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JAC Journal of Antimicrobial Chemotherapy (1998) 41, 163–169 Antibiotic-induced release of endotoxin: in-vitro comparison of meropenem and other antibiotics M. Trautmanna*, R. Zicka, T. Rukavinab, A. S. Crossc and R. Marrea a Department of Medical Microbiology and Hygiene, University of Ulm, Ulm, Germany; bDepartment of Microbiology, University of Rijeka, Croatia; cDivision of Infectious Diseases and Greenbaum Cancer Center, Department of Medicine, University of Maryland, Baltimore, MD, USA The influence of meropenem, a new carbapenem antibiotic, on cell morphology and in-vitro lipopolysaccharide (LPS) release from Escherichia coli was compared with that of imipenem, ceftazidime, tobramycin and ciprofloxacin. Free and cell-associated LPS was quantified by means of a capture ELISA method based on the recognition of E. coli LPS by monoclonal antibodies. Microscopically, meropenem was found to induce spheroplast formation similar to that seen with imipenem, while ceftazidime and ciprofloxacin induced filament formation. Free and cell-associated LPS levels were low in the presence of meropenem, imipenem, ciprofloxacin and tobramycin, but high in the presence of ceftazidime. Reduced endotoxin release appears to be a common property of carbapenem antibiotics. Morphological changes in bacteria in the presence of antibiotics do not predict their LPS-liberating effect since ciprofloxacin induced low levels of LPS despite causing filament formation. Introduction Systemic infections due to Gram-negative bacilli are frequently complicated by the development of septic shock and multiorgan failure. It is accepted that Gram-negative bacterial lipopolysaccharide (LPS, endotoxin) plays a significant role in the pathogenesis of sepsis-induced shock and tissue injury.1 While treatment with antibiotics is necessary to combat the infective process, antibiotics are also known to precipitate the release of endotoxins by disrupting the outer membrane of the organisms.2,3 When comparing different classes of antibiotics in vitro, Goto & Nakamura were the first to realize that bacteriostatic antibiotics did not liberate substantial quantities of endotoxin while bactericidal drugs induced the release of large amounts of free LPS.4 Later, it was shown that rapidly bactericidal agents exhibiting an intracellular killing mechanism such as the aminoglycosides cause a comparatively minor LPS release while cell-wall-active drugs may stimulate the release of major quantities of LPS.5 The carbapenem antibiotic imipenem differs from most of the -lactam antibiotics by causing reduced LPS release in vitro.6,7 Microscopic and electron-microscopic studies have shown that imipenem induces spheroplast formation while ceftazidime and other -lactams induce filament formation.8 This phenomenon has been explained by preferential binding of imipenem to penicillin-binding 2 (PBP2) in contrast to the third-generation cephalosporins which primarily bind to PBP3.9 PBP2 appears to be responsible for longitudinal cell growth (primarily inhibited by imipenem) while PBP3 is responsible for cell septation (primarily inhibited by the third-generation cephalosporins). Recently, clinical studies have shown that the differences in the rate and degree of LPS release induced by imipenem and third-generation cephalosporins may have clinical relevance.10,11 Meropenem is a new carbapenem which differs from imipenem in its stability against renal dehydropeptidase I and an enhanced activity against several species of Gramnegative enteric bacteria and Pseudomonas spp.12,13 The higher activity against Gram-negative bacilli has been related to an affinity of meropenem for both PBPs 2 and 3.14,15 Therefore, it was not clear whether the drug would primarily cause spheroplast or filament formation or whether meropenem would cause reduced LPS liberation. In this study, we analysed the influence of meropenem on LPS release by means of a serotype-specific, highly reproducible ELISA detection method of LPS based on O-antigen-specific monoclonal antibodies. Imipenem, ceftazidime, ciprofloxacin and tobramycin were compared. *Corresponding address: Department of Medical Microbiology and Hygiene, University of Ulm, Steinhövelstrasse 9, D-89075 Ulm, Germany. Tel: 49-731-5026951; Fax: 49-731-5026949. 163 © 1998 The British Society for Antimicrobial Chemotherapy M. Trautmann et al. Capture ELISA for E. coli LPS Materials and methods Bacteria E. coli strain Bort (O18:K1:H7), a CSF isolate originally obtained from a newborn, has been used previously in our laboratories.16,17 MICs of the antibiotics against this organism were measured by means of broth microdilution according to National Committee for Clinical Laboratory Standards (NCCLS) recommendations.18 Antibiotics Meropenem, imipenem, ceftazidime, ciprofloxacin and tobramycin were included in the study. Standard powders of known potency were obtained from the respective manufacturers. Stock solutions of 2560 mg/L were stored at 80°C. Growth conditions and preparation of test samples Bacterial cultures were grown in cation-supplemented Mueller–Hinton broth in a shaking waterbath at 37°C until they reached mid-log phase (3–4 h). The suspension was adjusted nephelometrically to a concentration of c. 108 organisms/mL which was confirmed by plate counts. One hundred microlitres of this suspension were added to 20 mL of fresh Mueller–Hinton broth containing twice or 50 times the MIC of the respective antibiotic or no antibiotic (growth control). Thus, the starting concentration of organisms in each vial was c. 15 105 organisms/mL. At the time points indicated, 100 L of the bacterial suspension were removed for the determination of viable bacterial cells by subculturing volumes of 100 L on Mueller–Hinton agar plates after serial ten-fold dilution in physiological saline. At the same time, a 1 mL sample was removed from each suspension, transferred to a 1.5 mL Eppendorf tube and centrifuged at 12,000g for 15 min. The supernatant was removed and frozen at 80°C until determination of free LPS. Cell-associated LPS was released from the cell pellet by the method of Hitchcock & Brown19 with minor modifications as follows: the pellets were solubilized in 50 L of lysis buffer and heated at 100°C for 10 min. After cooling, 10 L of DNase and 10 L of RNase (Boehringer Mannheim, Germany) were added to yield final concentrations of 100 and 25 g/mL, respectively. After incubation at 37°C for 2 h, 25 g of proteinase K (Boehringer Mannheim) solubilized in 10 L of lysis buffer, was added to the lysates which were incubated at 60°C for 1 h. After performing these lysis steps, the cell suspensions had completely cleared without residual debris. Finally, the enzymes were inactivated by heating the suspensions at 95°C for 10 min, and the solutions kept frozen at 80°C until determination of cellassociated LPS. LPS concentrations were determined by means of a monoclonal antibody (mAb)-based ELISA method. Ninety-sixwell flat-bottomed ELISA plates (Greiner, Nürtingen, Germany) were coated with 10 g/mL of a purified mouse mAb specific for E. coli O18 LPS (clone 26, mouse IgG 1 ).20 After overnight incubation at 4°C and removal of antigen, non-specific binding sites were blocked by the addition of a 1% casein–albumin solution as described previously.17 Standards of 0.5–64 ng/mL of purified E. coli O18 LPS and test samples containing unknown amounts of LPS were added to duplicate wells of the plate. After further overnight incubation at 4°C, plates were washed three times with phosphate-buffered saline (PBS). Bound LPS was traced with a second mouse mAb specific for E. coli O18 LPS (clone 141, mouse IgM 20) at a dilution of 1:2000. Plates were incubated at 4°C for 4 h and washed again. Bound mouse IgM was detected by adding alkaline phosphatase-conjugated goat anti-mouse IgM( ) antibody (Sigma, Deisenhofen, Germany) at appropriate dilution, and reactions developed as described previously.17 Examination of bacterial morphology Bacteria were grown in antibiotic-containing medium (twice and 50 times the MIC) as described above. After 4 h, samples were removed from the suspension and stained with Gram’s stain or methylene blue. Photographs were taken using a Zeiss microscope (Zeiss, Oberkochen, Germany). Statistics The Mann–Whitney U-test was used. A P value of was considered significant. 0.05 Results MIC determination MICs (mg/L) for E. coli strain Bort were as follows: ceftazidime, 0.125; meropenem, 0.015; imipenem, 0.125; tobramycin, 1.0; ciprofloxacin, 0.0078. Standardization of LPS capture ELISA The ELISA detection curve was log-linear at LPS concentrations of 4–64 ng/mL. Mean background optical density (OD) values 1 S.D. in the absence of LPS were 0.003 0.029 (n 27). The lower detection limit of the test, defined as the lowest standard concentration yielding mean ODs of 2 S.D. above background levels, was 2 ng/mL. The interassay coefficient of variation was 5.5% at 2 ng/mL, 4.9% at 16 ng/mL and 5.0% at 64 ng/mL (n 27). In order to determine whether the presence of 164 Meropenem-induced release of endotoxin antibiotics would influence LPS recovery, a 16 ng/mL LPS solution was mixed with various antibiotics at a concentration of 50 MIC before determining the LPS content of the mixture. No significant influence on LPS recovery could be detected (Table I). Influence of antibiotics on bacterial growth and LPS production There was no significant difference in the killing rate of the drugs with the exception of tobramycin which was very rapidly bactericidal at twice the MIC (Figure 1a and b). LPS levels determined during incubation with 2 MIC of the drugs were significantly different only for cellassociated LPS. Both ciprofloxacin and tobramycin produced significantly less cell-associated endotoxin than the -lactams (Table II). At 50 MIC, both free and cell-associated LPS concentrations were significantly lower for the carbapenems and ciprofloxacin than for ceftazidime (Table III). No significant differences between meropenem and imipenem were found. Morphological changes in bacteria Meropenem induced spheroplast formation similar to that induced by imipenem, while ceftazidime and ciprofloxacin induced filament formation. Filaments induced by ciprofloxacin, however, had a stouter appearance than those formed in the presence of ceftazidime (Figure 3). Discussion In most studies dealing with antibiotic-induced LPS release, bioreactive endotoxin has been measured by means of the Limulus amoebocyte lysate (LAL) assay which involves an enzymatic reaction triggered by the core region of LPS.6,11,21,22 Endotoxin levels determined by means of the LAL assay therefore reflect not only the Table I. LPS recovery by ELISA in the presence of antibiotics Antibiotic added None (control) Meropenem Imipenem Ceftazidime Ciprofloxacin Tobramycin LPS concentration (ng/mL) mean 1 S.D. % recovery 16.7 16.2 15.3 17.1 14.5 17.1 3.1 1.2 1.2 1.6 1.7 3.3 104 101 96 107 91 107 Drugs were tested at 50 MIC. Samples were spiked with LPS at a final concentration of 16 ng/mL. Values are from three independent experiments. Figure 1. Growth of E. coli Bort in the presence of 2 MIC (a) and 50 MIC (b) of the antibiotics. Data are geometric means of three to five experiments per time point. —— , control; , meropenem; , imipenem; - - - - -, ceftazidime; , ciprofloxacin; , tobramycin. concentration of LPS but also the accessibility of the inner core which may be unfolded during exposure to cell-wallactive drugs. 23 Conversely, aminoglycoside antibiotics are known to suppress the LAL reaction, although the mechanism of this suppression has not been elucidated.24 In the present study, we therefore used a mAb-based ELISA method for LPS quantification which is not influenced by the presence of antibiotics (Table I) and which has the additional advantage of not being prone to contamination with other LPSs. Using this test system, we found that both soluble and cell-associated LPS levels were similarly low during treatment of the cultures with meropenem and imipenem (Tables II and III). No significant differences between these drugs were found. A significantly lower release of free LPS and production of cell-associated LPS was found, at 50 MIC, for both carbapenems compared with ceftazidime (Table III). These findings show that, although meropenem is known to bind to both PBPs 2 and 3, it does not liberate more LPS than imipenem. 165 M. Trautmann et al. 166 Meropenem-induced release of endotoxin (a) (b) (c) (d) Figure 2. Morphological changes in bacteria in a culture of E. coli Bort incubated without antibiotic (control, a) and in the presence of 50 MIC of meropenem (b), ceftazidime (c) or ciprofloxacin (d). Changes induced by imipenem were identical to those seen with meropenem. Final magnification, 1000. In addition, we included a quinolone antibiotic since this group of drugs is increasingly used as a primary therapeutic option in Gram-negative sepsis. Earlier investigators found that higher total amounts of LPS were produced during exposure of bacteria to ciprofloxacin at 50 MIC compared with imipenem.3,9 A substantial endotoxin release following exposure of E. coli to ciprofloxacin and other quinolones has also been described by McConnell & Cohen.21 This stands in contrast to the clinical experience with ciprofloxacin which, for instance, has never been reported to produce Herxheimer reactions when used for treatment of typhoid fever in contrast to other drugs. 25–27 Our findings were at variance with those of the studies cited since we found that free LPS levels with ciprofloxacin were as low as those measured in the presence of the carbapenems at both 2 and 50 MIC, while total cell-associated LPS levels were even significantly lower at 2 MIC (Table II). The high levels of free LPS measured in other studies using the LAL test may have been due to an overestimation resulting from the peculiar ‘hypersensitivity’ of the LAL assay for free LPS which has been described by Mattsby-Baltzer et al.28 In their study, an ELISA detection method similar to the one described here measured LPS values much nearer to actual LPS levels determined by chemical analysis of LPS marker molecules. The lower amount of cell-bound LPS found in the presence of ciprofloxacin compared with ceftazidime cannot be explained by differences in the rate of bacterial killing since killing curves were nearly identical for ciprofloxacin and the -lactams. Rather, it may be assumed that the production and/or assembly of cell-bound LPS was inhibited by ciprofloxacin. LPS synthesis involves a number of enzymatic and transport processes encoded by the rfb gene cluster, and quinolones may interfere with coordinate transcription of these genes by inhibiting bacterial gyrase. Although animal experimental studies have shown that the varying potential of antibiotics to cause endotoxin release has implications for the outcome of experimental Gram-negative sepsis,29,30 doubts have been raised as to whether such findings may have clinical significance. However, two recent studies show that this may in fact be the case. In a carefully controlled, though small, study involving patients with urosepsis, Prins et al.11 were able to demonstrate that the use of imipenem as a primary antibiotic significantly shortened the time to defervescence compared with ceftazidime. Furthermore, there was a tendency for lower values of inflammatory parameters 167 M. Trautmann et al. such as serum tumour necrosis factor (TNF ) and interleukin 1 (IL-1) levels in the imipenem group. In a second report, Mock et al.10 performed a post hoc analysis of data from a previously conducted prospective, randomized trial designed to test the efficacy of -interferon in septic trauma patients treated with either PBP3-specific or nonPBP3-specific antibiotics. Mortality rates were 17% in the PBP3 group and 8% in the non-PBP3 group (P 0.02), supporting the concept that endotoxin-releasing properties of antibiotics affect the clinical outcome of sepsis.10 Our study shows that meropenem and ciprofloxacin also have low endotoxin-releasing potential which may deserve attention in future prospective, randomized clinical trials on this subject. Acknowledgement 10. Mock, C. N., Jurkovich, G. J., Dries, D. J. & Maier, R. V. (1995). Clinical significance of antibiotic endotoxin-releasing properties in trauma patients. Archives of Surgery 130, 1234–41. 11. Prins, J. M., van Deventer, S. J. H., Kuijper, E. J. & Speelman, P. (1994). Clinical relevance of antibiotic-induced endotoxin release. Antimicrobial Agents and Chemotherapy 38, 1211–8. 12. Catchpole, R. C., Wise, R., Thornber, D. & Andrews, J. M. (1992). In vitro activity of L-627, a new carbapenem. Antimicrobial Agents and Chemotherapy 36, 1928–34. 13. Hoban, D. J., Jones, R. N., Yamane, N., Frei, R., Trilla, A. & Pignatari, A. C. (1993). In vitro activity of three carbapenem antibiotics. Comparative studies with biapenem (L-627), imipenem, and meropenem against aerobic pathogens isolated worldwide. Diagnostic Microbiology and Infectious Disease 17, 299–305. 14. Sumita, Y., Fukasawa, M. & Okuda, T. (1990). Comparison of two carbapenems, SM-7338 and imipenem: affinities for penicillinbinding proteins and morphological changes. Journal of Antibiotics 43, 314–20. The authors thank Zeneca Ltd, Plankstadt, Germany, for supporting the study. 15. Pryka, R. D. & Haif, G. M. (1994). Meropenem: a new carbapenem antimicrobial. Annals of Pharmacotherapy 28, 1045–54. 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