INTRODUCTION

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The release of bacterial cell wall components into the cerebrospinal 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-) 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 neurons 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 phosphorylcholine (1). LTA additionally has a hydrophobic trihexosyldiacylglycerol residue. When injected into the subarachnoid space, TA and LTA cause profound meningeal inflammation (16).

In order to attenuate subarachnoid space inflammation, adjunctive 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, dexamethasone 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 immunosuppression 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 pneumococci (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 rifabutin, and the combination of streptogramins quinupristin and dalfopristin (RP59500) in comparison with the release during treatment with the cephalosporin and carbapenem antibiotics 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

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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). Trovafloxacin was a gift from Pfizer (Karlsruhe, Germany), and ceftriaxone was provided by Hoffmann-La Roche (Grenzach Wyhlen, Germany). Rifampin and meropenem were purchased from Grünenthal (Stolberg, Germany).

Release of proinflammatory components as a result of exposure to antibacterials. 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).

Sandwich ELISA for detection of pneumococcal LTA and TA. LTA was prepared 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 pressure 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 interaction chromatography on octyl-Sepharose (Pharmacia Biotech, Freiburg, Germany). The purification grade was determined by using the choline/phosphate ratio, which is 0.66 in pure LTA (1).

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 µg of ceftriaxone or rifampin per ml. Thereafter, the bacteria were transferred to poly-L-lysine-coated coverslips and fixed with 2% glutaraldehyde 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 Kingdom). After being mounted on stubs, the specimens were coated with gold-palladium in a cool sputter coater (Fisons Instruments, Uckfield, United Kingdom) and were examined in an electron microscope (DSM 960; Zeiss, Oberkochen, Germany).

Statistics. Ceftriaxone (10 µg/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

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The bactericidal rates during exposure to 10 µg of ceftriaxone (0.60 ± 0.08 log CFU/ml/h; r² = 0.98 ± 0.004), meropenem (0.87 ± 0.15 log CFU/ml/h; r² = 0.98 ± 0.02), quinupristin-dalfopristin (0.68 ± 0.22 log CFU/ml/h; r² = 0.93 ± 0.06), and trovafloxacin (0.79 ± 0.16 log CFU/ml/h; r² = 0.96 ± 0.08) per ml were slightly higher than those during exposure to 10 µg of rifabutin (0.45 ± 0.05 log CFU/ml/h; r² = 0.85 ± 0.05) and rifampin (0.37 ± 0.04 log CFU/ml/h; r² = 0.91 ± 0.05) per ml (values are means ± standard deviations). 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 × 10² and 2.6 × 10³/ml, respectively (Fig. 1).

View larger version (24K): [in this window] [in a new window]   FIG. 1.   Release of LTA and TA by S. pneumoniae type 3 during treatment with 10 µg of ceftriaxone (A), rifabutin (B), rifampin (C), quinupristin-dalfopristin (D), trovafloxacin (E), and meropenem (F) per ml and by untreated cultures (G) (closed circles). The means ± 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 ± standard errors of means).

A rapid and intense release of LTA and TA occurred in the first hours of exposure to 10 µg of ceftriaxone per ml (means ± standard deviations, 3,248 ± 1,688 ng/ml at 3 h and 3,827 ± 2,133 ng/ml at 12 h) and 10 µg of meropenem per ml (2,464 ± 1081 ng/ml at 3 h and 2,900 ± 1,364 ng/ml at 12 h) (the difference was not significant). Three hours after rifampin and rifabutin exposure (10 µg/ml each), mean LTA and TA concentrations of 166 ± 65 and 267 ± 121 ng/ml, respectively, were observed (for each group, P < 0.01 versus the concentrations after exposure to ceftriaxone). Three hours of exposure to 10 µg of quinupristin-dalfopristin per ml resulted in LTA and TA concentrations of 380 ± 285 ng/ml, and 10 µg of trovafloxacin per ml released 455 ± 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 µg of the various drugs per ml, the LTA and TA concentrations were 463 ± 126 ng/ml after exposure to rifampin, 669 ± 303 ng/ml after exposure to rifabutin, and 1,236 ± 772 ng/ml after exposure to quinupristin-dalfopristin (for each group, P < 0.05 versus the concentrations after exposure to ceftriaxone) and 1,745 ± 1,185 ng/ml after exposure to trovafloxacin (P = 0.12 versus the concentration after exposure to ceftriaxone) (Fig. 1). Meropenem at 10 µg/ml did not cause a significant reduction in the release of LTA and TA at any time (2,464 ± 1,081 ng/ml at 3 h and 2,900 ± 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 (1× the MIC and 1× the MBC), the LTA and TA levels in the supernatants were higher than those at higher concentrations of the antibacterial agents. At 1× the MIC and 1× the MBC, more LTA and TA were released from S. pneumoniae cultures in the presence of ceftriaxone, meropenem, trovafloxacin, and quinupristin-dalfopristin than from nontreated cultures, although ceftriaxone and meropenem were bactericidal and trovafloxacin and quinupristin-dalfoprisitin were bacteriostatic at these concentrations (Table 1; Fig. 2A). The LTA and TA concentrations released from bacteria exposed to rifampin at 1× the MIC and 1× the MBC and to rifabutin at 1× the MIC were close to the concentrations observed in the supernatants of untreated cultures (Table 1; Fig. 2B).

View larger version (19K): [in this window] [in a new window]   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 (1× the MIC and 1× the MBC), the concentrations of LTA and TA in the supernatant exceeded the levels observed in the untreated cultures.

Scanning electron microscopy revealed differences in bacterial morphology following treatment with ceftriaxone and rifampin (both at a concentration of 10 µg/ml) for 3 and 6 h. Rifampin induced no remarkable morphological changes. Occasionally, 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 surface (Fig. 3A). Furthermore, ceftriaxone-treated pneumococci developed filaments with lengths of up to 1 µm (Fig. 3A).

View larger version (84K): [in this window] [in a new window]   FIG. 3.   Representative scanning electron micrographs of S. pneumoniae type 3 after in vitro exposure to 10 µg of ceftriaxone (A) or rifampin (B) per ml for 6 h. The horizontal bar represents 500 nm. Note the numerous defects of the pneumococcal cell wall after exposure to ceftriaxone. Magnifications, ×15,600.

	DISCUSSION

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In the initial phase of treatment with -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 bacterial meningitis (8, 16). For gram-negative bacteria, the Limulus lysate assay is a sensitive method of measuring endotoxin activity in vitro and in vivo (3, 5, 11). To date, no method is capable of quantifying proinflammatory compounds of S. pneumoniae, the predominant pathogen in bacterial meningitis since the introduction of vaccination against H. influenzae type b. Although several approaches have been successful in detecting 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 components 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 µg/ml) and low (1× the MIC, 1× the MBC, and 0.5 and 2 µg/ml) concentrations.

At 10 µg/ml, all drugs investigated were rapidly bactericidal (Fig. 1). The mean bactericidal rate was slightly lower for rifampin and rifabutin than for ceftriaxone, meropenem, trovafloxacin, and quinupristin-dalfopristin (the differences in log 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 presence 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 pneumococci treated with 10 µg of rifampin per ml for 6 h were seen by scanning electron microscopy, whereas exposure to 10 µg of ceftriaxone per ml for an equal interval induced numerous 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- and interleukin-1 levels but did not reduce the final maximum concentrations (9). Treatment with rifabutin at high doses, in contrast, led to significantly lower concentrations of TNF- in CSF compared with those in CSF during treatment with ceftriaxone in experimental pneumococcal meningitis (12).

Although the mechanisms of release of LTA and TA and endotoxin likely differ, our data are in accordance with observations 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 recommended dose (5). In our in vitro study, however, for all antibacterial agents studied, the level of release of LTA and TA increased with decreasing drug concentrations. At low concentrations of antibacterial agents (1× the MIC and 1× 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). Similarly, concentrations of ceftazidime below and 2× 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 rifamycins 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 rifampin, rifabutin, and quinupristin-dalfopristin at concentrations of 10 µg/ml significantly decreased the amount of LTA and TA released during bacterial killing compared with the amount released after treatment with ceftriaxone. Further research 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 sequelae in patients with life-threatening pneumococcal infections. 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.