Iron-Modified Blood Culture Media Allow for the Rapid Diagnosis and Isolation of the Slow-Growing Pathogen Francisella tularensis.

The life-threatening disease tularemia is caused by Francisella tularensis, an intracellular Gram-negative bacterial pathogen. Due to the high mortality rates of the disease, as well as the low respiratory infectious dose, F. tularensis is categorized as a Tier 1 bioterror agent. The identification and isolation from clinical blood cultures of F. tularensis are complicated by its slow growth. Iron was shown to be one of the limiting nutrients required for F. tularensis metabolism and growth. Bacterial growth was shown to be restricted or enhanced in the absence or addition of iron. In this study, we tested the beneficial effect of enhanced iron concentrations on expediting F. tularensis blood culture diagnostics. Accordingly, bacterial growth rates in blood cultures with or without Fe2+ supplementation were evaluated. Growth quantification by direct CFU counts demonstrated significant improvement of growth rates of up to 6 orders of magnitude in Fe2+-supplemented media compared to the corresponding nonmodified cultures. Fe2+ supplementation significantly shortened incubation periods for successful diagnosis and isolation of F. tularensis by up to 92 h. This was achieved in a variety of blood culture types in spite of a low initial bacterial inoculum representative of low levels of bacteremia. These improvements were demonstrated with culture of either Francisella tularensis subsp. tularensis or subsp. holarctica in all examined commercial blood culture types routinely used in a clinical setup. Finally, essential downstream identification assays, such as matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS), immunofluorescence, or antibiotic susceptibility tests, were not affected in the presence of Fe2+. To conclude, supplementing blood cultures with Fe2+ enables a significant shortening of incubation times for F. tularensis diagnosis, without affecting subsequent identification or isolation assays. IMPORTANCE In this study, we evaluated bacterial growth rates of Francisella tularensis strains in iron (Fe)-enriched blood cultures as a means of improving and accelerating bacterial growth. The shortening of the culturing time should facilitate rapid pathogen detection and isolation, positively impacting clinical diagnosis and enabling prompt onset of efficient therapy.

An Improvement in Diagnostic Blood Culture Conditions Allows for the Rapid Detection and Isolation of the Slow Growing Pathogen Yersinia pestis.

Plague, caused by the human pathogen Yersinia pestis, is a severe and rapidly progressing lethal disease that has caused millions of deaths globally throughout human history and still presents a significant public health concern, mainly in developing countries. Owing to the possibility of its malicious use as a bio-threat agent, Y. pestis is classified as a tier-1 select agent. The prompt administration of an effective antimicrobial therapy, essential for a favorable patient prognosis, requires early pathogen detection, identification and isolation. Although the disease rapidly progresses and the pathogen replicates at high rates within the host, Y. pestis exhibits a slow growth in vitro under routinely employed clinical culturing conditions, complicating the diagnosis and isolation. In the current study, the in vitro bacterial growth in blood cultures was accelerated by the addition of nutritional supplements. We report the ability of calcium (Ca+2)- and iron (Fe+2)-enriched aerobic blood culture media to expedite the growth of various virulent Y. pestis strains. Using a supplemented blood culture, a shortening of the doubling time from ~110 min to ~45 min could be achieved, resulting in increase of 5 order of magnitude in the bacterial loads within 24 h of incubation, consequently allowing the rapid detection and isolation of the slow growing Y. pestis bacteria. In addition, the aerobic and anaerobic blood culture bottles used in clinical set-up were compared for a Y. pestis culture in the presence of Ca+2 and Fe+2. The comparison established the superiority of the supplemented aerobic cultures for an early detection and achieved a significant increase in the yields of the pathogen. In line with the accelerated bacterial growth rates, the specific diagnostic markers F1 and LcrV (V) antigens could be directly detected significantly earlier. Downstream identification employing MALDI-TOF and immunofluorescence assays were performed directly from the inoculated supplemented blood culture, resulting in an increased sensitivity and without any detectable compromise of the accuracy of the antibiotic susceptibility testing (E-test), critical for subsequent successful therapeutic interventions.

Treating Anthrax-Induced Meningitis in Rabbits.

Treatment of anthrax is challenging, especially during the advanced stages of the disease. Recently, the Centers for Disease Control and Prevention (CDC) updated its recommendations for postexposure prophylaxis and treatment of exposed populations (before and after symptom onset). These recommendations distinguished, for the first time, between systemic disease with and without meningitis, a common and serious complication of anthrax. The CDC considers all systemic cases meningeal unless positively proven otherwise. The treatment of patients suffering from systemic anthrax with suspected or confirmed meningitis includes the combination of three antibiotics, i.e., a fluoroquinolone (levofloxacin or ciprofloxacin), a ß-lactam (meropenem or imipenem), and a protein synthesis inhibitor (linezolid or clindamycin). In addition, treatment with an antitoxin (anti-protective antigen antibodies) and dexamethasone should be applied. Since the efficacy of most of these treatments has not been demonstrated, especially in animal meningitis models, we developed an anthrax meningitis model in rabbits and tested several of these recommendations. We demonstrated that, in this model, ciprofloxacin, linezolid, and meropenem were ineffective as single treatments, while clindamycin was highly effective. Furthermore, combined treatments of ciprofloxacin and linezolid or ciprofloxacin and dexamethasone failed in treating rabbits with meningitis. We demonstrated that dexamethasone actually hindered blood-brain barrier penetration by antibiotics, reducing the effectiveness of antibiotic treatment of anthrax meningitis in this rabbit model.