DOI: 10.17035/d.2016.0008716702
title: The redox-active drug metronidazole and thiol-depleting garlic compounds act synergistically in the protist parasite Spironucleus vortens

description: Spironucleus vortens is a protozoan parasite associated with significant mortalities in the freshwater angelfish, Pterophyllum scalare. Control of this parasite is especially problematic due to restrictions on the use of the drug of choice, metronidazole (MTZ), on fish farms. Use of garlic (Allium sativum) is undergoing a renaissance following experimental validations of its antimicrobial efficiency. Ajoene ((E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide), is a stable transformation product of allicin, the primary biologically active component of garlic. In the current study, an ajoene oil crude extract had a minimum inhibitory concentration (MIC) of 40 µg/ml against S. vortens. GC-MS and NMR spectroscopy revealed this ajoene extract contained a mixture of the (E) and (Z)-ajoene isomers along with diallyl disulphide (DADS) and diallyl trisulphide (DATS). The only component of the ajoene crude oil found to substantially inhibit S. vortens growth by optical density monitoring (Bioscreen C Reader) was (Z)-ajoene (MIC 16 µg/ml). Ajoene oil acted in synergy with MTZ in vitro, reducing the individual MIC of this drug (4 µg/ml) by 16-fold, and that of ajoene oil by 200-fold with a fractional inhibitory concentration (FIC) index of 0.263. This synergistic interaction was confirmed in vivo. S. vortens-infected P. scalare angelfish dosed orally with 0.5% (v/w) MTZ combined with 0.05% (v/w) ajoene displayed a significant reduction in faecal trophozoite count, whilst those fed on 0.5% MTZ flakes (half the recommended oral dose) alone did not. This study demonstrates for the first time the synergistic interaction between the synthetic drug MTZ and natural ajoene oil both in vitro and in vivo. Future work should evaluate the potential synergy of ajoene and MTZ against MTZ-resistant bacteria and protists.  Dataset 1: Optical density readings over time generated by the Biosceen C machine are given for each of the compounds tested (Untreated Control, Ajoene oil, Z-Ajoene, Allyl Alcohol. Allyl Methyl Disulphide, Diallyl Disulphide, Allicin and Metronidazole) against the growth of Spironucleus vortens. Each compound was tested in triplicate. Each triplicate value is adjusted to obtain a zero baseline value (denoted "Adj" in the Excel file). Finally the average of each triplicate value is also provided for each time point.  Dataset 2: As for Dataset 1, but using the Spironucleus vortens Sv1 isolate.  Dataset 3: Spironucleus vortens faecal trophozoite counts collected from angelfish (Pterophyllum scalare) in 4 different treatment groups (Untreated Control, Ajoene Treated, Metronidazole Treated and Metronidazole + Ajoene Treated). Faecal trophozoite counts were collected as follows: Briefly, faecal pellets were collected immediately after defecation and mounted as a squash preparation between a glass slide and coverslip. Squash preparations were viewed microscopically (400x magnification) for 15 min, analysing ca. 50 different fields of view, for the presence of S. vortens trophozoites displaying characteristic pyriform cell shape (10 µl in length) with rapid motility. The average trophozoite counts from two faecal samples were quantified in order to gain a more accurate estimation of the degree of infection due to differential host parasite shedding over time. Spironucleus vortens infections were quantified in this way at Day 0 (pre-treatment) and Day 5 (post-treatment) for the flake food treatment (Groups 1-4).