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Concept: Francisella tularensis


BACKGROUND: The intentional release of Bacillus anthracis in the United States in 2001 has heightened concern about the use of pathogenic microorganisms in bioterrorism attacks. Many of the deadliest bacteria, including the Class A Select Agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis, are highly infectious via the pulmonary route when released in aerosolized form. Hence, rapid, sensitive, and reliable methods for detection of these biothreats and characterization of their potential impact on the exposed population are of critical importance to initiate and support rapid military, public health, and clinical responses. METHODOLOGY/PRINCIPAL FINDINGS: We have developed microfluidic multiplexed PCR and sequencing assays based on the simultaneous interrogation of three pathogens per assay and ten loci per pathogen. Microfluidic separation of amplified fluorescently labeled fragments generated characteristic electrophoretic signatures for identification of each agent. The three sets of primers allowed significant strain typing and discrimination from non-pathogenic closely-related species and environmental background strains based on amplicon sizes alone. Furthermore, sequencing of the 10 amplicons per pathogen, termed “Rapid Focused Sequencing,” allowed an even greater degree of strain discrimination and, in some cases, can be used to determine virulence. Both amplification and sequencing assays were performed in microfluidic biochips developed for fast thermal cycling and requiring 7 µL per reaction. The 30-plex sequencing assay resulted in genotypic resolution of 84 representative strains belonging to each of the three biothreat species. CONCLUSIONS/SIGNIFICANCE: The microfluidic multiplexed assays allowed identification and strain differentiation of the biothreat agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis and clear discrimination from closely-related species and several environmental background strains. The assays may be extended to detect a large number of pathogens, are applicable to the evaluation of both environmental and clinical samples, and have the potential to be applied in military, public health, and clinical diagnostic settings.

Concepts: Bacteria, Microbiology, Vaccination, Pathogen, Bacillus, Yersinia pestis, Francisella tularensis, Bacillus anthracis


BACKGROUND: Tularemia 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. RESULTS: Cultivation 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. CONCLUSIONS: F. 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.

Concepts: DNA, Gene, Genetics, Bacteria, Evolution, Francisella tularensis, Hare, European Hare


Following a wide-area biological terror attack, numerous decontamination technologies, techniques, and strategies will be required for rapid remediation. Establishing an understanding of how disinfectants will perform under field conditions is of critical importance. The purpose of this study was to determine the efficacy of several liquid decontaminants, when used to inactivate vegetative biological agents on environmental surfaces. Aluminum, carpet, concrete, glass, and wood coupons were inoculated with 1×10(8)CFU of Burkholderia mallei, Francisella tularensis, Vibrio cholerae, or Yersinia pestis. Using spray-based application methods, decontamination was then attempted with pH-adjusted bleach, 1% citric acid, 70% ethanol, quaternary ammonia, or Pine-Sol®. Results indicated that decontamination efficacy varied significantly by decontaminant and organism. Materials such as wood are difficult to decontaminate, even when using sporicides. The data presented here will help responders develop efficacious remediation strategies following a large-scale contamination incident.

Concepts: Bacteria, Acid, Fungus, Vibrio cholerae, Vinegar, Yersinia pestis, Francisella tularensis, Burkholderia mallei


 Francisella novicida is a rare cause of human illness despite its close genetic relationship to F. tularensis, the agent of tularemia. During April-July 2011, three inmates at a Louisiana correctional facility developed F. novicida bacteremia; one died acutely.

Concepts: Health, Prison, Francisella tularensis


During the last decades, large tularemia outbreaks in humans have coincided in time and space with population outbreaks of common voles in northwestern Spain, leading us to hypothesize that this rodent species acts as a key spillover agent of Francisella tularensis in the region. Here, we evaluate for the first time a potential link between irruptive vole numbers and human tularemia outbreaks in Spain. We compiled vole abundance estimates obtained through live-trapping monitoring studies and official reports of human tularemia cases during the period 1997-2014. We confirm a significant positive association between yearly cases of tularemia infection in humans and vole abundance. High vole densities during outbreaks (up to 1000 voles/hectare) may therefore enhance disease transmission and spillover contamination in the environment. If this ecological link is further confirmed, the apparent multiannual cyclicity of common vole outbreaks might provide a basis for forecasting the risk of tularemia outbreaks in northwestern Spain.

Concepts: Time, Population ecology, Natural environment, Rodent, Francisella tularensis, Vole, Microtus, Common Vole


The occurrence of Francisella tularensis outside of endemic areas, such as North America and Eurasia, has been enigmatic. We report the metagenomic discovery and isolation of F. tularensis ssp. holarctica biovar japonica from diseased ringtail possums in Sydney, Australia. This finding confirms the presence of F. tularensis in the Southern Hemisphere.

Concepts: United States, Europe, Pacific Ocean, Australia, South America, Francisella tularensis, Possum, Southern Hemisphere


On June 27, 2016, the Minnesota Department of Health (MDH) Public Health Laboratory (PHL) was notified of a suspected Francisella tularensis isolate cultured at a hospital laboratory. The isolate was confirmed as F. tularensis type B at MDH PHL by reverse transcription-polymerase chain reaction, culture, and direct fluorescent antibody testing. Francisella tularensis subspecies tularensis (type A) and holarctica (type B) bacteria are the causative agents of tularemia.

Concepts: Immune system, Antibody, Bacteria, Causality, Culture, Proteobacteria, Penicillin, Francisella tularensis


Francisella tularensis is an intracellular pathogen that causes the fatal zoonotic disease tularaemia. Critical for its pathogenesis is the ability of the phagocytosed bacteria to escape into the cell cytosol. For this, the bacteria use a non-canonical type VI secretion system (T6SS) encoded on the Francisella pathogenicity island (FPI). Here we show that in F. novicida T6SS assembly initiates at the bacterial poles both in vitro and within infected macrophages. T6SS dynamics and function depends on the general purpose ClpB unfoldase, which specifically colocalizes with contracted sheaths and is required for their disassembly. T6SS assembly depends on iglF, iglG, iglI and iglJ, whereas pdpC, pdpD, pdpE and anmK are dispensable. Importantly, strains lacking pdpC and pdpD are unable to escape from phagosome, activate AIM2 inflammasome or cause disease in mice. This suggests that PdpC and PdpD are T6SS effectors involved in phagosome rupture.

Concepts: Immune system, Cell, Bacteria, Microbiology, Cytosol, Secretion, Cell biology, Francisella tularensis


The intracellular bacterial pathogen Francisella tularensis causes tularemia, a zoonosis that can be fatal. The type VI secretion system (T6SS) encoded by the Francisella pathogenicity island (FPI) is critical for the virulence of this organism. Existing studies suggest that the complete repertoire of T6SS effectors delivered to host cells is encoded by the FPI. Using a proteome-wide approach, we discovered that the FPI-encoded T6SS exports at least three effectors encoded outside of the island. These proteins share features with virulence determinants of other pathogens, and we provide evidence that they can contribute to intramacrophage growth. The remaining proteins that we identified are encoded within the FPI. Two of these FPI-encoded proteins constitute effectors, whereas the others form a unique complex required for core function of the T6SS apparatus. The discovery of secreted effectors mediating interactions between Francisella and its host significantly advances our understanding of the pathogenesis of this organism.

Concepts: Immune system, Cell, Bacteria, Microbiology, Golgi apparatus, Secretion, Cell biology, Francisella tularensis


The animals primarily infected by Francisella tularensis are rapidly consumed by scavengers, hindering ecologic investigation of the bacterium. We describe a 2012 natural tularemia epizootic among house mice in Switzerland and the assessment of infection of exposed humans. The humans were not infected, but the epizootic coincided with increased reports of human cases in the area.

Concepts: Human, Natural selection, Infection, Ecology, Natural environment, The Animals, Proteobacteria, Francisella tularensis