title: German Francisella tularensis isolates from European brown hares (Lepus europaeus) reveal genetic and phenotypic diversity.
abstract: BACKGROUND
  Tularemia is a zoonotic disease caused by Francisella tularensis that has been found in many different vertebrates. In Germany most human infections are caused by contact with infected European brown hares (Lepus europaeus). The aim of this study was to elucidate the epidemiology of tularemia in hares using phenotypic and genotypic characteristics of F. tularensis.
  
  
  RESULTS
  Cultivation of F. tularensis subsp. holarctica bacteria from organ material was successful in 31 of 52 hares that had a positive PCR result targeting the Ft-M19 locus. 17 isolates were sensitive to erythromycin and 14 were resistant. Analysis of VNTR loci (Ft-M3, Ft-M6 and Ft-M24), INDELs (Ftind33, Ftind38, Ftind49, RD23) and SNPs (B.17, B.18, B.19, and B.20) was shown to be useful to investigate the genetic relatedness of Francisella strains in this set of strains. The 14 erythromycin resistant isolates were assigned to clade B.I, and 16 erythromycin sensitive isolates to clade B.IV and one isolate was found to belong to clade B.II. MALDI-TOF mass spectrometry (MS) was useful to discriminate strains to the subspecies level.
  
  
  CONCLUSIONS
  F. tularensis seems to be a re-emerging pathogen in Germany. The pathogen can easily be identified using PCR assays. Isolates can also be identified within one hour using MALDI-TOF MS in laboratories where specific PCR assays are not established. Further analysis of strains requires genotyping tools. The results from this study indicate a geographical segregation of the phylogenetic clade B.I and B.IV, where B.I strains localize primarily within eastern Germany and B.IV strains within western Germany. This phylogeographical pattern coincides with the distribution of biovar I (erythromycin sensitive) and biovar II (erythromycin resistance) strains. When time and costs are limiting parameters small numbers of isolates can be analysed using PCR assays combined with DNA sequencing with a focus on genetic loci that are most likely discriminatory among strains found in a specific area. In perspective, whole genome data will have to be investigated especially when terrorist attack strains need to be tracked to their genetic and geographical sources.
authors.affiliation: Institute of Bacterial Infections and Zoonoses, Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Naumburger Str. 96A, Jena D-07743, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Müller
authors.forename: Helmut
authors.initials: H
authors.lastname: Hotzel
authors.forename: Peter
authors.initials: P
authors.lastname: Otto
authors.forename: Axel
authors.initials: A
authors.lastname: Karger
authors.forename: Barbara
authors.initials: B
authors.lastname: Bettin
authors.forename: Herbert
authors.initials: H
authors.lastname: Bocklisch
authors.forename: Silke
authors.initials: S
authors.lastname: Braune
authors.forename: Ulrich
authors.initials: U
authors.lastname: Eskens
authors.forename: Stefan
authors.initials: S
authors.lastname: Hörmansdorfer
authors.forename: Regina
authors.initials: R
authors.lastname: Konrad
authors.forename: Anne
authors.initials: A
authors.lastname: Nesseler
authors.forename: Martin
authors.initials: M
authors.lastname: Peters
authors.forename: Martin
authors.initials: M
authors.lastname: Runge
authors.forename: Gernot
authors.initials: G
authors.lastname: Schmoock
authors.forename: Bernd-Andreas
authors.initials: BA
authors.lastname: Schwarz
authors.forename: Reinhard
authors.initials: R
authors.lastname: Sting
authors.forename: Kerstin
authors.initials: K
authors.lastname: Myrtennäs
authors.forename: Edvin
authors.initials: E
authors.lastname: Karlsson
authors.forename: Mats
authors.initials: M
authors.lastname: Forsman
authors.forename: Herbert
authors.initials: H
authors.lastname: Tomaso
mesh_terms: D000825:Animal Structures / Q000382:microbiology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D024881:Drug Resistance, Bacterial; D004917:Erythromycin / Q000494:pharmacology; D005604:Francisella tularensis / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D014644:Genetic Variation; D005838:Genotype; D005858:Germany; D034621:Hares / Q000382:microbiology*; D008826:Microbial Sensitivity Tests; D018598:Minisatellite Repeats; D058889:Molecular Typing; D058974:Phylogeography; D016133:Polymerase Chain Reaction; D012376:Rodent Diseases / Q000382:microbiology*; D014406:Tularemia / Q000382:microbiology / Q000662:veterinary*
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D004917:Erythromycin