Variability and variation in drug susceptibility among Giardia duodenalis isolates and clones exposed to 5-nitroimidazoles and benzimidazoles in vitro

Argüello-García, Raúl; Cruz-Soto, Maricela; Romero-Montoya, Lydia; Ortega‐Pierres, Guadalupe

doi:10.1093/jac/dkh388

Cited by 49 publications

(34 citation statements)

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“…1) using subculture in liquid medium. Briefly, trophozoites (7.5 × 10 5 ) were incubated with the inhibitors for 30 min at 37 o C, washed twice with PBS and subcultured in fresh medium for 48 h at 37 o C. The cells were counted and their viability was determined by interpolation in a control curve obtained with different inocula of untreated trophozoites (Argüello-García et al 2004), and the experimental lethal concentrations at 50 and 20% (LC 50 and LC 20 respectively) were also calculated. The data obtained showed that E64 was a potent inhibitor with a LC 50 value of 7.47 µM.…”

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confidence: 99%

Giardia duodenalis: analysis of secreted proteases upon trophozoite-epithelial cell interaction in vitro

Rodríguez-Fuentes

Cedillo‐Rivera

Fonseca-Liñán

et al. 2006

Mem. Inst. Oswaldo Cruz

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Protease secretion by Giardia duodenalis trophozoites upon interaction with epithelial cells and its association with the parasite adhesion was studied in co-cultures of parasites with IEC6 epithelial cell monolayers in the presence or absence of protease inhibitors. Proteolytic activity in supernatants from trophozoites was enhanced when they were co-cultured with IEC6 cells. This activity was strongly inhibited by pre-incubation of live trophozoites with E-64 and TPCK and a concomitant inhibition of parasite adhesion to IEC6 cells was observed. These data suggest that trophozoites secrete cysteine-type proteases that play a role in the adhesion of G. duodenalis to epithelial cells.Key words: Giardia duodenalis -protease secretion -adhesion Giardiasis caused by the flagellated, intestinal protozoan parasite Giardia duodenalis (syn. G. lamblia, G. intestinalis) manifests as acute and chronic diarrhoea in communities of both developing and developed countries. It is also a leading cause of defined waterborne diarrhoea worldwide (Adam 1991). Infection results from ingestion of cysts, most commonly from faecal contamination of water and food whereas community-wide outbreaks result from contaminated water supplies. After excystation in the upper small intestine, adhesion of trophozoites to epithelial intestinal cells represents the first step in the pathogenesis of the disease. The use of IEC6, an epithelial cell line derived from rat small intestine, is a well validated model in which the inhibitory effect of both actin inhibitors and chelating agents on the attachment of G. duodenalis trophozoites has been previously demonstrated (McCabe et al. 1991). It is also well recognized that the adhesion process is a multi-factorial event that involves a suction force of the adhesive disk (Knaippe 1990), a mechanical process related to contractile proteins of the adhesive disk and the ventrolateral flange (Chávez et al. 1995) and surface molecules that allow the binding of trophozoites to receptors to host epithelial cells (Pegado & Souza 1994). It has been postulated that proteases may be involved in this process. Several studies have demonstrated protease activity in total extracts of Giardia trophozoites or in parasite vacuoles (Feely & Dyer 1987, Hare et al. 1989) including the Portland 1 (P1) strain cultured in vitro, with the predominance of cysteine, serine, and aspartic protease activities (Williams & Coombs 1995). It has also been shown that cysteine proteases of P1 strain degraded protein substrates such as gelatin, collagen, albumin, and azocasein while serine proteases were mainly active against haemoglobin (Coradi & Guimarães 2006). In addition, the presence of cysteine protease activities in excretory/secretory (E/S) products of P1 strain has also been reported (Jiménez et al. 2000). These E/S products are associated with mucosal injuries observed in murine giardiasis (Jiménez et al. 2004). However the possible relation between protease secretion and the adhesion of G. duodenalis to epithelial cells has not b...

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Giardia duodenalis: analysis of secreted proteases upon trophozoite-epithelial cell interaction in vitro

Rodríguez-Fuentes

Cedillo‐Rivera

Fonseca-Liñán

et al. 2006

Mem. Inst. Oswaldo Cruz

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“…Three of these hit compounds, benzalkonium chloride, paroxetine hydrochloride, and oxibendazole, were previously shown to have antibacterial activity (37)(38)(39). These three known antimicrobial compounds, as well as two additional hits, ethidium bromide (because it is associated with broad toxicity) and acetaminophen (because it was a false hit that was not confirmed in secondary screens), were not investigated further.…”

Section: Resultsmentioning

confidence: 99%

Characterization of a Francisella tularensis-Caenorhabditis elegans Pathosystem for the Evaluation of Therapeutic Compounds

Jayamani

Tharmalingam

Rajaraman

et al. 2017

Antimicrob Agents Chemother

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Francisella tularensis is a highly infectious Gram-negative intracellular pathogen that causes tularemia. Because of its potential as a bioterrorism agent, there is a need for new therapeutic agents. We therefore developed a whole-animal Caenorhabditis elegans-F. tularensis pathosystem for high-throughput screening to identify and characterize potential therapeutic compounds. We found that the C. elegans p38 mitogen-activate protein (MAP) kinase cascade is involved in the immune response to F. tularensis, and we developed a robust F. tularensis-mediated C. elegans killing assay with a Z= factor consistently of Ͼ0.5, which was then utilized to screen a library of FDA-approved compounds that included 1,760 small molecules. In addition to clinically used antibiotics, five FDA-approved drugs were also identified as potential hits, including the anti-inflammatory drug diflunisal that showed anti-F. tularensis activity in vitro. Moreover, the nonsteroidal anti-inflammatory drug (NSAID) diflunisal, at 4ϫ MIC, blocked the replication of an F. tularensis live vaccine strain (LVS) in primary human macrophages and nonphagocytic cells. Diflunisal was nontoxic to human erythrocytes and HepG2 human liver cells at concentrations of Ն32 g/ml. Finally, diflunisal exhibited synergetic activity with the antibiotic ciprofloxacin in both a checkerboard assay and a macrophage infection assay. In conclusion, the liquid C. elegans-F. tularensis LVS assay described here allows screening for anti-F. tularensis compounds and suggests that diflunisal could potentially be repurposed for the management of tularemia.

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“…Here, we also noted that trichomonad ATP readouts in this assay system are unreliable at concentrations below the MIC. Thus as previously commented (Argüello-García et al 2004;Upcroft and Upcroft 2001a), susceptibility assays and readouts can vary greatly within and between laboratories depending on the assay system. We also tested the parasite phosphatase activity assay described by MartinezGrueiro et al (2003), but the ID 90 readouts did not correlate with our MIC values for all drugs used (data not shown).…”

Section: Discussionmentioning

confidence: 98%

Susceptibility in vitro of clinically metronidazole-resistant Trichomonas vaginalis to nitazoxanide, toyocamycin, and 2-fluoro-2′-deoxyadenosine

et al. 2010

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This study investigates the susceptibility of a clinically metronidazole (Mz)-resistant isolate of Trichomonas vaginalis to alternative anti-trichomonal compounds. The microaerobic minimal inhibitory concentration (MIC) of the 5-nitroimidazole (NI) drug, Mz, against a typical Mz-susceptible isolate of T. vaginalis is around 3.2 microM Mz while the clinically, highly Mz-resistant isolate has an MIC of 50-100 microM. This isolate was cross-resistant to other members of the 5-NI family of compounds including tinidazole and other experimental compounds and maintained resistance under anaerobic conditions. In addition, this isolate was cross-resistant to the 5-nitrothiazole compound nitazoxanide and the 5-nitrofuran derivative, furazolidone. Adenosine analogues toyocamycin and 2-fluoro-2'-deoxyadenosine with no nitro group were also less effective against the clinically Mz-resistant isolate than a Mz-susceptible one. Three other isolates which were determined to be Mz-resistant soon after isolation lost resistance in the long term. One other isolate has maintained some level of permanent Mz resistance (MIC of 25 microM). A multi-drug resistance mechanism may be involved in these clinically Mz-resistant isolates.

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Variability and variation in drug susceptibility among Giardia duodenalis isolates and clones exposed to 5-nitroimidazoles and benzimidazoles in vitro

Abstract: Our observations support the idea that G. duodenalis cultures exhibit variations in their response to 5-nitroimidazoles and benzimidazoles as a result of a drug-independent competition between drug-susceptible and drug-resistant subpopulations when parasites are subcultured.

Cited by 49 publications

References 41 publications

Giardia duodenalis: analysis of secreted proteases upon trophozoite-epithelial cell interaction in vitro

Giardia duodenalis: analysis of secreted proteases upon trophozoite-epithelial cell interaction in vitro

Characterization of a Francisella tularensis-Caenorhabditis elegans Pathosystem for the Evaluation of Therapeutic Compounds

Susceptibility in vitro of clinically metronidazole-resistant Trichomonas vaginalis to nitazoxanide, toyocamycin, and 2-fluoro-2′-deoxyadenosine

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