Scientific Research Department, Armed Forces Radiobiology Research Institute, 8901 Wisconsin Avenue, Bethesda, MD 20889-5603, USA
Received 28 June 2005; returned 26 July 2005; revised 4 August 2005; accepted 13 September 2005
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Abstract |
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Methods: Ten-week-old B6D2F1/J female mice were either sham-irradiated or given a sublethal 7 Gy dose of 60Co-gamma-photon radiation 4 days prior to an intratracheal challenge with toxigenic B. anthracis Sterne spores. Mice were treated twice daily with 200 mg/kg clindamycin (subcutaneous or oral), 100 mg/kg moxifloxacin (oral), 50 mg/kg ciprofloxacin (subcutaneous) or a combination therapy (clindamycin + ciprofloxacin). Bacteria were isolated and identified from lung, liver and heart blood at five timed intervals after irradiation. Survival was recorded twice daily following intratracheal challenge.
Results: The use of clindamycin increased survival in gamma-irradiated and sham-irradiated animals challenged with B. anthracis Sterne in comparison with control mice (P < 0.001). Ciprofloxacin-treated animals had higher survival compared with clindamycin-treated animals in two experiments, and less survival in a third experiment, although differences were not statistically significant. Moxifloxacin was just as effective as clindamycin. Combination therapy did not improve survival of sham-irradiated animals and significantly decreased survival among gamma-irradiated animals (P = 0.01) in comparison with clindamycin-treated animals. B. anthracis Sterne was isolated from lung, liver and heart blood, irrespective of the antimicrobial treatment.
Conclusions: Treatment with clindamycin, ciprofloxacin or moxifloxacin increased survival in sham-irradiated and gamma-irradiated animals challenged intratracheally with B. anthracis Sterne spores. However, the combination of clindamycin and ciprofloxacin increased mortality associated with B. anthracis Sterne infection, particularly in gamma-irradiated animals.
Keywords: anthrax , irradiation , translocation , clindamycin , ciprofloxacin , moxifloxacin
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Introduction |
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Concomitant accidental or terrorism-related exposure to sublethal gamma or mixed-field (gamma and neutron) radiation would inevitably increase morbidity among individuals exposed to B. anthracis. Ionizing radiation damages the haematopoietic and gastrointestinal systems. Prompt, sublethal irradiation increases the susceptibility to bacterial infections by decreasing the number of circulating mature white blood cells and the number of epithelial cells in the intestine.7 In a previous study we demonstrated that sublethally irradiated mice have an increased susceptibility to the lethal B. anthracis Sterne infection that is associated with infections due to endogenous bacteria.8
Clindamycin is used to treat serious infections caused by susceptible organisms, including infections of the lungs and septicaemia. It is active against most aerobic and anaerobic Gram-positive cocci as well as Gram-negative anaerobic bacilli, but has poor activity against most Gram-negative facultative aerobes and Enterococcus strains.9 Clindamycin is also active against B. anthracis10,11 and it inhibits the production of -toxin by Clostridium perfringens12 and an M-protein by group A streptococcal species.13 Additionally, clindamycin may inhibit lipase activity by Staphylococcus aureus.14
Moxifloxacin has in vitro activity against a wide range of Gram-positive and Gram-negative microorganisms. It was approved by the US Food and Drug Administration (FDA) for the treatment of acute bacterial exacerbations of chronic bronchitis, acute bacterial sinusitis and mild to moderate community-acquired pneumonia. The bactericidal action of moxifloxacin against Gram-negative organisms is similar to that of ciprofloxacin, resulting from inhibition of the topoisomerase II (DNA gyrase) and topoisomerase IV required for bacterial DNA replication, transcription, repair and recombination. The C8-methoxy moiety of moxifloxacin contributes to enhanced activity, lower MICs and lower selection of resistant mutants of aerobic Gram-positive bacteria compared with the C8-H moiety present in ciprofloxacin.15 Its MIC for B. anthracis Sterne is similar to that of ciprofloxacin.10
Ciprofloxacin is one of three antimicrobial agents (ciprofloxacin, doxycycline and penicillin) approved by the FDA for the treatment of B. anthracis infections. The FDA approved it specifically for this indication in August 2000 based on the efficacy data found in rhesus monkeys.16 However, the in vitro resistance of B. anthracis Sterne to ciprofloxacin and doxycycline can increase following their serial inoculations in sub-inhibitory concentration.17 Resistance of B. anthracis to ciprofloxacin is the effect of a stepwise accumulation of GyrA and ParC mutations and increased efflux activity.18 Resistance of many Gram-positive and Gram-negative microorganisms to ciprofloxacin and other quinolones has also increased in recent years.19
In vitro studies using microorganisms other than B. anthracis have shown additive effects when clindamycin is combined with ciprofloxacin.20,21 Furthermore, the USA CDC recommends ciprofloxacin plus the use of another drug (i.e. clindamycin) that has in vitro activity against B. anthracis for the prophylaxis and treatment of inhalation anthrax.22 Their combination in vitro provides no indication of either synergy or antagonism against B. anthracis.23 However, drug interactions have never before been studied in an ionizing radiation-induced neutropenic animal model.
Because of the in vitro activity of clindamycin against B. anthracis, its ability to inhibit production of toxin in other bacterial species and the potential for combination therapy to achieve better survival than either drug individually, we evaluated the efficacy of clindamycin against an intratracheal challenge with toxigenic B. anthracis Sterne spores in gamma-irradiated and sham-irradiated mice. Survival and microbiological endpoints were determined and compared with similar endpoints associated with moxifloxacin, ciprofloxacin and combination (clindamycin and ciprofloxacin) therapy.
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Materials and methods |
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Ten-week-old female B6D2F1/J mice were supplied by Jackson Laboratories (Bar Harbor, ME, USA). They were quarantined and confirmed to be free of common murine pathogens by the Armed Forces Radiobiology Research Institute's (AFRRI's) veterinarian pathologist. For the duration of the experiment, the animals were housed in a segregated room in accordance with specifications outlined in the Guide for the Care and Use of Laboratory Animals.24 The animal facility and the programme are accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International, and all experimental procedures were performed in compliance with 9CFR, Animal Welfare Act and AFRRI's policies regarding animal care and use. The animals were housed with a 12:12 h light/dark cycle in polycarbonate cages with a filter cover (Micro-Isolator Lab Products, Maywood, NJ, USA) on hardwood chip bedding. Autoclaved commercial rodent feed and acidified water (pH 2.8) were provided ad libitum.
Bacteria
The bacteria used in this study were the B. anthracis Sterne strains (a gift from Colorado Serum Institute, Denver, CO, USA). Toxigenic B. anthracis Sterne spores were harvested from batch fermentations in Schaeffer's sporulation medium.25 Frozen spore stocks were kept in 10% glycerol/water at 70°C. The spore concentration was adjusted by diluting it with sterile water to achieve a concentration of 1.0 x 1010 cfu/mL. Prior to inoculation, the spore concentration was verified by tube dilution in sterile water and culture on tryptic soy agar (Becton Dickinson 221283).
Isolated microorganisms were identified by the Vitek identification system (bioMerieux Vitek, Hazelwood, MO, USA). B. anthracis Sterne was identified by colony morphology and MICs for recovered organisms were determined using standard CLSI methodology.26
Antimicrobials
Pharmacia & Upjohn Company (Kalamazoo, MI, USA) provided clindamycin as Cleocin Clindamycin® and clindamycin hydrochloride (batch K0300546, lot 40090) for subcutaneous and oral administration, respectively. Bayer Corporation (Mt Prospect, IL, USA; batch/lot no: 661093E) provided moxifloxacin in powder form for oral administration. Bayer (West Haven, CT, USA) provided 50 mg/kg ciprofloxacin in a 0.1 mL subcutaneous injection, BID as CIPRO® I.V. For oral administration, special 20-gauge, 1.5 inch, stainless steel feeding cannulas (Popper and Sons, Inc., New Hyde Park, NY, USA) were used, with small ball tips that were cleaned and sterilized in self-sealing pouches.
Experimental design
60Co-gamma-photon irradiation
A total of 656 mice were used in three experiments. The mice were divided into two groups: a sham-irradiated group and a 7 Gy dose of 60Co-gamma-photon radiation group (Table 1). Irradiated mice were placed in perforated acrylic plastic restrainers and irradiated to 7 Gy at 0.4 Gy/min mid-line tissue (MLT) dose from bilaterally positioned 60Co sources. Sham-irradiated mice were placed in identical restrainers for the same amount of time without irradiation.
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B. anthracis Sterne spore suspension was drawn from a serum bottle into a 1 mL tuberculin syringe. The syringe was then fitted with a blunt 1.5 inch 22-gauge pipetting needle bent at an angle of 135° at 1 inch from the tip. The tongue of the animal was gently pulled outward and laterally using the forceps. The blunt tip of the needle was inserted under the epiglottis to uncover the larynx and then lightly pushed between these into the tracheal lumen. The needle was inserted into the mid-trachea, a volume of 0.1 mL of spore suspension that contained 1.0 x 109 cfu B. anthracis Sterne spores was gently injected and the needle was withdrawn. Inspection of the pharynx was continued for a short time while the animal was kept on the board to make that sure no suspension was regurgitated.
Antimicrobial therapy
Sublethally irradiated and sham-irradiated mice were assigned to receive sterile water or 200 mg/kg clindamycin or 100 mg/kg moxifloxacin (oral) twice daily in Experiment 1. Mice were assigned to saline, 200 mg/kg clindamycin, 50 mg/kg ciprofloxacin or combination treatment groups (200 mg/kg clindamycin and 50 mg/kg ciprofloxacin) (subcutaneous) twice daily in Experiments 2 and 3. Sterile water and saline controlled for oral and subcutaneous administration of antimicrobials, respectively.
Mice were further assigned to sets to determine survival or for culturing bacteria in selected tissues in Experiments 1 and 2; survival was the only endpoint in Experiment 3. Those mice in survival sets were observed for signs of disease and survival for 40 days after initiating antimicrobial therapy. Lung, liver and heart blood tissues from mice in microbiological culture sets were assayed on days 3, 5, 7, 10 and 12 after spore challenge to assess the extent of B. anthracis Sterne infection.
Antimicrobial therapy was started 24 h after the intratracheal spore challenge and was continued, twice daily, for 21 days. At scheduled intervals, specimens of lung, liver and heart blood were removed aseptically from five mice in each microbiological culture set. Tissues were crushed with a sterile swab and spread onto Columbia sheep blood agar (SBA), Columbia colistin-nalidixic acid sheep blood agar (CNA) and xylose-lysine-desoxycholate agar (XLD).
Statistical analysis
The 45-day survival rates for the experimental groups of mice were compared with the Logrank Test (Prism 4, GraphPad Software, Inc.).
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Results |
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Both clindamycin and moxifloxacin increased the 45-day survival rate from 90% (27/30), found in sham-irradiated, sterile water-treated animals, to 100% (30/30) and 97% (29/30), respectively (P = 0.24). In gamma-irradiated animals, clindamycin increased the 45-day survival rate in sterile water-treated animals from 0% (0/20) to 25% (5/20) (P = 0.05) (Figure 1). An increase in survival was observed in mice given moxifloxacin, 55% (11/20), compared with those given clindamycin (P = 0.11).
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Experiment 2: antimicrobial treatment with clindamycin, ciprofloxacin and their combination in irradiated animals
All gamma-irradiated, saline-treated mice died within the first 2 weeks [0% survival (0/24)]. Twenty-one days of clindamycin therapy increased the 45-day survival rate to 50% (12/24) (P < 0.001; Figure 2), which was statistically similar to survival among ciprofloxacin-treated animals [67% (16/24), P = 0.38] and to combination-treated animals [46% (11/24), P = 1.0] (Figure 2).
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A survival of 36% (8/22) was observed in the sham-irradiated, intratracheally challenged mice treated with saline at 45 days post-sham-irradiation. Twenty-one days of clindamycin increased the survival rate to 100% (21/21) (P < 0.001) and did not differ statistically from 21 days of ciprofloxacin [95% (20/21), (P = 1.0)]. Combination therapy, however, decreased the survival rate to 86% (19/22) compared with clindamycin (P = 0.23; Figure 3).
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Antimicrobial susceptibility of B. anthracis Sterne
The MICs of clindamycin, ciprofloxacin and moxifloxacin for B. anthracis Sterne were 0.5, 0.08 and 0.08 mg/L, respectively. There was no change in the MIC of any antimicrobial when tested against B. anthracis Sterne cells recovered from lung, liver or heart blood tissue 40 days after initiating the 21-day treatment.
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Discussion |
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This study provides evidence that treatment with clindamycin can increase survival of gamma-irradiated and sham-irradiated mice challenged with toxigenic B. anthracis Sterne spores. All clindamycin treatment groups in our three experiments had significantly higher survival rates than their respective control groups. Although treatment with either moxifloxacin or ciprofloxacin was superior to clindamycinlargely in gamma-irradiated animalsthese differences were not statistically significant. In particular, moxifloxacin therapy in irradiated animals increased survival by 30% in comparison with clindamycin therapy.
Differences in survival between the treatment groups were not the result of increased resistance to B. anthracis Sterne. All drugs had MICs that were similar to reported MICs for other B. anthracis strains.10,11,17 Also, the lower survival among clindamycin-treated animals in the first experiment is probably a result of oral administration of clindamycin, which would decrease the bioavailability of the drug, versus the subcutaneous route. Three conditions that could have contributed to differences in survival, particularly among irradiated animals were: (i) the insufficient extermination of B. anthracis Sterne organisms; (ii) a low host response that was unable to keep pace with the replicating bacteria; and (iii) a lack of clindamycin, moxifloxacin or ciprofloxacin activity against a portion of the polymicrobial infection that emerged after exposure to ionizing radiation and B. anthracis Sterne challenge.
The doses of moxifloxacin and ciprofloxacin aimed to achieve a 24 h area under the concentrationtime curve (AUC) similar to that found in humans for the treatment of Streptococcus pneumoniae.29,30 In order to achieve a target 24 h AUC value (1219 h·mg/L), Onyeji et al.29 administered a single dose of 20 mg/kg ciprofloxacin, given subcutaneously to mice with renal impairment that had been induced with 5 mg/kg uranyl nitrate. We used a higher dose of ciprofloxacin since uranyl nitrate was not used. Similarly, Ernst et al.31 administered moxifloxacin, 100 mg/kg (oral) twice daily, to 2429 g Swiss-Webster mice with induced renal insufficiency (6 mg of uranyl nitrate). They determined the free murine 24 h AUC to be slightly higher than the human target. We therefore used 100 mg/kg (oral) because uranyl nitrate was not used.
Enteric bacteria translocation has been observed in chemotherapy-induced neutropenic mice,30 in lethally gamma-irradiated mice32 and in non-lethally gamma-irradiated mice infected with B. anthracis.8 We observed B. anthracis Sterne infection with enteric organisms in sham-irradiated mice infected with B. anthracis Sterne spores, which suggests the potential of B. anthracis toxins alone to induce translocation in the non-irradiated host. This conclusion is consistent with our previous study, which found a lack of translocation in sham-irradiated animals without anthrax challenge.8 Furthermore, translocation and mixed infection was observed in sham-irradiated mice challenged with toxigenic B. anthracis Sterne but not in gamma-irradiated mice challenged with non-toxigenic B. anthracis -Sterne-1.
Clindamycin is capable of inhibiting toxin production in C. perfringens, S. aureus and Streptococcus species,12,14 and it can protect a host from toxic shock syndrome induced by Escherichia coli lipopolysaccharide.33 The presence of bacterial translocation of enteric organisms in irradiated mice infected with B. anthracis Sterne and treated with clindamycin therefore suggest that clindamycin did not prevent toxin production by B. anthracis Sterne.
Equally remarkable was our finding that despite the clinical success of antimicrobial therapy, B. anthracis Sterne was still present in the organs of sham-irradiated and gamma-irradiated animals treated with antibiotics. Our previous studies have shown that gamma-irradiated mice infected with B. anthracis Sterne require therapy with antimicrobials that possess a broader spectrum of activity and are effective against B. anthracis Sterne as well as endogenous Gram-positive and Gram-negative translocated flora.34,35 Furthermore, initiating antimicrobial treatments within 24 h of challenge and continuing it for at least 21 days optimizes survival of the host because persistent inactive spores may germinate when antimicrobial therapy is completed.32,36 A period of 21 days following non-lethal gamma irradiation in mice also allows them time to begin recovering from the depression of bone marrow progenitor cells and from the depressed innate immune system. The persistence of B. anthracis Sterne underscores the practical difficulty in treating animals challenged with spores. This finding supports the rationale for prophylaxis and long-term therapy for inhalation anthrax that is recommended for humans, and validates this animal model.
Ciprofloxacin appeared to clear Gram-positive and Gram-negative infections among gamma-irradiated animals much better than clindamycin given that these microorganisms were isolated from a smaller proportion of ciprofloxacin-treated mice. In this study, the combination of clindamycin and ciprofloxacin was utilized in an attempt to broaden the antimicrobial spectrum of therapy and increase survival. This approach, however, revealed an adverse interaction between clindamycin and ciprofloxacin, particularly in gamma-irradiated animals, that reduced animal survival significantly. No such antagonism was previously observed in clinical trials in humans. An earlier study in healthy volunteers did not find any untoward interactions between ciprofloxacin and clindamycin or any changes in the pharmacokinetics of ciprofloxacin with the addition of clindamycin.37 We currently have no explanation for this observation. However, these findings underscore the importance of performing additional studies in order to further characterize this observation.
Evaluating and confirming this adverse relationship is of great importance as the combination of ciprofloxacin and clindamycin is suggested by the CDC for the treatment of infection due to B. anthracis22 and was given to 5 of the first 10 victims of the 2001 anthrax mail related epidemic.38 Of interest is that two of these five individuals did not survive. A potential yet unlikely cause for this phenomenon may be the ability of the antibiotic combination to alter the gastrointestinal flora and allow the outgrowth of a particular group of microorganisms that resulted in many of the observed infections. However, our data does not support this explanation as the organisms that were identified to cause infection in combination-treated animals were similar to those found to cause infection in animals treated with saline, clindamycin or ciprofloxacin alone (e.g. E. faecium, S. xylosus).
In conclusion, we found clindamycin therapy to be effective in the treatment of B. anthracis Sterne infections in sham-irradiated and gamma-irradiated hosts. However, the combination of clindamycin and ciprofloxacin therapy increased mortality associated with B. anthracis Sterne infection, particularly in gamma-irradiated animals. The mechanism responsible for the observed drug interaction is unknown and merits further study. Its predominance in gamma-irradiated animals suggests that radiation-induced damage influences the success of combination therapies.
Moxifloxacin therapy was very effective for the treatment of B. anthracis Sterne among sham-irradiated and gamma-irradiated animals. This may be due to its broader activity against many of the associated Gram-positive and Gram-negative organisms in comparison with ciprofloxacin and clindamycin.39 Furthermore, bacterial translocation was observed in sham-irradiated mice treated with clindamycin therapy, suggesting a lack of in vivo toxin suppression by this antibiotic. Additional studies may be required to assess the in vivo effectiveness of clindamycin to inhibit the production of toxin by B. anthracis.
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Acknowledgements |
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