. The natural inducers tried in our experiments did not lead to the formation of the dormant forms of salmonellae in this mutant strain.

The review discusses a role of mobile genetic elements in the evolution of pathogenic bacteria. Particularly great emphasis is placed on two recent thoroughly studied mobile genetic elements--the islets of pathogenicity and cassettes containing antibiotic resistant genes. The matters associated with the structural organization of these types of mobile elements, mechanisms of their translocations and involvement in the production of pathogenic bacteria are considered in detail. The data on the impact of macroorganisms on the horizontal dissemination of genetic information are discussed.

The gene of microbial lysozyme (lyz) of S. aureus 118 and the gene of lysostaphin (lzf) of S. aureus RN 3239 were cloned and their expression in B. subtilis cells was shown. Lysozyme production in B. subtilis recombinant clone pLF14-Lyz, obtained as the result of cloning, was 2.5-fold greater than lysozyme production in S. aureus wild strain 118. Lysostaphin production in B. subtilis recombinant strain pLF14-Lzf which had inherited the cloned genes was approximately equal to lysostaphin production observed in S. aureus initial strain RN 3239. The production of lysozyme and lysostaphin in the cells of B. subtilis recombinant strains was observed at 30 degrees C and pH 5.5, while in S. aureus initial strains 118 and RN 3239 bacteria produced lysozyme and lysostaphin at 37 degrees C and pH 7.5 respectively.

Differential gene expression in culturable and non-culturable forms of Salmonella typhimurium was studied by the molecular display method. Six fragments of differentially expressed gene cDNA, depending on culturable or non-culturable state of the cultures, were isolated, cloned, and sequenced. Identification of corresponding S. typhimurium differentially expressed genes was carried out by comparing the sequences of cDNA fragments with the bacterial genome data base.

Polymorphism of clinical manifestations in Toxoplasma infection and variegated disease patterns virtually rule out the diagnosis based solely on clinical symptoms, which makes modern laboratory tests particularly important. Amplification test system based on the polymerase chain reaction has been developed for the diagnosis of Toxoplasma infection. Computer analysis of nucleotide sequence of Toxoplasma gondii surface antigen gene P30 was analyzed, which helped choose and synthesize specific oligonucleotide primers. A method for biological material processing was selected, allowing sufficient DNA output. Optimal conditions for amplification reaction, ensuring absolute specificity and high (10-100 cells/sample) sensitivity, were determined.

The data on the development of a quantitative PCR technique are presented, including the synthesis of modified primers, the choice of conditions for the removal of reaction-mixture components from the amplified DNA, and the colorimetric detection of the PCR product. The application of this technique to the assessment of the level of gene expression in nonculturable bacterial forms characterized by a drastically reduced metabolism is described.

Approaches to the study of gene expression in the process of transition and prolonged stay of bacterial cells in the uncultivated state have been worked out with the use of the methods of reverse transcription and polymerase chain reaction. The differential expression of genes pqi and stn has been shown to occur in the uncultivated forms of S. typhimurium. The factor inducing the process of the transition of Salmonella cells into the uncultivated state has been found out.

The article deals with modern methods, viz. PCR, molecular display and genotherapy, which permit the new approach to the solution of problems connected with the identification of infective agents, the study of the mechanisms of the pathogenesis of infectious diseases and their treatment. In this article concrete examples, clearly demonstrating how each of the above-mentioned technologies makes it possible to broaden the circle of problems solved in infectious pathology of man, are presented.

Published reports about structural organization of genes coding for pathogenicity factors are reviewed. Many of such genes are often united into "virulence blocks" or "pathogenicity islands" and are surrounded by mobile genetic elements, promoting their transposition between related bacteria genomes and leading to changes in virulence in the course of evolution. Data on the similarity of nucleotide sequences of virulence genes in different bacteria are presented, despite differences in their localization in the relevant genomes. The role of rRNA genes in dissemination of virulence genes among different bacteria during transduction or conjugation is shown.

The data on the influence of mutation in genes, affecting vital functions of a bacterial cell, on the process of transition into the nonculturable state in S. typhimurium are presented. Four genes obtained by the authors have been identified. Mutations in these genes lead disturbances in the formation of S. typhimurium nonculturable forms. The nonculturable forms of salmonellae have been shown to retain their virulent properties and their capacity for recultivation, when inoculated into laboratory animals.

Presents a new method for making preparations of bacteria and their unculturable forms from suspensions with low concentrations of cells, from 1 x 108 to 1 x 104 per ml. to be examined under transmission electron microscope. The novelty of the method consists in the technique of concentrating bacteria by making a colloid solution consisting of bovine serum albumin and polyethyleneglycol. Associations of protein molecules, polyethyleneglycol, and bacteria, forming in the solution, are sedimented by centrifugation after 48-hour incubation at 4 degrees C. The solidity of the resultant macroscopic sediment is sufficient for studying the cells contained in it under transmission electron microscope.

Mutants of Salmonella typhimurium with an impaired process of transition to the nonculturable state were tainted. Mutants were divided into four phenotypic groups. In four mutants (representatives of each phenotypic group), genes with TnPhoA transposon insertions were cloned; these insertions caused a disturbance in the process of mutant cell transition to the nonculturable state. Nucleotide sequences of mutant gene fragments were determined. Comparison of nucleotide sequences obtained with a data bank on DNA nucleotide sequences of enterobacterial genomes allowed the identification of four genes involved in the control of nonculturable form generation in salmonellae.

The laboratory model for the induction of nonculturable forms in Salmonella typhimurium has been developed. S. typhimurium mutants with disturbances at different stages of their transfer from vegetative to nonculturable state have been obtained by insertion mutagenesis with the use of TnPhoA transposon. To identify the functions of genes involved in the process of the appearance of nonculturable forms, their cloning in the sequencing vector has been carried out.

A laboratory model of the induction of nonculturable forms in Salmonella typhimurium has been developed. Mutants of S. typhimurium were obtained using insertion mutagenesis via the TnPhoA transposon. These mutants were impaired in the cell transition from the vegetative to the nonculturable state assayed in this model. Mutants have various phenotypes and are located in different regions of the chromosome, as shown by the data obtained using pulsed-field electrophoresis of genomic DNA.

An immunosuppressive effect produced by live microbial cells of S. flexneri virulent plasmid-carrying strain 2a 516 (pSF 140-) was shown in relation to the formation of splenic cells producing antibodies to heterologous antigen (sheep red blood cells). This effect was most pronounced at an early period (days 4-7) after the infection of mice. On the contrary, avirulent plasmid-free variant (pSF 140-), genetically related to S. flexneri strain 2a 516, produced an immunostimulating effect at this period of observation. Such immunostimulating action of S. flexneri plasmid-free variant of the smooth type on immunocompetent effector T and B lymphocytes with the preservation of active hematopoiesis is indicative of the possibility of using these variants of shigellae as vector strains in constructing gene engineering (vector) vaccines.

The possibility to identify noncultivating forms of Salmonella by the polymerase chain reaction (PCR) has been shown. To do it the technique for Salmonella identification was elaborated, based on amplification of a 500 bp fragment of araC gene. Time course of populations of two Salmonella typhimurium strains during prolonged incubation in water was studied by the techniques of serial dilutions on solid nutrient media, acridine orange staining, and PCR. The strains differed in pathogenicity levels and genetic characteristics. Cells of nonvirulent strain were shown to loose gradually in the process of incubation the ability to grow on solid nutrient media and transform into noncultivating forms whose ability to proliferation can be restored under definite conditions. No difference in the dynamics identified by PCR or traditional microbiological techniques was found for population of virulent Salmonella typhimurium incubated in water indicating the possibility of fast degradation of cells having lost the ability to divide.

After oral immunization of lambs with S. typhimurium vaccine strain 274 these bacteria were characterized by low adhesion to the epithelial surface of the ileum, cecum, and colon, by very insignificant invasion into intestinal epithelium and then into lamina propria of the intestinal mucosa, mesenteric lymph nodes and the blood; the blood stream carried these bacteria further to the liver, spleen and other organs. Bacteria of strain 274 produced no damages, did not multiply and only persisted for 7-13 days in some lambs. No inflammation developed in response to oral immunization of the animals. The developing immunomorphological reaction presented as just a negligible increase in lymphocyte count in the intestinal epithelium, blast transformation and plasmatization in lymphatic follicles of Peyer's patches, hyperplastic mesenteric lymph nodes, blast and lymphocyte recirculation, moderate hyperplasia of white splenic pulp and insignificant lymphoid infiltration of portal interlayers of the liver.

As a result of screening among 11956 enterobacteria strains isolated from feces of normal children, grown-ups and lambs, seven active microcin-producing strains were obtained. The microcins were shown to be peptides or their derivatives with a low molecular weight (less than 10,000) and a broad spectrum of activity, mainly against gram-negative bacteria. According to cross immunity criteria the microcins studied belonged to two different types. Those of type I could be further classified into two subtypes on the account of difference in the spectrum of antibacterial activity. In 5 cases out of 7 the microcin-producing ability and immunity to microcins have been attributed to plasmids that the strains harboured. The effect of microcins on sensitive cells depended on ompR and ompF gene products.

The interaction of Shigella recombinant strains (with attenuating chromosomal mutations, with transposon-neutralized plasmid gene mutations, the hybrids of both strains), incapable of inducing keratoconjunctivitis in guinea pigs, with cells Hep-2 and enterocytes in the ligated loop of the small intestine of rabbits was studied. These strains retained, to varying extent, pronounced adhesiveness, but practically lost their invasiveness (though in Peyer's patches the translocation of bacteria by M-cells was observed) and cytotoxicity, as well as their capacity to multiply in epithelial cells and to cause destructive inflammation in the intestine. According to these criteria of evaluation, morphological investigations confirm the safety of the recombinant shigellae under study.

In 2-3 weeks after the oral immunization of rabbits, made in one or two administrations, with attenuated two-marker S. dysenteriae 1 strain VS-12 and recombinant S. dysenteriae VS-12/S. sonnei NR-18 and S. flexneri y433/S. sonnei NR-18 pronounced immunological reaction developed in the mucous membrane of the small intestine: blast transformation follicles of Peyer's patches, an increase in the number of lymphoblasts and plasmocytes in the cupolae of follicles and in intestinal villi, and an increase in the number of lymphocytes and macrophages in the intestinal epithelium with their release into the lumen of the intestine after challenge with virulent shigellae. The protective potency of these recombinants after challenge with massive doses of virulent shigellae was found to be high, which was shown by quantitative evaluation of the decrease of adhesion, invasiveness and cytotoxicity, suppression of epithelial lesions and development of inflammation in the intestinal mucosa.

Strains of Shigella flexneri isolated from patients in 4 geographic regions of the former USSR have been studied by plasmid analysis and fingerprinting techniques. It has been shown that these methods make possible to differentiate phenotypically identical strains for 9 genotypic patterns (7 different plasmid profiles and 3 specific hybridization patterns). Strains of Shigella flexneri isolated from each region possess the similar hybridization patterns independent of serovar and plasmid profiles. Similar hybridization patterns were found among the strains isolated in Erevan and Dushanbe. The strains isolated in Kiev are related to Erevan and Dushanbe strains, but they also possess the specific additional hybridization bands. Tashkent isolates are different from the strains isolated in other geographic regions. They are also of clonal origin in spite of the different serovars and plasmid profiles registered for these strains. Positive correlation has been found between serovar and plasmid profile characteristics of the strains. The clonal origin of the Tashkent isolates is different from the one of Erevan, Dushanbe and Kiev isolates, the latter having a similar clonal origin.