Mem Inst Oswaldo Cruz, Rio de Janeiro, 96(2) February 2001
Original Article

Mutations in the rpoB Gene of Rifampicin-resistant Mycobacterium tuberculosis Strains Isolated in Brazil and France

S Spindola de Miranda
+, AL KritskiI, I FilliolII, C MabilatII, G PanteixIII,
E DrouetIV

Departamento de Clínica Médica/Pneumologia, Faculdade de Medicina, Universidade Federal de Minas Gerais, Rua Alfredo Balena 190, 30130-100 Belo Horizonte, MG, Brasil
IHospital Universitário Clementino Fraga Filho, UFRJ, Rio de Janeiro, RJ, Brasil
IILaboratoire bioMerieux, Lyon, France
IIILaboratoire Marcel Mérieux, Lyon, France
IVFaculté de Pharmacie, Université Joseph-Fourier, Grenoble, France

Page: 247-250
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We evaluated the mutations in a 193bp of theu00a0rpoBu00a0gene by automated sequencing of rifampicin (RMP)-resistant and susceptibleu00a0Mycobacterium tuberculosisu00a0strains isolated from Brazil (25 strains) and France (37 strains). In RMP-resistant strains, mutations were identified in 100% (16/16) from France and 89% (16/18) from Brazil. No mutation was detected in the 28 RMP-susceptible strains. Among RMP-resistant or RMP-susceptible strains deletion was observed. A double point mutation which had not been reported before was detected in one strain from France. Among French resistant strains mutations were found in codons 531 (31.2%), 526, 513 and 533 (18.7% each). In Brazilian strains the most common mutations were in codons 531 (72.2%), 526 (11.1%) and 513 (5.5%). The heterogeneity found in French strains may be related to the fact that most of those strains were from African or Asian patients.

Resistance of Mycobacterium tuberculosis to antituberculous drugs has emerged as a major public health threat (Pablo-Mendes et al. 1998). The short course therapy with rifampicin (RMP), isoniazid (INH) and pyrazinamid (PZA) is the most effective regimen against tuberculosis. The efficacy of this regimen is hampered in regions where there is a high initial resistance to RMP. In those regions, the rapid detection of RMP-resistance is urgently needed. New methodologies have been proposed to cover this situation.

Development of resistance to RMP in M. tuberculosis follows a "single-step" high-level resistance pattern (Morris et al. 1995, Musser 1995). Mutants arise spontaneously in strains not exposed previously to the antibiotic at a rate of one mutation per 10-7 to 10-8 organisms (Cole 1994, Williams et al. 1994). Resistance has been attributed to changes in structural RNA polymerase (Watterson et al. 1998, Yuen et al. 1999). The structural and functional organization of RNA polymerase is conserved among bacteria (Hunt et al. 1994). Escherichia coli resistance to RMP is associated with specific nucleic acid substitutions in the gene encoding for RNA polymerase subunit-b (rpoB) (Miller et al. 1994, Kapur et al. 1995). Mutations associated with the RMP-resistant phenotype are generally localized in a 69bp "hot spot" region with mutations frequently in the codons: Ser-531, His-526 and Asp-516 (Telenti et al. 1993a, Williams et al. 1994, Hunt et al. 1994, Kapur et al. 1995, Musser 1995). Variations of mutations in rpoB gene have been described in M. tuberculosisstrains isolated from different regions of the world (Kapur et al. 1995, Morris et al. 1995, Whelen et al. 1995). In only 5% of RMP-resistant M. tuberculosis strains, mutations are not identified in a "hot spot" fragment; in these cases mutations at codons 381, 481, 505, 508, and 509 (Watterson et al. 1998) are sometimes reported. Commercial kits which cover the four most frequently observed mutations (D516V, H526Y, H528D and S531L) of the "hot spot" rpoB region have been proposed for clinical use (Inno-Lipa RifTB; Innogenetics, Belgium) (Goyal et al. 1997, Matsiota-Bernard et al. 1998, Hirano et al. 1999).

In France, the tuberculosis incidence is 17/100,000 inhabitants and resistance to anti-tuberculosis drugs has recently increased (Liard et al. 1994, Salomon et al. 1994). In Brazil, tuberculosis incidence is higher in urban areas such as the States of Rio de Janeiro (120/100.000) and São Paulo (80/100.00). Since 1981, the Brazilian National Tuberculosis Control Program adopted the daily self-administered short-course treatment regimen (2 RMP + INH + PZA/4 RMP + INH). The urban areas have reached 28% leading to an increase of tuberculosis abandon (Kritski et al. 1996, 1998). In Rio de Janeiro, drug-resistance surveillance performed in outpatient clinic showed a level of 1% of RMP primary resistance, but in patients attended in hospital, it increases to 7% (Pablo-Mendez et al. 1998).

In this study, we evaluate mutations in the rpoB gene of RMP-resistant and susceptible M. tuberculosis strains isolated from Brazil (25 strains) and France (37 strains).



Characterization of mycobacterial clinical isolates - Sixty two strains (34 RMP-resistant and 28 RMP-susceptible M. tuberculosis isolates) were recovered from Brazilian and French patients: Rio de Janeiro University, Brazil (14 strains), São Paulo University, Brazil (11 strains), France (13 strains), Africa (20 strains) and Asia (4 strains). One reference strain H37Ra was included.

Detection of rifampicin resistance - Strains were identified by standard biochemical methods in the Institut Pasteur of Lyon. RMP resistance was determined three times for each strain by the disk elution method using a 25 µg disk corresponding to 5 µg/ml final concentration (Sensi-Disk; BBL, Becton Dickinson Microbiology System, Cockeysville, MD).

Preparation of genomic mycobacterial DNA - Mycobacterial strains were cultured for three or four weeks at 37°C in 40 ml of Middlebrook 7H9 liquid medium with Dubos oleic albumen complex enrichment (Difco Laboratories, Detroit, MI) and 0.05% Tween 80. Cells were heat-killed at 80°C for 20 min, then harvested by centrifugation at 7,000 g for 30 min. Bacterial cells were resuspended in 10 mg/ml lysosyme in 10 mM Tris, 
1 mM EDTA (pH 8.0) for 2 h at 37°C, then lysed by sonication (Ultrasonic, BioBlock, Scientificâ- 88169), 12 min at 40°C. Finally, lysate was incubated for 1 h at 60°C with 0.5 mg/ml proteinase K, 1% sodium dodecyl sulphate in 50 mM Tris, 100 mM EDTA (pH 7.5). The DNA was purified by phenol-chloroform-isoamylalcohol extraction and isopropanol precipitation. The pellet was washed twice in 70% ethanol, then resuspended in 50 mM Tris, 100 mM EDTA (pH 8.0), and 5 µl of 1/500 dilution of purified DNA in water was used as a target in all polymerase chain reaction (PCR).

Sequencing analysis - Primers for PCR were synthesized with an Expedite Nucleic Acid Synthesis System (Perspective Biosystem). PCR reactions (50 µl) contained target DNA (10 µg), 15 pmol primers, 20 mM dNTP (Pharmacia Biotech), 2.5U AmpliTaq Gold polymerase (Perkin-Elmer Cetus, Norwalk, CT, USA), and KCl 500 mM, Tris HCl 100 mM, MgCl2 15 mM, gelatin 0.1%, pH 8.3. The reaction was performed in a Cyclogene Tech. thermal cycler (USA).

Primers and amplification conditions for the tuberculosis rpoB locus were as follows: upstream primer (rpo 105) 5'-CGTGGAGGCGATCACACC GCAGACGT-3'; downstream primer (rpo 273) 5'-GACCTCCAGCCCGGCACGCTCACG-3', which produced a 193-bp amplicon (Kapur et al. 1994). Amplification was performed during 40 cycles of 96°C (1 min), 55° (1 min) and 72°C (30 sec), after an initial 5 min at 96°C to denature input DNA, with a final 10 min at 72°C for strand extension.

Amplification products were detected by agarose gel electrophoresis in 2% agarose (Seakem GTG; FMC, Rockland, ME, USA) made up in 0.5x Tris-Borate-EDTA (TBE), with ethidium bromide. The products were not purified, and were sequenced by using primer rpo105 and rpo273 with Applied Biosystems 373A or 377 automated sequence protocol (ABI PRISMTM Dye Terminator).

Sequence alignment was done by using dedicated software (Villefranche Sur Mer, CNRS, Richard Christen).

Determination of minimal inhibitory concentration (MIC) - For one strain the MIC was determined by proportion agar dilution method. Middlebrook 7H10 agar, supplemented with Middlebrook OADC enrichement (DIFCO Laboratories, Detroit, MI) was used to prepare quadrant plates with different concentrations of drugs (Kent & Kubica 1985): RMP (0.004-40 µg/ml).

RFLP (restriction fragments length polymorphism) analysis - All the strains were analyzed in accordance with the protocol of Van Soolingen et al. (1992) for RFLP-IS6110.



Among the Brazilian strains from Rio de Janeiro, 12 (85.7%) were RMP resistant and 2 (14.3%) RMP-susceptible. Among the strains from São Paulo, 6 (54.5%) were RMP-resistant and 5 (45.5%) RMP-susceptible. Among the strains from Institut Pasteur of Lyon, France, 16 (43.2%) were RMP-resistant and 21 (56.7%) RMP-susceptible.

The presence of point mutations in the M. tuberculosis RMP-resistant strains was confirmed in 32/34 (94.1%) strains, within an amplicon representing a 193-bp region of the rpoB gene (Table I). The mutations in the RMP-resistant strains are demonstrated in the Table II; only two strains from Rio de Janeiro had no mutation. A double point mutation was detected in one strain from France, in two noncontiguous codons, but no insertion or deletion was observed (Table II).

In this study, the mutations within the rpoB gene of M. tuberculosis, which appear to be associated with the RMP-resistant phenotype identified, are consistent with other publications (Hunt et. al. 1994, Kapur et al. 1995, Kreiswirth & Musser 1995, Musser 1995). Point mutations within the Ser-531, His-526, Gly-513 or Asp-516 codons has been shown to lead to high-level resistance in E. coli (20 µg/ml)and M. tuberculosis (Jin & Gross 1988, Telenti et al. 1993a,b, Williams et al. 1994, Whelen et al. 1995). In our study, we found a change in codon 533 in four strains. This mutation have been reported for RMP-resistant and RMP-susceptible strains for Taniguchi et al. (1996). A double mutation in codons 516 (Asp) and 533 (Leu) was observed in our study (Gen Bank access number AF 292115), and presented a high level of resistance (MIC > 40 µg/ml). These data have not been reported before.

In Brazilian isolates, a predominance of point mutation was observed in codon 531. In French M. tuberculosis strains this figure did not occur. The variation observed in the French isolates may be due to the heterogeneity of the native countries of origin of the patients, as demonstrated in Table II. When we compared the RFLP profile with points mutation, no RFLP clustering was associated with mutations frequency described in other studies (Telenti et al. 1993b, Spindola de Miranda et al. 1996).

No point mutations were found in the RMP-susceptible strains in the sequenced region studied as previously described, but in Brazilian isolates, the absence of mutations in two RMP-resistant strains may be due to mutations outside therpoB region studied or to another resistance mechanism. These results suggest the usefulness of the rapid determination of the drug-susceptible characterization of M. tuberculosis using automated DNA sequencing methods.

The results obtained in our study must be validated with a great number of RMP-resistant strains for different region in Brazil. The MICs analysis of M. tuberculosis would be useful to evaluate the relationship between resistant phenotype and points mutation (Ohno et al. 1996).



Cole ST 1994. Mycobacterium tuberculosis: drug resistance mechanisms. Trends Microbiol 2: 411-415.

Goyal M, Shaw RJ, Banerjee DK, Coker RJ, Robertson BD, Young DB 1997. Rapid detection of multidrug-resistant tuberculosis. Eur Respir J 10: 1120-1124.

Hirano K, Abe C, Takahashi M 1999. Mutations in the rpoB gene of rifampin-resistant Mycobacterium tuberculosisstrains isolated mostly in Asian countries and their rapid detection by line probe assay. J Clin Microbiol 37: 2663-2666.

Hunt JM, Roberts G, Stockman L, Felmlee TA, Persing DH 1994. Detection of a genetic locus encoding resistance to rifampin in mycobacterial cultures and in clinical specimens. Diagn Microbiol Infect Dis 18: 219-227.

Jin DJ, Gross CA 1988. Mapping and sequencing of mutations in the Escherichia coli rpoB gene that lead to rifampicin resistance. J Mol Biol 202: 45-48.

Kapur V, Li LL, Hamrick MR, Plikaytis BB, Shinnick TM, Telenti A, Jacobs WR, Banerjee A, Cole S, Yuen KY, ClarridgeIII JE, Kreiswirth BN, Musser JM 1995. Rapid mycobacterium species assignment and unambiguous identification of mutations associated with antimicrobial resistance in Mycobacterium tuberculosis by automated DNA sequencing. Arch Pathol Lab Med 119: 131-138.

Kent PT, Kubica GP 1985. Public Health Mycobacte-riology. A Guide for the Level III Laboratory, Centers for Diseases Control, Atlanta.

Kreiswirth BN, Musser JM 1995. Rapid Mycobacterium species assignment and unumbiguous identification of mutations associated with antimicrobial resistance in Mycobacterium tuberculosis by automated DNA sequencing.Arch Pathol Lab Med 119:131-138.

Kritski AL, Lapa e Silva JR, Conde MB 1998. Tuberculosis and HIV: renewed challenge. Mem Inst Oswaldo Cruz 93: 417- 421.

Kritski AL, Marques MJO, Rabahi MF, Vieira MAS, Werneck Barroso E, Carvalho CES, Andrade GN, Bravo de Souza R, Gontijo PP, Riley LW 1996. Transmission of tuberculosis to close contacts of patients with multidrug resistant tuberculosis. Am J Respir Crit Care Med 153: 331-335.

Liard R, Harf R, Korobaeff M, Shwoebel V, Neukirch F 1994. HIV infection and tuberculosis: an epidemiological study from a French register. Tubercle Lung Dis 75: 291-296.

Matsiota-Bernard P, Vrioni G, Marinis E 1998. Characterization of rpoB mutations in rifampin-resistant clinicalMycobacterium tuberculosis isolates from Greece. J Clin Microbiol 36: 20-23.

Miller LP, Crawford J, Shinnick T 1994. The rpoB Gene of Mycobacterium tuberculosisAntimicrob Agents Chemother 38: 805-811.

Morris S, Bai GH, Suffys P, Portillo Gomez L, Fairchok M, Rouse D 1995. Molecular mechanism of multiple drug resistance in clinical isolates of Mycobacterium tuberculosisJ Infec Dis 171: 954-960.

Musser JM 1995. Antimicrobial agents resistance in mycobacteria: molecular genetic insights. Clin Microbiol Ver 8: 496-514.

Ohno H, Koga H, Kohno S, Tashiro T, Hara K 1996. Relationship between rifampin MICs and rpoB mutations ofMycobacterium tuberculosis strains isolated in Japan. Antimicrob Agents Chemother 40: 1053-1056.

Pablo-Mendez A, Raviglione MC, Laszlo A, Binkin N, Rieder HL, Bustreeo F, Cohn DL, Lambregts-van Weezenbeek CS, Kim SJ, Chaulet P, Nunn P 1998. Global surveillance for antituberculosis-drugs resistance, 1994-1997. N Engl J Med 338: 1641-1649.

Salomon N, Perlman DC, DePalo VA, Kolokathis A, Wilets I 1994. Drug resistant tuberculosis: factors associated with rise in resistance in an HIV infected urban population. Mt Sinai J Med 61: 341-348.

Spindola de Miranda S, Drouet E, Kritski AL, Peillon R, Panteix G 1996. Similar patterns of polymorphism between Brazilian and French Mycobacterium tuberculosis strains demonstrated by using IS-6110. Tubercle Lung Dis 77:1-140.

Taniguchi H, Aramaki H, Nikaido Y, Misuguchi M, Nakamura M, Koga T, Yoshida S 1996. Rifampicin resistance and mutation of the rpoB gene in Mycobacterium tuberculosisFEMS Microbiol Lett. 144: 103-108.

Telenti A, Imboden P, Marchesi F, Lowrie D, Cole S, Colston MJ, Matter L, Schopfer K, Bodmer T 1993a. Detection of rifampicin resistance mutations in Mycobacterium tuberculosis. Lancet 341: 647-650.

Telenti A, Imboden P, Marchesi F, Schmidheini T, Bodmer T 1993b. Direct, automated of rifampin resistantMycobacterium tuberculosis by polymerase chain reaction and single strand conformation polymorphism analysis.Antimicrob Agents Chemother 37: 2054-2058.

Van Soolingen D, Hermans PWM, De Haas PEW, Sool DR, Van Embden JD 1992. Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J Clin Microbiol 29: 2578-2586.

Watterson AS, Wilson SM, Yates MD, Drobneiewski FA 1998. Comparison of three molecular assays for rapid detection of rifampin resistance in Mycobacterium tuberculosisJ Clin Microbiol 36: 1969-1973.

Whelen AC, Felmlee TA, Hunt JM, Williams DL, Roberts GD, Stockman L, Persing DH 1995. Direct genotypic detection of Mycobacterium tuberculosis rifampin resistance in clinical specimens by using single tube heminested PCR. J Clin Microbiol 33: 556-561.

Williams DL, Waguespack C, Eisenach K, Crawford T, Portaels F, Salfinger M, Nolan CM, Abe C, Sticht Groh V, Gillis PT 1994. Characterization of rifampin resistance in phothogenic mycobacteria. Antimicrob Agents Chemother 38: 2380-2386.

Yuen LK, Leslie D, Coloe PJ 1999. Bacteriological and molecular analysis of rifampi-resistant Mycobacterium tuberculosis strains isolated in Australia. J Clin Microbiol 37: 3844-3850.

+Corresponding author. Fax: +55-31-3273.4985.
Received 16 November 1999
Accepted 4 October 2000

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