Tularemia: A rare cause of pediatric lymph nodes adenitis.

Adenopathy in pediatrics can have many different causes: infectious, tumoral, and inflammatory. We report the case of an 8-year-old patient with a febrile popliteal ulceration associated with an inflammatory satellite inguinal lymph node adenitis. Serological tests and polymerase chain reaction analyses confirmed the diagnosis of ulceroglandular tularemia. An appropriate antimicrobial therapy led to a full recovery. This case reminds us to consider tularemia as a potential emergent disease in children presenting with subacute to chronic lymphadenopathy and thereby to choose the correct diagnostic tool and appropriate antimicrobial therapy.

You're the Flight Surgeon.

Warneke J, Pavelites J. You're the flight surgeon: tularemia. Aerosp Med Hum Perform. 2020; 91(5):379-381.

Cardiac Electrical and Structural Changes During Bacterial Infection: An Instructive Model to Study Cardiac Dysfunction in Sepsis.

BACKGROUND: Sepsis patients with cardiac dysfunction have significantly higher mortality. Although several pathways are associated with myocardial damage in sepsis, the precise cause(s) remains unclear and treatment options are limited. This study was designed to develop a new model to investigate the early events of cardiac damage during sepsis progression. METHODS AND RESULTS: Francisella tularensis subspecies novicida (Ft.n) is a Gram-negative intracellular pathogen causing severe sepsis syndrome in mice. BALB/c mice (N=12) were sham treated or infected with Ft.n through the intranasal route. Serial electrocardiograms were recorded at multiple time points until 96 hours. Hearts were then harvested for histology and gene expression studies. Similar to septic patients, we illustrate both cardiac electrical and structural phenotypes in our murine Ft.n infection model, including prominent R' wave formation, prolonged QRS intervals, and significant left ventricular dysfunction. Notably, in infected animals, we detected numerous microlesions in the myocardium, previously observed following nosocomial Streptococcus infection and in sepsis patients. We show that Ft.n-mediated microlesions are attributed to cardiomyocyte apoptosis, increased immune cell infiltration, and expression of inflammatory mediators (tumor necrosis factor, interleukin [IL]-1ß, IL-8, and superoxide dismutase 2). Finally, we identify increased expression of microRNA-155 and rapid degradation of heat shock factor 1 following cardiac Ft.n infection as a primary cause of myocardial inflammation and apoptosis. CONCLUSIONS: We have developed and characterized an Ft.n infection model to understand the pathogenesis of cardiac dysregulation in sepsis. Our findings illustrate novel in vivo phenotypes underlying cardiac dysfunction during Ft.n infection with significant translational impact on our understanding of sepsis pathophysiology.

A 65-Year-Old Groundskeeper With High Fever, Pulmonary Nodules, and Thoracic Lymphadenopathy.

A 65-year-old man with treated latent tuberculous infection presented with 1 week of fevers (up to 39.6°C), chills, headache, lightheadedness, and malaise. He reported a chronic, nonproductive cough without hemoptysis but denied other localizing symptoms, sick contacts, or recent travel. He lived in an urban area in eastern Colorado and owned one healthy dog but otherwise denied known animal exposures. He was a retired oil driller who had worked in southern Arizona, New Mexico, and northern Mexico (Sonora region). Other travel included 3 years in the early 1970s working as a military aircraft mechanic in Vietnam, Laos, and Thailand. Six weeks prior to admission, he began work as a groundskeeper on a golf course that had experienced recent flooding, using a riding mower and exposing himself to airborne dust and organic debris. He smoked a pipe daily for 30 years but quit 2 months prior to presentation, although he continued to smoke marijuana weekly. He denied intravenous drug use.

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

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

Mouse models of aerosol-acquired tularemia caused by Francisella tularensis types A and B.

After preliminary assessment of virulence in AKR/J, DBA/1, BALB/c, and C57BL/6 mice, we investigated histopathologic changes in BALB/c and C57BL/6 mice infected with type A (strain SCHU S4) or type B (strain 425) Francisella tularensis by aerosol exposure. In mice exposed to type A infection, changes in histologic presentation were not apparent until day 3 after infection, when pyogranulomatous inflammation was detected in spleens and livers of BALB/c mice, and in lungs and spleens of C57BL/6 mice. Histopathologic changes were most severe and widespread in both mouse strains on day 5 after infection and seemed to completely resolve within 22 d of challenge. BALB/c mice were more resistant than C57BL/6 mice in lethal-dose calculations, but C57BL/6 mice cleared the infection more rapidly. Mice similarly challenged with type B F. tularensis also developed histopathologic signs of infection beginning on day 3. The most severe changes were noted on day 8 and were characterized by granulomatous or pyogranulomatous infiltrations of the lungs. Unlike type A infection, lesions due to type B did not resolve over time and remained 3 wk after infection. In type B, but not type A, infection we noted extensive inflammation of the heart muscle. Although no microorganisms were found in tissues of type A survivors beyond 9 d after infection, mice surviving strain 425 infection had a low level of residual infection at 3 wk after challenge. The histopathologic presentation of tularemia caused by F. tularensis types A and B in BALB/c and C57BL/6 mice bears distinct similarities to tularemia in humans.

A method for functional trans-complementation of intracellular Francisella tularensis.

Francisella tularensis is a highly infectious bacterial pathogen that invades and replicates within numerous host cell types. After uptake, F. tularensis bacteria escape the phagosome, replicate within the cytosol, and suppress cytokine responses. However, the mechanisms employed by F. tularensis to thrive within host cells are mostly unknown. Potential F. tularensis mutants involved in host-pathogen interactions are typically discovered by negative selection screens for intracellular replication or virulence. Mutants that fulfill these criteria fall into two categories: mutants with intrinsic intracellular growth defects and mutants that fail to modify detrimental host cell processes. It is often difficult and time consuming to discriminate between these two possibilities. We devised a method to functionally trans-complement and thus identify mutants that fail to modify the host response. In this assay, host cells are consistently and reproducibly infected with two different F. tularensis strains by physically tethering the bacteria to antibody-coated beads. To examine the efficacy of this protocol, we tested phagosomal escape, cytokine suppression, and intracellular replication for F. tularensis ΔripA and ΔpdpC. ΔripA has an intracellular growth defect that is likely due to an intrinsic defect and fails to suppress IL-1ß secretion. In the co-infection model, ΔripA was unable to replicate in the host cell when wild-type bacteria infected the same cell, but cytokine suppression was rescued. Therefore, ΔripA intracellular growth is due to an intrinsic bacterial defect while cytokine secretion results from a failed host-pathogen interaction. Likewise, ΔpdpC is deficient for phagosomal escape, intracellular survival and suppression of IL-1ß secretion. Wild-type bacteria that entered through the same phagosome as ΔpdpC rescued all of these phenotypes, indicating that ΔpdpC failed to properly manipulate the host. In summary, functional trans-complementation using bead-bound bacteria co-infections is a method to rapidly identify mutants that fail to modify a host response. Francisella tularensis is a facultative intracellular bacterial pathogen and is the causative agent of the disease tularemia. F. tularensis enters host cells through phagocytosis, escapes the phagosome, and replicates in the host cell cytosol while suppressing cytokine secretion [1]-[4]. Although substantial progress has been made in understanding the intracellular life cycle of F. tularensis, the F. tularensis proteins responsible for manipulating many host cell pathways are unknown. Identifying novel host-pathogen effector proteins is difficult because there is no rapid method to reliably distinguish between bacterial proteins that modify host processes and proteins that are involved in bacterial processes that are required for the bacteria to survive or replicate in the intracellular environment. The ability to identify mutants that are deficient for host-pathogen interactions is important because it can aid in prioritizing the investigation of genes of interest and in downstream experimental design. Moreover, certain mutant phenotypes, such as decreased phagosomal escape, hinder investigation of other potential phenotypes. A method to specifically complement these phenotypes would allow for further characterizations of certain F. tularensis mutants. Thus we sought to develop a method to easily identify and functionally complement mutants that are deficient for interactions with the host.

IL-12Rß2 is critical for survival of primary Francisella tularensis LVS infection.

Using a panel of vaccines that provided different degrees of protection, we previously identified the IL-12 receptor subunit ß2 as a mediator, whose relative expression correlated with strength of protection against secondary lethal challenge of vaccinated mice with an intracellular bacterium, the LVS of Francisella tularensis. The present study therefore tested the hypothesis that IL-12Rß2 is an important mediator in resistance to LVS by directly examining its role during infections. IL-12Rß2 KO mice were highly susceptible to LVS primary infection, administered i.d. or i.n. The LD50 of LVS infection of KO mice were 2 logs lower than those of WT mice, regardless of route. Five days after infection with LVS, bacterial organ burdens were significantly higher in IL-12Rß2 KO mice. IL-12Rß2 KO mice infected with lethal doses of LVS had more severe liver pathology, including significant increases in the liver enzymes ALT and AST. Despite decreased levels of IFN-γ, LVS-vaccinated IL-12Rß2 KO mice survived large lethal LVS secondary challenge. Consistent with in vivo protection, in vitro intramacrophage LVS growth was well-controlled in cocultures containing WT or IL-12Rß2 KO LVS-immune splenocytes. Thus, survival of secondary LVS challenge was not strictly dependent on IL-12Rß2. However, IL-12Rß2 is important in parenteral and mucosal host resistance to primary LVS infection and in the ability of WT mice to clear LVS infection and serves to restrict liver damage.

Tularaemia presenting as parapharyngeal abscess: case presentation.

OBJECTIVE: We report an extremely rare case of the oropharyngeal form of tularaemia, causing a parapharyngeal abscess. CASE REPORT: A 48-year-old woman presented with fever, sore throat, breathing difficulty and a right-sided neck swelling. This mass had previously been treated with penicillin without response, and had already been surgically drained once in another hospital. On physical examination, the tonsils were exudative and hypertrophic and the pharynx was hyperaemic. A fluctuant, 4 × 4 cm mass was seen on endoscopic examination, originating from the left parapharyngeal area and protruding towards the pyriform sinus, and partly obstructing the airway. Microagglutination test antibody titres for Francisella tularensis were positive (1/1280). The patient healed completely after definitive drainage of the abscess and antimicrobial therapy for 14 days (streptomycin, 2 × 1 g intramuscularly). CONCLUSION: Tularaemia should be considered in the differential diagnosis of patients presenting with tonsillopharyngitis, cervical lymphadenitis and parapharyngeal abscess who do not respond to treatment with penicillin, even if they do not live in an endemic region.

Francisella tularensis subsp. tularensis Schu S4 disulfide bond formation protein B, but not an RND-type efflux pump, is required for virulence.

Francisella tularensis subsp. tularensis is a highly virulent bacterium that is a CDC select agent. Despite advancements in the understanding of its biology, details pertaining to virulence are poorly understood. In previous work, we identified a transposon insertion mutant in the FTT0107c locus that was defective in intracellular survival in HepG2 and J77A.1 cells. Here, we report that this mutant was also highly attenuated in vivo. The FTT0107c locus is predicted to encode an ortholog of the disulfide bond formation B protein (DsbB). This designation was confirmed by complementation of an Escherichia coli dsbB mutant. This dsbB mutant of Schu S4 was highly attenuated in mice, but unlike what has been reported for Francisella novicida, intranasal immunization with a sublethal dose did not induce protection against wild-type challenge. dsbB was found to be transcribed in an operon with acrA and acrB, which encode an RND-type efflux pump. However, this pump did not make a significant contribution to virulence because strains with nonpolar deletions in acrA and acrB behaved like wild-type strain Schu S4 with respect to intracellular growth and in vivo virulence. This result is in contrast to a report that an acrB mutant of a live vaccine strain of F. tularensis has decreased virulence in mice. Overall, these results demonstrate key differences between the virulence requirements of Schu S4 and less virulent subspecies of Francisella. We have shown that DsbB is a key participant in intracellular growth and virulence, and our results suggest that there are critical virulence factors that contain disulfide bonds.

Initial delay in the immune response to Francisella tularensis is followed by hypercytokinemia characteristic of severe sepsis and correlating with upregulation and release of damage-associated molecular patterns.

"Francisella tularensis subsp. novicida" intranasal infection causes a rapid pneumonia in mice with mortality at 4 to 6 days with a low dose of bacteria (10(2) bacteria). The short time to death suggests that there is a failure of the innate immune response. As the neutrophil is often the first cell type to infiltrate sites of infection, we focused on the emigration of neutrophils in this infection, as well as cytokines involved in their recruitment. The results indicated that there was a significant delay in the influx of neutrophils into the bronchoalveolar lavage fluid of F. tularensis subsp. novicida-infected mice. The delay in neutrophil recruitment in F. tularensis subsp. novicida-infected mice correlated with a delay in the upregulation of multiple proinflammatory cytokines and chemokines, as well as a delay in caspase-1 activation. Strikingly, the initial delay in the upregulation of cytokines through 1 day postinfection was followed by profound upregulation of multiple cytokines and chemokines to levels consistent with hypercytokinemia described for severe sepsis. This finding was further supported by a bacteremia and the cellular relocalization and release of high-mobility group box-1 and S100A9, both of which are damage-associated molecular pattern molecules and are known to be mediators of severe sepsis.

Type IV pili in Francisella tularensis: roles of pilF and pilT in fiber assembly, host cell adherence, and virulence.

Francisella tularensis, a highly virulent facultative intracellular bacterium, is the causative agent of tularemia. Genome sequencing of all F. tularensis subspecies revealed the presence of genes that could encode type IV pili (Tfp). The live vaccine strain (LVS) expresses surface fibers resembling Tfp, but it was not established whether these fibers were indeed Tfp encoded by the pil genes. We show here that deletion of the pilF putative Tfp assembly ATPase in the LVS resulted in a complete loss of surface fibers. Disruption of the pilT putative disassembly ATPase also caused a complete loss of pili, indicating that pilT functions differently in F. tularensis than in model Tfp systems such as those found in Pseudomonas aeruginosa and Neisseria spp. The LVS pilF and pilT mutants were attenuated for virulence in a mouse model of tularemia by the intradermal route. Furthermore, although absence of pili had no effect on the ability of the LVS to replicate intracellularly, the pilF and pilT mutants were defective for adherence to macrophages, pneumocytes, and hepatocytes. This work confirms that the surface fibers expressed by the LVS are encoded by the pil genes and provides evidence that the Francisella pili contribute to host cell adhesion and virulence.

Francisella novicida LPS has greater immunobiological activity in mice than F. tularensis LPS, and contributes to F. novicida murine pathogenesis.

To further understand the role of LPS in the pathogenesis of Francisella infection, we characterized murine infection with F. novicida, and compared immunobiological activities of F. novicida LPS and the LPS from F. tularensis live vaccine strain (LVS). F. novicida had a lower intradermal LD(50) in BALB/cByJ mice than F. tularensis LVS, and mice given a lethal F. novicida dose intraperitoneally died faster than those given the same lethal F. tularensis LVS dose. However, the pattern of in vivo dissemination was similar, and in vitro growth of both bacteria in bone marrow-derived macrophages was comparable. F. novicida LPS stimulated very modest in vitro proliferation of mouse splenocytes at high doses, but F. tularensis LVS LPS did not. Murine bone marrow macrophages treated in vitro with F. novicida LPS produced IL12 and TNF-alpha, but did not produce detectable interferon-gamma, IL10, or nitric oxide; in contrast, murine macrophages treated with F. tularensis LVS LPS produced none of these mediators. In contrast to clear differences in stimulation of proliferation and especially cytokines, both types of purified LPS stimulated early protection against lethal challenge of mice with F. tularensis LVS, but not against lethal challenge with F. novicida. Thus, although LPS recognition may not be a major factor in engendering protection, the ability of F. novicida LPS to stimulate the production of proinflammatory cytokines including TNF-alpha likely contributes to the increased virulence for mice of F. novicida compared to F. tularensis LVS.

Tularaemia.

Tularaemia is a zoonotic bacterial disease of the Northern hemisphere. The causative agent, Francisella tularensis, is spread to humans by direct contact with infected rodents or lagomorphs, aerogenic exposure, ingestion of contaminated food or water, or by arthropod bites. The prevalence of tularaemia shows a wide geographic variation. In some endemic regions, outbreaks occur frequently, whereas nearby rural parts of a country may be completely free. F. tularensis is a facultative intracellular pathogen and its primary mammalian target cell is the mononuclear phagocyte. When tularaemia is acquired via the skin, a primary ulcer is often detected and in general, regional lymph nodes become prominently enlarged. When contracted by inhalation, the disease may present with pneumonia. Nearly as frequent, however, is the development of fever and general illness with no respiratory symptoms and no pulmonary radiological changes. When present, the changes vary widely and may sometimes include hilar enlargement indistinguishable from that of lymphoma. Within an outbreak, the first case of tularaemia is not always readily diagnosed. A decade may have lapsed since the disease was encountered and its existence may be more or less forgotten. The difficulty refers especially to the respiratory form, in which symptoms are less specific. In cases of atypical pneumonia or acute febrile disease with no local symptoms, a history of exposure to hares or rodents or merely living in an endemic region should be sufficient to include tularaemia among differential diagnoses. The microbiological diagnosis of tularaemia relies mainly on serology, and the treatment on broad-spectrum antibiotics. For decades, a live vaccine has been successfully used in risk groups but is presently not available due to difficulties in standardisation.

Cytokine expression in the liver during the early phase of murine tularemia.

Cytokine expression was determined in the livers of mice inoculated subcutaneously with Francisella tularensis LVS. During the first 48 h of infection, there was a logarithmic increase of bacteria in the liver, with a doubling time of 2.5 h. Within 48 h, tumor necrosis factor alpha (TNF-alpha), interleukin 10 (IL-10), IL-12, and gamma interferon (IFN-gamma) mRNAs were expressed, and production of TNF-alpha and IFN-gamma was demonstrated. There was no expression within 96 h of mRNA from IL-2, IL-3, or IL-4. After subcutaneous inoculation of heat-killed LVS, no expression of any of the cytokine mRNAs and no increase in the levels of TNF-alpha or IFN-gamma occurred. The expression of TNF-alpha, IL-12, and IFN-gamma is held to be important to evoke an early T-cell-independent host defense against F. tularensis as well as to drive the expansion of a protective Th1 cell response.