The lack of methods to identify Mycobacterium leprae with the resistance against multi-drugs quickly and specifically has hindered effective chemotherapy against M. leprae infection. To screen M. leprae with resistance against multi-drugs, the Touch-Down (TD)-PCR has been used in this study. Sequences of the folP, rpoA, B, and gyrA, B genes were analyzed for isolates of M. leprae from leprosy patients in Korea. We amplified designated region of several genes in M. leprae involved in drug resistance and could obtain the PCR products of each gene. The mutations in the particular region of folP, rpoB, and gyrB gene were certified by TD-PCR single-stranded conformational polymorphism and DNA sequencing, respectively.
Leprosy is a chronic infectious disease caused by Mycobacterium leprae and is still a major health problem globally. Although multi-drug therapy (MDT), introduced by the World Health Organization in 1981, has been very successful in reducing the prevalence of the disease, the annual reports of new cases indicate more than 500,000  and the emergence of the drug-resistant M. leprae had been reported [7,8,13,14]. The resistance pattern in M. leprae is comparable to drug resistance in Mycobacterium tuberculosis and raises major concern. For achievement of the elimination of leprosy, there are still many critical problems to be solved, such as identification and removal of source of infection, the route of transmission, and the detection of drug-resistant M. leprae as well as establishment of strategies of patients with drug-resistant bacilli.
The MDT regimen was based on rifampin, clofazimine, and dapsone. However, rifampin, ofloxacin, minocycline and clarithromycin were prescribed to patients who refused to take clofazimine fearing skin discoloration.
It is difficult to conduct the drug-resistance tests in short periods using clinical specimens, because M. leprae is not cultivable in artificial media. Therefore, M. leprae must be tested in mouse footpad assay to obtain drug susceptibility patterns . Because of the cumbersome nature of this drug screening method, comprehensive estimates of drug resistance in leprosy have been difficult to obtain. A widely used indirect measure of drug-resistant M. leprae is the radiometric analysis [3,4]. This method also has many disadvantages with regard to time, expense, and characterization of resistant mechanism for anti-leprosy drug. Therefore, it is necessary to establish rapid methods for detection of drug resistance in M. leprae to prevent and to identify existing cases of leprosy with drug resistance. Recently, the PCR was used to amplify various genomic sequences of M. leprae and other mycobacterial species to improve detection when low numbers of bacteria are present. The PCR has made it possible to clarify molecular changes in specific genes of M. leprae as well as other mycobacterial species with drug resistance [5–10].
A number of investigators reported the method for detection of M. leprae with drug resistance and the mutational changes in sequence of the three genes, folP[7,9,11], rpoB, and gyrA[13,14], which are responsible for resistance to dapsone, rifampin, and ofloxacin, respectively. However, because it is time-consuming to detect the mutation of the several genes in M. leprae with drug resistance, we tried to screen the several genes in short-step procedure.
Touch-Down (TD) PCR is a method for optimizing PCR by circumventing spurious priming during amplification even if the degree of primer–template complementary is not fully determined .
In this study, we analyzed the DNA sequences of particular regions of M. leprae folP, rpoB, and gyrB genes and applied the TD-PCR for screen of a single gene or multi-genes in M. leprae with resistance against drug from leprosy patients.
2 Materials and methods
2.1 Clinical specimens and bacterial isolates
Punch biopsies and isolates of M. leprae from seven leprosy patients were collected from the Institute of Hansen's Disease in Seoul. The patients had each shown a persistent or increased bacterial index in skin smears produced during or after routine treatment with MDT, including dapsone, rifampin, ofloxacin, and clarithromycin. A drug-susceptible strain of M. leprae, Thai 53, was used as a control.
2.2 Preparation of M. leprae DNA
After mincing half of the biopsy with No. 10 and No. 15 disposable scalpels in Petri dishes, 300 µl of PBS (phosphate buffered saline 10 mM, pH 7.2) was added and the sample was transferred to a micro-centrifuge tube. With three glass beads of 3 mm diameter, homogenization was performed by vortex for 3 min. To remove tissue debris, homogenates were centrifuged at 125×g for 10 min. After centrifugation of the supernatant at 15,000×g for 20 min, the pellet was resuspended in 50 µl of lysis buffer that contains proteinase K (10 mg) and Tween-80 (0.5%), and incubated at 60°C for 18 h, and was inactivated at 95°C for 10 min. M. leprae DNA was further extracted with phenol/chloroform/isoamyl alcohol (25:24:1) and chloroform/isoamyl alcohol (24:1), and then precipitated with 150 µl of ethanol. The precipitation was dried and dispensed with 20 µl of DW (Distilled Water) and incubated at 65°C for 5 min, and used as a template DNA.
Footpad granuloma was obtained from Thai 53 strain of M. leprae-infected nude mouse in the Institute of Hansen's Disease. Footpad was dissected, soaked in 1% iodine solution, and minced with scalpels. Homogenization was carried out in 2 ml of PBS with 25–30 glass beads by Mickle homogenizer (Mickle Laboratory Engineering Co., Surrey, UK). M. leprae DNA was isolated in the same manner as described above.
The nucleotide sequences of primers used in this study are listed in Table 1. A total 10 µl of mixture containing 1 µl template DNA and 100 pmol of each primer was overlaid with mineral oil in a reaction tube. A reaction mixture including 1.0 mM dNTPs, 0.5 U Taq DNA polymerase (Boehringer Mannheim, Mannheim, Germany), 50 mM KCl, 10 mM Tris–HCl (pH 8.0) and 1.5 mM MgCl2 was added. The reactants were put in the thermal cycler (GeneAmp PCR 9600, Applied Biosystems, Branchburg, CT, USA), which carried out the following TD-PCR program (Table 2). The amplified products were analyzed by electrophoresis in a 2% agarose gel and ethidium bromide staining.
The conditions of TD-PCR carried out in this study
94°C for 5 min, one cycle
TD program 1
45 s at 94°C, 45 s at 68°C, 90 s at 72°C, one cycle
45 s at 94°C, 45 s at 67°C, 90 s at 72°C, one cycle
45 s at 94°C, 45 s at 66°C, 90 s at 72°C, one cycle
45 s at 94°C, 45 s at 65°C, 90 s at 72°C, one cycle
45 s at 94°C, 45 s at 64°C, 90 s at 72°C, one cycle
45 s at 94°C, 45 s at 63°C, 90 s at 72°C, one cycle
45 s at 94°C, 45 s at 62°C, 90 s at 72°C, 35 cycles
72°C for 10 min, one cycle
↵TD means TD-PCR. The TD-PCR started with a high annealing temperature for the first primer-annealing step, and then reduced the annealing temperature by 1°C for each later cycle.
2.4 Single-stranded conformational polymorphism (SSCP) analysis by TD-PCR
To detect mutations in the genes of M. leprae involved in MDT resistance, SSCP analysis was employed using 0.5×MDE (Mutation Detection Enhancement) gel and autoradiography. Each PCR reaction was generally performed as described above except containing 0.5 µCi [32P]dCTP (Amersham Pharmacia Biotech, Buckinghamshire, UK). 10 µl of PCR-amplified fragment was mixed with 15 µl of 2×loading buffer solution containing 95% formamide, 10 mM NaOH, 20 mM EDTA, 0.02% bromophenol blue, and 0.02% xylene cyanol, and heated at 95°C for 5 min to denature the PCR product. Samples were quick-chilled on ice and 3 µl of reaction products were then immediately loaded onto a 0.5×MDE gel made with 0.6×TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA), 500 µl of ammonium persulfate, and 30 µl of TEMED. Electrophoresis was carried out for 18 h at 15 W. After electrophoresis, the gel was dried with vacuum dryer and then was subjected to autoradiography.
2.5 PCR-directed DNA sequencing
Aberrant bands extracted from the gel were reamplified and sequenced. To verify the sequence of the aberrant band, we used the same primers to amplify and sequence genomic DNA from each subject. M. leprae Thai 53 strain without drug resistance and without the aberrant band was also sequenced as control. The DNA sequences were determined using the AmpliCycle sequencing kit (Applied Biosystems, USA), which is a combination of PCR technique and Sanger's dideoxy sequence method. Sequencing was carried out according to the manufacturer's recommendations.
3.1 Identification of M. leprae
To identify M. leprae, we have used DNA-PCR with the primers for 18 kDa protein and RLEP sequence, which have M. leprae specificity. The result of PCR showed DNA bands with 362 bp and 129 bp, respectively (Fig. 1A).
TD-PCR for diagnosis of leprosy and detection of gene associated with MDT resistance of M. leprae Thai 53 obtained from nude mouse footpad. A: The result of PCR products for diagnosis of leprosy. Lane 1, 18 kDa (362 bp), lane 2, RLEP (129 bp). B: Reveals the amplifying results of the region for screen of mutant M. leprae with drug resistance. Lane 1, folP (177 bp); lanes 2 and 3, rpoB I (163 bp), II (179 bp); lanes 4 and 5, gyrA (187 bp), B (186 bp); lanes 6 and 7, 23S rRNA I (182 bp), II (180 bp); M is a DNA 100-bp ladder.
3.2 TD-PCR for the screen of drug-resistant M. leprae
In parallel, we amplified the region of folP, rpoB, gyrA, gyrB and 23S rRNA gene, involved with resistance for dapsone, rifampin, ofloxacin and clarithromycin , respectively, by TD-PCR and obtained the expected PCR products (Fig. 1B). These regions were targeted by anti-leprosy drug described above and showed frequency of mutation. The condition of PCR for primers is characterized in Table 3.
Characteristics of primers for diagnosis of leprosy and screening of MDT-resistant M. leprae used in this study
Screening of MDT resistance
3.3 Results of SSCP by TD-PCR and DNA sequencing
The results for each gene from clinical isolates are summarized in Table 4. Most of them showed a point mutation of a single gene, but the mutation with multi-drug resistance (dapsone and rifampin) was detected in only one case. Aberrantly migrating bands in folP, rpoB I, and gyrB genes could be observed on SSCP gel, whereas no mutation of 23S rRNA gene was detected in the tested M. leprae (Fig. 2). After isolation of DNA from aberrant bands, mutations of each gene were detected. All mutations were missense variants caused by single-nucleotide substitution. The mutations of folP, rpoB and gyrB were certified by TD-PCR SSCP and by direct DNA sequencing. The mutation of folP had a C to G transition at nucleotide 164 (Pro55Arg). The mutations of rpoB and gyrB had a C to T at nucleotide 1592 (Ser531Leu) and had a G to A at nucleotide 613 (Asp205Asn), respectively (Fig. 3).
TD-PCR followed by SSCP analysis for detection of mutant M. leprae with drug resistance. Bacteria were isolated from patients to be suspected with drug resistance. Aberrant bands are showed in only lanes 1, 2, and 5. Lane 1, folP; lanes 2 and 3, rpoB I,II; lanes 4 and 5, gyrA, B; lanes 6 and 7, 23S rRNA I,II, respectively. This figure shows the results from one-step PCR and one SSCP gel. The left band of each lane indicates Thai 53 strain, the wild-type of M. leprae.
The mutations in the region of folP, rpoB I, and gyrB genes in clinical isolates. SSCP (A–C) and sequencing analysis (D–F) of DNA from wild-type, Thai 53 (N) and mutant (M) strain of M. leprae. SSCP shows aberrant band (arrow-head) in mutant DNA when compared with SSCP from normal DNA. Cyclic sequencing analyses were performed using each of M. leprae DNA resulting from aberrant bands in SSCP analysis. D: Revealed a C to G transition (arrow) at codon 55 (CCC→CGC, Pro55Arg) in folP gene (A,D). In case of rpoB I (B,E) and gyrB (C,F), there were a C to T transition (arrow) at codon 531 (TCG→TTG, Ser531Leu) and a G to A transition (arrow) at codon 205 (GAT→AAT, Asp205Asn), respectively.
The principal goal of the present study was the detection of drug-resistant M. leprae from leprosy patients and the development of the method for a rapid detection of multi-gene-resistant M. leprae from leprosy patients. We suggest the TD-PCR is a method for detection and screen of M. leprae strain with resistance against multi-drugs, because it is possible to detect the mutation of several genes in a single amplification regimen.
It is general to do a susceptibility test before specific chemotherapy in bacterial infection, but MDT for 12 or 24 months against M. leprae infection was usually started without any information of drug resistance. For monitoring of drug resistance for M. leprae, the mouse footpad method and the radiometric analysis have been used. Neither the mouse footpad method, which takes up to 1 year, nor the radiometric analysis, which requires relatively large numbers of metabolically active M. leprae, is suitable for clinical specimen such as biopsy. Thus, the research on early detection and confirmation of drug-resistant M. leprae is still needed. Advances in the elucidation of molecular events responsible for drug resistance in mycobacteria have allowed the development of new tools for drug-resistance screening.
Recently, the method for simultaneous detection of M. leprae and its susceptibility to dapsone by using DNA heteroduplex analysis was reported . However it is difficult to screen the multi-drug resistance in M. leprae.
TD-PCR started with a high annealing temperature for the first primer-annealing step, and then reduced the annealing temperature by 1°C for each later cycle. TD-PCR can reduce nonspecific amplification and the time for optimized PCR reaction, and can improve specificity and product yield . The procedure involved programming a thermocycler so that the annealing temperature of the initial two cycles was well above the estimated melting temperature of the primer–template complexes. The annealing temperature was then decreased by 1°C every second cycle. TD-PCR programs usually contain a greater number of cycles than conventional PCR to compensate for the fact that the efficient amplification occurs first after a few cycles have been run. This method had been used in screen the mutation of only the rpoB gene of M. leprae. In the present study, we analyzed the DNA sequences of particular regions of M. leprae folP, rpoB, gyr and 23S rRNA genes, which are responsible for resistance to dapsone, rifampin, ofloxacin, and clarithromycin, respectively.
A simple DNA-based approach for the rapid screening of strains for the presence of resistant allele can be used. This could be done by means of SSCP analysis and direct sequencing. SSCP results by TD-PCR presented in Fig. 2 showed the variant bands by missense mutation, which was confirmed by direct DNA sequencing (Fig. 3), and suggested that it should be a relatively rapid and simple matter to extend this approach to multi-drug-resistant M. leprae isolates from clinical specimens.
We also report a new polymorphism of gyrB in M. leprae and the mutation is associated with ofloxacin resistance in leprosy. The mutation of folP and rpoB showed in this study were already demonstrated to be associated with drug resistance by animal study in other reports [9,11,14,18]. Mutations in the quinolone-resistance-determining region of gyrA were also reported for quinolone-resistant mycobacteria, M. tuberculosis and M. leprae. In this study, one mutation (Asp→Asn at position 205) in the region of gyrB was detected in isolate of M. leprae from the clinical specimen. Because the M. leprae strain was isolated from the patient who did not show any improvement of signs after treatment with ofloxacin, it is likely that the mutation is associated with drug resistance. However, it is not clear that such mutation is linked to drug resistance in M. leprae because there have been no reports on the positions of mutations which are responsible for ofloxacin resistance. We are investigating the relationship between genotype mutation and phenotypic resistance using M. leprae isolates in the mouse footpad assay.
Mycobacteria can be visualized by staining followed by microscopy, but this is not specific for M. leprae. DNA probes offer a route to the more sensitive detection and identification of M. leprae DNA through the application of the PCR using M. leprae specific primers. Therefore, we have used the AFB (Acid Fast Bacilli) staining (data not shown) and the PCR for identification of the DNA, which encodes M. leprae protein, 18 kDa  and the repetitive sequence of M. leprae, RLEP  (Fig. 1A), as a more sensitive and specific method than the empirical AFB examination.
In this study, a rapid TD-PCR-based assay was developed for the detection of M. leprae and of its resistance to multi-drugs from clinical specimens in a single experiment of PCR. Accordingly, TD-PCR may be useful for rapidly identifying patients infected with MDT-resistant M. leprae as well as the diagnosis of leprosy. Further investigation of the relation between our result of gyrB mutation and ofloxacin resistance will be certified by a drug-susceptibility test using mouse footpad assay.
This work was supported by grant from the Catholic Medical Center Research Foundation (2001).