Detection and Genotyping of Francisella tularensis in Animal Hosts and Vectors from Six Different Natural Landscape Areas, Gansu Province, China.

OBJECTIVE: Tularemia, also known as hare fever, is caused by the bacterium Francisella tularensis (F. tularensis) transmitted through diseased wild animals, blood sucking insects, or contaminated water or food, which is distributed worldwide. The purpose of this study was to investigate F. tularensis infection in animal hosts and vectors from six different natural landscape areas in Gansu Province and to identify the genotypes of the detected F. tularensis. METHODS: Rodents were captured by snap traps, and ticks were collected by dragging a cloth over the vegetation or from domestic animals. After species identification, DNA was isolated from the captured animals and detected by nested PCR assays targeting the F. tularensis fopA gene. The positive samples were further amplified to discriminate the species, and another two short-sequence tandem repeat regions (SSTR) were amplified to identify their genotypes. All positive fragments were sequenced and analyzed by ClustalX (5.0) and DNAClub software. RESULTS: A total of 407 rodents of 12 species were captured, among which six rodent species were positive for F. tularensis, with an overall prevalence of 3.93%. The geographical difference in infection rate was statistically significant. At the SSTR9 locus, there were 7 genotypes among positive rodent samples. A total of 1864 ticks were tested for evidence of tularemia by nested PCR assays, 69 of which were positive, with an average positive rate of 3.70% for F. tularensis in ticks. The positive rates were significantly different among different regions. Seven genotypes were identified at the SSTR9 locus, one of which seemed dominant in positive tick samples. All positive samples had the same genotype at the SSTR16 locus. CONCLUSION: There is natural infection of F. tularensis among animal vectors and hosts in Gansu Province, with diverse genotypes.

A study of innate immune kinetics reveals a role for a chloride transporter in a virulent Francisella tularensis type B strain.

Tularemia is a zoonotic disease of global proportions. Francisella tularensis subspecies tularensis (type A) and holarctica (type B) cause disease in healthy humans, with type A infections resulting in higher mortality. Repeated passage of a type B strain in the mid-20th century generated the Live Vaccine Strain (LVS). LVS remains unlicensed, does not protect against high inhalational doses of type A, and its exact mechanisms of attenuation are poorly understood. Recent data suggest that live attenuated vaccines derived from type B may cross-protect against type A. However, there is a dearth of knowledge regarding virulent type B pathogenesis and its capacity to stimulate the host's innate immune response. We therefore sought to increase our understanding of virulent type B in vitro characteristics using strain OR96-0246 as a model. Adding to our knowledge of innate immune kinetics in macrophages following infection with virulent type B, we observed robust replication of strain OR96-0246 in murine and human macrophages, reduced expression of pro-inflammatory cytokine genes from "wild type" type B-infected macrophages compared to LVS, and delayed macrophage cell death suggesting that virulent type B may suppress macrophage activation. One disruption in LVS is in the gene encoding the chloride transporter ClcA. We investigated the role of ClcA in macrophage infection and observed a replication delay in a clcA mutant. Here, we propose its role in acid tolerance. A greater understanding of LVS attenuation may reveal new mechanisms of pathogenesis and inform strategies toward the development of an improved vaccine against tularemia.

Using affinity propagation clustering for identifying bacterial clades and subclades with whole-genome sequences of Francisella tularensis.

By combining a reference-independent SNP analysis and average nucleotide identity (ANI) with affinity propagation clustering (APC), we developed a significantly improved methodology allowing resolving phylogenetic relationships, based on objective criteria. These bioinformatics tools can be used as a general ruler to determine phylogenetic relationships and clustering of bacteria, exemplary done with Francisella (F.) tularensis. Molecular epidemiology of F. tularensis is currently assessed mostly based on laboratory methods and molecular analysis. The high evolutionary stability and the clonal nature makes Francisella ideal for subtyping with single nucleotide polymorphisms (SNPs). Sequencing and real-time PCR can be used to validate the SNP analysis. We investigate whole-genome sequences of 155 F. tularensis subsp. holarctica isolates. Phylogenetic testing was based on SNPs and average nucleotide identity (ANI) as reference independent, alignment-free methods taking small-scale and large-scale differences within the genomes into account. Especially the whole genome SNP analysis with kSNP3.0 allowed deciphering quite subtle signals of systematic differences in molecular variation. Affinity propagation clustering (APC) resulted in three clusters showing the known clades B.4, B.6, and B.12. These data correlated with the results of real-time PCR assays targeting canSNPs loci. Additionally, we detected two subtle sub-clusters. SplitsTree was used with standard-setting using the aligned SNPs from Parsnps. Together APC, HierBAPS, and SplitsTree enabled us to generate hypotheses about epidemiologic relationships between bacterial clusters and describing the distribution of isolates. Our data indicate that the choice of the typing technique can increase our understanding of the pathogenesis and transmission of diseases with the eventual for prevention. This is opening perspectives to be applied to other bacterial species. The data provide evidence that Germany might be the collision zone where the clade B.12, also known as the East European clade, overlaps with the clade B.6, also known as the Iberian clade. Described methods allow generating a new, more detailed perspective for F. tularensis subsp. holarctica phylogeny. These results may encourage to determine phylogenetic relationships and clustering of other bacteria the same way.

Quick identification and epidemiological characterization of Francisella tularensis by MALDI-TOF mass spectrometry.

INTRODUCTION: Currently, Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) is being evaluated for its efficacy as a fast bacterial typing tool due to its great speed compared to other molecular methods. In this study, we evaluated MALDI-TOF as a tool for quick identification and typing of Francisella tularensis. MATERIALS AND METHODS: This study encompassed 86 strains from two different geographical origins (Spain and the Czech Republic), which were previously characterised by Pulsed-Field Gel Electrophoresis (PFGE) and Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA). The direct colony method was used for microbial identification. High-quality spectra of the 86 strains were obtained and their main spectra profiles (MSPs) were created for epidemiological typing using MALDI-TOF. Based on the MSPs, principal components were generated and a dendrogram was constructed. An in-house MALDI-TOF library entry was created for each group of PFGE and MLVA strains based on their high-quality spectra. Two dendrograms were obtained using these entries and the unique peaks in each entry were searched. RESULTS: All strains were correctly identified to the species level. No clear divisions were found in the 86-strain dendrogram; however, Spanish and Czech strains appeared separately in dendrograms created using MLVA and PFGE entries. Entries from our in-house MALDI-TOF library revealed 2-4 biomarker peaks for the detection of the five PFGE groups and 1-12 biomarker peaks for the detection of the seven MLVA groups. Finally, two and one specific biomarkers were found in the Czech and Spanish strains, respectively. CONCLUSION: MALDI-TOF can be used to accurately identify F. tularensis strains in less than 15 min. Moreover, data on geographical origin and PFGE and MLVA groups could be obtained in less than one hour after colony growing.

[Determination of the Subspecies of Francisella tularensis Isolated in Turkey by Molecular Methods]. / Türkiye'de Izole Edilen Francisella tularensis Alt Türlerinin Moleküler Yöntemlerle Belirlenmesi.

Francisella tularensis is a gram-negative, coccobasillus, facultative intracellular bacteria and causes a zoonotic disease, tularemia in humans. F.tularensis has four subspecies, which have different virulences for humans as F.tularensis subsp. tularensis, F.tularensis subsp. holarctica, F.tularensis subsp. mediasiatica and F.tularensis subsp. novicida. F.tularensis subsp. tularensis is the most virulent subspecies and mortality rate is high in human cases. F.tularensis subsp. holarctica, which has been reported in our country to date, has lower virulence than that of subsp. tularensis, and causes rare lethality among untreated patients. According to the erythromycin resistance and the properties of glucose-glycerol fermentation, F.tularensis subsp. holarctica has three biovar as biovar I, biovar II and biovar japonica. F.tularensis subsp. mediasiatica has been reported only in a few central asian countries and its virulence is similar to the F.tularensis subsp. holarctica F.tularensis subsp. novicida is avirulent for immunocompetent individuals but has been observed to cause infection in immunocompromised individuals. The aim of this study was to determine the F.tularensis subspecies in 259 F.tularensis strains isolated from clinical specimens, drinking water and a rodent sample and 517 F.tularensis PCR-positive DNA isolated from clinical specimens between years 2009 and 2014. Conventional PCR was performed using primers specific for the RD1 (Region Difference) region of F.tularensis. Subspecies were differentiated depending on the difference in PCR amplification product size. In our study, F.tularensis subsp. holarctica was detected in 764 samples yielding 922 base pair (bp) amplification product. The DNA samples obtained from one water and 11 lymph aspirates were determined as F.tularensis subsp. holarctica biovar japonica. The DNA sequence analysis of the amplification product of the RD1 region of the isolate from water sample was determined. The 1136 bp nucleotide sequence obtained from the DNA sequence analysis was 100% similar to F.tularensis subsp. holarctica biovar japonica (FCS075 strain-accesion number AF469618) when compared with GenBank data. The whole genome sequence of this isolate was also determined and recorded to GenBank with accesion number CP007148. None of the samples used in our study belonged to other sub-species. F.tularensis subsp. holarctica biovar japonica positive 11 lymph aspirate samples were sent to our center from Ankara (n= 1), Kayseri (n= 1) and Afyon (n= 9) provinces. The results of the current study revealed that F.tularensis subsp. holarctica biovar japonica caused a tularemia outbreak in a village in Afyon province at first time and it was observed sporadically in two other different provinces.

Genetic Diversity and Spatial Segregation of Francisella tularensis Subspecies holarctica in Germany.

Francisella tularensis is an intracellular pleomorphic bacterium and the causative agent of tularemia, a zoonotic disease with a wide host range. Among the F. tularensis subspecies, especially F. tularensis subsp. holarctica is of clinical relevance for European countries. The study presented herein focuses namely on genetic diversity and spatial segregation of F. tularensis subsp. holarctica in Germany, as still limited information is available. The investigation is based on the analysis of 34 F. tularensis subsp. holarctica isolates and one draft genome from an outbreak strain. The isolates were cultured from sample material being that of primarily human patients (n = 25) and free-living animals (n = 9). For six of 25 human isolates, epidemiological links between disease onset and tick bites could be established, confirming the importance of arthropod linked transmission of tularemia in Germany. The strains were assigned to three of four major F. tularensis subsp. holarctica clades: B.4, B.6, and B.12. Thereby, B.6 and B.12 clade members were predominantly found; only one human isolate was assigned to clade B.4. Also, it turned out that eight isolates which caused pneumonia in patients clustered into the B.6 clade. Altogether, eight different final subclades were assigned to clade B.6 (biovar I, erythromycin sensitive) and six to B.12 (biovar II, erythromycin resistant) in addition to one new final B.12 subclade. Moreover, for 13 human and 3 animal isolates, final subclade subdivisions were not assigned (B.12 subdivisions B.33 and B.34, and B.6 subdivision B.45) because official nomenclatures are not available yet. This gives credit to the genetic variability of F. tularensis subsp. holarctica strains in Germany. The results clearly point out that the given genetic diversity in Germany seems to be comparably high to that found in other European countries including Scandinavian regions. A spatial segregation of B.6 and B.12 strains was found and statistically confirmed, and B.12 clade members were predominantly found in eastern parts and B.6 members more in western to southern parts of Germany. The portion of B.12 clade members in northeastern parts of Germany was 78.5% and in southwestern parts 1.9%.

Phylogenetic Analysis of Francisella tularensis Group A.II Isolates from 5 Patients with Tularemia, Arizona, USA, 2015-2017.

We examined 5 tularemia cases in Arizona, USA, during 2015-2017. All were caused by Francisella tularensis group A.II. Genetically similar isolates were found across large spatial and temporal distances, suggesting that group A.II strains are dispersed across long distances by wind and exhibit low replication rates in the environment.

Phylogeographic Distribution of Human and Hare Francisella Tularensis Subsp. Holarctica Strains in the Netherlands and Its Pathology in European Brown Hares (Lepus Europaeus).

Sequence-based typing of Francisella tularensis has led to insights in the evolutionary developments of tularemia. In Europe, two major basal clades of F. tularensis subsp. holarctica exist, with a distinct geographical distribution. Basal clade B.6 is primarily found in Western Europe, while basal clade B.12 occurs predominantly in the central and eastern parts of Europe. There are indications that tularemia is geographically expanding and that strains from the two clades might differ in pathogenicity, with basal clade B.6 strains being potentially more virulent than basal clade B.12. This study provides information on genotypes detected in the Netherlands during 2011-2017. Data are presented for seven autochthonous human cases and for 29 European brown hares (Lepus europaeus) with laboratory confirmed tularemia. Associated disease patterns are described for 25 European brown hares which underwent post-mortem examination. The basal clades B.6 and B.12 are present both in humans and in European brown hares in the Netherlands, with a patchy geographical distribution. For both genotypes the main pathological findings in hares associated with tularemia were severe (sub)acute necrotizing hepatitis and splenitis as well as necrotizing lesions and hemorrhages in several other organs. Pneumonia was significantly more common in the B.6 than in the B.12 cases. In conclusion, the two major basal clades present in different parts in Europe are both present in the Netherlands. In hares found dead, both genotypes were associated with severe acute disease affecting multiple organs. Hepatitis and splenitis were common pathological findings in hares infected with either genotype, but pneumonia occurred significantly more frequently in hares infected with the B.6 genotype compared to hares infected with the B.12 genotype.

Isothermal DNA amplification combined with lateral flow dipsticks for detection of biothreat agents.

The recently developed methods of nucleic acids isothermal amplification are promising tools for point-of-care diagnostics and in the field detection of pathogenic microorganisms. However, application of these methods outside a laboratory faces some challenges such as the rapid and sensitive detection of amplified products and the absence of cross-reactivity with genetically related microorganisms. In the presented study we compared three methods of isothermal DNA amplification loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA) and thermophilic helicase-dependent isothermal DNA amplification (tHDA), for detection of highly dangerous pathogens, such as Bacillus anthracis, Francisella tularensis and Yersinia pestis, and combined them with lateral flow dipsticks for the rapid visualization of amplified products. We observed low specificity of the three methods for B. antharcis, medium for Y. pestis and high for F. tularensis detection. Sensitivity and the detection limit were high and comparable for all the methods. We concluded that the lateral flow dipsticks have been a very useful tool for product detection of the isothermal amplification methods and enable reading the results without the use of any equipment. However, our results showed that the use of isothermal amplification methods is strongly related to the risk of false positive results.

Phylogenetic Lineages of Francisella tularensis in Animals.

Tularemia is a zoonotic disease caused by the facultative intracellular bacterium Francisella tularensis. This microorganism can infect a plethora of animal species and its ecology is particularly complex. Much research was performed to understand its biology but many questions are still open, especially concerning the life cycle of this bacterium in the environment related to physical and biological parameters. Numerous animals are major hosts of F. tularensis but precise reservoir species are not yet well defined. Moreover, the exact range of species susceptible to tularemia is not clear and is complicated by the differences in virulence and ecology observed among the subspecies of F. tularensis. Indeed, different life cycles in nature, including the animal species concerned, were previously described for F. tularensis subsp. tularensis and F. tularensis subsp. holarctica. Recently, molecular techniques showing adequate discrimination between strains were developed, leading to the possibility to investigate links between phylogenetic lineages and infection in animals. New perspectives in research are now possible thanks to the information available and the simplicity of the molecular procedures. Current studies are unfolding the evolution of F. tularensis and these developments will lead to the elucidation of geographical and ecological differences observed by veterinarians, microbiologists and conservation biologists. However, systematic, coordinated collection of data and extensive sampling are important to efficiently assemble the findings of future research.

Subspecies differentiation and genotyping of Francisella tularensis strains isolated from clinical and environmental samples.

Molecular epidemiology is one of the most rapidly developing research area. In the light of past and modern technologies it has gained number of typing methods based on molecular biology techniques. In this report, the subspecies differentiation of Francisella tularensis and genotyping of strains isolated in Poland and other geographic locations were investigated using real-time PCR and multispacer typing (MST) methods respectively. In total, 49 strains of F. tularensis included 15 strains from Poland, for subspecies differentiation the real-time PCR method was applied. For molecular typing using MST method four intergenic spacers (IS) were sequenced and compared with those previously described and deposited in GenBank (NCBI). Phylogenetic testing was performed using with the UPGMA model using MEGA6 software. The real-time PCR enables to distinguish five strains belonged to type A and 44 to type B among deposited F. tularensis strains. The MST revealed previously described genotypes, as well as 23 new genotypes were detected. The use of real-time PCR and MST method are valuable in the analysis of F. tularensis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrate convenient molecular tools (real-time PCR and multispacer typing) for Francisella tularensis detection, differentiation and genotyping which can be applied for molecular epidemiological studies and providing useful information for scientific research and during natural tularaemia outbreaks.

ClpB mutants of Francisella tularensis subspecies holarctica and tularensis are defective for type VI secretion and intracellular replication.

Francisella tularensis, a highly infectious, intracellular bacterium possesses an atypical type VI secretion system (T6SS), which is essential for the virulence of the bacterium. Recent data suggest that the HSP100 family member, ClpB, is involved in T6SS disassembly in the subspecies Francisella novicida. Here, we investigated the role of ClpB for the function of the T6SS and for phenotypic characteristics of the human pathogenic subspecies holarctica and tularensis. The ∆clpB mutants of the human live vaccine strain, LVS, belonging to subspecies holarctica, and the highly virulent SCHU S4 strain, belonging to subspecies tularensis, both showed extreme susceptibility to heat shock and low pH, severely impaired type VI secretion (T6S), and significant, but impaired intracellular replication compared to the wild-type strains. Moreover, they showed essentially intact phagosomal escape. Infection of mice demonstrated that both ΔclpB mutants were highly attenuated, but the SCHU S4 mutant showed more effective replication than the LVS strain. Collectively, our data demonstrate that ClpB performs multiple functions in the F. tularensis subspecies holarctica and tularensis and its function is important for T6S, intracellular replication, and virulence.

Population Genomics of Francisella tularensis subsp. holarctica and its Implication on the Eco-Epidemiology of Tularemia in Switzerland.

Whole genome sequencing (WGS) methods provide new possibilities in the field of molecular epidemiology. This is particularly true for monomorphic organisms where the discriminatory power of traditional methods (e.g., restriction enzyme length polymorphism typing, multi locus sequence typing etc.) is inadequate to elucidate complex disease transmission patterns, as well as resolving the phylogeny at high resolution on a micro-geographic scale. In this study, we present insights into the population structure of Francisella tularensis subsp. holarctica, the causative agent of tularemia in Switzerland. A total of 59 Fth isolates were obtained from castor bean ticks (Ixodes ricinus), animals and humans and a high resolution phylogeny was inferred using WGS methods. The majority of the Fth population in Switzerland belongs to the west European B.11 clade and shows an extraordinary genetic diversity underlining the old evolutionary history of the pathogen in the alpine region. Moreover, a new B.11 subclade was identified which was not described so far. The combined analysis of the epidemiological data of human tularemia cases with the whole genome sequences of the 59 isolates provide evidence that ticks play a pivotal role in transmitting Fth to humans and other vertebrates in Switzerland. This is further underlined by the correlation of disease risk estimates with climatic and ecological factors influencing the survival of ticks.

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.

Comparison of virulence of Francisella tularensis ssp. holarctica genotypes B.12 and B.FTNF002-00.

BACKGROUND: Two main genetic groups (B.12 and B.FTNF002-00) of Francisella tularensis ssp. holarctica are endemic in Europe. The B.FTNF002-00 group proved to be dominant in Western European countries, while strains of the B.12 group were isolated mainly in Northern, Central and Eastern Europe. The clinical course of tularemia in the European brown hare (Lepus europaeus) also shows distinct patterns according to the geographical area. Acute course of the disease is observed in hares in Western European countries, while signs of sub-acute or chronic infection are more frequently detected in the eastern part of the continent. The aim of the present study was to examine whether there is any difference in the virulence of the strains belonging to the B.FTNF002-00 and B.12 genetic clades. RESULTS: Experimental infection of Fischer 344 rats was performed by intra-peritoneal injection of three dilutions of a Hungarian (B.12 genotype) and an Italian (B.FTNF002-00 genotype) F. tularensis ssp. holarctica strain. Moderate difference was observed in the virulence of the two genotypes. Significant differences were observed in total weight loss values and scores of clinical signs between the two genotypes with more rats succumbing to tularemia in groups infected with the B.FTNF002-00 genotype. CONCLUSIONS: Results of the experimental infection are consistent with previous clinical observations and pathological studies suggesting that F. tularensis ssp. holarctica genotype B.FTNF002-00 has higher pathogenic potential than the B.12 genotype.

A Molecular Survey for Francisella tularensis and Rickettsia spp. in Haemaphysalis leporispalustris (Acari: Ixodidae) in Northern California.

Francisella tularensis and Rickettsia spp. have been cultured from Haemaphysalis leporispalustris Packard, but their prevalence in this tick has not been determined using modern molecular methods. We collected H. leporispalustris by flagging vegetation and leaf litter and from lagomorphs (Lepus californicus Gray and Sylvilagus bachmani (Waterhouse)) in northern California. Francisella tularensis DNA was not detected in any of 1,030 ticks tested by polymerase chain reaction (PCR), whereas 0.4% of larvae tested in pools, 0 of 117 individual nymphs, and 2.3% of 164 adult ticks were PCR-positive for Rickettsia spp. Positive sites were Laurel Canyon Trail in Tilden Regional Park in Alameda Contra Costa County, with a Rickettsia spp. prevalence of 0.6% in 2009, and Hopland Research and Extension Center in Mendocino County, with a prevalence of 4.2% in 1988. DNA sequencing revealed R. felis, the agent of cat-flea typhus, in two larval pools from shaded California bay and live oak leaf litter in Contra Costa County and one adult tick from a L. californicus in chaparral in Mendocino County. The R. felis in unfed, questing larvae demonstrates that H. leporispalustris can transmit this rickettsia transovarially. Although R. felis is increasingly found in diverse arthropods and geographical regions, prior literature suggests a typical epidemiological cycle involving mesocarnivores and the cat flea, Ctenocephalides felis. To our knowledge, this is the first report of R. felis in H. leporispalustris. Natural infection and transovarial transmission of this pathogen in the tick indicate the existence of a previously undocumented wild-lands transmission cycle that may intersect mesocarnivore-reservoired cycles and collectively affect human health risk.

Possible misidentification of species in the Pseudomonas fluorescens lineage as Burkholderia pseudomallei and Francisella tularensis, and emended descriptions of Pseudomonas brenneri,Pseudomonas gessardii and Pseudomonas proteolytica.

Bacteria were isolated from an industrial water circuit in the Netherlands. These strains were identified using API 20 NE as possible, or likely, Burkholderia pseudomallei. With VITEK 2 some of these strains scored 'low discrimination' for Francisella tularensis, amongst others. A total of twenty-six strains were assigned to the species Pseudomonas brenneri, Pseudomonas gessardii or Pseudomonas proteolytica. Because of the possibility of misidentification of these environmental species as medical- and public-health relevant B. pseudomallei and F. tularensis, an emended description, based on tests results more customarily used in clinical laboratories, was suitable. For this reason, the strains in this study, including the type strains DSM 15294T, DSM 17152T and DSM 15321T, were subjected to a polyphasic identification procedure. This procedure consisted of multiple phenotypic tests, fatty acid analysis, 16S rDNA sequence analysis, matrix-assisted laser desorption ionization time-of-flight mass spectronomy and various species-specific molecular tests. Based on the results of the polyphasic procedures, the species descriptions of P. brenneri, P. gessardii and P. proteolytica have been emended.

Phylogeography of Francisella tularensis from Tibet, China: Evidence for an asian origin and radiation of holarctica-type Tularemia.

The geographical origin and radiation of holarctica-type tularemia, which has spread across the northern hemisphere, is open to scientific debate. Here, through phylogenetics, we show that five Tibetan Francisella tularensis isolates subsp. holarctica cluster between basal-positioned Japanese isolates and all other subspecies strains in the world, providing evidence for a previously unknown intermediate lineage next to the Japanese isolates. Importantly, identification of this new intermediate lineage complements current knowledge of tularemia epidemiology, supporting a geographical origin and radiation of the subsp. holarctica in Asia. In addition, thirteen Tibetan isolates belonging to a clade previously found only in North America and Scandinavia, further increases the diversity of holarctica strains in Asia. In summary, this study provides evidence for an Asian origin and radiation of holarctica-type tularemia.

A human macrophage-hepatocyte co-culture model for comparative studies of infection and replication of Francisella tularensis LVS strain and subspecies holarctica and mediasiatica.

BACKGROUND: Francisella tularensis, a gram-negative bacterium replicates intracellularly within macrophages and efficiently evades the innate immune response. It is able to infect and replicate within Kupffer cells, specialized tissue macrophages of the liver, and to modulate the immune response upon infection to its own advantage. Studies on Francisella tularensis liver infection were mostly performed in animal models and difficult to extrapolate to the human situation, since human infections and clinical observations are rare. RESULTS: Using a human co-culture model of macrophages and hepatocytes we investigated the course of infection of three Francisella tularensis strains (subspecies holarctica--wildtype and live vaccine strain, and mediasiatica--wildtype) and analyzed the immune response triggered upon infection. We observed that hepatocytes support the intracellular replication of Franciscella species in macrophages accompanied by a specific immune response inducing TNFα, IL-1ß, IL-6 and fractalkine (CX3CL1) secretion and the induction of apoptosis. CONCLUSIONS: We could demonstrate that this human macrophage/hepatocyte co-culture model reflects strain-specific virulence of Francisella tularensis. We developed a suitable tool for more detailed in vitro studies on the immune response upon liver cell infection by F. tularensis.

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.