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Background Tularemia is an important reemerging disease with a multimodal transmission pattern. Treatment outcomes of current recommended antibiotic regimens (including ciprofloxacin and doxycycline) remain unclear. In this retrospective cohort study, we report clinical, laboratory, geographical, and treatment outcomes of laboratory-confirmed tularemia cases over an 11-year period in Northern Sweden. Methods Data from reported tularemia cases (aged >10 years at time of study) in Norrbotten county between 2011 and 2021 were collected through review of electronic medical records and participant questionnaires; 415 of 784 accepted participation (52.9%). Of these, 327 were laboratory-confirmed cases (serology and/or polymerase chain reaction). A multivariable logistic regression model was used to investigate variables associated with retreatment. Results Median age of participants was 54 years (interquartile range [IQR], 41.5–65) and 49.2% were female. Although ulceroglandular tularemia was the predominant form (n = 215, 65.7%), there were several cases of pulmonary tularemia (n = 40; 12.2%). Inflammatory markers were largely nonspecific, with monocytosis frequently observed (n = 36/75; 48%). Tularemia was often misdiagnosed on presentation (n = 158, 48.3%), with 65 (19.9%) receiving initial inappropriate antibiotics and 102 (31.2%) retreated. Persistent lymphadenopathy was infrequent (n = 22, 6.7%), with 10 undergoing surgical interventions. In multivariable analysis of variables associated with retreatment, we highlight differences in time until receiving appropriate antibiotics (8 [IQR, 3.25–20.75] vs 7 [IQR, 4–11.25] days; adjusted P =.076), and doxycycline-based treatment regimen (vs ciprofloxacin; adjusted P =.084), although this was not significant after correction for multiple comparisons. Conclusions We comprehensively summarize clinical, laboratory, and treatment outcomes of type B tularemia. Targeting tularemia requires clinical awareness, early diagnosis, and timely commencement of treatment for an appropriate duration. [ABSTRACT FROM AUTHOR]

Background: Francisella tularensis, the bacterium that causes tularemia, has been a persistent and widespread pathogen in various regions of the world for centuries. Francisella tularensis can affect humans and various domestic and wild animals. The current study aimed to determine the epidemiological status of tularemia in countries of the WHO Eastern Mediterranean Region (EMRO) through a systematic review and meta-analysis. Methods: All included studies were identified through a systematic search of online databases, including Scopus, PubMed, Web of Science, and EMBASE, through July 26, 2022, using keywords and suitable combinations. We focused on cross-sectional studies investigating the prevalence of F. tularensis. The weighted pooled prevalence was calculated using a random-effects model. Results: A total of 206 studies were identified, of which 20 were finally included in the analysis. The human seroprevalence of tularemia in WHO-EMRO countries was 6.2% (95% CI, 4.2 9.2). In the subgroup analysis, anti-F. tularensis antibodies were found in 6.92% and 5.5% of the high-risk individuals and Iran, respectively. The pooled prevalence of F. tularensis in environmental samples (water and soil) from the WHO-EMRO countries was 5.8% (9.4% by PCR and 0.5% by culture). In addition, 2.5% (95% CI, 0.2 0.22.7) of ticks in WHO-EMRO countries were positive for F. tularensis. The pooled prevalence of F. tularensis in rodents is 2.0% (1.1% by PCR and 3.7% by serology). In addition, 0.6% of domestic ruminants (0.4% by PCR and 2.4% by serology) were positive for F. tularensis in WHO-EMRO countries. Conclusion: According to the results of the present study, tularemia is an endemic but neglected disease in the WHO-EMRO region. However, most studies on tularemia are limited to a few countries in this region. Studies on tularemia in human populations, reservoirs, and vectors have been conducted in all countries in the WHO-EMRO region to obtain more detailed information about the epidemiology of tularemia in these regions. Author summary: Tularemia is a bacterial zoonotic disease caused by Francisella tularensis. This bacterium can infect animals (domestic and wild vertebrates), invertebrates, and humans. Francisella tularensis is transferred to humans through contact with infected wild animals (, inhalation of infected aerosols, arthropod bites, consumption of contaminated water or contaminated food, and swimming and contact in polluted hydro-telluric environments. The mortality rate associated with this disease ranges from 2 to 60% in the absence of proper antibiotic treatment. The prevalence of F. tularensis varies significantly between countries, as these pathogens can infect a wide range of hosts and have multiple vectors. However, there needs to be complete data on the geographical distribution of different species of F. tularensis, as well as the status of reservoirs, vectors, and human cases in most parts of the world. Therefore, this study aimed to determine F. tularensis prevalence in the countries of the WHO Eastern Mediterranean Region (WHO-EMRO). Based on our study, the human seroprevalence of tularemia in WHO-EMRO countries was 6.2%. The pooled prevalence of F. tularensis in environmental samples (water and soil), ticks, rodents, and domestic ruminants were 5.8%, 2.5%, 2.0%, and 0.6% in the WHO-EMRO countries, respectively. [ABSTRACT FROM AUTHOR]

Tularemia is a zoonotic disease caused by the facultative intracellular gram-negative bacterium Francisella tularensis. F. tularensis has a very low infection dose by the aerosol route which can result in an acute, and potentially lethal, infection in humans. Consequently, it is classified as a Category A bioterrorism agent by the US Centers for Disease Control (CDC) and is a pathogen of concern for the International Biodefence community. There are currently no licenced tularemia vaccines. In this study we report on the continued assessment of a tularemia subunit vaccine utilising β-glucan particles (GPs) as a vaccine delivery platform for immunogenic F. tularensis antigens. Using a Fischer 344 rat infection model, we demonstrate that a GP based vaccine comprising the F. tularensis lipopolysaccharide antigen together with the protein antigen FTT0814 provided partial protection of F344 rats against an aerosol challenge with a high virulence strain of F. tularensis, SCHU S4. Inclusion of imiquimod as an adjuvant failed to enhance protective efficacy. Moreover, the level of protection afforded was dependant on the challenge dose. Immunological characterisation of this vaccine demonstrated that it induced strong antibody immunoglobulin responses to both polysaccharide and protein antigens. Furthermore, we demonstrate that the FTT0814 component of the GP vaccine primed CD4+ and CD8+ T-cells from immunised F344 rats to express interferon-γ, and CD4+ cells to express interleukin-17, in an antigen specific manner. These data demonstrate the development potential of this tularemia subunit vaccine and builds on a body of work highlighting GPs as a promising vaccine platform for difficult to treat pathogens including those of concern to the bio-defence community. [ABSTRACT FROM AUTHOR]

Recombinant Francisella tularensis universal stress protein with a C-terminal histidine-tag (rUsp/His6) was expressed in Escherichia coli. Endogenous F. tularensis Usp has a predicted molecular mass of 30 kDa, but rUsp/His6 had an apparent molecular weight of 33 kDa based on Western blot analyses. To determine the source of the higher molecular weight for rUsp/His6, post translational modifications were examined. Tryptic peptides of purified rUsp/His6 were subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) and fragmentation spectra were searched for acetylated lysines and polyaminated glutamines. Of the 24 lysines in rUsp/His6, 10 were acetylated (K63, K68, K72, K129, K175, K201, K208, K212, K233, and K238) and three of the four glutamines had putrescine, spermidine and spermine adducts (Q55, Q60 and Q267). The level of post-translational modification was substoichiometric, eliminating the possibility that these modifications were the sole contributor to the 3 kDa extra mass of rUsp/His6. LC-MS/MS revealed that stop codon readthrough had occurred resulting in the unexpected addition of 20 extra amino acids at the C-terminus of rUsp/His6, after the histidine tag. Further, the finding of polyaminated glutamines in rUsp/His6 indicated that E. coli is capable of transglutaminase activity. [ABSTRACT FROM AUTHOR]

Small multidrug resistance transporters efflux toxic compounds from bacteria and are a minimal system to understand multidrug transport. Most previous studies have focused on EmrE, the model SMR from Escherichia coli, finding that EmrE has a broader substrate profile than previously thought and that EmrE may perform multiple types of transport, resulting in substrate‐dependent resistance or susceptibility. Here, we performed a broad screen to identify potential substrates of three other SMRs: PAsmr from Pseudomonas aeruginosa; FTsmr from Francisella tularensis; and SAsmr from Staphylococcus aureus. This screen tested metabolic differences in E. coli expressing each transporter versus an inactive mutant, for a clean comparison of sequence and substrate‐specific differences in transporter function, and identified many substrates for each transporter. In general, resistance compounds were charged, and susceptibility substrates were uncharged, but hydrophobicity was not correlated with phenotype. Two resistance hits and two susceptibility hits were validated via growth assays and IC50 calculations. Susceptibility is proposed to occur via substrate‐gated proton leak, and the addition of bicarbonate antagonizes the susceptibility phenotype, consistent with this hypothesis. [ABSTRACT FROM AUTHOR]

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

Rodent populations often undergo large, regular fluctuations. We studied immunological condition and physiological stress in the common vole and how it varied with environmental factors linked with population fluctuations, namely flea and Francisella tularensis (the bacterium causing tularemia) prevalence. We used two leukocyte indices, neutrophil-to-lymphocyte ratio (N:L) and proportion of eosinophils, as physiological long-term stress indicators, and measured levels of immunoglobulins (Ig) as a generic index of immunological condition. Leukocyte indices showed a hump-shaped relationship with Ig levels, consistent with an interdependence between physiological stress and immunological condition. N:L was negatively associated with vole density only in males. Eosinophil proportion was explained by the interaction between flea burden and tularemia prevalence, with highest levels in flea-infested voles also infected with tularemia. Ig levels did not vary with sex or flea prevalence but increased in tularemia-infected voles. When Ig levels were included as covariates in our models, the associations between leukocyte indices and environmental factors remained significant. Results suggest sex-specific associations between physiological stress and population density, and an influence of tularemia infection depending on ectoparasite load. We recommend using immunological parameters complementarily to leukocyte indices when studying physiological stress and infection dynamics in wild populations. [ABSTRACT FROM AUTHOR]

Background Neuroinvasive infection with Francisella tularensis , the causative agent of tularemia, is rare. Establishing clinical suspicion is challenging if risk factors or clinical features classically associated with tularemia are absent. Tularemia is treatable with antibiotics; however, there are limited data to inform management of potentially fatal neuroinvasive infection. Methods We collected epidemiologic and clinical data on 2 recent US cases of neuroinvasive F. tularensis infection, and performed a literature review of cases of neuroinvasive F. tularensis infection published after 1950. Results One patient presented with focal neurologic deficits and brain lesions; broad-range molecular testing on resected brain tissue detected F. tularensis. The other patient presented with meningeal signs; tularemia was suspected based on animal exposure, and F. tularensis grew in cerebrospinal fluid (CSF) culture. Both patients received combination antibiotic therapy and recovered from infection. Among 16 published cases, tularemia was clinically suspected in 4 cases. CSF often displayed lymphocytic pleocytosis. Among cases with available data, CSF culture was positive in 13 of 16 cases, and F. tularensis antibodies were detected in 11 of 11 cases. Treatment typically included an aminoglycoside combined with either a tetracycline or a fluoroquinolone. Outcomes were generally favorable. Conclusions Clinicians should consider neuroinvasive F. tularensis infection in patients with meningitis and signs suggestive of tularemia or compatible exposures, lymphocyte-predominant CSF, unrevealing standard microbiologic workup, or lack of response to empiric bacterial meningitis treatment. Molecular testing, culture, and serologic testing can reveal the diagnosis. Favorable outcomes can be achieved with directed antibiotic treatment. [ABSTRACT FROM AUTHOR]

Background Francisella tularensis , the causative agent of tularemia, is endemic throughout the Northern Hemisphere and requires as few as 10 organisms to cause disease, making this potential bioterrorism agent one of the most infectious bacterial pathogens known. Aminoglycosides, tetracyclines, and, more recently, fluoroquinolones are used for treatment of tularemia; however, data on the relative effectiveness of these and other antimicrobial classes are limited. Methods Nine databases, including Medline, Global Health, and Embase, were systematically searched for articles containing terms related to tularemia. Articles with case-level data on tularemia diagnosis, antimicrobial treatment, and patient outcome were included. Patient demographics, clinical findings, antimicrobial administration, and outcome (eg, intubation, fatality) were abstracted using a standardized form. Results Of the 8878 publications identified and screened, 410 articles describing 870 cases from 1993 to 2023 met inclusion criteria. Cases were reported from 35 countries; more than half were from the United States, Turkey, or Spain. The most common clinical forms were ulceroglandular, oropharyngeal, glandular, and pneumonic disease. Among patients treated with aminoglycosides (n = 452 [52%]), fluoroquinolones (n = 339 [39%]), or tetracyclines (n = 419 [48%]), the fatality rate was 0.7%, 0.9%, and 1.2%, respectively. Patients with pneumonic disease who received ciprofloxacin had no fatalities and the lowest rates of thoracentesis/pleural effusion drainage and intubation compared to those who received aminoglycosides and tetracyclines. Conclusions Aminoglycosides, fluoroquinolones, and tetracyclines are effective antimicrobials for treatment of tularemia, regardless of clinical manifestation. For pneumonic disease specifically, ciprofloxacin may have slight advantages compared to other antimicrobials. [ABSTRACT FROM AUTHOR]

Background Tularemia, a potentially fatal zoonosis caused by Francisella tularensis , has been reported from nearly all US states. Information on relative effectiveness of various antimicrobials for treatment of tularemia is limited, particularly for newer classes such as fluoroquinolones. Methods Data on clinical manifestations, antimicrobial treatment, and illness outcome of patients with tularemia are provided voluntarily through case report forms to the US Centers for Disease Control and Prevention by state and local health departments. We summarized available demographic and clinical information submitted during 2006–2021 and evaluated survival according to antimicrobial treatment. We grouped administered antimicrobials into those considered effective for treatment of tularemia (aminoglycosides, fluoroquinolones, and tetracyclines) and those with limited efficacy. Logistic regression models with a bias-reduced estimation method were used to evaluate associations between antimicrobial treatment and survival. Results Case report forms were available for 1163 US patients with tularemia. Francisella tularensis was cultured from a clinical specimen (eg, blood, pleural fluid) in approximately half of patients (592; 50.9%). Nearly three-quarters (853; 73.3%) of patients were treated with a high-efficacy antimicrobial. A total of 27 patients (2.3%) died. After controlling for positive culture as a proxy for illness severity, use of aminoglycosides, fluoroquinolones, and tetracyclines was independently associated with increased odds of survival. Conclusions Most US patients with tularemia received high-efficacy antimicrobials; their use was associated with improved odds of survival regardless of antimicrobial class. Our findings provide supportive evidence that fluoroquinolones are an effective option for treatment of tularemia. [ABSTRACT FROM AUTHOR]

Tularemia is a disease caused by Francisella tularensis , a highly infectious bacteria that can be transmitted to humans by direct contact with infected animals. Because of the potential for zoonotic transmission of F. tularensis , veterinary occupational risk is a concern. Here, we report on a human case of tularemia in a veterinarian after an accidental needlestick injury during abscess drainage in a sick dog. The veterinarian developed ulceroglandular tularemia requiring hospitalization but fully recovered after abscess drainage and a course of effective antibiotics. To systematically assess veterinary occupational transmission risk of F. tularensis , we conducted a survey of veterinary clinical staff after occupational exposure to animals with confirmed tularemia. We defined a high-risk exposure as direct contact to the infected animal's body fluids or potential aerosol inhalation without use of standard personal protective equipment (PPE). Survey data included information on 20 veterinary occupational exposures to animals with F. tularensis in 4 states. Veterinarians were the clinical staff most often exposed (40%), followed by veterinarian technicians and assistants (30% and 20%, respectively). Exposures to infected cats were most common (80%). Standard PPE was not used during 80% of exposures; a total of 7 exposures were categorized as high risk. Transmission of F. tularensis in the veterinary clinical setting is possible but overall risk is likely low. Veterinary clinical staff should use standard PPE and employ environmental precautions when handling sick animals to minimize risk of tularemia and other zoonotic infections; postexposure prophylaxis should be considered after high-risk exposures to animals with suspected or confirmed F. tularensis infection to prevent tularemia. [ABSTRACT FROM AUTHOR]

Background Tularemia is caused by the gram-negative bacterium Francisella tularensis. Although rare, tularemia during pregnancy has been associated with pregnancy complications; data on efficacy of recommended antimicrobials for treatment are limited. We performed a systematic literature review to characterize clinical manifestations of tularemia during pregnancy and examine maternal, fetal, and neonatal outcomes with and without antimicrobial treatment. Methods We searched 9 databases, including Medline, Embase, Global Health, and PubMed Central, using terms related to tularemia and pregnancy. Articles reporting cases of tularemia with ≥1 maternal or fetal outcome were included. Results Of 5891 articles identified, 30 articles describing 52 cases of tularemia in pregnant patients met inclusion criteria. Cases were reported from 9 countries, and oropharyngeal and ulceroglandular tularemia were the most common presenting forms. A plurality (46%) of infections occurred in the second trimester. Six complications were observed: lymph node aspiration, lymph node excision, maternal bleeding, spontaneous abortion, intrauterine fetal demise, and preterm birth. No deaths among mothers were reported. Of 28 patients who received antimicrobial treatment, 1 pregnancy loss and 1 fetal death were reported. Among 24 untreated patients, 1 pregnancy loss and 3 fetal deaths were reported, including one where F. tularensis was detected in placental and fetal tissues. Conclusions Pregnancy loss and other complications have been reported among cases of tularemia during pregnancy. However, risk of adverse outcomes may be lower when antimicrobials known to be effective are used. Without treatment, transplacental transmission appears possible. These data underscore the importance of prompt recognition and treatment of tularemia during pregnancy. [ABSTRACT FROM AUTHOR]

Bacterial and mammalian cells are rich in putrescine, spermidine, and spermine. Polyamines are required for optimum fitness, but the biological function of these small aliphatic compounds has only been partially revealed. Known functions of polyamines include interaction with nucleic acids that alters gene expression and with proteins that modulate activity. Although polyamines can be incorporated into proteins, very few naturally occurring polyaminated proteins have been identified, which is due in part to the difficulty in detecting these adducts. In the current study, bovine albumin and the recombinant universal stress protein from Francisella tularensis were used as models for mass spectrometry analysis of polyaminated proteins. The proteins were covalently bound to putrescine, spermidine, or spermine by the action of carbodiimide or microbial transglutaminase. Tryptic peptides, subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS), were identified using Protein Prospector software. We describe the search parameters for identifying polyaminated peptides and show MS/MS spectra for adducts with putrescine, spermidine, and spermine. Manual evaluation led us to recognize signature ions for polyamine adducts on aspartate, glutamate, and glutamine, as well as neutral loss from putrescine, spermidine, and spermine during the fragmentation process. Mechanisms for the formation of signature ions and neutral loss are presented. Manual evaluation identified a false-positive adduct that had formed during trypsinolysis and resulted in peptide sequence rearrangement. Another false positive initially appeared to be a 71 kDa putrescine adduct on a cysteine residue. However, it was an acrylamide adduct on cysteine for a sample extracted from a polyacrylamide gel. The information presented in this report provides guidance and serves as a model for identifying naturally occurring polyaminated proteins. [ABSTRACT FROM AUTHOR]

Background Seroepidemiologic studies of human tularemia have been conducted throughout the northern hemisphere. The purposes of this study were (1) to provide an overview of Francisella tularensis seroprevalence data, and (2) to generate an estimate of the proportion of study participants whose infection remained subclinical. Methods We conducted a systematic review of F tularensis seroprevalence studies according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. We searched PubMed, Embase, and Web of Science covering the period from 1951 to 2023. Results The weighted pooled seroprevalence among 44 486 participants recruited in 52 studies was 3.7% (95% confidence interval [CI], 2.7–5.1). Reported seroprevalences ranged between 0.2% and 31.3%. Occupational activities associated with an increased likelihood of exposure (risk ratio, 3.51 [95% CI, 3.2–3.86]) and studies from North America versus Europe and Asia (4.53 [4.15–4.94]) were associated with significantly increased seropositive rates. Twenty-eight data sets (47%) reported clinical information on a total of 965 seropositive participants. The weighted pooled estimate for subclinical seropositivity was 84.4% (95% CI, 72.9%–991.7%). Studies from F tularensis type A areas (risk ratio, 0.37 [95% CI,.27–.51) and studies from sites where pulmonary tularemia prevailed (0.38 [.28–.51]) reported lower subclinical seropositivity rates than studies from type B areas and from areas of predominance of (ulcero)glandular or oropharyngeal tularemia, respectively. Conclusions Throughout the northern hemisphere, only a small proportion of study participants showed serologic evidence of exposure to F tularensis. Eight of 10 seropositive participants had no historical evidence of past clinical tularemia. [ABSTRACT FROM AUTHOR]

Background Seroepidemiologic studies of human tularemia have been conducted throughout the northern hemisphere. The purposes of this study were (1) to provide an overview of Francisella tularensis seroprevalence data, and (2) to generate an estimate of the proportion of study participants whose infection remained subclinical. Methods We conducted a systematic review of F tularensis seroprevalence studies according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. We searched PubMed, Embase, and Web of Science covering the period from 1951 to 2023. Results The weighted pooled seroprevalence among 44 486 participants recruited in 52 studies was 3.7% (95% confidence interval [CI], 2.7–5.1). Reported seroprevalences ranged between 0.2% and 31.3%. Occupational activities associated with an increased likelihood of exposure (risk ratio, 3.51 [95% CI, 3.2–3.86]) and studies from North America versus Europe and Asia (4.53 [4.15–4.94]) were associated with significantly increased seropositive rates. Twenty-eight data sets (47%) reported clinical information on a total of 965 seropositive participants. The weighted pooled estimate for subclinical seropositivity was 84.4% (95% CI, 72.9%–991.7%). Studies from F tularensis type A areas (risk ratio, 0.37 [95% CI,.27–.51) and studies from sites where pulmonary tularemia prevailed (0.38 [.28–.51]) reported lower subclinical seropositivity rates than studies from type B areas and from areas of predominance of (ulcero)glandular or oropharyngeal tularemia, respectively. Conclusions Throughout the northern hemisphere, only a small proportion of study participants showed serologic evidence of exposure to F tularensis. Eight of 10 seropositive participants had no historical evidence of past clinical tularemia. [ABSTRACT FROM AUTHOR]

Vaccine strains Yersinia pestis EV NIIEG at a dose of 103 CFU and Francisella tularensis 15 NIIEG at a dose of 102 CFU induced changes in the concentration of cyclic nucleotides in the thymus and spleen of white mice. Antigen-induced changes in the cAMP/cGMP ratio in immunocompetent organs had a phase or oscillatory character, which seems to be related to the regulation of postvaccination immunoreactivity in the body. Synthetic organoselenium compound 974zh stimulated an increase in the amplitude of cAMP/cGMP oscillations, indicating its stimulating effect on the immunogenic properties of vaccine strains at doses an order of magnitude below the standard doses. [ABSTRACT FROM AUTHOR]

For stochastic models with large numbers of states, analytical techniques are often impractical, and simulations time-consuming and computationally demanding. This limitation can hinder the practical implementation of such models. In this study, we demonstrate how neural networks can be used to develop emulators for two outputs of a stochastic within-host model of Francisella tularensis infection: the dose-dependent probability of illness and the incubation period. Once the emulators are constructed, we employ Markov Chain Monte Carlo sampling methods to parameterize the within-host model using records of human infection. This inference is only possible through the use of a mixture density network to emulate the incubation period, providing accurate approximations of the corresponding probability distribution. Notably, these estimates improve upon previous approaches that relied on bacteria counts from the lungs of macaques. Our findings reveal a 50% infectious dose of approximately 10 colony-forming units and we estimate that the incubation period can last for up to 11 days following low dose exposure. Author summary: Francisella tularensis is a highly infectious bacterium that remains in the top category of biothreat agents. Release of aerosolized bacteria could lead to many cases of acute and severe pneumonia over the days following. Mathematical modelling can contribute to the response to such an outbreak, combining dispersion models and disease models to identify the source of release and predict where cases are most likely to occur. However, these models can be computationally demanding and time consuming to run. In this article, we use neural networks to emulate the likelihood of disease and the duration of the incubation period from a stochastic within-host model. This enables rapid predictions to be made across a wide range of doses, thereby improving the practical applications of the model. [ABSTRACT FROM AUTHOR]

Background: Over 200 different animal species are susceptible to infection by tularemia, highly infectious disease caused by Francisella tularensis. Hares (Lepus sp.) and small rodents, such as common vole (Microtus arvalis), have been acknowledged as the most significant sources of human tularemia infection in most European countries. Our objective was to verify ability of these species in predicting incidence of human tularemia in a Central European country, the Czech Republic. Materials and Methods: We used 17 years of data on densities of European hare (Lepus europaeus) and common vole, and climate variability to test effects of these factors on temporal dynamics of tularemia incidence. The data were obtained from annual reports available from online e-repositories. Results: The analysis showed that 33% of the yearly variation in human tularemia incidence was explained solely by the abundance of European hare in the Czech Republic during 2007–2022. Density of common vole and North Atlantic Oscillation index, a measure of climate variability, did not significantly explain tularemia incidence. While hare population declined severely during 1993–2022, we did not detect any clear accompanied decrease in the prevalence of tularemia in humans and hares. Conclusion: Contrary to expectations, only hares proved capable in predicting yearly dynamics in human tularemia incidence in the Czech Republic. We call for continued monitoring of infection rates in hares and advocate the use of hunter estimates of hare abundance as a cheap and effective means of predicting the risk of tularemia. [ABSTRACT FROM AUTHOR]

Anthropogenic stressors are exacerbating the emergence and spread of pathogens worldwide. In regions like the Arctic, where ecosystems are particularly susceptible, marked changes are predicted in regional diversity, intensity, and patterns of infectious diseases. To understand such rapidly changing host‐pathogen dynamics and mitigate the impacts of novel pathogens, we need sensitive disease surveillance tools. We developed and validated a novel multiplexed, magnetic capture, and ddPCR tool for the surveillance of multiple pathogens in polar bears, a sentinel species that is considered susceptible to climate change and other stressors with a pan‐Arctic distribution. Through sequence‐specific magnetic capture, we concentrated five target template sequences from three zoonotic bacteria (Erysipelothrix rhusiopathiae, Francisella tularensis, and Mycobacterium tuberculosis complex) and two parasitic (Toxoplasma gondii and Trichinella spp.) pathogens from large quantities (<100 g) of host tissue. We then designed and validated two multiplexed probe‐based ddPCR assays for the amplification and detection of the low‐concentration target DNA. Validations used 48 polar bear tissues (muscle and liver). We detected 14, 1, 3, 4, and 22 tissue positives for E. rhusiopathiae, F. tularensis, M. tuberculosis complex, T. gondii, and Trichinella spp., respectively. These multiplexed assays offer a rapid, specific tool for quantifying and monitoring the changing geographical and host distributions of pathogens relevant to human and animal health. [ABSTRACT FROM AUTHOR]

Tularemia is increasing in Austria. We report Francisella tularensis subspecies holarctica isolated from 3 patients who had been bitten by arthropods. Next-generation sequencing showed substantial isolate similarity. Clinicians should consider bloodstream F. tularensis infections for patients with signs/symptoms of ulceroglandular tularemia, and surveillance of potential vectors should be intensified. [ABSTRACT FROM AUTHOR]

Route of exposure to pathogens can inform divergent disease pathogenesis and mortality rates. However, the features that contribute to these differences are not well established. Host metabolism has emerged as a critical element governing susceptibility and the metabolism of tissue exposure sites are unique. Therefore, specific metabolic niches may contribute to the course and outcome of infection depending on route of infection. In the current study, we utilized a combination of imaging and systems metabolomics to map the spatiotemporal dynamics of the host response to intranasal (i.n.) or intradermal (i.d.) infection of mice using the bacterium Francisella tularensis subsp tularensis (FTT). FTT causes lethal disease through these infection routes with similar inoculation doses and replication kinetics, which allowed for isolation of host outcomes independent of bacterial burden. We observed metabolic modifications that were both route dependent and independent. Specifically, i.d. infection resulted in early metabolic reprogramming at the site of infection and draining lymph nodes, whereas the lungs and associated draining lymph nodes were refractory to metabolic reprogramming following i.n. infection. Irrespective of exposure route, FTT promoted metabolic changes in systemic organs prior to colonization, and caused massive dysregulation of host metabolism in these tissues prior to onset of morbidity. Preconditioning infection sites towards a more glycolytic and pro-inflammatory state prior to infection exacerbated FTT replication within the lungs but not intradermal tissue. This enhancement of replication in the lungs was associated with the ability of FTT to limit redox imbalance and alter the pentose phosphate pathway. Together, these studies identify central metabolic features of the lung and dermal compartments that contribute to disease progression and identify potential tissue specific targets that may be exploited for novel therapeutic approaches. [ABSTRACT FROM AUTHOR]

Background: Brucellosis is a common zoonotic disease caused by Brucella, which causes enormous economic losses and public burden to epidemic areas. Early and precise diagnosis and timely culling of infected animals are crucial to prevent the infection and spread of Brucella. In recent years, RNA-guided CRISPR/Cas12a(Clustered Regularly Interspaced Short Palindromic Repeats and its associated protein 12a) nucleases have shown great promise in nucleic acid detection. This research aims to develop a CRISPR/CAST (CRISPR/Cas12a Test strip) package that can rapidly detect Brucella nucleic acid during on-site screening, especially on remote family pastures. The CRISPR/Cas12a system combined with recombinase polymerase amplification (RPA), and lateral flow read-out. Results: We selected the conserved gene bp26, which commonly used in Brucella infection detection and compared on Genbank with other Brucella species. The genomes of Brucella abortus 2308, Brucella suis S2, Brucella melitansis 16 M, and Brucella suis 1330, et al. were aligned, and the sequences were found to be consistent. Therefore, the experiments were only performed on B. melitensis. With the CRISPR/CAST package, the assay of Brucella nucleic acid can be completed within 30 min under isothermal temperature conditions, with a sensitivity of 10 copies/μl. Additionally, no antigen cross-reaction was observed against Yersinia enterocolitica O:9, Escherichia coli O157, Salmonella enterica serovar Urbana O:30, and Francisella tularensis. The serum samples of 398 sheep and 100 cattle were tested by the CRISPR/CAST package, of which 31 sheep and 8 cattle were Brucella DNA positive. The detection rate was consistent with the qPCR results and higher than that of the Rose Bengal Test (RBT, 19 sheep and 5 cattle were serum positive). Conclusions: The CRISPR/CAST package can accurately detect Brucella DNA in infected livestock within 30 min and exhibits several advantages, including simplicity, speed, high sensitivity, and strong specificity with no window period. In addition, no expensive equipment, standard laboratory, or professional operators are needed for the package. It is an effective tool for screening in the field and obtaining early, rapid diagnoses of Brucella infection. The package is an efficient tool for preventing and controlling epidemics. [ABSTRACT FROM AUTHOR]

Background:Francisella tularensis is a Gram-negative bacterium that causes tularemia in both human and animals. Tularemia is a potential serious zoonotic disease that is transmitted by different routes, including tick bites. Materials and Methods: This study deals with investigating the prevalence of F. tularensis in the ticks of local animal farms in Kurdistan region since the farmers are normally in close contact with livestock. We used molecular methods for this purpose. A total of 412 tick and 126 blood samples were gathered from goat, sheep, and cow flocks. The existence of F. tularensis 16Sr RNA gene was examined in the samples using nested-PCR technique. Results: In the animal blood specimens, no F. tularensis was found. The incidence of F. tularensis was 1.7% (7 out of 412) in the tick samples, representing a very lower possibility of tuleremia infection. Moreover, the two subspecies of F. tularensis novicida and holarctica were identified based on the sequencing of pdpD and RD genes, respectively. The F. tularensis subsp. novicida was isolated from four species of ticks, Hyalomma anatolicum, Rhipicephalus annulatus, Rhipicephalus sanguineus, and Ornithodoros spp., whereas the F. tularensis subsp. holarctica was isolated from Haemaphysalis parva and Hyalomma dromedarii species of ticks. Conclusion: Although its prevalence is very low, the isolation of F. tularensis subsp. holarctica from the ticks of farm animals suggests possible transmission of Tularemia through tick bite in Kurdistan region of Iraq. Ref: IR-UU-AEC-3/22. [ABSTRACT FROM AUTHOR]

Francisella tularensis is the causative agent of tularemia. We tested the susceptibility of 278 F. tularensis isolates from the United States received during 2009–2018 to 8 antimicrobial drugs (ciprofloxacin, levofloxacin, doxycycline, tetracycline, gentamicin, streptomycin, chloramphenicol, and erythromycin). All isolates were susceptible to all tested drugs. [ABSTRACT FROM AUTHOR]

Objective: We aim to investigate the species composition of ticks and the pathogen characteristics they carry in the Argun port area of the China–Russia border. Materials and Methods: Ticks were collected in surrounding grassland, mixed forest land, and other different habitats around the Argun port area at the Sino-Russian Border of Inner Mongolia in China in April 2019. The presence of 16 potential pathogens, including Yersinia Pestis, Francisella tularensis, Coxiella burnetii (Cb), Anaplasma sp. (Ap), spotted fever group rickettsiae (SFG Rk), Borrelia sp. (Bl), Leptospira, Bartonella spp., Babesia, Crimean-Congo hemorrhagic fever virus, tick-borne encephalitis virus, Bhanja virus, West Nile Virus, severe fever with thrombocytopenia syndrome bunyavirus, Hantaan virus, and bocavirus (boca) was analyzed by polymerase chain reaction. The DNA and amino acid sequences of tick-borne pathogens were compared for homology, and the phylogenetic trees were constructed by using Mega and Lasergene software. Results: A total of 210 ticks were collected and they belonged to three species: Dermacentor nuttalli, Ixodes persulcatus, and Haemaphysalis verticalis. Among them, 165 (78.57%) ticks tested positive for 5 pathogens, namely Ap, SFG Rk, Cb, Bl, and boca. Fifteen (7.14%) ticks were detected coinfection with two pathogens, and none were coinfected with three or more pathogens. Conclusion: This study shows the prevalence of at least five tick-borne pathogens in Argun, and there is a risk of coinfection by two pathogens in one tick. This study reveals the great importance of controlling tick-borne diseases in this region. [ABSTRACT FROM AUTHOR]

A previously healthy man in Austria had tularemia epididymo-orchitis develop, leading to unilateral orchiectomy. Francisella tularensis subspecies holartica was detected by 16S rRNA gene sequencing analysis of inflamed granulomatous testicular tissue. Clinicians should suspect F. tularensis as a rare etiologic microorganism in epididymo-orchitis patients with relevant risk factors. [ABSTRACT FROM AUTHOR]

Class II Fructose-1,6-bisphosphatases (FBPaseII) (EC: 3.1.3.11) are highly conserved essential enzymes in the gluconeogenic pathway of microorganisms. Previous crystallographic studies of FBPasesII provided insights into various inactivated states of the enzyme in different species. Presented here is the first crystal structure of FBPaseII in an active state, solved for the enzyme from Francisella tularensis (FtFBPaseII), containing native metal cofactor Mn2+ and complexed with catalytic product fructose-6-phosphate (F6P). Another crystal structure of the same enzyme complex is presented in the inactivated state due to the structural changes introduced by crystal packing. Analysis of the interatomic distances among the substrate, product, and divalent metal cations in the catalytic centers of the enzyme led to a revision of the catalytic mechanism suggested previously for class II FBPases. We propose that phosphate-1 is cleaved from the substrate fructose-1,6-bisphosphate (F1,6BP) by T89 in a proximal α-helix backbone (G88-T89-T90-I91-T92-S93-K94) in which the substrate transition state is stabilized by the positive dipole of the 〈-helix backbone. Once cleaved a water molecule found in the active site liberates the inorganic phosphate from T89 completing the catalytic mechanism. Additionally, a crystal structure of Mycobacterium tuberculosis FBPaseII (MtFBPaseII) containing a bound F1,6BP is presented to further support the substrate binding and novel catalytic mechanism suggested for this class of enzymes. [ABSTRACT FROM AUTHOR]

Bacterial pathogens have always been a part of the ecosystem in which we thrive. Some pathogens have caused deadly outbreaks in the past and have been exploited as an agent of threat. Natural hotspots for these biological pathogens are widely distributed throughout the world and hence they remain clinically important. Technological advancement and change in general lifestyle has driven the evolution of these pathogens into more virulent and resistant variants. There has been a growing concern over the development of multidrug-resistant bacterial strains that could be used as bioweapons. This rapid change in pathogens also propels the field of science to develop and innovate new strategies and methodologies which are superior and safer to the existing ones. Some bacterial agents like—Bacillus anthracis, Yersinia pestis, Francisella tularensis and toxins produced by strains of Clostridium botulinum, have been segregated as Category A substances as they pose imminent threat to public health with a history of life threatening and catastrophic disease. This review highlights some encouraging developments and value additions in the current plan of action for protection against these select biothreat bacterial pathogens. [ABSTRACT FROM AUTHOR]

Tick-borne infections, including those of bacterial origin, are significant public health issues. Antimicrobial resistance (AMR), which is one of the most pressing health challenges of our time, is driven by specific genetic determinants, primarily by the antimicrobial resistance genes (ARGs) of bacteria. In our work, we investigated the occurrence of ARGs in the genomes of tick-borne bacterial species that can cause human infections. For this purpose, we processed short/long reads of 1550 bacterial isolates of the genera Anaplasma (n = 20), Bartonella (n = 131), Borrelia (n = 311), Coxiella (n = 73), Ehrlichia (n = 13), Francisella (n = 959) and Rickettsia (n = 43) generated by second/third generation sequencing that have been freely accessible at the NCBI SRA repository. From Francisella tularensis, 98.9% of the samples contained the FTU-1 beta-lactamase gene. However, it is part of the F. tularensis representative genome as well. Furthermore, 16.3% of them contained additional ARGs. Only 2.2% of isolates from other genera (Bartonella: 2, Coxiella: 8, Ehrlichia: 1, Rickettsia: 2) contained any ARG. We found that the odds of ARG occurrence in Coxiella samples were significantly higher in isolates related to farm animals than from other sources. Our results describe a surprising lack of ARGs in these bacteria and suggest that Coxiella species in farm animal settings could play a role in the spread of AMR. [ABSTRACT FROM AUTHOR]

The suppressor of TCR signaling (Sts) proteins, Sts-1 and Sts-2, are a pair of closely related signaling molecules that belong to the histidine phosphatase (HP) family of enzymes by virtue of an evolutionarily conserved C-terminal phosphatase domain. HPs derive their name from a conserved histidine that is important for catalytic activity and the current evidence indicates that the Sts HP domain plays a critical functional role. Sts-1HP has been shown to possess a readily measurable protein tyrosine phosphatase activity that regulates a number of important tyrosine-kinase-mediated signaling pathways. The in vitro catalytic activity of Sts-2HP is significantly lower than that of Sts-1HP, and its signaling role is less characterized. The highly conserved unique structure of the Sts proteins, in which additional domains, including one that exhibits a novel phosphodiesterase activity, are juxtaposed together with the phosphatase domain, suggesting that Sts-1 and -2 occupy a specialized intracellular signaling niche. To date, the analysis of Sts function has centered predominately around the role of Sts-1 and -2 in regulating host immunity and other responses associated with cells of hematopoietic origin. This includes their negative regulatory role in T cells, platelets, mast cells and other cell types, as well as their less defined roles in regulating host responses to microbial infection. Regarding the latter, the use of a mouse model lacking Sts expression has been used to demonstrate that Sts contributes non-redundantly to the regulation of host immunity toward a fungal pathogen (C. albicans) and a Gram-negative bacterial pathogen (F. tularensis). In particular, Sts-/- animals demonstrate significant resistance to lethal infections of both pathogens, a phenotype that is correlated with some heightened anti-microbial responses of phagocytes derived from mutant mice. Altogether, the past several years have seen steady progress in our understanding of Sts biology. [ABSTRACT FROM AUTHOR]

Background: Bacterial epidemiology needs to understand the spread and dissemination of strains in a One Health context. This is important for highly pathogenic bacteria such as Bacillus anthracis, Brucella species, and Francisella tularensis. Whole genome sequencing (WGS) has paved the way for genetic marker detection and high-resolution genotyping. While such tasks are established for Illumina short-read sequencing, Oxford Nanopore Technology (ONT) long-read sequencing has yet to be evaluated for such highly pathogenic bacteria with little genomic variations between strains. In this study, three independent sequencing runs were performed using Illumina, ONT flow cell version 9.4.1, and 10.4 for six strains of each of Ba. anthracis, Br. suis and F. tularensis. Data from ONT sequencing alone, Illumina sequencing alone and two hybrid assembly approaches were compared. Results: As previously shown, ONT produces ultra-long reads, while Illumina produces short reads with higher sequencing accuracy. Flow cell version 10.4 improved sequencing accuracy over version 9.4.1. The correct (sub-)species were inferred from all tested technologies, individually. Moreover, the sets of genetic markers for virulence, were almost identical for the respective species. The long reads of ONT allowed to assemble not only chromosomes of all species to near closure, but also virulence plasmids of Ba. anthracis. Assemblies based on nanopore data alone, Illumina data alone, and both hybrid assemblies correctly detected canonical (sub-)clades for Ba. anthracis and F. tularensis as well as multilocus sequence types for Br. suis. For F. tularensis, high-resolution genotyping using core-genome MLST (cgMLST) and core-genome Single-Nucleotide-Polymorphism (cgSNP) typing produced highly comparable results between data from Illumina and both ONT flow cell versions. For Ba. anthracis, only data from flow cell version 10.4 produced similar results to Illumina for both high-resolution typing methods. However, for Br. suis, high-resolution genotyping yielded larger differences comparing Illumina data to data from both ONT flow cell versions. Conclusions: In summary, combining data from ONT and Illumina for high-resolution genotyping might be feasible for F. tularensis and Ba. anthracis, but not yet for Br. suis. The ongoing improvement of nanopore technology and subsequent data analysis may facilitate high-resolution genotyping for all bacteria with highly stable genomes in future. [ABSTRACT FROM AUTHOR]

Objective: As part of a research aiming at presenting an alternative approach for rapid determination of antimicrobial susceptibility by quantification of changes in expression levels of specific marker genes and gene sets, cultures of the virulent bacterial strain Francisella tularensis SchuS4 were grown in the presence of inhibitory/sub-inhibitory concentrations of either ciprofloxacin or doxycycline and their transcriptomic profiles were elucidated using differential expression analysis followed by functional annotation. Data description: RNA sequencing was performed to identify differentially expressed genes (DEGs) in response to exposure of F. tularensis SchuS4 to either ciprofloxacin or doxycycline, the antibiotics of choice for Tularemia therapy. Accordingly, RNA samples were collected 2 h post antibiotic exposure and subjected to RNA sequence analysis. Transcriptomic quantification of RNA representing duplicated samples generated highly similar gene expression data. Exposure to sub-inhibitory concentration [0.5 x MIC (minimal inhibitory concentration)] of doxycycline or ciprofloxacin modulated the expression of 237 or 8 genes, respectively, while exposure to an inhibitory concentration (1 x MIC) resulted in the modulation of 583 or 234 genes, respectively. Amongst the genes modulated upon doxycycline exposure upregulation of 31 genes encoding for translation-functions could be distinguished, as well as downregulation of 14 genes encoding for functions involved in DNA transcription and repair. Ciprofloxacin exposure impacted differently the RNA sequence profile of the pathogen, resulting in upregulation of 27 genes encoding mainly DNA replication and repair functions, transmembrane transporters and molecular chaperons. In addition, 15 downregulated genes were involved in translation processes. [ABSTRACT FROM AUTHOR]

Historically, Francisella tularensis (Ft) is endemic in Armenia. In Europe, specifically in Scandinavian countries (Finland and Sweden), therewereseveral outbreaks reported over the last 30 years. In these areas, the disease peaks are recorded between June and September, with more than 50 % of cases occurring during August, corresponding to mosquito emerging season. Although most of the recently reported (2017/23; 2018/5; 2019/4) cases inthree geographic clusters in Tavush marz without previously documented tularemia casesand with not detected source of infection and a mode of transmission. All patients with glandular and ulceroglandular forms of tularemia reported onset of symptoms between July and September.Тhe relationship between mosquito bites and tularemia is based on epidemiological and clinical data. [ABSTRACT FROM AUTHOR]

Ticks are obligatory hematophagous parasites that serve as vectors for a large amount of important human and livestock pathogens around the world, and their distribution and incidence of tick-associated diseases are currently increasing because of environmental biomass being modified by both climate change and other human activities. Hyalomma species are of major concern for public health, due to their important role as vectors of several diseases such as the Crimea–Congo hemorrhagic fever (CCHF) virus in humans or theileriosis in cattle. Characterizing the Hyalomma-associated microbiota and delving into the complex interactions between ticks and their bacterial endosymbionts for host survival, development, and pathogen transmission are fundamental, as it may provide new insights and spawn new paradigms to control tick-borne diseases. Francisella-like endosymbionts (FLEs) have recently gained importance, not only as a consequence of the public health concerns of the highly transmissible Francisella tularensis, but for the essential role of FLEs in tick homeostasis. In this comprehensive review, we discuss the growing importance of ticks associated with the genus Hyalomma, their associated tick-borne human and animal diseases in the era of climate change, as well as the role of the microbiome and the FLE in their ecology. We compile current evidence from around the world on FLEs in Hyalomma species and examine the impact of new molecular techniques in the study of tick microbiomes (both in research and in clinical practice). Lastly, we also discuss different endosymbiont-directed strategies for the control of tick populations and tick-borne diseases, providing insights into new evidence-based opportunities for the future. [ABSTRACT FROM AUTHOR]

Background:Brucella microti is a pathogen of rodents and wild mammals. Here, we report the first probable infection with B. microti in a mammalogist. Materials and Methods: In the study, we provided complete clinical description as well as laboratory analysis of probable human infection caused by B. microti. Results: Considering the clinical course of the infection, the obvious epidemiological link (a bite by an infected rodent), the isolation of a pathogen from a sick vole that was affected by clinical infection with B. microti, and the specific serological response (slow agglutination test) in human patient, we can conclude that the human disease described here was probably caused by B. microti, an emerging bacterial pathogen transmitted by rodents. Conclusion: Rodents and other wildlife need to be monitored not only for established zoonotic agents such as hantaviruses, lymphocytic choriomeningitis virus, Leptospira spp., Francisella tularensis, but also for Brucella microti and other atypical rodent-borne brucellae. [ABSTRACT FROM AUTHOR]

Due to suspicion of lymphoma or lung cancer 1 week later, an SP 18 sp FDG (B-D) plus a SP 68 sp Ga-labeled fibroblast activation protein inhibitor ( SP 68 sp Ga-FAPI) PET/CT scan (E-G) were performed. However, given an already known diagnosis of granulomatous disease like tularemia, SP 68 sp Ga-FAPI PET/CT might be suitable to assess extent and activity of chronic inflammation. [Extracted from the article]

IL-12p40 plays an important role in F. tularensis Live Vaccine Strain (LVS) clearance that is independent of its functions as a part of the heterodimeric cytokines IL-12p70 or IL-23. In contrast to WT, p35, or p19 knockout (KO) mice, p40 KO mice infected with LVS develop a chronic infection that does not resolve. Here, we further evaluated the role of IL-12p40 in F. tularensis clearance. Despite reduced IFN-γ production, primed splenocytes from p40 KO and p35 KO mice appeared functionally similar to those from WT mice during in vitro co-culture assays of intramacrophage bacterial growth control. Gene expression analysis revealed a subset of genes that were upregulated in re-stimulated WT and p35 KO splenocytes, but not p40 KO splenocytes, and thus are candidates for involvement in F. tularensis clearance. To directly evaluate a potential mechanism for p40 in F. tularensis clearance, we reconstituted protein levels in LVS-infected p40 KO mice using either intermittent injections of p40 homodimer (p80) or treatment with a p40-producing lentivirus construct. Although both delivery strategies yielded readily detectable levels of p40 in sera and spleens, neither treatment had a measurable impact on LVS clearance by p40 KO mice. Taken together, these studies demonstrate that clearance of F. tularensis infection depends on p40, but p40 monomers and/or dimers alone are not sufficient. [ABSTRACT FROM AUTHOR]

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known for its multifunctionality in several pathogenic bacteria. Our previously reported data suggest that the GAPDH homologue of Francisella tularensis, GapA, might also be involved in other processes beyond metabolism. In the present study, we explored GapA's potential implication in pathogenic processes at the host cell level. Using immunoelectron microscopy, we demonstrated the localization of this bacterial protein inside infected macrophages and its peripheral distribution in bacterial cells increasing with infection time. A quantitative proteomic approach based on stable isotope labeling of amino acids in cell culture (SILAC) combined with pull-down assay enabled the identification of several of GapA's potential interacting partners within the host cell proteome. Two of these partners were further confirmed by alternative methods. We also investigated the impact of gapA deletion on the transcription of selected cytokine genes and the activation of the main signaling pathways. Our results show that ∆gapA-induced transcription of genes encoding several cytokines whose expressions were not affected in cells infected with a fully virulent wild-type strain. That might be caused, at least in part, by the detected differences in ERK/MAPK signaling activation. The experimental observations together demonstrate that the F. tularensis GAPDH homologue is directly implicated in multiple host cellular processes and, thereby, that it participates in several molecular mechanisms of pathogenesis. [ABSTRACT FROM AUTHOR]

Succinic acid (SA), a key intermediate in the cellular tricarboxylic acid cycle (TCA), is a 4-carbon dicarboxylic acid of great industrial value. Actinobacillus succinogenes can ferment various carbon sources and accumulate relatively high concentrations of SA, but few reliable genetic engineering tools exist for A. succinogenes and this has hindered strain improvement to increase SA production for industrial application. Two different repressors, endonuclease-deactivated Cas9 (dCas9) from Streptococcus pyogenes and Cpf1 (dCpf1) from Francisella tularensis, were applied to construct a CRISPRi system in A. succinogenes. Codon-optimized Cas9 and native Cpf1 were successfully expressed in A. succinogenes, and the corresponding sgRNA and crRNA expression elements, promoted by the fumarate reductase promoter, frd, were introduced into the CRISPRi plasmid. The highest repression of the ackA gene (encoding acetate kinase) and thereby acetic acid production (~ eightfold) was achieved by the dCpf1-based CRISPRi system, in which the mutation site, E1006A acted at the start of the coding region of ackA, the gene which regulates acetic acid biosynthesis. Compared with the ackA gene knockout mutant, cell growth was moderately improved and SA production increased by 6.3%. Further, the SA titer and productivity in a 3 L fermenter reached 57.06 g/L and 1.87 g/L/h, and there was less acetic acid production. A dCpf1-based CRISPRi-mediated gene repression system was successfully established for the first time, providing a simple and effective tool for studying functional genomics in A. succinogenes and optimizing SA production. Keypoints: CRISPRi based on FnCpf1 and SpCas9opt was established in A. succinogenes. The target gene, ackA was repressed ~ eightfold by CRISPR-dCpf1. Repression of ackA increased succinate yield compared with ackA deletion. [ABSTRACT FROM AUTHOR]

BACKGROUND: Nicotinamide mononucleotide (NMN), a key intermediate of nicotinamide adenine dinucleotide, plays an important in anti‐aging and disease. Lactococcus lactis, an important probiotic lactic acid bacteria (LAB), has shown great potential for the biosynthesis of NMN, which will significantly affect the probiotic effects of the dairy products. RESULTS: We used the CRISPR/nCas9 technique to knockout nadR gene of L. lactis NZ9000 to enhance the accumulation of NMN by 61%. The nadE* gene from Francisella tularensis with codon optimization was heterologous in L. lactis NZ9000ΔnadR and has a positive effect on NMN production. Combined with optimization of the concentration of substrate nicotinamide, a final intracellular NMN titer was 2289 μmol L–1 mg–1 with 10 g L–1 nicotinamide supplement, which was 5.7‐fold higher than that of the control. The transcription levels of key genes (pncA, nadD and prs1) involved in NMN biosynthesis were up‐regulated by more than two‐fold, indicating that the increase of NMN titer was attributed to FtnadE* heterologous expression. CONCLUSION: Our study provides a better understanding of the NMN biosynthesis pathway in L. lactis, and can facilitate NMN production in LAB via synthetic biology approaches. © 2022 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Background/aim: According to Egyptian records, tularemia emerged in the Canaan region, where it was first identified and spread to Anatolia over the Euphrates. It was used as an active biological weapon for the first time in the Hittite-Arzawa War in 1320–1318 BC. This study aimed to investigate the seroprevalence of tularemia in the Inner Aegean Region, which is thought to be the region where this war was fought 3300 years ago. Materials and methods: Tularemia seropositivity in humans was investigated in 27 villages/neighborhoods in 3 districts in each of Manisa, Kütahya, and Uşak provinces. Before the study, the participants were informed about the disease via posters, and their blood samples were taken following filling out the questionnaire. Microagglutination tests were performed using in-house tularemia antigen and V plate for serological experiments. Rose-Bengal test was also performed on seropositive sera. Results: Of the total of 410 people, 226 (55.12%) were male. The mean age of the volunteers was 43.72 years. The highest participation was from Kütahya Province. According to the results of the tularemia microagglutination test, seropositivity was detected in 6 cases. It was determined that all of the seropositive volunteers were in Kütahya. When the tularemia antibody titers were examined, seropositivity was determined at 1/20–1/160 titers. No positivity was detected in the Rose-Bengal test for cross-reaction. Conclusion: Kütahya has been identified as a risky region in terms of tularemia in the Inner Aegean Region. In order to use the resources in the country economically, first of all, the risk areas in terms of tularemia should be determined by serological studies in all regions. In order to increase awareness about the disease, physicians and filiation teams should be trained in risky areas. Surveillance studies should be conducted to identify and monitor possible sources in areas identified as risky. [ABSTRACT FROM AUTHOR]

Aim: Francisella tularensis is a Gram-negative coccobacillus and is the causative agent of tularemia, which is endemic in our country. The most common clinical form in Turkey is the oropharyngeal form. Sensitive lymphadenopathy is the most important finding, and fever, fatigue, and muscle and joint pain may occur in all clinical forms. Rodents such as rabbits, mice, and squirrels are the main reservoirs for humans, and the transmission is through contact with infected animal secretions and organs, contaminated water, and food. This study aimed to examine the socio-demographic, epidemiological, and clinical features of cases diagnosed with tularemia. Materials and Methods: Among the 583 patients whose serum samples were sent with a preliminary diagnosis of tularemia between 2011 and 2021, tularemia microagglutination test result (MAT) =1/160 titer, 18 years and older cases were included in the study. Results: A total of 24 tularemia cases were detected, with a mean age of 43.3±17 years, 10 (41.7%) were male, and 14 (58.3%) were female. The most common symptoms and findings among the cases were lymphadenopathy (LAP) (95.8%), fatigue (66.7%), sore throat, and high fever (58.3%), and the most common epidemiological history was living in a rural area (91.7%) and dealing with animal husbandry (66.7%), and 18 (75%) cases were referred to as oropharyngeal tularemia. More than half of the cases were detected between October and March. Conclusion: Tularemia is one of the endemic diseases in our country, and the epidemiological history should be taken carefully and kept in mind in the differential diagnosis of lymphadenopathy. Since it is the first tularemia study conducted in Malatya, it shows the epidemiological characteristics of the region. [ABSTRACT FROM AUTHOR]

Tularemia is a zoonosis caused by the bacterium Francisella tularensis. Leporids are primary sources of human infections in the northern hemisphere. Africa is classically considered free of tularemia, but recent data indicate that this dogma might be wrong. We assessed the presence of this disease in wild leporids in Algeria. Between 2014 and 2018, we collected 74 leporids carcasses from spontaneously dead or hunted animals. Francisella tularensis DNA was detected by specific real-time PCR tests in 7/36 (19.44%) Cape hares (Lepus capensis) and 5/38 (13.15%) wild rabbits (Oryctolagus cuniculus). Known tularemia arthropod vectors infested half of the PCR-positive animals. At necropsy, F. tularensis-infected animals presented with an enlarged spleen (n = 12), enlarged adrenal glands (12), liver discoloration (12), hemorrhages (11), and pneumonia (11). Immunohistological examination of liver tissue from one animal was compatible with the presence of F. tularensis. Our study demonstrates the existence of tularemia in lagomorphs in Algeria. It should encourage investigations to detect this disease among the human population of this country. [ABSTRACT FROM AUTHOR]

Tularemia is a severe infectious disease caused by the Gram-negative bacteria Francisella tularensis. F. tularensis is currently divided into three subspecies, holarctica, tularensis, and mediasiatica, which differ in their virulence and geographic distribution. Subspecies mediasiatica is the least studied because of its very low documented virulence for humans and limited geographic distribution. It was discovered in sparsely populated regions of Central Asia. Since 2011, a new subsp. mediasiatica lineage was identified in Altai (Russia). In 2021, we isolated one subsp. mediasiatica strain in Krasnoyarsk Territory. In spite of its geographic origin, 500 km east from Altai, this strain belongs to the Altai lineage and contributes surprisingly little genetic diversity to previous knowledge. [ABSTRACT FROM AUTHOR]

Background: The spread of emerging tick-borne pathogens has steadily increased in Canada with the widespread establishment of tick vectors and vertebrate hosts. At present, Borrelia burgdorferi, the bacterium causing Lyme disease, is the most common tick-borne pathogen in Canada and primarily transmitted by Ixodes scapularis. A low prevalence of other emerging tick-borne pathogens, such as Anaplasma phagocytophilum, Babesia species, Borrelia miyamotoi, and Francisella tularensis have also been detected through surveillance efforts in Canada. Although Rickettsia rickettsii has been historically detected in Haemaphysalis leporispalustris in Canada, the current prevalence and geographic extent of this pathogen is unknown. Material and Methods: In this study, we assessed the presence and prevalence of several emerging tick-borne pathogens in ticks and hosts collected through tick dragging and small mammal trapping in Central Canada. Results: Nested PCR testing detected three pathogen species in ticks, with Babesia odocoilei and B. burgdorferi in I. scapularis in addition to R. rickettsii in H. leporispalustris. Three pathogen species were detected in small mammals by nested PCR including B. odocoilei in Blarina brevicauda, Babesia microti in Peromyscus leucopus, and a Hepatozoon species in P. leucopus and Peromyscus maniculatus. B. burgdorferi and Babesia species were the pathogens most often detected in our samples, suggesting they are widely distributed across Central Canada. We also detected B. odocoilei and R. rickettsii beyond their known geographic distribution. Conclusions: Our results provide evidence that emerging tick-borne pathogens may be present outside defined risk areas identified by current surveillance efforts in Canada. As a result, emerging tick-borne pathogens introduced by the dispersal of infected ticks by migratory birds or maintained by hosts and vectors through cryptic transmission cycles may go undetected. More comprehensive testing including all tick life stages and additional tick-borne pathogens will help detect the spread and potential risk of emerging or re-emerging tick-borne pathogens for human and wildlife populations throughout Canada. [ABSTRACT FROM AUTHOR]

Bacterial abortive agents such as Brucella spp., Salmonella spp., Campylobacter spp., Listeria spp. cause serious infections that lead to significant economic losses in cattle and sheep breeding. These bacteria can be easily cultured under in vitro laboratory conditions. Abortions caused by intracellular bacteria such as Coxiella burnetii (C. burnetii), Chlamydophila abortus (C. abortus) and Francisella tularensis (F. tularensis) are less prevalent in abortive cases and the diagnosis of these bacteria, many of which need to cell culture for cultivation and biosafety level-3 laboratory facilities for safe working, can be made by PCR. In this study, it was aimed to determine the molecular prevalence of C. burnetii, C. abortus and F. tularensis agents, which were neglected during the diagnosis of abortions, in cattle and sheep collected from different regions of Türkiye. A total of 395 clinical materials were analyzed via agent-specific commercial Real-Time PCR. As a result, the molecular prevalence of F. tularensis, C. burnetii and C. abortus was determined as 14%, 2.9% and 2.28%, respectively. F. tularensis in cattle clinical samples and C. burnetii in sheep clinical samples were not found. This is the first report that F. tularensis was found as an agent in an abortive material in Türkiye. Although it differs in terms of prevalence, it has been determined that these microorganisms, which are neglected in routine diagnosis, can be spread with aborted materials, especially vaginal discharge, which may pose a risk of transmission. [ABSTRACT FROM AUTHOR]

Objective: Tularemia is a rare and often overlooked zoonotic infection. While the ulceroglandular and glandular types are observed most frequently in epidemics originating in Europe, the oropharyngeal type is most common in Türkiye. The most common clinical findings are lymphadenopathy, skin rash, and tonsillitis. The aim of this study was to investigate the frequency of tularemia in Tokat, a province located in the Central Black Sea Region of Türkiye. Materials and Methods: The data of patients diagnosed with tularemia at Tokat Gaziosmanpaşa University Faculty of Medicine Training and Research Hospital between January 2011 and July 2021 were retrospectively analyzed. Laboratoryconfirmed cases were included in the study. Sociodemographic characteristics, risk factors, clinical and laboratory findings, treatments, and post-treatment follow-up of the cases were evaluated. Results: Tularemia was detected in 20 patients. A total of 80% patients lived in a village, 65% were engaged in agriculture as a profession, and 60% in animal husbandry. The most important possible sources of contamination were rodents near the house (40%) and non-chlorinated drinking water (50%). Among 20 cases, 57% were oropharyngeal tularemia, 95% were treated with monotherapy or combinations containing aminoglycosides, and 50% with surgical lymph node drainage. Conclusion: Tularemia is a rare infection in Tokat Province. But it should be considered in the differential diagnosis of patients living in rural areas who present with fever, sore throat, and cervical lymphadenopathy that is not responsive to beta-lactam agents. [ABSTRACT FROM AUTHOR]

Case: A case of Francisella tularensis finger proximal interphalangeal joint septic arthritis secondary to feral cat bite is presented. The patient underwent operative debridement on presentation. On postoperative day 5, a gram-negative rod resembling F. tularensis was identified. The patient received 4 weeks of gentamicin for culture-confirmed ulceroglandular tularemia. At the final follow-up, the infection had resolved, and full function of the digit had been regained. Conclusion: Francisella tularensis septic arthritis secondary to a feral cat bite is exceedingly rare but should be considered in the appropriate clinical context. Proper identification and treatment with antibiotics is essential for a positive outcome. [ABSTRACT FROM AUTHOR]

Tularemia is a highly contagious zoonotic infection caused by Francisella tularensis. Bacterial culture, serology and molecular methods are used in the diagnosis of tularemia. The agent is a dangerous pathogen, and the importance of serological tests in diagnosis has increased because of the difficulty in culturing the organism. In this study, a practical, fast and reliable lateral flow-based immunochromatographic test was planned to detect F. tularensis specific antibodies in the field. Partially purified lipopolysaccharide antigen obtained from the live vaccine strain of F. tularensis was used as antigen. To determine the sensitivity of the test, 17 true positive tularemia serum samples with known Microagglutination test results, and to determine of specificity of the test, 30 true negative serum samples were used. In this study, Brucella-positive patient sera of various titers from our laboratory's serum bank to determine possible cross-reactivity with Brucella antibodies were also tested. The sensitivity and specificity of the Lateral Flow Test (LFT) rapid diagnostic kit were evaluated in comparison to the standard microagglutination test. The sensitivity and specificity of the newly developed Lateral Flow Test (LFT) were found at 100% and 93.5%, respectively. LFT for tularemia revealed 5% cross-reaction with positive sera for brucellosis. Cross-reactions were observed at antibody titers of 1:20 and below. In conclusion, it was concluded that the newly developed lateral flow test is a fast, reliable, and practical alternative test for the serological diagnosis of tularemia and cross-reaction in the serological tests conducted for brucellosis and tularemia should always be considered. [ABSTRACT FROM AUTHOR]

CRISPR/Cas technology is a versatile tool for genome engineering in many organisms, including filamentous fungi. Cpf1 is a multi-domain protein of class 2 (type V) RNA-guided CRISPR/Cas endonuclease, and is an alternative platform with distinct features when compared to Cas9. However, application of this technology in filamentous fungi is limited. Here, we present a single CRISPR/Cpf1 plasmid system in Aspergillus aculeatus strain TBRC 277, an industrially relevant cell factory. We first evaluated the functionality of three Cpf1 orthologs from Acidaminococcus sp. BV3L6 (AsCpf1), Francisella tularensis subsp. novicida U112 (FnCpf1), and Lachnospiraceae bacterium (LbCpf1), in RNA-guided site-specific DNA cleavage at the pksP locus. FnCpf1 showed the highest editing efficiency (93 %) among the three Cpf1s. It was further investigated for its ability to delete a 1.7 kb and a 0.5 kb from pksP and pyr G genes, respectively, using two protospacers targeting these gene loci in a single crRNA array. Lastly, simultaneous editing of three sites within TBRC 277 genome was performed using three guide sequences targeting these two genes as well as an additional gene, kusA , which resulted in combined editing efficiency of 40 %. The editing of the NHEJ pathway by targeting kusA to generate a NHEJ-deficient strain of A. aculeatus TBRC 277 improved gene targeting efficiency and yielded more precise gene-editing than that of using wild-type strain. This promising genome-editing system can be used for strain improvement in industrial applications such as production of valuable bioproducts. [ABSTRACT FROM AUTHOR]