Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Noble, J. A. Articles by Crow, S. A. Search for Related Content PubMed PubMed Citation Articles by Noble, J. A. Articles by Crow, S. A., Jr.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, July 2002, p. 2069-2076, Vol. 46, No. 7
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.7.2069-2076.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Phagocytosis Affects Biguanide Sensitivity of Acanthamoeba spp.

Judith A. Noble,¹ Donald G. Ahearn,¹ Simon V. Avery,² and Sidney A. Crow Jr.^1*

Department of Biology, Georgia State University, University Plaza, Atlanta, Georgia 30303,¹ School of Life and Environmental Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom²

Received 7 September 2001/ Returned for modification 11 February 2002/ Accepted 2 April 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
The incidence of Acanthamoeba keratitis, a disease associated with contact lens wear, has been in apparent decline with the advent of multipurpose contact lens solutions. The concentrations of the biguanides chlorhexidine digluconate (CHX) and particularly polyhexamethylene biguanide (PHMB) included in multipurpose solutions (MPSs) are sublethal for amoebae. We evaluated by flow cytometry the effects of these two biguanides on phagocytosis of particles and the survival of trophozoites of Acanthamoeba castellanii and A. polyphaga. Trophozoites of A. castellanii and A. polyphaga (10⁶/ml) were exposed to solutions of 5 and 50 µg of PHMB and CHX per ml in the presence and absence of particles (i.e., heat-killed yeasts and bacteria and latex beads). In addition, trophozoites were exposed to particles treated with these concentrations of the two biguanides. In the absence of particles, trophozoites of A. polyphaga appeared to be more resistant to the biguanides than those of A. castellanii. In the presence of particles, the rates of survival of both species were decreased. In most instances, particles treated with sublethal concentrations of both biguanides that were adsorbed onto the particles reduced the incidence of phagocytosis. Particles present in MPSs in contact lens cases may be involved in the decreased incidence of Acanthamoeba keratitis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Acanthamoeba spp. are ubiquitous in aquatic habitats and colonize biofilms in swimming pools, hot tubs, domestic water taps, eyewash stations, contact lens disinfecting solutions, and air-conditioning systems in buildings and automobiles (2, 8, 34, 39, 40, 43, 48). The trophozoite is the active motile stage that feeds by phagocytosis and pinocytosis and divides by binary fission. A cyst stage is resistant to adverse environmental conditions such as desiccation (34), extreme temperatures (7), and antimicrobials (9, 32).

Acanthamoeba spp. may cause chronic granulomatous amoebic menigoencephalitis in immunocompromised individuals (27, 54, 58), but the clinical associations of the genus are mostly with keratitis (2, 4, 17, 28, 37, 41). Acanthamoeba keratitis is a rare disease that occurs most commonly among otherwise healthy individuals and seems to be associated with minor trauma to the eye and the use of contact lenses. The species detected in patients with keratitis, in order of decreasing frequency, are Acanthamoeba castellanii, A. polyphaga, A. rhysodes, A. culbertsoni, A. hatchetti, and A. griffini (2, 23, 27, 51, 58). Whether cysts or trophozoites, or both, are responsible for keratitis is unclear (2, 33). Primary contamination of the lens care system, particularly with gram-negative bacteria, seems to be a prerequisite for the establishment of infectious populations of Acanthamoeba (12). Approximately 50% of contact lens wearers have contaminating microorganisms in their contact lens cases at some time during their use of contact lenses (59). The bacteria that adhere to the surfaces of the lenses provide a substratum that facilitates the attachment, survival, and growth of Acanthamoeba spp., thus increasing the risk of Acanthamoeba keratitis (24, 25). The binding of Acanthamoeba spp. to unworn hydrogel lenses has also been related to water content, surface tensions, and the ionic charge of the lens (46).

Trophozoites of amoebae utilize various microorganisms, particularly gram-negative bacteria and yeasts, as nutrient sources (26, 45, 49, 57). Castellani (16) was the first to report the growth of Acanthamoeba in the presence of Cryptococcus pararoseus and Bacillus spp. Viable cells of Candida famata, Candida parapsilosis, Saccharomyces cerevisiae, and Cryptococcus neoformans are known to support the growth of A. castellanii (3, 10, 14, 42). Weekers et al. (56) found that the nonpigmented members of the family Enterobacteriaceae Escherichia coli and Klebsiella aerogenes appeared to be better food sources for A. castellanii, A. polyphaga, and Hartmanella vermiformis than the other indigenous soil bacteria tested. Wang and Ahearn (55) reported that the optimal ratio between bacteria and A. castellanii ranged from 10:1 to 1:1; however, densities of various bacteria to amoebae of 100:1 or greater in enrichment broth inhibited the growth and survival of A. castellanii. Amoebae also ingest silica, carbon particles, and latex beads. Uptake of beads is optimal at 30 to 35°C and is proportional to the concentration of beads and the incubation time. The internalization process involves the binding and accumulation of the beads at the cell surface until an optimum volume of beads is reached, at which time the beads are ingested until phagocytosis becomes saturated (3, 6, 11, 36, 57). Avery et al. (5) demonstrated that plasma membrane fluidity plays an important role in the phagocytic activity of A. castellanii. Increases in the unsaturation of membrane fatty acids that follow chilling of A. castellanii at 15°C coincide with increases in phagocytosis. In Acanthamoeba, bacteria are internalized in mass or individually and are then grouped in one vacuole. A phagolysosome is produced after vacuole fusion with a lysosome, and then acid pH and lysosomal enzymes lyse the internalized bacteria (21).

Of the many antimicrobials tested against Acanthamoeba spp. (12, 13, 18, 32, 38, 44, 47), the biguanides chlorhexidine digluconate (CHX) and polyhexamethylene biguanide (PHMB) appear to be the most effective. Various multipurpose contact lens solutions include PHMB at concentrations from 0.5 (0.00005%) to 5 µg/ml (0.0005%) (15, 35). These concentrations are significantly lower than the MIC of PHMB necessary to kill 10⁵ cysts of Acanthamoeba spp. per ml. Minimal amoebicidal concentrations of PHMB and CHX for 10⁵ trophozoites/ml range from 50 to 100 µg/ml after 24 h of exposure and are as low as 25 µg/ml after 72 h of exposure (22, 53). On the basis of the prevention of plaque formation on lawns of E. coli, Khunkitti et al. (29) reported that the minimal biocidal concentrations of CHX and PHMB for A. castellanii were 12.5 and 25 µg/ml for trophozoites and cysts, respectively. The overall disinfectant formulation, not the concentration of the inhibitor(s) such as the biguanides, determines the antimicrobial efficacy of a contact lens solution. Even so, the complete eradication of amoebae (10⁴ to 10⁶ cells) from current contact lens systems on the basis of the potency of the disinfectant against Acanthamoeba, particularly cysts, appears to be unlikely. Nevertheless, the subsidence of amoebic keratitis in Great Britain appears to have coincided with the greater use of multipurpose lens solutions with sublethal concentrations of inhibitors (52).

We undertook this study to determine whether the presence of particles including contaminating bacteria or yeasts would enhance the susceptibilities of Acanthamoeba spp. to biguanides. We evaluated the effects of sublethal levels of PHMB and CHX on phagocytosis and survival of selected Acanthamoeba spp. in the presence of various microorganisms and polystyrene latex beads by flow cytometry (FCM).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Culture maintenance and growth. Acanthamoeba spp. were obtained from the American Type Culture Collection (ATCC; Manassas, Va.). Working cultures of A. castellanii ATCC 30234 and A. polyphaga ATCC 30461 were maintained in a modified peptone glucose yeast extract medium (PYG). PYG contained 2.0% (wt/vol) proteose peptone (Difco Laboratories, Detroit, Mich.), 0.1% (wt/vol) yeast extract (Difco), and 1.8% (wt/vol) glucose (47). Subcultures were prepared weekly and maintained in PYG at 25°C with rotary aeration at 135 rpm. For the preparation of inocula, the medium (250 ml in 500-ml flasks) was inoculated with trophozoites from 48-h cultures to give an initial cell density of approximately 1.5 x 10⁵ to 2 x 10⁵/ml, and the mixture was incubated at 30°C on a rotary shaker at 135 rpm for 24 h. Trophozoites were pelleted by centrifugation at 750 x g and suspended in filter-sterilized phosphate-buffered saline (PBS; pH 7.4; Gibco BRL, Life Technologies, Gaithersburg, Md.) or 0.9% NaCl to a concentration of 2 x 10⁶/ml (at least 90% viable trophozoites), as determined from cell counts determined in a 0.1-ml hemacytometer counting chamber (Hausser Scientific Inc., Horsham, Pa.). C. albicans ATCC 10231 was maintained on Sabouraud dextrose agar (Difco) slants at 5°C. Working cultures were developed by transferring a loopful of the stock culture to 250-ml flasks containing 125 ml of Sabouraud dextrose broth (Difco). Cultures of C. albicans were incubated at 37°C on a rotary shaker at 135 rpm. The C. albicans yeasts were harvested by centrifugation at 3,000 x g, and the pellet was suspended in PBS. The broth cultures of C. albicans were filtered through a 5-µm-pore-size Isopore polycarbonate membrane filter (Millipore Corp., Bedford, Mass.). Yeasts less than 5 µm in size were used in the phagocytosis assays. Yeasts were killed by exposing them to heat at 80°C for 15 min and were centrifuged at 3,000 x g, and the pellet was suspended in 0.1 M NaHCO₃ to a concentration of 2 x 10⁸/ml. E. coli ATCC 6418 was maintained on tryptic soy agar (Difco) slants at 5°C and subcultured monthly on tryptic soy agar. Working cultures were prepared in 250-ml flasks containing 125 ml of tryptic soy broth (Difco) at 37°C for 18 h on a rotary shaker at 150 rpm. The bacteria were harvested by centrifugation at 3,000 x g and suspended in PBS. The bacteria were killed by exposing them to heat at 80°C for 10 min and were resuspended in 0.1 M NaHCO₃ to optical densities corresponding to concentrations of 2 x 10⁸ CFU/ml.

Fluorescent staining of yeasts and bacteria. Viable and heat-killed yeasts and bacteria (density, 2 x 10⁸/ml) were stained with 0.1 mg of fluorescein isothiocyanate (FITC; Sigma Chemical Co., St. Louis, Mo.) per ml in 0.1 M NaHCO₃ (pH 9.0) at 25°C for 2 to 4 h for the phagocytosis assays (20). Labeling uniformity and any loss of fluorescence from the labeled microorganisms were measured by FCM at up to 24 h after labeling. The maximal fluorescence labeling of heat-killed yeasts or bacteria with FITC was obtained with a minimum incubation time of 3 h at 37°C. The fluorescence from labeled cells was stable by storage at 5°C in the dark for up to 2 weeks. All phagocytosis assays were conducted in the dark to prevent photo bleaching of labeled cells that were no more than 1 week old.

Phagocytosis of particles. Procedures for determination of the rates of phagocytosis by trophozoites of Acanthamoeba spp. with fluorescence-labeled particles were adapted from those of Avery et al. (6). Fluoresbrite YG polystyrene latex microspheres (designed to match FITC filter settings of a maximum excitation wavelength of 458 nm and a maximum emission wavelength of 540 nm) were obtained from Polysciences Inc. (Warrington, Pa.). One-micrometer-diameter YG beads (300 µl; density, 2 x 10⁸/ml) were added to 2.7 ml of a cell suspension in PBS containing at least 90% viable trophozoites of Acanthamoeba (2 x 10⁶/ml) in a 50-ml flask. We used a bead-to-amoeba ratio of 100:1 in shake cultures at 150 rpm for 60 min at 30°C. After 10-min periods of incubation, 1.0 ml of the cell suspension was diluted 10-fold with cold (4°C) mannitol buffer (100 mM mannitol, 15 mM NaH₂PO₄ [pH 7.2]) and the suspensions were centrifuged at 750 x g for 5 min. Centrifugation separated trophozoites with associated beads (i.e., trophozoites with bound and internalized beads) from unbound beads. The mean number of beads bound to trophozoites was obtained from fluorescence histograms of amoebae and beads incubated in PBS containing 12.5 mM sodium azide. The maximal internalization of beads by trophozoites was determined during a 60-min incubation period at 30°C. Phagocytosis was quantified by use of previously described calculations (6). The number of internalized beads was the difference between the number of beads bound to trophozoites in the presence of sodium azide and the number internalized and bound in PBS. Heat-killed yeasts or bacteria (0.3 ml) were added to a suspension of trophozoites (2.7 ml) of Acanthamoeba spp. (2 x 10⁶/ml) in 50-ml flasks. The final concentration of yeasts or bacteria in suspension was 2 x 10⁷/ml. Phagocytosis assays with yeasts or bacteria were conducted in shake cultures at 150 rpm for up to 60 min at 30°C. Internalization of the particles was also determined from direct microscopic counts in representative tests.

Survival of trophozoites in solutions with various concentrations of biguanides. CHX (Sigma) and PHMB (SoftSwim; Bio-lab Inc., Decatur, Ga.) were prepared in distilled H₂O and stored in the dark at 22°C. Suspensions of trophozoites in PBS (4 x 10⁶/ml in 0.5 ml) were inoculated into solutions of CHX or PHMB (0.5 ml; final concentrations, 12.5, 25, 50, or 100 µg/ml) in polystyrene 24-well cell culture clusters (Corning Inc., Corning, N.Y.), and the mixtures were incubated for 1, 2, and 4 h at 22°C with rotary shaking. Trophozoite suspensions incubated in PBS served as controls. The suspensions of trophozoites (0.5 ml) were inoculated into 4.5 ml of Letheen broth (Difco) for 15 min at 22°C. After this neutralization step, the trophozoites (1.0 ml) were transferred to 5-ml round-bottom polystyrene tubes (Becton Dickinson Labware, Franklin Lakes, N.J.). The cells were stained with oxonol (Ox; final concentration, 5 µM; Molecular Probes, Eugene, Oreg.) or propidium iodide (PI; final concentration, 25 mg/liter; Sigma) for determination of viability by FCM. The suspensions were stained with Ox for 15 min and with PI for 1 min at 22°C. A mixture of heat-killed amoebae (80°C for 10 min) and live amoebae stained with Ox or PI served as the control for the establishment of the lower and upper fluorescence limits for FCM. The number of viable amoebae was also determined by microscopic examination of amoebae (methylene blue exclusion) in a counting chamber (9).

FCM analysis of phagocytosis and viability. Trophozoites were analyzed for fluorescence-labeled particles by methods based on those described by Ábel et al. (1) and Steinkamp et al. (50). FCM was performed with a FACSCalibur system (Becton Dickinson, Heidelberg, Germany). Illumination was from a 15-mW 488-nm argon ion laser. Trophozoites with internalized particles were detected by green fluorescence from FITC with a 530/30 bandpass filter (FL1-height [FL1-H]). At least 10,000 cells were analyzed per sampling. Setting of analysis gates (i.e., upper and lower thresholds for fluorescence), data handling, manipulation, and presentation were performed with CELLQuest software (Becton Dickinson). Nonviable amoebae were detected by the emission of red fluorescence from PI or the emission of green fluorescence from Ox with a 585/42 bandpass filter (FL2-H). Viable cells that excluded PI or Ox were detected by measurement of the background fluorescence with a 530/30 bandpass filter (FL1-H).

Effects of biguanide-treated particles on phagocytosis. YG beads (2 x 10⁹) and heat-killed C. albicans (2 x 10⁸) and E. coli (2 x 10⁸) cells were suspended in 1.0-ml suspensions of 5 µg of PHMB per ml, 50 µg of PHMB per ml, 5 µg of CHX per ml, or 50 µg of CHX per ml for 20 h in microcentrifuge tubes at 22°C. Suspensions were harvested by centrifugation at 4,000 rpm (Beckman Microfuge Lite), and the treated pellet was suspended in 1 ml of 0.9% NaCl. The level of phagocytosis of these treated particles (0.3 ml) by trophozoites (2 x 10⁶/ml in 2.7 ml) was compared with the level of phagocytosis of particles from solutions of the two concentrations of biocides by FCM.

Effects of biocides and particles on growth of amoebae. YG beads (final concentration, 2 x 10⁷/ml) were inoculated in 75 ml of PYG containing trophozoites of Acanthamoeba spp. (2 x 10⁵/ml) and 5 or 50 µg of PHMB per ml and 50 µg of CHX per ml. The cultures were incubated at 30°C with shaking at 135 rpm for 40 h. Periodically, 100 µl was taken from the cultures and added to 200 µl of 0.3% methylene blue solution in a 1.5-ml microcentrifuge tube. The number of viable amoebae was determined by microscopic examination of the amoebae (methylene blue exclusion) in a counting chamber (55).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Rate of internalization of particles by trophozoites of Acanthamoeba spp. in PBS without biguanides. The mean number of internalized beads per amoeba and the mean number of yeast per amoeba remained essentially constant during incubation times of 10 to 60 min, but the percentage of trophozoites of A. castellanii (60 to 70%) and A. polyphaga (90 to 96%) with internalized yeasts was highest after 60 min of incubation (Table 1). Approximately 35% of the trophozoites of both species of amoeba showed some internalized bacteria within 60 min, but the numbers per amoeba could not be determined accurately because of engulfment of clumps of bacteria followed by their rapid digestion.

View this table: [in this window] [in a new window]

TABLE 1. Internalization of polystyrene latex YG beads, yeasts, and bacteria by trophozoites of Acanthamoeba spp. after incubation for 60 min at 30°Ca

Survival of trophozoites in various concentrations of biguanides without particles. The numbers of viable trophozoites of A. castellanii and A. polyphaga decreased with exposure to increasing concentrations of the biguanides and generally with increased times of exposure (Table 2). Trophozoites of A. polyphaga tended to be more resistant than those of A. castellanii to both biguanides. As determined by staining with Ox, exposures to the biguanides with time, particularly at concentrations of 50 and 100 µg/ml, suggested that CHX was the more effective antimicrobial. FCM histogram plots of the shift in the fluorescence intensities of the trophozoites (stained with PI and Ox) after exposure to the biguanides are shown in Fig. 1 and 2. The results of FCM analyses with PI and Ox staining were in general agreement, but staining with PI indicated that more trophozoites were viable after exposure to both biguanides than staining with Ox. Staining with Ox indicated 7% viability after 2 h of exposure to 50 µg of PHMB per ml, whereas staining with PI indicated 22% viability.

View this table: [in this window] [in a new window]

TABLE 2. Log reductions of trophozoites of A. castellanii and A. polyphaga in different concentrations of PHMB or CHX as determined by viability staining with PI and Oxa

View larger version (43K): [in this window] [in a new window]

FIG. 1. Histogram plots (FL2-H) of the number of viable trophozoites of A. castellanii after exposure to PHMB in PBS and staining with PI. A total of 10,000 trophozoites were analyzed by FCM. Viable trophozoites exclude PI and have low levels of FL2 fluorescence. Dead cells fluoresce red and have high levels of FL2 fluorescence.

View larger version (49K): [in this window] [in a new window]

FIG. 2. Histogram plots (FL2-H) of the number of viable trophozoites of A. castellanii after exposure to PHMB in PBS and staining with Ox. A total of 10,000 trophozoites were analyzed by FCM. Viable trophozoites exclude Ox and have low levels of FL2 fluorescence. Dead cells fluoresce green and have high levels of FL2 fluorescence.

Effects of biguanide-treated particles on phagocytosis. The effects of PHMB- and CHX-treated particles and solutions of these biguanides on phagocytosis of particles by A. castellanii are indicated in Table 3. The trophozoites exposed to the treated particles encounter only traces of bound biguanide compared to the concentrations encountered during phagocytosis in the solutions. About 30% of the trophozoites internalized untreated latex beads, whereas only about 5 to 6% of the trophozoites internalized beads treated with PHMB and CHX. Phagocytosis of treated and treated yeasts was reduced by 6 to 30%. In contrast, phagocytosis of treated E. coli appeared to be enhanced by low levels of PHMB but appeared to be inhibited by CHX (Table 3).

View this table: [in this window] [in a new window]

TABLE 3. Effects of PHMB and CHX on phagocytosis of latex beads, C. albicans, and E. coli by trophozoites of A. castellanii

When untreated latex beads and trophozoites were simultaneously introduced into solutions of both biguanides, the incidence of trophozoite phagocytosis of latex beads was not reduced as markedly as it was with the treated beads. The incidence of phagocytosis decreased in instances in which trophozoites were introduced in biguanide solutions (particularly CHX) containing untreated yeasts and bacteria (Table 3).

Additional FCM analyses of trophozoite viability after phagocytosis of treated particles in PBS supported the earlier results that CHX has a greater inhibitory effect than PHMB (Fig. 3). Less than 10% of the trophozoites were viable after phagocytosis of yeasts and bacteria treated with 50 µg of CHX per ml, whereas 40% were viable after phagocytosis of yeasts and bacteria treated with PHMB (Fig. 3).

View larger version (43K): [in this window] [in a new window]

FIG. 3. Viabilities of A. castellanii trophozoites after phagocytosis of biguanide-treated particles. The percentages of viable trophozoites (2 x 106/ml) of A. castellanii after phagocytosis of treated YG beads (), C. albicans (), and E. coli () were determined by FCM analysis of 10,000 trophozoites. The error bars represent the standard error (n = 3).

Survival of trophozoites in the presence of biguanides and YG beads. Because CHX at 50 µg/ml was more inhibitory than PHMB to phagocytosis and because 50 µg of CHX per ml in PYG was biocidal after 6 to 18 h (data not given), we examined the survival of trophozoites in PYG broth containing PHMB with and without beads (Fig. 3). In contrast to the PYG control, growth did not occur over a 40-h incubation period in the presence of either 5 or 50 µg of PHMB per ml, and 50 µg/ml was biocidal between 15 and 20 h (data not shown). No mature cysts developed in the growth medium in the presence of PHMB. Trophozoites of A. castellanii but not trophozoites of A. polyphaga appeared to be more susceptible to 5 µg of PHMB per ml in the presence of beads than without beads (Fig. 4).

View larger version (28K): [in this window] [in a new window]

FIG. 4. Viabilities of A. castellanii (AC) (a) and A. polyphaga (AP) (b) in PYG growth medium containing PHMB (5 or 50 µg/ml). Trophozoites of Acanthamoeba spp. (105/ml) were inoculated into PYG containing latex beads (YG; 107/ml) or 50 µg/ml PHMB per ml and were incubated at 30°C with rotary shaking at 135 rpm for 40 h. The average percentage of viable amoebae per milliliter (n = 3) was determined from microscopic counts of cells stained with 0.3% methylene blue.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Acanthamoeba spp. bound and internalized more yeasts (and, seemingly, more bacteria) than latex beads. These observations are in general agreement with those of Bowers and Olszewski (11), who reported that A. castellanii preferentially ingested yeasts over latex beads. Also in agreement with Deere et al. (19), Ox staining indicated larger numbers of dead or damaged cells than PI staining did. In solutions of biguanides without particles, sublethal concentrations of CHX (5 µg/ml) were more inhibitory to amoebae than sublethal concentrations of PHMB, with A. polyphaga being more resistant than A. castellanii. Ludwig et al. (38) reported that treatment with a CHX (0.005%)-thimerosal (0.001%) solution killed trophozoites and cysts of A. castellanii but that those of A. polyphaga survived the treatment. However, some reports indicate variations in the sensitivities of both species (12, 44, 51). Khunkitti et al. (30) reported that the uptake of PHMB was rapid and caused more damage to trophozoites and cysts of A. castellanii than equivalent concentrations of CHX. A later report by Khunkitti et al. (31) indicated that concentrations of 6.125, 12.5, and 25 µg of CHX per ml were more biocidal for A. castellanii (1.75 x 10⁴ to 1.03 x 10⁵ cells/ml) at different stages of cell development than the same concentrations of PHMB. Contrasting susceptibility data in the literature may be related in part to life cycle variances as well as differences in the fragilities of cysts and trophozoites used in separate experiments (9, 29). Such factors were probably also involved in deviations in the response of our inocula to increased concentrations of biguanides. We used separate inoculum preparations in our experiments.

In our comparative susceptibility studies, we used log-phase cells composed of at least 90% viable trophozoites and cultures with similar susceptibilities to CHX. The biocidal activity of PHMB was enhanced in the presence of particles. After 20 h of incubation, 50% of the A. castellanii trophozoites in a solution of 5 µg of PHMB per ml were viable, whereas when YG beads were present, only 25% of the trophozoites survived. The concentrations of PHMB bound to treated particles were substantially less than the concentrations available to trophozoites in the solutions of PHMB; nevertheless, the incidence of phagocytosis of treated versus untreated particles was reduced.

The increased amoebicidal activity of PHMB in the presence of particles may be related to an alteration of the cytoplasmic membrane. During phagocytosis by amoebae, the attachment of a particle to the surface of the cell initiates and apparently stimulates a complex series of events in which the plasma membrane with the attached particle invaginates or the cell flows outward and surrounds the particle (57). Avery et al. (5) reported that unsaturation of membrane lipids of A. castellanii following chilling of late-exponential-phase or early-stationary-phase cells coincides with increases in phagocytosis, suggesting that the fluidity of the membrane dictates phagocytic activity. Particle-induced phagocytosis probably involves increased levels of uptake of a solution and the potential accumulation of an inhibitor to an injurious level. The decline in the level of phagocytosis and the viabilities of the trophozoites of A. castellanii exposed to sublethal concentrations of PHMB may reflect this hypothesis. An enhanced biocidal activity of CHX for trophozoites during phagocytosis of particles, particularly yeasts, by trophozoites was not as evident as the enhanced biocidal activity observed for PHMB, possibly because the greater susceptibility of trophozoites to CHX masked the effect. Alternatively, PHMB bound at low concentrations to the yeast cell wall was partially neutralized.

Our study suggests that CHX is more inhibitory to phagocytosis by Acanthamoeba than equivalent concentrations of PHMB. Furthermore, trophozoites of A. castellanii and A. polyphaga actively internalizing particles are more susceptible to CHX and PHMB than trophozoites in solutions of these compounds without particles. The presence of particle contaminants in PHMB multipurpose solutions in lens cases with the resultant enhanced uptake of PHMB may help explain, in part, the apparent subsidence of amoebic keratitis in recent years.

 
* Corresponding author. Mailing address: Department of Biology, Georgia State University, P.O. Box 4010, Atlanta, GA 30302-4010. Phone: (404) 651-3103. Fax: (404) 651-2509. E-mail: biosac{at}langate.gsu.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Ábel, G., J. Szollosi, and J. Fachet. 1991. Phagocytosis of fluorescent latex microbeads by peritoneal macrophages in different strains of mice: a flow cytometric study. Eur. J. Immunogen. 18:239-245.[Medline]
2
2. Ahearn, D. G., and M. M. Gabriel. 1997. Contact lenses, disinfectants, and Acanthamoeba keratitis. Adv. Appl. Microbiol. 43:35-56.[Medline]
3
3. Allen, P. G., and E. A. Dawidowicz. 1990. Phagocytosis in Acanthamoeba. I. A mannose receptor is responsible for the binding and phagocytosis of yeast. J. Cell. Physiol. 145:508-513.[CrossRef][Medline]
4
4. Auran, J. D., M. B. Starr, and F. A. Jakobiec. 1987. Acanthamoeba keratitis: a review of the Literature. Cornea 6:2-26.[Medline]
5
5. Avery, S. V., J. L. Harwood, and D. Lloyd. 1994. Low-temperature-induced adaptations in fatty acid metabolism of Acanthamoeba castellanii cultures of different ages: relationship to changes in cell division, oxygen uptake, and phagocytic activity. Microbiology 140:2423-2431.[Abstract/Free Full Text]
6
6. Avery, S. V., J. L. Harwood, and D. Lloyd. 1995. Quantification and characterization of phagocytosis in the soil amoeba Acanthamoeba castellanii by flow cytometry. Appl. Environ. Microbiol. 61:1124-1132.[Abstract]
7
7. Biddick, C. J., L. H. Rogern, and T. J. Brown. 1984. Viability of pathogenic and nonpathogenic free-living amoebae in long-term storage at a range of temperatures. Appl. Environ. Microbiol. 48:859-860.[Abstract/Free Full Text]
8
8. Bier, J. W., and T. K. Sawyer. 1990. Amoeba isolated from laboratory eyewash stations. Curr. Microbiol. 20:349-350.
9
9. Borazjani, R. N., L. L. May, J. A. Noble, S. V. Avery, and D. G. Ahearn. 2000. Flow cytometry for determination of the efficacy of contact lens disinfecting solutions against Acanthamoeba spp. Appl. Environ. Microbiol. 66:1057-1061.[Abstract/Free Full Text]
10
10. Bowen, I. D., W. T. Coakley, and C. J. James. 1979. The digestion of Saccharomyces cerevisiae by Acanthamoeba castellanii. Protoplasma 98:63-71.[CrossRef]
11
11. Bowers, B., and T. E. Olszewski. 1983. Acanthamoeba discriminates internally between digestible and indigestible particles. J. Cell Biol. 97:317-322.[Abstract/Free Full Text]
12
12. Brandt, F. H., D. A. Ware, and G. S. Visvesvara. 1989. Viability of Acanthamoeba cysts in ophthalmic solutions. Appl. Environ. Microbiol. 55:1144-1146.[Abstract/Free Full Text]
13
13. Buck, S. L., R. A. Rosenthal, and B. A. Schlech. 2000. Methods used to evaluate the effectiveness of contact lens care solutions and other compounds against Acanthamoeba: a review of the literature. Contact Lens Assoc. Ophthalmol. J. 26:72-84.
14
14. Bunting, L. A., J. B. Neilson, and G. S. Bulmer. 1979. Cryptococcus neoformans: gastronomic delight of a soil ameba. Sabouraudia 17:225-232.[Medline]
15
15. Burger, R. M., R. J. Franco, and K. Drlica. 1994. Killing acanthamoebae with polyaminopropyl biguanide: quantitation and kinetics. Antimicrob. Agents Chemother. 38:886-888.[Abstract/Free Full Text]
16
16. Castellani, A. 1930. An amoeba growing in cultures of a yeast. J. Trop. Med. Hyg. 33:188-191.
17
17. Centers for Disease Control. 1986. Acanthamoeba keratitis in soft-contact-lens wearers. Morb. Mortal. Wkly. Rep. 35:405-408.[Medline]
18
18. Davies, D. J. G., Y. Anthony, B. J. Meakin, S. Kilvington, and C. B. Anger. 1990. Evaluation of the anti-acanthamoebal activity of five contact lens disinfectants. Int. Contact Lens Clin. 17:14-20.[CrossRef]
19
19. Deere, D., J. Shen, G. Vesey, P. Bell, P. Bissinger, and D. Veal. 1998. Flow cytometry and cell sorting for yeast viability assessment and cell selection. Yeast 14:147-160.[CrossRef][Medline]
20
20. Drevets, D. A., and P. A. Campbell. 1991. Macrophage phagocytosis: use of fluorescence microscopy to distinguish between extracellular and intracellular bacteria. J. Immunol. Methods 142:31-38.[CrossRef][Medline]
21
21. Drozanski, W., and T. Chemielewski. 1979. Electron microscopic studies of Acanthamoeba castellanii infected with obligate intracellular bacterial parasite. Acta Microbiol. Polonica 28:476-511.
22
22. Elder, M. J., S. Kilvington, and J. K. G. Dart. 1994. A clinicopathologic study of in vitro sensitivity testing and Acanthamoeba keratitis. Investig. Ophthalmol. Vis. Sci. 35:1059-1064.[Abstract/Free Full Text]
23
23. Ferrante, A., B. Rowan-Kelly, and Y. H. Thong. 1984. In vitro sensitivity of virulent Acanthamoeba culbertsoni to a variety of drugs and antibiotics. Int. J. Parasitol. 14:53-56.[CrossRef][Medline]
24
24. Gorlin, A. I., M. M. Gabriel, L. A. Wilson, and D. G. Ahearn. 1996. Binding of Acanthamoeba to hydrogel contact lenses. Curr. Eye Res. 15:151-155.[Medline]
25
25. Gorlin, A. I., M. M. Gabriel, L. A. Wilson, and D. G. Ahearn. 1996. Effect of adhered bacteria on the binding of Acanthamoeba to hydrogel lenses. Arch. Ophthalmol. 114:576-580.[Abstract/Free Full Text]
26
26. Harf, C. 1993-1994. Free-living amoeba: interactions with environmental pathogenic bacteria. Endocytobiosis Cell Res. 10:167-183.
27
27. Hoffman, E. O., C. Garcia, J. Lunseth, P. McGarry, and J. Coover. 1978. A case of primary amebic meningoencephalitis. Light and electron microscopy, and immunological studies. Am. J. Trop. Med. Hyg. 27:29-38.
28
28. Jones, D. B., G. S. Visvesvara, and N. M. Robinson. 1975. Acanthamoeba polyphaga keratitis and Acanthamoeba uveitis associated with fatal meningoencephalitis. Trans. Ophthalmol. Soc. UK 95:221-232.[Medline]
29
29. Khunkitti, W., D. Lloyd, J. R. Furr, and A. D. Russell. 1996. The lethal effects of biguanide on cysts and trophozoites of Acanthamoeba castellanii. J. Appl. Bacteriol. 81:73-77.[Medline]
30
30. Khunkitti, W., D. Lloyd, J. R. Furr, and A. D. Russell. 1997. Aspects of the mechanisms of action of biguanides on trophozoites and cysts of Acanthamoeba castellanii. J. Appl. Microbiol. 82:107-114.[Medline]
31
31. Khunkitti, W., D. Lloyd, J. R. Furr, and A. D. Russell. 1998. Acanthamoeba castellanii: growth, encystment, excystment and biocide susceptibility. J. Infect. 36:43-48.[CrossRef][Medline]
32
32. Kilvington, S. 1990. Activity of water biocide chemicals and contact lens disinfectants on pathogenic free-living amoebae. Int. Biodeter. 26:127-138.[CrossRef]
33
33. Kilvington, S., and D. G. White. 1994. Acanthamoeba: biology, ecology, and human disease. Rev. Med. Microbiol. 5:12-20.
34
34. Kingston, D., and D. C. Warhurst. 1969. Isolation of amoebae from the air. J. Med. Microbiol. 2:27-36.[Abstract/Free Full Text]
35
35. Larkin, D. F. P., S. Kilvington, and J. K. G. Dart. 1992. Treatment of Acanthamoeba keratitis with polyhexamethylene biguanide. Ophthalmology 99:185-191.[Medline]
36
36. Lewis, M. B., and R. A. Weisman. 1976. Inhibition by theophylline of phagocytosis in Acanthamoeba castellanii. J. Protozool. 23:337-340.[Medline]
37
37. Lindquist, T. D., N. A. Sher, and D. J. Doughman. 1988. Clinical signs and medical therapy of early Acanthamoeba keratitis. Arch. Ophthalmol. 106:73-77.[Abstract/Free Full Text]
38
38. Ludwig, I. H., D. M. Meisler, I. Rutherford, F. E. Bican, R. H. Langston, and G. S. Visvesvara. 1986. Susceptibility of Acanthamoeba to soft contact lens disinfection systems. Investig. Ophthalmol. Vis. Sci. 27:626-628.[Abstract/Free Full Text]
39
39. Martinez, A. J. 1985. Free-living amebas: natural history, prevention, diagnosis, pathology and treatment of disease. CRC Press, Inc., Boca Raton, Fla.
40
40. Mergeryan, H. 1991. The prevalence of Acanthamoeba in the human environment. Rev. Infect. Dis. 13:410-412.
41
41. Moore, M. B., J. P. McCulley, C. Newton, L. M. Cobo, G. N. Foulks, D. M. O'Day, K. J. Johns, W. T. Driebe, L. A. Wilson, R. J. Epstein, and D. J. Doughman. 1987. Acanthamoeba keratitis: a growing problem in soft and hard contact lens wearers. Ophthalmology 94:1654-1661.[Medline]
42
42. Nero, L. C., M. G. Tarver, and L. R. Hedrick. 1964. Growth of Acanthamoeba castellanii with the yeast Torulopsis famata. J. Bacteriol. 87:220-225.[Abstract/Free Full Text]
43
43. Paszko-Kolva, C., H. Yamamoto, M. Shahamat, T. K. Sawyer, G. Morris, and R. R. Colwell. 1991. Isolation of amoebae, Pseudomonas and Legionella spp. from eyewash stations. Appl. Environ. Microbiol. 57:163-167.[Abstract/Free Full Text]
44
44. Penley, C. A., S. W. Willis, and S. G. Sickler. 1989. Comparative antimicrobial efficacy of soft and rigid gas permeable contact lens solutions against Acanthamoeba. Contact Lens Assoc. Ophthalmol J. 15:257-260.[CrossRef]
45
45. Schuster, F. L., M. Rahman, and S. Griffith. 1993. Chemotactic responses of Acanthamoeba castellanii to bacteria, bacterial components, and chemotactic peptides. Trans. Am. Microsc. Soc. 112:43-61.[CrossRef]
46
46. Seal, D. V., E. S. Bennett, A. K. McFadyen, E. Todds, and A. Tomlinson. 1995. Differential adherence of Acanthamoeba to contact lenses: effects of material characteristics. Optom. Vis. Sci. 72:23-28.[CrossRef][Medline]
47
47. Silvany, R. E., J. M. Dougherty, J. P. McCulley, T. S. Wood, R. W. Bowman, and M. B. Moore. 1990. The effect of currently available contact lens disinfection systems on Acanthamoeba castellanii and Acanthamoeba polyphaga. Ophthalmology 97:286-290.[Medline]
48
48. Simmons, R. B., L. J. Rose, S. A. Crow, and D. G. Ahearn. 1999. The occurrence and persistence of mixed biofilms in automobile air conditioning systems. Curr. Microbiol. 39:141-145.[CrossRef][Medline]
49
49. Steinert, M., K. Birkness, E. White, B. Fields, and F. Quinn. 1998. Mycobacterium avium bacilli grow saprozoically in coculture with Acanthamoeba polyphaga and survive within cyst walls. Appl. Environ. Microbiol. 64:2256-2261.[Abstract/Free Full Text]
50
50. Steinkamp, J. A., J. S. Wilson, G. C. Saunders, and C. C. Stewart. 1982. Phagocytosis: flow cytometric quantitation with fluorescent microspheres. Science 215:64.[Abstract/Free Full Text]
51
51. Stevens, A. R., and E. Willaert. 1980. Drug sensitivity and resistance of four Acanthamoeba species. Trans. R. Soc. Trop. Med. Hyg. 74:806-808.[CrossRef][Medline]
52
52. Stevenson, R. W. W., and D. V. Seal. 1998. Has the introduction of multi-purpose solutions contributed to a reduced incidence of Acanthamoeba keratitis in contact lens wearers?: a review. Contact Lens Anterior Eye 21:89-92.[CrossRef]
53
53. Tirado-Angel, J., M. M. Gabriel, L. A. Wilson, and D. G. Ahearn. 1996. Effects of polyhexamethylene biguanide and chlorhexidine on four species of Acanthamoeba in vitro. Curr. Eye Res. 15:225-228.[Medline]
54
54. Visvesvara, G. S. 1995. Pathogenic and opportunistic free-living amebae, 6th ed. American Society for Microbiology, Washington, D.C.
55
55. Wang, X., and D. G. Ahearn. 1997. Effect of bacteria on survival and growth of Acanthamoeba castellanii. Curr. Microbiol. 34:212-215.[CrossRef][Medline]
56
56. Weekers, P. H. H., P. L. E. Bodelier, J. P. H. Wijen, and G. D. Vogels. 1993. Effects of grazing by the free-living soil amoebae Acanthamoeba castellanii, Acanthamoeba polyphaga, and Hartmanella vermiformis on various bacteria. Appl. Environ. Microbiol. 59:2317-2319.[Abstract/Free Full Text]
57
57. Weisman, R. A., and E. D. Korn. 1967. Phagocytosis of latex beads by Acanthamoeba. I. Biochemical properties. Biochemistry 6:485-497.[CrossRef][Medline]
58
58. Willaert, E., A. R. Stevens, and G. Healy. 1978. Retrospective identification of Acanthamoeba culbertsoni in a case of amoebic meningoencephalitis. J. Clin. Pathol. 31:717-720.[Abstract/Free Full Text]
59
59. Wilson, L. A., A. D. Sawant, R. B. Simmons, and D. G. Ahearn. 1990. Microbial contamination of contact lens storage cases and solutions. Am. J. Ophthalmol. 110:193-198.[Medline]

---

Antimicrobial Agents and Chemotherapy, July 2002, p. 2069-2076, Vol. 46, No. 7
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.7.2069-2076.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Hughes, R., Heaselgrave, W., Kilvington, S. (2003). Acanthamoeba polyphaga Strain Age and Method of Cyst Production Influence the Observed Efficacy of Therapeutic Agents and Contact Lens Disinfectants. Antimicrob. Agents Chemother. 47: 3080-3084 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Noble, J. A. Articles by Crow, S. A. Search for Related Content PubMed PubMed Citation Articles by Noble, J. A. Articles by Crow, S. A., Jr.