Rifampicin Resistance and Mutation of The Rpob Gene in
Rifampicin Resistance and Mutation of The Rpob Gene in
Rifampicin Resistance and Mutation of The Rpob Gene in
Abstract
Using 39 clinical isolates of Mycobacterium tuberculosis strains with a broad range of susceptibility to rifampicin, we
examined the relationship between the degree of resistance to rifampicin and mutational sites of the rpoB gene. All rifampicin-
resistant strains had missense mutations. Twenty strains (95%) had a mutation in the cluster I region, which has also been
reported in Escherichia coli [Jin and Gross (1988) J. Mol. Biol. 202, 45-581, and the remaining one strain had a mutation at
codon 381 [Ala + Val] in the N-terminal region, which has not been reported in E. coli. Among 18 rifampicin-susceptible
strains, two had a mutation in the cluster I region and the other three strains had a mutation in the cluster III region. The
mutations at codons 513 (So/,), 526 (33%) or 531 (43%) in the cluster I region led to high level resistance to rifampicin (50 ug
ml-‘SMIC). The mutations at the other sites, in the cluster III region (codons 679 or 687) and even in the cluster I region
(codon 514, 521, or 533), showed low level (MIC = 12.5 ug ml-‘) or no (MIC < 0.39 ug ml-l) resistance to rifampicin. These
results suggest that mutations in the rpoB gene are, mostly, but not necessarily, associated with rifampicin resistance of
M. tuberculosis, and the sites of mutations on the rpoB gene will affect the level of resistance to rifampicin.
Keywords: Mycobacterium tuberculosis; Rifampicin resistance; rpoB gene; Mutational site; Level of resistance
037%1097/96/ $12.00 Copyright 0 1996 Federation of European Microbiological Societies. published by Elsevier Science B.V.
PIISO378-1097(96)00346-l
104 H. Taniguchi LJIul. IFEMS Microbiology Letters 144 (1996) 103-108
II, and III) [l] and one site at a 146 amino acid Ltd. Tokyo, Japan) containing 0, 10 and 50 pg ml-’
residue upstream of the cluster I region [5]. Further- rifampicin (Table 1). According to the instructions of
more, it has also been reported that a level of resist- the suppliers, a resistant level is shown by a percent
ance to rifampicin depended on the mutational sites degree comparing between the colony numbers
in the rpoB gene in E. cd, mutations in the cluster I grown on 1% Ogawa’s egg medium containing 10
region of the rpoB gene leading to especially high and 50 pg ml-’ of rifampicin and that on 1% Oga-
levels of resistance to rifampicin. wa’s egg medium not containing drug (control medi-
cation was performed for 30 cycles (1 min at 94”C, Among the 21 resistant strains, four showed a low
1 min at 55”C, 2 min at 72°C) by a model 480 ther- level of resistance to rifampicin (MIC = 12.5 ug ml-i).
mocycler (Perkin-Elmer Cetus). As shown in Fig. 1, In E. co/i, mutations of rifampicin-resistant strains
cluster I region was amplified using primers TR9 (5’- were located in three distinct clusters (cluster I, II,
TCGCCGCGATCAAGGAGT-3’) and TR8 (5’- and III regions) [l], and in the N-terminal region
TGCACGTCGCGGACCTCCA-3’), TRl (5’-TAC- (codon 146) [5] of the rpoB gene (Fig. 1). Most of
GGTCGGCGAGCTGATCC-3’) and TR2b (5’- the mutations leading to high level resistance were
Table 1
Sequence analysis of the rpoB gene of M. tuberculws clinical isolates
Strain no. Number MIC” ‘%,growth on” Mutational position’ Amino acid Nucleotide
of isolates tug ml-‘) change change
+RflO 50 N I II III
Resistant strains (n = 21 i
59 1 200 100 100 381’1 -1’ _ Ala + Val GCG + GTG
58 1 > 200 100 100 513 Gln + Leu CAA -+ CTA
hibited point mutations. Two of them had a muta- low level of resistance [l]. Because no strain having a
tion in the cluster I region (codon 521 or 533) and mutation in the cluster II region was present, we do
three strains showed a mutation in the cluster III not know whether a mutation at cluster II is in-
region (codon 679 or 687). This suggests that muta- volved in rifampicin resistance in M. tuberculosis.
tions in the cluster III region are not associated with It has been reported that in E. co& the sites of
rifampicin resistance or confer only a very low level mutation in the cluster I region will affect the level
of resistance in M. tuberculosis (MICc 0.39 ug of resistance to rifampicin [l]. However, the relation-
ml-‘). Also in E. coli, cluster III mutants showed a ship has not been reported between the degree of
H. Taniguchi et al. IFEMS Microbiology Letters 144 (1996) 103-108 107
rpoB ( /3 subunit)
Amino acid 1 1342
resistance to rifampicin and the mutational sites in don 381, 513, 514, 516, 526, 531, or 533 led to re-
IV. tuberculosis. So we summarized this relationship sistance to rifampicin. Among them, a mutation at
(Fig. 2). Our results showed that a mutation at co- codon 513, 526 or 531 led to high MICs (50 pg
N Region 1
El
CCC CCG ACC AAAGAGTCA EC: CAG ICC CTC TTG GAA AAC TTC TTC TTC AAG GAG
375 -Pro Pro Thr tys Glu Ser Ala Gin Thr teu Lou Glu Ass leu Phe Phe tyr Glu-392
1
I Region a
I I
a b
1 I bl
CTG ICC CAA TTC ATG GAC CAG AAC AAC CCC CTG TCG CCC TTG ACl: CAC AAG CGC CGA CTG TCG CCC CTG
Sll- leu Ser Glo Phe Met Asp Gin Asn Asn Pro teu Ser Gly Leu Tbr His lys Arg Arg teu Ser Ala teu-533
CTG AGC CAA TTC ATG GAC CAG AAC AAC CCC CTG TCG GGG TTG ACC CAC AAG CGC CGA CTG TCG CCC CTG
III Region
GCC AAC CGT CCC CTC ATG GCG GCA AAC ATG CAG CGC CAC GCG
616 -Ala Ass Arg Ala teu Met Gly Ala Asn Met Gin Ar CIn Ala- 689
2 1
K!l r El
Fig. 2. Mutational sites of M. tuberculosis rpoB gene. The mutational positions of rifampicin-resistant M. tuberculosis harboring 12.5 pg
ml-‘ SMICs or MICs < 0.39 pg ml-’ are illustrated above or below the wild-type sequence. The numbers on the left side above the boxes
are those of strains which harbored 50 pg ml-‘SMICs, and the numbers on the right side above the boxes are those of strains which har-
bored MIC= 12.5 kg ml-‘. ‘a’ and ‘b’ indicate a combination of two mutations harbored in one strain, corresponded to strains 20 and
72 in Table 1. We used the rpoB codon numbering system used by Telenti et al. [lo] for comparison between our data and those gener-
ated by them. The codon numbers are designated on the basis of alignment of the translated E. coli rpoB sequence, and are not the actual
M. tuberculosis rpoB codons.
108 H. Taniguchi et ul. I FEMS Microbiology Letters 144 (1996) 103-108