Identification of Mycobacterial Species by PCR Sequencing of Quinolone Resistance-Determining Regions of DNA Gyrase Genes

Abstract

The determination of the amino acid sequence of quinolone resistance-determining regions (QRDRs) in the A and B subunits of
DNA gyrase is the molecular test for the detection of fluoroquinolone resistance in mycobacteria. We looked to see if the
assignment of mycobacterial species could be obtained simultaneously by analysis of the corresponding nucleotide sequences.
PCR sequencing of gyrA and gyrB QRDRs was performed for 133 reference and clinical strains of 21 mycobacterial species commonly isolated in clinical laboratories.
Nucleotide sequences of gyrA and gyrB QRDRs were species specific, regardless of fluoroquinolone susceptibility.

Full text

JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 2003, p. 1311–1315 Vol. 41, No. 3
0095-1137/03/$08.00⫹0 DOI: 10.1128/JCM.41.3.1311–1315.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Identification of Mycobacterial Species by PCR Sequencing of
Quinolone Resistance-Determining Regions
of DNA Gyrase Genes
Jean-Noe¨l Dauendorffer, Isabelle Guillemin, Alexandra Aubry, Chantal Truffot-Pernot,
Wladimir Sougakoff, Vincent Jarlier, and Emmanuelle Cambau*
Laboratoire de Bacte´riologie-Hygie`ne, Faculte´deMe´decine Pitie´-Salpeˆtrie`re, Universite´ Paris VI, and Centre
National de Re´fe´rence pour la Re´sistance des Mycobacte´ries aux Antituberculeux, Groupe Hospitalier
Pitie´-Salpeˆtrie`re, Assistance publique-Hoˆpitaux de Paris, Paris, France
Received 10 September 2002/Returned for modification 25 October 2002/Accepted 9 December 2002
The determination of the amino acid sequence of quinolone resistance-determining regions (QRDRs) in the
A and B subunits of DNA gyrase is the molecular test for the detection of fluoroquinolone resistance in myco-
bacteria. We looked to see if the assignment of mycobacterial species could be obtained simultaneously by
analysis of the corresponding nucleotide sequences. PCR sequencing of gyrA and gyrB QRDRs was performed
for 133 reference and clinical strains of 21 mycobacterial species commonly isolated in clinical laboratories. Nu-
cleotide sequences of gyrA and gyrB QRDRs were species specific, regardless of fluoroquinolone susceptibility.
During the last few years, the emergence of infections caused
by nontuberculous mycobacteria and the report of tuberculosis
outbreaks has brought considerable interest in clinical myco-
bacteriology. It led to the development of new diagnosis tools
based on molecular technology for antibiotic susceptibility test-
ing and identification (24). Rapid identification of mycobacte-
ria to species level is recommended in clinical laboratories for
assessing the diagnosis of mycobacteriosis and also for making
decisions for effective therapy (2).
In the clinical laboratory, the differentiation of closely re-
lated species of mycobacteria by phenotypic and biochemical
tests remains difficult for some very common species. The
phenotypic methods are slow, require expertise, and often use
nonstandardized reagents (8). New biochemical methods such
as high-performance liquid chromatography of mycolic acids
are implemented only in specialized laboratories (4). Molecu-
lar methods were developed with enthusiasm because they are
rapid (no need to subgrow bacteria) and require a small quan-
tity of bacteria. The reference molecular method for identifi-
cation is the determination of sequences of 16S ribosomal
DNA (rDNA) (21), but identical sequences were reported for
some common species. Other DNA sequences or genes have
been described for the differentiation of mycobacterial species,
such as the internal transcribed spacer (ITS) 16S-23S (28),
recA (3), dnaJ (34), hsp65 encoding the 65-kDa heat shock
protein (27), rpoB encoding the B subunit of RNA polymerase
(20), the gene of the 32-kDa protein (30), sod encoding the
superoxide dismutase (41), and gyrB encoding the B subunit of
DNA gyrase (18). None of these genes can presently differen-
tiate all the mycobacterial species commonly isolated in the
clinical laboratory.
Fluoroquinolones are active against mycobacteria (23) and
are recommended for the treatment of drug-resistant tubercu-
losis, drug-resistant leprosy, and infections caused by some
nontuberculous mycobacteria (2, 9, 14). Fluoroquinolone re-
sistance is mainly due to alterations in DNA gyrase, the unique
type II topoisomerase of mycobacteria (1, 6, 7, 22, 35). These
alterations are substitutions in the quinolone resistance-deter-
mining regions (QRDRs) in the A subunit (region 67 to 106)
(numbering system used for Escherichia coli) and in the B
subunit (region 426 to 464), as described for other bacteria
(11). The detection of missense mutations at positions 83, 84,
and 87 in GyrA and positions 426, 447, and 464 in GyrB is a
rapid and efficient test for molecular detection of fluoroquin-
olone resistance in mycobacteria (5, 22, 26). This test is com-
plementary to the antibiotic susceptibility testing that is not
standardized yet for quinolones.
In the National Reference Center laboratory, we imple-
mented a few years ago the detection of fluoroquinolone re-
sistance in pathogenic mycobacteria by PCR sequencing of the
gyrA and gyrB QRDRs. Therefore, we were able to compare
the corresponding nucleotide sequences of different mycobac-
terial species. In a previous work, it was shown, for a few
mycobacterial species, that gyrA and gyrB sequences can dif-
ferentiate between some species and help in phylogenetic anal-
ysis (15). In the present study, PCR sequencing of gyrA and
gyrB QRDRs was tested to differentiate 21 mycobacterial spe-
cies commonly isolated in the clinical laboratory, out of which
17 were pathogenic and 4 were nonpathogenic. Reference and
clinical strains, wild-type strains for fluoroquinolone suscepti-
bility, and strains with acquired resistance to fluoroquinolones
were studied.
A total of 133 strains representing 21 mycobacterial species
have been studied: Mycobacterium tuberculosis (n⫽21; H37Rv
strain, 20 clinical strains of which 10 were fluoroquinolone-
resistant strains), M. bovis (n⫽7; ATCC 2001, five clinical
strains and M. bovis BCG), M. africanum (n⫽4; ATCC 30007
and three clinical strains), M. xenopi (n⫽6; ATCC 19250 and
five clinical strains), M. avium (n⫽8; ATCC 25291, six clinical
strains and one in vitro fluoroquinolone-resistant mutant), M.
* Corresponding author. Mailing address: Faculte´deMe´decine
Pitie´-Salpeˆtrie`re, 91, Bd de l’Hoˆpital, 75634 Paris Cedex 13, France.
Phone: 33 (0)1 40 77 97 46. Fax: 33 (0)1 45 82 75 77. E-mail: cambau
@chups.jussieu.fr.
1311

intracellulare (n⫽5; ATCC 13950 and four clinical strains), M.
gordonae (n⫽5; ATCC 14470 and four clinical strains), M.
kansasii (n⫽4; ATCC 12478 and three clinical strains), M.
gastri (n⫽3; ATCC 15754 and two clinical strains), M. mal-
moense (n⫽2; two clinical strains), M. szulgai (n⫽4; NCTC
10831 and three clinical strains), M. simiae (n⫽4; ATCC
25275 and three clinical strains), M. leprae (n⫽12; 12 clinical
strains, of which one is a fluoroquinolone-resistant strain),
M. marinum (n⫽5; ATCC 927 and four clinical strains),
M. ulcerans (n⫽6; ATCC 14188 and five clinical strains), M.
chelonae (n⫽3, ATCC 14472 and two clinical strains),
M. abscessus (n⫽6; ATCC 19977 and five clinical strains),
M. fortuitum (n⫽10; ATCC 6841 and one in vitro fluoroquin-
olone-resistant mutant and eight clinical strains, of which one
isafluoroquinolone-resistant strain), M. peregrinum (n⫽7;
ATCC 14467 and one in vitro fluoroquinolone-resistant mu-
tant and five clinical strains), M. smegmatis (n⫽8; ATCC
19420, mc
2
155 and three in vitro fluoroquinolone-resistant mu-
tants, and NCTC 53 and two in vitro fluoroquinolone-resistant
mutants), and M. aurum (n⫽3; ATCC 23366, CIPT
141210005 and one in vitro fluoroquinolone-resistant mutant).
Fluoroquinolone-resistant strains were described previously (5,
6, 7, 15, 16). All the clinical strains were identified by classical
phenotypic and biochemical tests (8) and molecular reference
tests. These molecular tests were DNA probes (13) (Accu-
probe and Genprobe; Biome´rieux, Marcy L’Etoile, France) for
M. tuberculosis complex, M. avium,M. intracellulare,M. gordo-
nae, and M. kansasii and sequencing of the 16S rDNA (21) or
of the hsp65 gene (27) for the other species. Extraction of
mycobacterial DNA and amplification of the DNA fragments
corresponding to the gyrA and gyrB QRDRs were performed as
previously described (15, 16) for gyrA by using the degenerated
oligonucleotides Pri9 (5⬘-CGCCGCGTGCTG/CATGCA/GA
TG-3⬘) and Pri8 (5⬘-C/TGGTGGA/GTCA/GTTA/GCCC/TG
GCGA-3⬘) and for gyrB by using GyrbA (5⬘-GAGTTGGTGC
GGCGTAAGAGC-3⬘) and GyrbE (5⬘-CGGCCATCAA/GCA
CGATCTTG-3⬘). The amplification reactions consisted of the
following steps: one denaturation cycle at 94°C for 10 min and
40 cycles of amplification at 94°C for 1 min, 55°C for 1 min, and
72°C for 1 min, followed by one elongation cycle at 72°C for 10
min. Sequencing of the gyrA and gyrB QRDRs was performed
as previously described (16).
Amino acid sequences of GyrA and GyrB QRDRs were
identical for all the mycobacterial species, except in two cases.
In the first case, 1 amino acid was different between the GyrA
sequences of M. fortuitum,M. peregrinum, and M. aurum, which
harbored a serine at position 83, and that of the other myco-
bacterial species, which harbored an alanine at position 83
(16). This difference, which we reported previously, has been
related to the intrinsic quinolone susceptibility of the former
three species. In the second case, 1 amino acid was different
between the GyrA sequences of different strains of M. tuber-
culosis, with either a serine or a threonine at position 88 (35).
The Ser88-Thr natural polymorphism has been related to the
phylogenetic origin of the strains. The strains with Thr88 are
ancestral to those with Ser88 and are more frequent (31).
Nucleotide sequences of the gyrA QRDR (120 bp) and of the
gyrB QRDR (117 bp) were, overall, highly conserved among all
the strains tested, the similarity values ranging between 75 and
100% for the gyrA QRDR and 79 to 100% for the gyrB QRDR
(Table 1). Such similarity values between mycobacterial spe-
cies have been reported for the genes used for molecular
identification to species level (31). The gyrA and gyrB nucleo-
tide sequences were compared for all the species (interspecies
similarity) and for all the strains within each species (intraspe-
cies similarity).
Interspecies comparison showed that nucleotide sequences
of the gyrA and gyrB QRDRs were species specific; i.e., they
were clearly different from one species to another (Fig. 1;
Table 1). Species that are closely related by either phenotypic
or biochemical characters or ribosomal sequences had differ-
ent gyrA and gyrB QRDR sequences. For instance, M. kansasii
TABLE 1. Highest similarity values (%) between the sequences of gyrA QRDR (lower left) and of gyrB QRDR (upper right)
from species of the Mycobacterium genus
Mycobacterial
species no. (name)
Similarity (% with species no.)
1 2 3 456789101112131415161718192021
1. (M. tuberculosis) 100 100 85.5 88.0 87.2 88.9 85.5 83.8 88.9 88.9 83.8 87.2 86.3 81.2 84.6 88.0 84.6 84.6 87.2 87.2
2. (M. bovis) 100 100 85.5 88.0 87.2 88.9 85.5 83.8 88.9 88.9 83.8 87.2 86.3 81.2 84.6 88.0 84.6 84.6 87.2 87.2
3. (M. africanum) 100 100 85.5 88.0 87.2 88.9 85.5 83.8 88.9 88.9 83.8 87.2 86.3 81.2 84.6 88.0 84.6 84.6 87.2 87.2
4. (M. xenopi) 90.8 90.8 90.8 91.4 94.0 90.6 91.4 93.2 93.2 94.9 89.7 88.9 88.9 82.9 87.2 94.0 92.3 95.7 90.6 95.7
5. (M. avium) 90.8 90.8 90.8 90.0 95.7 93.2 92.3 90.6 94.0 94.0 88.0 89.7 88.9 80.3 91.4 94.9 94.0 94.0 90.6 92.3
6. (M. intracellulare) 90.0 90.0 90.0 89.2 96.7 90.6 95.7 94.9 94.0 93.2 89.7 90.6 88.9 80.3 91.4 94.9 94.0 94.0 89.7 94.0
7. (M. gordonae) 92.5 92.5 92.5 90.8 90.8 93.3 92.3 91.4 92.3 95.7 88.0 88.9 88.9 80.3 85.5 92.3 89.7 89.7 95.7 91.4
8. (M. kansasii) 92.5 92.5 92.5 90.8 92.5 95.8 91.7 96.6 91.4 92.3 89.7 87.2 87.2 79.5 88.0 93.2 90.6 91.4 91.4 91.4
9. (M. gastri) 92.5 92.5 92.5 88.3 90.0 90.8 90.8 93.3 91.4 94.0 88.9 87.2 87.2 79.5 85.5 90.6 89.7 91.4 89.7 90.6
10. (M. malmoense) 90.0 90.0 90.0 90.8 91.7 93.3 94.2 92.5 90.8 94.9 87.2 90.6 89.7 80.3 90.6 94.0 94.0 93.2 90.6 94.0
11. (M. szulgai) 88.3 88.3 88.3 90.0 92.5 92.5 91.7 91.7 90.0 88.3 86.3 88.9 88.0 82.9 88.0 93.2 92.3 92.3 91.4 93.2
12. (M. simiae) 94.2 94.2 94.2 90.8 90.0 92.5 93.3 95.0 89.2 90.0 90.0 89.7 89.7 80.3 85.5 89.7 88.0 90.6 92.3 91.4
13. (M. marinum) 88.3 88.3 88.3 90.0 91.7 92.5 94.2 91.7 89.2 92.5 90.8 91.7 100 83.8 87.2 93.2 90.6 92.3 88.9 90.6
14. (M. ulcerans) 88.3 88.3 88.3 89.2 91.7 92.5 94.2 91.7 89.2 91.7 90.8 91.7 100 82.9 86.3 92.3 89.7 91.4 88.9 89.7
15. (M. leprae) 80.8 80.8 80.8 81.7 84.2 86.7 80.8 85.0 83.3 83.3 85.0 80.8 82.5 81.7 82.0 79.5 78.6 82.0 82.0 80.3
16. (M. chelonae) 90.8 90.8 90.8 86.7 86.7 88.3 90.0 90.0 86.7 89.2 86.7 88.3 87.5 87.5 80.0 93.2 93.2 92.3 88.0 89.7
17. (M. abscessus) 90.8 90.8 90.8 87.5 85.8 86.7 91.7 87.5 87.5 87.5 85.8 87.5 88.3 88.3 75.0 89.2 97.4 96.6 91.4 94.9
18. (M. fortuitum) 89.2 89.2 89.2 89.2 90.0 90.8 92.5 91.7 90.0 90.0 90.0 90.0 91.7 91.7 77.5 84.2 88.3 94.9 89.7 92.3
19. (M. peregrinum) 89.2 89.2 89.2 88.3 92.5 94.2 90.8 91.7 87.5 89.2 90.0 91.7 90.0 90.0 80.0 85.0 85.8 92.5 89.7 94.0
20. (M. smegmatis) 93.3 93.3 93.3 90.8 89.2 90.0 92.5 90.8 90.0 88.3 90.8 91.7 90.0 90.0 80.0 87.5 90.0 90.8 92.5 93.2
21. (M. aurum) 90.8 90.8 90.8 87.5 88.3 91.7 91.7 90.0 90.0 87.5 88.3 90.8 89.2 89.2 80.8 87.5 88.3 91.7 93.3 94.2
1312 NOTES J. CLIN.MICROBIOL.

and M. gastri, which have the same 16S and ITS rRNA se-
quences, had specificgyrA and gyrB QRDR sequences, with 8-
to 10-nucleotide differences (highest similarity value of 93.3%)
between the gyrA QRDRs and 4- or 5-nucleotide differences
(highest similarity value of 96.6%) between the gyrB QRDRs.
M. szulgai and M. malmoense, which are not differentiated by
their 16S rDNA sequences, were differentiated by 14 or 15
nucleotides (highest similarity value of 88.3%) between the
sequences of gyrA QRDRs and by 6 to 9 nucleotides (highest
similarity value of 94.9%) between the sequences of gyrB
QRDRs. M. avium and M. intracellulare, which belong to the
same complex, were differentiated by 4 to 7 nucleotides be-
tween the gyrA QRDRs and 5 to 9 nucleotides between the
gyrB QRDRs. Scotochromogen species, such as M. gordonae,
M. szulgai, and M. aurum, were differentiated with regard to
the gyrA QRDR sequences (10-, 14-, and 10-nucleotide differ-
ences, respectively) and with regard to the gyrB QRDR se-
quences (4- to 7-, 8- to 11-, and 10- or 11-nucleotide differ-
ences, respectively). Finally, gyrA and gyrB sequencing was
efficient for the differentiation of rapidly growing mycobacte-
rial species. Nucleotide sequences of gyrA and gyrB QRDRs
were clearly different not only between the M. chelonae group
(M. chelonae and M. abscessus) and the M. fortuitum group (M.
fortuitum and M. peregrinum) but also between the two species
within each group (Fig. 1). Precisely, the sequences of the gyrA
QRDRs from M. chelonae and M. abscessus differed by 13 or 14
nucleotides and the gyrB QRDR sequences differed by 8 to 14
nucleotides. The sequences of the gyrA QRDRs from the
strains of M. fortuitum and M. peregrinum differed by 9 nucle-
otides, and the gyrB QRDR sequences differed by 6 or 7 nu-
cleotides. However, species within the M. fortuitum group ap-
peared to form an homogeneous cluster and appeared well
differentiated from the M. chelonae group (Fig. 1; Table 1),
which is consistent with published data (39).
The nucleotide sequences of gyrA and gyrB QRDRs did not
discriminate with regard to the species that belong to the M.
FIG. 1. Alignment of the nucleotide sequences of the gyrA and gyrB QRDRs from the 21 mycobacterial species. Sequences of M. tuberculosis
were taken as the reference sequences, and dashes represent identical nucleotides. The nucleotide polymorphisms, i.e., a nucleotide difference that
has been observed between strains within the same species, were indicated by a letter in boldface type, and their meaning is the following: the letter
R when A or G was observed, Y for C or T, M for A or C, K for G or T, S for G or C, and W for A or T.
VOL. 41, 2003 NOTES 1313

tuberculosis complex, i.e., M. tuberculosis,M. africanum, and M.
bovis. This was expected, since so far the sequences of the
genes used for identification to species level do not differen-
tiate these species (31). The nucleotide sequences of gyrA and
gyrB QRDRs did not differentiate M. marinum from M. ulcer-
ans, either. Nearly all of the genes sequenced so far and used
for identification to species level were identical for M. ulcerans
and M. marinum (32).
Intraspecies similarity was studied by sequencing the gyrA
and gyrB QRDRs of 3 to 10 wild-type strains of each species.
Similarity ranged from 97.5 to 100%. Nucleotide differences
were observed rarely between the strains of a same species,
with the exception of fluoroquinolone-resistant mutants. The
intraspecies differences were considered a natural polymor-
phism of the sequence and are indicated in Fig. 1. Since only
some species were concerned, such as M. avium,M. intracellu-
lare,M. kansasii, and M. abscessus, it might be due to the
taxonomic heterogeneity of the species (38).
Molecular methods that were described for the identifica-
tion of mycobacteria often require PCR sequencing of long
DNA fragments. This can be circumvented by using PCR-
restriction fragment length polymorphism, and some laborato-
ries found it simple and inexpensive (10, 12, 17, 19, 29). How-
ever, they also reported disadvantages of PCR-restriction
fragment length polymorphism, such as a lack of specificity, the
need for a large panel of control species, and the fact that new
species are undetected (37, 40). Hybridization to oligonucleo-
tide probes (LiPA and Chip) is a simple and robust technique
but has been so far applied only to 16S rDNA and ITS rDNA
sequences (25, 33, 36). The work flow of the technique that we
described herein is simple: one PCR at a unique hybridization
temperature for the two genes followed by a short sequencing
(120 bp only for each gene). Nowadays, amplification and
sequence determination are implemented in most of the hos-
pitals or can be done outside for a low price.
Analysis of the nucleotide sequences of gyrA and gyrB
QRDRs can rapidly determine the mycobacterial species.
Rapid identification of nontuberculous mycobacteria to spe-
cies level is particularly useful, because the antibiotic suscep-
tibility pattern and the clinical interest vary depending on the
mycobacterial species (2). Moreover, for pathogenic mycobac-
teria, this test can simultaneously give an answer regarding
susceptibility to quinolones. The mutations that we have ob-
served in quinolone-resistant strains of M. tuberculosis com-
plex, M. fortuitum,M. leprae,M. avium,M. smegmatis, and M.
peregrinum did not result in a sequence specific to another
species. Conversely, nucleotide differences observed between
species were different from the mutations involved in fluoro-
quinolone resistance.
We thank Murielle Renard for technical assistance, Ve´ronique Vin-
cent for providing reference strains, and Miche`le Dailloux and Jean-
nette Maugein for providing clinical strains.
This study was supported by grants from the Association Franc¸aise
Raoul Follereau, the Association Claude Bernard, the Institut Na-
tional de La Sante´et de la Recherche Me´dicale (EMI 004), and the
University of Paris VI (research group UPRES EA 1541).
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