In vitro-induction of metronidazole-resistant Giardia duodenalis is not associated with nucleotide alterations in the genes involved in pro-drug activation

Giardiasis is an infectious disease caused by Giardia duodenalis. The pro-drug metronidazole (MTZ) is the first-line treatment for giardiasis. Parasite’s proteins as pyruvate:ferredoxin oxidoreductase (PFOR), ferredoxin (Fd), nitroreductase-1 (NR-1) and thioredoxin reductase (TrxR) participate in MTZ activation. Here, we showed Giardia trophozoites long-term exposed to MTZ presented higher IC50 than controls, showing the drug influenced the parasite survival. That reduction in MTZ’s susceptibility does not seem to be related to mutations in the genes pfor, fd, nr-1 or trxr. It points that different mechanism as alterations in other metabolic pathways can account for Giardia resistance to MTZ therapy.

Giardiasis is a waterborne disease caused by the protozoan Giardia duodenalis (syn. G. lamblia, G. intestinalis) that colonises the small intestine. The infection is worldwide distributed affecting around 280 million people each year most of them children. (1,2) Although the prevalence of giardiasis is high in low income regions, the transmission is also a concern in developed countries, especially as outbreaks. (2) G. duodenalis is a zoonotic parasite classified into eight assemblages (A to H). (3) The assemblages A and B are classically known to infect humans and dogs. In addition, the assemblage E infecting humans was also described in Brazil (4) and Australia. (5) The faecal-oral transmission along with the close interaction with pets increase the risk of G. duodenalis infection There are relatively few therapies available for treatment of giardiasis and 5-nitroimidazoles drugs, mainly metronidazole (MTZ), are first-line treatment. (6,7) MTZ is a pro-drug and need to be metabolised to exert biological activity. In G. duodenalis, the proteins pyruvate:ferredoxin oxidoreductase (PFOR) and ferredoxin (Fd) act together to activate MTZ. (8) However, two other proteins were also associated with MTZ activation: nitroreductase-1 (NR-1) (9) and thioredoxin reductase (TrxR). ( radical that causes oxidative damage to several cellular macromolecules, especially DNA and proteins, leading to the parasite's death. (7,11) The use of 5-nitroimidazoles is well established in clinical practice, the efficacy is high and the estimate cure rate can achieve up to 90% of the giardiasis treated cases. (6) However, increasing therapeutic failure has been observed in clinical practice. Studies conducted in Spain (12,13) and United Kingdom (14) evaluating travellers point to increasing rates of 5-nitroimidazole refractory Giardia infection. Moreover, in a Cuban cohort of 456 Giardia-microscopically positive individuals, 46% of them experienced therapeutic failure after the first course of MTZ treatment. (15) Indiscriminate use of antiparasitic drugs together with repeated treatments can increase the chances for rising MTZ-resistant G. duodenalis strains. These strains can spread into the environment, infect susceptible individuals and, consequently, make the treatment of giardiasis more difficult.
The resistance of microorganisms to different classes of drugs is a worldwide concern. Several mechanisms cause resistance to drugs, such as mutation, changes in gene expression and protein abundance, efflux pumps, epigenetic changes and others. Giardia resistance to 5-nitroimidazoles was extensively documented in vitro, but the mechanisms underlying this resistance is still largely unknown. (16)(17)(18)(19) Considering the role of PFOR, Fd, NR-1 and TrxR in the MTZ activation, nucleotide alterations in genes that encode these proteins could be associated with 5-nitroimidazoles parasitic resistance. Herein, we evaluated whether in vitro induced MTZ-resistant G. duodenalis strain presents mutations in these genes.
Four groups were defined where G. duodenalis was continuously exposed to different concentrations of MTZ as follow: 5 µM (MTZ5); 10 µM (MTZ10); 20 µM (MTZ20); and 80 µM (MTZ80). Two groups were used as controls: the group no exposed to MTZ (SMTZ) and the group exposed to 0.05% (V/V) of dimethylsulfoxide (Sigma, USA) (CDMSO), the vehicle in which MTZ was solubilised. After 16 weeks, the IC 50 was evaluated for all groups evaluated. (21) Briefly, 5 × 10 4 parasites from each experimental group were plated in a 96-well flat-bottom plate, in which a serial dilution of MTZ was carried out. The plates were incubated at 37°C for 72 h. Resazurin was added to evaluate the parasite's viability. After 4 h incubation, the reaction was quantified by fluorimeter (λex 560 nm -λem 590 nm). 21 The tests were performed in triplicate and the IC 50 results were analysed using the GrafPad Prism 6.0 software (www.grafpad.com/scientific-software/prism/). The results were expressed as IC 50 values and resistance factor, i.e, IC 50 fold change in relation to control. The ANOVA test was used for comparative analysis of the IC 50 of groups.
The DNA from experimental and control groups were extracted using DNAzol TM reagent (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The DNA was subjected to PCR to amplify gene fragments. The specific primers for fd (orf. GL50803_9662), nr-1 (orf.GL50803_6175) and trxr (orf.GL50803_9827) were designed using the NCBI Pick Primers Tool (https:// www.ncbi.nlm.nih.gov/tools/primer-blast/). The amplified fragments of nr-1 (953 base pairs), fd (251 base pairs) and tr (1,273 base pairs) comprised the complete sequence of these proteins. The amplified fragment of pfor (342 base pairs) (orf.GL50803_114608) contains the coding region of the catalytic site of the enzyme. (16) The primers sequences, the concentration of the mix reagents and amplification conditions are described in Table. The effectiveness and efficiency of PCR conditions were assessed by electrophoresis in agarose gel 1%.
The gene fragments obtained in the PCR reaction were purified using the QIAquick ® -PCR purification Kit (Qiagen, Germany), according to the manufacturer's instructions. The effectiveness and efficiency of PCR conditions were assessed by electrophoresis in agarose gel 1%. The purified fragments were subjected to sequencing using the BigDye ® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, USA). The precipitation and electrophoresis steps were per-formed at Sequencing Service DNA Sequencing Platform -Fundação Oswaldo Cruz. (22) The electropherograms were analysed and their quality was verified by the Chromas 2.4 program (http:// tecnelysium.com.au/wp/chormas/). The identity percentage was verified by the Basic Local Alignment Search Tool (BLASTn) (https: /blast.ncbi.nlm.nih.gov./ Blast.cgi). The obtained sequences were aligned with the nucleotide sequences of pfor, fd, nr-1 and trxr of G. duodenalis in GenBank using the CLUSTAL W algorithm (23) in the Mega 7.0 package (http://www.megasoftware.net) (24) and the consensus obtained by CAP3 Sequence Assembly Program (http://doua.prabi.fb/software/cap3).
To evaluate whether the MTZ exposition decreases the parasite's susceptibility to the drug, G. duodenalis trophozoites were continuously exposed to different concentrations of the MTZ during 16 weeks. Afterwards, the parasite's susceptibility to the different MTZ concentrations was evaluated by the rate of trophozoite replication determined by the IC 50 values and the resistance factor ( Figure). The IC 50 values of MTZ5, MTZ10 and MTZ20 groups were significantly higher in comparison to SMTZ and CDMSO groups. This result indicates that G. duodenalis exposition to MTZ induced a reduction of the parasite's susceptibility in a dose dependent manner. The IC 50 values of MTZ80 group could not be determined since the majority of parasites were dead at 16 weeks.
The reduced susceptibility of G. duodenalis trophozoites to MTZ could be caused by alterations in the nucleotide sequence of the genes associated with MTZ activation. Thus, nucleotide sequences of these genes were evaluated. All groups had their DNA amplified and sequenced. The length of the amplified fragments obtained by PCR are the expected for each amplified sequence. No alteration on the nucleotide sequence of pfor, fd, nr-1 and trxr was identified in the parasites from the exposed groups MTZ5, MTZ10, MTZ20, MTZ80 and controls. These results indicate that the reduction of MTZ susceptibility of Giardia trophozoites in vitro is not a consequence of pfor, fd, nr-1 and trxr nucleotide modifications.
Several factors can account for refractory cases of giardiasis after treatment with different drug classes, as inadequate use of drug doses, immunosuppression, reinfection and resistance of the parasite to drugs. In our study, we induced G. duodenalis-resistant trophozoites by in vitro exposition to different concentrations of MTZ and demonstrated that no nucleotide alteration occurred in genes previously associated with Giardia resistance. The in vitro exposure of G. duodenalis to MTZ significantly increased the IC 50 values of the groups exposed to this drug compared to controls, similar to the findings of other studies. (16,17,25) Herein, the IC 50 fold change values obtained from MTZ exposed parasites ranged from 2.01 to 3.51, as observed to other MTZresistant trophozoites strains. (17) However, our strategy of continuous MTZ exposition differed from other authors who used intermittent exposure of G. duodenalis to high concentrations of 5-nitroimidazoles or ultraviolet radiation followed by exposure to this drug. (25) As expected, the results demonstrated that the continuous exposure of G. duodenalis to MTZ decreased the parasite's susceptibility to this drug in vitro.
The mechanism responsible for G. duodenalis in vitro resistance to MTZ is still unknown. Since parasite's proteins such as PFOR, Fd, NR-1 and TrxR participate in the activation of MTZ, nucleotide changes in the genes that encode these proteins could prejudice the activation of the drug, leading to a reduction in Giardia susceptibility. This prompted us to evaluate the gene sequences of these proteins in strains of G. duodenalis resistant to MTZ in vitro. Unexpected, we did not find any nucleotide change in these analysed genes.
Analysis of transcriptome and proteome of resistant strains of G. duodenalis have shown great variability in gene expression and protein abundance of Fd and PFOR. (17,19) However, some studies have detected a decrease in the PFOR enzyme activity (10,26) or the Fd activity in MTZ-resistant G. duodenalis strains. (27) We extended previous studies that showed MTZ-resistant strains of G. duodenalis did not present any nucleotide changes in the gene region that encode PFOR catalytic site. (16) Fur-thermore, the expression of pfor mRNA was shown to be higher in the resistant strain of G. duodenalis compared to controls. (16) These findings evidence the complexity involving MTZ-G. duodenalis resistance process, which may include another mechanisms. Then, nucleotide changes only in PFOR and Fd are insufficient to elucidate the resistance of G. duodenalis to MTZ.
Like Fd and PFOR, gene expression and protein abundance of TrxR showed variable results when MTZresistant G. duodenalis strains were evaluated. (17)(18)(19) However, NR-1 was downregulated in three G. duodenalis strains resistant to MTZ, (17,19) with evidence of nonsense mutation in NR-1 transcripts in one of strains. (17) This fact demonstrates the importance of evaluating the nucleotide sequence of this gene in different in vitro MTZ-resistant G. duodenalis strains.
Indiscriminate use of antiparasitic drugs, a common practice in health care units, can select resistant strains of several microorganisms. Given the increase in therapeutic failure cases in the treatment of giardiasis with 5-nitroimidazoles, efforts must be made to elucidate which mechanisms may be responsible for the resistance of G. duodenalis to MTZ. In the present study, we demonstrated that the exposure of G. duodenalis to MTZ decreases the parasite's susceptibility to the drug; however, this decrease could not be attributed to nucleotide changes in the G. duodenalis genes linked to MTZ activation. Our results points that different mechanisms as alterations in other metabolic pathways can account for Giardia resistance to MTZ therapy.

ACKNOWLEDGEMENTS
To Dr Flavia Carneiro, for the critical review, to Prof Marlene Benchimol, who kindly provide us the cells of the G. duodenalis strain WB ATCC 50803, to the Trypanosomatid Biochemistry Laboratory-Fiocruz, for providing the MTZ used in this study. The samples were analysed by the Sequencing Service DNA Sequencing Platform-Fiocruz.