Isolation Of Francisella Tularensis Research Articles

Isolation of Francisella tularensis and Yersinia pestis from Blood Cultures by Plasma Purification and Immunomagnetic Separation Accelerates Antibiotic Susceptibility Determination.

The early symptoms of tularemia and plague, which are caused by Francisella tularensis and Yersinia pestis infection, respectively, are common to other illnesses, resulting in a low index of suspicion among clinicians. Moreover, because these diseases can be treated only with antibiotics, rapid isolation of the bacteria and antibiotic susceptibility testing (AST) are preferable. Blood cultures of patients may serve as a source for bacteria isolation. However, due to the slow growth rates of F. tularensis and Y. pestis on solid media, isolation by plating blood culture samples on proper agar plates may require several days. Thus, improving the isolation procedure prior to antibiotic susceptibility determination is a major clinically relevant need. In this study, we developed a rapid, selective procedure for the isolation of F. tularensis and Y. pestis from blood cultures. We examined drop-plating and plasma purification followed by immunomagnetic separation (IMS) as alternative isolation methods. We determined that replacing the classical isolation method with drop-plating is advantageous with respect to time at the expense of specificity. Hence, we also examined isolation by IMS. Sub-localization of F. tularensis within blood cultures of infected mice has revealed that the majority of the bacteria are located within the extracellular fraction, in the plasma. Y. pestis also resides within the plasma. Therefore, the plasma fraction was isolated from blood cultures and subjected to an IMS procedure using polyclonal anti-F. tularensis live vaccine strain (LVS) or anti-Y. pestis antibodies conjugated to 50-nm nano-beads. The time required to reach an inoculum of sufficient bacteria for AST was shortest when using the plasma and IMSs for both bacteria, saving up to 2 days of incubation for F. tularensis and 1 day for Y. pestis. Our isolation procedure provides a proof of concept for the clinical relevance of rapid isolation for AST from F. tularensis- and Y. pestis-infected patients.

Phylogeography of Francisella tularensis from Tibet, China: Evidence for an asian origin and radiation of holarctica-type Tularemia

The geographical origin and radiation of holarctica-type tularemia, which has spread across the northern hemisphere, is open to scientific debate. Here, through phylogenetics, we show that five Tibetan Francisella tularensis isolates subsp. holarctica cluster between basal-positioned Japanese isolates and all other subspecies strains in the world, providing evidence for a previously unknown intermediate lineage next to the Japanese isolates. Importantly, identification of this new intermediate lineage complements current knowledge of tularemia epidemiology, supporting a geographical origin and radiation of the subsp. holarctica in Asia. In addition, thirteen Tibetan isolates belonging to a clade previously found only in North America and Scandinavia, further increases the diversity of holarctica strains in Asia. In summary, this study provides evidence for an Asian origin and radiation of holarctica-type tularemia.

Francisella tularensis bacteria associated with feline tularemia in the United States.

Tularemia in the United States was examined by reviewing 106 Francisella tularensis isolates, mostly from Nebraska, collected during 1998–2012: 48% of Nebraska cases were cat-associated; 7/8 human cases were caused by subtype A.I. A vaccine is needed to reduce feline-associated tularemia, and cat owners should protect against bites/scratches and limit their pet’s outdoor access.

Development of a Novel Francisella tularensis Antigen Stained with Tetrazolium-blue for Tularemia Microagglutination Test

The isolation of Francisella tularensis in cultures is the reference method for the laboratory diagnosis of tularemia. However, due to the limitations such as the low sensitivity and need for high safety level and equipped laboratories, serologic methods are frequently used as diagnostic tools. F.tularensis-specific antibodies may be demonstrated by several methods, however microagglutination test (MA) remains the most common method with its high sensitivity and specificity. The aim of this study was to develop a novel MA test antigen prepared from whole F.tularensis cells and stained with tetrazolium-blue for more clear and easier evaluation. F.tularensis NCTC 10857 strain was cultured on the cysteine heart agar supplemented with 9% sheep blood and bacterial cells were harvested by scraping, collected in physiological saline (PS) and centrifuged at 1500 rpm for 10 minutes. For preparing stock antigen suspension cell concentration was adjusted to OD600=1.5, spectrophotometrically. Tetrazolium-blue solution (BTC [3,3'-(3,3'-Dimethoxy[1,1'-biphenyl]-4,4'-diyl) bis [2,5-diphenyl-2H-tetrazolium dichloride], T4375 Sigma-Aldrich) at the final concentration of 1% was added to cell suspension and incubated at 37°C for 5 hours for absorption. Then, the living cells were chemically inactivated by formaldehyde. Repeating centrifugations were performed to discard excess dye and formaline, then 0.4% formaline saline was added on the sediment. Optimum concentration of this novel antigen (BTC-Ag) for MA test was determined by plate titration method by using standard serum sample with a known MA titer (1/128). The performance of BTC-Ag in MA test was evaluated by using 100 patient sera positive for F.tularensis antibodies, and 100 tularemia negative patient sera (of them 50 were seropositive for brucellosis). All of the results were compared with standard MA test in which safranin-O stained antigen (SO-Ag) was used. There was 100% agreement between the two tests performed with BTC-Ag and SO-Ag in tularemia seropositive (in ≥ 1/20 titers and when ±1 dilution variation was accepted as normal) and seronegative sera. No significant cross reactivity with Brucella spp. was observed. Accuracy, sensitivity and specificity of BTC-Ag were found to be 100%. In conclusion, newly developed BTC-Ag for MA test provides better agglutination patterns resulting in a clear supernatant in wells, thus provides easy evaluation for the agglutination reaction, and is expected to facilitate tularemia serodiagnosis.

German Francisella tularensis isolates from European brown hares (Lepus europaeus)reveal genetic and phenotypic diversity

BackgroundTularemia is a zoonotic disease caused by Francisella tularensis that has been found in many different vertebrates. In Germany most human infections are caused by contact with infected European brown hares (Lepus europaeus). The aim of this study was to elucidate the epidemiology of tularemia in hares using phenotypic and genotypic characteristics of F. tularensis.ResultsCultivation of F. tularensis subsp. holarctica bacteria from organ material was successful in 31 of 52 hares that had a positive PCR result targeting the Ft-M19 locus. 17 isolates were sensitive to erythromycin and 14 were resistant. Analysis of VNTR loci (Ft-M3, Ft-M6 and Ft-M24), INDELs (Ftind33, Ftind38, Ftind49, RD23) and SNPs (B.17, B.18, B.19, and B.20) was shown to be useful to investigate the genetic relatedness of Francisella strains in this set of strains. The 14 erythromycin resistant isolates were assigned to clade B.I, and 16 erythromycin sensitive isolates to clade B.IV and one isolate was found to belong to clade B.II. MALDI-TOF mass spectrometry (MS) was useful to discriminate strains to the subspecies level.ConclusionsF. tularensis seems to be a re-emerging pathogen in Germany. The pathogen can easily be identified using PCR assays. Isolates can also be identified within one hour using MALDI-TOF MS in laboratories where specific PCR assays are not established. Further analysis of strains requires genotyping tools. The results from this study indicate a geographical segregation of the phylogenetic clade B.I and B.IV, where B.I strains localize primarily within eastern Germany and B.IV strains within western Germany. This phylogeographical pattern coincides with the distribution of biovar I (erythromycin sensitive) and biovar II (erythromycin resistance) strains. When time and costs are limiting parameters small numbers of isolates can be analysed using PCR assays combined with DNA sequencing with a focus on genetic loci that are most likely discriminatory among strains found in a specific area. In perspective, whole genome data will have to be investigated especially when terrorist attack strains need to be tracked to their genetic and geographical sources.

Isolation of Central Asian Subspecies of Tularemia Agent in the Altai Territory

Within the frames of activities attributed to the Reference Center for tularemia monitoring at SRC AMB, genetically identified are 4 isolates of Francisella tularensis , isolated in 2011 in the Altai Territory. These bacteria prove to be virulent for BALB/c mice, DCL being lower than 10 CFU. Using single-primer PCR-typing and MLVA assay distinguished have been the subspecies of the isolates. Three of them refer to the Central Asian subspecies, one – to the Holarctic, the former being isolated in the territory of the Russian Federation for the first time ever.

In vitro susceptibility of isolates of Francisella tularensis from Turkey

Tularemia is an infection caused by Francisella tularensis, which has a wide distribution in the northern hemisphere and diverse clinical manifestations. For decades, the drug of choice for the treatment of tularemia has been streptomycin, with tetracycline and chloramphenicol being used as alternatives. The purpose of the present study was to determine the in vitro antimicrobial susceptibility of a large panel of geographically diverse F. tularensis isolates from Turkey against traditional and newer antimicrobial agents. The antibiotic susceptibilities of 250 F. tularensis strains were examined using the Epsilometer test for 9 antimicrobial agents. Each isolate was identified by conventional and molecular techniques. All the strains were confirmed biochemically and using a combination of species- and subspecies-specific polymerase chain reaction (PCR) assays to be F. tularensis subsp. holarctica. One isolate was assigned to F. tularensis subsp. holarctica biovar japonica based on erythromycin susceptibility, an ability to ferment glycerol, and the nucleotide sequence of the region of difference 1 (RD1). All strains were susceptible to aminoglycosides (streptomycin and gentamicin), tetracyclines (tetracycline and doxycycline), chloramphenicol, 2 fluoroquinolones (ciprofloxacin and levofloxacin), and rifampicin. In addition, all isolates except 1 had a minimal inhibitory concentration (MIC) for erythromycin of > 256 μg/ml. Since the fluoroquinolones showed the lowest MIC values and have advantages such as excellent bioavailability and activity, availability of oral formulations, and lower toxicities, they represent candidate therapeutic options in the first-line treatment of tularemia. To the best of our knowledge, this is the first report of the presence of F. tularensis subsp. holarctica biovar japonica outside Japan.

Landscape Epidemiology of Tularemia Outbreaks in Sweden

Summer outbreaks of tularemia that occurred from 1995 through 2005 in 2 locations in Sweden affected 441 persons. We performed an epidemiologic investigation of these outbreaks using a novel strategy, involving high-resolution genotyping of Francisella tularensis isolates obtained from 136 patients (using 18 genetic markers developed from 6 F. tularensis genome sequences) and interviews with the patients. Strong spatial associations were found between F. tularensis subpopulations and the places of disease transmission; infection by some subpopulations occurred within areas as small as 2 km(2), indicating unidentified environmental point sources of tularemia. In both locations, disease clusters were associated with recreational areas beside water, and genetic subpopulations were present throughout the tularemia season and persisted over years. High-resolution genotyping in combination with patients' statements about geographic places of disease transmission provided valuable indications of likely sources of infection and the causal genotypes during these tularemia outbreaks.

Genetic diversity ofFrancisella tularensissubspeciesholarcticastrains isolated in Japan

The recently developed MLVA has high discriminatory power for the typing of individual strains or isolates of Francisella tularensis. In the present study, MLVA was applied to 33 Japanese F. tularensis subspecies holarctica strains to examine the genetic diversity of F. tularensis isolated. Among the seven VNTR loci analyzed, Ft-M2, Ft-M10, and Ft-M20 loci showed high genetic polymorphism in Japanese strains, whereas Ft-M3 was most variable in non-Japanese strains. These results provide novel extended information about the genomic diversity among the strains of F. tularensis ssp. holarctica distributed in Japan and enable determination of whether a given isolate is indigenous to Japan by examining these loci using MLVA.

Susceptibility of 71 French isolates of Francisella tularensis subsp. holarctica to eight antibiotics and accuracy of the Etest® method

Susceptibility of 71 French isolates of Francisella tularensis subsp. holarctica to eight antibiotics and accuracy of the Etest® method

Detection of Francisella tularensis subsp. holarctica in a European brown hare ( Lepus europaeus) in Thuringia, Germany

The isolation of Francisella tularensis subsp. holarctica biovar II (strain 06T0001) from a European brown hare ( Lepus europaeus) from Thuringia, Germany, is described for the first time. Identification of the microorganism was carried out by phenotypic characterisation, partial sequencing of the 16S rRNA gene and specific PCR using the primers TUL4-435/TUL4-863 and FtC1/FtC4. The epidemiology of tularemia in Germany is discussed and a risk assessment for humans is made.

Population Structure of Francisella tularensis

We have sequenced fragments of five metabolic housekeeping genes and two genes encoding outer membrane proteins from 81 isolates of Francisella tularensis, representing all four subspecies. Phylogenetic clustering of gene sequences from F. tularensis subsp. tularensis and F. tularensis subsp. holarctica aligned well with subspecies affiliations. In contrast, F. tularensis subsp. novicida and F. tularensis subsp. mediasiatica were indicated to be phylogenetically incoherent taxa. Incongruent gene trees and mosaic structures of housekeeping genes provided evidence for genetic recombination in F. tularensis.

Tularemia in a Kidney Transplant Recipient: An Unsuspected Case and Literature Review

Tularemia is a zoonotic infection that has rarely been reported in transplant recipients. The authors present a case of unsuspected tularemia in a kidney transplant patient that was diagnosed by isolation of Francisella tularensis in the blood. The patient was treated successfully with antibiotics. During diagnostic workup, a laboratory technician was exposed to tularemia by inhalation of the culture plate and received postexposure prophylaxis. This report emphasizes the importance of exposure history in the investigation of fever in an immunocompromised host and the special precautions needed when a virulent infectious organism is suspected.

In vitro susceptibility of field isolates of Francisella tularensis subsp. holarctica recovered in Spain to several antimicrobial agents

Forty-two recent (1997–1999) Spanish isolates of Francisella tularensis subsp. holarctica were tested in a broth microdilution method for their susceptibilities to 29 antimicrobial agents, including penicillins, cephalosporins, cephamicins, monobactams, penems, aminoglycosides, tetracyclines, macrolides, quinolones, chloramphenicol and fosfomycin. The isolates were resistant to β-lactam antibiotics and susceptible to chloramphenicol, ciprofloxacin, levofloxacin and norfloxacin.

Frequent isolation of Francisella tularensis from Dermacentor reticulatus ticks in an enzootic focus of tularaemia.

A total of 924 questing Dermacentor reticulatus (Fabricius), 504 Ixodes ricinus (L.), sixty Haemaphysalis concinna Koch and 718 mosquitoes (Aedes spp.) were examined in a floodplain forest ecosystem during the 1994-95 outbreak of tularaemia in South Moravia, Czech Republic. Francisella tularensis was not isolated from H.concinna ticks or Aedes spp. mosquitoes, whereas twenty-one isolates were recovered from the other haematophagous arthropods. Dermacentor reticulatus revealed a significantly higher infection rate (2.6%) than I.ricinus (0.2%). This tick species acts as principal vector for tularaemia in the enzootic focus. Monitoring of D.reticulatus for F.tularensis thus seems to be a very efficient approach in the surveillance of tularaemia in the flood-plain forest ecosystems of Europe.

Identification and classification of different isolates of Francisella tularensis.

The causative agent of tularemia, Francisella tularensis, occurs in two main biovars, the highly virulent F. t. biovar tularensis, found in North America; and the less virulent biovar palaearctica, found all over the northern hemisphere. Two other biovars have been proposed, F. t. biovar mediaasiatica and F. t. biovar palaearctica var. japonica. In Sweden tularemia is most frequently observed in man and varying hares (Lepus timidus), and occasionally in other species. Tularemia in hares is normally an acute fatal disease, although less fatal infections have been reported. The diagnosis of tularemia is routinely based on immunological reactions. We studied 10 different isolates of F. tularensis from varying hares, one isolate from an Ural owl (Strix uralensis), one vaccine strain, one strain of F. t. biovar japonica, and six isolates from a virulence study of F. tularensis, by biochemical tests and by hybridization experiments with probes complementary to 16S rRNA. All isolates, except the isolate F. t. biovar japonica, were characterized as F. t. biovar palaearctica by biochemical tests. In the 16S rRNA analysis all isolates were positive to the probe for Francisella tularensis and the probe for F. t. biovar palaearctica with the exception that F. t. biovar japonica reacted with the probe specific to F. t. biovar tularensis. To further confirm that the strains used belonged to F. t. biovar palaearctica virulence tests in rabbits were performed which disclosed this phenotype. The results presented in this work show that the isolated strains from the western part of Europe were F. t. biovar palaearctica, irrespective of animal origin or virulence.

Water- and airborne francisella tularensis biovar palaearctica isolated from human blood

The isolation of Francisella tularensis from human blood is extremely rare in Europe. We here describe two cases of septicemia caused by F. tularensis biovar palaearctica, where the causative bacterium was isolated from blood. One of our patients had ingested contaminated water; the other was probably infected by inhalation of contaminated dust. The isolation of the causative organism was essential for initiating the appropriate antibiotic treatment, which led to a rapid improvement. Since the responsive agent rarely is isolated from tularemia patients, blood cultures should be drawn more often in order to improve the diagnostic procedures.

宮城県川渡農場でのへい死ノウサギ寄生マダニからの野兎病菌分離例と放牧牛の血清抗体調査

宮城県川渡農場でのへい死ノウサギ寄生マダニからの野兎病菌分離例と放牧牛の血清抗体調査

Isolation of Francisella tularensis from infected frozen human blood

Francisella tularensis was isolated from human blood that was frozen for 3 months before it was examined. Before he became ill, the patient operated a "bush-hog" in an area thickly populated with rabbits. His illness was undiagnosed and untreated before his death. Portions of blood and tissue homogenates from necropsy were injected intraperitoneally into mice and inoculated onto glucose-cysteine-blood agar plates. F. tularensis did not grow from the culture plates, but mice inoculated with the blood died in 48 to 72 h. Fluorescent-antibody stains of mouse liver and spleen impression smears showed clumps of cells or amorphous masses of brightly staining envelope material around the cells. Tissue impressions of liver, spleen, and heart blood inoculated onto glucose-cysteine-blood agar yielded pure cultures of F. tularensis.