Characterization of Giardia lamblia WB C6 clones resistant to nitazoxanide and to metronidazole.
OBJECTIVE
The characterization of Giardia lamblia WB C6 strains resistant to metronidazole and to the nitro-thiazole nitazoxanide [2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide] as the parent compound of thiazolides, a novel class of anti-infective drugs with a broad spectrum of activities against a wide variety of helminths, protozoa and enteric bacteria.


METHODS
Issuing from G. lamblia WB C6, we have generated two strains exhibiting resistance to nitazoxanide (strain C4) and to metronidazole (strain C5) and determined their susceptibilities to both drugs. Using quantitative RT-PCR, we have analysed the expression of genes that are potentially involved in resistance formation, namely genes encoding pyruvate oxidoreductases (POR1 and POR2), nitroreductase (NR), protein disulphide isomerases (PDI2 and PDI4) and variant surface proteins (VSPs; TSA417). We have cloned and expressed PDI2 and PDI4 in Escherichia coli. Using an enzyme assay based on the polymerization of insulin, we have determined the activities of both enzymes in the presence and absence of nitazoxanide.


RESULTS
Whereas C4 was cross-resistant to nitazoxanide and to metronidazole, C5 was resistant only to metronidazole. Transcript levels of the potential targets for nitro-drugs POR1, POR2 and NR were only slightly modified, PDI2 transcript levels were increased in both resistant strains and PDI4 levels in C4. This correlated with the findings that the functional activities of recombinant PDI2 and PDI4 were inhibited by nitazoxanide. Moreover, drastic changes were observed in VSP gene expression.


CONCLUSIONS
These results suggest that resistance formation in Giardia against nitazoxanide and metronidazole is linked, and possibly mediated by, altered gene expression in drug-resistant strains compared with non-resistant strains of Giardia.
Müller
Joachim
J
Institute of Parasitology, University of Berne, Länggass-Strasse 122, CH-3012 Berne, Switzerland. joachim.mueller@ipa.unibe.ch
Sterk
Maaike
M
Hemphill
Andrew
A
Müller
Norbert
N
D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D002999:Clone Cells; D017931:DNA Primers; D004351:Drug Resistance; D005786:Gene Expression Regulation / Q000187:drug effects; D016829:Giardia lamblia / Q000187:drug effects*; D008795:Metronidazole / Q000494:pharmacology*; D009574:Nitro Compounds; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D011771:Pyruvate Oxidase / Q000235:genetics; D012313:RNA / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013844:Thiazoles / Q000494:pharmacology*
D000981:Antiprotozoal Agents; D017931:DNA Primers; D009574:Nitro Compounds; D011994:Recombinant Proteins; D013844:Thiazoles; D008795:Metronidazole; D012313:RNA; D011771:Pyruvate Oxidase; D019704:Protein Disulfide-Isomerases; C041747:nitazoxanide