The ultrastructural characteristics of the interaction of Legionella pneumophila with the infusorian protozoon Tetrahymena pyriformis

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.     

Inoculum size as a factor limiting success of inoculation for biodegradation

A study was conducted to determine the role of inoculum size of a bacterium introduced into nonsterile lake water in the biodegradation of a synthetic chemical. The test species was a strain of Pseudomonas cepacia able to grow on and mineralize 10 ng to 30 micrograms of p-nitrophenol (PNP) per ml in salts solution. When introduced into water from Beebe Lake at densities of 330 cells per ml, P. cepacia did not mineralize 1.0 microgram of PNP per ml. However, PNP was mineralized in lake water inoculated with 3.3 X 10(4) to 3.6 X 10(5) P. cepacia cells per ml. In lake water containing 1.0 microgram of PNP per ml, a P. cepacia population of 230 or 120 cells per ml declined until no cells were detectable at 13 h, but when the initial density was 4.3 X 10(4) cells per ml, sufficient survivors remained after the initial decline to multiply at the expense of PNP. The decline in bacterial abundance coincided with multiplication of protozoa. Cycloheximide and nystatin killed the protozoa and allowed the bacterium to multiply and mineralize 1.0 microgram of PNP, even when the initial P. cepacia density was 230 or 360 cells per ml. The lake water contained few lytic bacteria. The addition of KH2PO4 or NH4NO3 permitted biodegradation of PNP at low cell densities of P. cepacia. We suggest that a species able to degrade a synthetic chemical in culture may fail to bring about the same transformation in natural waters, because small populations added as inocula may be eliminated by protozoan grazing or may fail to survive because of nutrient deficiencies.     

Inoculum size as a factor limiting success of inoculation for biodegradation.

A study was conducted to determine the role of inoculum size of a bacterium introduced into nonsterile lake water in the biodegradation of a synthetic chemical. The test species was a strain of Pseudomonas cepacia able to grow on and mineralize 10 ng to 30 micrograms of p-nitrophenol (PNP) per ml in salts solution. When introduced into water from Beebe Lake at densities of 330 cells per ml, P. cepacia did not mineralize 1.0 microgram of PNP per ml. However, PNP was mineralized in lake water inoculated with 3.3 X 10(4) to 3.6 X 10(5) P. cepacia cells per ml. In lake water containing 1.0 microgram of PNP per ml, a P. cepacia population of 230 or 120 cells per ml declined until no cells were detectable at 13 h, but when the initial density was 4.3 X 10(4) cells per ml, sufficient survivors remained after the initial decline to multiply at the expense of PNP. The decline in bacterial abundance coincided with multiplication of protozoa. Cycloheximide and nystatin killed the protozoa and allowed the bacterium to multiply and mineralize 1.0 microgram of PNP, even when the initial P. cepacia density was 230 or 360 cells per ml. The lake water contained few lytic bacteria. The addition of KH2PO4 or NH4NO3 permitted biodegradation of PNP at low cell densities of P. cepacia. We suggest that a species able to degrade a synthetic chemical in culture may fail to bring about the same transformation in natural waters, because small populations added as inocula may be eliminated by protozoan grazing or may fail to survive because of nutrient deficiencies.     

Interactions of Pseudomonas Aeruginosa Hemagglutinins With Euglena Gracilis, Chlamydomonas Reinhardi, and Tetrahymena Pyriformis

SYNOPSIS The galactosephilic and mannosephilic hemagglutinins of Pseudomonas aeruginosa adsorbed onto Euglena gracilis, Chlamydomonas reinhardi , and Tetrahymena pyriformis . Furthermore, peroxidase binding to the 3 protozoan species was shown to be mediated by these lectins. Binding of Pseudomonas lectins to E. gracilis and C. reinhardi caused their specific agglutination, whereas no agglutination was observed with T. pyriformis , even after treatment by papain or by NaF. Added to the culture medium, the Pseudomonas hemagglutinins stimulated growth of E. gracilis and T. pyriformis due to their binding to these protozoa: this effect was partly inhibited by the specific sugar.     

Interactions of pseudomonas aeruginosa hemagglutinins with Euglena gracilis, Chlamydomonas reinhardi, and Tetrahymena pyriformis

The galactosephilic and mannosephilic hemagglutinins of Pseudomonas aeruginosa adsorbed onto Euglena gracilis, Chlamydomonas reinhardi, and Tetrahymena pyriformis. Furthermore, peroxidase binding to the 3 protozoan species was shown to be mediated by these lectins. Binding of Pseudomonas lectins to E. gracilis and C. reinhardi caused their specific agglutination, whereas no agglutination was observed with T. pyriformis, even after treatment by papain or by NaF. Added to the culture medium, the Pseudomonas hemagglutins stimulated growth of E. gracilis and T. pyriformis due to their binding to these protozoa; this effect was partly inhibited by the specific sugar.     

Survival of coliforms and bacterial pathogens within protozoa during chlorination.

The susceptibility of coliform bacteria and bacterial pathogens to free chlorine residuals was determined before and after incubation with amoebae and ciliate protozoa. Viability of bacteria was quantified to determine their resistance to free chlorine residuals when ingested by laboratory strains of Acanthamoeba castellanii and Tetrahymena pyriformis. Cocultures of bacteria and protozoa were incubated to facilitate ingestion of the bacteria and then were chlorinated, neutralized, and sonicated to release intracellular bacteria. Qualitative susceptibility of protozoan strains to free chlorine was also assessed. Protozoa were shown to survive and grow after exposure to levels of free chlorine residuals that killed free-living bacteria. Ingested coliforms Escherichia coli, Citrobacter freundii, Enterobacter agglomerans, Enterobacter cloacae, Klebsiella pneumoniae, and Klebsiella oxytoca and bacterial pathogens Salmonella typhimurium, Yersinia enterocolitica, Shigella sonnei, Legionella gormanii, and Campylobacter jejuni had increased resistance to free chlorine residuals. Bacteria could be cultured from within treated protozoans well after the time required for 99% inactivation of free-living cells. All bacterial pathogens were greater than 50-fold more resistant to free chlorine when ingested by T. pyriformis. Escherichia coli ingested by a Cyclidium sp., a ciliate isolated from a drinking water reservoir, were also shown to be more resistant to free chlorine. The mechanism that increased resistance appeared to be survival within protozoan cells. This study indicates that bacteria can survive ingestion by protozoa. This bacterium-protozoan association provides bacteria with increased resistance to free chlorine residuals which can lead to persistence of bacteria in chlorine-treated water. We propose that resistance to digestion by predatory protozoa was an evolutionary precursor of pathogenicity in bacteria and that today it is a mechanism for survival of fastidious bacteria in dilute and inhospitable aquatic environments.     

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.     

Nucleic Acid Enzyme Studies of Nonfermentative Gram-Negative Bacteria Using Thin-Layer Chromatography

A rapid and easy to perform procedure for determining the nucleic acid enzyme reactions of intact bacterial cells was developed. Overnight organism growth on triple sugar agar was tested for nucleoside phosphotransferase, nucleosidase, and nucleotidase activity. Reaction products were detected by means of thin-layer chromatography and fluorescence. Characteristic patterns were seen with certain strains of nonfermentative gram-negative bacteria, which indicated that these tests could aid in classification and identification of E0-1 group, Pseudomonas multivorans, P. cepacia, P. maltophilia, certain other Pseudomonas species, and Herellea vaginicola.     

Poliovirus and echovirus survival in Tetrahymena pyriformis culture in vivo

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)     

Predation of bacteria by the protozoa Tetrahymena pyriformis in toluene-degrading cultures

The influence of the toluene concentration on predation of toluene-degrading bacteria by the protozoa Tetrahymena pyriformis was investigated in suspended batch cultures continuously aerated with toluene-contaminated air. At gas phase concentrations of 0.035 to 0.74 g m–3, toluene did not significantly affected protozoan activity and the final bacteria concentration was reduced by growing protozoa by 98 to 99.9% compared to protozoa-free controls. As the toluene concentration was increased to 1.16–1.33 g m–3, the reduction of the bacteria cell concentration was 80%. At 3.35 g toluene m–3, growth of T. pyriformis was completely inhibited. Overall, the results presented herein demonstrate that protozoa grazing on bacteria play a major role in controlling bacterial cell concentration, but that the toxicity of the treated pollutants to the protozoa is an important factor that needs to be taken into account in biological treatment processes.     

Fluorescent acid-fast microscopy for measuring phagocytosis of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum by Tetrahymena pyriformis and their intracellular growth

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.     

Histone-antihistone interactions in the Escherichia coli-Tetrahymena pyriformis association

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.     

Antibiotic activity and siderophores of Pseudomonas cepacia

The ability of 46 strains of Pseudomonas cepacia to inhibit phytopathogenic fungi and the effect of iron on their antifungal activity were studied. The antifungal effect of the bacteria and the antimicrobial activity of their crude yellow and violet pigments showed a 4-5-fold decrease in the presence of Fe(III). The addition of 100 micrograms/ml of FeCl3 to the medium decreased the biosynthesis of violet and yellow pigments; the complex of the yellow pigment with Fe(III) promoted the growth of the P. cepacia producing strain under iron-deficient conditions. The data obtained suggest a participation of some P. cepacia pigments in iron transport. The resistance of the P. cepacia strains to the synthetic chelating agents hydroxyethylenediphosphonic and diethylenediaminepentaacetic acids was demonstrated, which may indicate a high Fe(III)-binding constant of P. cepacia siderophores.     

A mathematical model of the growth of a population of Legionella pneumophila in the presence of Tetrahymena pyriformis 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.     

Activation of the lac genes of Tn951 by insertion sequences from Pseudomonas cepacia.

We have identified three transposable gene-activating elements from Pseudomonas cepacia on the basis of their abilities to increase expression of the lac genes of the broad-host-range plasmid pGC91.14 (pRP1::Tn951). When introduced into auxotrophic derivatives of P. cepacia 249 (ATCC 17616), this plasmid failed to confer the ability to utilize lactose. The lac genes of Tn951 were poorly expressed in P. cepacia and were not induced by isopropyl-beta-D-thiogalactopyranoside. Lac+ variants of the pGC91.14-containing strains which formed beta-galactosidase at high constitutive levels as a consequence of transposition of insertion sequences from the P. cepacia genome to sites upstream of the lacZ gene of Tn951 were isolated. Certain of the elements also increased gene expression in other bacteria. For example, IS407 strongly activated the lacZ gene of Tn951 in Pseudomonas aeruginosa and Escherichia coli, and IS406 (but not IS407) did so in Zymomonas mobilis. The results indicate that IS elements from P. cepacia have potential for turning on the expression of foreign genes in a variety of gram-negative bacteria.     

The ultrastructural characteristics of Francisella tularensis interaction with Tetrahymena pyriformis

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.     

Trichloroethylene Degradation by Toluene-Oxidizing Bacteria Grown on Non-aromatic Substrates

The potential of trichloroethylene (TCE) to induce and non-aromatic growth substrates to support TCE degradation in five strains (Pseudomonas mendocina KR1, Ralstonia pickettii PKO1, Pseudomonas putida F1, Burkholderia cepacia G4, B. cepacia PR1) of toluene-oxidizing bacteria was examined. LB broth and acetate did not support TCE degradation in any of the wild-type strains. In contrast, fructose supported the highest specific levels of TCE oxidation observed in each of the strains tested, except B. cepacia G4. We discuss the potential mechanisms and implications of this observation. In particular, cells of P. mendocina KR1 degraded significant amounts of TCE during cell growth on non-aromatic substrates. Apparently, TCE degradation was not completely constrained by any given factor in this microorganism, as was observed with P. putida F1 (TCE was an extremely poor substrate) or B. cepacia G4 (lack of oxygenase induction by TCE). Our results indicate that multiple physiological traits are required to enable useful TCE degradation by toluene-oxidizing bacteria in the absence of aromatic cosubstrates. These traits include oxygenase induction, effective TCE turnover, and some level of resistance to TCE mediated toxicity.     

The phagosome containing Legionella pneumophila within the protozoan Hartmannella vermiformis is surrounded by the rough endoplasmic reticulum.

Legionella pneumophila is an intracellular parasite of protozoa and human phagocytes. To examine adaptation of this bacterium to parasitize protozoa, the sequence of events of the intracellular infection of the amoeba Hartmannella vermiformis was examined. The previously described uptake phenomenon of coiling phagocytosis by human monocytes was not detected. A 1 h postinfection with wild-type strain AA100, mitochondria were observed within the vicinity of the phagosome. At 2.5 h postinfection, numerous vesicles surrounded the phagosomes and mitochondria were in close proximity to the phagosome. At 5 h postinfection, the bacterium was surrounded by a ribosome-studded multilayer membrane. Bacterial multiplication was evident by 8 h postinfection, and the phagosome was surrounded by a ribosome-studded multilayer membrane until 15 h postinfection. The recruitment of organelles and formation of the ribosome-studded phagosome was defective in an isogenic attenuated mutant of L. pneumophila (strain AA101A) that failed to replicate within amoebae. At 20 h postinfection with wild-type strain AA100, numerous bacteria were present in the phagosome and ribosome were not detected around the phagosome. These data showed that, at the ultrastructural level, the intracellular infection of protozoa by L. pneumophila is highly similar to that of infection of macrophages. Immunocytochemical studies provided evidence that at 5 h postinfection the phagosome containing L. pneumophila acquired an abundant amount of the endoplasmic reticulum-specific protein (BiP). Similar to phagosomes containing heat-killed wild-type L. pneumophila, the BiP protein was not detectable in phagosomes containing the mutant strain AA101A. In addition to the absence of ribosomes and mitochondria, the BiP protein was not detected in the phagosomes at 20 h postinfection with wild-type L. pneumophila. The data indicated that the ability of L. pneumophila to establish the intracellular infection of amoebae is dependent on its capacity to reside and multiply within a phagosome surrounded by the rough endoplasmic reticulum. This compartment may constitute a rich source of nutrients for the bacteria and is probably recognized as cellular compartment. The remarkable similarity of the intracellular infections of macrophages and protozoa by L. pneumophila strongly supports the hypothesis that adaptation of the bacterium to the intracellular environment of protozoa may be the mechanism for its ability to adapt to the intracellular environment of human alveolar macrophages and causes pneumonia.     

松材线虫对其携带的细菌繁殖的影响

1.引言松材线虫病（Bursaphelenchus xylophilus）是松树的一种毁灭性病害,在日本、中国、韩国和北美、尼日利亚和葡萄牙等国家蔓延,造成了巨大经济损失,其中以日本和中国受害最重.一直认为松材线虫是引起该病的唯一病原,但近十几年来的研究发现,细菌在致病过程中可能起着重要作用,相继从病木和松材线虫体上分离到能对黑松苗有致萎活性的细菌.赵博光等首次根据实验提出松材线虫病是线虫和细菌共同侵染引起的复合侵染病害的假说,并在以后的试验中得到了验证.关于松材线虫对其细菌繁殖的影响研究鲜有报道.本试验采用从感病松树上分离并鉴定了的细菌菌株中选取假单胞属7株、其它属的细菌菌株3株,     

Protozoan growth rates on secondary-metabolite-producing Pseudomonas spp. correlate with high-level protozoan taxonomy

Different features can protect bacteria against protozoan grazing, for example large size, rapid movement, and production of secondary metabolites. Most papers dealing with these matters focus on bacteria. Here, we describe protozoan features that affect their ability to grow on secondary-metabolite-producing bacteria, and examine whether different bacterial secondary metabolites affect protozoa similarly. We investigated the growth of nine different soil protozoa on six different Pseudomonas strains, including the four secondary-metabolite-producing Pseudomonas fluorescens DR54 and CHA0, Pseudomonas chlororaphis MA342 and Pseudomonas sp. DSS73, as well as the two nonproducers P. fluorescens DSM50090(T) and P. chlororaphis ATCC43928. Secondary metabolite producers affected protozoan growth differently. In particular, bacteria with extracellular secondary metabolites seemed more inhibiting than bacteria with membrane-bound metabolites. Interestingly, protozoan response seemed to correlate with high-level protozoan taxonomy, and amoeboid taxa tolerated a broader range of Pseudomonas strains than did the non-amoeboid taxa. This stresses the importance of studying both protozoan and bacterial characteristics in order to understand bacterial defence mechanisms and potentially improve survival of bacteria introduced into the environment, for example for biocontrol purposes.