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We analysed diverse strains of Francisella tularensis subsp. holarctica to assess if its division into biovars I and II is associated with specific mutations previously linked to erythromycin resistance and to determine the distribution of this resistance trait across this subspecies.Three-hundred and fourteen F. tularensis subsp. holarctica strains were tested for erythromycin susceptibility and whole-genome sequences for these strains were examined for SNPs in genes previously associated with erythromycin resistance. Each strain was assigned to a global phylogenetic framework using genome-wide canonical SNPs. The contribution of a specific SNP to erythromycin resistance was examined using allelic exchange. The geographical distribution of erythromycin-resistant F. tularensis strains was further investigated by literature search.There was a perfect correlation between biovar II strains (erythromycin resistance) and the phylogenetic group B.12. Only B.12 strains had an A → C SNP at position 2059 in the three copies of the rrl gene. Introducing 2059C into an rrl gene of an erythromycin-susceptible F. tularensis strain resulted in resistance. An additional 1144 erythromycin-resistant strains were identified from the scientific literature, all of them from Eurasia.Erythromycin resistance in F. tularensis is caused by an A2059C rrl gene mutation, which exhibits a strictly clonal inheritance pattern found only in phylogenetic group B.12. This group is an extremely successful clone, representing the most common type of F. tularensis throughout Eurasia.

For many infections transmitting to humans from reservoirs in nature, disease dispersal patterns over space and time are largely unknown. Here, a reversed genomics approach helped us understand disease dispersal and yielded insight into evolution and biological properties of Francisella tularensis, the bacterium causing tularemia. We whole-genome sequenced 67 strains and characterized by single-nucleotide polymorphism assays 138 strains, collected from individuals infected 1947-2012 across Western Europe. We used the data for phylogenetic, population genetic and geographical network analyses. All strains (n=205) belonged to a monophyletic population of recent ancestry not found outside Western Europe. Most strains (n=195) throughout the study area were assigned to a star-like phylogenetic pattern indicating that colonization of Western Europe occurred via clonal expansion. In the East of the study area, strains were more diverse, consistent with a founder population spreading from east to west. The relationship of genetic and geographic distance within the F. tularensis population was complex and indicated multiple long-distance dispersal events. Mutation rate estimates based on year of isolation indicated null rates; in outbreak hotspots only, there was a rate of 0.4 mutations/genome/year. Patterns of nucleotide substitution showed marked AT mutational bias suggestive of genetic drift. These results demonstrate that tularemia has moved from east to west in Europe and that F. tularensis has a biology characterized by long-range geographical dispersal events and mostly slow, but variable, replication rates. The results indicate that mutation-driven evolution, a resting survival phase, genetic drift and long-distance geographical dispersal events have interacted to generate genetic diversity within this species. This work was supported by the Swedish Civil Contingencies Agency [grant number TA 014-2010-01;] and the US Department of Homeland Security’s Science and Technology Directorate [award number HSHQDC-10-C-00139;] pursuant to the agreement between the Kingdom of Sweden and the US government on Cooperation in Science and Technology for Homeland Security Matters. The authors R.E. and P.A. were supported by the Ministerio de Economía y Competitividad, Spain [grant number CGL2015-66962-C2-2-R]. We thank Johanna Thelaus, Per Stenberg, Andreas Sjödin, and Petter Lindgren for comments that improved the manuscript. We are grateful to the researchers, physicians, and veterinarians who contributed F. tularensis samples for this study.

The feasibility of using autonomous forest vehicles (which can be regarded as logical developments in the ongoing automation of forest machines), the systems that could be applied in them, their potential advantages and limitations (in the foreseeable future) are considered in this paper. The goals were to analyze: 1) the factors influencing the degree of automation in logging; 2) the technical principles that can be applied to autonomous forest machines, and 3) the feasibility of developing an autonomous path-tracking forest vehicle. A type of vehicle that is believed to have considerable commercial potential is an autonomous forwarder. The degree of automation is influenced by increased productivity, the machine operator as a bottle-neck, cost reduction, and environmental aspects. Technical principles that can be applied to autonomous forest vehicles are satellite navigation, wheel odometry, laser scanner, and radar. A new path-tracking algorithm has been developed to reduce deviations from the desired path by utilizing the driver’s steering commands. The presented system has demonstrated both possibilities and difficulties associated with autonomous forest machines. A field study has shown that it is quite possible for them to learn and track a path previously demonstrated by an operator with an accuracy of 0.1 m on flat ground and also to detect and avoid unexpected obstacles. Although the forest machine safely avoids obstacles, the study shows that further research in the field of obstacle avoidance is needed to optimize performance and ensure safe operation in a real forest environment.

We present the complete genome sequence of Francisella guangzhouensis strain 08HL01032 T , which consists of one chromosome (1,658,482 bp) and one plasmid (3,045 bp) with G+C contents of 32.0% and 28.7%, respectively.

Second generation sequencing has revolutionized genomic studies. However, most genomes contain repeated DNA elements that are longer than the read lengths achievable with typical sequencers, so the genomic order of several generated contigs cannot be easily resolved. A new generation of sequencers offering substantially longer reads is emerging, notably the Pacific Biosciences (PacBio) RS II system and the MinION system, released in early 2014 by Oxford Nanopore Technologies through an early access program. The latter has highly advantageous portability and sequences samples by measuring changes in ionic current when single-stranded DNA molecules are translocated through nanopores. We show that the MinION system produces long reads with high mapability that can be used for scaffolding bacterial genomes, despite currently producing substantially higher error rates than PacBio reads. With further development we anticipate that MinION will be useful not only for assembling genomes, but also for rapid detection of organisms, potentially in the field.

A strain of Francisella endociliophora was isolated from a laboratory culture of the marine ciliate Euplotes raikovi . Here, we report the complete genome sequence of the bacterial strain FSC1006 ( Francisella Strain Collection, Swedish Defence Research Agency, Umeå, Sweden).

The bacterium Francisella tularensis is recognized for its virulence, infectivity, genetic homogeneity, and potential as a bioterrorism agent. Outbreaks of respiratory tularemia, caused by inhalation of this bacterium, are poorly understood. Such outbreaks are exceedingly rare, and F. tularensis is seldom recovered from clinical specimens.A localized outbreak of tularemia in Sweden was investigated. Sixty-seven humans contracted laboratory-verified respiratory tularemia. F. tularensis subspecies holarctica was isolated from the blood or pleural fluid of 10 individuals from July to September 2010. Using whole-genome sequencing and analysis of single-nucleotide polymorphisms (SNPs), outbreak isolates were compared with 110 archived global isolates.There were 757 SNPs among the genomes of the 10 outbreak isolates and the 25 most closely related archival isolates (all from Sweden/Finland). Whole genomes of outbreak isolates were99.9% similar at the nucleotide level and clustered into 3 distinct genetic clades. Unexpectedly, high-sequence similarity grouped some outbreak and archival isolates that originated from patients from different geographic regions and up to 10 years apart. Outbreak and archival genomes frequently differed by only 1-3 of 1 585 229 examined nucleotides.The outbreak was caused by diverse clones of F. tularensis that occurred concomitantly, were widespread, and apparently persisted in the environment. Multiple independent acquisitions of F. tularensis from the environment over a short time period suggest that natural outbreaks of respiratory tularemia are triggered by environmental cues. The findings additionally caution against interpreting genome sequence identity for this pathogen as proof of a direct epidemiological link.

Summary: Advances in typing methodologies have recently reformed the field of molecular epidemiology of pathogens. The falling cost of sequencing technologies is creating a deluge of whole genome sequencing data that burdens bioinformatics resources and tool development. In particular, single nucleotide polymorphisms in core genomes of pathogens are recognized as the most important markers for inferring genetic relationships because they are evolutionarily stable and amenable to high-throughput detection methods. Sequence data will provide an excellent opportunity to extend our understanding of infectious disease when the challenge of extracting knowledge from available sequence resources is met. Here, we present an efficient and user-friendly genotype classification pipeline, CanSNPer, based on an easily expandable database of predefined canonical single nucleotide polymorphisms. Availability and implementation: All documentation and Python-based source code for the CanSNPer are freely available at http://github.com/adrlar/CanSNPer. Contact: adrian.larkeryd@foi.se

This thesis addresses the possibility of determining the exact volume of a small irregularshape using imaging methods. More specifically the suggested method is using a photogrammetric technique called Stereo Vision which makes it possible to extract 3-dimensional information from two or more digital images displaying different viewsof the same object.The motivation is to develop a non-destructive way for the Swedish mining companyLKAB to measure the porosity of their produced iron ore pellets. With known mass and absolute density the volume uniquely defines the porosity of a sample. With high accuracy volume measurements one can guarantee a certain quality of the porosity estimations.Techniques exist today that measure the porosity of individual pellets but they are either classified as environmentally unfriendly or affect the physical propertiesof the measured sample.The designed system is using the computer vision library OpenCV together with the mesh manipulation software MeshLab to generate an high accuracy model of the photographedobject. The volume of this model is then estimated using volume integrals according to already developed algorithms.Testing indicates that the final system can determine the volume with a relative error of approximately 1-2 %. As the targeted error is below 0.28 % this means that the system as it is designed today produces less accurate results than other already existing methods for porosity measurements. Future improvements are however suggested how to further increase the accuracy of this method to potentially outperformits competitors. Validerat; 20120821 (anonymous)

The feasibility of using autonomous forest vehicles (which can be regarded as logical developments in the ongoing automation of forest machines), the systems that could be applied in them, their potential advantages and their limitations (in the foreseeable future) are considered here. The aims were to analyse: (1) the factors influencing the degree of automation in logging; (2) the technical principles that can be applied to autonomous forest machines, and (3) the feasibility of developing an autonomous path-tracking forest vehicle. A class of such vehicles that are believed to have considerable commercial potential is autonomous wood shuttles (forwarders). The degree of automation is influenced by increased productivity, the machine operator as a bottle-neck, cost reduction, and environmental aspects. Technical principles that can be applied to autonomous forest vehicles are satellite navigation, laser odometry, wheel odometry, laser scanner and radar. The presented system has demonstrated both possibilities and difficulties associated with autonomous forest machines. It is in a field study shown that it is quite possible for them to learn and track a path previously demonstrated by an operator with an accuracy of 0.1m on flat ground. A new pathtracking algorithm has been developed to reduce deviations by utilizing the driver’s steering commands.

In 2011, a nationwide outbreak of tularaemia occurred in Norway with 180 recorded cases. It was associated with the largest peak in lemming density seen in 40 years. Francisella tularensis was isolated from 18 patients. To study the geographical distribution of F. tularensis genotypes in Norway and correlate genotype with epidemiology and clinical presentation, we performed whole genome sequencing of patient isolates. All 18 genomes from the outbreak carried genetic signatures of F. tularensis subsp. holarctica and were assigned to genetic clades using canonical single nucleotide polymorphisms. Ten isolates were assigned to major genetic clade B.6 (subclade B.7), seven to clade B.12, and one to clade B.4. The B.6 subclade B.7 was most common in southern and central Norway, while clade B.12 was evenly distributed between the southern, central and northern parts of the country. There was no association between genotype and clinical presentation of tularaemia, time of year or specimen type. We found extensive sequence similarity with F. tularensis subsp. holarctica genomes from high-endemic tularaemia areas in Sweden. Finding nearly identical genomes across large geographical distances in Norway and Sweden imply a life cycle of the bacterium without replication between the outbreaks and raise new questions about long-range migration mechanisms.

Waterborne epidemics of tularaemia caused by Francisella tularensis are increasingly reported in Turkey. We have used whole genome sequencing to investigate if F. tularensis isolated from patients could be traced back to drinking water sources. Tonsil swabs from 33 patients diagnosed with oropharyngeal tularaemia in three outbreaks and 140 water specimens were analysed. F. tularensis subsp. holarctica was confirmed by microagglutination and PCR in 12 patients and five water specimens. Genomic analysis of three pairs of patient and water isolates from outbreaks in Sivas, Çorum, and Kocaeli showed the isolates to belong to two new clusters of the F. tularensis B.12 genetic clade. The clusters were defined by 19 and 15 single nucleotide polymorphisms (SNPs) in a multiple alignment based on 507 F. tularensis genomes. One synonymous SNP was chosen as a new canonical SNP (canSNP) for each cluster for future use in diagnostic assays. No SNP was identified between the genomes from the patient–water pair of isolates from Kocaeli, one SNP between the pair of isolates from Sivas, whereas the pair from Çorum differed at seven SNPs. These results illustrate the power of whole genome sequencing for tracing F. tularensis patient isolates back to their environmental source.