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PubMed1998

Interactions of "Limax amoebae" and gram-negative bacteria: experimental studies and review of current problems.

Free-living amoebae can harbour bacteria inside their cysts giving them a microhabitat and protecting them from disinfectants. The aim of this study was to evaluate the potential importance of "Limax amoebae" as vectors for environmental and nosocomial bacteria in a hospital. It was shown that free-living amoebae are ubiquitous in the investigated hospital, occur syntopically with facultative human pathogens (Comamonas acidovorans and Pseudomonas aeruginosa) and may serve as hosts not only for these but also for bacteria isolated from clinical specimens (Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa); temperature is apparently of crucial importance for the interactions between these microorganisms. Recent studies have shown that "Limax amoebae" apart from acting as protective hosts, may also play a role for the thermotolerance, invasiveness and antibiotic-resistance of bacteria. Considering also the reduced immune-status of many patients, this "symbiosis" of free-living amoebae and bacteria might still be of underestimated hospital-hygienic importance.

Acanthamoeba↗

PubMed2001

[Number, activity and thermostability of the electrophoretic forms of acid phosphatase in Amoeba proteus, cultured at different temperatures].

In free-living amoebae (Amoeba proteus, strain B), cultured at 10 and 25 degrees C, we compared the number, activity, and thermostability of separate electromorphs of Triton-soluble acid phosphatase (AcP) revealed by disc-electrophoresis in polyacrylamide gel using 2-naphthyl phosphate (pH 4.0) as a substrate. No differences in the number of AcP electromorphs and their mobility were observed at both these temperatures. The total activity of AcP electromorphas per unit of cellular protein and their total thermostability were lower in amoebae acclimated to 10 degrees C than to 25 degrees C. The above decrease may be a consequence of a simultaneous decrease in the activity and thermostability of two tartrate-sensitive electromorphs, both being of lysosomal nature. The total activity and thermostability of tartrate-resistant AcP electromorphs did not differ in amoebae acclimated to the two above temperatures. In amoebae cultured at 10 degrees C the fall of activity and thermostability of lysosomal AcP correlates with the decrease in their primary cell thermoresistance and phagocytic activity. The obtained results confirm the earlier conclusion (Vysotskaya et al., 1994) that lysosomes may be involved in acclimation of electrothermal animals to changing environmental temperatures.

Acid Phosphatase↗

PubMed1990

Characterization of potentially pathogenic free-living amoebae in sewage samples of Calcutta, India.

1. It is widely accepted that foul or polluted environments are the principal sources of potentially pathogenic species of free-living amoebae. The present paper is the first report of occurrence of potentially pathogenic free-living amoebae in sewage samples of Calcutta, India. 2. We describe the occurrence, isolation, specific identification and comparative mouse pathogenicity test of two pathogenic amoebae, viz., Naegleria fowleri (N. aerobia) Carter, 1970, causing human meningoencephalitis and Acanthamoeba castellanii Douglas, 1930, causing granulomatous amoebic encephalitis, and one non-pathogenic amoeba, viz., A. astronyxis Ray and Hayes, 1959, in sewage samples of Calcutta, India. 3. The existence of both pathogenic and non-pathogenic amoebae living side by side is of considerable epidemiological relevance.

Acanthamoeba↗

PubMedDate not supplied

[Amoebae in the water supply: an epidemiological study (author's transl)].

Drinking water samples (836) of various origins have been examined for amoebae. For collector water, half of the samples are contamined without influence of the origin. So, occurrence of amoeba in underground waters is discussed. Filtration and disinfection are generally inefficient. Bottles of drinking water are very often polluted during bottling. International mineral waters of french origin are not so damaged during conditioning operation. Bacteria and amoebae populations are quite independant and can't be indicators one for another. Thermophilic amoebae strains are very rare and any pathogenic amoeba has been found during the study.

Amoeba↗

PubMed1994

Microtubule centers and the interphase microtubule cytoskeleton in amoebae of the cellular slime molds (Mycetozoans) Acytostelium leptosomum and Protostelium mycophaga.

We investigated the microtubule (MT) cytoskeleton and microtubule centers (MTC) in undifferentiated amoebae by indirect immunofluorescence with six monoclonal antitubulin antibodies, and by transmission electron microscopy and immunogold ultracytochemistry. Interphase amoebae of both species contain a distinct cytoplasmic complex of MTs, which is more elaborate in Protostelium mycophaga. In Acytostelium leptosomum amoebae a single MTC is attached to each interphase nucleus at its pointed end, as in the other dictyostelid cellular slime molds Dictyostelium discoideum and Polysphondylium violaceum. Ultrastructurally, MTCs of A. leptosomum also resemble those of these two species: They consist of an electron-opaque core shaped like a stout rod, which is embedded, together with nodules, in a fuzzy matrix. The nodules are the points of origin of the MTs. In most amoebae of P. mycophaga there are two MTCs on opposite sides of and close to the nucleus, but many amoebae also contain a variable number of MTCs that are remote from the nucleus. Nucleus-associated and "remote" MTCs are structurally identical. They consist of a ring-shaped core with inner and outer diameters of ca. 130 nm and 340 nm. A plug sits in the ring, and satellites are connected to the core by fine fibrils. The satellites are the points of origin of MTs. New MTCs are apparently formed during mitosis, the parent MTC probably serving as a template for the genesis of a new ring. The results support the notion that phylogenetically related organisms have similarly constructed MTCs and that these are dissimilar in less closely related organisms.

Antibodies, Monoclonal↗

PubMed1980

Conformation of ribosomes from the vegetative amoebae and spores of Dictyoistelium discoideum.

Ribosomes from two different cell types of D. discoideum--the undifferentiated amoebae and the differentiated spores--display considerable differences in protein composition (Ramagopal and Ennis 1979 and manuscript in preparation). These differences do not affect the three-dimensional structure of monosomes and large (60S) and small (40S) subunits from the two cell types to an extent detectable by sedimentation analysis or electron microscopy. High resolution electron microscopic images of ribosomal particles from the amoebae and the spores are similar and, in general, comparable to that of 80S, 60S, and 40S ribosomal particles from other eukaryotic sources. However, distinct differences in the conformation and stability of the two types of ribosomes are detectable by circular dichroic spectroscopy. The degree of the ordered secondary structure of rRNAs is similar in the 80S monosomes from the amoebae and the spores, but higher in the amoeba subunits. The results of thermal melting experiments show that the small subunit from the spores is more stable than that from the amoebae. The established differences in the conformation of rRNAs, most probably due to the interactions with cell-specific ribosomal proteins, can be responsible for the differences in stability of ribosomes from the two cell types of slime mold.

Cell Cycle↗

PubMed2005

Seasonal changes in free-living amoeba species in the root canopy of Zygophyllum dumosum in the Negev Desert, Israel.

The influence of seasonality and Zygophyllum dumosum root canopy on the species diversity of free-living amoebae at two soil depths (0-10 and 10-20 cm) was studied in a Negev Desert ecosystem in Israel. Free-living amoebae were extracted and identified after cultivation in non-nutritive agar plates. A total of 90 amoeba species were identified in the soil during the study period, with the most common genera present being Hartmannella, Platyamoeba, Vahlkampfia, Acanthamoeba, and Echinamoeba. Differences between the control soil and the soil under Z. dumosum were found mainly during the dry seasons, when 97% similarity was found between the two soil layers, which could be due to the effect of the shrub on the soil microenvironment. The amoeba community exhibited more species diversity in spring (reaching a value of 34 species) than in the winter (18 species) or summer and autumn (20 species), since the community has a time lag for becoming stabilized after the dry summer and autumn. This is one of the first studies on the amoeba population in the Negev Desert and elucidates the importance and the need for taking trophic and functional groups into consideration in order to understand biomineralization processes.

Amoebida↗

PubMed1984

Morphological changes and depressed phagocytic efficiency in Dictyostelium amoebae treated with toxic concentrations of cadmium.

The morphology and phagocytic efficiency of Dictyostelium discoideum amoebae exposed to cadmium was investigated at two Cd concentrations: a low toxic concentration--7 X 10(-5) M, and a high toxic concentration--2 X 10(-4) M. Both concentrations inhibited growth completely; however, only in the culture containing a high toxic concentration of cadmium were severe ultrastructural anomalies observed, notably, nucleolar changes and autophagic vacuolar formation. Using biological indices we conclude that the high concentration of cadmium was lethal and that morphological changes associated with this dose of cadmium may be secondary to cell death. In contrast, amoebae treated with a low toxic but nonlethal concentration of Cd showed an altered size distribution of cytoplasmic vacuoles and a decreased phagocytic efficiency. Cultures whose growth was completely inhibited with cobalt were also examined, as were untreated control cultures. By 24 hr Cd-treated amoebae showed a 20% decrease in the cytoplasmic mean-vacuolar diameter and a 69% decrease in phagocytic efficiency whereas Co and untreated controls showed no significant decrease in the cytoplasmic mean-vacuolar diameter. Phagocytic efficiency was only slightly diminished by Co. Changes in vacuolar profiles had been shown earlier to be related to membrane utilization in Dictyostelium amoebae. Cd at low toxic concentrations affects membrane function in Dictyostelium amoebae.

Cadmium↗

PubMedDate not supplied

Kinetics of endosomal acidification in Dictyostelium discoideum amoebae. 31P-NMR evidence for a very acidic early endosomal compartment.

We have examined the pH of the various endosomal compartments in the amoebae of the cellular slime mould Dictyostelium discoideum. This was accomplished both by fluorescence and by in vivo 31P-NMR methods. The fluid-phase marker, fluorescein-labeled dextran, was fed to the amoebae to report the average pH of their endocytic vesicles. During the progressive loading of successive endosomal compartments, we observed an early acidification down to a minimum value of pH < or = 5.3 after 30 min at 20 degrees C followed by an increase to an average pH of 5.8 when all the endosomal compartments were loaded by the fluid-phase marker. The weak fluorescence intensity of FITC-dextran at acidic pH precluded a more detailed investigation and we checked various phosphonate compounds as potential 31P-NMR pH probes for the endosomal compartments. Two molecules, aminomethylphosphonate and 2-aminoethylphosphonate, were selected for this study because of the large amplitudes of their chemical shift variation with pH (2 and 2.5 ppm, respectively) and their acidic pKs of 5.5 and 6.3, respectively. They were only moderately toxic (IC50% approximately 10 mM) towards both the axenic growth and the differentiation program of Dictyostelium amoebae. Internalization of the two aminophosphonates occurred only through the fluid-phase pinocytosis pathway as revealed by the full inhibition of their entry with 1 mM vanadate or 7.5 mM caffeine, two previously characterized inhibitors of endocytosis in Dictyostelium. We found that in vivo 31P-NMR of amoebae suspensions incubated with the aminophosphonates allowed the detection of three distinct intracellular compartments at pH 4.3, 5.8-6.0 and 7.3. Kinetics of aminophosphonate entry were analyzed and the results allowed us to reconstruct the time course for the acidification sequence during endocytosis. The data are consistent with the hypothesis that in Dictyostelium amoebae phosphonates occupy a highly acidic early endosomal compartment (t1/2 = 18 min; pH 4.3) before reaching a less acidic late endosomal/prelysosomal compartment (pH 5.8-6.0) from where they are immediately transported to, and trapped in, the cytoplasm (pH 7.3).

Acids↗

PubMed2004

Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals.

Knowledge that free-living amoebae are capable of causing human disease dates back some 50 years, prior to which time they were regarded as harmless soil organisms or, at most, commensals of mammals. First Naegleria fowleri, then Acanthamoeba spp. and Balamuthia mandrillaris, and finally Sappinia diploidea have been recognised as etiologic agents of encephalitis; Acanthamoeba spp. are also responsible for amoebic keratitis. Some of the infections are opportunistic, occurring mainly in immunocompromised hosts (Acanthamoeba and Balamuthia encephalitides), while others are non-opportunistic (Acanthamoeba keratitis, Naegleria meningoencephalitis, and cases of Balamuthia encephalitis occurring in immunocompetent humans). The amoebae have a cosmopolitan distribution in soil and water, providing multiple opportunities for contacts with humans and animals, as evidenced by antibody titers in surveyed human populations. Although, the numbers of infections caused by these amoebae are low in comparison to other protozoal parasitoses (trypanosomiasis, toxoplasmosis, malaria, etc.), the difficulty in diagnosing them, the challenge of finding optimal antimicrobial treatments and the morbidity and relatively high mortality associated with, in particular, the encephalitides have been a cause for concern for clinical and laboratory personnel and parasitologists. This review presents information about the individual amoebae: their morphologies and life-cycles, laboratory cultivation, ecology, epidemiology, nature of the infections and appropriate antimicrobial therapies, the immune response, and molecular diagnostic procedures that have been developed for identification of the amoebae in the environment and in clinical specimens.

Amebiasis↗

PubMed2006

[Intra-amoebal development of Legionella pneumophila and the potential role of amoebae in the transmission of Legionnaires' disease].

Legionnaires' disease is one of the major infectious risks related to hospital water systems. It is commonly accepted, that the disease is transmitted to man mostly by inhalation of water aerosols contaminated by Legionella pneumophila. The ability of L. pneumophila to multiply intracellularly within some amoebae better explains the ecology, the pathogenicity, and the virulence of this bacterium against human alveolar macrophages. The presence of these amoebae in water systems located where cases of Legionnaire's disease broke out, partly explains the difficulty in eradicating Legionella. Some studies also show that amoebae can play a major role in the transmission of the disease to man. Some other studies point out that inhaled amoebae could be involved in the pathogenesis of Legionnaire's disease. Future strategies to prevent the transmission of Legionella will probably have to include efficient treatments against amoebae.

Acanthamoeba↗

PubMed1990

Relationship between free amoeba and Legionella: studies in vitro and in vivo.

In 1980, Robowtham demonstrated that Legionella multiplies in free amoeba cytoplasm and hypothesized that the amoeba could act as a reservoir of virulent bacteria. In this paper we report various aspects of the relationship between amoeba and Legionella. A liquid medium co-culture method was applied to Acanthamoeba sp. and Legionella pneumophila serogroup 1. Within 4 days, Legionella growth increased by 2 log s CFU/ml. Using a direct immunofluorescence assay and electron microscopy, Legionella was shown to grow abundantly inside phagosomes, and bacteria and/or antigen were present on the cytoplasmic membrane of the amoeba. These aspects are very similar to those observed with Legionella-infected alveolar macrophages. The morphology and structure of Legionella cells were modified after 20 days of co-culture: - viable bacteria showed large fatty cytoplasmic inclusions, - gas liquid chromatography analysis demonstrated a decrease in the i16:0 fatty acid ratio. Cystic forms of amoeba were abundant but none contained viable Legionella. In an in-vivo study using a guinea-pig aerosol infection model, we compared the virulence of Legionella in co-culture with Legionella grown on charcoal dialysed yeast extract (CDYE) agar medium. The Legionella obtained by co-culture had an LD 50 (50% lethal dose) similar to that obtained for those grown on CDYE, showing that bacterial virulence is preserved in the cellular model.

Acanthamoeba↗

PubMed2000

Co-incubation of Acanthamoeba castellanii with strains of Pseudomonas aeruginosa alters the survival of amoeba.

Enhanced survival of Acanthamoeba castellanii has previously been reported following co-incubation with a single strain of Pseudomonas aeruginosa. The aim of this study was to evaluate the impact of different strains of P. aeruginosa on amoebae survival. Four contact lens solutions were challenged with A. castellanii for between 6 and 24 h, and survival rates of amoeba were calculated. Subsequently, A. castellanii was co-incubated with different strains of P. aeruginosa (strain 6294, an invasive isolate; 6206, a cytotoxic isolate; and Paer 001, a null isolate). Differences in amoeba survival over time between solutions for each bacterial strain were analysed. Non-neutralized hydrogen peroxide was the most effective system against A. castellani at all time points (P<0.05). Survival rates were not different between multipurpose solutions and neutralized hydrogen peroxide. Co-incubation with P. aeruginosa altered amoeba survival, and maximum survival occurred in the presence of the invasive strain of P. aeruginosa. Enhanced amoeba survival may occur in the presence of certain strains of Gram-negative bacteria, and with certain types of contact lens disinfection systems.

Acanthamoeba↗

PubMed2006

Detection of antibodies against free-living amoebae Balamuthia mandrillaris and Acanthamoeba species in a population of patients with encephalitis.

BACKGROUND: Balamuthia mandrillaris and Acanthamoeba species are 2 free-living amoebae responsible for granulomatous amoebic encephalitis in humans and animals. We have screened serum samples from hospitalized patients with encephalitis for antibodies against these 2 amoebae as a means of detecting a disease with few defining symptoms and a poor prognosis. METHODS: Indirect immunofluorescence antibody (IFA) staining of serum samples from patients with encephalitis was conducted over a period of 6 years to detect amoeba antibodies. More than 250 serum samples from patients hospitalized with encephalitis were screened. Most of the samples were from patients in California and were screened as part of the California Encephalitis Project, with a small number of specimens from other states. RESULTS: During the course of the study, 7 cases of Balamuthia encephalitis were detected; all cases were detected in Hispanic individuals, and all cases were fatal. Examination of hematoxylin-eosin-stained and immunostained sections of brain tissue obtained at biopsy or autopsy for amoebae confirmed balamuthiasis in all serum samples with positive IFA results. One case of Acanthamoeba encephalitis was detected in an immunocompromised individual with a normal antibody titer by identification of amoebae in immunostained brain tissue obtained at autopsy. CONCLUSIONS: IFA can be successfully used in screening for balamuthiasis and acanthamoebiasis in patients whose clinical presentation, laboratory results, and neuroimaging findings are suggestive of amoebic encephalitis. Ideally, this can lead to an earlier definitive diagnosis and earlier start of antimicrobial therapy. Without IFA staining, the balamuthiasis cases in our study would have been diagnosed as neurocysticercosis, tumor, tuberculosis, or viral encephalitis or would have been undiagnosed.

Adolescent↗

PubMed2006

Amoebae promote persistence of epidemic strains of MRSA.

The control of healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) infection is of concern worldwide. Given the evidence that several pathogenic species replicate within amoebae and emerge more virulent and more resistant and the abundance of amoebae in healthcare settings, we investigated interactions of Acanthamoeba polyphaga with epidemic MRSA isolates. MRSA proliferated in the presence of amoebae, attributable partly to intracellular replication. Following 24 h of co-culture, confocal microscopy revealed that c. 50% amoebae had viable MRSA within phago-lysosomes and 2% of amoebae were heavily infected with viable cocci throughout the cytoplasm. Infection control strategies should recognize the contribution of protozoa.

Acanthamoeba↗

PubMedDate not supplied

Identification of amoebae implicated in the life cycle of Pfiesteria and Pfiesteria-like dinoflagellates.

This study was undertaken to assess whether amoebae commonly found in mesohaline environments are in fact stages in the life cycles of Pfiesteria and Pfiesteria-like dinoflagellates. Primary isolations of amoebae and dinoflagellates were made from water and sediment samples from five tributaries of the Chesapeake Bay. Additional amoebae were also cloned from bioassay aquaria where fish mortality was attributed to Pfiesteria. Electron microscopy and small subunit (SSU) rRNA gene sequence analysis of these isolates clearly demonstrated that the commonly depicted amoeboid form of Pfiisteria is very likely a species of Korotnevella and is unrelated to Pfiesteria or Pfiesteria-like dinoflagellates. We have determined that the Pfiesteria and Pfiesteria-like dinoflagellates examined in this study undergo a typical homothallic life cycle without amoeboid stages. Furthermore, we have demonstrated that cloned amoebae sharing morphological characteristics described for stages in the life cycle of Pfiesteria do not transform into dinozoites. The strict clonal isolation and cultivation techniques used in this study substantially support the conclusion that the amoebae and some of the flagellates depicted in the life cycle of Pfiesteria are environmental contaminants of the Pfiesteria culture system and that the Ambush Predator Hypothesis needs to be rigorously reevaluated.

Animals↗

PubMed1977

Limax amoebae in public swimming pools of albany, schenectady, and rensselaer counties, new york: their concentration, correlations, and significance.

A survey was conducted on 30 halogenated public swimming pools, located in Albany, Schenectady, and Rensselaer counties, to determine their open-water limax amoeba densities. Six were outdoor pools. Other variables measured were the standard plate count, total seston, free residual chlorine or bromine, total alkalinity, total hardness, orthophosphate, total soluble phosphorus, specific conductance, pH, temperature, and several engineering parameters including the rate and type of filtration as well as a saturation index. Amoebae were isolated on agar plates at 37 degrees C using heat-killed bacterial suspensions of Enterobacter cloacae or Escherichia coli. Most probable number estimates of amoebic densities ranged from not detectable (<0.01) to 110 amoebae per liter. The median concentration of amoebae was 0.9/liter. Eighty percent of the pools examined had less than 5 amoebae per liter. Significant correlations (P < 0.05) were found between amoebic densities and the log(10) of the standard plate count, orthophosphate, and total soluble phosphorus. No significant difference was found between amoebic densities in outdoor and indoor pools. Preliminary tests for the presence of the human pathogen Naegleria fowleri were inconclusive.

Journal Article↗

PubMed1995

Cocultivation of the amoeba Naegleria fowleri and the amoebicin- producing strain Bacillus licheniformis M-4.

Antagonism between Bacillus licheniformis M-4 and the pathogenic amoeba Naegleria fowleri HB-1 during cocultivation was influenced by the composition of the medium and the initial amoeba/bacterium ratio. While a ratio of 50 caused complete lysis of amoebae in soil extract with 0.3% glucose (SEG) before 72 h, this ratio had to be at least 12-fold lower in order to obtain similar results in Cline medium. Sporulation of B. licheniformis M-4 took place much earlier in SEG. Amoebicin production was stimulated by the presence of amoebae by either shortening the time of production (as in SEG) or increasing the amount of amoebicins released (as in Cline medium). Electron microscopy showed that amoebae cocultivated in the Cline medium contained bacteria enclosed in digestive vacuoles, while amoebae from SEG cocultures did not.

Amebicides↗