Differential gene expression in a Bacteroides fragilis metronidazole-resistant mutant

Claudio Galuppo Diniz1,2, Luiz M. Farias2,*, Maria Auxiliadora R. Carvalho2, Edson R. Rocha1 and C. Jeffrey Smith1

1 Department of Microbiology and Immunology, The Brody School of Medicine, East Carolina University, Greenville, NC, USA; 2 Departamento de Microbiologia, ICB-UFMG, Belo Horizonte, MG, Brazil

Received 18 January 2004; returned 24 February 2004; revised 29 March 2004; accepted 4 April 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives: The current work focused on molecular changes in a spontaneous Bacteroides fragilismutant selected by low concentrations of metronidazole as an adaptive response to the drug.

Methods: A metronidazole-resistant strain derived from B. fragilis ATCC 25285 was selected by passage in the presence of drug using 0–4 mg/L gradient plates. Using a combination of proteomics for identification of differentially expressed proteins by two-dimensional electrophoresis and selected mutational analyses by single cross-over insertion and an allelic exchange, we have identified genes involved in the adaptive response to metronidazole.

Results: There are significant changes in the protein profiles of resistant strains. These changes appeared to affect a wide range of metabolic proteins including lactate dehydrogenase (up-regulated) and flavodoxin (down-regulated), which may be involved in electron transfer reactions. Also, the enzymic activity of the pyruvate–ferredoxin oxidoreductase (PorA) complex was impaired. Mutant strains lacking the genes for flavodoxin and PorA were less susceptible to metronidazole than the sensitive parent, and a double flavodoxin/PorA mutant had even less susceptibility but none of the mutants were as resistant as the spontaneous metronidazole-resistant strain.

Conclusions: Overall, the data indicated that there were global changes in the regulation of the physiology of the metronidazole-resistant strain. In addition, flavodoxin was identified as an important contributor to metronidazole sensitivity in B. fragilis.

Keywords: proteomics , anaerobes , drug resistance


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Species from the Bacteroides fragilis group are numerically predominant in the colon and they are the most clinically relevant anaerobes both in human and veterinary medicine.13 Treatment of B. fragilis infections requires antimicrobial treatment often in conjunction with surgical intervention but success is often compromised by the presence of antibiotic-resistant strains. Despite the widespread occurrence of antibiotic resistance in Bacteroides, metronidazole is still the drug of choice to treat serious infections in which the B. fragilis group is involved, but emerging resistance is being reported.4,5

The mechanism of action of 5-nitroimidazole drugs such as metronidazole is not entirely clear, but it is accepted that the nitro group is chemically reduced in the cell forming a nitro-radical anion intermediate which can damage DNA and other cell components.6,7 Recent reports suggest that metronidazole-dependent DNA breakage does occur in susceptible microbial cells.810 These findings were consistent with observations reporting the occurrence of mutations in metronidazole-resistant cells after drug exposure.11,12

Metronidazole resistance is generally associated with the inability to reduce the antimicrobial agent to its active form or with a decrease in intracellular accumulation.7 Available data indicate that enzymes which play a role in anaerobic electron transfer reactions might be responsible for the diminished ability to reduce the drug. The pyruvate–oxidoreductase (POR) complex is one key enzyme found to decrease in metronidazole-resistant strains.13 The inhibition of nitroimidazole activity may also be caused by aminothiol radical scavengers and radioprotectors, both of which are normally present in the cell.1416

In addition to the resistance mechanisms discussed above, some metronidazole-resistant Bacteroides strains have been shown to possess specific nitroimidazole resistance genes, the nim genes. The five reported nim genes (nimA, nimB, nimC, nimD and nimE) are carried on plasmids or in the chromosome although epidemiological studies have shown that 73% of resistant isolates possess the chromosomal genes.17 The nim genes appear to encode 5-nitroimidazole reductases, which serve to reduce the drugs to amine derivatives, which are not bactericidal.13 Thus this appears to be a unique resistance mechanism.

Interaction between metronidazole and the B. fragilis group leads to several changes that seem to go beyond the resistance phenomenon. Previous studies showed that metronidazole could induce phenotypic changes in B. fragilis strains with consequences for their biological and pathogenic properties.518 In fact, since the early 1940s, many studies have observed morphological changes in various microorganisms in the presence of different antimicrobial agents including metronidazole. These alterations are related to differences in the physiological patterns of these microorganisms.19

Metronidazole is used in different therapeutic situations, and often is the drug of choice to treat Bacteroides infection. Our objective in this study was to determine cellular alterations in B. fragilis selected by growth on low concentrations of drug. The characterization of such differences could help in the understanding of the resistance phenomenon and the pathogenic changes induced by metronidazole in this microbial group.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Bacterial strains

B. fragilis strain ATCC 25285 has a metronidazole MIC of 1 mg/L and is designated as the wild-type (WT) strain. A derivative of ATCC 25285 was selected by passage in the presence of drug using 0–4 mg/L gradient plates, as already described,20 and is designated as the MTZR strain. The MTZR strain has an MIC of 128 mg/L. A B. fragilis strain 638R, metronidazole-susceptible (MIC 0.5 mg/L), was the standard host strain for genetic manipulations.21 The MICs of metronidazole were determined when needed for the bacterial strains in this study, by the agar dilution technique recommended by the NCCLS, according to the methodology already established.1 Bacteroides strains were grown anaerobically in brain heart infusion broth supplemented with haemin, and cysteine (BHIS) for routine cultures and genetic procedures.22 Cysteine was omitted in some experiments where indicated, and 4 mg/L of metronidazole, 20 mg/L of rifampicin, 100 mg/L of gentamicin, 10 mg/L of erythromycin and 5 mg/L of tetracycline were added to the medium when required. Escherichia coli strains were grown in Luria-Bertani (LB) medium with 100 mg/L of ampicillin, 50 mg/L of spectinomycin, 5 mg/L of tetracycline and 50 mg/L of 5-bromo-4-chloro-3-indolyl ß-D-galactopyranoside (X-Gal) as indicated in the text.23

Identification of differentially expressed proteins by two-dimensional electrophoresis

According to the bacterial growth curve that was previously obtained for both strains, cell pellets from mid-log phase cultures of B. fragilis WT and MTZR strain (grown with metronidazole 4 mg/L) were washed in sterile PBS (NaCl 0.15 mM, Na2HPO4/NaH2PO4 50 mM, pH 7.2) and brought to a concentration of approximately 108 cells/mL (OD 0.3 at 550 nm). Crude cell extracts were obtained by cell disruption in a French press (American Instrument Co., Silver Spring, MD, USA) at 14000 lb/in2 followed by centrifugation for 30 min at 4°C in a table centrifuge and protein content was measured. Aliquots of 4 mL of crude extracts were concentrated and solubilized in 100 µL of lysis buffer [urea, Triton X-100, ß-mercaptoethanol, IPG buffer 4–7, phenylmethanesulphonyl fluoride (PMSF)] and to that solution was added 300 µL of sample buffer (urea, Triton X-100, ß-mercaptoethanol, IPG buffer 4–7). The crude extracts were submitted to two-dimensional (2D) gel electrophoresis. The first dimension, isoelectric focusing, was carried out using a dry immobilized pH gradient 4.0 to 7.0 (IPG) polyacrylamide strip (Immobiline DryStrip Kit, Amersham Pharmacia Biotech, Piscataway, NY, USA) loaded with 300 µg of crude cell extract and electrophoresed for 16.5 h at 20°C, 1 mA and 5 W using a gradient programming electrophoresis power supply EPS 3500 XL (Amersham Pharmacia Biotech, Piscataway, NY, USA), as follows: 300 V, 0.01 h; 300 V, 6 h; 3500 V, 5 h and 3500 V, 5.5 h. The second dimension was carried out in a SDS–PAGE 12% according to established procedures.24 After 2D electrophoresis the gels were stained with Coomassie Blue R250 for analyses or electroblotted to a polyvinylidene fluoride (PVDF) membrane using a transblot semidry transfer cell (Bio-Rad). The PVDF membranes were stained with Coomassie Blue 0.25% in 45% methanol, destained in 45% methanol and air-dried. The spots of interest were excised and the N-terminal amino acid sequence was determined by solid-phase, Edman degradation at the Protein Sequencing Facility, University of North Carolina, Chapel Hill, NC, USA.

DNA sequence analyses

The N-amino terminal sequences of differentially regulated proteins were used to query the B. fragilis genome sequence (http://www.sanger.ac.uk/Projects/B_fragilis/). In addition, the B. fragilis database was searched for the PorA (porA) and nitroreductase (rdx) genes by homology to the E. coli and Helicobacter pylori genes, respectively. Nucleotide sequences were analysed by comparisons using University of Wisconsin Genetics Computer Group DNA sequence analysis software,25 and the GenBank. Oligonucleotide sequences (NB01 to NB10) were used to generate internal fragments of the genes by PCR to be used as specific probes for northern blots (Table 1). Chromosomal DNA isolated from the WT strain or the MTZR strain were used as templates for PCR reactions according to established procedures.23 The thermocycling conditions were set as follows: 94°C, 5 min; 30 cycles of 94°C, 15 s; 60°C, 30 s; 72°C, 2 min and final extension at 72°C, 10 min.


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Table 1. Primers used in this study

 
RNA extraction and transcriptional analysis

WT and MTZR strains were grown to early, mid and late logarithmic phase. Total RNA was isolated by hot-phenol extraction as previously described.2 RNA concentration was measured and 40 µg of each sample was electrophoresed in a 1% formaldehyde–agarose gel. To ensure RNA quality and quantity, the gels were stained with ethidium bromide and visualized on a UV transilluminator. After destaining the gels, the RNA was transferred to nylon membranes and hybridized with the specific 32P-radiolabelled probes as described previously.2

Construction of single cross-over insertion mutants

Single cross-over insertion mutants derived from B. fragilis 638R were obtained by inactivating the genes for glutamate decarboxylase (gadA), PorA (porA), nitroreductase (rdx), flavodoxin (fldA) and thioredoxin peroxidase (tpx) using methods described previously.2 In brief, internal DNA fragments of genes of interest were amplified by PCR, using oligonucleotides containing sites for restriction enzymes and B. fragilis ATCC 25285 DNA as template. The nucleotide sequences of the primer sets used for these experiments, MT-01 to MT-05, are listed in Table 1. The amplified fragments [MT-01 (700 bp), MT-02 (760 bp), MT-03 (354 bp), MT-04 (443 bp), MT-05 (339 bp)] were then cloned into suicide vector pFD516, using the strain E. coli DH10B.22 The new plasmids carrying the fragments of genes of interest were mobilized from E. coliDH10B into B. fragilis 638R by triparental matings,26 and the new B. fragilis strains, exconjugants, carrying the single mutations, were selected anaerobically on BHIS agar plates containing 20 mg of rifampicin per L, 100 mg of gentamicin per L, and 10 mg of erythromycin per L. All mutant strains were verified by Southern hybridization analysis.

Construction of double mutants

Two double mutant strains derived from B. fragilis 638R were obtained by inactivating the genes which encode PorA (porA) and nitroreductase (rdx) or by inactivating the genes which encode PorA (porA) and flavodoxin (fldA). The first mutation was achieved using an allelic exchange system gene transfer to delete the porA gene into the chromosome of B. fragilis 638R as already described.2 Briefly, two DNA fragments of 1 kb corresponding to the 5' and 3' of the porA gene were amplified by PCR as described above using two sets of primers containing sites for different restriction enzymes to ensure their orientation. The nucleotide sequences of the primer sets used for these experiments, DM-01 and DM-02, are listed in Table 1. The amplified fragments were then cloned into suicide vector pFD516 using E. coli DH10B. After the purification of this construct a 2.6 kb blunt-ended SstI DNA fragment containing tet(Q) was inserted between the two porA fragments. The new construct carrying the two DNA fragments corresponding to the 5' and 3' of the porA gene separated by tet(Q) was mobilized from E. coli DH10B into B. fragilis 638R by triparental matings. The exconjugants were selected anaerobically on BHIS agar plates containing 20 mg of rifampicin per L, 100 mg of gentamicin per L, and 5 mg of tetracycline per L. The second mutation was obtained using the single cross-over insertion system described above by mobilizing the suicide vector pFD516 containing internal fragments of the genes rdx and fldA previously obtained, as described for single mutants, from E. coliDH10B into the B. fragilis 638R, {Delta}porA::tetQ. After this second transformation, the new B. fragilis strains exconjugants, carrying double mutations, were selected anaerobically on BHIS agar plates containing 20 mg of rifampicin per L, 100 mg of gentamicin per L, and 10 mg of erythromycin per L. In this system, the first mutation was selected by tetracycline due to the incorporation of tet(Q) and the second mutation was selected by erythromycin due to erm(F), which is codified by pFD516. All mutations were verified by Southern hybridization analysis.

Sequencing of porA

Seven overlapping DNA fragments were PCR amplified from WT and MTZR strain chromosomal DNA, using specific porA primers (SEQ01 to SEQ07; Table 1) derived from the B. fragilis ATCC 25285 genome sequence. The amplified fragments were then cloned into the pGEM-T vector for further nucleotide sequencing. Automated nucleotide sequencing was carried out on double-stranded DNA templates (Molecular Biology Resource Facility, University of Tennessee, Knoxville, TN, USA). Additional oligonucleotide primers were designed based on available sequence information to extend and confirm the existing sequence.

Enzyme assays

POR, and lactate dehydrogenase activity assays were carried out in crude extracts from WT and MTZR strains grown until mid-log-phase. Cell pellets were washed in PBS supplemented with 1 mM dithiothreitol (DTT) and crude extracts obtained as described above. Enzyme assays for POR, and lactate dehydrogenase were carried out according to established procedures.19,27 The data were compared by Student's t-test for individual analyses between mean values. Statistical analysis was carried out using the EPISTAT software (T.L. Gustafson, Round Rock, TX, USA) with the level of significance set at P<0.05.

Fermentation end-product analysis

Culture supernatants were collected from mid-log cultures and then filter sterilized. The samples were analysed for acid end-products at the National Center for Agricultural Utilization Research, USDA, Peoria, IL, USA. The VFA or methyl derivatives of non-volatile acids were quantified using a Hewlett Packard 6890 gas chromatograph with a flame ionization detector. Compounds were separated on a 30 m x 0.32 mm diameter (0.5 µm film thickness) Innowax PEG column using He carrier gas. Sterile BHIS was used as blank control. The results were corrected against blank samples. The data were compared by Student's t-test for individual analyses between mean values. Statistical analysis was carried out using the EPISTAT software (T.L. Gustafson, Round Rock, TX, USA) with the level of significance set at P<0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Comparison of protein expression patterns

The protein expression patterns were compared using 2D-PAGE with Coomassie staining to visualize the protein spots. As shown in Figure 1, there were significant differences between the two strains, relative to the parent and the MTZR strain. Both down-regulated and up-regulated peptides were observed and more than 10 differences were seen overall. The experiments were reproduced at least three times and the results shown here are typical. Only those spots that changed in every gel are marked in this figure. Protein spots that were altered more than two-fold were blotted to PVDF filters and the N-terminal amino acid sequences were determined for eight proteins. Attempts to determine the sequences of other proteins were not successful probably due to blocking at the N terminus. The genes for the eight proteins and their complete amino acid sequences were determined from the B. fragilis ATCC 25285 genome sequence database and are listed in Table 2. A wide range of different functional families were represented by the proteins but most seemed to be involved with either energy metabolism or with stress responses. Those proteins with a probable stress-related function include thioredoxin peroxidase and bacterioferritin involved in resistance to oxidative stress, glutamate decarboxylase involved in acid resistance, and peptidyl-prolyl isomerase involved in metabolism of mis-folded proteins.



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Figure 1. Electrophoretic pattern of cell-free extracts from WT parent B. fragilis ATCC 25285 (WT strain) and the metronidazole derivative strain (MTZR strain). Protein spots that were blotted to PVDF filters and the N-terminal amino acids sequences that were determined are surrounded by circles and squares. Circles indicate proteins that are down-regulated in the MTZR strain and squares indicate proteins that are up-regulated in the MTZR strain.

 

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Table 2. Differentially modulated proteins in metronidazole derivative strain (MTZR-strain)

 
Lactate dehydrogenase and flavodoxin were two proteins directly linked to energy metabolism, being up-regulated and down-regulated, respectively. Increased lactate dehydrogenase activity has been previously linked to metronidazole resistance,15,28 and this result was verified in our current study. Enzymic assays for lactate dehydrogenase activity showed 8.12 (±0.34) U/mg of protein in MTZR strain with 3.82 (±0.11) U/mg of protein in the parental strain, the results being statistically different (P<0.05). The flavodoxin may also be linked to metronidazole resistance since it is likely an electron acceptor in the POR enzyme complex which has been noted as a principal activator of metronidazole.4,28 Although we did not observe a protein spot that coincided with PorA in the 2D-PAGE gels, enzyme assays for the activity showed 259.50 (±20.68) U/mg of protein in the WT strain, but no activity in the MTZR strain. Several key fermentation products were measured in the MTZR and WT strains in order to determine whether there were differences consistent with the changes in enzyme activity. As shown in Table 3, there was a shift to higher levels of lactate production in the MTZR strain with a concomitant decrease in acetate. This is the result expected in a strain with low PorA activity and high lactate dehydrogenase activity.


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Table 3. Mean values of fermentation end-product analyses comparatively between WT and MTZR B. fragilis strains, and between parental B. fragilis 638R and PorA-deficient 638R strains

 
Transcriptional analysis of the MTZR strain

Control of transcription is one mechanism that may be used to modulate the levels of various proteins in the MTZR strain. In order to examine this possibility, mRNA levels for the genes encoding each of the modulated proteins were measured by northern hybridization. RNA was obtained from cultures at mid-logarithmic, late-logarithmic and stationary phase of growth and the results are shown in Figure 2. For the down-regulated proteins, there was generally good agreement between the northern analyses and the 2D-PAGE gels. That is, gadA, fkpA, tpx, bfr and fldA each showed a decrease in mRNA abundance during one or more of the growth phases tested. Of the three up-regulated proteins only the DKI isomerase clearly had higher mRNA levels in the MTZR strain (Figure 2). Based on similarity to other organisms, the DKI isomerase is likely involved in the metabolism of pectin. It is not clear why this might be transcriptionally up-regulated unless it is a response to the decreased energy levels that could result from a loss of PorA activity.



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Figure 2. Transcriptional analyses by northern hybridization of the genes encoding each of the modulated proteins by metronidazole and genes encoding the pyruvate–ferredoxin oxidoreductase (porA) and nitroreductase (rdx), during bacterial growth comparatively for the WT parent B. fragilis ATCC 25285 and the metronidazole derivative strain (MTZR strain).

 
The mRNA levels of the porA gene were also examined to determine whether there was a decrease that would correlate with the lack of PorA activity in the MTZR strain. As seen in Figure 2, porA was slightly down-regulated (about two-fold) in the MTZR strain but this was not sufficient to account for the lack of enzymic activity seen in the assays mentioned above. In order to gain more insight into this apparent discrepancy, the gene for porA was amplified from the MTZR strain chromosome and the nucleotide sequence was determined. There were two mutation sites in the MTZR protein that may have significant impact on PorA activity. A guanine to adenine mutation at position 3501 resulted in a premature TAG translation stop signal which would cause a truncation of the last 18 amino acids of the protein. Another site of mutations that might account for the reduced PorA activity was at positions 2597, 2600 and 2602 where three mutations resulted in the change in amino acid sequence from PAW to RVG. The PAW sequence is conserved in many POR enzymes in the NCBI database but its role in activity is not known. Taken together, these mutations are likely responsible for much of the reduced PorA activity.

Effect of targeted mutations on metronidazole resistance

In order to determine whether any of the modulated proteins in the MTZR strain were important for the resistance phenotype, a mutational approach was taken. As already known, genetic manipulation of B. fragilis ATCC 25285 is extremely difficult. Therefore, to test the effects of specific knockout mutations, we chose to use another B. fragilis strain, the 638R,21 for which the MIC of metronidazole is 0.5 mg/L. The focus in this part of the study was on the down-regulated proteins and mutations in several of the genes were constructed by insertional inactivation. Single mutations in gadA and tpx were made and verified by Southern hybridization. In neither case was there a significant effect on metronidazole susceptibility (Table 4). A different result was obtained when two genes encoding components involved with energy metabolism, porA and fldA, were inactivated. As shown in Table 4, both of these mutations resulted in a decrease in metronidazole susceptibility compared to the parent strain and the flavodoxin mutant was somewhat less susceptible than the PorA mutant. The porA mutant also was similar to the MTZR strain with respect to the production of fermentation end-products. As shown in Table 3, there was a decrease in acetate and an increase in lactate production in the porA mutant.


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Table 4. Effect of targeted mutations on metronidazole resistance in B. fragilis. MIC of metronidazole for selected single and double mutant stains

 
Neither the fldA nor the porA mutation in the strain 638R resulted in a metronidazole MIC as high as the MTZR strain. Further, both these mutations engendered different MICs and thus it seemed possible that their effect might be additive. To test this, a double mutant was constructed and as shown in Table 4, this mutant was significantly less susceptible than either of the single mutants. In fact, the double mutation resulted in the lowest susceptibility we have observed for any targeted mutation. This result suggests that multiple enzymes involved in oxidation/reduction and electron transfer reactions may be important in the activation of metronidazole. This idea prompted us to examine another oxidoreductase enzyme that has been implicated in metronidazole resistance in Helicobacter, the nitroreductase, Rdx. A gene encoding an Rdx homologue in the B. fragilis genome was identified and a mutation was engineered by insertional inactivation. As shown in Table 4, the rdx mutation resulted in a decrease in metronidazole susceptibility equal to that of the porA mutant. A double rdx and porA mutant was less susceptible to metronidazole than either single mutant.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In a previous study, it was shown that the phenotype of a metronidazole-resistant derivative of B. fragilis ATCC 25285 encompassed a broad range of traits including differences in metabolic and pathogenic properties.29 In order to determine the relationship between metronidazole resistance and the other properties, it is necessary to identify the basic genetic and physiological changes that have occurred in the MTZR strain. One approach used to study the changes was to compare the protein profiles of the MTZR strain and the parent grown under standard conditions.

It is known that antimicrobial agents influence both resident indigenous microbiota and pathogenic microorganisms when they are improperly used, incompletely absorbed after oral administration, and when they are excreted in saliva, bile or mucus.30 Even when most of the drug is systematically inactivated, residual antimicrobial agents are enough to disrupt the ecological balance in the bowel.31 It is generally thought that the mechanisms of acquired resistance result from selection of mutations that change cellular physiology or structure of a microorganism.3234 In this study, proteomics and targeted mutations were used as a tool to investigate the physiology and genetics of a metronidazole-resistant strain selected on low concentrations of the drug. Based on previous studies, the most widely accepted mechanism of resistance in anaerobic bacteria is one that prevents activation of the drug.2435 It is thought that electrons derived from the oxidative decarboxylation of pyruvate via pyruvate/ferredoxin oxidoreductase are required for reduction and activation of metronidazole, thus interference with this pathway will result in metronidazole resistance. Results from experiments reported in this study suggest an important but not an exclusive role of PorA in metronidazole reduction. The finding that the flavodoxin, FldA, was down-regulated at both the protein and transcriptional levels is consistent with the proposed mechanism of activation since FldA is thought to be an electron carrier in the PorA complex and could be used to donate electrons to metronidazole (Figures 1 and 2). There is the possibility that Bfr (which used to be known as cytochrome b1) may also be involved in pyruvate oxidation. This is taken from work that has shown that in E. coli Bfr can accept electrons from reduced flavin pyruvate oxidase and thus could be part of a similar pathway involving PorA, FldA and Bfr in B. fragilis.36,37 We also observed a complete loss of pyruvate–ferredoxin oxidoreductase activity in the MTZR strain which together with a two-fold decrease in porA transcription effectively removed this pathway from participation in metronidazole reduction. The loss of this pathway appeared to be compensated for by shunting electrons to lactate production (Figure 3). There was a clear shift in fermentation products from acetate to lactate in the MTZR strain (Table 3) and this was accompanied by an increase in the levels of the lactate dehydrogenase protein (Figure 1) and by a three-fold increase in lactate dehydrogenase activity. High activity of lactate dehydrogenase as well as high levels of lactate in metronidazole-resistant cells, have been reported by others and are thought to be a compensatory mechanism for the absence of the pyruvate–oxidoreductase complex.8,14



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Figure 3. Metabolic pathways thought to be shifted in MTZR strain. Metronidazole would be activated by pyruvate oxidation. Pyruvate–ferredoxin oxidoreductase complex (POR) catalyses oxidation of pyruvate to acetyl CoA. Electrons generated in this process are transferred to POR and metronidazole is activated when electrons flow from POR to its nitro drug, resulting in reduced metronidazole. The detection of increased levels of lactate dehydrogenase (Ldh) and lactate may suggest a shift in fermentation products from acetate to lactate in the MTZR strain.

 
Although these data indicate that metronidazole-resistant B. fragilis are minimizing electron flow through these low potential donors, this is clearly not the sole mechanism of resistance. The targeted porA, fldA and porA + fldA mutations while resulting in a decreased susceptibility did not approach the levels of metronidazole resistance displayed by the MTZR strain. We have also observed that the double porA + fldA mutant was less susceptible than either single mutation suggesting perhaps that electrons can follow multiple pathways and that likely that there are other potential electron donors. In H. pylori cells, a microaerophilic model, the metronidazole resistance was associated with inactivation of genes that encode for proteins related to the electron transfer such as a ferredoxin-like protein (FdxB) and a flavin oxidoreductase (FrxA) which may be needed for the metronidazole activation in susceptible cells.16,38,39 There are many such redox active proteins present in the Bacteroides and thus they may also be participating in the activation of metronidazole as suggested for H. pylori. Along these lines we did observe that a mutation in the rdx gene resulted in a decrease in susceptibility and when this was coupled with the porA mutation there was a further additional decrease in susceptibility. Obviously other enzymes and electron transfer components may act as electron donors in these anaerobes, but the primary reduction event seems to be carried out by the pyruvate–oxidoreductase complex, from which the nitroimidazoles might take electrons from the reduced ferredoxins or flavoproteins.

Data derived from the 2D-gels suggested that another functional group of proteins was modulated in the MTZR strain. These were the stress-related proteins Gad (acid stress), Tpx (peroxide stress), Bfr (iron stress) and FkpA (chaperone). It is not clear how this benefits the MTZR cell and in fact null mutations in the tpx and gadA genes did not significantly alter the metronidazole susceptibility profile. Besides, we do not want to over interpret the results and some other systems might be involved in this phenomenon. Further, this is somewhat at odds with a finding in the H. pylori system in which there was an increase in the levels of AhpC, a potent antioxidant stress-related enzyme.40 Currently we do not have a good explanation for this result but it is potentially important to note that three of these stress genes were down-regulated at both the protein and transcriptional levels. This suggests that their decrease may be part of some global regulatory response that is aimed at lowering the overall metabolic activity of the cell and hence reducing the need for stress resistance.

The comparison of protein expression patterns from parental (susceptible) and MTZR strains revealed a complex shift in the resistant strain resulting in both down- and up-regulation of proteins with different molecular weight and isoelectric points. Differences of this magnitude may not have resulted from the accumulation of single point mutations and thus may be part of a broader program of genetic control that alters the physiology to protect and prevent metronidazole activation. These global changes while not entirely understood did have other significant effects on the organisms. For example, we showed previously that the pathogenic potential of MTZR strains seems to be increased.5,29 It is known that virulence factors vary among individuals in the same microbial population and any agent interfering with their genetic profiles may influence the initial virulence properties of the population,41 so it is important that the mechanism of resistance be determined so that we can understand what aspects of the MTZR physiology impacts on virulence properties. The results reported here on the targeted mutations of several genes support the idea that there is no one specific gene for metronidazole resistance. In fact, by making several double mutations, it was possible to incrementally decrease susceptibility so it likely is the sum of these mutations that might confer metronidazole resistance. Others have also come to this same conclusion based on more biochemical approaches.42,43 This result may suggest that a global regulation could be involved in the metronidazole resistance phenomenon in this B. fragilis strain, and as an ecological consequence, this adaptive response could lead to the cellular alterations as shown with other studies in which metronidazole-resistant strains of Bacteroides had increased virulence and alterations in cell shape.5,29

This work represents one of the first studies using genetic and physiological approaches to understand the phenomenon of metronidazole resistance in the B. fragilis group. Many advances were made related to the changing patterns of gene/protein expression in the resistant strains, however there is still much to be undertaken regarding the precise mechanisms of resistance and how they might evolve in the environment of the host. In this regard, it may be useful to compare the patterns obtained with the MTZR strain and independently derived resistant strains or perhaps those obtained from clinical sources. Then it might be possible to identify the key changes that are responsible for resistance in nature.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We are grateful to Dr M. A. Cotta at the National Center for Agricultural Utilization Research, USDA, Peoria, IL, USA, for help with VFA analysis and to Luzia Rosa Rezende and José Sérgio Barros de Souza, for their technical help. This work was supported in part by PHS Grant AI28884 to C.J.S. and by CNPq and CAPES.


    Footnotes
 
* Corresponding author. Tel: +55-31-3499-27-59; Fax: +55-31-3499-27-30; Email: macedo{at}icb.ufmg.br


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Summanen, P., Baron, E. J., Citron, D. M., et al. (1993). Wadsworth Anaerobic Bacteriology Manual, p. 230. Star Publishing Company, Belmont, CA, USA.

2 . Rocha, E. R. & Smith, C. J. (1997). Regulation of Bacteroides fragilis katB mRNA expression by oxidative stress and carbon limitation. Journal of Bacteriology 179, 7033–9.[Abstract]

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