Phase Variation of LPS and Capsule Is Responsible for Stochastic Biofilm Formation in Francisella tularensis

Biofilms have been established as an important lifestyle for bacteria in nature as these structured communities often enable survivability and persistence in a multitude of environments. Francisella tularensis is a facultative intracellular Gram-negative bacterium found throughout much of the northern hemisphere. However, biofilm formation remains understudied and poorly understood in F. tularensis as non-substantial biofilms are typically observed in vitro by the clinically relevant subspecies F. tularensis subsp. tularensis and F. tularensis subsp. holarctica (Type A and B, respectively). Herein, we report conditions under which robust biofilm development was observed in a stochastic, but reproducible manner in Type A and B isolates. The frequency at which biofilm was observed increased temporally and appeared switch-like as progeny from the initial biofilm quickly formed biofilm in a predictable manner regardless of time or propagation with fresh media. The Type B isolates used for this study were found to more readily switch on biofilm formation than Type A isolates. Additionally, pH was found to function as an environmental checkpoint for biofilm initiation independently of the heritable cellular switch. Multiple colony morphologies were observed in biofilm positive cultures leading to the identification of a particular subset of grey variants that constitutively produce biofilm. Further, we found that constitutive biofilm forming isolates delay the onset of a viable non-culturable state. In this study, we demonstrate that a robust biofilm can be developed by clinically relevant F. tularensis isolates, provide a mechanism for biofilm initiation and examine the potential role of biofilm formation.

Structure of the Potential Virulence Factor from Francisella tularensis Shows Unexpected Presence of the SHS2 Motif and Similarity to Other Bacterial Virulence Factors

Pathogenic bacteria attack their host by secreting virulence factors that in various ways interrupt host defenses and damage their cells. Functions of many virulence factors, even from well-studied pathogens, are still unknown. Francisella tularensis is a class A pathogen and a causative agent of tularemia, a disease that is lethal without proper treatment. Here we report the three-dimensional structure and preliminary analysis of the potential virulence factor identified by the transcriptomic analysis of the F. tularensis disease models that is encoded by the FTT_1539 gene. The structure of the FTT_1539 protein contains two sets of three stranded antiparallel beta sheets, with a helix placed between the first and the second beta strand in each sheet. This structural motif, previously seen in virulence factors from other pathogens, was named the SHS2 motif and identified to play a role in protein-protein interactions and small molecule recognition. Sequence and structure analysis identified FTT_1539 as a member of a large family of secreted proteins from a broad range of pathogenic bacteria, such as Helicobacter pylori and Mycobacterium tuberculosis. While the specific function of the proteins from this class is still unknown, their similarity to the H. pylori Tip-α protein that induces TNF-a and other chemokines through NF-kB activation suggests the existence of a common pathogen-host interference mechanism shared by multiple human pathogens.

A Multidisciplinary Approach to the First Autochthonous Case of Tularemia Reported in Portugal

Francisella tularensis, a Gram-negative coccobacillus, is a highly virulent pathogen responsible for several zoonotic outbreaks in Europe in the last few decades. The authors report the case of a 46-year-old male who developed fever, myalgias and headache a week after having contact with animal feed contaminated by rodents. Serological tests were positive for Francisella tularensis. This first case of autochthonous tularemia in Portugal led to an intensive investigation involving several healthcare services and national governmental authorities. The authors address the possible underdiagnosis of this infection in the country.

Backbone chemical shift assignment and dynamics of the N-terminal domain of ClpB from Francisella tularensis type VI secretion system

The Hsp100 family member ClpB is a protein disaggregase which solubilizes and reactivates stress-induced protein aggregates in cooperation with the DnaK/Hsp70 chaperone system. In the pathogenic bacterium Francisella tularensis, ClpB is involved in type VI secretion system (T6SS) disassembly through depolymerization of the IglA-IglB sheath. This leads to recycling and reassembly of T6SS components and this process is essential for the virulence of the bacterium. Here we report the backbone chemical shift assignments and 15N relaxation-based backbone dynamics of the N-terminal substrate-binding domain of ClpB (1-156).

Diabetic Foot Osteomyelitis Caused by Francisella Tularensis

Purpose: A rare patient case of a diabetic foot osteomyelitis caused by Francisella tularensis is presented. Summary: A 69-year-old Caucasian female was admitted for the treatment of diabetic foot osteomyelitis. Her past medical history included type II diabetes mellitus, hypertension, chronic kidney disease, coronary artery disease, hypothyroidism, hyperuricemia and thyroidectomy. Empiric antimicrobial therapy consisting of clindamycin 600mg i.v. every 8hrs and impanel/cilastatin 200 mg i.v. every 6hrs hours was initiated immediately after admission. During her hospitalization, a pus sample from the infection site was taken for culture which showed a gram negative microorganism: Francisella tularensis. The strain was resistant to all the antibiotics tested with the exception of ciprofloxacin, ofloxacin, gentamicin, ceftazidime, cefepime, piperacillin/tazobactam and colistin. After culture results, the treatment regimen was changed to piperacillin/tazobactam 4.5g i.v. every 12hrs and ciprofloxacin 400mg i.v. every 12hrs. The patient continued to receive both antibiotics during hospitalization for 9 days with noted clinical improvement. The patient was discharged on piperacillin/tazobactam 4.5g i.v. every 12hrs and oral ciprofloxacin 500mg every 12hrs to complete a total duration of 6 weeks. Conclusion: This is the first reported case of a diabetic foot osteomyelitis caused by Francisella tularensis.

Cross-sectional Molecular Detection of Francisella tularensis in Domestic Rabbits in Sulaimani Province Kurdistan Region, Iraq

Tularemia is one of the diseases transmitted between humans and animals. It is caused by a Gram-negative bacterium Francisella tularensis. Recent serological studies suggested that tularemia can be an endemic bacterial zoonotic disease in some countries surrounding Iraq such as Iran and Turkey. The main objective of this study is to detect tularemia in Sulaimani province northeast Iraq near to Iran border. Sulaimani city also has contact with many Turkish cities. This study was conducted between Jun and October 2020. Blood samples were taken from one hundred local breed rabbits of different ages and sexes. A highly sensitive real-time PCR technique was used. Sixteen out of one hundred blood samples (16%) were positively taken from different local breed rabbits from four different places in Sulaimani province. All positive samples were detected in the center of Sulaimani city. No published documents have been reported yet about tularemia in Kurdistan Region. This paper documented molecular detection of F. tularensis in local breed rabbits in Sulaimani province Kurdistan Region-Iraq

Macrophages Demonstrate Guanylate-Binding Protein-Dependent and Bacterial Strain-Dependent Responses to Francisella tularensis

Francisella tularensis is a facultative intracellular bacterium and the etiological agent of tularemia, a zoonotic disease. Infection of monocytic cells by F. tularensis can be controlled after activation with IFN-γ; however, the molecular mechanisms whereby the control is executed are incompletely understood. Recently, a key role has been attributed to the Guanylate-binding proteins (GBPs), interferon-inducible proteins involved in the cell-specific immunity against various intracellular pathogens. Here, we assessed the responses of bone marrow-derived murine macrophages (BMDM) and GBP-deficient BMDM to F. tularensis strains of variable virulence; the highly virulent SCHU S4 strain, the human live vaccine strain (LVS), or the widely used surrogate for F. tularensis, the low virulent F. novicida. Each of the strains multiplied rapidly in BMDM, but after addition of IFN-γ, significant GBP-dependent control of infection was observed for the LVS and F. novicida strains, whereas there was no control of the SCHU S4 infection. However, no differences in GBP transcription or translation were observed in the infected cell cultures. During co-infection with F. novicida and SCHU S4, significant control of both strains was observed. Patterns of 18 cytokines were very distinct between infected cell cultures and high levels were observed for almost all cytokines in F. novicida-infected cultures and very low levels in SCHU S4-infected cultures, whereas levels in co-infected cultures for a majority of cytokines showed intermediate levels, or levels similar to those of F. novicida-infected cultures. We conclude that the control of BMDM infection with F. tularensis LVS or F. novicida is GBP-dependent, whereas SCHU S4 was only controlled during co-infection. Since expression of GBP was similar regardless of infecting agent, the findings imply that SCHU S4 has an inherent ability to evade the GBP-dependent anti-bacterial mechanisms.

Studies of ticks of the genus Dermacentor (Acari; Ixodidae) on the natural occurrence of tularemia pathogen in the conditions of the Central Pre-Caucasian region

The purpose of the research is the assessment of the Francisella tularensis occurrence in nature in ticks of the genus Dermacentor; understanding the physiological age in terms of tick infection with tularemia pathogen.Materials and methods. For the period from 2015 to 2019, we examined 8449 specimens of Dermacentor marginatus (916 pools), 8674 specimens of D. reticulatus (705 pools), and 109 specimens of D. niveus (40 pools) for tularemia infection. To assess the dependence of tularemia pathogen found in ticks of different physiological ages, we examined 2440 specimens of D. marginatus (360 pools), and 3349 specimens of D. reticulatus (412 pools) for the period from 2016 to 2019. Studies of ixodid ticks infected with tularemia pathogen were performed by the Natural Focal Infection Laboratory of the Stavropol Anti-Plague Institute. Pools of ixodid ticks were examined for the pathogen DNA of tularemia using reagent kits for identifying Francisella tularensis DNA by polymerase chain reaction with fluorescence hybridization of results recorded in real time.Results and discussion. The infection rate of the tularemia pathogen in ticks in the Central Pre-Caucasian region ranged from 0.044–1.127% in D. marginatus and 0.035–1.455% in D. reticulatus in different years. The greatest number of F. tularensis was isolated from the III physiological age ticks. For D. reticulatus ticks, no statistically significant dependence of the detected tularemia pathogen on physiological age was found.