A rapid, high-throughput viability assay for Blastocystis spp. reveals metronidazole resistance and extensive subtype-dependent variations in drug susceptibilities

Abstract

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, suggesting the existence of drug-resistant isolates. Furthermore, very little is known about Blastocystis susceptibility 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.

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 derived from two independent sets of experiments. The error bars represent standard errors.

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 of resazurin and XTT, denoted by higher RFU and RAU readings, respectively. Each point represents a mean of 6 values derived from two independent sets of experiments. The error bars represent standard errors.

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 μl of IMDM supplemented with 10% horse serum and 0.5% 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 based on these results. Each point represents a mean of 6 values derived from two independent experiments, with each experiment conducted in triplicate. The error bars represent standard errors.

FIG. 4.

Graph representing percent inhibition of Blastocystis subtype 4 and 7 cultures by Mz using the resazurin assay. The IC₅₀s 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 isolate B cultures at a concentration (conc.) of 32.5 ± 3.4 μg/ml, whereas isolate E cultures exhibited only minimal inhibition even at concentrations as high as 100 μg/ml. Each point represents a mean of six readings derived from two independent experiments. The error bars represent standard errors.

FIG. 5.

Chemical structures of position 1 and position 2 5-nitroimidazoles.

FIG. 6.

Confocal micrographs of Blastocystis stained with propidium iodide (arrow) and annexin V-FITC. (A) Mz^s ST-4 (WR-1) exhibited nuclear incorporation of PI and annexin V-FITC binding after 24 h of exposure to 12.5 μg/ml Mz. (B) Mz^r ST-7 (isolate E) did not exhibit these classical signs of cell death after Mz treatment. Both Mz^s and Mz^r isolates exhibited PI incorporation and annexin V-FITC binding after 24-h treatment with 12.5 μg/ml FUR, while no changes were observed in healthy parasites incubated with DMSO. Bars, 5 μm.