Roles of efflux pumps and nitroreductases in metronidazole-resistant Trichomonas vaginalis

Abstract

Trichomonas vaginalis infections significantly impact public health and are associated with increased likelihood of HIV infection, prostate cancer, and pregnancy complications. Current treatment relies almost exclusively on 5-nitroimidazoles, particularly metronidazole, raising concerns about drug resistance and treatment efficacy. This study is aimed at evaluating the effectiveness of metronidazole and tinidazole on metronidazole-resistant strains of T. vaginalis and at determining whether efflux pump inhibitors could reverse metronidazole resistance. Additionally, the roles of nitroreductases in metronidazole resistance were also studied. Metronidazole and tinidazole were tested on both metronidazole-sensitive and -resistant T. vaginalis strains. A checkerboard assay was conducted to assess the potential synergy between metronidazole or tinidazole and efflux pump inhibitors. Nitroreductase activity and ferric iron reduction assays were employed to study the functions of nitroreductases. Tinidazole demonstrated better effectiveness against metronidazole-resistant strains compared to metronidazole, with lower minimal lethal concentration levels. However, the tested efflux pump inhibitors did not significantly enhance the efficacy of metronidazole or tinidazole. Pyrimethamine showed some activity but did not improve the efficacy of the 5-nitroimidazoles in combination. Investigations into the role of nitroreductases and other enzymes in metronidazole resistance revealed no clear downregulation trend in resistant strains. Notably, nitroreductase 8 was capable of reducing ferric iron. While tinidazole remains a viable alternative for treating metronidazole-resistant T. vaginalis, efflux pump inhibitors do not effectively reverse resistance. The identification of nitroreductase's 8 iron-reducing activity suggests its involvement in metronidazole resistance mechanisms. This finding highlights the need for continued research to develop new treatment strategies and improve the management of trichomoniasis, ultimately reducing its public health burden.

Keywords: Trichomonas vaginalis; Antimicrobial resistance; Efflux pump inhibitors; Iron reduction; Metronidazole; Nitroreductase.

Fig. 1

Agarose gel electrophoresis (1%) of RT-PCR products of Ntrs, Ntr-like proteins, and OxRs from metronidazole-sensitive strains C1 (A), G3 (B), and JH31A (C)

Fig. 2

Agarose gel electrophoresis (1%) of RT-PCR products of Ntrs, Ntr-like proteins, and OxRs from metronidazole-resistant strains B7269 (A), CDC085 (B), and highly metronidazole-resistant C1 strain (C)

Fig. 3

Calibration curve of known concentrations of Fe₂SO₄ (0 – 60 µM) in presence of bipyridyl measured at OD₅₂₂ (A). Ferric iron reduction activity of recTvNtr8 determined through the quantification of complex formation of Fe²⁺ with bipyridyl (B). The absorption values from measurements obtained from recombinant protein and 100 or 500 µM FeCl₃ were matched against the calibration curve and presented as average values with error bars indicating standard deviation (SD ±). All measurements were performed in triplicates