Proteomic analysis of metronidazole resistance in the human facultative pathogen Bacteroides fragilis.

The anaerobic gut bacteria and opportunistic pathogen Bacteroides fragilis can cause life-threatening infections when leaving its niche and reaching body sites outside of the gut. The antimicrobial metronidazole is a mainstay in the treatment of anaerobic infections and also highly effective against Bacteroides spp. Although resistance rates have remained low in general, metronidazole resistance does occur in B. fragilis and can favor fatal disease outcomes. Most metronidazole-resistant Bacteroides isolates harbor nim genes, commonly believed to encode for nitroreductases which deactivate metronidazole. Recent research, however, suggests that the mode of resistance mediated by Nim proteins might be more complex than anticipated because they affect the cellular metabolism, e.g., by increasing the activity of pyruvate:ferredoxin oxidoreductase (PFOR). Moreover, although nim genes confer only low-level metronidazole resistance to Bacteroides, high-level resistance can be much easier induced in the laboratory in the presence of a nim gene than without. Due to these observations, we hypothesized that nim genes might induce changes in the B. fragilis proteome and performed comparative mass-spectrometric analyses with B. fragilis 638R, either with or without the nimA gene. Further, we compared protein expression profiles in both strains after induction of high-level metronidazole resistance. Interestingly, only few proteins were repeatedly found to be differentially expressed in strain 638R with the nimA gene, one of them being the flavodiiron protein FprA, an enzyme involved in oxygen scavenging. After induction of metronidazole resistance, a far higher number of proteins were found to be differentially expressed in 638R without nimA than in 638R with nimA. In the former, factors for the import of hemin were strongly downregulated, indicating impaired iron import, whereas in the latter, the observed changes were not only less numerous but also less specific. Both resistant strains, however, displayed a reduced capability of scavenging oxygen. Susceptibility to metronidazole could be widely restored in resistant 638R without nimA by supplementing growth media with ferrous iron sulfate, but not so in resistant 638R with the nimA gene. Finally, based on the results of this study, we present a novel hypothetic model of metronidazole resistance and NimA function.

Haemin deprivation renders Bacteroides fragilis hypersusceptible to metronidazole and cancels high-level metronidazole resistance.

BACKGROUND: Infections with Bacteroides fragilis are routinely treated with metronidazole, a 5-nitroimidazole antibiotic that is active against most anaerobic microorganisms. Metronidazole has remained a reliable treatment option, but resistance does occur, including in B. fragilis. OBJECTIVES: In this study we tested whether haemin, a growth supplement for B. fragilis in vivo and in vitro, had an influence on the susceptibility of resistant B. fragilis strains to metronidazole. We further tested whether haemin-deprived B. fragilis would be more susceptible to oxygen and oxidative stress. Metronidazole has been described to cause oxidative stress, which we argued would be exacerbated in haemin-deprived B. fragilis because the bacteria harness haemin, and the iron released from it, in antioxidant enzymes such as catalase and superoxide dismutase. METHODS: Haemin was omitted from growth media and the effect on metronidazole susceptibility was monitored in susceptible and resistant B. fragilis strains. Further, haemin-deprived B. fragilis were tested for resistance to aeration and hydrogen peroxide and the capacity for the removal of oxygen. RESULTS: Omission of haemin from the growth medium rendered metronidazole-resistant B. fragilis strains, including an MDR isolate from the UK, highly susceptible to metronidazole. Haemin deprivation further rendered B. fragilis highly susceptible to oxygen, which was further exacerbated in resistant strains. B. fragilis was incapable of scavenging oxygen when haemin was omitted. CONCLUSIONS: We propose that haemin deprivation overrules resistance mechanisms by rendering B. fragilis hypersusceptible to metronidazole due to a compromised antioxidant defence. Monitoring of haemin concentrations is imperative when conducting metronidazole susceptibility testing in B. fragilis.

Impact of Antioxidant Natural Compounds on the Thyroid Gland and Implication of the Keap1/Nrf2 Signaling Pathway.

BACKGROUND: Natural compounds with potential antioxidant properties have been used in the form of food supplements or extracts with the intent to prevent or treat various diseases. Many of these compounds can activate the cytoprotective Nrf2 pathway. Besides, some of them are known to impact the thyroid gland, often with potential side-effects, but in other instances, with potential utility in the treatment of thyroid disorders. OBJECTIVE: In view of recent data regarding the multiple roles of Nrf2 in the thyroid, this review summarizes the current bibliography on natural compounds that can have an effect on thyroid gland physiology and pathophysiology, and it discusses the potential implication of the Nrf2 system in the respective mechanisms. METHODS & RESULTS: Literature searches for articles from 1950 to 2018 were performed in PubMed and Google Scholar using relevant keywords about phytochemicals, Nrf2 and thyroid. Natural substances were categorized into phenolic compounds, sulfur-containing compounds, quinones, terpenoids, or under the general category of plant extracts. For individual compounds in each category, respective data were summarized, as derived from in vitro (cell lines), preclinical (animal models) and clinical studies. The main emerging themes were as follows: phenolic compounds often showed potential to affect the production of thyroid hormones; sulfur-containing compounds impacted the pathogenesis of goiter and the proliferation of thyroid cancer cells; while quinones and terpenoids modified Nrf2 signaling in thyroid cell lines. CONCLUSION: Natural compounds that modify the activity of the Nrf2 pathway should be evaluated carefully, not only for their potential to be used as therapeutic agents for thyroid disorders, but also for their thyroidal safety when used for the prevention and treatment of non-thyroidal diseases.