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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 2011, p. 637–648 Copyright 2011, American Society for Microbiology. All Rights Reserved.
A Rapid, High-Throughput Viability Assay for Blastocystis spp. Reveals Metronidazole Resistance and Extensive Subtype-Dependent Variations in Drug Susceptibilities䌤 Haris Mirza,1 Joshua D. W. Teo,1 Jacqui Upcroft,2 and Kevin S. W. Tan1* Laboratory of Molecular and Cellular Parasitology, Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117596, Singapore,1 and Queensland Institute of Medical Research, Brisbane, Queensland 4029, Australia2 Received 1 July 2010/Returned for modification 10 October 2010/Accepted 14 November 2010 Blastocystis is an emerging protistan parasite of controversial pathogenesis. Although metronidazole (Mz) is
standard therapy for Blastocystis infections, there have been accumulating reports of treatment failure, sug-
gesting the existence of drug-resistant isolates. Furthermore, very little is known about Blastocystis suscepti-
bility to standard antimicrobials. In the present study, we established resazurin and XTT viability microassays
for Blastocystis spp. belonging to subtypes 4 and 7, both of which have been suggested to represent pathogenic
zoonotic subtypes. The optimized resazurin assay was used to screen a total of 19 compounds against both
subtypes. Interestingly, subtype 7 parasites were resistant to Mz, a 1-position-substituted 5-nitroimidazole
(5-NI), while subtype 4 parasites were sensitive. Some cross-resistance was observed to tinidazole, another
1-position 5-NI. Conversely, subtype 4 parasites were resistant to emetine, while subtype 7 parasites were
sensitive. Position 2 5-NIs were effective against both subtypes, as were ornidazole, nitazoxanide, furazolidone,
mefloquine, quinicrine, quinine, cotrimoxazole (trimethoprim-sulfamethoxazole), and iodoacetamide. Both
subtypes were resistant to chloroquine, doxycycline, paromomycin, ampicillin, and pyrimethamine. This is the
first study to report extensive variations in drug sensitivities among two clinically important subtypes. Our
study highlights the need to reevaluate established treatment regimens for Blastocystis infections and offers
clear new treatment options for Mz treatment failures.
Blastocystis is an emerging enteric protistan parasite with no in vitro or in vivo data to support this hypothesis. Despite zoonotic potential (39, 57, 58). It is one of the most common these controversies, interest in the parasite has increased in parasites colonizing the human gut, with prevalences ranging recent years, as signified by the establishment of organizations between 10% of the population in developed countries and like the Blastocystis Research Foundation, which actively sup- 50% in developing countries (58). It frequently infects immu- port studies on subtype-dependent variations in Blastocystis nocompromised individuals (27, 40, 59) and has a high preva- pathobiology and treatment (6). The clinical significance of the lence in impoverished children (35) and HIV/AIDS (27) and intestinal parasite Giardia intestinalis was recognized only after cancer (59) patients. Individuals infected with Blastocystis it became possible to effectively eliminate it from the gut (33).
present with common intestinal symptoms, such as abdominal To understand the role of Blastocystis as a human pathogen, pain, vomiting, and bloating, as well as mucous and watery there is an urgent need to identify standardized and effective diarrhea (58). Blastocystis infections are commonly associated treatment options for various Blastocystis subtypes.
with dermatological disorders (25, 67) and irritable bowel syn- At least 9 out of the 11 subtypes of Blastocystis are known to colonize the human gut (57). The identification of antibiotic- Although metronidazole (Mz) treatment is considered first- resistant subtypes of the parasite and development of new line therapy for Blastocystis infections, therapeutic intervention therapeutic options to counter antimicrobial resistance require is equivocal because of the large number of asymptomatic a high-throughput screening tool. Conventional drug suscepti- carriers and frequent reports of treatment failure (3, 23, 37, 53, bility assays for Blastocystis (16, 68, 72, 75) are not suitable for 55). The confusion concerning the status of Blastocystis as a high-throughput drug screening (HTS) because they are ex- pathogen is primarily due to limitations of diagnostic tech- pensive, laborious, time-consuming, potentially hazardous, and niques, purported subtype-dependent variations in parasite prone to bias. Since the incidence of Blastocystis is higher in virulence, and variable host responses (55). The variation in developing countries (58), the cost and availability of sophis- treatment response suggests the presence of metronidazole- ticated equipment are also limitations for such screenings.
resistant (Mzr) subtypes of the parasite, but there are currently In this study, we evaluated two high-throughput viability assays and applied them to drug susceptibility microassays forBlastocystis. Resazurin (7-hydroxy-3H-phenoxazin-3-one 10- * Corresponding author. Mailing address: Laboratory of Molecular oxide) is the active compound of a propriety solution, Alamar and Cellular Parasitology, Department of Microbiology, Yong Loo Lin blue (41). The resazurin assay measures intrinsic cellular met- School of Medicine, National University of Singapore, 5 Science Drive abolic activity, which reduces resazurin and changes its color as 2, Singapore 117596, Singapore. Phone: 65-6516 6780. Fax: 65-6776- a measurable indicator of the number of viable cells that are 䌤 Published ahead of print on 22 November 2010.
present in a test sample (34, 47). Resazurin-based assays are MIRZA ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Sources of Blastocystis isolates metric measurements. For semiquantitative evaluation, the color change in eachwell was visually observed and recorded after 5 h.
Common hostsb Drug preparation. Compounds purchased from Sigma included Mz, ornida-
zole (Oz), ronidazole (Rz), furazolidone (FUR), mefloquine (MQ), quinacrine Symptomatic human, SGHa (QC), quinine (QN), chloroquine (CQ), emetine (EM), doxycycline (DOX), Symptomatic human, SGHa trimethoprim sulfate-sulfamethoxazole (TMP-SMZ), paromomycin (PAR), am- Wistar rat, animal surveyc picillin (AMP), pyrimethamine (PYR), and iodoacetamide (IA). Tinidazole (Tz) Sprague-Dawley rat, animal surveyc Humans, rats was purchased from AK Scientific, whereas nitazoxanide (NTZ) was purchased a Isolated from symptomatic patients presenting at the Singapore General from Romark Laboratory. C-17 is an experimental, chemically synthesized, 2-po- Hospital (SGH) (36).
sition 5-nitroimidazole (NI) compound (66). Stock solutions of each compound b Based on Tan (57).
to be tested were prepared fresh in dimethyl sulfoxide (DMSO). For drug c Isolated during an animal survey (11).
sensitivity determination, stock solutions were diluted in prereduced Blastocystismedium and transferred to 96-well plates. A total of 0.5 ⫻ 106 cells/well wereincubated for 24 h with different dilutions of the drugs ranging between 0 and 100 commonly used for drug susceptibility analysis of prokaryotic ␮g/ml. The final DMSO concentration was kept constant at 0.5%.
Confocal microscopy. Confocal micrographs of the parasites were taken in
(29) and eukaryotic (20, 34, 41, 46) cells. Much like resazurin, order to determine whether the alteration in Blastocystis redox activity under the tetrazolium salt 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)- drug tension observed in previous assays was also associated with morphological 2H-tetrazolium-5-carboxanilide (XTT) is reduced by mito- changes. Metronidazole-susceptible (Mzs) ST-4 (isolate WR-1) and Mzr ST-7 chondrial and cytoplasmic redox enzymes to a colored forma- (isolate E) were treated for 24 h with a 12.5-␮g/ml concentration of FUR and zan compound with a direct correlation with cell proliferation.
Mz. After drug exposure, the parasites were washed and resuspended in annexinV binding buffer (BioVision). Annexin V and propidium iodide (PI) (BioVision) Tetrazolium compounds have also been widely utilized for were then added to the cell suspension. Confocal imaging of cell suspensions was cytotoxic evaluation of both prokaryotic (14) and eukaryotic done using an Olympus Fluoview FV1000 (Japan) equipped with a dual filter set (5) organisms.
for fluorescein isothiocyanate (FITC) and rhodamine. Images were captured In the current study, we report that optimized resazurin and using Olympus Fluoview version 1.6b.
Statistical analysis and validation of reproducibility. Before a particular assay
XTT redox-based assays are suitable for viability studies of the was used for a full-scale HTS, smaller pilot screenings were used to predict its parasite. Blastocystis subtype 4 and subtype 7 isolates are most usefulness for large-scale applications. The Z⬘ factor predicts the robustness of commonly found in rats and birds, respectively (57). Both an assay for HTS by taking into account the mean and standard deviation of both subtypes are known to colonize the human gut, and studies positive and negative controls of the pilot screening (74). We calculated the Z⬘ suggest that both subtypes have pathogenic potential (54). We factors of both assays for Blastocystis drug screening using the following equation:Z⬘ factor ⫽ 1 ⫺ [(3␴ utilized the optimized assays to determine the susceptibility of c⫹ ⫹ 3␴c⫺)/ⱍ␮c⫹ ⫺ ␮c⫺ⱍ], where, c⫹ is the positive control (0.5% DMSO), c⫺ is the negative control (6.25 ␮g/ml FUR), ␴ is the standard Blastocystis isolates to a range of antimicrobial agents. We deviation, and ␮ is the mean.
observed extensive subtype-dependent variations in Blastocys- Assays having a Z⬘ factor score between 0.5 and 1 are considered excellent for tis susceptibility to a panel of conventional and experimental antiprotozoal agents and identified Mz- and emetine (EM)- Comparison of data sets with wide differences between their means should be made using the coefficient of variation (C ) instead of the standard deviation (␴).
resistant subtypes of the parasite. Importantly, we identified It represents the ␴ in the context of the mean (␮) and is another test used to several new and potentially effective treatment options for Mzr evaluate the robustness of an assay for HTS. We calculated the C s of both assays using the following formula (30): C ⫽ ␴/␮, where, C is the coefficient of variation, ␴ is the standard deviation of the positive control (5 ⫻ 105 parasites in200 ␮l culture medium plus 0.5% DMSO), and ␮ is the mean of the positive MATERIALS AND METHODS
control (5 ⫻ 105 parasites in 200 ␮l culture medium plus 0.5% DMSO).
Cell culture. Four axenized isolates of Blastocystis were used (Table 1). All
Assays with a C of ⬍1 are considered low variance and fit for HTS (30).
four isolates were subtyped previously by small-subunit rRNA gene analyses The final validation step was the screening of the dose-dependent antiproto- (39). Isolates WR-1 and S-1 belong to subtype 4, while isolates B and E belong zoal activity of Mz against 4 different isolates of Blastocystis repeated twice in to subtype 7, according to a recent Blastocystis sp. classification system (52).
triplicate. The results were statistically compared for reproducibility.
Cultures of all four isolates were maintained as described previously (36). In The statistical significance of variations between the drug susceptibility values brief, the parasites were maintained in 10 ml of prereduced Iscove's modified of 4 isolates was determined using one-way analysis of variance (ANOVA). A Dulbecco's medium (IMDM) containing 10% horse serum in an anaerobic jar one-way ANOVA test is ideal to test the statistical significance of the variations (Oxoid) with an AnaeroGen gas pack (Oxoid) at 37°C. The parasites were observed between means of three or more groups of data.
subcultured alternately at 72 and 96 h. Under these culture conditions, all fourparasites exhibited noncystic vacuolar morphology. This morphological state isadvantageous for assessment of MZ resistance because Blastocystis cysts are known to be resistant to the drug (73), complicating our study. Cultures wereharvested from log-phase in vitro cultures for viability studies in 96-well plates.
Resazurin and XTT result in fluorimetric and colorimetric
Microculture technique. In order to establish and validate the analytical meth-
reactions with Blastocystis in a cell density-dependent manner.
ods for Blastocystis viability determination, the microculture conditions wereoptimized for standard 96-well plates. Subtype 7 parasites (isolate B) were For semiquantitative analysis, visible color changes were ob- employed for the optimization experiments. Several parasite numbers between served after 5 h of incubation of resazurin and XTT with 103 and 106 cells were incubated in Blastocystis culture medium in a final volume Blastocystis sp. subtype 7 in 200 ␮l parasite culture medium.
of 200 ␮l/well in standard 96-well plates, unless otherwise stated. The 96-well Several shades of resazurin dye, ranging from blue to pink, plates were then incubated at 37°C under anaerobic conditions for 24 h unless developed with increasing cell density. Similarly, XTT devel- otherwise stated. After 24 h, the cultures were incubated with redox dyes for anadditional 3 h and 5 h for quantitative and semiquantitative evaluation, respec- oped shades ranging from yellow to deep orange with increas- tively. Unless otherwise stated, a 5% final dilution of the resazurin dye solution ing cell density. Minimums of 105 parasites/well were needed (Sigma) was used for resazurin assays, whereas XTT (Sigma) was used at a final to obtain visual evidence of color change for both dyes, al- concentration of 50 ␮g/ml. At the end of incubation, readings of resazurin though the color change was more obvious in the resazurin dye fluorescence were taken at 550-nm excitation and 570-nm emission wavelengths,while XTT assay measurements were made at an absorbance wavelength of 450 than with XTT.
nm. A Tecan Infinite M200 reader was used for both fluorimetric and colori- For quantitative analysis, fluorescence and absorbance mea-

BLASTOCYSTIS METRONIDAZOLE RESISTANCE AND SUSCEPTIBILITY FIG. 1. Correlation between the number of subtype 7 parasites and relative fluorescence units (RFU) (A) and relative absorbance units (RAU) (B) after 24 h of incubation and 3 h of development with resazurin and XTT, respectively. Each point represents an average of 6 values derivedfrom two independent sets of experiments. The error bars represent standard errors.
surements were taken for resazurin and XTT dyes, respec- linear range of cell density versus dye reduction for both assays tively, after 3 h of incubation. Negligible changes in absorbance (Fig. 1) and provides visible color changes in a short time.
and fluorescence measurements were observed between the Blastocystis requires 200-␮l/well volumes for optimal meta-
blank medium control and up to 104 parasites/well (Fig. 1), but bolic activity. For viability assays, cells should be at their op-
a linear increase in fluorimetric, as well as colorimetric, mea- timal metabolic activity. A recent study reported an increase in surements was noted from 104 parasites to 106 parasites/well metabolic activity of Acanthamoeba with a reduction of the (Fig. 1). The R2 values for resazurin and XTT dyes were cal- culture volume from 200 to 100 ␮l/well (34). In this study, a culated to be 0.995 and 0.983, respectively (Fig. 1; see Table 3).
decrease in volume per well resulted in a drop in Blastocystis A density of 5 ⫻ 105 parasites/well was chosen as the optimal metabolic activity (Fig. 2). Blastocystis, an anaerobic organism cell density for further experiments because it lies within the (57), should have higher metabolic activity in high well vol-

MIRZA ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 2. Correlation of total volume per well and dye concentration with relative fluorescence units (RFU) (A) and relative absorbance units (RAU) (B) for resazurin (res) and XTT dyes, respectively. Higher volumes per well and dye concentrations resulted in higher sensitivity ofresazurin and XTT, denoted by higher RFU and RAU readings, respectively. Each point represents a mean of 6 values derived from twoindependent sets of experiments. The error bars represent standard errors.
umes as opposed to Acanthamoeba, which is an aerobic pro- croplate growth conditions, exhibited an increasing degrada- tozoan (34). Therefore, a 200-␮l/well volume was used in all tion of resazurin over time, suggesting a rise in the redox subsequent experiments (Table 2 and Fig. 2).
activity of the culture (Fig. 3). This increase in redox activity Blastocystis exhibits exponential growth in microcultures.
could be due to an increase in either parasite numbers or Blastocystis sp. subtype 7, when incubated under optimal mi- metabolic activity. The redox activity of the parasite cultures

BLASTOCYSTIS METRONIDAZOLE RESISTANCE AND SUSCEPTIBILITY TABLE 2. Optimized parameters for resazurin and XTT assays TABLE 3. Statistical evaluation of the quality of resazurin and XTT assaysa Z⬘ factor C (%)b Growth medium .IMDM ⫹ 10% HSa ⫹ a Ideal HTS parameters are a Z⬘ factor of ⬎0.5 (74) and a C of ⬍10% (30).
b C %, coefficient of variance of cell controls.
Volume/well .200 ␮l Linearity of the dye reduction-versus-parasites/well curve.
Temperature .37°CCulture conditions.Anaerobic Contact time with dye (h) resazurin and XTT assays (Table 3). Both assays exhibited statistical reproducibility for dose-dependent activity assays ofantimicrobial agents against Blastocystis (Fig. 4).
Excitation/emission ␭ (nm) Blastocystis exhibits subtype-dependent variation in suscep-
tibility and resistance to Mz. Using the optimized resazurin
assay, the 50% inhibitory concentrations (IC s) of Mz against Optimal cell density (parasites/well).0.5 ⫻ 106 subtype 4 and subtype 7 isolates of Blastocystis were calculated.
Mz inhibited 50% of growth of subtype 4 isolates WR-1 and HS, heat-inactivated horse serum.
S-1 at concentrations of 5.5 ⫾ 2.89 ␮g/ml and 1.9 ⫾ 1.32 ␮g/ml,respectively (Table 4; Fig. 4 and 5). These values were within peaked at 24 h, followed by a drop, suggesting a slowing down the range of previously reported values of Mz susceptibility for of the culture growth or metabolism due to overcrowding. The Blastocystis (16, 75). The IC of Mz against isolate B (subtype 24-h time point was chosen for drug susceptibility assays (Table 7) was 32.5 ⫾ 3.4 ␮g/ml. This value is significantly higher than 2). The complete optimized parameters for both resazurin and of subtype 4 isolates (P ⬍ 0.01) and exceeds the XTT assays are summarized in Table 2.
average fecal Mz concentration of 9.5 ␮g/ml (26). Isolate E of Resazurin and XTT are suitable for HTS of antimicrobials
subtype 7 exhibited minimal susceptibility to Mz (Table 4 and against Blastocystis. HTS quality control parameters, i.e., a Z⬘
Fig. 4), even at concentrations as high as 100 ␮g/ml. These factor of ⬎0.5 (74) and a C of ⬍10% (30), were met by both results suggest that isolates B and E of subtype 7 are Mzr FIG. 3. Blastocystis subtype 7 exhibits a time-dependent increase in redox activity when cultured in a 96-well plate under the resazurin assay conditions described in this study. The starting parasite density was 0.5 ⫻ 106 cells in 200 ␮l of IMDM supplemented with 10% horse serum and0.5% DMSO. The redox activity of the culture peaked at 24 h, followed by a steady decline. A drug contact duration of 24 h was chosen basedon these results. Each point represents a mean of 6 values derived from two independent experiments, with each experiment conducted intriplicate. The error bars represent standard errors.

MIRZA ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 4. Graph representing percent inhibition of Blastocystis subtype 4 and 7 cultures by Mz using the resazurin assay. The IC s of Mz against subtype 4 isolates were found to be significantly lower than those of subtype 7 isolates (P ⬍ 0.01). Mz induced 50% inhibition of subtype 7 isolateB cultures at a concentration (conc.) of 32.5 ⫾ 3.4 ␮g/ml, whereas isolate E cultures exhibited only minimal inhibition even at concentrations ashigh as 100 ␮g/ml. Each point represents a mean of six readings derived from two independent experiments. The error bars represent standarderrors.
strains of Blastocystis. The XTT assay further confirmed these are impermeable to both PI and annexin V (71). Mzs ST-4 strains to be Mzr (Table 4).
(isolate WR-1) exhibited nuclear incorporation of PI and an- An Mzs isolate of Blastocystis exhibits typical morphological
nexin V binding after 24 h of exposure to a 12.5-␮g/ml con- features of cell death after exposure to Mz, as opposed to an
centration of Mz, suggesting a breach in the parasite cell mem- Mzr isolate. Our findings, based on resazurin and XTT assays,
brane (Fig. 6A). No changes were observed in Mzr ST-7 indicate suppression of parasite redox activity under drug ten- (isolate E) after Mz treatment (Fig. 6B). MZs and Mzr isolates sion. Concomitantly, to determine whether Blastocystis under- exhibited cell death morphology after treatment with a 12.5- goes morphological changes after drug exposure, parasites ␮g/ml concentration of FUR (Fig. 6A and B), whereas neither were stained with propidium iodide and annexin V-FITC. Both of the isolates incorporated PI or annexin V after treatment PI and annexin V stain only dying parasites (71). PI binds to with the DMSO control (Fig. 6A and B). These findings sug- the parasite nuclear material (71). Annexin V binds with high gest that after treatment with Mz, morphological alterations affinity to phosphatidylserine (PS). PS is located at the cytoso- typical of dying cells were observed in the Mzs isolate, while the lic face of the cell membrane and has access to annexin V only Mzr isolate remained unaffected.
when it becomes exposed at cell death (71). Healthy parasites Mzr isolates of Blastocystis exhibit cross-resistance with a
1-position-substituted 5-NI. Tz, a compound closely related to
Mz due to the presence of its side chain at position 1 of the
values of Blastocystis susceptibility to Mz imidazole ring (Fig. 5), was effective in killing both Mzr and Mzs isolates. Interestingly, Mzs subtype 4 isolates WR-1 and S 关␮g/ml (␮M)兴 exhibited IC s (0.51 ⫾ 0.02 and 0.3 ⫾ 0.1 ␮g/ml, respectively) Subtype 7 isolates Subtype 4 isolates of Tz lower than those of Mzr subtype 7 isolates B and E (5.13 ⫾ 0.16 and 9.33 ⫾ 0.45 ␮g/ml, respectively) (P ⬍ 0.01) 32.5 ⫾ 3.4 (189.8) 5.5 ⫾ 2.89 (32.16) 0.75 ⫾ 0.04 (4.38) (Table 5). Even within subtype 7, the IC of Tz for Mzr isolate 29 ⫾ 3.4 (169.36) 1.76 ⫾ 0.39 (10.27) 1.1 ⫾ 0.08 (6.4) E was significantly higher than that for isolate B (Table 5).
a NS, not susceptible to drug concentrations of ⱕ100 ␮g/ml.
These findings in Blastocystis suggest a cross-resistance pattern

BLASTOCYSTIS METRONIDAZOLE RESISTANCE AND SUSCEPTIBILITY ineffective against all four isolates of the parasite (data notshown).
Cysteine protease inhibition causes parasite death. The sig-
nificance of cysteine proteases in Blastocystis pathobiology iswell reported (36, 48, 58, 71). In this study, Inhibition of cys-teine protease activity of the parasite by IA resulted in com-plete inhibition of all four isolates with similar IC s, suggest- ing the importance of cysteine proteases in parasite survival.
We found both resazurin and XTT assays to be suitable for high-throughput analysis of drug susceptibility in Blastocystisisolates. The HTS parameters (a Z⬘ factor of ⬎0.5 and a C of ⬍10%) provide a highly conservative estimate of the sensitivityof an assay (30, 74). The high Z⬘ factor value, low C , and reproducibility of both resazurin and XTT assays suggest thatthey are robust and suitable for HTS. The option of semiquan-titative visual evaluation of color gives these assays the flexi- bility to be applied in the field without the need for sophisti- cated equipment. The suppression of metabolic activityobserved in these redox assays was also found to be associatedwith morphological signs of cell death (71), i.e., nuclear incor-poration of PI and annexin V binding to the cell membrane, similar to those exhibited by other parasites (7, 12). Oz, an- further validating these assays in determining drug susceptibil- other closely related 5-NI (Fig. 5), despite having a position 1 ities. Considering the large number of variant Blastocystis iso- side chain, was found to be equally effective against both Mzr lates and the predominance of the parasite in developing coun- and Mzs isolates. Interestingly, Mzr subtype 7 isolates exhibited tries (58) with limited research funding, these assays will be significantly higher susceptibility to the position 2 side chain particularly useful due to their low cost and high yield.
5-NIs Rz and C-17 (Fig. 5) than to position 1 5-NI. No signif- Subtype 7 isolates were shown to be resistant to Mz and icant subtype-dependent variation in Blastocystis susceptibility cross-resistant to Tz, the 1-position-substituted 5-NI of choice to position 2 5-NIs was observed.
to treat a wide variety of anaerobic organisms (4, 22). This is Blastocystis subtype 4 exhibits EM resistance. EM is an an-
consistent with previous reports of cross-resistance between tiamoebic agent with limited clinical use, reported to be effec- the two drugs in Trichomonas (12, 31) and Giardia (7, 61). In tive against Blastocystis in vitro (16, 75). Our study found EM to these organisms, resistance is proposed to be due to downregu- be effective against Mzr subtype 7 isolates (Table 6). Subtype 4 lation of the enzymes PFOR (65) and thioredoxin oxidoreduc- isolates S and WR-1, on the other hand, exhibited no inhibition tase (28), which in conjunction with the electron acceptor even at the highest test concentrations of 100 ␮g/ml, suggesting ferredoxin are believed to activate the 5-NI prodrugs to the EM resistance in subtype 4 isolates.
toxic radical states inside the parasite (28, 65). However, this Blastocystis exhibits subtype-dependent variations in suscep-
mechanism of activation has not been shown for Blastocystis, tibility to NTZ, MQ, and QC. NTZ, a well-documented pyru-
although PFOR and other oxidoreductase enzymes are present vate-ferredoxin oxidoreductase (PFOR) inhibitor (43), was in the organism (70). The subtype 4 isolates showed no con- found to be more effective against Mzr strains of the parasite in vincing uniformity in susceptibility to Mz and Tz, indicating this study (Table 6). Subtype 7 (avian) isolates were signifi- that new, unknown mechanisms of activation and/or resistance cantly more sensitive to NTZ than subtype 4 (rodent) isolates may be involved.
(P ⬍ 0.01). Similarly, the anti-malarial MQ and a closely re- All isolates were similarly susceptible to another 1-position lated drug, QC, were also found to be significantly more ef- 5-NI, Oz. Compared to Mz, the drug has significantly higher fective against subtype 7 isolates than subtype 4 (Table 6).
efficacy against Mzr isolates of Blastocystis (P ⬍ 0.01), as ob- No subtype-dependent variations in FUR and QN suscepti-
served in other parasites (10, 64) and also reported for Blas- bility. Both Mzr and Mzs isolates exhibited sensitivity to FUR
tocystis previously (16). However, its superior efficacy against and QN (Table 6), two well-known antiprotozoal agents.
Mzs isolates is not as obvious, again suggesting new, unknown Higher susceptibility of Blastocystis spp. to a TMP/SMZ
mechanisms of activation and/or resistance to 1-position 5-NIs ratio of 1:2 than to one of 1:5. SMZ and TMP are administered
in the parasite. Oz is frequently used to treat amoebiasis in in two different ratios for protozoan infections. TMP/SMZ India (21). Although the IC s of Oz against all four isolates ratios of 1:5 and 1:2 were tested for Blastocystis inhibition. All tested here (4.9 to 6.44 ␮M) were higher than the MIC of the isolates exhibited susceptibility to both combinations, but all drug against Entamoeba (0.25 ␮M) (10), its effectiveness four isolates were significantly more sensitive (P ⬍ 0.01) to a against both Mzr and Mzs isolates suggests the drug would be TMP/SMZ ratio of 1:2 than to one of 1:5 (Table 6).
a useful alternative to Mz to treat Blastocystis infections.
Nonsusceptibility of Blastocystis to broad-spectrum antibi-
Similarly to Oz, 2-position 5-NIs, the commercially available otics. PAR, PYR, CQ, DOX, and AMP were found to be
poultry drug Rz and the experimental drug C-17, were uni-

MIRZA ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 6. Confocal micrographs of Blastocystis stained with propidium iodide (arrow) and annexin V-FITC. (A) Mzs ST-4 (WR-1) exhibited nuclear incorporation of PI and annexin V-FITC binding after 24 h of exposure to 12.5 ␮g/ml Mz. (B) Mzr ST-7 (isolate E) did not exhibit theseclassical signs of cell death after Mz treatment. Both Mzs and Mzr isolates exhibited PI incorporation and annexin V-FITC binding after 24-htreatment with 12.5 ␮g/ml FUR, while no changes were observed in healthy parasites incubated with DMSO. Bars, 5 ␮m.
formly effective against the isolates of both subtypes tested.
1.54 ␮M, suggesting the potential of the drug as a broad- These 2-position 5-NIs exhibited significantly higher efficacy spectrum antiprotozoal agent against Mzr parasites. Two-po- against Mzr isolates than 1-position 5-NIs (P ⬍ 0.01), as ob- sition 5-NIs may prove to be effective alternatives to treat served in Giardia and Trichomonas (66). Again, the improved Blastocystis infections in cases of Mz treatment failure.
efficacy of 2-position 5-NIs against Mzs subtype 4 isolates is not The susceptibility of the Mzr subtype 7 isolates to NTZ and as obvious, suggesting a different mechanism of action in Blas- the reduced susceptibility to the Mzs subtype 4 isolates are also tocystis than in other organisms (66). The IC evidence for different mechanisms of action of NTZ in Blas- Giardia was recently reported to be 0.5 ␮M (17), whereas tocystis than in Giardia and Trichomonas, where cross-resis- against Trichomonas it exhibited a MIC of 6.3 ␮M (66). In this tance between Mz and NTZ is apparent (2). These data sug- of C-17 against Blastocystis ranged from 0.89 to gest that Mz treatment failures in blastocystosis may well BLASTOCYSTIS METRONIDAZOLE RESISTANCE AND SUSCEPTIBILITY values of Blastocystis for 5-NIs by resazurin assay 关␮g/ml (␮M)兴 Subtype 7 isolates Subtype 4 isolates 1-Position 5-NIsa 32.5 ⫾ 3.4 (189.8) 5.5 ⫾ 2.89 (32.16) 0.75 ⫾ 0.04 (4.38) 5.13 ⫾ 0.16 (20.52) 9.33 ⫾ 0.45 (37.32) 0.51 ⫾ 0.02 (2.04) 0.3 ⫾ 0.1 (1.2) 1.42 ⫾ 0.02 (6.44) 1.23 ⫾ 0.15 (5.58) 1.1 ⫾ 0.3 (4.9) 1.15 ⫾ 0.05 (5.22) 2-Position 5-NIsb 0.52 ⫾ 0.02 (2.6) 0.31 ⫾ 0.08 (1.55) 0.32 ⫾ 0.1 (1.6) 0.37 ⫾ 0.08 (1.85) 0.63 ⫾ 0.1 (1.56) 0.36 ⫾ 0.13 (0.89) 0.42 ⫾ 0.08 (1.04) 0.5 ⫾ 0.05 (1.24) a Side chain at position 1 of the imidazole ring of 5-NI.
b Side chain at position 2 of the imidazole ring of 5-NI.
c NS, not susceptible to drug concentrations of ⱕ100 ␮g/ml.
d FDA-approved antimicrobial agent.
e Veterinary antiparasitic agent.
f Experimental antiparasitic agent effective against Trichomonas and Giardia (66). respond to NTZ, as in the case of Cryptosporidium parvum parasites are not known, although they have been suggested to infections. C. parvum infections do not respond well to Mz act on protozoan cell membranes (62). The activity of QC (19), and NTZ is the treatment of choice, with in vitro IC s of against Blastocystis has been reported previously (16, 68), but ⬍10 ␮g/ml (60), similar to the IC s of the drug against both the current study is the first to report the potential usefulness Mzr and Mzs isolates of Blastocystis in this study. Recent in of MQ as an anti-Blastocystis drug.
vitro (68) and clinical data (55) also suggest the usefulness of EM is an effective antiamoebic agent with unpleasant side the drug in Blastocystis infections.
effects. It targets ribosomes and limits protein synthesis (43).
Another alternative to treat Mzr Blastocystis isolates is FUR, The in vitro activity of EM against Blastocystis has been eval- which was equally effective against all isolates in this study.
uated in two previous studies. While both studies suggested its FUR is a nitrofuran commonly used to treat giardiasis (49). It effectiveness against Blastocystis, Zierdt et al. reported strain- is activated inside the cell by NADH oxidase and generates to-strain variation in the susceptibility of the parasite to the toxic products that interfere with DNA processes in the para- drug (75). The multidrug resistance (MDR) phenotype of En- site (9). The IC s of FUR against both Mzr and Mzs isolates tamoeba histolytica exhibits resistance to a wide range of drugs, of Blastocystis were found to be similar to that against Giardia including EM, while responding to Mz (43), but no such MDR (2 ␮M) (5).
phenotypes have been reported in Blastocystis spp. Our study The prophylactic antimalarial MQ and a closely related describes the existence of EM resistance in Mzs isolates of drug, QC, were also found to be more effective against Mzr Blastocystis, suggesting that MDR phenotypes might be present subtype 7 isolates than Mzs subtype 4 isolates. These findings in the parasite. Clinically, however, EM has limited use be- are surprising because in Giardia, cross-resistance against QC cause of its severe side effects (32, 56).
has been observed between Mzr (8) and Tzr (63) strains, sug- TMP and SMZ are often prescribed in combination at a gesting a different mode of action of the drug in Blastocystis.
1:5 ratio as an alternative to Mz in Blastocystis infections.
The exact mechanisms of action of these drugs against luminal Clinical studies suggest that this drug combination success- TABLE 6. IC-50 values of anti-protozoal agents effective against Blastocystis isolates using the resazurin assay 关␮g/ml (␮M)兴 Subtype 7 (Mzr) isolates Subtype 4 (Mzs) isolates 0.62 ⫾ 0.07 (2.01) 1.14 ⫾ 0.49 (3.7) 4.15 ⫾ 0.41 (13.48) 0.65 ⫾ 0.05 (2.88) 1.06 ⫾ 0.4 (4.7) 0.49 ⫾ 0.01 (2.17) 0.475 ⫾ 0.05 (2.1) 1.49 ⫾ 0.83 (3.93) 1.85 ⫾ 0.88 (4.88) 4.7 ⫾ 0.35 (12.4) 5.1 ⫾ 0.58 (13.46) 1.9 ⫾ 0.2 (4.75) 5.1 ⫾ 0.47 (12.75) 4.9 ⫾ 0.53 (12.25) 5.1 ⫾ 1.1 (15.7) 4.3 ⫾ 2.4 (13.24) 3.2 ⫾ 0.52 (9.8) 5.4 ⫾ 1.4 (16.63) 1.03 ⫾ 0.4 (2.13) 1.32 ⫾ 0.9 (2.73) TMP:SMZ 1:2b TMP:SMZ 1:5b 0.34 ⫾ 0.05 (1.83) 0.2 ⫾ 0.03 (1.08) 0.33 ⫾ 0.06 (1.78) 0.26 ⫾ 0.02 (1.4) a N/S, not susceptible to ⱕ100-␮g/ml concentration of the drug.
b FDA-approved antimicrobial agent.
c Antiparasitic agent with adverse side effects; not currently used in clinical practice.
d Carcinogenic cysteine protease inhibitor; not clinically useful.
MIRZA ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
fully eradicates Blastocystis infections in 95% to 100% of of our findings across different life cycle stages of the par- cases (53, 54). There are no reports of the effectiveness of a asite. Despite this limitation, this is the first study suggesting 1:2 combination against Blastocystis. Our findings suggest subtype-dependent variation in the parasite response to che- the superiority of a 1:2 combination over a 1:5 combination with no subtype-dependent variation in susceptibility. We In conclusion, this study describes two cost-effective as- suggest that the 1:2 combination is likely to be more effec- says for high-throughput antimicrobial susceptibility analy- tive than the 1:5 combination in treatment of clinical infec- sis of Blastocystis. Using one of these assays, we demon- tions of Blastocystis.
strated for the first time subtype-dependent variations in the Cysteine proteases play an important role in the cell cycle susceptibility of Blastocystis to six different antiprotozoal and pathophysiology of protozoan parasites. Blastocystis cys- agents. We identified 4 new potential therapeutic options teine proteases have been reported to cleave human secretory against Blastocystis, namely, MQ, TMP-SMZ (1:2), Oz, and IgAs (58) and to induce upregulation of proinflammatory cy- FUR. Furthermore, we confirmed the antiprotozoal activi- tokines (48). A prosurvival role of legumain, a cysteine pro- ties of 10 compounds already reported to be effective against tease, has also been reported recently for Blastocystis (71).
Blastocystis. We also demonstrated in vitro Mz and EM Accumulating data in recent years suggest the therapeutic po- resistance in Blastocystis. By assessing the susceptibility of tential of protease inhibitors in parasitic infections (1, 42).
the parasite to different 5-NIs, we also demonstrated that Several cysteine protease inhibitors are being investigated as 5-NI resistance could be overcome in Blastocystis with more potential chemotherapeutic agents against parasites as diverse effective 5-NI compounds. Based on our findings, there is as Plasmodium (42, 44, 50), trypanosomes (18), and schisto- clearly a need to reevaluate currently established treatment somes (69). In this study, we found all four isolates to be highly regimens for Blastocystis infections.
susceptible to IA, a cysteine protease inhibitor, irrespective oftheir susceptibility to Mz. These findings suggest a potential role of cysteine protease inhibitors as a therapeutic option for This work was supported by a generous grant from the National Blastocystis isolates resistant to conventional antiprotozoal Medical Research Council (NMRC/1071/2006). H.M. and J.D.W.T.
are graduate students supported by National University of Singapore PAR is a broad-spectrum aminoglycoside (13). Although (NUS) research scholarships. This work was also supported in part byU01 Cooperative Research Agreement AI75527 from the National clinical studies suggest its effectiveness in the treatment of Institutes of Health. The study was facilitated by the commissioning of Blastocystis infections (3, 45, 67), in vitro data are equivocal (68, synthesis of C-17 by the NIH from the Southern Research Institute.
72). In this study, PAR was found to be ineffective against the We are grateful to Martin Lear and Oliver Simon for providing the isolates of both subtypes tested. The high clinical efficacy of the chemical structures of 5-nitroimidazoles.
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