Mutation of wbtJ, a N-formyltransferase involved in O-antigen synthesis, results in biofilm formation, phase variation and attenuation in Francisella tularensis.

Two clinically important subspecies, Francisella tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B) are responsible for most tularaemia cases, but these isolates typically form a weak biofilm under in vitro conditions. Phase variation of the F. tularensis lipopolysaccharide (LPS) has been reported in these subspecies, but the role of variation is unclear as LPS is crucial for virulence. We previously demonstrated that a subpopulation of LPS variants can constitutively form a robust biofilm in vitro, but it is unclear whether virulence was affected. In this study, we show that biofilm-forming variants of both fully virulent F. tularensis subspecies were highly attenuated in the murine tularaemia model by multiple challenge routes. Genomic sequencing was performed on these strains, which revealed that all biofilm-forming variants contained a lesion within the wbtJ gene, a formyltransferase involved in O-antigen synthesis. A ΔwbtJ deletion mutant recapitulated the biofilm, O-antigen and virulence phenotypes observed in natural variants and could be rescued through complementation with a functional wbtJ gene. Since the spontaneously derived biofilm-forming isolates in this study were a subpopulation of natural variants, reversion events to the wbtJ gene were detected that eliminated the phenotypes associated with biofilm variants and restored virulence. These results demonstrate a role for WbtJ in biofilm formation, LPS variation and virulence of F. tularensis.

Thoracic manifestations of tularaemia: a case series.

BACKGROUND: Tularaemia is a zoonotic disease caused by Francisella tularensis, a highly virulent bacterium that affects humans and small wild animals. It is transmitted through direct contact with infected animals or indirectly through contaminated soil, water or arthropod bites (e.g. ticks). Primary thoracic manifestations of tularaemia are infrequent and, therefore, a diagnostic challenge for clinicians. METHODS: We report six tularaemia cases with exclusively thoracic involvement diagnosed in a clinic for pulmonary diseases in Bavaria between 10/2020 and 02/2022. RESULTS: All patients lived or were active in rural areas, four reported a recent tick bite. All patients presented with thoracic lymphadenopathy and pulmonary tumours or consolidations; all underwent bronchoscopy with EBUS-TBNA of lymph nodes, three lung biopsies as well. Five patients showed inflammatory changes in the endobronchial mucosa. The main histological findings were necrotic epithelioid granulomas with remarkable granulocyte infiltration. All cases were identified by positive serology, five by PCR (here identification of F.t. ssp. Holarctica) from biopsy as well. As first-line therapy, oral ciprofloxacin was given (5/6); in 2/6 cases, a combination of quinolone-rifampicin was given. CONCLUSIONS: Pulmonary tularaemia may occur after tick bites and without extrathoracic manifestations. In patients who present with thoracic lymphadenopathy and pulmonary consolidations and who are exposed to increased outdoor activities, tularaemia should be included in the diagnostic pathway. Histologically, the presence of neutrophil-granulocyte infiltrations might help to distinguish tularaemia from other granulomatous infections, e.g. tuberculosis. The combination of quinolone-rifampicin rather than i.v. gentamicin reduced length of hospital stay in two patients.

Is clinical primary care surveillance for tularaemia a useful addition to laboratory surveillance? An analysis of notification data for Finland, 2013 to 2019.

BackgroundIn Finland, surveillance of tularaemia relies on laboratory-confirmed case notifications to the National infectious Diseases Register (NIDR).AimThe aim of the study was to assess the suitability and usefulness of clinical surveillance as an addition to laboratory notification to improve tularaemia surveillance in Finland.MethodsWe retrieved NIDR tularaemia surveillance and primary healthcare data on clinically diagnosed tularaemia cases in Finland between 2013 and 2019. We compared incidences, demographic distributions and seasonal trends between the two data sources.ResultsThe median annual incidence was 0.6 (range: 0.1-12.7) and 0.8 (range: 0.6-7.2) per 100,000 for NIDR notifications and primary healthcare notifications, respectively. Cases reported to NIDR were slightly older than cases reported to primary healthcare (median: 53 years vs 50 years, p = 0.04), but had similar sex distribution. Seasonal peaks differed between systems, both in magnitude and in timing. On average, primary healthcare notifications peaked 3 weeks before NIDR. However, peaks in NIDR were more pronounced, for example in 2017, monthly incidence per 100,000 of NIDR notifications peaked at 12.7 cases in September, while primary healthcare notifications peaked at 7.2 (1.8 ratio) in August.ConclusionsClinically diagnosed cases provide a valuable additional data source for surveillance of tularaemia in Finland. A primary healthcare-based system would allow for earlier detection of increasing incidences and thereby for early warning of outbreaks. This is crucial in order to implement targeted control and prevention measures as early as possible.

Tularaemia: a case report and review.

Tularaemia is a rare infectious disease caused by Francisella tularensis. In Poland, F. tularensis infections are caused by F. tularensis subspecies holarctica (type B). The disease is widespread among multiple animal species. Humans are usually infected via insect bites and less commonly by other routes (contact with animals, inhalation of contaminated aerosol or dust, or oral route). In recent years, the prevalence of tularaemia in Poland was slightly more than dozen cases per year. Depending on the route of infection, the disease has various clinical presentations, of which the most common is the ulceroglandular form. We present a typical case of this clinical form, along with information on epidemiology, clinical presentation, diagnosis, and treatment of this rare disease. Because of a low prevalence and miscellaneous clinical features, the diagnosis is often delayed. Tularaemia should be included in the differential diagnosis of fever with local lymph node enlargement as well as atypical cases of upper airway infections and pneumonia.

Francisella tularensis as the cause of protracted fever.

BACKGROUND: Tularemia, a re-emerging, potential life threatening infectious disease, can present itself with nonspecific clinical symptoms including fever, chills and malaise. Taking a detailed history of exposure and a highly raised index of clinical suspicion are necessary to take the appropriate diagnostic and therapeutic steps in this setting. Here, a case report of typhoid tularaemia is presented. CASE PRESENTATION: A 53-year old male forester and farmer with protracted fever, abdominal pain, diarrhoea and loss of weight, who experienced productive cough and a pulmonary infiltrate later in the course of disease, was admitted for further investigation. Tularaemia was suspected only owing to history and confirmed by serologic testing more than three weeks after the beginning of the symptoms. The initial antibiotic therapy with ceftriaxone/doxycycline was switched to ciprofloxacin, resulting in the resolution of fever and symptoms. CONCLUSION: Tularaemia has to be considered as a differential diagnosis in febrile patients, even more in cases with protracted fever. Since tularaemia is expanding geographically, involving more animal hosts and causing larger outbreaks, clinicians have to be aware of this potentially fatal disease.

Bacterial lipoproteins and other factors released by Francisella tularensis modulate human neutrophil lifespan: Effects of a TLR1 SNP on apoptosis inhibition.

Francisella tularensis infects several cell types including neutrophils, and aberrant neutrophil accumulation contributes to tissue destruction during tularaemia. We demonstrated previously that F. tularensis strains Schu S4 and live vaccine strain markedly delay human neutrophil apoptosis and thereby prolong cell lifespan, but the bacterial factors that mediate this aspect of virulence are undefined. Herein, we demonstrate that bacterial conditioned medium (CM) can delay apoptosis in the absence of direct infection. Biochemical analyses show that CM contained F. tularensis surface factors as well as outer membrane components. Our previous studies excluded roles for lipopolysaccharide and capsule in apoptosis inhibition, and current studies of [14 C] acetate-labelled bacteria argue against a role for other bacterial lipids in this process. At the same time, studies of isogenic mutants indicate that TolC and virulence factors whose expression requires FevR or MglA were also dispensable, demonstrating that apoptosis inhibition does not require Type I or Type VI secretion. Instead, we identified bacterial lipoproteins (BLPs) as active factors in CM. Additional studies of isolated BLPs demonstrated dose-dependent neutrophil apoptosis inhibition via a TLR2-dependent mechanism that is significantly influenced by a common polymorphism, rs5743618, in human TLR1. These data provide fundamental new insight into pathogen manipulation of neutrophil lifespan and BLP function.

Francisella tularensis in Swedish predators and scavengers.

Tularaemia is a zoonotic disease, in Europe caused by Francisella tularensis subsp. holarctica. Many lagomorphs and a variety of small rodents are wildlife species prone to develop clinical disease, while predators and scavengers are relatively resistant and may serve as sentinels. Blood samples from 656 Swedish wild predators and scavengers were serologically investigated using slide agglutination and microagglutination. In the slide agglutination test, 34 seropositive animals were detected, and they were found among all species investigated: brown bear (Ursus arctos), Eurasian lynx (Lynx lynx), raccoon dog (Nyctereutes procyonoides), red fox (Vulpes vulpes), wild boar (Sus scrofa), wolf (Canis lupus) and wolverine (Gulo gulo). Due to haemolysis the microagglutination test was more difficult to read at low titres, and only 12 animals were classified as seropositive. F. tularensis subsp. holarctica was detected by a polymerase chain reaction in lymphatic tissues of the head in one brown bear, one red fox and one wolf. The significance of this finding regarding possible latency of infection is not clear. In conclusion, the results of this study indicate that all predator and scavenger species included in this study may serve as sentinels for tularaemia in Sweden. Their role as reservoirs is unclear.

Large outbreak of tularaemia, central Sweden, July to September 2019.

On 31 of July 2019, the Public Health Agency of Sweden was alerted about an increasing number of tularaemia cases in Gävleborg, a county in central Sweden. The number of cases increased thereafter peaking at about 150 reports of illnesses every week. As at 6 October, a total of 979 cases (734 laboratory-confirmed) have been reported, mainly from counties in central Sweden. The outbreak is now considered over (as at 14 October).

Molecular identification of the source of an uncommon tularaemia outbreak, Germany, autumn 2016.

BackgroundIn 2016, an uncommon outbreak of oropharyngeal tularaemia involving six human cases occurred in Germany, caused by drinking contaminated fresh must after a grape harvest.AimWe describe the details of laboratory investigations leading to identification of the outbreak strain, its characterisation by next generation sequencing (NGS) and the finding of the possible source of contamination.MethodsWe incubated wine samples in different media and on agar plates. NGS was performed on DNA isolated from young wine, sweet reserve and an outbreak case's lymph node. A draft genome of the outbreak strain was generated. Vertebrate-specific PCRs using primers targeting the mitochondrial cytochrome b gene and product analyses by blast search were used to identify the putative source of must contamination.ResultsNo bacterial isolate could be obtained. Analysis of the draft genome sequence obtained from the sweet reserve attributed this sequence to Francisella tularensis subsp. holarctica, belonging to the B.12/B.34 phylogenetic clade (erythromycin-resistant biovar II). In addition, the DNA sequence obtained from the case's isolate supported our hypothesis that infection was caused by drinking contaminated must. The vertebrate-specific cytochrome b sequence derived from the young wine and the sweet reserve could be assigned to Apodemus sylvaticus (wood mouse), suggesting that a wood mouse infected with F. tularensis may have contaminated the must.ConclusionThe discovered source of infection and the transmission scenario of F. tularensis in this outbreak have not been observed previously and suggest the need for additional hygienic precautionary measures when processing and consuming freshly pressed must.

[Tularemia: A case report]. / Un cas de tularémie.

BACKGROUND: Tularaemia is a zoonotic disease caused by inoculation with the Gram-negative coccobacillus Francisella tularensis. It was first described in the United States in 1911 and is a rare disease with an annual reported incidence in France between 2002 and 2012 of 0.07 cases per 100,000 habitants. Reporting of the disease in humans has been mandatory in France since 2003. PATIENTS AND METHODS: Herein we report a case of tularaemia following a tick bite in a patient in the north of France. DISCUSSION: Tularaemia is a rare form of zoonosis that should be sought in the event of unexplained adenitis. Clinical presentations vary, and in certain cases only dermatological signs are manifest. Diagnosis is confirmed by bacterial serology. Rapid initiation of suitable antibiotics produces a favourable and benign outcome in most cases. However, the offending organism, which is potentially lethal, is classed as a potential bioterrorism agent.

Subspecies differentiation and genotyping of Francisella tularensis strains isolated from clinical and environmental samples.

Molecular epidemiology is one of the most rapidly developing research area. In the light of past and modern technologies it has gained number of typing methods based on molecular biology techniques. In this report, the subspecies differentiation of Francisella tularensis and genotyping of strains isolated in Poland and other geographic locations were investigated using real-time PCR and multispacer typing (MST) methods respectively. In total, 49 strains of F. tularensis included 15 strains from Poland, for subspecies differentiation the real-time PCR method was applied. For molecular typing using MST method four intergenic spacers (IS) were sequenced and compared with those previously described and deposited in GenBank (NCBI). Phylogenetic testing was performed using with the UPGMA model using MEGA6 software. The real-time PCR enables to distinguish five strains belonged to type A and 44 to type B among deposited F. tularensis strains. The MST revealed previously described genotypes, as well as 23 new genotypes were detected. The use of real-time PCR and MST method are valuable in the analysis of F. tularensis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrate convenient molecular tools (real-time PCR and multispacer typing) for Francisella tularensis detection, differentiation and genotyping which can be applied for molecular epidemiological studies and providing useful information for scientific research and during natural tularaemia outbreaks.

Francisella tularensis prevalence and load in Dermacentor reticulatus ticks in an endemic area in Central Europe.

A total of 7778 host-seeking adult Dermacentor reticulatus (Ixodida: Ixodidae) ticks were examined for the prevalence of Francisella tularensis holarctica (Thiotrichales: Francisellaceae) in a natural focus of tularaemia in the floodplain forest-meadow ecosystem along the lower reaches of the Dyje (Thaya) river in South Moravia (Czech Republic) between 1995 and 2013. Ticks were pooled (10 specimens per pool) and their homogenates inoculated subcutaneously in 4-week-old specific pathogen-free mice. Dead mice were sectioned, their spleens cultivated on thioglycollate-glucose-blood agar and impression smears from the spleen, liver and heart blood were Giemsa-stained. Sixty-four pools were positive for F. tularensis: the overall minimum infection rate (MIR) was 0.82%. Overall MIRs for the 4714 female and 3064 male D. reticulatus examined were 0.89 and 0.72%, respectively; MIRs fluctuated across years between 0.0 and 2.43%. The estimated bacterial load in infected ticks varied from 0.84 to 5.34 log10 infectious F. tularensis cells per tick (i.e. from about seven to 220 000 cells). Ticks with low loads were more prevalent; more than 1000 infectious cells were detected in 24 ticks (0.3% of all ticks and 37.5% of infected ticks). Monitoring of D. reticulatus for the presence and cell numbers of F. tularensis may be a valuable tool in the surveillance of tularaemia.

Environmental surveillance during an outbreak of tularaemia in hares, the Netherlands, 2015.

Tularaemia, a disease caused by the bacterium Francisella tularensis, is a re-emerging zoonosis in the Netherlands. After sporadic human and hare cases occurred in the period 2011 to 2014, a cluster of F. tularensis-infected hares was recognised in a region in the north of the Netherlands from February to May 2015. No human cases were identified, including after active case finding. Presence of F. tularensis was investigated in potential reservoirs and transmission routes, including common voles, arthropod vectors and surface waters. F. tularensis was not detected in common voles, mosquito larvae or adults, tabanids or ticks. However, the bacterium was detected in water and sediment samples collected in a limited geographical area where infected hares had also been found. These results demonstrate that water monitoring could provide valuable information regarding F. tularensis spread and persistence, and should be used in addition to disease surveillance in wildlife.

Multiple-locus variable-number tandem-repeat analysis of Francisella tularensis from Quebec, Canada.

UNLABELLED: Francisella tularensis is ubiquitous in the Northern Hemisphere. Yet, little is known about the disease and its ecology within Canada as few serological studies have shown exposure to the disease and fewer case studies have been reported. This report is the first to describe the molecular subtyping of F. tularensis isolates within eastern Canada using multiple-locus variable-number tandem-repeat analysis. From 1998 to 2011, a total of 73 specimens were isolated from unique human and animal sources. As expected, F. tularensis subsp. tularensis AI and F. tularensis subsp. holarctica subtypes were observed, corresponding to the known geographical division within this species. The majority of human isolates (78%) and all animal (hare) isolates were of the more virulent, AI type. Half of the B isolates were isolated from patients living in a region of Quebec where muskrat densities are known to be high. A relatively high level of marker diversity was found, suggestive of multiple introductions of the organism to the region, or more likely ongoing endemicity. There was no evidence of ongoing outbreaks or transmission, and the bulk of cases were likely due to interaction between human activity and the environment (e.g. hunting/trapping activities). SIGNIFICANCE AND IMPACT OF THE STUDY: This study reveals the diversity of Francisella tularensis in eastern Canada using multiple-locus variable-number tandem-repeat analysis. It was initiated to further the understanding of the species within North America as previous studies elucidating the diversity and phylogeography of the species have consisted mostly of specimens from the United States. Type A tularaemia, the most life-threatening subtype of the species and a Category A biothreat agent, is restricted to North America, and this study serves to broaden the knowledge of the epidemiology and diversity of the organism.

Pilot Cross-Sectional Study of Three Zoonoses (Lyme Disease, Tularaemia, Leptospirosis) among Healthy Blood Donors in Eastern Slovakia.

OBJECTIVE: The aim of the study was to determine the seroprevalence of three zoonotic infections among healthy blood donors/volunteers in Eastern Slovakia. METHODS: Sera from 124 blood donors were investigated for the presence of antibodies against Borrelia burgdorferi, Francisella tularensis and Leptospira pomona. The participants also completed the questionnaire about demographic, exposure and epidemiological characteristics. Two serological methods were used for the diagnosis: the enzyme linked protein A/G assay (ELPAGA) and the Western blot (WB). First, sera were screened by ELPAGA (except for leptospirosis). RESULTS: The observed seroprevalence was 15% for Lyme borreliosis (LB) and 4% for tularaemia (TUL). The results were confirmed by WB. Positive IgG antibodies (WB method) were detected only in 1.6% of examined for LB and 0.8% for TUL. Our results did not identify any antibodies against Leptospira pomona agent in the examined healthy blood donors group. CONCLUSIONS: ELPAGA seroprevalence for TUL was significantly higher in blood donors working in the agricultural area in the direct contact with hay, straw, manure, and agricultural land. Our outputs determine tick bite as a significant risk factor for LB. The study confirms the explosion of tick-borne diseases in the healthy population of people. The exposure risk for leptospirosis seems to be minimal.

Complement sensitivity and factor H binding of European Francisella tularensis ssp. holarctica strains in selected animal species.

Francisella tularensis is a Gram-negative bacterium, the causative agent of the zoonotic disease tularaemia. The bacterium has developed several extracellular and intracellular strategies to evade the hosts' innate and adaptive immune responses. The aims of the study were to examine complement sensitivity of wild and attenuated F. tularensis ssp. holarctica strains in animal hosts of distinct sensitivity to the bacterium, to compare the complement-evading ability of wild strains of different phylogeographic background, and to examine the role of factor H in the host-pathogen interactions. Complement sensitivity assays were carried out on various F. tularensis ssp. holarctica wild strains and on the attenuated live vaccine strain (LVS) with sera of the highly sensitive house mouse (Mus musculus), the moderately sensitive European brown hare (Lepus europaeus) and the relatively resistant cattle (Bos taurus). Specific binding of complement regulator factor H to bacterial membrane proteins was examined by Western blot assays. All wild strains interacted with the hosts' complement system and showed no significant differences in their survivability. The attenuated LVS was resistant to serum killing in mouse, but was lysed in the sera of hare and cattle. Direct binding of factor H to F. tularensis membrane proteins was not detected.

Evolution toward high-level fluoroquinolone resistance in Francisella species.

OBJECTIVES: Francisella tularensis, a CDC class A potential bioterrorism agent, is a Gram-negative bacterium responsible for tularaemia. Understanding the mechanisms of resistance to antibiotics used as first-line treatment is of major security relevance. METHODS: We propagated the three parental reference strains Francisella tularensis subsp. holarctica live vaccine strain, Francisella novicida and Francisella philomiragia with increasing concentrations of ciprofloxacin, a fluoroquinolone used as curative and prophylactic treatment for tularaemia. This evolution procedure provided us with high-level ciprofloxacin-resistant mutants and all evolutionary intermediates towards high-level resistance. We determined the resistance levels to other fluoroquinolones (levofloxacin and moxifloxacin) and other antibiotic families (aminoglycosides, tetracyclines and macrolides) and characterized the genetic changes in the fluoroquinolone target genes encoding DNA gyrase and topoisomerase IV. RESULTS: All high-level resistant mutants shared cross-resistance to the tested fluoroquinolones, while some also revealed striking levels of cross-resistance to other clinically relevant antibiotic classes. High-level resistant mutants carried one to three mutations, including some not previously reported. We mapped all mutations onto known topoisomerase three-dimensional structures. Along the pathways towards high-level resistance, we identified complex evolutionary trajectories including polymorphic states and additional resistance mechanisms likely to be associated with efflux processes. CONCLUSIONS: Our data demonstrated the efficiency and speed of in vitro production of mutants highly resistant to fluoroquinolones in Francisella species. They emphasize the urgent need to identify all antibiotic resistance mechanisms in these species, develop molecular tools for their detection and design new therapeutic alternatives for tularaemia.

Characterization of three Francisella tularensis genomes from Oklahoma, USA.

Francisella tularensis , the causative agent for tularaemia, is a Tier 1 select agent, and a pan-species pathogen of global significance due to its zoonotic potential. Consistent genome characterization of the pathogen is essential to identify novel genes, virulence factors, antimicrobial resistance genes, for studying phylogenetics and other features of interest. This study was conducted to understand the genetic variations among genomes of F. tularensis isolated from two felines and one human source. Pan-genome analysis revealed that 97.7 % of genes were part of the core genome. All three F. tularensis isolates were assigned to sequence type A based on single nucleotide polymorphisms (SNPs) in sdhA. Most of the virulence genes were part of the core genome. An antibiotic resistance gene coding for class A beta-lactamase was detected in all three isolates. Phylogenetic analysis showed that these isolates clustered with other isolates reported from Central and South-Central USA. Assessment of large sets of the F. tularensis genome sequences is essential in understanding pathogen dynamics, geographical distribution and potential zoonotic implications.

Pathogenicity and virulence of Francisella tularensis.

Tularaemia is a zoonotic disease caused by the Gram-negative bacterium, Francisella tularensis. Depending on its entry route into the organism, F. tularensis causes different diseases, ranging from life-threatening pneumonia to less severe ulceroglandular tularaemia. Various strains with different geographical distributions exhibit different levels of virulence. F. tularensis is an intracellular bacterium that replicates primarily in the cytosol of the phagocytes. The main virulence attribute of F. tularensis is the type 6 secretion system (T6SS) and its effectors that promote escape from the phagosome. In addition, F. tularensis has evolved a peculiar envelope that allows it to escape detection by the immune system. In this review, we cover tularaemia, different Francisella strains, and their pathogenicity. We particularly emphasize the intracellular life cycle, associated virulence factors, and metabolic adaptations. Finally, we present how F. tularensis largely escapes immune detection to be one of the most infectious and lethal bacterial pathogens.

Case report: tularaemia in a white-handed gibbon (Hylobates lar), Germany.

In 2021, a white-handed gibbon (Hylobates lar) succumbed to illness shortly after transfer from one zoo to another in Germany, due to Francisella tularensis subsp. holarctica infection. To determine the source of infection, whole genome sequencing of the gibbon-derived isolate was performed and wild pest rodents (and captive squirrels) from both zoos were screened for F. tularensis. The F. tularensis whole genome sequence obtained from the gibbon was closely related to previous subclade B.281 sequences obtained from hares from Baden-Wuerttemberg, the same region where the gibbon was first housed. However, F. tularensis DNA was detected in one Norway rat from the receiving zoo. Therefore, neither zoo can be excluded as the source of infection.