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Tularemia is caused by subspecies of Francisella tularensis and can manifest in a variety of disease states, with the pneumonic presentation resulting in the greatest mortality. Despite decades of research, there are no approved vaccines against F. tularensis in the United States. Traditional vaccination strategies, such as live-attenuated or subunit vaccines, are not favorable due to inadequate protection or safety concerns. Because of this, novel vaccination strategies are needed to combat tularemia. Here we discuss the current state of and challenges to the tularemia vaccine field and suggest novel vaccine approaches going forward that might be better suited for protecting against F. tularensis infection. [ABSTRACT FROM AUTHOR]

Adenopathies are a frequent cause of recourse in internal medicine. When histological analysis reveals the presence of granuloma, multiple infectious or non-infectious etiologies are considered. If diagnoses of lymphoma, sarcoidosis or tuberculosis are easily mentioned, tularemia should also be considered in the differential diagnosis. A 54-year-old patient had a fever at the evening with night sweats and a cough resistant to two lines of antibiotics. A thoraco-abdomino-pelvic CT scan revealed hilar and mediastinal adenopathies that appeared hypermetabolic with PET-TDM, as well as pulmonary nodules. A PCR performed on lymph node biopsy and serology allowed the diagnosis of tularemia. The evolution was favourable after antibiotic treatment. The association of fever, night sweats, altered general state and mediastinal adenopathies should be considered as a diagnosis of tularemia. Ganglionic biopsy, combined with molecular biology techniques and serology, can confirm the diagnosis. [ABSTRACT FROM AUTHOR]

Tularemia is a rare zoonotic disease caused by the two predominant subspecies of Francisella tularensis, namely subspecies tularensis and subspecies holarctica. The latter is less virulent than the former, is endemic in Europe, and usually has a mild disease course, although respiratory involvement and bacteraemia can occur. Tularemia in Belgium is rare, but the incidence seems to be increasing. It therefore seems prudent to raise awareness among clinicians for this potentially severe disease. We report the first case of pneumonic tularemia with bacteraemia from Belgium, and want to recommend including Francisella tularensis in the differential diagnosis of pneumonia when an unfavorable evolution is seen with standard treatment. [ABSTRACT FROM AUTHOR]

• Tularemia can be transmitted through the ingestion of contaminated grape products. • F. tularensis can be introduced into the food chain via mechanical harvesting of grapes and presumably infected rodents. • The clinical course of tularemia can be unexpectedly long and require long antibiotic treatment durations. Tularemia is caused by Francisella tularensis and can occasionally establish foodborne transmission. Patients were identified by active case detection through contact with the treating physicians and consent for publication was obtained. Clinical data were accumulated through a review of the patient charts. Serology, culture, and PCR methods were performed for confirmation of the diagnosis. A 46-year-old patient was hospitalised in the University Hospital Frankfurt (a tertiary care hospital) for pharyngitis and cervical lymphadenitis with abscess. A diagnosis of tularemia was made serologically, but treatment with ciprofloxacin initially failed. F. tularensis was detected in pus from the lymph node using a specific real-time PCR. The use of RD1 PCR led to the identification of the subspecies holarctica. Antibiotic therapy with high-dose ciprofloxacin and gentamicin was administered and was subsequently changed to ciprofloxacin and rifampicin. During a must-tasting, five other individuals became infected with tularemia by ingestion of contaminated must. All patients required treatment durations of more than 14 days. Mechanically harvested agricultural products, such as wine must, can be a source of infection, probably due to contamination with animal carcasses. The clinical course of tularemia can be complicated and prolonged and requires differentiated antibiotic treatment. [ABSTRACT FROM AUTHOR]

Abstract Alveolar macrophages resident in the lung are prominent phagocytic effector cells of the pulmonary innate immune response, and paradoxically, are attractive harbors for pathogens. Consequently, facultative intracellular bacteria, such as Francisella tularensis , can cause severe systemic disease and sepsis, with high morbidity and mortality associated with pulmonary infection. Current clinical treatment, which involves exhaustive oral or intravenous antibiotic therapy, has limitations such as systemic toxicity and off-target effects. Pulmonary administration represents a promising alternative to systemic dosing for delivering antibiotics directly to the lung. Here, we present synthesized mannosylated ciprofloxacin polymeric prodrugs for efficient pulmonary delivery, targeting, and subsequent internalization by alveolar macrophages. We demonstrate significant improvement in efficacy against intracellular infections in an otherwise uniformly lethal airborne Francisella murine model (F. novicida). When administered to the lungs of mice in a prophylactic regimen, the mannosylated ciprofloxacin polymeric prodrugs led to 50% survival. In a treatment regimen that was concurrent with infection, the survival of mice increased to 87.5%. Free ciprofloxacin antibiotic was ineffective in both cases. This significant difference in antibacterial efficacy demonstrates the impact of this delivery platform based on improved physiochemical, pharmacokinetic, and pharmacodynamic properties of ciprofloxacin administered via our glycan polymeric prodrug. This modular platform provides a route for overcoming the limitations of free drug and increasing efficacy in treatment of intracellular infection. Graphical abstract Image 1 [ABSTRACT FROM AUTHOR]

A β-class carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium Francisella tularensis (FtuβCA) was cloned and purified, and the anion inhibition profile was investigated. Based on the measured kinetic parameters for the enzyme catalyzed CO 2 hydration reaction (k cat of 9.8 × 10 5 s −1 and a k cat /K M of 8.9 × 10 7 M −1 s −1 ), FtuβCA is a highly effective enzyme. The activity of FtuβCA was not inhibited by a range of anions that do not typically coordinate Zn(II) effectively, including perchlorate, tetrafluoroborate, and hexafluorophosphate. Surprisingly, some anions which generally complex well with many cations, including Zn(II), also did not effectively inhibit FtuβCA, e.g., fluoride, cyanide, azide, nitrite, bisulphite, sulfate, tellurate, perrhenate, perrhuthenate, and peroxydisulfate. However, the most effective inhibitors were in the range of 90–94 µM (sulfamide, sulfamic acid, phenylarsonic and phenylboronic acid). N,N -Diethyldithiocarbamate (K I of 0.31 mM) was a moderately potent inhibitor. As Francisella tularensis is the causative agent of tularemia, the discovery of compounds that can interfere with the life cycle of this pathogen may result in novel opportunities to fight antibiotic drug resistance. [ABSTRACT FROM AUTHOR]

Background Tularemia is caused by Francisella tularensis , a gram-negative bacterium that has been weaponized as an aerosol. For protection of personnel conducting biodefense research, the United States Army required clinical evaluation of a new lot of tularemia live vaccine strain manufactured in accordance with Current Good Manufacturing Practices. Methods A phase 2 randomized clinical trial compared the new lot (DVC-LVS) to the existing vaccine that has been in use for decades (USAMRIID-LVS). The vaccines were delivered by scarification to 228 participants. Safety, reactogenicity, take and/or antibody levels were assessed on days 0, 1, 2, 8, 14, 28, 56, and 180. Principal Results Both vaccines were safe and had acceptable reactogenicity profiles during six months of follow-up. There were no serious or grade 3 and 4 laboratory adverse events. Moderate systemic reactogenicity (mostly headache or feeling tired) was reported by ∼23% of participants receiving either vaccine. Injection site reactogenicity was mostly mild itchiness and pain. The frequencies of vaccine take skin reactions were 73% (95% CI, 64, 81) for DVC-LVS and 80% (95% CI, 71, 87) for USAMRIID-LVS. The 90% CI for the difference in proportions was −6.9% (−16.4, 2.6). The rates of seroconversion measured by microagglutination assay on days 28 or 56 were 94% (95% CI, 88, 98; n = 98/104) for DVC-LVS and 94% (95% CI, 87, 97; n = 103/110) for USAMRIID-LVS (p = 1.00). Day 14 sera revealed more rapid seroconversion for DVC-LVS relative to USAMRIID-LVS: 82% (95% CI, 73, 89) versus 55% (95% CI, 45, 65), respectively (p < 0.0001). Major conclusions The DVC-LVS vaccine had similar safety, reactogenicity, take and antibody responses compared to the older USAMRIID vaccine, and was superior for early (day 14) antibody production. Vaccination take was not a sensitive surrogate for seroconversion in a multi-center study where personnel at five research clinics performed assessments. ClinicalTrials.gov identifier NCT01150695 [ABSTRACT FROM AUTHOR]

Tularemia is a rare zoonotic disease in Germany. Francisella tularensis has been isolated previously from ticks in southern Germany underscoring the importance of ticks ( Ixodes ricinus ) in tularemia transmission, but there have been only few reports from this region with single cases or small case series of tick-borne transmissions of tularemia. We report five cases of non-game animal associated tularemia diagnosed from 2010 to 2016 in southwestern Germany – Baden-Wuerttemberg. Our case series and molecular typing (MLVA) results add published clinical experience to this underdiagnosed disease and consolidate previous findings regarding tick-borne transmission of tularemia and phylogenetic diversity in Germany. [ABSTRACT FROM AUTHOR]

A new β-class carbonic anhydrase (CA, EC 4.2.1.1) has been cloned, purified and characterized in the genome of the pathogenic bacterium Francisella tularensis responsible of the febrile illness tularemia. This enzyme, FtuβCA, showed a k cat of 9.8 × 10 5 s −1 and a k cat /K M of 8.9 × 10 7 M −1 s −1 for the CO 2 hydration, physiological reaction, being one of the most effective β-CAs known to date, with a catalytic activity only 1.68-times lower than that of the human(h) isoform hCA II. A panel of 39 simple aromatic and heterocyclic sulfonamides, as well as clinically used drugs incorporating sulfonamide/sulfamate zinc-binding groups, was used to investigate the inhibition profile of FtuβCA with these classes of derivatives. The enzyme generally showed a weaker affinity for these inhibitors compared to other α- and β-CAs investigated earlier, with only acetazolamide and its deacetylated precursor having inhibition constant <1 µM. Indeed, the two compounds acetazolamide AAZ and its deacetylated precursor 13 (K I s of 655–770 nM), as well as metanilamide and methazolamide (K I s of 2.53–2.92 µM), were the best FtuβCA inhibitors detected so far. As the physiological role of bacterial β-CAs is poorly understood for the virulence/life cycle of these pathogens, the present study may constitute a starting point for the design of effective pathogenic bacteria CA inhibitors with potential use as antiinfectives. [ABSTRACT FROM AUTHOR]

Francisella tularensis subsp. tularensis strain SchuS4 (Ftt) is a highly virulent intracellular bacterium. Inhalation of 10 or fewer organisms results in an acute and potentially lethal disease called pneumonic tularemia. Ftt infections occur naturally in the U.S. and Ftt was developed as a bioweapon. Thus, there is a need for vaccines that protect against this deadly pathogen. Although a live vaccine strain of Francisella tularensis (LVS) exists, LVS fails to generate long-lived protective immunity against modest challenge doses of Ftt. We recently identified an important role for high avidity CD4 + T cells in short-term protection and hypothesized that expanding this pool of cells would improve overall vaccine efficacy with regard to longevity and challenge dose. In support of our hypothesis, application of a prime/boost vaccination strategy increased the pool of high avidity CD4 + T cells which correlated with improved survival following challenge with either increased doses of virulent Ftt or at late time points after vaccination. In summary, we demonstrate that both epitope selection and vaccination strategies that expand antigen-specific T cells correlate with superior immunity to Ftt as measured by survival. [ABSTRACT FROM AUTHOR]

Tularemia or rabbit fever is a transmissible disease from animals, rodents, and insects to human populations that is caused by Francisella tularensis. Epidemiological studies showed that tularemia is endemic throughout most different regions of the world. Recent evidence documented the transmission of the F. tularensis in a different part of Asia. Because there is no updated review information for tularemia in Iran, we performed this systematic review. In this study, we systematically explored biomedical databases (Google Scholar, Scopus, PubMed, and Web of sciences) to identify epidemiology, reservoirs, and carriers of Francisella in animal and human clinical specimens from 2010 to 2020, either in English or in Persian. Different studies have shown the different frequencies of F. tularensis among human and animal resources in eighteen provinces of Iran. In total, 1242 human clinical specimens, 1565 animal samples, and 355 environmental water samples were investigated to find F. tularensis in different provinces of Iran. According to the collected documents, 94 human clinical samples, 69 water samples, and 26 animal specimens were introduced as positive samples for the F. tularensis. According to studies, thirteen species of rodent and hare presented as an inter-epizootic reservoir. Only one species of tick (D. marginatus) was introduced as a vector for Francisella in Iran. According to these results, it is essential for exclusive attention to the prevalence of F. tularensis in different provinces of Iran. Furthermore, special planning should be done for prevention, control of the outbreak, and proper treatment of the tularemia. [ABSTRACT FROM AUTHOR]

Francisella tularensis is the cause of the zoonotic disease tularemia. In Sweden and Scandinavia, epidemiological studies have implicated mosquitoes as a vector. Prior research has demonstrated the presence of Francisella DNA in infected mosquitoes but has not shown definitive transmission of tularemia from a mosquito to a mammalian host. We hypothesized that antimicrobial peptides, an important component of the innate immune system of higher organisms, may play a role in mosquito host-defense to Francisella . We established that Francisella sp. are susceptible to two cecropin antimicrobial peptides derived from the mosquito Aedes albopictus as well as Culex pipiens . We also demonstrated induced expression of Aedes albopictus antimicrobial peptide genes by Francisella infection C6/36 mosquito cell line. We demonstrate that mosquito antimicrobial peptides act against Francisella by disrupting the cellular membrane of the bacteria. Thus, it is possible that antimicrobial peptides may play a role in the inability of mosquitoes to establish an effective natural transmission of tularemia. [ABSTRACT FROM AUTHOR]

In Slovenia, a small Central European country, where tularemia cases are very rare and mostly sporadic, six cases of ulceroglandular tularemia were recognised in 2012–2013 in patients residing in or visiting a small geographical area of <6 km 2 . Epidemiological data indicated transmission by a tick bite in at least 3/6 patients. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Francisella tularensis ( Ft ) is a Category A biothreat agent for which there currently is no FDA-approved vaccine. Thus, there is a substantial effort underway to develop an effective tularemia vaccine. While it is well established that gender can significantly impact susceptibility to primary infection, the impact of gender on vaccine efficacy is not well established. Thus, development of a successful vaccine against tularemia will require an understanding of the impact gender has on vaccine-induced protection against this organism. In this study, a role for gender in vaccine-induced protection following Ft challenge is identified for the first time. In the present study, mucosal vaccination with inactivated Ft (i Ft ) LVS elicited gender-based protection in C57BL/6Tac mice against respiratory challenge with Ft LVS. Specifically, vaccinated male mice were more susceptible to subsequent Ft LVS challenge. This increased susceptibility in male mice correlated with increased bacterial burden, increased tissue inflammation, and increased proinflammatory cytokine production late in post-challenge infection. In contrast, improved survival of i Ft -vaccinated female mice correlated with reduced bacterial burden and enhanced levels of Ft -specific Abs in serum and broncho-alveolar lavage (BAL) fluid post-challenge. Furthermore, vaccination with a live attenuated vaccine consisting of an Ft LVS superoxide dismutase (SodB) mutant, which has proven efficacious against the highly virulent Ft SchuS4 strain, demonstrated similar gender bias in protection post- Ft SchuS4 challenge. Of particular significance is the fact that these are the first studies to demonstrate that gender differences impact disease outcome in the case of lethal respiratory tularemia following mucosal vaccination. In addition, these studies further emphasize the fact that gender differences must be a serious consideration in any future tularemia vaccine development studies. [ABSTRACT FROM AUTHOR]

Tularaemia is a zoonotic disease caused by Francisella tularensis, a Gram-negative, facultative intracellular bacterium. Typically, human and animal infections are caused by F tularensis subspecies tularensis (type A) strains mainly in Canada and USA, and F tularensis subspecies holarctica (type B) strains throughout the northern hemisphere, including Europe. In the past, the epidemiological, clinical, therapeutic, and prognostic aspects of tularaemia reported in the English medical literature were mainly those that had been reported in the USA, where the disease was first described. Tularaemia has markedly changed in the past decade, and a large number of studies have provided novel data for the disease characteristics in Europe. In this Review we aim to emphasise the specific and variable aspects of tularaemia in different European countries. In particular, two natural lifecycles of F tularensis have been described in this continent, although not fully characterised, which are associated with different modes of transmission, clinical features, and public health burdens of tularaemia. [ABSTRACT FROM AUTHOR]

Francisella tularensis is a human zoonotic pathogen and the causative agent of tularemia, a severe infectious disease. Given the extreme infectivity of F. tularensis and its potential to be used as a biological warfare agent, a fast and sensitive detection method is highly desirable. Herein, we construct a novel detection platform composed of two units: (1) Magnetic beads conjugated with multiple capturing antibodies against F. tularensis for its simple and rapid separation and (2) Genetically-engineered apoferritin protein constructs conjugated with multiple quantum dots and a detection antibody against F. tularensis for the amplification of signal. We demonstrate a 10-fold increase in the sensitivity relative to traditional lateral flow devices that utilize enzyme-based detection methods. We ultimately envision the use of our novel nanoprobe detection platform in future applications that require the highly-sensitive on-site detection of high-risk pathogens. [ABSTRACT FROM AUTHOR]

Tularemia is a zoonotic disease that has been reported in many countries of the Northern Hemisphere. However, in some countries, such as Iran, this disease has been neglected by the health care system, and it is under-reported. Here we report an unusual case of ulceroglandular tularemia occurring in a 35-year-old woman who presented with a skin lesion of the left flank, inguinal lymphadenopathy, and an abdominal abscess. The serological and real-time PCR tests for tularemia were positive for this patient, and infection by Francisella tularensis subsp. holarctica was confirmed. It is necessary to implement various educational programs to increase the awareness of physicians with tularemia. [ABSTRACT FROM AUTHOR]

Tularemia is a zoonotic disease caused by Francisella tularensis , which is transmitted to humans most commonly by contact with infected animals, tick bites, or inhalation of aerosolized bacteria. F. tularensis is highly infectious via the aerosol route; inhalation of as few as 10–50 organisms can cause pneumonic tularemia. Left untreated, the pneumonic form has more than >30% case-fatality rate but with early antibiotic intervention can be reduced to 3%. This study compared tularemia disease progression across three species of nonhuman primates [African green monkey (AGM), cynomolgus macaque (CM), and rhesus macaque (RM)] following aerosolized F. tularensis Schu S4 exposure. Groups of the animals exposed to various challenge doses were observed for clinical signs of infection and blood samples were analyzed to characterize the disease pathogenesis. Whereas the AGMs and CMs succumbed to disease following challenge doses of 40 and 32 colony forming units (CFU), respectively, the RM lethal dose was 276,667 CFU. Following all challenge doses that caused disease, the NHPs experienced weight loss, bacteremia, fever as early as 4 days post exposure, and tissue burden. Necrotizing-to-pyogranulomatous lesions were observed most commonly in the lung, lymph nodes, spleen, and bone marrow. Overall, the CM model consistently manifested pathological responses similar to those resulting from inhalation of F. tularensis in humans and thereby most closely emulates human tularemia disease. The RM model displayed a higher tolerance to infection and survived exposures of up to 15,593 CFU of aerosolized F. tularensis . [ABSTRACT FROM AUTHOR]

Tularemia is a bacterial zoonotic infection. The disease is endemic in most parts of the world, has been reported through the northern hemisphere between 30 and 71°N latitude. Francisella tularensis causes infection in a wide range of vertebrates (rodents, lagomorphs) and invertebrates (ticks, mosquitoes and other arthropods). Humans can acquire this infection through several routes including; a bite from an infected tick, deerfly or mosquito, contact with an infected animal or its dead body. It can also be spread to human by drinking contaminated water or breathing contaminated dirt or aerosol. Clinical manifestation of this disease varies depending on the biotype, inoculum and port of entry. Infection is potentially life threatening, but can effectively be treated with antibiotics. Travelers visiting rural and agricultural areas in endemic countries may be at greater risk. Appropriate clothing and use of insect repellants is essential to prevent tick borne illness. Travelers also should be aware of food and waterborne disease; avoid consuming potentially contaminated water and uncooked meat. Physicians should be aware of any clinical presentation of tularemia in the patients returning from endemic areas. [ABSTRACT FROM AUTHOR]

Francisella tularensis is the causative agent of tularemia an infectious zoonotic disease. We attempted the molecular detecting of F. tularensis in small ruminants and ticks attached to these animals in Kurdistan province (the west of Iran). In this study, 250 blood and 244 tick samples were collected from sheep and goats and were tested for F. tularensis IS Ftu2 gene detection using the Real Time-TaqMan PCR method. The collected ticks were morphologically classified as Dermacentor marginatus (67.2%), Rhipicephalus turanicus (12.30%), Rhipicephalus sanguineus (10.66%), and Haemaphysalis concinna (9.83%). No positive F. tularensis were identified in animal blood samples. F. tularensis was detected in 2 (0.82%) ticks samples. Positive samples were identified as F. tularensis subsp. holarctica and collected from D. marginatus ticks and Divandareh county. In this study, the presence of F. tularensis in ticks of Kurdistan province was confirmed, the possible role of ticks in the transmission to livestock and human through tick bites in this region should be considered. • F. tularensis was detected in 0.82% of small ruminant ticks in western Iran. • F. tularensis was detected in Dermacentor marginatus ticks. • Positive samples were identified as F. tularensis subsp. holarctica. • F. tularensis could be endemic in Iran. [ABSTRACT FROM AUTHOR]

Due to the high infectivity and fatal effect on human population, Francisella tularensis (F. tularensis) is classified as a potential biological warfare agent. The interaction between host and pathogen behind the successful establishment of F. tularensis infection within the human host is largely unknown. In our present work, we have studied the molecular level interactions between the host cellular components and F. tularensis genes to understand the interplay between the host and pathogen. Interestingly, we have identified the pathways associated with the pathogen offensive strategies that help in invasion of host defensive systems. The F. tularensis genes purL, katG, proS, rpoB and fusA have displayed high number of interactions with the host genes and thus play a crucial role in vital pathogen pathways. The pathways identified were involved in adaptation to different stress conditions within the host and might be crucial for designing new therapeutic interventions against tularemia. A total number of 33 whole genome sequences available for various F. tularensis strains were downloaded from NCBI database. The phylogenetic tree was constructed using ParSNP tool and visualized using iTOL tool. The representative strain F. tularensis SUH4 was highlighted in yellow color. (A) Phylogenetic tree in rooted tree layout, and (B) Circular tree view of phylogenetic tree. [Display omitted] • Phylogenetic tree of 33 whole genome sequences of Francisella strains constructed. • Gene interaction network between 377 host and 237 pathogen genes was constructed. • Clustering analysis of gene networks resulted densely inter connected gene clusters. • FEA has resulted with key pathways which play critical role in host and pathogen. • Important pathogen genes were identified and reported as new potential drug targets. [ABSTRACT FROM AUTHOR]

B Methods and materials: b Two searchers were conducted using: «tularemia outbreak» and «francisella tularensis outbreak» in title and/or abstract using PubMed database for articles that had been published until 01.10.2018. B Background: b Tularemia is a highly contagious, multi-systemic zoonotic disease caused by francisella tularensis that can give rise to outbreaks. Tularemia outbreaks can occur via several transmission ways; evidence shows that even though a certain group, for example one family, is infected with the same source, individuals can develop different forms of the disease according to the port of entry. [Extracted from the article]

In Europe, tularemia is caused by Francisella tularensis subsp. holarctica and is a sporadic disease affecting mainly wildlife animals and humans. Classification of this species relies on canonical single nucleotide polymorphisms (canSNPs). Four main clades have been described for F. tularensis subsp. holarctica : B.4, B.6, B.12 and B.16. Phylogeographic studies have shown that clade B.6 is predominant in Western Europe and B.12 in Eastern and Central Europe. Based on this global phylogeny, we aimed to design a molecular typing assay for all genetic subclades of subclade B.11, which is the predominant subclade in clade B.6. We designed high-resolution melting (HRM) primers for the screening of 109 canSNPs divided in seven orders of discrimination for the molecular epidemiology analysis and tracking of Francisella tularensis subsp. holarctica in Western Europe. • Canonical single nucleotide polymorphisms canSNPs are used to classify Francisella tularensis. • Discrimination assays designed for 113 markers identified in Western Europe. • It enables discrimination of Francisella tularensis isolates in Western Europe for epidemiological surveillance and outbreaks. [ABSTRACT FROM AUTHOR]

• There is some evidence about the circulation of F. tularensis in livestock and human populations in western Iran. • We tested the exposure to F. tularensis using the standard tube agglutination method, ELISA, and TaqMan real-time PCR. • Of the 51 surveyed human sera, two samples were positive and one sample had a borderline status. However, all animal sera and spleen samples were negative for tularemia. • Surface water, arthropod bite, and rodent activity are probable transmission routes of F. tularensis in human populations at these areas. Tularemia is a zoonotic disease that transmitted to humans and domestic animals by wildlife, especially rodents. There are some evidences of the circulation of F. tularensis in rodents, livestock, human populations, and surface waters in western parts of Iran. In this study, we investigated the exposure of livestock and ranchers to F. tularensis in the endemic regions of western Iran. Blood samples were collected from 289 sheep, 103 cattle, and 51 ranchers in 2018. Animal sera were tested by standard tube agglutination method. The specific IgGs against F. tularensis were evaluated by ELISA in human sera. Moreover, the extracted DNAs from 50 sheep spleen samples were evaluated using TaqMan real-time PCR for the presence of ISFtu2 and FopA genes. All animal sera and spleen samples were negative for tularemia. Of the 51 human samples, two samples were seropositive and one sample showed a borderline status for tularemia. Serologic evidence of F. tularensis in the ranchers but negative results in the livestock indicates different transmission routes in human populations and domestic animals in western Iran. Therefore, drinking contaminated water, contact to wildlife or rodents and arthropod bite should be considered as probable routes in the suspicious areas. [ABSTRACT FROM AUTHOR]

• Fluoroquinolone resistance in Francisella tularensis is rapidly selected in vitro • Mutations in all genes encoding type II topoisomerases are first selected • Mutations in FupA/B, a protein implicated in iron transport, are often combined • Mutations in genes coding for transport systems are also possibly involved • Fluoroquinolone resistance in F. tularensis involves complex mechanisms Objectives: Fluoroquinolone (FQ)-resistant mutants were previously selected from the live vaccine strain (LVS) of Francisella tularensis (F. tularensis) subsp. holarctica. This study further characterised all genetic changes that occurred in these mutants during the evolutionary trajectory toward high-level FQ resistance, and their potential impact on F. tularensis antibiotic resistance and intracellular fitness. Methods: The whole genomes of FQ-resistant mutants were determined and compared with those of their parental strain. All detected mutations were evaluated for their potential impact on FQ resistance and intracellular multiplication of F. tularensis. Results: As compared with the parental LVS genome, 28 mutations were found in the derived FQ-resistant mutants. These mutations involved all genes encoding type II topoisomerases (i.e. gyrA, gyrB, parC , and parE). Interestingly, some of them were not previously associated with FQ resistance, warranting further characterisation. Mutations associated with FQ resistance were also found in other genes, including three encoding proteins involved in transport processes. Most of the detected mutations did not alter multiplication of the corresponding mutants in J774 cells. In contrast, all mutations at locus FTL_0439 encoding FupA/B, a membrane protein involved in iron transport, were associated with FQ resistance and fitness loss. Conclusion: FQ resistance in F. tularensis is complex and may involve single or combined mutations in genes encoding type II topoisomerases, transport systems and FupA/B. In vivo studies are now required to assess the potential role of these mutations in FQ treatment failures. Image, graphical abstract [ABSTRACT FROM AUTHOR]

Francisella tularensis is the causative agent for the potentially fatal disease tularemia. The lipoprotein Flpp3 has been identified as a virulence determinant of tularemia with no sequence homology outside the Francisella genus. We report a room temperature structure of Flpp3 determined by serial femtosecond crystallography that exists in a significantly different conformation than previously described by the NMR-determined structure. Furthermore, we investigated the conformational space and energy barriers between these two structures by molecular dynamics umbrella sampling and identified three low-energy intermediate states, transitions between which readily occur at room temperature. We have also begun to investigate organic compounds in silico that may act as inhibitors to Flpp3. This work paves the road to developing targeted therapeutics against tularemia and aides in our understanding of the disease mechanisms of tularemia. • X-ray free-electron laser unveils alternative protein structure of lipoprotein • Advanced molecular dynamics explore conformational space among solved structures • Virtual ligand screening shows possible lead fragments for potential drug therapies Using X-ray free-electron laser (XFEL) technology, Zook et al. describe an alternative protein conformation of the virulence determinant Flpp3 and use molecular dynamics (MD) to investigate conformational landscapes between these conformations. MD identifies several intermediate conformations that allow these conformational transitions to occur at physiologically relevant temperatures and provide insight to potential future drug therapies for the potentially deadly disease tularemia. [ABSTRACT FROM AUTHOR]

• Isolation of F. tularensis from surface water specifies the disease cycle in the environment and help us to control the disease in the endemic areas. • Isolation of F. tularensis from surface water is associated with a very low success rate and depends on various environmental parameters. • Many considerations should be addressed to improve the chance of isolation of F. tularensis from environmental samples. The etiologic agent of tularemia, Francisella tularensis , is transmitted to humans via ingestion of contaminated water or food, arthropods bite, respiratory aerosols, or direct contact with infected animals body fluids or tissues. In the current study, due to the importance of water in transmitting the disease and the report of the disease in different regions of Iran, surface water of Kurdistan province were evaluated for the presence of F.tularensis. Sampling was carried out in five-counties of Kurdistan province. Sixty-six specimens of surface water were collected. The detection was carried out by targeting ISFtu2 and fopA genes using TaqMan real-time PCR. Moreover, the samples were both cultured and inoculated into NMRI inbreed mice. Spleens of inoculated mice and bacterial isolates were tested by TaqMan real-time PCR. Despite the lack of isolation of F. tularensis , the results of the molecular testing indicate the presence of bacteria in surface water. Molecular positivity of one sample (1.51%) was confirmed using a real-time PCR for both ISFtu2 and fopA genes. Moreover, 4.54% of the samples were positive for ISFtu2. Since the in vitro isolation of bacteria from environmental samples is associated with a very low success rate and depends on various environmental parameters, the use of molecular techniques for monitoring of the bacteria in the contaminated areas is fully recommended. [ABSTRACT FROM AUTHOR]

• The present study aimed to investigate the infection of rodents, as potential reservoirs of tularemia in Iran. • The most frequent trapped species belonged to Meriones persicus (75.5%). • The tube agglutination test was performed on rodent serum, and three out of 407 serum samples were positive (0.74%) for Francisella tularensis. • Five out of 433 (1.15%) extracted DNA samples were PCR positive (1.15%). • Considering that all the seropositive rodents belonged to jirds, which are widespread in the Iranian Plateau, the risk of human infections acquired from these animals is high in the studied region. Tularemia is a zoonotic disease, the most important hosts of which are rodents. Endemic regions and reservoirs of F. tularensis are not well-researched areas in Iran. The present study aimed to study F. tularensis infection in the rodent populations of western Iran. Samples were collected in different areas of Kabudar Ahang County in Hamadan province (west of Iran) from 2014 to 2017. Tularemia serological and molecular tests were conducted using the tube agglutination test and Real-time PCR method tracking the ISFtu2 gene. Positive serum samples were evaluated for cross-reactivity with brucellosis. A total of 433 rodents, collected from 33 localities, were included in the study. The most abundant species belonged to the Persian jird (Meriones persicus ; 75.5%), and Libyan jird (Meriones libycus ; 10.1%). Among the studied samples, three (0.74 %) were seropositive and five (1.15%) were PCR positive. Seropositive samples were two M. persicus and one M. libycus , and PCR positive rodents were four M. persicus and one M. vinogradovi. Tularemia seropositive samples showed no cross-reactivity with brucellosis. Given the presence of infection in rodents with tularemia agent in the studied area, it is crucial to elucidate the risks of rodent exposure to tularemia for physicians, health personnel and the general population. [ABSTRACT FROM AUTHOR]

Methods used to prepare bone marrow-derived macrophages (BMDMs) may influence the outcomes of immunological assays in which they are used. Supernatant conditioned by growth of L929 cells has often been used to generate mouse macrophages from bone marrow in vitro but is subject to lot-to-lot variability. To reduce experimental variability and to standardize techniques across laboratories, we investigated recombinant M-CSF (rM-CSF) as an alternative supplement for BMDM maturation in the context of macrophage infection, using the intracellular bacterium Live Vaccine Strain (LVS) of Francisella tularensis as a prototype. We compared rM-CSF with L929 supernatant in terms of their effects on mouse and rat macrophage growth, maturation patterns, surface marker expression, and the expression of selected genes. Further, we compared macrophage infectivity and bacterial replication using LVS. Finally, we compared the in vitro function of BMDMs co-cultured with splenocytes from vaccinated animals in terms of their control of intramacrophage bacterial replication, as well as production of cytokines and nitric oxide. We demonstrated that rM-CSF produced BMDMs with similar, or minimal, phenotypic and gene expression outcomes compared to those generated with media containing L929 supernatant. Most importantly, functional outcomes were similar. Taken together, our data support the use of the rM-CSF in cell culture media as an alternative to L929-supplemented media for functional bioassays that use C57BL/6J mouse or Fischer 344 rat BMDMs to study intracellular infections. This comparison therefore facilitates future protocol standardization. Unlabelled Image • L929 supernatants, used to differentiate bone marrow-derived macrophages, are variable. • We comprehensively compared mouse and rat macrophages grown with L929 or rM-CSF. • Macrophages were analyzed in the context of Francisella tularensis LVS infection. • rM-CSF supplementation resulted comparable but more consistent outcomes. • rM-CSF can therefore replace L929 supernatant during studies of intracellular bacteria. [ABSTRACT FROM AUTHOR]

Francisella tularensis, which causes potentially fatal tularemia, has been considered an attractive agent of bioterrorism and biological warfare due to its low infectious dose, reported environmental persistence, and ability to be transmitted to humans via multiple exposure routes. Due to slow growth on even selective culture media, detection of viable F. tularensis from environmental and drinking water samples, usually takes >3 days. Therefore, a rapid viability polymerase chain reaction (RV-PCR) method was developed to detect and identify viable F. tularensis cells in environmental samples. The method uses a change in PCR response during high throughput (48-well) sample incubation in Brain Heart Infusion/Vitox/Fildes/Histidine growth medium to detect viable F. tularensis presence, which is at least two times faster than the current plate culture-based method. Using the method, 101 to 102 live F. tularensis cells were detected in simulated complex sample matrices containing chemical and biological interferences. • Rapid detection of viable F. tularensis is critical during a high-consequence tularemia incident. • Pre- and post-enrichment differential PCR analysis-based method provides more timely results than culture-based methods. • Features include high-throughput sample processing, a small foot-print, reduced labor, and less biohazardous waste generated. [ABSTRACT FROM AUTHOR]

Francisella tularensis is the causative agent of the human disease referred to as tularemia. Other Francisella species are known but less is understood about their virulence factors. The role of environmental amoebae in the life-cycle of Francisella is still under discussion. Francisella sp. strain W12-1067 (F-W12) is an environmental Francisella isolate recently identified in Germany which is negative for the Francisella pathogenicity island, but exhibits a putative alternative type VI secretion system. Putative virulence factors have been identified in silico in the genome of F-W12. In this work, we established a "scatter screen", used earlier for pathogenic Legionella , to verify experimentally and identify candidate fitness factors using a transposon mutant bank of F-W12 and Acanthamoeba lenticulata as host organism. In these experiments, we identified 79 scatter clones (amoeba sensitive), which were further analyzed by an infection assay identifying 9 known virulence factors, but also candidate fitness factors of F-W12 not yet described as fitness factors in Francisella. The majority of the identified genes encoded proteins involved in the synthesis or maintenance of the cell envelope (LPS, outer membrane, capsule) or in the metabolism (glycolysis, gluconeogenesis, pentose phosphate pathway). Further 13C-flux analysis of the Tn5 glucokinase mutant strain revealed that the identified gene indeed encodes the sole active glucokinase in F-W12. In conclusion, candidate fitness factors of the new Francisella species F-W12 were identified using the scatter screen method which might also be usable for other Francisella species. [ABSTRACT FROM AUTHOR]