Efficacy of single and combined antibiotic treatments of Anthrax in rabbits 1

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1 AAC Accepted Manuscript Posted Online 21 September 2015 Antimicrob. Agents Chemother. doi: /aac Copyright 2015, American Society for Microbiology. All Rights Reserved. Efficacy of single and combined antibiotic treatments of Anthrax in rabbits 1 Shay Weiss, Zeev Altboum, Itai Glinert, Josef Schlomovitz, Assa Sittner, Elad Bar-David, David Kobiler and 3 Haim Levy * 4 Department of Infectious Diseases, Israel Institute for Biological Research, Ness-Ziona, Israel 5 Short title: Efficacy of antibiotic treatment of Anthrax 7 Corresponding author: 9 Haim Levy 10 Department of Infectious Diseases 11 Israel Institute for Biological Research 12 P.O.Box 19, Ness-Ziona, Israel 13 Tel: haiml@iibr.gov.il

2 2 Abstract 18 Respiratory Anthrax is a fatal disease in the absence of early treatment with antibiotics. Rabbits are highly 19 susceptible to infection with Bacillus anthracis spores by intranasal instillation succumbing within 2 to 4 day 20 post infection. This study aims to test the efficiency of antibiotic therapy to treat systemic Anthrax in this 21 relevant animal model. Delaying the initiation of antibiotic administration to over 24 hours post infection 22 resulted in treatment of animals with systemic Anthrax in varying degrees of bacteremia and toxemia. As the 23 onset of symptoms in humans was reported to start on day 1-7 post exposure, delaying the initiation of 24 treatment by hours (time frame for mass distribution of antibiotics) may result in treating sick 25 populations. We evaluated the efficacy of antibiotic administration as a function of bacteremia levels at the 26 time of treatment initiation. Here we compare the efficacy of treatment with Clarithromycin, Augmentin, 27 Imipenem, Vancomycin, Rifampin, and Linezolid to the previously reported efficacy of Doxycycline and 28 Ciprofloxacin. We demonstrate that treatment with Augmentin, Imipenem, Vancomycin, and Linezolid were 29 as affective as Doxycycline and Ciprofloxacin, curing rabbits exhibiting bacteremia levels of up to CFU/ml. Clarithromycin and Rifampin were shown to be effective only as a post exposure prophylactic 31 treatment but failed to treat the systemic (bacteremic) phase of Anthrax. Furthermore we evaluate the 32 contribution of combined treatment of Clindamycin and Ciprofloxacin demonstrating improvement in efficacy 33 when compared to Ciprofloxacin alone

3 3 Introduction 37 Bacillus anthracis, the causative agent of Anthrax, is a Gram-positive aerobic spore-forming bacillus (1). The 38 exact disease caused by this virulent bacterium depends on the route of infection (2, 3). Cutaneous exposure 39 causes a local disease with typical lesions that without treatment might develop into a systemic disease. Oral 40 consumption of spores can lead to an oropharyngeal or gastrointestinal infection, which can be fatal. In 41 contrast, respiratory exposure to spores leads directly to a fulminant systemic disease which is fatal without 42 treatment (4, 5). In respiratory Anthrax in humans, the incubation time from infection till onset of symptoms 43 is estimated to be 1 to 7 days. The disease begins with non-specific flu-like symptoms lasting 2-3 days, with a 44 sudden progression to severe respiratory distress and shock leading to death within hours. In 45 experimental animal models there are no preceding physiological symptoms, even with bacteremia, until the 46 development of severe respiratory distress at the late stage in close proximity to death (6). Therefore, disease 47 progression is characterized by levels of blood bacteremia and PA concentrations, two closely correlated 48 markers considered a reliable measure of disease severity in animal models (7). 49 The importance of timely and effective treatment was demonstrated in the 2001 Anthrax bioterrorism attack in 50 USA, when 5 of the 10 inhalational Anthrax patients died in spite of massive antibiotic administration, 51 probably due to the use of ineffective antibiotic therapy and/or delayed treatment initiation (i.e. during the 52 fulminating stage of the disease) (8, 9). Effective post exposure therapy protocols, preventing the 53 establishment of a fatal Anthrax disease have been described in the literature. Post exposure treatment with 54 Penicillin (10-12), Doxycycline (10), Ciprofloxacin (10, 13) and Levofloxacin (13) efficiently protected 55 rhesus monkeys following inhalation of lethal doses of virulent B. anthracis spores. In guinea pigs, treatment 56 with Penicillin (14), Doxycycline(15), Tetracycline (16), Ciprofloxacin (15, 16) and Erythromycin (16) 57 protected the animals, but upon treatment termination the animals died due to Anthrax relapse (12, 15, 16). To 58 develop improved/alternative treatment protocols for systemic Anthrax, we previously evaluated the efficacy 59 of antibiotic treatment at different stages based on bacteremia levels (17). In rabbits, treatment with 60 Ciprofloxacin (17), Levofloxacin (18) or Doxycycline (17) was efficient in preventing the death of animals 61 with systemic Anthrax. Furthermore, combination of anti-pa antibodies with either Ciprofloxacin or 62

4 4 Levofloxacin therapy demonstrated improved efficacy when compared to the single-antibiotic treatment (17, 63 19). 64 Recently, the Centers for Disease Control and Prevention (CDC) updated the guidelines for prevention and 65 treatment of Anthrax, designating three treatment protocols for the disease: post exposure prophylaxis, 66 systemic Anthrax without meningitis and systemic Anthrax with suspected or confirmed meningitis (20). The 67 current version emphasizes combining antimicrobial therapy for systemic Anthrax, with three or more drugs. 68 Optional antimicrobials include: Ciprofloxacin, Levofloxacin, Moxifloxacin, Doxycycline, Amoxicillin, 69 Clindamycin, Meropenem, Penicillin or Ampicillin, Linezolid, Chloramphenicol, Rifampicin, and 70 Vancomycin. It should be mentioned that only in-vitro sensitivity data is available for the majority of the 71 suggested antimicrobial drugs (21) while there is a substantial knowledge gap regarding their individual in- 72 vivo efficacy, as well as the efficacy of combined therapy. 73 Herein, we tested single or dual antibiotic administration in order to evaluate their efficacy, in a relevant 74 animal model, both as post exposure prophylaxis as well as systemic Anthrax therapy (characterized by the 75 presence of both bacteria and toxins in the circulation)

5 5 Materials and Methods 78 B. anthracis strain. The strain used in this study was ATCC (Vollum) (Tox+ Cap+) from the IIBR 79 collection (22). 80 Animals. New Zealand white rabbits ( kg) were obtained from Charles River (USA). The animals 81 received food and water ad libitum. The animals were inoculated via the respiratory route by intranasal 82 instillation (i.n.). The estimation of i.n. 50% lethal dose (LD 50 ) in rabbits is 2x10 4 CFU (23). 83 This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of 84 Laboratory Animals of the National Research Council. The protocols were approved by the Committee on the 85 Ethics of Animal Experiments of the IIBR. 86 Exposure and treatment regimens. Groups of rabbits were inoculated i.n. with 2-6x10 6 (100LD 50 ) 87 Vollum spores. For single antibiotic treatments, 32 hours post inoculation, blood samples were drawn from 88 the rabbits' ear vein to determine the level of bacteremia and the animals were immediately treated with 89 antibiotics. For dual antibiotic treatment, with ciprofloxacin and clindamycin, blood samples were drawn from 90 the ear vein starting at 30 hours, and every 2 hours afterwards for determination of serum PA levels by 91 immune-assay. For each animal the individual treatment initiation time was defined according to the blood PA 92 (>40 ng/ml). Single antibiotic treatment continued twice daily for a period of 14 days and combined treatment 93 continued for 3 days (due to known toxicity of Clindamycin to rabbits) followed by single treatment with 94 ciprofloxacin alone for 11 days. Afterwards the animals were monitored for survival for additional 30 days. 95 To test the effect of treatment on the development of specific protective immune response, the surviving 96 animals were tested for acquired protective immunity by subcutaneous injection of 2x10 4 (100LD 50 ) Vollum 97 spores (23). 98 ELISA for PA. Serum levels of PA were determined as previously reported (17). Briefly, PA levels were 99 determined by direct ELISA in 96-well microtiter plates (Nunc, Rosklide, Denmark), using purified PA as the 100 reference standard. Plates were coated with 100 µg/ml of diluted rabbit serum α-pa in NaHCO 3 buffer 101 (50mM, ph 9.6) and subsequently blocked with 5% skim milk (Becton Dickinson, Sparks, MD). The plates 102 were washed with phosphate-buffered saline containing 0.05% Tween 20 (PBST), and incubated with the 103

6 6 tested sera (diluted 1:2 in 0.5% skim milk) for 1h at 37oC. For the standard curve, known concentrations of 104 purified PA in 50% serum were used. The plates were washed with PBST, and incubated with diluted rabbit 105 α-pa serum. Following a PBST rinse, plates were developed with alkaline phosphatase-conjugated goat α- 106 rabbit immunoglobulin G (IgG) (Sigma, St. Louis, MO) as the detecting reagent and p-nitrophenyl phosphate 107 (Sigma, St. Louis, MO) as the substrate. Absorbance at 405nm was determined using a Spectramax 190 micro 108 plate reader (Molecular Devices, Sunnyvale, CA). The end point was defined as the highest dilution at which 109 the absorbance was >3 standard deviations above that of the negative control. The sensitivity of this assay was 110 determined as 10ng/ml PA. 111 In vitro toxin secretion assays. The level of PA secretion in response of different concentrations of 112 Ciprofloxacin and Clindamycin, was determine by a broth microdilution test. The antibiotic was diluted in 113 NBY-HCO 3 medium (8gr Nutrient Broth, 3gr Yeast extract, 5gr Glucose, 9gr NaHCO 3, in 1L medium), and 114 the Vollum vegetative bacteria were added to a final concentration of 5x10 5 CFU/ml. The plates were 115 incubated at 37 o C in 10% CO 2 atmosphere for 16h. The PA concentration was determined by ELISA and the 116 bacterial concentration by serial dilution and colony counts on tryptose agar plates. 117 Determination of bacteremia. Bacterial levels in the blood were determined as previously reported (16). 118 Briefly, each blood sample was plated undiluted and after serial dilutions in saline. The plates were incubated 119 for 16h at 37 o C and the bacteremia was determined by colony counting. The lower limit of detection was CFU/ml. 121 Pharmacokinetics of the antibiotics in rabbits. The pharmacokinetics of each antibiotic used in this work is 122 presented in Table 1. Blood samples were drawn from rabbits at various time points after antibiotic 123 administration and the concentration of the antibiotic in the serum was defined by determining the highest 124 serum dilution that inhibits the growth of VollumΔpXO1ΔpXO2. The serum inhibitory concentration (SIC) is 125 presented as the reciprocal of the maximal inhibitory dilution. All the antibiotics tested, administered by 126 different routes, showed significant serum inhibitory concentration, lasting for several hours. 127

7 7 Statistical analysis. The significance of the differences in survival rates between treated groups and untreated 128 controls and differences in bacteremia and time to treatment, was determined by Fisher's exact test, two tailed, 129 using Prism 6 software (Graphpad, USA). 130

8 8 Results 131 Efficacy of single antibiotic treatments 132 In this study we evaluated the efficiency of different antibiotic agents, which are available in hospitals or in 133 community health care centers, to treat experimentally-induced respiratory Anthrax. The antibiotics tested 134 were selected according to their mechanism of action. Augmentin (Amoxicillin-Clavulanate) (β-lactams), 135 Vancomycin (Glycopeptides) and Imipenem (Carbapenems) represent the cell-wall synthesis inhibitory 136 agents. Rifampin was tested as an RNA inhibitor, and from the protein synthesis inhibitors, clarithromycin 137 (Macrolids) and Linezolid (Oxazolidinone) were used. Treatment efficacy was studied using our previously 138 reported rabbit model (17), in which bacteremia levels serve as a marker for systemic disease progression 139 following intranasal instillation of Vollum spores. Based on our experience we defined 32 hours post exposure 140 as the time point to determine the bacteremia level and administer antibiotics, allowing for a wide range of 141 bacteremia levels for treatment. In all experiments inoculation dose was LD 50 of Vollum spores and 142 antibiotic treatment started 32 hours post-injection and continued twice-daily for 14 days. To ensure rapid 143 systemic antibiotic dispersion, the first dose was administered i.v. (when the adequate product was available) 144 and the following doses were administrated s.c. 145 Cell wall synthesis inhibitors treatment regimens initiated with an i.v. antibiotic administration and followed 146 by s.c. antibiotic injections (Table 1). The antibiotic treatment was given to groups of 21, 23 and 19 rabbits 147 (for Augmentin, Vancomycin and Imipenem, respectively). As demonstrated in Fig.1A (upper panels), all the 148 cell-wall inhibitors were very effective as post-exposure prophylaxis, preventing the development of Anthrax 149 when the treatment started before the onset of bacteremia (blue symbols). Those antibiotics were also very 150 effective in curing highly bacteremic rabbits (red symbols) with bacteremia loads of up to 10 6 CFU/ml 151 (Augmentin and Imipenem) and 10 5 CFU/ml (Vancomycin). After the cessation of antibiotic treatment, all the 152 surviving animals were monitored for an additional period of 14 days. Our findings demonstrate that except 153 for one animal (treated with Imipenem) all the animals were cured (Fig 1A, lower panels). Serum conversion 154 was observed in all the bacteremic animals that were treated with Vancomycin and Imipenem (data not 155 shown). It should be mentioned that after cessation of treatment with Augmentin the rabbits developed severe, 156 non-anthrax related diarrhea that caused death in 5 animals. 157

9 9 Treatment with the RNA inhibitor Rifampin was conducted on 20 infected rabbits, and was administered p.o. 158 twice daily (Table 1). Prophylactic treatment was given to five rabbits, whereas the rest were bacteremic at 159 treatment initiation. As can be clearly seen from the data presented in Fig 1B (upper panel and lower panel), 160 rifampin failed to cure bacteremic animals or even delay time to death, and was effective only as a 161 prophylactic treatment. No relapse of the disease was observed in the prophylactic group of rifampin (Fig 1B, 162 lower panel). 163 The treatment regimen for protein synthesis inhibitors included p.o. administration of Clarithromycin or 164 Linezolid (Table 1). Our data demonstrates that these two antibiotics were effective as prophylactic treatments 165 (Fig 2, blue symbols and lines) and that Linezolid was very efficient in curing highly bacteremic animals, with 166 bacteremia levels of 10 5 to 10 6 CFU/ml (Fig 2. red symbols and lines). Clarithromycin, on the other hand, 167 failed to cure animals even at very low bacteremia levels (Fig 2. red symbols and lines), and was effective 168 only as a prophylactic treatment (Fig 2. blue symbols and lines). 169 Efficacy of combined antibiotic treatment. 170 In a previous study (17), we had demonstrated that Ciprofloxacin treatment was effective for treating animals 171 with bacteremia levels of up to 10 5 CFU/ml. We were also able to demonstrate the added benefit of combined 172 treatment of Ciprofloxacin with anti-pa antibodies in animals that had bacteremia levels in the range between CFU/ml. Therefore, we decided to evaluate the efficacy of combined treatment with Ciprofloxacin 174 and Clindamycin, a combination that was recommended by the CDC (20), in animals with bacteremia levels 175 of 10 5 to 10 7 CFU/ml. In order to determine when each individual animal was in the required bacteremia range 176 for treatment initiation, levels of PA in the sera were used to predict the level of bacteremia (7). For that 177 purpose, 32 animals were inoculated with LD 50 Vollum spores. Blood was drawn from the ear vein 178 and serum PA levels were determined by a rapid immuno-assay, starting 30 hours post infection and every hours thereafter. Antibiotic treatment was initiated when the infected animals had serum PA levels in the 180 range of higher than 40 ng/ml. The initial combined treatment, with Ciprofloxacin (60 mg/kg, p.o.) and 181 Clindamycin (60 mg/kg, i.v), was given to 15 animals and was compared to single antibiotic treatment (only 182 Ciprofloxacin) that was given to 16 animals (one animal died before antibiotics were administered). The 183

10 10 combined treatment was continued for 3 days (with Clindamycin administered subcutaneously), and from 184 days 4-14 both groups were treated with Ciprofloxacin only. As seen in 2 Figure 3, both groups were 185 statistically identical, exhibiting similar bacteremia ranges and treated at similar time points after infection. 186 Whereas the Ciprofloxacin treatment cured only 33% of the animals (4/12), the combined treatment of 187 animals with initial bacteremia of up to 10 6 CFU/ml, exhibited an impressive statistically significant 188 improvement (P=0.0102) by curing 100% of the treated animals (10/10). No mono treatment experiment 189 using clindamycin was preformed due to known toxicity to rabbits. 190 In an attempt to elucidate the mechanisms underlying this observation, we studied the effect of the two 191 antibiotics on the proliferation of the bacteria and on the release of the toxins (PA), both in vitro and in vivo. 192 As can be seen in Fig. 4A (upper panels, in vitro experiments) Ciprofloxacin exhibits strong inhibition of 193 bacterial proliferation, accompanied by a significant increase in toxin concentration. In contrast, Clindamycin 194 showed a strong reduction in toxin secretion, even at concentrations sub-inhibitory for proliferation. 195 Therefore, we can assume that Clindamycin's contribution to the increased curative effect of the combined 196 treatment relies on its ability to inhibit the increased toxin secretion. This conclusion is substantiated by the in 197 vivo studies (Fig 4B lower panels), where our data suggest an increased effect of the combined antibiotic 198 treatment (purple lines) on the toxin secretion, but not on bacterial proliferation in comparison to single 199 antibiotic treatments with ciprofloxacin or clindamycin (red and blue lines, respectively)

11 11 Discussion 202 Rapid initiation of antimicrobial therapy is crucial in the management of severe infection and sepsis. Proper 203 selection of an antimicrobial treatment regimen against the infectious agent, with optimal pharmacokinetic 204 and pharmacodynamic characteristics, is highly important (24). The same principles are important in the case 205 of systemic Anthrax, for which the delay in antibiotic treatment is tightly associated with increased mortality. 206 The recommendations for antibiotic treatment of inhalational Anthrax are mainly based on in vitro 207 susceptibility testing, in-vivo animal studies, and fragmented clinical experience, as no clinical human studies 208 are available on these infections. Previous studies in animal models and the reports on the treatment of 209 systemic Anthrax in humans have demonstrated that the Ciprofloxacin treatment is highly efficient (25). 210 Current CDC guidelines recommend single treatment with a fluoroquinolone or a tetreacycline as 211 postexposure prophylaxis, and combination therapy for the established disease. For patients with proven or 212 suspected meningitis, the recommended combination includes a fluoroquinolone (Ciprofloxacin or 213 Levofloxacin), a bactericidal cell-wall acting agent (e.g. Meropenem) and a protein synthesis inhibitor (e.g. 214 Linezolid). If meningitis is ruled out, treatment can include a fluoroquinolone and a protein synthesis inhibitor 215 only (e.g. Linezolid or Clindamycin). Combinational antimicrobial treatment is not usually recommended for 216 most infectious diseases due to possible unexpected antagonistic effects of the antibiotics. It has, however, an 217 important role for specific indications, including broad coverage of polymicrobial infections, empiric 218 coverage for agents with suspected antibacterial resistance, and in cases of synergistic effects of the individual 219 components. Limited in-vitro testing has been done to evaluate combination therapy for B. anthracis. In a 220 study by Athamna et al (26), most combinations have shown no synergism, including the recommended 221 combinations of ciprofloxacin with linezolid, clindamycin, and rifampicin, and some combinations were even 222 antagonistic. Only the combination of rifampicin and clindamycin was shown to be synergistic (26). Brook et 223 al (27) have examined the effect of combination treatment in an irradiated mouse model with B. anthracis 224 Sterne infection. The addition of clindamycin to ciprofloxacin actually increased mortality (27). Clinically, 225 retrospective studies have shown that combination treatment was associated with decreased mortality (28). 226 In this manuscript we report our studies in rabbits, determining the efficacy of treatment with single 227 antibiotics on Anthrax in different stages of the disease, from post-exposure prophylaxis to advanced systemic 228

12 12 disease (highly bacteremic animals). As can be seen, all the antibiotics tested were effective as prophylactic 229 treatments for as long as the drugs were given. Prophylactic treatment with Vancomycin and Clarithromycin 230 failed to prevent disease upon cessation of treatment. Antibiotic treatment does not eliminate all the spores 231 from the lungs and residual spores can initiate the development of the disease. This can be prevented by either 232 prolonged treatment or combining treatment with the administration of active PA based vaccination. 233 In previous publications, we and others have shown in several animal models that established systemic 234 Anthrax can be treated with antibiotics. In this study, we tested additional antibiotics demonstrating their 235 relative efficacy. Whereas the cell-wall synthesis inhibitors, Augmentin, Vancomycin or Imipenem, were very 236 efficient in treating systemic Anthrax, the RNA inhibitor, Rifampin, failed to cure rabbits. Imipenem and 237 Rifampin, with similar SICs and kinetics, achieve a completely different treatment outcome, indicating a 238 possible difference between their in vivo and in vitro efficacies. The protein synthesis inhibitors show 239 variations in their efficacy, as Linezolid did cure bacteremic rabbits, while Clarithromycin completely failed 240 to save them. A possible reason for the discrepancy in this case relies on the fact that Clarithromycin's MIC 241 for B. anthracis ( μg/ml) is in the upper borderline of the sensitive range (as determined for 242 staphylococcus) ( whereas all the others 243 protein synthesis inhibitors are in the sensitive range. 244 The beneficial effect of the combination of antibiotics with different mechanisms of action is demonstrated in 245 the Ciprofloxacin-Clindamycin experiments. The reduction in toxin secretion induced by Clindamycin seems 246 to contribute to the increased curative effect of the combined antibiotic treatment. Further studies should be 247 carried out to demonstrate that the suggested mechanism underlies the additive effect. 248 Herein we report the efficacy of single antibiotic treatments on different stages of systemic Anthrax. This data 249 can contribute to the building of a robust experimental base for crafting better recommendations for efficient 250 prevention and treatment of Anthrax

13 13 Acknowledgments 254 We would like to thank Nili Rothschild for her excellent technical assistance. Special thanks to Dr. Avigdor 255 Shafferman, Dr. Arie Ordentlich, Dr. Shmuel Yitzhaki and Dr. Tal Brosh-Nissimov (MD) for their fruitful 256 discussions. 257

14 14 Table 1. Pharmacokinetics of tested antibiotics in rabbits 258 Trade name Antibiotic agent Administration route Dose (mg/kg) Cmax (SIC) Tmax (h) Tmin (h) Augmentin Amoxycillin and Clavulanic acid i.v. s.c. 150 (Amoxy) Vancomycin i.v Vancomycin 40 Mylan s.c Tienam Imipenem-Cilastatin i.v. s.c Rifadin Rifampin p.o Kalcid Clarithromycin p.o. 80 8(29) 2(29) 12(29) Zyvoxid Linezolid p.o

15 15 Figure legend 262 Fig 1. Efficacy during and after treatment Cell wall synthesis inhibitors (A), Nucleic acids synthesis 264 inhibitors (B). The rabbits were infected by i.n. instillation of Vollum spores and treatment was initiated 32 h 265 post infection. Upper panel: Bacteremia level of each individual rabbit at treatment initiation and the outcome 266 of the treatment (during the antibiotic administration: live (circle) or dead (x). The animals that received 267 prophylactic treatment (sterile at treatment initiation) are marked in blue. Rabbits with bacteremia in the rage 268 of 10 to 10 6 CFU/ml are marked in red. Rabbits with bacteremia higher than 10 6 CFU/ml are marked in black. 269 Lower panel: survival rate with time. Gray untreated, blue prophylactics, red treatment of bacteremic 270 animals with bacteremia of CFU/ml. Gray boxes indicate the duration of antibiotic treatment. 271 Fig 2. Efficacy during and after treatment Protein synthesis inhibitors. The rabbits were infected by i.n. 273 instillation of Vollum spores and treatment was initiated 32 h post infection. Upper panel: Bacteremia level of 274 each individual rabbit at treatment initiation and the outcome of the treatment (during the antibiotic 275 administration: live (circle) or dead (x). The animals that received prophylactic treatment (sterile at treatment 276 initiation) are marked in blue. Rabbits with bacteremia in the rage of 10 to 10 6 CFU/ml are marked in red. 277 Rabbits with bacteremia higher than 10 6 CFU/ml are marked in black. Lower panel: survival rate with time. 278 Gray untreated, blue prophylactics, red treatment of bacteremic animals with bacteremia of CFU/ml. Gray boxes indicate the duration of antibiotic treatment. 280 Fig 3. Efficacy during and after treatment combined treatment. Upper panel: Bacteremia level of each 282 individual rabbit at treatment initiation and the outcome of the treatment: live (circle) or dead (x). Rabbits 283 with bacteremia in the rage of 10 to 10 6 CFU/ml are marked in red. Rabbits with bacteremia higher than CFU/ml are marked in black. Lower panel: survival rate with time. Gray untreated, red treatment of 285 bacteremic animals with bacteremia of CFU/ml. Gray boxes indicate the duration of antibiotic 286 treatment. 287 Fig 4. Pharmacodynamics of ciprofloxacin and clindamycin in-vitro and In-vivo. A: In vitro effect of 288 increasing concentrations of Ciprofloxacin or Clindamycin on bacterial growth (red) or PA secretion (blue). 289 B: Pharmacodynamics of bacteremia (right) or serum PA (left) following the initiation of antibiotic treatment. 290 Ciprofloxacin (red), Clindamycin (blue) and the combined treatment of Ciprofloxacin and Clindamycin 291 (purple)

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