Relevance. Specific epidemiology of sapronotic (soilborne, waterborn) bacteria is characterized from the ecological point of view. The characteristic feature of soil-borne, water-born pathogens is an ability to exist autonomously in the environment. Aims. This analytical review is focused on hydrobionts and crops as alternative hosts for several soilborne and waterborne pathogenic bacteria (Vibrio, Legionella, Yersinia, Listeria monocytogenes). Conclusions. Published experimental results evidence capabilities of human and animal pathogens to colonize protozoan and plant tissues. Novel approaches are discussed to minimize risks of infection spreading.

The natural foci of sapronoses are characterized from the ecological point of view. Many pathogenic bacteria (Vibrio, Yersinia, Salmonella, Listeria, Escherichia, etc.), as well as conditionally pathogenic enterobacteria, are connected by a common ecological feature, the ability to exist autonomously in the external environment. The role of pathogen associations with other organisms in the formation of pathogen reservoirs is discussed. This analytical review of the current literature focuses on the role of crops and wild plants as alternative hosts for a number of pathogenic bacteria (Vibrio, Yersinia, Salmonella, Listeria, Escherichia, etc.). Experimental evidence shows that the causative agents of human and animal diseases can penetrate and multiply within tissues of many cultivated plants, the consumption of which contributes to outbreaks of foodborne infections. Novel approaches to the future minimization of this global epidemiological issue are discussed. These approaches include the search for wild plants that are resistant to enteropathogens and the construction of transgenic plant crops based on them that express antimicrobial peptides. Studies of wild plants of medical importance are described. With the use of nature-like biotechnologies, they can be used to produce new, highly active compounds that are effective against the multiresistant properties of a number of antibiotics. The results of the author’s research on the biological activity of wild-plant extracts with respect to Listeria and toxin-producing Echerichia are presented.

Цель. Определить вклад природных вариантов фактора инвазии Listeria monocytogenes InlB в вероятность развития перинатальной инфекции на модели интрагастрального заражения беременных мышей. Материалы и методы. Мышам на 12-16 день беременности интрагастрально вводили 10 КОЕ изогенных рекомбинантных штаммов L. monocytogenes EGDeΔinlB::InlB9 и EGDeΔinlB::InlB14, экспрессирующих отличающиеся природные варианты InlB9 и InlB14. Через 72 час мышей подвергали эвтаназии, вскрывали и оценивали микробную нагрузку во внутренних органах. Результаты. Перинатальная инфекция наблюдалась только при инфекции штаммом, экспрессирующим InlB14. Также микробная нагрузка этого штамма превышала нагрузку штамма EGDeΔinlB::InlB9 в 715, 315 и 70 раз для печени, селезенки и Пейеровых бляшек соответственно (p<0,01). Микробные нагрузки в эпителии тонкого кишечника были сравнимы для двух штаммов. Раздельные высевы плацент и плодов одной мыши, инфицированной EGDeΔinlB::InlB14, выявили возбудитель в 5 плацентах и 2 плодах из 9. Заключение. Природные варианты InlB отличаются по своей способности способствовать развитию перинатальной инфекции при интрагастральном пути введения L. monocytogenes.

Aim. Using the model of intragastric Listeria monocytogenes infection in pregnant mice to compare an input of found in nature variants of the invasion factor InlB in perinatal listeriosis. Materials and methods. Mice on 12-16 days of pregnancy were injected intragastrically with 108 CFU of isogenic recombinant L. monocytogenes strains EGDeAinlB::InlB9 and EGDeAinlB:: InlB14. The strains expressed naturally occurring InlB variants, InlB9 and InlB14. In 72 h, mice were subjected to euthanasia to evaluate bacterial loads in the internal organs. Results. Only the strain, which expressed InlB14, caused perinatal infection. Microbial loads in the liver, spleen and Peyer’s patches was 715, 315 and 70 times higher for this strain than for the strain EGDeAinlB:: InlB9 (p

Specific epidemiology of sapronotic (soil-borne) bacteria is characterized from the ecological point of view. The characteristic feature of soil-borne pathogens is an ability to exist autonomously in the environment. This analytical review is focused on crops as alternative hosts for a number of soil-borne pathogenic bacteria (Yersinia, Salmonella, Listeria, Escherichia etc). Published experimental results evidence capabilities of human and animal pathogens to colonize plant tissues. Novel approaches are discussed to minimize risks of infection spreading with crops. These approaches include an analysis of wild plant natural resistance to pathogenic bacteria and a construction of transgenic plant crops expressing antimicrobial peptides. Multiple studies are cited that established wild plants used in traditional medicine as a source for obtaining molecules effective against resistant pathogens. The review includes recent author results on activity of wild plant extracts against Listeria and toxin-producing Escherichia.

Проблема бактериальных кишечных инфекций, в частности, инфекций, ассоциированных с энтерогеморрагическими Escherichia coli (EHEC), носит глобальный характер и для своего решения требует комплексного подхода, включающего, в том числе, экспериментальные исследования в области экологии возбудителей. На моделях вегетирующих растений базилика Ocimum basilicum бактериологическими методами показано длительное существование многочисленных популяций токсин-продуцирующих E. coli O157:H7 внутри растительных тканей. Полимеразная цепная реакция подтвердила сохранение гена токсина stxl на протяжении опытов. На каллусах пекинской капусты Brassica rapa subsp. pekinensis и листового салата Lactuca sativa изучены стадии формирования биопленок E. coli. Методами световой и сканирующей электронной микроскопии выявлено, что бактериальные биопленки на поверхности каллусов после контаминации формируются чрезвычайно быстро (6-24 часа), обеспечивая жизнеспособность популяций возбудителей. Гистологические исследования инфицированных каллусов не выявили выраженного фитопатогенного воздействия на модельные растения. В статье обсуждается экстраполяция результатов лабораторных экспериментов в полевые условия с выявлением путей циркуляции патогенных эшерихий в агроценозе с точки зрения обеспечения пищевой безопасности населения.

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.

Sapronosis is an infectious disease caused by pathogenic microorganisms that are natural inhabitants of soil and/or aquatic ecosystems. They are capable of exiting in the environment outside of the host for a long time. Causative agents of sapronosis are polypathogenic, i.e., they cause infections in a wide range of hosts. The aim of the work was to establish a place in the clonal species structure for Listeria monocytogenes and Yersinia pseudotuberculosis isolated from both patients in hospitals and wild rodents and hydrobionts in natural foci. A total of 42 L. monocytogenes and 29 Y. pseudotuberculosis isolates were studied. 17 L. monocy-togenes and 29 Y. pseudotuberculosis isolates were obtained from patients; 25 L. monocytogenes isolates were obtained from wild animals and soil. The isolates were characterized using Multilocus Sequence Typing (MLST). Previously published MLST schemes were applied, including an analysis of the sequences of internal regions of seven marker genes for general metabolism. All Y. pseudotuberculosis isolates were shown to belong to the clonal complex occurred in the Russian Far East, Japan, and China, while L. monocytogenes isolates were referred to several clonal complexes, some of which have a worldwide distribution. The genetic similarity between L. monocytogenes isolates that cause invasive infections in wild animals in natural listeriosis foci and isolates related to listeriosis outbreaks has been demonstrated. These results suggested that high virulence could be a selective trait that enables the efficient reproduction of sapronosis causative agents in natural ecosystems. The variability of virulence factors of L. monocytogenes isolated from the same natural focus and related to MLST type was indicated. A hypothesis has been suggested that links the polyhostality of sapronosis causative agents and the genetic variability in virulence factors. Based on this hypothesis, a correlation has been suggested between the sequences of virulence factors and the source of isolation. A model of the formation of epidemiologically significant strains of the causative agents in natural foci of sapronosis was constructed.

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.

Сапронозы - это инфекционные заболевания, вызываемые патогенными микроорганизмами, ко- торые являются сочленами природных почвенных и/или водных экосистем, способными длитель- ное время существовать в окружающей среде вне организма хозяина. Возбудители сапронозов ха- рактеризуются полипатогенностью - способностью вызывать патологический процесс в широком круге организмов. Целью работы было установление места в клональной структуре вида для изо- лятов возбудителей сапронозов Listeria monocytogenes и Yersinia pseudotuberculosis, выделенные от больных в клинике и от диких грызунов и гидробионтов в природных очагах. Всего исследо- вано 42 изолята L. monocytogenes и 29 изолятов Y. pseudotuberculosis, включая изоляты, выделен- ные от больных (17 изолятов L. monocytogenes, выделенных из биопсийного материала случаев пренатального листериоза, 29 изолятов Y. pseudotuberculosis, выделенных от больных с диагнозом дальневосточной скарлатиноподобной лихорадки) и изоляты, выделенные из природных очагов (25 изолятов L. monocytogenes, выделенных из внутренних органов инфицированных животных и из почвы). Все изоляты были охарактеризованы методом мультилокусного сиквенс-типирова- ния (MLST). Использованы ранее опубликованные схемы MLST, включающие анализ последова- тельностей внутренних областей семи маркерных генов общего метаболизма,на основе которых созданы международные базы данных. Показано, что все изученные изоляты Y. pseudotuberculosis относятся к одному клональному комплексу, встречающемуся в Дальневосточном регионе РФ, Японии и Китае, в то время как изоляты L. monocytogenes относятся к нескольким клональным комплексам, некоторые из которых распространены на разных континентах. Показано генетиче- ское сходство между изолятами L. monocytogenes, вызывающими инвазивную инфекцию у диких животных в природных очагах листероиза, и изолятами, связанными с эпидемическими вспышка- ми листериоза. Полученные результаты позволяют предположить, что повышенная вирулентность может быть селективным признаком, обеспечивающим более эффективное размножение возбуди- телей сапронозов в природных экосистемах. Выявлена вариабельность факторов патогенности у изолятов L. monocytogenes, выделенных из одного природного очага и относящихся к MLST типу. Предлагается гипотеза, связывающая полигостальность возбудителей сапронозов с вариабельно- стью факторов патогенности как проявлением индивидуалной изменчивости. С точки зрения этой гипотезы рассматривается корреляция последовательностей факторов патогенности с источником выделения. На основе полученных результатов предлагается модель формирования в природных очагах сапронозов эпидемиологически значимых штаммов возбудителя.

С популяционно-экологических позиций рассмотрены данные о паразитизме ряда возбудителей природноочаговых сапронозов в гидробионтах (простейшие, ракообразные, черви, моллюски, иглокожие, рыбы). Симбиотические отношения между популяциями патогенных бактерий и простейших строятся по типу “паразит–хозяин”. Ультраструктурный анализ демонстрирует незавершенный характер фагоцитоза как в планктонных формах, так и в биопленках, формируемых протозойно-бактериальными сообществами. Это обеспечивает возбудителям длительное существование в окружающей среде. Экспериментально воспроизведена миграция возбудителя псевдотуберкулеза по разным трофическим цепям от нижнего уровня сообщества к высшему. Длительное сохранение возбудителей сапронозных инфекций различными гидробионтами, возможность миграции бактерий по пищевым цепям трофической пирамиды, с последующим заражением наземных животных и человека, определяют высокую экологическую и эпидемиологическую актуальность всестороннего изучения путей циркуляции патогенов в экосистемах окружающей среды.

Data on parasitism of the infectious agents of sapronoses in hydrobionts (protozoans, crustaceans, worms, mollusks, echinoderms, and fishes) are considered from the population-ecological viewpoint. The symbiotic relationships between populations of pathogenic bacteria and protozoans are of the host-parasite type. An ultrastructural analysis demonstrates that phagocytosis is incomplete both in planktonic forms and in biofilms formed by bacteria and protozoa. This provides for long-term survival of infectious agents in the environment. The migration of Yersinia pseudotuberculosis along trophic chains from the lowest to the highest level has been simulated experimentally. The long-term survival of pathogenic bacteria in aquatic animals and the ability of bacteria to migrate along trophic chains, reaching soil animals and humans, provide evidence that comprehensive studies on the routes of circulation of pathogens in natural ecosystems are highly relevant from the ecological and epidemiological viewpoints.

The postgenomic stage of biotechnology allows bioinformation approaches to be used for revealing previously unknown factors participating in different processes of the vital the functions of organisms. In this work, bioinformation approaches have been applied to the analysis of factors involved in the virulence of grampositive bacterium Listeria monocytogenes . Several open reading frames (ORF) have been identified in the L. monocytogenes genome, which encode proteins with a high level of homology to bacterial peptidases and meet the developed criteria. The effect of ORFs on virulence has been proved experimentally by intravenous infection of mice with the constructed L. monocytogenes strains carrying site-specific insertion mutations in the studied genes. The obtained strains have been characterized by growth rate, morphology, and production of surface proteins.

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.

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.

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.

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.