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Genetic genealogy and epidemiology of Francisella
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases. (Tularemia)
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
Keyword [en]
Francisella tularensis, tularemia, genotyping, epidemiology, GIS
National Category
Infectious Medicine
Research subject
Infectious Diseases
URN: urn:nbn:se:umu:diva-22452ISBN: 978-91-7264-803-6OAI: diva2:217570
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
List of papers
1. Evolution of subspecies of Francisella tularensis
Open this publication in new window or tab >>Evolution of subspecies of Francisella tularensis
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2005 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 187, no 11, 3903-3908 p.Article in journal (Refereed) Published
Abstract [en]

Analysis of unidirectional genomic deletion events and single nucleotide variations suggested that the four subspecies of Francisella tularensis have evolved by vertical descent. The analysis indicated an evolutionary scenario where the highly virulent F. tularensis subsp. tularensis (type A) appeared before the less virulent F. tularensis subsp. holarctica (type B). Compared to their virulent progenitors, attenuated strains of F. tularensis exhibited specific unidirectional gene losses.

Evolution; Molecular, Francisella tularensis/classification/*genetics/pathogenicity, Gene Deletion, Genome; Bacterial, Molecular Sequence Data, Oligonucleotide Array Sequence Analysis, Phylogeny, Virulence
urn:nbn:se:umu:diva-2382 (URN)10.1128/JB.187.11.3903-3908.2005 (DOI)15901721 (PubMedID)
Available from: 2007-05-11 Created: 2007-05-11 Last updated: 2016-03-01Bibliographically approved
2. Canonical insertion-deletion markers for rapid DNA typing of Francisella tularensis
Open this publication in new window or tab >>Canonical insertion-deletion markers for rapid DNA typing of Francisella tularensis
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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.

urn:nbn:se:umu:diva-21151 (URN)18217558 (PubMedID)
Available from: 2009-04-03 Created: 2009-04-03 Last updated: 2016-03-01Bibliographically approved
3. A real-time PCR array for hierarchical identification of environmental and human pathogenic Francisella isolates
Open this publication in new window or tab >>A real-time PCR array for hierarchical identification of environmental and human pathogenic Francisella isolates
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(English)Manuscript (Other academic)
National Category
Microbiology in the medical area
urn:nbn:se:umu:diva-22864 (URN)
Available from: 2009-05-19 Created: 2009-05-19 Last updated: 2012-02-02Bibliographically approved
4. A high-resolution landscape epidemiology of tularemia exposed by genetic analysis of the causative agent isolated from infected humans
Open this publication in new window or tab >>A high-resolution landscape epidemiology of tularemia exposed by genetic analysis of the causative agent isolated from infected humans
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(English)Manuscript (Other academic)
urn:nbn:se:umu:diva-22869 (URN)
Available from: 2009-05-19 Created: 2009-05-19 Last updated: 2016-03-01

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