Monoxenic and axenic cultivation of carrier and patient strains of Entamoeba histolytica.

All of five strains of Entamoeba histolytica, isolated from symptomatic cases of amoebiasis, could be adapted to axenic growth on the TP-S-1 medium of Diamond (1968). Four axenic strains were started from amoeba-Crithidia cultures; one could be axenized directly after isolation from a case of cutaneous amoebiasis. Attempts to monoxenize, resp. axenize strains, isolated from Dutch, asymptomatic carriers, were less successful. Only three out of ten strains could be submitted to bacteria-free growth. These three strains, however, originated probably from a recent case of intestinal amoebiasis. The results, suggesting that highly virulent strains can be easier cultivated bacteria-free than those with low or no virulence, are further discussed. The yield of axenic amoebae per tube fluctuates largely depending on many factors such as the strain, the number of transfers (i.e. degree of establishment), the quality of Panmede liver digest and serum in the TP-S-1 medium, and the care of manipulating the cultures. For optimal growth, a more acid medium was required in an amoeba-Crithidia culture than in an axenic culture. Multinucleated, giant amoebae were frequently observed in axenic cultures.     

Entamoeba histolytica: concurrent irreversible loss of infectivity-pathogenicity and encystment potential after prolonged maintenance in axenic culture in vitro

The NIH-200 strain of Entamoeba histolytica became avirulent after more than 2 yr maintenance in axenic culture in vitro. In an attempt to restore virulence to the amoeba, it was transferred to Locke's egg rice-flour medium with various combinations of the following bacteria: Bacteroides sp., Clostridium perfringens, Escherichia coli, and Streptococcus faecalis. Similar cultures were established with a mixed bacterial flora (comprising many unknown species), with and without rice flour, and an attempt was made to induce encystation. Subsequent inoculation of amoebae from the various amoeba-bacteria cultures into the cecum of germfree and exgermfree guinea pigs harboring the same bacteria, as the culture-produced inoculum did not in any instance produce amoebic lesions or prolonged amoebic infections of the enteric lumen. All attempts to induce encystation were unsuccessful; the amoeba had lost its encystment potential, and this was believed to be intimately related to the irreversible loss of virulence.     

Survival of Entamoeba and related amoebae at low temperature-II. Viability of amoebae and cysts stored in liquid nitrogen

It was found that E. histolytica, E. histolytica-like, E. hartmanni, E. invadens, E. terrapinae, E. moshkovskii grown with a mixed bacterial flora, could be recovered after prolonged storage in liquid nitrogen. The longest period yet tested for E. histolytica is 382 weeks (7·3 years). Storage of amoebae of E. ranarum and E. coli was less successful. E. histolytica amoebae grown axenically or monoxenically were less easily stored than those amoebae grown with a mixed bacterial flora. Cysts were non-viable after freezing.E. histolytica amoebae showed the same virulence to rats and sensitivity to emetine after storage in liquid nitrogen, as was observed before freezing.A summary of a recommended procedure for freezing Entamoeba and related amoebae is given.     

Effect of phosphatidylcholine-cholesterol liposomes on Entamoeba histolytica virulence

Trophozoites of Entamoeba histolytica HM-1:IMSS become less virulent after long-term maintenance in axenic cultures. The factors responsible for the loss of virulence during in vitro cultivation remain unclear. However, it is known that in vitro cultivation of amoeba in culture medium supplemented with cholesterol restores their virulence. In this study, we analyzed the effect of adding phosphatidylcholine-cholesterol (PC-Chol) liposomes to the culture medium and evaluated the effect of this lipid on various biochemical and biological functions of E. histolytica HM-1:IMSS in terms of its virulence. The addition of PC-Chol liposomes to the culture medium maintained the virulence of these parasites against hamster liver at the same level as the original virulent E. histolytica strain, even though these amoebae were maintained without passage through hamster liver for 18 months. The trophozoites also showed increased endocytosis, erythrophagocytosis, and carbohydrate residue expression on the amoebic surface. Protease activities were also modified by the presence of cholesterol in the culture medium. These findings indicate the capacity of cholesterol to preserve amoeba virulence and provide an alternative method for the maintenance of virulent E. histolytica trophozoites without the need for in vivo procedures.     

Events in the amoebal-plasmodial transition ofPhysarum polycephalum studied by enrichment for committed cells

Summary Amoebae of strain CLof Physarum polycephalum undergo apogamic development to form multinucleate plasmodia. During the amoebalplasmodial transition, large uninucleate cells become irreversibly committed to plasmodium development. In developing cultures, amoebae lose the ability to flagellate before they become committed. Enriched suspensions of committed cells can be obtained by inducing asynchronous differentiating cultures to flagellate and passing the cells through a glass bead column. Committed cells can be cultured to form plasmodia on bacterial lawns or in axenic liquid medium but cannot be cultured on axenic agar medium. Uninucleate committed cells express tubulin isotypes characteristic of amoebae, but after culture in axenic liquid medium, the cells express plasmodial specific tubulin isotypes.Abbrevations SDM Semi-defined medium - DSDM Dilute semidefined medium - LIA Liver infusion agar - SBS Standard bacterial suspension - IEF Isoelectric focussing - SDS Sodium dodecyl sulphate - PAUF Precommitted amoebae unable to flagellate (for the explanation of these cells see text).     

Pathogenic and Nonpathogenic Acanthamoeba spp. in Thermally Polluted Discharges and Surface Waters1

During spring and autumn, the total number of amoebae and the number of Acanthamoeba species able to grow at 37°C were determined in six thermally polluted factory discharges and the surrounding surface waters. The isolated Acanthamoeba strains were studied for growth in axenic medium, cytopathic effect in Vero cell cultures, and virulence in mice. Although more amoebae were isolated in autumn, the number of Acanthamoeba species was lower than in spring, when the percent of pathogenic strains among the isolates was highest. Higher concentrations of amoebae were found in warm discharges, and more virulent strains occurred in thermal discharges than in surface waters.     

Entamoeba histolytica: virulence potential and sensitivity to metronidazole and emetine of four isolates possessing nonpathogenic zymodemes

The pathogenic potential of four Entamoeba histolytica isolates obtained from asymptomatic carriers and possessing nonpathogenic zymodemes was compared to four E. histolytica strains obtained from invasive cases of amebiasis and having pathogenic zymodemes. Both xenic and axenic cultures of a number of strains were tested. Determinations of cytopathogenicity were done in vitro by measuring the rates of destruction of tissue cultured monolayers of baby hamster kidney cells by intact amebae or by its cell-free extracts. The in vivo virulence was tested by assessing their capacity to form hepatic abscesses in hamsters or cecal ulcerations in rats. The results obtained show that two of the isolates from asymptomatic carriers (strains SAW 1734R clAR and WI:0385:191) were as virulent as three of the invasive ones (HM-1:IMSS, 200:NIH, and SAW 408). Two other isolates from asymptomatic carriers and one from a dysentery case were avirulent. All the E. histolytica isolates tested were similarly sensitive to metronidazole and emetine (IC50 1-10 micrograms/ml). The results indicate that the pathogenic potential of E. histolytica varies between isolates and can be affected by culture conditions and by the presence or absence of bacterial cells. These findings suggest that virulence does not necessarily correlate with a pathogenic zymodeme.     

Transfer of the cecal flora of the hamster to the germfree C3H mouse: use of this model to study the flora of the anti-Clostridium difficile barrier

The purpose of this work was the research and development of an experimental model to study anti-Clostridium difficile caecal microflora in the hamster. First the existence of this "barrier" was verified in conventional hamsters. Then, the caecal flora from these animals was orally transferred to C3H germfree mice. The barrier effect was maintained in the axenic mice. The comparative bacteriological analysis of hamster and mouse feces did not reveal important variations in the dominant anaerobic flora (P less than 0.01). After treatment with erythromycin, the barrier effect was maintained and while the disappearance of Escherichia coli was observed, the dominant anaerobic flora persisted. After dilution (10(-2] and subsequent heating (70 degrees C, 10 min) of caecal contents, the inhibitory activity against C. difficile was maintained although the number of aerobic and aerotolerant bacteria was reduced. The isolation from caecal microflora of anaerobic strains implicated in the resistance to colonization is presently underway in Freter anaerobic chambers.     

Changes in the DNA content of amoebae of Dictyostelium discoideum during growth and development.

A fluorimetric assay has been used to determine the DNA content of amoebae of Dictyostelium discoideum during growth and development. Amoebae grown in axenic culture tended to be multinucleate and had a greater DNA content than amoebae grown with a bacterial substrate, which were mononucleate. During the first 10 h of development there was little change in the DNA content of amoebae grown with a bacterial substrate, but the average DNA content per cell in amoebae grown axenically decreased as the amoebae became virtually mononucleate. Amoebae at 10 h development that had been harvested during exponential axenic growth were divided into two populations by countercurrent distribution in a polymer two-phase system. DNA content indicated that one population was largely in the G2-phase of the cell cycle, whereas the other population was largely in the G1-phase. Similar results were obtained at 10 h development with amoebae harvested during the stationary phase of axenic growth, although these amoebae start development all in the G2-phase of the cell cycle. Spores had a low DNA content, indicating that they were in G1-phase. It is proposed that all amoebae in G2-phase after early development differentiate, after mitosis, into spores and that stalk cells are formed from amoebae that remain in G1-phase after 10 h development.     

Entamoeba histolytica: Cysteine proteinase activity and virulence. Focus on cysteine proteinase 5 expression levels

Cysteine proteinase (CP) activity and CP5 mRNA levels were analyzed in eleven samples of Entamoeba histolytica isolated from patients presenting different clinical profiles. The virulence degree of the isolates, determined in hamster liver, correlated well with the clinical form of the patient and culture conditions. CP5 mRNA levels were also determined in sample freshly picked up directly from liver amoebic abscess. Differences were not observed in the levels of CP5 mRNA and CP specific activity among the cultured samples. However, different levels of CP5 mRNA were observed in trophozoite freshly isolated from hepatic amoebic lesions. These results reinforce the importance of CP5 for the virulence of amoebae and the need for studies with the parasite present in lesions to validate mechanisms involved in pathogenesis of amoebiasis.     

Uptake and replication of Salmonella enterica in Acanthamoeba rhysodes

The ability of salmonellae to become internalized and to survive and replicate in amoebae was evaluated by using three separate serovars of Salmonella enterica and five different isolates of axenic Acanthamoeba spp. In gentamicin protection assays, Salmonella enterica serovar Dublin was internalized more efficiently than Salmonella enterica serovar Enteritidis or Salmonella enterica serovar Typhimurium in all of the amoeba isolates tested. The bacteria appeared to be most efficiently internalized by Acanthamoeba rhysodes. Variations in bacterial growth conditions affected internalization efficiency, but this effect was not altered by inactivation of hilA, a key regulator in the expression of the invasion-associated Salmonella pathogenicity island 1. Microscopy of infected A. rhysodes revealed that S. enterica resided within vacuoles. Prolonged incubation resulted in a loss of intracellular bacteria associated with morphological changes and loss of amoebae. In part, these alterations were associated with hilA and the Salmonella virulence plasmid. The data show that Acanthamoeba spp. can differentiate between different serovars of salmonellae and that internalization is associated with cytotoxic effects mediated by defined Salmonella virulence loci.     

Axenic cultivation of Entamoeba dispar in newly designed yeast extract-iron-gluconic acid-dihydroxyacetone-serum medium

Yeast extract-iron-gluconic acid-dihydroxyacetone-serum medium that allows axenic cultivation of Entamoeba dispar was designed based on casein-free yeast extract-iron-serum (YI-S) medium, and the usefulness of the medium was assessed. The main differences from YI-S medium are replacement of glucose by gluconic acid, addition of dihydroxyacetone and D-galacturonic acid monohydrate, and sterilization by filtration. This medium promoted the axenic growth of 5 strains of E. dispar (2 strains of nonhuman primate isolates and 3 strains of human isolates). In addition, to clarify the biological basis for the growth of E. dispar in this medium, analyses of relevant enzymes on the glycolytic pathway of the amoebae as well as of the protozoans that are the best culture supplement for amoebae are being performed.     

Induction of trap formation in nematode-trapping fungi by a bacterium

Three soil bacterial strains were identified as Chryseobacterium sp. TFB on the basis of their 16S rRNA gene sequences. Conidia of Arthrobotrys oligospora produced a few mycelial traps (MT) and conidial traps (CT) when cultured with bacterial cells that they did not produce when cultured with a bacterial cell-free culture filtrate. However, co-culture of A. oligospora with bacterial cells and bacteria-free filtrate simultaneously induced MT and CT in large amounts. With the increased concentration of bacteria-free filtrate, the number of typical CT increased, but conidial germination was progressively inhibited. Scanning electron microscopy of A. oligospora co-cultured with bacteria revealed that bacterial attachment to hyphae was a prerequisite to trap formation and that bacteria-free filtrate facilitated bacterial attachments to hyphae. The results that the addition of nutrients in co-culture medium decreased the number of traps suggest that this type of trap formation may be favoured at a low nutrient status. Eight fungi tested were able to form MT and CT when co-cultured with bacterial cells and bacteria-free culture filtrate, but the abilities varied among species. This study provides novel evidence that under laboratory conditions, soil bacteria attaching to hyphae could induce traps in nematode-trapping fungi.     

Ampicillin inactivation in the caecum of axenic, gnotoxenic and conventional lambs: interaction with resistant or sensitive Escherichia coli

The fate of orally administered ampicillin was studied in axenic lambs, in gnotoxenic lambs given a complex microflora and a mixture of ampicillin resistant and/or sensitive strains of Escherichia coli, and in conventional lambs. In axenic lambs or animals with a sensitive microflora, antibiotic concentrations of 500-1600 micrograms ml-1 were detected in the intestine, and most of the ampicillin passed through the small intestine and entered the large intestine, within 12-15 h of administration. These antibiotic concentrations were sufficient to decrease the numbers of ampicillin-sensitive E. coli from 10(8)-10(9) bacteria ml-1 to about 10(5)-10(6) bacteria ml-1 by 8 h after ampicillin administration. Second and third doses of antibiotic had no further effect on the bacterial count. Administration of ampicillin to animals hosting ampicillin-resistant E. coli resulted in a significant inactivation of the antibiotic in the intestine. As might be expected there was little reduction in the numbers of these organisms. These results are similar to those observed in conventional lambs hosting resistant E. coli as the dominant colibacillary flora.     

Elimination of microbial flora from conventionally raised Syrian hamsters by antimicrobial agents

An attempt was made to identify and eliminate the normal endogenous microflora from conventionally-raised Syrian hamsters. A total of 14 bacterial and 2 yeast species were identified using enrichment, differential and selective media. The elimination of endogenous flora was carried out in two steps. In each step, hamsters were given a filter-sterilized mixture of two or more antibiotics for 10 weeks continuously through drinking water. During the second week, the hamsters were immersed once into a germicidal solution then housed in horizontal laminar-flow units. They were monitored at weekly intervals for the presence or absence of microorganisms in their fecal samples and fur swabs which were cultured on media as mentioned above. In step I, up to four resistant bacteria were cultured. On the basis of the antibiotic sensitivity profile of the surviving bacteria, step II procedure was started. The antibiotic mixture containing Kanamycin and Amphotericin B was fatal for the hamsters in both steps. Within 4 to 5 weeks of step II, all groups of hamsters tested bacteria-free and remained so for the duration of the step. Two weeks following withdrawal of antibiotics from drinking water, only one group of hamsters showed the presence of Escherichia coli. The other groups of hamsters tested bacteria-free for the 5 week duration of the experiment. The autopsies of these hamsters revealed greatly enlarged and thin-walled cecums, a characteristic of the germfree state.     

Modulation of Complement Resistance and Virulence of Naegleria fowleri Amoebae by Alterations in Growth Media

ABSTRACT. Highly-pathogenic, mouse-passaged Naegleria fowleri amoebae are complement resistant. The present study evaluates the effect of complement on N. fowleri and the virulence of the amoebae after animal passage and growth in two different axenic media. Pathogenic N. fowleri maintained in "enriched" Cline medium are virulent for mice and resistant to complement lysis. A rapid decline in resistance to complement and virulence for mice is observed when highly-pathogenic N. fowleri are grown in Nelson medium lacking hemin. N. fowleri maintained in Nelson medium can be rendered complement-resistant by shifting the amoebae to growth in Cline medium for 2 h prior to the addition of complement. Cycloheximide treatment of N. fowleri maintained in Nelson medium blocks the transition to a complement-resistant phenotype following a shift in growth medium. Proteins were radiolabeled with [³⁵S] during a shift from Nelson to Cline medium to identify specific polypeptides which may be associated with the functional activities related to virulence and resistance to complement.     

Cocultivation of Legionella pneumophila and free-living amoebae.

Studies of the interaction of Legionella pneumophila with free-living amoebae showed that Naegleria lovaniensis and Acanthamoeba royreba could use L. pneumophila as a sole food source. However, growth of the amoebae on nonnutrient agar plates seeded with L. pneumophila was slower than growth on nonnutrient agar plates seeded with Escherichia coli. On inoculation of L. pneumophila into axenic cultures of N. lovaniensis and A. royreba, 99.9% of the L. pneumophila was destroyed within 24 h. After several weeks, however, some amoeba cultures became chronically infected and supported the growth of L. pneumophila. Amoebae exposed to L. pneumophila and containing adhered L. pneumophila, L. pneumophila antigens, or both, showed no increased pathogenic potential on intranasal inoculation of weanling mice. Similarly, L. pneumophila propagated in chronically infected amoeba cultures showed no increase in virulence on intraperitoneal inoculation of guinea pigs relative to L. pneumophila grown in yeast extract broth.     

Isolation and identification of pathogenic Naegleria australiensis (Amoebida, Vahlkampfiidae) from a spa in northern Italy

Samples from therapeutic swimming pools and mud basins were cultured for free-living amoebae. Seven strains of pathogenic Naegleria species were isolated. Although some of the strains were as virulent as Naegleria fowleri, the etiological agent of primary amoebic meningoencephalitis, they were identified as Naegleria australiensis with the indirect fluorescent-antibody technique. The virulence of the isolates for mice corresponded with the cytopathic effect for Vero cells. The N. australiensis strains were isolated from swimming pools with water temperatures ranging from 32 to 35 degrees C and from mud with temperatures from 25 to 43 degrees C. The presence of pathogenic N. australiensis in the swimming pools did not correlate with bacterial indicators.     

Isolation and identification of pathogenic Naegleria australiensis (Amoebida, Vahlkampfiidae) from a spa in northern Italy.

Samples from therapeutic swimming pools and mud basins were cultured for free-living amoebae. Seven strains of pathogenic Naegleria species were isolated. Although some of the strains were as virulent as Naegleria fowleri, the etiological agent of primary amoebic meningoencephalitis, they were identified as Naegleria australiensis with the indirect fluorescent-antibody technique. The virulence of the isolates for mice corresponded with the cytopathic effect for Vero cells. The N. australiensis strains were isolated from swimming pools with water temperatures ranging from 32 to 35 degrees C and from mud with temperatures from 25 to 43 degrees C. The presence of pathogenic N. australiensis in the swimming pools did not correlate with bacterial indicators.     

Molecular Changes in Entamoeba histolytica in Response to Bacteria1

Entamoeba histolytica, the protozoan parasite, is the causative agent of amoebiasis. The degree of virulence, as inferred from invasiveness, of potentially pathogenic strains may be regulated by both host and parasite factors that determine the gut environment. One such factor that plays an important role is the bacterial flora in the gut. Previous studies have clearly shown that bacterial flora is an important determinant of virulence in E. histolytica. However, the exact nature of changes induced in E. histolytica in response to bacteria and their role in virulence is not clear. In this study the levels of a number of molecules potentially important in virulence mechanisms were determined in E. histolytica cells grown with and without normal human bacterial flora, using enzyme-linked immunosorbent assay. Significant changes were observed only after the E. histolytica cells had been adapted to grow with bacterial flora for a number of generations, and not in short term culture.