Rapid differentiation of Francisella species and subspecies by fluorescent in situ hybridization targeting the 23S rRNA

<p>Abstract</p> <p>Background</p> <p><it>Francisella (F.) tularensis </it>is the causative agent of tularemia. Due to its low infectious dose, ease of dissemination and high case fatality rate, <it>F. tularensis </it>was the subject in diverse biological weapons programs and is among the top six agents with high potential if misused in bioterrorism. Microbiological diagnosis is cumbersome and time-consuming. Methods for the direct detection of the pathogen (immunofluorescence, PCR) have been developed but are restricted to reference laboratories.</p> <p>Results</p> <p>The complete 23S rRNA genes of representative strains of <it>F. philomiragia </it>and all subspecies of <it>F. tularensis </it>were sequenced. Single nucleotide polymorphisms on species and subspecies level were confirmed by partial amplification and sequencing of 24 additional strains. Fluorescent In Situ Hybridization (FISH) assays were established using species- and subspecies-specific probes.</p> <p>Different FISH protocols allowed the positive identification of all 4 <it>F. philomiragia </it>strains, and more than 40 <it>F. tularensis </it>strains tested. By combination of different probes, it was possible to differentiate the <it>F. tularensis </it>subspecies <it>holarctica, tularensis, mediasiatica </it>and <it>novicida</it>. No cross reactivity with strains of 71 clinically relevant bacterial species was observed. FISH was also successfully applied to detect different <it>F. tularensis </it>strains in infected cells or tissue samples. In blood culture systems spiked with <it>F. tularensis</it>, bacterial cells of different subspecies could be separated within single samples.</p> <p>Conclusion</p> <p>We could show that FISH targeting the 23S rRNA gene is a rapid and versatile method for the identification and differentiation of <it>F. tularensis </it>isolates from both laboratory cultures and clinical samples.</p

Investigating an Airborne Tularemia Outbreak, Germany

Infectious aerosols can contribute to the transmission of tularemia during processing of dead hares

Re-emergence of tularemia in Germany: Presence of <it>Francisella tularensis </it>in different rodent species in endemic areas

<p>Abstract</p> <p>Background</p> <p>Tularemia re-emerged in Germany starting in 2004 (with 39 human cases from 2004 to 2007) after over 40 years of only sporadic human infections. The reasons for this rise in case numbers are unknown as is the possible reservoir of the etiologic agent <it>Francisella (F.) tularensis</it>. No systematic study on the reservoir situation of <it>F. tularensis </it>has been published for Germany so far.</p> <p>Methods</p> <p>We investigated three areas six to ten months after the initial tularemia outbreaks for the presence of <it>F. tularensis </it>among small mammals, ticks/fleas and water. The investigations consisted of animal live-trapping, serologic testing, screening by real-time-PCR and cultivation.</p> <p>Results</p> <p>A total of 386 small mammals were trapped. <it>F. tularensis </it>was detected in five different rodent species with carrier rates of 2.04, 6.94 and 10.87% per trapping area. None of the ticks or fleas (n = 432) tested positive for <it>F. tularensis</it>. We were able to demonstrate <it>F. tularensis-</it>specific DNA in one of 28 water samples taken in one of the outbreak areas.</p> <p>Conclusion</p> <p>The findings of our study stress the need for long-term surveillance of natural foci in order to get a better understanding of the reasons for the temporal and spatial patterns of tularemia in Germany.</p

Phylogeography of Francisella tularensis subsp. holarctica, Europe

Francisella tularensis subsp. holarctica isolates from Austria, Germany, Hungary, Italy, and Romania were placed into an existing phylogeographic framework. Isolates from Italy were assigned to phylogenetic group B.FTNF002–00; the other isolates, to group B.13. Most F. tularensis subsp. holarctica isolates from Europe belong to these 2 geographically segregated groups

Treatment of Tularemia in Patient with Chronic Graft-versus-Host Disease

We describe a case of human tularemia caused by Francisella tularensis subsp. holarctica in a stem cell transplant recipient with chronic graft-versus-host disease who was receiving levofloxacin prophylaxis. The infection was characterized by pneumonia with septic complications. The patient was successfully treated with doxycycline. Tularemia is a zoonotic infection caused by the gramnegative bacterium Francisella tularensis. Humans are accidental hosts; infection occurs after contact with infected animals, contaminated water or soil, or invertebrate vectors (1). Strains of the 2 subspecies F. tularensis subsp. tularensis and F. tularensis subsp. holarctica account for virtually all infections in humans. Only rarely have strains of the subspecies F. tularensis novicida or the closely related species F. philomiragia or F. hispaniensis been cultured from clinical specimens (2). F. tularensis subsp. tularensis, also referred to as type A, is found almost exclusively in North America and is the most virulent subspecies. F. tularensis subsp. holarctica, also referred to as type B, is found predominantly in Asia and Europe, but also in North America (3). Patients infected with F. tularensis have abrupt onset of fever, chills, headache, and malaise after an incubation period of 2–21 days. Additional signs and symptoms may develop, depending on the portal of entry. The most common signs and symptoms are lymphadenopathy, fever, pharyngitis, appearance of ulcers/eschars/papules, nausea and vomiting, and hepatosplenomegaly. Antimicrobial drug therapy should be administered to patients with this suspected or confirmed diagnosis, even though spontaneous resolution may occur in 50%–95 % o

Rapid high resolution genotyping of Francisella tularensis by whole genome sequence comparison of annotated genes ("MLST+").

The zoonotic disease tularemia is caused by the bacterium Francisella tularensis. This pathogen is considered as a category A select agent with potential to be misused in bioterrorism. Molecular typing based on DNA-sequence like canSNP-typing or MLVA has become the accepted standard for this organism. Due to the organism's highly clonal nature, the current typing methods have reached their limit of discrimination for classifying closely related subpopulations within the subspecies F. tularensis ssp. holarctica. We introduce a new gene-by-gene approach, MLST+, based on whole genome data of 15 sequenced F. tularensis ssp. holarctica strains and apply this approach to investigate an epidemic of lethal tularemia among non-human primates in two animal facilities in Germany. Due to the high resolution of MLST+ we are able to demonstrate that three independent clones of this highly infectious pathogen were responsible for these spatially and temporally restricted outbreaks