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

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

Molecular Characterization of German Acinetobacter baumannii Isolates and Multilocus Sequence Typing (MLST) Analysis Based on WGS Reveals Novel STs

Acinetobacter baumannii (A.&nbsp;baumannii) is a major cause of severe nosocomial infections worldwide. The emergence of infections associated with A.&nbsp;baumannii poses a significant health risk in Germany. A. baumannii is part of the ACB complex and is difficult to distinguish from other species phenotypically, necessitating its reliable identification. The current study analyzed 89 A. baumannii strains from human and non-human origins by matrix-assisted laser desorption/ionization (MALDI–TOF) and PCR detection of intrinsic blaOXA-51-like carbapenemase, blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and ISAba 1 genes. Whole-genome sequencing (WGS) was applied for species confirmation and strain type determination. Combining the molecular detection of the intrinsic blaOXA-51-like carbapenemase gene together with MALDI–TOF with a score value of &gt;2.300 proved to be a suitable tool for A. baumannii identification. WGS data for all of the sequenced strains confirmed the identity of all A. baumannii strains. The Pasteur scheme successfully assigned 79.7% of the strains into distinct STs, while the Oxford scheme succeeded in allocating only 42.7% of isolates. Multilocus sequence typing (MLST) analysis based on the Pasteur scheme identified 16 STs. ST/241 was the most prevalent in samples from non-human origin, whereas ST/2 was predominant in human samples. Furthermore, eight isolates of non-human origin were allocated to seven new STs (ST/1410, ST/1414, ST/1416, ST/1417, ST/1418, ST/1419, and ST/1421). Ten isolates from non-human origin could not be typed since new alleles were observed in the loci Pas_cpn60, Pas_rpoB, and Pas_gltA. MLST analysis based on the Pasteur scheme was more appropriate than the Oxford scheme for the current group of A. baumannii