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awska,2 Ewa Augustynowicz-Kope
,3 Zofia Zwolska,3 Stefan Niemann,4 Jarosaw Dziadek,2* and Doris Hillemann4
Department of Genetics of Microorganisms, University of 
ód
,1
Center for Medical Biology, Polish Academy of Sciences, ód,2
Department of Microbiology, National Research Institute of Tuberculosis and Lung Diseases, Warsaw, Poland,3
National Reference Center for Mycobacteria, Forschungszentrum Borstel, Borstel, Germany4
Received 7 November 2003/ Returned for modification 15 December 2003/ Accepted 11 March 2004
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ABSTRACT |
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T in the regulatory region of inhA. A full correlation between the DNA sequence analysis and real-time PCR data was obtained. We conclude that the real-time PCR method is fast and reliable for the detection of RMP and INH resistance-associated mutations in M. tuberculosis clinical isolates. |
INTRODUCTION |
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Early diagnosis of the disease and the rapid identification of resistance to primary anti-TB agents are essential for the efficient treatment and control of MDR strains. It is known that resistance to INH and RMP is a key factor in determining the effectiveness of the currently recommended standard treatment regimens. The elucidation of the mechanism of action of these drugs, which was accomplished only recently, has led to the development of new rapid diagnostic methods (4, 7, 14, 19, 22).
The rapid detection of RMP resistance is of particular importance, since it also represents a valuable surrogate marker for MDR resistance, which is a tremendous obstacle to TB therapy (10). Collectively, DNA sequencing studies have demonstrated that >95% of RMP-resistant (Rmpr) M. tuberculosis strains have a mutation within the 81-bp hot-spot region (codons 507 to 533) of the RNA polymerase ß subunit (rpoB) gene (23). The prevalence of the mutations determined so far varies for M. tuberculosis strains obtained from different countries. Thus, it is important to determine the distribution of resistance mutations at the level of each country prior to molecular tests being introduced for routine diagnostics (4).
Unlike RMP resistance, INH resistance is apparently controlled by a more complex genetic system that involves several genes (21). However, extensive studies have demonstrated that INH resistance is most frequently associated with a specific mutation in katG (codon 315), a gene that encodes the catalase-peroxidase enzyme in M. tuberculosis (18). Also, InhA (enoyl-ACP-reductase), an enzyme involved in mycolic acid biosynthesis, was identified as a main target of INH, and mutations in the regulatory region of the inhA gene have been linked to INH resistance (12).
The aim of the present study was to determine the drug resistance profiles of 105 drug-resistant M. tuberculosis strains isolated in Poland and to detect mutations present in the rpoB and katG genes as well as in the regulatory region of the inhA gene. Two molecular methods were used. In the first one, mutations that have been associated with RMP and/or INH resistance were determined by automated DNA sequence analysis. The results were compared with those obtained by real-time PCR technology.
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MATERIALS AND METHODS |
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Primary isolation, differentiation, and drug susceptibility testing were performed with Löwenstein-Jensen medium and the BACTEC 460-TB system (Becton-Dickinson, Sparks, Md.), as reported earlier (1).
The relatedness of all resistant strains was investigated by studying restriction fragment length polymorphisms (RFLPs) of the IS6110 element (28). The spoligotyping (spacer oligotyping) method (11) was additionally used to confirm the relationships of the strains.
DNA preparation. A rapid DNA extraction procedure for the direct testing of M. tuberculosis on Löwenstein-Jensen slants was performed. A loopful of organisms was suspended in 1 ml of sterile water and the bacteria were lysed by boiling for 20 min. The cells were centrifuged (12,000 x g for 5 min) and then frozen overnight at –20°C. The supernatant was used as a template for amplification.
Standard PCR. The DNA extract was amplified with the primers listed in Table 1. Primer pairs TR8-TR9 and TB86-TB87 were used to amplify the regions within the rpoB and katG genes, respectively (22). Primers TB92 and TB93 were applied for the amplification of a fragment containing the regulatory region of the inhA locus (8). Prior to PCR, a preincubation step (95°C for 15 min) was performed to activate the HotStart Taq DNA polymerase (Qiagen GmbH, Hilden, Germany). The PCR was carried out for 35 cycles of denaturation at 94°C for 30 s, annealing at 62°C (for rpoB), 67°C (for katG), or 64°C (for inhA) for 30 s, and extension at 72°C for 45 s in an MJ Research thermocycler. PCR products were examined by gel electrophoresis and purified by use of a QIAquick PCR purification kit (Qiagen).
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Probes for detection of mutations by real-time PCR. The sequences of the probes used for the present study are listed in Table 1. For the detection of mutations in the rpoB gene, two pairs of fluorescence resonance energy transfer (FRET) probes, the rpo (25) and RPO1 (7) probes, were used. Both pairs of probes were labeled with fluorescein (at the 3' ends) and Red 640 (at the 5' ends) and were homologous to the wild-type (wt) sequence. Thus, mutations in the DNA region covered by the probes led to different melting temperatures (Tms) for the probes when they were hybridized to the target.
For katG, a set of standard FRET probes and three TaqMan minor groove binder (MGB) probes were applied. The FRET probes covered the region containing codon 315, which is the most frequent target for mutations related to INH resistance. This pair of probes was labeled with fluorescein (TB anchor) and Red 640 (TB sensor) (25). Additionally, three katG-specific TaqMan MGB probes were designed to be homologous to wt codon 315 (AGC) or to one of the most frequent substitutions, ACC or ACA. These oligonucleotides contained a reporting fluorescent dye (FAM or VIC) at the 5' end and a quencher dye (TAMRA) at the 3' end. During PCR, the 5'3' exonuclease activity of Taq DNA polymerase cleaves the downstream probe as it extends. As the probe is degraded, the reporter dye is separated from the quencher. This results in increased fluorescence as amplification proceeds. Because the cleavage occurs only if the probe hybridizes to the target, the fluorescence detected originates from specific amplification.
For the detection of mutations in the inhA locus, a pair of conventional FRET probes was used (anchor-sensor design) (26). The 5'-Red 640-labeled sensor (TB ATT) covered the segment containing the regulatory region (Table 1).
All primers and FRET probes were synthesized by TIB MOLBIOL (DNA Synthesis Service, Berlin, Germany), and TaqMan MGB probes were synthesized by Applied Biosystems (Weiterstadt, Germany).
Real-time PCR assay. The PCR mixtures, in a final volume of 20 µl, contained 2.5 mM MgCl2, a 200 µM concentration of each deoxynucleoside triphosphate, a 1 µM concentration of each primer, a 0.2 µM concentration of each probe (except for the RPO1 and TaqMan MGB probes, which were added to final concentrations of 0.04 and 0.1 µM, respectively), 1 U of HotStart Taq DNA polymerase (Qiagen), and 2 µl of template DNA. Additionally, the reaction mixture for rpoB amplification included 8 µl of Q solution (Qiagen). For preparation of the components for the katG gene PCR with TaqMan MGB probes, 10 µl of a commercial ready-to-use reaction mix (universal PCR master mix; Roche) was used.
The cycling parameters used for experiments with FRET probes were as follows: 95°C for 10 min and 35 to 40 amplification cycles at 94°C for 15 s; 62, 67, or 64°C (annealing temperatures for rpoB, katG, and inhA, respectively) for 20 s; and 72°C for 30 s. PCR cycling was followed by a melting-curve analysis from 50 to 85°C, with fluorescence readings taken every 1°C. The Tm of each of the FRET probes for the wt sequence was calculated empirically as the average value of the Tms obtained in at least three independent experiments. When a probe binds to a mismatched sequence, the Tm deviates with respect to that of a perfectly matched wt sequence. The standard deviations (SD) of the Tms for probes rpo, RPO1, TB (katG), and TB (inhA) were 0.10, 0.33, 1.20, and 0.36°C. In all cases in which the deviations in the Tm were more than two times the SD, a mutation was suspected.
For PCRs with TaqMan MGB probes, the conditions for cycling were 94°C for 10 min followed by 40 to 45 cycles of 92°C for 15 s and 60°C for 1 min. All reactions were performed in a Rotor-Gene 2000 instrument (Corbett Research, Mortlake, Australia).
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RESULTS |
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GTG (Asp-to-Val) mutation in codon 516, and in the other strain codon 517 had the mutation CAACTA (Gln to Leu). In total, five new alleles were identified in the present study, with all involving changes in one or two codons. Two Rmpr strains (3.1%) contained no mutations in the sequenced region, although these strains were phenotypically resistant to RMP. None of the three RMP-sensitive control isolates had any mutation.
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TTG) was detected in 24 strains as an increase in the Tm of the probe (from 1.92 to 2.62°C; Table 2), whereas for the 5 strains with three different mutations at codon 526, the Tm of the probe was 1.78 to 3.38°C lower (depending on the mutation) than that of the wt strain (Table 2). With the RPO1 probes (7), mutations in the 5' half of the core in 29 strains were also detected by reductions in the Tms of the probes (from –2.33 to –7.33°C; Table 2). The real-time PCR assay results were in full accordance with the nucleotide sequencing data.
Detection of mutations in the katG gene.
Eighty-three M. tuberculosis strains that were resistant to INH [alone or in combination with another drug(s)] were investigated by DNA sequencing of a 209-bp katG fragment including codon 315, the codon at which mutations are most frequently associated with INH resistance. Fifty-seven (68.7%) Inhr isolates had mutations at codon 315 (Table 3). The wt codon, AGC (Ser), was altered to ACC (Thr) in 50 strains, ACT (Thr) in 4 strains, ACA (Thr) in 1 strain, AAC (Asn) in 1 strain, and ATC (Ile) in 1 strain. One strain harbored a double mutation affecting codon 279 (GGCACC [GlyThr]). In 25 (30.1%) cases, no mutation was found in the analyzed fragment. Four INH-susceptible control isolates possessed the wt sequence.
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Detection of mutations in the inhA regulatory region. Twenty-six Inhr M. tuberculosis strains that had no mutations at katG codon 315 (including one strain carrying a mutation at codon 279) were investigated in a real-time PCR test. A set of standard FRET probes was used to detect changes in a 22-bp fragment upstream of the start site of inhA (26). For 4 INH-susceptible control isolates and 14 Inhr strains (including one strain with a mutation at codon 279 of katG), the Tms were within the range obtained for the reference strain, and therefore they were not considered mutants (Table 3). For the remaining 12 (14.5%) Inhr strains, deviations in the Tms of the probes suggested mismatches, as there were increases in the Tms of the probes (from 4.85 to 5.25°C; Table 3) compared to those for the wt reference strain.
To confirm the alterations in the inhA regulatory region in the 12 Inhr strains, we determined the DNA sequence of the 248-bp fragment of this locus. We found that all of the strains carried a single point mutation, CT, at position 15 upstream of the start site of the inhA gene (Table 3). This mutation has previously been reported by other groups. The present understanding of this mutation is that it probably confers resistance by a drug titration effect. Overall, 58 (69.9%) Inhr M. tuberculosis strains investigated in this study harbored mutations in the katG gene, 12 (14.5%) strains carried a mutation in the regulatory region of the inhA gene, and the remaining 13 (15.7%) isolates had no detectable changes in the two targets analyzed. In all cases, full concordance was obtained between the real-time PCR results and DNA sequencing data.
DNA fingerprinting analysis. Two standardized DNA fingerprinting techniques, spoligotyping (11) and IS6110 RFLP typing (28), were used to differentiate the strains in order to assess possible genetic similarities between them. The numbers of IS6110 copies per isolate varied from 5 to 20 (A. Sajduda, A. Brzostek, and J. Dziadek, unpublished results). A total of 73 different IS6110 fingerprint patterns were identified for the 105 analyzed strains. Fifty-nine strains showed unique fingerprints, which indicates their epidemiologic independence. However, 46 strains (44%) clustered into 14 groups consisting of two to seven isolates with identical IS6110 RFLP patterns, representing possible cases of recent transmission. Direct epidemiologic links could not be established for most of the cases in clusters. However, the transmission of the same drug-resistant M. tuberculosis strain among the patients of one cluster or their infection by contact with one or more index patients is further supported by the observation that in most clusters all of the strains exhibited the same polymorphism in the Rmpr and/or Inhr-associated genes and identical or very similar drug resistance profiles (data not shown). Also, only five of the clusters had isolates with different Rmpr and/or Inhr mutations, indicating an independent acquirement of resistance to these drugs. Although the epidemiologic data obtained in the present study are preliminary, they show that the transmission of drug-resistant M. tuberculosis strains may have occurred in Poland and seems to have contributed to the emergence of drug-resistant TB in this country.
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DISCUSSION |
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Our findings of mutations in the rpoB and katG genes were comparable to those reported for strains from other parts of the world, especially the common mutations, which reflect a global pattern (16). The rpoB codons 531, 526, and 516 are the most frequently mutated codons worldwide, although variations in the relative frequencies of mutations in these codons have been described for M. tuberculosis isolates from different geographic locations (reviewed in reference 4). Also, in our study most of the strains with the Rmpr phenotype (69%) contained missense mutations which led to amino acid substitutions at the Ser531 (41%), Asp516 (16%), and His526 (9%) residues. A comparison of these results with the results of other studies showed that the mutations at codon 531 also predominate in Poland (4, 23, 27). In contrast with previously published data, the frequency of mutations at codon 516 was found to be high in Poland (4, 23). A high frequency of changes at this codon was also described recently for MDR M. tuberculosis isolates from East Hungary (3), Latvia (27), North India (20), and East Asia (15). A characteristic finding was the high frequency of double mutations occurring in two separate codons (20%). Altogether, we found five novel alleles, which broadens the range of known mutations in the rpoB gene. The high incidence of these less common changes in Polish strains can be explained by either geographic variations in the frequencies of particular rpoB mutations or a sample bias. Sequence analysis identified no mutation in two (3.1%) of the strains tested, although they were resistant to RMP as determined by the proportion method. A recent study revealed that a mutation associated with RMP resistance can also be located outside the 81-bp rpoB core region, although this does not occur frequently (9). Other possibilities are that in these resistant strains, other rare rpoB mutations, heteroresistance (a mixture of susceptible and resistant subpopulations), or less likely, another mechanism of resistance may be involved (3, 10).
In Inhr strains, neither insertions nor deletions (complete or partial) of katG were found, which is evidence of the rare occurrence of these mutations in clinical isolates, although they were reported previously by other authors (20, 24). We observed that almost 70% of the strains studied carried the Ser315Thr substitution, which is in concordance with the bulk of the published data (16). However, the frequency of this mutation was lower in Polish strains than the 93.6% of strains in northwestern Russia (13), 91% of strains in Latvia (26), and 85.7% of strains in Lithuania (2). In accordance with previous reports, the most frequent mutation was AGC to ACC at katG codon 315 (60%). A novel double mutation, AGC to ACT, which also leads to an amino acid change from Ser to Thr, was found in four strains. However, three of these isolates seemed to be epidemiologically related, according to their identical patterns in the IS6110 RFLP analysis. We also identified one strain with a double mutation at codon 279 (GGCACC) that has not been described previously. This strain carried wt katG codon 315 and showed no mutation in the regulatory region of inhA (see below). We assume that this mutation is involved in the INH resistance of this strain.
Since 31% of the Inhr strains in this study had no mutation at katG codon 315, it is likely that mutations in other genes, such as the inhA locus, significantly contribute to resistance. Previous studies have shown that mutations in the upstream region of the inhA locus result in increased levels of InhA (NADH-dependent enoyl-acyl carrier protein reductase) expression, thereby elevating the drug target levels and producing INH resistance via a titration mechanism (16). We searched for mutations in the inhA regulatory region of 26 Inhr M. tuberculosis strains with a wt codon 315 in katG and found a substitution at position 15 upstream of the start codon in 12 (14.5%) isolates. This frequency was similar to the 13.5% frequency in strains from Lithuania (2) but higher than the 4.3% frequency in strains from Seville, Spain (24). Since a polymorphism in the inhA regulatory region accounted for almost 15% of all Inhr strains examined in this study, we suggest screening for such a polymorphism in all isolates from Poland that have no detectable changes at codon 315 of the katG gene. Fourteen (16.5%) strains showed a lack of mutations in both the katG gene and the inhA locus fragments analyzed here. According to previous studies, there are some 18 additional genes that have been implicated in INH resistance, including kasA, ndh, and the oxyR-ahpC intergenic region (17). These genes either are involved in mycolic acid biosynthesis or are overexpressed as a response to the buildup or cellular toxicity of INH. Therefore, the alterations responsible for INH resistance in these 14 strains may be associated with any of these loci.
The prevalence of Rmpr mutations within the rpoB core region (97%) as well as Inhr mutations at katG codon 315 and the inhA locus (69 and 14.5%, respectively) in M. tuberculosis strains from Poland indicated the potential for a rapid diagnostic test for the detection of drug-resistant TB. Different genotypic assays have been proposed for the detection of mutations involved in drug resistance in M. tuberculosis, including a recent real-time PCR assay coupled to fluorescence detection. Formats that utilize molecular beacons (6) and biprobes (5) for the detection of RMP resistance have been reported. However, as many as five different beacons are required to encompass the entire rpoB core region, and three biprobes described by Edwards et al. (5) detected mutations in only four codons of the rpoB gene. We evaluated a standard FRET probe set that was previously described by Torres et al. (25) for the detection of Rmpr mutations at codons 526 and 531 of the rpoB gene and the RPO1 dual-sensor FRET probes that were recently described by García de Viedma et al. (7) to detect changes in the 5' region of the rpoB core. Using both probe sets, we were able to detect all 17 different mutations in nine different codons. The majority of these alterations were not represented in previous reports (7, 25), thus further proving the efficiency of the method.
Also, the INH probe (TB sensor) utilized by Torres et al. (25) proved to be efficient for detecting mutations at katG codon 315. Using this probe, we were able to detect not only the previously described substitutions (ACC and AAC) but also three additional changes (ACT, ACA, and ATC) that were not present in the authors' collection. Analysis of the melting profiles of the three probes allowed the determination of the codon positions of mutations or even the distinction of different nucleotide substitutions at the same codon. As an alternative to FRET probes, we propose a set of three TaqMan MGB probes designed to detect the wt sequence AGC and the two most frequent substitutions, ACC and ACA. These short oligonucleotides are perfectly suited to search for mutations at a particular position, as in the case of katG codon 315. Compared to FRET probes, the TaqMan MGB probes offer the advantage of detecting a particular mutation that may have further implications, since different mutations may be associated with different levels of resistance.
Since almost 15% of the strains studied had a CT nucleotide substitution at position 15 upstream of the start site of the inhA gene, we applied a pair of standard FRET probes to search for mutations in the regulatory region of inhA (26). Mutations in this region cause the overexpression of this gene. The inhA-specific probe sequence was designed to bind to a mutant sequence at position –15 in the putative promoter site (A instead of C). In all cases, the Tms for the mutants were ca. 5°C higher than the Tm for the wt reference strain, proving this test efficient for detecting mutations in the regulatory region of inhA. We emphasize that in all cases studied, the results obtained by real-time PCR amplification corresponded to the nucleotide sequence data.
In the present study, we determined the occurrence and frequencies of different kinds of mutations at various target loci in drug-resistant clinical isolates of M. tuberculosis from Poland. We developed a basis for the detection of mutations underlying RMP and INH resistance in M. tuberculosis in Poland by the application of real-time PCR technology. This molecular method is fast and reliable and could be directly applied for examinations of clinical material.
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ACKNOWLEDGMENTS |
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We thank Olfert Landt (TIB MOLBIOL SyntheseLabor, Berlin, Germany) for designing the primers and FRET probes and for providing us with some of these as a gift for testing.
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FOOTNOTES |
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ód
, Poland. Phone: 48 42 6771250. Fax: 48 42 6771230. E-mail: jdziadek{at}cmiwpan.lodz.pl. 
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REFERENCES |
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baþ, Z., T. Kocagöz, A. Alp, and A. Günalp. 2003. Rapid detection of rifampin resistance in Mycobacterium tuberculosis isolates by heteroduplex analysis and determination of rifamycin cross-resistance in rifampin-resistant isolates. J. Clin. Microbiol. 41:816-818.
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