- Email: [email protected]
Molecular analysis of isoniazid resistance in Mycobacterium tuberculosis isolates recovered from South Korea
Diagnostic Microbiology and Infectious Disease 47 (2003) 497–502
www.elsevier.com/locate/diagmicrobio
Mycobacteriology
Molecular analysis of isoniazid resistance in Mycobacterium tuberculosis isolates recovered from South Korea Soo-Young Kima, Yeon-Joon Parkb, Won-Il Kimc,*, Sun-Hwa Leed, Chulhun Ludgerus Change, Seok-Jin Kanga, Chang-Suk Kangc a
Department of Clinical Pathology, College of Medicine, the Catholic University of Korea, St. Vincent’s Hospital, 93 Chi-dong, Paldal-ku, Suwon 442-723, South Korea b Department of Clinical Pathology, College of Medicine, the Catholic University of Korea, Kangnam St. Mary’s Hospital, 505 Banpo-dong, Seocho-ku, Seoul 137-040, South Korea c Department of Clinical Pathology, College of Medicine, the Catholic University of Korea, St. Mary’s Hospital, 62 Youido-dong, Youngdeungpo-ku, Seoul 150-713, South Korea d Neodin Medical Institute, 2-3 Yongdap-dong, Sungdong-ku, Seoul 133-847, South Korea e Department of Clinical Pathology, College of Medicine, Pusan National University College of Medicine, 1-10 Ami-dong, Seo-ku, Pusan 602-739, South Korea Received 6 January 2003; received in revised form 19 May 2003
Abstract The katG, inhA and ahpC genes, in 71 isoniazid (INH)-resistant and 26 INH-susceptible Mycobacterium tuberculosis isolates, from South Korea were examined by sequencing and MspI restriction enzyme analysis. Mutations in the katG 315 alone, katG 315 plus inhA, katG 315 plus ahpC, katG 309 alone, katG 309 plus inhA, inhA alone, and ahpC alone, were detected in 54.9, 2.8, 1.4, 1.4, 1.4, 19.7, and 5.6% of the 71 INH-resistant isolates, respectively. There was no statistically significant difference (p ⬎ 0.05) in the frequencies of these mutations for the INH-monoresistant compared with the multidrug-resistant isolates. Mutations in the katG codon 315 were associated with the high-level of INH resistance (MIC, ⬎1 g/ml), whereas the mutation in the inhA promoter region was associated with the low-level of INH resistance (MIC, ⬎0.2 to 1 g/ml). The previously undescribed GGT3 GAT (Gly3 Asp) mutation in the katG codon 309 was found in two rifampin, including-multidrug-resistant isolates, but we cannot assess if this is predictive of INH resistance. The sensitivity and specificity of molecular analysis of the katG codon 315 and/or the inhA promoter region were 80.3 and 100%, respectively. Therefore, mutations in these regions are highly predictive of INH resistance in South Korea. © 2003 Elsevier Inc. All rights reserved. Keywords: Mycobacterium tuberculosis; Isoniazid resistance; katG; inhA; ahpC
1. Introduction From 1995 to 2002, the prevalence of radiologically diagnosed active pulmonary tuberculosis decreased from 1 to 0.5% of total South Korean population (Ministry of Health & Welfare, & Korean National Tuberculosis Association, 1995; unpublished data). From 1996 to 2000, the resistance to isoniazid (INH) and rifampin (RIF) decreased from 34.2 to 14.7% and 35.9 to 9.7%, respectively, and the resistance to them in combination dropped from 25.3 to 7.3% (Lee et al., 2001). Improved living conditions, in* Corresponding author. Tel.: ⫹82-2-3779-1296; fax: ⫹82-2-7836648. E-mail address: [email protected] (W.-I. Kim). 0732-8893/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0732-8893(03)00132-9
creased national medical insurance rate, South Korea’s universal BCG vaccination policy, and advanced antituberculosis chemotherapy contributed to these decreases, but the prevalence of tuberculosis and the resistance to primary antituberculosis drugs, is still much higher in South Korea than in developed countries. Therefore, the rapid detection of drug resistance is essential for continued decreases in the prevalence of tuberculosis and drug resistance in South Korea. The determination of drug resistance in M. tuberculosis using conventional culture methods routinely takes 6 to 10 weeks. In order to shorten this period, targeted molecular approaches have been performed in Europe and the United States. Genomic mutations frequently associated with resistance to each of the primary antituberculosis drugs have
498
S.-Y. Kim et al. / Diagnostic Microbiology and Infectious Disease 47 (2003) 497–502
been identified. More than 95% of RIF-resistant isolates are associated with mutations within an 81-bp region of the rpoB gene; thus, investigation of RIF resistance is relatively straightforward. In contrast, resistance to INH is associated with a variety of mutations that affect several genes, including: those encoding the catalase-peroxidase (katG), which converts INH to an active form; the enoyl acyl carrier protein reductase (inhA), which is involved in mycolic acid biosynthesis; the alkyl-hydroperoxide reductase (ahpC), which is involved in the cellular response to oxidative stress; the ketoacyl acyl carrier protein synthase (kasA), which is important in fatty acid elongation; and the NADH dehydrogenase (ndh) (Lee et al., 2001; Ramaswamy & Musser, 1998). The katG gene is the most commonly altered, with the majority of mutations occurring in codon 315 (Haas et al., 1997; Musser et al., 1996). Mutations in the katG codon 315 and the promoter region of the inhA have been identified in INH-resistant, but not INH-susceptible isolates (Dobner et al., 1997; Kiepiela et al., 2000; Nachamkin et al., 1997). With a few exceptions, most ahpC promoter mutations have been identified in INH-resistant isolates (Rinder et al., 1998; Sreevatsan et al., 1997). Mutations in the kasA gene have been frequently identified in INH-susceptible isolates (Lee et al., 1999; Piatek et al., 2000). Mutations occurring in the katG codon 315, together with those in the promoter regions of the inhA and ahpC, may account for most of isolates resistant to INH. However, because of considerable variance in the reported data, more information is required on the molecular mechanisms of INH resistance. The prevalence of INH-resistant tuberculosis in South Korea is known, but no serious efforts have been made to identify the INH resistance genotypes or their prevalence in the community. The purpose of our study was to determine the frequencies of mutations in the katG codon 315 and promoter regions of the inhA and ahpC, in the 71 INH-resistant and 26 INH-susceptible isolates from South Korea, and their association with multidrug resistance and the level of INH resistance.
2. Materials and methods 2.1. Mycobacterial isolates Ninety-seven clinical M. tuberculosis isolates were collected over 3 years between 2000 and 2002 from 3 clinical laboratories in South Korea. The 42 INH-resistant isolates were obtained from the Korean Institute of Tuberculosis, the 19 INH-resistant and 15 INH-susceptible isolates from the Neodin Medical Institute and the 10 INH-resistant and 11 INH-susceptible isolates from the Kangnam St. Mary’s Hospital. Susceptibility testing was performed by the 1% proportion method with Lo¨ wenstein-Jensen (L-J) medium, with the following critical drug concentrations: INH, 0.2
Table 1 Names and sequences of primers Target
Primer
katG 315
TB86, TB87, TB92, TB93, TB90, TB91,
inhA ahpC
Product size (bp) 5⬘-GAAACAGCGGCGCTGGATCGT 5⬘-GTTGTCCCATTTCGTCGGGG 5⬘-CCTCGCTGCCCAGAAAGGGA 5⬘-ATCCCCCGGTTTCCTCCGGT 5⬘-CCGATGAGAGCGGTGAGCTG 5⬘-ACCACTGCTTTGCCGCCACC
210 248 237
g/ml; RIF, 40 g/ml; streptomycin, 4 and 10 g/ml; ethambutol, 2 g/ml; kanamycin, 40 g/ml; enviomycin, 40 g/ml; prothionamide, 40 g/ml; cycloserine, 30 g/ml; paraaminosalicylic acid, 1 g/ml; ofloxacin, 2 g/ml. Fiftyseven isolates were further tested with 1 g/ml INH to determine the high-level of INH resistance. Twenty-five isolates were INH monoresistant, 33 were RIF-including-multidrug resistant and 13 were RIF-excluding-multidrug resistant. RIF-including-multidrug resistance was defined as resistance to at least INH and RIF according to the World Health Organization. RIF-excluding-multidrug resistance was defined as resistance to INH and other drugs, excluding RIF in this study. Twenty-six INH-susceptible isolates were susceptible to all the other antituberculosis drugs tested. 2.2. Polymerase chain reaction (PCR) amplification and sequencing M. tuberculosis cultures were grown on Ogawa or L-J medium at 37°C for 4 to 6 weeks and stored at room temperature in the dark, ready for use. Two to six mycobacterial colonies were collected from the agar surface, and the DNA extracted using a QIAamp DNA mini kit (QIAGEN, Hilden, Germany). Primers for the amplification of the katG codon 315, inhA and ahpC are listed in Table 1 (Telenti et al., 1997). The amplification reactions were performed in a final volume of 50 L, containing: 0.2 g of genomic DNA, 20 pmol of each primer, 0.2 mM of deoxynucleotide, 1X PCR buffer (10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, pH 8.3) and 2.5 U of Taq polymerase (Roche Boehringer Mannheim, Indianapolis, USA). The PCR was performed in a PTC-200 Peltier thermal cycler (MJ Research, Watertown, USA) using the following conditions: initial denaturation step at 94°C for 3 min; 30 cycles of denaturation at 94°C for 45 sec, annealing at 65°C for 30 sec, and extension at 72°C for 30 sec; and a final extension at 72°C for 5 min. The PCR products were purified with a QIAquick PCR purification kit (QIAGEN, Hilden, Germany), and directly sequenced using a BigDye Terminators sequencing kit and an ABI PRISM 3700 automated sequencer (Applied Biosystems, Foster city, USA).
S.-Y. Kim et al. / Diagnostic Microbiology and Infectious Disease 47 (2003) 497–502
499
Table 2 Frequencies of mutations associated with INH resistance in the regions examined Isolates
INH-resistant (n ⫽ 71) INH only (n ⫽ 25) INH ⫹ RIFa (n ⫽ 33) INH ⫹ otherb (n ⫽ 13) INH-susceptible (n ⫽ 26) a b
Localization of mutations [no. (%)] katG 315 alone
katG 315/inhA
katG 315/ahpC
katG 309 alone
katG 309/inhA
inhA alone
ahpC alone
Other
39 (54.9) 14 (56) 16 (48.5) 9 (69.2) 0
2 (2.8) 2 (8) 0 0 0
1 (1.4) 1 (4) 0 0 0
1 (1.4) 0 1 (3) 0 0
1 (1.4) 0 1 (3) 0 0
14 (19.7) 4 (16) 8 (24.2) 2 (15.4) 0
4 (5.6) 0 3 (9.1) 1 (7.7) 1 (3.9)
9 (12.7) 4 (16) 4 (12.1) 1 (7.7) 25 (96.2)
RIF, rifampin; resistant to at least INH and RIF (RIF-including-multidrug resistant). resistant to INH and other drugs, excluding RIF (RIF-excluding-multidrug resistant).
2.3. Restriction fragment length polymorphism (RFLP) MspI restriction enzyme analysis of the 210 bp fragment of the katG gene was undertaken to confirm the Ser315Thr mutation as follows: PCR-amplicons (10 L) were incubated with 7 L of deionized water, 2 L of 10X buffer (10 mM Tris-HCl, 10 mM MgCl2, 1 mM dithioerythritol, pH 7.5) and 1 L of MspI (10 U/l) (Roche Boehringer Mannheim, Indianapolis, USA) at 37°C for 1hr. The resulting restricted products were electrophoresed at 100V for 1.5 hr into a 10% polyacylamide gel by using 0.5X TBE (Trisborate-EDTA) buffer, and the bands were visualized by UV transillumination. 2.4. Statistical analysis Statistical comparisons were made using Chi-squared tests, with p ⬍ 0.05 considered as statistically significant.
3. Results Mutations in the katG codon 315 alone, katG codon 315 plus inhA, katG codon 315 plus ahpC, katG codon 309 alone, katG codon 309 plus inhA, inhA alone, and ahpC alone, were detected in 54.9, 2.8, 1.4, 1.4, 1.4, 19.7, and 5.6% of the 71 INH-resistant isolates, respectively (Table 2), but with no statistically significant difference (p ⬎ 0.05) in the frequencies of these mutations for the INH-monoresistant compared with the RIF-including- or excluding-multidrug-resistant isolates. Mutations in the katG codon 315 and/or the inhA promoter region and/or the ahpC promoter region were detected in 61 (85.9%), and mutations in the katG codon 315 and/or the inhA promoter region in 57 (80.3%) of the 71 INH-resistant isolates. Mutations in the katG codon 315 and/or inhA were not detected in all INHsusceptible isolates, but one INH-susceptible isolate had a mutation in the ahpC promoter region, a G3 A substitution at position -46 relative to the ahpC mRNA start site (Table 2 & 3). Therefore, if mutations in the codon 315 of the katG and the promoter regions of the inhA and ahpC were considered, the sensitivity and specificity were 85.9 and 96.2%,
respectively. If mutations in codon 315 of the katG and the promoter region of the inhA were considered, the corresponding values were 80.3 and 100%, respectively. Of the 42 INH-resistant isolates with mutations in the katG codon 315, 37 had a single AGC3 ACC (Ser3 Thr) mutation, and 2 INH-monoresistant isolates had both this mutation and one in the inhA promoter region. Two RIFexcluding-multidrug-resistant isolates had a single AGC3 AAC (Ser3 Asn) mutation in the katG codon 315, and 1 INH-monoresistant isolate had both an AGC3 CGC (Ser3 Arg) mutation in the katG codon 315 and a mutation in the ahpC promoter region, a C3 T substitution at position -39. Thus, mutations in the katG codon 315 were detected in 42 (59.2%) of the 71 INH-resistant isolates (Table 2 & 3). The RFLP analysis of the katG detected all the Ser315Thr mutations, but no other mutations. Therefore, the RFLP analysis detected 39 (54.9%) of the 71 INHresistant isolates. Two RIF-including-multidrug-resistant isolates had an GGT3 GAT (Gly3 Asp) in the katG codon 309. Of these two isolates, one had a single mutation, and the other had a mutation in the inhA promoter region also (Table 2 & 3). Seventeen (23.9%) of the 71 INH-resistant isolates had a C3 T substitution on the 5⬘ side of the presumed ribosomal binding site in the inhA promoter region. Of the 4 isolates exhibiting resistance to both INH and ethionamide, 3 showed this mutation, whereas 14 (20.9%) of the 67 isolates exhibiting resistance to INH only or INH and other drugs, excluding ethionamide, showed this mutation (p ⬍ 0.05). Five (7%) of the 71 INH-resistant isolates had mutations in 5 different locations of their ahpC promoter regions (Table 3). Table 4 shows the association between the level of INH resistance and the location of mutations detected in 57 INH-resistant isolates, with these being tested at concentrations of 0.2 and 1 g/ml INH. Mutations in the katG codon 315 were significantly more common in the isolates with the high-level of INH resistance (MIC, ⬎1 g/ml) (76.2%) than in those with the low-level of INH resistance (MIC, ⬎0.2 to 1 g/ml) (40%; p ⬍ 0.05). In contrast, mutations in the inhA promoter region were significantly more common in the isolates with the low-level of INH resistance (46.7%) than
500
S.-Y. Kim et al. / Diagnostic Microbiology and Infectious Disease 47 (2003) 497–502
Table 3 Mutations identified in 71 INH-resistant and 26 INH-susceptible isolates Gene
Positiona
Mutation
Mutation type
Amino acid change
Isolatesb
katG
315
AGC3ACC AGC3AAC AGC3CGC GGT3GAT C3T G3A C3T G3T C3T G3A
Reported Reported Reported Novel Reported Reported Reported Reported Reported Reported
Ser3Thr Ser3Asn Ser3Arg Gly3Asp
16 Ir, 16 Mr, 7 Or 2 Or 1 Ir 2 Mr 6 Ir, 9 Mr, 2 Or 1 Mr, 1 S 1 Ir 1 Mr 1 Or 1 Mr
inhA ahpC
309 Ribosomal binding site -46 -39 -12 -10 -9
a
Numbering of the ahpC promoter region nucleotide in relation to the ahpC mRNA start site. I , resistant to INH only; Mr, resistant to at least INH and RIF (RIF-including-multidrug resistant); Or, resistant to INH and other drugs, excluding RIF (RIF-excluding-multidrug resistant); S, susceptible. b r
in those with the high-level of INH resistance (4.8%; p ⬍ 0.05).
4. Discussion Genotypic testing of antimycobacterial drug resistance is an attractive alternative to conventional susceptibility testing due to its ability to provide rapid and accurate results. Although a variety of mutations are associated with INH resistance, mutations in the katG codon 315 and the promoter regions of the inhA and ahpC have been shown to be more prevalent than other mutations (Piatek et al., 2000; Telenti et al., 1997). This study has identified the frequencies of mutations in these loci in M. tuberculosis isolates from South Korea, and mutations in the katG codon 315 and the inhA promoter region should be assayed for predicting INH resistance in South Korea. We found mutations in the katG codon 315 in 59.2% of the INH-resistant isolates, and therefore concluded that this mutation to have the highest predictive power for INH resistance in M. tuberculosis. Most investigators found these mutations in 34.6 to 62.2% of INH-resistant isolates, for which MICs of INH were at least ⬎0.2 g/ml (Gonzalez et al., 1999; Piatek et al., 2000; Table 4 Association between the level of INH resistance and the location of mutations detected in 57 INH-resistant isolates Mutation
katG codon 315 alone inhA alone ahpC alone katG codon 315 plus inhA katG codon 309
No. (%) of isolates High-levela (n ⫽ 42)
Low-levelb (n ⫽ 15)
32 (76.2) 2 (4.8) 3 (7.1) 2 (4.8) 0
6 (40) 7 (46.7) 0 0 1 (6.7)
MIC of INH was ⬎1 g/ml for the 42 isolates. MIC of INH was ⬎0.2 to 1 g/ml for the 15 isolates. c NS, not significant. a
b
P
⬍0.05 ⬍0.05 NSc NS NS
Telenti et al., 1997), whereas other investigators found these mutations in 78.1 to 97.4% of INH-resistant isolates, for which MICs of INH were at least ⬎1 g/ml (Abate et al., 2001; Kiepiela et al., 2000; Marttila et al., 1998). These findings suggest that mutations in the katG codon 315 are associated with the high-level of INH resistance. This was also observed in our study; of the 42 isolates exhibiting the high-level of INH resistance (MIC, ⬎1 g/ml), 32 (76.2%) showed these mutations, whereas 6 (40%) of the 15 isolates exhibiting the low-level of INH resistance (MIC, ⬎0.2 to 1 g/ml) showed these mutations (p ⬍ 0.05). Geographical differences in the frequencies of these mutations were also apparent in the analysis of data from other studies: mutations in the katG codon 315 were detected in 34.6% of the INH-resistant isolates from Madrid, but in 62.2% from New York (Piatek et al., 2000). Therefore, the documented information regarding the frequencies and types of mutaions in one country or geographical region may not be generally applicable. Previous investigators reported that mutations in the katG codon 315 were associated with multidrug resistance (Piatek et al., 2000; van Soolingen et al., 2000). In this study, however, no significant differences were found in the frequencies of these mutations between the INH-monoresistant and the RIF-including- or excluding-multidrug-resistant isolates. Heteroresistance has been observed in M.tuberculosis (Rinder et al., 2001), and isolates were cultured on media containing both 0.2 and 0.5 g/ml INH to increase detection of these mutations. However, repeated tests with colonies taken from the INH-containing media produced identical results (data not shown). The RFLP analysis detected all the Ser315Thr mutations, which were 92.9% of the katG codon 315 mutations, and is regarded as relatively simple, rapid and inexpensive technique to screen these mutations in a clinical laboratory. Mutations in the katG codon 309 seem to be rare, and have not previously been reported. In our sample, two mutations were encountered in this position. Because no other mutations were observed in one isolate, we suggest
S.-Y. Kim et al. / Diagnostic Microbiology and Infectious Disease 47 (2003) 497–502
that the katG codon 309 was involved in the resistance mechanism of this isolate. However, information on the mutation in the katG codon 309 was not available for many of the isolates. This association will be explored in future studies. With regard to the promoter region of the inhA, this mutation was found in 23.9% of the INH-resistant isolates. Most investigators found these mutations in 10 to 34.2% of the INH-resistant isolates (Gonzalez et al., 1999; Kiepiela et al., 2000; Lee et al., 1999; Telenti et al., 1997). Of the 4 isolates exhibiting resistance to both INH and ethionamide, 3 showed this mutation, whereas 14 (20.9%) of 67 isolates exhibiting resistance to INH only or INH and other drugs, excluding ethionamide, showed this mutation (p ⬍ 0.05). Therefore, we confirmed that a C3 T substitution in the inhA promoter region occurred at a disproportionately higher rate in isolates resistant to both INH and ethionamide, than in isolates resistant to INH only or INH and other drugs, excluding ethionamide (Lee et al., 2000). In the inhA promoter region, a C3 T substitution on the 5⬘end of the presumed ribosomal binding site was the only mutation detected in this study, and M.tuberculosis isolates recovered from Korea and Spain also showed only this single mutation in the inhA promoter region (Lee et al., 2000; Telenti et al., 1997). However, our data were at variance with the study of Kiepiela et al. (2000), who reported that most isolates from Africa had the T3 A nucleotide substitution. In the case of the inhA, discrepancies in the results between studies seem to reflect the different geographical prevalences of specific genotypes. In contrast to the katG codon 315 mutations, the mutation in the inhA promoter region was associated with the low-level of INH resistance (p ⬍ 0.05). Mutations in the ahpC promoter region occurred at a low frequency (7%) among the INH-resistant isolates and were not obligatory in the INH-resistant isolates. Most investigators found these mutations in 4.8 to 24.2% of the INHresistant isolates (Kelly et al., 1997; Lee et al., 2001; Rinder et al., 1998; Telenti et al., 1997). Simple nucleotide substitutions were identified at positions -9, -10, -12, -39, and -46 (designated relative to the ahpC mRNA start site). Of these, the nucleotide substitution at position -46 was observed in a susceptible isolate, which was in line with the study of Sreevatsan et al. (1997), who reported that the nucleotide substitutions at position -46 and ⫹33 were not associated with INH resistance. Although other mutations in the ahpC promoter region appeared to be predictive of INH resistance, analysis of this target contributed minimally to the diagnostic strategy due to the low frequency. Mutations in the ahpC promoter region were identified in katG-defective, catalase-negative tubercle bacilli only to compensate for the loss of catalase-peroxidase activity (Kelly et al., 1997). However, our analysis showed that most isolates with the katG codon 315 missense mutations had no mutation in the ahpC promoter region and hence confirmed there was no simple relationship between these two mutations (Rinder et al., 1998; Sreevatsan et al., 1997; Telenti et al., 1997).
501
In the management of mycobacterial infections, the rapid detection and appropriate treatment with susceptible drugs is crucial. These goals can be achieved by improving the diagnosis of drug-resistant tuberculosis using molecular methods. In this study, if mutations in the katG codon 315 and the promoter region of the inhA were considered, the sensitivity and specificity were 80.3 and 100%, respectively. If mutations in the katG codon 315 and the promoter regions of the inhA and ahpC were considered, the corresponding values were 85.9 and 96.2%, respectively. Therefore, molecular analysis of the katG codon 315 and the promoter region of the inhA might be a good alternative to conventional susceptibility testing for the detection of INH resistance in South Korea.
Acknowledgments The authors thank Dong Hwa Han for excellent technical assistance. This study was supported in part by the Clinical Research Fund of St. Vincent’s Hospital.
References Abate, G., Hoffner, S. E., Thomsen, V. O., & Miorner, H. (2001). Characterization of isoniazid-resistant strains of Mycobacterium tuberculosis on the basis of phenotypic properties and mutations in katG. Eur J Clin Microbiol Infect Dis 20, 329 –333. Dobner, P., Rusch-Gerdes, S., Bretzel, G., Feldmann, K., Rifai, M., Loscher, T., & Rinder, H. (1997). Usefulness of Mycobacterium tuberculosis genomic mutations in the genes katG and inhA for the prediction of isoniazid resistance. Int J Tuberc Lung Dis 1, 365–369. Gonzalez, N., Torres, M. J., Aznar, J., & Palomares, J. C. (1999). Molecular analysis of rifampin and isoniazid resistance of Mycobacterium tuberculosis clinical isolates in Seville, Spain. Tuber Lung Dis 79, 187–190. Haas, W. H., Schilke, K., Brand, J., Amthor, B., Weyer, K., Fourie, P. B., Bretzel, G., Sticht-Groh, V., & Bremer, H. J. (1997). Molecular analysis of katG gene mutations in strains of Mycobacterium tuberculosis complex from Africa. Antimicrob Agents Chemother 41, 1601–1603. Kelley, C. L., Rouse, D. A., & Morris, S. L. (1997). Analysis of ahpC gene mutations in isoniazid-resistant clinical isolates of Mycobacterium tuberculosis. Antimicrob Agents Chemother 41, 2057–2058. Kiepiela, P., Bishop, K. S., Smith, A. N., Roux, L., & York, D. F. (2000). Genomic mutations in the katG, inhA and ahpC genes are useful for the prediction of isoniazid resistance in Mycobacterium tuberculosis isolates from Kwazulu Natal, South Africa. Tuber Lung Dis 80, 47–56. Lee, A. S., Lim, I. H., Tang, L. L., Telenti, A., & Wong, S. Y. (1999). Contribution of kasA analysis to detection of isoniazid-resistant Mycobacterium tuberculosis in Singapore. Antimicrob Agents Chemother 43, 2087–2089. Lee, A. S., Teo, A. S., & Wong, S. Y. (2001). Novel mutations in ndh in isoniazid-resistant Mycobacterium tuberculosis isolates. Antimicrob Agents Chemother 45, 2157–2159. Lee, H., Cho, S. N., Bang, H. E., Lee, J. H., Bai, G. H., Kim, S. J., & Kim, J. D. (2000). Exclusive mutations related to isoniazid and ethionamide resistance among Mycobacterium tuberculosis isolates from Korea. Int J Tuberc Lung Dis 4, 441– 447. Lee, S. H., Kim, S. Y., Kea, H. K., & Hwang, Y. S. (2001). The results of surveys of resistance to antituberculous drugs. Korean J Clin Microbiol 4, S98 –S99.
502
S.-Y. Kim et al. / Diagnostic Microbiology and Infectious Disease 47 (2003) 497–502
Marttila, H. J., Soini, H., Eerola, E., Vyshnevskaya, E., Vyshnevskiy, B. I., Otten, T. F., Vasilyef, A. V., & Viljanen, M. K. (1998). A Ser315Thr substitution in katG is predominant in genetically heterogeneous multidrug-resistant Mycobacterium tuberculosis isolates originating from the St. Petersburg area in Russia. Antimicrob Agents Chemother 42, 2443–2445. Ministry of Health & Welfare, & Korean National Tuberculosis Association. (1995). Report on the 7th tuberculosis prevalence survey in Korea. Musser, J. M., Kapur, V., Williams, D. L., Kreiswirth, B. N., van Soolingen, D., & van Embden, J. (1996). Characterization of the catalaseperoxidase gene (katG) and inhA locus in isoniazid-resistant and -susceptible strains of Mycobacterium tuberculosis by automated DNA sequencing: restricted array of mutations associated with drug resistance. J Infect Dis 173, 196 –202. Nachamkin, I., Kang, C., & Weinstein, M. P. (1997). Detection of resistance to isoniazid, rifampin, and streptomycin in clinical isolates of Mycobacterium tuberculosis by molecular methods. Clin Infect Dis 24, 894 –900. Piatek, A. S., Telenti, A., Murray, M. R., El-Hajj, H., Jacobs, W. R. Jr., Kramer, F. R., & Alland, D. (2000). Genotypic analysis of Mycobacterium tuberculosis in two distinct populations using molecular beacons: implications for rapid susceptibility testing. Antimicrob Agents Chemother 44, 103–110.
Ramaswarmy, S., & Musser, J. M. (1998). Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis: 1998 update. Tuber Lung Dis 79, 3–29. Rinder, H., Thomschke, A., Rusch-Gerdes, S., Bretzel, G., Feldmann, K., Rifai, M., & Loscher, T. (1998). Significance of ahpC promoter mutations for the prediction of isoniazid resistance in Mycobacterium tuberculosis. Eur J Clin Microbiol Infect Dis 17, 508 –511. Rinder, H., Mieskes, K., & Loscher, T. (2001). Heteroresistance in Mycobacterium tuberculosis. Int J Tuberc Lung Dis 5, 339 –345. Sreevatsan, S., Pan, X., Zhang, Y., Deretic, V., & Musser, J. M. (1997). Analysis of the oxyR-ahpC region in isoniazid-resistant and -susceptible Mycobacterium tuberculosis complex organisms recovered from diseased humans and animals in diverse localities. Antimicrob Agents Chemother 41, 600 – 606. Telenti, A., Honore, N., Bernasconi, C., March, J., Ortega, A., Heym, B., Takiff, H., & Cole, S. T. (1997). Genotypic assessment of isoniazid and rifampin resistance in Mycobacterium tuberculosis: a blind study at reference laboratory level. J Clin Microbiol 35, 719 –723. Van Soolingen, D., de Haas, P. E., van Doorn, H. R., Kuijper, E., Rinder, H., & Borgdorff, M. W. (2000). Mutations at amino acid position 315 of the katG gene are associated with high-level resistance to isoniazid, other drug resistance, and successful transmission of Mycobacterium tuberculosis in the Netherlands. J Infect Dis 182, 1788 –1790.
www.elsevier.com/locate/diagmicrobio
Mycobacteriology
Molecular analysis of isoniazid resistance in Mycobacterium tuberculosis isolates recovered from South Korea Soo-Young Kima, Yeon-Joon Parkb, Won-Il Kimc,*, Sun-Hwa Leed, Chulhun Ludgerus Change, Seok-Jin Kanga, Chang-Suk Kangc a
Department of Clinical Pathology, College of Medicine, the Catholic University of Korea, St. Vincent’s Hospital, 93 Chi-dong, Paldal-ku, Suwon 442-723, South Korea b Department of Clinical Pathology, College of Medicine, the Catholic University of Korea, Kangnam St. Mary’s Hospital, 505 Banpo-dong, Seocho-ku, Seoul 137-040, South Korea c Department of Clinical Pathology, College of Medicine, the Catholic University of Korea, St. Mary’s Hospital, 62 Youido-dong, Youngdeungpo-ku, Seoul 150-713, South Korea d Neodin Medical Institute, 2-3 Yongdap-dong, Sungdong-ku, Seoul 133-847, South Korea e Department of Clinical Pathology, College of Medicine, Pusan National University College of Medicine, 1-10 Ami-dong, Seo-ku, Pusan 602-739, South Korea Received 6 January 2003; received in revised form 19 May 2003
Abstract The katG, inhA and ahpC genes, in 71 isoniazid (INH)-resistant and 26 INH-susceptible Mycobacterium tuberculosis isolates, from South Korea were examined by sequencing and MspI restriction enzyme analysis. Mutations in the katG 315 alone, katG 315 plus inhA, katG 315 plus ahpC, katG 309 alone, katG 309 plus inhA, inhA alone, and ahpC alone, were detected in 54.9, 2.8, 1.4, 1.4, 1.4, 19.7, and 5.6% of the 71 INH-resistant isolates, respectively. There was no statistically significant difference (p ⬎ 0.05) in the frequencies of these mutations for the INH-monoresistant compared with the multidrug-resistant isolates. Mutations in the katG codon 315 were associated with the high-level of INH resistance (MIC, ⬎1 g/ml), whereas the mutation in the inhA promoter region was associated with the low-level of INH resistance (MIC, ⬎0.2 to 1 g/ml). The previously undescribed GGT3 GAT (Gly3 Asp) mutation in the katG codon 309 was found in two rifampin, including-multidrug-resistant isolates, but we cannot assess if this is predictive of INH resistance. The sensitivity and specificity of molecular analysis of the katG codon 315 and/or the inhA promoter region were 80.3 and 100%, respectively. Therefore, mutations in these regions are highly predictive of INH resistance in South Korea. © 2003 Elsevier Inc. All rights reserved. Keywords: Mycobacterium tuberculosis; Isoniazid resistance; katG; inhA; ahpC
1. Introduction From 1995 to 2002, the prevalence of radiologically diagnosed active pulmonary tuberculosis decreased from 1 to 0.5% of total South Korean population (Ministry of Health & Welfare, & Korean National Tuberculosis Association, 1995; unpublished data). From 1996 to 2000, the resistance to isoniazid (INH) and rifampin (RIF) decreased from 34.2 to 14.7% and 35.9 to 9.7%, respectively, and the resistance to them in combination dropped from 25.3 to 7.3% (Lee et al., 2001). Improved living conditions, in* Corresponding author. Tel.: ⫹82-2-3779-1296; fax: ⫹82-2-7836648. E-mail address: [email protected] (W.-I. Kim). 0732-8893/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0732-8893(03)00132-9
creased national medical insurance rate, South Korea’s universal BCG vaccination policy, and advanced antituberculosis chemotherapy contributed to these decreases, but the prevalence of tuberculosis and the resistance to primary antituberculosis drugs, is still much higher in South Korea than in developed countries. Therefore, the rapid detection of drug resistance is essential for continued decreases in the prevalence of tuberculosis and drug resistance in South Korea. The determination of drug resistance in M. tuberculosis using conventional culture methods routinely takes 6 to 10 weeks. In order to shorten this period, targeted molecular approaches have been performed in Europe and the United States. Genomic mutations frequently associated with resistance to each of the primary antituberculosis drugs have
498
S.-Y. Kim et al. / Diagnostic Microbiology and Infectious Disease 47 (2003) 497–502
been identified. More than 95% of RIF-resistant isolates are associated with mutations within an 81-bp region of the rpoB gene; thus, investigation of RIF resistance is relatively straightforward. In contrast, resistance to INH is associated with a variety of mutations that affect several genes, including: those encoding the catalase-peroxidase (katG), which converts INH to an active form; the enoyl acyl carrier protein reductase (inhA), which is involved in mycolic acid biosynthesis; the alkyl-hydroperoxide reductase (ahpC), which is involved in the cellular response to oxidative stress; the ketoacyl acyl carrier protein synthase (kasA), which is important in fatty acid elongation; and the NADH dehydrogenase (ndh) (Lee et al., 2001; Ramaswamy & Musser, 1998). The katG gene is the most commonly altered, with the majority of mutations occurring in codon 315 (Haas et al., 1997; Musser et al., 1996). Mutations in the katG codon 315 and the promoter region of the inhA have been identified in INH-resistant, but not INH-susceptible isolates (Dobner et al., 1997; Kiepiela et al., 2000; Nachamkin et al., 1997). With a few exceptions, most ahpC promoter mutations have been identified in INH-resistant isolates (Rinder et al., 1998; Sreevatsan et al., 1997). Mutations in the kasA gene have been frequently identified in INH-susceptible isolates (Lee et al., 1999; Piatek et al., 2000). Mutations occurring in the katG codon 315, together with those in the promoter regions of the inhA and ahpC, may account for most of isolates resistant to INH. However, because of considerable variance in the reported data, more information is required on the molecular mechanisms of INH resistance. The prevalence of INH-resistant tuberculosis in South Korea is known, but no serious efforts have been made to identify the INH resistance genotypes or their prevalence in the community. The purpose of our study was to determine the frequencies of mutations in the katG codon 315 and promoter regions of the inhA and ahpC, in the 71 INH-resistant and 26 INH-susceptible isolates from South Korea, and their association with multidrug resistance and the level of INH resistance.
2. Materials and methods 2.1. Mycobacterial isolates Ninety-seven clinical M. tuberculosis isolates were collected over 3 years between 2000 and 2002 from 3 clinical laboratories in South Korea. The 42 INH-resistant isolates were obtained from the Korean Institute of Tuberculosis, the 19 INH-resistant and 15 INH-susceptible isolates from the Neodin Medical Institute and the 10 INH-resistant and 11 INH-susceptible isolates from the Kangnam St. Mary’s Hospital. Susceptibility testing was performed by the 1% proportion method with Lo¨ wenstein-Jensen (L-J) medium, with the following critical drug concentrations: INH, 0.2
Table 1 Names and sequences of primers Target
Primer
katG 315
TB86, TB87, TB92, TB93, TB90, TB91,
inhA ahpC
Product size (bp) 5⬘-GAAACAGCGGCGCTGGATCGT 5⬘-GTTGTCCCATTTCGTCGGGG 5⬘-CCTCGCTGCCCAGAAAGGGA 5⬘-ATCCCCCGGTTTCCTCCGGT 5⬘-CCGATGAGAGCGGTGAGCTG 5⬘-ACCACTGCTTTGCCGCCACC
210 248 237
g/ml; RIF, 40 g/ml; streptomycin, 4 and 10 g/ml; ethambutol, 2 g/ml; kanamycin, 40 g/ml; enviomycin, 40 g/ml; prothionamide, 40 g/ml; cycloserine, 30 g/ml; paraaminosalicylic acid, 1 g/ml; ofloxacin, 2 g/ml. Fiftyseven isolates were further tested with 1 g/ml INH to determine the high-level of INH resistance. Twenty-five isolates were INH monoresistant, 33 were RIF-including-multidrug resistant and 13 were RIF-excluding-multidrug resistant. RIF-including-multidrug resistance was defined as resistance to at least INH and RIF according to the World Health Organization. RIF-excluding-multidrug resistance was defined as resistance to INH and other drugs, excluding RIF in this study. Twenty-six INH-susceptible isolates were susceptible to all the other antituberculosis drugs tested. 2.2. Polymerase chain reaction (PCR) amplification and sequencing M. tuberculosis cultures were grown on Ogawa or L-J medium at 37°C for 4 to 6 weeks and stored at room temperature in the dark, ready for use. Two to six mycobacterial colonies were collected from the agar surface, and the DNA extracted using a QIAamp DNA mini kit (QIAGEN, Hilden, Germany). Primers for the amplification of the katG codon 315, inhA and ahpC are listed in Table 1 (Telenti et al., 1997). The amplification reactions were performed in a final volume of 50 L, containing: 0.2 g of genomic DNA, 20 pmol of each primer, 0.2 mM of deoxynucleotide, 1X PCR buffer (10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, pH 8.3) and 2.5 U of Taq polymerase (Roche Boehringer Mannheim, Indianapolis, USA). The PCR was performed in a PTC-200 Peltier thermal cycler (MJ Research, Watertown, USA) using the following conditions: initial denaturation step at 94°C for 3 min; 30 cycles of denaturation at 94°C for 45 sec, annealing at 65°C for 30 sec, and extension at 72°C for 30 sec; and a final extension at 72°C for 5 min. The PCR products were purified with a QIAquick PCR purification kit (QIAGEN, Hilden, Germany), and directly sequenced using a BigDye Terminators sequencing kit and an ABI PRISM 3700 automated sequencer (Applied Biosystems, Foster city, USA).
S.-Y. Kim et al. / Diagnostic Microbiology and Infectious Disease 47 (2003) 497–502
499
Table 2 Frequencies of mutations associated with INH resistance in the regions examined Isolates
INH-resistant (n ⫽ 71) INH only (n ⫽ 25) INH ⫹ RIFa (n ⫽ 33) INH ⫹ otherb (n ⫽ 13) INH-susceptible (n ⫽ 26) a b
Localization of mutations [no. (%)] katG 315 alone
katG 315/inhA
katG 315/ahpC
katG 309 alone
katG 309/inhA
inhA alone
ahpC alone
Other
39 (54.9) 14 (56) 16 (48.5) 9 (69.2) 0
2 (2.8) 2 (8) 0 0 0
1 (1.4) 1 (4) 0 0 0
1 (1.4) 0 1 (3) 0 0
1 (1.4) 0 1 (3) 0 0
14 (19.7) 4 (16) 8 (24.2) 2 (15.4) 0
4 (5.6) 0 3 (9.1) 1 (7.7) 1 (3.9)
9 (12.7) 4 (16) 4 (12.1) 1 (7.7) 25 (96.2)
RIF, rifampin; resistant to at least INH and RIF (RIF-including-multidrug resistant). resistant to INH and other drugs, excluding RIF (RIF-excluding-multidrug resistant).
2.3. Restriction fragment length polymorphism (RFLP) MspI restriction enzyme analysis of the 210 bp fragment of the katG gene was undertaken to confirm the Ser315Thr mutation as follows: PCR-amplicons (10 L) were incubated with 7 L of deionized water, 2 L of 10X buffer (10 mM Tris-HCl, 10 mM MgCl2, 1 mM dithioerythritol, pH 7.5) and 1 L of MspI (10 U/l) (Roche Boehringer Mannheim, Indianapolis, USA) at 37°C for 1hr. The resulting restricted products were electrophoresed at 100V for 1.5 hr into a 10% polyacylamide gel by using 0.5X TBE (Trisborate-EDTA) buffer, and the bands were visualized by UV transillumination. 2.4. Statistical analysis Statistical comparisons were made using Chi-squared tests, with p ⬍ 0.05 considered as statistically significant.
3. Results Mutations in the katG codon 315 alone, katG codon 315 plus inhA, katG codon 315 plus ahpC, katG codon 309 alone, katG codon 309 plus inhA, inhA alone, and ahpC alone, were detected in 54.9, 2.8, 1.4, 1.4, 1.4, 19.7, and 5.6% of the 71 INH-resistant isolates, respectively (Table 2), but with no statistically significant difference (p ⬎ 0.05) in the frequencies of these mutations for the INH-monoresistant compared with the RIF-including- or excluding-multidrug-resistant isolates. Mutations in the katG codon 315 and/or the inhA promoter region and/or the ahpC promoter region were detected in 61 (85.9%), and mutations in the katG codon 315 and/or the inhA promoter region in 57 (80.3%) of the 71 INH-resistant isolates. Mutations in the katG codon 315 and/or inhA were not detected in all INHsusceptible isolates, but one INH-susceptible isolate had a mutation in the ahpC promoter region, a G3 A substitution at position -46 relative to the ahpC mRNA start site (Table 2 & 3). Therefore, if mutations in the codon 315 of the katG and the promoter regions of the inhA and ahpC were considered, the sensitivity and specificity were 85.9 and 96.2%,
respectively. If mutations in codon 315 of the katG and the promoter region of the inhA were considered, the corresponding values were 80.3 and 100%, respectively. Of the 42 INH-resistant isolates with mutations in the katG codon 315, 37 had a single AGC3 ACC (Ser3 Thr) mutation, and 2 INH-monoresistant isolates had both this mutation and one in the inhA promoter region. Two RIFexcluding-multidrug-resistant isolates had a single AGC3 AAC (Ser3 Asn) mutation in the katG codon 315, and 1 INH-monoresistant isolate had both an AGC3 CGC (Ser3 Arg) mutation in the katG codon 315 and a mutation in the ahpC promoter region, a C3 T substitution at position -39. Thus, mutations in the katG codon 315 were detected in 42 (59.2%) of the 71 INH-resistant isolates (Table 2 & 3). The RFLP analysis of the katG detected all the Ser315Thr mutations, but no other mutations. Therefore, the RFLP analysis detected 39 (54.9%) of the 71 INHresistant isolates. Two RIF-including-multidrug-resistant isolates had an GGT3 GAT (Gly3 Asp) in the katG codon 309. Of these two isolates, one had a single mutation, and the other had a mutation in the inhA promoter region also (Table 2 & 3). Seventeen (23.9%) of the 71 INH-resistant isolates had a C3 T substitution on the 5⬘ side of the presumed ribosomal binding site in the inhA promoter region. Of the 4 isolates exhibiting resistance to both INH and ethionamide, 3 showed this mutation, whereas 14 (20.9%) of the 67 isolates exhibiting resistance to INH only or INH and other drugs, excluding ethionamide, showed this mutation (p ⬍ 0.05). Five (7%) of the 71 INH-resistant isolates had mutations in 5 different locations of their ahpC promoter regions (Table 3). Table 4 shows the association between the level of INH resistance and the location of mutations detected in 57 INH-resistant isolates, with these being tested at concentrations of 0.2 and 1 g/ml INH. Mutations in the katG codon 315 were significantly more common in the isolates with the high-level of INH resistance (MIC, ⬎1 g/ml) (76.2%) than in those with the low-level of INH resistance (MIC, ⬎0.2 to 1 g/ml) (40%; p ⬍ 0.05). In contrast, mutations in the inhA promoter region were significantly more common in the isolates with the low-level of INH resistance (46.7%) than
500
S.-Y. Kim et al. / Diagnostic Microbiology and Infectious Disease 47 (2003) 497–502
Table 3 Mutations identified in 71 INH-resistant and 26 INH-susceptible isolates Gene
Positiona
Mutation
Mutation type
Amino acid change
Isolatesb
katG
315
AGC3ACC AGC3AAC AGC3CGC GGT3GAT C3T G3A C3T G3T C3T G3A
Reported Reported Reported Novel Reported Reported Reported Reported Reported Reported
Ser3Thr Ser3Asn Ser3Arg Gly3Asp
16 Ir, 16 Mr, 7 Or 2 Or 1 Ir 2 Mr 6 Ir, 9 Mr, 2 Or 1 Mr, 1 S 1 Ir 1 Mr 1 Or 1 Mr
inhA ahpC
309 Ribosomal binding site -46 -39 -12 -10 -9
a
Numbering of the ahpC promoter region nucleotide in relation to the ahpC mRNA start site. I , resistant to INH only; Mr, resistant to at least INH and RIF (RIF-including-multidrug resistant); Or, resistant to INH and other drugs, excluding RIF (RIF-excluding-multidrug resistant); S, susceptible. b r
in those with the high-level of INH resistance (4.8%; p ⬍ 0.05).
4. Discussion Genotypic testing of antimycobacterial drug resistance is an attractive alternative to conventional susceptibility testing due to its ability to provide rapid and accurate results. Although a variety of mutations are associated with INH resistance, mutations in the katG codon 315 and the promoter regions of the inhA and ahpC have been shown to be more prevalent than other mutations (Piatek et al., 2000; Telenti et al., 1997). This study has identified the frequencies of mutations in these loci in M. tuberculosis isolates from South Korea, and mutations in the katG codon 315 and the inhA promoter region should be assayed for predicting INH resistance in South Korea. We found mutations in the katG codon 315 in 59.2% of the INH-resistant isolates, and therefore concluded that this mutation to have the highest predictive power for INH resistance in M. tuberculosis. Most investigators found these mutations in 34.6 to 62.2% of INH-resistant isolates, for which MICs of INH were at least ⬎0.2 g/ml (Gonzalez et al., 1999; Piatek et al., 2000; Table 4 Association between the level of INH resistance and the location of mutations detected in 57 INH-resistant isolates Mutation
katG codon 315 alone inhA alone ahpC alone katG codon 315 plus inhA katG codon 309
No. (%) of isolates High-levela (n ⫽ 42)
Low-levelb (n ⫽ 15)
32 (76.2) 2 (4.8) 3 (7.1) 2 (4.8) 0
6 (40) 7 (46.7) 0 0 1 (6.7)
MIC of INH was ⬎1 g/ml for the 42 isolates. MIC of INH was ⬎0.2 to 1 g/ml for the 15 isolates. c NS, not significant. a
b
P
⬍0.05 ⬍0.05 NSc NS NS
Telenti et al., 1997), whereas other investigators found these mutations in 78.1 to 97.4% of INH-resistant isolates, for which MICs of INH were at least ⬎1 g/ml (Abate et al., 2001; Kiepiela et al., 2000; Marttila et al., 1998). These findings suggest that mutations in the katG codon 315 are associated with the high-level of INH resistance. This was also observed in our study; of the 42 isolates exhibiting the high-level of INH resistance (MIC, ⬎1 g/ml), 32 (76.2%) showed these mutations, whereas 6 (40%) of the 15 isolates exhibiting the low-level of INH resistance (MIC, ⬎0.2 to 1 g/ml) showed these mutations (p ⬍ 0.05). Geographical differences in the frequencies of these mutations were also apparent in the analysis of data from other studies: mutations in the katG codon 315 were detected in 34.6% of the INH-resistant isolates from Madrid, but in 62.2% from New York (Piatek et al., 2000). Therefore, the documented information regarding the frequencies and types of mutaions in one country or geographical region may not be generally applicable. Previous investigators reported that mutations in the katG codon 315 were associated with multidrug resistance (Piatek et al., 2000; van Soolingen et al., 2000). In this study, however, no significant differences were found in the frequencies of these mutations between the INH-monoresistant and the RIF-including- or excluding-multidrug-resistant isolates. Heteroresistance has been observed in M.tuberculosis (Rinder et al., 2001), and isolates were cultured on media containing both 0.2 and 0.5 g/ml INH to increase detection of these mutations. However, repeated tests with colonies taken from the INH-containing media produced identical results (data not shown). The RFLP analysis detected all the Ser315Thr mutations, which were 92.9% of the katG codon 315 mutations, and is regarded as relatively simple, rapid and inexpensive technique to screen these mutations in a clinical laboratory. Mutations in the katG codon 309 seem to be rare, and have not previously been reported. In our sample, two mutations were encountered in this position. Because no other mutations were observed in one isolate, we suggest
S.-Y. Kim et al. / Diagnostic Microbiology and Infectious Disease 47 (2003) 497–502
that the katG codon 309 was involved in the resistance mechanism of this isolate. However, information on the mutation in the katG codon 309 was not available for many of the isolates. This association will be explored in future studies. With regard to the promoter region of the inhA, this mutation was found in 23.9% of the INH-resistant isolates. Most investigators found these mutations in 10 to 34.2% of the INH-resistant isolates (Gonzalez et al., 1999; Kiepiela et al., 2000; Lee et al., 1999; Telenti et al., 1997). Of the 4 isolates exhibiting resistance to both INH and ethionamide, 3 showed this mutation, whereas 14 (20.9%) of 67 isolates exhibiting resistance to INH only or INH and other drugs, excluding ethionamide, showed this mutation (p ⬍ 0.05). Therefore, we confirmed that a C3 T substitution in the inhA promoter region occurred at a disproportionately higher rate in isolates resistant to both INH and ethionamide, than in isolates resistant to INH only or INH and other drugs, excluding ethionamide (Lee et al., 2000). In the inhA promoter region, a C3 T substitution on the 5⬘end of the presumed ribosomal binding site was the only mutation detected in this study, and M.tuberculosis isolates recovered from Korea and Spain also showed only this single mutation in the inhA promoter region (Lee et al., 2000; Telenti et al., 1997). However, our data were at variance with the study of Kiepiela et al. (2000), who reported that most isolates from Africa had the T3 A nucleotide substitution. In the case of the inhA, discrepancies in the results between studies seem to reflect the different geographical prevalences of specific genotypes. In contrast to the katG codon 315 mutations, the mutation in the inhA promoter region was associated with the low-level of INH resistance (p ⬍ 0.05). Mutations in the ahpC promoter region occurred at a low frequency (7%) among the INH-resistant isolates and were not obligatory in the INH-resistant isolates. Most investigators found these mutations in 4.8 to 24.2% of the INHresistant isolates (Kelly et al., 1997; Lee et al., 2001; Rinder et al., 1998; Telenti et al., 1997). Simple nucleotide substitutions were identified at positions -9, -10, -12, -39, and -46 (designated relative to the ahpC mRNA start site). Of these, the nucleotide substitution at position -46 was observed in a susceptible isolate, which was in line with the study of Sreevatsan et al. (1997), who reported that the nucleotide substitutions at position -46 and ⫹33 were not associated with INH resistance. Although other mutations in the ahpC promoter region appeared to be predictive of INH resistance, analysis of this target contributed minimally to the diagnostic strategy due to the low frequency. Mutations in the ahpC promoter region were identified in katG-defective, catalase-negative tubercle bacilli only to compensate for the loss of catalase-peroxidase activity (Kelly et al., 1997). However, our analysis showed that most isolates with the katG codon 315 missense mutations had no mutation in the ahpC promoter region and hence confirmed there was no simple relationship between these two mutations (Rinder et al., 1998; Sreevatsan et al., 1997; Telenti et al., 1997).
501
In the management of mycobacterial infections, the rapid detection and appropriate treatment with susceptible drugs is crucial. These goals can be achieved by improving the diagnosis of drug-resistant tuberculosis using molecular methods. In this study, if mutations in the katG codon 315 and the promoter region of the inhA were considered, the sensitivity and specificity were 80.3 and 100%, respectively. If mutations in the katG codon 315 and the promoter regions of the inhA and ahpC were considered, the corresponding values were 85.9 and 96.2%, respectively. Therefore, molecular analysis of the katG codon 315 and the promoter region of the inhA might be a good alternative to conventional susceptibility testing for the detection of INH resistance in South Korea.
Acknowledgments The authors thank Dong Hwa Han for excellent technical assistance. This study was supported in part by the Clinical Research Fund of St. Vincent’s Hospital.
References Abate, G., Hoffner, S. E., Thomsen, V. O., & Miorner, H. (2001). Characterization of isoniazid-resistant strains of Mycobacterium tuberculosis on the basis of phenotypic properties and mutations in katG. Eur J Clin Microbiol Infect Dis 20, 329 –333. Dobner, P., Rusch-Gerdes, S., Bretzel, G., Feldmann, K., Rifai, M., Loscher, T., & Rinder, H. (1997). Usefulness of Mycobacterium tuberculosis genomic mutations in the genes katG and inhA for the prediction of isoniazid resistance. Int J Tuberc Lung Dis 1, 365–369. Gonzalez, N., Torres, M. J., Aznar, J., & Palomares, J. C. (1999). Molecular analysis of rifampin and isoniazid resistance of Mycobacterium tuberculosis clinical isolates in Seville, Spain. Tuber Lung Dis 79, 187–190. Haas, W. H., Schilke, K., Brand, J., Amthor, B., Weyer, K., Fourie, P. B., Bretzel, G., Sticht-Groh, V., & Bremer, H. J. (1997). Molecular analysis of katG gene mutations in strains of Mycobacterium tuberculosis complex from Africa. Antimicrob Agents Chemother 41, 1601–1603. Kelley, C. L., Rouse, D. A., & Morris, S. L. (1997). Analysis of ahpC gene mutations in isoniazid-resistant clinical isolates of Mycobacterium tuberculosis. Antimicrob Agents Chemother 41, 2057–2058. Kiepiela, P., Bishop, K. S., Smith, A. N., Roux, L., & York, D. F. (2000). Genomic mutations in the katG, inhA and ahpC genes are useful for the prediction of isoniazid resistance in Mycobacterium tuberculosis isolates from Kwazulu Natal, South Africa. Tuber Lung Dis 80, 47–56. Lee, A. S., Lim, I. H., Tang, L. L., Telenti, A., & Wong, S. Y. (1999). Contribution of kasA analysis to detection of isoniazid-resistant Mycobacterium tuberculosis in Singapore. Antimicrob Agents Chemother 43, 2087–2089. Lee, A. S., Teo, A. S., & Wong, S. Y. (2001). Novel mutations in ndh in isoniazid-resistant Mycobacterium tuberculosis isolates. Antimicrob Agents Chemother 45, 2157–2159. Lee, H., Cho, S. N., Bang, H. E., Lee, J. H., Bai, G. H., Kim, S. J., & Kim, J. D. (2000). Exclusive mutations related to isoniazid and ethionamide resistance among Mycobacterium tuberculosis isolates from Korea. Int J Tuberc Lung Dis 4, 441– 447. Lee, S. H., Kim, S. Y., Kea, H. K., & Hwang, Y. S. (2001). The results of surveys of resistance to antituberculous drugs. Korean J Clin Microbiol 4, S98 –S99.
502
S.-Y. Kim et al. / Diagnostic Microbiology and Infectious Disease 47 (2003) 497–502
Marttila, H. J., Soini, H., Eerola, E., Vyshnevskaya, E., Vyshnevskiy, B. I., Otten, T. F., Vasilyef, A. V., & Viljanen, M. K. (1998). A Ser315Thr substitution in katG is predominant in genetically heterogeneous multidrug-resistant Mycobacterium tuberculosis isolates originating from the St. Petersburg area in Russia. Antimicrob Agents Chemother 42, 2443–2445. Ministry of Health & Welfare, & Korean National Tuberculosis Association. (1995). Report on the 7th tuberculosis prevalence survey in Korea. Musser, J. M., Kapur, V., Williams, D. L., Kreiswirth, B. N., van Soolingen, D., & van Embden, J. (1996). Characterization of the catalaseperoxidase gene (katG) and inhA locus in isoniazid-resistant and -susceptible strains of Mycobacterium tuberculosis by automated DNA sequencing: restricted array of mutations associated with drug resistance. J Infect Dis 173, 196 –202. Nachamkin, I., Kang, C., & Weinstein, M. P. (1997). Detection of resistance to isoniazid, rifampin, and streptomycin in clinical isolates of Mycobacterium tuberculosis by molecular methods. Clin Infect Dis 24, 894 –900. Piatek, A. S., Telenti, A., Murray, M. R., El-Hajj, H., Jacobs, W. R. Jr., Kramer, F. R., & Alland, D. (2000). Genotypic analysis of Mycobacterium tuberculosis in two distinct populations using molecular beacons: implications for rapid susceptibility testing. Antimicrob Agents Chemother 44, 103–110.
Ramaswarmy, S., & Musser, J. M. (1998). Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis: 1998 update. Tuber Lung Dis 79, 3–29. Rinder, H., Thomschke, A., Rusch-Gerdes, S., Bretzel, G., Feldmann, K., Rifai, M., & Loscher, T. (1998). Significance of ahpC promoter mutations for the prediction of isoniazid resistance in Mycobacterium tuberculosis. Eur J Clin Microbiol Infect Dis 17, 508 –511. Rinder, H., Mieskes, K., & Loscher, T. (2001). Heteroresistance in Mycobacterium tuberculosis. Int J Tuberc Lung Dis 5, 339 –345. Sreevatsan, S., Pan, X., Zhang, Y., Deretic, V., & Musser, J. M. (1997). Analysis of the oxyR-ahpC region in isoniazid-resistant and -susceptible Mycobacterium tuberculosis complex organisms recovered from diseased humans and animals in diverse localities. Antimicrob Agents Chemother 41, 600 – 606. Telenti, A., Honore, N., Bernasconi, C., March, J., Ortega, A., Heym, B., Takiff, H., & Cole, S. T. (1997). Genotypic assessment of isoniazid and rifampin resistance in Mycobacterium tuberculosis: a blind study at reference laboratory level. J Clin Microbiol 35, 719 –723. Van Soolingen, D., de Haas, P. E., van Doorn, H. R., Kuijper, E., Rinder, H., & Borgdorff, M. W. (2000). Mutations at amino acid position 315 of the katG gene are associated with high-level resistance to isoniazid, other drug resistance, and successful transmission of Mycobacterium tuberculosis in the Netherlands. J Infect Dis 182, 1788 –1790.