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Vibrio cholerae interacts with many organisms in the environment, including heterotrophic protists (protozoa). Several species of protozoa have been reported to release undigested bacteria in expelled food vacuoles (EFVs) when feeding on some pathogens. While the production of EFVs has been reported, their biological role as a vector for the transmission of pathogens remains unknown. Here we report that ciliated protozoa release EFVs containing V. cholerae. The EFVs are stable, the cells inside them are protected from multiple stresses, and large numbers of cells escape when incubated at 37 °C or in the presence of nutrients. We show that OmpU, a major outer membrane protein positively regulated by ToxR, has a role in the production of EFVs. Notably, cells released from EFVs have growth and colonization advantages over planktonic cells both in vitro and in vivo. Our results suggest that EFVs facilitate V. cholerae survival in the environment, enhancing their infectious potential and may contribute to the dissemination of epidemic V. cholerae strains. These results improve our understanding of the mechanisms of persistence and the modes of transmission of V. cholerae and may further apply to other opportunistic pathogens that have been shown to be released by protists in EFVs.

Endosymbiotic interactions are frequently found in nature, especially in the group of protists. Even though many endosymbioses have been studied in detail, little is known about the mechanistic origins and physiological prerequisites of endosymbiont establishment. A logical step towards the development of endocytobiotic associations is evading digestion and escaping from the host's food vacuoles. Surface properties of bacteria are probably involved in these processes. Therefore, we chemically modified the surface of a transformant strain of Escherichia coli prior to feeding to Tetrahymena pyriformis. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide allows any substance carrying amino- or carboxyl groups to be bound covalently to the bacterial surface by forming a peptide bond, thus, altering its properties biochemically and biophysically in a predictable manner. The effect of different traits on digestion of T. pyriformis was examined by fluorescence and transmission electron microscopy. The efficiency of digestion differs considerably depending on the coupled substances. Alkaline substances inhibit digestion partially, resulting in incomplete digestion and slightly enhanced escape rates. Increasing hydrophobicity leads to much higher escape frequencies. Both results point to possible mechanisms employed by pathogenic bacteria or potential endosymbionts in evading digestion and transmission to the host's cytoplasm., (© 2018 The Author(s) Journal of Eukaryotic Microbiology © 2018 International Society of Protistologists.)

Mycobacteria are widespread microorganisms that live in various environments, including man-made water systems where they cohabit with protozoa. Environmental mycobacterial species give rise to many opportunistic human infections and can infect phagocytic protozoa. Protozoa such as amoebae and ciliates feeding on bacteria can sometimes get rid of non-digestible or pathogenic material by packaging it into secreted fecal pellets. Usually, packaged bacteria are still viable and are protected against chemical and physical stresses. We report here that mycobacteria can be packaged into pellets by ciliates. The model bacterium Mycobacterium smegmatis survived digestion in food vacuoles of the ciliate Tetrahymena pyriformis and was included in expelled fecal pellets. LIVE/DEAD® staining confirmed that packaged M. smegmatis cells preserved their viability through the process. Scanning and transmission electron microscopy revealed that bacteria are packaged in undefined filamentous and/or laminar substances and that just a thin layer of material seemed to keep the pellet contents in a spherical shape. These results imply that packaging of bacteria is more common than expected, and merits further study to understand its role in persistence and dissemination of pathogens in the environment., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Species recovery after disturbances depends on the strength and duration of disturbance, on the species traits and on the biotic interactions with other species. In order to understand these complex relationships, it is essential to understand mechanistically the transient dynamics of interacting species during and after disturbances. We combined microcosm experiments with simulation modelling and studied the transient recovery dynamics of a simple microbial food web under pulse and press disturbances and under different predator couplings to an alternative resource., Results: Our results reveal that although the disturbances affected predator and prey populations by the same mortality, predator populations suffered for a longer time. The resulting diminished predation stress caused a temporary phase of high prey population sizes (i.e. prey release) during and even after disturbances. Increasing duration and strength of disturbances significantly slowed down the recovery time of the predator prolonging the phase of prey release. However, the additional coupling of the predator to an alternative resource allowed the predator to recover faster after the disturbances thus shortening the phase of prey release., Conclusions: Our findings are not limited to the studied system and can be used to understand the dynamic response and recovery potential of many natural predator-prey or host-pathogen systems. They can be applied, for instance, in epidemiological and conservational contexts to regulate prey release or to avoid extinction risk of the top trophic levels under different types of disturbances.

Campylobacter jejuniis the leading cause of bacterial gastroenteritis worldwide. Transmission to humans occurs through consumption of contaminated food or water. The conditions affecting the persistence of C. jejuniin the environment are poorly understood. Some protozoa package and excrete bacteria into multilamellar bodies (MLBs). Packaged bacteria are protected from deleterious conditions, which increases their survival. We hypothesized that C. jejuni could be packaged under aerobic conditions by the amoeba Acanthamoeba castellanii or the ciliate Tetrahymena pyriformis, both of which are able to package other pathogenic bacteria.A. castellanii did not produce MLBs containing C. jejuni In contrast, when incubated with T. pyriformis,C. jejuni was ingested, packaged in MLBs, and then expelled into the milieu. The viability of the bacteria inside MLBs was confirmed by microscopic analyses. The kinetics of C. jejuni culturability showed that packaging increased the survival of C. jejuniup to 60 h, in contrast to the strong survival defect seen in ciliate-free culture. This study suggests that T. pyriformis may increase the risk of persistence of C. jejuniin the environment and its possible transmission between different reservoirs in food and potable water through packaging., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Endosymbiosis in ciliates is a common and highly diverse phenomenon in nature, but its development at the mechanistic level and the origins are not easy to understand, since these associations may have arisen at any time during evolution. Therefore a laboratory model is helpful. It could be provided by the interaction of Tetrahymena pyriformis and Escherichia coli. Microscopic analyses with a genetically manipulated fluorescent strain of E. coli show single bacteria leaving food vacuoles and escaping digestion, an important prerequisite for further experiments. Under selective conditions, beneficial for T. pyriformis, the ciliate was shown to internalize E. coli cells. After feeding, bacteria, transformed with the plasmids pBS-neoTet or pNeo4, provide T. pyriformis with the ability to handle toxic conditions, caused by the aminoglykoside antibiotic paromomycin. Axenic cultures or cocultures with untransformed bacteria show lower cell numbers and survival rates compared to cocultures with transformed bacteria after transfer to paromomycin containing media. PCR detects bacterial DNA inside T. pyriformis cells. Additionally, microscopical analysis of selectively grown cocultures reveals fluorescing particles in the cytoplasm of T. pyriformis containing DNA and lipids, corresponding in size to E. coli. This system could be a reasonable model for understanding mechanisms of endosymbiosis establishment in ciliates., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Background: The gram-positive pathogenic bacterium Listeria monocytogenes is widely spread in the nature. L. monocytogenes was reported to be isolated from soil, water, sewage and sludge. Listeriolysin O (LLO) is a L. monocytogenes major virulence factor. In the course of infection in mammals, LLO is required for intracellular survival and apoptosis induction in lymphocytes. In this study, we explored the potential of LLO to promote interactions between L. monocytogenes and the ubiquitous inhabitant of natural ecosystems bacteriovorous free-living ciliate Tetrahymena pyriformis., Results: Wild type L. monocytogenes reduced T. pyriformis trophozoite counts and stimulated encystment. The effects were observed starting from 48 h of co-incubation. On the day 14, trophozoites were eliminated from the co-culture while about 5 x 104 cells/ml remained in the axenic T. pyriformis culture. The deficient in the LLO-encoding hly gene L. monocytogenes strain failed to cause mortality among protozoa and to trigger protozoan encystment. Replenishment of the hly gene in the mutant strain restored toxicity towards protozoa and induction of protozoan encystment. The saprophytic non-haemolytic species L. innocua transformed with the LLO-expressing plasmid caused extensive mortality and encystment in ciliates. During the first week of co-incubation, LLO-producing L. monocytogenes demonstrated higher growth rates in association with T. pyriformis than the LLO-deficient isogenic strain. At latter stages of co-incubation bacterial counts were similar for both strains. T. pyriformis cysts infected with wild type L. monocytogenes caused listerial infection in guinea pigs upon ocular and oral inoculation. The infection was proved by bacterial plating from the internal organs., Conclusions: The L. monocytogenes virulence factor LLO promotes bacterial survival and growth in the presence of bacteriovorous ciliate T. pyriformis. LLO is responsible for L. monocytogenes toxicity for protozoa and induction of protozoan encystment. L. monocytogenes entrapped in cysts remained viable and virulent. In whole, LLO activity seems to support bacterial survival in the natural habitat outside of a host.

The natural reservoir of Francisella tularensis, the causative agent of tularaemia, is yet to be identified. We investigated the possibility that Francisella persists in natural aquatic ecosystems between outbreaks. It was hypothesized that nutrient-rich environments, with strong protozoan predation, favour the occurrence of the tularaemia bacterium. To investigate the differences in adaptation to aquatic environments of the species and subspecies of Francisella, we screened 23 strains for their ability to survive grazing by the ciliate Tetrahymena pyriformis. All the Francisella strains tested were consumed at a low rate, although significant differences between subspecies were found. The survival and virulence of gfp-labelled F. tularensis ssp. holarctica were then studied in a microcosm experiment using natural lake water, with varying food web complexities and nutrient availabilities. High nutrient conditions in combination with high abundances of nanoflagellates were found to favour F. tularensis ssp. holarctica. The bacterium was observed both free-living and within the cells of a nanoflagellate. Francisella tularensis entered a viable but nonculturable state during the microcosm experiment. When studied over a longer period of time, F. tularensis ssp. holarctica survived in the lake water, but loss of virulence was not prevented by either high nutrient availability or the presence of predators.

Modeling of interaction Yersinia pestis-Tetrahymena pyriformis in artificial soil ecosystem (ASE) containing soil of burrows of main carrier from Gorno-Altayski natural plague reservoir, as well as in physiological solution (PS) and in Hottinger broth (HB). Optimal proportion of bacterial and protozoa cells was possible to obtain and depended from virulence of Y. pestis and environmental conditions. In ASE at 18-22 degrees C association was the most stable under the microbial burden of 100 microbial cells (m.c.) per infusorian. Resistance of plague agent to phagocytosis by T. pyriformis was determined by strain's virulence. Avirulent strain Y. pestis [cyrillic letter: see text]-2377 was rapidly eliminated by protozoan in HB, PS and in ASE under the burden of 10 m.c per infusorian. Y. pestis [cyrillic letter: see text]-3443 with selective virulence compared with [cyrillic letter: see text]-2377 preserved in association longer in any tested medium. Highly virulent Y. pestis [cyrillic letter: see text]-3448 was the most resistant to phagocytosis by T. pyriformis.

Using the Escherichia coli-Tetrahymena pyriformis system, we revealed the involvement of bacterial antihistone activity and protozoan histones in interactions between pro- and eukaryotic microorganisms. Antihistone activity enhanced the viability of E. coli in association with T. pyriformis, according to our data on the dynamics of E. coli cell numbers. The strain with antihistone activity induced incomplete phagocytosis in the infusorians, resulting in cytological changes and ultrastructural alterations that indicated the retention of bacterial cells in phagosomes. Bacteria with antihistone activity located in the T. pyriformis cytoplasm influenced the eukaryotic nucleus. This manifested itself in macronucleus decompactization and a decrease in the average histone content in the population of infusorians. The data obtained suggest that protozoan histone inactivation by bacteria is one of the mechanisms involved in prokaryote persistence in associations with eukaryotic microorganisms.

Humans play little role in the epidemiology of Escherichia coli O157:H7, a commensal bacterium of cattle. Why then does E. coli O157:H7 code for virulence determinants, like the Shiga toxins (Stxs), responsible for the morbidity and mortality of colonized humans? One possibility is that the virulence of these bacteria to humans is coincidental and these virulence factors evolved for and are maintained for other roles they play in the ecology of these bacteria. Here, we test the hypothesis that the carriage of the Stx-encoding prophage of E. coli O157:H7 increases the rate of survival of E. coli in the presence of grazing protozoa, Tetrahymena pyriformis. In the presence but not the absence of Tetrahymena, the carriage of the Stx-encoding prophage considerably augments the fitness of E. coli K-12 as well as clinical isolates of E. coli O157 by increasing the rate of survival of the bacteria in the food vacuoles of these ciliates. Grazing protozoa in the environment or natural host are likely to play a significant role in the ecology and maintenance of the Stx-encoding prophage of E. coli O157:H7 and may well contribute to the evolution of the virulence of these bacteria to colonize humans.

The failure to reduce the Campylobacter contamination of intensively reared poultry may be partially due to Campylobacter resisting disinfection in water after their internalization by waterborne protozoa. Campylobacter jejuni and a variety of waterborne protozoa, including ciliates, flagellates, and alveolates, were detected in the drinking water of intensively reared poultry by a combination of culture and molecular techniques. An in vitro assay showed that C. jejuni remained viable when internalized by Tetrahymena pyriformis and Acanthamoeba castellanii for significantly longer (up to 36 h) than when they were in purely a planktonic state. The internalized Campylobacter were also significantly more resistant to disinfection than planktonic organisms. Collectively, our results strongly suggest that protozoa in broiler drinking water systems can delay the decline of Campylobacter viability and increase Campylobacter disinfection resistance, thus increasing the potential of Campylobacter to colonize broilers.

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.

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.

Fluorescence in situ hybridization (FISH) was applied to detect ingested natural bacteria within the food vacuoles of ciliates harvested from the natural sediment. In addition to this important qualitative aspect, FISH was also successfully used to measure the bacterivory of a culture of the ciliate Tetrahymena pyriformis on natural field sediment bacteria. In this feeding experiment, we compared the FISH technique with the only available alternative technique using fluorescently stained sediment (FS-sediment). The ingestion rate of unstained sediment bacteria determined by FISH was 4.6 bacteria per ciliate and hour. In contrast, Tetrahymena pyriformis cells that fed on bacteria from FS-sediment ingested 12.7 bacteria per ciliate and hour. Bacterial abundances in the sediment were equal in both sediment types (4 x 10(8) cells g sediment dry weight(-1)) when determined by DAPI counts. However, when analyzed using DTAF-counts, the number of bacteria in the FS-sediment increased to 9.7 x 10(8) cells g sediment dry weight(-1). From our findings we conclude that bacterivory by ciliates is overestimated when FS-sediment is used because DTAF stains bacteria as well as protein-containing detritus particles, which are also ingested by many ciliates. In contrast, FISH is a direct, a posteriori method that specifically stains phylogenetic lineages, e.g. eubacteria, after ingestion and thereby avoids a false determination of the number of ingested bacteria. Thus this method can also be used for the study of natural ciliate bacterivory in benthic systems.

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.

A method was developed that enabled real-time monitoring of the uptake and survival of bioluminescent Escherichia coli O157 within the freshwater ciliate Tetrahymena pyriformis. Constitutively bioluminescent E. coli O157 pLITE27 was cocultured with T. pyriformis in nutrient-deficient (Chalkley's) and in nutrient-rich (proteose peptone, yeast extract) media. Non-internalised bacteria were inactivated by addition of colistin, indicated by a decline in bioluminescence. Protozoa were subsequently lysed with Triton X-100 which lead to a further drop in bioluminescence, consistent with release of live internal bacteria from T. pyriformis into the colistin-containing environment. Bioluminescence measurements for non-lysed cultures indicated that internalised E. coli O157 pLITE27 cells were only slowly digested by T. pyriformis, in both media, over the time period studied. The results suggest that bioluminescent bacteria are useful tools in the study of bacterial intra-protozoan survival.

The study of the duration of the insecticidal activity of a mixture of infusoria with each of these bacteria revealed differences in the pattern of their relations during their long contact. T. pyriformis may be used for the prolongation of its insecticidal action if Bti is applied. An increase in the duration of this action depends on the viability of tetrahymena in the experimental flasks and on the presence of free spores and crystals of Bti. The prolongation of the action may be associated with the multiplication of this type of bacteria in the digestive vacuoles of infusoria. The use of Bsph in the mixture with T. pyriformis caused no increase in the insecticidal activity of bacteria. Tetrahymena increased the duration of Bsph and even reduced the period of its larvicidal activity. Tetrahymena used a sporocrystalline complex of Bsph as food and reduced its water concentration.

Fluorescent acid-fast microscopy (FAM) was used to enumerate intracellular Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum in the ciliated phagocytic protozoan Tetrahymena pyriformis. There was a linear relationship between FAM and colony counts of M. avium cells both from cultures and within protozoa. The Ziehl-Neelsen acid-fast stain could not be used to enumerate intracellular mycobacteria because uninfected protozoa contained acid-fast, bacterium-like particles. Starved, 7-day-old cultures of T. pyriformis transferred into fresh medium readily phagocytized M. avium, M. intracellulare, and M. scrofulaceum. Phagocytosis was rapid and reached a maximum in 30 min. M. avium, M. intracellulare, and M. scrofulaceum grew within T. pyriformis, increasing by factors of 4- to 40-fold after 5 days at 30 degrees C. Intracellular M. avium numbers remained constant over a 25-day period of growth (by transfer) of T. pyriformis. Intracellular M. avium cells also survived protozoan encystment and germination. The growth and viability of T. pyriformis were not affected by mycobacterial infection. The results suggest that free-living phagocytic protozoa may be natural hosts and reservoirs for M. avium, M. intracellulare, and M. scrofulaceum.

Lysozyme and antilysozyme activities present in a wide range of microorganisms determine the so-called lysozyme-antilysozyme system of hydrobionts, which greatly contribute to the formation of aquatic biocenoses. However, the mechanism of the functioning of this system in natural freshwater communities remains obscure. The experimental investigation of lysozyme-antilysozyme interactions in a model Tetrahymena--Escherichia community showed that the antilysozyme activity of Escherichia coli leads to incomplete phagocytosis, thus enhancing bacterial survival in a mixed culture with infusoria. The selection and reproduction of bacterial cells resistant to grazing by infusoria determine the character of host-parasite interactions and allow bacteria to survive. It was demonstrated that the antilysozyme activity of microorganisms, which is responsible for bacterial persistency in natural biocenoses, is involved in the maintenance of protozoa-bacteria communities in bodies of water.

The electron-microscopic study of the interaction of F. tularensis virulent and attenuated strains with infusoria of the species T. pyriformis was dynamically studied. In this study the structural changes of F. tularensis and T. pyriformis cells, as well as their capacity for survival, were revealed. The data on their ultrastructure correlated with the dynamics of the number of both F. tularensis and T. pyriformis: during the whole term of observation the tendency to a slow decrease in the number of F. tularensis was registered with the concentration of T. pyriformis remaining stable. The interaction of F. tularensis with T. pyriformis may be regarded as a variant of commensal, but not antagonistic interactions.

Spores of Bacillus thuringiensis subsp. israelensis and their toxic crystals are bioencapsulated in the protozoan Tetrahymena pyriformis, in which the toxin remains stable. Each T. pyriformis cell concentrates the spores and crystals in its food vacuoles, thus delivering them to mosquito larvae, which rapidly die. Vacuoles containing undigested material are later excreted from the cells. The fate of spores and toxin inside the food vacuoles was determined at various times after excretion by phase-contrast and electron microscopy as well as by viable-cell counting. Excreted food vacuoles gradually aggregated, and vegetative growth of B. thuringiensis subsp. israelensis was observed after 7 h as filaments that stemmed from the aggregates. The outgrown cells sporulated between 27 and 42 h. The spore multiplication values in this system are low compared to those obtained in carcasses of B. thuringiensis subsp. israelensis-killed larvae and pupae, but this bioencapsulation represents a new possible mode of B. thuringiensis subsp. israelensis recycling in nontarget organisms.

In this work isogenic E.coli strains K-12J53 and J53pAlz60, differing in the presence of the antilysozyme characteristic, were used. In the process of joint cultivation the degradation of lysozyme in protozoa by antilysozyme-active E.coli was established. The initial culture of antilysozyme-active E.coli K-12pAlz60 was found to be slightly heterogeneous with respect to the antilysozyme characteristic; this heterogeneity increased after the interaction of E.coli with protozoa. As the result of their joint cultivation with Tetrahymena, clones with low (2 micrograms/ml) antilysozyme activity (ALA) disappeared from the population and clones with medium and high values of ALA (3, 4, 5, 6 micrograms/ml) accumulated. The dynamics of the interaction of infusoria with Escherichia cells (ALA+) on the ultrastructural level in 1-6 and 24 hours revealed the gradual increase of processes leading to the destruction of most of the bacteria (up to their complete digestion) and, at the same time, the presence, observed also at an early period, of intact bacteria, resistant to lysis, whose survival was ensured by their antilysozyme characteristic.

In situ hybridization using fluorescent oligonucleotide probes complementary to unique regions of 16S rRNA molecules provides a way of identifying the food vacuole contents of bactivorous protists. Laboratory experiments with Tetrahymena showed rRNAs in food vacuoles are degraded slowly enough to permit their use as hybridization targets for such probes. A probe specific for a hypervariable region of the small subunit rRNA of an unnamed proteobacterium abundant in a local lake was then synthesized. It was used to probe the food vacuoles of the ciliates present in fixed water samples collected from the same lake. The vacuoles of several filter-feeding ciliates bound the probe, indicating that such probes can be used to identify the food vacuole contents of ciliates collected from natural samples.

All Legionella longbeachae strains, both serogroups of L. bozemanii, and three strains of L. anisa reproducibly infected washed Tetrahymena pyriformis at 30 degrees C. L. pneumophila serogroup 1 strains infected T. pyriformis less reproducibly than did L. longbeachae. Low-level concentrations of nutrients in cocultures inhibited infection. Four L. micdadei strains and L. anisa ATCC 35292 failed to infect T. 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.

Bacteria of the family Legionellaceae form a monophyletic group within the gamma-subclass of Proteobacteria. Based on comparative sequence analysis we constructed two oligonucleotide probes complementary to regions of 16S rRNA characteristic for Legionellaceae. Probe specificities were tested by whole-cell or dot-blot hybridization against 14 serogroups of Legionella pneumophila, 22 different Legionella spp. and 72 non-legionellae reference strains. Using optimized conditions both probes hybridized to all tested strains of L. pneumophila. Probes LEG226 and LEG705 hybridized to 71% and 90% of the Legionella species tested, respectively. With the exception of Methylomonas alba none of the non-target strains showed complete sequence homology within the target molecule. In a preliminary evaluation the results of classical techniques employing selective media, immunofluorescence and the probe assay were in good accordance for routine environmental and clinical isolates. L. pneumophila suspended in drinking water at approximately 10(3)-10(4) c.f.u. ml-1 could be rapidly detected by a combination of membrane filtration on polycarbonate filters and whole-cell hybridization. Even after incubation for 1 year a proportion of the released cells was still detectable. In situ hybridization also facilitated visualization of Legionella spp, cells in model biofilms. A combination of in situ hybridization and confocal laser scanning microscopy (CLSM) was used to analyse the three-dimensional arrangement of L. pneumophila within cells of the ciliated protozoan Tetrahymena pyriformis. Whole-cell probing with 16S rRNA-targeted oligonucleotides could, in the future, complement established techniques like immunofluorescence and PCR in ecological and epidemiological studies of Legionellaceae.

Types of interaction between P. aeruginosa and T. pyriformis have been studied. The study has revealed that phagocytizing protozoa are killed by P. aeruginosa in the process of their joint cultivation. The data obtained in this study indicate that in the process of interaction of these species phagocytosis-resistant cells are selected from heterogeneous microbial population. The use of this model is believed to be promising in the study of the fate of P. aeruginosa in abiotic and biotic environment.

Legionella pneumophila is an intracellular parasite of freshwater protozoa and human macrophages. Recent studies determined that the macrophage infectivity potentiator (Mip) surface protein, a prokaryotic homolog of the FK506-binding proteins, is required for optimal infection of macrophages. To determine whether Mip is also involved in L. pneumophila infection of protozoa, we examined the ability of a strain lacking Mip to parasitize Hartmannella amoebae and Tetrahymena ciliates. After 3 days of incubation, approximately 1000-fold fewer bacteria were recovered from protozoan cocultures infected with the Mip- strain than from those cocultures infected with an isogenic Mip+ strain. The mip mutant was, however, not impaired in its ability to bind to amoebae cell surfaces, indicating that Mip is involved in bacterial resistance to intracellular killing and/or intracellular multiplication. These data suggest that L. pneumophila employs similar genes and mechanisms to infect human cells and protozoa. Furthermore, they support the hypothesis that the ability of L. pneumophila to parasitize macrophages and hence to cause human disease is a consequence of its prior adaptation to intracellular growth within protozoa.

The mathematical model describing the dynamics of the growth of L. pneumophila in aqueous environment in the presence of protozoa has been worked out. The model has demonstrated considerable heterogeneity of the initial population of virulent L. pneumophila strains. The number of bacteria capable of multiplication in Infusoria is no more than 0.1% of the initial population. The time of the generation of the infective agent inside Tetrahymena pyriformis is 2.8 hours.

Y. enterocolitica and Y. pseudotuberculosis have been experimentally found to penetrate into plants (cabbage, salad, pea, oat) from infected soil and water and to remain in different plant organs (roots, seeds, leaves) up to 30 days (the term of observation). The processes of S-R dissociation in Yersinia cultures isolated from plants, as well as changes in some enzymatic properties, have been established. In the test on Infusoria the cytotoxic action of strains isolated from plants has been noted. The possible role of plants in carrying pathogenic bacteria from the soil to the surface ecological systems and the subsequent infection of animals and humans is discussed.

Between October 1987 and March 1989, we tested 144 water samples obtained from the plumbing and cooling tower systems of 5 Paris hospitals for the presence of legionellae and amoebae. Of the samples tested for Legionella, 67 out of 144 (46.5%) were positive, and 82 out of 116 tested for amoebae (70.7%) were positive. The ability of protozoa to support the multiplication of legionella was shown by incubating samples at 35.5 degrees C for 7-15 days. Prior to determining the presence of legionellae and amoebae, 51 of the 144 samples were incubated. After incubation, 22 out of 25 (88%) samples which were positive for the presence of both Legionella and amoebae showed multiplication of Legionella. In 3 out of the 25 (12%) samples containing Legionella and amoebae, Legionella failed to multiply. Six out of the 51 (11.8%) samples which were negative in direct culture for Legionella but positive for amoebae, became positive after incubation. Legionella did not multiply in samples negative for amoebae, nor was there proliferation in samples after filtration through a 1.2-microns membrane followed by incubation for the same period and temperature. Strains of Legionella pneumophila serogroup 1 and serogroup 6 (SG1 and SG6), including 3 patient isolates and 2 environmental isolates, were cocultured with 2 strains of amoebae and Tetrahymena pyriformis. Plate counts, Gimenez staining and electron microscopy demonstrated that intracellular legionellae proliferation occurred.

Selective changes in Y. enterocolitica virulence have been made in experiments on eukaryotic cells, Infusoria, and peritoneal macrophages, used as models. In parallel experiments with the passage of Yersinia through Infusoria and mammalian macrophages an analogy to the processes of phagocytosis has been established.

In this work the morphological features of the interaction of L. pneumophila virulent strain and T. pyriformis have been studied on the submicroscopic level in the time course of the process. The study has shown the process of the destruction of the bacterial population and the penetration of individual intact Legionella cells from the phagosome into the endoplasm of T. pyriformis after 6-9 hours of interaction in the form of the budding of the phagosome and further multiplication of Legionella in the endoplasm. As revealed in this study, T. pyriformis have two types of phagosomes characterized by different variants of the destruction of Legionella. In T. pyriformis lysosomes-like granules, mitochondria and the granular endoplasmatic network take part in the process of interaction. The process of interaction has been found to end by day 7 in the death of all protozoal cells taking part in interaction.

L. pneumophila avirulent strains have been shown to lose their capacity for multiplication in T. pyriformis, while the concentration of the virulent strain increases 1000-fold. The loss of the hemolytic activity of L. pneumophila virulent strain leads to the loss of its capacity for multiplication in infusorians.

The decisive role of biotic factors in the preservation of the viability of Y. pseudotuberculosis is shown. The results of experiments suggest that protozoa may serve as reservoir in the spread of Y. pseudotuberculosis infection.

In a series of experiments, we have determined that Legionella pneumophila will proliferate as an intracellular parasite of the ciliated holotrich Tetrahymena pyriformis in sterile tap water at 35 degrees C. After 7 days of incubation, serpentine chains of approximately 10(3) L. pneumophila cells were observed throughout the cytoplasm of the protozoan infected initially with 1 to 30 L. pneumophila cells. The overall L. pneumophila population increased from ca. 1.0 X 10(2) to ca. 5.0 X 10(4) cells per ml in the coculture within this time frame. The interactions between the protozoan and the bacterium appear to depend upon their concentrations as well as temperature of incubation. L. pneumophila did not multiply in sterile tap water alone, in suspensions of lysed T. pyriformis, or in cell-free filtrates of a T. pyriformis culture. In addition to establishing an ecological model, we found that addition of T. pyriformis to environmental specimens served as an enrichment method that improved isolation of legionella from the specimens.

Axenic cultures of Tetrahymena pyriformis, strain I MT IV, grown in a defined medium at room temperature, were used to study interactions of these protozoa with vaccination strain L Sc 2ab of poliovirus type 1, vaccination strain P 712 of poliovirus type 2 and with type 30 echovirus, strain 480/78. T. pyriformis cultures in media containing 10(3.0) TCD50/1 ml of type poliovirus, 10(3.0) TCD50/1 ml of type 2 poliovirus or 10(2.5) TCD50/1 ml echovirus 30 and in virus-free medium did not differ one from another in their growth and die-away kinetics during the 21 days of observation. Two-day T. pyriformis cultures were infected with poliovirus 1 (initial concentration 10(3.2) TCD50/1 ml), and poliovirus 2 and echovirus 30 (initial concentrations 10(3.0) TCD50/1 ml). Viruses were titrated in test tube cultures of BGM cells. The supernatant fluid, standardized sediment and samples of control virus suspension free of protozoa were titrated after 0, 2, 6, 10, 13, 18, 28 and 30 days. Most of the virus in culture was found associated with the sediment, both in the period of active growth and during the die-away phase of T. pyriformis protozoa. The virus in sediment was present at higher titres and its survival time was longer than in virus in liquid phase. Thirteen days after the first contact between T. pyriformis and virus the sediment and supernatant fluid of the old protozoan culture and the T. pyriformis-free control viral suspension were taken and used as inocula for new two-day T. pyriformis cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Newborn mice were infected intracerebrally with abacterial cultures of the GL strain of Tetrahymena pyriformis, induced experimentally six years ago in vitro with Coxsackie B-5 virus. In the brain of the test animals there were pathologic changes similar to those found in the primary investigation of the pathogenicity of this strain, i.e., after 96 h of contact with the virus. Thus, the pathogenicity acquired by T. pyriformis, as well as the persistence of Coxsackie B-5 virus in this ciliate, can be considered stable. Despite such specific changes in the biological properties of T. pyriformis, the changes were not reflected in the morphology of the protozoon, which was investigated by means of light and electron microscopy.