Journal of Antimicrobial Chemotherapy (1999) 44, 477-481
© 1999 The British Society for Antimicrobial Chemotherapy
Protective effect of trovafloxacin, ciprofloxacin and ampicillin against Streptococcus pneumoniaein a murine sepsis model
a Department of Pharmacy Practice, School of Pharmacy, The University of Kansas Medical Center, Kansas City, KS 66160-7231 b Department of Pharmacy Research, Hartford Hospital, Hartford, CT 06102-5037, USA c Division of Infectious Diseases, Hartford Hospital, Hartford, CT 06102-5037, USA d Office of Research Administration, Hartford Hospital, Hartford, CT 06102-5037, USA
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Abstract |
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Trovafloxacin is a new fluoroquinolone that has potent microbiological activity against the pneumococcus, including penicillin-resistant strains. To evaluate the protective effect of trovafloxacin, ciprofloxacin and ampicillin against penicillin-susceptible, -intermediate and -resistant strains of Streptococcus pneumoniae, an intraperitoneal, immunocompetent mouse model of sepsis was used. The minimum lethal dose (MLD) for each isolate was determined in duplicate. A single sc dose of each antibiotic was administered over a wide range of doses 1 h after the ip inoculation of the test isolate at the MLD. The assessment of the protective dose for 50% of the population (PD50) for each antimicrobial/bacteria combination was performed in triplicate and the PD50 value was calculated at the end of 5 days. Results showed that trovafloxacin provided PD50 values that were significantly lower than those of ciprofloxacin for all isolates. For the penicillin-susceptible and -intermediate isolates, the PD50 values of ampicillin were significantly lower than those for either of the fluoroquinolones studied; however, trovafloxacin was statistically superior to both ciprofloxacin and ampicillin against the penicillin-resistant strain. Therefore, regardless of penicillin susceptibility, trovafloxacin has potent activity against Streptococcus pneumoniae and may be a viable alternative for the treatment of penicillin-resistant isolates.
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Introduction |
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Streptococcus pneumoniaeremains the leading bacterial aetiology of community-acquired pneumonia, acute sinusitis and bacterial meningitis.1,2,3,4 While the prevalence of this organism has long been recognized, new concern has been generated by the increasing worldwide frequency of penicillin non-susceptible isolates.5,6,7 Currently in the USA the reported frequency of S. pneumoniae with reduced sensitivity to penicillin is around 24–34%.5,8 As a result of the prevalence of this pathogen and the change in susceptibility to routinely used antimicrobials, there may be a need for a change in current treatment practices.9
Fluoroquinolone antibiotics are typically known for their excellent activity against Gram-negative pathogens. However, extended-spectrum fluoroquinolones with potent Gram-positive activity, such as trovafloxacin, have only recently become available. Trovafloxacin, a unique, synthetic fluoroquinolone with a long half-life, is characterized by its microbiological activity against anaerobes, Gram-negative and Gram-positive pathogens, including penicillin non-susceptible pneumococci.10 Typical MIC values of trovafloxacin for S. pneumoniaeare between four- and 32-fold lower than those reported for other fluoroquinolones such as ciprofloxacin and ofloxacin.11,12
The purpose of this study was to compare the protective effects of trovafloxacin, ciprofloxacin and ampicillin against penicillin-susceptible, -intermediate and -resistant isolates of S. pneumoniae using an intraperitoneal, immunocompetent mouse model of sepsis.
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Materials and methods |
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Approval for all study methods and procedures was obtained from the Institutional Animal Care Use Committee prior to study initiation.
Bacterial strains and susceptibility testing
Thirteen S. pneumoniae clinical isolates from Hartford Hospital and three isolates from Pfizer Laboratories were screened for susceptibility to penicillin, ampicillin, ciprofloxacin and trovafloxacin. MICs were determined in duplicate according to the National Committee for Clinical Laboratory Standards (NCCLS) using a microdilution technique and antimicrobial standard powders with known potencies.13 Cation-adjusted Mueller–Hinton broth (CAMHB, Becton Dickinson, Cockeysville, MD, USA) supplemented with 2.5–5% lysed horse blood (LHB, Remel, Lenexa, KS, USA) was used as the bacterial growth medium in all in-vitro studies. Three isolates were selected which displayed a range of susceptibility for penicillin and ampicillin: one strain each of penicillin-susceptible, -intermediate and -resistant S. pneumoniae.
Antibiotics
The following antibiotics were used in all in-vivo studies: ampicillin sodium salt standard powder (Sigma Chemical Company, St Louis, MO, USA (lot 076H0373)), ciprofloxacin iv injection, 40 g/L (Bayer Corporation, West Haven, CT, USA (lot 6LCA)) and trovafloxacin standard powder (Pfizer Laboratories, New York, NY, USA (lot 31075-199-1F)).
Preparation of bacterial suspensions
Fresh colonies of the test isolates were taken from tryptocasein-soy agar plates (100 mm) with 5% sheep blood (Becton Dickinson) after overnight growth and inoculated into CAMHB with LHB. The bacterial suspension was placed into a 5% CO2 incubator for 4 h before the start of all experiments. Colony forming unit (cfu) counts were verified by plating serially diluted aliquots on agar plates.
Animals
Immunocompetent Swiss Webster mice (Taconic Laboratory Animals and Services, Germantown, NY, USA) weighing approximately 25 g were used throughout this study. Animals were allowed, at least 72 h after delivery, to acclimatize to laboratory surroundings before experimentation.
Minimum lethal dose determination
The minimum lethal dose (MLD) was determined in duplicate for each study isolate. Groups of mice (n = 10) were injected ip with 0.5 mL of inocula ranging from 101 to 107 cfu/mL. The MLD inoculum was selected on the basis of 100% mortality at the end of 48 h. For each MLD determination, a control group of 10 mice received normal saline instead of the bacterial suspension.
Protective dose determination
The protective dose for 50% of the population (PD50) was determined in triplicate for each isolate over a 5 day study period. Groups of mice (n = 10) were infected ip with 0.5 mL of the test isolate MLD bacterial suspension. The mean weight of the animal population was determined within the immediate 24 h period before bacterial infection. Using the mean population weight, dose-ranging studies were performed. Antibiotic solutions were serially diluted and prepared such that 0.2 mL would contain the following range of concentrations: trovafloxacin 0.5–32 mg/kg, ciprofloxacin 1–64 mg/kg and ampicillin 0.25–128 mg/kg. Antibiotics were administered as a single sc dose at the dorsal base of the neck 1 h after inoculation with bacteria. A group of 10 mice served as control for each experiment and received 0.2 mL of normal saline instead of antibiotic.
Determination of activity
Animals were observed a minimum of three times daily over a 5-day period after inoculation. The PD50 was calculated from the plot of survivors versus dose administered for each experiment using the Sigmoid Emax model.14
Statistical analysis
Protective dose data were compared between antimicrobials for each isolate using an analysis
of variance method followed by the Scheffe test for multiple comparisons. A P-value of
0.05 was considered significant.
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Results |
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Susceptibility testing
The Table shows the pre-experimental MIC results of ampicillin, trovafloxacin and ciprofloxacin for the three selected S. pneumoniae isolates. The screening MIC values for penicillin for each isolate were as follows: SP22, 0.06; SP45, 0.125 and SP46, 4.
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Minimum lethal dose determinations
Mortality was not observed in any of the control groups at 48 h during any of the MLD determinations. The virulent study isolates had MLD values ranging from 102 (SP22 and SP45) to 104 (SP46) cfu/mL as shown in the Table.
Protective dose determinations
Plots of percentage survival versus dose (mg/kg) are shown in Figures 1, 2 and 3 and PD50 experimental results are presented in the Table. These data show that ampicillin had significantly lower PD50 values (range 1.0–2.7 mg/kg) for the PCN-S and PCN-I isolates when compared with either trovafloxacin (range 11.5–22.8 mg/kg) or ciprofloxacin (>64 mg/kg). Trovafloxacin gave PD50 values that were significantly lower than those of ciprofloxacin for all strains (10.8–22.8 mg/kg versus >64 mg/kg). For the penicillin-resistant isolate, trovafloxacin (10.8 mg/kg) was statistically superior to both ampicillin (>128 mg/kg) and ciprofloxacin (>64 mg/kg). Mortality was noted for all animals in the control groups at day 5, the majority having died within the immediate 48 h period following infection.
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) was 1.0 ± 0.5
mg/kg and of trovafloxacin (•) was 22.8 ± 3.6 mg/kg. The value for ciprofloxacin
was >64 mg/kg.
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Discussion |
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The dose required to produce a survival rate of 50% with ampicillin against both the penicillin-susceptible and -intermediate strains was significantly lower when compared with trovafloxacin and ciprofloxacin. However, against the penicillin-resistant isolate ampicillin displayed poor activity, as was expected by its MIC value of 16 mg/L. Ciprofloxacin displayed poor in-vivo activity against all study isolates as shown by the significantly higher PD50 values compared with both ampicillin and trovafloxacin. Trovafloxacin produced the desired protective value with significantly lower dose when compared with ciprofloxacin against all tested isolates. Additionally, it displayed more potent activity against the penicillin-resistant strains compared with both ampicillin and ciprofloxacin. The trovafloxacin PD50 value for SP22 was approximately twice that for either SP45 or SP46. This may be explained by the slightly higher MIC values for SP22 (MIC 0.125 mg/L) which was one dilution higher than the MIC value for SP45 or SP46 (MIC 0.06 mg/L).
The observed differences in microbiological activity between trovafloxacin and ciprofloxacin against S. pneumoniaemay be due to an additional enzyme target for trovafloxacin.10 Trovafloxacin has been found to inhibit both DNA gyrase and another type II topoisomerase, topoisomerase IV. The inhibition of topoisomerase IV is thought to confer enhanced potency against Gram-positive pathogens, such as S. pneumoniae.
Our group has previously shown that significantly lower PD50 values are observed for penicillin G compared with ciprofloxacin when using a similar murine model of pneumococcal sepsis.15 In this study, a penicillin-susceptible strain of S. pneumoniae was used with MICs of penicillin G and ciprofloxacin of 0.03 mg/L and 1.0 mg/L, respectively. The reported PD50 of penicillin G was 0.307 ± 0.325 mg/kg, and for ciprofloxacin it was 25.52 ± 1.95 mg/kg. The PD50 of ciprofloxacin was lower than the values observed in our present study, but in the study of Sullivan and colleagues, the animals received ciprofloxacin doses every 3 h for a total of eight doses. Additionally, in our study, all of the test strains had ciprofloxacin MIC values of 2 mg/L, which was higher than the MIC value of 1 mg/L used in the multidose ciprofloxacin study.
The activity of trovafloxacin and ciprofloxacin against S. pneumoniae has been evaluated in other in-vivo models including a rabbit model of meningitis,16,17 a systemic (intraperitoneal) sepsis model18 and a murine pneumonia model.18,19 Regardless of the model investigated, trovafloxacin has consistently been shown to be more active against pneumococcus than other fluoroquinolones, including ciprofloxacin.
Our results seem to correlate well with previously published reports of trovafloxacin efficacy and ciprofloxacin failure against S. pneumoniae in other in-vivo models.16,17,18,19 The effects of trovafloxacin against penicillin-resistant strains of S. pneumoniae may add to its clinical usefulness as penicillin resistance increases worldwide.
In summary, using a murine model of intraperitoneal sepsis, we have demonstrated that trovafloxacin has potent microbiological activity against S. pneumoniae compared with ciprofloxacin, regardless of penicillin susceptibility. However, when considering penicillin-susceptible and -intermediate strains, ampicillin displayed superior activity compared with trovafloxacin and ciprofloxacin.
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Acknowledgments |
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The authors thank the members of the animal care facility at our institution for the care of the animals through the study period. This work was supported by a grant from Pfizer Laboratories, Inc. This work was presented at the Thirty-seventh Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Ontario, Canada, 28 September–1 October 1997.20
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Notes |
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* Correspondence address. Hartford Hospital, Division of Infectious Diseases, 80 Seymour Street, PO Box 5037, Hartford, CT 06102-5037, USA. Tel: +1-860-545-3941; Fax: +1-860-545-5112; E-mail: dnicola{at}harthosp.org
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Received 4 January 1999; returned 13 April 1999; revised 10 May 1999; accepted 27 May 1999
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