A new-generation 5-nitroimidazole can induce highly metronidazole-resistant Giardia lamblia in vitro
Introduction
Metronidazole (MTR) (Fig. 1) belongs to the family of 5-nitroimidazole (NI) drugs that are the mainstay for treating infections caused by the clinically important anaerobic protozoa Giardia lamblia (synonymous with G. intestinalis and G. duodenalis), Trichomonasvaginalis, Entamoeba histolytica[1] and Blastocystis sp. (reviewed in [2]) and the anaerobic bacteria, particularly Helicobacter pylori, Clostridium difficile and Bacteroides fragilis[3]. MTR has a side chain in the 1-position of the imidazole ring, with the all important nitro group in the 5-position (Fig. 1). The nitro group is activated by low redox potential reactions in anaerobes, producing toxic nitro radicals that ultimately cause death of the anaerobic organism [4]. In G. lamblia, T. vaginalis and E. histolytica, this has been proposed to occur via reduction by the low electron potential couple, pyruvate:ferredoxin oxidoreductase (PFOR) and ferredoxin (FDOX) [5], [6]. This proposal has recently been challenged by Leitsch et al. [7], [8] who claim that in T. vaginalis and E. histolytica thioredoxin reductase (TrxR) has nitroreductase activity and that NADPH is the primary source of reducing power for MTR and tinidazole activation.
1-Position-modified 5-NIs commonly in clinical use include tinidazole, recommended in cases of MTR treatment failures of T. vaginalis (reviewed in [9]), secnidazole and ornidazole, whilst ronidazole, a veterinary drug, and satranidazole are among the few commercially available 5-NIs with 2-position side chains. Some experimental 5-NI compounds with 2-position modifications (Fig. 1) demonstrate ca. 40-fold [10] to 80-fold (compound 29 in [10]) greater efficacy against G. lamblia than MTR, a 1-position 5-NI, and 5-NIs with 4-position side chains have also been reported to be more effective than MTR against T. vaginalis[11]. MTR treatment failures and clinical MTR resistance have been documented in cases of giardiasis [12]. In G. lamblia, the mechanism of MTR resistance has been proposed to involve downregulation of PFOR activity and the FDOX protein [13], [14], [15].
This study, as well as our previous reports [10], [11], [16], [17], point towards the potential for developing a clinically safe, highly effective 5-NI that, similarly to MTR, is effective against a wide range of anaerobes but is far more potent and therefore likely to overcome MTR resistance. However, the limit of resistance that these parasites can develop must be tested—will new, more potent 5-NIs eventually induce more highly resistant organisms? In this study, we explore the potential of G. lamblia to develop resistance against one highly effective 2-position 5-NI compound.
Section snippets
Isolates and culture
The following G. lamblia isolates were used: BRIS/83/HEPU/106 (106) and BRIS/87/HEPU/713 (713) [18]; the laboratory-induced MTR-resistant (MTRR) lines 713-M3 [19] and 106-2ID10[20] derived from the above isolates, respectively; the laboratory-induced C17-resistant (C17R) line 106-17A; and MTRRC17R line 713-M3-C17 (see Section 2.3).
Giardia lamblia was cultured axenically in modified TYI-S-33 medium [21] as previously described [22]. MTRRGiardia lines 713-M3 and 106-2ID10 were maintained in the
Drug susceptibility of Giardia parent lines
Consistent with previous reports [10], [17], [23], we have shown that 5-NIs [C14, C17 and C18 (see [17] and Fig. 1) and commercially available ronidazole] modified in the 2-position of the imidazole ring display greater activity against G. lamblia than the more traditional 1-position 5-NIs (MTR, tinidazole and ornidazole), with MTR consistently displaying the lowest activity (Table 1). The RA of C17 compared with MTR was 17.5 and 14.3 for G. lamblia MTRS isolates 106 and 713, respectively (
Discussion
The 2-position 5-NI compound C17 is extremely effective against G. lamblia in vitro, with ID90 values 14.3–17.7-fold more effective than MTR. These ID90 values are consistent with our previous reports using different assay readouts [10], [17]. Also consistent with C17 being highly effective was the difficulty in developing C17RG. lamblia lines. Many unsuccessful attempts were made to grow a range of isolates and lines in low levels of C17, including quinacrine- and albendazole-resistant lines
Acknowledgment
The authors thank Marc S. Ayers from Romark Laboratories, L.C. (Tampa, FL) for the nitazoxanide.
Funding: This work was supported by the National Health and Medical Research Council of Australia, the Australian Research Council and by U01 Cooperative Research Agreement AI75527 from the US National Institutes of Health (NIH). This study was facilitated by the commissioning of synthesis of C17 by NIH from Southern Research Institute (USA).
Competing interests: None declared.
Ethical approval: Not
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