Resistance towards metronidazole in Blastocystis sp.: A pathogenic consequence

Abstract

Blastocsytis sp. is a protozoan parasite that has been linked to common gastrointestinal illnesses. Metronidazole, the first line therapy, was reported to show frequent inefficacy. Previously, Blastocystis sp. isolated from different population showed varying metronidazole resistance. However, the effect of metronidazole treatment on pathogenic potentials of Blastocystis sp. isolated from different populations, which is known to have different gut environment, is unclear. This study investigates the in vitro effect of metronidazole on the pathogenic potentials of Blastocystis sp. isolated from urban and orang asli individuals. Blastocystis sp. ST 3 isolated from symptomatic and asymptomatic individuals were treated with a range of metronidazole concentration. The parasites' growth characteristics, apoptotic rate, specific protease activity and the ability to proliferate cancer cells were analyzed upon treatment with 0.001 mg/l metronidazole. The study demonstrates that Blastocystis sp. isolates showed increase in the parasite numbers especially the amoebic forms (only in urban isolates) after treating with metronidazole at the concentration of 0.001 mg/ml. High number of cells in post-treated isolates coincided with increase of apoptosis. There was a significant increase in cysteine protease of Blastocystis sp. isolates upon treatment despite the initial predominance of serine protease in asymptomatic isolates. Metronidazole resistant Blastocystis sp. also showed significant increase in cancer cell proliferation. Resistance to metronidazole did not show significant different influence on the pathogenicity between Blastocystis sp. isolated from urban and orang asli individual. However, an increase in parasite numbers, higher amoebic forms, cysteine protease and ability to proliferate cancer cells implicates a pathogenic role. The study provides evidence for the first time, the effect of metronidazole towards enhancing pathogenic potentials in Blastocystis sp. when isolated from different gut environment. This necessitates the need for reassessment of metronidazole treatment modalities.

Fig 1. Treatment of Blastocystis sp. with various concentration of metronidazole.

Growth profile of symptomatic and asymptomatic isolates from urban and orang asli showed increased proliferation of cells at 0.001 mg/ml of metronidazole. Values are expressed as mean ± SD of 5 isolates. Cells were counted in 1 ml. Control experiment was done using PBS to replace metronidazole. *P<0.05 in Students t-test for comparison of means of peak cell count between different metronidazole concentrations.

Fig 2. Generation time of Blastocystis sp. for control (Pre-treatment) and treated with 0.001 mg/ml metronidazole (Post-treatment).

Symptomatic and asymptomatic isolates and represented by black and blue circles respectively. Isolates obtained from symptomatic urban individual showed significant decrease in generation time. The black lines represents mean of 5 isolates. *P = 0.005 for comparisons of mean between control and metronidazole treated Blastocystis sp. isolated from symptomatic urban individual.

Fig 3. Amoebic forms seen in Blastocystis sp. isolated from urban symptomatic individuals.

(A) Amoebic forms (arrow) seen under microscope at 40X magnification coexisting with vacuolar forms. Amoebic forms are seen with protrusion of the cytoplasm. Bar = 10μm (B) Increase in the number of amoebic morphology upon 0.001 mg/ml metronidazole treatment (Post-treatment) compared to the control (Pre-treatment). Amoebic forms were only observed in isolates from symptomatic urban individuals. Values are expressed as mean ± SD of 5 isolates. *represent P<0.001 in Students t-test comparing means of peak amoebic formation count in control and metronidazole treated isolates.

Fig 4. Staining of Blastocystis sp. cells to identify apoptosis and rate of apoptosis in control (Pre-treatment) and 0.001 mg/ml metronidazole treated (Post-treatment) Blastocystis sp. isolated from urban and orang asli individuals.

(i) Light microscopy image of 0.001 mg/ml drug treated Blastocystis sp.(ii) Fluorescence image of parasite cells stained blue with Hoechst stain binding to DNA and staining nuclei in the periphery of the viable parasite(V). (iii) Fluorescence image of parasite stained with fluorescein isothiocyanate (FITC) labeled Annexin V identifying the apoptotic cells (A) through the binding with phosphatidylserine at the outer cell surface. The scatterplots show percentage of apoptotic forms in five Blastocystis sp. isolates. Black and blue circles represent symptomatic and asymptomatic isolates respectively. The black and blue lines represent the mean of respective data. Significantly higher rate of apoptosis was seen in isolates obtained from orang asli individuals. *represents P = 0.001 in Students t-test for comparison of percentage of apoptotic forms between pre- and post-treatment.

Fig 5. Specific protease activities in control (Pre-treatment) and 0.001 mg/ml metronidazole treated (Post-treatment) Blastocystis sp. isolated from urban and orang asli individual.

Black circles and red circles represent protease activity of pre-treated and post-treated isolates respectively. The black line represent the mean(SD) of the 5 isolates tested. Note the predominance of cysteine protease upon treatment in Blastocystis sp. isolated from urban and orang asli individuals. *represents P<0.001 for the comparison with the mean of control. ^#represents P<0.001 in Students t-test for comparison of protease activity pre- and post treatment.

Fig 6. Effect of antigens from control (Pre-treatment) and 0.001 mg/ml metronidazole treated (Post-treatment) Blastocystis sp. on the proliferative ability of colon cancer cell, HCT116.

The black and blue circles represent antigens extracted from symptomatic and asymptomatic isolates respectively. The black lines represent the mean of 5 isolates. Note the increased proliferative ability in antigens of Blastocystis sp. isolated from urban individuals upon metronidazole treatment. *represents P<0.001 in Students t-test for comparison of mean between pre- and post-treatment.

Fig 7. Schematic diagram depicting the major findings in the current study.

Metronidazole treatment at a certain concentration (0.001 mg/ml) heightens the parasite number. (1), (2) The parasite cells forms high number if granular forms that in turn produce viable vacuolar forms. (3) When the condition turn unfavourable, the parasites may encyst and excreted in large numbers as reported in a case study previously. (4) It could be that some vacuolar forms that is metronidazole resistant Blastocystis sp. would retain. (5) This resistant vacoular forms and increased amoebic forms of Blastocystis sp. exhibit increased pathogenic potentials (as shown by the thunder symbols) in terms of greater cysteine protease activity, amoebic forms and ability to proliferate cancer cells.