A new-generation 5-nitroimidazole can induce highly metronidazole-resistant Giardia lamblia in vitro

doi:10.1016/j.ijantimicag.2010.03.004

International Journal of Antimicrobial Agents

Volume 36, Issue 1, July 2010, Pages 37-42

https://doi.org/10.1016/j.ijantimicag.2010.03.004 Get rights and content

Abstract

The 5-nitroimidazole (NI) compound C17, with a side chain carrying a remote phenyl group in the 2-position of the imidazole ring, is at least 14-fold more active against the gut protozoan parasite Giardialamblia than the 5-NI drug metronidazole (MTR), with a side chain in the 1-position of the imidazole ring, which is the primary drug for the treatment of giardiasis. Over 10 months, lines resistant to C17 were induced in vitro and were at least 12-fold more resistant to C17 than the parent strains. However, these lines had ID₉₀ values (concentration of drug at which 10% of control parasite ATP levels are detected) for MTR of >200 μM, whilst lines induced to be highly resistant to MTR in vitro have maximum ID₉₀ values around 100 μM (MTR-susceptible isolates typically have an ID₉₀ of 5–12.8 μM). The mechanism of MTR activation in Giardia apparently involves reduction to toxic radicals by the activity of pyruvate:ferredoxin oxidoreductase (PFOR) and the electron acceptor ferredoxin. MTR-resistant Giardia have decreased PFOR activity, which is consistent with decreased activation of MTR in these lines, but C17-resistant lines have normal levels of PFOR. Therefore, an alternative mechanism of resistance in Giardia must account for these super-MTR-resistant cells.

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 (MTR^R) lines 713-M3 [19] and 106-2ID₁₀[20] derived from the above isolates, respectively; the laboratory-induced C17-resistant (C17^R) line 106-17A; and MTR^RC17^R line 713-M3-C17 (see Section 2.3).

Giardia lamblia was cultured axenically in modified TYI-S-33 medium [21] as previously described [22]. MTR^RGiardia 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 MTR^S 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 C17^RG. 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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A new-generation 5-nitroimidazole can induce highly metronidazole-resistant Giardia lamblia in vitro

Abstract

Introduction

Section snippets

Isolates and culture

Drug susceptibility of Giardia parent lines

Discussion

Acknowledgment

Drug Resist Updat

Eur J Med Chem

Mol Biochem Parasitol

Mol Biochem Parasitol

Acta Trop

Trans R Soc Trop Med Hyg

Trans R Soc Trop Med Hyg

Infect Genet Evol

Drug Resist Updat

Drug targets and mechanisms of resistance in the anaerobic protozoa

Clin Microbiol Rev

New insights on classification, identification, and clinical relevance of Blastocystis spp.

Clin Microbiol Rev

Nitroimidazole drugs—action and resistance mechanisms. I. Mechanisms of action

J Antimicrob Chemother

A purified ferredoxin from Giardiaduodenalis

Eur J Biochem

Mode of action of metronidazole on anaerobic bacteria and protozoa

Surgery

Nitroimidazole action in Entamoebahistolytica: a central role for thioredoxin reductase

PLoS Biol

Trichomonasvaginalis: metronidazole and other nitroimidazole drugs are reduced by the flavin enzyme thioredoxin reductase and disrupt the cellular redox system. Implications for nitroimidazole toxicity and resistance

Mol Microbiol

Drug resistance in the sexually transmitted protozoan Trichomonasvaginalis

Cell Res