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Background: The zoonotic bacterium Francisella tularensis, the causative agent of tularaemia, can be transmitted to humans via multiple routes, including through contact with infected animals, contaminated water or arthropod vectors. Ticks have not previously been described as transmitting the disease in Sweden. Recently, Ixodid tick species have expanded their latitudinal and altitudinal range in Sweden to areas where the disease is endemic. Tularaemia is a cause of growing concern, spreading to new areas in Sweden and infecting hares and humans.

Objectives: To establish whether ticks could be a potential arthropod vector in the transmission of F. tularensis subsp. holarctica in Sweden.

Methods: Ticks were collected from northern Sweden and screened for F. tularensis. A follow-up study with ticks collected from F. tularensis-positive hares was performed. Ticks were analysed using real-time PCR and a pathological examination was performed on the hares.

Results: F. tularensis subsp. holarctica was identified in ticks from one cat and three F. tularensis-infected hares. Two hares had skin lesions associated with tick bites with intralesional F. tularensis bacteria.

Conclusions: F. tularensis subsp. holarctica was isolated from ticks collected from the hares and cat, the first such reports in ticks in Sweden. Identification of the bacteria at the tick bite site and the more chronic character of the skin lesions compared to those of inner organs suggest that the ticks infected the hares. The cat showed no clinical signs of disease, suggesting that its tick was indeed the vector. These new findings suggest that ticks play a role in the transmission of F. tularensis to human and animal hosts in Sweden.

Background: Tularemia is a re-emerging disease in Sweden, frequently affecting working-age individuals and often resulting in prolonged recovery times. The disease-related economic impact has not previously been investigated outside bioterrorism scenarios. In this study we assess the economic burden associated with endemic tularemia in Sweden.  

Method: Data on primary care visits, hospital admissions, and sick leaves were collected from participants with serology-confirmed tularemia through questionnaires and electronic medical records in Northern Sweden from 2011 to 2021. The dataset was enhanced with national cost-of-care data for tularemia in primary and specialist care from NordDRG (Diagnosis Related Group; 2021-2023), and sickness benefit data from the Social Insurance Agency (2011-2023). Total direct and indirect costs were estimated by integrating these data sources and adjusted for inflation to 2025 levels. Average salary and labor productivity (Gross Domestic Product [GDP] per person employed) was assumed.

Results: Among participants (n=294), the mean age was 52 years; 68.1% were employed or job-seeking, with 71.3% of these reporting sick leave during illness. Healthcare costs were primarily driven by general practitioner visits (mean 1.74 visits; 5,125 SEK per participant [p.p.]) and hospital admissions (15.6% of participants; mean 4.7 days; 11,268 SEK p.p.) with relatively low complexity (mean DRG weight 0.8), while antibiotic treatment and diagnostics were less costly (figure 1). Indirect costs included sick pay (≤14 days; 70.0%; 4,106 SEK p.p.), sickness benefit (>14 days; 30.0%; 5,835 SEK p.p.), and lost GDP-based productivity, and made up 75.7% of total costs (78,503 SEK p.p.).

A mean of 394 (range 87-1,048) tularemia cases per year were reported to the Swedish Public Health Agency between 2011-2023. The estimated annual societal cost of human tularemia infection was 30.9 million SEK (range 6.8-82.3 million SEK). 

Conclusion: Tularemia imposes a significant socioeconomic burden on society primarily through morbidity and prolonged recovery. Regional outbreaks could have detrimental effects on local economy and public services. Further evaluation of the cost-effectiveness of primary and secondary preventive measures is required. 

Background: Tularaemia is a geographically widespread disease affecting animals and humans. In Sweden, transmission patterns are complex, occurring mainly through mosquito vectors. We investigated human exposure and whether passive tularaemia surveillance (reported by the public) of deceased wild hares could be used to temporally and geographically predict outbreaks among humans. 

Methods: A survey was sent to the 830 cases of reported tularaemia in Norrbotten county, Sweden, between 2011-2021; and 313/415 (75.4%) respondents with laboratory-evidence of tularaemia were included. Geographic data from human infections in 2019 (n=54) and 2020 (n=77) was compared to data on deceased forest hares from the Swedish Veterinary Agency, matched by year and region.

Results: Respondents (n=313) rarely reported direct exposure to hares (8,6%) and/or other rodents (3.8%) during the 2-weeks prior to illness; while recreational activities (forest hiking 61.6%; mushroom/berry-picking 24.0%; fishing 11.5%; and hunting 3.8%) were more common. Peak incidence of reported deceased hares in 2019 and 2020 (n=84 and n=66; 11/15 [73.3%] and 19/21 [90.4%] PCR-positive for tularaemia, respectively) corresponded to peak incidence of symptom onset of human cases (median difference +6 days [2019] and -2 days [2020]; p=0.066 and p=0.695, respectively). Distribution of reported hares corresponded with municipalities with highest incidence of human tularaemia and location of self-reported suspected infection (Figure 1). Most reported their location of infection to be within their residential municipality (n=92/106, 86.8%).

Conclusion: Passive surveillance of tularaemia using deceased hares correlates with symptom onset in humans and could predict geographical outbreaks in the community. Surveillance of other affected/reservoir species should be considered. 

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.

Antimicrobial resistance in Neisseria gonorrhoeae is threatening the treatment and control of gonorrhea globally, and new treatment options are imperative. Utilizing our dynamic in vitro hollow fiber infection model (HFIM), we examined the pharmacodynamics of the first-in-class spiropyrimidinetrione (DNA gyrase B inhibitors), zoliflodacin, against the N. gonorrhoeae reference strains World Health Organization F (susceptible to all relevant antimicrobials) and WHO X (extensively drug resistant, including resistance to ceftriaxone) over 7 days. Dose-range experiments with both strains, simulating zoliflodacin single oral dose regimens of 0.5–8 g, and dose-fractionation experiments with WHO X, simulating zoliflodacin oral dose therapy with 1–4 g administered as q12 h and q8 h for 24 h, were performed. A kill-rate constant that reflected a rapid bacterial kill during the first 6.5 h for both strains and all zoliflodacin doses was identified. In the dose-range experiments, the zoliflodacin 2–8 g single-dose treatments successfully eradicated both WHO strains, and resistance to zoliflodacin was not observed. However, zoliflodacin as a single 0.5 g dose failed to eradicate both WHO strains, and a 1 g single dose failed to eradicate WHO X in one of two experiments. The zoliflodacin 1 g/day regimen also failed to eradicate WHO X when administered as two and three divided doses given at q12 h and q8 h in the dose-fractionation studies, respectively. All failed regimens selected for zoliflodacin-resistant mutants. In conclusion, these data demonstrate that zoliflodacin should be administered at >2 g as a single oral dose to provide effective killing and resistance suppression of N. gonorrhoeae. Future studies providing pharmacokinetic data for zoliflodacin (and other gonorrhea therapeutic antimicrobials) in urogenital and extragenital infection sites, particularly in the pharynx, and evaluation of gonococcal strains with different gyrB mutations would be important.

Treatment options for gonorrhoea are scarce. Drug repurposing of bioactive molecules approved for other conditions might therefore be of value. We developed a method for wide-scale, systematic drug repurposing screen to identify molecules with activity against Neisseria gonorrhoeae and screened the Prestwick Chemical Library (1200 FDA-approved drugs). As a proof-of-concept, we further examined one promising and interesting screening hit (auranofin; antirheumatic agent). Three WHO gonococcal reference strains (WHO F, O, P) were used for the Library screening. The strains were grown in presence of a fixed concentration of the library drugs in 384-well plates for 12 h, and the remaining bacterial respiration, to reflect growth, was then quantitatively measured using optical density (OD) 450 nm and a resazurin assay. The activity of auranofin was further examined using in vitro susceptibility testing (minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)) against genetically diverse antimicrobial-resistant N. gonorrhoeae strains and time-kill assays. Sixty-eight molecules significantly inhibited bacterial growth of WHO F, O and P. Auranofin showed potent in vitro bactericidal activity (in MIC-, MBC- and time-kill assays) against four WHO reference strains. No cross-resistance between auranofin and any antimicrobial currently or previously used for gonorrhoea treatment was found when examining 51 selected antimicrobial-resistant gonococcal strains. In conclusion, this is the first wide-scale systematic screening effort for repurposing drugs for future treatment of gonorrhoea. Additional studies examining mechanism(s) of action, resistance development, in vivo anti-gonococcal activity and pharmacokinetics/pharmacodynamics for gonococcal infections of auranofin and several other significant screening hits would be valuable.

Energetic nutrients are oxidized to sustain high intracellular NADPH/NADP(+) ratios. NADPH-dependent reduction of thioredoxin-1 (Trx1) disulfide and glutathione disulfide by thioredoxin reductase-1 (TrxR1) and glutathione reductase (Gsr), respectively, fuels antioxidant systems and deoxyribonucleotide synthesis. Mouse livers lacking both TrxR1 and Gsr sustain these essential activities using an NADPH-independent methionine-consuming pathway; however, it remains unclear how this reducing power is distributed. Here, we show that liver-specific co-disruption of the genes encoding Trx1, TrxR1, and Gsr (triplenull) causes dramatic hepatocyte hyperproliferation. Thus, even in the absence of Trx1, methionine-fueled glutathione production supports hepatocyte S phase deoxyribonucleotide production. Also, Trx1 in the absence of TrxR1 provides a survival advantage to cells under hyperglycemic stress, suggesting that glutathione, likely via glutaredoxins, can reduce Trx1 disulfide in vivo. In triple-null livers like in many cancers, deoxyribonucleotide synthesis places a critical yet relatively low-volume demand on these reductase systems, thereby favoring high hepatocyte turnover over sustained hepatocyte integrity.

We describe a method for the quantification of proteins in a biological matrix through digestion with pepsin. Pepsin is a gastric protease that efficiently cleaves proteins in an acidic environment. In this study, it has been used to generate peptides used for the quantification of physiologically relevant thioredoxin proteins in a lysate of Bacillus anthracis-the causative agent of anthrax. Carefully selected signature peptides for proteins that were digested with pepsin were immobilized on agarose gel. Filtered samples were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) and by two-dimensional liquid chromatography tandem mass spectrometry (LC/LC-MS/MS) when additional selectivity was needed. Some important incubation parameters were adjusted to get the highest possible peptide yield. Escherichia coli was used as a surrogate matrix for the method development. The method was validated at a low nM range for selectivity, accuracy and precision. Validation showed that signature peptides were selective for the proteins, and that the method accuracy varied between 89 and 115 % with a precision of less than 12 %. The results from using pepsin in analyzing samples from Bacillus anthracis were similar to those previously obtained using western blot, and they validate pepsin as a suitable protease to generate signature peptides in a complex biological matrix as an alternative to trypsin.

Background: Tularemia, which is caused by the intracellular bacterium Francisella tularensis, exist in several different forms: ulceroglandular, oculoglandular, typhoid (septic), pulmonary and oropharyngeal. Sweden experiences recurrent epidemics with irregular intervals and geographical localizations, and the infection is classified as notifiable. The infection is usually treated using ciprofloxacin or doxycycline although aminoglycosides is an option.

Methods: Cases were extracted from the County of Norrbotten database of notifiable diseases and were analyzed with respect to geographic localization, clinical presentation, and a number of other parameters.

Results: A total of 406 cases in the county were reported between July and November of 2015, with a peak in late August. This is a 100-fold increase compared to 2014 and a 10-fold increase compared to 2013.The incidence differed considerably between communities in the county and were up to 330 cases per 100,000 with a mean incidence of 160 cases per 100,000 inhabitants. Cases tended to cluster around bodies of water, where mosquitoes are more prevalent. The ulceroglandular form was dominant with 80% of reported cases, although typhoid, pulmonary, and oropharyngeal cases were reported. Most cases were diagnosed using PCR and/or serology, although some ulceroglandular cases were diagnosed presumptively based on a typical ulcer, palpable nodes, and fever. Most cases were treated by primary care physicians using ciprofloxacin with a high success rate, although doxycycline was sometimes used.

Conclusion: The epidemic, which is the biggest in the County of Norrbotten so far, and one of the biggest in Sweden ever, demonstrates that tularemia is a disease that needs to receive more attention, at least in the northern parts of the country.

Background: Bacillus anthracis is the causative agent of anthrax, a disease associated with a very high mortality rate in its invasive forms. Methods: We studied a number of ebselen analogs as inhibitors of B. anthracis thioredoxin reductase and their antibacterial activity on Bacillus subtilis, Staphylococcus aureus, Bacillus cereus and Mycobacterium tuberculosis. Results: The most potent compounds in the series gave IC50 values down to 70 nM for the pure enzyme and minimal inhibitory concentrations (MICs) down to 0.4 mu M (0.12 mu g/ml) for B. subtilis,1.5 mu M (0.64 mu g/ml) for S. aureus, 2 mu M (0.86 mu g/ml) for B. cereus and 10 mu g/ml for M. tuberculosis. Minimal bactericidal concentrations (MBCs) were found at 1-1.5 times the MIC, indicating a general, class-dependent, bactericidal mode of action. The combined bacteriological and enzymological data were used to construct a preliminary structure-activity-relationship for the benzoisoselenazol class of compounds. When S. aureus and B. subtilis were exposed to ebselen, we were unable to isolate resistant mutants on both solid and in liquid medium suggesting a high resistance barrier. Conclusions: These results suggest that ebselen and analogs thereof could be developed into a novel antibiotic class, useful for the treatment of infections caused by B. anthracis, S. aureus, M. tuberculosis and other clinically important bacteria. Furthermore, the high barrier against resistance development is encouraging for further drug development. General significance: We have characterized the thioredoxin system from B. anthracis as a novel drug target and ebselen and analogs thereof as a potential new class of antibiotics targeting several important human pathogens.