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Antimicrobial Agents and Chemotherapy, July 2000, p. 1842-1845, Vol. 44, No. 7
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Roles of gyrA Mutations in Resistance of
Clinical Isolates and In Vitro Mutants of Bacteroides
fragilis to the New Fluoroquinolone Trovafloxacin
Rafik
Bachoual,1
Luc
Dubreuil,2
Claude-James
Soussy,1 and
Jacques
Tankovic1,*
Service de
Bactériologie-Virologie-Hygiène, Hôpital Henri
Mondor, Créteil,1 and
Faculté de Pharmacie, Lille,2
France
Received 1 February 2000/Returned for modification 30 March
2000/Accepted 21 April 2000
 |
ABSTRACT |
We determined whether gyrA mutations were present in
fluoroquinolone-resistant laboratory mutants derived from the
Bacteroides fragilis reference strain ATCC 25285 and in
clinical isolates of B. fragilis. The two first-step
mutants selected on ciprofloxacin (CIP) were devoid of gyrA
mutations, whereas two of the three CIP-selected second-step mutants
studied presented the same gyrA mutation leading to a
Ser82Phe change. Unusual GyrA alterations, Asp81Asn or Ala118Val, were
detected in two of the three first-step mutants selected on
trovafloxacin (TRO), Mt3 and Mt1, respectively. The Ala118Val change
had no effect on the susceptibility of Mt1 to CIP. No second-step
mutant could be obtained with TRO as a selector. For the 12 clinical
isolates studied, a Ser82Phe change in GyrA was found only in the 3 strains which showed the highest levels of TRO resistance (MIC, 4 µg/ml). Thus, the resistance phenotypes and genotypes observed in
fluoroquinolone-resistant clinical isolates of B. fragilis
were similar to those found in CIP-selected laboratory mutants, whereas
peculiar mutational events could be selected in vitro with TRO.
| |
INTRODUCTION |
Bacteroides fragilis is
the anaerobic organism most frequently isolated from patients with
bacteremia (with an attributable mortality of 19.3%
[11]) and intra-abdominal infections (8). Older fluoroquinolones, including ciprofloxacin and ofloxacin, show
little activity against anaerobes and B. fragilis in
particular, but some of the newer compounds, notably clinafloxacin,
moxifloxacin, and trovafloxacin, are much more active in vitro (1,
5) as well as in vivo (13, 14).
Fluoroquinolones block DNA replication by forming a stable ternary
complex with DNA and type II DNA topoisomerases: DNA gyrase and DNA
topoisomerase IV (4). Gyrase is a tetrameric enzyme consisting of two A and two B subunits encoded by the gyrA
and gyrB genes, respectively. It catalyzes ATP-dependent
negative supercoiling of double-stranded DNA. Topoisomerase IV, also a heterodimer encoded by the parC and parE genes,
is involved in chromosomal partitioning. Mutational alterations of the
subunits of DNA gyrase or topoisomerase IV, as well as altered
permeation mechanisms, have been shown to be related to quinolone
resistance (4).
The prevalence of acquired resistance of B. fragilis to the
new fluoroquinolones, in particular trovafloxacin, appears to be low
worldwide, less than 5% (12; L. Dubreuil, E. Singer, S. Bland, and A. Sedallian, Prog. Abstr. 9th Eur. Cong. Clin. Microbiol. Infect. Dis., abstr. 793, p. 297, 1999). However, two recent
reports from the United States found surprisingly high frequencies of
resistance to trovafloxacin (MIC, 
4 µg/ml): 16% (D. R. Snydman, L. A. MacDermott, and N. V. Jacobus, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 906, p. 218, 1999) and 48% (D. R. Gustafson, L. M. Sloan, and J. E. Rosenblatt, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother.,
abstr. 905, p. 218, 1999). The gyrA and gyrB
genes from B. fragilis have recently been cloned and
sequenced (9), and levofloxacin-selected second-step but not
first-step in vitro mutants were found to carry a gyrA
mutation leading to the replacement of Ser82 (equivalent to resistance
hot spot Ser83 of GyrA of Escherichia coli) with Phe
(9). In addition, active efflux of norfloxacin, resembling that produced by the NorA/Bmr-type transporter in gram-positive bacteria (7), may also exist in B. fragilis
(6).
Our aims were to determine the prevalence of resistance to
trovafloxacin in clinical strains of B. fragilis isolated
from our hospital and to look for the role of gyrA mutations
in the resistance of clinical isolates as well as of laboratory mutants selected with either ciprofloxacin or trovafloxacin.
| |
MATERIALS AND METHODS |
Bacterial strains.
One hundred fifty strains of B. fragilis isolated from clinical samples between 1995 and 1999 and
one reference strain, B. fragilis ATCC 25285, were studied.
The origins of the strains were as follows: cutaneous suppurations, 34;
intra-abdominal suppurations, 30; suppurations of unspecified origin,
20; blood, 29; stools, 1; unknown, 36. To evaluate the role of
gyrA in fluoroquinolone resistance, we randomly selected 9 fluoroquinolone-resistant isolates, with various resistance levels,
among the 150 clinical isolates, and we also studied 3 resistant
clinical strains isolated in 1996 in eastern France: L1, L2 (Nancy),
and L3 (Annecy). Identification to the species level was performed with
the API 20A system (bioMérieux, Marcy l'Etoile, France).
Susceptibility testing.
MICs were determined by the agar
dilution technique using Wilkins-Chalgren medium (Oxoid, Lyon, France)
supplemented with 5% sheep blood and an inoculum of 105
CFU per spot. MICs were read after 48 h of incubation at 37°C in
an anaerobic atmosphere. Breakpoints used for ciprofloxacin and
trovafloxacin were those recommended by the Comité de
l'Antibiogramme de la Société Française de
Microbiologie (CA-SFM) (2). The quinolone agents tested were
provided by the manufacturers as powders suitable for susceptibility
testing. We also tested the effects of reserpine (Sigma, Saint-Quentin
Fallavier, France) at a concentration of 20 µg/ml on the MICs of
norfloxacin and trovafloxacin.
Frequency of mutation.
The reference strain B. fragilis ATCC 25285 was used as a parental strain to select
quinolone-resistant in vitro mutants in two successive steps by plating
an inoculum of approximately 1010 bacteria onto
Wilkins-Chalgren agar (Oxoid) supplemented with 5% sheep blood and
containing either ciprofloxacin or trovafloxacin at two times the MIC.
After 48 h of anaerobic incubation at 37°C, the colonies were
counted and the frequencies of mutation were determined relative to the
total viable count of organisms plated.
Amplification by PCR of the QRDR of the gyrA
gene.
The quinolone resistance-determining region (QRDR) of
gyrA was PCR amplified with oligonucleotide primers
5'-TGGAACTGGGAAATACGTCAG and 5'-GCATCACTTTGGGTTCCATC,
generating a DNA fragment of 320 bp corresponding to nucleotide
positions 152 to 471 of the gyrA gene of B. fragilis (9). Chromosomal DNA was prepared using the
QiaAmp Kit (Qiagen, Courtaboeuf, France) according to the manufacturer's recommendations. PCR was carried out in a 100-µl volume containing 250 µM deoxynucleoside triphosphates (Pharmacia Biotech, Uppsala, Sweden), 10 µl of 10-fold diluted DNA extract, 100 pmol of each primer, and 2.5 U of Taq DNA polymerase
(Boehringer Mannheim, Meylan, France). Amplification was performed over
40 cycles of 1 min at 95°C, 1 min at 50°C, and 1 min at 72°C.
Restriction fragment length polymorphism (RFLP) and nucleotide
sequence analysis of PCR-amplified DNA.
Amplification products
were digested with HinfI restriction endonuclease
(Pharmacia) and subjected to electrophoresis in a 2% agarose gel in
order to detect a mutation at codon 81 or 82 of gyrA of
B. fragilis. For nucleotide sequencing, amplification products were purified with the microspin columns S-400 HR (Pharmacia) and analyzed using the Taq-Dye-Deoxy-Terminator sequencing
kit (Perkin-Elmer Biosystems, Courtaboeuf, France) and the automatic DNA sequencer ABI Prism 310 (Perkin-Elmer Biosystems). The
oligonucleotide primers used were identical to those for DNA amplification.
| |
RESULTS AND DISCUSSION |
Prevalence of resistance to trovafloxacin and of high-level
resistance to ciprofloxacin among isolates of B. fragilis
from our hospital (1995 through 1999).
The MICs of trovafloxacin
and ciprofloxacin at which 50% of the isolates were inhibited
(MIC50s) were 0.25 and 4 µg/ml, respectively (Fig.
1). By use of the breakpoint
concentrations recommended by the CA-SFM (2), 87% (131 of
150), 3% (4 of 150), and 10% (15 of 150) of the strains investigated
were classified as susceptible (MIC 
1 µg/ml), intermediate
(MIC = 2 µg/ml), and resistant (MIC 4 µg/ml) to
trovafloxacin, respectively. Furthermore, 25% (38 of 150) of the
strains studied required a trovafloxacin MIC greater than or equal to 1 µg per ml and had high-level resistance to ciprofloxacin (MIC 32 µg/ml). Although the MICs of trovafloxacin for half of these
isolates were not sufficiently increased for the strains to be
considered intermediate or resistant by standard criteria, they most
probably had already acquired at least one mechanism of resistance to
fluoroquinolones. These high-level ciprofloxacin-resistant strains were
more frequent in 1995 (5 of 16; 31%) and 1996 (5 of 15; 33%) than in
1997 (14 of 57; 25%), 1998 (11 of 47; 23%), and 1999 (3 of 15; 20%).
This decrease probably parallels a decreased usage of fluroquinolones
in our hospital, from 
12 kg/year in 1995 to 7 kg/year in 1998.
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FIG. 1.
Distribution of trovafloxacin and ciprofloxacin MICs
against 150 clinical strains of B. fragilis.
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|
The prevalence of resistance to trovafloxacin in our hospital was
unexpectedly high compared to findings of less than 5% intermediate
or
resistant strains reported from previous studies in Europe
and the
United States (
12; Dubreuil et al., 9th Eur. Cong.
Clin.
Microbiol. Infect. Dis.). However, two recent reports from the
United States found even higher frequencies of trovafloxacin resistance
in clinical isolates of
B. fragilis (MIC
4 µg/ml)
than that
reported here: 16% (87 of 530 isolates from 1996 through
1997)
in a multicenter study (Snydman et al., 39th ICAAC) and 48%
(43
of 90 isolates from 1998 through 1999) in a report from the Mayo
Clinic (Gustafson et al., 39th ICAAC). Taken together, these data
suggest that the prevalence of fluoroquinolone resistance among
B. fragilis clinical isolates may vary substantially from
one
hospital to another. These variations may correlate with the level
of fluoroquinolone usage, but this remains to be
determined.
Presence of gyrA mutations in fluoroquinolone-resistant
clinical isolates.
We studied 12 strains for which the MICs of
ciprofloxacin ranged from 16 to 128 µg/ml. Compared to trovafloxacin,
sparfloxacin was two- to eightfold less active, moxifloxacin was
approximately as active, and clinafloxacin was either as active or two-
to fourfold more active. Thus, acquired resistance to fluoroquinolones
in clinical isolates of B. fragilis does not modify the
relative activities of the new fluoroquinolones.
We determined the nucleotide sequence of the QRDR of
gyrA
from codon 55 to 138. The same mutational alteration, Ser82Phe,
was
found for isolates C5, C14, and L1, which showed the highest
level of
resistance to trovafloxacin (MIC = 4 µg/ml [Table
1]).
The Ser82 residue in the GyrA of
B. fragilis is equivalent to
resistance hot spot Ser83 in
the GyrA of
E. coli. Analysis of
the restriction map of the
PCR-amplified 320-bp fragment of
gyrA of
B. fragilis showed that this
gyrA mutation was able to
abolish
one of the two existing
HinfI sites (the restriction
site GANTC
was changed to AANTC in
gyrA mutants). This was
confirmed by RFLP
analysis (data not shown).
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TABLE 1.
Susceptibilities of clinical isolates of B. fragilis to fluoroquinolones and amino acid substitutions in
the A subunit of DNA gyrase
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|
Presence of gyrA mutations in fluoroquinolone-resistant
in vitro mutants.
The reference strain B. fragilis ATCC
25285 (ciprofloxacin and trovafloxacin MICs, 4 and 0.25 µg/ml,
respectively) was plated onto agar containing either ciprofloxacin or
trovafloxacin at two times the MIC. With ciprofloxacin, first-step
mutants were obtained at a frequency of 3 · 10
8.
Two of these, Mc1 and Mc2, were selected at random for further study.
The activity of trovafloxacin against these mutants was unchanged,
whereas that of ciprofloxacin was reduced twofold. Mc1 and Mc2 were
plated again at two times the MIC of ciprofloxacin. Second-step mutants
were obtained at a mean frequency of 2 · 109.
Three second-step mutants were further studied: Mc3, deriving from Mc1,
and Mc4 and Mc5, deriving from Mc2. The relative activities of
clinafloxacin, moxifloxacin, sparfloxacin, and trovafloxacin against
these mutants (Table 2) were similar to
those against the clinical isolates described above (Table 1).
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TABLE 2.
Susceptibilities of in vitro mutants of B. fragilis to fluoroquinolones and amino acid substitutions in
the A subunit of DNA gyrase
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|
The same
gyrA mutation that was found in clinical isolates
(derived substitution Ser82Phe) was found only for second-step
mutants
Mc4 and Mc5, for which the MICs of ciprofloxacin and trovafloxacin
showed the highest increase (16-fold) and which were classified
as
trovafloxacin resistant (MICs, 4 µg/ml [Table
2]). The
fluoroquinolone
resistance phenotypes of these two laboratory mutants
were similar
to those of the above-described clinical isolates carrying
gyrA mutations. Thus, with ciprofloxacin as a selector, two
consecutive
mutational events, of which the second is a typical
gyrA mutation,
can confer trovafloxacin resistance in
B. fragilis. Similar results
have been obtained with
levofloxacin as a selector: levofloxacin-selected
first-step mutants
were devoid of
gyrA or
gyrB mutations, but
a
gyrA mutation identical to that described above occurred in
second-step mutants (
9). Taken together, these data suggest
that DNA gyrase of
B. fragilis could be only a secondary
target
of ciprofloxacin and levofloxacin, similar to what is observed
in gram-positive bacteria (
4), topoisomerase IV being the
primary
target. This remains to be verified by amplification and
sequencing
of the critical regions of the
parC and
parE genes from first-step
in vitro mutants and
low-level-resistant clinical
isolates.
With trovafloxacin at two times the MIC, first-step mutants were
obtained at a frequency of 1 · 10
9. Three of
these, Mt1, Mt2, and Mt3, were selected at random for
further study.
Despite repeated attempts, we were not able to
obtain second-step
mutants with trovafloxacin at two times the
MIC as a selector
(frequency of mutation, <8 · 10
11). Sharp
increases in the MICs of trovafloxacin, 8- to 16-fold,
were observed
for the three trovafloxacin-selected first-step
mutants studied,
whereas the activity of ciprofloxacin was only
slightly reduced (2- to
4-fold) in two cases and was even unchanged
for mutant Mt1. Our
findings confirm those of a previous study
that had shown that
ciprofloxacin and trovafloxacin selected for
mutants of
B. fragilis with specific phenotypes (L. J. V. Piddock
and
V. Ricci, Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother.,
abstr. C180, p. 121, 1998). Furthermore, these data suggest that
DNA
gyrase may be the primary target of trovafloxacin in
B. fragilis.
This may represent another example of the fact that
different
fluoroquinolones can have different primary targets in the
same
bacterial species, as has already been shown for
Streptococcus pneumoniae (
10).
Mutant Mt1 was as susceptible as the parental strain to ciprofloxacin
and carried an unusual
gyrA mutation leading to an Ala118Val
substitution. Mutant Mt3 carried a novel, yet undescribed
gyrA mutation leading to an Asp81Asn substitution; Asp81 is
equivalent
to Asp82 of the GyrA of
E. coli. Thus,
trovafloxacin appears to
be able to select for unusual
gyrA
mutations that diminish the
activity of trovafloxacin selectively. This
is particularly striking
for the Ala118Val substitution in mutant Mt1,
which is associated
with an eightfold increase in the MIC of
trovafloxacin, whereas
the MICs of ciprofloxacin, sparfloxacin,
moxifloxacin, and clinafloxacin
are unchanged (Table
2). The reason for
that dissociated resistance
could be the bulkiness of both the side
chain of valine (three
methyl groups instead of one for alanine) and
the N-1 substituent
of trovafloxacin (a difluorophenyl instead of a
cyclopropyl for
the other drugs tested). An identical substitution in
GyrA has
been found in an in vitro mutant of
Salmonella
enterica serovar
Typhimurium selected on nalidixic acid (mutant
L30
nalA), and
the MIC of ciprofloxacin for that mutant
remained low (0.03 µg/ml)
(
3).
Effects of reserpine on the MICs of norfloxacin and
trovafloxacin.
It has been shown that norfloxacin is actively
pumped out in wild-type B. fragilis and that norfloxacin
efflux is decreased in the presence of reserpine (6). In
addition, a one-step norfloxacin-selected mutant with threefold
increases in the MICs of norfloxacin and ethidium bromide and no change
in the MIC of sparfloxacin has been described (6). This
suggests that enhanced active efflux with a specificity similar to that
of the NorA/Bmr-type transporter of gram-positive bacteria could be
involved in the fluoroquinolone resistance of B. fragilis.
To detect such a mechanism, we determined the MICs of norfloxacin and
trovafloxacin with or without 20 µg of reserpine/ml
for reference
strain ATCC 25285, the 12 fluoroquinolone-resistant
clinical isolates
described above, and the resistant in vitro
mutants. For all strains
tested, the MICs of trovafloxacin were
unaffected by the presence of
reserpine whereas the activity of
norfloxacin was reduced only twofold
in the presence of reserpine
(data not shown). Thus, it was unlikely
that a NorA/Bmr-type efflux
was implicated in the resistance of the
strains
studied.
In conclusion, the resistance phenotypes and genotypes observed in
clinical isolates were similar to those obtained in vitro
with
ciprofloxacin. In contrast, trovafloxacin selected for peculiar
mutants. This raises the possibility of similar phenomena occurring
in
vivo, either with trovafloxacin or with other new
fluoroquinolones.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Service de
Bactériologie-Virologie-Hygiène, Hôpital Henri
Mondor, 51 Avenue du Maréchal de Lattre de Tassigny, 94 010 Créteil, France. Phone: 33-1-49812828. Fax: 33-1-49812839. E-mail: jacques.tankovic{at}hmn.ap-hop-paris.fr.
| |
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Antimicrobial Agents and Chemotherapy, July 2000, p. 1842-1845, Vol. 44, No. 7
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
