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Canonical insertion-deletion markers for rapid DNA typing of Francisella tularensis
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
FOI, Umeå (Swedish Defence Research Agency).
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2007 (English)In: Emerging Infectious Diseases, ISSN 1080-6040, E-ISSN 1080-6059, Vol. 13, no 11, 1725-1732 p.Article in journal (Refereed) Published
Abstract [en]

To develop effective and accurate typing of strains of Francisella tularensis, a potent human pathogen and a putative bioterrorist agent, we combined analysis of insertion-deletion (indel) markers with multiple-locus variable-number tandem repeat analysis (MLVA). From 5 representative F. tularensis genome sequences, 38 indel markers with canonical properties, i.e., capable of sorting strains into major genetic groups, were selected. To avoid markers with a propensity for homoplasy, we used only those indels with 2 allelic variants and devoid of substantial sequence repeats. MLVA included sequences with much diversity in copy number of tandem repeats. The combined procedure allowed subspecies division, delineation of clades A.I and A.II of subspecies tularensis, differentiation of Japanese strains from other strains of subspecies holarctica, and high-resolution strain typing. The procedure uses limited amounts of killed bacterial preparations and, because only 1 single analytic method is needed, is time- and cost-effective.

Place, publisher, year, edition, pages
2007. Vol. 13, no 11, 1725-1732 p.
URN: urn:nbn:se:umu:diva-21151PubMedID: 18217558OAI: diva2:210672
Available from: 2009-04-03 Created: 2009-04-03 Last updated: 2016-03-01Bibliographically approved
In thesis
1. Genetic genealogy and epidemiology of Francisella
Open this publication in new window or tab >>Genetic genealogy and epidemiology of Francisella
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis is about analyzing genetic differences among isolates of Francisella tularensis – the tularemia-causing bacterium. To elucidate how these bacterial isolates are related, and their geographical and genetic origins, I have developed typing assays for Francisella and used them to study the epidemiology of tularemia.

Tularemia is an infectious disease of humans and other mammals found throughout the Northern Hemisphere. The severity of the disease depends on the type of F. tularensis causing the infection. In Sweden, as in other countries of Europe and Eurasia, tularemia is caused by F. tularensis subsp. holarctica, while other varieties of the bacterium occur in Middle Asia and North America. It is important to identify a tularemia infection promptly in order to initiate the correct antibiotic treatment. A rapid identification of the causative F. tularensis variety gives additional clinical information. In recent years, several genomes of various Francisella strains have been sequenced, and in this thesis, I have utilized these genomes to identify genetic markers.

In studies reported in the first paper (I) appended to the thesis, we identified and analyzed insertion/deletion mutations (INDELs) inferred to have resulted from a sequence repeat-mediated excision mechanism. We found eight new Regions of Difference (RDs) among Francisella strains. Using RDs together with single nucleotide polymorphisms (SNPs), we were able to predict an evolutionary scenario for F. tularensis in which Francisella novicida was the oldest variety while F. tularensis subsp. holarctica was the youngest. We also found that all virulence-attenuated isolates analyzed had deletions at two specific genetic regions - denoted RD18 and RD19 – suggesting that repeat-mediated excision is a mechanism of attenuation in F. tularensis.

In subsequent studies (presented in paper II), we developed a combined analysis of INDELs lacking flanking repeats and variable number of tandem repeats (VNTRs). Both markers could be assayed using the same analytical equipment. The inclusion of INDELs provided increased phylogenetic robustness compared with the use of VNTRs alone, while still maintaining a high level of genetic resolution.

In analyses described in the next paper (III), we selected INDELs from paper (II) and discovered novel SNPs by DNA comparisons of multiple Francisella strains. Thirty-four phylogenetically informative genetic markers were included in a hierarchical real-time PCR array for rapid and robust characterization of Francisella. We successfully used the assay to genotype 14 F. tularensis isolates from tularemia patients and DNA in six clinical ulcer specimens.

Finally, in paper (IV) we demonstrated a strategy to enhance epidemiological investigations of tularemia by combining GIS-mapping of disease-transmission place collected from patient interviews, with high-resolution genotyping of F. tularensis subsp. holarctica isolates recovered from tularemia patients. We found the geographic distributions of specific F. tularensis subsp. holarctica sub-populations to be highly localized during outbreaks (infections by some genotypes being restricted to areas as small as 2 km2), indicative of a landscape epidemiology of tularemia with distinct point sources of infection.

In conclusion, the results acquired during the studies underlying this thesis contribute to our understanding of the genetic genealogy of tularemia at both global and local outbreak scales.

Place, publisher, year, edition, pages
Umeå: Department of Clinical Microbiology, Umeå University, 2009. 47 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1270
Francisella tularensis, tularemia, genotyping, epidemiology, GIS
National Category
Infectious Medicine
Research subject
Infectious Diseases
urn:nbn:se:umu:diva-22452 (URN)978-91-7264-803-6 (ISBN)
Infektionssjukdomar, 901 85, Umeå
Public defence
2009-06-05, 933, building 3A, NUS, 13:00 (English)
Available from: 2009-05-19 Created: 2009-05-11 Last updated: 2016-03-01Bibliographically approved

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