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Antimicrobial Agents and Chemotherapy, October 2000, p. 2719-2727, Vol. 44, No. 10
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Cloning and Nucleotide Sequence of the DNA Gyrase
(gyrA) Gene from Mycoplasma hominis and
Characterization of Quinolone-Resistant Mutants Selected In Vitro
with Trovafloxacin
C. M.
Bébéar,*
O.
Grau,
A.
Charron,
H.
Renaudin,
D.
Gruson, and
C.
Bébéar
Laboratoire de Bactériologie,
Université Victor Segalen Bordeaux 2, 33076 Bordeaux, France
Received 27 March 2000/Returned for modification 30 May
2000/Accepted 8 July 2000
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ABSTRACT |
We report the cloning and characterization of the gyrA
gene of the Mycoplasma hominis DNA gyrase, which was
previously shown to be associated with quinolone resistance in this
organism. The 2,733-bp gyrA gene encodes a protein of 911 amino acids with a calculated molecular mass of 102.5 kDa. As expected,
M. hominis GyrA exhibits higher homology with the GyrA
subunits of the gram-positive bacteria Clostridium
acetobutylicum, Bacillus subtilis,
Streptococcus pneumoniae, and Staphylococcus
aureus than with its Escherichia coli counterpart.
Knowing the entire sequence of the gyrA gene of M. hominis could be very useful for confirming the role of the GyrA
subunit in fluoroquinolone resistance. Twenty-nine mutants of M. hominis were selected stepwise for resistance to trovafloxacin, a
new potent fluoroquinolone, and their gyrA,
gyrB, parC, and parE quinolone
resistance-determining regions were characterized. Three rounds of
selection yielded 3 first-step, 12 second-step, and 14 third-step
mutants. The first-step mutants harbored a single substitution,
Glu460
Lys (E. coli coordinates), in ParE. GyrA changes,
Ser83Leu, Glu87Lys, and Ala119Glu or Val, were found only in
the second round of selection. At the third step, additional substitutions, at ParC Ser80, Ser81, and Glu84 and ParE Leu440, associated with high-level resistance to fluoroquinolones, appeared. Thus, high-level resistance to trovafloxacin required three steps and
was associated with alterations in both fluoroquinolone targets. According to these genetic data, in M. hominis, as in
Staphylococcus aureus and Streptococcus
pneumoniae, topoisomerase IV seems to be the primary target of trovafloxacin.
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INTRODUCTION |
The intracellular targets of
fluoroquinolones in bacteria are considered to be the type II
topoisomerases, DNA gyrase and topoisomerase IV (23). DNA
gyrase is composed of two A and two B subunits, encoded by the
gyrA and the gyrB genes, respectively. This
tetrameric enzyme catalyzes ATP-dependent negative supercoiling of DNA.
Topoisomerase IV, a C2E2 tetramer encoded by
the parC and parE genes, is essential for
chromosome partitioning. Mutations in the quinolone
resistance-determining regions (QRDRs) of GyrA and ParC mainly and GyrB
and ParE less frequently have been described as the major mechanism for
quinolone resistance (10, 23).
Mycoplasma hominis is a cause of urogenital tract infections
and has been implicated in extragenital infections as well, especially in immunocompromised patients (46). We recently reported in vitro and in vivo fluoroquinolone-resistant mutants of M. hominis associated with alterations in GyrA, ParC, and ParE QRDRs
(3, 5, 7). Furthermore, previous genetic studies showed that topoisomerase IV was the primary target of pefloxacin, ofloxacin, and
ciprofloxacin, whereas DNA gyrase was the primary target of sparfloxacin (5, 25).
Concerning the target genes of fluoroquinolones in M. hominis, the gyrB, parC, and parE
genes and only the QRDR sequence of gyrA have been cloned
and sequenced (3, 4, 28). Here we report the cloning,
sequencing, and organization of the complete M. hominis gyrA
gene, as well as a detailed analysis of the gyrA, gyrB, parC, and parE QRDRs from
M. hominis mutants selected in a stepwise-manner for
resistance to trovafloxacin.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and DNA manipulations.
The
M. hominis reference strain PG21 (ATCC 23114) was grown in
Hayflick modified broth medium supplemented with arginine
(17). The Escherichia coli strain JM109 and the
vector pGEM3zf(+) (Promega) were used to construct libraries and to
subclone DNA inserts. Chromosomal DNA from M. hominis PG21
was obtained as previously described (47). Manipulations of
DNA, including electrophoresis, Southern blotting, and in situ colony
hybridization, were carried out by standard procedures (40).
For Southern and colony hybridization, DNA was radiolabeled with 50 µCi of [
-32P]dCTP (3,000 Ci/mmol) using a NonaPrimer
kit from Appligene. Plasmid DNA was amplified in E. coli by
using a QIAprep spin miniprep kit (Qiagen).
Restriction mapping and cloning procedures for the
gyrA locus.
Genomic DNA was singly or doubly digested
with various restriction enzymes. Restriction fragments were separated
by electrophoresis, blotted to nylon membranes, and hybridized to
-32P-labeled probes under standard stringent conditions.
Two DNA probes, MH3-MH4 and 321-322, corresponding to the 5' and 3'
regions of gyrA, respectively, were generated by PCR
amplification of the M. hominis genomic DNA with primers MH3
and MH4 (3) and with primers 321 and 322 (this study; see
below), respectively. A restriction map was constructed from the
hybridization patterns of genomic DNA obtained with each of the two probes.
M. hominis PG21 genomic DNA was then digested with
EcoRI, BglII, or
BglII-HindIII. The fragments were ligated to
the linearized vector dephosphorylated and digested with
EcoRI, BamHI, or
BamHI-HindIII, respectively. After
transformation of E. coli by ligation mixtures, recombinant
clones containing the gyrA sequences were selected by colony
hybridization with the [-32P]dCTP-labeled MH3-MH4 or
321-322 DNA fragments. Hybridization-positive clones were selected, and
their plasmid content was determined. Three recombinant plasmids,
pMH3.1, pMH14.1, and pMH2.20, containing M. hominis DNA
inserts of 4.2, 3.7, and 0.95 kbp, respectively, were selected for
sequencing studies.
PCR amplification.
PCRs were carried out with a Perkin-Elmer
Cetus thermal cycler with 100 ng of template DNA for M. hominis PG21 or with 2 µl of a broth culture for
trovafloxacin-resistant mutants and 1 µM each primer as described
elsewhere (5). Primer sets MH3 and MH4, MH6 and MH7, MH11
and MH13, and MH27 and MH28, previously described (5), were
used to amplify the gyrA, gyrB, parC,
and parE QRDRs, respectively. The gyrA 3'-end
probe from M. hominis PG21 was generated with primers 321 (5'-TTAACAAGCGATGGTGTTGC-3') and 322 (5'-GATAATTTTCTGTCATTGTCTTC-3'). Amplification was achieved with an initial denaturation step of 10 min at 94°C; 40 cycles of 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C; and a final 10-min
extension step at 72°C.
DNA sequence analysis.
Double-stranded DNA was sequenced on
both strands by using an ABI PRISM dRhodamine terminator cycle
sequencing ready reaction kit with AmpliTaq DNA polymerase FS and an
ABI PRISM 377 sequencer (Perkin-Elmer Applied Biosystems) according to
the manufacturer's instructions. Forward and reverse primers, flanking
the multiple-cloning-site polylinker of the pGEM3zf(+) vector, as well
as internal primers were used to obtain the complete sequences of the
DNA inserts of the three recombinant plasmids. PCR products of the
quinolone-resistant strains were directly sequenced after purification
with a Wizard PCR Preps DNA purification system (Promega). Pairwise and
multiple sequence alignments were done with ALIGNp, CLUSTAL W
(Infobiogen), and BLAST (National Center for Biotechnology Information) software.
PFGE.
Pulsed-field gel electrophoresis (PFGE) was performed
as previously described by using restriction endonucleases
BamHI, SalI, SmaI, and XhoI
(4, 28). Fragments containing the gyrA gene were
identified by using the [-32P]dCTP-labeled MH3-MH4 DNA
fragment as a probe.
Antimicrobial agents and determination of MICs.
Antibiotics
were purchased from the following manufacturers: norfloxacin, Merck
Sharp & Dohme, Roma, Italy; pefloxacin and sparfloxacin,
Rhône-Poulenc-Rorer, Vitry-sur-Seine, France; ofloxacin, Hoechst
Marion Roussel, Romainville, France; ciprofloxacin, Bayer-Pharma, Puteaux, France; and trovafloxacin, Pfizer, Orsay, France. The MICs of
the fluoroquinolones were determined by the agar dilution method as
previously described (2).
In vitro selection of trovafloxacin-resistant mutants.
Stepwise selection of trovafloxacin-resistant mutants was performed by
plating approximately 2 × 107 color-changing units of
strain PG21 onto Hayflick modified agar medium containing increasing
inhibitory concentrations of trovafloxacin. After 48 h of
incubation at 37°C, resistant colonies were grown in broth medium
without antibiotic and used for the next round of selection. The
frequency of mutation was determined as the number of colonies
appearing on the plate with antibiotic divided by the number of
colonies in the inoculum.
Nucleotide sequence accession number.
The DNA sequence
corresponding to the gyrA-encompassing fragment has been
assigned GenBank accession no. AF242654.
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RESULTS AND DISCUSSION |
Cloning and organization of the gyrA locus.
Southern blot hybridization of M. hominis genomic DNA with
probes MH3-MH4 and 321-322 revealed that most of the gyrA
gene was contained in a 4.2-kbp EcoRI fragment. By using
combinations of single and double digests of the DNA with the enzymes
BglII, EcoRI, HindIII, and
NsiI, we established the restriction map of the
gyrA gene region. As indicated in Fig.
1, the 3.7-kbp
BglII-HindIII fragment overlapping the
4.2-kbp EcoRI fragment was found to contain the 5' end of
gyrA. These two fragments were recovered from genomic libraries of M. hominis by in situ colony hybridization, and
the respective recombinant plasmids, pMH3.1 and pMH14.1, obtained were
sequenced (Fig. 1). DNA probe 321-322, corresponding to the 3' end of
the M. hominis sequenced fragment, was chosen to clone the
3' end of the gyrA gene and its flanking regions. The
0.95-kbp BglII fragment containing the gyrA 3'
end was cloned in E. coli, and recombinant plasmid pMH2.20
(Fig. 1) was sequenced.
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FIG. 1.
Restriction map and organization of the M. hominis PG21 gyrA locus. E, EcoRI; H,
HindIII; B, BglII; N, NsiI. ?,
hypothetical gene. ORFs are numbered 1 to 5. Arrowheads indicate the
transcription sense of the ORFs. Sizes of DNA inserts are indicated in
parentheses.
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Sequencing of the inserts of the three recombinant plasmids allowed the
characterization of a 6,243-bp genomic DNA fragment
of
M. hominis (Fig.
1). This sequence was found to contain five
putative
open reading frames (ORFs) (ORF1 to ORF5). Three ORFs
(ORF1, ORF4, and
ORF5) were functionally assigned, based on significant
sequence
similarities to genes encoding proteins with known functions
from other
organisms (Fig.
1). ORF4, nucleotides 2798 to 5530,
was assigned as the
gyrA gene of
M. hominis (see
below).
Sequence analysis of the M. hominis GyrA subunit.
The predicted GyrA polypeptide contains 911 amino acids (aa) and has a
calculated molecular mass of 102.5 kDa. It contains only one
UGA-encoded tryptophan residue and has a G+C content of 31%. AT-rich
sequences characteristic of putative 
10 and 35 promoter sequences
and a putative ribosome binding site were found upstream of the ATG
initiation codon. The M. hominis GyrA subunit, with 911 residues, seems to be the largest GyrA subunit sequenced so far among
organisms related to gram-positive bacteria. Only three gram-negative
bacteria have a larger GyrA protein, Neisseria gonorrhoeae
(916 aa) (8), Aeromonas salmonicida (922 aa)
(33), and Pseudomonas aeruginosa (923 aa)
(27). It is noteworthy that M. hominis ParC, 866 aa long, is also the largest topoisomerase IV ParC subunit known
(4). Compared to other GyrA subunits, the M. hominis protein contains an additional stretch of 58 aa at the
N-terminal end (see Fig. 3).
Compared to the GyrA and ParC proteins of
M. genitalium
(
16),
M. pneumoniae (
22),
Ureaplasma urealyticum (accession no.
AF222894),
Bacillus subtilis (
26),
Staphylococcus
aureus (
13,
30),
Streptococcus pneumoniae
(
1,
34),
Clostridium acetobutylicum
(
44), and
E. coli (
38,
42), the
M. hominis GyrA polypeptide exhibits a higher percentage of
identity with
the GyrA subunits than with the ParC subunits. The
identity of
M. hominis GyrA with the other GyrA proteins
varies between 39.2%
(
E. coli) and 47.8% (
U. urealyticum), while its identity with
the ParC proteins ranges
from 28.3% (
M. pneumoniae) to 35.4% (
B. subtilis). Like the topoisomerase IV ParC and ParE subunits
(
4),
M. hominis GyrA shows higher homology with
its counterpart in
the gram-positive bacteria
B. subtilis
(45% identity),
S. pneumoniae (44.1%), and
S. aureus (42.9%) than with that in the gram-negative
bacterium
E. coli (39.2%).
Among the eubacteria, the best identity score, found with
C. acetobutylicum GyrA, is in agreement with the phylogenetic origin
of the
Mollicutes, believed to have arisen from ancestors of
low-G+C-content
gram-positive bacteria, such as
Clostridium
(
48). In a comparison
with other human mycoplasmas,
M. hominis GyrA was found to share
47.8, 45.3, and 43.9% identical
amino acids with the GyrA subunits
of
U. urealyticum,
M. genitalium, and
M. pneumoniae, respectively.
From these data, we assigned the ORF4-encoded polypeptide as the
GyrA
subunit of
M. hominis. An overall identity of 33.3% was
found
between the GyrA and ParC peptide sequences of
M. hominis. This
percentage is lower than that of the GyrB-ParE
comparison (44.2%)
(
4).
A protein tree was constructed from the GyrA and ParC sequences of the
nine bacteria listed above. As shown in Fig.
2, the
GyrA and ParC sequences clearly
clustered in two groups. The topoisomerase
II sequences of the
gram-positive bacteria with low G+C contents
(
B. subtilis,
S. aureus,
S. pneumoniae, and
C. acetobutylicum)
and of the class
Mollicutes (
M. pneumoniae,
M. genitalium,
U. urealyticum,
and
M. hominis) formed differentiated clusters, as
previously shown by Huang (
24). For mycoplasmas,
phylogenetic
data obtained with topoisomerase II are in good agreement
with
those obtained with 16S rRNA (
29). Indeed,
M. pneumoniae GyrA
and
M. genitalium GyrA belong to the
same phylogenetic group,
while
M. hominis GyrA and
U. urealyticum GyrA form two distinct
groups. As in the 16S rRNA
tree,
U. urealyticum formed on group
and
M. pneumoniae and
M. genitalium formed another group
arising
from the same branch.
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FIG. 2.
Protein tree for full-length GyrA and ParC subunits from
nine bacteria: M. hominis (4; this
study), M. genitalium (16), M. pneumoniae (22), U. urealyticum, B. subtilis (26), S. aureus (13,
30), S. pneumoniae (1, 34), C. acetobulylicum (44), and E. coli (38,
42). The tree was compiled by using the CLUSTAL W
multiple-alignment program.
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In Fig.
3, the GyrA amino
acid sequence of
M. hominis was compared to those of
M. genitalium,
M. pneumoniae,
B. subtilis,
S. aureus,
S. pneumoniae,
C. acetobutylicum, and
E. coli. The
highest homology among
the GyrA proteins of all eight bacteria
is located at the N-terminal
moiety, while the C-terminal region
is much less conserved. Amino acid
residues Ser153, Ser154, and
Glu157 of
M. hominis are the
equivalents of Ser83, Ser84, and
Glu87 of
E. coli GyrA,
which have been shown to be hot spots for
quinolone resistance. Indeed,
we have reported substitutions of
these three amino acids in
fluoroquinolone-resistant mutants of
M. hominis selected in
vivo and in vitro (
3,
5,
7).
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FIG. 3.
Alignment of the M. hominis (Mh) GyrA amino
acid sequence with those of its counterparts in M. genitalium (Mg) (16), M. pneumoniae (Mp)
(22), B. subtilis (Bs) (26), S. aureus (Sa) (30), S. pneumoniae (Sp)
(1), C. acetobutylicum (Ca) (44), and
E. coli (Ec) (42). An asterisk indicates a
residue identical in all eight proteins. Residues involved in quinolone
resistance in M. hominis are indicated by diamonds. Dashes
indicate gaps.
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Location of the DNA gyrase (gyrA) gene on the genomic
map of M. hominis PG21.
PFGE and Southern blot
hybridization with probe MH3-MH4 containing the gyrA QRDR of
M. hominis confirmed that the gyrA gene was
located within SmaI, BamHI, XhoI, and
SalI genomic DNA fragments of 100, 84.5, 124, and 410 kbp,
respectively (4, 28). Hence, the gyrA gene is
located within the 74-kpb region where these restriction fragments
overlap. It is noteworthy that this region is quite distant from the
topoisomerase II genes gyrB and parC-parE.
In many bacteria,
gyrB lies close to the origin of
replication, where genes are organized in the following order:
dnaA,
dnaN,
recF,
gyrB, and
gyrA. A similar gene organization has been described
for
M. pneumoniae (
22) and
M. genitalium
(
16). In
U. urealyticum,
for which the complete
genomic sequence is now available (
http://genome.microbio.uab.edu/uu/),
gyrB and
gyrA are contiguous and are located
between
dnaN and
recA but about 100 kbp
downstream from
dnaA. However, in
M. hominis,
gyrA and
gyrB were shown not to be coupled.
Instead,
gyrA mapped
at least 35 kbp downstream of
gyrB (
28). These results were
confirmed by our
PFGE data showing a
gyrA gene 47 and 31 kbp distant
from
gyrB and
parE-parC, respectively (data not
shown). Furthermore,
we found the following gene organization around
the
gyrA gene;
ftsH homolog, hypothetical
MG347-ORF homolog,
gyrA homolog, and
dnaE
homolog. In
M. genitalium and
M. pneumoniae,
ftsH and
dnaE homologs are located within 30- and
40-kbp regions, respectively,
surrounding the origin of replication. In
addition, the
M. hominis DNA primase motif homolog found
downstream from
gyrA shared homology
with only the 3'-end
parts of other bacterial
dnaE or
dnaG genes.
These genes encode DNA primases that synthesize small RNA primers
at
replication forks during DNA synthesis. In
M. genitalium
(
16)
and
M. pneumoniae (
22), they are
located close to the origin
of replication. Such a situation in
M. hominis, with
gyrA being
found between
ftsH and
dnaE homologs but not downstream from
the
gyrB and
dnaA regions, could be explained by
chromosomal rearrangement.
Thus, it is tempting to speculate that
M. hominis gyrB and
gyrA were first contiguous
and located in the vicinity of the initiation
site of replication,
before being separated by chromosomal rearrangement
during evolution.
It should be noted that
M. hominis oriC has
not yet been
identified.
Characterization of trovafloxacin-resistant mutants selected in a
stepwise manner.
Trovafloxacin-resistant mutants were selected
stepwise in vitro to further examine the role of topoisomerase IV and
DNA gyrase in the development of resistance. The scheme used for the
selection of trovafloxacin-resistant mutants is summarized in Fig.
4. Following this procedure, three
independent sets of first- and second-step mutants and four independent
sets of third-step mutants were obtained with mutation frequencies
ranging from 10
7 to 104. Except for the
104 frequency obtained with mutant IT3 on a trovafloxacin
concentration corresponding to the MIC, mutation frequencies were
similar for all steps and all concentrations used for selection (one to
four times the MIC). These frequencies were relatively high (1 × 107 to 2.5 × 106) and were equivalent
to those found for the selection of ofloxacin- and
sparfloxacin-resistant mutants (5, 25), confirming the high
rate of mutation of M. hominis (12). The 29 trovafloxacin-resistant strains obtained were characterized for their
susceptibilities to six fluoroquinolones and for the QRDR status of
their gyrA, parC, gyrB, and
parE genes (Table 1).
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FIG. 4.
Relationships between M. hominis PG21 and
fluoroquinolone-resistant mutants IT1 to IIIT2F11 selected by stepwise
exposure to trovafloxacin. The numbers outside the boxes indicate the
trovafloxacin concentrations (in micrograms per milliliter) used in the
selection steps. The superscripts +, °, and * indicate the presence
of mutations in GyrA, ParC, and ParE, respectively (Table 1).
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For each of the first-step mutants, IT1 to IT3, there was only a ParE
Glu466
Lys change associated with a fourfold significant
increase in
the MICs of trovafloxacin, ciprofloxacin, and norfloxacin,
and there
was no significant increase (one- to twofold) in the
MICs of
sparfloxacin, ofloxacin, and pefloxacin. A twofold increase
usually is
considered not significant within experimental error
for the twofold
dilution method used for MIC
determinations.
When first-step mutants IT2 and IT3 were used as parental strains, 12 second-step mutants were selected; all had acquired
an additional GyrA
substitution. Six of them, IIT2B to IIT2F,
bearing a Ser153
Leu
substitution, and IIT3C, bearing a Glu157
Lys
substitution, showed
four- to eightfold increases in the MICs
of trovafloxacin,
sparfloxacin, and ciprofloxacin but no changes
in the MICs of
ofloxacin, norfloxacin, and pefloxacin. The six
remaining mutants,
IIT2A, IIT3A, IIT3B, and IIT3D to IIT3F, all
carried an amino acid
change at Ala189 in GyrA. For second-step
mutants harboring the
Ala
Glu change, the MICs of trovafloxacin,
sparfloxacin, and
ciprofloxacin were two- to fourfold higher than
those for their
parental strains. Surprisingly, mutant IIT2A did
not shown any
significant fluoroquinolone MIC increase even though
it had acquired a
Ala
Val substitution at the same position,
189.
Finally, 14 third-step mutants generated from two different parental
strains, IIT2D and IIT2F (ParE Glu466
Lys and GyrA Ser153
Leu),
all
had acquired an additional alteration in topoisomerase IV
subunits,
either ParC or ParE. Ten of them, IIIT2D2, IIIT2F2 to
IIIT2F4, and
IIIT2F6 to IIIT2F11, were found to carry the ParC
Ser91
Ile
substitution, while mutants IIIT2D1 and IIIT2D3 had
Ser92
Pro and
Glu95
Gln changes, respectively. Except for strain
IIIT2D3, all
of these third-step mutants were characterized by
significant increases
in the MICs of trovafloxacin (8-fold), sparfloxacin
(32- to
64-fold), and ciprofloxacin (
4-fold) and especially by
dramatic
increases in the MICs of ofloxacin (32-fold and pefloxacin
(8-fold).
Strain IIIT2D3, bearing the Glu95
Gln substitution,
exhibited
globally two- to eightfold smaller increases in the
MICs of the
quinolones tested, compared to the other third-step
parC
mutant strains. Two mutants, IIIT2F1 and IIIT2F5, harbored
an
additional substitution in ParE, corresponding to a Leu446
Phe
change
and associated with the same fluoroquinolone MIC increases
as those
seen with the ParC Ser91-mutated third-step mutants.
It should be noted
that the increased ofloxacin and pefloxacin
MICs were found associated
only with ParC and ParE amino acid
changes at position 446 and not with
the ParE substitution at
position 466, contained in first-step
mutants.
In summary, the development of a high level of resistance to
trovafloxacin (MIC,
16 µg/ml) in
M. hominis occurred in
three
steps, each associated with a mutation in the topoisomerase gene,
beginning with a ParE alteration and involving alternating changes
in
DNA gyrase and topoisomerase
IV.
Recent studies with the gram-positive bacteria
S. aureus and
S. pneumoniae indicated that different quinolones can have
different
preferential targets, depending on the bacterial species and
on
whether the studies are based on genetic or biochemical enzymatic
data (
9,
13,
15,
18,
20,
31,
32,
35-37,
43,
45).
Trovafloxacin was reported to initially target topoisomerase IV
by
several genetic and enzymatic data for both
S. aureus
(
14,
20) and
S. pneumoniae (
18,
19,
39,
45). For
M. hominis,
a low-G+C-content organism
related to gram-positive bacteria,
we and others showed by genetic
studies that the primacy for fluoroquinolones
of the target enzyme
seemed to be drug specific (
5,
25).
DNA gyrase is the
primary target of sparfloxacin, whereas topoisomerase
IV is the primary
target of pefloxacin, ofloxacin, and ciprofloxacin.
In this study, we
have determined the target specificity of trovafloxacin
in
M. hominis through analysis of mutants selected in a stepwise
manner.
All the first-step trovafloxacin-resistant mutants harbored
a change in
the ParE QRDR, while
gyrA mutations were detected
only in
second-step mutants. These results indicate that, in
M. hominis, as in
S. pneumoniae or
S. aureus,
topoisomerase IV is
the primary target of
trovafloxacin.
GyrA and ParC mutations selected by trovafloxacin in
M. hominis were predominantly those described previously for other
fluoroquinolones.
GyrA positions Ser153 (Ser83) and Glu157 (Asp87)
(
E. coli coordinates)
and ParC positions Ser91 (Ser80) and
Ser92 (Ala81) were found
to be hot spots for quinolone resistance in
many bacteria (
23)
and were already described as being
mutated in
M. hominis (
5,
7,
25). In contrast,
GyrA Ala189 (Ala119), ParC Glu95 (Glu84),
and ParE Leu446 (Leu440) and
Glu466 (Glu460) substitutions are
novel. The GyrA Ala119
Val or Glu
substitution was previously
described for quinolone-resistant isolates
of
Salmonella enterica serovar Typhimurium (
21).
In
M. hominis, only the Ala
Glu amino
acid change was
associated with significant increases in fluoroquinolone
MICs. One
explanation could be the charge difference induced by
the amino acid
change. Indeed, the Ala
Val substitution does not
lead to a change in
the residue charge (Ala and Val are both nonpolar),
while the Ala
Glu
change substitutes a nonpolar residue with a
larger, negatively charged
one. The new Glu84
Gln substitution
in ParC has also been found to
occur at the same position in the
GyrA subunit of
S. pneumoniae clinafloxacin-resistant mutants
(
36).
In contrast to the GyrA and ParC changes, to our knowledge, the ParE
mutations acquired in the first- and third-step mutants
have never been
reported. First, the Glu466
Lys change in the
first-step mutants does
not occur in the EGDSA and PLRGK stretches
designed as the GyrB QRDR
(
49). However, this position is located
in a motif already
associated with fluoroquinolone resistance.
An Asn470
Asp mutation, 2 aa upstream from Glu466, has been described
for
S. aureus
ParE (
15). Moreover, the Glu474
Lys substitution
recently
described for
S. pneumoniae GyrB (
36) corresponds
to
the amino acid position just before Glu466 in
M. hominis.
The
ParE Glu466 alteration, occurring in all
M. hominis
mutants, may
be significant in quinolone resistance. A provocative
experiment
would be to point mutagenize back to wild type the ParE
position
466 Lys mutants to see if the effect on the ParE subunit
affects
trovafloxacin resistance. The second mutation found in ParE,
Leu446
Phe,
lies in the second QRDR motif, PLRGK. Two ParE
alterations have
already been described for this motif; they concern
the proline
residue
Pro451
Ser or Gln in
S. aureus
(
20,
41) and Pro454
Ser
in
S. pneumoniae
(
36). These data confirm that GyrB or ParE
QRDR limits may
require extension compared to the first QRDR,
described for
E. coli (
49). It is interesting that, while the
Glu466
substitution was associated with resistance to trovafloxacin,
ciprofloxacin, and norfloxacin, only the substitution of Leu446
led to
significant increases in the MICs of ofloxacin and pefloxacin.
The
functional role of the ParE QRDR is unknown, but it is tempting
to
speculate that some mutations could interfere with quinolone
action,
depending on the molecule and the mutated
position.
The results described here indicate that only one mutation in
parE or
parC is necessary to reach a high level
of resistance
(MIC,
8 µg/ml) of
M. hominis to
ciprofloxacin, norfloxacin, and
pefloxacin. In contrast, for
sparfloxacin and trovafloxacin, at
least three sequential mutational
events, two in topoisomerase
IV and one in DNA gyrase, are required to
lead to high-level resistance
in
M. hominis, as previously
described for trovafloxacin resistance
in
S. aureus
(
14) and coagulase-negative staphylococci (
11).
However, for sparfloxacin, the presence of GyrA Ser83 and ParC
Ser80 or
Glu84 mutations was shown to be associated with high-level
resistance
in sparfloxacin-selected mutants of
M. hominis (
5,
25). Furthermore, as previously reported for
M. hominis in vitro
and clinical isolates resistant to
fluoroquinolones (
6), our
data suggest that trovafloxacin
could retain activity against
parE and
parE-gyrA
mutants (MICs, 0.12 to 2 µg/ml).
In conclusion, these data clearly confirm the enhanced activity of new
fluoroquinolones, such as trovafloxacin, against mycoplasmas
and
indicate that susceptibility testing with ciprofloxacin or
ofloxacin
would not suffice to evaluate the activity of this antimicrobial
class
against these microorganisms. Furthermore, knowing the complete
sequences of the four topoisomerase genes of
M. hominis is
the
starting point for further enzymatic studies of DNA gyrase or
topoisomerase IV preferential targeting of different
fluoroquinolones.
| |
ACKNOWLEDGMENTS |
This study was supported in part by a grant from Pfizer and a
grant from Pôle Aquitaine Santé.
We thank John Glass and Gail Cassell from the University of Alabama at
Birmingham for kindly providing gyrase and topoisomerase IV sequences
of U. urealyticum serovar 3. The complete genome sequence is
available at the following website:
http://genome.microbio.uab.edu/uu/. We thank Joel Renaudin for
helpful comments and critical reading of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Bactériologie, Université Victor Segalen Bordeaux 2, 146 Rue Léo Saignat, 33076 Bordeaux Cedex, France. Phone: (33)
5.57.57.16.25. Fax: (33) 5.56.93.29.40. E-mail:
cecile.bebear{at}u-bordeaux2.fr.
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Abstract
Introduction
