[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.

[Evolution of epidemiology]. / Evolutsiia épidemiologii.

The emergence, formation and stages of the development of today epidemiology of infectious diseases are analyzed. Differences in views concerning the essence of epidemiology as science between Russian and foreign schools are indicated. The brief description of the main stages in rendering basic concepts of Russian epidemiology more extensive and profound, including the gradual alteration of the view on epidemiology as the science dealing only with the epidemic process, is presented. In this connection new data on the epidemiological specificity of zoonotic, and especially sapronotic infections, leading to an essenmtal correction of the main postulates of epidemiology, are analyzed. Broader notions of the reservoirs of the causative agents of infections are given, the discrete character of the epidemic (epizootic) process, the role of the latent forms of bacteria in their preservation both in the environment and in the human body in the course of the infectious process are postulated. The complexity of the term "emerging infections" is discussed and 3 variants of its interpretation are proposed. The increasing role of anthropogenic factors, including technogenic effects, is emphasized; in future these factors may be capable of accelerating the man-made evolution of epidemiology.

[Integrative processes in modern epidemiology]. / Integrativnye protsessy v sovremennoi épidemiologii.

The integration of achievements in population ecology and the molecular genetics of pathogenic microorganisms into nowadays epidemiology of infectious diseases and the formation of its corresponding trends are discussed. The decisive role of highly complicated key problems of general epidemiology, such as the regularities of the autochthonous existence of pathogenic bacteria in the environment, the mechanisms of the pathogenicity regulation of infective agents and the formation of their epidemic variants, the mechanisms of the prolonged reservation of pathogenic bacteria at interepidemic (interepizootic) periods, the mechanisms of transformation of active (vegetative) and dormant forms in bacterial populations at different stages of epidemic and epizootic cycles, is demonstrated. The detection and analysis of such processes, and especially their latent mechanisms, become possible with the use of new concepts and methodological approaches, highly productive for the progress of epidemiology.

[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.

[Strategies of adaptive changes in Vibrio cholerae in natural water reservoirs]. / Strategiia adaptivnoi izmenichivosti kholernykh vibrionov v prirodnykh vodoemakh.

The currently available data on different variations in cholera vibrio habiting in water reservoirs are summarized. The pattern of variations of the main signs of vibrions is discussed in the context of "typical-atypical-inactive-resting (uncultivable) forms". Emphasis is placed on the reversible pattern and adaptive essence of variation whose basis is clonal and selective processes in the heterogeneous bacterial population. Possible populational mechanisms responsible for the development of epidemic types of cholera vibrions in the aqueous ecosystems as a prerequisite for human morbidity are discussed. The efficiency of ecological and molecular genetic studies for the epidemiology of cholera is recognized.

[Natural-focus infections: the key questions and new vantage points]. / Prirodnoochagovye infektsii: kliuchevye voprosy i novye pozitsii.

A short history of the concept of natural focal infections is presented: the idea put forward by D. K. Zabolotnyi, E. I. Pavlovskii's teaching, 3 stages of its development. A number of fundamental questions and the modern content of the concept are considered. The natural foci of infections are a combination of surface, soil and/or water ecosystems, including the population of the causative agent of infection. In contrast to surface ecosystems, in soil and water ecosystems the hosts of the causative agents of sapronotic infections ae soil invertebrates and hydrobios, in which these agents may circulate in biocenotic trophic chains. The circulation of the causative agents in natural foci is a discrete process; the mechanisms and forms of the existence of pathogenic bacteria during seasonal and prolonged periods between epidemics is considered. Special attention is given to latent (nonculturable) forms of bacteria. The complex character of the status of the causative agents of natural focal infections is discussed.

[Natural foci of diseases: the development of the concept at the close of the century]. / Prirodnaia ochagovost' boleznei: razvitie kontseptsii k iskhodu veka.

E. N. Pavlovskii's concept of natural focality of diseases and the development of general knowledge about natural foci and their structural (components), functional (mechanisms of pathogen maintenance), and ecosystem-related organization (assortment and interrelations of ecosystems) are reviewed from principal (in authors' opinion) aspects. The 60-year history of this theory includes three stages at which its scope and contents differed. At the first stage, it concerned transmissible zoonoses. It had been assumed that structurally, natural foci necessarily include the pathogen-vector-host triad, and the functioning of the focus is provided for by only pathogen circulation in terrestrial ecosystems. At the second stage, it became clear that vector is not a necessary structural component of any focus (an example of nontransmissible diseases), although the functioning of foci remained to be unequivocally attributed to the continuous pathogen circulation among animals of terrestrial ecosystems. The third stage is characterized by an understanding that, in general, the presence of a warm-blooded host in the focus is also unnecessary for pathogen survival, and natural foci can be represented by soil and aquatic ecosystems. The only necessary and specific component of any natural focus is the pathogen population. In this context, modern views on natural focality of diseases are reviewed, and the essence of the terms "natural focus" and "epizootic process" is defined. It is proposed to distinguish the phases of pathogen reservation and epizootic spread (circulation) in ecosystems of any type. The current state of this concept provides evidence that, in general biological terms, studies on natural focality of diseases belong to one of the fields of symbiotology.

[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 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.

[Nonculturable forms of Yersinia pseudotuberculosis in the soils of a natural focus of pseudotuberculosis]. / Nekul'tiviruemye formy Yersinia pseudotuberculosis v pochvakh prirodnogo ochaga psevdotuberkuleza.

Soil samples taken from the natural focus of pseudotuberculosis, characterized by the persistent presence of Y. pseudotuberculosis, were studied. Bacteria were detected by means of polymerase chain reaction with the use of 2 test systems; one of them was specific to the chromosomal invasiveness gene (inv) and the other, to gene yopA, localized on plasmid pCad. In 10 out of 24 sample of genetic material of the total soil microflora, Y. pseudotuberculosis gene inv was detected, two of these samples being also containing gene yopA. The calculated concentration of Y. pseudotuberculosis varied 10(2)-10(4) microbial cells per 1 g of soil. Bacteriological studies yielded negative results, which is indicative of the existence of Y. pseudotuberculosis in the soil of the natural focus in the nonculturable form.