Previous Article | Next Article 
Journal of Clinical Microbiology, August 2000, p. 3119-3122, Vol. 38, No. 8
Laboratório Central do Rio Grande do
Sul, Fundação Estadual de Produção e Pesquisa
em Saúde, Porto Alegre
Received 21 March 2000/Returned for modification 16 April
2000/Accepted 29 May 2000
Mutations in a 69-bp region of the rpoB gene associated
with rifampin resistance (Rifr) in 100 isolates (82 Rifr) from three states of Brazil were studied. Twenty-one
different kinds of mutations were identified in the Rifr
isolates, and six new alleles are described.
According to the World Health
Organization, 8 million cases of tuberculosis (TB) occur each year,
resulting in 3 million deaths (12). Strains of
Mycobacterium tuberculosis resistant to at least two drugs,
such as rifampin and isoniazid, are considered multidrug
resistant (MDR-TB). These multidrug-resistant strains arise by
sequential acquisition of resistance-conferring mutations in several
genes as a consequence of antibiotic selection. This situation causes
great concern worldwide because of the prolonged infectivity, which
increases the risk of transmission (1).
Rifampin is one of the most important chemotherapeutic agents used to
combat infections by M. tuberculosis and can be assumed to
be a surrogate marker for MDR-TB (3, 11). Resistance to this
drug has been shown to be due to alteration of the target molecule, the
In this study, we have used DNA sequencing to characterize mutations in
the 69-bp region of the rpoB gene. We analyzed 82 rifampin-resistant (Rifr) M. tuberculosis
isolates from three states of Brazil (Rio Grande do Sul [RS],
São Paulo [SP], and Rio de Janeiro [RJ]). SP and RJ are
located in the southeast region, and RS is located in the southern region.
Isolates used.
One hundred M. tuberculosis isolates
from different states of Brazil were analyzed by sequencing. From RS,
38 Rifr and 16 rifampin-susceptible (Rifs)
clinical isolates were isolated in the Laboratório Central do Rio
Grande do Sul. From RJ, 26 samples were obtained from the Centro de
Referência Professor Hélio Fraga (25 Rifr and 1 Rifs). Twenty isolates were provided by Instituto Adolfo
Lutz and Faculdade de Ciências Farmacêuticas Araraquara,
both in SP (19 Rifr and 1 Rifs). The samples
were collected from 1996 to 1998.
Culture and susceptibility testing.
Cultures were grown on
Ogawa medium. Rifampin susceptibility was determined on
Löwenstein-Jensen medium by the proportional method of Canetti et
al. (2). The isolates were also tested for susceptibility to
isoniazid, ethambutol, pyrazinamide, and streptomycin by the same
method (Table 1).
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Mutations in the rpoB Gene of
Multidrug-Resistant Mycobacterium tuberculosis Isolates
from Brazil
Rio Grande do Sul CEP
90130-001,1 and Centro de Biotecnologia,
Universidade Federal do Rio Grande do Sul, Porto AlegreRio Grande
do Sul CEP 91501-970,2 Brazil
ABSTRACT
Top
Abstract
Text
References
TEXT
Top
Abstract
Text
References
subunit of RNA polymerase (5, 15). This putative rifampin resistance is associated with mutations that occur within a
69-bp region of the rpoB gene, which encodes the subunit
of RNA polymerase. The types of mutations include single-nucleotide changes and in-frame deletions and insertions (8, 15, 18).
TABLE 1.
Resistance patterns of MDR-TB isolates from Brazil
Sequencing of rpoB. To detect the mutations associated with Rifr, a 157-bp region of the rpoB gene was sequenced. A loopful of bacteria was suspended in 500 µl of TE (10 mM Tris, 1 mM EDTA, pH 8), and the DNA was extracted using cetyltrimethyl ammonium bromide as described previously (17). Purified M. tuberculosis DNA from the clinical isolates and reference strain H37RV was used to produce a 157-bp fragment of the rpoB gene, from nucleotide 1846 to 2002 (GenBank accession no. U12205) by using the primers TR9 (5'-TCGCCGCGATCAAGGAGT) and TR8 (5'-TGCACGTCGCGGACCTCCA) by the protocol described by Telenti et al. (15). The unincorporated nucleotides and primers were separated from the amplified DNA using MicroSpin columns (Pharmacia Biotech). Manual sequencing was carried out with the Thermo Sequenase Radiolabeled Terminator Cycle Sequencing kit (Amersham) according to the manufacturer's instructions and using the primers described above. For each sample the sequence was examined twice in one direction, using as a template the products of two independent amplification reactions. The isolates that showed new mutations were sequenced again using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) on an ABI Prism 310 genetic analyzer (Perkin-Elmer).
Mutations in the rpoB gene of Rifr M. tuberculosis isolates from RS.
In this group, DNA sequence
analysis of 38 resistant isolates revealed 9 different kinds of
missense mutations within a 157-bp region of the rpoB gene.
All isolates had a single-point mutation, and the highest frequency of
mutation was observed in the codon Ser-531 (55%). Point mutations in
codons 526 (29%), 516 (8%), 511 (3%), and 522 (3%) were also
observed (Table 2). We found no mutations
in the 157-bp rpoB segment sequenced from one
Rifr isolate and 16 Rifs isolates.
|
Mutations in the rpoB gene of Rifr M. tuberculosis isolates from SP. Twenty M. tuberculosis isolates (19 Rifr and one Rifs) were analyzed. Single-point mutations were detected in 18 of the isolates with the resistant phenotype. Seven different kinds of nucleotide substitution were revealed in four codons of the rpoB gene. The mutations were located in codon 531 (58%) and in codon 526 (26%). Two samples had changes of two bases in the same codon. Mutations in codons 522 (5%) and 533 (5%) were also detected (Table 2). One M. tuberculosis Rifr isolate contained no mutation within the region of the rpoB gene examined. Two new alleles (526CTG [encoding Leu] and 533CCT [encoding Pro]) were identified in this group of isolates (isolates SP28 and SP22). No mutations were observed in the Rifr isolate.
Mutations in the rpoB gene of Rifr M. tuberculosis isolates from RJ. The analysis of 26 M. tuberculosis isolates (25 Rifr and 1 Rifs) from RJ showed 10 mutations within a 157-bp region of the rpoB gene, affecting 12 amino acids in this region. Sixteen isolates presented one point mutation in codons 531 (52%), 516 (8%), and 513 (4%), while one isolate (4%) had a 4-codon deletion (515 to 518) and another isolate contained a point mutation in one codon (513) and a 3-codon deletion (514 to 516). Four Rifr isolates (16%) of M. tuberculosis contained point mutations in two separate codons, resulting in two amino acid substitutions for each isolate (513 and 514; 511 and 515; 531 and 526; and 511 and 516). One isolate had point mutations in three separate codons (524, 525, and 526) and a one-codon deletion (527) (Table 2). One M. tuberculosis Rifr isolate contained no mutations within the 157-bp region of the rpoB gene. Four new alleles were identified in this group, all involving changes in two or more codons. Isolate RJ37 showed changes in codons 514 and 531. In isolate RJ48, the codons 524, 525, and 526 changed, together with a deletion of codon 527. Isolate RJ49 showed a deletion of codons 514 to 516 associated with a change in codon 513, and isolate RJ55 showed a deletion of four codons (515 to 518).
General analysis. Twenty-one different types of mutations were identified in 82 Rifr M. tuberculosis clinical isolates, and six new alleles were identified (Table 2). Most of them were single-nucleotide mutations (81%) involving seven codons. Eleven isolates (13%) exhibited more complex mutations. No silent substitutions were observed in the rpoB gene region examined for any of the M. tuberculosis isolates analyzed in this study. The codons most frequently affected by point mutations were 531, 526, and 516, with frequencies of 54%, 21%, and 7%, respectively. Although codon 526 was the second most affected, in Rifr isolates from RJ no mutation restricted to this codon was observed. No mutations were revealed in the rpoB segment sequenced from 18 Rifs isolates. Three Rifr isolates (4%) contained no mutations in this sequenced region, although these isolates were resistant to rifampin as determined by the proportional method.
The presence of mutations in a restricted region of the rpoB gene has been found in more than 96% of M. tuberculosis strains with various levels of rifampin resistance (4, 6, 10, 15, 18). While more than 20 distinct missense mutations within the 69-bp hypervariable region of rpoB accounting for rifampin resistance in M. tuberculosis have been reported (9), two of these mutations (Ser531
Leu and
His526Tyr) account for 
65% (61% in our study) of
rifampin resistance (6, 15, 16). These substitutions were
considered important to the acquisition of rifampin resistance, and it
is clear that these two amino acids are critical sites for this characteristic.
In our study, we observed that 82% of the M. tuberculosis
isolates with the Rifr phenotype contained missense
mutations which led to amino acid substitutions at the
Ser531 (54%), His526 (21%), and
Asp516 (7%) residues. Similar mutations and
frequencies of codon substitution in Rifr
M. tuberculosis have been reported previously (6, 7,
13, 14, 15). Other missense mutations as well as deletions were found in 21% of the Rifr M. tuberculosis strains. A characteristic finding was the high frequency of double mutations occurring in two separate codons (16%) in the RJ isolates and more complex mutations, such as a combination of point mutations with a deletion of one or more codons in two cases [CAA(Gln513)CAC(His), with a
deletion of codons encoding Lys514, Tyr515,
and Leu516; TTG(Leu524)TGG(Trp), AAC(Tyr525)CCC(Pro), and
CAC(His526)CAG(Gln), with a deletion of the codon
for Phe527] and deletion of four codons (encoding Tyr515, Leu516, Val517, and
Leu518) in one isolate.
In general, isolates from RS and SP showed some similarity in the
mutation frequencies. Samples from RJ showed a wider range of the
mutations described above. Some of the mutations observed in RJ
isolates were not found in other isolates analyzed in this work and in
isolates from other published studies. Further studies must be done to
find an explanation for such differences.
Sequence analysis identified no mutation in three of the isolates
tested, although these isolates were resistant to rifampin. Similar
observations have been reported by others (6, 7, 15) and
suggest that mutations located outside the region of analysis can
result in rifampin resistance. Another possibility is that in these
resistant strains, changes have occurred in genes whose products
participate in antibiotic permeation or metabolism (6). No
silent mutations were observed in the sequenced regions of
rifampin-susceptible or -resistant M. tuberculosis isolates in this analysis.
No association was found between particular mutations in the
rpoB gene and drug susceptibility patterns of MDR-TB
isolates, supporting the view that the mutations leading to rifampin
resistance are independent events unrelated to these mutations
affecting the development of resistance to the others antibiotics
tested (18).
In this study, we identified mutations in rifampin-resistant M. tuberculosis strains from Brazil that were commonly found in
strains from other parts of the world. We found six new alleles, four
of them in isolates from Rio de Janeiro.
Nucleotide sequence accession numbers. The new alleles found in this study have been deposited in GenBank under accession no. AF143771, AF146567, AF147030, AF147031, AF147033, and AF147034.
| |
ACKNOWLEDGMENTS |
|---|
We thank Angela M. W. Barreto from Instituto Prof. Hélio Fraga, Maria Alice Telles from Instituto Adolfo Lutz, and Clarice Q. F. Leite from USP-Araraquara for kindly providing the M. tuberculosis isolates and for availability to help. We also thank Lee Riley for his invaluable advice.
This investigation received financial support from FAPERGS CNPq. Part of this work was also supported by Fogarty International Center, National Institutes of Health (TW 00905).
| |
FOOTNOTES |
|---|
*
Corresponding author. Mailing address: Centro de
Biotecnologia/UFRGS, Av. Bento Gonçalves 9500, Prédio
43421, CEP 91501-970 Porto AlegreRS, Brazil. Phone: 55 51 316-6070. Fax: 55 51 319-1079. E-mail:
zaha{at}dna.cbiot.ufrgs.br.
| |
REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. |
Bifani, P. J.,
B. B. Plikaytis,
V. Kapur,
K. Stockbauer,
X. Pan,
M. L. Lutfey,
S. L. Moghazeh,
W. Eisner,
T. M. Daniel,
M. H. Kaplan,
J. T. Crawford,
J. M. Musser, and B. N. Kreiswirth.
1996.
Origin and interstate spread of New York City multidrug-resistant Mycobacterium tuberculosis clone family.
JAMA
275:452-457 |
| 2. | Canetti, G., W. Fox, A. Khomenko, H. T. Mahler, N. K. Menon, D. A. Mitchison, N. Rist, and N. A. Smeley. 1969. Advances in techniques of testing mycobacterial drug sensitivity, and the use of sensitivity tests in tuberculosis control programmes. Bull. W. H. O. 41:21-43[Medline]. |
| 3. |
Drobniewski, F. A., and S. M. Wilson.
1998.
The rapid diagnosis of isoniazid and rifampicin resistance in Mycobacterium tuberculosisa molecular story.
J. Med. Microbiol.
47:189-196 |
| 4. | Felmlee, T. A., Q. Liu, A. C. Whelen, D. Williams, S. S. Sommer, and D. H. Persing. 1995. Genotypic detection of Mycobacterium tuberculosis rifampin resistance: comparison of single-strand conformation polymorphism and dideoxy fingerprinting. J. Clin. Microbiol. 33:1617-1623[Abstract]. |
| 5. |
Honore, N., and S. T. Cole.
1993.
Molecular basis of rifampin resistance in Mycobacterium leprae.
Antimicrob. Agents Chemother.
37:414-418 |
| 6. |
Kapur, V.,
L. L. Li,
S. Iondanescu,
M. R. Hamrick,
A. Wanger,
B. N. Kreiswirth, and J. M. Musser.
1994.
Characterization by automated DNA sequencing of mutations in the gene (rpoB) encoding the RNA polymerase subunit in rifampin-resistant Mycobacterium tuberculosis strains from New York City and Texas.
J. Clin. Microbiol.
32:1095-1098 |
| 7. |
Matsiota-Bernard, P.,
G. Vrioni, and E. Marinis.
1998.
Characterization of rpoB mutations in rifampin-resistant clinical Mycobacterium tuberculosis isolates from Greece.
J. Clin. Microbiol.
36:20-23 |
| 8. |
Miller, L. P.,
J. T. Crawford, and T. M. Shinnick.
1994.
The rpoB gene of Mycobacterium tuberculosis.
Antimicrob. Agents Chemother.
38:805-811 |
| 9. | Musser, J. M. 1995. Antimicrobial agent resistance in mycobacteria: molecular genetic insights. Clin. Microbiol. Rev. 8:496-514[Abstract]. |
| 10. | Ohno, H., H. Koga, T. Kuroita, K. Tomono, K. Ogawa, K. Yanagihara, Y. Yamamoto, J. Miyamoto, T. Tashiro, and S. Kohno. 1997. Rapid prediction of rifampin susceptibility of Mycobacterium tuberculosis. Am. J. Respir. Crit. Care Med. 155:2057-2063[Abstract]. |
| 11. | Rattan, A., A. Kalia, and N. Ahmad. 1998. Multidrug-resistant Mycobacterium tuberculosis: molecular perspectives. Emerg. Infect. Dis. 42:195-207. |
| 12. |
Raviglione, M. C.,
D. E. Sinder, and A. Kochi.
1995.
Global epidemiology of tuberculosismorbidity and mortality of a worldwide epidemic.
JAMA
273:220-226 |
| 13. | Scarpellini, P., S. Braglia, A. M. Brambilla, M. Dalessandro, P. Cichero, A. Gori, and A. Lazzarin. 1997. Detection of rifampin resistance by single-strand conformation polymorphism analysis of cerebrospinal fluid of patients with tuberculosis of the central nervous system. J. Clin. Microbiol. 35:2802-2806[Abstract]. |
| 14. |
Telenti, A.,
F. Imboden,
F. Marchesi,
T. Schmidheini, and T. Bodmer.
1993.
Direct, automated detection of rifampin-resistant Mycobacterium tuberculosis by polymerase chain reaction and single-strand conformation polymorphism analysis.
Antimicrob. Agents Chemother.
37:2054-2058 |
| 15. | 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[CrossRef][Medline]. |
| 16. | Telenti, A., N. Honoré, C. Bernasconi, J. March, A. Ortega, B. Heym, H. E. Takiff, and T. Cole. 1997. Genotypic assessment of isoniazid and rifampin resistance in Mycobacterium tuberculosis: a blind study at reference laboratory level. J. Clin. Microbiol. 35:719-723[Abstract]. |
| 17. |
Van Embden, J. D. A.,
M. D. Cave,
J. T. Crawford,
J. W. Dale,
K. D. Eisenach,
B. Gicquel,
P. W. M. 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 |
| 18. |
Williams, D. L.,
C. Waguespack,
K. Eisenach,
J. T. Crawford,
F. Portaels,
M. Salfinger,
C. M. Nolan,
C. Abe,
V. Stich-Groh, and T. P. Gillis.
1994.
Characterization of rifampin resistance in pathogenic mycobacteria.
Antimicrob. Agents Chemother.
38:2380-2386 |
Journal of Clinical Microbiology, August 2000, p. 3119-3122, Vol. 38, No. 8
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
O'Sullivan, D. M., McHugh, T. D., Gillespie, S. H.
(2005). Analysis of rpoB and pncA mutations in the published literature: an insight into the role of oxidative stress in Mycobacterium tuberculosis evolution?. J Antimicrob Chemother
55: 674-679
[Abstract] [Full Text] -
Lee, A. S. G., Lim, I. H. K., Tang, L. L. H., Wong, S. Y.
(2005). High Frequency of Mutations in the rpoB Gene in Rifampin-Resistant Clinical Isolates of Mycobacterium tuberculosis from Singapore. J. Clin. Microbiol.
43: 2026-2027
[Full Text] -
Jou, R., Chen, H.-Y., Chiang, C.-Y., Yu, M.-C., Su, I.-J.
(2005). Genetic Diversity of Multidrug-Resistant Mycobacterium tuberculosis Isolates and Identification of 11 Novel rpoB Alleles in Taiwan. J. Clin. Microbiol.
43: 1390-1394
[Abstract] [Full Text] -
Yam, W. C., Tam, C. M., Leung, C. C., Tong, H. L., Chan, K. H., Leung, E. T. Y., Wong, K. C., Yew, W. W., Seto, W. H., Yuen, K. Y., Ho, P. L.
(2004). Direct Detection of Rifampin-Resistant Mycobacterium tuberculosis in Respiratory Specimens by PCR-DNA Sequencing. J. Clin. Microbiol.
42: 4438-4443
[Abstract] [Full Text] -
Silva, M. S. N., Senna, S. G., Ribeiro, M. O., Valim, A. R. M., Telles, M. A., Kritski, A., Morlock, G. P., Cooksey, R. C., Zaha, A., Rossetti, M. L. R.
(2003). Mutations in katG, inhA, and ahpC Genes of Brazilian Isoniazid-Resistant Isolates of Mycobacterium tuberculosis. J. Clin. Microbiol.
41: 4471-4474
[Abstract] [Full Text] -
Yue, J., Shi, W., Xie, J., Li, Y., Zeng, E., Wang, H.
(2003). Mutations in the rpoB Gene of Multidrug-Resistant Mycobacterium tuberculosis Isolates from China. J. Clin. Microbiol.
41: 2209-2212
[Abstract] [Full Text] -
Fan, X.-Y., Hu, Z.-Y., Xu, F.-H., Yan, Z.-Q., Guo, S.-Q., Li, Z.-M.
(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
[Abstract] [Full Text] -
Van Der Zanden, A. G. M., Te Koppele-Vije, E. M., Vijaya Bhanu, N., Van Soolingen, D., Schouls, L. M.
(2003). Use of DNA Extracts from Ziehl-Neelsen-Stained Slides for Molecular Detection of Rifampin Resistance and Spoligotyping of Mycobacterium tuberculosis. J. Clin. Microbiol.
41: 1101-1108
[Abstract] [Full Text] -
Hwang, H.-Y., Chang, C.-Y., Chang, L.-L., Chang, S.-F., Chang, Y.-H., Chen, Y.-J.
(2003). Characterization of rifampicin-resistant Mycobacterium tuberculosis in Taiwan. J Med Microbiol
52: 239-245
[Abstract] [Full Text] -
Cavusoglu, C., Hilmioglu, S., Guneri, S., Bilgic, A.
(2002). Characterization of rpoB Mutations in Rifampin-Resistant Clinical Isolates of Mycobacterium tuberculosis from Turkey by DNA Sequencing and Line Probe Assay. J. Clin. Microbiol.
40: 4435-4438
[Abstract] [Full Text] -
Qian, L., Abe, C., Lin, T.-P., Yu, M.-C., Cho, S.-N., Wang, S., Douglas, J. T.
(2002). rpoB Genotypes of Mycobacterium tuberculosis Beijing Family Isolates from East Asian Countries. J. Clin. Microbiol.
40: 1091-1094
[Abstract] [Full Text] -
Bartfai, Z., Somoskovi, A., Kodmon, C., Szabo, N., Puskas, E., Kosztolanyi, L., Farago, E., Mester, J., Parsons, L. M., Salfinger, M.
(2001). Molecular Characterization of Rifampin-Resistant Isolates of Mycobacterium tuberculosis from Hungary by DNA Sequencing and the Line Probe Assay. J. Clin. Microbiol.
39: 3736-3739
[Abstract] [Full Text] -
Mani, C., Selvakumar, N., Narayanan, S., Narayanan, P. R.
(2001). Mutations in the rpoB Gene of Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolates from India. J. Clin. Microbiol.
39: 2987-2990
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»