JAC Advance Access originally published online on April 18, 2007
Journal of Antimicrobial Chemotherapy 2007 59(6):1057-1064; doi:10.1093/jac/dkm086
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Molecular characteristics of rifampicin- and isoniazid-resistant Mycobacterium tuberculosis isolates from the Russian Federation
1 Research Institute for Physical–Chemical Medicine of Ministry of Public Health of Russian Federation, Malaya Pirogovskaya St, 1a, 119992 Moscow, Russia 2 National Research Centre for Antibiotics, Moscow, Russia 3 Central Tuberculosis Research Institute, Russian Academy of Medical Sciences, Moscow, Russia 4 Ural Research Institute for Phthisiopulmonology, Ekaterinburg, Russia 5 Novosibirsk Tuberculosis Research Institute of Ministry of Public Health of Russian Federation, Novosibirsk, Russia
* Corresponding author. Tel: +7-495-246-45-70; Fax: +7-495-246-45-01; E-mail: afanasev_max{at}mail.ru
Received 6 December 2006; returned 29 January 2007; revised 9 February 2007; accepted 27 February 2007
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Abstract |
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Objectives: Three Mycobacterium tuberculosis genetic loci—rpoB and katG genes and the fabG1(mabA)–inhA operon promoter region—were studied to reveal the mutations associated with rifampicin and isoniazid resistance.
Methods: Four hundred and twelve isolates of M. tuberculosis from different regions of the Russian Federation were collected during 1997–2005. A matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry (MALDI–TOF MS)-based minisequencing method was used for the detection of mutations.
Results: Thirteen different variants of single mutations in codons 533, 531, 526, 516, 513 and 511 of the rifampicin resistance-determining region of the rpoB gene as well as the TTG insertion in the 514a position were found among the rifampicin-resistant isolates. Single nucleotide substitutions in codons 531, 526 and 516 (64.8%, 10.3% and 7.7%, respectively) were the most prevalent mutations. Codon 526 was shown to be the most variable of all. No mutations were detected in rpoB genes for 29 (10.7%) of the rifampicin-resistant isolates. 76.9% of the isoniazid-resistant isolates carried single mutations in codon 315 of the katG gene. For another 12.9% of them, double mutations in the katG gene and the fabG1(mabA)–inhA promoter region were revealed. No mutations were detected in 8.2% of the isoniazid-resistant isolates.
Conclusions: Molecular analysis of the loci of rpoB and katG genes and the inhA promoter region of 412 M. tuberculosis clinical isolates from various parts of the Russian Federation was carried out. The new MALDI-TOF MS-based method may be used for rapid and accurate monitoring of the spread of drug resistance.
Keywords: drug resistance , tuberculosis , SNPs , MALDI–TOF mass spectrometry
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Introduction |
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Tuberculosis (TB) still represents a serious health problem in several parts of the world. In Russia, TB morbidity was about 80 cases per 100 000 of population in 2004. Although TB morbidity decreased slightly in recent years, the situation remains alarming due to a major increase in the incidence and prevalence of multidrug-resistant tuberculosis (MDR-TB). The MDR phenotype is defined as resistance at least to isoniazid and rifampicin— the two most effective drugs recommended by the WHO for the first-line treatment of TB.1–3 The prevalence of primary resistance to both of them reaches up to 45% in several regions of the Russian Federation.4 As rifampicin and isoniazid are the first-line drugs recommended by the WHO for the treatment of TB,3 resistance to both medications substantially increases the cost and duration of treatment, while decreasing the efficacy.
Susceptibility testing of Mycobacterium tuberculosis isolates by phenotypic methods requires a minimum of 14 days from a primary specimen. This can be reduced significantly if molecular analysis is used.5
At present, drug resistance-associated point mutations, deletions or insertions have been described for 11 genes of M. tuberculosis.6
Numerous studies have determined that up to 95% of rifampicin-resistant M. tuberculosis strains have point mutations within a small 81 bp hot-spot region, named the rifampicin resistance-determining region (RRDR). This region is located between codons 507 and 533 of the RNA polymerase ß subunit gene (rpoB). The most common mutations (up to 86%) in this region are observed in Ser-531, His-526 and Asp-516 codons.7
Unlike rifampicin, resistance to isoniazid is related to several genes. As a rule, the isoniazid-resistant strains have mutations in the catalase-peroxidase (katG) gene.
The promoter of the fabG1(mabA)–inhA operon (inhA promoter) is the second significant region for isoniazid resistance. This operon includes two genes, fabG1(mabA) and inhA, coding for ß-ketoacyl reductase and the fatty-acid enoyl–acyl carrier protein reductase involved in mycolic acid biosynthesis. Independent nucleotide substitutions in two positions of the promoter (–15T or –8C) lead to overexpression of the target for isoniazid active radical forms and, therefore, to isoniazid resistance. The role of the other genes, kasA and ndh, in isoniazid resistance is not completely resolved and needs further study.6
The aim of this study was to evaluate the prevalence of known mutations associated with resistance to rifampicin and isoniazid for 412 isolates of M. tuberculosis, collected in the Russian Federation during 1997–2005. Three genetic loci—rpoB and katG genes and the promoter region of the fabG1(mabA)–inhA operon—were analysed. We used a minisequencing reaction followed by matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry (MALDI–TOF MS) of the reaction products for the detection of expected substitutions. This method was based on primer extension reaction to one, two or three nucleotides at the point of interest. Subsequent MALDI–TOF MS analysis of the reaction products allows determination of the nucleotide sequence in the investigated site. Previously, this approach was successfully used for hepatitis C virus genotyping8 and for detection of fluoroquinolone resistance single nucleotide polymorphisms in gyrA and parC genes of Neisseria gonorrhoeae.9
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Materials and methods |
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Bacterial isolates
The 412 isolates of M. tuberculosis from sputum of epidemiologically unlinked TB patients from central Russia (Moscow and Moscow region; n = 98), Western Siberian (Novosibirsk; n = 154) and Ural regions (Yekaterinburg; n = 160) were collected in a non-randomized way from the reference laboratories. Species identification and drug susceptibility testing were performed according to the WHO recommendation-based10 Ministry of Health of Russian Federation protocols.11 Rifampicin and isoniazid susceptibility tests were carried out by the absolute concentration method on Löwenstein–Jensen medium containing 40 mg/L rifampicin or 1 mg/L isoniazid, respectively. All the drugs were manufactured by Sigma, USA. The results of microbiological tests were recorded 21 days after the inoculation. Isolates were considered resistant in the case when more than 20 colonies grew on the drug-containing medium.
DNA of M. tuberculosis was isolated using the ‘DNA-express’ kit (Lytech Ltd, Russia) in accordance with the manufacturer's instructions.
DNA extracts were dispensed into 96-well PCR plates (Simport, Canada) at 3 µL per well by means of an automatic workstation Biomec 2000 (Becman Coulter Inc., USA). Prepared plates were stored at –20°C.
The standard PCR was carried out in 10 µL of reaction mixture. The reaction mixture contained 66 mM Tris–HCl (pH 9.0), 16.6 mM (NH4)2SO4, 2.5 mM MgCl2, 250 µM of each dNTP, 1 U of Taq DNA polymerase (Promega, USA) and 10 pmol of each primer. The 321 bp fragment of the rpoB gene (including the RRDR), the 168 bp fragment of the katG gene and the 320 bp fragment of the inhA promoter region were amplified with the following primers sets: MtrpoBf (5'-GAGGCGATCACCGCAGAC-3') and MtrpoBr (5'-GGTACGGCGTTTCGATGAAC-3'); MtkatGf (5'-ACCCGAGGCTGCTCCGCTGG-3') and MtkatGr (5'-CAGCTCCCACTCGTAGCCGT-3') and MtfabGf (5'-GCCTCGCTGGCCCAGAAAGG-3') and MtfabGr (5'-CTCCGGATCCACGGTGGGT-3'). All primers were designed using Oligo_6.31 software (Molecular Biology Insights Inc., USA) and sequences of M. tuberculosis H37Rv strain (GenBank accession number NC000962).
A programmed thermocycler TETRAD DNA ENGINE (MJ Research, Inc.) was used. A universal amplification profile was worked out for all three loci: the initial heating step was at 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 61°C for 15 s and 72°C for 20 s and a final step at 72°C for 5 min.
The dephosphorylation step was carried out in the same 96-well reaction plates. The 5 µL reaction mixture contained 66 mM Tris–HCl (pH 9.0), 16.6 mM (NH4)2SO4, 2.5 mM MgCl2 and 0.5 U of shrimp alkaline phosphatase (Fermentas, Lithuania) and was added to each PCR product and incubated at 37°C for 20 min to dephosphorylate the 5'-end phosphate groups of deoxynucleoside triphosphates. At the end, the enzyme was deactivated by heating at 85°C for 10 min.
Primer extension reaction (minisequencing)
Primer extension reactions were carried out in the same 96-well PCR plate. The 7 µL of the reaction mixture contained 66 mM Tris–HCl (pH 9.0), 16.6 mM (NH4)2SO4, 2.5 mM MgCl2 and 2 U of TermiPol DNA polymerase (Solis Biodyne, Estonia), 20 pmol of oligonucleotide primer, 0.2 mM dNTP and 0.2 mM ddNTP, (in accordance with Table 1) were added to PCR products of rpoB, katG and fabG1 genes. Thermocycling protocols were developed to optimize the output of the extended primers: generally, the protocol was 70 cycles of 94°C for 20 s, Ta for 20 s and 72°C for 15 s. Here, Ta is a temperature that is optimal for the working of all oligonucleotide primers in the reaction mixture (Table 1).
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Products of the primer extension reaction were purified on a cation-exchange resin (SpectroClean Resin, Sequenom, USA). Resin (6–8 mg) was suspended in 15 µL of ultrapure water (Merck, Germany) and added into the reaction volume. Then, the contents of the test tubes were thoroughly mixed and incubated at room temperature for at least 1 h. After centrifugation for 5 min at 1000 rpm, the supernatant was used for MS analysis.
MS analysis was performed as described previously.8 Briefly, the sample aliquot (0.2–1 µL) was spotted onto a matrix, which was preliminarily dried on the AnchorChipTM (Bruker Daltonics, Germany) 400 µm targets. The matrix employed was a saturated solution of 3-hydroxypicolinic acid (Fluka, Germany) in a 1:1 acetonitrile/water mixture (Merck) mixed with 0.4 M dibasic ammonium citrate (Fluka) in a 9:1 (v/v) ratio. All the solvents utilized were MS grade. Mass spectra were collected using the Reflex IV MALDI–TOF MS (Bruker Daltonics), which was operated in the linear mode. A 337 nm nitrogen laser with 9 Hz pulse frequency and mode of positive ions was used. MS parameters were optimized for the range of m/z values from 1000 to 10 000, using the mass spectra of peptides for calibration. Each mass spectrum was collected at 30 laser pulses at constant laser power and constant threshold value, in order to enhance the resolution.
Registration and analysis of the obtained spectra were carried out using XMASS and XT of v.1.1 (Bruker Daltonics) software.
Conclusions about the nucleotide composition of extended primers were inferred on the basis of the appearance of peaks of calculated m/z ratio in mass spectra. If the unforeseen peaks arise, we could determine the nucleotide sequence by calculating the mass difference between neighbouring lines in spectra (Figure 1).
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DNA sequencing of rpoB, katG and fabG1 gene PCR fragments
Cycle sequencing reactions were performed using an ABI Prism® BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit and an ABI Prism® 3100 Genetic Analyzer (Applied Biosystems, USA; Hitachi, Japan) according to the manufacturers' instructions.
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Susceptibility testing
Among 412 M. tuberculosis isolates included in the study, 260 (63.1%) were identified as MDR, (i.e. resistant to both rifampicin and isoniazid), 84 (20.4%) were susceptible to rifampicin and isoniazid and 11 (2.7%) and 57 (13.8%) isolates were resistant, respectively, to either rifampicin or isoniazid.
Detection of mutations in the rpoB gene
Mutations in the RRDR of the rpoB gene were detected in 241 (88.9%) of 271 rifampicin-resistant M. tuberculosis isolates. Thirteen different types of mutations were identified in Leu-533, Ser-531, His-526, Asp-516, Gln-513 and Leu-511 codons and one variant insertion between codons 514 and 515.
No substitutions in Ser-522 were detected for M. tuberculosis isolates investigated. Mutation TCG 
TTG (Ser Leu) in codon 531 was the most prevalent; it was detected in 171 (41.5% of total number) isolates. Five different types of point nucleotide substitutions were found in the His-526 codon of 28 (6.8%) isolates. Twenty (4.85%) isolates revealed three variants of mismatches in Asp-516. Four (1%), one (0.25%) and two (0.5%) isolates carried mutations in Leu-533, Gln-513 and Leu-511 codons, respectively. Insertion Phe-514a was found for two M. tuberculosis isolates.
Eight isolates carried point mutations in two separated codons: Ser-531 and His-526 (seven isolates) and Asp-516 and Leu-511 (one isolate).
Detection of mutation in the Ser-315 codon of the katG gene and in the inhA promoter
Substitutions in the Ser-315 codon of katG and/or the inhA promoter region were found in 291 (91.7%) of 317 isoniazid-resistant M. tuberculosis isolates. Most of the M. tuberculosis isolates—243 (59%)—had mutation Ser-315 Thr (AGC ACC). Mutation Ser-513 Thr, caused by double nucleotide alteration AGC ACA, was revealed in one isolate only.
Forty-seven (11.4%) isolates had point nucleotide mutations in the –15 and –8 positions of the inhA promoter region. In 44 of them, substitutions in Ser-315 of katG and the inhA promoter were detected.
In one isolate, a new substitution (T C) in the –8 position of the inhA promoter region was found.
Direct DNA sequencing of gene fragments obtained as a result of PCR was carried out for one hundred isolates and confirmed perfectly the results of minisequencing analysis.
All genetic variants revealed in the present study are shown in Table 2.
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Discussion |
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Comparative analysis of genotypes and phenotypes of M. tuberculosis isolates is shown in Table 3. Resistance to rifampicin was correctly identified by MALDI–TOF MS analysis in 90.4% of MDR isolates and in 54.5% of isolates demonstrating isolated phenotypical resistance to this antibiotic; no mutations were detected for phenotypically susceptible isolates.
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No substitutions in the studied loci of RRDR were observed for 29 (11.1%) rifampicin-resistant M. tuberculosis isolates. It has been previously observed that
10% of rifampicin-resistant strains do not have any mutations in the RRDR.7 For isoniazid, resistance was predicted correctly in 94.25% of MDR isolates and in 80.7% of isolates with isolated phenotypical resistance to isoniazid. Twenty-six (8.2%) isoniazid-resistant isolates had no detectable changes in the two targets analysed.
The sequencing procedure of the RRDR of the rpoB gene, fragments of the katG gene and the inhA promoter region from isolates that had a resistant phenotype, but had no mutations in the studied loci, confirmed the results of minisequencing analysis and confirmed the absence of mutations in the 81 bp RRDR of rpoB, fragments of the katG gene and the inhA promoter. Probably, in these cases, resistance might be concerned with changes in other loci of the M. tuberculosis genome.
The codons most frequently affected by point mutations were Ser-531, His-526 and Asp-516, with frequencies of 64.8%, 10.3% and 7.7%, respectively, in agreement with previous data.12–14 Mutations in the Leu-533 codon were revealed for four rifampicin-resistant isolates. The wild-type Leu-511 was altered in two isolates, and the Gln-513 codon was altered in one isolate.
Contrary to our data, Toungoussova et al.15 detected mutations in codon Gln-513 more frequently, for 10% of rifampicin-resistant isolates. The insertion Phe-514a (TTC) was identified for two isolates. Eight (3.3%) rifampicin-resistant isolates had point mutations in two separate codons.
Most isoniazid-resistant isolates—244 (76.97%)—contained nucleotide substitutions in the Ser-315 codon of the katG gene. This agrees with the fact that the frequency of mutations in this codon is 93% for isoniazid-resistant strains in the Russian Federation.12,14 Only three isoniazid-resistant isolates had changes in the inhA promoter and an intact Ser-315 codon.
Mutations in two loci (katG and inhA promoter) were found simultaneously in 44 (13.88%) samples.
Previously, it was noted that the structure and frequency of occurrence of mutant codons for M. tuberculosis varied for different regions of the world.16,17 By comparison of the presented data with those in the literature, it was observed that high prevalence mutations in Ser-531 also occurred in strains from Germany (75.7%),18 Peru (68.1%)19 and Italy (63.3%).20 His-526 mutations are predominantly observed in the USA (44%)21 and China (40%)22 and Asp-516 in Hungary (37.9%),23 whereas in our samples, the frequency of these mutations was much lower.
Mutations in Ser-315 of katG also have geographical variations, for example, they were not detected in Equatorial Guinea24 but were detected in a substantial 93.6% of isolates from Northwest Russia.25 Moreover, in our samples, only three (1%) isoniazid-resistant strains had isolated inhA promoter mutations, whereas in some world regions, prevalence of these mutations can reach up to 80%.24
In our work, we made an attempt to compare groups of drug-resistant isolates among themselves, using their mutation profiles. The spread of resistance to rifampicin and isoniazid was analysed for Central, West Siberian and Ural regions of the Russian Federation. The 79, 82 and 110 rifampicin-resistant isolates and 81, 104 and 132 isoniazid-resistant isolates were collected from these three regions, respectively. It is significant that previously12,13,25,26 mutation profiles of resistant M. tuberculosis strains were studied mainly for the European part of the Russian Federation (Moscow and Moscow Oblast, Samara Oblast, Archangel Oblast, St Petersburg etc.); strains from the Eastern part (Ural, Siberian and War West) were poorly investigated.
Comparison analysis reveals that strains circulating in different geographical regions possess different mutation profiles (Table 4). However, future monitoring studies are needed to confirm the reliability of observed differences for evaluation of the epidemiological features of circulating drug-resistant strains. It is evident that the frequency and type of mutations in one country or geographical region are not applicable more generally.
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In the present study, molecular analysis of the loci of rpoB and katG genes and the inhA promoter region of 412 M. tuberculosis clinical isolates from various parts of the Russian Federation was carried out. Unfortunately, mutations which lead to high-level resistance were prevalent for all regions from where isolates were collected.
For M. tuberculosis mutation identification, a new method by means of a minisequencing approach based on MALDI–TOF MS was used. We succeeded in realization of the so-called ‘one-tube’ technique, when all analysis steps are implemented in one reaction tube, without transferring material between tubes. It decreases the probability of error and chances of cross-contamination. Furthermore, the cost of analysis can be compared practically with the cost of two standard PCR reactions, but the accuracy is similar to direct sequencing of gene fragments. Execution time for the analysis is several hours (but is possible within a working day).
Thus, this method may be interesting and useful for rapid and accurate monitoring of the spread of drug resistance. At the same time, this approach may be suitable for timely correction of antituberculosis treatment in accordance with the mutation profile of M. tuberculosis strains. This should help avoid the generation and transmission of drug-resistant strains in the community.
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Transparency declarations |
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None to declare.
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Acknowledgements |
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We thank V. A. Karpov for the synthesis of oligonucleotide primers. This work was supported by Bruker Daltonics, Germany (Development Contract No. BDALIPCM 270505).
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References |
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Nikolayevsky V, Brown T, Balabanova Y, et al. Detection of mutations associated with isoniazid and rifampin resistance in Mycobacterium tuberculosis isolates from Samara Region, Russian Federation. J Clin Microbiol (2004) 42:4498–502.
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Toungoussova O, Sandven P, Mariandyshev A, et al. Spread of drug-resistant Mycobacterium tuberculosis strains of the Beijing genotype in the Archangel Oblast, Russia. J Clin Microbiol (2002) 40:1930–7.
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Cavusoglu C, Hilmioglu S, Guneri S, et al. Characterization of rpoB mutations in rifampin-resistant clinical isolates of Mycobacterium tuberculosis from Turkey by DNA sequencing and line probe assay. J Clin Microbiol (2002) 40:4435–8.
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Van Der Zanden A, Te Koppele-Vije E, Vijaya Bhanu N, et al. Use of DNA extracts from Ziehl-Neelsen-stained slides for molecular detection of rifampin resistance and spoligotyping of Mycobacterium tuberculosis. J Clin Microbiol (2003) 41:1101–8.
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Hillemann D, Weizenegger M, Kubica T, et al. Use of the genotype MTBDR assay for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis complex isolates. J Clin Microbiol (2005) 43:3699–703.
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Miotto P, Piana F, Penati V, et al. Use of genotype MTBDR assay for molecular detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis clinical strains isolated in Italy. J Clin Microbiol (2006) 44:2485–91.
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Jun Y, Shi W, Xie J, et al. Mutations in the rpoB gene of multidrug-resistant Mycobacterium tuberculosis isolates from China. J Clin Microbiol (2003) 41:2209–12.
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