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Antimicrobial Agents and Chemotherapy, August 1998, p. 2032-2035, Vol. 42, No. 8
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Prevalence of a Putative Efflux Mechanism among
Fluoroquinolone-Resistant Clinical Isolates of
Streptococcus pneumoniae
Nigel P.
Brenwald,1
Martin J.
Gill,2 and
Richard
Wise1,*
Department of Microbiology, City Hospital NHS
Trust, Birmingham B18 7QH,1 and
Department of Infection, University of Birmingham Medical
School, Birmingham B15 2TT,2 United Kingdom
Received 2 February 1998/Returned for modification 16 March
1998/Accepted 28 May 1998
 |
ABSTRACT |
Twenty-three norfloxacin-selected first-step mutants of
Streptococcus pneumoniae showed low-level fluoroquinolone
resistance. Their susceptibility to norfloxacin in the presence or
absence of reserpine and known efflux pump substrates was determined by an agar dilution method. Five mutants showed four- to eightfold increases in their susceptibility to norfloxacin in the presence of
reserpine and four- to eightfold decreases in their susceptibility to
acriflavine and ethidium bromide. This phenotype is suggestive of an
efflux mechanism of resistance. A representative of these mutants,
1N27, accumulated significantly less ethidium bromide than the parent
strain; reserpine abolished these differences. No changes in the
quinolone resistance-determining regions of parC,
parE, gyrA, or gyrB were found in
this mutant. By our validated agar dilution method, the efflux
phenotype was sought in clinical isolates of S. pneumoniae.
Of 1,037 clinical isolates examined from the United Kingdom, 273 showed
reduced susceptibility to norfloxacin or ciprofloxacin. Of these,
45.4% showed the efflux phenotype. Our findings suggest that an efflux
mechanism may be a frequent cause of clinically significant
fluoroquinolone resistance in pneumococci.
| |
INTRODUCTION |
Streptococcus pneumoniae
is an important pathogen and a major etiologic agent of
community-acquired pneumonia. Effective treatment of pneumococcal
infections has until recently relied upon the use of beta-lactam
antibiotics, but with the emergence of antibiotic resistance, their use
is now increasingly compromised (2, 4, 9). There is
considerable interest in the use of alternative antimicrobials, such as
the fluoroquinolones. Although presently available fluoroquinolones,
such as ciprofloxacin, are limited in their effectiveness, newer
developmental compounds show greater promise (6, 8).
A problem associated with the use of fluoroquinolones is the selection
of spontaneous resistant mutants. Several studies with pneumococci have
shown that low-level resistance can result from mutations in
topoisomerase IV (10, 13, 16, 17). Increased levels of
resistance occur following the acquisition of additional mutations in
gyrA, which encodes the A subunit of DNA gyrase (10, 13, 16).
Recently, studies in our laboratories and elsewhere have described an
efflux mechanism as a further cause of low-level resistance in
pneumococci (7, 20). However, these descriptions are
restricted to laboratory-generated mutants, and no data are available
on the occurrence of this form of fluoroquinolone resistance in
clinical isolates. Therefore, we undertook a study to determine the
prevalence of the efflux mechanism of resistance in clinical strains of
S. pneumoniae. In order to examine a large number of
clinical strains a phenotypic method was used. This was based upon the
effect of reserpine, a known efflux pump inhibitor (12, 15),
on the activity of norfloxacin, and on the susceptibility of the
strains to unrelated efflux pump substrates. The effectiveness of this method for the detection of the efflux mechanism was validated with an
in vitro mutant which had been examined for antimicrobial susceptibility, altered levels of ethidium bromide accumulation, and
changes in topoisomerase IV and DNA gyrase.
| |
MATERIALS AND METHODS |
Antibiotics and chemicals.
Norfloxacin was supplied by Merck
& Co Inc., Rahway, N.J.; ciprofloxacin and moxifloxacin (BAY 12-8039)
were supplied by Bayer AG, Wuppertal, Germany; and sparfloxacin was
supplied by Rhône Poulonc Rorer, Vitry-sur-Seine, France. All
chemicals were from Sigma-Aldrich Company Ltd., Poole, United Kingdom.
Selection and susceptibility of resistant mutants.
First-step mutants of S. pneumoniae ATCC 49619 were selected
by inoculating 108 CFU onto Columbia agar (Oxoid,
Basingstoke, United Kingdom) supplemented with 5% horse blood and
containing norfloxacin at a concentration of 4× the MIC. Following
incubation at 35 to 37°C in an atmosphere of 4 to 6% CO2
for 3 days, mutants were subcultured onto antimicrobial agent-free
medium. Their susceptibilities to norfloxacin (in the presence or
absence of 10 µg of reserpine/ml), ciprofloxacin, sparfloxacin,
moxifloxacin (BAY 12-8039), ethidium bromide, and acriflavine were
determined by an agar dilution method with Iso-Sensitest agar (Oxoid)
supplemented with 5% horse blood with an inoculum of 104
CFU/spot (18). Reserpine was freshly prepared before use,
and media containing it were used immediately. After incubation at 35 to 37°C in 4 to 6% CO2 for 18 to 20 h, the MIC was
recorded as the lowest antibiotic concentration inhibiting growth. The norfloxacin susceptibility of several strains, in the presence or
absence of reserpine, was also determined by a broth MIC method (18).
Accumulation of ethidium bromide.
Measurement of the level
of ethidium bromide accumulation and efflux in S. pneumoniae
ATCC 49619 (parent strain) and strain 1N27 (norfloxacin-selected
mutant) was based on a previously described method (5, 14,
15). Briefly, for measurement of the level of accumulation,
bacterial suspensions with an optical density at 550 nm of 0.2 were
prepared in uptake buffer (NaCl, 110 mM; KCl, 7 mM; NH4Cl,
50 mM; Na2HPO4, 0.4 mM; Tris base, 52 mM;
glucose, 0.2% adjusted to pH 7.5 with HCl) and were then exposed to
ethidium bromide at a concentration of 2 µg/ml. The increase in
fluorescence as ethidium bromide entered the cells was recorded
fluorometrically with a Perkin-Elmer model LS50 spectrofluorimeter
(excitation 
, 530 nm; emission , 600 nm) at 30°C. The effect of
reserpine on the level of accumulation was determined in a similar way, except that reserpine was added to the uptake buffer at a concentration of 10 µg/ml.
For determining ethidium bromide loss, bacterial suspensions were first
exposed to ethidium bromide (2 µg/ml) in the presence of reserpine
(10 µg/ml) for 20 min at 37°C. The cells were then pelleted by
centrifugation and were resuspended in fresh uptake buffer. The loss of
ethidium bromide from the cells was measured as a decrease in
fluorescence.
DNA amplification and sequencing.
Chromosomal DNA was
extracted from the parent strain and mutant 1N27 by a standard
procedure (3). Using primers and reaction conditions
described previously (16), the quinolone
resistance-determining regions (QRDRs) of parC,
parE, gyrA, and gyrB were amplified by PCR with chromosomal DNA used as the template. PCR products were sequenced by automated fluorescence sequencing.
Detection of efflux phenotype in clinical isolates of S. pneumoniae.
A total of 1,037 clinically significant nonreplicated
strains of S. pneumoniae, which had been isolated during
1996 and 1997 from patients in the United Kingdom, were studied. Their
susceptibilities to norfloxacin and ciprofloxacin were determined by an
agar dilution method. Strains for which norfloxacin MICs were >8
µg/ml and ciprofloxacin MICs were >1 µg/ml were further examined
for the effect of reserpine (10 µg/ml) on the activity of norfloxacin
and their susceptibilities to ethidium bromide and acriflavine. The
activities of several other fluoroquinolones were also determined.
| |
RESULTS |
Antimicrobial susceptibilities of fluoroquinolone-resistant
mutants.
A total of 23 resistant mutants were selected with
norfloxacin. For these mutants, four- to eightfold increases in the
MICs of norfloxacin and ciprofloxacin compared with those for the
parent strain were found (Table 1).
However, the activity of norfloxacin against five of the mutants was
increased four- to eightfold in the presence of reserpine. For these
same five mutants the MICs of ethidium bromide and acriflavine
increased four- to eightfold compared with the MICs for the parent
strain. This reduction in susceptibility to ethidium bromide was also
reversed by reserpine (data not shown). The activities of sparfloxacin
and moxifloxacin against these mutants were unchanged. Reserpine alone
did not show any inhibitory activity.
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TABLE 1.
Susceptibilities of S. pneumoniae ATCC 49619 (parent strain) and norfloxacin-selected mutants to fluoroquinolones
and efflux pump substrates
|
|
Sequencing of QRDRs of parC, parE,
gyrA, and gyrB.
No sequence differences were
found between the parent strain and the presumptive efflux mutant,
1N27, at any previously described position within the QRDRs of
parC, parE, gyrA, and gyrB.
Accumulation and efflux of ethidium bromide.
Figure
1 compares the levels of accumulation of
ethidium bromide in the parent strain and mutant 1N27. The rate of
accumulation in the mutant was significantly slower, and after 20 min
the level of accumulation in the mutant was 69.5% less than that in
the parent strain. In the presence of reserpine there was no
significant difference between the levels of accumulation in the parent
strain and the mutant, although the overall level of ethidium bromide accumulation increased in both strains. The rate of ethidium bromide loss from 1N27 was significantly increased compared with that from the
parent strain (Fig. 2).
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FIG. 1.
Accumulation of ethidium bromide by the parent strain
( ) and a mutant strain, 1N27 ( ), of S. pneumoniae in
the absence of reserpine (A) and in the presence of reserpine (B). Each
point is the mean of at least three experiments. Bars represent
standard deviations.
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FIG. 2.
Efflux of ethidium bromide from the parent strain ()
and a mutant strain, 1N27 (), of S. pneumoniae. Each
point is the mean of at least three experiments. Bars represent
standard deviations.
|
|
Antimicrobial susceptibilities of the clinical isolates.
Of
the 1,037 clinical isolates, 273 strains showed reduced susceptibility
to norfloxacin or ciprofloxacin (MIC of norfloxacin, >8 µg/ml; MIC
of ciprofloxacin, >1 µg/ml). These strains could be divided into two
groups on the basis of the effect of reserpine on their susceptibility
to norfloxacin. Group A contained strains for which the MIC of
norfloxacin was reduced only twofold or less by reserpine (Table
2). Group B contained the 45.4% of
strains for which the MIC of norfloxacin was reduced fourfold or
greater by reserpine. Ninety-eight of the group B strains had
susceptibility patterns identical to those of the norfloxacin-selected
efflux mutants, and for the 98 group B strains the MICs of ethidium
bromide and acriflavine were 
16 µg/ml. However, the remaining 25 strains showed variations in their susceptibilities to ethidium bromide and acriflavine. For 8 strains the MICs of ethidium bromide and acriflavine were 
8 µg/ml, for 11 strains the MICs of acriflavine were 16 µg/ml but the MICs of ethidium bromide were 8 µg/ml, and for 6 strains the MICs of ethidium bromide were 16 µg/ml and
the MICs of acriflavine were 8 µg/ml.
| |
DISCUSSION |
Several efflux proteins are well described in gram-positive
bacteria, including Bmr in Bacillus subtilis (1)
and NorA in Staphylococcus aureus (15). These can
mediate low-level resistance to hydrophilic fluoroquinolones and a
variety of unrelated compounds; both are inhibited by reserpine
(14, 15). More recently, reserpine was shown to inhibit the
level of accumulation of ethidium bromide in an ethidium
bromide-selected mutant of S. pneumoniae which showed
cross-resistance to fluoroquinolones (5). For this reason we
used reserpine to detect mutants resistant to fluoroquinolones by an
efflux mechanism.
We showed that reserpine significantly increased the activity of
norfloxacin against several norfloxacin-selected mutants. These
presumptive efflux mutants also showed increased levels of resistance
to ethidium bromide and acriflavine, which agrees with the description
by Zeller et al. (20) of the presence of an efflux pump in
pneumococci. Ethidium bromide accumulation in strain 1N27, a
presumptive efflux mutant, was significantly reduced compared with that
in the parent strain; these differences were abolished by reserpine.
Increased loss of ethidium bromide was shown from strain 1N27,
suggesting the presence of an efflux mechanism of resistance. The
reduced activity of norfloxacin and ciprofloxacin against strain 1N27
occurred in the absence of changes in the QRDR of topoisomerase IV or
DNA gyrase. Interestingly, the level of accumulation in the parent
strain in the presence of reserpine was significantly higher than that
in uptake medium alone. This increase in the level of accumulation may
be the result of reserpine inhibiting low levels of a constitutively
expressed efflux protein in the parent strain.
We used an agar dilution method to detect the effect of reserpine on
the activity of norfloxacin. Studies with Bmr have suggested that
reserpine may be inactivated by agar (14). We found no difference in the effect of reserpine on the MIC of norfloxacin when
MICs were determined by a broth or an agar dilution method. However, we
did find that reserpine is labile and should be used immediately once
it is in solution.
Having demonstrated that our agar dilution method was able to detect
laboratory mutants with an efflux phenotype, we determined the
prevalence of an efflux mechanism of fluoroquinolone resistance among
clinical isolates of S. pneumoniae. Reserpine significantly increased the activity of norfloxacin against 45.4% of the clinical strains with reduced susceptibility to norfloxacin or ciprofloxacin. Although the majority of these strains had susceptibility patterns identical to those of the efflux mutants, 20% of the strains showed variations in their susceptibilities to ethidium bromide and
acriflavine. It is possible that these strains may be resistant by
further efflux mechanisms which have different substrate profiles or
that mutations in the efflux pump have changed the spectrum of
cross-resistance. Both the mutants and the clinical strains with the
efflux phenotype remained susceptible to the more hydrophobic compounds
sparfloxacin and moxifloxacin. This suggests that hydrophobic
fluoroquinolones are poor substrates for the pneumococcal efflux pump,
as they are for NorA (11, 19).
Most studies investigating the mechanism of fluoroquinolone resistance
in clinical strains of S. pneumoniae have shown that alterations in the target sites are responsible for resistance. We have
presented data that suggest that an efflux mechanism may be a common
cause of clinically significant fluoroquinolone resistance. Our study
is the first to screen large numbers of clinical isolates for an efflux
mechanism of antibiotic resistance. Further work is required to
establish the genetic basis of efflux pump resistance in pneumococci.
| |
ACKNOWLEDGMENT |
This work was supported by a grant from Bayer AG.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, City Hospital NHS Trust, Dudley Road, Birmingham B18 7QH, United Kingdom. Phone: 44 121 507 4255. Fax: 44 121 551 7763. E-mail:
r.wise{at}bham.ac.uk.
| |
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Antimicrobial Agents and Chemotherapy, August 1998, p. 2032-2035, Vol. 42, No. 8
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
