AAC Accepts, published online ahead of print on 30 July 2012 Antimicrob. Agents Chemother. doi:10.1128/aac.01104-12 Copyright 2012, American Society for Microbiology. All Rights Reserved. 1 2 Consequences of delayed ciprofloxacin and doxycycline treatment regimens against F. tularensis airway infection 3 4 Running title: Delayed antibiotic treatments of tularemia 5 6 7 Shahar Rotem $, Erez Bar-Haim $, Hila Cohen, Uri Elia, Raphael Ber, Avigdor Shafferman # and Ofer Cohen 8 9 10 Department of Biochemistry and Molecular Genetics, Israel Institute for Biological research, Ness-Ziona, Israel 11 12 13 $ Equal contribution # Address correspondence to Avigdor Shafferman, avigdors@iibr.gov.il
14 15 16 17 18 19 This study examines the efficacy, bacterial load and humoral response of an extensively delayed ciprofloxacin or doxycycline treatments following airway exposure of mice to F.tularensis holarctica (LVS) or to the highly virulent F.tularensis tularensis (SchuS4) strains. A delay in onset of both antibiotic treatments allowed the rescue of all LVS infected animals. Yet, for animals infected with SchuS4, only ciprofloxacin was efficacious and prolongation of treatment rescued all animals. 20
21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Francisella tularensis, a Gram-negative facultative intra-cellular bacterium, is the etiological agent of tularemia and is classified as a category A biological warfare agent by the CDC (6, 13). Human infections are mainly caused by F. tularensis holarctica (type B) or by tularensis (type A) subspecies (19). The latter, especially when inhaled, is highly infectious and virulent in humans, (7, 19). Antibiotics usually provide curative therapy for tularemia. Mortality depends on the infection type, patient's health and treatment onset. Aminoglycosides offer good bactericidal properties and low relapse rates, yet due to their toxicity and the need for parenteral administration, they are not the drug of choice for prophylactic treatment. According to the CDC guidelines, in cases of F. tularensis epidemics or a bio-terror scenario, ciprofloxacin and doxycycline represent the treatment of choice (6). Tetracycline and doxycycline treatment have documented relapses (~ 10%), however, others indicate tetracycline to be as effective as streptomycin when a prolonged treatment is given (8, 16). Ciprofloxacin and other quinolones have been shown to be effective against experimental tularemia (14, 15, 20), as well as in clinical treatment of type B tularemia (1, 2, 10, 17). 37 38 39 40 41 42 Efficacy of different ciprofloxacin and doxycycline treatment regimens against F. tularensis airway infection. Mice (female Balb/c) were inoculated intranasally with 100LD 50 of either F. tularensis LVS (10 5 CFU) or SchuS4 (10 2 CFU), a dose that initiates a rapidly fatal disease, resulting in death of all animals within 5-7 days (3). Treatments with ciprofloxacin (50mg/kg b.i.d, i.p.) or doxycycline (40mg/kg b.i.d, i.p.)
43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 were initiated either at 24, 48 or 72 hours post-infection (p.i.), and consisted for 7 and 14 days respectively. Ciprofloxacin or doxycycline treatment rescued all LVS infected mice regardless of initiation time of treatment (Table 1). In the case of delayed treatment starting 72 hours p.i., clear clinical signs (weight loss, scruffy appearance, lethargy) were observed. At the end of the treatment all LVS exposed animals exhibited no clinical signs and significant anti-f.tularensis antibody titers were measured 45 days p.i. (Table 1). When ciprofloxacin treatment was initiated 72 hours post SchuS4 infection, ~30% of the mice appeared to be morbid and 4-5 days after cessation of treatment died, while treatment starting 24 or 48 hours p.i., rescued all animals. None of the SchuS4 infected mice that were treated with doxycycline 72 hours following infection survived this airway infection (Table 1), while survival of mice treated 24 or 48 hours p.i. were 90% and 30% respectively. During the treatment periods described above all mice survived irrespective of the antibiotic used. Similar systematic study performed by Russell et. al, (15) found that following SchuS4 intra-peritoneal exposure, treatment with either doxycycline or ciprofloxacin is efficient when initiated 24 hours p.i.. These results, of relatively early treatment p.i. are in complete agreement with our results, although we used a different route of infection airway exposure. However, when the onset of treatment was delayed to 48 or even 72 hours p.i., a scenario which could be relevant to bio-terror, we find a clear advantage of ciprofloxacin over doxycycline (Table 1). We quantified the bacterial loads in the lungs, liver and spleen of airway infected mice, during antibiotic treatment and following its cessation (as described in (4, 11)). As seen in Figure 1, in LVS exposed mice, all organs tested were clear of bacteria 4-7 days
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 after ciprofloxacin administration. Doxycycline reduced the bacterial load during the 14 days of treatment, albeit at a lower rate (Figure 1). Unlike the case for ciprofloxacin, two days after cessation of doxycycline (day 19 p.i.) bacteria re-emerged in all organs, reaching a level of approximately 10 3 CFU in the lungs, liver or spleen (note that the LD 50 for intranasal administration with LVS is 10 3 CFU and ~1 CFU for intra-peritoneal administration). The bacterial load in the organs remained for 7 days post-treatment, declining gradually below the limit of detection (Figure 1). No morbidity was observed, and all animals survived. The bacterial loads of SchuS4 in organs from animals that were treated with ciprofloxacin or doxycycline 72 hours p.i., was determined immediately after cessation of treatment and 2-3 days thereafter (Figures 2 and 3). Ten days p.i. (the last day of ciprofloxacin treatment), all the animals were devoid of bacteria (Figure 2). However, 3 days later (13 days p.i.) 30% of the animals exhibited considerable levels of bacteria in the lungs, liver and spleen (ca. 10 3 CFU equivalent to 10 3 LD 50 i.n. or i.p.), in agreement with the observation of survival shown in Table 1. Doxycycline treatment initiated 72 hours post SchuS4 infection was efficient as long as the animals received the antibiotic, yet the disease relapsed after treatment secession. At this stage approximately 10 2 CFU were found in the lungs, spleen and liver which further increased to 10 3-10 4 CFU in all these organs two days later (Figure 3A). These results are in accordance with the inability to rescue animals from SchuS4 i.n. infection if treatment is initiated 72 hours p.i. (Table 1). The occurrence of infection relapse was documented in the treatment of human cases of tularemia, particularly when tetracycline, or doxycycline, were used (8, 12, 16, 17) underlying the poorly understood "depot effect" of F. tularensis infection (4, 5, 9, 18). Of
89 90 91 92 93 94 95 note, SchuS4 and LVS infected mice eventually exhibited similar levels of humoral response following doxycycline treatment, yet only LVS infected animals survived the high level of reemerging bacteria (equivalent lethal doses of over 10-100 LD 50, see Figure 1). The data underline the importance of timely onset of antibiotic treatment as an efficient countermeasure against F. tularensis tularensis infection and clearly demonstrate the advantage of ciprofloxacin in cases of delayed onset of treatment. 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 Consequences of further extension of the period of antibiotic treatments of SchuS4 infected animals. To reduce and eventually eliminate bacterial relapse after termination of treatment, we examined the extension of ciprofloxacin and doxycycline treatment periods in the SchuS4 airway infection murine model. An extended treatment consisting of ten days of ciprofloxacin administration (instead of 7 days), increased survival (to 100%) even when treatment was initiated 72 hours p.i. without any disease relapse (Table 2). Conversely, prolongation of doxycycline treatment to 21 days yielded a marginal improvement, when the treatment was initiated 72 hours p.i. (Table 2). Following the extended treatment, no bacteria were found (limit of detection <5 CFU) in all inspected tissues (Figure 3B), however, 2 days after cessation of treatment a relapse occurred with bacterial counts reaching 10 4 CFU in all organs. The findings of this study establish that both ciprofloxacin and doxycycline are effective in preventing the development of tularemia in the mouse model following airway infection, but in the case of bacteriostatic antibiotic such as doxycycline there is a significant failure to achieve complete recovery unless treatment is initiated within a
112 113 114 115 116 117 short time window (24 hours p.i.). At later stages of the disease (72 hours p.i), prolonging ciprofloxacin treatment (to 10 days) but not doxycycline (to 21 days) appeared to be an effective strategy for successful therapy against the highly virulent F. tularensis SchuS4 strain. These observations have important implications for designing efficient therapeutic approaches for treatment of tularemia in various scenarios, where early treatment is not possible. 118 119 120 121 122 Acknowledgments: We thank Dr. T. Chitlaru, Dr. G. Zaide, I. Inbar, G. Fridman, S. Eherlich and S. Maoz for their assistance in performing some of the experiments and Dr. N. Ariel for fruitful discussions and critical reading of the manuscript. 123
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179 Figure legends 180 181 182 183 184 185 186 Figure 1. Bacterial counts of LVS during and following antibiotic treatment. LVS bacterial counts in the lungs (A) and liver (B) during and post antibiotic treatment with ciprofloxacin (gray diamonds) or doxycycline (black rectangle); Arrows indicate the onset and termination of the indicated treatment. The limit of detection was 5 CFU/organ. Values represent an average and standard-deviations of results from 3 animals per time point. 187 188 189 190 191 192 193 194 195 196 Figure 2. Bacterial counts of SchuS4 after cessation of ciprofloxacin treatment. Counts of residual bacteria were determined when ciprofloxacin treatment was initiated 72 hours p.i. and continued for 7 days (note that when treatment initiated 48 hours p.i. with ciprofloxacin, bacteria were not detected in any organ after cessation of treatment, see text). Bacterial counts, were measured in 10 animals, in the lungs (black bar), liver (grey bar) and spleen (white bar) at the end of the treatment (day 10 p.i.) and 3 days later (day 13 p.i.) in additional 10 different animals. The limit of detection was 5 CFU/organ. For survival data after ciprofloxacin treatment regimen, see Table 2. Values represent an average and standard-deviations of 3-10 (as indicated) animals per time point. 197 198 199 200 201 Figure 3. Bacterial counts of SchuS4 after cessation of doxycycline treatment. Bacterial counts following SchuS4 infection when doxycycline treatment was initiated 72 hours p.i. for a period of 14 days (upper panel) or 21 days (lower panel) days. Bacterial counts in the lungs (black bar), liver (grey bar) and spleen (white bar) where measured at
202 203 204 205 the end of the treatment (day 18 and 25 p.i. respectively) and 2 days later (day 20 and 27 p.i. respectively). The limit of detection was 5 CFU/organ. For survival data after doxycycline treatment regimen see Table 2. Values represent an average and standarddeviations of 3 animals per time point.
206 207 208 209 210 Table 1: Comparative efficacy of different regimens of treatment by ciprofloxacin and doxycycline, initiated at different times post airway (i.n.) infection of LVS or SchuS4. Treatment Onset of treatment (hours post infection) % Survival a (n=10) LVS infection SchuS4 infection Ab Titer of surviving animals b (GMT) LVS infection None n.a. 0 0 n.a. n.a. SchuS4 infection Ciprofloxacin (7 days treatment) 24 100 100 800 80 48 100 100 840 200 72 100 70 1680 2500 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 Doxycycline (14 days treatment) 24 100 90 12600 7500 48 100 30 29000 21000 72 100 0 29000 n.a. a Significance from control was determined by the Fisher exact test (p<0.05) b Different animals were used for monitoring survival and for immunological parameters. Antibody (Ab) titer limit of detection<40. Antibody titers were calculated as reciprocal geometric mean titers (GMT), with geometric standard deviations not greater than 1.8. n.a: not applicable.
235 236 237 238 239 Table 2: The effect of duration of antibiotic treatment initiated 72 hours post airway infection by SchuS4. Treatment Onset of treatment (hours post infection) Duration of treatment (days) None - 0 0 % Survival SchuS4 infection a (n=10) Ciprofloxacin 72 7 70 10 100 240 241 242 243 14 0 Doxycycline 72 21 10 a Significance from control was determined by the Fisher exact test (p<0.05), except for the 21 days treated doxycycline group.