Failure of oily chloramphenicol depot injection to treat plague in a murine model

Lila Rahalisona, Annie Guiyouleb, Stephane P. Bonacorsic, Ivan Slacanind, Suzanne Chanteaua and Elisabeth Carnielb,*

a Laboratoire de la Peste, Institut Pasteur de Madagascar, BP 1274 Antananarivo, Madagascar; b National Reference Laboratory and WHO Collaborating Center for Yersinia, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15; c Hôpital Robert Debré, Laboratoire de Bactériologie, 75019 Paris, France; d Laboratoire Central, Rue du Canal 1, 2502 Bienne, Switzerland


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effective low-cost single-dose therapy would be invaluable in treating human plague. The efficacy of single- or two-dose injections of oily chloramphenicol (OCm) was compared with that of standard multiple injections of reference drugs (streptomycin or chloramphenicol) in a murine plague model. A single injection of OCm was ineffective. Two doses cleared bacteraemia and limited bacterial growth in the mouse spleen but were less effective in reducing mortality than standard therapy. However, because of the marked pharmacokinetic differences between mice and humans, the failure of depot injection of OCm in murine plague treatment is not indicative of its ineffectiveness in human plague.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Plague has been one of the most devastating diseases of human history, but the development of antibiotics significantly reduced the morbidity and mortality associated with this infection. The number of human cases remained low for several decades, but since the 1990s, plague is considered as a re-emerging disease.1 A total of 28,570 human plague cases have been reported to WHO over the last 15 years (1983–1997).2 Except for one recent isolate,3 Yersinia pestis, the plague bacillus, is usually sensitive to all antibacterial agents used against Gram-negative infections. The antibiotics recommended for plague treatment (streptomycin, chloramphenicol and tetracycline)4 should be administered at least twice daily for several days. However, in practice, because of the remote context of most endemic plague foci, the number of daily doses and the treatment duration achieved are often drastically reduced. There is a real need for a low-cost treatment, easily administered under most field conditions. Since a single- or two-dose regimen of oily chloramphenicol (OCm) has been shown to be a highly effective depot preparation for the treatment of meningococcal meningitis in Africa,57 we wondered whether this therapy could also be used to treat plague. In this study, the efficacy of one or two injections of OCm versus repeated injections of streptomycin or chloramphenicol to treat Y. pestis infection was compared in a murine experimental model.8


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strain

Mice were infected with the fully virulent Y. pestis strain 6/69. The MICs of chloramphenicol and streptomycin for strain 6/69, determined by the dilution method in Mueller– Hinton agar, were 4 mg/L and 2 mg/L, respectively.

Pharmacokinetic assays of OCm and chloramphenicol in mouse serum

After im injection into mice of 100 mg/kg chloramphenicol (Sigma, Paris, France) or 200 mg/kg OCm (Astrapin, Pfaffen-Schwabenhein, Germany), the serum of three mice was collected at 0, 0.30, 1, 2 and 5 h post-injection. The serum concentrations of these antibiotics were evaluated after extraction with ethyl acetate, using a high performance liquid chromatography method with a diode-array apparatus (HP-1090).

Establishment of mouse infection and therapy

An inoculum of 1000 ± 200 bacteria (>100 LD50) was injected iv into groups of five pathogen-free female OF1 mice (age 5 weeks, weight 25 ± 2 g, from Iffa Credo, L'Arbresle, France). Treatment was commenced 24 h after initiation of infection. The untreated control group received im or sc injections of physiological saline instead of antibiotic. For the streptomycin-treated group, mice received sc injections of 30 mg/kg bodyweight of streptomycin (Panpharma, Fougères, France), tid for 2 days, as previously described.8 Chloramphenicol was injected im tid for 2 days at a dose of 100 mg/kg in order to mimic the early mean serum concentration found in humans.7 Following the protocol used to treat meningitis, the OCm-treated group received either a single im injection at 24 h post-infection, or two im injections of 200 mg/kg at 24 and 48 h post-infection.

Assessment of infection and therapy

Five animals in each group were killed at 8, 24, 32, 56 and 72 h post-infection. Blood was sampled by heart puncture with a heparinated syringe. Spleens were aseptically removed and homogenized in 1.5 mL of physiological saline. Bacteriological counts were performed by plating 10-fold serial dilutions of the biological samples in duplicate on to trypto-casein soy agar supplemented with 0.025% haemin. The detection limit was 10 cfu. Bacterial isolates from blood or spleen were screened by the disc diffusion test in Mueller–Hinton agar for the emergence of resistance following antibiotic selection. Absence of carryover effect8 with streptomycin, chloramphenicol and OCm in the spleen homogenates was checked. To follow the survival of the animals over a longer period of time, groups of five mice infected with either 100 or 1000 cfu were treated from 24 h post-infection either with three daily injections of streptomycin or chloramphenicol for 2, 3 or 4 days, or with one or two injections of OCm. Mice were observed for 15 days and mortality was checked daily.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pharmacokinetic assays of OCm and chloramphenicol in mice

The first step in the establishment of the therapeutic protocol for chloramphenicol and OCm was to determine the pharmacokinetic parameters of these two drugs in the mouse serum. For chloramphenicol, the early mean concentration measured 30 min after im administration of 100 mg/kg of the antibiotic was 26.1 mg/L and the half-life in the mouse serum was 43 min. The early mean concentration and half-life of OCm after im injection of 200 mg/kg of the antibiotic were 74.6 mg/L and 60 min, respectively.

Growth kinetics of Y. pestis in the blood of treated or untreated infected mice

The results of the growth of Y. pestis in animal blood are shown in Figure 1Go. In all experiments, the untreated group was characterized by a rapid increase in the number of circulating Y. pestis starting 24 h post-infection, and the death of all animals within 3 days of infection. As expected, the groups treated with streptomycin or chloramphenicol were able to clear bacteria from their blood after 2 days of treatment. One injection of OCm resulted in a transient decrease in the number of circulating bacteria at 32 h, followed by further multiplication of bacteria in the blood. In contrast, two injections of this antibiotic at 24 h intervals resulted in the clearance of the bacteria from the bloodstream, suggesting that a single injection of OCm was not sufficient to treat plague but that two injections were as effective as the reference antibiotics in clearing the bacteria from the blood.



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Figure 1. Growth kinetics of Y. pestis in the blood of infected mice. The animals received either (•), physiological saline (untreated), or ({blacktriangledown}) one of the following antibiotics: (a) streptomycin, (b) chloramphenicol and (c) one dose or (d) two doses of oily chloramphenicol. The arrows below the horizontal axes represent the time points of antibiotic injections. The vertical bars indicate s.d. and the horizontal dotted line the limit of detection.

 
Growth kinetics of Y. pestis in the spleen of treated or untreated infected mice

A bacterial count was also performed on the spleen of infected animals. As shown in Figure 2Go, exponential growth of Y. pestis in the spleens of untreated mice started 8 h post-infection. Two days of treatment with three doses per day of streptomycin resulted in the disappearance of Y. pestis from the spleen of infected animals while the same therapeutic protocol only transiently decreased the number of bacteria in the spleen of chloramphenicol-treated mice. These results suggest that chloramphenicol is not as effective as streptomycin for the treatment of plague. One dose of OCm was not sufficient to clear splenic bacteria. Two injections led to a significant decrease in the number of splenic bacteria, although it did not eliminate them completely. Therefore, a single injection of OCm cannot control Y. pestis infection but two injections of this antibiotic appear to be as efficient as multiple injections of chloramphenicol in reducing the bacterial load in the spleen of infected animals.



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Figure 2. Growth kinetics of Y. pestis in the spleen of infected mice. The animals received either (•), physiological saline (untreated), or ({blacktriangledown}) one of the following antibiotics: (a) streptomycin, (b) chloramphenicol and (c) one dose or (d) two doses of oily chloramphenicol. The arrows below the horizontal axes represent the time points of antibiotic injections. The vertical bars indicate s.d. and the horizontal dotted line the limit of detection.

 
Effect of the different treatments on mice survival

Since a transient decrease in the number of bacteria in the spleen or blood of infected animals was sometimes followed by a secondary bacterial multiplication, the survival of the animals was checked over a longer period of time. Groups of five mice were infected as previously described and were subsequently treated from 24 h post-infection either with three daily injections of streptomycin or chloramphenicol for 2, 3 or 4 days, or with one or two injections of OCm. Streptomycin was found to be very active against plague in the experimental model of murine infection. After 2 days of treatment with this antibiotic, only one of five mice died, as previously observed,8 and after 3 or 4 days of treatment, all mice survived. The lower efficacy of chloramphenicol was confirmed by the observation that 4 days of treatment were necessary to cure four of five mice. In the OCm-treated group, a single injection of the antibiotic was completely ineffective and all mice died. Since our experimental infection was quite severe (>100 LD50), the efficacy of one injection of OCm against a lower initial bacterial inoculum (100 cfu) was tested. Although the spleen and the blood of the animals were sterilized (data not shown), the mortality rate remained high (seven of 10 mice). We are therefore unable to show satisfactory efficacy for single OCm injection in plague therapy. After two injections of OCm, only two of five animals survived the infection. This result, along with the fact that spleens of infected animals were not completely sterilized (Figure 2Go), indicates that although two injections of OCm can significantly reduce bacterial numbers, the reduction is temporary and insufficient to clear the microorganism. Secondary growth of persisting bacteria in lymphoid tissue probably led to relapsing disease and death.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of this work was to assess the efficiency of low-cost depot injection of OCm, in a reproducible murine experimental model of plague.8 Our results indicate that both one and two injections of OCm are less effective than repeated injections of the reference antibiotics, streptomycin and chloramphenicol, for the treatment of murine plague. These results may be explained partially by our observation that the half-life of OCm in the mouse serum is not much higher than that of chloramphenicol. Moreover, the major predictive pharmacokinetic parameter for bacteriostatic antibiotics is the time during which the drug concentration is greater than the MIC. For OCm, an inhibitory concentration was maintained for only 4 h, which is close to that achieved with chloramphenicol (1.5 h three times per day) and is much shorter than the 24 h observed in humans.7 Therefore, because of the marked differences in the pharmacokinetic parameters between mice and humans, the failure of one or even two injections of OCm in the therapy of murine plague may not signify that these regimens would be ineffective in treating human plague.


    Acknowledgments
 
We thank Michael Prentice for careful reading of this manuscript and Astrapin laboratory (Pfaffen-Schwabenhein, Germany) for the gift of oily chloramphenicol. This work was supported in part by funds from the Programme National de Lutte contre la Peste à Madagascar (subsidized by the World Bank). Lila Rahalison received a grant from the Actions Concertées des Instituts Pasteur.


    Notes
 
* Corresponding author. Tel: +33-1-45-68-83-26; Fax: +33-1-40-61-30-01; E-mail: carniel2{at}pasteur.fr Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Schrag, S. J. & Wiener, P. (1995). Emerging infectious diseases: what are the relative roles of ecology and evolution? Trends in Evolution and Ecology 10, 319–24.

2 . WHO. (1999). Human plague in 1997. Weekly Epidemiological Records 74, 340–4.

3 . Galimand, M., Guiyoule, A., Gerbaud, G., Rasoamanana, B., Chanteau, S., Carniel, E. et al. (1997). Multidrug resistance in Yersinia pestis mediated by a transferable plasmid. New England Journal of Medicine 337, 677–80.[Free Full Text]

4 . Barnes, A. M. & Quan, T. J. (1992). Plague. In Infectious Diseases, (Sherwood, L., Bartlett, J. G. & Blacklow, N. R., Eds), pp. 1285–91. W. B. Saunders Company, Philadelphia, PA.

5 . Pécoul, B., Varaine, F., Keita, M., Soga, G., Djibo, A., Soula, G. et al. (1991). Long-acting chloramphenicol versus intravenous ampicillin for treatment of bacterial meningitis. Lancet 338, 862–6.[ISI][Medline]

6 . Saliou, P., Ouedraogo, L., Muslin, D. & Rey, M. (1977). L'injection unique de chloramphénicol dans le traitement de la méningite cérébrospinale en Afrique tropicale. Médecine Tropicale 37, 189–93.

7 . Wali, S. S., Macfarlane, J. T., Weir, W. R. C., Cleland, P. G., Ball, P. A. J., Hassa-King, M. et al. (1979). Single injection treatment of meningococcal meningitis. 2. Long-acting chloramphenicol. Transactions of the Royal Society of Tropical Medicine and Hygiene 73, 698–702.[ISI][Medline]

8 . Bonacorsi, S. P., Scavizzi, M. R., Guiyoule, A., Amouroux, J. H. & Carniel, E. (1994). Assessment of a fluoroquinolone, three beta-lactams, two aminoglycosides, and a cycline in treatment of murine Yersinia pestis infection. Antimicrobial Agents and Chemotherapy 38, 481–6.[Abstract]

Received 6 July 1999; returned 27 October 1999; revised 8 November 1999; accepted 22 November 1999