Daptomycin versus vancomycin in the treatment of methicillin-resistant. Staphylococcus aureus meningitis in experimental rabbit model.

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1 AAC Accepts, published online ahead of print on 14 January 2013 Antimicrob. Agents Chemother. doi: /aac Copyright 2013, American Society for Microbiology. All Rights Reserved. 1 2 Daptomycin versus vancomycin in the treatment of methicillin-resistant Staphylococcus aureus meningitis in experimental rabbit model Daptomycin vs vancomycin in rabbit MRSA meningitis model Selin Bardak-Ozcem*, Tuncer Turhan**, Oguz Resat Sipahi*, Bilgin Arda*, Husnu Pullukcu*, Tansu Yamazhan*, Meltem Isikgoz-Tasbakan*, Hilal Sipahi***, Sercan Ulusoy* (Izmir, TR) *Ege University Faculty of Medicine Department of Infectious Diseases and Clinical Microbiology, Izmir, Turkey **Ege University Faculty of Medicine Department of Neurosurgery, Izmir, Turkey *** Bornova Public Health Center, Izmir, Turkey Corresponding author: Oguz Resat Sipahi Ege University Faculty of Medicine Department of Infectious Diseases and Clinical Microbiology, Izmir, Turkey Tel: Fax: oguz.resat.sipahi@ege.edu.tr 1

2 In this study, it was aimed to compare the antibacterial activity of daptomycin and vancomycin in the treatment of methicillin-resistant Staphylococcus aureus (MRSA) meningitis (induced by ATCC MRSA strain) in experimental rabbit meningitis model. After an eight h period time of treatment, bacterial count decreased significantly in both treatment groups compared to control group (p<0.05). However, there was no statistically significant difference between treatment groups. Our results suggest that the antibacterial activity of daptomycin is similar to vancomycin in the treatment of experimental MRSA meningitis model of rabbits. 2

3 Staphylococcus aureus may be found causing community-acquired and nosocomial bacterial meningitis and is associated with significant mortality. Methicillin-resistant S. aureus (MRSA) is one of the major etiologic agents in hospital-acquired central nervous system infections. Since the available treatment options are limited, therapy of meningitis, is problematic (1). The aim of this study was to compare the efficacy of vancomycin and daptomycin in MRSA meningitis in experimental rabbit meningitis model. Bacterial strain S.aureus ATCC (vancomycin MIC: 1 mg/l, daptomycin MIC: mg/l measured in duplicate using the Etest; AB BIODISK, Solna, Sweden) was used as the infecting bacteria (2). Bacterial solution was prepared in 0.9% NaCl with a concentration of 10 6 colonyforming units (CFU)/ml as described elsewhere (2,3). Antimicrobial agents Drugs used were vancomycin (Lilly, Indianapolis, USA) and daptomycin (Novartis Pharma AG, Basel, Switzerland). Rabbit meningitis model New Zealand white rabbits weighing kg were anaesthetized by intramuscular injections of ketamine (35 mg/kg) and xylazine (5 mg/kg) before each intraventricular intervention including induction of meningitis and CSF sampling (2). The duration of anaesthesia was min. Then 0.5 ml of the bacterial solution of MRSA was injected directly into the cisterna magna of each rabbit using a 22 G spinal needle (Hayat Ticaret, Istanbul, Turkey). 3

4 Animals were not anaesthetised after the primary inoculation and between the CSF sampling procedures. In addition, they were kept in their cages except intraventricular interventions Sixteen hours after the inoculation; meningitis criteria were investigated. CSF white cell count more than 1000/mm 3 (counted by Thoma slide) and a bacterial count 10 3 cfu/ml were accepted as the indications of meningitis. Then, rabbits were separated into three groups daptomycin (D) received one dose of 15 mg/kg, vancomycin (V) group received 20 mg/kg vancomycin two times (4h apart) through peripheral ear vein. Control (C) group did not receive any treatment (4). Quantitative bacterial cultures were performed in CSF samples which were obtained at the beginning and the 8 th hour of the treatment. CSF and serum drug levels were measured by bioassay technique in samples obtained at the 8 th hour of the treatment (2,4). At the end of the study period (8 h after the initiation of treatment), animals were humanely killed by intravenous infusion of high dose nembutal. Measurement of bacterial concentrations Bacterial concentrations in CSF were measured at the end of 16 th h (End of incubation period and before the first dosage of vancomycin or daptomycin) and 8 hours after initiation of treatment by plating undiluted and serial 10-fold and 100-fold dilutions of CSF (20 μl) (5) on 5% sheep blood agar and incubated at 37 C for 24 h. Bacterial response was evaluated in three categories; full response, sterilization of CSF; partial response, any decrease in bacterial count; and bacteriological failure, an increased bacterial count (2). Antibiotic assay 4

5 Levels of daptomycin were measured twice by a bioassay technique using ATCC 9341 Kuceria rhizophilia and by ATCC 6633 Bacillus subtilllis. Standards were prepared fresh on the day of use in pooled rabbit serum for serum daptomycin levels and in 5% rabbit serum for CSF daptomycin levels (4). Assay curves were produced using standard dilutions including 0.5, 1, 2, 4, 8, 16, 32 and 64 mg/l daptomycin and vancomycin. A concentration of 20 mg/l daptomycin or vancomycin including control rabbit sera was used for each test (4,6). The sensitivity of the assay was 1 mg/l for daptomycin and 2 mg/l for vancomycin. The assay had a good reproducibility (±10%). Statistical analysis Data were evaluated by SPSS 13.0 package program using Mann-Whitney U test, Kruskal Wallis test and Fisher s x 2 test. A p-value less than 0.05 was considered significant. Ethics The study was approved by the local ethical committee on animal studies (Approval no: ). In vitro killing assays The study strain ATCC was grown in sterile human serum to an optical density of 0.3 at 590 nm and then diluted 4000-fold to 10 4 cfu corresponding approximately to bacterial concentrations in the CSF of rabbits before initiation of therapy (4). Daptomycin and vancomycin were added at concentrations corresponding to 1, 5 and 10 MIC. Bacterial titres were determined at 0, 2, 4, 6 and 8 h by serial dilution of samples, plated on agar plates containing 5% sheep blood and incubated at 37 C for 24 h. Experiments were performed in triplicate and results are expressed as mean log10 cfu/ml ± standard deviation. 5

6 The antibacterial efficacy of daptomycin and vancomycin against the study strain ATCC is demonstrated in Figure 1. Neither daptomycin, nor vancomycin resulted in killing rates meeting bactericidal effect criterion ( 3 log decrease) or lead to sterilization in the agar plates during the 8 h period in x5 or x10 MIC concentrations (Figure 1). However, x5 or x10 MIC daptomycin concentrations resulted in a log 10 cfu/ml better killing rate when compared with x1 MIC level. At the beginning of the in vivo study, 32 rabbits were inoculated with bacteria, of which 29 were alive and all had developed meningitis at the end of 16 h incubation time. Three rabbits were excluded, since they died during the 16 h incubation time. These animals were separated into three groups (Vancomycin:9, daptomycin: 10, control group:10 rabbits). Mean bacterial concentrations of these three groups were similar as (Table 1, Kruskal Wallis Test, p>0,05). Eight hours after initiation of treatment, all rabbits in both vancomycin or daptomycin groups had partial response, bacterial count decreased significantly in both treatment groups vs control group (Table 1, Mann Whitney U test, p<0.05). However, there was no statistically significant difference between D and V groups (Mann Whitney U test, p>0.05). Daptomycin could be measured in all but one rabbit which could not be punctured adequate CSF sample. Mean CSF daptomycin level was 1.87 ± 0.39 mg/l whereas mean serum level was 57.5±6.2 mg/l. CSF/serum daptomycin ratio ranged between %. Vancomycin serum level was over the detection limit of bioassay (2 mg/l) in five rabbits and their mean vancomycin concentration was 19.2±17.9 mg/l. Vancomycin 6

7 CSF level was over the detection limit of bioassay (2 mg/l) in three rabbits. Their vancomycin concentrations were 2.2 and 3.2 mg/l, respectively During the antibiotherapy period of 8 h, mortality was similar between treatment groups and control group (0/9 in vancomycin, 1/10 in daptomycin and 1/10 in control group, Fisher s x 2 test, p>0.05). MRSA meningitis occurs mostly after central nervous system operations. Treatment is challenging. The first line treatment in MRSA meningitis is vancomycin (7,8). Although vancomycin does not penetrate at all to CSF without inflammation of meninges, it penetrates to CSF in a limited amount during meningitis (2,7,8). There may be treatment failure in the strains with MIC> 1 mg/dl which may also theoretically be associated with 20 mg/kg q12 h dosage (9). Other treatment modalities are teicoplanin, linezolid, vancomycin combined with rifampicin and intrathecal vancomycin (1,7-9). Daptomycin is a cyclic lipopeptide with rapid concentration dependent bactericidal activity without cell lysis. It is highly effective against gram-positive multidrugresistant bacteria. The published experience with daptomycin in the treatment of meningitis is anecdotal (1). There are also limited data related to pharmacokinetics of daptomycin in the experimental rabbit meningitis model. Gerber et al. (4) compared the efficacy of daptomycin with vancomycin in the experimental rabbit MSSA meningitis model. Due to the fact that these dosages led to serum levels in concordance with humans receiving standard dosages. Daptomycin was given as a single dose of 15 mg/kg and vancomycin as 20 mg/kg at 0. and 4. hour. At the 8 th 7

8 hour of daptomycin treatment the bacterial count decreased 4.54 ± 1.12 log 10 cfu/ml whereas vancomycin led to a 3.43 ± 1.17 log 10 cfu/ml decrease (p<0.05). They concluded that daptomycin was more effective than vancomycin in the treatment of MSSA meningitis of rabbits. In our study, although daptomycin was given as the same as 15 mg/kg single dose, at the 8 th hour of the treatment the bacterial count decreased 3.610±0.677 log 10 cfu/ml. Hence, in MRSA meningitis model daptomycin decreased bacterial count around 1 log less than MSSA model. In the presented study, time kill assay also resulted in a steeper line for daptomycin with no sterility in the plate during 8 h period, when compared with results of Gerber et al. Both results can be speculated to be due to difference in intrinsic properties of strains and/or bacterial virulence. Another explanation may be about one log 10 CFU/ml higher start inoculum in the CSF during the start of therapy and time-kill assay.. In the presented study, daptomycin was given, 15 mg/kg because this amount produced serum and CSF levels comparable to those described corresponded to levels obtained in humans with a dose of 6 mg/kg (4). Vancomycin, which is a time dependent bactericidal, was given at 0. and 4 h, according to its pharmacokinetic properties as previous studies (4,10). In our experimental study, the bioassay method was used instead of HPLC due to economical issues. However, daptomycin level was over the level of detection in the all CSF samples by bioassay. Even though we could have checked only the trough levels, the trough CSF daptomycin levels were above the MIC. For daptomycin, bioassay seems sufficient to detect the drug levels in CSF and blood. Unlike daptomycin, vancomycin levels could not be measured in all rabbits. Hence four and 8

9 six rabbits had less vancomycin levels than the lowest detection limit of bioassay (2 mg/l) in serum and CSF, respectively. Gerber et al (4) reported trough CSF vancomycin levels of 1.9 mg/l after 8 h using the same vancomycin dosage in the presented study. Our findings are also in concordance with their results. The relatively short time of observation, the bolus administration of the vancomycin instead of continuous infusion, the absence of peak drug concentrations and the usage of the bioassay to detect of drug levels which led to detection of vancomycin levels in only a few rabbits are limitations of the presented study. The first treatment option in the MRSA meningitis is vancomycin. To our knowledge there is no clinical or experimantal study comparing the efficacy of vancomycin and daptomycin in MRSA meningitis and this is the first study to compare daptomycin and vancomycin. Our results suggest that daptomycin is at least as effective as vancomycin in the treatment of MRSA meningitis in an experimental meningitis model in rabbits. Additional data should confirm our experiments in advance of clinical trials to assess efficacy in humans. Acknowledgement: This study was supported by a grant from Ege University. Conflict of interest: SU and ORS received speaker s honorarium from Novartis. References 1) Bardak-Ozcem S, Sipahi OR. (2012) Approach to hospital-acquired and methicillin-resistant Staphylococcus aureus meningitis. Mediterr. J. Infect. Microb. Antimicrob. 1: 13. 9

10 ) Sipahi OR, Arda B, Yurtseven T, Sipahi H, Ozgiray E, Suntur BM, Ulusoy S Vancomycin versus teicoplanin in the therapy of experimental methicillinresistant Staphylococcus aureus (MRSA) meningitis. Int. J. Antimicrob. Agents. 26: ) Bilgehan H Klinik Mikrobiyolojik Tanı. 3th. ed, Fakülteler Barıs Yayınları. 4) Gerber P, Stucki A, Acosta F, Cottagnoud M, Cottagnoud P Daptomycin is more efficacious than vancomycin against a methicillin-susceptible Staphylococcus aureus in experimental meningitis. J. Antimicrob. Chemother, 57: ) Stucki A, Cottagnoud M, Acosta F, Egerman U, Läuffer J, Cottagnoud P Evaluation of ceftobiprole activity against a variety of gram-negative pathogens, including Escherichia coli, Haemophilus influenzae (β-lactamase positive and β-lactamase negative), and Klebsiella pneumoniae, in a rabbit meningitis model. Antimicrob. Agents Chemother. 56(2): ) Mook-Kanamori BB, Rouse MS, Kang CI, van de Beek D, Steckelberg JM, Patel R Daptomycin in experimental murine pneumococcal meningitis BMC Infectious Diseases 9:50 7) Tunkel AR, Hartman BJ, Kaplan SL, Kaufman BA, Roos KL, Scheld WM, Whitley RJ Practice guidelines for the management of bacterial meningitis. Clin. Infect. Dis. 1;39(9): ) Tunkel AR, van de Beek D, Scheld WM Acute meningitis, p In Mandell GL, Bennett JE, Dolin R, (ed). Principles and practice of infectious diseases, 7th ed, volume 1. Churchill Livingstone, Elsevier, New York. 9) Sipahi OR, Bardak S, Turhan T, Arda B, Pullukcu H, Ruksen M, Aydemir S, Dalbasti T, Yurtseven T, Zileli M, Ulusoy S Linezolid in the treatment of 10

11 methicillin-resistant staphylococcal post-neurosurgical meningitis: a series of 17 cases. Scand. J. Infect. Dis. 43(10): ) Cottagnoud P, Pfister M, Acosta F, Cottagnoud M, Flatz L, Kühn F, Müller HP, Stucki A Daptomycin is highly efficacious against penicillin-resistant and penicillin- and quinolone-resistant pneumococci in experimental meningitis. Antimicrob. Agents Chemother. 48: Downloaded from on November 18, 2018 by guest 11

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13 Table 1. Bacteriological results of cerebrospinal culture results. Bacteria n* Treatment 0. h 8. h Difference Control 3.448± ± ±0.682 Daptomycin 3.958± ± ±0.677 Vancomycin 3.703± ± ±0.697 *Mean±standard deviation number of bacteria as log10 cfu/ml Downloaded from on November 18, 2018 by guest