JAC

Journal of Antimicrobial Chemotherapy (2001) 47, 261270

Original articles
Effect of hydrophobicity and molecular mass on the accumulation
of fluoroquinolones by Staphylococcus aureus
Laura J. V. Piddock*, Yu Fang Jin and Deborah J. Griggs
Antimicrobial Agents Research Group, Division of Immunity and Infection, The Medical School,
University of Birmingham, Birmingham B15 2TT, UK
Ten novel fluoroquinolones, with similar chemical structures but differing antibacterial
activities and hydrophobicities, were studied to evaluate the role of the physical properties of
quinolones on their accumulation and antibacterial activity for Staphylococcus aureus. Six of
the 10 agents and tosufloxacin were more active against quinolone-susceptible and -resistant
S. aureus than the remaining four agents and several piperazinyl fluoroquinolones. Changes to
the side chains of the pyrollidinyl substituent at the R7 position alone made little difference
to the MICs. Comparison of MICs of agents that were structurally identical apart from the R1
substituents, confirmed that a t-butyl group confers enhanced activity against S. aureus over a
cyclopropyl or ethyl group at this position. The steady-state concentrations (SSCs) of the 10
novel quinolones accumulated by wild-type S. aureus did not correlate with their MICs or
chemical structures. There was no apparent relationship between logP of the quinolone and
accumulation by S. aureus F77; however, accumulation was positively correlated with molecular mass for 9/10 agents (r = 0.745) confirming that high molecular mass is not a barrier to
accumulation in S. aureus. For all 10 agents, the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) increased the concentration of quinolone accumulated by SA-1199,
suggesting that NorA was inhibited. The fold increase of the SSC in the presence of CCCP did
not correlate with hydrophobicity, but the SSC of agents with either an ethyl or cyclopropyl
group at R1 was increased two- to three-fold in the presence of CCCP, suggesting that affinity
for the NorA efflux pump may be influenced by quinolone structure.

Introduction
For an antibacterial agent to be effective, it must penetrate
the bacterial cell to reach its target. The primary target of
fluoroquinolones is DNA gyrase, an enzyme essential for
DNA supercoiling, which is encoded by the gyrA and gyrB
genes. Topoisomerase IV (encoded by the grlA/parC and
grlB/parE genes) is also involved in quinolone action, and
may be the dominant target in Staphylococcus aureus.1 The
susceptibility of an organism to a particular quinolone is
determined by both the ability of the agent to penetrate the
cell envelope and the inhibitory activity of the agent against
its target.2
The factors affecting the accumulation of quinolones by
Escherichia coli and other Gram-negative bacteria have
been studied, but less is known about accumulation by

S. aureus. The physical properties of quinolones (e.g. relative hydrophobicity, charge or molecular mass) are important for penetration into the Gram-negative bacterial cell.
Increasing molecular mass, hydrophobicity and negative
charge hinder penetration of antibiotics into Gramnegative organisms through the porin channels, although
hydrophobic molecules appear to enter via the lipopolysaccharide or across the lipid bilayer.3,4 Gram-positive bacteria do not possess an outer membrane, and so lack outer
membrane proteins and lipopolysaccharide. Accumulation
by S. aureus is thought to take place by simple diffusion
across the cytoplasmic membrane5,6 but an active efflux
mechanism has been identified in staphylococci which
reduces accumulation. The norA locus codes for the protein NorA, which appears to straddle the cell membrane
and act as an active efflux pump of hydrophilic quinolones.7

*Corresponding author. Tel: 44-121-414-6966; Fax: 44-121-414-6966; E-mail: l.j.v.piddock@bham.ac.uk

261
2001 The British Society for Antimicrobial Chemotherapy

L. J. V. Piddock et al.
NorA-mediated increased efflux has been described for
quinolone-resistant S. aureus.8 Mutations in regulatory
genes controlling the expression of NorA have been shown
to result in overexpression of the gene,9,10 and a regulatory
locus, arlR-arlS, has recently been described that is involved in the control of norA expression.11
Asuquo & Piddock12 compared the kinetics of quinolone
accumulation in S. aureus with that into E. coli and Pseudomonas aeruginosa. They concluded that accumulation was
more rapid, and that quinolones were accumulated to a
higher concentration by S. aureus than by Gram-negative
organisms. No correlation was found between the antibacterial activity of each agent and the amount of quinolone accumulated in S. aureus, which confirmed earlier
findings.13,14 Takenouchi et al.15 investigated the relationship between the structure and hydrophobicity of quinolones and their activity against a NorA-overproducing
mutant of S. aureus and its parent strain. They found that
decreased activity of the quinolones correlated with the
bulkiness of the C7 substituent and the bulkiness and
hydrophobicity of the C8 substituent, but not with hydrophobicity. It has been proposed that for Gram-positive
organisms, high molecular weight is not a barrier to penetration.2,12
The proportion of charged and uncharged microspecies
of quinolones has been shown to affect accumulation in
the bacterial cell.16 Quinolone species with no net charge
(uncharged HQ0 and zwitterion HQ forms) penetrate the
cytoplasmic membrane of S. aureus more readily than
charged species (HQ+ and HQ).17,18 Alovero et al.19 found
that accumulation of sulphanilyl fluoroquinolones into
S. aureus was decreased three-fold in the presence of magnesium ions and increased at lower pH. They suggested that
compounds like the sulphanilyl fluoroquinolones, which
have only one ionizable group, are accumulated in the cell
more favourably than are zwitterionic quinolones,18 and
that this may be important for their enhanced in vitro activity against S. aureus, since the inhibition of DNA gyrase is
similar for both groups of agents.
The aim of this study was to determine whether the
physical properties of quinolones affect their accumulation
and play a role in antibacterial activity against S. aureus, by
comparing the accumulation of 10 structurally similar
fluoroquinolone molecules, with differing antibacterial
activities and hydrophobicities. The effect of the NorA
efflux pump inhibitor, carbonyl cyanide m-chlorophenylhydrazone (CCCP) on accumulation of the 10 agents was
also determined.

atory mutant derived from F77, which was ciprofloxacin

resistant but susceptible to nalidixic acid. SA-1199 (wild
type), SA-1199B (grlA mutation and constitutive overproducer of NorA) and SA-1199-3 (inducible overproducer
of NorA) were kindly donated by G. Kaatz, Washington
State University.20 Ten structurally related fluoroquinolones were provided by Parke DavisWarner Lambert
(Ann Arbor, MI, USA). All other antibacterial agents
were provided as gifts from the respective manufacturers.
Bacteriological media were supplied by Unipath (Basingstoke, UK) and chemicals by BDH or Sigma (Poole, UK).

Materials and methods

Results

Bacterial strains and media

Comparison of quinolone structures

S. aureus F77 (NCTC type strain 8532) and E. coli I113

(NCTC 10418) were obtained from the National Collection
of Type Cultures, Colindale, UK. S. aureus F145 was a labor-

The chemical structures of the 10 PD quinolones were compared with those of common fluoroquinolones and nalidixic acid (Table I) for similarities in the R1, R7 and R8

Antibiotic susceptibility testing

The MICs of the 10 related fluoroquinolones (PD compounds) were determined by the microbroth doubling
dilution method; MICs of all other agents were determined by the agar doubling dilution technique as described
previously.12,21

Hydrophobicity and fluorescence of PD quinolones

The partition coefficient between 1-octanol and 0.1 M
sodium phosphate buffer pH 7 was determined for each
quinolone as described by Asuquo & Piddock.12 The
excitation (ex) and emission (em) maxima of the 10 PD
compounds were determined in 0.1 M glycine pH 3 and in
50 mM phosphate buffer at pH 6, 7 and 8 (with and without
boiling for 10 min) using a scanning spectrophotometer,
and compared with other quinolones. The amount of fluorescence emitted by 0.1 mg/L quinolone in 0.1 M glycine
pH 3 and in 50 mM phosphate buffer at pH 6, 7 and 8 was
measured in a LS30 luminescence spectrophotometer
(Perkin Elmer, Beaconsfield, UK).

Quinolone accumulation
The accumulation of each quinolone was determined
between three and six times, using the modified fluorescence method.12 To investigate the role of the NorA efflux
pump on accumulation of the PD agents by SA-1199,
CCCP was added to a parallel set of tubes to a final concentration of 100 M. In addition, the accumulation assays
were repeated and, instead of using 0.1 M glycine pH 3 to
lyse the cells, the cell pellet was resuspended in 50 mM
phosphate buffer pH 7 and boiled for 10 min. The concentration of quinolone accumulated was determined by spectrofluorimetry using the appropriate excitation and emission
maxima for the agent.

262

Nucleus

NA

Agent

PD112956

PD114456

Norfloxacin

263

Nalidixic acid

PD124979

PD130390

Ciprofloxacin

C2H5

C2H5

C2H5

C2H5

R1

CH3

R7

Substituent

R8

331.4

485.39

408.78

232

319

356.4

319.33

Molecular
mass

0.344

0.611

1.301

0.031b 1.509

0.453

0.245

20.02b

0.5

0.25

0.12

128

0.022b 1.657

0.514

logP

S. aureus F77

E. coli I113

96 (ND)

97 (127)

172 (125)

54 (ND)

72 (ND)

134 (112)

156 (89)

79 (90)

ND

0.03

74 (79)

ND

ND

38 (86)

84 (79)

0.06

0.06

0.03

0.5

0.12

MIC accumulationa MIC accumulationa

(mg/L) (ng/mg)
(mg/L) (ng/mg)

0.924

0.119

0.306

Papp

Hydrophobicity

Table I. Chemical structures, physical properties, antibacterial activity and accumulation by S. aureus and E. coli for 10 PD agents and common quinolones

Quinolone accumulation by S. aureus

264
H

Temafloxacin A

C(CH3)3

PD135671

C(CH3)3

R8

PD164534

PD135498

R7

Sparfloxacin

R1

PD138086

Nucleus

Agent

Substituent

419

480.95

485.08

415.08

415.85

Molecular
mass

Table I. (Continued)

0.807

0.433

0.221

0.022

logP

0.746b 0.127

0.156

0.369

0.601

1.052

ND

Papp

Hydrophobicity

E. coli I113

0.06

0.12

0.12

0.12

0.12

84 (ND)

46 (121)

165 (121)

91 (57)

130 (116)

ND

0.25

0.03

0.03

0.06

0.06

0.06

ND

84 (44)

35 (42)

63 (50)

48 (54)

ND

MIC accumulationa MIC accumulationa

(mg/L) (ng/mg)
(mg/L) (ng/mg)

S. aureus F77

L. J. V. Piddock et al.

PD164413

PD164532

Ofloxacin

oxazine ring linkage

of N1 and C8

CH(CH3)2

see R1

360

391.9

386.94

403

0.830

0.518

0.228

0.088b 1.055

0.148

0.303

1.69b

0.5

0.06

67 (ND)

118 (124)

326 (102)

152 (ND)

0.12

0.5

0.5

0.06

ND

81 (80)

111 (76)

ND

Nucleus B is a stereoisomer of nucleus A. Papp, partition coefficient in 1-octanol0.1 M sodium phosphate buffer pH 7; NA, not applicable (the nucleus of nalidixic acid resembles A but is
non-fluorinated at R6 position); ND, not done.
a
Accumulation was measured using two methods of cell lysis: using 0.1 M glycine pH 3 and, in brackets, by boiling in 50 mM phosphate buffer pH 7 for 10 min.
b
Data for fluoroquinolones other than the PD agents from Asuquo & Piddock.12

Tosufloxacin

Quinolone accumulation by S. aureus

265

L. J. V. Piddock et al.
Table II. Susceptibility to quinolones and accumulation of PD agents by mutant S. aureus
SA-1199 steady-state
concentration (ng/mg)

MIC (mg/L)
Agent
PD112956
PD114456
PD124979
PD130390
PD135498
PD135671
PD138086
PD164413
PD164532
PD164534
Ciprofloxacin
Nalidixic acid
Norfloxacin
Sparfloxacin
Temafloxacin
Tosufloxacin

F77

F145

SA-1199

1
1
0.12
0.25
0.12
0.12
0.12
2
4
0.06
0.5
128
1
0.12
1
0.06

4
4
1
1
0.12
0.25
0.25
4
8
0.12
4
16
16
0.12
2
0.5

0.25
0.5
0.12
0.12
0.03
0.06
0.03
0.25
1
0.03
0.3
512
0.5
0.1
0.25
0.12

SA-1199B SA-1199-3
16
16
2
1
1
1
0.5
16
32
0.5
16
512
16
0.2
4
2

0.25
0.5
0.12
0.12
0.06
0.06
0.03
1
0.5
0.06
1.6
512
4
0.1
0.25
0.12

CCCP

CCCP

fold increase

14
54
40
62
52
42
30
39
87
25

43
147
71
105
66
58
51
63
107
34

3.1
2.7
1.8
1.7
1.3
1.4
1.6
1.6
1.2
1.4

CCCP and CCCP, concentration of quinolone accumulated in the absence and presence of 100 M CCCP, respectively. Bacterial cells were
lysed in 0.1 M glycine pH 3 following accumulation assay.

substituents. All 10 PD compounds had only hydrogen at

the R8 position (like ciprofloxacin, norfloxacin, temafloxacin and tosufloxacin), and all had a pyrrolidinyl group
at position R7 similar to that of tosufloxacin. At the R1
position, PD112956 and PD114456 had an ethyl substituent
like norfloxacin, and PD124979 and PD130390 had a
cyclopropyl ring like ciprofloxacin and sparfloxacin. Both
PD138086 and PD164534 had a 2,4-difluorophenyl substituent at the R1 position like temafloxacin and tosufloxacin. PD135498 and PD135671 had a t-butyl substituent
at R1, PD164413 a cyclobutyl and PD164532 an isopropyl
group (Table I).

Antibacterial activity
The antibacterial activity of the 10 PD agents against the
five strains of S. aureus was compared. Six PD agents
(PD124979, PD130390, PD135498, PD135671, PD138086
and PD164534) and tosufloxacin were more active against
each S. aureus strain than the other four PD agents
(PD112956, PD114456, PD164413 and PD164532), nalidixic
acid, ciprofloxacin, norfloxacin and temafloxacin (Table
II). The MICs of pairs of agents that had identical groups at
the R1 position but that differed only in the side chains on
the pyrrolidinyl ring at position R7, were very similar:
PD112956 and PD114456 (ethyl group at R1); PD124979
and PD130390 (cyclopropyl ring at R1); PD135498 and
135671 (t-butyl group at R1), PD138086 and PD164534

(2,4-difluorophenyl group at R1) (Tables I and II). The

structures of PD112956, PD135498 and PD164532 differed
only in the R1 substituent and showed varying activities
against S. aureus. PD135498 was the most active of these
three agents with a t-butyl group at R1 (MICs 0.031 mg/L),
PD112956 was less active (ethyl group; MICs 0.2516
mg/L), while PD164532 was the least active with an isopropyl group at R1 (MICs 0.532 mg/L).
F145 was two- to eight-fold less susceptible than its
parent F77, to all the PD quinolones except PD135498. The
MICs of the PD agents were similar for SA-1199-3
(inducible NorA overproducer) and its parent SA-1199;
however, SA-1199B (constitutive NorA overproducer) was
eight- to 64-fold less susceptible than SA-1199.

Physical properties of quinolones

The partition coefficient (Papp) determined for each PD
agent (Table I) ranged from 0.119 (logP 0.924; PD114456)
to 1.052 (logP 0.022; PD13548). The fluorescence excitation
and emission maxima at pH 3, 6, 7 and 8 were determined
for each PD agent and the comparative fluorescence at
each pH was measured in arbitrary units. The excitation
maxima differed by no more than 6 nm at different pH
values for any of the PD compounds. The emission maxima
for all the PD agents shifted to a shorter wavelength by
approximately 2550 nm, as the pH increased. Six of the
PD agents emitted more than twice the fluorescence at

266

Quinolone accumulation by S. aureus

pH 7 than at pH 3 (PD114456, PD124979, PD130390,
PD138086, PD164413 and PD164534). The remaining four
agents (PD112956, PD135498, PD135671 and PD164532)
fluoresced equally well at both pH values.

Quinolone accumulation
The steady-state concentrations (SSCs) of the PD quinolones accumulated by wild-type S. aureus SA-1199 did not
correlate with the MICs of the agents, e.g. PD138086 (MIC
0.03 mg/L) was accumulated to approximately one-third
the concentration of PD164532 (MIC 1 mg/L) by SA-1199
(Table II). Similarly, the SSCs of drugs accumulated by SA1199 or F77 bore no correlation with the structure of the R1
or R7 substituent. For example, the SSCs of some agents
with identical substituents at R1 accumulated by SA-1199
were broadly similar (e.g. for PD138086 and PD164534,
which have a 2,4-difluorophenyl group at R1, the SSCs
were 30 and 25 ng/mg, respectively; and for PD135498 and
PD135671, which have a t-butyl group, the SSCs were 52
and 42 ng/mg). In contrast, other agents, such as PD112956
and PD114456, which have an ethyl group at R1, have SSCs
of 14 and 54 ng/mg, while PD124979 and PD130390 (cyclopropyl group) have SSCs of 40 and 62 ng/mg, respectively.
The concentrations of PD112956, PD135498 and PD164532 accumulated (which were structurally identical apart
from the substituents at the R1 position), were quite different: 14, 52 and 87 ng/mg, respectively.
For all 10 PD agents, the presence of CCCP increased
the concentration of quinolone accumulated by S. aureus
SA-1199 (Table II). The accumulation of agents with an
ethyl group at R1 (PD112956 and PD114456) and with a
cyclopropyl group at this position (PD124979 and PD130390), was increased by CCCP approximately three- and
two-fold, respectively. Although the SSC was increased
2.7-fold by the presence of the NorA efflux inhibitor CCCP
for the least hydrophobic agent (PD114456; logP 0.924)
compared with 1.3-fold for the most hydrophobic agent
(PD135498; logP 0.022), there was no apparent correlation
between logP and the fold increase of the SSC for the other
agents in the presence of CCCP.
Six of the 10 PD agents emitted 50% less fluorescence
at pH 3 than at pH 7. However, only for PD164534 was
the accumulation by F77 significantly lower after cell lysis
at pH 3 (46 ng/mg) than at pH 7 (121 ng/mg) (Table I).
Accumulation of the 10 PD agents by SA-1199 was determined only using cell lysis at pH 3, but none of the six
agents that emitted less fluorescence at pH 3 were accumulated to lower concentrations compared with the other
agents.
There was no apparent relationship between the accumulation of the 10 PD agents, measured following cell lysis
in pH 3 glycine or in pH 7 phosphate buffer, with boiling,
and the logP of each agent for S. aureus F77 (r  0.042 and
r  0.320, respectively, where r is the correlation coefficient) or E. coli I113 (r  0.162, r  0.339; data not shown).

Figure 1. Relationship between molecular mass and accumulation of nine PD quinolones by S. aureus F77. Accumulation was
determined by the modified fluorescence method12 and cells were
lysed in 50 mM phosphate buffer pH 7 with boiling for 10 min
(SSC, steady-state concentration).

A weak positive correlation (r  0.399) was shown between

accumulation into S. aureus F77 and molecular mass after
cell lysis at pH 7. However, the omission of the data point
for PD135671 yielded a correlation equation of coefficient
of 0.745 (Figure 1). However, there was no apparent
relationship between accumulation and molecular mass for
S. aureus following lysis in pH 3 glycine (r  0.361), or
E. coli I113 following cell lysis at either pH 3 or pH 7
(r  0.151, r  0.492), nor between accumulation and MIC
for S. aureus F77 (r  0.290, r  0.115) or E. coli I113
(r  0.233, r  0.534) with lysis at either pH (data not
shown).
The relationship between the accumulation of the 10 PD
agents by S. aureus F77 and SA-1199 and logP, molecular
mass and MIC was compared and was similar for the two
strains, although SA-1199 accumulated each fluoroquinolone to a lower concentration than F77.

Discussion
The comparison of MICs of the 10 PD compounds and
their different structures suggested that changes in the side
chains of the R7 pyrrolidinyl ring alone had little effect on
the activity of these agents against S. aureus, but that the
substituent at the R1 position was more important. For this
series of fluoroquinolones, the t-butyl substituent was the
most active group at the R1 position, and other workers
have shown this substitution to be important for activity
against Gram-positive bacteria.22,23 Although S. aureus
F145 (ciprofloxacin-resistant mutant) and SA-1199B (constitutive NorA overproducer) were two- to 64-fold less susceptible to the PD agents than their respective wild-type
parent strains, all the strains were more susceptible to the
six most active PD agents and tosufloxacin, than to nalidixic acid, ciprofloxacin, norfloxacin and temafloxacin. This
confirms the observation that quinolones with an aminopyrrolidine ring at R7 tend to be more active against Grampositive organisms than piperazinyl quinolones.24,25

267

L. J. V. Piddock et al.
The concentration of quinolone accumulated did not
correlate with the MIC for S. aureus F77 or SA-1199, as
some agents that were accumulated to a high concentration
also had a high MIC for S. aureus. This is in agreement with
earlier studies.2,12,26 While the MIC data demonstrated that
the R1 group was important for antibacterial activity, there
was no clear relationship between the chemical structure
and the concentration accumulated for the 10 agents. The
data concur with earlier studies which have concluded that
while quinolone structure does affect the interaction with
the target, it has little influence on the concentration of the
agent accumulated.2,14
The concentration of all the PD agents accumulated by
SA-1199 was increased in the presence of CCCP, suggesting that NorA was inhibited. However, the concentration
of those agents with an ethyl or cyclopropyl group at the R1
position accumulated by SA-1199 was two- to three-fold
higher in the presence of CCCP, which may suggest a link
between quinolone structure and the affinity for the NorA
efflux pump. Piddock et al.26 reported variation in the fold
increase of the concentration of quinolone accumulated by
E. coli in the presence of CCCP for different agents, and
proposed that the variance could reflect different affinities
of an efflux pump for each quinolone. McCaffrey et al.6
found that CCCP had little effect on the accumulation of
ofloxacin and fleroxacin by S. aureus, while others have
shown increased activity or accumulation of the hydrophilic quinolones, norfloxacin and ciprofloxacin, in the
presence of efflux inhibitors.7,8,27,28 Takenouchi et al.15
demonstrated an increase in norfloxacin accumulation by
SA-1199 and SA-1199B in the presence of CCCP, while the
accumulation of CS-940 was not affected. The variability in
the reported effects upon efflux by CCCP and other efflux
inhibitors appears to be dependent on the quinolone and
the bacterial strain used and may reflect the affinity of the
quinolone as a substrate for NorA, where those quinolones
that are a poor substrate for the carrier protein are expelled
from the bacterial cell less efficiently.15
Although NorA has been proposed to have a greater
affinity for hydrophilic quinolones,7 in this study, the fold
increase of the SSC for SA-1199 in the presence of CCCP
did not correlate with logP. Some studies have found a
correlation between the concentration of fluoroquinolone
accumulated and molecular hydrophobicity for S. aureus,2,6,7
but none was observed in the present study or others.12 It
remains to be established whether accumulation is determined primarily by hydrophobicity or by quinolone structure. Further investigation of how the quinolone structure
affects affinity for the NorA pump would be interesting.
In previous studies in this laboratory it had been observed that some agents were accumulated to low concentrations in bacteria, and that some of these agents also
fluoresced poorly. To address the question of whether the
low SSCs were an artefact of the method using lysis at pH 3,
the fluoresence was determined at a range of pHs. In
addition, the SSC was determined for S. aureus F77 after

cell lysis at both pH 3 and pH 7. Although six of the 10 PD

quinolones emitted less than half the fluorescence at pH 3
than at pH 7, the differences observed in the fluorescence
were not reflected in differences in the measured accumulation using the two methods of cell lysis. This is as would be
expected, as standard solutions used for calibration were
prepared in the same pH medium used for cell lysis for each
method. The preparation of quinolone standard solutions
in the lysis buffer should negate any differences due to low
fluorescence at pH 3. However, any errors in measuring the
concentration of quinolone in the lysed cell solution would
be greater where less fluorescence is emitted. Therefore,
assuming cell lysis was complete under both conditions
used, the measurement of accumulation should be more
accurate at pH 7, where 6/10 PD agents were more fluorescent, than at pH 3.
The accumulation of the PD agents by S. aureus bore no
relationship to either logP or the MIC. However, previous
workers have reported a positive correlation between
quinolone accumulation and logP for S. aureus.2,6 Asuquo
& Piddock12 found no direct relationship between hydrophobicity or molecular mass and accumulation or with
antibacterial activity. In addition, Bazile et al.2 found that a
more bulky and hydrophobic quinolone (BMY 40397; logP
1.05) was accumulated to a high concentration by S. aureus,
demonstrating that molecular mass is not a limiting factor
for accumulation. In the present study, a positive correlation (r  0.745) was shown between accumulation of 9/10
PD agents in S. aureus F77 and molecular mass following
cell lysis at pH 7. The cause of this apparent association
observed when cell lysis was carried out at pH 7 and not pH
3, is not clear, but may be a consequence of the differential
fluorescence of the agents at the two pHs.
Recently, it has been proposed that the activity of
quinolones against S. aureus is dependent on the hydrophobicity and/or bulkiness of the substituents at R7 and
R8, and not the molecular hydrophobicity.15 Bulkiness of
the R7 substituents of the PD agents used in this study was
estimated by comparison with the values determined by
Takenouchi et al.15 for identical or similar substituents. No
correlation was observed between either the bulkiness of
the R7 group and SSCs in F77, or the MIC increment ratio
of SA-1199B and SA-1199 for this range of quinolones
(data not shown).
Nikaido & Thanassi18 discussed the dynamics of quinolone accumulation during the initial rapid phase and suggested that for resistant strains that actively efflux fluoroquinolones from the cell (such as S. aureus expressing
norA), a higher rate of influx may enhance antibacterial
activity. Re-analysis of the data from Asuquo & Piddock12
shows that for S. aureus there is a direct relationship
between the rate of accumulation of fluoroquinolones during the initial rapid phase and hydrophobicity (r  0.699;
Figure 2), suggesting that the initial rate of influx is dependent on hydrophobicity. Further study of the complex
relationship of quinolone accumulation by S. aureus,

268

Quinolone accumulation by S. aureus

8. Kaatz, G. W., Seo, S. M. & Ruble, C. A. (1991). Mechanisms of
fluoroquinolone resistance in Staphylococcus aureus. Journal of
Infectious Diseases 163, 10806.
9. Kaatz, G. W., Seo, S. M. & Ruble, C. A. (1993). Efflux-mediated
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134555.

Figure 2. Re-analysis of data for initial rate of accumulation of

fluoroquinolones into S. aureus F77 and logP.12

particularly during the initial rapid phase, with hydrophobicity, molecular mass and structure is warranted, and
will aid the development of new quinolone antibacterial
agents with enhanced activity against S. aureus.

Acknowledgements
We acknowledge the assistance of Dr Tom Renau (previously of Parke DavisWarner Lambert, currently Microcide, Mountain View, CA) for providing information on
the PD compounds. This study was supported by a grant
from the Medical Research Council ROPA scheme.

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multidrug resistance transporter NorA from Staphylococcus aureus
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Received 25 May 2000; returned 6 September 2000; revised 23

October 2000; accepted 12 November 2000

270