Cytotoxic Activity of N-Chlorotaurine on Acanthamoeba spp.

Abstract

Acanthamoeba spp. are the causative agents of Acanthamoeba keratitis (AK), which mainly occurs in contact lens wearers, and of skin lesions, granulomatous amoebic encephalitis (GAE), and disseminating diseases in the immunocompromised host. AK therapy is complex and irritating for the eye, skin lesions are difficult to treat, and there is no effective treatment for GAE. Therefore, new anti-Acanthamoeba drugs are needed. We investigated the anti-Acanthamoeba activity of N-chlorotaurine (NCT), an endogenous mild antiseptic. It was shown that NCT has amoebicidal qualities, both in phosphate-buffered saline (PBS) and in amoebic culture medium. After 6 h of treatment with 10 mM NCT in PBS, the levels of trophozoites of all strains investigated already showed at least a 2-log reduction. When the trophozoites were treated with 20 mM NCT in culture medium, they showed a 2-log reduction after 24 h. The addition of NH₄Cl to NCT led to a faster decrease in the numbers of living cells, if tests were carried out in PBS. A delay of excystation was observed when cysts were treated with 55 mM (1%) NCT in culture medium. A complete failure of excystment was the result of treatment with 1% NCT plus 1% NH₄Cl in PBS. Altogether, NCT clearly demonstrated amoebicidal activity at concentrations well tolerated by human tissues and might be useful as a topical drug for the treatment of Acanthamoeba infections. The addition of ammonium chloride can be considered to enhance the activity.

Free-living amoebae of the genus Acanthamoeba are the causative agents of Acanthamoeba keratitis (AK), and they have also been associated with infections that occur predominantly in the immunocompromised host, namely, granulomatous amoebic encephalitis, cutaneous lesions, pneumonitis, and sinusitis (5, 38).

In industrialized countries Acanthamoeba keratitis is associated with the use of contact lenses (3, 31). However, AK is also common among patients with a history of trauma and exposure to contaminated water (4, 11, 12, 36, 37). AK is estimated to affect 1 in 250,000 individuals in the United States, and in the United Kingdom ∼400 cases have been diagnosed since 1957 (16). In the United Kingdom, Europe, and other countries with similar contact lens use and hygiene conditions, the incidence is estimated to be 0.33 per 10,000 soft contact lens wearers per year (34). AK is usually treated with a combination of polyhexamethylene biguanide (PHMB) or chlorhexidine and propamidine isethionate (Brolene). However, the therapeutic regimen is difficult to handle, as these disinfectants have to be applied every hour during the first weeks and altogether for up to 6 months (32). PHMB is toxic for human keratocytes at the minimal cysticidal concentrations of 5.62 μg/ml for 24 h and 2.37 μg/ml for 48 h (17). Moreover, it is responsible for complications concerning coinfections with bacteria, which are suppressed by PHMB during therapy but which recrudesce after the termination of therapy. The failure of PHMB therapy is also reported, and among the causes of treatment failure is resistance to propamidine isethionate (6). To date there is no effective treatment for granulomatous amoebic encephalitis (35). Successful therapy for disseminated Acanthamoeba infections is dependent on early diagnosis and the initiation of therapy before involvement of the central nervous system (5).

A general problem in the therapy of Acanthamoeba infections is the ability of the parasite to form cysts in the case of nutrient deprivation, desiccation, and changes in temperature and pH (18). These cysts show high levels of resistance to hydrochloric acid as well as to biocides (15). One remaining cyst can theoretically cause a relapse (41). To date PHMB and chlorhexidine are the most effective cysticidal agents known (16); however, both of them are also toxic to the eye.

N-Chlorotaurine (NCT; Cl—HN—CH₂—CH₂—SO₃—), the hydrophilic derivative of the amino acid taurine, is a long-lived oxidant produced by human granulocytes and monocytes from hypochlorite and taurine during the oxidative burst. It is more stable but considerably less toxic than hypochlorite (24). Long-lived oxidants in vivo may participate in both the elimination of pathogens and the limitation of inflammatory tissue damage (2); the latter occurs by the downregulation of proinflammatory cytokines (19).

After the vermicidal activity of NCT had been demonstrated (45), the solution of the chemically synthesized sodium salt of NCT (8) was shown to possess viricidal, bactericidal, and fungicidal (22-24) activities. Clinical studies with human and rabbit eyes, the human skin, and the external ear canal, among others, showed the very good tolerability and efficacy of 55 mM (1%) NCT (25-27, 29, 39). The oxidative activity of NCT has been detected for a period of time sufficient to inactivate pathogens in vivo in those studies. At 37°C the oxidation capacity decreases to zero within 3 weeks (20). The fact that the aqueous solution of NCT shows a decomposition rate of only 0.43% per day at room temperature (RT), which declines to ∼0.03% per day (10% per year) if it is kept at 2 to 4°C (8), demonstrates the sufficient stability of NCT for clinical application. Thus, this substance may have a high potential as a topical drug for the treatment of AK and Acanthamoeba skin lesions.

The aim of the present study was to characterize the in vitro susceptibility to NCT of trophozoites and cysts of different strains of Acanthamoeba spp. that varied in their virulence. Tests were carried out in phosphate-buffered saline (PBS) as well as in culture medium to consider the influence of organic material. Moreover, the influence of β-alanine, as well as that of NH₄Cl, on the amoebicidal activity was investigated, because previous studies have revealed a high potential for these substances to increase the efficacy of NCT (20, 24). Furthermore, the influence of an acidic pH on the activity of NCT was investigated, as this had been described to decrease the killing times of bacteria (22).

MATERIALS AND METHODS

Culture of amoebae.

Different strains of Acanthamoeba spp. were used in this study: one highly virulent strain (Acanthamoeba hatchetti 11DS, genotype T6, morphological group II) isolated from the cornea of an AK patient (42) and two nonvirulent strains, one derived from the contact lens case of an asymptomatic individual (Acanthamoeba castellanii 4CL, genotype T4, morphological group II) and one derived from the contact lens case of a non-AK patient (Acanthamoeba polyphaga 5SU, genotype T4, morphological group II) (44). One veterinary isolate from the necrotic tissue of a lizard (Basiliscus plumifrons) with undefined clinical relevance (A. castellanii 40AB, genotype T4, morphological group II) (43) was also included in the study.

Trophozoites were cultured axenically in peptone-yeast extract-glucose (PYG) medium (40) at RT in 75-cm² tissue culture flasks. They were subcultured once a week by shaking the culture flasks to detach the cells adhering to the bottom, centrifugation at 800 × g for 10 min, resuspension in 25 ml PYG medium, and transfer to a new flask.

For encystment the PYG medium was decanted and replaced by Hirukawa encystment medium (10), followed by incubation at RT for several days. After 14 days a pure cyst culture was harvested by centrifugation at 800 × g for 10 min.

General test procedures.

The white crystalline sodium salt (molecular weight, 181.57 g/mol) of NCT was dissolved in either 0.01 M PBS (pH 6.5) or PYG (pH 6.8) to reach the following concentrations: 10 mM (0.18%), 20 mM (0.36%), 55 mM (1%), and 160 mM (3%).

The trophozoites were centrifuged, washed with PBS, and resuspended in either PYG or PBS; they were then stained with trypan blue (0.4%) and counted in a Bürker-Türk hemocytometer. The experiments were carried out in six-well microtiter plates. Two milliliters of PYG or PBS with the desired concentration of NCT (10 mM in PBS, 20 mM in PYG) was placed into each well, and the wells were seeded with 10⁵ trophozoites/ml.

After 1, 6, and 24 h, the dead and living trophozoites were counted in a hemocytometer by phase-contrast microscopy and trypan blue staining. The killing times were calculated by the following formula, based on the exposure times of the last wells with living cells (t_n) and the first wells with dead cells only (t_{n + 1}): (t_n + t_{n + 1})/2.

All experiments were carried out in triplicate assays and were repeated in three independent setups. The killing times were averaged, and the 50% and the 90% effective concentrations (EC₅₀s and EC₉₀s, respectively) after 1, 6, and 24 h of incubation were calculated.

The cysts were counted in a Bürker-Türk hemocytometer and adjusted to 10⁴ and 10⁵ cells/ml. Ten milliliters of these adjusted cells was then incubated with NCT at a final concentration of 55 mM (1%) in PYG or PBS.

In experiments with cysts, NCT was neutralized with sodium thiosulfate (3% to neutralize 1% NCT) after 6 or 24 h, and then the cysts were resuspended in PYG medium and incubated at RT for up to 14 days. The excystation times of the treated and the nontreated cysts were observed by microscopic screening and counting on days 1, 4, 6, 11, and 14. The failure of excystment was proven by plating 100 μl of the respective treated cyst suspension on an agar plate overlaid with Escherichia coli. These plates were monitored daily for up to 1 week for the possible occurrence of trophozoites.

For each test a control series was included. This consisted of trophozoites incubated in PBS or PYG, each without NCT. Untreated cysts were used as negative controls and were resuspended in PYG on the same day that NCT was neutralized. Neutralization controls, i.e., cysts treated with NCT that had previously been inactivated with sodium thiosulfate, revealed no inhibition of excystation.

Influences of β-alanine, NH₄Cl, and pH.

Studies by Nagl and colleagues has revealed a 20-fold enhancement of the bactericidal effect of NCT when it was coincubated with β-alanine in a molar ratio of 1:16 (20). Furthermore, a 50- to 300-fold reduction in the killing times of bacteria was observed when NH₄Cl was added (in a NCT/NH₄Cl molar ratio of 1:3.4) (7, 20). In order to investigate the influence of either β-alanine or NH₄Cl on the amoebicidal effectiveness of NCT, trophozoites of strains 11DS and 5SU were cotreated with NCT and β-alanine at a molar ratio of 1:16 and with NCT and NH₄Cl at molar ratios of 1:3.4 and 1:29. Cysts were cotreated with 1% NCT and 1% NH₄Cl (molar ratio, 1:3.4).

Test procedures were carried out as described above; and the killing or excystation times, as well as the EC₅₀s and EC₉₀s, were evaluated and compared to the excystation and killing times of treatment without additives.

To test the influence of the pH on the effectiveness of NCT against trophozoites, the pH of the mixtures of NCT in PBS and NCT in PYG was adjusted to 5. Tests were carried out as described above, and the killing times and EC₅₀s and EC₉₀s were compared to the killing times and ECs at pH 7.

Control experiments with NH₄Cl or β-alanine only, as well as PBS or PYG only at pH 5, proved the absence of any amoebicidal effect of these agents when they were used alone.

Statistics.

Single-factor analysis of variance and the Tukey honestly significant difference procedure (SPSS, version 14.0) were used for statistical analyses. P values of <0.05 were considered significant.

RESULTS

Susceptibility of trophozoites.

Trophozoites of Acanthamoeba spp. showed high degrees of susceptibility to NCT when NCT was used in both PBS and PYG. Experiments with NCT alone were carried out with all four strains; the cotreatment experiments and experiments investigating the influence of pH were carried out with strains 11DS and 5SU.

In PBS, 10 mM NCT was sufficient for a 2-log reduction in the numbers of amoebae within 6 h (Fig. 1A). The killing times were 15 h for strain 5SU, 30 h for strain 40AB and strain 11DS, and 36 h for strain 4CL. There were no statistically significant differences in the cell counts between the four strains.

FIG. 1.

Treatment of Acanthamoeba spp. with NCT in PBS at pH 7 (A) and cotreatment with NH₄Cl and β-alanine and consequences of pH lowered to pH 5 (B1 and B2). (A) Effects of 10 mM NCT on strains 11DS (♦), 5SU (□), 4CL (×), and 40AB (▴); effects of 55 mM NCT on strains 11DS (▪) and 5SU (⋄); and effects of no treatment (control; *); (B1) results for strain 11DS with 10 mM NCT and 34 mM NH₄Cl (○), 290 mM NH₄Cl (−), or 160 mM β-alanine (•) and at pH 5 (+); (B2) results for strain 5SU with 10 mM NCT and 34 mM NH₄Cl (○) or 160 mM β-alanine (•) and at pH 5 (+).

In PYG, a 2-log reduction was observed after 24 h of treatment with 20 mM NCT (Fig. 2A). In these experiments, the survival curve for trophozoites of highly pathogenic strain 11DS showed a steeper decline within the first 6 h of treatment (P = 0.001); however, killing times of about 36 h were obtained for all strains investigated (data not shown).

FIG. 2.

Treatment of Acanthamoeba spp. with NCT in PYG at pH 7 (A) and cotreatment of 20 mM NCT with 68 mM NH₄Cl and 320 mM β-alanine and the consequences of lowering of pH to 5 (10 mM NCT) (B1 and B2). (A) Effects of 20 mM NCT on strains 11DS (♦), 5SU (□), 4Cl (×), and 40AB (▴); effects of 55 mM NCT on strains 11DS (▪) and 5SU (⋄); and effects of no treatment (control; *); (B1) results for strain 11DS with NH₄Cl (○) or β-alanine (•) and at pH 5 (+); (B2) results for strain 5SU with NH₄Cl (○) or β-alanine (•) and at pH 5 (+).

NCT at 55 mM in PBS led to a rounding of the cells within 15 min and to complete killing after 30 min for strain 11DS. For strain 5SU the killing time was 1 h; however, sporadic cysts could be observed (Fig. 1A). When trophozoites of strains 11DS and 5SU were treated with 55 mM NCT in PYG, they rounded up after 30 min and were killed within 3 h (Fig. 2A). Incubation of trophozoites of strain 11DS with 0.16 M NCT led to a rounding already after a few seconds and to complete killing within 1 h. The trophozoites of strain 5SU were killed by 0.16 M NCT within 30 min (data not shown).

Influences of NH₄Cl, β-alanine, and pH.

Cotreatment and pH tests were carried out with strains 11DS and 5SU. Cotreatment of strain 11DS with 10 mM NCT and 34 mM NH₄Cl (molar ratio, 1:3.4) in PBS did not lead to a shorter killing time; however, cotreatment with 160 mM β-alanine led to a decrease in the killing time from 30 h to 15 h. After 24 h trophozoites cotreated with NCT and 34 mM NH₄Cl showed a higher survival rate compared to that for trophozoites treated with NCT alone, NCT at pH 5, and NCT with β-alanine. In contrast to this result, cotreatment with 290 mM NH₄Cl led to a 2-log reduction in the numbers of viable cells within 1 h and to complete killing within 24 h (Fig. 1B1).

After 1 h of incubation with 10 mM NCT plus 34 mM NH₄Cl in PBS, strain 5SU showed statistically lower cell counts than those obtained after treatment with 10 mM NCT alone and after cotreatment with 10 mM NCT plus 160 mM β-alanine (P < 0.001). However, after 6 h there was no statistically significant difference between the treatment with NCT plus NH₄Cl, NCT alone, and NCT at pH 5. Cotreatment with 10 mM NCT and 160 mM β-alanine in PBS did not reveal any reduction in the overall killing time; in fact, the killing of strain 5SU was even decelerated compared to the time of killing after treatment with NCT alone, which was significant after 1 and 6 h (P < 0.001) (Fig. 1B2).

Cotreatment with 55 mM NCT and 190 mM NH₄Cl in PBS led to a rounding of the cells within 15 min, but the killing time was not reduced compared to that after treatment with 55 mM NCT alone. After 1 h 75% of the trophozoites of strain 5SU were killed, whereas almost all trophozoites of strain 11DS were still alive at the same time point. However, after 3 h the complete killing of both strains was achieved (data not shown).

In PBS, the best results in killing the trophozoites of strain 11DS were achieved with NCT plus NH₄Cl in a molar ratio of 1:29. For strain 5SU, cotreatment with NCT and NH₄Cl at a molar ratio of 1:3.4 led to an increase in the efficacy of NCT within the first hour of treatment, but the enhancing effect was less the longer that the experiment lasted.

In PYG, treatment with 20 mM NCT plus 68 mM NH₄Cl showed a slightly enhanced effectiveness compared to treatment with 20 mM NCT alone after 6 h in strain 11DS. In strain 5SU, cotreatment with NCT and 68 mM NH₄Cl did not lead to faster killing (Fig. 2B1 and 2B2). Surprisingly, for strain 11DS cotreatment with NCT and 580 mM NH₄Cl (molar ratio, 1:29) did not augment the enhancing effect but reduced the effectiveness of NCT at all time points investigated (data not shown). For strain 5SU there was no difference between cotreatment with NCT and 580 mM NH₄Cl and cotreatment with NCT and 68 mM NH₄Cl (data not shown).

Acidification of the pH from 7 to 5 led to hardly any accelerated killing either in PBS or in PYG. For both strains investigated, the only effect of a lower pH was a lower cell count after 1 h in PBS, which was statistically significant (P < 0.001) (Fig. 1B1 and B2). In PYG, no statistically significant enhancing effect was observable (Fig. 2B1 and 2B2).

The EC₅₀s and EC₉₀s for strains 11DS and 5SU are shown in Table 1. The EC₅₀s and EC₉₀s of NCT in PYG were more than twice as high as the EC₅₀s and EC₉₀s of NCT in PBS. For strain 11DS, the EC₅₀s and EC₉₀s at pH 5 in PBS (data not shown) were similar to those of NCT plus NH₄Cl in PBS after 6 h and 24 h. After treatment with 10 mM NCT at pH 5 for 1 h, the mean EC₅₀s and EC₉₀s were calculated to be 6.67 mM and 12.3 mM, respectively. These effective concentrations were 66% less than the concentrations needed for the killing of 50% and 90% of the trophozoites with NCT at pH 7 (Table 1). For strain 5SU, a decrease from pH 7 to pH 5 had effects on the EC₅₀s and EC₉₀s similar to those of cotreatment with NCT and 34 mM NH₄Cl at all time points.

TABLE 1.

EC₅₀s and EC₉₀s of strains 11DS and 5SU in PBS and PYG after 1, 6, and 24 h of incubation with 10 mM NCT (in PBS) and 20 mM NCT (in PYG)^a

Incubation time (h)	Treatment	Cotreatment	Strain 11DS		Strain 5SU
			EC50 (mM)	EC90 (mM)	EC50 (mM)	EC90 (mM)
1	PBS	NCT	15.55	29.40	29.26	58.85
	PBS	NCT + NH4Cl	12.90	23.13	5.31	9.50
	PYG	NCT	10.10	18.78	23.06	42.18
	PYG	NCT + NH4Cl	13.92	25.00	16.57	29.70
6	PBS	NCT	3.89	7.71	4.00	7.95
	PBS	NCT + NH4Cl	5.19	9.13	5.00	9.08
	PYG	NCT	9.15	17.43	11.67	23.59
	PYG	NCT + NH4Cl	10.40	18.89	12.92	23.47
24	PBS	NCT	3.65	7.56	3.47	7.38
	PBS	NCT + NH4Cl	5.11	9.13	4.96	9.03
	PYG	NCT	7.38	15.57	8.10	16.23
	PYG	NCT + NH4Cl	10.17	18.12	10.25	18.26

^aValues are the means of three independent experiments. All experiments were carried out at pH 7. Cotreatment experiments show EC₅₀s and EC₉₀s of 10 mM NCT plus 34 mM NH₄Cl (in PBS) and 20 mM NCT plus 68 mM NH₄Cl (in PYG). For strain 5SU, NH₄Cl increased the effectiveness of NCT significantly (P < 0.001) after 1 h of cotreatment in PBS. However, NH₄Cl had no additive effects on treatment with NCT for strain 11DS in general and for strain 5SU after 6 and 24 h. In PYG, coincubation with NCT and NH₄Cl decreased the EC₅₀s and EC₉₀s for strain 5SU after 1 h, but this result was not statistically significant.

In PYG, a lower pH did not lead to an increase in the efficacy of NCT, except for that against strain 5SU after 1 h of incubation, where a decrease in the pH led to a decrease in the mean EC₅₀ and EC₉₀ of about 10 mM (EC₅₀ = 13 mM, EC₉₀ = 32 mM) compared to the mean EC₅₀ and EC₉₀ of NCT at pH 7.

Susceptibility of cysts.

Incubation of the cysts with 55 mM NCT in PYG for 24 h and the ensuing neutralization caused a delay of excystation. All strains showed lower excystation rates after treatment with NCT during the first 6 days compared to the rates for the nontreated controls. Trophozoites of strain 4CL in the treated group showed slightly higher counts than those in the nontreated group after 11 days, but this result was not significant (Table 2).

TABLE 2.

Comparison of excysted trophozoites treated with 55 mM NCT for 24 h and nontreated controls after 1, 4, 6, and 11 days^a

Strain and treatment	Trophozoite count
	1 day	4 days	6 days	11 days
4CL
Control	2.7 × 103	1.4 × 104	7.4 × 104	9.6 × 104
55 mM NCT	0	2 × 103	7.2 × 103	1 × 105
40AB
Control	1.5 × 103	2.6 × 104	7.8 × 104	3.1 × 105
55 mM NCT	0	1.3 × 104	3.8 × 104	1.3 × 105
11DS
Control	4.3 × 104	2.5 × 105	4.7 × 105	9.4 × 105
55 mM NCT	4.6 × 103	1 × 105	2.1 × 105	4.1 × 105
5SU
Control	8 × 102	2 × 104	3.8 × 104	2.4 × 105
55 mM NCT	1 × 103	8 × 103	1.8 × 104	2 × 105

^aValues are the means of three independent experiments. Nontreated acanthamoebae showed higher excystment rates than treated ones. This observation was statistically significant for strains 4CL, 40AB, and 11DS on days 1, 4, and 6 (P < 0.05) and for strain 5SU on day 4 (P = 0.035).

Cotreatment of cysts was performed with strains 11DS and 5SU. Incubation of the cysts with 55 mM (1%) NCT plus 190 mM (1%) NH₄Cl for 24 h in PBS led to a complete loss of viability for both strains. This was also proven by incubating the treated cysts on xenic agar, where no growth or even excystation was observed, even after 14 days (data not shown).

DISCUSSION

NCT has already been proven to be highly bactericidal, viricidal, fungicidal, and vermicidal (22-24, 45). The current study appends amoebicidal activity to this long list of microbicidal capacities of NCT.

All Acanthamoeba strains investigated were susceptible to NCT, which was shown to have at least amoebostatic effects. Generally, the effectiveness of NCT was higher in PBS than in culture medium, which might be due to the presence of ingredients in PYG medium that lower the effectiveness by trans-halogenation and the consumption of active chlorine by reaction above all with thiols (22). Surprisingly, in PYG (but not in PBS) a steeper decline in the survival curve of the highly pathogenic strain 11DS within the first 6 h of treatment was observed. This increased killing could be due to the faster growth rates observed for pathogenic Acanthamoeba strains (44). A faster growth rate, which is associated with a higher rate of metabolism, might lead to the faster uptake of NCT and therefore the faster oxidation of intracellular proteins, leading to death. However, this phenomenon fades with time and did not lead to a shorter time for complete killing. In PBS, however, free-living amoebae showed hardly any metabolism, as can be seen in Fig. 1 (the controls did not multiply) and as has already been described by Page (30); therefore, the killing of all strains is synchronized.

Nagl et al. have already described the killing of bacteria and viruses within 30 to 60 min by 55 mM NCT (20, 21, 23), whereas the killing of fungi required longer incubation times at this concentration of NCT (24). The results presented in the current study point out that the killing times of NCT for Acanthamoeba spp. are comparable to the killing times for other eukaryotic cells, like fungi.

Organic components of the human tissue or the culture medium are important factors, as they are able to lead to trans-halogenation (22). Nagl et al. have described the positive influence of defined organic material on the microbicidal potential of NCT (20). In that study they tested the effects of NH₄Cl and β-alanine on the effectiveness of NCT. Those tests revealed promising results, as the bactericidal and fungicidal activities of NCT were not diminished but enhanced in the presence of NH₄Cl, whereby the more lipophilic and therefore more microbicidal monochloramine (NH₂Cl) was formed via chlorine transfer (NH₄Cl + NCT-Na↔ NH₂Cl + taurine + Na⁺ + Cl⁻) (20, 24) and the killing times were thus reduced.

Similarly, the activity of NCT against strain 11DS was increased when the strain was coincubated with NCT and 290 mM NH₄Cl in PBS. However, no more enhancing effect was observable when the experiments were carried out in PYG. The ineffectiveness of NH₄Cl in PYG might be due to the ingredients of the culture medium, as, e.g., proteose peptone, one of the main components, binds to the NH₂Cl that is formed and therefore cancels out the killing time-decreasing factor (8, 20). Furthermore, in PYG, the idiom “less is more” is valid. Cotreatment with 20 mM NCT and 68 mM NH₄Cl revealed lower EC₅₀s and EC₉₀s than cotreatment with 20 mM NCT and 580 mM NH₄Cl in the case of strain 11DS; nevertheless, NCT was more effective when it was administered alone. This might be due to the more rapid decay of NCT plus NH₄Cl because of high molar ingredients in this experiment.

In strain 5SU, cotreatment with NH₄Cl revealed the same results, irrespective of whether NCT and NH₄Cl were used at molar ratios 1:3.4 and 1:29 in PBS and in PYG, respectively.

However, there was a strong amoebicidal effect against strain 11DS treated with 10 mM NCT plus 290 mM NH₄Cl in PBS for 1 h. At this time point, a 2-log reduction was observable for strain 11DS. This nearly 10-fold higher concentration of NH₄Cl lowers the concentration of NCT needed to kill 50% and 90% of the amoebae within 1 h by about 40%. The penetration of active chlorine through the cornea has been shown to be higher for NCT plus NH₄Cl than for NCT alone, and highly microbicidal concentrations of the combination did not lead to any overt signs of ocular toxicity in the rabbit eye (33). These facts suggest the need for the further investigation of NCT plus NH₄Cl as a formulation for the treatment of Acanthamoeba infections.

In our study a lower pH did not enhance the efficacy of NCT in total. However, in PBS as well as in culture medium, the efficacy was increased during the first hour of treatment, but after 6 and 24 h, NCT was as effective at pH 7 as it was at pH 5. These results differ from the observation by Nagl et al. of decreased killing times for bacteria at a lower pH (22). Interestingly, this phenomenon was not observed when the experiments were carried out with suspensions of molds (90% hyphae and 10% conidiophores) and yeasts (60% pseudohyphae and 40% blastoconidiae) instead of bacteria. These variations in susceptibility can be explained by the differences in the cell wall and the membrane compositions (24), which could also be an explanation for the results observed in our study. Another explanation could be the fact that the pathogenic Acanthamoeba trophozoites are able to grow at pHs ranging from 4 to 12 (13) and will therefore not show any signs of membrane rupture or higher permeability that could simplify the penetration of NCT into the cytoplasm.

The treatment of cysts with 55 mM NCT in PYG for 24 h resulted in a delay of excystation in all strains investigated. This delay is comparable to the lag of regrowth found in bacteria (21). In that study incubation with 55 mM NCT for 1 min caused a lag of regrowth of 3 h in Staphylococcus aureus.

In two of the strains investigated (strain 11DS and strain 5SU), incubation of the cysts with 55 mM NCT plus 190 mM NH₄Cl led to the complete failure of excystation. Although these cysts showed no obvious differences from the nontreated ones as determined by light microscopy, it must be assumed that the lipophilic NH₂Cl that was formed penetrated the cyst wall and led to the 100% killing of the cysts. This correlates well with the rapid killing of bacteria, fungal spores, and even mycobacteria by NCT plus ammonium chloride, which has also been explained by the penetration of monochloramine (7, 20, 24, 27). As a hydrophilic molecule, NCT without ammonium chloride needs at least a few minutes to pass the lipid bilayers of all types of microbes to reach concentrations of active chlorine inside the microbes that induce an irreversible impact on the intracellular targets (1). It has been proven that chlorination of the surface of bacteria and fungi by incubation with NCT for sublethal incubation times can affect their virulence but does not kill them (9, 21, 22). The penetration of NCT into the cornea can be improved significantly by ammonium chloride (33), which might become important for the treatment of Acanthamoeba eye infections. As most cysticidal agents provoke only a 2-log reduction in the numbers of viable cysts (14) or do not show 100% killing, as some cysts reside in deeper corneal strata (41), a therapy with NCT combined with NH₄Cl might be promising.

To estimate the potential of NCT for use as a drug, it can be stated that it is active against Acanthamoeba spp. at concentrations well tolerated by tissues of different body sites, e.g., the eye, the skin, and mucous membranes (25, 26, 28, 29, 39). As NCT is an endogenous amino acid derivative, neither allergic nor toxic side effects should occur. Moreover, the development of resistance is highly unlikely, because of the quite unspecific mode of action, which is based on the oxidation of the SH residues of proteins (8).

In conclusion, it was shown that NCT possesses a high level of activity against pathogenic Acanthamoeba trophozoites, with EC₅₀s and EC₉₀s between 3 mM and 30 mM in PBS and between 7 mM and 25 mM in PYG. The EC₅₀s and EC₉₀s were even lower when NH₄Cl was used as an additive. Moreover, cotreatment with 55 mM NCT and 190 mM NH₄Cl was also shown to be highly effective against Acanthamoeba cysts, resulting in the complete loss of viability. Thus, NCT might be a very promising drug for the treatment of Acanthamoeba infections.