The effects of identical nim gene-insertion sequence combinations on the expression of the nim genes and metronidazole resistance in Bacteroides fragilis strains.

In this study we examined whether the same nim gene-insertion sequence (IS) element combinations give rise to the same expression levels as they harbor shared IS element-borne promoters. From our quantitative analysis, we found that the expressions of the nimB and nimE genes with their cognate IS elements were similar, but the metronidazole resistance of these strains were more diverse.

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.

An unusual thioredoxin system in the facultative parasite Acanthamoeba castellanii.

The free-living amoeba Acanthamoeba castellanii occurs worldwide in soil and water and feeds on bacteria and other microorganisms. It is, however, also a facultative parasite and can cause serious infections in humans. The annotated genome of A. castellanii (strain Neff) suggests the presence of two different thioredoxin reductases (TrxR), of which one is of the small bacterial type and the other of the large vertebrate type. This combination is highly unusual. Similar to vertebrate TrxRases, the gene coding for the large TrxR in A. castellanii contains a UGA stop codon at the C-terminal active site, suggesting the presence of selenocysteine. We characterized the thioredoxin system in A. castellanii in conjunction with glutathione reductase (GR), to obtain a more complete understanding of the redox system in A. castellanii and the roles of its components in the response to oxidative stress. Both TrxRases localize to the cytoplasm, whereas GR localizes to the cytoplasm and the large organelle fraction. We could only identify one thioredoxin (Trx-1) to be indeed reduced by one of the TrxRases, i.e., by the small TrxR. This thioredoxin, in turn, could reduce one of the two peroxiredoxins tested and also methionine sulfoxide reductase A (MsrA). Upon exposure to hydrogen peroxide and diamide, only the small TrxR was upregulated in expression at the mRNA and protein levels, but not the large TrxR. Our results show that the small TrxR is involved in the A. castellanii's response to oxidative stress. The role of the large TrxR, however, remains elusive.

Characterization of the components of the thioredoxin system in Bacteroides fragilis and evaluation of its activity during oxidative stress.

OBJECTIVES: Bacteroides fragilis has a pronounced ability to survive prolonged exposure to atmospheric oxygen. The major objective of this study was to biochemically characterize the components of the thioredoxin system in B. fragilis. The nitroreductase activity of TrxR was also assayed. METHODS: Components of the thioredoxin system were expressed in E. coli and used in a disulfide reductase activity assay. Activity of TrxR was measured with purified recombinant enzyme or with cell extracts after or without exposure to oxygen or hydrogen peroxide, respectively. RESULTS: Of all six thioredoxins tested, only thioredoxins A, D, and F were reduced by recombinant TrxR and natural TrxR present in B. fragilis cell extracts. Exposure to oxygen and hydrogen peroxide increased the activity of TrxR. Further, B. fragilis TrxR acts as a nitroreductase with furazolidone or 1-Chloro-2,4-dinitrobenzene as substrates but cannot reduce metronidazole. CONCLUSION: TrxR shows an increase in activity under the conditions of oxidative stress and exerts nitroreductase activity.

Initial expression levels of nimA are decisive for protection against metronidazole in Bacteroides fragilis.

OBJECTIVES: In the genus Bacteroides, the nim genes are resistance determinants for metronidazole, a nitroimidazole drug widely used against anaerobic pathogens. The Nim proteins are considered to act as nitroreductases. However, data from several studies suggest that the expression levels of Nim do not increase with increasing resistance which is conflicting with this notion. The impact of Nim protein levels on low-level metronidazole resistance, however, representing the early stage of induced resistance in the laboratory, has not been assessed as yet. METHODS: The nimA gene was cloned into two different plasmids and introduced into B. fragilis strain 638R. Expression levels of nimA mRNA were measured by RT-qPCR and compared to those in strain 638R harbouring plasmid pI417, the original clinical plasmid harbouring IS element IS1168 with the nimA gene. Further, metronidazole susceptibility was assessed by Etest and the activity of pyruvate:ferredoxin oxidoreductase (PFOR) was measured in all strains after induction of high-level metronidazole resistance. RESULTS: The level of protection against metronidazole by nimA correleated with the level of expression of nimA mRNA. Further, the activity of PFOR in highly-resistant B. fragilis 638R was only preserved when expression levels of nimA were high. CONCLUSIONS: Although the development of high-level metronidazole resistance in B. fragilis strains with a nimA gene is not caused by an increase of nimA expression as compared to the less resistant parent strains, nimA expression levels might be of decisive importance in the early stage of resistance development. This has potential implications for metronidazole resistance in clinical isolates.

A powerful qPCR-high resolution melting assay with taqman probe in plasmodium species differentiation.

BACKGROUND: The use of highly sensitive molecular tools in malaria diagnosis is currently largely restricted to research and epidemiological settings, but will ultimately be essential during elimination and potentially eradication. Accurate diagnosis and differentiation down to species levels, including the two Plasmodium ovale species and zoonotic variants of the disease, will be important for the understanding of changing epidemiological patterns of the disease. METHODS: A qPCR-high resolution melting (HRM) method was to detect and differentiate all human Plasmodium species with one forward and one reverse primer set. The HRM detection method was further refined using a hydrolysis probe to specifically discriminate Plasmodium falciparum. RESULTS: Out of the 113 samples tested with the developed HRM-qPCR- P. falciparum probe assay, 96 (85.0 %) single infections, 12 (10.6 %) mixed infections, and 5 (4.4 %) were Plasmodium negative. The results were concordant with those of the nested PCR at 98.2 %. The assay limit of detection was varied from 21.47 to 46.43 copies /µl, equivalent to 1-2.11 parasites/µl. All P. falciparum infections were confirmed with the associated Taqman probe. CONCLUSIONS: Although the dependence on qPCR currently limits its deployment in resource-limited environments, this assay is highly sensitive and specific, easy to perform and convenient for Plasmodium mono-infection and may provide a novel tool for rapid and accurate malaria diagnosis also in epidemiological studies.

Molecular characterization of metronidazole resistant Bacteroides strains from Kuwait.

Eleven metronidazole resistant Bacteroides and one newly classified Phocaeicola dorei strain from Kuwait were investigated for their resistance mechanisms and the emergence of their resistant plasmids. All but one strain harbored nimE genes on differently sized plasmids. Of the 11 nimE genes, 9 were preceded by full copies of the prototype ISBf6 insertion sequence element, one carried a truncated ISBf6 and one was activated by an additional copy of IS612B. Nucleotide sequencing results showed that the nimE ISBf6 distances were constant and all five different plasmids shared a common region, suggesting that (i) the nimE-ISBf6 configuration was inserted into an undisclosed common genetic element, (ii) over time, this common element was mutated by insertions and deletions, spreading the resultant plasmids. Of the 10 B. fragilis strains in this collection, 6 were also cfiA-positive, one with full imipenem resistance, indicating a tendency for multidrug resistance (MDR) among such isolates. The significant number of metronidazole resistant Bacteroides spp. and P. dorei strains with the MDR phenotype warns of difficulties in treatment and suggests promoting adherence to antibiotic stewardship recommendations in Kuwait.

Identification of the NADH-oxidase gene in Trichomonas vaginalis.

The microaerophilic human parasite Trichomonas vaginalis causes infections in the urogenital tract and is one of the most often sexually transmitted pathogens worldwide. Due to its anaerobic metabolism, it has to quickly remove intracellular oxygen in order to avoid deactivation of essential metabolic enzymes such as oxygen-sensitive pyruvate:ferredoxin oxidoreductase (PFOR). Two major enzyme activities which are responsible for the removal, i.e. reduction, of molecular oxygen have been identified in T. vaginalis flavin reductase, formerly designated NADPH oxidase, which indirectly reduces oxygen to hydrogen peroxide via flavin mononucleotide (FMN), and NADH oxidase which reduces oxygen to water. Flavin reductase has been identified and characterized at the gene level as well as enzymatically, but NADH oxidase has so far only been characterized enzymatically with enzyme isolated from T. vaginalis cell extracts. In this study, we identified NADH oxidase by mass spectrometry after isolation of the enzyme from gel bands positively staining for NADH oxidase activity. In strain C1 (ATCC 30001) which is known to lack NADH oxidase activity completely, the NADH oxidase gene has a deletion at position 1540 of the open reading frame leading to a frame shift and, as a consequence, to premature termination of the encoded polypeptide.

A review on metronidazole: an old warhorse in antimicrobial chemotherapy.

The 5-nitroimidazole drug metronidazole has remained the drug of choice in the treatment of anaerobic infections, parasitic as well as bacterial, ever since its development in 1959. In contrast to most other antimicrobials, it has a pleiotropic mode of action and reacts with a large number of molecules. Importantly, metronidazole, which is strictly speaking a prodrug, needs to be reduced at its nitro group in order to become toxic. Reduction of metronidazole, however, only takes place under very low concentrations of oxygen, explaining why metronidazole is exclusively toxic to microaerophilic and anaerobic microorganisms. In general, resistance rates amongst the pathogens treated with metronidazole have remained low until the present day. Nevertheless, metronidazole resistance does occur, and for the treatment of some pathogens, especially Helicobacter pylori, metronidazole has become almost useless in some parts of the world. This review will give an account on the current status of research on metronidazole's mode of action, metronidazole resistance in eukaryotes and prokaryotes, and on other 5-nitroimidazoles in use.

Trichomonas vaginalis flavin reductase 1 and its role in metronidazole resistance.

The enzyme flavin reductase 1 (FR1) from Trichomonas vaginalis, formerly known as NADPH oxidase, was isolated and identified. Flavin reductase is part of the antioxidative defence in T. vaginalis and indirectly reduces molecular oxygen to hydrogen peroxide via free flavins. Importantly, a reduced or absent flavin reductase activity has been reported in metronidazole-resistant T. vaginalis, resulting in elevated intracellular oxygen levels and futile cycling of metronidazole. Interestingly, FR1 has no close homologue in any other sequenced genome, but seven full-length and three truncated isoforms exist in the T. vaginalis genome. However, out of these, only FR1 has an affinity for flavins, i.e. FMN, FAD and riboflavin, which is high enough to be of physiological relevance. Although there are no relevant changes in the gene sequence or any alterations of the predicted FR1-mRNA structure in any of the strains studied, FR1 is not expressed in highly metronidazole-resistant strains. Transfection of a metronidazole-resistant clinical isolate (B7268), which does not express any detectable amounts of FR, with a plasmid bearing a functional FR1 gene nearly completely restored metronidazole sensitivity. Our results indicate that FR1 has a significant role in the emergence of metronidazole resistance in T. vaginalis.

A study on Nim expression in Bacteroides fragilis.

Members of the genus Bacteroides, mainly Bacteroides fragilis, can cause severe disease in man, especially after intestinal perforation in the course of abdominal surgery. Treatment is based on a small number of antibiotics, including metronidazole, which has proved to be highly reliable throughout the last 40 to 50 years. Nevertheless, metronidazole resistance does occur in Bacteroides and has been mainly attributed to Nim proteins, a class of proteins with a suggested nitroreductase function. Despite the potentially high importance of Nim proteins for human health, information on the expression of nim genes in B. fragilis is still lacking. It was the aim of this study to demonstrate expression of nim genes in B. fragilis at the protein level and, furthermore, to correlate Nim levels with the magnitude of metronidazole resistance. By the application of 2D gel electrophoresis, Nim proteins could be readily identified in nim-positive strains, but their levels were not elevated to a relevant extent after induction of resistance with high doses of metronidazole. Thus, the data herein do not provide evidence for Nim proteins acting as nitroreductases using metronidazole as a substrate, because no correlation between Nim levels and levels of metronidazole resistance could be observed. Furthermore, no evidence was found that Nim proteins protect B. fragilis from metronidazole by sequestering the activated antibiotic.

Entamoeba histolytica: identification of thioredoxin-targeted proteins and analysis of serine acetyltransferase-1 as a prototype example.

Entamoeba histolytica, the causative agent of amoebiasis, possesses the dithiol-containing redox proteins Trx (thioredoxin) and TrxR (Trx reductase). Both proteins were found to be covalently modified and inactivated by metronidazole, a 5-nitroimidazole drug that is commonly used to treat infections with microaerophilic protozoan parasites in humans. Currently, very little is known about enzymes and other proteins participating in the Trx-dependent redox network of the parasite that could be indirectly affected by metronidazole treatment. On the basis of the disulfide/dithiol-exchange mechanism we constructed an active-site mutant of Trx, capable of binding interacting proteins as a stable mixed disulfide intermediate to screen the target proteome of Trx in E. histolytica. By applying Trx affinity chromatography, two-dimensional gel electrophoresis and MS, peroxiredoxin and 15 further potentially redox-regulated proteins were identified. Among them, EhSat1 (E. histolytica serine acetyltransferase-1), an enzyme involved in the L-cysteine biosynthetic pathway, was selected for detailed analysis. Binding of Trx to EhSat1 was verified by Far-Western blot analysis. Trx was able to restore the activity of the oxidatively damaged EhSat1 suggesting that the TrxR/Trx system protects sensitive proteins against oxidative stress in E. histolytica. Furthermore, the activity of peroxiredoxin, which is dependent on a functioning TrxR/Trx system, was strongly reduced in metronidazole-treated parasites.

Protein profiles and immunoreactivities of Acanthamoeba morphological groups and genotypes.

Acanthamoeba is a free-living protozoan found in a wide variety of habitats. A classification of Acanthamoeba into currently eighteen genotypes (T1-T18) has been established, however, data on differences between genotypes on the protein level are scarce. The aim of this study was to compare protein and immunoreactivity profiles of Acanthamoeba genotypes. Thirteen strains, both clinical and non-clinical, from genotypes T4, T5, T6, T7, T9, T11 and T12, representing three morphological groups, were investigated for their protein profiles and IgG, IgM and IgA immunoreactivities. It was shown that protein and immunoreactivity profiles of Acanthamoeba genotypes T4, T5, T6, T7, T9, T11 and T12 are clearly distinct from each other, but the banding patterns correlate to the morphological groups. Normal human sera revealed anti-Acanthamoeba antibodies against isolates of all investigated genotypes, interestingly, however only very weak IgM and virtually no IgA immunoreactivity with T7 and T9, both representing morphological group I. The strongest IgG, IgM and IgA immunoreactivities were observed for genotypes T4, T5 and T6. Differences of both, protein and immunological patterns, between cytopathic and non-cytopathic strains, particularly within genotype T4, were not at the level of banding patterns, but rather in expression levels.

Comparative proteomic analysis of metronidazole-sensitive and resistant Trichomonas vaginalis suggests a novel mode of metronidazole action and resistance.

The microaerophilic parasite Trichomonas vaginalis occurs worldwide and causes inflammation of the urogenital tract, especially in women. With 156 million infections annually, trichomoniasis is the most prevalent non-viral sexually transmitted disease. Trichomoniasis is treated with 5-nitroimidazoles, especially metronidazole, which are prodrugs that have to be reduced at their nitro group to be activated. Resistance rates to metronidazole have remained comparably low, but they can be higher in certain areas leading to an increase of refractory cases. Metronidazole resistance in T. vaginalis can develop in vivo in clinical isolates, or it can be induced in the laboratory. Both types of resistance share certain characteristics but differ with regard to the dependence of ambient oxygen to become manifest. Although several candidate factors for metronidazole resistance have been described in the past, e.g. pyruvate:ferredoxin oxidoreductase and ferredoxin or thioredoxin reductase, open questions regarding their role in resistance have remained. In order to address these questions, we performed a proteomic study with metronidazole-sensitive and -resistant laboratory strains, as well as with clinical strains, in order to identify factors causative for resistance. The list of proteins consistently associated with resistance was surprisingly short. Resistant laboratory and clinical strains only shared the downregulation of flavin reductase 1 (FR1), an enzyme previously identified to be involved in resistance. Originally, FR1 was believed to be an oxygen scavenging enzyme, but here we identified it as a ferric iron reductase which produces ferrous iron. Based on this finding and on further experimental evidence as presented herein, we propose a novel mechanism of metronidazole activation which is based on ferrous iron binding to proteins, thereby rendering them susceptible to complex formation with metronidazole. Upon resolution of iron-protein-metronidazole complexes, metronidazole radicals are formed which quickly react with thiols or proteins in the direct vicinity, leading to breaks in the peptide backbone.

A Comparison of Bottom-Up Proteomic Sample Preparation Methods for the Human Parasite Trichomonas vaginalis.

Bottom-up proteomic approaches depend on the efficient digestion of proteins into peptides for mass spectrometric analysis. Sample preparation strategies, based on magnetic beads, filter-aided systems, or in-solution digests, are commonly used for proteomic analysis. Time-intensive methods like filter-aided sample preparation (FASP) have led to the development of new, more time-efficient filter-based strategies like suspension trappings (S-Traps) or magnetic bead-based strategies like SP3. S-Traps have been reported as an alternative proteomic sample preparation method as they allow for high sodium dodecyl sulfate (SDS) concentrations to be present in the sample. In this study, we compare the efficiency of different protocols for FASP, SP3, and S-Trap-based digestion of proteins after extraction from Trichomonas vaginalis. Overall, we found a high number of protein IDs for all tested methods and a high degree of reproducibility within each method type. However, FASP with a 3 kDa cutoff filter unit outperformed the other methods analyzed, referring to the number of protein IDs. This is the first work providing the direct comparison of four different bottom-up proteomic approaches regarding the most efficient proteomic sample preparation protocol for the human parasite T. vaginalis.

Proteomics-Based RT-qPCR and Functional Analysis of 18 Genes in Metronidazole Resistance of Bacteroides fragilis.

Previously, we reported that metronidazole MICs are not dependent on the expression levels of nim genes in B. fragilis strains and we compared the proteomes of metronidazole-resistant laboratory B. fragilis strains to those of their susceptible parent strains. Here, we used RT-qPCR to correlate the expression levels of 18 candidate genes in a panel of selected, clinical nim gene-positive and -negative B. fragilis strains to their metronidazole MICs. Metronidazole MICs were correlated with the expression of certain tested genes. Specifically, lactate dehydrogenase expression correlated positively, whereas cytochrome fumarate reductase/succinate dehydrogenase, malate dehydrogenase, phosphoglycerate kinase redox and gat (GCN5-like acetyltransferase), and relA (stringent response) regulatory gene expressions correlated negatively with metronidazole MICs. This result provides evidence for the involvement of carbohydrate catabolic enzymes in metronidazole resistance in B. fragilis. This result was supported by direct substrate utilization tests. However, the exact roles of these genes/proteins should be determined in deletion-complementation tests. Moreover, the exact redox cofactor(s) participating in metronidazole activation need to be identified.

Evaluating the amoeba thioredoxin reductase selenoprotein as potential drug target for treatment of Acanthamoeba infections.

The genus Acanthamoeba comprises facultative pathogens, causing Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). In both diseases, treatment options are limited, and drug development is challenging. This study aimed to investigate the role of the large thioredoxin reductase selenoprotein of Acanthamoeba (AcTrxR-L) as a potential drug target assessing the effects of the thioredoxin reductase inhibitors auranofin, TRi-1, and TRi-2 on AcTrxR-L activity and on the viability of Acanthamoeba trophozoites. Recombinant expression and purification of AcTrxR-L as a selenoprotein allowed assessments of its enzymatic activity, with reduction of various substrates, including different thioredoxin isoforms. Auranofin demonstrated potent inhibition towards AcTrxR-L, followed by TRi-1, and TRi-2 exhibiting lower effectiveness. The inhibitors showed variable activity against trophozoites in culture, with TRi-1 and TRi-2 resulting in strongly impaired trophozoite viability. Cytotoxicity tests with human corneal epithelial cells revealed lower susceptibility to all compounds compared to Acanthamoeba trophozoites, underscoring their potential as future amoebicidal agents. Altogether, this study highlights the druggability of AcTrxR-L and suggests it to be a promising drug target for the treatment of Acanthamoeba infections. Further research is warranted to elucidate the role of AcTrxR-L in Acanthamoeba pathogenesis and to develop effective therapeutic strategies targeting this redox enzyme.

A reprofiled drug, auranofin, is effective against metronidazole-resistant Giardia lamblia.

Giardiasis is one of the most common causes of diarrheal disease worldwide. Treatment is primarily with 5-nitro antimicrobials, particularly metronidazole. Resistance to metronidazole has been described, and treatment failures can occur in up to 20% of cases, making development of alternative antigiardials an important goal. To this end, we have screened a chemical library of 746 approved human drugs and 164 additional bioactive compounds for activity against Giardia lamblia. We identified 56 compounds that caused significant inhibition of G. lamblia growth and attachment. Of these, 15 were previously reported to have antigiardial activity, 20 were bioactive but not approved for human use, and 21 were drugs approved for human use for other indications. One notable compound of the last group was the antirheumatic drug auranofin. Further testing revealed that auranofin was active in the low (4 to 6)-micromolar range against a range of divergent G. lamblia isolates representing both human-pathogenic assemblages A and B. Most importantly, auranofin was active against multiple metronidazole-resistant strains. Mechanistically, auranofin blocked the activity of giardial thioredoxin oxidoreductase, a critical enzyme involved in maintaining normal protein function and combating oxidative damage, suggesting that this inhibition contributes to the antigiardial activity. Furthermore, auranofin was efficacious in vivo, as it eradicated infection with different G. lamblia isolates in different rodent models. These results indicate that the approved human drug auranofin could be developed as a novel agent in the armamentarium of antigiardial drugs, particularly against metronidazole-resistant strains.

Unexpected properties of NADP-dependent secondary alcohol dehydrogenase (ADH-1) in Trichomonas vaginalis and other microaerophilic parasites.

Our previous observation that NADP-dependent secondary alcohol dehydrogenase (ADH-1) is down-regulated in metronidazole-resistant Trichomonas vaginalis isolates prompted us to further characterise the enzyme. In addition to its canonical enzyme activity as a secondary alcohol dehydrogenase, a pronounced, so far unknown, background NADPH-oxidising activity in absence of any added substrate was observed when the recombinant enzyme or T. vaginalis extract were used. This activity was strongly enhanced at low oxygen concentrations. Unexpectedly, all functions of ADH-1 were efficiently inhibited by coenzyme A which is a cofactor of a number of key enzymes in T. vaginalis metabolism, i.e. pyruvate:ferredoxin oxidoreductase (PFOR). These observations could be extended to Entamoeba histolytica and Tritrichomonas foetus, both of which have a homologue of ADH-1, but not to Giardia lamblia which lacks an NADP-dependent secondary alcohol dehydrogenase. Although we could not identify the substrate of the observed background activity, we propose that ADH-1 functions as a major sink for NADPH in microaerophilic parasites at low oxygen tension.

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.