a Welsh School of Pharmacy and b School of Biosciences, Cardiff University, Cardiff CF10 3XF, UK
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Abstract |
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Introduction |
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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 46 h, 2024 h and 27 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.
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Materials and methods |
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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 FolinCiocalteu 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. 024 and 1236 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.
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Results |
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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 I). 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 II
).
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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 3a and Table III
). 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 3b
and Table III
).
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Discussion |
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Since resistance on short-term exposure to all the biocides and moist heat developed after incubation of cells for 12 h (Table III), 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 (02 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 2a). 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 (6.12525 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 II and Figure II
), 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 12.525 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 2b). 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 III. The means (± S.D.), of all results obtained during the 1236 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 III
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.
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Notes |
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References |
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Received 5 November 1999; returned 17 January 2000; revised 2 February 2000; accepted 14 February 2000