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Antimicrobial Agents and Chemotherapy, December 1998, p. 3317-3319, Vol. 42, No. 12
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Examination of Methicillin-Resistant and
Methicillin-Susceptible Staphylococcus aureus Mutants with
Low-Level Fluoroquinolone Resistance
Mark C.
Sulavik
and
Neil L.
Barg*
Department of Medicine, Division of
Infectious Diseases, University of Michigan Medical School, and
Department of Veterans Affairs Medical Center, Ann Arbor, Michigan
Received 12 January 1998/Returned for modification 25 February
1998/Accepted 17 September 1998
 |
ABSTRACT |
For Staphylococcus aureus, stepwise mutations result in
high-level quinolone resistance. Methicillin-resistant and -susceptible quinolone-resistant, first-step mutants generated in vitro were obtained and found to be no different than those recovered from murine
abscesses. Approximately 10% of all first-step mutants were resistant
to ethidium bromide, and selected strains had mutations that mapped to
flqB. NorA-mediated resistance among first-step mutants may
be more prevalent than previously reported.
| |
TEXT |
Resistance to fluoroquinolones
occurs in a stepwise fashion (24). The mutants initially
obtained are resistant to low concentrations of quinolones (first-step
mutants), and subsequent mutations lead to higher-level resistance. The
most common mutation conferring low-level resistance occurs in the
flqA locus (3, 4, 25). DNA topoisomerase IV,
encoded at the flqA locus, is the primary target of
fluoroquinolones (3, 4, 16, 26) for Staphylococcus aureus but not necessarily for other bacterial species. Some
flqA mutants are also hypersusceptible to novobiocin
(25) (Novhs), and mutations of selected strains
map to grlB (6). Second-step mutations have been
identified in gyrA. Mutations in gyrA, plus a
mutation in topoisomerase IV (grlA/flqA) (3, 16,
26), confer high-level resistance to quinolone antibiotics.
A mutation only in gyrA has no effect on susceptibility to
quinolones (16, 26); thus, such mutations have not been
found among first-step mutants (3, 4, 7, 8, 9, 19, 25).
Low-level quinolone resistance due to increased expression of the
NorA multidrug efflux pump are due to mutations in the flqB
locus (17) and possibly other loci (10).
However, it is unclear when, or at what stage in resistance, altered
NorA expression occurs. Here we present data implicating a role for
NorA in first-step quinolone resistance.
There are limited data regarding the effect of the passage of strains
in vivo on the ability to detect first-step mutants from common
staphylococcal infections such as those of the skin. In vivo passage
without quinolone selection might result in positive or negative
selection for different classes of fluoroquinolone-resistant mutants. In the present study, we found that passage in vivo did not alter the distribution of different classes of first-step mutants for both methicillin-resistant S. aureus (MRSA) and
methicillin-susceptible S. aureus (MSSA) clinical strains.
Methods and reagents.
Tetracycline and erythromycin were used
at 10 and 5 µg/ml, respectively. Resistance to fusidic acid
(Fusr), ciprofloxacin (Cfxr), or ethidium
bromide (Ebrr) was determined when growth was evident
on brain heart infusion agar (BHA) containing fusidic acid (10 µg/ml), ciprofloxacin (1 µg/ml), or ethidium bromide (20 µg/ml).
Broth microdilution MIC determinations were done with
cation-adjusted Mueller-Hinton broth (1).
BHA-novobiocin gradient plate assays were performed
according to the method of Szybalski (21).
Strains were considered Novhs when the length of
confluent growth across a gradient plate containing novobiocin (0 to
0.25 µg/ml) was <66% of that of the parental strain. Identification
to the species level was performed and clonality of mutant
colonies was confirmed with an Acustaph kit (Carr-Scarborough
Microbiologicals, Inc., Decatur, Ga.) and by pulsed-field gel
electrophoresis of SmaI-digested genomic DNA (22), respectively.
Three-day murine abscess formation was performed by the method of
Bunce et al. (
2) using an initial inoculum of
10
7 CFU of
S. aureus.
High-molecular-weight DNA was obtained and transformations
were done as described previously (
20), with genetic
mapping
strains (
18).
Results.
Using a modification of published methods (3, 8,
23, 25), we isolated quinolone-resistant mutants from broth
cultures (in vitro mutants) and from murine abscesses (in vivo-passaged mutants). Consistent with previous reports (5, 12, 14), more quinolone-resistant mutants were recovered by using
ciprofloxacin than by using either ofloxacin or levofloxacin. For
in vitro mutants, 108 CFU of 38 independent brain
heart infusion broth cultures of the laboratory strain RN4220
(13) were inoculated onto BHA containing 1 µg of
ciprofloxacin per ml (two times the MIC). A mean of 11 colonies/108 CFU were obtained after 24 h of
incubation at 37°C. Selection with ofloxacin at the same
concentration (two times the MIC) yielded fivefold fewer mutants
than those obtained by ciprofloxacin selection. Data similar to those
above were obtained for MSSA (NB1720 and NB1722) and MRSA (NB1725 and
NB1726) clinical strains. Except for a single mutant obtained from
strain NB1726, no mutants were obtained with levofloxacin selection at
two times the MIC (0.75 µg/ml).
Low-level-quinolone-resistant mutants were obtained from murine
abscesses (in vivo-passaged mutants) infected with
S. aureus RN4220 or MSSA or MRSA clinical strains at frequencies
similar
to those obtained for in vitro mutants (Table
1). It is possible
that first-step
mutants existed at the time of infection and persisted
within the
abscess because of the inoculum used to produce the
abscess. It is also
possible that some first-step mutants emerged
during infection and were
not differentially eliminated during
growth.
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TABLE 1.
Isolation of quinolone-resistant mutants derived from
quinolone-susceptible strains present in murine abscesses
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For all first-step mutants obtained, the mean MIC of
ciprofloxacin increased approximately fourfold above the mean MIC
for
parental strains. For ofloxacin and levofloxacin, the
increases
were two- to threefold. Thus, compared to ciprofloxacin, the
relative
potency of levofloxacin and ofloxacin increased with
single-step
resistance
a result consistent with those of Thomson et
al. (
23).
Next, the phenotypes of first-step mutants were characterized.
Ebr
r is an indicator of increased
norA
expression (
10), and Nov
hs is an indicator of
mutant alleles of
flqA (topoisomerase IV).
Both phenotypes
were found among the in vitro- and the in vivo-passaged
mutants. In 38 independent experiments, RN4220 was inoculated
onto solid media
containing 1 µg of ciprofloxacin per ml, and
one resistant
mutant per experiment was selected. Of the 38 chosen,
10 were
Nov
hs and 1 was Ebr
r. A different sampling
procedure yielded a higher percentage of
Ebr
r mutants.
Instead of sampling one mutant from many in an individual
experiment,
all mutants obtained on a plate in each of six experiments
were
examined. When all first-step mutants were sampled in each
experiment,
a mean of 13% of mutants were Ebr
r. For in vivo-passaged
mutants, of 54 obtained from 18 mouse abscesses
infected with RN4220,
30 were Nov
hs while 8 (derived from 5 abscesses) were
Ebr
r. Of 27 MSSA isolates (NB1721) obtained from 8 abscesses, 8 were
Nov
hs while none were
Ebr
r, and for 27 MRSA isolates (NB1725) derived from
10 abscesses,
6 were Nov
hs and 3 were Ebr
r.
Interestingly, although first-step mutants were heterogeneous
based on
susceptibilities to Ebr, MICs of the three quinolones
tested were
equivalent for all Ebr
s and Ebr
r mutants. The
data suggest that for first-step mutants, resistance
may be mediated by
mutations affecting either topoisomerase IV
or
NorA.
Quinolone resistance caused by increased expression of
norA (
16) is detected by cross-resistance
to other NorA substrates,
such as ethidium bromide and cetrimide
(
10). Resistance to these
substrates is reduced by the NorA
inhibitor, reserpine (
11,
15). Among the quinolone-resistant
strains in Table
2, only
the
Ebr
r strains showed fourfold increases in resistance to
cetrimide.
In our hands, reserpine did not differentiate
quinolone-resistant
strains that were Ebr
r from those that
were Ebr
s, because it reduced resistance levels in all
strains tested.
For EbrR first-step mutants, the data support the
argument for
mediation of quinolone resistance by NorA.
Mutations in the
flqB locus, and possibly in other loci
(
10), result in the increased expression of
norA
(
25), which confers
ciprofloxacin, Ebr, and cetrimide
resistance (
11,
15). Selected
Cfx
r
Ebr
r VA476 and VA331 strains were found to have mutations
that mapped
by genetic transformation to the
flqB locus
(Table
3). The data
suggest a gene order
as follows:
fus, 1108 locus, Cfx
r
Ebr
r locus. The cotransformation frequencies and gene
order are similar
to those reported for 1108 and
flqB (4%)
by Ng et al. (
17).
Thus, the Cfx
r
Ebr
r locus maps to a location similar to that for
flqB. In addition,
the absence of discordance between
Cfx
r and Ebr
r phenotypes in genetic
crosses suggests that the same locus is
responsible for both
phenotypes. Mutations in the
grlA/flqA locus
have been shown
to confer resistance to ciprofloxacin (
25).
Here, mutations
in our Cfx
r Ebr
s strains, VA3 and VA352, mapped
to the
flqA locus (Table
3).
The ~50 and 11%
cotransformation frequencies of (Tn
917lac)
2 and
(Tn
917lac)
1 with the Cfx
r locus
are consistent with the 43 and 10% cotransduction frequencies
with
flqA, respectively, identified by Trucksis et al.
(
25).
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TABLE 3.
Linkage of Cfxr Ebrr
S. aureus with
(chr::Tn916)1108 and
Cfxr S. aureus with
(chr::Tn917lac)2
and (chr::Tn917lac)1
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|
We conclude that first-step quinolone-resistant MRSA and MSSA
strains
whether obtained in vitro or via in vivo passage
harbor
either mutations in topoisomerase IV or mutations that alter NorA
multidrug efflux pump
activity.
| |
ACKNOWLEDGMENTS |
We thank David Hooper and Terri Kenney for their helpful comments.
This work was supported in part by a grant from the Robert Wood Johnson
Pharmaceutical Research Institute.
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FOOTNOTES |
*
Corresponding author. Present address: Internal
Medicine Associates of Yakima, 316 Holton Ave., Yakima, WA 98902. Phone: (509) 575-7666. Fax: (509) 576-4370. E-mail:
nbarg{at}umich.edu.
Present address: Genome Therapeutics Corporation, Waltham, MA 02453.
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Antimicrobial Agents and Chemotherapy, December 1998, p. 3317-3319, Vol. 42, No. 12
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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