Genotypic analysis of multidrug-resistant Mycobacterium tuberculosis isolates from Monterrey, Mexico

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1 Journal of Medical Microbiology (2004), 53, DOI /jmm Genotypic analysis of multidrug-resistant Mycobacterium tuberculosis isolates from Monterrey, Mexico Srinivas V. Ramaswamy, 1 Shu-Jun Dou, 1 Adrian Rendon, 2 Zhenhua Yang, 3,4 M. Donald Cave 3,5 and Edward A. Graviss 1 Correspondence Edward A. Graviss egraviss@bcm.tmc.edu 1 Houston Tuberculosis Initiative, Department of Pathology, Baylor College of Medicine, Houston, TX, USA 2 Pulmonary Services and Clinical Pathology Laboratory, University Hospital of Monterrey, Universidad Autonomy de Nuevo Leon, Nuevo Leon, Mexico 3 Regional Tuberculosis Genotyping Laboratory, Central Arkansas Veterans Healthcare System, AR, USA 4 Department of Medicine and 5 Department of Anatomy, University of Arkansas for Medical Sciences, Little Rock, AR, USA Received 10 June 2003 Accepted 13 November 2003 Thirty-seven multidrug-resistant and 13 pan-susceptible isolates of Mycobacterium tuberculosis were analysed for the diversity of genotypes associated with known drug-resistance mechanisms. The isolates were obtained from patients attending a university tuberculosis clinic in Monterrey, Mexico. A total of 25 IS6110-RFLP patterns were obtained from the multidrug-resistant tuberculosis (MDR-TB) isolates. Approximately 65 % of the MDR-TB isolates were attributed to secondary resistance. Different drug-susceptibility patterns were seen with the clustered isolates. The percentage of isolates resistant to isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and streptomycin (STR) was 100, 97.3, 48.7 and 67.6, respectively. The most common resistanceassociated polymorphisms for the four drugs were as follows: INH, Ser315Thr (67. 6%)inkatG; RIF, Ser450Leu (41. 7%)inrpoB; EMB, Met306Ile/Val/Leu (66. 7%)inembB; and STR, Lys43Arg (24 %) in rpsl. Drug-resistance-associated mutations were similar to changes occurring in isolates from other areas of the world, but unique, previously unreported, mutations in katg (n ¼ 5), rpob (n ¼ 1) and rrs (n ¼ 3) were also identified. INTRODUCTION Present address: Epidemiology Department, School of Public Health, University of Michigan at Ann Arbor, MI, USA. Abbreviations: EMB, ethambutol; INH, isoniazid; MDR-TB, multidrugresistant tuberculosis; PZA, pyrazinamide; RIF, rifampicin; STR, streptomycin. The emergence and widespread dissemination of multidrugresistant (MDR) strains of Mycobacterium tuberculosis pose a serious threat to tuberculosis (TB) control in the new millennium (Raviglione et al., 1995). The lack of new therapeutic agents to treat MDR-TB along with a need to develop quick and efficient molecular diagnostic tools has stimulated research in the past few years to delineate the molecular genetic basis of drug resistance in M. tuberculosis. Drug resistance in M. tuberculosis is due to the acquisition of mutations in chromosomally encoded genes and the generation of multidrug resistance is a consequence of serial accumulation of mutations primarily due to inadequate therapy. Several studies have shown that resistance to isoniazid (INH) is due to mutations in the katg gene and about 50 % of isolates with katg mutations have an amino acid replacement at codon 315 (Zhang et al., 1992; Heym et al., 1995; Marttila et al., 1998; Ramaswamy & Musser, 1998). The rpob gene, which encodes the â-subunit of RNA polymerase, harbours a mutation in an 81 bp region in about 95 % of rifampicin (RIF)-resistant M. tuberculosis strains recovered globally (Ramaswamy & Musser, 1998; Telenti et al., 1993; Jin & Gross, 1988; Kapur et al., 1994). Streptomycin (STR) resistance is due to mutations in the rrs and rpsl genes which encode the 16S rrna and ribosomal protein S12, respectively (Ramaswamy & Musser, 1998; Nair et al., 1993; Meier et al., 1994; Sreevatsan et al., 1996). Mutations in the pnca gene have been shown to develop pyrazinamide (PZA) resistance in approximately 70 % of clinical isolates of M. tuberculosis resistant to PZA and & 2004 SGM Printed in Great Britain IP:

2 S. V. Ramaswamy and others approximately 65 % of clinical isolates resistant to ethambutol (EMB) have a mutation in the embb gene (Ramaswamy & Musser, 1998; Ramaswamy et al., 2000; Telenti et al., 1997b; Sreevatsan et al., 1997b). A global surveillance programme initiated in 1994 by the World Health Organization and the International Union Against TB and Lung Disease to monitor drug resistance has provided information on the prevalence of drug resistance in three states of Mexico, which included Baja California, Oaxaca and Sinaloa (World Health Organization, 1997; Anonymous, 1998a, b). This was the first population-based TB drug-resistance study in Mexico which reported both high and medium TB incidence in 1994 (World Health Organization, 1997). Recently, a clinic-based molecular epidemiological study of TB in Monterrey, Mexico, determined the diversity of RFLP patterns and the extent of drug resistance of M. tuberculosis isolates from patients who attended the clinic (Yang et al., 2001). Based on both IS6110 and ptbn12 characterization, 39 % of 166 isolates were shown to belong to 22 clusters, indicating extensive recent transmission. The study also showed that the prevalence of drug-resistant TB was high, with 32 % of the 186 isolates testing drug-resistant and 18 % MDR (Yang et al., 2001). The present investigation was undertaken to identify resistance-associated mutations in the MDR-TB isolates recovered from patients who attended the Jose E. Gonzalez University Hospital TB clinic in Monterrey, Mexico. It has been suggested that mutations conferring drug resistance may vary geographically (Rinder et al., 1997). Thus, the information gained by genotyping drug-resistant isolates helps not only to identify genetic markers in M. tuberculosis strains unique to a particular geographical niche, but also in the evaluation of molecular screening tests to identify MDR- TB. METHODS Bacterial isolates. Fifty MDR (n ¼ 37) and susceptible (n ¼ 13) M. tuberculosis isolates recovered from patients suffering from pulmonary tuberculosis were studied. Isolates were collected between January 1996 and March 1998 and are a subset of 186 strains initially isolated from patients attending the Jose E. Gonzalez University Hospital in Monterrey, Mexico (Yang et al., 2001). Specimens were cultured on Löwenstein Jensen slants and were identified as M. tuberculosis on the basis of a positive niacin test. Drug susceptibility testing was done using the proportion method with INH (0. 2 ìgml 1 ), RIF (40 ìgml 1 ), STR (4 ìgml 1 ) and EMB (2 ìgml 1 ). Resistance to any of the four drugs tested was defined as >1 % growth on drugcontaining medium compared to a control medium (National Committee for Clinical Laboratory Standards, 1995). Isolates resistant to at least INH and RIF were considered MDR. DNA isolation, characterization and genetic group analysis. Isolation of DNA and IS6110-RFLP analysis were performed by using an internationally standardized method (van Embden et al., 1993). The molecular profiles were analysed by computer-assisted analysis using the BIOIMAGE software WHOLE BAND ANALYSER, version 3.4. Of the 34 samples analysed, the number of IS6110 copies ranged from two to 14. Ten isolates had less than six copies of IS6110 and were defined as lowcopy isolates. Twenty isolates had unique IS6110 profiles and the remaining 14 isolates shared five different band patterns. Isolates with the same IS6110-RFLP pattern or low-copy isolates were subjected to a secondary typing method using ptbn12 as probe, which contains the polymorphic GC-rich sequences (Chaves et al., 1996). Of the nine isolates that gave results with ptbn12-typing, one cluster containing two isolates was identified. The isolates in the cluster could be further distinguished based on their susceptibility patterns (Table 1). All isolates were assigned to one of three principal genetic groups based on polymorphisms present at gyra codon 95 and katg codon 463 (Sreevatsan et al., 1997a). Twenty isolates (54 %) were group 3, and 17 isolates (46 %) were group 2. There were no group 1 isolates. Isolates with the same IS6110, ptbn12 profiling patterns and genetic grouping were considered to be clonally related. PCR amplification and DNA sequencing. The major resistancedetermining regions of the katg, embb, rpsl and rpob genes were amplified using oligonucleotide primers and PCR conditions described previously (Heym et al., 1995; Kapur et al., 1994; Sreevatsan et al., 1996; Ramaswamy et al., 2000; Escalante et al., 1998). In addition to rpsl for determining STR resistance-associated mutations, the entire rrs gene encoding the 16S rrna was amplified using two sets of primers: F1 (59- GTCAGGATATTTCTAAATACCTTTGG-39), R1 (59-CACCTCAGCG TCAGTTACTG-39), F2 (59-CAGTAACTGACGCTGAGGAG-39) and R2 (59-GTTTTCGTGGTGCTCCTTAG-39). A GeneAmp System 9700 thermocycler (Applied Biosystems) was used for targeted DNA amplification. Unincorporated nucleotides and primers were removed by filtration using Microcon 100 microconcentrators (Amicon). DNA sequencing reactions were performed with the BigDye Terminator Cycle Sequencing kit (Applied Biosystems) with appropriate primers and purified PCR-amplified DNA as the template. The sequencing reactions were cleaned using Centrisep spin columns (Princeton Separations) and run on an ABI PRISM 377 DNA Sequencer (Applied Biosystems). The sequence data generated were assembled and edited electronically with the ALIGN and EDITSEQ programs (DNASTAR) and compared with the H37Rv genome database as well as with corresponding sequences from the susceptible M. tuberculosis strains (Cole et al., 1998). RESULTS AND DISCUSSION This study was undertaken to describe the drug-resistanceassociated alleles in MDR-TB isolates recovered from Monterrey, Mexico. Although individual drug-resistant isolates have been studied previously (Viader-Salvadó et al., 2003), this is the first description of mutations identified by sequencing in a collection of MDR-TB isolates from Mexico. Correlation between susceptibility testing and genotypic data Drug susceptibility data for the MDR-TB isolates are shown in Table 1. All 37 isolates were resistant to INH and RIF, except one strain (1531), which was susceptible to RIF. Twenty-five isolates (67. 6 %) were resistant to STR, and 18 isolates (48. 7 %) were resistant to EMB. Thirteen isolates (35. 1 %) were resistant to all four drugs tested and 16 isolates (43. 2 %) were resistant to three drugs. No correlation could be found between drug susceptibility patterns and molecular characterization data. The drug susceptibility profile correlated well with the observed and previously reported frequencies of mutations found in katg and inha (91. 8 %), 108 IP: Journal of Medical Microbiology 53

3 Genotyping MDR-TB isolates from Monterrey Table 1. Genetic group, IS6110, ptbn12 and comparison between the susceptibility test data and genotype data of MDR-TB isolates Isolate Genetic group* No. of bands FP ptbn12 Susceptibility patternk Nature of resistance Additional genotypic resistance# M007 I, R, S, E M I, R, S, E M I, R, S, E M016 I, R, E M I, R, S, E M023 I, R, S, E M026 I, R 2 E M035 I, R M036 I, R M I, R 1 S M023 I, R, S, E M084 I, R, S, E M088 I, R, S, E M093 I, R, S 2 E M110 I, S, E M071 I, R, S 2 E M117 I, R, S M120 I, R, S M I, R, S M125 I, R, S M I, R, S, E M049 NA I, R, E 1 S ND I, R, S, E M I, R, S, E M150 I, R, S, E M154 I, R, E M084 I, R, S M187 I, R, E 2 S ND I, R M200 I, R, S, E ND I, R, S M205 I, R, S M026 I, R, S M I, R, S M227 I, R M026 I, R M I, R 2 *Genetic group designation based on polymorphisms in katg codon 463 and gyra codon 95. Number of hybridizing bands by IS6110-typing. ND, Not determined. IS6110 fingerprinting code. Data not shown for pan-susceptible isolates. Polymorphic GC-rich sequence fingerprinting code. NA, Not available. ksusceptibility testing done by proportion method in Monterrey. I, isoniazid; R, rifampicin; S, streptomycin; E, ethambutol. Susceptible isolates with genotypic resistance are shown. 1, Primary resistance; 2, secondary resistance. #Additional genotypic alterations were identified in the designated isolates. IP:

4 S. V. Ramaswamy and others embb (83. 3 %) and rpob (83. 8 %) (Ramaswamy & Musser, 1998; Kapur et al., 1994; Ramaswamy et al., 2000; Telenti et al., 1997a). Eleven isolates (44 %) with streptomycin resistance had a resistance-associated mutation. One MDR- TB isolate (1674) had no mutations identified in the target regions. Four MDR-TB isolates had additional genotypic resistance, three of which were identified in embb and the other change was found in rrs. Isolates in the same cluster had different drug susceptibilities and resistance-associated mutations. This indicates that a majority of the isolates had acquired mutations independently. This rules out the presence and dissemination of a highly successful MDR-TB clone in the Monterrey community based on the recovered isolates. Although all MDR-TB isolates described by Yang et al. (2001) were used in this study and the sample size is small, it is reasonable to think that genotypes described in this study are circulating in the Monterrey and surrounding communities. rrs gene. Isolate 1258, believed to be sensitive to STR by drug susceptibility testing, had a C!T substitution at position 516 of rrs, which has been previously reported to be associated with drug resistance. Three other drug-resistant isolates had ac!t change at position 491 of rrs, but this change was also observed in three drug-sensitive isolates, suggesting that this polymorphism is not associated with drug resistance. A recent study also showed that this change is not associated with STR resistance, but is deeply rooted within an evolutionary clade of isolates from a suburb of Cape Town in South Africa (Victor et al., 2001). Our data corroborate with their findings and show that this change is associated only with major genetic group 3 isolates recovered from the Monterrey region in Mexico. Also, three more undescribed nucleotide substitutions in rrs of STR-resistant isolates were identified in our study. These changes were located at nucleotide positions 189 (G!A), 426 (G!T) and 1238 (T!C). Analysis of mutations in the target regions Resistance-associated mutations in katg, inha and rpob, markers for INH and RIF resistance, respectively, were found in 30 isolates (81. 1 %). The nucleotide and amino acid changes identified in the drug-resistant isolates are shown in Table 2. The study shows that MDR-TB strains from Monterrey not only have mutations in regions of genes previously shown to be involved in drug resistance, but also have mutations not described previously (Ramaswamy & Musser, 1998). For example, two common substitution mutations found in codons 450 and 445 of rpob were also seen in strains from this study. Fifteen isolates (40. 1 %) had a mutation in codon 450 and 27 % (n ¼ 10) of RIF-resistant isolates had a substitution in codon 445. Two isolates, 616 and 1142, had two different mutations each in rpob and no changes were seen in any of the susceptible isolates. One isolate had a missense change in codon 480 (Ile!Val) that has not been described previously (Table 2). Among the INH-resistant strains, 25 isolates (67. 6 %) had a substitution mutation () at codon 315 of katg, which is the most common mutation described in INH-resistant strains. Recently, Viader-Salvadó et al. (2003) showed that 53. 7% of INH-resistant isolates showed a mutation in codon 315 of katg and 86 % of RIF-resistant isolates had a resistanceassociated mutation in rpob of M. tuberculosis isolates recovered from north-east Mexico. New mutations were also identified in codons 249 (Arg!Cys), 275 (Thr!Ser), 307 (Gly!Glu) and 727 (Ala!Asp) of katg in INH-resistant isolates. The entire rpsl and rrs genes, encoding the ribosomal protein S12 and 16S rrna, respectively, were sequenced for mutations associated with STR resistance. Only 11 isolates (44 %) harboured mutations that were not found in susceptible isolates, indicating that genes other than rpsl and rrs are involved in STR resistance. Six isolates had a substitution in codon 43 of rpsl and no corresponding changes were found in the drug-sensitive isolates. Three isolates had a T!C change at nucleotide position 1238 of the Twelve of the 18 (66. 7 %) EMB-resistant isolates had mutations in the 461 bp embb region sequenced. In addition, three isolates judged to be sensitive to EMB by susceptibility testing showed resistance-associated mutations in embb. This discrepancy is probably due to heteroresistance involving mixed cultures. A total of 12 isolates had a substitution mutation in codon 306 and the remaining three isolates had amino acid replacements in codon 406. Two isolates, 730 and 1498, were part of a cluster with identical IS6110 (4 bands, profile M059) and ptbn12 characterization (147), but differed in their drug susceptibility profiles. Both isolates had identical alleles in rpob, katg and rpsl. However, isolate 1498 differed from 730 by its susceptibility to EMB, suggesting that 730 arose from 1498 by acquiring additional resistance to EMB. Mutations associated with high-level resistance to fluoroquinolones (FQs) are generally clustered in a 40 amino acid stretch around codon 95 of the gyra gene (Ramaswamy & Musser, 1998). Although the susceptibility testing was not done for FQs, the resistance-determining region in gyra was sequenced for all the isolates to determine the major genetic grouping. No mutations associated with FQ resistance were detected. In conclusion, the genotypic analysis of MDR-TB isolates from Monterrey, Mexico, identified that commonly found mutations in drug-resistant isolates from different regions of the world are also found in this region (Ramaswamy & Musser, 1998). Molecular strategies used to rapidly detect resistance-associated mutations would be applicable to isolates in Monterrey and other parts of Mexico. The new mutations identified in this study illustrate that, in spite of several genotypic studies done on drug-resistant isolates, there still remains a number of resistance-associated mutations to be discovered in M. tuberculosis. It remains to be seen if any of the new mutations identified in this study can also be found in other parts of Mexico based on a larger sample size and an epidemiologically independent group of isolates. 110 IP: Journal of Medical Microbiology 53

5 Genotyping MDR-TB isolates from Monterrey Table 2. Mutations identified in drug-resistant isolates Automated DNA sequencing of the most common resistance-determining regions of katg (390 bp, codons ), inha promoter (395 bp, 199 to +196 bases), rpob (494 bp, codons ), embb (461 bp, codons ) and the entire rpsl (375 bp plus 104 bp upstream region) and rrs genes (1537 bp plus 53 bp upstream region). The rpob codon numbering is based on the H37Rv genome and not on the Escherichia coli rpob numbering system as described previously. Both the nucleotide and the amino acid changes are shown. Asterisks indicate novel mutations., Wild-type; Nt., nucleotide. Isolate katg changes inha changes rpob changes rpsl changes rrs changes embb changes ; AGC!ACC, 306; ATG!ATA, 480; ATC!GTC, Ile!Val* ; AGC!ACA, ; AGC!ACC, 435; GAC!GAG, Asp!Glu 306; ATG!GTG, Met!Val 445; CAC!AAC, His!Asn ; AGC!ACC, 306; ATG!ATA, ; AGC!ACC, 435; GAC!GTC, Asp!Val Nt. 1238; T!C* 306; ATG!CTG, Met!Leu ; AGC!ACC, 441; TCG!TTG, 306; ATG!ATA, ; AGC!ACC, 406; GGC!GAC, Gly!Asp ; ACC!TCC, 445; CAC!GAC, Thr!Ser* His!Asp ; AGC!ACC, ups; C!T 450; TCG!TTG, Nt. 1238; T!C* ; AGC!ACC, 306; ATG!ATA, ; CGC!TGC, Arg!Cys* 445; CAC!TAC, 306; ATG!ATT, ; AGC!ACC, 406; GGC!GAC, Gly!Asp ups; C!T 406; GGC!GCC, Gly!Ala ; GCC!GAC, Ala!Asp* Nt. 491; C!T 306; ATG!ATA, ups; C!T Nt. 189; G!A* ; AGC!ACC, 445; CAC!GAC, His!Asp ; AGC!ACC, 445; CAC!TGC, His!Cys ; GGA!GAA, Gly!Glu* ; deletion* Nt. 1238; T!C* ; CGC!TGC, Arg!Cys* 445; CAC!TAC, 306; ATG!ATT, IP:

6 S. V. Ramaswamy and others Table 2. cont. Isolate katg changes inha changes rpob changes rpsl changes rrs changes embb changes ; AGC!ACC, 306; ATG!ATC, ; AGC!ACC, 445; CAC!TAC, ; AGC!ACC, 445; CAC!GAC, His!Asp 306; ATG!ATA, ; AGC!ACC, Nt. 426; G!T* 1258 Nt. 516; C!T 306; ATG!ATA, ; AGC!ACC, 445; CAC!TAC, ; AGC!ACC, 441; TCG!TTG, ; AGC!ACC, 441; TCG!TTG, ; AGC!ACC, Nt. 491; C!T ; AGC!ACC, 445; CAC!TAC, ; AGC!ACC, ; AGC!ACC, Nt. 491; C!T ; AGC!ACC, 452; CTG!CCG, Leu!Pro ; AGC!ACC, 435; GAC!GTC, Asp!Val ACKNOWLEDGEMENTS This study was funded in part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, under control no. R01-AI The study used resources and facilities at the Central Arkansas Veterans Health Services Center in Little Rock, AR, USA. REFERENCES Anonymous (1998a). Population-based survey for drug resistance of tuberculosis Mexico, MMWR Morb Mortal Wkly Rep 47, Anonymous (1998b). Guidelines for surveillance of drug resistance in tuberculosis. WHO Geneva/IUATLD Paris. International Union Against Tuberculosis and Lung Disease. Int J Tuberc Lung Dis 2, Chaves, F., Yang, Z. H., El Hajj, H., Alonso, M., Burman, W. J., Eisenach, K. D., Dronda, F., Bates, J. H. & Cave, M. D. (1996). Usefulness of the secondary probe ptbn12 in DNA fingerprinting of Mycobacterium tuberculosis. J Clin Microbiol 34, Cole, S. T., Brosch, R., Parkhill, J. & 39 other authors. (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, Escalante, P., Ramaswamy, S., Sanabria, H., Soini, H., Pan, X., Valiente-Castillo, O. & Musser, J. M. (1998). Genotypic characterization of drug-resistant Mycobacterium tuberculosis isolates from Peru. Tuber Lung Dis 79, Heym, B., Alzari, P. M., Honoré, N. & Cole, S. T. (1995). Missense mutations in the catalase-peroxidase gene, katg, are associated with isoniazid resistance in Mycobacterium tuberculosis. Mol Microbiol 15, Jin, D. J. & Gross, C. A. (1988). Mapping and sequencing of mutations in the Escherichia coli rpob gene that lead to rifampicin resistance. J Mol Biol 202, Kapur, V., Li, L. L., Iordanescu, S., Hamrick, M. R., Wanger, A., Kreiswirth, B. N. & Musser, J. M. (1994). Characterization by automated DNA sequencing of mutations in the gene (rpob) encoding the RNA polymerase â subunit in rifampin-resistant Mycobacterium tuberculosis strains from New York City and Texas. J Clin Microbiol 32, 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, Meier, A., Kirschner, P., Bange, F.-C., Vogel, U. & Böttger, E. C. (1994). Genetic alterations in streptomycin-resistant Mycobacterium tuberculosis: mapping of mutations conferring resistance. Antimicrob Agents Chemother 38, Nair, J., Rouse, D. A., Bai, G.-H. & Morris, S. L. (1993). The rpsl gene and streptomycin resistance in single and multiple drug-resistant strains of Mycobacterium tuberculosis. Mol Microbiol 10, National Committee for Clinical Laboratory Standards (1995). Anti- 112 IP: Journal of Medical Microbiology 53

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