Emergence of resistance to biocides during differentiation of Acanthamoeba castellanii

N. A. Turnera, A. D. Russella,*, J. R. Furra and D. Lloydb

a Welsh School of Pharmacy and b School of Biosciences, Cardiff University, Cardiff CF10 3XF, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A synchronous encystment method was used to study the order of development of resistance of Acanthamoeba castellanii to a range of biocides. The emerging resistance during encystation to short-term exposure to the minimum amoebicidal concentrations of each biocide tested was recorded during the first 36 h of the differentiation process. Hydrochloric acid and moist heat were tested as possible resistance markers. Development of the acid-insoluble, proteincontaining, ectocyst wall and the cellulose endocyst wall was followed by quantification of the acid- and alkali-insoluble residues of cell samples removed from synchronous encystment cultures up to 36 h. Resistance to chemical agents (polyhexamethylene biguanide, benzalkonium chloride, propamidine isethionate, pentamidine isethionate, dibromopropamidine isethionate, hydrogen peroxide) and to moist heat was seen to develop between 14 and 24 h after trophozoites were inoculated into the encystment media. Resistance to hydrochloric acid developed between 0 and 2 h and to chlorhexidine diacetate between 24 and 36 h. Levels of acid-insoluble residues began to increase after 8 h and alkali-insoluble residues (cellulose) were detected after 16 h and coincided with the emergence of resistance to all the agents tested except hydrochloric acid. The results suggest that resistance to the biocides tested probably results largely from the physical barrier of the cyst walls rather than as a consequence of a metabolically dormant cyst.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Free-living amoebae of the genus Acanthamoeba are ubiquitous in natural environments. A number of pathogenic strains have been associated with cases of primary amoebic meningoencephalitis (although Naegleria fowleri is the main aetiological agent), amoebic encephalitis and the potentially sight-threatening eye infection Acanthamoeba keratitis which is mainly associated with contact lens wear.1

During periods of stress, trophozoites of Acanthamoeba undergo a cellular differentiation process (termed encystment) resulting in the formation of double-walled cysts with their associated increased in vitro resistance to antimicrobial agents used both in therapy and in contact lens disinfection systems.2 The outer layer (ectocyst wall) consists of an acid-insoluble protein-containing material, and the inner wall (endocyst wall) is composed mainly of cellulose.3 Three phases have been recognized during the application of synchronous encystment methods, viz. the induction stage, the wall-synthesis stage (which is divided into two marker events as each wall is synthesized) and finally a dormant stage with reduced metabolic activity. These phases are sequential and the duration of each stage is thought to be approximately 4–6 h, 20–24 h and 2–7 days, respectively.4

We were interested in the emergence of resistance to commonly used antimicrobial agents during the encystation process and how this relates to (i) the known order of cyst wall development, and (ii) other known ultrastructural and physiological changes. Various biocides used in the treatment of Acanthamoeba keratitis and in contact lens disinfection systems were tested using a synchronous encystation method in order to determine a possible sequential order of resistance of the organism to these agents. These included the aromatic diamidines (propamidine, pentamidine and dibromopropamidine isethionates), cationic disinfectants (chlorhexidine diacetate, polyhexamethylene biguanide and benzalkonium chloride) and hydrogen peroxide. Cysts of Acanthamoeba demonstrate a relatively high resistance to hydrochloric acid5 as well as to moist heat,6 a key resistance marker used in similar studies of bacterial endospore formation.7 These two agents were tested as possible resistance markers during the encystation process. An attempt was also made to follow the development of the cyst walls by quantification of the alkali-insoluble cellulose component and acid-insoluble component (believed to be mainly protein) of cell samples taken during encystation.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals

Chlorhexidine diacetate (CHA), phosphate-buffered saline (PBS) tablets, potassium chloride, 2-amino-2-methyl-1,3-propanediol, dibasic sodium phosphate, azolectin, Tween 80, sodium carbonate, sodium citrate, sodium carbonate and Folin–Ciocalteu phenol reagent were purchased from Sigma Chemicals Co. (Poole, Dorset, UK). Benzalkonium chloride (BZK) was purchased from Thorn and Ross (Huddersfield, UK). Polyhexamethylene biguanide (PHMB) was provided by AstraZeneca Ltd (Macclesfield, Cheshire, UK), and propamidine isethionate (PROP), pentamidine isethionate (PENT) and dibromopropamidine isethionate (DBPI) by Rhone-Poulenc (Dagenham, UK). Hydrogen peroxide, hydrochloric acid, sulphuric acid, sodium chloride, sodium hydroxide and sodium thiosulphate were purchased from Fisher Scientific Ltd (Loughborough, UK). Magnesium sulphate, calcium chloride, potassium dihydrogen orthophosphate, sodium hydrogen carbonate and cupric sulphate were purchased from BDH Ltd (Poole, Dorset, UK).

Organism and culture

Acanthamoeba castellanii (Neff strain), was grown axenically in a growth medium (PYG) containing proteose peptone 0.75% (w/v; Difco, Becton Dickinson UK Ltd, Oxford, UK), yeast extract 0.75% (w/v; Oxoid Ltd, London, UK) and glucose 1.5% (w/v; Fisher). Five millilitres from a stationary phase culture was inoculated into 50 mL PYG medium and incubated at 30°C in a shaking water-bath (Haake SWB 20, Karlsruhe, Germany) agitated at 90 strokes/min. Inoculum cultures for transfer to encystment media were grown axenically in PYG prepared as described by Neff et al.,8 except that the vitamins were eliminated; this medium contains 0.001 M MgSO4.7H2O, 0.00005 M CaCl2.2H2O and 0.002 M KH2PO4, adjusted to pH 7.0. Encystment cultures to be used in minimum cysticidal concentration (MCC) assays were prepared as follows: trophozoites grown as described above to stationary phase for 72 h were washed twice in encystment medium and inoculated into 100 mL of this medium in a 250 mL flask, incubated at 30°C and agitated at 90 strokes/min in a shaking water-bath for 7 days. The encystment medium8 consisted of 0.1 M KCl, 0.02 M 2-amino-2-methyl-1,3-propanediol, 0.008 M MgSO4.7H2O, 0.0004 M CaCl2.2H2O and 0.001 M NaHCO3. The pH was adjusted to 8.8 before autoclaving. All cell counts were performed with a haemocytometer slide (Fuchs-Rosenthal, Fisher).

Standard plaque assay

Standard plaque assays were adapted from Khunkitti et al.9 Samples (0.1 mL) were spread on the surface of an SM/5 plate consisting of (w/v): proteose peptone, 0.2%; yeast extract, 0.2%; glucose, 0.2%; MgSO4, 0.02%; Bacto-agar (Difco), 2.0%. A 30 µL aliquot of an overnight culture of Escherichia coli 8545 was then mixed with 3 mL of molten top agar containing (w/v): tryptone (Oxoid, L42), 0.01%; sodium chloride, 0.05%; Bacto-agar, 0.7% at 45°C and poured on to the plate. The plates were incubated at 30°C for 10 days. Plaques were counted daily after 2 days' incubation.

Determination of minimum trophocidal concentration (MTC), MCC and resistance to moist heat

Double-washed cell suspensions (0.1 mL) in Page's amoeba saline [PAS, containing (g/L); NaCl 0.12, MgSO4.7H2O 0.004, Na2HPO4 0.142, KH2PO4 0.136, CaCl2.2H2O 0.004] were added to 0.9 mL of biocide solution to give the required final concentration (c. 1.5 x 105 cells/mL) and held at 24 ± 2°C for 1 h. Samples (0.1 mL) were then neutralized for 10 min with 0.9 mL of neutralizer, which consisted of 0.75% w/v azolectin in 5% Tween (polysorbate) 80 for CHA, PHMB, BZK, PROP, PENT and DBPI. The time for 0.5% w/v sodium thiosulphate to neutralize hydrogen peroxide was 30 min. HCl-treated suspensions were immediately washed twice in deionized water. After neutralization, the suspensions were centrifuged (MSE, Micro Centaur, Loughborough, UK) at 800g for 5 min, washed in deionized water and serially diluted in PAS before performing the standard plaque assay as described above. The lowest biocide concentration preventing plaque formation after incubation for 10 days at 30°C was taken as the MTC for the trophozoites and as the MCC for the cysts.

Resistance to moist heat was determined as follows: double-washed cell suspensions (0.1 mL) in PAS (c. 1.5 x 105 cells/mL) were added to 0.9 mL of pre-equilibrated deionized water and held for 30 min at a range of temperatures before transferring the bottle to cold water (4°C) for 5 min in order to quench the reaction. Samples (0.1 mL) were then diluted in PAS before performing the standard plaque assay as described above. The lowest temperature preventing plaque formation after incubation for 10 days at 30°C was taken as the minimum effective temperature.

Synchronous encystment

Trophozoites from a 72 h culture grown as described above were washed twice in encystment media (800g for 5 min) and inoculated into a 1 L bench-top fermenter (LH Fermentation 502D, LH Engineering Co. Ltd, Stoke Poges, UK) containing 800 mL of encystment medium to give a final cell concentration of c. 1.5 x 105 cells/mL. The fermenter vessel was maintained at 30°C, agitated at 200 rpm and aerated at 1800 cm3/min/L of encystment medium during the assessment periods.

Measurement of cyst resistance

For convenience, two separate assessment periods were carried out, viz. 0–24 and 12–36 h. After inoculation (0 h), samples were taken every 2 h either from 0 to 12 h or from 12 to 24 h. A final sample was taken 12 h after the two assessment periods (at 24 and 36 h, respectively). Differential cell counts were performed every 2 h, as described above. Samples (1 mL) containing c. 1.5 x 105 cells/mL were washed and resuspended in 0.1 mL of PAS, 0.9 mL of each test solution added and held at 24 ± 2°C for 1 h (control samples were resuspended in PAS only). The concentration of each test solution represented the MTC value previously determined for each biocide and HCl. Resistance to moist heat (46°C) was tested by resuspending the washed pellet in 0.1 mL of PAS. To this, 0.9 mL of pre-equilibrated deionized water was added and held for 30 min before transferring the bottle to cold water in order to quench the reaction, as described above. Neutralization of the biocides, serial dilution and standard plaque assays were carried out as described above for the determination of MTC and MCC. All assays were performed in triplicate and repeated on at least two occasions to check the results and order of resistance development.

Quantification of alkali-insoluble cellulose and acid-insoluble components of cysts during encystment

Encystation was initiated and samples (totalling 90 mL) were removed periodically from the fermenter vessel during the separate assessment periods as described previously. Samples were immediately frozen at –20°C and processed within 7 days.

The procedure for quantification of alkali-insoluble cellulose was adapted from Griffiths & Hughes.10 Samples (60 mL) were centrifuged (MSE) at 800g for 10 min then washed three times in deionized water. The pellet was resuspended in 2 M NaOH and autoclaved at 15 psi (120°C) for 1 h. The residue was washed in 2 M HCl and finally five times in deionized water. A standard curve was produced and quantification of the cellulose-containing residue was determined turbidimetrically at 400 nm using a LKB Biochrom, Ultrospec II spectrophotometer (Cambridge, UK).

The procedure for producing acid-insoluble residues was adapted from Saeman et al.11 and Neff & Neff.4 Samples (30 mL) removed from the encystment cultures were centrifuged at 800g for 10 min at 0°C and resuspended in 5 mL PBS before sonication for 3 min in a sonicating water-bath (PUL60, Kerry Ultrasonics Ltd, Hitchin, UK). This was necessary in order to lyse any trophozoites present in the sample. The suspension was washed three times in deionized water (800g for 10 min at 0°C). Samples were kept on ice between the procedures above. The pellet was resuspended in 0.3 mL of 72% v/v sulphuric acid. After 1 h incubation at 30°C the suspension was diluted with 9 mL of deionized water and centrifuged at 2000g for 20 min followed by repeated washing in deionized water (2000g for 15 min) until the supernatant was neutral to litmus paper. Finally the pellet was resuspended in 0.1 mL of 1 M NaOH, and dissolved by heating to 90°C for 30 min before the Lowry12 assay was carried out. There was a linear relationship between dry weight of the acid-insoluble residue and the colour development of the folin phenol reagent at 750 nm.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
For the purposes of this study, the attainment of resistance was defined as the time required for 1% of the differentiating cells to survive the short-term exposure treatment in question, as estimated by standard plaque assays. This is an arbitrary value which has been assigned during similar studies using bacterial endospores.7,13,14

The values obtained during the determination of the MTC and MCC of the biocides tested demonstrated that trophozoites were sensitive to all the agents tested (Table IGo). Chlorhexidine diacetate (8 mg/L) and PHMB (2.5 mg/L) were the most, and DBPI (60 mg/L) the least, effective against trophozoites. Cysts were sensitive to CHA, BZK and hydrogen peroxide at higher concentrations but not to the diamidines (PROP, PENT and DBP) or PHMB at the highest concentrations tested (500 mg/L). Trophozoites were sensitive to HCl at concentrations of 0.08 M and moist heat temperatures of 46°C, whereas cysts were more resistant but were sensitive to 3 M HCl and moist heat temperatures of 56°C (Table IIGo).


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Table I. Minimum trophocidal concentration (MTC) and minimum cysticidal concentration (MCC) of various biocide solutions for A. castellanii
 

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Table II. Sensitivity of A. castellanii to hydrochloric acid and moist heat
 
During the first assessment period (0–24 h, Figure 1a and bGo) resistance to all the biocides tested developed between 12 h and 24 h as did resistance to moist heat. Resistance to 0.08 M HCl developed between 0 and 2 h and continued to rise throughout the assessment period, closely matching the observed percentage encystation (determined from differential cell counts of trophozoites and induced cysts).



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Figure 1. Emergence of resistance to (a) biguanides and benzalkonium chloride, (b) diamidines and hydrogen peroxide during the first 24 h of encystation of A. castellanii at 30°C. (a) CHA 8 mg/L (x), PHMB 2.5 mg/L ({circ}), BZK 12 mg/L ({blacktriangleup}). (b) PROP 18 mg/L ({circ}), PENT 15 mg/L ({diamondsuit}), DBPI 60 mg/L ({triangleup}) and hydrogen peroxide 0.6% v/v (x). Resistance markers: 0.08 M HCl ({blacksquare}), moist heat 46°C ({square}). Induced cysts (•) are expressed as a percentage of total cell count, and resistance as percentage survivors.

 
Results for the second assessment period (12–36 h) are presented in Figure 2a and bGo. Figure 2aGo illustrates the emergence of resistance during differentiation of A. castellanii to BZK (12 mg/L), PHMB (2.5 mg/L), CHA (8 mg/L) and moist heat (46°C). Resistance to PHMB developed between 14 and 16 h. Resistance to BZK developed between 16 and 18 h as did resistance to moist heat. CHA resistance was the last to emerge and developed between 24 and 36 h.



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Figure 2. Emergence of resistance to (a) biguanides and benzalkonium chloride, (b) diamidines and hydrogen peroxide during 12–36 h of encystation of A. castellanii at 30°C. (a) CHA 8 mg/L (x), PHMB 2.5 mg/L ({circ}), BZK 12 mg/L ({blacktriangleup}). (b) PROP 18 mg/L ({circ}), PENT 15 mg/L ({diamondsuit}), DBPI 60 mg/L ({triangleup}) and hydrogen peroxide 0.6% v/v (x). Resistance markers: 0.08 M HCl ({blacksquare}), moist heat 46°C ({square}). Induced cysts (•) are expressed as percentage of total cell count, and resistance as percentage survivors.

 
The emergence of resistance to hydrogen peroxide (0.6% v/v), PROP (18 mg/L), PENT (15 mg/L) and DBPI (60 mg/L) is illustrated in Figure 2bGo. Resistance to PROP developed at 12–14 h, to PENT at 14–16 h and to moist heat and hydrogen peroxide at 16–18 h. Finally, resistance to DBPI emerged between 20 and 22 h.

During the first assessment period, the quantity of the acid-insoluble residue obtained from samples taken during encystation was seen to rise after 8 h (Figure 3aGo and Table IIIGo). Cellulose levels (measured turbidimetrically as the alkali-insoluble residue of samples) increased between 12 and 24 h. Values for the levels of the acid-insoluble residue increased throughout the second assessment period while cellulose levels increased only after c. 16 h (Figure 3bGo and Table IIIGo).



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Figure 3. Changes in the levels of acid-insoluble residue ({blacksquare}) and the alkali-insoluble cellulose residue ({blacktriangleup}) of samples during encystation of A. castellanii at 30°C. (a) Represents changes during the first 24 h and (b) 12–36 h after trophozoites are placed in the encystment medium. Induced cysts ({blacksquare}) are expressed as a percentage of total cell count.

 

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Table III. Summary of results: emergence of resistance in Acanthamoeba castellanii to chemical agents and moist heat during 12–36 h of encystmenta
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
From the time that trophozoites are first placed into encystment medium a number of biochemical, physiological and ultrastructural changes are believed to occur.3 During the first 4–6 h (induction phase) trophozoites begin to lose their amoeboid appearance and become rounded.4 Although no cyst walls are discernible at this stage with light microscopy, transmission electron microscopy studies of encysting Acanthamoeba have shown the appearance of a discontinuous layer of amorphous material at the cell surface. This becomes a continuous layer by the time the cells become rounded and represents early ectocyst wall development.15 During the present study, the levels of acid-insoluble residues of samples from encysting cultures showed a slight rise after 8 h which continued throughout both the assessment periods (0–24 and 12–36 h) which substantiates the above observations. It should be noted, however, that a certain level of maturity would probably need to be reached before immature cysts are not lost in the processing of the samples.

Since resistance on short-term exposure to all the biocides and moist heat developed after incubation of cells for 12 h (Table IIIGo), it is likely that changes taking place during the induction phase as well as probably the ectocyst synthesis sub-phase do not result in resistance of the cell to these agents at this time. Resistance to HCl was seen to develop during this period (0–2 h). This observed resistance may be transient since increases in the rate of endogenous respiration during the early stages of encystation of Acanthamoeba (up to 10 h) with non-nutrient encystment methods have been noted.10,16 This is followed by a decline to a negligible value during cyst maturation.10 Proton transport mechanisms are believed to be intimately linked with oxidative phosphorylation as well as the transport of other ions17 and this may explain the early resistance to HCl. If this is so, the mechanism of resistance to 0.08 M HCl during early encystation is unlikely to be the same as the extreme acid tolerance up to 3 M HCl observed in mature metabolically dormant cysts, the nature of which is unclear.

A substantial difference was observed in the MCC values obtained for CHA and PHMB, which was reflected in the time taken for resistance to these compounds to emerge (Figure 2aGo). Khunkitti et al.18 investigated the sensitivity to PHMB of trophozoites in the exponential growth phase, pre-encystment trophozoites (induction phase) and mature cysts using an asynchronous encystment method over a range of PHMB concentrations (6.125–25 mg/L). Pre-encystment trophozoites were slightly more sensitive to CHA than exponential phase trophozoites although the reverse was true for PHMB-treated cells. If differentiating cells have a greater sensitivity to CHA this would explain the difference observed between the times at which resistance to these agents was seen to develop.

From the results presented (Tables I and IIGoGo and Figure IIGo), it would appear that PHMB, even at high concentrations, is not cysticidal, which differs from the findings of other workers.1922 This study, however, was carried out in a medium in which the pH decreases with increasing concentration of PHMB, which would be a contributory factor in this apparent lack of a cysticidal effect. Previous studies from this laboratory9,18 in which PBS, or Tris or borate buffers were employed have shown PHMB to be cysticidal at concentrations of 12.5–25 mg/L. Furthermore, only short-term exposure (1 h, 24 ± 2°C) to biocides was undertaken in the present investigation, as we wished to compare the responses to antimicrobial agent, of trophozoites and developing cysts in relation to cell wall composition. The short-term exposure could also account for the comparatively poor cysticidal activity of the diamidines in this investigation.

The order of emerging resistance of the diamidines was PROP, PENT and finally DBPI (Figure 2bGo). DBPI was shown to have an MTC value over three times higher than both PROP and PENT. Results of in vitro testing of various clinical isolates of Acanthamoeba spp. with PROP and PENT revealed little overall difference between the effectiveness of the two compounds against trophozoites or cysts.19,23,24 Differences in the effectiveness of DBPI against Acanthamoeba isolates when compared with either PENT or PROP have been noted.2527 Concentration values required for inhibition of growth (with a 3 day exposure time)26 and MCCs (48 h contact time)25 have been shown to be more than double. However, in this study resistance to DBPI developed later than to both PROP and PENT. This indicates that differentiating cells might be more sensitive to DBPI than exponential growth phase cells.

Resistance to BZK and hydrogen peroxide emerged at around the same time as to PHMB and the diamidines. Although the mechanism(s) of action of hydrogen peroxide is/are likely to be different from that of the cationic biocides (including BZK) the results suggest that the mechanism of resistance of Acanthamoeba may be similar.

Resistance to all the biocides and moist heat developed between 12 and 36 h and this coincided with a rise in the levels of cellulose determined turbidimetrically as the alkali-insoluble component of cell samples. Similar increases in the level of cellulose have been demonstrated in other studies. Griffiths & Hughes10 detected cellulose in measurable quantities c. 14 h after placement of trophozoites into a comparable non-nutrient encystment medium. Neff & Neff4 detected measurable amounts of cellulose after c. 16 h as an alkali-insoluble anthrone-reactive carbohydrate. While it is reasonable to suppose that the development and thickening of the endocyst wall reduces permeability to chemical agents, the thermal-buffering capacity of even a mature cyst wall is likely to be small. It is generally agreed that resistance to moist heat in bacterial spores is caused principally by partial dehydration of the protoplast with its associated lowering of water activity.28 Bowers & Korn15 observed an increase in water expulsion vesicles after synthesis of the outer cyst wall of A. castellanii was complete and suggested that dehydration occurred either before or during synthesis of the endocyst wall. This would account for the wrinkled appearance of the mature cyst as the cytoplasm was drawn away from the ectocyst wall.15 The partially dehydrated state could be maintained by the presence of the thick osmotically inextensible endocyst wall. Similar mechanisms of heat resistance have been suggested for other organisms, e.g. endospore-forming bacteria28 and spores of Actinomyces spp.29

A summary of results obtained during this present study is provided in Table IIIGo. The means (± S.D.), of all results obtained during the 12–36 h assessment period are shown. Experiments with biocides, HCl and moist heat were performed in triplicate, and all experiments were carried out on at least two different occasions. Although the S.D. values may be high in some instances, the trend in each experiment was the same, and as such the results depicted in Table IIIGo provide an invaluable insight into cell wall composition and the responses to biocides of cells at different stages in their development. In the absence of cyst wall-less mutants or protoplasts of Acanthamoeba it is difficult to assess the role played by the cyst walls in the overall resistance of a mature cyst. However, the results of the present study suggest that the synthesis of the proteinaceous ectocyst wall is unlikely to protect the differentiating cell from the lethal effects of moist heat and the biocides tested. By contrast, resistance to the biocides tested commenced with the synthesis of the cellulosecontaining endocyst wall and probably results largely from the physical barrier of the cyst walls rather than as a consequence of a metabolically dormant cyst.


    Notes
 
* Corresponding author. Tel. +44-02920-875812; Fax: +44-02920-874149; E-mail: russelld2{at}Cardiff.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 5 November 1999; returned 17 January 2000; revised 2 February 2000; accepted 14 February 2000