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Journal of Clinical Microbiology, April 1999, p. 998-1003, Vol. 37, No. 4
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Molecular Evidence for Heterogeneity of the
Multiple-Drug-Resistant Mycobacterium tuberculosis
Population in Scotland (1990 to 1997)
Z.
Fang,1,
C.
Doig,2
A.
Rayner,2
D. T.
Kenna,1
B.
Watt,2 and
K. J.
Forbes1,*
Medical Microbiology, Aberdeen University,
Foresterhill, Aberdeen, AB25 2ZD,1 and
Scottish Mycobacteria Reference Laboratory, The City
Hospital, Edinburgh, EH10 5SB,2 United Kingdom
Received 1 October 1998/Returned for modification 6 November
1998/Accepted 30 December 1998
 |
ABSTRACT |
Multiple-drug-resistant Mycobacterium tuberculosis
(MDR-MTB) has been well studied in hospitals or health care
institutions and in human immunodeficiency virus-infected populations.
However, the characteristics of MDR-MTB in the community have not been well investigated. An understanding of its prevalence and
circulation within the community will help to estimate the problem
and optimize the strategies for control and prevention of its
development and transmission. In this study, MDR-MTB isolates from
Scotland collected between 1990 and 1997 were characterized, along with
non-drug-resistant isolates. The results showed that they were
genetically diverse, suggesting they were unrelated to each other and
had probably evolved independently. Several new alleles of
rpoB, katG, and ahpC were
identified: rpoB codon 525 (ACC
AAC; Thr525Asn);
katG codon 128 (CGGCAG; Arg128Gln) and codon 291 (GCTCCT; Ala291Pro); and the ahpC synonymous
substitution at codon 6 (ATTATC). One of the MDR-MTB isolates
from an Asian patient had an IS6110 restriction fragment
length polymorphism pattern very similar to that of the MDR-MTB W
strain and had the same drug resistance-related alleles but did not
have any epidemiological connection with the W strains. Additionally, a
cluster of M. tuberculosis isolates was identified in our
collection of 715 clinical isolates; the isolates in this cluster had
genetic backgrounds very similar to those of the W strains, one of
which had already developed multiple drug resistances. The diverse
population of MDR-MTB in Scotland, along with a low incidence of
drug-resistant M. tuberculosis, has implications for
the control of the organism and prevention of its spread.
| |
INTRODUCTION |
Multiple-drug-resistant
Mycobacterium tuberculosis (MDR-MTB), which is defined as
isolates resistant to both isoniazid (INH) and rifampin
(RIF)
the frontline antitubercular agents and the backbone of
current antituberculosis treatment regimensis worsening the global
tuberculosis emergency and has caused deep concern worldwide.
Understanding the causation, genetic mechanisms, and transmission of
MDR-MTB will be of great value in optimizing the strategies to control
and prevent its development and transmission. Recent molecular-genetic
analyses of drug-resistant M. tuberculosis have disclosed a
number of resistance mechanisms. For example, more than 95% of
RIF-resistant M. tuberculosis isolates have mutations in
rpoB, the gene encoding the RNA polymerase 
-subunit
(25). Three genes have been implicated in INH resistance:
katG, encoding catalase-peroxidase, which transforms INH
into its active form (41); inhA, encoding a
putative mycolic acid synthesis enzyme involved in cell wall synthesis
(2); and ahpC, encoding alkyl hydroperoxidase, which functions as a component of antioxidant reductase (32). The W strain of M. tuberculosis,
first isolated in New York City in 1992, has resistance to INH,
ethambutol (EMB), RIF, and streptomycin (STR) and has a characteristic
IS6110 restriction fragment length polymorphism (RFLP)
banding pattern; subsequently, variants with a few
IS6110 RFLP differences were isolated (4, 13, 24)
while other variants were found which were susceptible to
antituberculosis drugs.
Unlike many bacterial species which can acquire antibiotic resistance
genes by transduction, conjugation, or transformation, there is no
evidence so far of the mobilization of genes between M. tuberculosis isolates; indeed, all resistances so far
characterized have been genomically based. As a result, analysis of the
alleles of drug resistance genes and of their associations will provide useful information on the generation and transmission of resistant isolates and will also provide insight into the population
dynamics of M. tuberculosis in response to
the selective pressure of antimycobacterial agents. So far the great
majority of investigations of MDR-MTB have been of hospital- or health
care institute-based outbreaks (references 1, 3, 4,
15, and 16 and reference
14 and references therein), and these have provided
valuable information on MDR-MTB transmission and its vulnerable
subjects. The few non-outbreak- or community-based investigations have
tended to use IS6110 RFLP alone as a genetic marker
for the characterization of the drug-resistant populations (27,
28, 38).
In order to have a genetic insight into the MDR-MTB population in
Scotland, we have studied some of the drug resistance genes of the
MDR-MTB isolates collected in Scotland between 1990 and 1997, including
rpoB, katG, inhA, and ahpC.
These isolates were also characterized by IS6110 RFLP
and other genetic markers, together with drug-sensitive
isolates collected at the same time. The results indicated that
the MDR-MTB isolates in Scotland are genetically diverse. This, in
combination with a low rate of primary drug resistance in
Scotland, lead to some thoughts on the global control of MDR-MTB.
| |
MATERIALS AND METHODS |
Isolates.
A total of 715 isolates of M. tuberculosis, including 10 MDR-MTB isolates, were studied; all of
them were received by the Scottish Mycobacteria Reference Laboratory in
Edinburgh, Scotland, from 1990 to 1997.
Antimicrobial susceptibility test.
Drug sensitivity testing
was performed with a Bactec radiometric system (Becton Dickinson,
Paramus, N.J.) with the following antimicrobial agents: INH, 0.1 µg/ml; EMB, 2.0 µg/ml; RIF, 0.5 µg/ml; pyrazinamide (PZA), 100 µg/ml; ciprofloxacin, 2.0 µg/ml; rifabutin, 0.2 µg/ml; amikacin,
2.0 µg/ml; STR, 4.0 µg/ml; capreomycin, 8.0 µg/ml; clofazimine,
0.8 µg/ml; prothionamide, 2.0 µg/ml; clarithromycin, 4.0 µg/ml;
and sparfloxacin, 2.0 µg/ml (33). All 715 isolates were
tested for susceptibility to INH, RIF, EMB, and PZA. In the case of
isolates resistant to any of these, further drug susceptibility tests
were performed.
PCR-based single-strand conformational polymorphism (PCR-SSCP)
analysis.
Conventional PCR was used to amplify rpoB,
katG, inhA, and ahpC genes, and the
products were checked on agarose gels. One microliter of the PCR
product was mixed with 2 µl of denaturing buffer (95% formamide,
0.05% bromophenol blue, 0.05% xylene cyanol, 20 mM EDTA), and the
mixture was heated in a heated-lid thermocycler (WellTemp, Cambridge,
United Kingdom) for 5 min at 95°C and then immediately cooled in ice
water for 5 min. The denatured PCR products were separated via
electrophoresis on a PhastSystem (Pharmacia Biotech AB, Uppsala,
Sweden) on PhastGel 8 to 25% gradient gel (Pharmacia Biotech) with
PhastGel DNA buffer strips (Pharmacia Biotech). The separation program
has two steps: a sample application step (100 V; 4 mA; 1.0 W; 15°C;
10 Vh) and a sample separation step (400 V; 10 mA; 2.0 W; 15°C; 500 Vh). After separation, the gels were stained with PhastGel DNA
silver-staining kit (Pharmacia Biotech) and air dried for about 3 h, according to the manufacturer's instructions.
DNA sequencing and DNA sequence analysis.
DNA was sequenced
with an Applied Biosystems (Warrington, United Kingdom) 377A automated
DNA sequencer with a Prism-Ready mix kit based on Ampli-Taq CS and
polymerase. The programs in the Genetics Computer Group package
(version 8.1) used in this study for DNA sequence analysis were GAP
(26) and BESTFIT (34).
IS6110 RFLP analysis.
IS6110 RFLP
analysis was performed according to the recommended method with some
modifications (11, 37). Briefly, the M. tuberculosis genomic DNA was digested with PvuII,
subjected to agarose gel electrophoresis, and subsequently blotted onto a nylon membrane. After hybridization with a digoxigenin (DIG)-labelled IS6110 DNA probe, the hybridization bands were detected by
the DIG detection procedure. The IS6110 probe was labelled
in a PCR with DIG-dUTP (Boehringer Mannheim GmbH, Mannheim,
Germany). PvuII-digested supercoiled DNA ladder
(Gibco-BRL, Life Technologies Ltd. Paisley, United Kingdom) and
X174-HaeIII DNA (Advanced Biotechnologies, London, United
Kingdom) were DIG labelled with a random-primed DNA-labelling method.
Pairwise similarities of IS6110 fingerprint patterns were
calculated by the Dice coefficient of similarity with GelCompar
software (version 4.0; Applied Maths, Kortrijk, Belgium).
Polymorphism analysis of codon 463 in katG.
The
polymorphism analysis was conducted by PCR-based RFLP with primers K5
and K6 and endonuclease restriction enzyme NciI (Boehringer
Mannheim GmbH).
| |
RESULTS |
Susceptibility patterns.
Ten of the 12 MDR-MTB isolates and
their susceptibility patterns are shown in Table
1.
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TABLE 1.
Patterns of antimicrobial susceptibility of MDR-MTB
isolates and corresponding genetic mutations detected
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|
IS6110 RFLP patterns.
A total of 715 clinical
isolates of M. tuberculosis collected from 1990 to
1997, including the 10 MDR-MTB isolates, were subjected to
IS6110 RFLP analysis. The patterns of the MDR-MTB isolates were diverse (Fig. 1): two pairs of
isolates (isolates 9208 and 9219 and isolates 9511 and 9604) had more
than 80% similarity by the Dice coefficient (8) but still
had three and five differences in IS6110-hybridizing bands
within each pair, respectively. IS6110 RFLP patterns can be
used to efficiently group together genetically related isolates of
M. tuberculosis (11). A dendrogram based on
the RFLP pattern similarities of the 715 isolates also indicated that the 10 MDR-MTB isolates did not cluster together but were generally scattered throughout the dendrogram, except isolates 9511 and
9604, suggesting that they were indeed not genetically closely related
strains but had evolved independently. Using the published
IS6110 pattern of the W strain (4, 24) as a
reference, isolate 9511 was found to be very similar, and this isolate
was also resistant to INH, EMB, RIF, and STR (Table 1). In the
715-isolate dendrogram, isolates 9511 and 9604 clustered together with
11 other isolates at greater than 75% similarity (Fig.
2).
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FIG. 2.
Schematic presentation of the IS6110 RFLP
patterns and dendrogram of the subcluster containing isolates 9511 and
9604 from the database containing 715 M. tuberculosis
isolates. Isolate 92/4029 from an adjacent subcluster was used as an
out group.
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|
rpoB polymorphism.
The causative mutation of many
RIF-resistant mutants has been mapped to the rpoB gene, the
vast majority of these being in the region from codon 505 to codon 353 (25). Accordingly, the DNA sequence of this region was
determined for all 10 of the isolates from a PCR product amplified
across the region (primers TR7 and RpoB). Subsequent sequencing
revealed that all 10 of the MDR-MTB isolates had missense mutations in
the region (Tables 2 and
3).
Polymorphism in katG, inhA and
ahpC.
PCR-SSCP was performed on all of the PCR products from
katG, inhA, and ahpC to identify
mutant sequences, which were then subjected to DNA sequencing. An
example of the PCR-SSCP polymorphic pattern from inhA is
illustrated in Fig. 3.
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FIG. 3.
PCR-SSCP patterns of the partial inhA locus
of isolates with primers InhA1 and InhA2. Lane 1, no denaturing DNA of
drug-susceptible isolate 8 as nondenatured DNA control; lane 2, denaturing DNA of isolate 8 as denatured DNA control; lanes 3 to 9, DNA
from isolates 9002, 9202, 9208, 9214, 9219, 9310, and 9511. Clear
shifts of single-stranded DNA can be seen in lanes 5 and 7, which were
confirmed by DNA sequencing to be due to single-nucleotide mutations
(see the text for details).
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|
Four pairs of primers (Table
3) were designed to span the region of the
katG coding region that has been reported to harbor
most of
the mutations. The mutations found in the isolates had
several features
(Table
4). Firstly, 5 of the 10 isolates
were
found to have mutations in
katG; all of them were
missense substitutions
and three of them were at codon 315, a
preponderance which has
been noted by others (references
9 and
25 and references
therein)
and which has been demonstrated to confer high INH resistance
(
29). Secondly, two new mutations were identified, one of
them
at codon 128 (CGG
CAG; Ala128Gln) and the other at codon 291 (GCT
CCT;
Ala291Pro).
The Arg-Leu polymorphism at codon 463 of the
katG gene has
been demonstrated not to confer INH resistance but has been proposed
as
an evolutionarily stable genetic marker (
36). Two isolates
(isolates 9511 and 9604) had the codon for leucine; the others
had the
arginine codon when the 10 isolates were screened by the
PCR-based
RFLP.
inhA was examined with one primer pair (InhA1-InhA2)
amplifying across the putative
inhA regulatory region and
another primer
pair (InhA3-InhA4) amplifying across codon 280. Among
the 10 isolates,
none was found to have a mutation at codon 280. Two
isolates (isolates
9208 and 9219) were identified that had a
single-nucleotide substitution
1 nucleotide upstream of the putative
ribosome binding site (nucleotide
148 [EMBL accession no.
U41388]),
and these two isolates did
not have any mutations in their
katG genes.
Mutations were found at the
ahpC locus in 2 of the 10 isolates (isolates 9202 and 2022 [Table
1]). Both isolates had
substitutions
in the intergenic region between
ahpC and
oxyR, at position
9
and position
46, respectively
(positions are as designated in
reference
40).
Interestingly, a previously unreported synonymous
substitution at codon
6 (ATT
ATC) was also found to be present
in isolate
9202.
| |
DISCUSSION |
Resistance to RIF.
Seven different mutations were identified
among the 10 isolates (Table 2); several features of the RIF resistance
phenotype and their genetic bases were disclosed by this study.
Firstly, isolate 9002 had substitutions at both codon 511 (CTGCCG; Leu511Pro) and codon 516 (GACGCC; Asp516Ala);
although each of these has been reported previously, this is the first
report of them in combination. Secondly, codon 531 is known to be a hot
spot in rpoB for mutational change in M. tuberculosis (25), and half (five) of the isolates here
also carried this mutation. Thirdly, like RIF, rifabutin is a
derivative of rifamycin B (17), and it is normally used for
prophylaxis against Mycobacterium avium complex infection in
patients with AIDS (31). However, since a proportion of
RIF-resistant M. tuberculosis isolates are susceptible to it (6), and as it can achieve a relatively high
concentration in human lung tissue (12), it can be a useful
alternative to treatment with RIF. The relationship between
rpoB mutations and resistance to RIF and rifabutin was
examined. While all 10 isolates were resistant to RIF by definition, 2 (isolates 1238 and 9219) of them were susceptible to rifabutin (Table
2). As noted by Bodmer et al. (6), who examined the
susceptibility of 14 different rpoB mutants to several
rifamycin B derivatives, it seems likely that the mutations in codons
511, 516, and 531 confer cross-resistance to both RIF and rifabutin,
and in this list we would also include the codon 505 mutation. The
codon 533 mutation only rendered isolate 9219 resistant to RIF and not
to rifabutin, and this mutation is also in addition to those
investigated by Bodmer et al. (6). More intriguingly,
although various mutations have been noted which render resistance to
both RIF and rifabutin (6) as in the His526Tyr substitution,
this seems not to be the case for the His526Leu substitution,
suggesting that different substitutions of a codon may have different effects.
Resistance to INH.
Although katG, inhA,
and ahpC have been associated with INH resistance in
M. tuberculosis, the mechanism(s) of INH action and of
resistance to it are not completely understood. For instance, it is
uncertain whether ahpC mutations confer resistance to INH directly or only secondarily as compensation for the decreased ability
of the organism to survive oxidative-stress environments due to KatG
alterations (7, 19, 22, 32, 36). In the 10 isolates of
MDR-MTB studied here, 1 had no detectable mutations in the three genes
examined, implying either that there was a resistance mutation outside
the investigated regions of these three genes or that change was
located elsewhere in the genome. Of the other nine isolates of MDR-MTB
examined, all had a mutation in one, but not more than one, of these
three genes, providing further supporting evidence that these genes are
implicated in the majority of INH resistances. The absence of double
katG-ahpC mutations here supports the hypothesis that
mutation of ahpC independently confers INH resistance rather
than as a secondary response to the mutation in katG.
Clones and drug resistance in M. tuberculosis.
Isolate 9511 is particularly interesting in that it was isolated from a
patient of Pakistani nationality and has an IS6110 RFLP pattern very similar to that of the W strain. In addition, it had
the same alleles of katG and inhA as the W
strain, i.e., katG polymorphism at codon 463, a
katG drug resistance mutation at Ser315Thr, and no
mutations in inhA. However, it had a different mutation in
rpoB; the W strain has mutations in rpoB of
either His526Tyr or Ser531Leu (4), while isolate 9511 had a
Ser531Trp mutation. This all suggests that isolate 9511 is closely
related to the W strains isolated in the United States. No
IS6110 patterns very similar to isolate 9511 could be found
in our database, and as it was isolated from a Pakistani patient who
had never been in the United States before he was diagnosed, it is
likely that it is derived from Asia (20). Isolate 9604 was
recovered from a Scottish patient; it is located in the same
IS6110 RFLP subcluster as isolate 9511 (Fig. 2) and had the
same drug resistance-related mutations and katG 463 polymorphism as isolate 9511. As several closely related isolates from
Scottish patients have been identified in our database (Fig. 2), this
suggests that this isolate is also related to the W strain but has
probably evolved in Scotland.
Taken together these results suggest that there is a worldwide cluster
of
M. tuberculosis isolates which includes the W
strain,
isolate 9511, and isolate 9604. The isolates of this cluster
have
much genomic similarity (common IS
6110 RFLPs, Leu codon
463 in
katG). Not all isolates in these different lineages
have antimicrobial
resistances; some W strains are pansusceptible to
the antimycobacterial
agents (
4), and all but one of the
isolates closely related
to 9511 and 9604 were pansusceptible, too
(Fig.
2). The drug resistance
mutations seen in this cluster are not
unique to it and are often
found in other unrelated isolates, such as
codon 315 of
katG in
isolate 1238, codon 526 of
rpoB in isolate 9310, codon 531 of
rpoB in
isolate 9202, and other mutations reported elsewhere (reference
25 and references therein). It would be interesting
to perform
a more extensive genetic survey of isolates in this cluster
from
around the world to chart the evolutionary links among them and
their acquisition of resistances. Along these lines, we have identified
a large cluster of isolates with very similar genetic makeups
in our
collection, many, but not all, of which have developed
INH
resistance (our unpublished data). As a result, we speculate
that some
clones of
M. tuberculosis which occur worldwide have
a
tendency to become drug
resistant.
Heterogeneity of the MDR-MTB isolates and its implications.
Since the late 1980s there have been outbreaks of MDR-MTB infection
around the world, especially in association with patients with AIDS;
epidemiological analysis of these MDR-MTB infection outbreaks has
suggested that MDR-MTB is transmitted more rapidly and that this is of
itself contributing to the increased incidence of tuberculosis in the
world (1, 14-16, 27, 28). However, our study of MDR-MTB
isolates from Scotland, at both genomic and resistance gene levels,
suggests that rather than the epidemic spread of one strain there
is a heterogeneous MDR-MTB population. This observation is
consistent with two other facts.
Firstly, the incidence of primary resistance (the presence of a
drug-resistant strain in a patient who has never received
antituberculosis treatment), which is widely used as a parameter
to
evaluate the efficacy of a tuberculosis control program (
10,
39), is low in Scotland. The Scottish incidence of isolates
with
resistance to one or more antimycobacterial drugs, which
was 4.0% from
1990 to 1997, contrasts with the much higher rates
of 8.4% in Germany
in 1995, 11% in Korea in 1994, 14% in the United
States in 1991, and
24% in Morocco in 1995. Indeed, the primary
MDR-MTB isolation rate of
0.3% in Scotland during the same period
contrasts with 3.5% in the
United States in 1991 (
5,
10,
19,
27). Secondly, the
incidence of tuberculosis in Scotland
has steadily declined up to the
mid-1980s; since then, although
it has leveled out (
30),
there have been few recorded significant
outbreaks of the disease.
Despite human immunodeficiency virus
infection in Scotland since this
period, there is a very low tuberculosis
incidence in these patients
(
21) and there was no overrepresentation
of MDR-MTB cases in
this group. The causes of the low incidences
of tuberculosis, both of
sensitive and resistant strains, in Scotland
are presently under study.
Conceivably, more rigorous control
measures or perhaps differences in
the human population in Scotland
have prevented such an outbreak to
date.
In conclusion, it is apparent that the general incidence of MDR-MTB in
Scotland is low, paralleling the low incidence of single-drug
resistances in Scotland, and that the origins of these MDR-MTB
isolates
are diverse. Some have apparently been acquired from
outside Scotland,
while others have apparently evolved within
Scotland, probably over a
number of decades. It is important to
remember that drug-resistant
tuberculosis first became prevalent
when chemotherapy was introduced in
the 1940s and that by the
late 1950s and early 1960s many
industrialized countries had isolates
resistant to both INH and STR. It
was only with a combination
of enhanced implementation of tuberculosis
control programs and
the introduction of RIF-containing short-course
chemotherapy (
35)
that this situation was alleviated. While
the incidence of drug-resistant
tuberculosis is currently low in
Scotland, it is apparent that
such isolates can be acquired from both
exogenous and endogenous
sources. Tight controls must be maintained for
the treatment of
patients with tuberculosis in Scotland, most
particularly to prevent
the emergence of a multiple-drug-resistant
strain derived from
isolates which may be more host adapted to the
Scottish population,
as may be the case with the W strain in the United
States, than
one derived from outside this country. Past treatment
failures
may have left a legacy of future potential MDR-MTB.
| |
ACKNOWLEDGMENTS |
We thank P. Carter, and K. Reay for DNA sequencing and synthesis
of the oligonucleotide primers. DNA sequence analysis benefited from
SEQNET, the BBSRC facility (Daresbury, United Kingdom). We also thank
three anonymous referees for their critical comments and suggestions to
improve this paper.
This study was financially supported by The Scottish Office Department
of Health; Chest, Heart and Stroke Scotland; and a Milner Scholarship
from the University of Aberdeen.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Medical
Microbiology, Aberdeen University, Foresterhill, Aberdeen,
AB25 2ZD, United Kingdom. Phone: 44 1224 663123, ext. 54953. Fax:
44 1224 685604. E-mail: mmb001{at}abdn.ac.uk.
Present address: Dept. of Biomedical Sciences, University of
Bradford, West Yorkshire, BD7 1DP, United Kingdom.
| |
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Journal of Clinical Microbiology, April 1999, p. 998-1003, Vol. 37, No. 4
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
