Download Journal of Antimicrobial Chemotherapy (2000) 45, 483

Journal of Antimicrobial Chemotherapy (2000) 45, 483-488
Levofloxacin penetrates human monocytes and enhances
intracellular killing of Staphylococcus aureus and Pseudomonas
aeruginosa
Raymond P. Smitha,b*, Aldona L. Baltcha,b, Mary A. Frankea, Phyllis B. Michelsena and
Lawrence H. Boppa
a
Infectious Disease Section, Stratton VA Medical Center, 113 Holland Avenue; bAlbany Medical College,
Albany, NY 12208, USA
Intracellular bacteria often cause relapsing and refractory infections. However, these infections
can be treated effectively with antibiotics such as ofloxacin which penetrate into the cells
containing bacteria. As levofloxacin, the levorotatory isomer of ofloxacin, has enhanced antibacterial activity, we tested the levofloxacin concentration in human monocytes and the effects
of intracellular levofloxacin on monocyte killing of Staphylococcus aureus strain ATCC 29213
and Pseudomonas aeruginosa strain PA1348A. Human monocytes were incubated with levofloxacin at various pH values and temperatures. Following incubation, the monocytes were
separated from incubation media, and intracellular (C) and extracellular (E) levofloxacin concentrations were determined. Mean CIE ratios after 15 min of incubation with 6 and 12 mg/L
levofloxacin at pH 7.4 were 6.4 and 7.1, respectively. CIE ratios were similar at pH 7.4 and 8.0,
but decreased at lower pH values. To study the effects of levofloxacin on intracellular killing of
S. aureus and P. aeruginosa, opsonized bacteria were added to monolayers of monocytes. Following phagocytosis, monocytes were incubated with various concentrations of levofloxacin,
ciprofloxacin and rifampicin, alone or in combination. Levofloxacin (2.5 and 4 mg/L) significantly reduced the survival of cell-associated S. aureus and was more effective than ciprofloxacin at similar concentrations (P< 0.01). Enhanced killing of cell-associated P. aeruginosa
by levofloxacin (0.5 and 1.0 mg/L) was also observed. Activities of levofloxacin and ciprofloxacin
against cell-associated P. aeruginosa were similar. Addition of rifampicin did not augment the
bactericidal activity of levofloxacin. Since levofloxacin is concentrated in human monocytes
and increases their bactericidal activity against intracellular bacteria, it should be considered
for treatment of infections caused by susceptible intracellular bacteria.
Introduction
Infections caused by intracellular bacterial pathogens are
common and frequently cause serious diseases in compromised hosts. These infections may relapse and are difficult
to treat unless the antimicrobial agent used has intracellular activity.1,2 Examples of important infections involving
intracellular bacteria include legionellosis, salmonellosis3
and certain infections caused by Staphylococcus aureus*
The importance of using antibiotics with intracellular
antibacterial activity has been shown for some of these
infections, i.e. legionellosis.2,5,6
Levofloxacin, a fluoroquinolone antibiotic, is the levo-
rotatory component of ofloxacin, a racemic mixture. It
is highly active in vitro against Gram-negative and Grampositive bacteria, including Legionella pneumophila,
S. aureus, Pseudomonas aeruginosa, salmonellae and
others.7'8 We studied the ability of human monocytes to
concentrate levofloxacin under various conditions of temperature and pH, as well as the effect of levofloxacin on the
killing of S. aureus and P. aeruginosa associated with
human monocytes. We also compared the effects of
levofloxacin, ciprofloxacin and rifampicin (which is often
given with fluoroquinolones in the treatment of severe
infections) on the bactericidal activity of human monocytes.
•Corresponding author. Tel: +1-518-462-3311 ext. 3080; Fax: +1-518-462-3350; E-mail: [email protected]
483
© 2000 The British Society for Antimicrobial Chemotherapy
R. P. Smith et al.
Materials and methods
cell-associated extracellular water were determined using
3
H-labelled water and 14C-labelled polyethylene glycol as
described previously.10
Bacterial strains, antibiotics and susceptibility testing
P. aeruginosa strain PA1348A, isolated from the blood of a
bacteraemic patient9 and S. aureus strain ATCC 29213 ,
were used in this study. Levofloxacin was obtained from the
R. W. Johnson Pharmaceutical Research Institute (Raritan,
NJ, USA), ciprofloxacin was obtained from Bayer AG
(West Haven, CT, USA) and rifampicin was obtained from
Sigma Chemical Co. (St Louis, MO, USA). Antibiotic solutions were prepared on the day of use according to the
manufacturers' instructions. MICs were determined by the
macrodilution method. The MICs (mg/L) for P. aeruginosa
strain PA1348A were as follows: levofloxacin, 0.5; ciprofloxacin, 0.125 and rifampicin, 64. For S. aureus strain
ATCC 29213 the MICs (in mg/L) were: levofioxacin, 0.25;
ciprofloxacin, 0.5 and rifampicin, 0.008.
Preparation of monocytes
Monocytes were isolated from the heparinized blood of
healthy human donors by centrifugation using Histopaque
1077 (Sigma Chemical Co.)- The cells were tested for
viability using the trypan blue exclusion test, counted and
resuspended at a final concentration of 107 cells/mL in
either Hanks' balanced salt solution without Ca2+ or Mg2+
(HBSS) (for drug uptake studies) or HBSS + 10% fetal calf
serum (for bactericidal studies). The separated monocytes
were resuspended at the appropriate pH values and temperatures in individual experiments as described below.
Bactericidal assays
Bacteria were grown overnight in Mueller-Hinton (MH)
broth (BBL, Cockeysville, MD, USA). Following centrifugation of the broth culture at 2000g for 15 min, the resulting
bacterial pellet was resuspended in HBSS. Bacteria were
then opsonized for 60 min at 37°C with constant agitation
using 20% pooled, heat-inactivated normal human serum.
Following opsonization, the bacteria were washed and
resuspended in HBSS at a final concentration of 1 X 107
cfu/mL and held at 4°C until use. Using a 10:1 ratio of
opsonized bacteria to monocytes and appropriate concentrations of levofloxacin, ciprofloxacin and rifampicin,
samples were incubated at 37°C on a rotator at 10 rpm.
Sampling times were 0,1,2,3,4 and 24 h for P. aeruginosa
and 0, 20, 45, 90 and 180 min, and 24 h for S. aureus. Samples (100 -L) were diluted in 2.5 mL of phosphate-buffered
saline (PBS) and centrifuged for 5 min at 160g. The resulting pellets were washed twice in PBS using 2.5 mL each
time. Monocytes were lysed with distilled water. Serially
diluted 25 -L samples were plated on MH agar plates
(BBL). Colonies were counted after plates had been incubated overnight at 37°C. These cell-associated bacteria are
reported as cfu/mL. The percentage of viable counts =
(geometric mean cfu/mL at each time point/geometric
mean cfu/mL at time 0) x 100.
Statistical analysis
Concentration of levofioxacin in human monocytes
The ability of human monocytes to concentrate levofioxacin was tested at extracellular levofioxacin concentrations
of 6 and 12 mg/L. The effects of temperature were tested at
pH 7.4 and temperatures of 4, 37 and 42°C. The effects of
pH were tested at 37°C and pH 4.0, 5.0 and 8.0. Treated
monocytes were separated from the incubation medium
after 2,5,10 and 20 min by velocity gradient centrifugation
through silicon oil (Dow-Corning, Corning, NY, USA) at
12000g.10 The resulting monocyte pellets were then resuspended in 2 mL of 0.1 M glycine buffer, pH 3.0 and incubated for 2 h at room temperature in order to lyse the cells.
The lysate was then centrifuged at 10000g to remove cell
debris. Intracellular (from the cleared lysate) and extracellular (from the incubation medium remaining on top
of the silicon oil) levofioxacin concentrations were then
measured by fluorescence spectroscopy10 using an SP
Model 500C Fluorometer (SLM Aminco, Inc., St Louis,
MO, USA) at excitation and emission wavelengths of 262
and 496 nm, respectively. Levofloxacin concentrations
were expressed as CIE ratios, where C is the intracellular
concentration and E is the extracellular concentration. For
each experiment, the total cellular volume and volume of
Statistical analyses of the results of the monocyte bactericidal assays were performed using Iog10 of the cfu/mL and
analysis of variance.11 The level of significance was 0.05.
Results
Intracellular concentration of levofloxacin
Levofloxacin concentrations within viable human monocytes were in the range 18-40 mg/L, and were up to six
times the extracellular concentration following incubation
for 15 min at pH 7.4 and 37°C. Peak concentrations were
reached in 5 to 15 min (Figure 1). The intracellular levofloxacin concentration following incubation at pH 4 or 5
(data combined for pH 4 and 5) was lower than at pH 7.4.
At pH 8.0 the intracellular levofloxacin concentration was
similar to that at pH 7.4 (Figure 1). Figure 1 shows the
effect of temperature on levofloxacin uptake by human
monocytes. During the first 10 min of incubation the intracellular levofloxacin concentration rose more rapidly at
42°C than at 37°C. However, at 42°C the intracellular concentration decreased after 10 min, while at 37°C it continued to rise. After 15 min the intracellular levofloxacin
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Levofloxacin and human monocytes
Figure 1. Uptake of levofloxacin by human monocytes. (a) Effect
of pH: , pH 7.4; , pH 8.0; , pH 4, 5. (b) Effect of
temperature:  ,37°C; ,42°C; ,4°C.
concentrations at 37°C and 42°C were similar. In contrast,
at 4°C and pH 7.4 the intracellular and extracellular levofloxacin concentrations were similar, suggesting that little if
any drug penetration into monocytes had occurred at that
temperature. Complete washout of levofloxacin from
monocytes occurred within 2 to 5 min following removal of
the drug from the incubation medium (data not shown).
Bactericidal activity in human monocytes
The intracellular antibacterial activities of levofloxacin and
ciprofloxacin were studied singly and in combination with
rifampicin. Activities against P. aeruginosa strain PA1348A
and S. aureus strain ATCC 29213 were determined.
The intracellular activities of levofloxacin and ciprofloxacin against P. aeruginosa strain PA1348A were both
concentration and time dependent (Figure 2). The killing
rates of effective doses were greatest during the first hour
for both drugs, but maximum killing and differences among
doses were seen at 3-4 h for all concentrations tested (P <
0.01). Figure 2 shows the activities of levofloxacin and
ciprofloxacin against P. aeruginosa strain PA1348A singly
at 1 X MIC (0.5 mg/L of levofloxacin, 0.125 mg/L of cipro-
Figure 2. Bactericidal activities of levofloxacin, ciprofloxacin and
rifampicin against intracellular P. aeruginosa.  , Control, (a)
Levofloxacin: ,0.5 X MIC (0.25 mg/L); , 1 X MIC (0.5
mg/L); , 2 x MIC (1.0 mg/L). (b) Ciprofloxacin: ,0.5 X MIC
(0.0625 mg/L); , 1 X MIC (0.125 mg/L); , 2 X MIC (0.25
mg/L). (c)  , Ciprofloxacin (0.125 mg/L) alone; ,
levofloxacin (0.5 mg/L) alone; , rifampicin (8 mg/L) alone; ,
levofloxacin (0.5 mg/L) and rifampicin (8 mg/L) in combination;
,ciprofloxacin (0.125 mg/L) and rifampicin (8 mg/L) in
combination.
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R. P. Smith et al.
floxacin) and in combination with rifampicin (8 mg/L).
When tested individually, all three drugs showed rapid
antibacterial activity at 1 h, with maximum total activity at
3-4 h. The differences in the activities of either levofloxacin
or ciprofloxacin when usedin combination with rifampicin
were not statistically significant. The percentage of bacterial survival at 24 h was 0.2-0.6% for each of the three
drugs and for the combinations.
Figure 3 shows the intracellular activity of levofloxacin
against S. aureus strain ATCC 29213. After 20 min at 2.5
and 4 mg/L (average and high concentrations achievable in
human serum) the differences in survival percentages
between the treated and untreated cells were not statistically significant. However, at both 45 and 90 min the bacterial survival percentages were significantly lower than
those of the controls (P < 0.01). There was no significant
difference in bacterial survival between the two drug concentrations. When levofloxacin, ciprofloxacin and rifampicin were tested individually against 5. aureus strain ATCC
29213 at concentrations achievable in human serum (Figure 3), rifampicin demonstrated the greatest antibacterial
activity after 45 min of incubation. However, there was no
significant difference in the survival of ciprofloxacintreated and control cells. At 90 min of exposure, all three
drugs exhibited antibacterial activity, with rifampicin and
levofloxacin more effective than ciprofloxacin (P < 0.01).
At 180 min, levofloxacin had significantly greater antibacterial activity than ciprofloxacin or rifampicin alone
or either fluoroquinolone in combination with rifampicin
(P < 0.05). Survival at 24 h was <5% for cells treated with
levofloxacin or ciprofloxacin alone or in combination with
rifampicin.
Discussion
A variety of antimicrobial agents, including macrolides,
ketolides, lincosamines, rifamycins and fluoroquinolones,
are known to be concentrated inside mammalian phagocytes, reaching greater intracellular concentrations than
their extracellular concentrations. 10,12-26 Because of their
bactericidal activities against both Gram-positive and Gramnegative intracellular pathogens the fluoroquinolones are
potentially the most important of these agents.
The uptake kinetics and intracellular concentrations of
levofloxacin in human monocytes found in this study are
similar to those reported for ciprofloxacin.16,27 Our study
indicates that intracellular concentrations of levofloxacin
in monocytes are also similar to those found in polymorphonuclear leucocytes (PMNs).28 Several fluoroquinolone
antibiotics have been shown to penetrate and be concentrated in human PMNs,10,17,18,22,25,27,28 macrophages22,29 and
monocytes.26 In previous studies of the ability of human
phagocytic cells to concentrate fluoroquinolones, a range
of four-fold to >40-fold concentration has been
observed.10,14,17,18,22,25,26,28,29 Methodological differences, including
source of cells, type of cells and detection assays,
90 Time
(min)
Figure 3. Bactericidal activities for S. aureus of levofloxacin,
ciprofloxacin and rifampicin, alone and in combination, at concentrations achievable in human serum. , Control, (a) Levofloxacin: , 2.5 mg/L; O, 4.0 mg/L. (b) , Levofloxacin (4 mg/L)
and rifampicin (8 mg/L); , ciprofloxacin (5 mg/L); ,
rifampicin (8 mg/L); , ciprofloxacin (5 mg/L) and rifampicin (8
mg/L); , levofloxacin (4 mg/L).
could account for some of the differences among the drugs
tested. For fluoroquinolones in general, there appear to be
no energy-dependent mechanisms that determine intracellular drug concentrations.30 For most of the fluoroquinolones studied to date, elution of drug from phagocytic
cells is rapid following removal of the drug from the extracellular environment.28 However, one study showed that
fluoroquinolones are more readily retained by phagocytic
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Levofloxacin and human monocytes
cells containing ingested L. pneumophila than by the same
cells in the absence of ingested bacteria.13 This suggests
fluoroquinolone trapping in the presence of ingested bacteria that have escaped from phagolysosomes. When
studied with PMNs and measured by the same method used
in our studies10 levofloxacin reached intracellular concentrations approximately six times the extracellular concentration.28 The responses to lowered temperature and
changes in pH were similar to those seen with other fluoroquinolones.28 Studies have suggested that fluoroquinolones, including levofloxacin, are taken up more slowly by
human macrophages than by PMNs.22 However, some of
these studies have used mononuclear cells harvested from
peritoneal dialysate and the effect of this medium on cell
behaviour is not known.
The role of antibacterial activity within phagocytic cells
and the outcome of experimental and clinical infections are
well established, especially for infections caused by L.
pneumophila8,29 However, relatively little is known about
the augmentation of intracellular bacterial killing by human
monocytes. Antibacterial activity in phagocytic cells depends upon the intracellular site of drug localization (which
is unknown for fluoroquinolones) relative to the intracellular location of the bacteria, the stability of the drug in the
intracellular environment and the susceptibility of the
microorganism to the drug while within the cell. The latter
can vary with the metabolic activity or growth rate of the
ingested organism. The present study provided evidence
that the rate and degree of intracellular killing of P. aeruginosa are affected equally by levofloxacin and ciprofloxacin
at concentrations with equivalent effectiveness (reflected
in the MICs of the individual drugs).
Because of concern that S. aureus will develop resistance
when exposed to low (sub-MIC) concentrations of fluoroquinolones, we studied the effects of levofloxacin and
ciprofloxacin at (extracellular) concentrations similar to
achievable serum concentrations. Since rifampicin has
been shown to be the most active agent against intraphagocytic staphylococci,26 we included rifampicin both as a comparative agent and to investigate its possible additive or
antagonistic effects in combination with each of the fluoroquinolones studied. There was no dose effect for levofloxacin, but levofloxacin caused significant killing of
intracellular 5. aureus after as little as 45 min incubation.
Although ciprofloxacin was active against 5. aureus by 180
min of incubation, levofloxacin demonstrated the best
activity, even when compared with rifampicin alone. When
compared with the individual fluoroquinolones alone,
addition of rifampicin to either fluoroquinolone did not increase bacterial killing. This suggests that the antibacterial
activity of levofloxacin against S. aureus is greater than that
of rifampicin in the intracellular environment.
Previous studies with 5. aureus have shown that drugs
with a very high degree of penetration into phagocytic
cells, such as erythromycin, clarithromycin, azithromycin,
roxithromycin and clindamycin, act in an inhibitory but
not bactericidal fashion in experiments lasting up to
24 h.12,15,19,21,24,26,31 At appropriate concentrations ciprofloxacin has
been shown to enhance the activity of phagocytic cells in
killing S. aureus.16 Our study demonstrates the rapid
bactericidal activity of levofloxacin against both P.
aeruginosa and S. aureus. The activity of levofloxacin
against P. aeruginosa was equivalent to that of ciprofloxacin, the most active anti-pseudomonal fluoroquinolone in
current clinical use. In contrast, levofloxacin was more
active than ciprofloxacin against S. aureus.
Levofloxacin remains in human mononuclear phagocytes as long as extracellular levofloxacin is present, and
reaches concentrations within the cells approximately sixfold greater than the external concentration. More importantly, the presence of levofloxacin enhances the ability of
human monocytes to kill both 5. aureus and P. aeruginosa,
two organisms that can persist inside mammalian cells.
Since other antibacterial agents either lack bactericidal
capability or do not reach adequate concentrations within
phagocytes, levofloxacin offers an advantage in the treatment of serious infections caused by microorganisms that
can persist inside phagocytic cells.
Acknowledgements
This work was supported by a grant from the Pharmaceutical Research Institute of R. W. Johnson, Inc. and in
part by the Medical Research Service of the Department of
Veterans' Affairs.
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Received 2 July 1999; returned 28 September 1999; revised 14
October 1999; accepted 1 November 1999
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