JAC Advance Access published online on June 24, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn259
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Original research |
The effect of oxidative stress on the mutation rate of Mycobacterium tuberculosis with impaired catalase/peroxidase function
1 Centre for Medical Microbiology, Department of Infection, Hampstead Campus, University College London, Rowland Hill Street, Hampstead, London NW3 2PF, UK 2 Health Protection Agency, Regional Microbiology Network, Holborn Gate, London WC1V 7PP, UK
* Correspondence address. Department of Infectious and Tropical Disease, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK. Tel: +44-20-7927-2468; Fax: +44-20-7637-4314; E-mail: denise.o'sullivan{at}lshtm.ac.uk
Received 20 December 2007; returned 9 April 2008; revised 13 May 2008; accepted 2 June 2008
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Abstract |
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Objectives: To determine the effect of oxidative stress on isoniazid-resistant Mycobacterium tuberculosis deficient in catalase/peroxidase activity to varying degrees through mutation in katG.
Methods: The mutation rate was determined for a set of isogenic strains with different katG alleles giving different catalase and/or peroxidase activities following exposure to the oxidizing agent, hydrogen peroxide. Mutants were selected on rifampicin, and the location and nature of the mutation were identified by sequencing the rpoB gene.
Results: No evidence was found to suggest that strains that had impaired catalase/peroxidase activity were hypermutable, and the presence of excess hydrogen peroxide had no effect on the mutation rate. An unusual pattern of mutations in rpoB was observed in catalase-deficient strains with only 3 of 66 having mutations within the rifampicin resistance-determining region.
Conclusions: The mutation rate of M. tuberculosis in response to oxidative stress is not increased in strains with significant deficits in catalase and peroxidase activity. Our data suggest that isoniazid-resistant strains compensate for their reduced ability to detoxify oxidative stress effectively. Interestingly, mutations were found in unusual locations at positions similar to those found in clinical isoniazid-resistant strains.
Key Words: oxidative stress response , isoniazid , M. tuberculosis
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Introduction |
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Mycobacterium tuberculosis resides in a macrophage phagolysosome, where the organism must protect itself against reactive oxygen and nitrogen intermediates.1 Nucleic acid and sugar moieties are susceptible to the oxygen-free radicals produced, leading to base degradation, single-strand breakage and cross-linking to proteins.2 Catalase/peroxidase protects M. tuberculosis by detoxifying hydrogen peroxide and reactive nitrogen intermediates and reducing organic peroxides.3 Catalase, encoded by katG, has both catalase and peroxidase activities, of which the peroxidase activity seems to be necessary for the activation of isoniazid.4 Resistance to isoniazid occurs most frequently (40% to 50%) through mutations in katG,5,6 most commonly Ser-315
Thr. With this mutation, the enzyme cannot activate isoniazid, but still protects against host antibacterial radicals.7 As intramacrophage survival of M. tuberculosis depends on its oxidative stress defence, it is surprising that oxyR, a central regulator of this response, is inactivated by multiple mutations.8 The oxyR is transcribed from ahpC, which contributes to the reduction of peroxides.9 In isoniazid-resistant katG-deficient strains and isoniazid-susceptible strains that are exposed to isoniazid, enhanced expression of ahpC is observed, so there is a functional overlap between katG and ahpC activity.10,11
Isoniazid monoresistance is significantly more common than rifampicin monoresistance, and it has a higher mutation rate, 10–8 mutations per cell division compared with 10–10 mutations per cell division.12 It is usually assumed that multidrug resistance emerges first to isoniazid followed by rifampicin, although direct evidence for this sequence of events is lacking.13 We propose the hypothesis that mutation in katG could lead to an elevated spontaneous mutation rate due to a reduction in an organism's capacity to deal with oxidative stress. This would explain why isoniazid resistance is observed before rifampicin resistance and could be important in the accumulation of multiple mutations and in the genesis of multidrug-resistant tuberculosis. Co-existing mutations in rpsL conferring resistance to streptomycin and rpoB to rifampicin have been confirmed to be antagonistic in Mycobacterium smegmatis14 and Escherichia coli.15 Mutations in gyrA and/or parC leading to quinolone resistance act in synergy to induce further mutation in Streptococcus pneumoniae.16 To test this hypothesis, we investigated the rate, location and sequence of spontaneous mutants to rifampicin, with and without the addition of hydrogen peroxide, of a panel of isoniazid-resistant M. tuberculosis strains with defined impairment in peroxidase and catalase activity.5
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Strain characteristics
Isogenic strains with different katG genotypes described previously (Table 1)5 were used and were a gift from Professor Stewart Cole (Institut Pasteur, Paris).
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Determination of the mutation rate by the Poisson distribution (p0) method
The mutation rate was determined using the p0 method as described previously, as this is the most appropriate method when low mutation rates are expected.17,18 Briefly, strains were cultured in 4 mL of Middlebrook 7H9 broth (BD) containing 10% albumin dextrose catalase (ADC) (BD) and 0.2% Tween 80 (BDH) broth to a turbidity equivalent to that of a 0.5 McFarland growth standard. The volume was adjusted to give 
5x103 cells/mL, and an aliquot of 1 mL was distributed into 28 microcentrifuge tubes and incubated for 2 weeks at 37°C with daily gentle agitation. Mutants were selected on Middlebrook 7H10 agar plates, which were prepared containing 0.25% glycerol (Sigma-Aldrich), 10% oleic acid ADC (OADC) enrichment (BD) and 2 mg/L (2x MIC) rifampicin (Sigma-Aldrich). At the end of the incubation period, a Miles and Misra19 plate count was performed in triplicate on three broth cultures. The remaining 25 broth cultures were harvested at 13 000 g for 5 min, the supernatant discarded and the pellet plated onto the antibiotic-containing plates. The proportion of cultures with no mutants (p0) was used to derive the mutation rate, as described previously.18 These experiments were performed on at least two occasions for each strain.
Mutation rate of isoniazid-resistant strains after exposure to hydrogen peroxide
The strains INH34pAP01, INH34pAP23 and H37Rv were exposed to a stringent oxidative stress by adding 8 mM H2O2 (Sigma-Aldrich) for 24 h, and the mutation rate was determined as described previously.
Identification of the genotype of rifampicin-resistant colonies
Colonies growing on rifampicin (2 mg/L) were suspended in a microcentrifuge tube containing 400 µL of TE buffer and heat-killed.20 The supernatant was stored at 4°C for subsequent PCR, which was performed with primers from Telenti et al.21 and Jenkins et al.22 and designed to amplify three overlapping fragments (positions 451–1735 of the rpoB gene sequence). The products were sequenced as described previously.22
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Results |
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Mutation rate of M. tuberculosis to rifampicin
Hypermutability was defined as a 10-fold difference in mutation rates, but all were in the range between 1.3x10–7 and 3.3x10–6 mutations per cell generation (Table 1). Additionally, there was no significant difference between the rates obtained when comparing strains treated or untreated with hydrogen peroxide. Also, the individual katG genotype had no impact on the mutation rate. When compared with H37Rv, only INH34pAP22, a strain with the mutation Ala-139Val with catalase/peroxidase activity, had a mutation rate >10-fold.
Of the 66 resistant mutants, 3 contained a mutation in the rifampicin resistance-determining region (RRDR). No other recognized resistance mutations were found (Table 2).
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Discussion |
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We tested the hypothesis that strains containing katG mutations were likely to have an elevated mutation rate due to their compromised catalase/peroxidase activity. The mutation rates of strains with no catalase/peroxidase activity, INH34pAP01 and INH34pAP21, were within an order of magnitude of those of strains that had enzymic activity, INH34pAP21, INH34pAP23, INH34pPD28 and H37Rv (Table 1). Also, there was no difference in the mutation rate for any of the strains compared with INH34pPD28, a strain with a wild-type katG and a functional catalase and peroxidase. It was interesting to note that INH34pAP01, which contained a deletional interruption of katG, did not have an elevated mutation rate. Thus, using Werngren and Hoffner's definition,23 these differences are not significant. Our methods are not sensitive enough to determine <10-fold differences reliably, but smaller changes may be biologically significant. Even when stressed with H2O2 at a dose twice that shown to cause mutation in M. tuberculosis,24 there was no evidence that such treatments had a significant effect on the mutation rate of the INH34 strains, compared with H37Rv (Table 1). This implies that compensatory mutations may counterbalance the loss of katG.25 The clinical isolate INH34 has an up-regulated promoter region of ahpC, so this may have compensated for catalase/peroxidase deficiencies as a result of mutations in katG.10,26 Other genes involved in isoniazid resistance may be ndh, in which single nucleotide polymorphisms have been reported in M. tuberculosis.27
An important finding of this study was that only 4.5% of the rifampicin-resistant isolates had mutations within the RRDR, compared with 90% of the rifampicin-resistant clinical isolates.7,28 This surprising result is similar to our previous data from a clinical study that showed a different pattern of mutations in rpoB among outbreak isoniazid-resistant strains in North London.22 None of the strains in that study had the commonly reported mutations in the RRDR; three had rare mutations in the RRDR (two with Ser-531Trp and one with His-526Arg) and three had mutations outside the RRDR (Val-146Phe), which has previously been described in clinical isolates.22,29
The mutants were isolated at 2 mg/L rifampicin (twice the MIC for M. tuberculosis H37Rv). A relationship between susceptibility to rifampicin and the pattern of mutations in rpoB has been observed. Rifampicin-resistant strains with an MIC of 
32 mg/L have been shown to have 11% of mutations occurring in the RRDR, and all strains with an MIC of 
64 mg/L have mutations within the RRDR.30 The colonies sequenced here, therefore, may have had low MICs, and this could have affected the distribution of locations of mutations. However, Morlock et al.31 used 2 mg/L for isolating rifampicin-resistant colonies and found a pattern similar to that observed in rifampicin-resistant clinical isolates. The contribution of drug efflux pumps with drug resistance has been reported in M. tuberculosis; in particular, efpA32 and a multidrug tap-like pump.33 Changes in the efflux pumps may render the mycobacterial cell wall resistant to the accumulation of rifampicin.34 Rifampicin-resistant mutants with no rpoB mutation might arise from enhanced efflux, mediating low-level rifampicin resistance, although Morlock et al. did not find evidence of efflux mechanisms in their study.
In conclusion, M. tuberculosis strains with an impaired oxidative stress response do not have an increased mutation rate and were able to compensate, following the addition of hydrogen peroxide. An unusual pattern of mutation was observed in rifampicin-resistant mutants, of which some were isoniazid-resistant, with only 4.5% occurring within the RRDR.
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This work was part funded by a grant from the British Society for Antimicrobial Chemotherapy.
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None to declare.
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Acknowledgements |
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We would like to thank Professor Stewart Cole (Institut Pasteur, Paris) for providing strains.
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References |
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