[The role of technogenic and biological factors in spread of foodborne bacterial infections].

Epidemiologic aspects of ecology of foodborne infection causative agents that have taken root in contemporary technogenic loci created by humans in urbocenoses which include agrocomplexes of animal breeding, vegetable growing in open and closed ground are discussed. Soil and water sources with a wide transport network where microorganisms dwell in the environment under favorable for vital functions and growth conditions (humus deposits, temperature regimen, optimal pH, associations with hydrobionts, biofilm formation) are potent secondary reservoirs of infection causative agents. Some molecular-genetic mechanisms of polyhostality of Gram negative bacteria and listeria are examined.

[Presence of Yersinia enterocolitica in conditions of agro complex].

AIM: Detection of duration of existence of Yersinia enterocolitica in substrates ofagro complex and formation of biofilms by causative agent during artificial semination of forage and meat products. MATERIALS AND METHODS: Y. enterocolitica 09 strain and its rifampicin-resistant (Rmr) mutant were used. Microbial landscape of samples was studied by seeding on selective media (HiMedia), biochemical properties of isolates were controlled on API test-systems (Bio-Merieux). The presence ofyopA gene localized on virulence plasmid pCad was determined in PCR. Vital staining of biofilms was carried out by Live/Dead stain (Invitrogen, USA). Visualization of the data was registered by using GMS-510 (USA) microscope with digital camera and Skope Photo software (USA). Formation ofyersinia bacterial biofilms was confirmed by using scanning electron microscope (SEM) JSM6380 (Japan). RESULTS: Prolonged duration of existence of Y. enterocolitica in substrates of agro complex with conservation of pCad virulence plasmid by causative agent was detected. SEM demonstrated stages of biofilm formation during artificial semination of animal forage, meat products and materials of food equipment in a wide range of temperatures from 10 to 30 degrees C, and vital stain detected viable yersinia in mature biofilms. CONCLUSION: Agro complexes are a variant oftechnogenic foci where ecological conditions for prolonged existence of sapronosis are formed.

[Identification of proteins determining the virulence of Listeria monocytogenes using the bioinformation analysis].

The postgenomic stage of biotechnology allows the bioinformation approaches to be used for revealing previously unknown factors involved in different processes. In this work, bioinformation approaches were applied to analysis of factors involved in the L. monocytogenes virulence. Several open reading frames (ORFs) were identified in L. monocytogenes genome, which encode proteins with high level homology to bacterial peptidases and which meets the developed criteria. The influence of the ORFs on virulence was demonstrated by infection of mice with L. monocytogenes strains mutated in the studied genes. Growth rates, morphology, and production of surface proteins characterized the obtained strains.

[Burkholderia cepacia under different ecological conditions: the amount and variability of the bacterial population].

In a series of prolonged experiments with the use of the bacteriological method and PCR analysis the amount and state of B. cepacia population, associated and not associated with infusoria Tetrahymena pyriformis, were dynamically evaluated under different conditions: in water, brain heart broth, soil extract and at different temperature (4 degrees C and 25 degrees C). In soil extract at 25 degrees C B. cepacia existed in the vegetative state for the period of up to 3 months, while at 4 degrees C, in the absence of protozoa, the transition of these microorganisms into the uncultivable forms occurred in 9 days, and they could be detected only with the use of PCR. Protozoa maintained the existence of the vegetative bacteria for as long as 2 months, and in 3-4 months uncultivable forms of B. cepacia cells were registered. In water at low temperature B. cepacia disappeared in 2 months, evidently, eaten up by infusoria. The population variability of B. cepacia under different conditions of their existence was established: S-R dissociation, a decrease in biochemical activity, growth deceleration. A high level of cytopathogenicity in B. cepacia pigment-forming clones was noted. In the process of transition into the uncultivable state pigment formation in B. cepacia population decreased up. The ecological plasticity and multi-pathogenicity of B. cepacia as phytopathogens and the causative agents of human diseases are discussed.

[Variability of the clonal structure of Yersinia pseudotuberculosis population under different conditions]. / Izmenchivost' klonal'noi struktury populiatsii Yersinia pseudotuberkulosis v raznykh usloviiakh sushchestvovaniia.

In a series of experiments the dynamics of the clonal structure of Y. pseudotuberculosis population was evaluated by cytopathogenicity in soil extract, as well as in associations with blue-green algae (cyanobacteria) and infusoria, under different temperature conditions. In all variants of experiments made at low environmental temperature (10 degrees C) a considerable part of Y. pseudotuberculosis clones (25-40%) was found to be cytopathogenic, while at 22 degrees C such clones were absent or had low cytopathogenicity. At the same time experiments made under the same temperature conditions (10 degrees C) showed the variability of the clonal structure of the bacterial population in different associations and sterile soil extract, as well as at different periods of the experiments. At low temperatures Y. pseudotuberculosis virulent (cytopathogenic) clones, in contrast to avirulent ones, were characterized by the presence of virulence plasmid p45, as well as high urease and catalase activity. The results of the experiments are discussed from the viewpoint of the clonal concept of bacterial populations and their pathogenicity.

[Biocenosis of the natural foci of sapronotic infections (the results of 15-year observations)]. / Biotsenoticheskie osnovy prirodnoi ochagovosti sapronozov (itogi 15-letnikh nabliudenii).

The summarized results of original studies, carried out in 1988-2003 by scientists of the Gamaleya Research Institute of Epidemiology and Microbiology, are discussed. The part dealing with the regularity and mechanisms of the circulation of pathogenic bacteria in the biocenosis of soil and water reservoirs includes the following subjects: population and intracellular interactions with protozoa; the evaluation of different representatives of hydrobios as hosts and the transmission along trophic biocenotic chains; the effect of algae and their metabolic products on bacterial populations; the possibility of the colonization of higher plants from the soil and the subsequent infection of rodents; the variability and clonal structure of bacterial population in its interaction with protozoa and plants; the chain of Yersinia circulation in agrocenosis. The part dealing with the mechanisms of the prolonged reservation of causative agents includes such subjects as the reversible transition of bacteria into the latent (uncultivable) state in soils and water reservoirs, as well as the biological inductors of this process; the prolonged preservation of latent bacteria in the cysts of protozoa and blue-green algae; the indication of causative agents in the natural foci of plague and pseudotuberculosis. On the basis of the original investigations and the data of literature new theoretical review in the field of the natural foci and the epidemiology of sapronotic infections have been formulated.

[Experimental evaluation of interaction between Yersinia pestis and soil infusoria and possibility of prolonged preservation of bacteria in the protozoan oocysts]. / Eksperimental'naia otsenka vzaimodeistvii Yersinia pestis EV s pochvennymi infuzoriiami i vozmozhnosti dlitel'nogo sokhraneniia bakterii v tsistakh prosteishikh.

The character and outcome of interactions between Y. pestis (vaccine strain and soil infusoria Tetrahymena pyriformis (axenic culture) were under experimental study. The parallel use of the bacteriological method and PCR test systems made it possible to follow the dynamics of Y. pestis cells (strain EV) with different plasmid profiles in their interaction with infusoria, as well as their passage into the protozoa cysts. The study revealed the complete utilization of Y. pestis cells lacking virulence factors by infusoria. The presence of plasmids of virulence influenced only the duration of complete bacterial phagocytosis. A drop in the temperature of cultivation to 2 degrees C induced the mass and rapid encystment of infusoria. In the PCR analysis specific DNA fragments of Y. pestis cells, preserved in the latent (uncultivable) state, were detected in the cysts of protozoa within the period of up to 14 months, while the results of bacteriological studies were negative. The data thus obtained are discussed with regard to the possible mechanisms of the existence and prolonged reservation of Y. pestis in the soils of natural foci with participation of protozoa.

[Ultrastructure of Yersinia pseudotuberculosis in the process of their reversible transition into the dormant (non-culturable) state in association with blue-green algae]. / Ul'trastruktura Yersinia pseudotuberculosis v protsesse obratimogo perekhoda v pokoiashcheesia (nekul'tiviruemoe) sostoianie v assotsiatsii s sine-zelenymi vodorosliami.

The ultrastructural organization of Y. pseudotuberculosis in the process of the transition of vegetative cells into the dormant (noncultivable) state in interaction with blue-green algae of the species Anabaena variabilis was studied by the method of transmission electron microscopy. The use of type specific Y. pseudotuberculosis serum made it possible to identify Y. pseudotuberculosis cells in the bacterial association and to find out whether their antigenic properties remained intact in time. The dormant forms of Y. pseudotuberculosis, recultivated by passage through the axenic culture of unfusoria (Tetrahymena pyryformis), were also studied with the use of electron microscopy. The revertants were found to be at different stages of restoration of their typical morphological characteristics and antigenic properties were partially retained. The fine structure of Y. pseudotuberculosis cells in the initial culture was shown to be similar to that of the revertants of dormant forms, morphological criteria of the dormant cell ultrastructure were established. The cyclic processes of reversible transition from vegetative forms to dormant ones in bacterial populations under the influence of hydrobios is regarded as an adaptive mechanism of their existence in the environment.

[Effect of blue-green alga (Cyanobacteria) and their exometabolites on formation of resting forms and variability of Yersinia pseudotuberculosis]. / Obrazovanie pokoiashchiksia form i izmenchivost' Yersinia pseudotuberculosis pod vozdeistviem sine-zelenykh vodoroslei (tsianobacterii) i ikh ékzometabolitov.

Research, carried out with the use of bacteriological methods and polymerase chain reaction, revealed that the transformation of Y. pseudotuberculosis, associated with blue-green algae Anabaena variabilis, into resting (noncultivable) forms took shorter time than in soil extract containing no algae. The exometabolites of "old" cultures of these algae sharply accelerated the formation of resting Y. pseudotuberculosis forms. The influence of the algae and the products of their metabolism was manifested far more intensively at 22 degrees C than at 4 degrees C. After passage through infusoria resting Y. pseudotuberculosis forms, preserved in the mucous covering of cyanobacteria, partially reverted into vegetative forms, capable of growing on solid culture media. The revertants essentially differed from the initial vegetative forms by having lower enzymatic activity, agglutinability and cytopathogenicity, as well as by the loss of plasmid p45. The probable role of blue-green algae, widely spread in soils and water reservoirs, in the processes of reversible transformation of Y. pseudotuberculosis vegetative and resting forms, closely connected with seasonal changes of temperature conditions.

[Ecological-genetic mechanisms of the transition of Salmonella typhimurium to the dormant state in the environment]. / Ekologo-geneticheskie mekhanizmy perekhoda Salmonella typhimurium v pokoiashcheesia sostoianie v okruzhaiushchei srede.

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.

[Dormant forms of Yersinia pseudotuberculosis during interaction with green algae and their exometabolites (population dynamics and ultrastructure)]. / Pokoiashchiesia formy Yersinia pseudotuberculosis pri vzaimodeistvii s zelenymi vodorosliami i ikh ékzometabolitami (populiatsionnaia dinamika i ul'trastruktura).

The dynamics of the transition of Y.pseudotuberculosis to the latent (noncultivable) state in sterile soil extraction and in soil extraction, containing Scenedesmus algae or their exometabolites, was evaluated by the bacteriological method and with the use of polymerase chain reaction. The formation of Y.pseudotuberculosis latent forms achieved its highest rate under the action of algal exometabolites (on day 11), while in the presence of algal cells such formation was delayed to 3-5 months. The partial reversion of Y.pseudotuberculosis nonculativable forms to vegetative, bacteriologically detectable forms occurred under the action of fetal serum, as well as live or killed infusoria. Transmission electron microscopy demonstrated that noncultivable Y.pseudotuberculosis had a round form, diminished cell size, denser cytoplasm and enlarged cytoplasmic space. The reversible transition of bacteria to the latent state is regarded as their adaptation to unfavorable factors during their existence in soils and water reservoirs.

[The population dynamics of Salmonella typhi and Tetrahymena pyriformis during joint cultivation]. / Dinamika populiatsii Salmonella typhi i Tetrahymena pyriformis pri sovmestnom kul'tivirovanii.

The data on changes in the number, as well as cultural and biological properties of S. typhi and free-living T. pyriformis in the course of their joint cultivation at 2 degrees C and 25 degrees C are presented. As suggested on the basis of the results of this experiment, the interaction of S. typhi with T. pyriformis may facilitate the preservation of the bacteria in the environment and be a stage in their migration along trophic chains.

[The possibility of preserving the causative agent of plague in soil in resting (nonculturable) form]. / O vozmozhnosti sokhraneniia vozbuditelia chumy v pochve v pokoiashcheisia (nekul'tiviruemoi) forme.

The parallel study of soil specimens from the burrows of great gerbils, taken in the natural focus of infection at the period between epidemics, by the bacteriological method and with the use of polymerase chain reaction (PCR) demonstrated that, simultaneously with the negative results of inoculations into nutrient media, PCR revealed the presence of Y. pestis in 4 out of 72 soil specimens. The serological study (in the indirect hemagglutination test) revealed the presence of Y. pestis capsular antigen (fraction l) in 16 out of 75 soil specimens. The transition of Y. pestis (strain EV) into the nonculturable state was experimentally shown in sterile soil extract and in association with protozoa, algae and Euglena. Bacteriologically detected Y. pestis disappeared or were represented by a few cells after day 7, while PCR could register the presence of Y. pestis at a concentration of 10(4)-10(5) microbial cells per ml until the end of the term of observation (30 days). After the enrichment of the nutrient medium with fetal serum the complete reversion of Y. pestis nonculturable forms was observed: the concentration of bacteria grown on agar was 10(5) colony-forming units per ml. Transition into the nonculturable state was also observed in distilled water. The results of this investigation are discussed with special emphasis to the possibility of the preservation of Y. pestis in the nonculturable from during the period between epidemics as applied to the problem of plague epizootic.

[Pathogenic Listeria in the soil and in association with algae: a reversible transition into a nonculturable state]. / Patogennye listerii v pochve i v assotsiatsii s vodorosliami: obratimyi perekhod v nekul'tiviruemoe sostoianie.

The analysis of the dynamics of the amount of L. monocytogenes in soil extract in association with green algae and in their absence, carried out in parallel by the bacteriological method and in polymerase chain reaction (PCR), revealed the mass transition of L. monocytogenes into the nonculturable state. The proportion of vegetative (bacteriologically detected) forms in the bacterial population rapidly decreased, while its total amount remained unchanged due to the formation of inactive (nonculturable) L. monocytogenes forms. Algae or their metabolites considerable accelerated this process: by day 26 L. monocytogenes could not be isolated, but in PCR they could be registered at their initial concentration: 10(6) microbial cells/ml. Inactive L. monocytogenes forms were shown to revert into the vegetative (culturable) state under the action of fetal serum, live and killed infusoria, auxin. The concentration of revertants grown on a solid culture medium was high: 10(4)-10(5) colony-forming units/ml.

[Listeria in plants: an experimental study of its colonization, numbers and variability]. / Listerii v rasteniiakh: éksperimental'noe izuchenie kolonizatsii, chislennosti i izmenchivosti.

The possibility for Listeria monocytogenes to penetrate into plants from the soil via the root system was experimentally proved. Listeria were shown to continuously persist for 30 days (the term of observation) in the vegetative organs of wheat (roots, stems and leaves). The concentration of Listeria was 10(9) CFU/g in the environment (soil extract) and the roots of wheat, 10(6)-10(7) CFU/g in stems, 10(8)-10(9) CFU/g in leaves. Six days later the dissociation of colonies in the S-form into small (up to 1 mm) and large (3-4 mm) was observed; in contrast to Listeria in large colonies, those in small colonies had high catalase activity and pronounced cytopathogenic action. The problem of the possible role of plants as the natural reservoir of bacteria, pathogenic for humans and animals, is discussed.

[The interrelationships between Vibrio cholerae and the infusorian Tetrahymena pyriformis]. / Izuchenie vzaimootnoshenii Vibrio cholerae s infuzoriiami Tetrahymena pyriformis.

The results of the study of interaction between V. cholerae of different virulence and T. pyriformis are presented. The study has revealed the heterogeneity of V. cholerae population: alongside easily phagocytized vibrios, there are vibrios resistant to the digestive action of T. pyriformis. An increase in the number of V. cholerae in association with T. pyriformis has been evaluated, taking into account the selective multiplication of vibrios resistant to phagocytosis. The data on changes in the agglutinative, phagolytic and virulent properties of V. cholerae cultivated together with T. pyriformis are presented. The suggestion has been made that protozoa can function as hosts of pathogenic vibrios supporting their existence in water.

[Bacterial pathogenicity factors: their functions in the environment]. / Faktory patogennosti bakterii: funktsii v okruzhaiushchei srede.

On the basis of literature data and the results of experimental studies the concept of the universal character of the main pathogenicity factors of bacteria (adhesion, enzymes, toxins, resistance to phagocytosis) as the adaptive mechanisms of bacterial populations under different ecological conditions, including their existence in the environment, has been put forward. Resistance of bacteria to phagocytosis as the mechanism of their protection from predators (protozoa, etc.), necessary for the existence bacteria in the ecological systems of water and soil, is considered in detail.

[Potential hosts and routes of the circulation of Yersinia pseudotuberculosis in an aqueous ecosystem]. / Potentsial'nye khoziaeva i puti tsirkuliatsii Yersinia pseudotuberculosis v vodnoi ekosisteme.

The interaction between Y. pseudotuberculosis and different representatives of the aqueous and, partially, soil biocenosis was studied. The study revealed that invertebrates of the plankton and benthos (water fleas, cyclops, mollusks, Oligochaeta, larvae of insects), as well as fish and higher plants, ensured the prolonged existence of Yersinia in water. Thus hydrobiotic organisms were to be regarded as potential natural hosts of Y. pseudotuberculosis. A suggestion was made that fish, the final link of some alimentary chains, could present epidemic danger as a food product for human consumption.