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Francisella tularensis is the causative agent of tularemia. We tested the susceptibility of 278 F. tularensis isolates from the United States received during 2009-2018 to 8 antimicrobial drugs (ciprofloxacin, levofloxacin, doxycycline, tetracycline, gentamicin, streptomycin, chloramphenicol, and erythromycin). All isolates were susceptible to all tested drugs., Competing Interests: Potential conflicts of interest. The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2024.)

Tularemia is a bacterial zoonosis caused by Francisella tularensis . We conducted a serosurvey of black-tailed prairie dogs ( Cynomys ludovicianus ) in Devils Tower National Monument, Wyoming, US, following an epizootic in voles ( Microtus spp.) due to F. tularensis . Only 1 of 44 (2%) sampled prairie dogs was seropositive for F. tularensis , providing evidence of survival and potentially limited spread among free-ranging prairie dogs.

In July 2017, fever and sepsis developed in 3 recipients of solid organs (1 heart and 2 kidneys) from a common donor in the United States; 1 of the kidney recipients died. Tularemia was suspected only after blood cultures from the surviving kidney recipient grew Francisella species. The organ donor, a middle-aged man from the southwestern United States, had been hospitalized for acute alcohol withdrawal syndrome, pneumonia, and multiorgan failure. F. tularensis subsp. tularensis (clade A2) was cultured from archived spleen tissue from the donor and blood from both kidney recipients. Whole-genome multilocus sequence typing indicated that the isolated strains were indistinguishable. The heart recipient remained seronegative with negative blood cultures but had been receiving antimicrobial drugs for a medical device infection before transplant. Two lagomorph carcasses collected near the donor's residence were positive by PCR for F. tularensis subsp. tularensis (clade A2). This investigation documents F. tularensis transmission by solid organ transplantation.

A total of 6,255 ticks belonging to three genera and six species (Haemaphysalis flava Neumann, Haemaphysalis longicornis Neumann, Haemaphysalis phasiana Saito, Ixodes nipponensis Kitaoka & Saito, Ixodes persulcatus Schulze, and Amblyomma testudinarium Koch) collected from May-August, 2013, at four southwestern provinces in the Republic of Korea (ROK) were submitted to the Armed Forces Research Institute of Medical Sciences and assayed for selected tick-borne pathogens. One pool each of H. flava and H. phasiana was positive by PCR and sequencing for a Francisella-like endosymbiont, while all pools were negative for Francisella tularensis, the causative agent of tularemia., (Published by Oxford University Press on behalf of Entomological Society of America 2017. This work is written by US Government employees and is in the public domain in the US.)

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.

Francisella tularensis is the causative agent of the acute disease tularemia. Due to its extreme infectivity and ability to cause disease upon inhalation, F. tularensis has been classified as a biothreat agent. Two subspecies of F. tularensis, tularensis and holarctica, are responsible for tularemia in humans. In comparison, the closely related species F. novicida very rarely causes human illness and cases that do occur are associated with patients who are immune compromised or have other underlying health problems. Virulence between F. tularensis and F. novicida also differs in laboratory animals. Despite this varying capacity to cause disease, the two species share ~97% nucleotide identity, with F. novicida commonly used as a laboratory surrogate for F. tularensis. As the F. novicida U112 strain is exempt from U.S. select agent regulations, research studies can be carried out in non-registered laboratories lacking specialized containment facilities required for work with virulent F. tularensis strains. This review is designed to highlight phenotypic (clinical, ecological, virulence, and pathogenic) and genomic differences between F. tularensis and F. novicida that warrant maintaining F. novicida and F. tularensis as separate species. Standardized nomenclature for F. novicida is critical for accurate interpretation of experimental results, limiting clinical confusion between F. novicida and F. tularensis and ensuring treatment efficacy studies utilize virulent F. tularensis strains.

Background: The use of prototypic strains is common among laboratories studying infectious agents as it promotes consistency for data comparability among and between laboratories. Schu S4 is the prototypic virulent strain of Francisella tularensis and has been used extensively as such over the past six decades. Studies have demonstrated virulence differences among the two clinically relevant subspecies of F. tularensis, tularensis (type A) and holarctica (type B) and more recently between type A subpopulations (A1a, A1b and A2). Schu S4 belongs to the most virulent subspecies of F. tularensis, subspecies tularensis., Methods: In this study, we investigated the relative virulence of Schu S4 in comparison to A1a, A1b, A2 and type B strains using a temperature-based murine model of infection. Mice were inoculated intradermally and a hypothermic drop point was used as a surrogate for death. Survival curves and the length of temperature phases were compared for all infections. Bacterial burdens were also compared between the most virulent type A subpopulation, A1b, and Schu S4 at drop point., Results: Survival curve comparisons demonstrate that the Schu S4 strain used in this study resembles the virulence of type B strains, and is significantly less virulent than all other type A (A1a, A1b and A2) strains tested. Additionally, when bacterial burdens were compared between mice infected with Schu S4 or MA00-2987 (A1b) significantly higher burdens were present in the blood and spleen of mice infected with MA00-2987., Conclusions: The knowledge gained from using Schu S4 as a prototypic virulent strain has unquestionably advanced the field of tularemia research. The findings of this study, however, indicate that careful consideration of F. tularensis strain selection must occur when the overall virulence of the strain used could impact the outcome and interpretation of results.

The study of infectious agents, their pathogenesis, the host response and the evaluation of newly developed countermeasures often requires the use of a living system. Murine models are frequently used to undertake such investigations with the caveat that non-biased measurements to assess the progression of infection are underutilized. Instead, murine models predominantly rely on symptomology exhibited by the animal to evaluate the state of the animal's health and to determine when euthanasia should be performed. In this study, we used subcutaneous temperature as a non-subjective measurement to follow and compare infection in mice inoculated with Francisella tularensis, a Gram-negative pathogen that produces an acute and fatal illness in mice. A reproducible temperature pattern defined by three temperature phases (normal, febrile and hypothermic) was identified in all mice infected with F. tularensis, regardless of the infecting strain. More importantly and for the first time a non-subjective, ethical, and easily determined surrogate endpoint for death based on a temperature, termed drop point, was identified and validated with statistical models. In comparative survival curve analyses for F. tularensis strains with differing virulence, the drop point temperature yielded the same results as those obtained using observed time to death. Incorporation of temperature measurements to evaluate F. tularensis was standardized based on statistical models to provide a new level of robustness for comparative analyses in mice. These findings should be generally applicable to other pathogens that produce acute febrile disease in animal models and offers an important tool for understanding and following the infection process.

This study evaluated commercial automated and manual DNA extraction methods for the isolation of Francisella tularensis DNA suitable for real-time polymerase chain reaction (PCR) analysis from cell suspensions and spiked cotton, foam, and polyester swabs. Two automated methods, the MagNA Pure Compact and the QIAcube, were compared to 4 manual methods, the IT 1-2-3 DNA sample purification kit, the MasterPure Complete DNA and RNA purification kit, the QIAamp DNA blood mini kit, and the UltraClean Microbial DNA isolation kit. The methods were compared using 6 F. tularensis strains representing the 2 subspecies which cause the majority of reported cases of tularemia in humans. Cell viability testing of the DNA extracts showed that all 6 extraction methods efficiently inactivated F. tularensis at concentrations of ≤10⁶ CFU/mL. Real-time PCR analysis using a multitarget 5' nuclease assay for F. tularensis revealed that the PCR sensitivity was equivalent using DNA extracted by the 2 automated methods and the manual MasterPure and QIAamp methods. These 4 methods resulted in significantly better levels of detection from bacterial suspensions and performed equivalently for spiked swab samples than the remaining 2. This study identifies optimal DNA extraction methods for processing swab specimens for the subsequent detection of F. tularensis DNA using real-time PCR assays. Furthermore, the results provide diagnostic laboratories with the option to select from 2 automated DNA extraction methods as suitable alternatives to manual methods for the isolation of DNA from F. tularensis., (Published by Elsevier Inc.)

The American dog tick, Dermacentor variabilis (Say) (Acari: Ixodidae), has been implicated as a potential bridging vector to humans of Francisella tularensis, the etiological agent of tularemia. Since the initial studies evaluating vector competency of D. variabilis were conducted, F. tularensis has been subdivided into subspecies and clades that differ in their geographical distribution in the United States and in the severity of infections caused in humans. Here, we demonstrate that D. variabilis nymphs efficiently acquire, transtadially maintain, and transmit each of the strains tested (clades A1b and A2, and type B). Transmission efficiency by adult females was similarly high among infection groups and ranged from 58% for type B to 89% for A2 infections. In addition, we demonstrated that transmission can occur shortly after tick attachment. These findings support the concept that D. variabilis adults may play a significant role in epizootic transmission of F. tularensis, and as a bridging vector to humans.

Francisella tularensis is a facultative, intracellular, zoonotic pathogen and the causative agent of tularemia. Historically, F. tularensis has been subdivided into subspecies on the basis of phenotypic traits, including biochemical reactivity and virulence. More recently, a number of genotypic methods, ranging from relatively insensitive methods to full genome sequencing, have been used to investigate genetic diversity within F. tularensis. These analyses indicate that F. tularensis is a pathogen of low sequence diversity with pair-wise average nucleotide identities > 99.2% across subspecies. Nonetheless, genomic rearrangements and sequence deletions exist between and within F. tularensis subspecies, creating polymorphisms detectable by genotyping methods. Genetic subpopulations intermediate to the subspecies and strain level have been identified within F. tularensis subsp. tularensis and F. tularensis subsp. holarctica by several different typing methods. These genetic subpopulations have been associated with differences in disease severity, geographic distribution, and transmission patterns. For example, one F. tularensis subsp. tularensis subpopulation has been found to be significantly associated with mortality in humans. Additionally, genotypic analyses of Francisella spp. have provided information for use in the rational design of strain panels for validation of F. tularensis diagnostic tests. This review provides a guide to the various F. tularensis genotyping methods.

Francisella tularensis, the etiological agent of tularemia, is regarded as a potential bioterrorism agent. The advent of bioterrorism has heightened awareness of the need for validated methods for processing environmental samples. In this study we determined the optimal method for processing environmental swabs for the recovery and subsequent detection of F. tularensis by the use of real-time PCR assays. Four swab processing recovery methods were compared: heat, sonication, vortexing, and the Swab Extraction Tube System (SETS). These methods were evaluated using cotton, foam, polyester and rayon swabs spiked with six pathogenic strains of F. tularensis. Real-time PCR analysis using a multi-target 5'nuclease assay for F. tularensis showed that the use of the SETS method resulted in the best limit of detection when evaluated using multiple strains of F. tularensis. We demonstrated also that the efficiency of F. tularensis recovery from swab specimens was not equivalent for all swab processing methodologies and, thus, that this variable can affect real-time PCR assay sensitivity. The effectiveness of the SETS method was independent of the automated DNA extraction method and real-time PCR platforms used. In conclusion, diagnostic laboratories can now potentially incorporate the SETS method into specimen processing protocols for the rapid and efficient detection of F. tularensis by real-time PCR during laboratory bioterrorism-related investigations., (Published by Elsevier B.V.)

In the United States, the American dog tick, Dermacentor variabilis (Say) is considered an important biological vector of Francisella tularensis, the etiologic agent of tularemia. In this study, we evaluated the vector efficiency of nymphal D. variabilis infected as larvae with differing clades and subspecies (A1b, A2, and type B) of F. tularensis. In all cases, D. variabilis larvae were able to acquire, maintain, and transstadially transmit F. tularensis. Significant replication of the bacteria also occurred in infected nymphs. Transmission of F. tularensis to Swiss Webster mice was not observed with A1b, and low rates were observed with A2 (8.0%) and type B (13.5%). Negative effects on tick survivorship were also observed for A1b, A2, and type B infections. Our results provide evidence of a high fitness cost and low transmission rates during the immature stages, suggesting that D. variabilis may play a limited role in enzootic maintenance of F. tularensis.

Two subspecies of Francisella tularensis are recognized: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holartica (type B). Type A has been subdivided further into A1a, A1b, and A2, which differ geographically and clinically. The aim of this work was to determine whether or not differences among subspecies and clades translate into distinct ecological niches. We used 223 isolates from humans and wildlife representing all six genotypes (type A, B, A1, A2, A1a, or A1b). Ecological-niche models were built independently for each genotype, using the genetic algorithm for rule-set prediction. The resulting models were compared using a non-parametric multivariate analysis-of-variance method. A1 and A2 are ecologically distinct, supporting the previously observed geographic division, whereas ecological niches for types A and B overlapped notably but A1a and A1b displayed no appreciable differences in their ecological niches.

Francisella tularensis subspecies tularensis (type A) and holarctica (type B) are of clinical importance in causing tularemia. Molecular typing methods have further separated type A strains into three genetically distinct clades, A1a, A1b and A2. Epidemiological analyses of human infections in the United States suggest that A1b infections are associated with a significantly higher mortality rate as compared to infections caused by A1a, A2 and type B. To determine if genetic differences as defined by molecular typing directly correlate with differences in virulence, A1a, A1b, A2 and type B strains were compared in C57BL/6 mice. Here we demonstrate significant differences between survival curves for infections caused by A1b versus A1a, A2 and type B, with A1b infected mice dying earlier than mice infected with A1a, A2 or type B; these results were conserved among multiple strains. Differences were also detected among type A clades as well as between type A clades and type B with respect to bacterial burdens, and gross anatomy in infected mice. Our results indicate that clades defined within F. tularensis subsp. tularensis by molecular typing methods correlate with virulence differences, with A1b strains more virulent than A1a, A2 and type B strains. These findings indicate type A strains are not equivalent with respect to virulence and have important implications for public health as well as basic research programs.

Tularemia is primarily caused by two subspecies of Francisella tularensis worldwide, ssp. tularensis (type A) and ssp. holarctica (type B), which were originally delineated by phenotypic differences. Application of molecular typing methods to investigate population structure of F. tularensis has confirmed that categorizing the two subspecies via phenotypic characteristics corresponds with genotypic differentiation. In addition, genotyping methods have demonstrated that both subspecies, type A and type B, can be further distinguished into subpopulations and, in some cases, biological relevance has been ascribed to these identified subpopulations. Genetic variation among both type A and type B subpopulations has been shown to correlate with differences in geographic distribution and has also been coupled to distinct ecological niches, animal hosts and replication foci. Among type A subpopulations, strain variation is linked to differing clinical manifestations in humans and virulence in mice. This article will highlight our current understanding of F. tularensis subpopulations, including methods for their detection, their observed epidemiologic differences, implications for public health and basic research programs, as well as future challenges yet to be solved.

Background: A low genetic diversity in Francisella tularensis has been documented. Current DNA based genotyping methods for typing F. tularensis offer a limited and varying degree of subspecies, clade and strain level discrimination power. Whole genome sequencing is the most accurate and reliable method to identify, type and determine phylogenetic relationships among strains of a species. However, lower cost typing schemes are necessary in order to enable typing of hundreds or even thousands of isolates., Results: We have generated a high-resolution phylogenetic tree from 40 Francisella isolates, including 13 F. tularensis subspecies holarctica (type B) strains, 26 F. tularensis subsp. tularensis (type A) strains and a single F. novicida strain. The tree was generated from global multi-strain single nucleotide polymorphism (SNP) data collected using a set of six Affymetrix GeneChip resequencing arrays with the non-repetitive portion of LVS (type B) as the reference sequence complemented with unique sequences of SCHU S4 (type A). Global SNP based phylogenetic clustering was able to resolve all non-related strains. The phylogenetic tree was used to guide the selection of informative SNPs specific to major nodes in the tree for development of a genotyping assay for identification of F. tularensis subspecies and clades. We designed and validated an assay that uses these SNPs to accurately genotype 39 additional F. tularensis strains as type A (A1, A2, A1a or A1b) or type B (B1 or B2)., Conclusion: Whole-genome SNP based clustering was shown to accurately identify SNPs for differentiation of F. tularensis subspecies and clades, emphasizing the potential power and utility of this methodology for selecting SNPs for typing of F. tularensis to the strain level. Additionally, whole genome sequence based SNP information gained from a representative population of strains may be used to perform evolutionary or phylogenetic comparisons of strains, or selection of unique strains for whole-genome sequencing projects.

Francisella tularensis subsp. tularensis (type A) is subdivided into clades A1 and A2. Human tularemia infections caused by A1 and A2 differ with respect to clinical outcome; A1 infections are associated with a higher case fatality rate. In this study, we develop and evaluate TaqMan polymerase chain reaction (PCR) assays for identification of A1 and A2. Both assays were shown to be specific to either A1 or A2, with sensitivities of 10 genomic equivalents. Real-time PCR results for identification of A1 and A2 were in complete agreement with results obtained by pulsed field gel electrophoresis analysis or conventional PCR when specimens from sporadic tularemia cases and a tularemia outbreak involving both A1 and A2 were tested. In addition, outbreak samples not previously typed to the clade level could be classified as A1 or A2. The assays described here provide new diagnostic tools with a level of sensitivity not previously available for identification of A1 and A2 infections.

Francisella tularensis contains several highly pathogenic subspecies, including Francisella tularensis subsp. holarctica, whose distribution is circumpolar in the northern hemisphere. The phylogeography of these subspecies and their subclades was examined using whole-genome single nucleotide polymorphism (SNP) analysis, high-density microarray SNP genotyping, and real-time-PCR-based canonical SNP (canSNP) assays. Almost 30,000 SNPs were identified among 13 whole genomes for phylogenetic analysis. We selected 1,655 SNPs to genotype 95 isolates on a high-density microarray platform. Finally, 23 clade- and subclade-specific canSNPs were identified and used to genotype 496 isolates to establish global geographic genetic patterns. We confirm previous findings concerning the four subspecies and two Francisella tularensis subsp. tularensis subpopulations and identify additional structure within these groups. We identify 11 subclades within F. tularensis subsp. holarctica, including a new, genetically distinct subclade that appears intermediate between Japanese F. tularensis subsp. holarctica isolates and the common F. tularensis subsp. holarctica isolates associated with the radiation event (the B radiation) wherein this subspecies spread throughout the northern hemisphere. Phylogenetic analyses suggest a North American origin for this B-radiation clade and multiple dispersal events between North America and Eurasia. These findings indicate a complex transmission history for F. tularensis subsp. holarctica.

Background: In the United States, tularemia is caused by Francisella tularensis subsps. tularensis (type A) and holarctica (type B). Molecular subtyping has further divided type A into 2 subpopulations, A1 and A2. Significant mortality differences were previously identified between human infections caused by A1 (14%), A2 (0%) and type B (7%). To verify these findings and to further define differences among genotypes, we performed a large-scale molecular epidemiologic analysis of F. tularensis isolates from humans and animals., Methods: Pulsed-field gel electrophoresis with PmeI was performed on 302 type A and 61 type B isolates. Pulsed-field gel electrophoresis pattern and epidemiologic analyses were performed. Logistic regression was used to assess factors associated with human mortality., Results: Pulsed-field gel electrophoresis typing identified 4 distinct type A genotypes, A1a, A1b, A2a, and A2b, as well as type B. Genotypic and geographic divisions observed among isolates from humans were mirrored among isolates from animals, specifically among animal species that are linked to human infection and to enzootic maintenance of tularemia. Significant differences between human infections caused by different genotypes were identified with respect to patient age, site of organism recovery, and mortality. Human infections due to A1b resulted in significantly higher mortality (24%) than those caused by A1a (4%), A2 (0%), and type B (7%)., Conclusions: Three type A genotypes, A1a, A1b, and A2, were shown to be epidemiologically important. Our analysis suggests that A1b strains may be significantly more virulent in humans than A1a, A2, or type B strains. These findings have important implications for disease progression, disease prevention, and basic research programs.

Arthropod transmission of tularemia occurs throughout the northern hemisphere. Few pathogens show the adaptability of Francisella tularensis to such a wide array of arthropod vectors. Nonetheless, arthropod transmission of F. tularensis was last actively investigated in the first half of the 20th century. This review will focus on arthropod transmission to humans with respect to vector species, modes of transmission, geographic differences and F. tularensis subspecies and clades.

Tularemia is a tick-borne zoonotic bacterial disease. In the United States, human tularemia infections are caused by Francisella tularensis subspecies tularensis (Type A, clades A1 and A2) or F. tularensis subspecies holarctica (Type B). We developed a mouse model that can be used to study the ability of ticks to acquire and transmit fully virulent strains of F. tularensis (A1, A2, and Type B). We showed that 1) bacteremia was evident by 2 days post-infection (dpi) for A1, A2, and B, 2) bacteremia was expected to reach levels of > 10(8) cfu/mL by 3 dpi for A1 and A2 but not until 4 dpi for Type B, and 3) illness onset was delayed for mice exposed to Type B compared with A1 and A2. To maximize the likelihood of ticks acquiring infection from laboratory-infected mice before they become moribund and must be euthanized, ticks should be placed on mice so that periods of rapid engorgement occur 3-4 dpi for A1 and A2 and 4-5 dpi for Type B. Rigorous experimental studies of tick vector competence and efficiency conducted under standardized conditions are required to address several significant public health issues related to preventing and controlling tularemia. Our study provides the basis for a mouse model needed as the starting point to address these questions.

In July 2007, a deer fly-associated outbreak of tularemia occurred in Utah. Human infections were caused by 2 clades (A1 and A2) of Francisella tularensis subsp. tularensis. Lagomorph carcasses from the area yielded evidence of infection with A1 and A2, as well as F. tularensis subsp. holarctica. These findings indicate that multiple subspecies and clades can cause disease in a localized outbreak of tularemia.

Due to concern that Francisella tularensis, the causative agent of tularemia, may be used as a bioterrorist weapon, the Clinical and Laboratory Standards Institute recently provided a susceptibility testing method with breakpoints. Here, 169 isolates (92 type A and 77 type B) from North America were tested against seven antimicrobial agents (streptomycin, gentamicin, tetracycline, doxycycline, ciprofloxacin, levofloxacin, and chloramphenicol) used for the treatment of tularemia. The MICs for all of the isolates fell within the susceptible range. In addition, all isolates had MICs for erythromycin of 0.5 to 4 microg/ml, in contrast to an MIC of >256 microg/ml for the common laboratory strain LVS (live vaccine strain).

Tularemia is caused by two subspecies of Francisella tularensis, F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B). F. tularensis subsp. tularensis is further subdivided into two genetically distinct populations (A.I and A.II) that differ with respect to geographical location, anatomical source of recovered isolates, and disease outcome. Using two human clinical isolates, suppression subtractive hybridization was performed to identify 13 genomic regions of difference between A.I and A.II strains. Two PCR assays, one to identify A.I and A.II as well as to discriminate between F. tularensis subsp. holarctica and F. novicida and another specific for A.I, were developed. This is the first report to identify and characterize conserved genomic differences between A.I and A.II.

Motivation: An important application of protein microarray data analysis is identifying a serodiagnostic antigen set that can reliably detect patterns and classify antigen expression profiles. This work addresses this problem using antibody responses to protein markers measured by a novel high-throughput microarray technology. The findings from this study have direct relevance to rapid, broad-based diagnostic and vaccine development., Results: Protein microarray chips are probed with sera from individuals infected with the bacteria Francisella tularensis, a category A biodefense pathogen. A two-step approach to the diagnostic process is presented (1) feature (antigen) selection and (2) classification using antigen response measurements obtained from F.tularensis microarrays (244 antigens, 46 infected and 54 healthy human sera measurements). To select antigens, a ranking scheme based on the identification of significant immune responses and differential expression analysis is described. Classification methods including k-nearest neighbors, support vector machines (SVM) and k-Means clustering are applied to training data using selected antigen sets of various sizes. SVM based models yield prediction accuracy rates in the range of approximately 90% on validation data, when antigen set sizes are between 25 and 50. These results strongly indicate that the top-ranked antigens can be considered high-priority candidates for diagnostic development., Availability: All software programs are written in R and available at http://www.igb.uci.edu/index.php?page=tools and at http://www.r-project.org., Supplementary Information: Supplementary data are available at Bioinformatics online.

The presence of Francisella-like endosymbionts in tick species known to transmit tularemia poses a potential diagnostic problem for laboratories that screen tick samples by PCR for Francisella tularensis. Tick samples initially considered positive for F. tularensis based on standard 16S rRNA gene PCR were found to be positive only for Francisella-like endosymbionts using a multitarget F. tularensis TaqMan assay (ISFtu2, tul4, and iglC) and 16S rRNA gene sequencing. Specificity of PCR-based diagnostics for F. tularensis should be carefully evaluated with appropriate specimen types prior to diagnostic use.

Francisella tularensis, the causative agent of tularemia, is a highly infectious pathogen of humans and animals, yet little is known about the surface proteins of this organism that mediate mechanisms of pathogenicity. lambdaTnphoA was used to generate random alkaline phosphatase gene fusions in a F. tularensis subsp. tularensis (strain Schu S4) genomic library to identify genes encoding exported extracytoplasmic proteins. Eleven genes encoding membrane-associated proteins were identified by this method and their respective signal peptides were characterized. Three of the genes encoded conserved 'housekeeping' enzymes, while the other eight genes were unique to F. tularensis, encoding proteins with molecular masses ranging from 11 to 78kDa as deduced from the amino acid sequences. Two genes putatively encoded lipoproteins based on the presence of characteristic signal peptidase II cleavage sites. Four selected proteins were found associated with outer membranes from Schu S4 and LVS strains by Western blotting. Indirect immunofluorescence of strain Schu S4 cells also showed evidence of protein localization to the outer membrane. Protein database searches produced significant alignments with proteins from other bacteria involved in carbohydrate transport, lipid metabolism, and cell envelope biogenesis, thereby providing clues for putative functions. These findings demonstrated that TnphoA mutagenesis can be used in conjunction with F. tularensis genome sequence data to provide a foundation for studies to identify and define cellular surface protein virulence factors of this pathogen.

Francisella tularensis is found in a wide variety of hosts and extrahost environments, making culture recovery a diagnostic challenge. Here we demonstrate improved recovery times and good sensitivity (90%) when cultures were inoculated on the site of an investigation using fresh tissues. For contaminated specimens, antibiotic supplementation of enriched cysteine heart agar blood culture medium improved recovery of F. tularensis by 81.1%. For transport of tissues, immediate freezing yielded culture recovery rates as high as 94%.

A tularemia outbreak, caused by Francisella tularensis type B, occurred among wild-caught, commercially traded prairie dogs. F. tularensis microagglutination titers in one exposed person indicated recent infection. These findings represent the first evidence for prairie-dog-to-human tularemia transmission and demonstrate potential human health risks of the exotic pet trade.

An immunohistochemical assay was developed and tested for detection of Francisella tularensis lipopolysaccaride antigen in tissues of captive prairie dogs (Cynomys ludovicianus). Tissues from 59 cases of F. tularensis were examined by this technique, which was corroborated by direct fluorescent antibody assay and direct isolation of the organism. In infected prairie dogs, studies indicated multiple, severe, necroprurulent foci occurring in the liver, lung, spleen, terminal ileum, and mandibular lymph node. Immunohistochemical analysis of the same formalin-fixed tissues indicated the presence of F. tularensis antigen in neutrophils and macrophages of these lesions and occurring extracellularly in areas of necrosis. This report demonstrates that immunohistochemical analysis is a rapid procedure that can be used to determine the pathogenesis of F. tularensis in rodent populations.

Oropharyngeal tularemia was identified as the cause of a die-off in captured wild prairie dogs at a commercial exotic animal facility in Texas. From this point source, Francisella tularensis-infected prairie dogs were traced to animals distributed to the Czech Republic and to a Texas pet shop. F. tularensis culture isolates were recovered tissue specimens from 63 prairie dogs, including one each from the secondary distribution sites. Molecular and biochemical subtyping indicated that all isolates were F. tularensis subsp. holarctica (Type B). Microagglutination assays detected antibodies against F. tularensis, with titers as great as 1:4,096 in some live animals. All seropositive animals remained culture positive, suggesting that prairie dogs may act as chronic carriers of F. tularensis. These findings demonstrate the need for additional studies of tularemia in prairie dogs, given the seriousness of the resulting disease, the fact that prairie dogs are sold commercially as pets, and the risk for pet-to-human transmission.

Tularemia is the zoonotic disease caused by the gram-negative coccobacillus Francisella tularensis. Its wide distribution in the environment poses a challenge for understanding the transmission, ecology, and epidemiology of the disease. F. tularensis is also considered a potential biological weapon due to its extreme infectivity. We have developed a multitarget real-time TaqMan PCR assay capable of rapidly and accurately detecting F. tularensis in complex specimens. Targeted regions included the ISFtu2 element and the 23kDa, fopA, and tul4 genes. Analysis of the four TaqMan assays demonstrated that three (ISFtu2, 23kDa, and tul4) performed within our established criterion of a detection limit of one organism. The combined use of the three assays was highly specific, displaying no cross-reactivity with the non-Francisella bacteria tested and capable of differentially diagnosing both F. tularensis and Francisella philomiragia. When the multitarget TaqMan assay (ISFtu2, 23kDa, and tul4) was compared to culturing, using environmentally contaminated specimens, the TaqMan PCR assay was significantly more sensitive than culturing (P

Francisella tularensis is found throughout the Northern Hemisphere, where it is associated with the disease of tularaemia in animals and humans. The isolation and identification is reported of a novicida-like subspecies of F. tularensis from a foot wound sustained in brackish water in the Northern Territory of Australia.

In July 2017, fever and sepsis developed in 3 recipients of solid organs (1 heart and 2 kidneys) from a common donor in the United States; 1 of the kidney recipients died. Tularemia was suspected only after blood cultures from the surviving kidney recipient grew Francisella species. The organ donor, a middle-aged man from the southwestern United States, had been hospitalized for acute alcohol withdrawal syndrome, pneumonia, and multiorgan failure. F. tularensis subsp. tularensis (clade A2) was cultured from archived spleen tissue from the donor and blood from both kidney recipients. Whole-genome multilocus sequence typing indicated that the isolated strains were indistinguishable. The heart recipient remained seronegative with negative blood cultures but had been receiving antimicrobial drugs for a medical device infection before transplant. Two lagomorph carcasses collected near the donor's residence were positive by PCR for F. tularensis subsp. tularensis (clade A2). This investigation documents F. tularensis transmission by solid organ transplantation.

In July 2017, fever and sepsis developed in 3 recipients of solid organs (1 heart and 2 kidneys) from a common donor in the United States; 1 of the kidney recipients died. Tularemia was suspected only after blood cultures from the surviving kidney recipient grew Francisella species. The organ donor, a middle-aged man from the southwestern United States, had been hospitalized for acute alcohol withdrawal syndrome, pneumonia, and multiorgan failure. F. tularensis subsp. tularensis (clade A2) was cultured from archived spleen tissue from the donor and blood from both kidney recipients. Whole-genome multilocus sequence typing indicated that the isolated strains were indistinguishable. The heart recipient remained seronegative with negative blood cultures but had been receiving antimicrobial drugs for a medical device infection before transplant. Two lagomorph carcasses collected near the donor's residence were positive by PCR for F. tularensis subsp. tularensis (clade A2). This investigation documents F. tularensis transmission by solid organ transplantation. [ABSTRACT FROM AUTHOR]

Francisella tularensis is a highly virulent zoonotic pathogen that causes tularemia and, because of weaponization efforts in past world wars, is considered a tier 1 biothreat agent. Detection and surveillance of F. tularensis may be confounded by the presence of uncharacterized, closely related organisms. Through DNA-based diagnostics and environmental surveys, novel clinical and environmental Francisella isolates have been obtained in recent years. Here we present 7 new Francisella genomes and a comparison of their characteristics to each other and to 24 publicly available genomes as well as a comparative analysis of 16S rRNA and sdhA genes from over 90 Francisella strains. Delineation of new species in bacteria is challenging, especially when isolates having very close genomic characteristics exhibit different physiological features--for example, when some are virulent pathogens in humans and animals while others are nonpathogenic or are opportunistic pathogens. Species resolution within Francisella varies with analyses of single genes, multiple gene or protein sets, or whole-genome comparisons of nucleic acid and amino acid sequences. Analyses focusing on single genes (16S rRNA, sdhA), multiple gene sets (virulence genes, lipopolysaccharide [LPS] biosynthesis genes, pathogenicity island), and whole-genome comparisons (nucleotide and protein) gave congruent results, but with different levels of discrimination confidence. We designate four new species within the genus; Francisella opportunistica sp. nov. (MA06-7296), Francisella salina sp. nov. (TX07-7308), Francisella uliginis sp. nov. (TX07-7310), and Francisella frigiditurris sp. nov. (CA97-1460). This study provides a robust comparative framework to discern species and virulence features of newly detected Francisella bacteria. [ABSTRACT FROM AUTHOR]

Background The use of prototypic strains is common among laboratories studying infectious agents as it promotes consistency for data comparability among and between laboratories. Schu S4 is the prototypic virulent strain of Francisella tularensis and has been used extensively as such over the past six decades. Studies have demonstrated virulence differences among the two clinically relevant subspecies of F. tularensis, tularensis (type A) and holarctica (type B) and more recently between type A subpopulations (A1a, A1b and A2). Schu S4 belongs to the most virulent subspecies of F. tularensis, subspecies tularensis. Methods In this study, we investigated the relative virulence of Schu S4 in comparison to A1a, A1b, A2 and type B strains using a temperature-based murine model of infection. Mice were inoculated intradermally and a hypothermic drop point was used as a surrogate for death. Survival curves and the length of temperature phases were compared for all infections. Bacterial burdens were also compared between the most virulent type A subpopulation, A1b, and Schu S4 at drop point. Results Survival curve comparisons demonstrate that the Schu S4 strain used in this study resembles the virulence of type B strains, and is significantly less virulent than all other type A (A1a, A1b and A2) strains tested. Additionally, when bacterial burdens were compared between mice infected with Schu S4 or MA00-2987 (A1b) significantly higher burdens were present in the blood and spleen of mice infected with MA00-2987. Conclusions The knowledge gained from using Schu S4 as a prototypic virulent strain has unquestionably advanced the field of tularemia research. The findings of this study, however, indicate that careful consideration of F. tularensis strain selection must occur when the overall virulence of the strain used could impact the outcome and interpretation of results. [ABSTRACT FROM AUTHOR]

Background: Francisella tularensis is an intracellular pathogen that causes tularemia in humans and the public health importance of this bacterium has been well documented in recent history. Francisella philomiragia, a distant relative of F. tularensis, is thought to constitute an environmental lineage along with Francisella novicida. Nevertheless, both F. philomiragia and F. novicida have been associated with human disease, primarily in immune-compromised individuals. To understand the genetic relationships and evolutionary contexts among different lineages within the genus Francisella, the genome of Francisella spp. strain TX07-7308 was sequenced and compared to the genomes of F. philomiragia strains ATCC 25017 and 25015, F. novicida strain U112, and F. tularensis strain Schu S4. Results: The size of strain ATCC 25017 chromosome was 2,045,775 bp and contained 1,983 protein-coding genes. The size of strain TX07-7308 chromosome was 2,035,931 bp and contained 1,980 protein-coding genes. Pairwise BLAST comparisons indicated that strains TX07-7308 and ATCC 25017 contained 1,700 protein coding genes in common. NUCmer analyses revealed that the chromosomes of strains TX07-7308 and ATCC 25017 were mostly collinear except for a few gaps, translocations, and/or inversions. Using the genome sequence data and comparative analyses with other members of the genus Francisella (e.g., F. novicida strain U112 and F. tularensis strain Schu S4), several strain-specific genes were identified. Strains TX07-7308 and ATCC 25017 contained an operon with six open reading frames encoding proteins related to enzymes involved in thiamine biosynthesis that was absent in F. novicida strain U112 and F. tularensis strain Schu S4. Strain ATCC 25017 contained an operon putatively involved in lactose metabolism that was absent in strain TX07-7308, F. novicida strain U112, and F. tularensis strain Schu S4. In contrast, strain TX07-7308 contained an operon putatively involved in glucuronate metabolism that was absent in the genomes of strain ATCC 25017, F. novicida strain U112, and F. tularensis strain Schu S4. The polymorphic nature of polysaccharide biosynthesis/modification gene clusters among different Francisella strains was also evident from genome analyses. Conclusions: From genome comparisons, it appeared that genes encoding novel functions have contributed to the metabolic enrichment of the environmental lineages within the genus Francisella. The inability to acquire new genes coupled with the loss of ancestral traits and the consequent reductive evolution may be a cause for, as well as an effect of, niche selection of F. tularensis. Sequencing and comparison of the genomes of more isolates are required to obtain further insights into the ecology and evolution of different species within the genus Francisella. [ABSTRACT FROM AUTHOR]

Tularemia in the United States is caused by 2 subspecies of Francisella tularensis, subspecies tularensis (type A) and subspecies holarctica (type B). We compared clinical and demographic features of human tularemia cases from 1964 to 2004 from 39 states in which an isolate was recovered and subtyped. Our data indicate that type A and type B infections differ with respect to affected populations, anatomic site of isolation, and geographic distribution. Molecular subtyping with pulsed-field gel electrophoresis further defined 2 subpopulations of type A (type A-east and type A-west) that differ with respect to geographic distribution, disease outcome, and transmission. Our data suggest that type A-west infections are less severe than either type B or type A-east infections. Through a combined epidemiologic and molecular approach to human cases of tularemia, we provide new insights into the disease for future investigation. [ABSTRACT FROM AUTHOR]

In July 2017, fever and sepsis developed in 3 recipients of solid organs (1 heart and 2 kidneys) from a common donor in the United States; 1 of the kidney recipients died. Tularemia was suspected only after blood cultures from the surviving kidney recipient grew Francisella species. The organ donor, a middle-aged man from the southwestern United States, had been hospitalized for acute alcohol withdrawal syndrome, pneumonia, and multiorgan failure. F. tularensis subsp. tularensis (clade A2) was cultured from archived spleen tissue from the donor and blood from both kidney recipients. Whole-genome multilocus sequence typing indicated that the isolated strains were indistinguishable. The heart recipient remained seronegative with negative blood cultures but had been receiving antimicrobial drugs for a medical device infection before transplant. Two lagomorph carcasses collected near the donor's residence were positive by PCR for F. tularensis subsp. tularensis (clade A2). This investigation documents F. tularensis transmission by solid organ transplantation. [ABSTRACT FROM AUTHOR]