Biguanide-induced changes in Acanthamoeba castellanii: an electron microscopic study

Trophozoites and cysts of Acanthamoeba castellanii were exposed to chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB); changes in cell ultrastructure and surface structure were examined by both transmission and scanning electron microscopy. PHMB caused a greater degree of structural and membrane damage; the cytoplasmic contents were severely depleted and there were clusters of densely stained precipitates on the cell surface. Concentrations of CHA greater than 100 μg ml⁻¹ produced shrinkage from the cyst wall. At high concentrations, PHMB induced a slight withdrawal of the cytoplasm from the wall and, unlike CHA, induced swelling of the cysts. These findings do not define the mechanisms of action of CHA and PHMB, but provide evidence that a major target site for both agents is the plasma membrane. However, additional intracellular damage undoubtedly contributes to the lethal effects. The greater resistance of cysts may be associated with reduced biguanide uptake.     

Biguanide-induced changes in Acanthamoeba castellanii: an electron microscopic study

Trophozoites and cysts of Acanthamoeba castellanii were exposed to chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB); changes in cell ultrastructure and surface structure were examined by both transmission and scanning electron microscopy. PHMB caused a greater degree of structural and membrane damage; the cytoplasmic contents were severely depleted and there were clusters of densely stained precipitates on the cell surface. Concentrations of CHA greater than 100 microg ml(-1) produced shrinkage from the cyst wall. At high concentrations, PHMB induced a slight withdrawal of the cytoplasm from the wall and, unlike CHA, induced swelling of the cysts. These findings do not define the mechanisms of action of CHA and PHMB, but provide evidence that a major target site for both agents is the plasma membrane. However, additional intracellular damage undoubtedly contributes to the lethal effects. The greater resistance of cysts may be associated with reduced biguanide uptake.     

Aspects of the mechanisms of action of biguanides on trophozoites and cysts of Acanthamoeba castellanii

A non-radioactive method was used to investigate the uptake by Acanthamoeba castellanii of chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB). Based on the Giles et al. (1974) hypothesis, the uptake of CHA by trophozoites appeared to be of the L3 pattern whereas that of cysts was C2. Unlike CHA, trophozoites took up PHMB with an L2 pattern at low concentrations followed by a C-type pattern at higher concentrations, the uptake by cysts was found to be of the C2 pattern with a plateau effect at high concentrations. A diphasic leakage effect was found in trophozoites whereas a relatively low peak of maximal leakage occurred from cysts treated with high biocide concentrations. The amount of pentose release depended on the formulation ingredients. No correlation between pentose leakage and trophozoicidal or cysticidal activity was found.     

X-ray microanalysis of chlorine and phosphorus content in biguanide-treated Acanthamoeba castellanii

Energy dispersive analysis of X-rays (EDAX) was used to study the effects of chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB) on Acanthamoeba castellanii. A high variation of elements occurred in untreated individual cells and only two elements, Cl (a biocide marker) and P, were investigated. X-ray dot mapping of untreated trophozoites and cysts revealed that Cl in cells was uniformly distributed throughout the cytoplasm, whereas P was less dense in the vacuoles. X-ray dots of Cl in biocide-treated trophozoites and cysts appeared denser and evenly distributed within the cells as the biguanide concentration increased. Quantitative analysis of either CHA or PHMB within the cells using Cl as an elemental marker was unsatisfactory because of the high Cl levels in untreated cells. The apparent increases of P in some experiments with treated cells might be associated with reduced permeability, protein coagulation or aggregation of phospholipids.     

Resistance, biguanide sorption and biguanide-induced pentose leakage during encystment of Acanthamoeba castellanii

AIMS: This study investigates the effects of biguanides during encystment of Acanthamoeba castellanii. METHODS AND RESULTS: A non-nutrient encystment system was used to investigate the changes in the levels of sorption (uptake) of three non-cysticidal concentrations (10, 20 and 50 microg ml(-1)) of chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB) as well as their effects on viability and leakage of pentose sugars during the first 36 h of encystment. Trophozoites treated with CHA or PHMB were more sensitive and generally sorbed more of each biocide than cysts. During encystment, the largest increases in resistance developed between 18 and 36 h for both biguanides with the resistance emerging to biguanide concentrations of 10 or 20 microg ml(-1) between 18 and 24 h. At 50 microg ml(-1) resistance emerged between 24 and 36 h. There was a general decrease in biocide sorption during encystment between 0-24 and 0-21 h for CHA and PHMB, respectively, at a concentration of 50 microg ml(-1). The greatest decline in biguanide-induced pentose leakage was between 0 and 12 h. CONCLUSIONS: The results suggest that during encystment some of the changes in the susceptibility to CHA or PHMB may be related to decreases in the levels of biocide sorption, which is limited by the developing cyst wall. SIGNIFICANCE AND IMPACT OF THE STUDY: During encystation, changes occur in biguanide sensitivity. The physical barrier of the cyst wall may be an important factor in limiting biocide sorption.     

Relationship between Legionella pneumophila and Acanthamoeba polyphaga: physiological status and susceptibility to chemical inactivation.

Survival studies were conducted on Legionella pneumophila cells that had been grown intracellularly in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells grown under iron depletion; in contrast, iron-depleted conditions increased the susceptibilities of cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1 x MIC) gave 99.99% reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure to PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.     

Relationship between Legionella pneumophila and Acanthamoeba polyphaga: physiological status and susceptibility to chemical inactivation.

Survival studies were conducted on Legionella pneumophila cells that had been grown intracellularly in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells grown under iron depletion; in contrast, iron-depleted conditions increased the susceptibilities of cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1 x MIC) gave 99.99% reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure to PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.     

Resistance of Acanthamoeba castellanii cysts to physical,chemical, and radiological conditions

Resistance of Acanthamoeba castellanii cysts to disinfection agents, antimicrobial agents, heat, freeze-thawing, ultraviolet radiation (UV), gamma irradiation, and cellulase were evaluated in vitro. Following exposure to different agents, the cysts were removed and cultured for A. castellanii trophozoites for 3-14 days. Solutions containing 20% isopropyl alcohol or 10% formalin effectively killed A. castellanii cysts. Hydrogen peroxide (3%, AOSept Disinfectant) effectively killed A. castellanii cysts after 4 hr of exposure. Polyhexamethylene biguanide (0.02%), clotrimazole (0.1%), or propamidine isethionate (Brolene) were effective in killing A. castellanii cysts in vitro. Acanthamoeba castellanii cysts were resistant to both 250 K rads of gamma irradiation and 800 mJ/cm2 of UV irradiation. Excystment of trophozoites was accelerated after exposure to 10, 100, and, 1,000 units of cellulase. These results suggest that A. castellanii cysts benefit by enhanced survival because of their resistance to very harsh environmental conditions.     

Acanthamoeba royreba sp. n. from a human tumor cell culture

A new species of Acanthamoeba was isolated from a culture of an established line of human choriocarcinoma cells. The identification of this strain, originally called the Oak Ridge strain, and the establishment of a new species for it were based on morphologic, serologic, and immunochemical studies. In general, the structure of the trophozoite did not differ significantly from that of other species of Acanthamoeba, except that a body which more closely resembled a centriole than material described previously as centriolar satellites was observed in trophozoites examined with the electron microscope. The dimensions of the trophozoite were the smallest among the species of Acanthamoeba. The cyst was typical of the genus, but differed from those of other species by its smaller size and the presence of numerous ostioles. Studies of the Oak Ridge strain by immunofluorescence using antisera developed against the isolate and Acanthamoeba culbertsoni, A. castellanii, A. polyphaga, A. rhysodes, A. astronyxis, and A. palestinensis revealed the antigenic uniqueness of the Oak Ridge strain. It was demonstrated by immunoelectrophoretic analyses of the soluble proteins of the Oak Ridge strain that shared approximately 1/2 of its antigenic structure with A. castellanii and A. culbertsoni. The antigenic differences of the isolate from other species of Acanthamoeba were deduced from comparison of the antigenic constitution of these species and the Oak Ridge strain with A. culbertsoni and A. castellanii. Although the strain was initially recognized by its cytopathogenicity for cultures, it did not produce acute infections in mice after intranasal inoculation of 1 X 10(4) ameba/mouse. The foregoing results constituted the basis for the establishment of the Oak Ridge strain as a new species, A. royreba sp. n., in the genus Acanthamoeba.     

In vitro shock response to different stressors in free living and pathogenic Acanthamoeba

Three stresses, viz heat, oxidative and pH shocks, were applied to cultures of three species of Acanthamoeba, free-living Acanthamoeba rhysodes and pathogenic Acanthamoeba castellanii and Acanthamoeba culbertsoni. The effect of each stressor on trophozoite integrity was evaluated by the amount of heat shock protein (HSP)60 and HSP70 produced and by exclusion of 0.2% Congo Red. HSP60 and HSP70 levels were estimated using Western blotting and subsequent densitometric analyses. Unstimulated trophozoites from A. rhysodes produced the lowest background levels of HSP60 and HSP70 and were the amoebae most affected by (mammalian-type) stresses as judged by their enhanced HSP production and decreased viability upon exposure to such conditions. In contrast, unstimulated Acanthamoeba of the pathogenic variety had relatively high background levels of test HSPs and seemed undisturbed by the types of stresses they must deal with when entering their hosts. These studies suggest that high HSP levels in amphizoic acanthamoebae may indicate their involvement in (i) tolerance induction to hosts' stressors and/or (ii) in species' virulence.     

Antigens of selected Acanthamoeba species detected with monoclonal antibodies

Acanthamoeba species are ubiquitous soil and freshwater protozoa that have been associated with infections of the human brain, skin, lungs and eyes. Our aim was to develop specific antibodies to aid in rapid and specific diagnosis of clinically important isolates. Mice were variously immunised with live mixtures of Acanthamoeba castellanii strain 112 (AC112) trophozoites and cysts, or with sonicated, formalin-fixed or heat-treated trophozoites, or with a trophozoite membrane preparation. Eight hybridoma cell lines secreting monoclonal antibodies reactive with A. castellanii epitopes were generated. Seven of the new antibodies (designated AMEC1-3 and MTAC1-4) were isotyped as IgMkappa and one (MTAC5) as IgG1kappa. All of the novel antibodies bound to AC112 cysts, and MTAC4 and MTAC5 also bound to trophozoites as measured by flow cytometry on unfixed cells. Single chain antibody fragments that retained parental antibody binding characteristics were engineered from three of the hybridomas (AMEC1, MTAC3 and MTAC4). Four monoclonal antibodies (AMEC1, AMEC3, MTAC1, MTAC3) bound reliably to unfixed cysts of clinical isolates of A. castellanii (two strains) and Acanthamoeba polyphaga (two strains), belonging to Pussard-Pons morphological group II, and to Acanthamoeba lenticulata and Acanthamoeba culbertsoni, belonging to Pussard-Pons morphological group III. None of the antibodies bound to cysts or trophozoites of the environmental group I species, Acanthamoeba tubiashi. Antibodies AMEC1, MTAC3, MTAC4 and MTAC5 reacted with buffered formalin-fixed AC112 by immunohistochemistry, and also stained Acanthamoeba in sections of infected rat cornea and buffered formalin-fixed, paraffin-embedded infected human cornea. These antibodies may be useful in diagnosing pathogenic Acanthamoeba species in clinical specimens, provided that cysts are present.     

Protease activities of Acanthamoeba polyphaga and Acanthamoeba castellanii

Acanthamoeba spp. are free-living amoebae that cause amoebic granulomatous encephalitis, skin lesions, and ocular amoebic keratitis in humans. Several authors have suggested that proteases could play a role in the pathogenesis of these diseases. In the present work, we performed a partial biochemical characterization of proteases in crude extracts of Acanthamoeba spp. and in conditioned medium using 7.5% SDS-PAGE copolymerized with 0.1% m/v gelatin as substrate. We distinguished a total of 17 bands with proteolytic activity distributed in two species of Acanthamoeba. The bands ranged from 30 to 188 kDa in A. castellanii and from 34 to 144 kDa in A. polyphaga. Additionally, we showed that the pattern of protease activity differed in the two species of Acanthamoeba when pH was altered. By using protease inhibitors, we found that the proteolytic activities belonged mostly to the serine protease family and secondly to cysteine proteases and that the proteolytic activities from A. castellanii were higher than those in A. polyphaga. Furthermore, aprotinin was found to inhibit crude extract protease activity on Madin-Darby canine kidney (MDCK) monolayers. These data suggest that protease patterns could be more complex than previously reported.     

Acanthamoeba castellanii: gene profile of encystation by ESTs analysis and KOG assignment

The trophozoite of Acanthamoeba transforms into a cyst, the resistant form under harmful environments such as starvation, cold and certain chemicals used in medical treatment. To investigate the factors mediating encystation, ESTs of encystation-induced A. castellanii were analysed and compared to those of trophozoites. Each EST was compared by the predicted proteins from the ESTs, to the cyst and the trophozoite by reciprocal BLAST analysis, KOG assignment, and gene annotation. In addition to the genes previously reported to encystation mediate such as cyst specific protein 21, protein kinase C, proteasome and heat shock protein, several genes like cullin 4, autophage protein 8 and ubiquitin-conjugating enzymes were identified to be related to encystation. Five kinds of proteinase genes were detected in cyst ESTs. The information of the genes expressed during encystation may open the way to further study on differentiation and resistance of cyst-forming pathogenic protozoa.     

In vitro amoebicidal activity of Origanum syriacum and Origanum laevigatum on Acanthamoeba castellanii cysts and trophozoites

In some patients, complete treatment of amoebic keratitis is difficult because of the resistance of cysts to therapeutic agents. The aim of this study was to evaluate the in vitro amoebicidal activity of methanolic extracts of Origanum syriacum and Origanum laevigatum. In the presence of methanolic extracts (ranging from 1.0 to 32.0mg/ml), numbers of the viable Acanthamoeba castellanii trophozoites and cysts were decreased. Both extracts showed a time and dose dependent amoebicidal action on the trophozoites and cysts. Of the extracts tested, O. syriacum showed the stronger amoebicidal effect on the trophozoites and cysts. In the presence of 32 mg/ml extract, no viable trophozoites were observed within third hour. The extract was also found effective against the cysts within 24th hour. In the case of O. laevigatum, no viable trophozoites were observed within 72nd hour at the concentrations of 16 and 32 mg/ml. As expected, cysts were found more resistant to the extracts than the trophozoites.     

Changes in Transfer Ribonucleic Acids Accompanying Encystment in Acanthamoeba castellanii

Transfer ribonucleic acid (tRNA) from exponentially growing cells (trophozoites) and from precysts of Acanthamoeba castellanii were examined by reversed-phase column (RPC-2) chromatography. This system gave excellent resolution of isoaccepting species of tRNA. The tRNAs for 12 amino acids were studied. A comparison of trophozoite and precyst tRNA elution profiles revealed no apparent differences in the number of isoaccepting species of alanyl-, arginyl-, asparaginyl-, glycyl-, leucyl-, lysyl-, methionyl-, phenylalanyl-, tryptophanyl-, or valyl-tRNAs. Seryl-tRNAs from trophozoites were eluted as three components, whereas precyst seryl-tRNAs were eluted as only two components. Precharged trophozoite and precyst isoleucyl-tRNAs were both eluted as single components; however, post-chromatography charging of trophozoite tRNA resulted in three components of activity for tRNA(Ile) and only one component for precyst tRNA(Ile). None of the observed changes could be attributed to differences in synthetases or to the presence of altered tRNA lacking the CCA terminus or partially degraded by nucleases. The possible significance of these observations is discussed.     

Weak cytotoxic activity of miltefosine against clinical isolates of Acanthamoeba spp

Hexadecylphosphocholine (miltefosine) is an anticancer drug active in vitro against various protozoan parasites, and recently used for the treatment of disseminated Acanthamoeba infection. In the present study, we present results of weak cytotoxic activity of this potential amoebicidal agent for 2 of 3 clinical isolates of Acanthamoeba spp. Although the inhibition effect for all tested concentrations was apparent, and showed 100% eradication of trophozoites of Acanthamoeba castellanii strain at a concentration of 62.5 μM after 24 hr, the strains Acanthamoeba sp. and Acanthamoeba lugdunensis exhibited low sensitivity to hexadecylphosphocholine, even in high concentrations. The determined minimal trophocidal concentrations were 250 μM for Acanthamoeba sp. and 500 μM for A. lugdunensis after 24 hr of exposure. Although hexadecylphosphocholine is a potential agent for treatment of Acanthamoeba keratitis and systemic infections, in clinical practice the possible insusceptibility of the amoebic strain should be considered for optimizing therapy.     

Purification from Acanthamoeba castellanii of proteins that induce gelation and syneresis of F-actin.

From Acanthamoeba castellanii, we have purified four proteins each of which alone causes a solution of F-actin to gel. The four active proteins have subunit molecular weights of about 23,000, 28,000, 32,000 and 38,000, respectively; the last three may be dimers in their native proteins. Together, these four proteins account for about 97% of the gelation activity of the whole extract; not more than about 3% of the total activity of the unfractionated extract can be due to a 250,000-dalton polypeptide. Another protein fraction, purified by agarose chromatography, induces shrinking (syneresis) of gels formed from F-actin and any of the gelation factors. That fraction contains a high Ca2+-, low (K+,EDTA)-ATPase and a major polypeptide of 170,000 daltons both of which bind to actin in the shrunken gel pellet. The active fraction does not contain the previously described Acanthamoeba myosin (Pollard, T. D., and Korn, E. D. (1973) J. Biol. Chem. 248, 4682-4690).     

Growth, encystment and survival of Acanthamoeba castellanii grazing on different bacteria

Free-living amoebae of the genus Acanthamoeba are widely distributed in soil and water collections, where trophozoites (vegetative, multiplicative stages) feed mainly by phagocytosis and thus control bacterial populations in the environment. Here, we examined the growth, encystment and survival of Acanthamoeba castellanii receiving different bacteria (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Bacillus subtilis, Bacillus megaterium, Micrococcus luteus, and Staphylococcus aureus) in nonnutrient saline. All bacteria assayed induced a dose-dependent proliferative response, in most cases maximized with a bacterial dose of 1 x 10(9) mL(-1); except for M. luteus, trophozoites grew better with viable than with heat-killed bacteria. In addition, Acanthamoeba growth was improved by adding bacteria on alternate days. Single-dose experiments indicated a temporal association between the growth of trophozoite and bacterial consumption, and higher consumption of M. luteus, E. coli and P. aeruginosa, bacterial species that allowed the highest trophozoite yields. Long-term Acanthamoeba-bacteria incubation revealed that encystment was significantly delayed by almost all the bacteria assayed (including S. aureus, which elicited a poor growth response) and that the presence of bacteria markedly increased cyst yield; final cyst recovery clearly depended on both the dose and the type of the bacterium given, being much higher with E. coli, M. luteus and P. aeruginosa.     

Effect of Biocides on Biofilm Bacteria from Dental Unit Water Lines

Microbial biofilm formation in dental unit water lines (DUWL) is a phenomenon that has been recognized for nearly four decades. Water delivered by DUWL can harbor high numbers of bacteria, including opportunistic pathogens. Biofilms on tubing within DUWL may serve as a reservoir for these microorganisms and should therefore be controlled. In this study, the effects of eight biocides were monitored on DUWL biofilms individually and in combination by epifluorescence microscopy and total viable counts (TVC). The effects of sodium dodecyl sulphate (SDS), hydrogen peroxide (H₂O₂), sodium hypochlorite (NaOCl), phenol (Phe), Tween 20 (Tw 20), ethylenediaminetetraacetic acid (EDTA), chlorohexidine gluconate (CHX), and povidine iodine (PI) were tested on DUWL biofilms alone and in combination. PI was found to have negligible effects on biofilm removal either applied alone or in combined form with CHX. Applying all biocides simultaneously did not completely eliminate viable bacteria nor did they remove biofilm. Overall, when combined, the biocides performed better than singly applied products. The most effective biocides were NaOCl and Phe (both alone and in combination).     

Induced encystment improves resistance to preservation and storage of Acanthamoeba castellanii

Several conditions that allow the preservation, storage and rapid, efficient recovery of viable Acanthamoeba castellanii organisms were investigated. The viability of trophozoites (as determined by time to confluence) significantly declined over a period of 12 months when stored at -70 degrees C using dimethyl sulfoxide (DMSO; 5 or 10%) as cryopreservant. As A. castellanii are naturally capable of encystment, studies were undertaken to determine whether induced encystment might improve the viability of organisms under a number of storage conditions. A. castellanii cysts stored in the presence of Mg2+ at 4 degrees C remained viable over the study period, although time to confluence was increased from approximately 8 days to approximately 24 days over the 12-month period. Storage of cysts at -70 degrees C with DMSO (5 or 10%) or 40% glycerol, but not 80% glycerol as cryopreservants increased their viability over the 12-month study period compared with those stored at room temperature. Continued presence of Mg2+ in medium during storage had no adverse effects and generally improved recovery of viable organisms. The present study demonstrates that A. castellanii can be stored as a non-multiplicative form inexpensively, without a need for cryopreservation, for at least 12 months, but viability is increased by storage at -70 degrees C.