| 1. |
- Rydén, Patrik, 1969-, et al.
(författare)
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Outbreaks of tularemia in a boreal forest region depends on mosquito prevalence
- 2012
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Ingår i: Journal of Infectious Diseases. - : Oxford University Press (OUP). - 0022-1899 .- 1537-6613. ; 205:2, s. 297-304
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Tidskriftsartikel (refereegranskat)abstract
- Background. We aimed to evaluate the potential association of mosquito prevalence in a boreal forest area with transmission of the bacterial disease tularemia to humans, and model the annual variation of disease using local weather data.Methods. A prediction model for mosquito abundance was built using weather and mosquito catch data. Then a negative binomial regression model based on the predicted mosquito abundance and local weather data was built to predict annual numbers of humans contracting tularemia in Dalarna County, Sweden.Results. Three hundred seventy humans were diagnosed with tularemia between 1981 and 2007, 94% of them during 7 summer outbreaks. Disease transmission was concentrated along rivers in the area. The predicted mosquito abundance was correlated (0.41, P < .05) with the annual number of human cases. The predicted mosquito peaks consistently preceded the median onset time of human tularemia (temporal correlation, 0.76; P < .05). Our final predictive model included 5 environmental variables and identified 6 of the 7 outbreaks.Conclusions. This work suggests that a high prevalence of mosquitoes in late summer is a prerequisite for outbreaks of tularemia in a tularemia-endemic boreal forest area of Sweden and that environmental variables can be used as risk indicators.
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| 2. |
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| 3. |
- Desvars, Amélie, 1980-, et al.
(författare)
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Epidemiology and Ecology of Tularemia in Sweden, 1984-2012
- 2015
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Ingår i: Emerging Infectious Diseases. - : Centers for Disease Control and Prevention (CDC). - 1080-6040 .- 1080-6059. ; 21:1, s. 32-39
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Tidskriftsartikel (refereegranskat)abstract
- The zoonotic disease tularemia is endemic in large areas of the Northern Hemisphere, but research is lacking on patterns of spatial distribution and connections with ecologic factors. To describe the spatial epidemiology of and identify ecologic risk factors for tularemia incidence in Sweden, we analyzed surveillance data collected over 29 years (1984-2012). A total of 4,830 cases were notified, of which 3,524 met all study inclusion criteria. From the first to the second half of the study period, mean incidence increased 10-fold, from 0.26/100,000 persons during 1984-1998 to 2.47/100,000 persons during 1999 2012 (p<0.001). The incidence of tularemia was higher than expected in the boreal and alpine ecologic regions (p<0.001), and incidence was positively correlated with the presence of lakes and rivers (p<0.001). These results provide a comprehensive epidemiologic description of tularemia in Sweden and illustrate that incidence is higher in locations near lakes and rivers.
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| 4. |
- Edin, Alicia, et al.
(författare)
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Development and Laboratory Evaluation of a Real-Time PCR Assay for Detecting Viruses and Bacteria of Relevance for Community-Acquired Pneumonia
- 2015
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Ingår i: Journal of Molecular Diagnostics. - : Elsevier BV. - 1525-1578 .- 1943-7811. ; 17:3, s. 315-324
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Tidskriftsartikel (refereegranskat)abstract
- Community-acquired pneumonia may present with similar clinical symptoms, regardless of viral or bacterial cause. Diagnostic assays are needed to rapidly discriminate between causes, because this will guide decisions on appropriate treatment. Therefore, a quantitative real-time PCR (qPCR) assay with duplex reactions targeting eight bacteria and six viruses was developed. Technical performance was examined with linear plasmids. Upper and Lower respiratory tract specimens were used to compare the qPCR assay with standard microbiological methods. The limit of detection was 5 to 20 DNA template copies with approximately 1000-fold differences in concentrations of the two competing templates. SDs for positive controls were <5%. The use of the qPCR assay resulted in 113 positive identifications in 94 respiratory specimens compared with 38 by using standard diagnostics. Diagnostic accuracy of the qPCR assay varied between 60% positive agreement with standard tests for Streptococcus pneumoniae and 100% for Mycoplasma pneumoniae, Moraxella catarrhalis, and Staphylococcus aureus. Negative percentage of agreement was >95% for M. pneumoniae, Streptococcus pyogenes, respiratory syncytial virus, and influenza A virus; whereas it was only 56% for Haemophilus influenzae. Multiple microbial agents were identified in 19 of 44 sputum and 19 of 50 nasopharynx specimens. We conclude that in parallel qPCR detection of the targeted respiratory bacteria and viruses is feasible. The results indicate good technical performance of the assay in clinical specimens.
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| 5. |
- Edin, Alicia, 1985-
(författare)
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Improved diagnosis and prediction of community-acquired pneumonia
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Community-acquired pneumonia (CAP) is a major cause of morbidity and mortality worldwide. Although there is wide variation in the microbial etiology, CAP may manifest with similar symptoms, making institution of proper treatment challenging. Therefore, etiological diagnosis is important to ensure that correct treatment and necessary infection control measures are instituted. This provides a challenge for conventional microbial diagnostic methods, typically based on culture and direct antigen tests. Moreover, existing molecular biomarkers have poor prognostic value. Few studies have investigated the global metabolic response during infection and virtually nothing is known about early responses after the start of antimicrobial treatment. The aim of this work was to improve diagnostic and predictive methods for CAP.In paper I, a qPCR panel targeting 15 pathogens known to cause CAP was developed and evaluated. It combined identification of bacterial pathogens and viruses in the same diagnostic platform. The method proved to be robust and the results consistent with those obtained by standard methods. The panel approach, compared to conventional, selective diagnostics, detected a larger number of pathogens. In Paper II, whole blood samples from 65 patients with bacteremic sepsis were analyzed for metabolite profiles. Forty-nine patients with symptoms of sepsis, but later attributed to other diagnoses, were matched according to age and sex and served as a control group. Six metabolites were identified, all of which predicted growth of bacteria in blood culture. One of the metabolites, myristic acid, alone predicted bacteremic sepsis with a sensitivity of 100% and a specificity of 95%. Paper III and IV were based on a clinical study enrolling 35 patients with suspected CAP in need of hospital care. The aim was to study the metabolic response during the early phase of acute infection. The qPCR panel developed in Paper I was used to obtain the microbial etiological diagnosis. Paper IV focused on the global metabolic response and highlighted the dynamics of changes in major metabolic pathways during early recovery. A specific metabolite pattern for M. pneumoniae etiology was found. Four metabolites accurately predicted all but one patient as either M. pneumoniae etiology or not. Paper III looked at phospholipid levels during the first 48 hours after hospital admission. It was found that all major phospholipid species, especially the lysophosphatidyl-cholines, were pronouncedly decreased during acute infection. Levels started to increase the day after admission, reaching statistical significance at 48 hours. Paper II-IV showed that metabolomics might be used to study a number of different aspects of infection, such as etiology, disease progress and recovery. Knowledge of the metabolic profiles of patients may not only be utilized for biomarker discovery, as proposed in this work, but also for the future development of targeted therapies and supportive treatment.
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| 6. |
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| 7. |
- Johansson, Anders, 1966-, et al.
(författare)
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Francisella tularensis : Tularemia
- 2012
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Ingår i: BSL3 and BSL4 agents. - Weinheim, Germany : Wiley-VCH Verlagsgesellschaft. - 9783527317158 - 9783527645114 ; , s. 71-84
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Bokkapitel (refereegranskat)abstract
- Francisella tularensis is the causative agent of tularemia. This emerging zoonosis shows several clinical manifestations complicating its diagnosis. The chapter focus on the characteristics of this bacterium, the several forms of the disease, its diagnosis and molecular epidemiology.
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| 8. |
- Johansson, Anders, 1966-, et al.
(författare)
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Francisella tularensis : Tularemia
- 2012
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Ingår i: BSL3 and BSL4 agents. - Weinheim, Germany : Wiley-VCH Verlagsgesellschaft. - 9783527317158 - 9783527645114 ; , s. 309-311
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Bokkapitel (refereegranskat)abstract
- This practical guide gives some starting points to manage biological safety issues with Francisella tularensis.
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| 9. |
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| 10. |
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| 11. |
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| 12. |
- Kauppi, Anna M., et al.
(författare)
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Metabolites in Blood for Prediction of Bacteremic Sepsis in the Emergency Room
- 2016
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Ingår i: PLOS ONE. - : Public Library Science. - 1932-6203. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- A metabolomics approach for prediction of bacteremic sepsis in patients in the emergency room (ER) was investigated. In a prospective study, whole blood samples from 65 patients with bacteremic sepsis and 49 ER controls were compared. The blood samples were analyzed using gas chromatography coupled to time-of-flight mass spectrometry. Multivariate and logistic regression modeling using metabolites identified by chromatography or using conventional laboratory parameters and clinical scores of infection were employed. A predictive model of bacteremic sepsis with 107 metabolites was developed and validated. The number of metabolites was reduced stepwise until identifying a set of 6 predictive metabolites. A 6-metabolite predictive logistic regression model showed a sensitivity of 0.91(95% CI 0.69-0.99) and a specificity 0.84 (95% CI 0.58-0.94) with an AUC of 0.93 (95% CI 0.89-1.01). Myristic acid was the single most predictive metabolite, with a sensitivity of 1.00 (95% CI 0.85-1.00) and specificity of 0.95 (95% CI 0.74-0.99), and performed better than various combinations of conventional laboratory and clinical parameters. We found that a metabolomics approach for analysis of acute blood samples was useful for identification of patients with bacteremic sepsis. Metabolomics should be further evaluated as a new tool for infection diagnostics.
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| 13. |
- Kelk, Peyman, et al.
(författare)
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Cellular and molecular response of human macrophages exposed to Aggregatibacter actinomycetemcomitans leukotoxin.
- 2011
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Ingår i: Cell death & disease. - : Nature Publishing Group. - 2041-4889. ; 2, s. e126-
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Tidskriftsartikel (refereegranskat)abstract
- Aggregatibacter (Actinobacillus) actinomycetemcomitans is a facultative anaerobic gram-negative bacterium associated with severe forms of periodontitis. A leukotoxin, which belongs to the repeats-in-toxin family, is believed to be one of its virulence factors and to have an important role in the bacterium's pathogenicity. This toxin selectively kills human leukocytes by inducing apoptosis and lysis. Here, we report that leukotoxin-induced cell death of macrophages proceeded through a process that differs from the classical characteristics of apoptosis and necrosis. A. actinomycetemcomitans leukotoxin-induced several cellular and molecular mechanisms in human macrophages that led to a specific and excessive pro-inflammatory response with particular secretion of both interleukin (IL)-1β and IL-18. In addition, this pro-inflammatory cell death was inhibited by oxidized ATP, which indicates involvement of the purinergic receptor P2X(7) in this process. This novel virulence mechanism of the leukotoxin may have an important role in the pathogenic potential of this bacterium and can be a target for future therapeutic agents.
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| 14. |
- Kelk, Peyman, 1976-, et al.
(författare)
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IL-1beta secretion induced by Aggregatibacter (Actinobacillus) actinomycetemcomitans is mainly caused by the leukotoxin
- 2008
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Ingår i: International Journal of Medical Microbiology. - : Elsevier BV. - 1438-4221 .- 1618-0607. ; 298:5/6, s. 529-541
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Tidskriftsartikel (refereegranskat)abstract
- Aggregatibacter (Actinobacillus) actinomycetemcomitans forms a leukotoxin that selectively lyses primate neutrophils, monocytes and triggers apoptosis in promyeloic cells and degranulation of human neutrophils. Recently, we showed that the leukotoxin causes activation of caspase-1 and abundant secretion of bio-active IL-1beta from human macrophages. In this study, we show that high levels of IL-beta correlated with a high proportion of A. actinomycetemcomitans in clinical samples from a patient with aggressive periodontitis. To determine the relative contribution of leukotoxin to the overall bacteria-induced IL-1beta secretion, macrophages were isolated from peripheral blood and exposed to different concentrations of live A. actinomycetemcomitans strains with either no, low or high production of leukotoxin. Cell lysis and levels of IL-1beta, IL-6, TNF-alpha and caspase-1 were measured by ELISA and flow cytometry. Leukotoxin was the predominant cause of IL-1beta secretion from macrophages, even in the A. actinomycetemcomitans strain with low leukotoxin production. Macrophages exposed to non-leukotoxic bacteria accumulated cytosolic pro-IL-1beta, which was secreted by a secondary exposure to leukotoxic bacteria. In conclusion, the present study shows for the first time that A. actinomycetemcomitans-induced IL-1beta secretion from human macrophages in vitro is mainly caused by leukotoxin.
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| 15. |
- Kelk, Peyman, 1976-, et al.
(författare)
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Inflammatory cell death of human macrophages in response to Aggregatibacter actinomycetemcomitans leukotoxin
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Aggregatibacter (Actinobacillus) actinomycetemcomitans is a facultative anaerobic gram-negative bacterium associated with severe forms of periodontitis. A leukotoxin, which belongs to the Repeats in Toxin (RTX) family, is believed to be one of its virulence factors and to play an important role in the bacterium's pathogenicity. This toxin selectively kills human leukocytes by inducing apoptosis and lysis. Here we report that leukotoxin-induced cell death of macrophages proceeded through a process that differs from the classical characteristics of apoptosis and necrosis. Interestingly, this process resembled pyroptosis, and resulted in an extensive leukotoxin-induced interleukin-1β (IL-1β) secretion. This activation was mainly mediated by caspase-1 activation, while the levels of mRNA for IL-1β were not affected by the leukotoxin. A similar pattern was seen for IL-18, but the level of that cytokine was about 30 times lower. Both of these cytokines are synthesized as biologically inactive precursors and need active caspase-1 for their activation and secretion. In conclusion, A. actinomycetemcomitans leukotoxin induces a pyroptosis-like cell death in human macrophages and that leads to a specific and excessive pro-inflammatory response. This novel virulence mechanism of the leukotoxin may play an important role in the pathogenic potential of this bacterium.
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| 16. |
- Kolnes, Anders Jensen, et al.
(författare)
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TGFBR3L is associated with gonadotropin production in non-functioning gonadotroph pituitary neuroendocrine tumours
- 2023
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Ingår i: Pituitary. - : Springer. - 1386-341X .- 1573-7403. ; 26:2, s. 227-236
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Tidskriftsartikel (refereegranskat)abstract
- Purpose Transforming growth factor-beta receptor 3-like (TGFBR3L) is a pituitary enriched membrane protein selectively detected in gonadotroph cells. TGFBR3L is named after transforming growth factor-beta receptor 3 (TGFBR3), an inhibin A co-receptor in mice, due to sequence identity to the C-terminal region. We aimed to characterize TGFBR3L detection in a well-characterized, prospectively collected cohort of non-functioning pituitary neuroendocrine tumours (NF-PitNETs) and correlate it to clinical data.Methods 144 patients operated for clinically NF-PitNETs were included. Clinical, radiological and biochemical data were recorded. Immunohistochemical (IHC) staining for FSH beta and LH beta was scored using the immunoreactive score (IRS), TGFBR3L and TGFBR3 were scored by the percentage of positive stained cells.Results TGFBR3L staining was selectively present in 52% of gonadotroph tumours. TGFBR3L was associated to IRS of LH beta (median 2 [IQR 0-3] in TGFBR3L negative and median 6 [IQR 3-9] in TGFBR3L positive tumours, p < 0.001), but not to the IRS of FSH beta (p = 0.32). The presence of TGFBR3L was negatively associated with plasma gonadotropin concentrations in males (P-FSH median 5.5 IU/L [IQR 2.9-9.6] and median 3.0 [IQR 1.8-5.6] in TGFBR3L negative and positive tumours respectively, p = 0.008) and P-LH (median 2.8 IU/L [IQR 1.9-3.7] and median 1.8 [IQR 1.1-3.0] in TGFBR3L negative and positive tumours respectively, p = 0.03). TGFBR3 stained positive in 22% (n = 25) of gonadotroph tumours with no correlation to TGFBR3L.Conclusion TGFBR3L was selectively detected in half (52%) of gonadotroph NF-PitNETs. The association to LH beta staining and plasma gonadotropins suggests that TGFBR3L may be involved in hormone production in gonadotroph NF-PitNETs.
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| 17. |
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| 18. |
- Kroca, Michal, et al.
(författare)
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Vγ9Vδ2 T cells in human legionellosis
- 2001
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Ingår i: Clinical and diagnostic laboratory immunology. - 1071-412X. ; 8, s. 949-954
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Tidskriftsartikel (refereegranskat)
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| 19. |
- Müller, Daniel C., et al.
(författare)
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Phospholipid Levels in Blood during Community-Acquired Pneumonia
- 2019
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Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 14:5
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Tidskriftsartikel (refereegranskat)abstract
- Phospholipids, major constituents of bilayer cell membranes, are present in large amounts in pulmonary surfactant and play key roles in cell signaling. Here, we aim at finding clinically useful disease markers in community-acquired pneumonia (CAP) using comprehensive phospholipid profiling in blood and modeling of changes between sampling time points. Serum samples from 33 patients hospitalized with CAP were collected at admission, three hours after the start of intravenous antibiotics, Day 1 (at 12–24 h), Day 2 (at 36–48 h), and several weeks after recovery. A profile of 75 phospholipid species including quantification of the bioactive lysophosphatidylcholines (LPCs) was determined using liquid chromatography coupled to time-of-flight mass spectrometry. To control for possible enzymatic degradation of LPCs, serum autotaxin levels were examined. Twenty-two of the 33 patients with a clinical diagnosis of CAP received a laboratory-verified CAP diagnosis by microbial culture or microbial DNA detection by qPCR. All major phospholipid species, especially the LPCs, were pronouncedly decreased in the acute stage of illness. Total and individual LPC concentrations increased shortly after the initiation of antibiotic treatment, concentrations were at their lowest 3h after the initiation, and increased after Day 1. The total LPC concentration increased by a change ratio of 1.6–1.7 between acute illness and Day 2, and by a ratio of 3.7 between acute illness and full disease resolution. Autotaxin levels were low in acute illness and showed little changes over time, contradicting a hypothesis of enzymatic degradation causing the low levels of LPCs. In this sample of patients with CAP, the results demonstrate that LPC concentration changes in serum of patients with CAP closely mirrored the early transition from acute illness to recovery after the initiation of antibiotics. LPCs should be further explored as potential disease stage biomarkers in CAP and for their potential physiological role during recovery.
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| 20. |
- Plymoth, Martin, et al.
(författare)
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Targeting tularemia : clinical, laboratory, and treatment outcomes from an 11-year retrospective observational cohort in northern sweden
- 2024
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Ingår i: Clinical Infectious Diseases. - : Oxford University Press. - 1058-4838 .- 1537-6591. ; 78:5, s. 1222-1231
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Tidskriftsartikel (refereegranskat)abstract
- Background: Tularemia is an important re-emerging 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 2011-2021 were collected through review of electronic medical records and participant questionnaires; with 415 out of 784 accepting participation (52.9%). Of these, 327 were laboratory-confirmed cases (serology and/or PCR). A multivariable logistic regression model was used to investigate variables associated with re-treatment.Results: Median age of participants was 54 years (IQR 41.5-65) and 49.2% were female. While 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 non-specific, with monocytosis frequently observed (n=36/75; 48%). Tularemia was often misdiagnosed upon presentation (n=158, 48.3%), with 65 (19.9%) receiving initial inappropriate antibiotics, and 102 (31.2%) re-treated. Persistent lymphadenopathy was infrequent (n=22, 6.7%), with 10 undergoing surgical interventions. In multivariable analysis of variables associated with re-treatment, we highlight differences in time until receiving appropriate antibiotics (8 [IQR 3.25-20.75] vs. 7 [IQR 4-11.25] days; adjusted p=0.076), and doxycycline-based treatment regimen (vs. ciprofloxacin; adjusted p=0.084), although not significant after correction for multiple comparisons.Conclusion: 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.
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| 21. |
- Rydén, Patrik, 1969-, et al.
(författare)
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Effects of climate change on tularemia disease activity in Sweden
- 2009
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Ingår i: Global Health Action. - 1654-9880. ; 2
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Tidskriftsartikel (refereegranskat)abstract
- Tularaemia is a vector-borne infectious disease. A large majority of cases transmitted to humans by bloodfeeding arthropods occur during the summer season and is linked to increased temperatures. Therefore, the effect of climate change is likely to have an effect on tularaemia transmission patterns in highly endemic areas of Sweden. In this report, we use simulated climate change scenario data and empirical data of temperatures critical to tularaemia transmission to forecast tularaemia outbreak activity. The five high-endemic counties: Dalarna, Gävleborg, Norrbotten, Värmland and Örebro represent only 14.6% of the total population of Sweden, but have recorded 40.1-81.1% of the number of annual human tularaemia in Sweden from 1997 until 2008. We project here earlier starts and a later termination of future tularaemia outbreaks for the time period 2010-2100. For five localised outbreak areas; Gagnef (Dalarna), Ljusdal (Gävleborg), Harads (Norrbotten), Karlstad (Värmland) and Örebro municipality (Örebro), the climate scenario suggests an approximately 2°C increase in monthly average summer temperatures leading to increases in outbreak durations ranging from 3.5 weeks (Harads) to 6.6 weeks (Karlstad) between 2010 and 2100. In contrast, an analysis of precipitation scenarios indicates fairly stable projected levels of precipitation during the summer months. Thus, there should not be an increased abundance of late summer mosquitoes that are believed to be main vectors for transmission to humans in these areas. In conclusion, the results indicate that the future climate changes will lead to an increased burden of tularaemia in high-endemic areas of Sweden during the coming decades.
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| 22. |
- Rzhepishevska, Olena, et al.
(författare)
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The antibacterial activity of Ga3+ is influenced by Llgand complexation as well as the bacterial carbon source
- 2011
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Ingår i: Antimicrobial Agents and Chemotherapy. - Washington, D. C. : American Society for Microbiology. - 0066-4804 .- 1098-6596. ; 55:12, s. 5568-5580
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Tidskriftsartikel (refereegranskat)abstract
- Gallium ions have previously been shown to exhibit antibacterial and antibiofilm properties. In this study we report differential bactericidal activity of two gallium complexes; gallium desferrioxamine B (Ga-DFOB) and gallium citrate (Ga-cit). Modeling of gallium speciation in growth medium showed that DFOB and citrate both can prevent precipitation of Ga(OH)(3), but some precipitation can occur above pH 7 with citrate. Despite this, Ga-cit inhibitory concentrations (IC(90)) were lower than those of Ga-DFOB for clinical isolates of Pseudomonas aeruginosa, and several reference strains of other bacterial species. Treatment with Ga compounds mitigated damage inflicted on murine J774 macrophage-like cells infected with P. aeruginosa PAO1. Again, Ga-cit showed more potent mitigation than did Ga-DFOB. Ga was also taken up more efficiently by P. aeruginosa in the form of Ga-cit than in the form of Ga-DFOB. Neither Ga-cit nor Ga-DFOB was toxic to several human cell lines tested and no pro-inflammatory activity was detected in human lung epithelial cells after exposure in vitro. Metabolomic analysis was used to delineate the effects of Ga-cit on the bacterial cell. Exposure to Ga resulted in lower concentrations of glutamate, a key metabolite for P. aeruginosa, and of many amino acids, indicating that Ga affects various biosynthesis pathways. Altered protein expression profile in presence of Ga-cit suggested that some compensatory mechanisms were activated in the bacterium. Furthermore, the antibacterial effect of Ga was shown to vary depending on the carbon source, which has importance in the context of medical applications of gallium.
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| 23. |
- Sun, Kun, et al.
(författare)
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Screening for inhibition of Vibrio cholerae VipA-VipB interaction identifies small-molecule compounds active against type VI secretion
- 2014
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Ingår i: Antimicrobial Agents and Chemotherapy. - 0066-4804 .- 1098-6596. ; 58:7, s. 4123-4130
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Tidskriftsartikel (refereegranskat)abstract
- The type VI secretion system (T6SS) is the most prevalent bacterial secretion system and an important virulence mechanism utilized by Gram-negative bacteria, either to target eukaryotic cells or to combat other microbes. The components show much variability, but some appear essential for the function, and two homologues, denoted VipA and VipB in Vibrio cholerae, have been identified in all T6SSs described so far. Secretion is dependent on binding of an alpha-helical region of VipA to VipB, and in the absence of this binding, both components are degraded within minutes and secretion is ceased. The aim of the study was to investigate if this interaction could be blocked, and we hypothesized that such inhibition would lead to abrogation of T6S. A library of 9,600 small-molecule compounds was screened for their ability to block the binding of VipA-VipB in a bacterial two-hybrid system (B2H). After excluding compounds that showed cytotoxicity toward eukaryotic cells, that inhibited growth of Vibrio, or that inhibited an unrelated B2H interaction, 34 compounds were further investigated for effects on the T6SS-dependent secretion of hemolysin-coregulated protein (Hcp) or of phospholipase A(1) activity. Two compounds, KS100 and KS200, showed intermediate or strong effects in both assays. Analogues were obtained, and compounds with potent inhibitory effects in the assays and desirable physicochemical properties as predicted by in silico analysis were identified. Since the compounds specifically target a virulence mechanism without affecting bacterial replication, they have the potential to mitigate the virulence with minimal risk for development of resistance.
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| 24. |
- Twine, Susan, et al.
(författare)
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A mutant of Francisella tularensis strain SCHU S4 lacking the ability to express a 58-kilodalton protein is attenuated for virulence and is an effective live vaccine
- 2005
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Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 73:12, s. 8345-8352
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Tidskriftsartikel (refereegranskat)abstract
- Francisella tularensis subsp. tularensis (type A) strain SCHU S4 is a prototypic strain of the pathogen that is highly virulent for humans and other mammals. Its intradermal (i.d.) 50% lethal dose (LD50) for mice is <10 CFU. We discovered a spontaneous mutant, designated FSC043, of SCHU S4 with an i.d. LD50 of >10(8) CFU. FSC043 effectively vaccinated mice against challenge with a highly virulent type A strain, and the protective efficacy was at least as good as that of F. tularensis LVS, an empirically attenuated strain which has been used as an efficacious human vaccine. Comparative proteomics was used to identify two proteins of unknown function that were identified as defective in LVS and FSC043, and deletion mutants of SCHU S4 were created for each of the two encoding genes. One mutant, the DeltaFTT0918 strain, failed to express a 58-kDa protein, had an i.d. LD50 of approximately 10(5) CFU, and was found to be less capable than SCHU S4 of growing in peritoneal mouse macrophages. Mice that recovered from sublethal infection with the DeltaFTT0918 mutant survived when challenged 2 months later with >100 LD50s of the highly virulent type A strain FSC033. This is the first report of the generation of defined mutants of F. tularensis subsp. tularensis and their use as live vaccines.
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| 25. |
- Alam, Athar, et al.
(författare)
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ClpB mutants of Francisella tularensis subspecies holarctica and tularensis are defective for type VI secretion and intracellular replication
- 2018
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Francisella tularensis, a highly infectious, intracellular bacterium possesses an atypical type VI secretion system (T6SS), which is essential for the virulence of the bacterium. Recent data suggest that the HSP100 family member, ClpB, is involved in T6SS disassembly in the subspecies Francisella novicida. Here, we investigated the role of ClpB for the function of the T6SS and for phenotypic characteristics of the human pathogenic subspecies holarctica and tularensis. The Delta clpB mutants of the human live vaccine strain, LVS, belonging to subspecies holarctica, and the highly virulent SCHU S4 strain, belonging to subspecies tularensis, both showed extreme susceptibility to heat shock and low pH, severely impaired type VI secretion (T6S), and significant, but impaired intracellular replication compared to the wild-type strains. Moreover, they showed essentially intact phagosomal escape. Infection of mice demonstrated that both Delta clpB mutants were highly attenuated, but the SCHU S4 mutant showed more effective replication than the LVS strain. Collectively, our data demonstrate that ClpB performs multiple functions in the F. tularensis subspecies holarctica and tularensis and its function is important for T6S, intracellular replication, and virulence.
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| 26. |
- Alam, Athar, et al.
(författare)
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Dissociation between the critical role of ClpB of Francisella tularensis for the heat shock response and the DnaK interaction and its important role for efficient type VI secretion and bacterial virulence
- 2020
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Ingår i: PLoS Pathogens. - : Public Library of Science. - 1553-7366 .- 1553-7374. ; 16:4, s. 1-27
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Tidskriftsartikel (refereegranskat)abstract
- Author summary Type VI secretion systems (T6SSs) are essential virulence determinants of many Gram-negative pathogens, including Francisella tularensis. This highly virulent bacterium encodes an atypical T6SS lacking ClpV, the ATPase crucial for prototypic T6SS sheath disassembly. It, however, possesses ClpB, a protein critical for heat shock survival via its interaction with DnaK. Since ClpB possesses ATPase activity, it has been hypothesized to provide a compensatory function for the absence of ClpV, a hypothesis supported by the recent findings from us and others. Here, we investigated how F. tularensis ClpB controls T6S. In silico modelling of the ClpB-DnaK complex identified key interactions that were experimentally verified. For example, mutating one of the DnaK-interacting residues rendered the bacterium exquisitely susceptible to heat shock, but had no effect on T6S and virulence. In contrast, removing the N-terminal of ClpB only had a slight effect on the heat shock response, but strongly compromised both T6S and virulence. Intriguingly, the Escherichia coli ClpB could fully complement the function of F. tularensis ClpB. The data demonstrate that the two critical roles of ClpB, mediating heat shock survival and effective T6S, are dissociated and that the N-terminal is crucial for T6S and virulence. Francisella tularensis, a highly infectious, intracellular bacterium possesses an atypical type VI secretion system (T6SS), which is essential for its virulence. The chaperone ClpB, a member of the Hsp100/Clp family, is involved in Francisella T6SS disassembly and type VI secretion (T6S) is impaired in its absence. We asked if the role of ClpB for T6S was related to its prototypical role for the disaggregation activity. The latter is dependent on its interaction with the DnaK/Hsp70 chaperone system. Key residues of the ClpB-DnaK interaction were identified by molecular dynamic simulation and verified by targeted mutagenesis. Using such targeted mutants, it was found that the F. novicida ClpB-DnaK interaction was dispensable for T6S, intracellular replication, and virulence in a mouse model, although essential for handling of heat shock. Moreover, by mutagenesis of key amino acids of the Walker A, Walker B, and Arginine finger motifs of each of the two Nucleotide-Binding Domains, their critical roles for heat shock, T6S, intracellular replication, and virulence were identified. In contrast, the N-terminus was dispensable for heat shock, but required for T6S, intracellular replication, and virulence. Complementation of the Delta clpB mutant with a chimeric F. novicida ClpB expressing the N-terminal of Escherichia coli, led to reconstitution of the wild-type phenotype. Collectively, the data demonstrate that the ClpB-DnaK interaction does not contribute to T6S, whereas the N-terminal and NBD domains displayed critical roles for T6S and virulence.
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| 27. |
- Alam, Athar, et al.
(författare)
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The Role of ClpB in Bacterial Stress Responses and Virulence
- 2021
-
Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media S.A.. - 2296-889X. ; 8
-
Forskningsöversikt (refereegranskat)abstract
- Bacterial survival within a mammalian host is contingent upon sensing environmental perturbations and initiating an appropriate counter-response. To achieve this, sophisticated molecular machineries are used, where bacterial chaperone systems play key roles. The chaperones are a prerequisite for bacterial survival during normal physiological conditions as well as under stressful situations, e.g., infection or inflammation. Specific stress factors include, but are not limited to, high temperature, osmolarity, pH, reactive oxidative species, or bactericidal molecules. ClpB, a member of class 1 AAA+ proteins, is a key chaperone that via its disaggregase activity plays a crucial role for bacterial survival under various forms of stress, in particular heat shock. Recently, it has been reported that ClpB also regulates secretion of bacterial effector molecules related to type VI secretion systems. In this review, the roles of ClpB in stress responses and the mechanisms by which it promotes survival of pathogenic bacteria are discussed.
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| 28. |
- Andersson, Henrik, 1972-
(författare)
-
A microarray analysis of the host response to infection with Francisella tularensis
- 2006
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Francisella tularensis is a gram-negative bacterium that is the cause of the serious and sometimes fatal disease, tularemia, in a wide range of animal species and in humans. The response of cells of the mouse macrophage cell line J774 to infection with Francisella tularensis LVS was analyzed by means of a DNA microarray. It was observed that the infection conferred an oxidative stress upon the target cells and many of the host defense mechanisms appeared to be intended to counteract this stress. The infection was characterized by a very modest inflammatory response. Tularemia caused by inhalation of F. tularensis subspecies tularensis is one of the most aggressive infectious diseases known. We used the mouse model to examine in detail the host immune response in the lung. After an aerosol challenge all mice developed clinical signs of severe disease, showed weight loss by day four of infection, and died the next day. Gene transcriptional changes in the mouse lung samples were examined on day one, two, and four of infection. Genes preferentially involved in host immune responses were activated extensively on day four but on day one and two, only marginally or not at all. Several genes upregulated on day four are known to depend on IFN-gamma or TNF-alpha for their regulation. In keeping with this finding, TNF-alpha and IFN-gamma levels were found to be increased significantly in bronchoalveolar lavage on day four. We undertook an analysis of the transcriptional response in peripheral blood during the course of ulceroglandular tularemia by use of Affymetrix microarrays. Samples were obtained from seven individuals at five occasions during two weeks after the first hospital visit and convalescent samples three months later. In total 265 genes were differentially expressed. The most prominent changes were noted in samples drawn on days 2-3 and a considerable proportion of the upregulated genes appeared to represent an IFN-gamma-induced response and also a pro-apoptotic response. Genes involved in the generation of innate and acquired immune responses were found to be downregulated, presumably a pathogen-induced event. A logistic regression analysis revealed that seven genes were good predictors of the early phase of tularemia. Recently, a large number of methods for the analysis of microarray data have been proposed but there are few comparisons of their relative performances. We undertook a study to evaluate established and novel methods for filtration, background adjustment, scanning, and censoring. For all analyses, the sensitivities at low false positive rates were observed together with a bias measurement. In general, there was a trade off between the analyses ability to identify differentially expressed genes and their ability to obtain unbiased estimators of the desired ratios. A commonly used standard analysis using background adjustment performed poorly. Interestingly, the constrained model combining data from several scans resulted in high sensitivities. For experiments where only low false discovery rates are acceptable, the use of the constrained model or the novel partial filtration method are likely to perform better than some commonly used standard analyses.
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| 29. |
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| 30. |
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| 31. |
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| 32. |
- Asnafi, Nader, 1960-, et al.
(författare)
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HYFO98 : Slutrapport
- 1998
-
Rapport (övrigt vetenskapligt/konstnärligt)
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| 33. |
- Binesse, Johan, et al.
(författare)
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Roles of Reactive Oxygen Species-Degrading Enzymes of Francisella tularensis SCHU S4
- 2015
-
Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 83:6, s. 2255-2263
-
Tidskriftsartikel (refereegranskat)abstract
- Francisella tularensis is a facultative intracellular bacterium utilizing macrophages as its primary intracellular habitat and is therefore highly capable of resisting the effects of reactive oxygen species (ROS), potent mediators of the bactericidal activity of macrophages. We investigated the roles of enzymes presumed to be important for protection against ROS. Four mutants of the highly virulent SCHU S4 strain with deletions of the genes encoding catalase (katG), glutathione peroxidase (gpx), a DyP-type peroxidase (FTT0086), or double deletion of FTT0086 and katG showed much increased susceptibility to hydrogen peroxide (H2O2) and slightly increased susceptibility to paraquat but not to peroxynitrite (ONOO-) and displayed intact intramacrophage replication. Nevertheless, mice infected with the double deletion mutant showed significantly longer survival than SCHU S4-infected mice. Unlike the aforementioned mutants, deletion of the gene coding for alkyl-hydroperoxide reductase subunit C (ahpC) generated a mutant much more susceptible to paraquat and ONOO- but not to H2O2. It showed intact replication in J774 cells but impaired replication in bone marrow-derived macrophages and in internal organs of mice. The live vaccine strain, LVS, is more susceptible than virulent strains to ROS-mediated killing and possesses a truncated form of FTT0086. Expression of the SCHU S4 FTT0086 gene rendered LVS more resistant to H2O2, which demonstrates that the SCHU S4 strain possesses additional detoxifying mechanisms. Collectively, the results demonstrate that SCHU S4 ROS-detoxifying enzymes have overlapping functions, and therefore, deletion of one or the other does not critically impair the intracellular replication or virulence, although AhpC appears to have a unique function.
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| 34. |
- Borgström, Anna, et al.
(författare)
-
Wetlands as a potential multifunctioning tool to mitigate eutrophication and brownification
- 2024
-
Ingår i: Ecological Applications. - 1051-0761 .- 1939-5582.
-
Tidskriftsartikel (refereegranskat)abstract
- Eutrophication and brownification are ongoing environmental problems affecting aquatic ecosystems. Due to anthropogenic changes, increasing amounts of organic and inorganic compounds are entering aquatic systems from surrounding catchment areas, increasing both nutrients, total organic carbon (TOC), and water color with societal, as well as ecological consequences. Several studies have focused on the ability of wetlands to reduce nutrients, whereas data on their potential to reduce TOC and water color are scarce. Here we evaluate wetlands as a potential multifunctional tool for mitigating both eutrophication and brownification. Therefore, we performed a study for 18 months in nine wetlands allowing us to estimate the reduction in concentrations of total nitrogen (TN), total phosphorus (TP), TOC and water color. We show that wetland reduction efficiency with respect to these variables was generally higher during summer, but many of the wetlands were also efficient during winter. We also show that some, but not all, wetlands have the potential to reduce TOC, water color and nutrients simultaneously. However, the generalist wetlands that reduced all four parameters were less efficient in reducing each of them than the specialist wetlands that only reduced one or two parameters. In a broader context, generalist wetlands have the potential to function as multifunctional tools to mitigate both eutrophication and brownification of aquatic systems. However, further research is needed to assess the design of the generalist wetlands and to investigate the potential of using several specialist wetlands in the same catchment.
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| 35. |
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| 36. |
- Broman, T, et al.
(författare)
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Molecular Detection of Persistent Francisella tularensis Subspecies holarctica in Natural Waters
- 2011
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Ingår i: International Journal of Microbiology. - : Hindawi Publishing Corporation. - 1687-918X .- 1687-9198. ; 2011, s. Article ID 851946-
-
Tidskriftsartikel (refereegranskat)abstract
- Tularemia, caused by the bacterium Francisella tularensis, where F. tularensis subspecies holarctica has long been the cause of endemic disease in parts of northern Sweden. Despite this, our understanding of the natural life-cycle of the organism is still limited. During three years, we collected surface water samples (n = 341) and sediment samples (n = 245) in two areas in Sweden with endemic tularemia. Real-time PCR screening demonstrated the presence of F. tularenis lpnA sequences in 108 (32%) and 48 (20%) of the samples, respectively. The 16S rRNA sequences from those samples all grouped to the species F. tularensis. Analysis of the FtM19InDel region of lpnA-positive samples from selected sampling points confirmed the presence of F. tularensis subspecies holarctica-specific sequences. These sequences were detected in water sampled during both outbreak and nonoutbreak years. Our results indicate that diverse F. tularensis-like organisms, including F. tularensis subsp. holarctica, persist in natural waters and sediments in the investigated areas with endemic tularemia.
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| 37. |
- Bruhn, Anders Dalhoff, et al.
(författare)
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Terrestrial Dissolved Organic Matter Mobilized From Eroding Permafrost Controls Microbial Community Composition and Growth in Arctic Coastal Zones
- 2021
-
Ingår i: Frontiers in Earth Science. - : Frontiers Media SA. - 2296-6463. ; 9
-
Tidskriftsartikel (refereegranskat)abstract
- Climate warming is accelerating erosion along permafrost-dominated Arctic coasts. This results in the additional supply of organic matter (OM) and nutrients into the coastal zone. In this study we investigate the impact of coastal erosion on the marine microbial community composition and growth rates in the coastal Beaufort Sea. Dissolved organic matter (DOM) derived from three representative glacial deposit types (fluvial, lacustrine, and moraine) along the Yukon coastal plain, Canada, were used as substrate to cultivate marine bacteria using a chemostat setup. Our results show that DOM composition (inferred from UV-Visible spectroscopy) and biodegradability (inferred from DOC concentration, bacterial production and respiration) significantly differ between the three glacial deposit types. DOM derived from fluvial and moraine types show clear terrestrial characteristics with low aromaticity (Sr: 0.63 ± 0.02 and SUVA254: 1.65 ± 0.06 L mg C−1 m−1 & Sr: 0.68 ± 0.01 and SUVA254: 1.17 ± 0.06 L mg C−1 m−1, respectively) compared to the lacustrine soil type (Sr: 0.71 ± 0.02 and SUVA254: 2.15 ± 0.05 L mg C−1 m−1). The difference in composition of DOM leads to the development of three different microbial communities. Whereas Alphaproteobacteria dominate in fluvial and lacustrine deposit types (67 and 87% relative abundance, respectively), Gammaproteobacteria is the most abundant class for moraine deposit type (88% relative abundance). Bacterial growth efficiency (BGE) is 66% for DOM from moraine deposit type, while 13 and 28% for DOM from fluvial and lacustrine deposit types, respectively. The three microbial communities therefore differ strongly in their net effect on DOM utilization depending on the eroded landscape type. The high BGE value for moraine-derived DOM is probably caused by a larger proportion of labile colorless DOM. These results indicate that the substrate controls marine microbial community composition and activities in coastal waters. This suggests that biogeochemical changes in the Arctic coastal zone will depend on the DOM character of adjacent deposit types, which determine the speed and extent of DOM mineralization and thereby carbon channeling into the microbial food web. We conclude that marine microbes strongly respond to the input of terrestrial DOM released by coastal erosion and that the landscape type differently influence marine microbes.
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| 38. |
- Bröms, Jeanette E, 1974-, et al.
(författare)
-
A conserved α-helix essential for a type VI secretion-like system of Francisella tularensis
- 2009
-
Ingår i: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 191:8, s. 2431-2446
-
Tidskriftsartikel (refereegranskat)abstract
- Francisella tularensis harbors genes with similarity to genes encoding components of a type VI secretion system (T6SS) recently identified in several gram-negative bacteria. These include iglA and iglB, the homologues of which are conserved in most T6SSs. We used a yeast two-hybrid system to study the interaction of the Igl proteins of F. tularensis LVS. We identified a region of IglA, encompassing residues 33-132, necessary for efficient binding to IglB as well as for IglAB protein stability and intra-macrophage growth. In particular, residues 103-122, overlapping with a highly conserved alpha-helix, played an absolutely essential role. Point mutations within this domain caused modest defects in IglA-IglB binding in yeast, but markedly impaired intra-macrophage replication and phagosomal escape, resulting in severe attenuation of LVS in mice. Thus, IglA-IglB complex formation is clearly crucial for Francisella pathogenicity. This interaction may be universal to T6S, since IglAB homologues of Yersinia pseudotuberculosis, Pseudomonas aeruginosa, Vibrio cholerae, Salmonella typhimurium and Escherichia coli were also shown to interact in yeast and the interaction was dependent on the preservation of the same alpha-helix. Heterologous interactions formed between non-native IglAB proteins further supported the notion of a conserved binding site. Thus, IglA-IglB complex formation is clearly crucial for Francisella pathogenicity and the same interaction is conserved in other human pathogens.
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| 39. |
- Bröms, Jeanette E., et al.
(författare)
-
A functional VipA-VipB interaction is required for the type VI secretion system activity of Vibrio cholerae O1 strain A1552
- 2013
-
Ingår i: BMC Microbiology. - London, England : BioMed Central. - 1471-2180. ; 13, s. 96-
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Many Gram-negative bacteria rely on a type VI secretion system (T6SS) to infect eukaryotic cells or to compete against other microbes. Common to these systems is the presence of two conserved proteins, in Vibrio cholerae denoted VipA and VipB, which have been shown to interact in many clinically relevant pathogens. In this study, mutagenesis of a defined region within the VipA protein was used to identify residues important for VipB binding in V. cholerae O1 strain A1552. Results: A dramatically diminished interaction was shown to correlate with a decrease in VipB stability and a loss of hemolysin co-regulated protein (Hcp) secretion and rendered the bacterium unable to compete with Escherichia coli in a competition assay. Conclusions: This confirms the biological relevance of the VipA-VipB interaction, which is essential for the T6SS activity of many important human pathogens.
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| 40. |
- Bröms, Jeanette E., et al.
(författare)
-
A mutagenesis-based approach identifies amino acids in the N-terminal part of Francisella tularensis IglE that critically control type VI system-mediated secretion
- 2017
-
Ingår i: Virulence. - : TAYLOR & FRANCIS INC. - 2150-5594 .- 2150-5608. ; 8:6, s. 821-847
-
Tidskriftsartikel (refereegranskat)abstract
- The Gram-negative bacterium Francisella tularensis is the etiological agent of the zoonotic disease tularemia. Its life cycle is characterized by an ability to survive within phagocytic cells through phagosomal escape and replication in the cytosol, ultimately causing inflammasome activation and host cell death. Required for these processes is the Francisella Pathogenicity Island (FPI), which encodes a Type VI secretion system (T6SS) that is active during intracellular infection. In this study, we analyzed the role of the FPI-component IglE, a lipoprotein which we previously have shown to be secreted in a T6SS-dependent manner. We demonstrate that in F. tularensis LVS, IglE is an outer membrane protein. Upon infection of J774 cells, an Delta iglE mutant failed to escape from phagosomes, and subsequently, to multiply and cause cytopathogenicity. Moreover, Delta iglE was unable to activate the inflammasome, to inhibit LPS-stimulated secretion of TNF-alpha, and showed marked attenuation in the mouse model. In F. novicida, IglE was required for in vitro secretion of IglC and VgrG. A mutagenesis-based approach involving frameshift mutations and alanine substitution mutations within the first similar to 38 residues of IglE revealed that drastic changes in the sequence of the extreme N-terminus (residues 2-6) were well tolerated and, intriguingly, caused hyper-secretion of IglE during intracellular infection, while even subtle mutations further downstream lead to impaired protein function. Taken together, this study highlights the importance of IglE in F. tularensis pathogenicity, and the contribution of the N-terminus for all of the above mentioned processes.
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| 41. |
- Bröms, Jeanette E, et al.
(författare)
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Dissection of the Functions of the IglC Protein of Francisella tularensis
- 2010
-
Ingår i: The challenge of highly pathogenic microorganisms. - Dordrecht : Springer. - 9789048190539 - 9789048190546 ; , s. 67-75
-
Konferensbidrag (refereegranskat)abstract
- Francisella tularensis harbors genes with similarity to genes encoding components of a type VI secretion system (T6SS). These include iglA and iglB, the homologues of which are conserved in T6SSs. They are part of the igl operon, also encompassing the iglC and iglD genes. We have used a yeast two-hybrid system to study the interaction of the Igl proteins of E tularensis LVS. Previously, we identified a region of IglA necessary for efficient binding to IglB as well as for IglAB protein stability and intra-macrophage growth with an essential role for a conserved alpha-helical region. Thus, IglA-IglB complex formation is clearly crucial for Francisella pathogenicity and the same interaction is conserved in other human pathogens. Herein, the interaction of IglC with other members of the operon was investigated. It showed no binding to the other members in the yeast two-hybrid assay and we found also that two cysteine residues, C191 and C192, predicted to be putative prenylation sites, played no role for the important contribution of IglC to the intracellular replication of E tularensis although C191 was important for the stability of the protein.
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| 42. |
- Bröms, Jeanette E., et al.
(författare)
-
DotU and VgrG, core components of type VI secretion systems, are essential for Francisella LVS pathogenicity
- 2012
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:4
-
Tidskriftsartikel (refereegranskat)abstract
- The Gram-negative bacterium Francisella tularensis causes tularemia, a disease which requires bacterial escape from phagosomes of infected macrophages. Once in the cytosol, the bacterium rapidly multiplies, inhibits activation of the inflammasome and ultimately causes death of the host cell. Of importance for these processes is a 33-kb gene cluster, the Francisella pathogenicity island (FPI), which is believed to encode a type VI secretion system (T6SS). In this study, we analyzed the role of the FPI-encoded proteins VgrG and DotU, which are conserved components of type VI secretion (T6S) clusters. We demonstrate that in F. tularensis LVS, VgrG was shown to form multimers, consistent with its suggested role as a trimeric membrane puncturing device in T6SSs, while the inner membrane protein DotU was shown to stabilize PdpB/IcmF, another T6SS core component. Upon infection of J774 cells, both Delta vgrG and Delta dotU mutants did not escape from phagosomes, and subsequently, did not multiply or cause cytopathogenicity. They also showed impaired activation of the inflammasome and marked attenuation in the mouse model. Moreover, all of the DotU-dependent functions investigated here required the presence of three residues that are essentially conserved among all DotU homologues. Thus, in agreement with a core function in T6S clusters, VgrG and DotU play key roles for modulation of the intracellular host response as well as for the virulence of F. tularensis.
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| 43. |
- Bröms, Jeanette E, 1974-, et al.
(författare)
-
IglG and IglI of the Francisella pathogenicity island are important virulence determinants of Francisella tularensis LVS
- 2011
-
Ingår i: Infection and Immunity. - : American Society for Microbiology. - 0019-9567 .- 1098-5522. ; 79:9, s. 3683-3696
-
Tidskriftsartikel (refereegranskat)abstract
- The Gram-negative bacterium Francisella tularensis is the causative agent of tularemia, a disease intimately associated with the multiplication of the bacterium within host macrophages. This in turn requires the expression of Francisella pathogenicity island (FPI) genes, believed to encode a type VI secretion system. While the exact functions of many of the components have yet to be revealed, some have been found to contribute to the ability of Francisella to cause systemic infection in mice as well as to prevent phagolysosomal fusion and facilitate escape into the host cytosol. Upon reaching this compartment, the bacterium rapidly multiplies, inhibits activation of the inflammasome, and ultimately causes apoptosis of the host cell. In this study, we analyzed the contribution of the FPI-encoded proteins IglG, IglI, and PdpE to the aforementioned processes in F. tularensis LVS. The ΔpdpE mutant behaved similarly to the parental strain in all investigated assays. In contrast, ΔiglG and ΔiglI mutants, although they were efficiently replicating in J774A.1 cells, both exhibited delayed phagosomal escape, conferred a delayed activation of the inflammasome, and exhibited reduced cytopathogenicity as well as marked attenuation in the mouse model. Thus, IglG and IglI play key roles for modulation of the intracellular host response and also for the virulence of F. tularensis.
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| 44. |
- Bröms, Jeanette E, 1974-, et al.
(författare)
-
The role of the Francisella Tularensis pathogenicity island in type VI secretion, intracellular survival, and modulation of host cell signaling
- 2010
-
Ingår i: Frontiers in microbiology. - : Frontiers Media SA. - 1664-302X. ; 1, s. 136-
-
Tidskriftsartikel (refereegranskat)abstract
- Francisella tularensis is a highly virulent gram-negative intracellular bacterium that causes the zoonotic disease tularemia. Essential for its virulence is the ability to multiply within host cells, in particular monocytic cells. The bacterium has developed intricate means to subvert host immune mechanisms and thereby facilitate its intracellular survival by preventing phagolysosomal fusion followed by escape into the cytosol, where it multiplies. Moreover, it targets and manipulates numerous host cell signaling pathways, thereby ameliorating the otherwise bactericidal capacity. Many of the underlying molecular mechanisms still remain unknown but key elements, directly or indirectly responsible for many of the aforementioned mechanisms, rely on the expression of proteins encoded by the Francisella pathogenicity island (FPI), suggested to constitute a type VI secretion system. We here describe the current knowledge regarding the components of the FPI and the roles that have been ascribed to them.
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| 45. |
- Bröms, Jeanette E., et al.
(författare)
-
Unique substrates secreted by the type VI secretion system of Francisella tularensis during intramacrophage infection
- 2012
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:11
-
Tidskriftsartikel (refereegranskat)abstract
- Gram-negative bacteria have evolved sophisticated secretion machineries specialized for the secretion of macromolecules important for their life cycles. The Type VI secretion system (T6SS) is the most widely spread bacterial secretion machinery and is encoded by large, variable gene clusters, often found to be essential for virulence. The latter is true for the atypical T6SS encoded by the Francisella pathogenicity island (FPI) of the highly pathogenic, intracellular bacterium Francisella tularensis. We here undertook a comprehensive analysis of the intramacrophage secretion of the 17 FPI proteins of the live vaccine strain, LVS, of F. tularensis. All were expressed as fusions to the TEM beta-lactamase and cleavage of the fluorescent substrate CCF2-AM, a direct consequence of the delivery of the proteins into the macrophage cytosol, was followed over time. The FPI proteins IglE, IglC, VgrG, IglI, PdpE, PdpA, IglJ and IglF were all secreted, which was dependent on the core components DotU, VgrG, and IglC, as well as IglG. In contrast, the method was not directly applicable on F. novicida U112, since it showed very intense native beta-lactamase secretion due to FTN_1072. Its role was proven by ectopic expression in trans in LVS. We did not observe secretion of any of the LVS substrates VgrG, IglJ, IglF or IglI, when tested in a FTN_1072 deficient strain of F. novicida, whereas IglE, IglC, PdpA and even more so PdpE were all secreted. This suggests that there may be fundamental differences in the T6S mechanism among the Francisella subspecies. The findings further corroborate the unusual nature of the T6SS of F. tularensis since almost all of the identified substrates are unique to the species.
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| 46. |
- Bönquist, Linda, 1974-, et al.
(författare)
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MglA and Igl proteins contribute to the modulation of Francisella tularensis live vaccine strain-containing phagosomes in murine macrophages
- 2008
-
Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 76:8, s. 3502-3510
-
Tidskriftsartikel (refereegranskat)abstract
- The Francisella tularensis live vaccine strain (LVS), in contrast to its iglC mutant, replicates in the cytoplasm of macrophages. We studied the outcome of infection of the murine macrophagelike cell line J774A.1 with LVS and with iglC, iglD, and mglA mutants, the latter of which is deficient in a global regulator. Compared to LVS, all of the mutants showed impaired intracellular replication up to 72 h, and the number of the mglA mutant bacteria even decreased. Colocalization with LAMP-1 was significantly increased for all mutants compared to LVS, indicating an impaired ability to escape into the cytoplasm. A lysosomal acidity-dependent dye accumulated in approximately 40% of the vacuoles containing mutant bacteria but not at all in vacuoles containing LVS. Preactivation of the macrophages with gamma interferon inhibited the intracellular growth of all strains and significantly increased acidification of phagosomes containing the mutants, but it only slightly increased the LAMP-1 colocalization. The intracellular replication and phagosomal escape of the iglC and iglD mutants were restored by complementation in trans. In conclusion, the IglC, IglD, and MglA proteins each directly or indirectly critically contribute to the virulence of F. tularensis LVS, including its intracellular replication, cytoplasmic escape, and inhibition of acidification of the phagosomes.
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| 47. |
- Champion, Mia D, et al.
(författare)
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Comparative genomic characterization of Francisella tularensis strains belonging to low and high virulence subspecies
- 2009
-
Ingår i: PLoS pathogens. - : Public Library of Science (PLoS). - 1553-7374. ; 5:5, s. e1000459-
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Tidskriftsartikel (refereegranskat)abstract
- Tularemia is a geographically widespread, severely debilitating, and occasionally lethal disease in humans. It is caused by infection by a gram-negative bacterium, Francisella tularensis. In order to better understand its potency as an etiological agent as well as its potential as a biological weapon, we have completed draft assemblies and report the first complete genomic characterization of five strains belonging to the following different Francisella subspecies (subsp.): the F. tularensis subsp. tularensis FSC033, F. tularensis subsp. holarctica FSC257 and FSC022, and F. tularensis subsp. novicida GA99-3548 and GA99-3549 strains. Here, we report the sequencing of these strains and comparative genomic analysis with recently available public Francisella sequences, including the rare F. tularensis subsp. mediasiatica FSC147 strain isolate from the Central Asian Region. We report evidence for the occurrence of large-scale rearrangement events in strains of the holarctica subspecies, supporting previous proposals that further phylogenetic subdivisions of the Type B clade are likely. We also find a significant enrichment of disrupted or absent ORFs proximal to predicted breakpoints in the FSC022 strain, including a genetic component of the Type I restriction-modification defense system. Many of the pseudogenes identified are also disrupted in the closely related rarely human pathogenic F. tularensis subsp. mediasiatica FSC147 strain, including modulator of drug activity B (mdaB) (FTT0961), which encodes a known NADPH quinone reductase involved in oxidative stress resistance. We have also identified genes exhibiting sequence similarity to effectors of the Type III (T3SS) and components of the Type IV secretion systems (T4SS). One of the genes, msrA2 (FTT1797c), is disrupted in F. tularensis subsp. mediasiatica and has recently been shown to mediate bacterial pathogen survival in host organisms. Our findings suggest that in addition to the duplication of the Francisella Pathogenicity Island, and acquisition of individual loci, adaptation by gene loss in the more recently emerged tularensis, holarctica, and mediasiatica subspecies occurred and was distinct from evolutionary events that differentiated these subspecies, and the novicida subspecies, from a common ancestor. Our findings are applicable to future studies focused on variations in Francisella subspecies pathogenesis, and of broader interest to studies of genomic pathoadaptation in bacteria.
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| 48. |
- Chin, Chui-Yoke, et al.
(författare)
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Francisella FlmX broadly affects lipopolysaccharide modification and virulence
- 2021
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Ingår i: Cell Reports. - : Elsevier. - 2211-1247. ; 35:11
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Tidskriftsartikel (refereegranskat)abstract
- The outer membrane protects Gram-negative bacteria from the host environment. Lipopolysaccharide (LPS), a major outer membrane constituent, has distinct components (lipid A, core, O-antigen) generated by specialized pathways. In this study, we describe the surprising convergence of these pathways through FlmX, an uncharacterized protein in the intracellular pathogen Francisella. FlmX is in the flippase family, which includes proteins that traffic lipid-linked envelope components across membranes. flmX deficiency causes defects in lipid A modification, core remodeling, and O-antigen addition. We find that an F. tularensis mutant lacking flmX is >1,000,000-fold attenuated. Furthermore, FlmX is required to resist the innate antimicrobial LL-37 and the antibiotic polymyxin. Given FlmX's central role in LPS modification and its conservation in intracellular pathogens Brucella, Coxiella, and Legionella, FlmX may represent a novel drug target whose inhibition could cripple bacterial virulence and sensitize bacteria to innate antimicrobials and antibiotics.
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| 49. |
- Conlan, J Wayne, et al.
(författare)
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Differential ability of novel attenuated targeted deletion mutants of Francisella tularensis subspecies tularensis strain SCHU S4 to protect mice against aerosol challenge with virulent bacteria : effects of host background and route of immunization
- 2010
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Ingår i: Vaccine. - : Elsevier BV. - 0264-410X .- 1873-2518. ; 28:7, s. 1824-1831
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Tidskriftsartikel (refereegranskat)abstract
- Francisella tularensis subspecies tularensis is a highly virulent facultative intracellular pathogen of humans and a potential biological weapon. A live vaccine strain, F. tularensis LVS, was developed more than 50 years ago by pragmatic attenuation of a strain of the less virulent holarctica subspecies. LVS was demonstrated to be highly effective in human volunteers who were exposed to intradermal challenge with fully virulent subsp. tularensis, but was less effective against aerosol exposure. LVS faces regulatory hurdles that to date have prevented its licensure for general use. Therefore, a better defined and more effective vaccine is being sought. To this end we have created gene deletion mutants in the virulent subsp. tularensis strain and tested them for their ability to elicit a protective immune response against systemic or aerosol challenge with the highly virulent wild-type subsp. tularensis strain, SCHU S4. Both oral and intradermal (ID) primary vaccination routes were assessed in BALB/c and C3H/HeN mice as was oral boosting. One SCHU S4 mutant missing the heat shock gene, clpB, was significantly more attenuated than LVS whereas a double deletion mutant missing genes FTT0918 and capB was as attenuated as LVS. In general mice immunized with SCHU S4DeltaclpB were significantly better protected against aerosol challenge than mice immunized with LVS. A single ID immunization of BALB/c mice with SCHU S4DeltaclpB was at least as effective as any other regimen examined. Mice immunized with SCHU S4Delta0918DeltacapB were generally protected to a similar degree as mice immunized with LVS. A preliminary examination of immune responses to vaccination with LVS, SCHU S4DeltaclpB, or SCHU S4Delta0918DeltacapB provided no obvious correlate to their relative efficacies.
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| 50. |
- Conlan, J. Wayne, et al.
(författare)
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Modern Development and Production of a New Live Attenuated Bacterial Vaccine, SCHU S4 Delta clpB, to Prevent Tularemia
- 2021
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Ingår i: Pathogens. - : MDPI. - 2076-0817. ; 10:7
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Tidskriftsartikel (refereegranskat)abstract
- Inhalation of small numbers of Francisella tularensis subspecies tularensis (Ftt) in the form of small particle aerosols causes severe morbidity and mortality in people and many animal species. For this reason, Ftt was developed into a bona fide biological weapon by the USA, by the former USSR, and their respective allies during the previous century. Although such weapons were never deployed, the 9/11 attack quickly followed by the Amerithrax attack led the U.S. government to seek novel countermeasures against a select group of pathogens, including Ftt. Between 2005-2009, we pursued a novel live vaccine against Ftt by deleting putative virulence genes from a fully virulent strain of the pathogen, SCHU S4. These mutants were screened in a mouse model, in which the vaccine candidates were first administered intradermally (ID) to determine their degree of attenuation. Subsequently, mice that survived a high dose ID inoculation were challenged by aerosol or intranasally (IN) with virulent strains of Ftt. We used the current unlicensed live vaccine strain (LVS), first discovered over 70 years ago, as a comparator in the same model. After screening 60 mutants, we found only one, SCHU S4 Delta clpB, that outperformed LVS in the mouse ID vaccination-respiratory-challenge model. Currently, SCHU S4 Delta clpB has been manufactured under current good manufacturing practice conditions, and tested for safety and efficacy in mice, rats, and macaques. The steps necessary for advancing SCHU S4 Delta clpB to this late stage of development are detailed herein. These include developing a body of data supporting the attenuation of SCHU S4 Delta clpB to a degree sufficient for removal from the U.S. Select Agent list and for human use; optimizing SCHU S4 Delta clpB vaccine production, scale up, and long-term storage; and developing appropriate quality control testing approaches.
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