ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1998, p. 277–281 Vol. 42, No.

2
0066-4804/98/$04.0010
Copyright © 1998, American Society for Microbiology

Differential Release of Lipoteichoic and Teichoic Acids from

Streptococcus pneumoniae as a Result of Exposure to
b-Lactam Antibiotics, Rifamycins, Trovafloxacin,
and Quinupristin-Dalfopristin
K. STUERTZ,1 H. SCHMIDT,1 H. EIFFERT,2 P. SCHWARTZ,3 M. MÄDER,1 AND R. NAU1*
1 2 3
Departments of Neurology, Medical Microbiology, and Morphology, University of Göttingen, Göttingen, Germany
Received 11 April 1997/Returned for modification 14 October 1997/Accepted 15 November 1997

The release of lipoteichoic acid (LTA) and teichoic acid (TA) from a Streptococcus pneumoniae type 3 strain

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during exposure to ceftriaxone, meropenem, rifampin, rifabutin, quinupristin-dalfopristin, and trovafloxacin
in tryptic soy broth was monitored by a newly developed enzyme-linked immunosorbent assay. At a concen-
tration of 10 mg/ml, a rapid and intense release of LTA and TA occurred during exposure to ceftriaxone
(3,248 6 1,688 ng/ml at 3 h and 3,827 6 2,133 ng/ml at 12 h) and meropenem (2,464 6 1,081 ng/ml at 3 h and
2,900 6 1,364 ng/ml at 12 h). Three hours after exposure to rifampin, rifabutin, quinupristin-dalfopristin, and
trovafloxacin, mean LTA and TA concentrations of less than 460 ng/ml were observed (for each group, P < 0.01
versus the concentrations after exposure to ceftriaxone). After 12 h of treatment, the LTA and TA concentra-
tions were 463 6 126 ng/ml after exposure to rifampin, 669 6 303 ng/ml after exposure to rifabutin, and
1,236 6 772 ng/ml after exposure to quinupristin-dalfopristin (for each group, P < 0.05 versus the concen-
trations after exposure to ceftriaxone) and 1,745 6 1,185 ng/ml after exposure to trovafloxacin (P 5 0.12 versus
the concentration after exposure to ceftriaxone). At 10 mg/ml, bactericidal antibacterial agents that do not
primarily affect cell wall synthesis reduced the amount of LTA and TA released during their cidal action
against S. pneumoniae in comparison with the amount released after exposure to b-lactams. Larger quantities
of LTA and TA were released after treatment with low concentrations (13 the MIC and 13 the minimum
bactericidal concentration) than after no treatment for all antibacterial agents except the rifamycins. This does
not support the concept of using a low first antibiotic dose to prevent the release of proinflammatory cell wall
components.

The release of bacterial cell wall components into the cere-
brospinal fluid (CSF) during antibiotic-induced bacterial lysis
may cause a burst of meningeal inflammation subsequent to
the initiation of antibiotic therapy (8, 16).
Pneumococcal cell wall components stimulate the synthesis
of tumor necrosis factor alpha (TNF-a) and interleukin-6 by
human monocytes (4). Heat-inactivated pneumococci and
pneumococcal cell walls exert a cytotoxic effect in cultures of
microglia and astrocytes. No neuronal damage was observed
when neurons were cultured alone, whereas coculture of neu-
rons and glial cells exposed to pneumococcal cell walls resulted
in the death of neurons (6). These findings raise the possibility
that pneumococcal cell wall components mediate the damage
of brain tissue during meningitis not only by inducing the
invasion of leukocytes but also by directly stimulating glial
cells.
Teichoic acid (TA) and lipoteichoic acid (LTA) are the most
potent proinflammatory constituents of the membrane and the
cell wall of Streptococcus pneumoniae. They are composed of
repetitive oligosaccharide units conjugated to phosphorylcho-
line (1). LTA additionally has a hydrophobic trihexosyldiacyl-
glycerol residue. When injected into the subarachnoid space,
TA and LTA cause profound meningeal inflammation (16).
In order to attenuate subarachnoid space inflammation, ad-
* Corresponding author. Mailing address: Department of Neurol-
ogy, University of Göttingen, Robert-Koch-Str. 40, D-37075 Göttin-
gen, Germany. Phone: 49-551-398455 or 49-551-396684. Fax: 49-551-
398405. E-mail: rnau@gwdg.de.
junctive anti-inflammatory drugs have been introduced into
the therapy for bacterial meningitis. Yet, although different
regimens have been proven to be effective in animal models,
only dexamethasone therapy has been successfully established
in clinical practice. Dexamethasone, however, has been shown
to decrease the concentrations of hydrophilic antibiotics in
CSF and to affect CSF sterilization for those pathogens with a
decreased sensitivity to antibiotics (10). Furthermore, dexa-
methasone may promote neuronal damage in the dentate gyrus
of the hippocampal formation by inhibiting the uptake of the
neurotoxic excitatory amino acid glutamate by astrocytes (17).
A therapeutic approach which circumvents immunosuppres-
sion and the associated problems is to decrease the release of
bacterial proinflammatory cell wall products into the CSF. This
may be possible by using bactericidal antibacterial agents that
do not directly affect cell wall synthesis. Such compounds may
terminate the ability of bacteria to grow, replicate, and release
cell wall components several hours before bacterial lysis occurs
(7). Quinolones, rifamycins, and pristinamycins are promising
in this regard: they are rapidly bactericidal against pneumo-
cocci (2, 9, 12). At lower concentrations quinolones inhibit the
topoisomerase II, and at high concentrations they also affect
protein synthesis and RNA synthesis. Rifamycins inhibit RNA
synthesis, and pristinamycins affect protein synthesis.
In the present study we addressed the question of whether
different modes of action of antibacterial agents influence the
amount of proinflammatory cell wall components released
from S. pneumoniae. For this purpose we studied the release of
LTA and TA from pneumococci during in vitro treatment with
the quinolone trovafloxacin, the rifamycins rifampin and ri-

277
278 STUERTZ ET AL.
fabutin, and the combination of streptogramins quinupristin
and dalfopristin (RP59500) in comparison with the release
during treatment with the cephalosporin and carbapenem an-
tibiotics ceftriaxone and meropenem using a newly developed
enzyme-linked immunosorbent assay (ELISA).
(The study has been presented in part at the 8th European
Congress of Clinical Microbiology and Infectious Diseases,
Lausanne, Switzerland, 25 to 28 May 1997.)
MATERIALS AND METHODS
Reagents and bacterial strains. Rifabutin was kindly provided by Pharmacia
Upjohn (Milano, Italy), and RP59500, consisting of 30% quinupristin and 70%
dalfopristin, was provided by Rhone-Poulenc Rorer (Cologne, Germany). Trova-
floxacin was a gift from Pfizer (Karlsruhe, Germany), and ceftriaxone was pro-
vided by Hoffmann-La Roche (Grenzach Wyhlen, Germany). Rifampin and
meropenem were purchased from Grünenthal (Stolberg, Germany).
The mouse immunoglobulin A (IgA) monoclonal antibody (MAb) TEPC-15,
which recognizes the phosphorylcholine residues of pneumococcal LTA and TA,
was purchased from Sigma, Deisenhofen, Germany.
Bacteria were grown at 37°C in tryptic soy broth (Difco, Detroit, Mich.). The
unencapsulated R6 strain (a gift from W. Fischer, Erlangen, Germany) was used
for the preparation of LTA, and a penicillin-sensitive S. pneumoniae type 3 strain
(15) was used to investigate the release of cell wall components. The MICs and
minimal bactericidal concentrations (MBCs) for this strain were determined by
the broth macrodilution method according to the guidelines of the National
Commitee for Clinical Laboratory Standards, and the respective values were as
follows: ceftriaxone, 0.03, and 0.06 mg/ml; meropenem, 0.008 and 0.03 mg/ml;
quinupristin-dalfopristin, 0.06 and 0.12 mg/ml; rifabutin, 0.008 and 0.06 mg/ml;
rifampin, 0.008m and 0.06 mg/ml; and trovafloxacin, 0.06 and 0.12 mg/ml.
Release of proinflammatory components as a result of exposure to antibac-
terials. S. pneumoniae type 3 was grown to an optical density at 578 nm of
approximately 0.1. Cells were collected by centrifugation and were resuspended
in fresh tryptic soy broth to a final concentration of approximately 108 CFU/ml.
Control cultures were grown after resuspension without antibacterial agents. The
resuspension in fresh broth ensured growth in the logarithmic phase for at least
3 h, i.e., drugs were added during the logarithmic phase of growth. At the end of
the logarithmic phase, bacterial titers approximated 1010 CFU/ml (see Fig. 1G).
In the first set of experiments, ceftriaxone, rifampin, rifabutin, quinupristin-
dalfopristin, trovafloxacin, and meropenem at concentrations of 10 mg/ml each
were added to 15-ml aliquots of the bacterial suspension. The uniform concen-
tration of 10 mg/ml was used to ensure a maximum bactericidal effect for all drugs
studied. Samples (1 ml) for the detection of free LTA and free TA were collected
before and at 1, 2, 3, 4, 6, 8, 10, and 12 h after the addition of drugs. For each
antibacterial agent, this experiment was repeated five times on separate days.
In the second set of experiments, the release of LTA and TA after 1, 2, 3, 4,
6, 8, 10, and 12 h of exposure to ceftriaxone, rifampin, rifabutin, quinupristin-
dalfopristin, trovafloxacin, and meropenem was studied at four different concen-
trations: the MIC and the MBC, as determined previously, and 0.5 and 2 mg/ml
(n 5 3 for each antibacterial agent at each concentration).
Samples were centrifuged at 10,000 3 g for 10 min, and the supernatants were
frozen at 220°C for the measurement of LTA and TA. Bacterial counts were
determined at 0, 3, 6, 9, and 12 h by plating 10-ml samples of 10-fold dilutions of
bacteria onto blood agar plates. The bactericidal rates (dlog CFU per milliliter
per hour) were determined by log-linear regression analysis of bacterial titers
(CFU per milliliter) versus time, and the coefficient of total determination (r2)
was given in brackets.
Sandwich ELISA for detection of pneumococcal LTA and TA. LTA was pre-
pared from S. pneumoniae R6 (1). Bacteria were grown in 10 liters of tryptic soy
broth overnight at 37°C. The bacterial cells were disrupted with a French pres-
sure cell and were then extracted with methanol and chloroform at different
concentration ratios. LTA was detected in the aqueous layer. Following the
removal of methanol, the suspension was further purified by hydrophobic inter-
action chromatography on octyl-Sepharose (Pharmacia Biotech, Freiburg, Ger-
many). The purification grade was determined by using the choline/phosphate
ratio, which is 0.66 in pure LTA (1).
Polyclonal antibodies were raised in two New Zealand White rabbits subcu-
taneously immunized with 500 mg of LTA mixed with an equal volume of
incomplete Freund’s adjuvant. Immunization was repeated every 4 weeks until
high titers ($1:32,000) were obtained. The sera were preserved at 220°C.
The ELISA developed for the quantification of LTA and TA release from
bacterial cultures used MAb TEPC-15 as the capture antibody and the polyclonal
antiserum raised against LTA as the detector antibody. MAb TEPC-15 was
coated overnight on 96-well microtiter plates (Nunc GmbH, Wiesbaden, Ger-
many) at a concentration of 5 mg/ml. Blocking as well as the following steps were
carried out with 5% fetal calf serum in phosphate-buffered saline. Appropriate
dilutions of samples or twofold dilutions of purified LTA as a standard were
incubated for 2 h, followed by a 2-h incubation with polyclonal antiserum (1:
4,000). Bound rabbit antibodies were quantified with peroxidase-conjugated goat
anti-rabbit IgG antibodies (1:8,000; Dianova, Hamburg, Germany). Enzyme ac-
ANTIMICROB. AGENTS CHEMOTHER.
tivity was determined with a 1-mg/ml solution of 2,29-azino-di(3-ethylbenzthia-
zolinsulfonate in 50 mM phosphate-citrate-buffer (pH 4.4) containing 3 mM
sodium perborate. Absorption was determined after 30 min of incubation in an
ELISA reader (Titertek Multiskan Plus MK II; Flow Laboratories GmbH, Meck-
enheim, Germany) at 405 nm.
Standard curves were constructed for each assay. Intra-assay and interday
coefficients of variation determined by repeatedly measuring quality control
samples spiked with LTA were 8.4 and 10.9%, respectively, at 300 ng/ml and 7.1
and 7.1%, respectively, at 1,800 ng/ml. No cross-reactions were observed with
supernatants from cultures of Listeria monocytogenes, Haemophilus influenzae,
Staphylococcus epidermidis, viridans group streptococci, Enterococcus faecalis,
Escherichia coli, Pseudomonas aeruginosa, Corynebacterium xerosis, Salmonella
enteritidis, Proteus spp., Enterobacter sakazakii, and Enterobacter cloacae grown in
tryptic soy broth and killed by exposure to ceftriaxone or vancomycin. The
ELISA did recognize components released by Staphylococcus aureus. This reac-
tion could easily be eliminated by adding human serum or solutions containing
human IgG and was assumed to be due to protein A, a cell wall component of
S. aureus known to bind specifically to the Fc portion of IgG (14). The addition
of IgG in excess did not diminish the ELISA reaction in response to purified
LTA or samples containing pneumococcal cell wall products. Ceftriaxone, mero-
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penem, quinupristin-dalfopristin, rifabutin, rifampin, and trovafloxacin did not
bind to TA or LTA or interfere with the assay.
Electron microscopy. For scanning electron microscopy, S. pneumoniae type 3
was grown to the late logarithmic phase in tryptic soy broth and was then treated
for 3 or 6 h with 10 mg of ceftriaxone or rifampin per ml. Thereafter, the bacteria
were transferred to poly-L-lysine-coated coverslips and fixed with 2% glutaral-
dehyde in 0.1 M cacodylate buffer for 24 h. After dehydration in graded ethanol,
samples were dried in a critical-point dryer (Polaron, Watford, United King-
dom). After being mounted on stubs, the specimens were coated with gold-
palladium in a cool sputter coater (Fisons Instruments, Uckfield, United King-
dom) and were examined in an electron microscope (DSM 960; Zeiss,
Oberkochen, Germany).
Statistics. Ceftriaxone (10 mg/ml) was used as the standard antibacterial agent,
and the results obtained with ceftriaxone were compared with those obtained
with the other drugs at the same concentration by unpaired analysis of variance.
The P values were adjusted for repeated testing by the Bonferroni method.
RESULTS
The bactericidal rates during exposure to 10 mg of ceftriax-
one (20.60 6 0.08 Dlog CFU/ml/h; r2 5 0.98 6 0.004), mero-
penem (20.87 6 0.15 Dlog CFU/ml/h; r2 5 0.98 6 0.02),
quinupristin-dalfopristin (20.68 6 0.22 Dlog CFU/ml/h; r2 5
0.93 6 0.06), and trovafloxacin (20.79 6 0.16 Dlog CFU/ml/h;
r2 5 0.96 6 0.08) per ml were slightly higher than those during
exposure to 10 mg of rifabutin (20.45 6 0.05 Dlog CFU/ml/h;
r2 5 0.85 6 0.05) and rifampin (20.37 6 0.04 Dlog CFU/ml/h;
r2 5 0.91 6 0.05) per ml (values are means 6 standard devi-
ations). The differences in the bactericidal rates did not reach
statistical significance. Twelve hours after the initiation of
treatment, the concentrations of viable bacteria were below the
detection limit of 102 CFU/ml after exposure to ceftriaxone,
meropenem, quinupristin-dalfopristin, and trovafloxacin,
whereas in the rifabutin- and rifampin-treated cultures the
mean bacterial counts were 4.8 3 102 and 2.6 3 103/ml, re-
spectively (Fig. 1).
A rapid and intense release of LTA and TA occurred in the
first hours of exposure to 10 mg of ceftriaxone per ml (means 6
standard deviations, 3,248 6 1,688 ng/ml at 3 h and 3,827 6
2,133 ng/ml at 12 h) and 10 mg of meropenem per ml (2,464 6
1081 ng/ml at 3 h and 2,900 6 1,364 ng/ml at 12 h) (the
difference was not significant). Three hours after rifampin and
rifabutin exposure (10 mg/ml each), mean LTA and TA con-
centrations of 166 6 65 and 267 6 121 ng/ml, respectively,
were observed (for each group, P , 0.01 versus the concen-
trations after exposure to ceftriaxone). Three hours of expo-
sure to 10 mg of quinupristin-dalfopristin per ml resulted in
LTA and TA concentrations of 380 6 285 ng/ml, and 10 mg of
trovafloxacin per ml released 455 6 89 ng of LTA and TA per
ml during the first 3 h (P , 0.01 versus the concentrations after
exposure to ceftriaxone). After 12 h of treatment with 10 mg of
the various drugs per ml, the LTA and TA concentrations were
463 6 126 ng/ml after exposure to rifampin, 669 6 303 ng/ml
VOL. 42, 1998 RELEASE OF LIPOTEICHOIC AND TEICHOIC ACIDS 279

TABLE 1. Release of LTA and TA by S. pneumoniae type 3 during

treatment with antibacterial agents at different concentrations
Concn (ml/ml) of
Antibacterial Concn Bactericidal rate LTA at the
agent (mg/ml) (Dlog CFU/ml) following times:

3h 12 h

Ceftriaxone MIC 20.29 4,640 7,382

MBC 20.47 4,625 6,693
0.5 20.55 2,282 2,313
2 20.68 2,217 1,916

Rifabutin MIC 20.21 482 2,673

MBC 20.37 319 627
0.5 20.39 507 756
2 20.40 378 803

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Rifampin MIC 20.13 623 3,050
MBC 20.36 470 3,641
0.5 20.36 352 542
2 20.49 332 536

Trovafloxacin MIC 0.00 2,138 9,512

MBC 0.01 2,341 13,732
0.5 20.42 1,551 7,357
2 20.50 729 1,176

Quinupristin- MIC 20.03 607 4,063

dalfopristin
MBC 20.12 801 5,191
0.5 20.37 453 2,777
2 20.63 415 800

Meropenem MIC 20.17 1,796 8,022

MBC 20.47 3,312 5,354
0.5 20.54 1,807 2,416
2 20.69 1,799 2,213

No antibacterial 0.34 720 3,433

agent

treated cultures, although ceftriaxone and meropenem were

FIG. 1. Release of LTA and TA by S. pneumoniae type 3 during treatment
with 10 mg of ceftriaxone (A), rifabutin (B), rifampin (C), quinupristin-dalfo-
pristin (D), trovafloxacin (E), and meropenem (F) per ml and by untreated
cultures (G) (closed circles). The means 6 standard errors of the means of five
experiments are shown. The asterisks denote significant differences versus the
results for ceftriaxone-treated bacteria. The open squares represent the bacterial
titers (means 6 standard errors of means).
after exposure to rifabutin, and 1,236 6 772 ng/ml after expo-
sure to quinupristin-dalfopristin (for each group, P , 0.05
versus the concentrations after exposure to ceftriaxone) and
1,745 6 1,185 ng/ml after exposure to trovafloxacin (P 5 0.12
versus the concentration after exposure to ceftriaxone) (Fig.
1). Meropenem at 10 mg/ml did not cause a significant reduc-
tion in the release of LTA and TA at any time (2,464 6 1,081
ng/ml at 3 h and 2,900 6 1,363 ng/ml at 12 h) when compared
to the release resulting from exposure to ceftriaxone.
For all antibacterial agents studied, the release of LTA and
TA from S. pneumoniae type 3 was dose dependent. At low
concentrations (13 the MIC and 13 the MBC), the LTA and
TA levels in the supernatants were higher than those at higher
concentrations of the antibacterial agents. At 13 the MIC and
13 the MBC, more LTA and TA were released from S. pneu-
moniae cultures in the presence of ceftriaxone, meropenem,
trovafloxacin, and quinupristin-dalfopristin than from non-
bactericidal and trovafloxacin and quinupristin-dalfoprisitin
were bacteriostatic at these concentrations (Table 1; Fig. 2A).
The LTA and TA concentrations released from bacteria ex-
posed to rifampin at 13 the MIC and 13 the MBC and to
rifabutin at 13 the MIC were close to the concentrations
observed in the supernatants of untreated cultures (Table 1;
Fig. 2B).
Scanning electron microscopy revealed differences in bacte-
rial morphology following treatment with ceftriaxone and ri-
fampin (both at a concentration of 10 mg/ml) for 3 and 6 h.
Rifampin induced no remarkable morphological changes. Oc-
casionally, blebs were seen on the bacterial surface, and blebs
were also found on a few bacteria receiving no treatment (Fig.
3B). In contrast, treatment with ceftriaxone resulted in the
abundant formation of blebs of different sizes on the cell sur-
face (Fig. 3A). Furthermore, ceftriaxone-treated pneumococci
developed filaments with lengths of up to 1 mm (Fig. 3A).
DISCUSSION
In the initial phase of treatment with b-lactam antibiotics, a
brisk increase in meningeal inflammation occurs. This increase
is caused by the release of proinflammatory bacterial cell wall
components and may be responsible, in part, for the early
mortality and long-term sequelae seen in patients with bacte-
280 STUERTZ ET AL. ANTIMICROB. AGENTS CHEMOTHER.

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FIG. 2. Release of LTA and TA by S. pneumoniae type 3 during treatment with ceftriaxone (A) and rifampin (B) at different concentrations. Note that at low
ceftriaxone concentrations (13 the MIC and 13 the MBC), the concentrations of LTA and TA in the supernatant exceeded the levels observed in the untreated
cultures.

rial meningitis (8, 16). For gram-negative bacteria, the Limulus

lysate assay is a sensitive method of measuring endotoxin ac-
tivity in vitro and in vivo (3, 5, 11). To date, no method is
capable of quantifying proinflammatory compounds of S. pneu-
moniae, the predominant pathogen in bacterial meningitis
since the introduction of vaccination against H. influenzae type
b. Although several approaches have been successful in detect-
ing antigens of S. pneumoniae in patients with meningitis and
respiratory tract infections (13, 14), these assays are unsuitable
for quantitative measurements.
The ELISA that we developed with MAb TEPC-15, which
recognizes phosphorylcholine residues, is able to quantitate
both TA and LTA, the most active proinflammatory compo-
nents of the cell wall of S. pneumoniae (16). The assay was
calibrated with a standard LTA preparation (1) and does not
differentiate between LTA and TA. It was used to measure the
release of LTA and TA in response to several antibacterial
agents with divergent modes of action at high (10 mg/ml) and
low (13 the MIC, 13 the MBC, and 0.5 and 2 mg/ml) concen-
trations.
At 10 mg/ml, all drugs investigated were rapidly bactericidal
(Fig. 1). The mean bactericidal rate was slightly lower for
rifampin and rifabutin than for ceftriaxone, meropenem, trova-
floxacin, and quinupristin-dalfopristin (the differences in dlog
CFU per milliliter per hour were not significant). Despite
similar bactericidal rates, the release of LTA and TA from the
bacteria was slower in the presence of rifampin, rifabutin,
trovafloxacin, and quinupristin-dalfopristin than in the pres-
ence of ceftriaxone and meropenem (Fig. 1). The rifamycins
and quinupristin-dalfopristin led to distinct reductions in LTA
and TA release. After 12 h of exposure to trovafloxacin and
ceftriaxone, however, the LTA and TA concentrations were
not significantly different. No morphological changes in pneu-
mococci treated with 10 mg of rifampin per ml for 6 h were
seen by scanning electron microscopy, whereas exposure to 10
mg of ceftriaxone per ml for an equal interval induced numer-
ous defects in the pneumococcal cell wall (Fig. 3).
In the rabbit model of pneumococcal meningitis, high-dose
trovafloxacin delayed the antibiotic-induced increase in TNF-a
and interleukin-1b levels but did not reduce the final maximum
concentrations (9). Treatment with rifabutin at high doses, in
FIG. 3. Representative scanning electron micrographs of S. pneumoniae type
contrast, led to significantly lower concentrations of TNF-a in 3 after in vitro exposure to 10 mg of ceftriaxone (A) or rifampin (B) per ml for
CSF compared with those in CSF during treatment with ceftri- 6 h. The horizontal bar represents 500 nm. Note the numerous defects of the
axone in experimental pneumococcal meningitis (12). pneumococcal cell wall after exposure to ceftriaxone. Magnifications, 315,600.
VOL. 42, 1998
Although the mechanisms of release of LTA and TA and
endotoxin likely differ, our data are in accordance with obser-
vations with E. coli and the measurement of endotoxin levels.
At high concentrations of antibacterial agents, the absolute
amount of endotoxin released by ciprofloxacin was similar to
that set free by ceftazidime. In serial investigations, however,
ciprofloxacin released only 12.7% of the endotoxin within the
first hour of exposure, whereas ceftazidime released 61.9% (3).
In an attempt to reduce the level of release of endotoxin
from gram-negative bacteria, some physicians have advocated
the initial use of bactericidal antibiotics below their recom-
mended dose (5). In our in vitro study, however, for all anti-
bacterial agents studied, the level of release of LTA and TA
increased with decreasing drug concentrations. At low concen-
trations of antibacterial agents (13 the MIC and 13 the
MBC), the levels of LTA and TA in S. pneumoniae cultures
treated with ceftriaxone, meropenem, trovafloxacin, and
quinupristin-dalfopristin were higher than those in cultures not
treated with the antibacterial agents (Table 1; Fig. 2A). Simi-
larly, concentrations of ceftazidime below and 23 above the
MIC induced a strong lipopolysaccharide release in vitro, and
these levels exceeded the levels released by untreated controls
(5). In our study, only in the presence of low doses of rifamy-
cins were the quantities of LTA and TA released into the
medium not higher than those observed in the supernatants of
untreated cultures (Table 1; Fig. 2B).
In conclusion, bactericidal antibacterial agents without a
primary influence upon cell wall synthesis delayed the release
of LTA and TA in comparison with the time of release after
treatment with ceftriaxone. Furthermore, treatment with ri-
fampin, rifabutin, and quinupristin-dalfopristin at concentra-
tions of 10 mg/ml significantly decreased the amount of LTA
and TA released during bacterial killing compared with the
amount released after treatment with ceftriaxone. Further re-
search with animal models may help in evaluating whether a
delayed or decreased level of release of these proinflammatory
compounds may reduce overall mortality or long-term se-
quelae in patients with life-threatening pneumococcal infec-
tions. At low antibacterial agent concentrations close to the
MIC, larger quantities of LTA and TA were released during
treatment with all drugs except the rifamycins than during no
treatment. For this reason, our data do not support the concept
of using a low first antibiotic dose to prevent the release of
proinflammatory cell wall components.
ACKNOWLEDGMENT
This work was supported by the Deutsche Forschungsgemeinschaft
(grant Na 165/2-2).
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