rpoB Gene Mutations and Molecular Characterization of Rifampin-Resista
http://www.100md.com
《微生物临床杂志》
Katharine Hsu International Research Center of Human Infectious Diseases, Shandong Provincial Tuberculosis Control Center, Jinan, Shandong, China
Department of Pathology, Baylor College of Medicine, Houston, Texas
Center for Human Molecular and Translational Infectious Diseases Research, The Methodist Hospital Research Institute, Houston, Texas
ABSTRACT
Sixty rifampin (RIF)-resistant and 75 RIF-susceptible Mycobacterium tuberculosis isolates from Shandong Province, China, were analyzed for rpoB gene mutations and genotyped. Mycobacterial interspersed repetitive unit (MIRU) genotype 223325173533 was overrepresented among RIF-resistant isolates. MIRU combined with IS6110 restriction fragment length polymorphism analysis as the second-line genotyping method may reflect epidemiologic links more reliably than each method alone.
TEXT
Tuberculosis (TB) remains a major public health concern in China, causing 150,000 deaths per year and making China second only to India in TB mortality (22). A significant challenge for TB control in China is the rapid dissemination and high prevalence of drug-resistant Mycobacterium tuberculosis (12). Rifampin (RIF), one of the principal first-line antituberculosis drugs, inhibits DNA-directed RNA synthesis of M. tuberculosis by binding to the subunit of RNA polymerase (10). Mutations in the rpoB gene, encoding the subunit of RNA polymerase, have been shown to be strongly associated with RIF-resistant phenotypes in multiple study populations (6, 9, 14, 17). rpoB mutations are more likely segregated in an 81-bp region called the RIF resistance-determining region (RRDR). Because up to 90% of RIF-resistant strains carry RRDR mutations within codons 516, 526, and 531, these mutational "hotspots" are being used to rapidly identify RIF-resistant isolates (8, 15).
The Beijing family, a dominant M. tuberculosis genotype in China and the rest of Asia, has been associated with increased frequencies of drug resistance in some populations, but not in others (5). More-reliable genotyping strategies are required to refine the Beijing family and identify possible subgroups, which may explain the drug resistance scenario. With few exceptions (7, 13), there have been few investigations into the molecular characterization of RIF-resistant M. tuberculosis isolates from mainland China, reflecting a general lack of understanding of this important topic.
In this study, 60 RIF-resistant and 75 RIF-susceptible M. tuberculosis isolates were collected through an M. tuberculosis sentinel surveillance network in Shandong Province, China (21). These isolates were obtained from different patients with no familial ties. Drug susceptibility testing was performed by the proportion method on Lwenstein-Jensen medium using the critical drug concentrations for RIF (40 μg/ml), isoniazid (0.2 μg/ml), streptomycin (4 μg/ml), and ethambutol (EMB; 2 μg/ml). The drug resistance profiles of 60 RIF-resistant isolates are shown in Table 1. Seventy-five M. tuberculosis isolates were susceptible to all first-line antituberculosis drugs.
Extraction of M. tuberculosis genomic DNA was performed by standard methods (20). A 495-bp region of the rpoB gene including the RRDR was amplified by PCR with forward primer 5' GACGACATCGACCACTTC and reverse primer 5' GGTCAGGTACACGATCTC. PCR fragments were sequenced with an ABI 377 DNA sequencer (Applied Biosystems, Inc., Foster City, CA). The new alleles were confirmed by further PCR and resequencing from the original DNA. Sequence data were independently analyzed by two biologists for quality control purposes.
Three independent genotyping methods were applied to molecularly characterize the 135 M. tuberculosis isolates. IS6110 restriction fragment length polymorphism (RFLP) characterization was conducted by following the standard protocol of van Embden et al. (20), with data analyzed by Whole Band Analysis software (version 3.2; BioImage, Ann Arbor, MI). Spoligotyping was carried out by using commercial kits from Isogen Bioscience BV (Maarssen, The Netherlands). Mycobacterial interspersed repetitive unit (MIRU) typing was performed by PCR amplification of the 12 MIRU loci following the protocol described previously, and samples were given a 12-digit MIRU identification number (3).
Among the 60 RIF-resistant M. tuberculosis isolates, 55 (91.7%) were found to have mutations in the rpoB RRDR with 10 different genotypes at six codons (Table 2). Codon 531 had the highest mutational frequency (33/55; 60.0%) followed by codons 526 (16/55; 29.1%), 516 (4/55; 7.3%), 511 (2/55; 3.6%), 515 (1/55; 1.8%), and 518 (1/55; 1.8%). Codon 526 showed the highest variability of mutations, with five different nucleotide substitutions. One multidrug-resistant isolate (resistant to four first-line drugs) presented rare multiple mutations at three codons, 511, 515, and 518. In the 75 RIF-susceptible isolates, 3 (4.0%) presented with mutations in the RRDR of rpoB, and they exclusively occurred at codon 533 with a novel nucleotide substitution, CTG (Leu)ATG (Met). Together, the overall agreement rate between the RDDR mutation of ropB and the RIF-resistant phenotype was 94.1% (127/135), which was consistent with previous studies of isolates from China (4, 13, 24). The relevance between codon 533 mutation and RIF resistance phenotype has been argued in previous studies, in which M. tuberculosis isolates with codon 533 mutations showed RIF susceptibility (MIC, 1.0 mg/liter) (11), low-level resistance (MIC, 12.5 mg/liter) (16), or high-level resistance (MIC, 512 mg/liter) (24). Our limited data suggest that codon 533 mutations are not associated with RIF resistance. The variability seen in RIF-resistant phenotypes in the codon 533 mutant isolates may thus be caused by other unknown mutations.
IS6110 RFLP identified seven RFLP clusters and 117 unique patterns. The IS6110 copy numbers ranged from 8 to 24. The largest cluster consisted of six epidemiologically linked RIF-susceptible isolates. Among 60 RIF-resistant isolates, there were 8 (13.3%) isolates falling into four IS6110 RFLP clusters; similarly, 10 (13.3%) of 75 RIF-susceptible isolates were identified in three clusters (Table 3). Spoligotyping determined four clusters and 12 unique patterns. Of the 135 isolates, 116 (85.9%) belonged to the Beijing family. Beijing family isolates were found more often among RIF-resistant isolates (90.0%) than among RIF-susceptible isolates (82.7%), but the difference was not statistically significant. Fifty-seven MIRU genotypes were found from all isolates in this study, including 40 unique patterns and 17 clusters (including 95 isolates) (Table 3). The largest cluster of 44 (32.6%) isolates shared MIRU genotype 223325173533 and are referred to as the "Shandong cluster" in this study. Interestingly, all isolates from the "Shandong cluster" were members of the Beijing family. The "Shandong cluster" isolates were found more often among RIF-resistant isolates (41.7%; 25/60) than among RIF-susceptible isolates (25.3%; 19/75) (P = 0.04). A comparative analysis using the Houston Tuberculosis Initiative database showed that print 33 from Houston, Texas, and strain 210 from Los Angeles, California (25), had the same MIRU genotype as the "Shandong cluster." Previously, strain 210 has been reported to be more virulent (25) with clonal expansion observed in several U.S. populations (1, 23). These findings suggest that the "Shandong cluster" may be an important subcluster of the Beijing family.
Among all molecular typing methods for M. tuberculosis, IS6110 RFLP has been considered the most discriminatory method and standardized for extensive applications in tracking M. tuberculosis transmission. However, the reliability of IS6110 RFLP reflecting epidemiological links between TB patients has been a concern in some studies (2, 18, 19). In this study, the rpoB genotypes and drug resistance phenotypes, together with the sentinel surveillance sites of the clustered and rpoB mutant M. tuberculosis isolates, are presented in Table 4. Four IS6110 RFLP clusters (A to D) carried rpoB mutations in the RRDR. Isolates from clusters A and B showed the intracluster consistency between the sites, MIRU and rpoB genotypes, and drug resistance profiles, except for a slight difference of the EMB resistance phenotype in cluster A. In contrast, isolates in clusters C and D presented intracluster inconsistency between sites, MIRU and rpoB genotypes, and drug resistance profiles (Table 4). Besides the "Shandong cluster," five MIRU clusters (clusters a to e) had rpoB mutations. Four (clusters a, b, d, and e) of the five MIRU clusters showed intracluster consistency between rpoB genotypes, drug resistance profiles, and sites. However, none of them showed intracluster identity in IS6110 RFLP patterns. These observations suggest the complexity of M. tuberculosis genomic variation and in agreement with a recent study (19) suggest that MIRU typing may more likely reflect epidemiologic links between TB patients than IS6110 RFLP. However, "Shandong cluster" isolates presented with a high prevalence in our study (32.6%), as well as a recent study from Hong Kong, China (21.6%) (7), indicating that MIRU alone does not provide enough discriminatory power for differentiating the "Shandong cluster" unless IS6110 RFLP is used as the second-line genotyping method.
In general, our data provide additional molecular insights into RIF-resistant M. tuberculosis strains in China. The newly identified "Shandong cluster," showing an association with the RIF resistance phenotype and strain 210, justifies further investigations into its virulence, transmissibility, and molecular mechanisms of drug resistance. Additionally, our results also provided important supplemental evidence for a recent strategy of subdividing the Beijing family, which suggested that the addition of RFLP to MIRU offered a higher discriminatory value than the addition of MIRU to RFLP (7).
ACKNOWLEDGMENTS
We thank Katharine Hsu, The Katharine Hsu Foundation, and Hopson Medical Education and Development System for supporting this international collaboration.
FOOTNOTES
Corresponding author. Mailing address: Department of Pathology (206E), Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Phone: (713) 798-8022. Fax: (713) 798-8895. E-mail: xma@bcm.edu.
REFERENCES
Beggs, M. L., K. D. Eisenach, and M. D. Cave. 2000. Mapping of IS6110 insertion sites in two epidemic strains of Mycobacterium tuberculosis. J. Clin. Microbiol. 38:2923-2928.
Braden, C. R., G. L. Templeton, M. D. Cave, S. Valway, I. M. Onorato, K. G. Castro, D. Moers, Z. Yang, W. W. Stead, and J. H. Bates. 1997. Interpretation of restriction fragment length polymorphism analysis of Mycobacterium tuberculosis isolates from a state with a large rural population. J. Infect. Dis. 175:1446-1452.
Cowan, L. S., L. Mosher, L. Diem, J. P. Massey, and J. T. Crawford. 2002. Variable-number tandem repeat typing of Mycobacterium tuberculosis isolates with low copy numbers of IS6110 by using mycobacterial interspersed repetitive units. J. Clin. Microbiol. 40:1592-1602.
Fan, X. Y., Z. Y. Hu, F. H. Xu, Z. Q. Yan, S. Q. Guo, and Z. M. Li. 2003. Rapid detection of rpoB gene mutations in rifampin-resistant Mycobacterium tuberculosis isolates in Shanghai by using the amplification refractory mutation system. J. Clin. Microbiol. 41:993-997.
Glynn, J. R., J. Whiteley, P. J. Bifani, K. Kremer, and D. van Soolingen. 2002. Worldwide occurrence of Beijing/W strains of Mycobacterium tuberculosis: a systematic review. Emerg. Infect. Dis. 8:843-849.
Huang, H., Q. Jin, Y. Ma, X. Chen, and Y. Zhuang. 2002. Characterization of rpoB mutations in rifampicin-resistant Mycobacterium tuberculosis isolated in China. Tuberculosis (Edinburgh) 82:79-83.
Kam, K. M., C. W. Yip, L. W. Tse, K. L. Wong, T. K. Lam, K. Kremer, B. K. Au, and D. van Soolingen. 2005. Utility of mycobacterial interspersed repetitive unit typing for differentiating multidrug-resistant Mycobacterium tuberculosis isolates of the Beijing family. J. Clin. Microbiol. 43:306-313.
Kocagoz, T., Z. Saribas, and A. Alp. 2005. Rapid determination of rifampin resistance in clinical isolates of Mycobacterium tuberculosis by real-time PCR. J. Clin. Microbiol. 43:6015-6019.
Mani, C., N. Selvakumar, S. Narayanan, and P. R. Narayanan. 2001. Mutations in the rpoB gene of multidrug-resistant Mycobacterium tuberculosis clinical isolates from India. J. Clin. Microbiol. 39:2987-2990.
McClure, W. R., and C. L. Cech. 1978. On the mechanism of rifampicin inhibition of RNA synthesis. J. Biol. Chem. 253:8949-8956.
Ohno, H., H. Koga, S. Kohno, T. Tashiro, and K. Hara. 1996. Relationship between rifampin MICs for and rpoB mutations of Mycobacterium tuberculosis strains isolated in Japan. Antimicrob. Agents Chemother. 40:1053-1056.
Pablos-Mendez, A., M. C. Raviglione, A. Laszlo, N. Binkin, H. L. Rieder, F. Bustreo, D. L. Cohn, C. S. Lambregts-van Weezenbeek, S. J. Kim, P. Chaulet, P. Nunn, et al. 1998. Global surveillance for antituberculosis-drug resistance, 1994-1997. N. Engl. J. Med. 338:1641-1649.
Qian, L., C. Abe, T. P. Lin, M. C. Yu, S. N. Cho, S. Wang, and J. T. Douglas. 2002. rpoB genotypes of Mycobacterium tuberculosis Beijing family isolates from East Asian countries. J. Clin. Microbiol. 40:1091-1094.
Spindola de Miranda, S., A. Kritski, I. Filliol, C. Mabilat, G. Panteix, and E. Drouet. 2001. Mutations in the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis strains isolated in Brazil and France. Mem. Inst. Oswaldo Cruz 96:247-250.
Tang, X., S. L. Morris, J. J. Langone, and L. E. Bockstahler. 2005. Microarray and allele specific PCR detection of point mutations in Mycobacterium tuberculosis genes associated with drug resistance. J. Microbiol. Methods 63:318-330.
Taniguchi, H., H. Aramaki, Y. Nikaido, Y. Mizuguchi, M. Nakamura, T. Koga, and S. Yoshida. 1996. Rifampicin resistance and mutation of the rpoB gene in Mycobacterium tuberculosis. FEMS Microbiol. Lett. 144:103-108.
Telenti, A., P. Imboden, F. Marchesi, D. Lowrie, S. Cole, M. J. Colston, L. Matter, K. Schopfer, and T. Bodmer. 1993. Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis. Lancet 341:647-650.
van Deutekom, H., S. P. Hoijng, P. E. de Haas, M. W. Langendam, A. Horsman, D. van Soolingen, and R. A. Coutinho. 2004. Clustered tuberculosis cases: do they represent recent transmission and can they be detected earlier Am. J. Respir. Crit. Care Med. 169:806-810.
van Deutekom, H., P. Supply, P. E. de Haas, E. Willery, S. P. Hoijng, C. Locht, R. A. Coutinho, and D. van Soolingen. 2005. Molecular typing of Mycobacterium tuberculosis by mycobacterial interspersed repetitive unit-variable-number tandem repeat analysis, a more accurate method for identifying epidemiological links between patients with tuberculosis. J. Clin. Microbiol. 43:4473-4479.
van Embden, J. D., M. D. Cave, J. T. Crawford, J. W. Dale, K. D. Eisenach, B. Gicquel, P. Hermans, C. Martin, R. McAdam, T. M. Shinnick, and P. M. Small. 1993. Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J. Clin. Microbiol. 31:406-409.
Wang, S., F. Zhang, and H. Duanmu. 1999. Study on epidemic of drug-resistant tuberculosis in Shandong province, China. Zhonghua Jie He He Hu Xi Za Zhi 22:728-730. (In Chinese.)
World Health Organization. 2002. WHO report 2002. Global tuberculosis control. Surveillance, planning, financing. World Health Organization, Geneva, Switzerland.
Yang, Z., P. F. Barnes, F. Chaves, K. D. Eisenach, S. E. Weis, J. H. Bates, and M. D. Cave. 1998. Diversity of DNA fingerprints of Mycobacterium tuberculosis isolates in the United States. J. Clin. Microbiol. 36:1003-1007.
Yue, J., W. Shi, J. Xie, Y. Li, E. Zeng, and H. Wang. 2003. Mutations in the rpoB gene of multidrug-resistant Mycobacterium tuberculosis isolates from China. J. Clin. Microbiol. 41:2209-2212.
Zhang, M., J. Gong, Z. Yang, B. Samten, M. D. Cave, and P. F. Barnes. 1999. Enhanced capacity of a widespread strain of Mycobacterium tuberculosis to grow in human macrophages. J. Infect. Dis. 179:1213-1217.(Xin Ma, Haiying Wang, Yunfeng Deng, Zhim)
Department of Pathology, Baylor College of Medicine, Houston, Texas
Center for Human Molecular and Translational Infectious Diseases Research, The Methodist Hospital Research Institute, Houston, Texas
ABSTRACT
Sixty rifampin (RIF)-resistant and 75 RIF-susceptible Mycobacterium tuberculosis isolates from Shandong Province, China, were analyzed for rpoB gene mutations and genotyped. Mycobacterial interspersed repetitive unit (MIRU) genotype 223325173533 was overrepresented among RIF-resistant isolates. MIRU combined with IS6110 restriction fragment length polymorphism analysis as the second-line genotyping method may reflect epidemiologic links more reliably than each method alone.
TEXT
Tuberculosis (TB) remains a major public health concern in China, causing 150,000 deaths per year and making China second only to India in TB mortality (22). A significant challenge for TB control in China is the rapid dissemination and high prevalence of drug-resistant Mycobacterium tuberculosis (12). Rifampin (RIF), one of the principal first-line antituberculosis drugs, inhibits DNA-directed RNA synthesis of M. tuberculosis by binding to the subunit of RNA polymerase (10). Mutations in the rpoB gene, encoding the subunit of RNA polymerase, have been shown to be strongly associated with RIF-resistant phenotypes in multiple study populations (6, 9, 14, 17). rpoB mutations are more likely segregated in an 81-bp region called the RIF resistance-determining region (RRDR). Because up to 90% of RIF-resistant strains carry RRDR mutations within codons 516, 526, and 531, these mutational "hotspots" are being used to rapidly identify RIF-resistant isolates (8, 15).
The Beijing family, a dominant M. tuberculosis genotype in China and the rest of Asia, has been associated with increased frequencies of drug resistance in some populations, but not in others (5). More-reliable genotyping strategies are required to refine the Beijing family and identify possible subgroups, which may explain the drug resistance scenario. With few exceptions (7, 13), there have been few investigations into the molecular characterization of RIF-resistant M. tuberculosis isolates from mainland China, reflecting a general lack of understanding of this important topic.
In this study, 60 RIF-resistant and 75 RIF-susceptible M. tuberculosis isolates were collected through an M. tuberculosis sentinel surveillance network in Shandong Province, China (21). These isolates were obtained from different patients with no familial ties. Drug susceptibility testing was performed by the proportion method on Lwenstein-Jensen medium using the critical drug concentrations for RIF (40 μg/ml), isoniazid (0.2 μg/ml), streptomycin (4 μg/ml), and ethambutol (EMB; 2 μg/ml). The drug resistance profiles of 60 RIF-resistant isolates are shown in Table 1. Seventy-five M. tuberculosis isolates were susceptible to all first-line antituberculosis drugs.
Extraction of M. tuberculosis genomic DNA was performed by standard methods (20). A 495-bp region of the rpoB gene including the RRDR was amplified by PCR with forward primer 5' GACGACATCGACCACTTC and reverse primer 5' GGTCAGGTACACGATCTC. PCR fragments were sequenced with an ABI 377 DNA sequencer (Applied Biosystems, Inc., Foster City, CA). The new alleles were confirmed by further PCR and resequencing from the original DNA. Sequence data were independently analyzed by two biologists for quality control purposes.
Three independent genotyping methods were applied to molecularly characterize the 135 M. tuberculosis isolates. IS6110 restriction fragment length polymorphism (RFLP) characterization was conducted by following the standard protocol of van Embden et al. (20), with data analyzed by Whole Band Analysis software (version 3.2; BioImage, Ann Arbor, MI). Spoligotyping was carried out by using commercial kits from Isogen Bioscience BV (Maarssen, The Netherlands). Mycobacterial interspersed repetitive unit (MIRU) typing was performed by PCR amplification of the 12 MIRU loci following the protocol described previously, and samples were given a 12-digit MIRU identification number (3).
Among the 60 RIF-resistant M. tuberculosis isolates, 55 (91.7%) were found to have mutations in the rpoB RRDR with 10 different genotypes at six codons (Table 2). Codon 531 had the highest mutational frequency (33/55; 60.0%) followed by codons 526 (16/55; 29.1%), 516 (4/55; 7.3%), 511 (2/55; 3.6%), 515 (1/55; 1.8%), and 518 (1/55; 1.8%). Codon 526 showed the highest variability of mutations, with five different nucleotide substitutions. One multidrug-resistant isolate (resistant to four first-line drugs) presented rare multiple mutations at three codons, 511, 515, and 518. In the 75 RIF-susceptible isolates, 3 (4.0%) presented with mutations in the RRDR of rpoB, and they exclusively occurred at codon 533 with a novel nucleotide substitution, CTG (Leu)ATG (Met). Together, the overall agreement rate between the RDDR mutation of ropB and the RIF-resistant phenotype was 94.1% (127/135), which was consistent with previous studies of isolates from China (4, 13, 24). The relevance between codon 533 mutation and RIF resistance phenotype has been argued in previous studies, in which M. tuberculosis isolates with codon 533 mutations showed RIF susceptibility (MIC, 1.0 mg/liter) (11), low-level resistance (MIC, 12.5 mg/liter) (16), or high-level resistance (MIC, 512 mg/liter) (24). Our limited data suggest that codon 533 mutations are not associated with RIF resistance. The variability seen in RIF-resistant phenotypes in the codon 533 mutant isolates may thus be caused by other unknown mutations.
IS6110 RFLP identified seven RFLP clusters and 117 unique patterns. The IS6110 copy numbers ranged from 8 to 24. The largest cluster consisted of six epidemiologically linked RIF-susceptible isolates. Among 60 RIF-resistant isolates, there were 8 (13.3%) isolates falling into four IS6110 RFLP clusters; similarly, 10 (13.3%) of 75 RIF-susceptible isolates were identified in three clusters (Table 3). Spoligotyping determined four clusters and 12 unique patterns. Of the 135 isolates, 116 (85.9%) belonged to the Beijing family. Beijing family isolates were found more often among RIF-resistant isolates (90.0%) than among RIF-susceptible isolates (82.7%), but the difference was not statistically significant. Fifty-seven MIRU genotypes were found from all isolates in this study, including 40 unique patterns and 17 clusters (including 95 isolates) (Table 3). The largest cluster of 44 (32.6%) isolates shared MIRU genotype 223325173533 and are referred to as the "Shandong cluster" in this study. Interestingly, all isolates from the "Shandong cluster" were members of the Beijing family. The "Shandong cluster" isolates were found more often among RIF-resistant isolates (41.7%; 25/60) than among RIF-susceptible isolates (25.3%; 19/75) (P = 0.04). A comparative analysis using the Houston Tuberculosis Initiative database showed that print 33 from Houston, Texas, and strain 210 from Los Angeles, California (25), had the same MIRU genotype as the "Shandong cluster." Previously, strain 210 has been reported to be more virulent (25) with clonal expansion observed in several U.S. populations (1, 23). These findings suggest that the "Shandong cluster" may be an important subcluster of the Beijing family.
Among all molecular typing methods for M. tuberculosis, IS6110 RFLP has been considered the most discriminatory method and standardized for extensive applications in tracking M. tuberculosis transmission. However, the reliability of IS6110 RFLP reflecting epidemiological links between TB patients has been a concern in some studies (2, 18, 19). In this study, the rpoB genotypes and drug resistance phenotypes, together with the sentinel surveillance sites of the clustered and rpoB mutant M. tuberculosis isolates, are presented in Table 4. Four IS6110 RFLP clusters (A to D) carried rpoB mutations in the RRDR. Isolates from clusters A and B showed the intracluster consistency between the sites, MIRU and rpoB genotypes, and drug resistance profiles, except for a slight difference of the EMB resistance phenotype in cluster A. In contrast, isolates in clusters C and D presented intracluster inconsistency between sites, MIRU and rpoB genotypes, and drug resistance profiles (Table 4). Besides the "Shandong cluster," five MIRU clusters (clusters a to e) had rpoB mutations. Four (clusters a, b, d, and e) of the five MIRU clusters showed intracluster consistency between rpoB genotypes, drug resistance profiles, and sites. However, none of them showed intracluster identity in IS6110 RFLP patterns. These observations suggest the complexity of M. tuberculosis genomic variation and in agreement with a recent study (19) suggest that MIRU typing may more likely reflect epidemiologic links between TB patients than IS6110 RFLP. However, "Shandong cluster" isolates presented with a high prevalence in our study (32.6%), as well as a recent study from Hong Kong, China (21.6%) (7), indicating that MIRU alone does not provide enough discriminatory power for differentiating the "Shandong cluster" unless IS6110 RFLP is used as the second-line genotyping method.
In general, our data provide additional molecular insights into RIF-resistant M. tuberculosis strains in China. The newly identified "Shandong cluster," showing an association with the RIF resistance phenotype and strain 210, justifies further investigations into its virulence, transmissibility, and molecular mechanisms of drug resistance. Additionally, our results also provided important supplemental evidence for a recent strategy of subdividing the Beijing family, which suggested that the addition of RFLP to MIRU offered a higher discriminatory value than the addition of MIRU to RFLP (7).
ACKNOWLEDGMENTS
We thank Katharine Hsu, The Katharine Hsu Foundation, and Hopson Medical Education and Development System for supporting this international collaboration.
FOOTNOTES
Corresponding author. Mailing address: Department of Pathology (206E), Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Phone: (713) 798-8022. Fax: (713) 798-8895. E-mail: xma@bcm.edu.
REFERENCES
Beggs, M. L., K. D. Eisenach, and M. D. Cave. 2000. Mapping of IS6110 insertion sites in two epidemic strains of Mycobacterium tuberculosis. J. Clin. Microbiol. 38:2923-2928.
Braden, C. R., G. L. Templeton, M. D. Cave, S. Valway, I. M. Onorato, K. G. Castro, D. Moers, Z. Yang, W. W. Stead, and J. H. Bates. 1997. Interpretation of restriction fragment length polymorphism analysis of Mycobacterium tuberculosis isolates from a state with a large rural population. J. Infect. Dis. 175:1446-1452.
Cowan, L. S., L. Mosher, L. Diem, J. P. Massey, and J. T. Crawford. 2002. Variable-number tandem repeat typing of Mycobacterium tuberculosis isolates with low copy numbers of IS6110 by using mycobacterial interspersed repetitive units. J. Clin. Microbiol. 40:1592-1602.
Fan, X. Y., Z. Y. Hu, F. H. Xu, Z. Q. Yan, S. Q. Guo, and Z. M. Li. 2003. Rapid detection of rpoB gene mutations in rifampin-resistant Mycobacterium tuberculosis isolates in Shanghai by using the amplification refractory mutation system. J. Clin. Microbiol. 41:993-997.
Glynn, J. R., J. Whiteley, P. J. Bifani, K. Kremer, and D. van Soolingen. 2002. Worldwide occurrence of Beijing/W strains of Mycobacterium tuberculosis: a systematic review. Emerg. Infect. Dis. 8:843-849.
Huang, H., Q. Jin, Y. Ma, X. Chen, and Y. Zhuang. 2002. Characterization of rpoB mutations in rifampicin-resistant Mycobacterium tuberculosis isolated in China. Tuberculosis (Edinburgh) 82:79-83.
Kam, K. M., C. W. Yip, L. W. Tse, K. L. Wong, T. K. Lam, K. Kremer, B. K. Au, and D. van Soolingen. 2005. Utility of mycobacterial interspersed repetitive unit typing for differentiating multidrug-resistant Mycobacterium tuberculosis isolates of the Beijing family. J. Clin. Microbiol. 43:306-313.
Kocagoz, T., Z. Saribas, and A. Alp. 2005. Rapid determination of rifampin resistance in clinical isolates of Mycobacterium tuberculosis by real-time PCR. J. Clin. Microbiol. 43:6015-6019.
Mani, C., N. Selvakumar, S. Narayanan, and P. R. Narayanan. 2001. Mutations in the rpoB gene of multidrug-resistant Mycobacterium tuberculosis clinical isolates from India. J. Clin. Microbiol. 39:2987-2990.
McClure, W. R., and C. L. Cech. 1978. On the mechanism of rifampicin inhibition of RNA synthesis. J. Biol. Chem. 253:8949-8956.
Ohno, H., H. Koga, S. Kohno, T. Tashiro, and K. Hara. 1996. Relationship between rifampin MICs for and rpoB mutations of Mycobacterium tuberculosis strains isolated in Japan. Antimicrob. Agents Chemother. 40:1053-1056.
Pablos-Mendez, A., M. C. Raviglione, A. Laszlo, N. Binkin, H. L. Rieder, F. Bustreo, D. L. Cohn, C. S. Lambregts-van Weezenbeek, S. J. Kim, P. Chaulet, P. Nunn, et al. 1998. Global surveillance for antituberculosis-drug resistance, 1994-1997. N. Engl. J. Med. 338:1641-1649.
Qian, L., C. Abe, T. P. Lin, M. C. Yu, S. N. Cho, S. Wang, and J. T. Douglas. 2002. rpoB genotypes of Mycobacterium tuberculosis Beijing family isolates from East Asian countries. J. Clin. Microbiol. 40:1091-1094.
Spindola de Miranda, S., A. Kritski, I. Filliol, C. Mabilat, G. Panteix, and E. Drouet. 2001. Mutations in the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis strains isolated in Brazil and France. Mem. Inst. Oswaldo Cruz 96:247-250.
Tang, X., S. L. Morris, J. J. Langone, and L. E. Bockstahler. 2005. Microarray and allele specific PCR detection of point mutations in Mycobacterium tuberculosis genes associated with drug resistance. J. Microbiol. Methods 63:318-330.
Taniguchi, H., H. Aramaki, Y. Nikaido, Y. Mizuguchi, M. Nakamura, T. Koga, and S. Yoshida. 1996. Rifampicin resistance and mutation of the rpoB gene in Mycobacterium tuberculosis. FEMS Microbiol. Lett. 144:103-108.
Telenti, A., P. Imboden, F. Marchesi, D. Lowrie, S. Cole, M. J. Colston, L. Matter, K. Schopfer, and T. Bodmer. 1993. Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis. Lancet 341:647-650.
van Deutekom, H., S. P. Hoijng, P. E. de Haas, M. W. Langendam, A. Horsman, D. van Soolingen, and R. A. Coutinho. 2004. Clustered tuberculosis cases: do they represent recent transmission and can they be detected earlier Am. J. Respir. Crit. Care Med. 169:806-810.
van Deutekom, H., P. Supply, P. E. de Haas, E. Willery, S. P. Hoijng, C. Locht, R. A. Coutinho, and D. van Soolingen. 2005. Molecular typing of Mycobacterium tuberculosis by mycobacterial interspersed repetitive unit-variable-number tandem repeat analysis, a more accurate method for identifying epidemiological links between patients with tuberculosis. J. Clin. Microbiol. 43:4473-4479.
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