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Antimicrobial Agents and Chemotherapy, April 2009, p. 1624-1627, Vol. 53, No. 4
0066-4804/09/$08.00+0     doi:10.1128/AAC.00186-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Efficacy of Ciprofloxacin in an Experimental Model of Escherichia coli Chorioamnionitis in Rabbits

Elise Launay,¹ Nicolas Joram,¹ Cédric Jacqueline,² Anne Francoise Miegeville,² Jocelyne Caillon,^2,3 Gilles Potel,² Jean Christophe Roze,¹ and Christèle Gras-Le Guen^1,2*

Département de Périnatologie, Hôpital Mère Enfant CHU Nantes, Nantes 44 093, France,¹ Université de Nantes, Nantes Atlantique Universités, Thérapeutiques Cliniques et Expérimentales des Infections, EA3826, UFR Médecine, 1 Rue G. Veil, Nantes F-44035, France,² Laboratoire de Microbiologie CHU Nantes, Nantes 44 093, France³

Received 10 February 2008/ Returned for modification 11 May 2008/ Accepted 3 December 2008

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Pregnant rabbits were treated with ciprofloxacin alone or with gentamicin in a model of Escherichia coli chorioamnionitis, and the results were compared with those for untreated rabbits. The survival rate increased and the bacteremia decreased significantly in treated fetuses in comparison to controls (P = 0.003). Nevertheless, rapid selection of resistant mutants is a major limit to ciprofloxacin applications.

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Maternal-fetal infection is a frequent problem in neonatology (2) and has deleterious consequences, such as prematurity (10), periventricular leukomalacia (6), and bronchopulmonary dysplasia (17). Maternal antibiotherapy is not always effective. Clinicians are divided between continuing the pregnancy under antibiotherapy, with the risk of leaving the fetus in a potentially deleterious environment, and stopping the pregnancy and treating the child ex utero with the risk of prematurity complications (9, 12).

In this context, it seemed interesting to study the efficacy of antibiotics known for their effective activity in purulent conditions, such as fluoroquinolones. The purpose of this study was to determine in an experimental model of chorioamnionitis (11) whether maternal treatment with ciprofloxacin associated with gentamicin constitutes an effective treatment for an infected fetus.

We used a previously described model of chorioamnionitis (11) with 10⁶ CFU Escherichia coli K1 inoculated into one uterine horn. The MICs of ciprofloxacin and gentamicin were 0.006 mg/liter and 0.5 mg/liter, respectively. The 30 pregnant rabbits used in this study were randomly assigned to one of the following three groups: (i) untreated animals (n = 6) sacrificed 12 or 36 h after inoculation or kept alive for follow-up; (ii) animals treated with ciprofloxacin alone (n = 12) for 24, 48, or 72 h; or (iii) animals treated with ciprofloxacin and gentamicin (n = 12) and sacrificed after 24, 48, or 72 h of treatment. Antibiotic treatment began 12 h after the inoculation. A dose of 30 mg/kg of body weight twice a day of ciprofloxacin (Ciflox; Pfizer, Paris, France) was administered to pregnant rabbits through a catheter inserted into a marginal vein of the ear (18), and 10 mg/kg of gentamicin was administered once a day (Schering-Plough, NJ) using an intramuscular method.

The pharmacokinetic objectives for both drugs were comparable to those observed in human cases: the area under the concentration-time curve (AUC)/MIC was >125 and the AUC from 1 to 24 h was 49 µg/h/ml for ciprofloxacin (8); for gentamicin, the maximum concentration of drug in serum (C_max)/MIC was >10, the C_max was 20 mg/liter, and the half-life was 1.5 h (4). The protein binding in rabbit serum was similar to that of human serum (14). Ciprofloxacin and gentamicin (in mothers and in four fetuses) were assayed using a reference microbiological method (13).

The number of living fetuses relative to the total number of fetuses extracted was noted. After euthanasia, blood was withdrawn from mothers and pups, and the placentas, lungs, livers, and brains from four to five fetuses/group were crushed with 500 µl of sterile physiological water and then cultured on tryptic soy agar, eosin-methylene blue, and charcoal agar for 24 h (11).

The surviving bacteria were identified with Api20E (Biomerieux), and a test for susceptibility to nalidixic acid, ofloxacin, and ciprofloxacin was carried out. The MICs of ciprofloxacin were obtained by using an Etest tip. Pulsed-field gel electrophoresis was performed on surviving bacteria and inoculum bacteria in order to compare strains (1).

Bacterial counts were subjected to one-way analysis of variance, followed by Scheffe's test for intergroup comparisons (Statistica software). Percentages were compared by using the chi-square test with Yates's correction.

All fetuses were alive 36 h after inoculation with E. coli, but the fetal survival rate (number of living fetuses relative to the total number of extracted fetuses) without treatment decreased, and no fetuses survived at 60 h after inoculation (Fig. 1). With ciprofloxacin treatment, fetal survival was 50%, with a significant difference from survival in the untreated group appearing after a 48-h treatment (14/30 versus 0/20, respectively [P = 0.02]). Treatment with ciprofloxacin and gentamicin resulted in an improvement of fetal survival, with a significant increase in the survival rates after a 24-h treatment (23/30 fetuses surviving versus 1/18 in the untreated group and versus 10/21 in the monotherapy group [P = 0.03]) and after a 48-h treatment (23/32 versus 14/30 in the monotherapy group [P = 0.04] and versus 0/20 in the untreated group [P = 0.0003, respectively]).

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FIG. 1. Fetal survival rates with different treatments (Kaplan-Meier curves). The survival rate is the number of living fetuses relative to the total number of fetuses extracted from each mother rabbit. The best survival rate is observed with the ciprofloxacin-and-gentamicin treatment as soon as 24 h after the beginning of the treatment.

As soon as 12 h after inoculation, 50% of maternal blood cultures were positive. All the maternal blood cultures were sterile after 48 h of monotherapy and 24 h of bitherapy treatment. A 24-h bitherapy treatment resulted in sterilization of the blood and amniotic fluid cultures of surviving fetuses (Fig. 2a and b). The investigation of tissular cultures (placenta, brain, lung, and liver) (Fig. 2c to f) revealed significant reductions of bacterial growth following mono- and bitherapy, faster in the bitherapy groups, although we observed a light bacterial regrowth. The pharmacokinetic parameters showed that objectives were obtained, with a mean ciprofloxacin AUC from 0 to 12 h of 20.74 mg/liter; a mean AUC/MIC of 3,460, and a mean gentamicin C_max of 20.1 ± 1.6 mg/liter (mean ± standard deviation) with a half-life of 1.5 h. We found high ciprofloxacin residual concentrations in fetal blood and amniotic fluid (more than 10 times the MIC). We estimated a transplacental diffusion of around 35%. The E. coli strains isolated from placenta and brain cultures (two different mothers) after a 48-h treatment in the bitherapy group became resistant to nalidixic acid (MIC, >16 mg/liter) and exhibited an intermediate response to ofloxacin (MIC, 2 mg/liter) but remained sensitive to ciprofloxacin (MIC, 0.125 mg/liter; a 16-fold increase). The E. coli strains isolated were similar to the initial bacteria (similitude of 98%) as determined by pulsed-field gel electrophoresis (Fig. 3).

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FIG. 2. Quantitative fetal tissue culture results for different treatments. (a to f) Blood (a), amniotic fluid (b), placenta (c), brain (d), lung (e), liver (f). The error bars represent standard deviations. Asterisks represent significant differences. The arrow on the y axis represents the CFU amount shown by quantitative culture 12 h after inoculation (before treatment was started). *, P < 0.05 versus results with no treatment; **, P < 0.05 versus results with monotherapy; ***, P < 0.05 versus results with bitherapy. For each group, the number of animals studied varied from 3 to 22.

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FIG. 3. Dendrogram from comparison of different strains by pulsed-field gel electrophoresis. 1, Source strain; 2, strain from brain culture after 72 h of bitherapy; 3, strains from placenta culture after 72 h of bitherapy; 4, strains from blood culture of a dead fetus.

The use of ciprofloxacin is an effective treatment for E. coli chorioamnionitis in this experimental model because it improved fetal survival and resulted in an early sterilization of the blood cultures of all the surviving fetuses. In comparison to results obtained previously with ceftriaxone in the same experimental model (11), ciprofloxacin resulted in a greater and faster decrease from the amount of the inoculum in fetal blood. Ciprofloxacin seemed to be of interest because of its greater activity in purulent pus (3), smaller inoculum effect (7), activity when bacteria were in a stationary state of growth, and antibiotic effect (5). Moreover, an association with gentamicin resulted in a faster and more intense bactericidal effect with a significantly better survival rate than ciprofloxacin alone. The absence of a significant benefit in fetal survival compared to ceftriaxone-plus-gentamicin treatment may reflect the limitations of the model, in which dead fetuses constitute a persistent source of bacteria and toxins.

Moreover, we noticed some regrowth after 2 days with the bitherapy treatment, paradoxically not observed for the monotherapy group. The possibility of a gentamicin impact (antagonism) on ciprofloxacin pharmacokinetics is unlikely, especially when considering that some studies even obtained evidence of a slight synergic effect (5). The selection of quinolone-resistant mutants appears to be a valuable hypothesis, because ciprofloxacin is known to have a resistance selection effect in E. coli strains (15). Robicsek et al. described a new adaptation of a common aminoglycoside acetyltransferase [AAC(6')-1b] that is able to modify ciprofloxacin (16). This enzyme confers reduced susceptibility to ciprofloxacin. However, the presence of this enzyme was unlikely because the susceptibility of the E. coli strain to gentamicin was unchanged after bitherapy.

The application of such a therapeutic scheme is not necessarily a first choice in clinical practice because if bacteriological results concerning fetal septicemia are hopeful, the fast selection of resistant strains is a major barrier to the use of this form of therapy.

 
* Corresponding author. Mailing address: Département de Périnatologie, Hôpital Mère Enfant CHU Nantes, 38 Bd. Jean Monnet, 44093 Nantes CEDEX 1, France. Phone and fax: 33 240 083 483. E-mail: christele.grasleguen{at}chu-nantes.fr

Published ahead of print on 2 January 2009.

Top ABSTRACT INTRODUCTION REFERENCES

 

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Antimicrobial Agents and Chemotherapy, April 2009, p. 1624-1627, Vol. 53, No. 4
0066-4804/09/$08.00+0     doi:10.1128/AAC.00186-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Launay, E. Articles by Gras-Le Guen, C. Search for Related Content PubMed PubMed Citation Articles by Launay, E. Articles by Gras-Le Guen, C.