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Antimicrobial Agents and Chemotherapy, May 1998, p. 1005-1011, Vol. 42, No. 5
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Characteristics and Dynamics of Bacterial
Populations during Postantibiotic Effect Determined by Flow
Cytometry
Magnús
Gottfredsson,1,2,3,*
Helga
Erlendsdóttir,4
Ásbjörn
Sigfússon,1 and
Sigurdur
Gudmundsson2,3,4
Departments of
Immunology,1
Internal
Medicine,2 and
Microbiology,4 Landspítalinn
National University Hospital, and
University of Iceland
Medical School,3 Reykjavik, Iceland
Received 8 September 1997/Returned for modification 8 February
1998/Accepted 24 February 1998
 |
ABSTRACT |
Changes in bacterial ultrastructure after antibiotic exposure and
during the postantibiotic effect (PAE) have been demonstrated by
electron microscopy (EM). However, EM is qualitative and subject to
individual interpretation. In contrast, flow cytometry gives qualitative and quantitative information. The sizes and nucleic acid
contents of Escherichia coli and Pseudomonas
aeruginosa were studied during antimicrobial exposure as well as
during the PAE period by staining the organisms with propidium iodide
and analyzing them with flow cytometry and fluorescence microscopy. The
effects of ampicillin, ceftriaxone, ciprofloxacin, gentamicin, and
rifampin were studied for E. coli, whereas for P. aeruginosa imipenem and ciprofloxacin were investigated. After
exposure of E. coli to ampicillin, ceftriaxone, and
ciprofloxacin, filamentous organisms were observed by fluorescence
microscopy. These changes in morphology were reflected by increased
forward light scatter (FSC) and nucleic acid content as measured by
flow cytometry. For the
-lactams the extent of filamentation
increased in a dose-dependent manner after drug removal, resulting in
formation of distinct subpopulations of bacteria. These changes peaked
at 20 to 35 min, and bacteria returned to normal after 90 min after
drug removal. In contrast, the subpopulations induced by ciprofloxacin
did not return to normal until >180 min after the end of the
classically defined PAE. Rifampin resulted in formation of small
organisms with low FSC, whereas no distinctive characteristics were
noted after gentamicin exposure. For P. aeruginosa an
identifiable subpopulation of large globoid cells and increased nucleic
acid content was detected after exposure to imipenem. These changes
persisted past the PAE, as defined by viability counting. Swollen
organisms with increased FSC were detected after ciprofloxacin
exposure, even persisting during bacterial growth. In summary, for
-lactam antibiotics and ciprofloxacin, the PAE is characterized by
dynamic formation of enlarged cell populations of increased nucleic
acid content, whereas rifampin induces a decrease in size and nucleic
acid content in the organisms. Flow cytometry is an ideal method for
future studies of bacterial phenotypic characteristics during the PAE.
 |
INTRODUCTION |
The presence of a temporary
inhibition of bacterial growth after previous exposure to
antimicrobials, or postantibiotic effect (PAE), was initially described
in 1944 (4, 7), but interest in this phenomenon was
revitalized more than three decades later (30). The clinical
significance of a PAE has been attributed to its impact on
antimicrobial dosing, most markedly reflected in the increased use of
once-daily aminoglycosides both in normal and in immunocompromised
hosts (1, 2, 22, 24, 31). However, despite its potential
clinical significance, studies on metabolic events during PAE are
limited, and the mechanisms remain poorly understood (3, 9,
14-16, 35). It has been hypothesized that multiple mechanisms
may be involved, since patterns of bacterial DNA synthesis and
ultrastructure are dependent on the organism-antibiotic combination
studied (14, 15). However, most studies on
the mechanisms of PAE only measure average values of antibacterial
effects or PAEs (3, 5, 9, 14, 16, 17, 33, 35) in organisms
which potentially respond in a heterogeneous fashion to antimicrobial
agents (15, 27). Therefore, studies which quantitate the
potential variability of bacterial populations after exposure to
antibiotics are warranted. Flow cytometry is an ideal methodology for
study of this phenomenon, since it allows for rapid and sensitive
description of physical and biochemical characteristics of individual
bacteria or fungi, which already may be of value in antibiotic
susceptibility testing (8, 11, 25, 26, 36, 38-41). Although
flow cytometry has been used to study several different organisms
during continuous exposure to antibiotics, studies on bacteria during
the PAE using this technique are lacking. It is therefore of interest
to study whether specific physical or biochemical characteristics and
variability of bacterial populations during PAE can be detected by flow
cytometry. We used this method to make serial measurements of sizes and
nucleic acid contents of Escherichia coli and
Pseudomonas aeruginosa immediately after antimicrobial
exposure and during the PAE after exposure to several commonly used
antimicrobial agents.
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TABLE 1.
MICs, multiples of MICs, and duration of PAEs for
organism-antibiotic combinations studied by flow cytometry
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MATERIALS AND METHODS |
Organisms.
E. coli ATCC 25922 and P. aeruginosa ATCC 27853 (American Type Culture Collection,
Rockville, Md.) were used in this study.
Antibiotics.
Ampicillin was supplied by Astra
(Södertälje, Sweden), ceftriaxone by F. Hoffmann-La Roche
Ltd. (Basel, Switzerland), rifampin by Ciba-Geigy (Basel, Switzerland),
ciprofloxacin by Bayer AG (Leverkusen, Germany), gentamicin by Roussel
Laboratories Ltd. (Uxbridge, United Kingdom), imipenem by Merck Sharp & Dohme International (Rahway, N.J.), and tobramycin by Eli Lilly & Co.
(Indianapolis, Ind.). Stock solutions were prepared in sterile saline,
except for imipenem and rifampin, for which phosphate-buffered saline and methanol, respectively, were used, and solutions were stored at
20°C until use. MICs were determined by a standard microtiter dilution method (32).
Chemicals and media.
RPMI 1640 (with glutamine), Dulbecco's
minimal medium (DMM), and Hanks' balanced salt solution were purchased
from Gibco (Paisley, United Kingdom). Propidium iodide was from Sigma
(St. Louis, Mo.), and Mueller-Hinton agar was from Difco (Detroit,
Mich.).
Organism-antibiotic combinations.
Before each experiment,
three to four colonies of the test organism were transferred to 5 ml of
DMM, diluted serially, and grown overnight at 35.5°C to reach a
logarithmic-growth phase. Subsequently, the culture was adjusted to an
inoculum of ~107 CFU/ml by a 0.5 McFarland standard. The
bacteria were exposed to the antibiotics for 1 h in prewarmed RPMI
1640 at 37°C. The following combinations were used (concentrations
are given in parentheses): E. coli and ampicillin (2, 4, or
8 times the MIC), ceftriaxone (2, 4, or 8 times the MIC), gentamicin
(equivalent to or twice the MIC), ciprofloxacin (equivalent to or twice
the MIC), or rifampin (equivalent to or twice the MIC), and P. aeruginosa and imipenem (2, 4, or 8 times the MIC) or
ciprofloxacin (twice the MIC).
Drug removal.
After 1 h of antibiotic exposure, the
antibiotics were removed by spinning the bacterial culture twice for 5 min at 1,500 × g in DMM. The bacteria were
subsequently resuspended in prewarmed RPMI 1640.
PAE.
Viable counts were estimated immediately after drug
removal and then at 90-min intervals by serial dilution in ice-cold
normal saline and plating on Mueller-Hinton agar. The PAE was defined as previously described (7), as the difference in time
required for the exposed organisms and unexposed controls to grow 1 log10 unit (in CFU per milliliter). A negative value
therefore indicates a growth rate faster than that of control
organisms, whereas a positive value indicates a delay in growth.
Fixation and staining for nucleic acids.
Immediately after
drug removal, aliquots were taken from the bacterial culture at regular
intervals (at 0, 20, 35, 50, 70, and 90 min, and at 90-min intervals
thereafter) (14), fixed in formaldehyde buffer (final
concentration, 2%), and stored at 4°C. The samples were subsequently
centrifuged at 1,500 × g for 12 min. The organisms in
the pellet were stained with propidium iodide (final concentration, 200 µg/ml) in 20% ethanol for 4 h in the dark at 37°C. After
staining, the organisms were washed twice in Hanks' balanced salt
solution for 5 min, resuspended in normal saline, and analyzed within
2 h.
Flow cytometry and microscopy.
The samples were analyzed in
a flow cytometer (FACScan; Becton Dickinson, Sunnyvale, Calif.). Five
thousand to 10,000 bacteria were analyzed at each time point with a
blue argon laser (488-nm wavelength at 500 mW). The instrument was set
at 10-fold linear amplification of narrow-angle forward light scatter
(FSC) and at logarithmic amplification of orange (or propidium iodide)
fluorescence 2 (FL2). The threshold was adjusted for particles smaller
than bacteria. At each time point, the size and nucleic acid content were measured (by FSC and FL2, respectively). Samples were also examined with a fluorescence microscope (Leitz Laborlux D) at selected
time points and photographed (with a Nikon FX-35A camera with Kodak
Ektachrome 800 ASA film). Each experiment (organism-antibiotic combination) was performed two to five times on separate days, and each
combination was analyzed by flow cytometry.
Data analysis and statistics.
Gating on particles that
stained for double-stranded nucleic acids (FL2) was performed with Cell
Quest software (Becton Dickinson) as shown in Fig. 2 (left panels). The
gates were based on samples from the control culture during the
logarithmic-growth phase and adjusted so that the lower limits in FSC
were identical to the threshold to avoid the gating out of small
organisms. Three size intervals, based on the size distribution of
control organisms, were defined as within 2 standard deviations (SDs),
between 2 and 4 SDs and >4 SDs, representing the 97.4th, 97.4th to
99.2th, and >99.2th percentiles of the control size distribution,
respectively (see Fig. 2, right panels). Comparisons between control
organisms and antibiotic-exposed bacteria were based on these
predetermined intervals. The Kolmogorov-Smirnov nonparametric test was
used for statistical comparisons between control bacteria and organisms in the PAE phase. The level of significance was set at a P
value of P <0.01.
 |
RESULTS |
PAE.
The organism-antimicrobial combinations, with their
respective MICs and PAEs, are shown in Table 1. Growth curves from
typical PAE experiments with ceftriaxone and ciprofloxacin (each at a concentration equivalent to twice the MIC) against E. coli
are shown in Fig. 1A.

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FIG. 1.
(A) A typical PAE experiment for E. coli ATCC
25922 after exposure to ceftriaxone or ciprofloxacin (each at a
concentration equivalent to twice the MIC), as determined by viability
counting. As shown, no PAE was seen after ceftriaxone exposure, but
ciprofloxacin induced a PAE of 1.9 h. The organisms were stained
with propidium iodide and examined by fluorescence microscopy. (B
through D) Photomicrographs of untreated control organisms (B),
ceftriaxone-exposed organisms 35 min after drug removal (C), and
ciprofloxacin-exposed organisms 270 min after drug removal (D). Both
antibiotics induced filamentation, but this morphological form
persisted past the classically defined PAE in organisms exposed to
ciprofloxacin. Magnification, ×880.
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Fluorescence microscopy.
Examples of bacterial morphology as
seen through the fluorescence microscope are shown in Fig. 1B through
D. Normal-appearing rods are shown in Fig. 1B. The PAE phase was
characterized by filament formation after exposure to ceftriaxone and
ciprofloxacin. The filaments seen 35 min after ceftriaxone exposure
(Fig. 1C) reverted to normal morphology within 90 min, whereas the
changes induced by ciprofloxacin, seen 270 min after exposure (Fig.
1D), persisted past resumption of regrowth.
Flow cytometry.
Typical scattergrams showing FSC and FL2 for
the untreated control organisms E. coli and P. aeruginosa are shown in Fig. 2 (left
panels). FSC is an indicator of size, whereas FL2 represents double-stranded nucleic acid (DNA and RNA) content. As shown, viable
organisms in the logarithmic-growth phase had fairly uniform normally
distributed size characteristics and nucleic acid contents. Debris
outside R1 and R2 was gated out. Three size intervals were defined
based on the sizes of the control organisms in the logarithmic-growth phase; these represent the 97.4th, 97.4th to 99.2th, and >99.2th percentile of the control size distribution (Fig. 2, right panels).

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FIG. 2.
(Left panels) Dot plots of FSC peak height (shown as
FSC-H), indicative of size, and FL2 peak height (shown as FL2-H),
indicative of double-stranded nucleic acid content, for the untreated
control organisms E. coli ATCC 25922 and P. aeruginosa ATCC 27853 in the logarithmic-growth phase. The gates
used are also shown (R1 for E. coli and R2 for P. aeruginosa). (Right panels) Histograms for bacterial size (FSC-H),
with three intervals, representing 2 SDs (97.4th percentile), 2 to 4 SDs (97.4th to 99.2th percentile), and >4 SDs (>99.2th percentile) of
the control size distribution. These intervals were used for
comparisons between controls and antibiotic-exposed organisms.
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Effects of antibiotics.
Five antibiotics with different
mechanisms of action were tested against E. coli, and the
sizes and nucleic acid contents of the organisms were compared to the
control distribution. The
-lactams ampicillin and ceftriaxone do not
induce a PAE in E. coli, as determined by viability counting
(Table 1). Still, a profound effect on bacterial size and nucleic acid
content was observed after exposure to these antibiotics. An example of
the effects induced by ampicillin is given in Fig.
3 (upper panels). As shown, the bacterial
population was characterized by enlarged organisms which continued to
increase in size even after drug removal. At 20 to 35 min after drug
removal, two populations of bacteria were identified, with large
bacteria dominating the culture (Fig. 3, upper left panel), as distinct
from the control (P < 0.001). Parallel to the increase
in bacterial size, an increase in nucleic acid content occurred (Fig.
3, upper middle panel). Thus, after 35 min, less than 10% of the
bacterial population was within 2 SDs of the size of the control
population; the remainder showed increased size and increased nucleic
acid staining. These changes are summarized in Fig. 3 (upper right
graph); rapid convergence towards normal bacterial characteristics was
observed during the initial 90 min of the experiment. Similar changes
were noted after ceftriaxone exposure (data not shown). For the
-lactams, the extent of the changes in FSC and FL2 was not dependent
on the multiple of the MIC tested.

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FIG. 3.
Histograms showing a comparison of the size distribution
(FSC-H) (left panels) and nucleic acid content (FL2-H) (middle panels)
of E. coli during the PAE after exposure to ampicillin at a
concentration equivalent to twice the MIC at 35 min after drug removal
and after exposure to rifampin at a concentration equivalent to the MIC
(lower panels) at 90 min after drug removal. Dotted-and-dashed lines,
control organisms; solid lines, organisms previously exposed to the
antibiotics. (Upper right graph) Progressive changes in size, compared
to sizes of control organisms, as a function of time after previous
exposure to ampicillin. (Lower right graph) Summary of the minimal
changes in size that were noted after previous exposure to rifampin.
The sizes of the antibiotic-treated organisms were compared to three
size intervals derived from the control, which are described in text
and shown in Fig. 2. Open circles, bacteria in the PAE phase which were
within 2 SDs of control size; solid squares, bacteria within 2 to 4 SDs; open squares, organisms >4 SDs from the control distribution.
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The PAE phase after rifampin exposure (Fig.
3, lower panels) was
characterized by a fairly uniform population of small organisms
(lower
left panel) with low nucleic acid content (lower middle
panel). These
growth-suppressed organisms were significantly smaller
than control
bacteria (
P < 0.001). As with ciprofloxacin, these
changes outlasted the PAE, as determined by viability counting
(270 versus 90 min), and were followed by reversal to normal characteristics
during late regrowth.
Filamentation was also observed after ciprofloxacin exposure (Fig.
1D).
However, in contrast to results with the

-lactams,
this
morphological alteration did not appear until after drug
removal but
continued to increase in an apparently dose-dependent
manner. An
example of these effects, observed 270 min after drug
removal, is shown
in Fig.
4 (left panels). The sizes of the
antibiotic-exposed
organisms were clearly different from those of the
control population
(
P < 0.001). In parallel, an
increase in nucleic acid content
was also seen (Fig.
4, middle panels).
The subpopulation of filaments
continued to increase for 180 to 360 min
after drug removal (Fig.
4, right panels), thus outlasting the
classically defined PAE.
Similarly, the increased nucleic acid content
was still noted
at the end of the experiment (data not shown).

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FIG. 4.
Histograms showing a comparison of the size
distributions (FSC-H; left panels) and nucleic acid contents (FL2-H;
middle panels) of E. coli during the PAE after exposure to
ciprofloxacin at a concentration equivalent to the MIC and at a
concentration equivalent to twice the MIC at 270 min after drug
removal. Dotted-and-dashed lines, control organisms; solid lines,
bacteria previously exposed to ciprofloxacin. Graphs (right panels)
show progressive changes in size compared to sizes of controls. The
sizes of antibiotic-treated organisms were compared to three control
size intervals described in the text and shown in Fig. 2. Open circles,
bacteria within 2 SDs of control size; solid squares, bacteria within 2 to 4 SDs; open squares, organisms >4 SDs from the control
distribution.
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Gentamicin did not induce any measurable change in bacterial
characteristics during PAE (data not shown).
For
P. aeruginosa, two antibiotics with different mechanisms
of action were tested. Two distinct subpopulations of bacteria
were
identified during PAE after exposure to imipenem, a

-lactam
well
known to induce PAE: small organisms and globoid cells of
increased
size (significantly different from the control;
P <
0.001). In contrast to results seen with the
E. coli-

-lactam
combinations, the large globoid cells persisted
for >180 min (Fig.
5, upper left panel).
A corresponding increase in nucleic acid
content was also seen (Fig.
5,
upper middle panel). The subpopulation
of globoid organisms persisted
past the classically defined PAE
(>180 versus 85 min) (Fig.
5, upper
right panel).

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FIG. 5.
Histograms showing the size distributions (FSC-H; left
panels) and nucleic acid contents (FL2-H; middle panels) of P. aeruginosa during the PAE after exposure to imipenem at a
concentration equivalent to twice the MIC and to ciprofloxacin at a
concentration equivalent to the MIC at 180 and 70 min after drug
removal, respectively. Dotted-and-dashed lines, control organisms;
solid lines, antibiotic-exposed organisms. The graphs (right panels)
show progressive changes in size compared to sizes of controls. The
sizes of antibiotic-treated organisms were compared to three control
size intervals described in text and shown in Fig. 2. Open circles,
bacteria within 2 SDs of control size; solid squares, bacteria within 2 to 4 SDs; open squares, organisms >4 SDs from the control
distribution.
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Ciprofloxacin produced changes in
P. aeruginosa which were
characterized by organism swelling (increased diameter and similar
length) (Fig.
5, lower left panel) and marginally but consistently
increased nucleic acid content (Fig.
5, lower middle panel), but
filamentation was not observed at the concentrations tested.
 |
DISCUSSION |
In this study we analyzed bacterial characteristics during the PAE
by flow cytometry. This method has previously been well described for
measurements of antibiotic susceptibility in bacteria and fungi
(11, 13, 26, 28, 35, 36, 39-41). It is also ideal for
studies on physical and chemical characteristics of bacteria, since it
allows for rapid analysis of a large number of individual organisms
with high accuracy (8). By performing our sampling at short
time intervals, we were able to demonstrate dynamic changes in the
organisms after removal of the antibiotics.
We studied the effects of five different antibiotics on flow-cytometric
findings for E. coli during PAE: ampicillin, ceftriaxone, ciprofloxacin, rifampin, and gentamicin. Of interest, the
-lactams ampicillin and ceftriaxone, which do not induce a PAE in this species
as defined by viability counting, induced progressive bacterial
elongation for 20 to 35 min after removal of the antibiotics. This
observation was confirmed by fluorescence microscopy (Fig. 1C). It has
previously been shown that at low concentrations, ampicillin binds
preferentially to penicillin binding protein-3 (PBP-3), causing
filament formation (37). Our results thus suggest that the
period immediately following drug removal may be characterized by
persistent inhibition of PBP-3. Therefore, although it has been claimed
that
-lactams do not induce a PAE in gram-negative bacteria
(7), with the exception of imipenem (6, 18, 19, 34), this observation suggests persistent intracellular
antimicrobial action. Similarly, it has been shown that the PAE in
streptococci after penicillin exposure can be induced by inhibition of
PBP activity (42).
Rifampin, an inhibitor of DNA-dependent RNA polymerase, caused a
uniform and consistent decrease in bacterial size and nucleic acid
content which persisted past the duration of the PAE, as determined by
viability counting. This antibiotic has previously been shown to result
in a profound metabolic suppression in bacteria during the PAE
(14). Our current results do not explain why this abnormal
morphology was so prevalent in bacteria during the PAE after rifampin
exposure. Persistence of the antibiotic, with inhibition in RNA
transcription and a subsequent block in protein translation and growth
arrest, could potentially account for the presence of these small
bacteria. In contrast to the other antimicrobials tested, gentamicin
did not induce any discernible changes in bacterial characteristics, as
measured by our methodology.
Ciprofloxacin, a quinolone antimicrobial agent which inhibits DNA
gyrase and blocks DNA replication, has been well documented to cause
filamentation in E. coli (10, 27). In contrast to the relatively short-lived effects of the
-lactams, the effects seen
after ciprofloxacin exposure were characterized by a progressive increase in filament formation, lasting past the classically defined PAE (Fig. 4). In fact, at 6 h after drug removal, close to half the bacterial population previously exposed to a concentration of the
drug equivalent to twice the MIC was still exhibiting filamentation. These results are in accordance with those of Lorian et al., who observed discrepancy between the PAE as defined by viability counting and the PAE as defined by morphology (27). Due to the
progressive increase in filamentation after drug removal, these
observations suggest that the aberrant morphology may be a result of
persistent intracellular antimicrobial action. We have previously shown
that the PAE phase after ciprofloxacin exposure is characterized by a
progressive increase in DNA synthesis (14), which could be due to an increase in DNA repair as a result of persistent
antimicrobial action during the PAE. Alternatively, the increase in DNA
could be due to continued attempts at DNA replication, since DNA
polymerase activity is not hampered, but this replication is abortive
because the circular DNA can not be separated as a result of gyrase
inhibition.
We studied the effects of two antibiotics, imipenem and ciprofloxacin,
on PAE in P. aeruginosa. Imipenem primarily inhibits PBP-2
(21). It has previously been shown that this interaction results in the formation of ovoid or globoid cells (15, 20, 37). Imipenem is unique among
-lactams for its ability to
induce a PAE in gram-negative bacteria (6, 18, 19, 34). We
were able to demonstrate a progressive increase in formation of a
subpopulation of globoid cells which persisted past the classically
defined PAE. Competitive inhibition of the D2 porin, which facilitates the uptake of basic amino acids and imipenem through the outer membrane
(23), has been shown to reduce the duration of PAE after
imipenem exposure in P. aeruginosa (5),
suggesting that adequate uptake and PBP-2 binding may be prerequisite
for PAE induction. Our observations thus suggest that intracellular
persistence of this antibiotic could account for its PAE.
At the concentrations tested, ciprofloxacin did not induce
filamentation in P. aeruginosa. The size was slightly
increased, however, probably as a result of bacterial swelling that has
been described previously (15). Similarly, a small but
consistent parallel increase in nucleic acid content, which has
previously been reported from a study using another methodology
(14), was also observed.
In summary, PAE measured by viability counts represents only a mean of
the alterations observed for different populations of bacteria. We have
shown that the PAE is characterized by formation of bacterial
subpopulations of remarkable heterogeneity. Formation of these
populations is a dynamic process and can be quantitated by flow
cytometry. In some cases, as for
-lactams, the morphological changes
induced by continuous exposure to the antibiotics continue to increase
for a variable amount of time, whereas ciprofloxacin induces a
progressive increase in filamentation which lasts past the classically
defined PAE. Future studies could further identify other changes, such
as those that measure membrane potential, respiratory activity
(29), or lipopolysaccharide surface antigen expression
(12).
 |
ACKNOWLEDGMENTS |
This study was supported in part by the Icelandic Science
Foundation (to S.G.), the University of Iceland Research Fund (to S.G.)
and the Icelandic Immunology Society Science Fund (to M.G.).
We thank S. A. Desai and J. R. Perfect for helpful comments
on the manuscript.
 |
FOOTNOTES |
*
Corresponding author. Present address: Department of
Internal Medicine, Division of Infectious Diseases, P.O. Box 3824, Duke University Medical Center, Durham, NC 27710. Phone: (919) 684-2660 or
(919) 681-5055. Fax: (919) 684-8902. E-mail:
gottf003{at}mc.duke.edu.
 |
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Antimicrobial Agents and Chemotherapy, May 1998, p. 1005-1011, Vol. 42, No. 5
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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