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Journal of Clinical Microbiology, April 2001, p. 1591-1594, Vol. 39, No. 4
Academic Medical Center, Department of
Medical Microbiology,1 and Free
University of Amsterdam, Department of Cell Biology, Faculty of
Medicine,2 Amsterdam, and National
Institute of Public Health and Environment, Diagnostic Laboratory for
Infectious Disease and Perinatal Screening,
Bilthoven,3 The Netherlands
Received 9 October 2000/Returned for modification 14 December
2000/Accepted 4 February 2001
A mutation (CCG Tuberculosis is the leading cause of
death due to infectious diseases worldwide (4), although
various drugs against Mycobacterium tuberculosis are
available. One of the mainstay drugs for the treatment of tuberculosis
is isoniazid (INH). Its effectiveness against M. tuberculosis was initially reported in 1952 (3, 15).
Today, INH-resistant M. tuberculosis organisms are not rare
anymore; prevalence was reported to be 7% in a recent study performed
in The Netherlands (25). The emergence of
multidrug-resistant strains (resistant to at least INH and rifampin)
(7, 11, 12, 20) has further complicated the treatment of
tuberculosis. Therefore, and because of the organism's slow growth
rate, rapid methods for detecting drug resistance in clinical isolates
of M. tuberculosis are required. The primary mechanism of
resistance in M. tuberculosis is the accumulation of
mutations in genes coding for drug targets or drug-converting enzymes
(16).
In the last decade, mutations in katG (24, 26)
and inhA (2) have been found to account for 60 to 70% and 10 to 15% of INH resistance cases among M. tuberculosis isolates, respectively (11). The two
predominant mutations of katG, and those most referred to,
are found within codons 315 and 463 (17).
The mutation at codon 315 has been found to be an important indicator
for INH resistance as well as for multidrug resistance among isolates
of M. tuberculosis organisms recovered from patients in The
Netherlands (25).
The aims of this study were to assess whether the Arg463Leu mutation is
also predictive of INH resistance and, if so, to develop a diagnostic
PCR-based screening method for this type of INH resistance.
(This work was presented in part at the 40th Interscience Conference on
Antimicrobial Agents and Chemotherapy, Toronto, Canada, 17 to 20 September 2000.)
M. tuberculosis isolates and assessment of INH
resistance.
M. tuberculosis isolates from 395 patients
who were diagnosed with tuberculosis in The Netherlands in the period
of 1993 to 1997 were used in this study. The isolates were sent by
medical microbiology laboratories in The Netherlands to the National
Institute of Public Health and the Environment (RIVM, Bilthoven, The
Netherlands) for routine typing and susceptibility tests.
Susceptibility to INH was measured with the MIC method using 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, and 50 µg of INH/ml in Middlebrook 7H10 medium
(8). Isolates were considered resistant if more than 1%
of the bacteria in the inoculum grew in the presence of INH
concentrations of DNA isolation.
M. tuberculosis isolates were grown
on Löwenstein-Jensen solid medium or Middlebrook 7H9 liquid
medium for 7 days to an optical density corresponding to
108 bacteria/ml and were harvested by centrifugation
(4,500 × g for 15 min). Chromosomal DNA was isolated
as described by Ausubel et al. (1). Briefly, the bacteria
were killed by heating at 80°C for 20 min and then incubated with 1 mg of lysozyme/ml at 37°C for 1 h. The bacterial suspension was
further incubated with 1% sodium dodecyl sulfate and 0.1 mg of
proteinase K/ml at 65°C for 10 min. Lysis was completed by incubating
the suspension with 1% N-cetyl-N,N,N-trimethyl
ammonium bromide at 65°C for 10 min. DNA was extracted from the lysed
bacteria by chloroform-isoamyl alcohol and subsequently precipitated
with isopropanol.
PCR.
The 25-µl reaction mixture for PCR contained 100 ng of
chromosomal DNA as template, 0.2 mM concentrations of each
deoxynucleoside triphosphate (Amersham Pharmacia Biotech, Piscataway,
N.J.), 0.5 pM primer 1.1 (5'-CTGCTCCGCTGGAGCAGATG-3'), 0.5 pM primer 1.2 (5'-CCGACTTGGGCTGCAGGCG-3'), 1.25 U of
Taq polymerase (Perkin-Elmer, Norwalk, Conn.), and 2 mM
MgCl2 in PCR buffer B (Promega, Madison, Wis.), with final
concentrations of 10 mM Tris-HCl (pH 9.0), 50 mM KCl, and 0.1% Triton
X-100. The thermocycling protocol was 95°C for 1 min, 66°C for 1 min and 72°C for 1 min for 8 cycles, followed by 32 cycles of 95°C
for 1 min, 58°C for 1 min, and 72°C for 1 min.
Restriction endonuclease analysis (REA).
To detect the
Arg463Leu mutation of katG (463-REA), PCR products were
digested with NciI according to the instructions of the
manufacturer (New England Biolabs, Beverly, Mass.). NciI cut the wild-type amplicon at two positions but cut an amplicon with the
Arg463Leu (CGG
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.4.1591-1594.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
The Susceptibility of Mycobacterium
tuberculosis to Isoniazid and the Arg
Leu Mutation at Codon 463 of katG Are Not Associated
ABSTRACT
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Abstract
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CTG [ArgLeu]) in codon 463 of
katG (catalase peroxidase) of Mycobacterium
tuberculosis has been found in isoniazid (INH)-resistant strains.
A PCR restriction endonuclease analysis to detect this mutation was
applied to 395 M. tuberculosis isolates from patients in
The Netherlands. The proportion of isolates with a detectable mutation
was 32% (32 out of 100) and 29% (85 out of 295) among INH-susceptible
isolates and INH-resistant or -intermediate isolates, respectively.
Sequencing of five INH-susceptible isolates with such mutations showed
that all five had the Arg463Leu mutation. We conclude that the
Arg463Leu mutation of katG of M. tuberculosis
is not a reliable indicator of INH resistance.
TEXT
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1 µg/ml. If growth of more than 1% of the
inoculum in the presence of 0.5 µg of INH/ml occurred, then the
isolates were classified as having intermediate susceptibility.
CTG) mutation at only one position (Fig.
1). In theory, an Arg463Pro (CGGCCG)
mutation would remain undetected with this assay. Furthermore, it is
possible that a mutation outside codon 463, but within the recognition
site of NciI comprising codon 463, would prevent
NciI from cutting. However, in the literature, no mention of
such mutations was found.
View larger version (24K):
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FIG. 1.
(A and B) REA of a 643-bp amplicon of M. tuberculosis katG. (A) In order to detect a mutation at codon 315, the amplicon was digested by MspA1I. A wild-type (wt)
amplicon was cut at three positions, but an amplicon with a mutation in
the recognition sequence encompassing codon 315 was cut at only two.
(B) NciI was used to detect the mutation at codon 463. NciI cut a wild-type amplicon at two positions, but an
amplicon with a mutation in the recognition sequence containing codon
463 was cut at only one. (C and D) Gel electrophoresis of digested
fragments derived from five different isolates (1 to 5) of
M. tuberculosis allows discrimination of wild-type and
mutant isolates for codons 315 (C) and 463 (D). Arrows indicate mutant
DNA fragments. Lane M, 100-bp ladder with bands at 0.1-kb intervals,
starting at 0.1 kb.
ACC or ACA
[Ser315Thr], AGCAAC [Ser315Asn], AGCATC [Ser315Ile]
[9, 10, 17]) at two positions (Fig. 1). An
amplicon with an AGCCGC (Ser315Arg) mutation, which has been
described once (9), was cut where the wild type was cut and was therefore not detected with this assay.
The DNA restriction fragments were analyzed on 1% agarose, as
described earlier (19).
Fluorescence-based sequencing and analysis. The katG region comprising the mutation at codon 463 was amplified using primers 1.12 (5'-CAAGCAGACCCTGCTGTGGC-3') and 2.0 (5'-TGCTGCTTTCTCTATGGCGG-3'). The DNA sequences of these amplicons were determined by a PCR-based sequence reaction using the ABI PRISM Dye Terminator Cycle Sequencing Core Kit (Perkin-Elmer, Gouda, The Netherlands) according to the instructions supplied by Applied Biosystems Incorporated (Foster City, Calif.). The sequences were analyzed on an automatic sequenator (model 370A; Applied Biosystems Incorporated).
Prevalence of mutations at codon 315 and 463 of katG in the Netherlands. In total, 395 patient isolates of M. tuberculosis were tested for INH susceptibility and analyzed with 463-REA. Of these isolates, 225 were resistant and 70 were of intermediate susceptibility, while 100 were INH susceptible. In order to detect whether the Arg463Leu mutation could be present, a 643-bp region of katG was amplified by PCR using primers 1.1 and 1.2. For detection of the mutation at codon 315, the same amplicon was used but was digested with MspA1I. 463-REA showed that among the 225 resistant isolates, 64 (28%) had a mutation in the NciI recognition sequence that includes codon 463. Of the 70 isolates with an intermediate susceptibility to INH and the 100 INH-susceptible isolates, 21 (30%) and 32 (32%) isolates also carried a mutation in that recognition sequence, respectively (Table 1).
|
T mutation at the
second base pair position of codon 463, resulting in a putative
ArgLeu change.
315-REA showed that among 100 INH-susceptible isolates, none were found
to have a mutation in the MspA1I recognition sequence that
includes codon 315.
INH resistance of M. tuberculosis organisms is associated
with mutations in or deletions of katG (60 to 70%) or
mutations in inhA (10 to 20%). The genetic mechanism of INH
resistance remains unknown for 10 to 15% of the INH-resistant
isolates. The mutations in katG occur in 50 to 60% of the
INH-resistant isolates at codon 315 and in 25 to 45% of these isolates
at codon 463 (5, 16, 17).
Our results, in conjunction with those of an earlier study
(25), show that there is a strict relationship between the
presence of a mutation at codon 315 and INH resistance of M. tuberculosis isolates.
In contrast, the Arg463Leu mutation and INH resistance are not as
strictly associated. katG encoding Leu at codon 463, either as the prevalent allele or as a polymorphism, is also present in
Mycobacterium intracellulare, Mycobacterium bovis, M. bovis BCG, Mycobacterium africanum, and Mycobacterium
microti isolates. These mycobacterial species are in general less
susceptible to INH (9, 10). For M. bovis BCG,
there is a strong association between MICs of INH and the presence of
the CGGCTG mutation at codon 463 (for 463R, MIC < 0.05 µg/ml, for 463L, MIC > 2 µg/ml).
However, in previous studies, the mutation at codon 463 was found in 3 to 61% of INH-susceptible M. tuberculosis isolates (5, 6, 13, 17, 18, 23). Also, it was found that the
activity of catalase, a katG-encoded enzyme, did not differ among isolates having either Arg or Leu at codon 463 (21,
22). Furthermore, complementation of katG-negative
INH-resistant M. tuberculosis strains with katG
having the CGGCTG (Arg463Leu) mutation fully restored the virulence
and catalase activity of these strains (14, 21). Hence,
there was no biochemical support for the observation that CGGCTG
(Arg463Leu) was associated with resistance to INH. The results of our
study support this observation since the distribution of a mutation in
the recognition site of NciI comprising codon 463 among
INH-resistant isolates, isolates with intermediate susceptibility, and
INH-susceptible isolates was similar. Sequencing of five randomly
selected INH-susceptible isolates that had a mutation in this
recognition site confirmed the presence of the CGGCTG (Arg463Leu)
mutation. These results show that the Arg463Leu mutation in
katG of M. tuberculosis does not, at least not by
itself, confer resistance to INH.
In addition, we assessed whether the presence or absence of the
CGGCTG (Arg463Leu) mutation was associated with differences in the
distribution of MICs, resistance to drugs other than INH, or the
probability of being in a restriction fragment length polymorphism cluster, similarly as reported by van Soolingen et al.
(25). However, virtually no differences were found (data
not shown).
Our findings have three implications. First, the presence of the
CGGCTG (Arg463Leu) mutation in katG in M. tuberculosis is neither biochemically nor epidemiologically
associated with INH resistance, with intermediate INH susceptibility,
or with multidrug resistance in M. tuberculosis in The
Netherlands. Therefore, this mutation should be considered a
polymorphism unrelated to the selective pressure of drug treatment in
M. tuberculosis. Second, the percentage of isolates with INH
resistance which is attributable to mutations in katG has
been overestimated. Third, the CGGCTG (Arg463Leu) mutation is not
indicative of INH resistance of an M. tuberculosis isolate.
Therefore, the development of a diagnostic PCR for detection of INH
resistance should not be based upon the katG CGGCTG
(Arg463Leu) mutation.
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FOOTNOTES |
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* Corresponding author. Mailing address: Academic Medical Center, Dept. of Medical Microbiology, P.O. Box 22700, 1100DE Amsterdam, The Netherlands. Phone: 31 20 5664860. Fax: 31 20 6979271. E-mail: h.r.vandoorn{at}amc.uva.nl.
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Journal of Clinical Microbiology, April 2001, p. 1591-1594, Vol. 39, No. 4
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.4.1591-1594.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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