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Antimicrobial Agents and Chemotherapy, May 1999, p. 1013-1019, Vol. 43, No. 5
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Pharmacodynamic Effects of Subinhibitory
Concentrations of Rufloxacin on Bacterial Virulence
Factors
Pier Carlo
Braga,*
Maria Teresa
Sala, and
Monica
dal Sasso
Department of Pharmacology, School of
Medicine, University of Milan, Milan, Italy
Received 22 December 1997/Returned for modification 6 January
1999/Accepted 14 February 1999
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ABSTRACT |
It has been reported that subinhibitory concentrations (sub-MICs)
of some fluoroquinolones are still capable of affecting the topological
characteristics of DNA (inhibition DNA-gyrase) and that this leads to a
reduction in some of the factors responsible for bacterial virulence
(by means of the disruption of protein synthesis and alterations in
phenotype expression), even though the microorganisms themselves are
not killed. The present study investigated the ability of sub-MICs of
rufloxacin, an orally absorbed monofluorinated quinolone with a long
half-life (28 to 30 h), to interfere with the bacterial virulence
parameters of adhesiveness, hemagglutination, hydrophobicity, motility,
and filamentation, as well as their interactions with host neutrophilic defenses such as phagocytosis, killing, and oxidative bursts. It was
observed that Escherichia coli adhesiveness was
significantly reduced at rufloxacin concentrations of 1/32 MIC,
hemagglutination and hydrophobicity were significantly reduced at
concentrations of, respectively, 1/4 MIC and 1/8 MIC, and motility was
significantly reduced at concentrations of 1/16 MIC; filamentation was
still present at concentrations of 1/4 MIC. Phagocytosis was not
affected, but killing significantly increased from 1/2 MIC to 1/8 MIC;
oxidative bursts measured by means of chemiluminescence were not
affected. The fact that sub-MICs are still effective in interfering
with the parameters of bacterial virulence is useful information that needs to be correlated with pharmacokinetic data in order to extend our
knowledge of the most effective concentrations that can be used to
optimize treatment schedules, for example, single administrations, particularly in noncomplicated lower urinary tract infections.
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INTRODUCTION |
Bacterial virulence reflects the
ability of infecting microorganisms to produce pathological effects in
an invaded host. The extent and severity of these effects depend on a
number of bacterial cell functions, such as their invasive and adhesive
capacities, fimbriation, their outermost surface characteristics and
motility, their interaction with host defenses (phagocytosis and
killing), the role of metabolism and the release of exocellular and
endocellular products, and rates of replication.
In cases of bacterial infection antibiotic therapy is usually adopted,
on the assumption that the drug concentrations at the site of infection
reach the minimum bactericidal concentration, thus eliminating the
virulence of the microorganisms by killing them. In contrast, although
the microorganisms do not necessarily die, MICs can inhibit the growth
of bacteria and, in most cases, significantly reduce their virulence
(49).
With intermittent antibiotic administration, commonly used to treat
bacterial infections, pharmacokinetic curves show concentrations that
fluctuate on the basis of the dosing schedule. At the site of
infection, these concentrations may exceed the in vitro MIC for the
invading microorganism for a certain period of time, subsequently decrease more or less rapidly to values corresponding to the MIC, and
finally drop to subinhibitory concentrations (sub-MICs), generally between doses. This is particularly true in the case of tissue infections, because tissue antibiotic concentrations are frequently lower than those in the blood.
A growing body of evidence (9, 13, 35, 39, 42, 50, 51)
strongly suggests that antibiotic concentrations of less than the
conventionally determined MICs may still be effective in reducing
bacterial virulence by interfering with bacterial cell functions. These
findings have generated interest in the effects of exposing bacteria to
low concentrations of antibiotics (13, 34), so that
pharmacodynamic and pharmacokinetic data can be correlated and therapy
can thus be optimized. Rufloxacin is an orally absorbed monofluorinated
quinolone with a long half-life (28 to 30 h), consistently high
bactericidal concentrations at the site of infection (especially in
urine), and good penetration into infected tissues (53, 54).
Although some scattered information exists in the literature (5,
7, 17), the effects of sub-MICs of rufloxacin have not yet been
fully investigated. The aim of the present study was therefore to
investigate the activity of such concentrations on various bacterial
cell functions in order to evaluate their ability to reduce bacterial virulence.
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MATERIALS AND METHODS |
Adhesion assay.
Two Escherichia coli strains from
clinical isolates (urinary infections) and lyophilized E. coli ATCC 25922 (D.I.D., Milan, Italy) were used. The MICs were
determined in Mueller-Hinton broth by using the tube macrodilution
method (38). Each tube contained twofold dilutions of the
antibiotic and a final bacterial inoculum of 106 CFU/ml.
The tubes were incubated for 18 h at 37°C. The MIC was defined
as the lowest concentration of antibiotic that prevented turbidity in
the test tube after incubation.
For the adhesion assay, cell suspensions of each organism were prepared
from overnight cultures (18 h) in tryptic soy broth (Sigma) at 37°C
under static conditions. The organisms were harvested, washed three
times in phosphate-buffered saline (PBS), and adjusted to 3 × 108 organisms/ml as determined by direct microscopic counts
(interference contrast microscopy) in a Petroff-Hausser bacterial
counting chamber.
Human periurethral epithelial cells were collected from the sediment of
fresh urine (50 ml) of apparently healthy females
and resuspended in 20 ml of PBS. The suspensions obtained from
three to five subjects were
pooled. The epithelial cells were
then passed through a needle (0.3 mm
diameter) to disrupt cell
aggregates and washed three to four times to
free them from debris
and nonadherent bacteria by using low-speed
differential centrifugation
(240 ×
g for 10 min at
21°C).
PBS was added to the washed epithelial cell suspensions to give
10
5 cells/ml as determined by means of a direct microscopic
count
(interference contrast microscopy) in a Bürker chamber. The
ability
of the bacteria to adhere to epithelial cells was investigated
by mixing together 1:1 volumes of standardized suspensions of
bacteria
(3 × 10
8/ml) and epithelial cells (3 × 10
5/ml) in polystyrene tubes. The tubes were rotated end
over end
at 10 rpm for 60 min at 37°C. The epithelial cells were
separated
from the nonadherent bacteria by means of differential
centrifugation
at 100 ×
g for 5 min. The final
epithelial cell pellet was resuspended
in a small quantity of PBS,
placed on a round microscope coverslip,
and
dried.
The coverslip with the cells was fixed in 2.5% glutaraldehyde in 0.1 M
cacodylate buffer (pH 7.1) for 60 min at 4°C. After
several
dehydrations in alcohol, the coverslips underwent critical-point
drying, were coated with 200-Å gold, and counted by using a scanning
electron microscope (SEM). The adhesiveness of bacterial to epithelial
cells was determined by counting the number of epithelial cells
with
40 adhering bacteria per 100 cells (
47). Each test was
performed twice. Control epithelial cell suspensions were always
included to provide data on the number of bacteria that were already
attached (natural acquisition) when the cells were
collected.
For the inhibition test, the bacteria were grown in the presence of
subinhibitory concentrations of rufloxacin (37°C for 18
h), from
1/2 to 1/128 MIC (1/2, 1/4, 1/8, 1/16, 1/32, 1/64, and
1/128), or with
the same amount of medium without any antibiotic
(control). The
bacteria were harvested by centrifugation at 470
×
g
and resuspended in PBS at a final concentration of 3 × 10
8 bacteria/ml. They were challenged with epithelial cells
as in
the adherence assay, and the samples were prepared for SEM
analyses.
Hemagglutination assay.
Anticoagulated guinea pig and human
group A erythrocytes were collected 1:1 in Alsever solution (2.05%
glucose, 0.8% sodium citrate, 0.42% NaCl, 0.055% citric acid [pH
6.1]), washed three times in PBS, and finally suspended in saline; the
washed erythrocytes were then stored at 4°C and used within 5 days.
The hemagglutination tests were performed by using a slightly modified
version of the procedure of Evans et al. (
22). Twenty
microliters of a 5% (vol/vol) suspension of erythrocytes in saline
and
20 µl of the bacterial suspension (10
9 cells/ml) were
pipetted onto microscopy slides. The slides were
gently rotated at room
temperature for 5 min and read after 15
min.
To test for mannose sensitivity, 20 µl of 1% mannose solution was
added to a duplicate slide containing undiluted bacteria.
The
hemagglutination test was applied to samples of each bacterial
strain
grown with the different sub-MICs of rufloxacin. The results
were
recorded as grade 3 when hemagglutination occurred in a very
short
period of time and was complete (coarse clumping); a reduction
in
clumping that gave only different degrees of fine granularity
was
recorded as 2, 1, or negative (
22).
Hydrophobicity assay.
The hydrophobicity of the bacterial
cell surfaces was measured by using the salt aggregation test (SAT)
(29). Briefly, a bacterial suspension of 25 µl (5 × 109 bacterial cells/ml in 0.002 M sodium phosphate buffer
[pH 6.8]) was mixed with an equal volume of ammonium sulfate
[(NH4)2 SO4] ranging from 3.2 to
0.2 M (pH 6.8). The bacteria-salt solution was gently rocked for 2 min
at 20°C, and aggregation was visually read against a black background.
A reaction causing optimal aggregation was regarded as positive (i.e.,
when most of the bacteria aggregated to give a clear
solution and white
aggregates with a diameter of approximately
1/10 mm). A reaction with
no aggregates or only a few aggregates,
i.e., not modifying the overall
view of the sample, was regarded
as negative. All the readings were
compared with the reaction
at the highest salt molarity (positive
control).
Bacterial suspensions mixed with 0.0002 M sodium phosphate (pH 6.8)
without the addition of salt were used as a negative control.
The SAT
value represented the lowest concentration of ammonium
sulfate at which
aggregation was observed. The test was repeated
for each strain and for
each sub-MIC of
rufloxacin.
Motility.
The bacteria were grown overnight in tryptic soy
broth (Sigma) at 37°C, and a 5-µl aliquot of the cell suspension
(3 × 108 cells) was placed on the agar surface of the
semisolid swarming medium (agar motility) (1% tryptone, 0.5% NaCl,
0.25% agar dissolved in distilled water [pH 7.1]) (9).
After inoculation, the assay plates (petri dishes [diameter, 9 cm],
containing 10 ml of medium) were placed in a water-saturated
Plexiglas
incubator at 37°C. The plates were illuminated obliquely
and viewed
against a dark background, and the diameter of the
swarming zones was
measured with a ruler at regular time
intervals.
Assays were performed on each strain grown overnight with the different
sub-MICs of rufloxacin and inoculated onto plates
with the swarming
medium containing the same sub-MICs. The control
plates contained no
antibiotic.
Filamentation.
Changes in the morphology of bacilli do not
occur at the same time or in the same ratios for all the organisms in a
given population of bacteria exposed to a given concentration of an antibiotic (33), so in order to verify the exact kinetics at the time of morphological change, a 700-µl aliquot was withdrawn from
the cultures at 0.5, 1, 2, 4, and 8 h and processed for SEM observation. This procedure (8) was adopted after control
samples showed that the 700-µl extraction did not interfere with
growth, so it was possible to compare the evolution of the changes in the same culture.
For each determination, the 700-µl aliquot was added to 2 ml of broth
and centrifuged at 450 ×
g; the final pellet was
resuspended
in 100 µl of PBS (0.02 M phosphate and 0.15 M NaCl [pH
7.3]),
placed on a round microscope coverslip, and dried. The
coverslip
was then fixed in 2.5% glutaraldehyde in 0.1 M cacodylate
buffer
(pH 7.1) for 60 min at 4°C. After dehydration in graded
alcohol,
the coverslip underwent critical-point drying, was coated with
200-Å gold and observed in a
SEM.
The microscopic fields to be counted, bacterial size, and the
morphology recorded were selected by means of random
scanning.
The morphological characteristics before and after incubation with the
different sub-MICs of rufloxacin were classified and
quantitated, with
the observer unaware of the concentration of
antibiotic or the duration
of incubation. The lengths of the filaments
and their proportions in
the total number of microorganisms per
100 randomly observed bacteria
were recorded; a minimum of eight
different fields were examined for
each sub-MIC and time. The
normal length of
E. coli can vary
from 1.2 to 2.3 µm; during the
mid-division cycle it is about 5 µm.
Organisms of up to 15 µm were classified as short filaments, and
those longer than 15 µm were classified as long filaments
(
33).
Phagocytosis and killing. (i) Collection of human PMNs.
Peripheral venous blood was drawn from healthy adult donors into
heparinized (5 U/ml) syringes. The blood (5 ml) was stratified on 3 ml
of Polymorphoprep (Nycomed Pharma), and the polymorphonuclear leukocytes (PMNs) were separated by density gradient centrifugation.
When necessary, any residual erythrocytes in the granulocyte
preparation were lysed with a 0.15-mol/liter of NH
4Cl
solution
(pH 7.4). The PMNs were collected and washed in
glutamine-containing
RPMI 1640 (Sigma) after being passed through a
150-µm-internal-diameter
needle in order to disrupt cell aggregates.
They were then tested
for viability by trypan blue exclusion. The final
cell suspension
was adjusted to the cell numbers needed for each test
by counting
in a Bürker chamber (interference contrast
microscopy).
(ii) Phagocytosis and bacterial killing.
The phagocytic and
bacterial killing capacities of the PMNs were determined by using a
fluorochrome assay (acridine orange stain), which distinguishes viable
and dead microorganisms intracellularly. The procedure of
Bellinati-Pires et al. (4) was adopted with slight
modifications. Equal volumes of PMNs (2 × 106
cell/ml) and preopsonized bacteria (2 × 107
bacteria/ml) were mixed in tubes at a ratio of 1:10 (PMN/bacteria) in a
final volume of 0.5 ml. The tubes were incubated at 37°C and rotated
end over end (6 rpm) for 30 min.
Phagocytosis was stopped by placing each tube in an ice bath and adding
0.5 ml of ice-cold medium to the bacteria-PMN suspension.
Noningested
bacteria were removed by means of differential centrifugation
(100 ×
g for 7 min) and two washes. The pellet was
stained with
200 µl of 14.4-mg/liter acridine orange (pH 7.2) (Sigma)
in medium
for 1 min. Immediately after staining, 1 ml of ice-cold
Hanks'
balanced salt solution (HBSS) was added to the PMN suspension,
which was then centrifuged at 160 ×
g for 7 min at
4°C. The cells
were washed twice with ice-cold HBSS and kept in an
ice bath until
microscopic
examination.
Acridine orange makes bacteria fluorescent, so phagocytized bacteria
can be easily counted; killed bacteria are also clearly
visible because
they become yellow-red under UV light, whereas
living bacteria are
green. After staining with acridine orange
to avoid overestimating
phagocytosis on the basis of the
E. coli CFU attached to the
surface of the PMNs but not yet internalized,
the techniques of Hed
(
27) and Goldner et al. (
24) were used
to quench
the extracellular membrane-adherent microorganism fluorescence
by
crystal violet (500 µg/ml for 20 min). Since crystal violet
does not
penetrate PMNs, it does not alter the fluorescence of
ingested
microorganisms. Just before the observation of each cell
sample, a drop
of the cell suspension was wet mounted on a microscope
slide and sealed
with nail varnish. It was then immediately examined
under oil immersion
with a UV epifluorescence microscope (Leitz)
equipped with an
excitation filter at 450 to 490 nm, a beam split
mirror at 510 nm, and
a cut-off filter at 520
nm.
A short time interval lasting no longer than 10 min was established for
each slide reading, and if this was not sufficient,
another slide was
prepared from the ice-cold suspension. A total
of 100 PMNs was observed
for each slide. The number of cells phagocytizing
at least three
bacteria/100 PMNs gave the percent of phagocytosis,
whereas the average
number of bacteria in each phagocytizing cell
gave the phagocytic
index. The percentage of killed bacteria was
obtained by using the
following formula: number of dead bacteria/number
of dead + live
bacteria × 100. The killing index was the average
number of dead
bacteria per PMN. The same procedure was followed
to determine the
effect of the exposure of PMNs to different sub-MICs
of
rufloxacin.
Measurement of oxidative burst response by
chemiluminescence.
Luminol-amplified chemiluminescence (LACL) was
investigated by using a slightly modified version of the procedure of
Robinson et al. (43) for pathogenic organisms. In brief, 0.1 ml of PMN suspension (106 cells/ml) and 0.30 ml of HBSS
with Ca2+ and Mg2+ plus 0.05 ml
10
4 M luminol (diluted from a first stock solution in
dimethyl sulfoxide; Sigma) were put into a 3-ml flat-bottomed
polystyrene vial. The vial was placed in a lightproof chamber of the
Luminometer 1250 (Bio Orbit), and the carousel was rotated to bring the
sample in line with the photomultiplier tube in order to record
background activity. A suspension of preopsonized killed Candida
albicans cells (2 × 107 cells/ml) in a final
volume of 0.05 ml was added as a stimulus for oxidative bursts, and the
resulting light output in millivolts was continuously recorded on a
chart recorder and, simultaneously, by means of a digital printout set
for 1- or 10-s recording integrals.
All the constituents of the mixture were kept at 37°C during the
reaction by means of thermostatically controlled water passing
through
a polished hollow metal sample holder. No mixing took
place during the
recordings. The gain control was set to give
a reading of 10 mV for a
built-in standard. A background subtraction
control zeroed the
instrument prior to the addition of the opsonized
cells. The patterns
of LACL responses were determined by calculating
the initial slopes,
peaks (in millivolts), times to peak, slopes
of declining response, and
areas under the curves. Because the
peaks correlate well with the other
parameters, a first analysis
is presented simply in terms of peak
counts. However, since oxidant
radical production has its own time
course, the simple peak (which
freezes measuring at a single time) does
not completely characterize
the phenomenon over time, so the data are
also expressed as a
curve. The analysis was completed by investigating
the effects
of sub-MICs of rufloxacin on PMN oxidative
bursts.
Data analysis.
Each test was performed six to nine times for
each strain and for each sub-MIC and control. Values are expressed as
the means of all data ± standard errors of the means. The
statistical significance of the differences was calculated by using the
t test and, when necessary, analysis of variance between
treatments, followed by multiple pair comparisons according to Dunnett
when the differences were statistically significant. Differences were
considered statistically significant when the test yielded a value of
0.05.
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RESULTS |
During the control test performed before antibiotic challenge, all
of the bacterial strains adhered well to human epithelial cells, with a
certain degree of variability in the number of bacteria per cell. The
mean of the rufloxacin MICs was 1 µg/ml, and after incubation with
sub-MICs, bacterial adhesiveness decreased. The data are summarized in
Table 1. To normalize the
susceptibilities of the different strains, the data were also expressed
as percentages of inhibition versus control, as shown in Fig.
1. As expected, peak inhibition was
observed at 1/2 MIC, but inhibition was statistically significant at a
MIC as low as 1/32.
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TABLE 1.
Effects of various subinhibitory concentrations of
rufloxacin on E. coli adhesiveness to human
epithelial cells
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FIG. 1.
Percent inhibition of E. coli adherence to
human epithelial cells after incubation with different sub-MICs of
rufloxacin.
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The inhibition of hemagglutination and hydrophobicity were significant
as low as 1/4 and 1/8 MIC, respectively (Fig.
2). The different radii of the swarming
zone read at different times are a measure of the motility of E. coli strains. Incubation with 1/2 and 1/4 MICs completely
inhibited bacterial motility, and this inhibition was significant as
low as 1/16 MIC (Table 2).
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FIG. 2.
Effects of various rufloxacin sub-MICs of E. coli hemagglutination and hydrophobicity (S.A.T., salt aggregation
test).
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TABLE 2.
Mean values of swarming zone diameter (mm) at different
hours for E. coli incubated with different sub-MICs
of rufloxacin
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The morphological changes induced by the sub-MICs of rufloxacin
consisted of filamentation. The exposure of E. coli to 1/2 MIC and, to a lesser extent, even to 1/4 MIC was capable of inducing filamentation within 2 to 4 h (Table
3). The above parameters relate to the
bacteria themselves, but there are also host-bacteria interaction
parameters, such as phagocytosis and killing. These were investigated
by using fluorescence microscopy and acridine orange fluorochrome.
Figure 3A and B show neutrophils with
phagocytized E. coli. The green bacteria are still alive,
whereas those that are orange-red have been killed. Rufloxacin sub-MICs
did not change the percentage of phagocytosis or the phagocytic index
but significantly increased the killing of bacteria as low as 1/8 MIC
(Table 4).
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TABLE 3.
Ratios of normal-length bacteria, short filaments, long
filaments, and ghosts for every 100 bacteria at different times
and concentrations
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FIG. 3.
Microphotography (epifluorescence) of PMN phagocytosis
and killing. (A) Unexposed E. coli. (B) E. coli
after exposure to 1/2 MIC rufloxacin (living bacteria are green; dead
bacteria are orange-red).
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Possible interference with oxidative bursts was investigated by
exposing the neutrophils to different sub-MICs of rufloxacin. The
findings reported in Table 5 and Fig.
4 show that the rufloxacin sub-MICs did
not interfere with this important feature.
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TABLE 5.
Means ± standard deviations of LACL (millivolts) of
PMNs (peaks) before and after incubation with different sub-MICs
of rufloxacin
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FIG. 4.
LACL curves of PMNs before and after incubation with
different sub-MICs of rufloxacin (curves are shown only down to 1/16
MIC to avoid a crowded figure).
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DISCUSSION |
Bacterial virulence is a result of the specific properties of both
bacterial and host cells and their reciprocal interactions. Adhesion is
considered to be the first step in the sequence of events leading to
colonization and subsequent infection (1, 3). The ability to
adhere is an important determinant of virulence (21), and
the exposure of bacteria to sub-MICs of antibiotics generally weakens
this ability (14, 30, 45, 47). This effect has been observed
in the case of both quinolones and fluoroquinolones; sub-MICs of
oxolinic acid (26), ciprofloxacin (52),
pefloxacin (18), enoxacin (6, 49), rufloxacin
(7), and lomefloxacin (6) lead to a significant
decrease in the adhesion of E. coli.
Our rufloxacin findings are similar to those found with other
quinolones, but they add further information about the extent of the
effect. We observed significantly reduced adhesion at concentrations ranging from 1/2 to 1/32 MIC (corresponding to 0.03 µg/ml), whereas the majority of previous studies considered only 1/2 or 1/4 MIC.
Sub-MICs of antibiotics can exert their antiadhesive effects in
different ways. They may inhibit the synthesis or expression of
adhesins on the bacterial cell surface, lead to the formation of
functionally aberrant adhesins, cause the release of adhesins from the
surface of bacterial cells, or modify bacterial shape in a such way as
to interfere with the ability of the microorganisms to approach
receptors on animal cell surfaces (31, 32).
It has been reported that the fluoroquinolones pefloxacin and enoxacin
inhibit adhesion on the basis of the first mechanism of adhesion
inhibition (10, 18), so it is likely that rufloxacin also
reduces the number of adhesins and thus the possibility of bacterial
anchorage (or junction). This is also partially correlated with
hemagglutination (an indirect measure of fimbriation) and hydrophobicity.
At 1/2 MIC (and to a lesser extent also at 1/4 MIC), rufloxacin is
capable of inducing morphological changes in E. coli, such as different levels of filamentation. Maximum filamentation (
40%) was achieved after 2 h of incubation of bacteria with the 1/2 MIC
of rufloxacin (7). Quinolone-induced filamentation has also
been observed with the use of pefloxacin (18), enoxacin (25, 49), lomefloxacin (49), ciprofloxacin
(16, 25, 52), oxolinic acid (2, 26), and
nalidixic acid (26).
The mechanism of filamentation differs from the well-known
penicillin-binding protein inhibition induced by 
-lactam
antibiotics, probably because initial DNA-gyrase inhibition followed by
alterations in DNA topology and synthesis (12, 20, 48, 49)
provide the signal necessary for the induction of an SOS response
pathway that inhibits cell division (28, 41, 46). In
addition to filamentation, the SOS response can involve vacuolation and
the leakage of intracellular material (28), which is
probably a secondary mechanism of death (19).
The fact that rufloxacin sub-MICs affect some important functions of
E. coli is also confirmed by the significant reduction in
swarming observed after treatment with 1/2 to 1/16 MIC. Together with
adhesiveness, this function is correlated with the pathogenicity of
bacteria, because the inhibition of motility also reduces the possibility of the formation of new colonies and the spread of infection from the first point of contact.
It has been shown that the sub-MICs of various antimicrobial agents
induce morphological and biochemical changes in bacteria, thus making
them more susceptible to phagocytosis and killing (36).
There are some data in the literature concerning the effect of
quinolone sub-MICs on neutrophil phagocytosis and the killing of
gram-positive bacteria (23, 40), but only a few
investigations have explored their effects on E. coli.
Pre-exposure of E. coli-encapsulated strains to 1/2 MICs of
fleroxacin and ciprofloxacin has been found to enhance phagocytosis
(6), and sub-MICs of ciprofloxacin can increase the killing
of E. coli by neutrophils (37). As for
rufloxacin, none of the three strains investigated showed any
modification in phagocytosis, but killing was significantly increased
in two strains for which the MIC was as low as 1/8 and in one strain
for which the MIC was as low as 1/4.
Table 6 summarizes the known data
concerning the interactions of quinolone sub-MICs with different
virulence parameters. This aspect of nonfluorinated quinolones has not
been investigated. The ciprofloxacin, enoxacin, and pefloxacin
monofluorinated quinolones have been studied in terms of some
parameters, but rufloxacin has been better characterized. The only
difluorinated quinolone that has been partially studied is lomefloxacin
(11), and trifluorinated quinolones need to be further
investigated. Comparison of the data reveals some interesting
similarities in effects. Adhesiveness and fimbriation are reduced, and
filamentation is increased; phagocytosis remains almost unaffected, but
killing is increased; finally, neutrophil oxidative bursts do not vary.
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TABLE 6.
Overall data on the activity of sub-MICs of different
quinolones and fluoroquinolones on various bacterial virulence factors
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Previous studies have demonstrated that exposure to low concentrations
of quinolones may reduce the ability of pathogens to cause clinical
symptoms by decreasing the level of production of virulence factors
(10, 15). The success of quinolone treatment in patients
with sub-MIC levels in their blood (44) supports the
clinical significance of our findings. These data must be interpreted
in relation to pharmacokinetic behavior when the effectiveness of
concentrations reaching mucosal surfaces and other tissues during
therapy is considered.
The usual therapeutic dose of rufloxacin is 400 mg, and our MIC for
E. coli was 1 µg/ml. The pharmacokinetic curve of a single dose of 400 mg (53) indicates that sub-MICs occur after
about 48 h (Fig. 5). Given that the
average overall effects of sub-MICs of rufloxacin can still be
considered significant at levels as low as 1/8 MIC (which corresponds
to 0.12 µg/ml), interpolation of this value with the pharmacokinetic
curve shows that the effects of sub-MICs may last as long as
(approximately) 132 h after drug administration (rufloxacin has a
half-life of 28 to 30 h).
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FIG. 5.
Mean plasma levels after a single 400-mg oral dose of
rufloxacin and interpolation with 1 and 1/8 MIC of rufloxacin.
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Such information can be used to optimize treatment schedules; for
example, single administrations for the treatment of noncomplicated lower urinary tract infections such as cystitis.
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ACKNOWLEDGMENTS |
The technical collaboration of G. Poli and T. Zuccotti is greatly
appreciated. We gratefully acknowledge the helpful comments made by V. Gianelle of the Centro Microscopia Elettronica, Azienda U.S.S.L. 38, Milan, Italy. We also gratefully acknowledge Bracco, Milan, Italy,
for the kind gift of rufloxacin.
This study was partially supported (60%) by a grant from MURST.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dept. of
Pharmacology, Via Vanvitelli 32, 20129 Milano, Italy. Phone:
39-2-70146363. Fax: 39-2-70146371. E-mail: bragapc{at}unimi.it.
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Antimicrobial Agents and Chemotherapy, May 1999, p. 1013-1019, Vol. 43, No. 5
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
