Vol. 8, No. 5 Printed in U.S.A.

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1975, p. 591-65 Copyright © 1975 American Society for Microbiology

In Vitro Effects of Amphotericin B on Growth and Ultrastructure of the Amoeboflagellates Naegleria gruberi and Naegleria fowleri F. L. SCHUSTER* AND EMANUEL RECHTHAND

Department of Biology, Brooklyn College, Brooklyn, New York 11210 Received for publication 1 August 1975

In vitro effects of the polvene antibiotic amphotericin B (AmB) on growth, viability, and ultrastructure of amoeboflagellates of the genus Naegleria were examined. The strains studied were the nonpathogenic Naegleria gruberi EGB and the Carter and TY strains of the pathogenic Naegleria fowleri. AmB was amoebicidal at all concentrations used (0.25, 0.50, and 1.0 ,ug/ml) when the drug was added to cultures in lag phase, as determined by viability testing, but was mainly inhibitory when added to log-phase cultures. The drug produced ultrastructural modifications at all concentrations (0.05 to 1.0 gg/ml). These changes included distortion of nuclear shape, increase in cytoplasmic membranes (both rough and smooth endoplasmic reticulum), decrease in number of food vacuoles, absence of pseudopod formation, mitochondrial abnormalities, increase in autophagic vacuoles, and blebbing of the plasma membrane. These alterations of amoebic ultrastructure became more pronounced with increased time in AmB and with increase in AmB concentration in the growth medium.

The genus Naegleria comprises a group of amoebae or, more correctly, amoeboflagellates, found widely dispersed in soil and freshwater habitats. The life cycle of Naegleria includes a feeding or trophic amoeboid stage, a transitory flagellate stage, and, under certain conditions, a dormant cyst stage (17, 35, 36). Once studied primarily for its morphogenetic capacities, members of the genus have gained attention over the past decade as causal agents of a particularly virulent disease of humans termed primary amoebic meningoencephalitis (7, 11, 38). Naegleria gruberi is a presumably nonpathogenic species readily isolated from nature, where it feeds on bacteria; Naegleria fowleri, the pathogenic species, has been isolated from humans suffering from primary amoebic meningoencephalitis but with difficulty, if at all, from natural habitats. The two species are distinguished from one another on the basis of temperature tolerance (23), immunochemistry (1, 40), pathogenicity (6), and morphological differences in the cyst stage (28, 37). Based on in vitro studies employing a variety of drugs (5, 9, 13, 14), as well as a small number of clinical applications (4, 7, 16), the polyene antibiotic amphotericin B (AmB) has been regarded as the drug of choice in treating primary amoebic meningoencephalitis. Though used in some instances against protozoa (27, 30), AmB has had its major application in systemic fungal

infections of humans (25, 30). Evidence supports the site of action of AmB at the plasma membrane of the cell, where the lipophilic drug combines with sterols, possibly creating pores in the membrane (2, 3, 26) and causing leakage of cellular constituents across the membrane (18, 22). Electron microscope observations on the effects of AmB (and other polyenes) on fungi have appeared in the literature (21, 31, 33); there are, however, no published reports on effects of this drug on Naegleria ultrastructure, either in vitro or in vivo. This report, therefore, presents: (i) data on the effect of AmB on in vitro growth of trophic forms of the nonpathogenic N. gruberi and two strains of the pathogenic N. fowleri, and (ii) electron microscopic observations on AmB-mediated changes in ultrastructure of the same Naegleria spp. in vitro.

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MATERIALS AND METHODS

Three strains of Naegleria were employed in this study, encompassing forms from diverse geographic locations and having different optimal growth temperatures: (i) N. gruberi EGB, from a California soil sample, used as a representative of the nonpathogenic forms from nature, (ii) N. fowleri, Carter's 1966 isolate, from an Australian victim of primary amoebic meningoencephalitis (6), and (iii) N. fowleri TY, isolated from a victim of primary amoebic meningoencephalitis in Virginia (16). Growth. Amoebae were grown axenically in phos-

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phate-buffered yeast-peptone-liver medium made up in dilute saline (Pringsheim solution) to which was added 10% heat-inactivated fetal calf serum (pH 6). N. gruberi EGB was grown at 21 C; the two N. fowleri strains were grown at 30 (TY) and 37 C (Carter's strain). For testing of AmB, amoebae were grown in screw-capped micro-Fernbach flasks (25 ml) or screw-capped Erlenmeyer flasks (125 ml) containing 10 ml of culture fluid; for growth determinations in the presence of AmB, Erlenmeyer flasks (125 ml) containing 20 ml of culture fluid were used. Drug treatment. Working stock dilutions (50 ,g/ml) of AmB (Fungizone; E. R. Squibb & Sons, Inc., Princeton, N.J.) were prepared from powdered drug, using Pringsheim solution for hydration and dilution. After hydration, AmB stock solutions were stored in the freezer and generally used within 1 week of hydration to minimize loss of activity. Both drug stocks and growth vessels containing diluted AmB were exposed to light for minimal periods of time (inoculation, sampling, etc.) to prevent photooxidation of the AmB. The two controls employed in the experiments consisted of growth vessels to which were added either Pringsheim solution or sodium deoxycholate (0.25 and 0.50 ug/ml, respectively; Mann Research Labs) in place of AmB. The latter control was necessary since sodium deoxycholate is incorporated as a solubilizing agent for AmB in Fungizone. Final AmB concentrations employed for growth and electron microscope studies were: 0.05, 0.10, 0.25, and 1.0 ,g/ml. Growth and viability of amoebae. To evaluate the effect of AmB on growth, 24-h-old cultures of amoebae were inoculated into growth vessels to give cell populations of 103 to 104 cells/ml. (Inoculum size was similar in all cases, ranging from 104 to 105 cells/ml from 24-h-old cultures, in order to give the desired number of amoebae per milliliter for growth determinations and viability testing.) Flasks were incubated at appropriate growth temperatures, and 1-ml samples were removed daily over a 7-day period for counting, etc. Cell counts were made on a Coulter counter (model ZF)- Viability tests were performed at the time of counting by inoculating 0.1 ml of medium from control and drug-treated cultures into 5 ml of yeast-peptone-liver medium (1:50 dilution) in screw-capped test tubes; tubes were slanted and examined over several days for evidence of growth of amoebae. A second type of viability testing was employed in which AmB was added to 2.5-day-old log-phase cultures of amoebae (ca. 5 x 105 cells/ml) at a final concentration of 0.5 ,ug/ml. Samples (0.1 ml) were removed at intervals of 1, 3, 24, and 48 h. Light microscopy and photomicrography. Samples from control and drug-containing flasks were examined and photographed using a Zeiss microscope equipped with Nomarski optics. Growth of amoebae for electron microscopy. Amoeba populations to be fixed for electron microscopy were handled in two ways. (i) AmB was added to 2.5- to 3-day-old log-phase cultures, which were then fixed 48 h after addition of the antibiotic, or (ii) AmB was added to 2.5- to 3-day-old log-phase cultures, which were then fixed 1, 24, and 48 h after addition of the antibiotic. In addition to the regular

ANTIMICROB. AGENTS CHEMOTHER.

control population of amoebae, sodium deoxycholate-containing cultures were fixed for electron microscopy. Electron microscopy. Amoebae were harvested from growth vessels, concentrated by centrifugation, and fixed in 2% glutaraldehyde in either veronal-acetate or collidine buffer, postfixed in veronal-acetate-buffered OSO4 and, after dehydration through alcohols, embedded in Maraglas epoxy resin. Thin sections of embedded materials were stained with lead citrate and examined in a Philips 300 electron microscope (80 kV).

RESULTS Relative toxicity of AmB for amoebae. Appropriate concentrations of AmB used in this study were determined after initial screening to select drug levels that would produce ultrastructural changes in amoebae without being obviously amoebicidal. An amoebicidal drug concentration was taken as a drug level that would cause immediate rounding of trophic amoebae, ballooning of the plasma membrane, and subsequent rupturing of the membrane with release of cellular contents. Of the two species studied, N. gruberi and N. fowleri, the latter appeared more susceptible to AmB at all concentrations in terms of changes visible by light (rounding of trophic amoebae, swelling, lysis) and electron microscopic (alterations of ultrastructure) examination of amoebae. Effect of AmB on growth. Comparisons of control and drug-treated populations are seen in Fig. 1. When added to growth vessels at the same time as the amoeba inocula, all concentrations of AmB employed (0.25 to 1.0 ,ug/ml) were amoebicidal. No recovery from drug inhibition occurred through the duration of growth determination; if any decay of AmB occurred at growth temperatures employed in this study, as has been reported by Hamilton and Elliott (24), the initial effects of the drug on sparse amoeba populations were irreversible. Viability testing. Inocula removed from growth vessels at the time of sampling for counting in the experiments illustrated in Fig. 1 indicated that none of the amoebae from AmBtreated cultures was viable. Trophozoites in viability tests remained rounded, did not settle on the test tube wall, and were, presumably, dead. This could probably not be attributed to AmB carryover from the growth vessel, since, with AmB-treated, log-phase cultures, the degree of dilution was sufficient to permit growth to occur. In contrast to the effect of AmB on newly inoculated populations, when added to cultures 2.5 days old (ca. 5 x 105 amoebae/ml), viability tests made after 1, 3, and 24 h all showed recovery and growth of amoebae. Recovery results for 48-h exposure of log cells to AmB varied;

SUSCEPTIBILITY OF NAEGLERIA TO AmB

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FIG. 1. Growth curves for N. gruberi EGB and N. fowleri TY and Carter, showing control populations (-), sodium deoxycholate-control populations (-), and populations treated with AmB at concentrations of 0.25 jig/ml (v), 0.5 pg/ml (U), and 1.0 pglml (0). These curves are based on data derived from two experiments.

cultures sometimes showed recovery and sometimes did not. Effect of sodium deoxycholate. Because of the possibility that the AmB solubilizing agent

might be affecting the amoebae, sodium deoxycholate-containing controls were used in both growth and ultrastructural studies. Concentrations of sodium deoxycholate used in this study,

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SCHUSTER AND RECHTHAND

comparable to levels that would be present in the diluted Fungizone, had minimal effect on growth and no apparent effect on ultrastructure of amoebae. In growth studies (Fig. 1), sodium deoxycholate had a slight inhibitory effect on amoebae, but this was non-existent by day 7. None of the ultrastructural observations reported below as due to AmB were evident in amoebae grown in the presence ofsodium deoxycholate. Light microscopic observations. Naegleria amoebae have a pronounced polarity; the anterior end of the cell is characterized by a clear ectoplasmic pseudopod and the posterior end is characterized by the contractile vacuole and a variable number of sticky, filamentous pseudopods. This normal appearance is seen in the amoebae in Fig. 2 through 4. Cells treated with subamoebicidal concentrations of AmB were rounded and nonmotile and showed no tendency to form food vacuoles (Fig. 5) as did control cells. Ultrastructural effects of AmB. Most of the ultrastructural features reported here were observed in all three of the naeglerias studied, even though illustrations might not be included of all the strains. Figure 6 shows a typical N. fowleri (Carter's strain) from a control culture. The nucleus was the most striking and obvious feature of these cells, being about 5 ,m in diameter and containing a central nucleolus. Control cells were actively motile, as can be inferred from the electron micrograph by the presence of a large ectoplasmic pseudopod free of the usual cellular organelles (cf. Fig. 6 with Fig. 2-4). Vesicles of endoplasmic reticulum were sparse and scattered irregularly through the endoplasm; characteristically dense mitochondria were similarly scattered. As evident at the light microscopic level, control cells were filled with large numbers of vacuoles, presumably food vacuoles formed by fluid uptake (with a small amount of debris, derived from the liver or fetal calf serum, that was present in the medium). Figure 7, in comparison, shows an amoeba (Carter's strain) after 24 h in 0.5 gg of AmB per ml. Ultrastructural changes included alteration of nuclear shape, increase in rough and smooth endoplasmic reticulum, swelling and degeneration of mitochondria, appearance of autophagic vacuoles, and a decrease in the number of food vacuoles over that normally seen in control cells. This decrease in vacuoles was consistent with light microscopic observations of drug-treated cells (Fig. 5). No evidence of ectoplasmic pseudopod formation was found, and amoebae from drug-treated populations had a rounded shape.

ANTiMICROB. AGENTS CHEMOTHER.

Nuclei of amoebae exposed to AmB were decidedly irregular in outline (Fig. 8-10), contrasting with the rounded nuclei of control cells. This distortion was more evident in cells exposed to high (1 ,g/ml) AmB concentrations. Bulges (Fig. 7 and 10) and comma-shaped projections (Fig. 9) were frequently observed in these cells; the nucleolar region, however, appeared unmodified. In spite of the highly distended shapes (Fig. 8), no evidence of blebbing of the nuclear projections was ever seen in thin sections. Multinucleate trophozoites were frequently encountered in AmB-treated populations. Cytoplasmic membrane proliferation, involving both rough and smooth endoplasmic reticulum (ER), was characteristic of all amoeba strains in all AmB concentrations. Large rosettes (Fig. 11) and whorls (Fig. 12 and 13) of rough ER were commonly found in the cytoplasm of AmB-treated cells, in contrast to the sparse ER of control cells. These ER configurations in a three-dimensional view would be formed of membrane sheets, and such sheets were often seen in association with the nuclear envelope, which they resembled in general appearance. Lipid globules, usually found in proximity to the nuclear envelope, were seen to be enclosed in these membrane sheets (Fig. 8). Smooth ER, barely evident in control amoebae, was present in highly organized configurations, often in conjunction with rough ER, in cells from AmB-containing cultures (Fig. 7, 14, and 15). These complexes were pronounced in cells that had been in contact with AmB for 48 h (Fig. 14 and 15) and somewhat less developed in cells in contact with AmB for 24 h (Fig. 7). The origin of the smooth ER is not clear. In favorable sections, continuity was evident between vesicles of rough and smooth ER, and it is suggested that smooth vesicles arise from the rough ER. This is supported by the appearance, after 1 h in contact with AmB, of rough ER vesicles at the periphery of amoebae (Fig. 16) and their continuity with smooth ER. The area about the nucleus of AmB-treated amoebae appeared to have an abundance of vesicles of both the rough and smooth variety (Fig. 9 and 10). Mitochondrial modifications ofboth quantitative and qualitative nature were observed in amoebae, particularly the TY and Carter strains, treated with AmB (Fig. 7, 17-19). In some instances, there appeared to be an increase in numbers of mitochondria in drugtreated cells. This is evident in Fig. 17, where sheets of ER intercalate with numerous, small mitochondria. Some of the qualitative changes in mitochondrial ultrastructure are seen in Fig.

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FIG. 2-5. Light micrographs (Nomarski optics) of control and AmB-treated amoebae. FIG. 2. N. fowleri Carter's strain. Trophic amoeba from control culture. Note numerous vacuoles in cytoplasm and presence of clear ectoplasmic pseudopods at the anterior (lower) end of the amoeba. (x2,200). FIG. 3. N. fowleri TY. Trophic amoeba from control culture. Cytoplasmic vacuoles and filamentous pseudopods occur in abundance. Ectoplasmic pseudopod denotes anterior end of trophozoite. (x2,200). FIG. 4. N. gruberi EGB. Elongate trophozoite from control culture. Nucleus indicated by arrow. This extended shape is typical ofactively motile trophozoite. (x2,200). FIG. 5. N. fowleri, Carter's strain. Cell from culture exposed to AmB (1 pg/ml) for 24 h. Amoeba appears rounded, number of food vacuoles is reduced (cf. Fig. 2), and cytoplasm has a granular aspect. Nucleus seen at arrow. (x5,500).

18 and 19. Because of mitochondrial swelling, (Fig. 8, 13, 16). Electron-dense inclusions were the cristae, which were usually obscured in also observed in mitochondria (Fig. 18). Autocontrol cells by the dense mitochondrial matrix, phagosomal vacuoles containing mitochondria were readily seen. Numerous examples of in varying stages of degeneration were fregreatly swollen mitochondria were observed quently seen in drug-treated amoebae (Fig. 19).

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FIG. 8. N. fowleri, Carter's strain, from cultures exposed to AmB (1 pg/ml) for24 h. Nucleus (N) of trophic amoeba showing gross distortion. Nucleolar region appears unaffected. Cytoplasm contains swollen mitochondria (M), as well as a membrane-bound cluster of lipid globules (L). (x16,500). FIG. 9. N. gruberi EGfi, from culture exposed to AmB (1 pg/ml) for 48 h. Nucleus with irregular outline. Nuclear region enclosed by sheets of rough ER, which also enclose proximal areas containing vesicles of ER. Lipid globule (L). (xll ,500). FIG. 10. N. fowleri TY, from culture exposed to AmB (0.25 pg/ml) for 48 h. Nucleus with lobate projections. Note presence of rough ER in proximity to nuclear envelope. Arrow points to vesicle in contact with lamella of nuclear envelope, suggestive of origin of cytoplasmic vesicles from nuclear membrane. (X18,500). 597

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SCHUSTER AND RECHTHAND

ANTiMICROB. AGzNTs CHZMOTHZR.

FIG. 11. N. fowleri, Carter's strain, from culture exposed to AmB (1 pg/ml) for 48 h. Rosette is made up of rough ER membranous sheets. Note presence in center of rosette of cluster of rough ER vesicles . (X20,O00). FIG. 12. N. gruberi EGB, from culture exposed to AmB (05 pg/ml) for 48 h. Rough ER sheets are formed into a membranous whorl. (x35,000). FIG. 13. N. fowleri TY, from culture exposed to AmB (0.1 pg/ml) for48 h. Whorl ofrough ER membranes. Note swollen, apparently degenerating mitochondria (M) in vicinity. (x33,000).

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601

These mitochondrial modifications were less ob- have been shown to be susceptible to AmB (4, 6, vious in N. gruberi amoebae. In the TY and 15, 16), a point of particular interest since these Carter strains, mitochondrial degeneration oc- organisms are capable of invading the central curred in all of the AmB concentrations em- nervous system of humans, causing an almost ployed. invariably fatal meningoencephalitis. This Another feature seen in AmB-treated amoe- study has examined the effects of AmB on bae was the presence of small cytoplasmic blebs growth and ultrastructure of some representa(ca. 0.5 gm in diameter) at the cellular periph- tive strains of free-living and pathogenic Naeery (Fig. 20 and 21). These usually contained gleria spp. in vitro. only cytoplasm but sometimes included vesicles Growth and viability. According to most of rough and smooth ER. Presumably, these published accounts, Naegleria trophozoites are projections pinched off from the cell and passed inhibited by concentrations of AmB _1 ,ug/ml. into the growth medium. In vitro inhibitory concentrations of AmB were reported as: 0.6 and 0.075 ,g/ml as minimum immobilizing and minimum growth inhibitory DISCUSSION levels, respectively (5); 0.1 ,ug/ml (9); 1 or 2 Polyene antibiotics, a group of compounds of ,ug/ml (10); 5 ,ug/ml (14); 0.625 Ag/ml over 6 h of which AmB is a member, bind to the plasma exposure'(15); 0.06 to 0.25 ,ug/ml (29); 0.001 membrane of cells, disturbing its role as a selec- ,ug/ml (32); 0.07 and 0.7 ,g/ml for two different tively permeable barrier (25, 30). AmB is re- strains (34). Amoebicidal drug levels were reported to combine with membrane sterols, par- ported as: 2 ,ug/ml in successive transfers (10); ticularly cholesterol and ergosterol, causing 25 ,ug/ml (14); 0.313 ,ug/ml over 24 h of exposure leakage of cellular constituents into the me- (15). In contrast to the sensitivity of Naegleria dium. The major application for AmB has been amoebae, Acanthomoeba (=Hartmannella) against systemic fungal infections, but it also spp., a soil amoeba whose pathogenicity for has activity against protozoans (22, 27). In re- humans has not been confirmed (11), is incent years, amoebae of the genus Naegleria hibited by drug levels 100 to 125 jig/ml (8, 9, 14).

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FIG. 20. N. fowleri, Carter's strain, from culture exposed to AmB (05 pg/ml) for 24 h. Apparent bleb formation at edge of rounded amoeba. Plasma membrane itself has normal 'unit membrane" appearance. Some vesicles are visible within the bleb. (x 89,500). FIG. 21. N. fowleri, Carter's strain, from culture exposed to AmB (1 pg/ml) for 48 h. Another example of

bleb formation. (x54,000).

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Some of the variation noted in susceptibility of Naegleria to AmB could be due to strain differences and method of cultivation of the amoebae. These variables would include the use of bacterized medium for drug testing, pH of the test medium, duration of exposure to AmB, and growth phase of the amoeba population. All of these factors probably influence response to some degree. Chang (10) noted that Naegleria amoebae exposed to AmB showed a decrease in motility, with cells rounding up. This was also observed in the present study. Chang applied the drug, at concentrations of 1 or 2 Ag/ml, to the maintenance medium and found that two to eight transfers were required before amoebae were killed at the lower concentration, with pathogenic naeglerias appearing more susceptible to AmB than the nonpathogenic N. gruberi. When Chang introduced AmB into amoeba cultures at peak growth, little effect was observed beyond rounding of the trophic amoebae. In the present study, incorporation of AmB into the growth medium (0.25 to 1.0 gg/ml) resulted in an amoebicidal effect, as determined by viability testing, for both pathogenic and nonpathogenic strains. As Chang found, our results indicate that actively growing populations of amoebae exposed to AmB will round up but are not necessarily killed by the drug. Evidently AmB produced irreversible effects in amoebae during an early stage of growth ("lag" phase) that are less pronounced in cells from log-phase cultures. Electron microscopic examination of these log-phase cells indicated ultrastructural changes leading ultimately to lysis of amoebae, but these changes were apparently reversible as indicated by recovery of amoebae in viability testing. Duma (15) reported rounding, granulation, and disintegration of Naegleria amoebae within 3 h after exposure to AmB concentrations of _1.25 ,g/ml. Carter (5) used rounding of amoebae and loss of cytoplasmic movement as criteria for AmB susceptibility in immobilization testing. The hemoflagellate Trypanosoma cruzi exhibited granularity and loss of motility after in vitro exposure to AmB concentrations as low as 0.125 jig/ml (27). Ultrastructural effects of AmB. All concentrations of AmB employed in this study caused organellar changes evident in thin-sectioned material. These changes were more obvious in amoebae exposed to maximal concentrations (1 ug/ml) than in amoebae exposed to minimal drug levels (0.05 gg/ml); some changes were evident after exposure for 1 h to the drug (the shortest period of drug exposure prior to fixation of cells for electron microscopy) but

ANTiMICROB. AGENTS CHEMOTHER.

were pronounced after exposure for 48 h to AmB (the longest period of drug exposure prior to fixation). In general, AmB-induced alterations included enhanced nuclear plasticity, increase in cytoplasmic membranes (both rough and smooth ER), decrease in number of food vacuoles, and mitochondrial degeneration. These changes were more obvious in the pathogenic strains (TY and Carter) of N. fowleri than they were in the nonpathogenic N. gruberi. Because of its presumed action at the plasma membrane of sterol-containing cell types, membranes of amoebae exposed to AmB were examined carefully for defects. None were found, but thin sectioning would probably not be the best technique to employ in a search for membrane modifications. Freeze etching, a more suitable technique permitting examination of the cleaved inner surface of the plasma membrane, was employed by Nozawa et al. (33) to study the

pathogenic fungus Epidermophyton floccosum exposed to AmB. These investigators observed aggregation of 8.5-nm particles and the formation of depressions within the plasma membrane, suggesting lipid rearrangement as a result of drug-sterol interaction. This same study also revealed vesiculation at the plasma membrane, presumably induced by AmB, as did AmB treatment of other pathogenic fungi (Histoplasma capsulatum andBlastomyces dermatitidis) examined in the electron microscope by Lane et al. (31). Vesiculation was evident in naeglerias treated with AmB. This occurred as blebs developing on the plasma membrane of the cells. Unlike the fungi, where the cell membrane is enclosed within a cell wall which would trap any forming vesicles, blebs produced by amoebae would pass into the growth medium. Under normal conditions, amoebae exhibit a high degree of membrane turnover, brought about by formation of pseudopods and food and pinocytotic and osmoregulatory vacuoles. This turnover appeared reduced in AmB-treated amoebae, as indicated by reduced motility and decreased food vacuole formation. Nozawa et al. (33) have suggested that AmB causes a withdrawal of sterol from membrane phospholipid, leading to altered membrane fluidity. The same restriction on membrane fluidity might occur in Naegleria. The production of membrane defects can be assumed from the appearance of various cellular constituents in the growth medium after AmB exposure. E. F. Gale (18) monitored the effect of amphotericin methyl ester on Candida albicans using release of K+ as a measure of membrane alteration. Ghosh and Ghosh (22)

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noted a pH-dependent release of amino acids and ribonucleic acid, but not deoxyribonucleic acid, from Candida after exposure to AmB. Neither study employed microscopy to describe any possible mechanism at the ultrastructural level whereby such release might be facilitated. G. R. Gale (21), however, in a study that involved electron microscopy, observed no effect on the cytoplasmic membrane of Candida from 5 to 120 min after exposure of cells to AmB. Though no defects of the plasma membrane, other than the production of blebs, were noted in the present study, electron microscopic examination of AmB-treated amoebae revealed an increase of cytoplasmic membranes. This increase was, in fact, the most striking structural change that occurred in drug-treated cells. Two types of membranes were involved: rough and smooth ER. The proximity of sheets of rough ER to the nuclear envelope, the envelopment of lipid globules (usually found outside of the nuclear envelope) within these sheets, and the similar appearance of the nuclear membranes and rough ER all support the origin of rough ER from the nuclear envelope. Smooth ER, occurring in the cytoplasm as large masses of interconnected tubules, was seen to be in continuity with elements of rough ER. The complexity of these various membrane configurations increased with time in AmB, but initiation of membrane proliferation was found in amoebae after only 1 h in the drug. The significance of the proliferation of the membranous networks is not known. Does any portion of the mass of cytoplasmic membranes become incorporated into the plasma membrane of the amoeba? Is this a synthetic mechanism for replacement of plasma membrane lost through bleb formation? These questions can be answered by a more dynamic means of following membrane synthesis and turnover. Relevant to the production of cytoplasmic membranes is the unusual appearance of nuclei of cells exposed to AmB. Distortion of nuclear shape may be responsible, in part, for the increase in cytoplasmic membranes, though evidence of nuclear blebbing was difficult to confirm. What often looked to be blebs in the cytoplasm originating from the nucleus turned out to be extensions of a second nucleus found in successive serial sections. Ghosh and Ghosh (22) observed no loss of deoxyribonucleic acid from Candida after AmB treatment, though amino acids and ribonucleic acid were being released. This is consistent with the nucleus retaining its integrity. In the present study, rupture of the plasma membrane was followed by release of cellular contents, but even then

603

the nuclei remained intact. Mitochondrial alterations were commonly observed in drug-treated amoebae. These included increase in numbers of mitochondria, appearance of mitochondrial inclusions, incorporation of mitochondria into autophagosomal vacuoles, and mitochondrial swelling leading to complete ultrastructural degeneration. Lane et al. (31) observed mitochondrial swelling in B. dermatitidis and H. capsulatum after in vitro exposure to AmB concentrations of 1 pkg/ml. This damage was viewed as a secondary consequence of action of AmB on the fungal cells; the primary effect was at the cell membrane where changes in permeability to ions were occurring, which led to production of ultrastructural alterations in the mitochondria. This presumed indirect effect of the drug on mitochondria is supported by G. R. Gale's observation (20) on Candida that, although AmB suppressed 02 uptake in the presence of substrate in intact cells, no comparable respiratory inhibition was noted using cell-free systems. The presence of dense inclusions within mitochondria of amoebae exposed to the drug is suggestive of some disturbance of ionic balance. Factors influencing AmB response. A number of factors may contribute to variability of cellular response to AmB. Ghosh and Ghosh (22) reported maximal release of amino acids from Candida at pH 3 and minimal release at pH 6 to 7. E. F. Gale (18), however, observed increased K+ release from Candida, with increase of pH to a value of 8. The pH of the yeastpeptone-liver medium used in the present study was 6, and no attempt was made to vary pH either above or below this level to evaluate such changes on ultrastructural modifications induced by AmB. Serum was found to have a neutralizing effect on AmB-mediated inhibition of deoxyribonucleic acid synthesis in lymphocytes according to Tarnvik and Ans6hn (39); the range of serum concentrations used in their study was 5 to 45%. Fetal calf serum (10%) was used as a nutritional supplement in Naegleria growth medium. Judging from the immobilization of trophic amoebae in drug-containing growth vessels, it seems unlikely that drug action was being blocked by serum levels in the medium. Susceptibility of Candida exposed to amphotericin methyl ester showed a decrease with phase of growth (19). This loss of susceptibility was reported to be due to the neutral lipid content of the cell wall, which would cause a binding of the drug. Spheroplasts of Candida did not show the same loss of susceptibility. Log-phase Naegleria amoebae (2 to 3 days old),

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though exhibiting ultrastructural modifications with time in AmB, appeared less susceptible to the drug than amoebae in the lag phase of growth (.1 day old). For lag-phase cells, all drug concentrations employed in this study were amoebicidal; log-phase cells, as indicated by viability testing, could recover even after exposure to AmB of up to 48 h. AmB-induced membrane "defects" in lag-phase cells may be too extreme for recovery to occur, whereas logphase cells, which probably have substantial reserves of membrane-precursor molecules, might survive drug exposure by extensive replacement of defective membranes. Carter (5) noted that there was an almost 10-fold difference in susceptibility in AmB levels that would immobilize 7-day-old trophozoites (0.6 ug/ml) and would inhibit growth in bacterized cultures (0.075 ,ug/ml). Presumably growing cells were more susceptible to the drug than stationaryphase amoebae. Inoculum size is another potential variable. Jamieson and Anderson (29) found that the minimal inhibitory dose of AmB varied directly with the size of the Naegleria inoculum. This may be a factor in the variation found in viability testing of log-phase amoebae exposed to AmB for 48 h; variations in inoculum size would determine whether or not recovery would occur. Hamilton and Elliott (24) reported complete destruction of AmB after 24 h at 37 C in drug testing (in vitro) against Cryptococcus neoformans. Temperatures employed in the present study were those optimal for growth of the three amoeba strains: 21 C for EGB, 30 C for TY, and 37 C for Carter's isolate. Lag-phase cultures were very susceptible to all drug concentrations employed so that, even if breakdown of AmB was taking place in the growth medium, no recovery of amoebae was found. For log-phase cultures exposed to the drug, viability testing indicated recovery of amoebae from effects of AmB after exposure of 1 to 24 h. This recovery, however, was erratic with cultures exposed to AmB for 48 h; even when recovery of such 48-h cultures did occur, growth was slow as compared to control cultures and cultures treated with AmB for 1, 3, and 24 h. In log-phase cultures, the magnitude of ultrastructural change increased with increasing time in AmB-containing medium until, after 48 h, evidence of cellular lysis was common in thinsectioned populations of amoebae. It is hoped that these in vitro studies of AmBtreated Naegleria amoebae can serve as a basis for evaluating effects of the drug on pathogenic N. fowleri inoculated into mice. AmB has been

ANTIMICROB. AGENTS CHEMOTHER.

shown by several investigators (5, 12, 13) to be effective in protecting mice and other experimental animals from amoebic infection and, in spite of its potential toxicity, has promise in clinical treatment of humans suffering from primary amoebic meningoencephalitis (4, 7, 16). The occurrence of a reproducible set of ultrastructural changes in pathogenic amoebae may be useful in monitoring effectiveness of in vivo drug susceptibility in chemotherapeutic studies involving Naegleria. ACKNOWLEDGMENTS We thank Betty Hershenov and Avrom Pollak for their much appreciated assistance with portions of this study. This work was supported by Public Health Service grant Al 12058 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Anderson, K., and A. Jamieson. 1972. Agglutination test for the investigation of the genus Naegkria. Pathology 4:273-278. 2. Andreoli, T. E. 1973. On the anatomy of amphotericin B-cholesterol pores in lipid bilayer membranes. Kidney Int. 4:337-345. 3. Andreoli, T. E. 1974. The structure and function of amphotericin B-cholesterol pores in lipid bilayer membranes. Ann. N.Y. Acad. Sci. 235:448-468. 4. Apley, J., S. K. R. Clarke, A. P. C. H. Roome, S. A. Sandry, G. Saygi, B. Silk, and D. C. Warhurst. 1970. Primary amoebic meningoencephalitis in Britain. Br. Med. J. 1:596-599. 5. Carter, R. F. 1969. Sensitivity to amphotericin B of a Naegleria sp. isolated from a case of primary amoebic meningoencephalitis. J. Clin. Pathol. 22:470-474. 6. Carter, R. F. 1970. Description of Naegleria sp. isolated from two cases ofprimary amoebic meningoencephalitis, and of the experimental pathological changes induced by it. J. Pathol. 100:217-244. 7. Carter, R. F. 1972. Primary amoebic meningo-encephalitis. An appraisal of present knowledge. Trans. R. Soc. Trop. Med. Hyg. 66:193-213. 8. Casemore, D. P. 1970. Sensitivity of Hartmannella (Acanthamoeba) to 5-fluorocytosine, hydroxystilbamidine, and other substances. J. Clin. Pathol. 23:649652. 9. (Cerva, L. 1972. In vitro drug resistance of pathogenic Naegleria gruberi strains, p. 431-432. In M. Hejplar. M. Semonsky, and S. Masak (ed.), Advances in antimicrobial and antineoplastic chemotherapy, vol I. University Park Press, Baltimore. 10. Chang, S. L. 1971. Small, free-living amebas: cultivation, quantitation, identification, classification, pathogenesis, and resistance, p. 201-254. In T. C. Cheng (ed.), Current topics in comparative pathobiology, vol. 1. Academic Press Inc., New York. 11. Culbertson, C. G. 1971. The pathogenicity of soil amebas. Annu. Rev. Microbiol. 25:231-254. 12. Culbertson, C. G., W. M. Overton, and P. W. Ensminger. 1972. Pathogenic Hartmannella (Acanthamoeba) and Naegkria: studies on experimental chemotherapy and pathology, p. 433-434. In M. Hejtlar, M. Semonsky, and S. Masaik (ed.), Advances in antimicrobial and antineoplastic chemotherapy, vol. 1. University Park Press, Baltimore. 13. Das, S. R. 1971. Chemotherapy of experimental amoebic meningoencephalitis in mice infected with Naegleria aerobia. Trans. R. Soc. Trop. Med. Hyg.

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