Efficacy of Doxycycline and Ciprofloxacin for Treatment of Pneumonic Tularemia in Cynomolgus Macaques.

BACKGROUND: The incidence of pneumonic tularemia is very low; therefore, it is not feasible to conduct clinical efficacy testing of tularemia medical countermeasures (MCMs) in humans. The US Food and Drug Administration's Animal Model Qualification Program under the Drug Development Tools Program is a regulatory pathway for animal models used in MCM efficacy testing and approval under the Animal Rule. The National Institute of Allergy and Infectious Diseases and Biomedical Advanced Research and Development Authority worked together to qualify the cynomolgus macaque model of pneumonic tularemia. METHODS: Using the model parameters and end points defined in the qualified model, efficacy of the antibiotics doxycycline and ciprofloxacin was evaluated in separate studies. Antibiotic administration, aimed to model approved human dosing, was initiated at time points of 24 hours or 48 hours after onset of fever as an indicator of disease. RESULTS: Upon aerosol exposure (target dose of 1000 colony-forming units) to Francisella tularensis SchuS4, 80% of vehicle-treated macaques succumbed or were euthanized. Ciprofloxacin treatment led to 10 of 10 animals surviving irrespective of treatment time. Doxycycline administered at 48 hours post-fever led to 10 of 10 animals surviving, while 9/10 animals survived in the group treated with doxycycline 24 hours after fever. Selected surviving animals in both the placebo and doxycycline 48-hour group showed residual live bacteria in peripheral tissues, while there were no bacteria in tissues from ciprofloxacin-treated macaques. CONCLUSIONS: Both doxycycline and ciprofloxacin were efficacious in treatment of pneumonic tularemia, although clearance of bacteria may be different between the 2 drugs.

Systematic Review: Clinical Features, Antimicrobial Treatment, and Outcomes of Human Tularemia, 1993-2023.

BACKGROUND: Francisella tularensis, the causative agent of tularemia, is endemic throughout the Northern Hemisphere and requires as few as 10 organisms to cause disease, making this potential bioterrorism agent one of the most infectious bacterial pathogens known. Aminoglycosides, tetracyclines, and, more recently, fluoroquinolones are used for treatment of tularemia; however, data on the relative effectiveness of these and other antimicrobial classes are limited. METHODS: Nine databases, including Medline, Global Health, and Embase, were systematically searched for articles containing terms related to tularemia. Articles with case-level data on tularemia diagnosis, antimicrobial treatment, and patient outcome were included. Patient demographics, clinical findings, antimicrobial administration, and outcome (eg, intubation, fatality) were abstracted using a standardized form. RESULTS: Of the 8878 publications identified and screened, 410 articles describing 870 cases from 1993 to 2023 met inclusion criteria. Cases were reported from 35 countries; more than half were from the United States, Turkey, or Spain. The most common clinical forms were ulceroglandular, oropharyngeal, glandular, and pneumonic disease. Among patients treated with aminoglycosides (n = 452 [52%]), fluoroquinolones (n = 339 [39%]), or tetracyclines (n = 419 [48%]), the fatality rate was 0.7%, 0.9%, and 1.2%, respectively. Patients with pneumonic disease who received ciprofloxacin had no fatalities and the lowest rates of thoracentesis/pleural effusion drainage and intubation compared to those who received aminoglycosides and tetracyclines. CONCLUSIONS: Aminoglycosides, fluoroquinolones, and tetracyclines are effective antimicrobials for treatment of tularemia, regardless of clinical manifestation. For pneumonic disease specifically, ciprofloxacin may have slight advantages compared to other antimicrobials.

Treatment Outcome of Severe Respiratory Type B Tularemia Using Fluoroquinolones.

BACKGROUND: Fluoroquinolones lack approval for treatment of tularemia but have been used extensively for milder illness. Here, we evaluated fluoroquinolones for severe illness. METHODS: In an observational study, we identified case-patients with respiratory tularemia from July to November 2010 in Jämtland County, Sweden. We defined severe tularemia by hospitalization for >24 hours and severe bacteremic tularemia by Francisella tularensis subsp. holarctica growth in blood or pleural fluid. Clinical data and drug dosing were retrieved from electronic medical records. Chest images were reexamined. We used Kaplan-Meier curves to evaluate time to defervescence and hospital discharge. RESULTS: Among 67 case-patients (median age, 66 years; 81% males) 30-day mortality was 1.5% (1 of 67). Among 33 hospitalized persons (median age, 71 years; 82% males), 23 had nonbacteremic and 10 had bacteremic severe tularemia. Subpleural round consolidations, mediastinal lymphadenopathy, and unilateral pleural fluid were common on chest computed tomography. Among 29 hospitalized persons with complete outcome data, ciprofloxacin/levofloxacin (n = 12), ciprofloxacin/levofloxacin combinations with doxycycline and/or gentamicin (n = 11), or doxycycline as the single drug (n = 6) was used for treatment. One disease relapse occurred with doxycycline treatment. Treatment responses were rapid, with median fever duration 41.0 hours in nonbacteremic and 115.0 hours in bacteremic tularemia. Increased age-adjusted Charlson comorbidity index predicted severe bacteremic tularemia (odds ratio, 2.7 per score-point; 95% confidence interval, 1.35-5.41). A 78-year-old male with comorbidities and delayed ciprofloxacin/gentamicin treatment died. CONCLUSIONS: Fluoroquinolone treatment is effective for severe tularemia. Subpleural round consolidations and mediastinal lymphadenopathy were typical findings on computed tomography among case-patients in this study.

XFEL and NMR Structures of Francisella Lipoprotein Reveal Conformational Space of Drug Target against Tularemia.

Francisella tularensis is the causative agent for the potentially fatal disease tularemia. The lipoprotein Flpp3 has been identified as a virulence determinant of tularemia with no sequence homology outside the Francisella genus. We report a room temperature structure of Flpp3 determined by serial femtosecond crystallography that exists in a significantly different conformation than previously described by the NMR-determined structure. Furthermore, we investigated the conformational space and energy barriers between these two structures by molecular dynamics umbrella sampling and identified three low-energy intermediate states, transitions between which readily occur at room temperature. We have also begun to investigate organic compounds in silico that may act as inhibitors to Flpp3. This work paves the road to developing targeted therapeutics against tularemia and aides in our understanding of the disease mechanisms of tularemia.

Nanoparticle Formulation of Moxifloxacin and Intramuscular Route of Delivery Improve Antibiotic Pharmacokinetics and Treatment of Pneumonic Tularemia in a Mouse Model.

Francisella tularensis causes a serious and often fatal infection, tularemia. We compared the efficacy of moxifloxacin formulated as free drug vs disulfide snap-top mesoporous silica nanoparticles (MSNs) in a mouse model of pneumonic tularemia. We found that MSN-formulated moxifloxacin was more effective than free drug and that the intramuscular and subcutaneous routes were markedly more effective than the intravenous route. Measurement of tissue silica levels and fluorescent flow cytometry assessment of colocalization of MSNs with infected cells revealed that the enhanced efficacy of MSNs and the intramuscular route of delivery was not due to better delivery of MSNs to infected tissues or cells. However, moxifloxacin blood levels demonstrated that the nanoparticle formulation and intramuscular route provided the longest half-life and longest time above the minimal inhibitory concentration. Thus, improved pharmacokinetics are responsible for the greater efficacy of nanoparticle formulation and intramuscular delivery compared with free drug and intravenous delivery.

Case Report: Atypical Cutaneous Manifestations of Tularemia after Horsefly Bite.

Tularemia is an infectious zoonosis caused by Francisella tularensis, an aerobic, noncapsulated, Gram-negative coccobacillus. It is more common in the northern hemisphere, and there are sporadic reports in non-endemic areas. The bacterium is usually transmitted by the bite or feces of a tick or other arthropods such as mosquitoes and horseflies. We report a case of an Italian patient with tularemia after a horsefly bite.

Antibiotic susceptibility of Francisella tularensis subsp. holarctica strains isolated from tularaemia patients in France between 2006 and 2016.

Objectives: To determine the in vitro susceptibility to 18 antibiotics of human strains of Francisella tularensis isolated in France between 2006 and 2016, to check the absence of acquired resistance and to evaluate potential therapeutic alternatives. Methods: Fifty-nine clinically unrelated F. tularensis subsp. holarctica strains identified at the French National Reference Centre for Francisella as belonging to the phylogenetic subclade B.FTNF002-00 were used. MICs were determined in CAMHB medium supplemented with 2% PolyViteX®, using the CLSI broth microdilution method. Results: All strains were susceptible to fluoroquinolones (ofloxacin, ciprofloxacin, levofloxacin and moxifloxacin; MIC range: 0.016-0.25 mg/L), aminoglycosides (gentamicin and tobramycin; MIC range: ≤0.03-0.25 mg/L), doxycycline (MIC range: 0.125-0.25 mg/L) and chloramphenicol (MIC range: 0.5-2 mg/L). The erythromycin MIC range (0.5-2 mg/L) confirmed that all isolates belonged to biovar I of F. tularensis subsp. holarctica. Azithromycin and telithromycin displayed lower MIC ranges (0.25-1 and 0.03-0.5 mg/L, respectively). The tigecycline MIC range (0.25-1 mg/L) was slightly higher than that of doxycycline. All strains were resistant to ampicillin, meropenem, daptomycin, clindamycin and linezolid. Conclusions: F. tularensis strains isolated in France remain susceptible to antibiotic classes recommended for tularaemia treatment. Because fluoroquinolones display the lowest MIC90, have bactericidal activity and have lower therapeutic failure rates compared with doxycycline, they may be advocated as first-line treatment of mild cases of tularaemia, predominant in Europe. MIC data also indicate that azithromycin or telithromycin may be possible therapeutic options against biovar I strains from Western Europe in case of contraindication to first-line antibiotics.

In vitro and in vivo evaluation of fluoroquinolone resistance associated with DNA gyrase mutations in Francisella tularensis, including in tularaemia patients with treatment failure.

Fluoroquinolones (FQs) are highly effective for treating tularaemia, a zoonosis caused by Francisella tularensis, but failures and relapses remain common in patients with treatment delay or immunocompromised status. FQ-resistant strains of F. tularensis harboring mutations in the quinolone-resistance determining region (QRDR) of gyrA and gyrB, the genes encoding subunits A and B of DNA gyrase, have been selected in vitro. Such mutants have never been isolated from humans as this microorganism is difficult to culture. In this study, the presence of FQ-resistant mutants of F. tularensis was assessed in tularaemia patients using combined culture- and PCR-based approaches. We analyzed 42 F. tularensis strains and 82 tissue samples collected from 104 tularaemia cases, including 32 (30.7%) with FQ treatment failure or relapse. Forty F. tularensis strains and 55 clinical samples were obtained before any FQ treatment, while 2 strains and 15 tissue samples were collected after treatment. FQ resistance was evaluated by the minimum inhibitory concentration (MIC) for the bacterial strains, and by newly developed PCR-based methods targeting the gyrA and gyrB QRDRs for both the bacterial strains and the clinical samples. None of the F. tularensis strains displayed an increased MIC compared with FQ-susceptible controls. Neither gyrA nor gyrB QRDR mutation was found in bacterial strains and tissue samples tested, including those from patients with FQ treatment failure or relapse. Further phenotypic and genetic resistance traits should be explored to explain the poor clinical response to FQ treatment in such tularaemia patients.

The Fluorocycline TP-271 Is Efficacious in Models of Aerosolized Francisella tularensis SCHU S4 Infection in BALB/c Mice and Cynomolgus Macaques.

TP-271 is a novel, fully synthetic fluorocycline in development for complicated bacterial respiratory infections. TP-271 was active in vitro against a panel of 29 Francisella tularensis isolates, showing MICs against 50% and 90% of isolates of 0.25 and 0.5 µg/ml, respectively. In a mouse model of inhalational tularemia, animals were exposed by aerosol to 91 to 283 50% lethal doses (LD50)/mouse of F. tularensis SCHU S4. Following 21 days of once-daily intraperitoneal dosing with TP-271 at 3, 6, 12, and 18 mg/kg of body weight/day, initiating at 24 h postchallenge, survival was 80%, 100%, 100%, and 100%, respectively. When treatment was initiated at 72 h postchallenge, survival was 89%, 100%, 100%, and 100% in the 3-, 6-, 12-, and 18-mg/kg/day TP-271 groups, respectively. No mice treated with the vehicle control survived. Surviving mice treated with TP-271 showed little to no relapse during 14 days posttreatment. In a nonhuman primate model of inhalational tularemia, cynomolgus macaques received an average aerosol exposure of 1,144 CFU of F. tularensis SCHU S4. Once-daily intravenous infusion with 1 or 3 mg/kg TP-271, or vehicle control, for 21 days was initiated within 6 h of confirmed fever. All animals treated with TP-271 survived to the end of the study, with no relapse during 14 days after the last treatment, whereas no vehicle control-treated animals survived. The protection and low relapse afforded by TP-271 treatment in these studies support continued investigation of TP-271 for use in the event of aerosolized exposure to F. tularensis.

Redox-Triggered Release of Moxifloxacin from Mesoporous Silica Nanoparticles Functionalized with Disulfide Snap-Tops Enhances Efficacy Against Pneumonic Tularemia in Mice.

Effective and rapid treatment of tularemia is needed to reduce morbidity and mortality of this potentially fatal infectious disease. The etiologic agent, Francisella tularensis, is a facultative intracellular bacterial pathogen which infects and multiplies to high numbers in macrophages. Nanotherapeutics are particularly promising for treatment of infectious diseases caused by intracellular pathogens, whose primary host cells are macrophages, because nanoparticles preferentially target and are avidly internalized by macrophages. A mesoporous silica nanoparticle (MSN) has been developed functionalized with disulfide snap-tops that has high drug loading and selectively releases drug intracellularly in response to the redox potential. These nanoparticles, when loaded with Hoechst fluorescent dye, release their cargo exclusively intracellularly and stain the nuclei of macrophages. The MSNs loaded with moxifloxacin kill F. tularensis in macrophages in a dose-dependent fashion. In a mouse model of lethal pneumonic tularemia, MSNs loaded with moxifloxacin prevent weight loss, illness, and death, markedly reduce the burden of F. tularensis in the lung, liver, and spleen, and are significantly more efficacious than an equivalent amount of free drug. An important proof-of-principle for the potential therapeutic use of a novel nanoparticle drug delivery platform for the treatment of infectious diseases is provided.

Mesoporous Silica Nanoparticles with pH-Sensitive Nanovalves for Delivery of Moxifloxacin Provide Improved Treatment of Lethal Pneumonic Tularemia.

We have optimized mesoporous silica nanoparticles (MSNs) functionalized with pH-sensitive nanovalves for the delivery of the broad spectrum fluoroquinolone moxifloxacin (MXF) and demonstrated its efficacy in treating Francisella tularensis infections both in vitro and in vivo. We compared two different nanovalve systems, positive and negative charge modifications of the mesopores, and different loading conditions-varying pH, cargo concentration, and duration of loading-and identified conditions that maximize both the uptake and release capacity of MXF by MSNs. We have demonstrated in macrophage cell culture that the MSN-MXF delivery platform is highly effective in killing F. tularensis in infected macrophages, and in a mouse model of lethal pneumonic tularemia, we have shown that the drug-loaded MSNs are much more effective in killing F. tularensis than an equivalent amount of free MXF.

Hunting for tularaemia - a review of cases in North Carolina.

Human infections with Francisella tularensis can be acquired via numerous routes, including ingestion, inhalation, arthropod bite or direct contact with infected animals. Since 1991, there have been 25 reported cases of tularaemia in North Carolina, most of which were associated with rabbit hunting or cat bites. We present two adults cases of pulmonary and oropharyngeal tularaemia and review the reported cases since 1991-2013. We also present the fifth case of pulmonary empyema. While cavitary pneumonias are primarily treated with drainage, we illustrate a case of cavitary pneumonia associated with tularaemia successfully treated with oral ciprofloxacin after drainage. Tularaemia should be considered in patients with a perplexing radiographic image, animal exposure and lack of response to conventional empiric broad-spectrum antibiotics. Even in serious cases of pneumonic tularaemia, fluoroquinolones may provide a suitable alternative to aminoglycosides.

The potential of liposome-encapsulated ciprofloxacin as a tularemia therapy.

Liposome-encapsulation has been suggested as method to improve the efficacy of ciprofloxacin against the intracellular pathogen, Francisella tularensis. Early work with a prototype formulation, evaluated for use against the F. tularensis live vaccine strain, showed that a single dose of liposomal ciprofloxacin given by the intranasal or inhalational route could provide protection in a mouse model of pneumonic tularemia. Liposomal ciprofloxacin offered better protection than ciprofloxacin given by the same routes. Liposomal ciprofloxacin has been further developed by Aradigm Corporation for Pseudomonas aeruginosa infections in patients with cystic fibrosis and non-cystic fibrosis bronchiectasis. This advanced development formulation is safe, effective and well tolerated in human clinical trials. Further evaluation of the advanced liposomal ciprofloxacin formulation against the highly virulent F. tularensis Schu S4 strain has shown that aerosolized CFI (Ciprofloxacin encapsulated in liposomes for inhalation) provides significantly better protection than oral ciprofloxacin. Thus, liposomal ciprofloxacin is a promising treatment for tularemia and further research with the aim of enabling licensure under the animal rule is warranted.

Tularemia in central Anatolia.

BACKGROUND: Tularemia is a bacterial zoonosis with diverse clinical manifestations depending on bacterial subspecies and the route of the infection. METHODS: We collected data prospectively of cases diagnosed and treated for tularemia in our institution during the epidemics from December 2009 to August 2011. Specific antibodies were screened by a microagglutination test. Throat swab and lymph node aspirate cultures were obtained and polymerase chain reaction (PCR) was performed on these specimens. Lymph nodes were characterized on the basis of ultrasound reports. RESULTS: A total of 139 patients were confirmed with tularemia. The age range of the patients was 6-83 years (mean: 43) and 84 (60.4 %) of them were females. Patients had clinical presentations compatible with oropharyngeal (74 %), glandular (15.8 %), and oculoglandular (5.0 %) tularemia. Ultrasonography (US) was performed in 108 patients. Antibiotics (aminoglycosides, quinolones, and doxycycline) were used in 138 patients. Fine-needle aspiration (FNA) or surgical drainage of fluctuant lymph nodes were performed in 51 (39 %) patients. Therapeutic failure was observed in 43 (30.9 %) patients. Elevation of erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels were observed to be significantly higher in patients with therapeutic failures (p = 0.003 and 0.004, respectively). The success rate was significantly higher in patients with early treatment (p = 0.004). No difference was found between the effectiveness of aminoglycoside or quinolone treatments. The increase in the short and long axes, and the characteristics of lymph nodes detected on US were significantly associated with treatment failures (p < 0.001). Intranodal necrosis was found in 45 patients. The treatment success rate was 40 % in patients with intranodal necrosis. CONCLUSION: To the best of our knowledge, this is the first study defining the US findings of patients with tularemia and its association with treatment success. Ciprofloxacin is an effective and convenient choice in epidemics of tularemia and early treatment is still the cornerstone of successful therapies.

Consequences of delayed ciprofloxacin and doxycycline treatment regimens against Francisella tularensis airway infection.

This study examines the efficacy, bacterial load, and humoral response of extensively delayed ciprofloxacin or doxycycline treatments following airway exposure of mice to Francisella tularensis subsp. holarctica (strain LVS) or to the highly virulent F. tularensis subsp. tularensis (strain SchuS4). A delay in onset of both antibiotic treatments allowed the rescue of all LVS-infected animals. However, for animals infected with SchuS4, only ciprofloxacin was efficacious and prolongation of treatment rescued all animals.

Substituted diphenyl ethers as a broad-spectrum platform for the development of chemotherapeutics for the treatment of tularaemia.

OBJECTIVES: The National Institute of Allergy and Infectious Disease classifies Francisella tularensis as a Category A priority pathogen. Despite the availability of drugs for treating tularaemia, the mortality in naturally acquired cases can still approach 30%. In addition, the usefulness of existing drugs for treatment in response to exposure or for prophylaxis is limited because of toxicity and delivery concerns. The aim of this study was to assess the efficacy of the lead alkyl-substituted diphenyl ether, SBPT04, in the F. tularensis murine model of infection. METHODS: SBPT04 was delivered by intraperitoneal (ip) and oral (po) routes, and mice were monitored for morbidity, mortality and relapse of disease. Pharmacokinetic studies were performed to evaluate bioavailability. Phase I and Phase II metabolism of SBPT04 was assessed in mouse and human microsomes. RESULTS: SBPT04, a potent inhibitor of the enoyl-ACP reductase enzyme ftuFabI, has efficacy against F. tularensis in the murine model of infection when delivered by both ip and po routes. SBPT04 delivered ip cleared infection by day 4 of treatment, and SBPT04 delivered po resulted in delayed dissemination. Importantly, SBPT04 delivered ip or po demonstrated efficacy with no signs of relapse of disease. Pharmacokinetic studies show increased serum concentrations following ip delivery compared with po delivery, which correlates with the observed survival rate of 100%. CONCLUSIONS: In addition to being a potent lead, this work substantiates substituted diphenyl ethers as a platform for the development of novel broad-spectrum chemotherapeutics to other bacterial agents in addition to F. tularensis.

Tularemia, a reemerging disease in northwest Turkey: epidemiological investigation and evaluation of treatment responses.

An outbreak of tularemia occurred in three provinces in Turkey in February 2004 and reemerged in the same provinces in February 2005. A total of 61 cases, 54 of which were confirmed with the micro-agglutination test, were diagnosed with oropharyngeal tularemia. No culture for Francisella tularensis was attempted, but PCR for F. tularensis was positive in aspiration material of suppurated lymphadenitis of 7 patients. F. tularensis detection with PCR was negative in water samples, but epidemiologic and environmental findings suggested that contaminated water or food was the cause of the outbreaks. Late initiation antibiotic therapy could not prevent suppuration and draining of the involved lymph nodes.

Treatment of murine pneumonic Francisella tularensis infection with gatifloxacin, moxifloxacin or ciprofloxacin.

The efficacies of gatifloxacin and moxifloxacin were assessed in a BALB/c mouse model of pneumonic tularemia and compared with the efficacy of ciprofloxacin. The rate of relapse following dexamethasone treatment was also investigated. Mice were given 100 mg/kg of the antibiotic by oral administration twice daily for 14 days following an aerosol challenge. All three fluoroquinolones prevented disease during the treatment period, but significant failure rates occurred after the cessation of therapy. Both gatifloxacin and moxifloxacin were more effective than ciprofloxacin at reducing late mortality. Fluoroquinolones may therefore be considered useful candidates for the treatment of pneumonic tularemia.

In vivo efficacy of fluoroquinolones against systemic tularaemia infection in mice.

OBJECTIVES: The in vivo efficacy of ciprofloxacin, gatifloxacin and moxifloxacin were assessed in an experimental Francisella tularensis Schu S4 infection in the BALB/c mouse model. METHODS: Mice were given 100 mg/kg of antibiotic by oral administration twice daily commencing at 6, 24 or 48 h post-exposure and continued for 14 days post-exposure. All mice were challenged subcutaneously with 1 x 10(6) cfu F. tularensis Schu S4 and observed for a period of 56 days. RESULTS: Treatment initiated 6 h post-exposure resulted in 94, 100 and 100% survival for ciprofloxacin, gatifloxacin and moxifloxacin, respectively. When treatment was delayed until 24 h post-exposure the survival rates were ciprofloxacin 67%, gatifloxacin 96% and moxifloxacin 100%. Treatment initiated at 48 h post-exposure resulted in a significant reduction in the survival rate of the ciprofloxacin-treated mice, with 0% survival compared with 84 and 62% for gatifloxacin and moxifloxacin, respectively. Non-treated infected control mice died within 96 h post-exposure. Dexamethasone given at day 42 for 7 days to suppress the animals' immune system caused relapse in all of the treatment groups. CONCLUSIONS: Both gatifloxacin and moxifloxacin were more effective at preventing mortality than ciprofloxacin and could be considered as alternative antibiotics in the treatment of systemic F. tularensis infection.