Kerstin Myrtennäs

German Francisella tularensis isolates from European brown hares (Lepus europaeus)reveal genetic and phenotypic diversity

BackgroundTularemia 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.ResultsCultivation 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.ConclusionsF. 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.

Eight New Genomes and Synthetic Controls Increase the Accessibility of Rapid Melt-MAMA SNP Typing of Coxiella burnetii

The case rate of Q fever in Europe has increased dramatically in recent years, mainly because of an epidemic in the Netherlands in 2009. Consequently, there is a need for more extensive genetic characterization of the disease agent Coxiella burnetii in order to better understand the epidemiology and spread of this disease. Genome reference data are essential for this purpose, but only thirteen genome sequences are currently available. Current methods for typing C. burnetii are criticized for having problems in comparing results across laboratories, require the use of genomic control DNA, and/or rely on markers in highly variable regions. We developed in this work a method for single nucleotide polymorphism (SNP) typing of C. burnetii isolates and tissue samples based on new assays targeting ten phylogenetically stable synonymous canonical SNPs (canSNPs). These canSNPs represent previously known phylogenetic branches and were here identified from sequence comparisons of twenty-one C. burnetii genomes, eight of which were sequenced in this work. Importantly, synthetic control templates were developed, to make the method useful to laboratories lacking genomic control DNA. An analysis of twenty-one C. burnetii genomes confirmed that the species exhibits high sequence identity. Most of its SNPs (7,493/7,559 shared by >1 genome) follow a clonal inheritance pattern and are therefore stable phylogenetic typing markers. The assays were validated using twenty-six genetically diverse C. burnetii isolates and three tissue samples from small ruminants infected during the epidemic in the Netherlands. Each sample was assigned to a clade. Synthetic controls (vector and PCR amplified) gave identical results compared to the corresponding genomic controls and are viable alternatives to genomic DNA. The results from the described method indicate that it could be useful for cheap and rapid disease source tracking at non-specialized laboratories, which requires accurate genotyping, assay accessibility and inter-laboratory comparisons.

Genomic analyses of Francisella tularensis strains confirm disease transmission from drinking water sources, Turkey, 2008, 2009 and 2012.

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.

Hare-to-Human Transmission of Francisella tularensis subsp. holarctica, Germany

In November 2012, a group of 7 persons who participated in a hare hunt in North Rhine-Westphalia, Germany, acquired tularemia. Two F. tularensis subsp. holarctica isolates were cultivated from human and hare biopsy material. Both isolates belonged to the FTN002–00 genetic subclade (derived for single nucleotide polymorphisms B.10 and B.18), thus indicating likely hare-to-human transmission.

Phylogeography of Francisella tularensis subspecies holarctica in Finland, 1993-2011.

Abstract Background: Finland repeatedly reports some of the highest incidences of tularaemia worldwide. To determine genetic diversity of the aetiologic agent of tularaemia, Francisella tularensis, a total of 76 samples from humans (n = 15) and animals (n = 61) were analysed. Methods: We used CanSNPs and canINDEL hydrolysis or TaqMan MGB probes for the analyses, either directly from the clinical tissue samples (n = 21) or from bacterial isolates (n = 55). Results: The genotypes of the strains were assigned to three previously described basal subspecies holarctica clades. The majority of strains (n = 67) were assigned to B.12, a clade reported to dominate in Scandinavia and Eastern Europe. A single strain was assigned to clade B.4, previously reported from North America, Europe and China. The remaining strains (n = 8) were members of clade B.6. Importantly, new diversity was discovered in clade B.6. We describe two newly designed TaqMan MGB probe assays for this new B.6 subclade B.70, and its previously identified sister clade B.11, a clade dominantly found in Western Europe. Conclusions: The high genetic diversity of F. tularensis subspecies holarctica present in Finland is consistent with previous findings in Sweden. The results suggest a northern and southern division of the B.6 subclade B.10, where B.11 predominates in Western and Central Europe and B.70 is found in Fennoscandia. Further research is required to define whether the vast diversity of genotypes found is related to different habitats or reservoir species, their different postglacial immigration routes to Fennoscandia, or dynamics of the reservoir species.

Francisella tularensis subsp. tularensis Group A.I, United States

We used whole-genome analysis and subsequent characterization of geographically diverse strains using new genetic signatures to identify distinct subgroups within Francisella tularensis subsp. tularensis group A.I: A.I.3, A.I.8, and A.I.12. These subgroups exhibit complex phylogeographic patterns within North America. The widest distribution was observed for A.I.12, which suggests an adaptive advantage.

Phylogeography of Francisella tularensis subspecies holarctica in Finland, 1993-2011

Abstract Background: Finland repeatedly reports some of the highest incidences of tularaemia worldwide. To determine genetic diversity of the aetiologic agent of tularaemia, Francisella tularensis, a total of 76 samples from humans (n = 15) and animals (n = 61) were analysed. Methods: We used CanSNPs and canINDEL hydrolysis or TaqMan MGB probes for the analyses, either directly from the clinical tissue samples (n = 21) or from bacterial isolates (n = 55). Results: The genotypes of the strains were assigned to three previously described basal subspecies holarctica clades. The majority of strains (n = 67) were assigned to B.12, a clade reported to dominate in Scandinavia and Eastern Europe. A single strain was assigned to clade B.4, previously reported from North America, Europe and China. The remaining strains (n = 8) were members of clade B.6. Importantly, new diversity was discovered in clade B.6. We describe two newly designed TaqMan MGB probe assays for this new B.6 subclade B.70, and its previously identified sister clade B.11, a clade dominantly found in Western Europe. Conclusions: The high genetic diversity of F. tularensis subspecies holarctica present in Finland is consistent with previous findings in Sweden. The results suggest a northern and southern division of the B.6 subclade B.10, where B.11 predominates in Western and Central Europe and B.70 is found in Fennoscandia. Further research is required to define whether the vast diversity of genotypes found is related to different habitats or reservoir species, their different postglacial immigration routes to Fennoscandia, or dynamics of the reservoir species.

An improved multiple-locus variable-number of tandem repeat analysis (MLVA) for the fish pathogen Francisella noatunensis using capillary electrophoresis

BackgroundFrancisellosis, caused by the bacterium Francisella noatunensis subsp. noatunensis, remains a serious threat to Atlantic cod (Gadhus morhua) farming in Norway and potentially in other countries. As outbreak strains appear clonal in population structure, access to highly discriminatory typing tools is critical for understanding the epidemiology of francisellosis infections in aquaculture. In this study, a simplified multiple-locus variable-number of tandem repeat analysis (MLVA) targeting five highly polymorphic variable number of tandem repeat (VNTR) loci in a single multiplex PCR was developed to rapidly discriminate between outbreak strains.ResultsThe assay resulted in identification of at least 13 different allelic profiles or subpopulations among 91 F. noatunensis isolates from farmed cod in Norway. The VNTR loci appear relatively stable, with isolates originating from individual outbreaks showing identical MLVA profiles following repeated passage. MLVA displayed greater discriminatory power than pulse-field gel electrophoresis (PFGE). Both MLVA and PFGE show good epidemiological concordance by their abilities to separate outbreak strains from epidemiologically unrelated isolates.ConclusionsThe MLVA method presented here is robust, easy to perform and provides a good alternative to other typing systems for F. noatunensis subsp. noatunensis and epidemiological study of francisellosis in cod.

Antibiotic susceptibility in vitro of Francisella tularensis subsp. holarctica isolates from Germany

Background: Tularaemia is a zoonotic disease caused by the bacterium Francisella tularensis. In Germany, the disease is still rare (e.g. 34 human cases reported in 2015). There is a lack of data about the susceptibility of F. tularensis strains to antibiotics, because many cases are diagnosed using serological assays only. Objectives: The antibiotic susceptibility in vitro of F. tularensis subsp. holarctica strains isolated in Germany was assessed to determine whether the currently recommended empirical therapy is still adequate. Methods: A total of 128 F. tularensis strains were investigated that were collected between 2005 and 2014 in Germany from wild animals, ticks and humans. All isolates were genotyped using real‐time PCR assays targeting canonical SNPs, and antibiotic susceptibility was tested using MIC test strips on agar plates. MIC values were interpreted using CLSI breakpoints. Results: The strains were susceptible to antibiotics commonly recommended for tularaemia therapy, i.e. aminoglycosides (MIC90 values: gentamicin 1 mg/L; streptomycin 4.0 mg/L), tetracyclines (MIC90 values: tetracycline 0.5 mg/L; doxycycline 1.5 mg/L) and quinolones (MIC90 value: ciprofloxacin 0.064 mg/L). Chloramphenicol (MIC90 value: 3.0 mg/L) may be of value in treatment of tularaemia meningitis. Ninety‐four isolates were susceptible to erythromycin, which defines biovar I (genotypes B.4 and B.6); 34 were resistant (biovar II; genotype B.12). Conclusions: The F. tularensis isolates investigated in this study showed the typical antibiotic susceptibility pattern that was previously observed in other countries. Therefore, recommendations for empirical antibiotic therapy of tularaemia can remain unchanged. However, antibiotic susceptibility testing of clinical isolates should be performed whenever possible.

Long-range dispersal moved Francisella tularensis into Western Europe from the East

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.