Successful treatment of glandular tularemia with azithromycin in a pregnant woman in Austria.

Treatment of tularemia during pregnancy is challenging due to toxicity of standard treatment regimens. Here, we report a 31-year-old woman with glandular tularemia who was successfully treated with intravenous azithromycin. Follow-up examinations over a 6-month period showed a sustained response to treatment. She later gave birth to a healthy child.

Type VI Secretion System and Its Effectors PdpC, PdpD, and OpiA Contribute to Francisella Virulence in Galleria mellonella Larvae.

Francisella tularensis causes the deadly zoonotic disease tularemia in humans and is able to infect a broad range of organisms including arthropods, which are thought to play a major role in Francisella transmission. However, while mammalian in vitro and in vivo infection models are widely used to investigate Francisella pathogenicity, a detailed characterization of the major Francisella virulence factor, a noncanonical type VI secretion system (T6SS), in an arthropod in vivo infection model is missing. Here, we use Galleria mellonella larvae to analyze the role of the Francisella T6SS and its corresponding effectors in F. tularensis subsp. novicida virulence. We report that G. mellonella larvae killing depends on the functional T6SS and infectious dose. In contrast to other mammalian in vivo infection models, even one of the T6SS effectors PdpC, PdpD, or OpiA is sufficient to kill G. mellonella larvae, while sheath recycling by ClpB is dispensable. We further demonstrate that treatment by polyethylene glycol (PEG) activates Francisella T6SS in liquid culture and that this is independent of the response regulator PmrA. PEG-activated IglC secretion is dependent on T6SS structural component PdpB but independent of putative effectors PdpC, PdpD, AnmK, OpiB1, OpiB2, and OpiB3. The results of larvae infection and secretion assay suggest that AnmK, a putative T6SS component with unknown function, interferes with OpiA-mediated toxicity but not with general T6SS activity. We establish that the easy-to-use G. mellonella larvae infection model provides new insights into the function of T6SS and pathogenesis of Francisella.

VOC fingerprints: metabolomic signatures of biothreat agents with and without antibiotic resistance.

Category A and B biothreat agents are deemed to be of great concern by the US Centers for Disease Control and Prevention (CDC) and include the bacteria Francisella tularensis, Yersinia pestis, Burkholderia mallei, and Brucella species. Underscored by the impact of the 2020 SARS-CoV-2 outbreak, 2016 Zika pandemic, 2014 Ebola outbreak, 2001 anthrax letter attacks, and 1984 Rajneeshee Salmonella attacks, the threat of future epidemics/pandemics and/or terrorist/criminal use of pathogenic organisms warrants continued exploration and development of both classic and alternative methods of detecting biothreat agents. Volatile organic compounds (VOCs) comprise a large and highly diverse group of carbon-based molecules, generally related by their volatility at ambient temperature. Recently, the diagnostic potential of VOCs has been realized, as correlations between the microbial VOC metabolome and specific bacterial pathogens have been identified. Herein, we describe the use of microbial VOC profiles as fingerprints for the identification of biothreat-relevant microbes, and for differentiating between a kanamycin susceptible and resistant strain. Additionally, we demonstrate microbial VOC profiling using a rapid-throughput VOC metabolomics method we refer to as 'simultaneous multifiber headspace solid-phase microextraction' (simulti-hSPME). Finally, through VOC analysis, we illustrate a rapid non-invasive approach to the diagnosis of BALB/c mice infected with either F. tularensis SCHU S4 or Y. pestis CO92.

Interferon Gamma Reprograms Host Mitochondrial Metabolism through Inhibition of Complex II To Control Intracellular Bacterial Replication.

The mechanisms by which interferon gamma (IFN-γ) controls the replication of cytosolic pathogens independent of responses, such as the generation of reactive oxygen species/reactive nitrogen species (ROS/RNS), have not been fully elucidated. In the current study, we developed a model using Francisella tularensis, the causative agent of tularemia, in which pathways triggered by IFN-γ commonly associated with bacterial control were not required. Using this model, we demonstrated that IFN-γ-mediated production of itaconate and its ability to impair host mitochondrial function, independent of activity on the pathogen, were central for the restriction of bacterial replication in vitro and in vivo We then demonstrate that IFN-γ-driven itaconate production was dispensable, as directly targeting complex II using cell membrane-permeable metabolites also controlled infection. Together, these findings show that while reprogramming of mitochondrial metabolism is a key factor in IFN-γ control of intracellular bacteria, the development of antimicrobial strategies based on targeting host mitochondrial metabolism independent of this cytokine may be an effective therapeutic approach.

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

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

Francisella tularensis: FupA mutation contributes to fluoroquinolone resistance by increasing vesicle secretion and biofilm formation.

Francisella tularensis is the causative agent in tularemia for which the high prevalence of treatment failure and relapse is a major concern. Directed-evolution experiments revealed that acquisition of fluoroquinolone (FQ) resistance was linked to factors in addition to mutations in DNA gyrase. Here, using F. tularensis live vaccine strain (LVS) as a model, we demonstrated that FupA/B (Fer-Utilization Protein) expression is linked to FQ susceptibility, and that the virulent strain F. tularensis subsp. tularensis SCHU S4 deleted for the homologous FupA protein exhibited even higher FQ resistance. In addition to an increased FQ minimal inhibitory concentration, LVSΔfupA/B displayed tolerance toward bactericidal compounds including ciprofloxacin and gentamicin. Interestingly, the FupA/B deletion was found to promote increased secretion of outer membrane vesicles (OMVs). Mass spectrometry-based quantitative proteomic characterization of vesicles from LVS and LVS∆fupA/B identified 801 proteins, including a subset of 23 proteins exhibiting differential abundance between both strains which may therefore contribute to the reduced antibiotic susceptibility of the FupA/B-deleted strain. We also demonstrated that OMVs are key structural elements of LVSΔfupA/B biofilms providing protection against FQ. These results provide a new basis for understanding and tackling antibiotic resistance and/or persistence of Francisella and other pathogenic members of the Thiotrichales class.

Disulfiram, an alcohol dependence therapy, can inhibit the in vitro growth of Francisella tularensis.

Disulfiram (DSF) can help treat alcohol dependency by inhibiting aldehyde dehydrogenase (ALDH). Genomic analysis revealed that Francisella tularensis, the causative agent of tularemia, has lost all but one ALDH-like domain and that this domain retains the target of DSF. In this study, minimum inhibitory concentration (MIC) assays demonstrated that both DSF and its primary metabolite diethyldithiocarbamate (DDC) have strong antimicrobial activity against F. tularensis strain SCHU S4, with the MIC of DSF determined as 2 µg/mL in comparison with 8 µg/mL for DDC. The activity of DSF was further confirmed using an in vitro human macrophage infection assay. Francisella tularensis bacteria in DSF-treated cells were reduced in comparison with untreated and DDC-treated cells, comparable with that observed in doxycycline-treated cells. This suggests that DSF may be suitable for further investigation as an in vivo therapy for tularemia.

Contributions of TolC Orthologs to Francisella tularensis Schu S4 Multidrug Resistance, Modulation of Host Cell Responses, and Virulence.

Francisella tularensis is a Gram-negative, facultative intracellular pathogen and the causative agent of tularemia. Previous studies with the attenuated live vaccine strain (LVS) identified a role for the outer membrane protein TolC in modulation of host cell responses during infection and virulence in the mouse model of tularemia. TolC is an integral part of efflux pumps that export small molecules and type I secretion systems that export a range of bacterial virulence factors. In this study, we analyzed TolC and its two orthologs, FtlC and SilC, present in the fully virulent F. tularensis Schu S4 strain for their contributions to multidrug efflux, suppression of innate immune responses, and virulence. We found that each TolC ortholog participated in multidrug efflux, with overlapping substrate specificities for TolC and FtlC and a distinct substrate profile for SilC. In contrast to their shared roles in drug efflux, only TolC functioned in the modulation of macrophage apoptotic and proinflammatory responses to Schu S4 infection, consistent with a role in virulence factor delivery to host cells. In agreement with previous results with the LVS, the Schu S4 ΔtolC mutant was highly attenuated for virulence in mice by both the intranasal and intradermal routes of infection. Unexpectedly, FtlC was also critical for Schu S4 virulence, but only by the intradermal route. Our data demonstrate a conserved and critical role for TolC in modulation of host immune responses and Francisella virulence and also highlight strain- and route-dependent differences in the pathogenesis of tularemia.

Nanoparticle Formulation of Moxifloxacin and Intramuscular Route of Delivery Improve Antibiotic Pharmacokinetics and Treatment of Pneumonic Tularemia in a Mouse Model.

Francisella tularensis causes a serious and often fatal infection, tularemia. We compared the efficacy of moxifloxacin formulated as free drug vs disulfide snap-top mesoporous silica nanoparticles (MSNs) in a mouse model of pneumonic tularemia. We found that MSN-formulated moxifloxacin was more effective than free drug and that the intramuscular and subcutaneous routes were markedly more effective than the intravenous route. Measurement of tissue silica levels and fluorescent flow cytometry assessment of colocalization of MSNs with infected cells revealed that the enhanced efficacy of MSNs and the intramuscular route of delivery was not due to better delivery of MSNs to infected tissues or cells. However, moxifloxacin blood levels demonstrated that the nanoparticle formulation and intramuscular route provided the longest half-life and longest time above the minimal inhibitory concentration. Thus, improved pharmacokinetics are responsible for the greater efficacy of nanoparticle formulation and intramuscular delivery compared with free drug and intravenous delivery.

Polymer-augmented liposomes enhancing antibiotic delivery against intracellular infections.

Pulmonary intracellular infections, such as tuberculosis, anthrax, and tularemia, have remained a significant challenge to conventional antibiotic therapy. Ineffective antibiotic treatment of these infections can lead not only to undesired side effects, but also to the emergence of antibiotic resistance. Aminoglycosides (e.g., streptomycin) have long been part of the therapeutic regiment for many pulmonary intracellular infections. Their bioavailability for intracellular bacterial pools, however, is limited by poor membrane permeability and rapid elimination. To address this challenge, polymer-augmented liposomes (PALs) were developed to provide improved cytosolic delivery of streptomycin to alveolar macrophages, an important host cell for intracellular pathogens. A multifunctional diblock copolymer was engineered to functionalize PALs with carbohydrate-mediated targeting, pH-responsive drug release, and endosomal release activity with a single functional polymer that replaces the pegylated lipid component to simplify the liposome formulation. The pH-sensing functionality enabled PALs to provide enhanced release of streptomycin under endosomal pH conditions (70% release in 6 hours) with limited release at physiological pH 7.4 (16%). The membrane-destabilizing activity connected to endosomal release was characterized in a hemolysis assay and PALs displayed a sharp pH profile across the endosomal pH development target range. The direct connection of this membrane-destabilizing pH profile to model drug release was demonstrated in an established pyranine/p-xylene bispyridinium dibromide (DPX) fluorescence dequenching assay. PALs displayed similar sharp pH-responsive release, whereas PEGylated control liposomes did not, and similar profiles were then shown for streptomycin release. The mannose-targeting capability of the PALs was also demonstrated with 2.5 times higher internalization compared to non-targeted PEGylated liposomes. Finally, the streptomycin-loaded PALs were shown to have a significantly improved intracellular antibacterial activity in a Francisella-macrophage co-culture model, compared with free streptomycin or streptomycin delivered by control PEGylated liposomes (13× and 16×, respectively). This study suggests the potential of PALs as a useful platform to deliver antibiotics for the treatment of intracellular macrophage infections.

Newly emerging ulceroglandular tularaemia in Western Austria.

Tularaemia is a rare zoonotic disease caused by Francisella tularensis in humans. In Europe infections of humans and animals are mainly caused by F. tularensis subspecies holarctica. We report the first three documented cases of tularaemia in humans in Western Austria.

Determination of absolute configuration and binding efficacy of benzimidazole-based FabI inhibitors through the support of electronic circular dichroism and MM-GBSA techniques.

We have previously reported benzimidazole-based compounds to be potent inhibitors of FabI for Francisella tularensis (FtFabI), making them promising antimicrobial hits. Optically active enantiomers exhibit markedly differing affinities toward FtFabI. The IC50 of benzimidazole (-)-1 is ∼100× lower than the (+)-enantiomer, with similar results for the 2 enantiomers. Determining the absolute configuration for these optical compounds and elucidating their binding modes is important for further design. Electronic circular dichroism (ECD) quantum calculations have become important in determining absolute configurations of optical compounds. We determined the absolute configuration of (-)/(+)-1 and (-)/(+)-2 by comparing experimental spectra and theoretical density functional theory (DFT) simulations of ECD spectra at the B3LYP/6-311+G(2d, p) level using Gaussian09. Comparison of experimental and calculated ECD spectra indicates that the S configuration corresponds to the (-)-rotation for both compounds 1 and 2, while the R configuration corresponds to the (+)-rotation. Further, molecular dynamics simulations and MM-GBSA binding energy calculations for these two pairs of enantiomers with FtFabI show much tighter binding MM-GBSA free energies for S-1 and S-2 than for their enantiomers, R-1 and R-2, consistent with the S configuration being the more active one, and with the ECD determination of the S configuration corresponding to (-) and the R configuration corresponding to (+). Thus, our computational studies allow us to assign (-) to (S)- and (+) to (R)- for compounds 1 and 2, and to further evaluate structural changes to improve efficacy.

Francisella tularensis D-Ala D-Ala Carboxypeptidase DacD Is Involved in Intracellular Replication and It Is Necessary for Bacterial Cell Wall Integrity.

D-alanyl-D-alanine carboxypeptidase, product of dacD gene in Francisella, belongs to penicillin binding proteins (PBPs) and is involved in remodeling of newly synthetized peptidoglycan. In E. coli, PBPs are synthetized in various growth phases and they are able to substitute each other to a certain extent. The DacD protein was found to be accumulated in fraction enriched in membrane proteins from severely attenuated dsbA deletion mutant strain. It has been presumed that the DsbA is not a virulence factor by itself but that its substrates, whose correct folding and topology are dependent on the DsbA oxidoreductase and/or isomerase activities, are the primary virulence factors. Here we demonstrate that Francisella DacD is required for intracellular replication and virulence in mice. The dacD insertion mutant strain showed higher sensitivity to acidic pH, high temperature and high osmolarity when compared to the wild-type. Eventually, transmission electron microscopy revealed differences in mutant bacteria in both the size and defects in outer membrane underlying its SDS and serum sensitivity. Taken together these results suggest DacD plays an important role in Francisella pathogenicity.

Zinc Acquisition Mechanisms Differ between Environmental and Virulent Francisella Species.

Zinc is an essential nutrient for bacterial growth. Because host cells can restrict pathogen access to zinc as an antimicrobial defense mechanism, intracellular pathogens such as Francisella must sense their environment and acquire zinc in response. In many bacteria, the conserved transcription factor Zur is a key regulator of zinc acquisition. To identify mechanisms of zinc uptake in Francisella novicida U112, transcriptome sequencing of wild-type and putative zur mutant bacteria was performed. Only three genes were confirmed as directly regulated by Zur and zinc limitation by quantitative reverse transcription-PCR. One of these genes, FTN_0879, is predicted to encode a protein with similarity to the zupT family of zinc transporters, which are not typically regulated by Zur. While a putative znuACB operon encoding a high-affinity zinc transporter was identified in U112, expression of this operon was not controlled by Zur or zinc concentration. Disruption of zupT but not znuA in U112 impaired growth under zinc limitation, suggesting that ZupT is the primary mechanism of zinc acquisition under these conditions. In the virulent Francisella tularensis subsp. tularensis Schu S4 strain, zupT is a pseudogene, and attempts to delete znuA were unsuccessful, suggesting that it is essential in this strain. A reverse TetR repression system was used to knock down the expression of znuA in Schu S4, revealing that znuA is required for growth under zinc limitation and contributes to intracellular growth within macrophages. Overall, this work identifies genes necessary for adaptation to zinc limitation and highlights nutritional differences between environmental and virulent Francisella strains.IMPORTANCEFrancisella tularensis is a tier 1 select agent with a high potential for lethality and no approved vaccine. A better understanding of Francisella virulence factors is required for the development of therapeutics. While acquisition of zinc has been shown to be required for the virulence of numerous intracellular pathogens, zinc uptake has not been characterized in Francisella This work characterizes the Zur regulon in F. novicida and identifies two transporters that contribute to bacterial growth under zinc limitation. In addition, these data identify differences in mechanisms of zinc uptake and tolerance to zinc limitation between F. tularensis and F. novicida, highlighting the role of znuA in the growth of Schu S4 under zinc limitation.

Antibiotic susceptibility of Francisella tularensis subsp. holarctica strains isolated from tularaemia patients in France between 2006 and 2016.

Objectives: To determine the in vitro susceptibility to 18 antibiotics of human strains of Francisella tularensis isolated in France between 2006 and 2016, to check the absence of acquired resistance and to evaluate potential therapeutic alternatives. Methods: Fifty-nine clinically unrelated F. tularensis subsp. holarctica strains identified at the French National Reference Centre for Francisella as belonging to the phylogenetic subclade B.FTNF002-00 were used. MICs were determined in CAMHB medium supplemented with 2% PolyViteX®, using the CLSI broth microdilution method. Results: All strains were susceptible to fluoroquinolones (ofloxacin, ciprofloxacin, levofloxacin and moxifloxacin; MIC range: 0.016-0.25 mg/L), aminoglycosides (gentamicin and tobramycin; MIC range: ≤0.03-0.25 mg/L), doxycycline (MIC range: 0.125-0.25 mg/L) and chloramphenicol (MIC range: 0.5-2 mg/L). The erythromycin MIC range (0.5-2 mg/L) confirmed that all isolates belonged to biovar I of F. tularensis subsp. holarctica. Azithromycin and telithromycin displayed lower MIC ranges (0.25-1 and 0.03-0.5 mg/L, respectively). The tigecycline MIC range (0.25-1 mg/L) was slightly higher than that of doxycycline. All strains were resistant to ampicillin, meropenem, daptomycin, clindamycin and linezolid. Conclusions: F. tularensis strains isolated in France remain susceptible to antibiotic classes recommended for tularaemia treatment. Because fluoroquinolones display the lowest MIC90, have bactericidal activity and have lower therapeutic failure rates compared with doxycycline, they may be advocated as first-line treatment of mild cases of tularaemia, predominant in Europe. MIC data also indicate that azithromycin or telithromycin may be possible therapeutic options against biovar I strains from Western Europe in case of contraindication to first-line antibiotics.

Benzoxazoles, Phthalazinones, and Arylurea-Based Compounds with IMP Dehydrogenase-Independent Antibacterial Activity against Francisella tularensis.

Francisella tularensis is the causative agent of tularemia and a potential biowarfare agent. The virulence of F. tularensis is decreased by deletion of guaB, the gene encoding IMP dehydrogenase (IMPDH), suggesting that this enzyme is a target for antibacterial design. Here we report that F. tularensis growth is blocked by inhibitors of bacterial IMPDHs. Seventeen compounds from two different frameworks, designated the D and Q series, display antibacterial activities with MICs of <1 µM. These compounds are also active against intracellular infections. Surprisingly, antibacterial activity does not correlate with IMPDH inhibition. In addition, the presence of guanine does not affect the antibacterial activity of most compounds, nor does the deletion of guaB These observations suggest that antibacterial activity derives from inhibition of another target(s). Moreover, D compounds display antibacterial activity only against F. tularensis, suggesting the presence of a unique target or uptake mechanism. A ΔguaB mutant resistant to compound D73 contained a missense mutation (Gly45Cys) in nuoB, which encodes a subunit of bacterial complex I. Overexpression of the nuoB mutant conferred resistance to D73 in both wild-type and ΔguaB strains. This strain was not resistant to Q compounds, suggesting that a different off-target mechanism operates for these compounds. Several Q compounds are also effective against Mycobacterium tuberculosis, in which a second target has also been implicated, in addition to IMPDH. The fortuitous presence of multiple targets with overlapping structure-activity relationships presents an intriguing opportunity for the development of robust antibiotics that may avoid the emergence of resistance.

In vitro and in vivo evaluation of fluoroquinolone resistance associated with DNA gyrase mutations in Francisella tularensis, including in tularaemia patients with treatment failure.

Fluoroquinolones (FQs) are highly effective for treating tularaemia, a zoonosis caused by Francisella tularensis, but failures and relapses remain common in patients with treatment delay or immunocompromised status. FQ-resistant strains of F. tularensis harboring mutations in the quinolone-resistance determining region (QRDR) of gyrA and gyrB, the genes encoding subunits A and B of DNA gyrase, have been selected in vitro. Such mutants have never been isolated from humans as this microorganism is difficult to culture. In this study, the presence of FQ-resistant mutants of F. tularensis was assessed in tularaemia patients using combined culture- and PCR-based approaches. We analyzed 42 F. tularensis strains and 82 tissue samples collected from 104 tularaemia cases, including 32 (30.7%) with FQ treatment failure or relapse. Forty F. tularensis strains and 55 clinical samples were obtained before any FQ treatment, while 2 strains and 15 tissue samples were collected after treatment. FQ resistance was evaluated by the minimum inhibitory concentration (MIC) for the bacterial strains, and by newly developed PCR-based methods targeting the gyrA and gyrB QRDRs for both the bacterial strains and the clinical samples. None of the F. tularensis strains displayed an increased MIC compared with FQ-susceptible controls. Neither gyrA nor gyrB QRDR mutation was found in bacterial strains and tissue samples tested, including those from patients with FQ treatment failure or relapse. Further phenotypic and genetic resistance traits should be explored to explain the poor clinical response to FQ treatment in such tularaemia patients.

In Vitro Antibiotic Susceptibilities of Francisella tularensis Determined by Broth Microdilution following CLSI Methods.

In vitro susceptibilities for 47 antibiotics were determined in 30 genetic diverse strains of Francisella tularensis by the broth microdilution method following Clinical and Laboratory Standards Institute (CLSI) methods. The F. tularensis strains demonstrated susceptibility to aminoglycosides, fluoroquinolones, and tetracyclines. There was a distinct difference in macrolide susceptibilities between A and B type strains, as has been noted previously. The establishment and comparison of antibiotic susceptibilities of a diverse but specific set of F. tularensis strains by standardized methods and the establishment of population ranges and MIC50/90 values provide reference information for assessing new antibiotic agents and a baseline to monitor any future emergence of resistance, whether natural or intentional.

Pneumonic tularaemia: experience of 58 cases from 2000 to 2012 in Northern Finland.

BACKGROUND: Pneumonic tularaemia is less common clinical form of tularaemia compared with the ulceroglandular form, with only a limited number of case reports and case series in Europe. In Finland, Northern Ostrobothnia is an endemic area of tularaemia with occasional seasonal outbreaks. METHODS: In our study, a consecutive series of 58 pneumonic tularaemia cases diagnosed and treated in Oulu University Hospital in 2000-2012 were retrospectively analysed in terms of epidemiology, clinical course, and prognosis. RESULTS: The incidence of pneumonic tularaemia showed peaks in cycles of a few years and most cases were diagnosed in late summer or early autumn. Respiratory symptoms were absent in 47% of patients, and 7% had normal chest X-ray. The chest computed tomography (CT) was performed in 81% of patients, demonstrating variable findings associated with pneumonic tularaemia. Bronchoscopy was performed for 22 (38%) patients and four (18%) of these also proceeded into mediastinoscopy. Moreover, thoracoscopy was performed for one (2%) patient. Two (3%) patients were treated shortly in the intensive care unit (ICU) during their stay in hospital. No mortality was observed. CONCLUSIONS: Most cases of pneumonic tularaemia are diagnosed during the seasonal outbreaks. The lack of specific symptoms often complicates the diagnosis and leads to unnecessarily invasive examinations.