Francisella Tularensis Research Articles

ANATOMY OF THE IMMUNE RESPONSE TO FRANCISELLA TULARENSISLVS INFECTION IN THE LUNGS.

Abstract The respiratory mucosa is under constant immune surveillance because of its vulnerability to infectious diseases. Infection of the lungs with the live vaccine strain (LVS) derived from Francisella tularensis (Ft) subspecies holarctica models pulmonary tularaemia-like disease in mice. Current evidence suggest many immune cells and cytokines respond to Ft LVS infection, but how these various immune system components are integrated to mount a protective response remains unclear. Hence, in a transcriptomics study at single cell resolution, we characterized the acute immune landscape in the lungs of C57BL/6 mice at day 0 and day 7 post intranasal inoculation with Ft LVS. Defining features of the immune response include a robust type 1 immune response characterized by the accumulation of inflammatory neutrophils and the expansion of innate-like effector lymphocytes, primarily interferon-γ producing NKT1, NK, and effector CD8 +T cells. Increased accumulation of MAIT17 over MAIT1 cells is another feature, which is in line with previous findings linking MAIT cells and IL-17 to LVS immune response. Surprisingly, a significant proportion of MAIT17 cells either maintained or upregulated type 1 inflammatory markers. Similarly, MAIT1 cells maintained or upregulated Type 3 inflammatory markers. Additionally, a highly active MAIT cell subset has increased Nr4a1 expression, which encodes Nur77, suggesting a T cell receptor-mediated activation. By contrast, NKT1 cells poorly upregulated Nr4a1 expression but induced Il18r1 expression suggesting, a cytokine-mediated activation. Consequently, we predict that unconventional NKT & MAIT cells integrate innate cues to control tularaemia-like disease caused by Ft LVS infection in the mouse. VUSM MSTP NIGMS of the National Institutes of Health T32GM007347 (GDO); IO1 BX001444, BX001610, BX00xxx, IK6 BX004595 & RO1 AI137082; IBX000915A (HMA)

Disparate natural killer T cell subsets exhibit opposing regulatory effects on the cytokine storm.

Abstract Inflammation is both a critical component of the immune system for effective clearance of microbial challenges and a mechanism by which some pathogens cause disease, e.g., tularemia, dengue fever, and COVID-19. The bacterium Francisella tularensis (Ft), the causative agent of tularemia, elicits a cytokine storm in which the immune system over-responds to the bacterium, releasing massive amounts of proinflammatory cytokines. Untreated, tularemia can reach a mortality rate as high as 30% in healthy adults with inhalation of as few as 10 colony forming units inducing respiratory tularemia. While Ftis spread from zoonotic sources in nature, the ease of aerosol spread and high mortality led to its weaponization and continued concern for bioterrorism applications. Natural Killer T (NKT) cells are important immune cells with the capability of modulating the inflammatory response. NKT cells are activated early following Ftinfection suggesting a role in the innate immune response in tularemia. NKT cells are categorized into two distinct subsets, type I and type II based on their antigen recognition, or not, of α-galactosylceramide. Literature suggests that these subsets have opposing regulatory phenotypes, however, a clear understanding of their roles in regulating inflammation remains unknown and an emerging frontier in NKT research. Our lab has observed opposing roles in suppressing the cytokine storm by NKT subtypes. Ongoing studies will elucidate the mechanism utilized to influence the cytokine storm. This project will yield a better understanding of NKT cell-mediated inflammation during Ftinfection and, more broadly, characterize their involvement and potential for therapeutic targeting for a number of NIH priority pathogens. Supported by grants from National Institute of General Medical Sciences (NIGMS) Research Initiative for Scientific Enhancement (RISE) grant R25 GM 06921-18

Disease Surveillance of Cougars (Puma concolor) in Utah, USA.

Adult, free-ranging cougars (Puma concolor) were sampled in three regions of Utah, US, from 2018 to 2021. A total of 68% (23/34) of the sampled cougars had antibodies to feline parvovirus, 15% (5/33) to canine distemper virus, 18% (6/34) to calicivirus, and 22% (8/37) to Yersinia pestis. Forty-one percent (13/32) had IgG antibodies to Toxoplasma gondii and 6% (2/33) to feline immunodeficiency virus, and 3% (1/32) were positive for Dirofilaria immitis (heartworm) antigen. All were seronegative for Toxoplasma gondii IgM, feline enteric coronavirus, SARS-CoV-2, feline leukemia virus, feline herpesvirus, and Francisella tularensis. Tapeworms and Toxascaris leonina eggs were detected in the feces. The disease exposures detected were similar to what has been reported from cougar populations in other western US states, and the current level of exposures is unlikely to have a negative impact on the state's population.

Efficacy of Intravenously Administered Gepotidacin in Cynomolgus Macaques following a Francisella tularensis Inhalational Challenge.

Francisella tularensis (F. tularensis) is a Centers for Disease Control (CDC) category "A" Gram-negative biothreat pathogen. Inhalation of F. tularensis can cause pneumonia and respiratory failure and is associated with high mortality rates without early treatment. Gepotidacin is a novel, first-in-class triazaacenaphthylene antibiotic that inhibits bacterial DNA replication by a distinct mechanism of action. Gepotidacin selectively inhibits bacterial DNA replication via a unique binding mode, has activity against multidrug-resistant target pathogens, and has demonstrated in vitro activity against diverse collections of F. tularensis isolates (MIC90 of 0.5 to 1 μg/mL). Gepotidacin was evaluated in the cynomolgus macaque model of inhalational tularemia, using the SCHU S4 strain, with treatment initiated after exposure and sustained fever. Macaques were dosed via intravenous (i.v.) infusion with saline or gepotidacin at 72 mg/kg/day to support a human i.v. infusion dosing regimen of 1,000 mg three times daily. The primary study endpoint was survival, with survival duration and bacterial clearance as secondary endpoints. Gepotidacin treatment resulted in 100% survival compared to 12.5% in the saline-treated control group (P < 0.0001) at Day 43 postinhalational challenge. All gepotidacin-treated animals were blood and organ culture negative for F. tularensis at the end of the study. In contrast, none of the saline control animals were blood and organ culture negative. Gepotoidacin's novel mechanism of action and the efficacy data reported here (aligned with the Food and Drug Administration Animal Rule) support gepotidacin as a potential treatment for pneumonic tularemia in an emergency biothreat situation.

The impact of lipid A modification on biofilm and related pathophysiological phenotypes, endotoxicity, immunogenicity, and protection of Salmonella Typhimurium

This study presents the engineering of a less endotoxic Salmonella Typhimurium strain by manipulating the lipid-A structure of the lipopolysaccharide (LPS) component. Salmonella lipid A was dephosphorylated by using lpxE from Francisella tularensis. The 1-phosphate group from lipid-A was removed selectively, resulting in a close analog of monophosphoryl lipid A. We observed a significant impact of ∆pagL on major virulence factors such as biofilm formation, motility, persistency, and immune evasion. In correlation with biofilm and motility retardation, adhesion and invasion were elevated but with reduced intracellular survival, a favorable phenotype prospect of a vaccine strain. Western blotting and silver staining confirmed the absence of the O-antigen and truncated lipid-A core in the detoxified Salmonella mutant. In vitro and in vivo studies demonstrated that the dephosphorylated Salmonella mutant mediated lower pro-inflammatory cytokine secretion than the wild-type strain. The vaccine strains were present in the spleen and liver for five days and were cleared from the organs by day seven. However, the wild-type strain persisted in the spleen, liver, and brain, leading to sepsis-induced death. Histological evaluations of tissue samples further confirmed the reduced endotoxic activity of the detoxified Salmonella mutant. The detoxification strategy did not compromise the level of protective immunity, as the vaccine strain could enhance humoral and cellular immune responses and protect against the wild-type challenge in immunized mice.

Deletion of FTL_1199 to determine the role of this gene in erythrocyte invasion by Francisella tularensis.

Francisella tularensis is a bacterium that induces the zoonotic disease tularemia. In the course of infection, F. tularensis bacteria invade erythrocytes, a phenomenon that heightens the colonization of ticks after a blood meal. To better understand the mechanism of erythrocyte invasion, we hypothesized that transcription of bacterial genes significant in erythrocyte invasion would be upregulated upon exposure to these host cells. An RNA-seq unveiled that transcription of 7% of F. tularensis genes augment when in erythrocyte presence. Of these, we pinpointed a putative transcriptional regulator, FTL_1199. The goal was to delete FTL_1199 in F. tularensis LVS. SOE PCR amplified and duplicated the up and downstream regions of the target gene in tandem into a shuttle vector that is insecure within F. tularensis. This newly generated plasmid, pDEL1199, was mobilized inside of F. tularensis by conjugation. Merodiploid strains generated by homologous recombination were isolated and transformed with pGUTS. Expression of I-Sce1 within the merodiploid produces a double-stranded break. This breakage resulted in a second recombination that either ensued to wild-type or deletion of FTL_1199 deduced through a PCR. Finally, in DFTL_1199 strains, pGUTS was cured by successive cultivation in the absence of selection followed by replica-plating on chocolate II agar ± kanamycin. Gentamicin protection assays showed reduced levels of erythrocyte invasion for F. tularensis DFTL_1199 compared to wild type bacteria. However, complementation of FTL_1199 to the deletion mutant restored this strain’s ability to invade red blood cells. These findings demonstrate that FTL_1199 is important for erythrocyte invasion by F. tularensis.&#x0D; &#x0D; (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence, R15HL14735 from NHLBI, and funds from the NASA West Virginia Space Grant Consortium).

Screening putative transcriptional regulators in Francisella tularensis for attenuation using a multifaceted approach.

Francisella tularensis is a gram-negative intracellular pathogen that produces a severe infection known as Tularemia. RNA-Seq data revealed that in the presence of erythrocytes, several genes encoding putative transcriptional regulators were modulated: FTL_0671, FTL_1199, and FTL_1665. Gene deletion strains were constructed using Francisella tularensis LVS. The objective of this project was to screen these gene deletion strains for attenuation. A multifaceted approach was utilized to determine the level of replication within macrophages and overall attenuation in vivo. Transforming the bacteria with green fluorescent protein allowed a plate reader to visualize and quantify intracellular growth in macrophages. However, inconsistencies in the data from these experiments led to the utilization of a gentamicin protection assay. This protocol provided a more accurate and reliable method of determining intracellular replication. The results of this experiment revealed a significant increase in the replication of FTL_1665 within macrophages. We sought to determine if these results would translate to hypervirulence in a live model. A chicken embryo infection model confirmed that FTL_1665 was significantly hypervirulent in vivo. In the future, we plan to experiment with the upregulation of the target gene to produce an attenuated strain. This gene may also serve as a potential drug target.

The Efficacy, Toxicity &amp; Pharmacology of Dillapiole: a potential new treatment for Tularemia

Francisella tularensis, a highly infectious bacterium, is the causative agent of Tularemia (rabbit fever). Categorized by the Center for Disease Control and Prevention as a Category A bioterrorism agent, Francisella tularensis is of the highest level of concern. Previously, we identified that dillapiole, a compound extracted from fennel, dampens F. tularensis virulence gene expression. While having no apparent effect on the viability of F. tularensis, treatment with this compound leads to reduced bacterial viability during in vitro infection of THP-1 monocytes and RAW 264.7 macrophages. In this study, we sought to determine if dillapiole exhibited a therapeutic effect in vivo, and to characterize the toxicity and pharmacology of this compound. In a murine tularemia model, female mice treated with dillapiole trended toward increased survival compared to those treated with the vehicle. However, dillapiole- or vehicle-treated male mice showed increased mortality compared to the females, suggesting gender-specific differences in the murine immune response to F. tularensis. Dillapiole was not toxic to HEK-293 cells in vitro, nor was this compound toxic to primary human hepatocytes when tested up to a concentration of 11 mg/ml (50 mM). Dillapiole was shown to be relatively stable in human, rat, and mouse plasma with a half-life greater than 120 minutes in all cases. However, this compound showed moderately high binding to plasma proteins (86% in human plasma and 75% in mouse plasma). In addition, while dillapiole showed moderate clearance by human and rat liver microsomes, mouse liver microsomes exhibited high clearance. Collectively, these data could explain the minimal efficacy observed in vivo. Therefore, future investigations should involve the rat infection model to determine the potential efficacy of dillapiole as a novel treatment for tularemia.

Role of LpnA in Francisella tularensis Susceptibility to Resazurin.

Francisella tularensis is defined by the Centers for Disease Control and Prevention as a Category A bioterrorism agent due to its low infectious dose, high mortality rate, and ease of aerosolization. Given there is no licensed tularemia vaccine in the United States and the possible development and release of antibiotic-resistant F. tularensis strains, there is an urgent need for new treatments against this bacterium. We determined the phenoxazine dye resazurin (Rz) exhibits antimicrobial activity against F. tularensis and other gram-negative bacteria. The mode of action of this compound is not understood, but potential targets of Rz were identified in a high throughput screen for resistant isolates. Ninety-three percent of the Rz-resistant (Rzr) isolates sequenced contained mutations within the coding regions of FTL_0421 (lpnA), FTL_0895, and FTL_1504 (katG). To confirm mutation of lpnA was contributing to Rz resistance, we introduced a wild-type copy of lpnA into Rzr1 and then assessed the susceptibility of the resulting complemented strain (Rzr1/pABST-lpnA). The minimum inhibitory concentration (MIC) of Rzr1 and Rzr1/pABST-lpnA were similar, therefore, we wanted to confirm we were restoring expression of LpnA in the complemented strain via Western blot analysis. As expected, LpnA was expressed in wild-type LVS but not in Rzr1 due to the genetic mutation. In the Rzr1 strain transformed with pABST-lpnA, we still did not observe expression of LpnA suggesting our complementation strategy was ineffective. In the future, we plan to take an alternative approach to investigate the role of LpnA in resazurin resistance by creating a lpnA deletion mutant.

Role of KatG in Francisella tularensis Susceptibility to Resazurin.

The CDC classifies Francisella tularensis as a Category A bioterrorism agent. Due to the risk of potential release of antibiotic-resistant F. tularensis strains, new therapeutics against F. tularensis must be developed. Resazomycins are resazurin (Rz)-based compounds that exhibit antimicrobial activity against F. tularensis and other gram-negative bacteria. The mode of action of resazomycins is not understood, but potential targets of the antibiotic were identified in a high throughput screen for Rz-resistant isolates. The FTL_1504 gene was identified as being mutated in all of the Rz-resistant (Rzr) strains sequenced. FTL_1504 encodes for the catalase KatG which catalyzes the decomposition of hydrogen peroxide to water and oxygen. Moreover, KatG has also been shown to play an essential role in the activity of a different antibiotic, isonazid. Four different coding mutations were characterized in katG that included single base pair substitutions and a deletion with all resulting in the introduction of a premature stop codon. All Rzr mutants tested had reduced catalase activity compared to wild-type F. tularensis LVS with three of the four mutations resulting in loss of KatG expression. Based on this data, we hypothesize KatG plays a role in the susceptibility of F. tularensis to resazurin. To address this, we are generating a katG deletion mutant in F. tularensis LVS and measuring its Rz susceptibility via agar dilution assays. Investigation of the contribution of KatG may unveil a unique role for the oxidative stress response in the mode of action of this family of antibiotics

Characterization of two predicted lipid metabolism genes (FTL_1569 and FTL_1570) in the intracellular pathogen, Francisella tularensis LVS.

Many species of bacteria enter a viable but non-culturable (VBNC) state when exposed to stressful conditions. The VBNC state acts as an adaptive strategy that allows for long-term survival of bacteria in unfavorable environments. VBNC bacteria are difficult to detect in the environment due to an inability to grow on nutrient agar. Although the ability to enter the VBNC state may be advantageous for bacteria, it poses a risk to human health. Francisella tularensis, transitions rapidly and spontaneously to the VBNC state, giving it the potential to be an excellent model organism for the study of this phenomenon. Microscopic observations reveal that changes in cell morphology are coincident with the VBNC state. To characterize these morphological changes, fluorescence microscopy imaging using membrane lipid staining has been used in the live vaccine strain of F. tularensis. Microscopy imaging suggested that lipid metabolism may be important for the transition into the VBNC state, and the change in morphology. To investigate further, genetic deletion mutants have been constructed from two predicted lipid metabolism genes, FTL_1569 (phosphoglycolate phosphatase) and FTL_1570 (phospholipase D family protein). Gentamicin protection assays with immune cells and red blood cells have been conducted to investigate the role of these genes in virulence of F. tularensis. Our preliminary results suggest that lipid metabolism may play roles in both persistence and in invasion in F. tularensis. (This work was supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence).

Screening Resorufin Pentyl Ether Analogs for Enhanced Antimicrobial Activity Against Francisella tularensis and Neisseria gonorrhoeae

Antibiotic resistance is an urgent public health threat. The CDC estimates there are approximately 2.8 million new cases of antibiotic-resistant infections annually resulting in 35,000 deaths and billions of dollars in health care costs. The development of new drugs is imperative to combat this crisis and prevent the loss of additional lives from once “curable” diseases. Resazomycins, a novel family of antibiotics, have bactericidal activity against Francisella tularensis and Neisseria gonorrhoeae. One resazomycin, resorufin pentyl ether (RPE), significantly reduces vaginal colonization by N. gonorrhoeae in a mouse model of infection. Repeated administration of RPE, however, fails to clear the infection, in contrast to a single dose of ceftriaxone, an antibiotic commonly used to treat gonorrhea, which clears the infection within 24 hours. Further characterization of resazomycins revealed the efficacy of these compounds is limited by interaction with serum albumin and reduced oxygen concentrations found within mammalian tissues. Therefore, we hypothesize that novel resazurin analogs that maintain antimicrobial activity in the presence of serum albumin and low oxygen will have improved therapeutic efficacy in vivo. To date, two different derivatives of RPE have been synthesized and tested for antimicrobial activity against F. tularensis and N. gonorrhoeae – 1-methyl RPE and 4-methyl RPE. Neither of these compounds inhibited the growth of F. tularensis or N. gonorrhoeae. Next, we plan to prepare a series of ketone derivatives of resazurin to alter the electrophilicity and reduction potential of these compounds and test their efficacy against F. tularensis and N. gonorrhoeae.

Analyzing the contribution to pathogenesis by three putative transcriptional regulators of Francisella tularensis using the chicken embryo infection model.

SHANIA DAVIS, MARIAH CASHBAUGH, ELIO DELATORE &amp; JOSEPH HORZEMPA Dept of Biomedical Sciences, West Liberty University, West Liberty, WV, 26074. Analyzing the contribution to pathogenesis by three putative transcriptional regulators of Francisella tularensis using the chicken embryo infection model.&#x0D; Francisella tularensis is a highly pathogenic bacterium that can be used as a biological weapon. This bacterium causes tularemia which is also known as Rabbit Fever. Previously, our laboratory identified three F. tularensis loci predicted to encode transcriptional regulators (FTL_0671, FTL_1199, and FTL_1665) that exhibited transcriptional changes in the presence of human erythrocytes. We hypothesized that because transcriptional modulations were associated with the presence of host cues, these potential transcriptional regulators may be important for pathogenesis. Therefore, mutants of these three loci and the isogenic wild-type bacterium (strain LVS) were used to infect chicken embryos. Healthy chicken embryos were injected with 104, 105, or 106 colony forming units of each strain, and chicken embryoys were candled daily for viability. The chicken embryos were housed in an egg incubator (~37°C with occasional rocking) and were observed over a span of 10 days. Neither FTL_0671 nor FTL_1199 affected the pathogenesis of F. tularensis. However, chicken embryos infected with the FTL_1665 deletion strain exhibited mortality more rapidly compared to those infected with wild-type bacteria suggesting that this locus may be repressing pathogenesis. Future studies will evaluate the function of FTL_1665 to determine if the protein encoded by this locus truly mediates transcriptional regulation.

Role of FTL_0895 in Francisella tularensis Susceptibility to Resazurin

Resistance to antibiotic treatments coupled with the decline in antibiotic discovery has resulted in a steady increase in deaths caused by once “curable” bacterial infections. Developing new drugs is crucial to prevent more loss of life in the future. We discovered the compound resazurin exhibits antimicrobial activity against gram-negative bacteria including Francisella tularensis (Ft), however, certain strains of Ft have developed resistance to resazurin. Understanding how Ftdevelops resistance to resazurin will help with defining the mechanism by which resazurin elicits its antimicrobial effect. Whole genome sequencing of resazurin-resistant (Rzr) Ft LVS mutants revealed 93% of the isolates sequenced possessed mutations in the coding regions of FTL_0421, FTL_0895, and FTL_1504. The focus of my project was to explore the role of FTL_0895 in resazurin susceptibility. To confirm this gene plays a role in the reduced susceptibility of the Rzr strains to resazurin, a wild-type copy of FTL_0895 was cloned into the Francisella shuttle vector pABST and thenelectroporated into one of the Rzr mutants, Rzr1. Complementation with FTL_0895 did not restore sensitivity of the Rzr1 strain to resazurin suggesting that mutation of this gene alone is not responsible for resistance to resazurin. Therefore, we have cloned FTL_0895 into a different Ft shuttle vector pMQ2 so we can complement back Rzr1 with this gene in combination with either FTL_1504 or FTL_0421. Agar dilution and time kill assays will be conducted on the resulting Rzr-complemented strains to determine their susceptibility to resazurin.

The Effect of Dillapiole on the pathogenesis of Escherichia coli

Dillapiole, a compound isolated from the plant, Foeniculum vulgare (Fennel, Hinojo), was previously discovered to have been effective at diminishing virulence factor expression of Francisella tularensis, a gram-negative bacterium with bioterror potential. The purpose of this work is to examine the therapeutic potential of dillapiole on other pathogenic bacteria such as Escherichia coli. E. coli is a motile, rod-shaped, gram-negative bacterium that is typically found in the intestines of animals and humans and can be an opportunistic pathogen. With the increase of multi-drug resistance among various bacteria, it is important for further research on new therapeutic agents. As in previous studies involving F. tularensis, data presented here suggest that dillapiole does not directly diminish the viability of E. coli. Dubia cockroach infection assays were used to evaluate the ability of dillapiole to dampen the pathogenesis of E. coli. Roaches infected with E. coli that were subsequently treated with dillapiole exhibited similar survival to those treated with tetracycline. However, cockroaches that were treated with the vehicle (DMSO) exhibited reduced levels of survival. This finding suggests that dillapiole might be dampening E. coli pathogenesis or could be modulating cockroach immunity. Ongoing studies are focusing on the effect of dillapiole on E. coli virulence gene expression (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence).&#x0D;

Role of FTL_0445 in Francisella tularensis Susceptibility to Resazurin

Tularemia is a potentially fatal disease caused by the Category A bioterrorism agent Francisella tularensis. Aminoglycosides, fluoroquinolones, and tetracyclines can be used to treat tularemia; however, there is a high incidence of relapse and treatment failures when using these drugs. Furthermore, there is no tularemia vaccine licensed for use in the United States. Therefore, new antibiotics that target F. tularensis are being investigated. A novel family of resazurin-based antibiotics called resazomycins exhibit antimicrobial activity against F. tularensis and other Gram-negative pathogens including Neisseria gonorrhoeae. The mode of action of resazomycins has yet to be determined. To elucidate potential targets of resazurin (Rz), we screened for spontaneous Rz-resistant (Rzr) F. tularensis LVS mutants. Through the screen, 93% of all Rzr mutants sequenced contained mutations within the coding regions of FTL_0421, FTL_0895, and FTL_1504. In addition, 100% of all Rzr mutants sequenced contained a mutation approximately 50bp upstream of the gene FTL_0445. To understand the effect the mutation has on transcription of FTL_0445, RNA was isolated from wild-type LVS and an Rzr mutant (Rzr1). Quantitative reverse transcription PCR revealed FTL_0445 was upregulated in Rzr1 compared to wild-type LVS. To determine the role of FTL_0445 in Rz resistance, a FTL_0445 null deletion mutant is being generated in Rzr1 using standard molecular genetic techniques. Upon completion, the Rz sensitivity of the deletion mutant will be assessed using agar dilution assays. Understanding the role of FTL_0445 in Rz susceptibility would facilitate further development of these compounds as potential treatments for tularemia and gonorrhea.

Applying RNA-Seq to investigate the transition into a Viable But Non Culturable State (VBNC) for the intracellular pathogen, Francisella tularensis LVS.

Many species of bacteria, under conditions of stress or nutrient limitation, enter a state of dormancy referred to as viable but non-culturable (VBNC). VBNC bacteria persist in the environment, are difficult to detect and identify by many standard laboratory methods and can be altered in their susceptibility to antibiotics. Entry into the VBNC state is often accompanied by morphological changes; however, the mechanisms underlying this are poorly understood. Francisella transitions rapidly and spontaneously to a VBNC state, and therefore has the potential to be an excellent model organism for the study of this phenomenon. To investigate the transcriptome of VBNC F. tularensis LVS we extracted RNA from culturable and VBNC cells and carried out an RNA-Seq analysis using both Long-read Nanopore and Illumina Sequencing. Differentially expressed genes (DEGs) were identified using a DE-Seq pipeline. Over 300 genes were significantly upregulated and ~100 genes were down regulated in VBNC cells. Amongst the upregulated genes were some involved in the transport of metals or small molecules, and we have also identified putative transcriptional regulators that may be master controllers of the VBNC process. Our data represents the first transcriptomic analysis of F. tularensis LVS as it transitions into the VBNC state. Identifying genes that are involved in this transition is a critical first step in understanding the mechanisms that drive F. tularensis LVS into the VBNC state and will help us identify factors that allow the resuscitation of these bacteria. This will have important implications for understanding both environmental persistence and pathogenicity of Francisella species. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence)

Passive collection of ticks in New Hampshire reveals species-specific patterns of distribution and activity.

Ticks and tick-borne diseases are increasing in the United States, including New Hampshire (NH). We report on the findings of an ongoing free crowdsourcing program spanning four years within NH. The date of tick's submission was recorded along with species, sex, stage, location they were collected (translated into latitude and longitude), the activity the individual was doing when the tick was found, and host species. A total of 14,252 ticks belonging to subclass Acari, family Ixodidae and genera Ixodes, Dermacentor, Amblyomma, and Haemaphysalis was recorded from the period 2018-2021 throughout NH. A total of 2,787 Ixodes scapularis and 1,041 Dermacentor variabilis, were tested for the presence of Borrelia sp. (Spirochaetales: Spirochaetaceae), B. burgdorferi sensu lato, B. miyamotoi, B. mayonii, Babesia microti (Piroplasmida: Babesiidae), Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), Francisella tularensis (Thiotrichales: Francisellaceae), and Rickettsia rickettsii (Rickettsiales: Rickettsiaceae) by PCR. For the I. scapularis ticks tested, the pathogen prevalence was 37% B. burgdorferi s.l. 1% B. miyamotoi, 6% A. phagocytophilum, and 5% Ba. microti. Only one D. variabilis resulted positive to F. tularensis. We created state-wide maps informing the differences of ticks as detailed by administrative divisions. Dermacentor variabilis peaked in June and I. scapularis peaked in May and October. The most reported activity by people with tick encounters was while walking/hiking, and the least was biking. Using the reported distribution of both species of ticks, we modeled their climate suitability in the target territory. In NH, I. scapularis and D. variabilis have distinct patterns of emergence, abundance, and distribution. Tick prevention is important especially during April-August when both tick species are abundant and active.

Liver manifestations of tick-borne diseases.

Liver manifestations of tick-borne diseases.

A rare cause requiring consideration in the differential diagnosis of neck masses: tularemia.

Tularemia is a zoonotic disease caused by the Gram-negative coccobacillus Francisella tularensis. It is frequently overlooked in the differential diagnosis of neck masses because of its rarity. The purpose of this study is to report cases diagnosed with tularemia among patients presenting to our clinic with neck masses and to share our experience. Patients presented to our hospital with cervical masses and diagnosed with tularemia were included in this retrospective study. Medical files of all patients were evaluated, and physical examination findings, titration values, date of diagnosis, location of the abscess or mass, place of residence, occupation, drinking water sources, sedimentation (SED), C-reactive protein (CRP), and white blood cell (WBC) values were recorded. Seventy-six patients were included in the study. Forty patients (52.6%) were living in rural villages and 36 (47.4%) in urban areas. Thirty-one (40.8%) were engaged in animal husbandry and 29 (38.2%) in agriculture. In terms of drinking water sources, 59 patients (73.6%) obtained water from the mains, while 10 (13.32%) used well water. The most frequently observed clinical findings were swelling in the neck, sore throat, lethargy, and fever. Neck swelling frequently occurred in levels II and III. Since tularemia is rare and there are no specific clinical findings, diagnosis may be problematic. Ear, nose and throat (ENT) specialists should be familiar with the clinical symptoms of tularemia in the head and neck region and should consider a preliminary diagnosis of tularemia in the differential diagnosis of persistent neck masses.