The first one has been achieved by growing ZnO nanowires, nanorods, and nanobelts on the flexible polyethersulfone or polyethylene terephthalate (PET) substrate via a chemical solution method [6, 7]. The other one was an alternative way in which zig-zag-shaped or network electrodes (consisting of patterned

noble metals, carbon nanotubes, or graphene) were employed as a top electrode to efficiently bend the ZnO nanostructures for transmitting the external mechanical energy as well as possible [8, 9]. However, these kinds of top electrodes needed a somewhat sophisticated fabrication process for the preparation of patterned electrodes or synthesis of carbon-based nanomaterials. On the other hand, one-dimensional (1D) ZnO nanostructures including nanowires or nanorods provide an effective deformation (i.e., stretch and compression) under external Sotrastaurin clinical trial mechanical energy due to their high aspect ratio which generates the piezoelectric charges [10]. Additionally, they have been reliably synthesized and vertically integrated on various flexible substrates with ZnO seed coating by hydrothermal or electrochemical deposition (ED) method [11–14]. find more Particularly, the ED method has many advantages for growing 1D ZnO nanostructures because the electric

energy enables a short time process at low temperature [15]. In this work, we prepared ZnO nanorod

arrays (NRAs) on an indium tin oxide (ITO)-coated PET substrate (i.e., ITO/PET) using the ED method and fabricated ZnO NRA-based NGs with an efficient top electrode TSA HDAC molecular weight which was obtained by evaporating gold (Au) onto the surface of silica spheres. SPTLC1 Herein, the multilayer of silica spheres was facilely deposited on the PET substrate by rolling the colloidal solution of silica spheres. Methods Figure 1 shows the schematic diagram for the fabrication of the Au-coated silica sphere array as a top electrode of ZnO NRA-based NGs: (i) preparation of colloidal solution (i.e., dispersed by silica spheres) on the PET substrate, (ii) rolling and drying the colloidal solution, and (iii) e-beam evaporation of Au onto the silica sphere array. Silica spheres were synthesized using a modified Stober process [16]. After the mixture solution with 200 ml of ethanol, 40 ml of ammonia, and 40 ml of de-ionized (DI) water was kept at 60°C, 20 ml of tetraethyl orthosilicate (TEOS) was slowly dropped for 2 h using a burette. Here, all the chemicals were of analytical grade (Sigma-Aldrich, St. Louis, MO, USA). Then, the silica sphere powder was obtained by centrifugation and drying at 70°C. After that, the powder was mixed with ethanol at a concentration of 50 g/l. To increase the viscosity of the colloidal solution, 0.2% weight of poly-4-vinylphenol was added [17].

pseudomallei),

albeit loosely related. Further work that includes prophages derived from environmental and clinical isolates from other Burkholderia species as well as from other microbes is needed to refine these relationships. Burkholderia Mocetinostat spp. are responsible for a number of potentially devastating infectious diseases for which no vaccines currently exist. The presence of a wide variety of bacteriophages within these bacteria opens the possibility that phage therapy may be developed to augment present antibiotic treatments. We present here a detailed comparative analysis of gene content within and between groups of bacteriophages, putative prophages, and prophage-like regions in BMS202 purchase various Burkholderia species and strains. Several interesting genes and gene groups associated with pathogenicity and various metabolic functions were identified within specific groups. This study provides the first estimate of the relative contribution of prophages to the vast phenotypic diversity found among the Burkholderiae. Acknowledgements This research was sponsored by the Medical Biological Defense Research Program, U.S. Army Medical Research and Materiel Command (project 02-4-5X-026). This project was also funded with federal

funds from the National Institute Poziotinib mouse of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services under contract number N01-AI-30071. We thank Kathy Abiraterone Kuehl for assistance with electron microscopy. The opinions, interpretations, conclusions, and recommendations expressed here are those of the author and

are not necessarily endorsed by the U.S. Army in accordance with AR 70-31. Electronic supplementary material Additional file 1: Additional tables. This file contains Tables S1 and S2 that describe the host range of phiE202 and all the strains that were used to search for prophages. Table S1. Bacterial strains used to examine the host range of bacteriophage phiE202. Table S2. Burkholderia strains searched for putative prophage. (PDF 191 KB) References 1. Rotz LD, Khan AS, Lillibridge SR, Ostroff SM, Hughes JM: Public health assessment of potential biological terrorism agents. Emerg Infect Dis 2002,8(2):225–230.PubMedCrossRef 2. Vietri N, DeShazer D: Melioidosis. In Medical Aspects of Biological Warfare. Edited by: Dembek Z. Washington, DC: Dept of the Army, Office of the Surgeon General, Borden Institute; 2007:225–230. 3. Holden MT, Titball RW, Peacock SJ, Cerdeno-Tarraga AM, Atkins T, Crossman LC, Pitt T, Churcher C, Mungall K, Bentley SD, et al.: Genomic plasticity of the causative agent of melioidosis, Burkholderia pseudomallei . Proc Natl Acad Sci USA 2004,101(39):14240–14245.PubMedCrossRef 4. Tuanyok A, Leadem BR, Auerbach RK, Beckstrom-Sternberg SM, Beckstrom-Sternberg JS, Mayo M, Wuthiekanun V, Brettin TS, Nierman WC, Peacock SJ, et al.: Genomic islands from five strains of Burkholderia pseudomallei . BMC Genomics 2008, 9:566.PubMedCrossRef 5.

The position of the codon immediately upstream of the transposon insertion site is indicated in brackets. Two additional experiments were performed to complete the physiological characterization of these mutants with respect to arsenite oxidation. First, arsenic species were quantified by HPLC-ICP-AES on filtered culture supernatants. H. arsenicoxydans was grown in liquid medium supplemented selleck chemicals with 1.33 mM arsenite and showed 100% transformation of As(III) into As(V) after 48 h, whereas M1 (aoxA) and M2 (aoxB) mutants used as controls were not able to transform As(III) into As(V). The same loss of arsenite

oxidase activity was measured in Ha482 (aoxS), Ha483 (aoxR), Ha2646 (dnaJ) and Ha3109 (rpoN) mutants. In contrast to the results obtained on agar plates, Ha3437 (modC) and Ha3438 (modB) strains showed 100% transformation of arsenite (Table 1, Figure 1A). Previous studies have demonstrated that the bioavailability of metals or trace elements considerably varies according to the type of matrix used for microbial Nutlin-3a nmr growth [18]. We therefore assumed that Mo might be partly sequestred on CDM agar medium, resulting in a lack of arsenite oxidase activity Wortmannin clinical trial on plate. To test this hypothesis, As(III) oxidase tests were performed on CDM agar plates supplemented

with 50 μM Mo. The addition of Mo to the solid medium restored As(III) oxidase activity in both Ha3437 (modC) and Ha3438 (modB) mutants while it had no effect on other mutant strains (Figure 1B). Table 1 Determination of arsenic speciation in H. arsenicoxydans wild-type and mutant strains. Strain Mutated gene Arsenic species Ergoloid identifieda     As(III) As(V) ULPAs1 / – + M1b aoxA + – M2b aoxB + – Ha482 aoxS + – Ha483 aoxR + – Ha2646 dnaJ + – Ha3109 rpoN + – Ha3437 modC – + Ha3438 modB – + Determined by HPLC-ICP-AES after 48 h growth in CDM medium containing 100 mg.liter of arsenite. b [9] Second, we have previously demonstrated that the polar flagellum-dependent motility of H. arsenicoxydans is

increased in the presence of As(III), suggesting that arsenite oxidation may result in a gain of energy [6]. The motility of mutant strains was therefore tested on plates containing different concentrations of As(III), i. e. 0.66 mM, 1.33 mM and 2 mM. The diameter of the swarming rings was measured after 72 h. As shown in Figure 3, the disruption of aoxA, aoxB, aoxR, aoxS or rpoN genes abolished the improvement of swarming performances in the presence of As(III). Unlike those mutants, a disruption in dnaJ completely abolished the motility of H. arsenicoxydans in the presence or the absence of As(III). DnaJ is known to be essential for the expression of the flhDC flagellar master operon in Escherichia coli [19]. The lack of motility observed in the dnaJ mutant suggests the existence of a similar flhDC-dependent regulation of flagellar genes in H. arsenicoxydans.

Preparation of A-MNCs and HA-MRCAs A-MNCs were fabricated using the nano-emulsion method [23]. First, 10 mg of MNCs was dissolved in 4 mL of n-hexane (organic phase). The organic phase was injected into 30 mL of de-ionized water (aqueous phase) containing 100 mg of aminated P80. After mutual saturation, the solution was emulsified for 20 min under ultrasonification (ULH700S, Ulssohitech, Cheongwon-gun, South Korea) at 450 W. The mixture was kept overnight at room temperature to remove the volatile organic solvent. The products were purified using a centrifugal filter (Centriprep

YM-3, 3-kDa molecular weight cutoff (MWCO), Amicon, Millipore Corporation, Billerica, MA, USA) in triplicate at 3,000 rpm for 30 min. HA-MRCAs with different molar ratios of HA were fabricated by EDC-NHS chemistry. buy Z-DEVD-FMK First, the pH of the A-MNC solution was adjusted to neutral condition by the addition of 0.1 N HCl solution. Then, various amounts of HA (0.43, 1.7, and 6.8 μmol) were dissolved in the 40 mL of de-ionized water followed by the addition of EDC and sulfo-NHS. Each HA solution was added to A-MNC solution containing 5 mg of MNCs. The HA and A-MNCs were reacted for 2 h at room temperature. Finally, EDC, sulfo-NHS, and unbound HA were removed using dialysis (MWCO, 25, 000) against excess de-ionized water. Characterization of

A-MNCs and HA-MRCAs The size distributions and zeta potential values of A-MNCs and HA-MRCAs were measured using laser scattering

(ELS-Z, Otsuka Electronics, Osaka, Japan). The inorganic selleck ratios (%) and the crystallinities of magnetic nanocrystals in A-MNCs and HA-MRCAs were analyzed using a thermo-gravimetric analyzer (SDT-Q600, TA Instruments, Newcastle, DE, USA) and X-ray diffraction (X-ray diffractometer Ultima3, Rigaku, Tokyo, Japan) at 25°C, respectively. The magnetic properties of A-MNCs and HA-MRCAs were also detected by a vibration sample magnetometer (model 9407, Lake Shore Cryotronics, Inc., Westerville, OH, USA) at 25°C. Cell viability assay for A-MNCs and HA-MRCAs The cytotoxic effect of A-MNCs and HA-MRCAs against MDA-MB-231 cells (CD44-abundant cancer cell line) was analyzed by measuring the inhibition of cell growth using an assay for WST-1 ((4-(3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio)-1,3-benzene P-type ATPase disulfonate)). MDA-MB-231 cells were maintained in RPMI containing 10% FBS and 1% antibiotics at 37°C in a humidified atmosphere with 5% CO2. MDA-MB-231 cells were harvested at a density of 1.0 × 104 cells/100 μL in a 96-well plate and incubated at 37°C in 5% CO2 atmosphere overnight. The cells were then treated with various concentrations of A-MNCs and HA-MRCAs for 24 h. After incubation, the cells were rinsed with 100 μL PBS (pH 7.4, 1 mM), and then 10 μL of WST-1 solution was added to each well. The absorbance was measured at 450 nm with a https://www.selleckchem.com/products/mm-102.html reference wavelength of 600 nm.

Discussion The mycobacterial cell

BI 6727 order envelope is a lipid-rich complex structure that surrounds the bacillus and is thought to play a critical role in the pathogenicity of Mycobacterium tuberculosis. Nearly 2.5% of the M. tuberculosis H37Rv proteome is predicted to consist of lipoproteins [17]. A large number of these Momelotinib mycobacterial lipoproteins have been suggested to be important components for the synthesis of the mycobacterial cell envelope, as well as for sensing processes, protection from stressful factors and host-pathogen interactions; nevertheless, the function and localization of a considerable number of putative lipoproteins remains yet unknown [41]. Lipoproteins are translocated across the cytoplasmic membrane and then anchored to either the periplasm or the outer membrane and have been suggested to play important roles related to virulence

because they are predicted to participate in intracellular transport, cell-wall metabolism, cell adhesion, signaling and protein degradation [42]. Rv0679c was initially classified as a hypothetical membrane protein of M. tuberculosis [9] and was later suggested to be a putative lipoprotein [29]. It is a 165-amino-acid-long protein with a theoretical NVP-BGJ398 price molecular mass of 16.6 kDa, whose function has not been fully characterized yet. In this study, PCR and RT-PCR techniques were used to examine the distribution of the Rv0679c gene in the MTC, as well as in mycobacteria other than tuberculosis (which included saprophytic and environmental species), with the aim of establishing a preliminary relationship between the presence of the protein encoding gene in a particular mycobacterial species and its virulence, considering that to develop a subunit antituberculous vaccine, it would be better to select peptides (more specifically Thymidylate synthase HABPs) from

M. tuberculosis proteins involved in host cell invasion that are exclusively present in MTC or in mycobacterium species related to invasive processes or causing disease, such as Rv0679c. The results of this study indicate that the gene encoding Rv0679c is present in the MTC, as shown by the PCR amplification of a 346-bp band from genomic DNA of M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. africanum, M. bovis, M. bovis BCG and M. microti; but no amplification was detected in Mycobacterium spp. strains outside the complex. Nevertheless, it is worth noting that Rv0679c homologues have been recently reported in different Mycobacterium genomes (e.g. M. smegmatis, M. marinum and M. avium), which indicates that such primers are specific for the MTC strains assessed in this study. Furthermore, reverse transcription assays indicate that the gene is actively transcribed in M. tuberculosis H37Rv, M. tuberculosis H37Ra and M. africanum. Intriguingly, although expression of Rv0679c homologous protein in M. bovis BCG was described by Matsuba et al. [29], gene transcription was not detected in M. bovis nor in M.

Hence, SBE-��-CD two questions arise: (i) Are RNA helicases truly involved in the Giardia RNAi pathway? (ii) What is the minimal protein repertoire for post-transcriptional gene silencing in eukaryotic cells? In the present study, we identified the complete set of SF2 helicases

in this anaerobic flagellated protozoan by searching the G. lamblia genome database of the WB isolate, which allowed the selleck Identification of 22 DEAD-box, 6 DEAH-box and 4 Ski2p putative RNA helicases, along with seven helicases of family Swi2/Snf2, 3 helicases from family RecQ and 4 helicases from family Rad3. These sequences were used to analyze the relationship between the composition of the SF2 helicases in Giardia and their corresponding homologs in yeast and humans. In addition, the level of expression during antigenic variation and encystation was analyzed, demonstrating both differential and variable expression of individual RNA helicases in these processes. We also discuss the potential role of the RNA helicase domain

Selleck Autophagy Compound Library in Dicer enzymes of higher eukaryotes. Results Identification of SF2 helicases in Giardia lamblia By using the human eIF4A (Eukaryotic Initiation Factor 4A) amino acid sequence as the DEAD-box helicase prototype [27] and the human ATP-dependent RNA-helicase DHX8 amino acid sequence as the DEAH-box helicase prototype [27], we performed an extensive analysis of the Giardia assemblage A, isolate WB, genome database [28] and detected 22 and 6 orthologs, respectively. We were also able to obtain the sequences of 4 putative RNA helicases belonging to the Ski2 family, which is generally classified inside the DExH-box family; and a previously described UPF1 homolog from SF1 [29]. These helicases belong to three of the nine families Meloxicam described from SF2. Therefore, in an attempt to identify any other helicase from this superfamily we performed a PSI-BLASTP search within the Giardia genome using the sequences described from humans, yeast and Escherichia coli, following Fairman-Williams [8]. Using this approach, we were able to recognize 14 additional putative helicases from three different families, 3 helicases from the RecQ

family, 7 helicases from the Swi2/Snf2 family, and 4 helicases from the Rad3 family. The sequences from the remaining three families of SF2 helicases present in humans, yeast and E. coli (RecG-like, RIG-I-like and NS3/NPH-II) do not have significant homology with any gene of G. lamblia. The Giardia Database gene number, the Contig number and position, and the gene length and codified protein molecular weight for each one of the SF2 helicases studied in this work are summarized in Additional file 1: Table S1. The HCD is virtually conserved in length between the three RNA helicases families, ranging from 361 to 425 amino acids, whereas the greatest differences found, as expected, were in the N- and C-terminal regions of each helicase family (see Additional file 2: Table S2).

These relationships carry evolutionary relevance, since our proteomic analyses, combined with the phylogenetic studies [100], suggest that the Myoviridae are mainly influenced by vertical evolution rather than by horizontal gene transfer. As observed in AZD9291 mouse the Cluster dendrogram, the clusters are populated unevenly – several include only one phage while two, the largest, include dozens phages. This reflects the fact that past phage research has focused on coliphages, and suggests that

we should broaden our research to include phages from a broader range of bacteria. Table 4 Concordance of classifications Classification ICTV Proteomic Tree 2 —- Phage_Finder This work Reference ICTV VIIIth Report, 2005 Edwards and Rohwer, 2005 Serwer et al., 2004 Fouts, 2006  

Approach Traditional NCT-501 mw Signature genes Large terminase   CoreGenes Phage or phage group T4, Aeh1, KVP40, RB43, RB49, 25, 31 44RR2.8t, 65 T4 T4, KVP40, RB49   T4, Aeh1, KVP40, RB43, RB49, 25, 31 44RR2.8t, selleck chemical 65   P1     P1 P1   P2, Fels-2, HP1, HP2, K139, φCTX, 186 P2. HP1, HP2, φCTX P2, Fels-2, HP1, HP2, L413-C, 186; Mu P2, φCTX, 186 (HP1 occupies a separate position) P2, Fels-2, HP1, HP2, K139, L-413C, φCTX, 186   Mu Mu     Mu   SPO1 K   P100, Twort SPOl, K, P100, Twort   ΦH         Comparison of our results with those of the ICTV (ICTV VIIIth Report, 2005), Proteomic Tree 2 (Edwards and Rohwer, 2005), Phage_Finder (Fouts, 2006) and phylogeny of terminases (Serwer et al., 2004). Among the 102 analyzed Myoviridae, phage Mu displayed the most significant evidence of horizontal gene exchange. This tuclazepam virus is related to three members of pilus-specific Siphoviridae infecting Pseudomonas aeruginosa (DMS3, D3112, B3 [59, 60, 101]), sharing 20 to 40% of its genes with each of them. These phages can be viewed as true hybrids, produced by recombination of different ancestors and, like the couple lambda/P22 (to be described in a future paper), cross family boundaries based on tail morphology. Nonetheless, the majority of Myoviridae, when forced to

cluster, do so in a logical manner: upgrading of the ICTV genus “”P2 phages”" to the Pduovirinae with two genera (“”P2 viruses”" and “”HP1 viruses”") is a straightforward proposal and the same is true for the Spounavirinae (SPO1 viruses and Twort viruses). Relationships among T4-like phages are more complicated. We reject the postulated inclusion of the cyanophages since their overall similarity to T4 is too low for consideration, at least according to our criteria. Comeau and Krisch [29] have recently recognized three groups of T4-related phages. The “”Near T4″” group containing the T-evens, Pseudo T-evens, and Schizo T-evens; the “”Far T4″” clade including Exo-T4 phage RM378; and, the “”Cyano T4″” assemblage. We believe that the latter are sufficiently different from the other T4 viruses to be excluded from the Teequatrovirinae at this time.

JCR and ADC supervised the work and revised the manuscript. All authors read and approved the final manuscript.”
“Background Enteropathogenic Escherichia coli (EPEC) is a leading cause of infantile diarrhoea in developing countries [1]. Net secretory diarrhoea results from altered host cell signaling events, loosening of tight junctions and is exacerbated by the destruction of absorptive tissue, the host intestinal microvilli [2]. These phenotypes are mediated by a type III secretion

system, a molecular Selleckchem NVP-HSP990 syringe that secretes bacterial proteins into host cells, and is a common feature of many gram-negative pathogens [3]. The mechanism of EPEC diarrhoeal disease is similar to that of enterohemorrhagic E. coli (EHEC), and thus EPEC can be used as a surrogate for investigating disease caused by this more serious threat to selleck chemicals public health. While, by learn more definition, EPEC, possesses no diffusible toxins, EHEC in contrast produces Shiga toxin, causing bloody diarrhoea or hemorrhagic colitis. The production of Shiga toxin also can lead to the life-threatening complication, hemolytic uremic syndrome (HUS), which occurs in approximately 10% of reported cases of EHEC infection [4]. In developing countries, studies have shown that administering zinc to children with diarrhoea reduces the severity of disease [5, 6]. It was initially hypothesized that this effect was due to correction of zinc deficiencies

often seen in impoverished and malnourished children in these regions of the world. Certainly zinc is an important nutrient due to its fundamental role as a cofactor – over 300 zinc-depedent enzymes have been identified from all forms of life, with many of these such as carbonic anhydrase forming a basic part of human metabolism. Zinc is also found in non-enzymatic

contexts in humans, for example its structural role in the ubiquitous zinc finger transcriptional regulators [7]. Zinc is also important for immune function, and zinc deficiency adversely affects the health and development of children [8, 9]. However, a double-blind, Clomifene randomized, controlled study involving 937 children with acute diarrhoea conducted in New Delhi, India demonstrated that zinc supplementation benefited children in the experimental group irrespective of the child’s initial plasma zinc level [5]. Thus beyond being an important co-factor necessary for immune and enzyme function in children, zinc also reduces the duration and severity of diarrhoeal disease caused by E. coli. For an initial study conducted in Calcutta, many, but not all of the reported cases were caused by EPEC. Thus some researchers have argued for greater use of zinc supplementation to treat bacterial diarrhoeal disease in children in the developing world [10]. In EPEC, zinc causes a reduction in net protein secretion via the type III secretion system [11], encoded within the pathogenicity island termed the locus of enterocyte effacement or LEE.

Proliferation rates were determined at day 1, 2, 3, 4 post-transfection, and quantification was done on a microtiter plate reader (Spectra Rainbow, Tecan) according to the manufacturer’s protocol. Meanwhile, the mimic-transfected cells were trypsinized and replated at 200 cells per well in 6-well plates, cultured for 7 days, then fixed with methanol and stained with 0.1% crystal violet in 20% methanol for 15 min. Western blotting Whole-cell lysate or nuclear extract

was subjected to Western blot analysis as described previously [21]. The following antibodies were used for Western blot: GAPDH (10494-1-AP, Proteintech), PTEN (22034-1-AP, Proteintech). Statistics The statistical HDAC cancer analyses for miR-19a expression in clinical samples, correlation of miR-19a expression with patients’ clinicopathological variables were conducted using the Bonferroni multiple-comparison test. The other statistical analyses were evaluated by independent samples T test (two-tailed). P ≤ 0.05 was considered statistically significant. Results miR-19a is up-regulated in bladder cancer cells To analyze the expression of miR-19a in bladder cancer, q-PCR using Taqman probes

was conducted to measure the levels of miR-19a. We firstly examined the expression of mature miR-19a in immortalized human bladder epithelium (HCV29 and HU609) cells and five human bladder cancer cell lines (J82, HT1376, RT4, T24 and TCCSUP). The expression level of miR-19a in bladder cancer cell lines was significant higher than that in the normal bladder epithelium cells. Expression level of miR-19a in RT4 was a little lower GANT61 mw than that in the four other bladder cancer cell lines (Figure 1A). These data Blebbistatin in vivo demonstrated that the up-regulation of miR-19a might be relevant to the genesis and development of bladder cancer. second Figure 1 miR-19a is significantly up-regulated in bladder

cancer cell lines and in bladder cancer tissues. (A) The expression level of miR-19a in two immortalized human bladder epithelium cells (HCV29 and HU609) and five bladder cancer cell lines (J82, HT1376, RT4, T24 and TCCSUP). Data are shown as mean + s.d. (n = 3); * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001. (B) The relative expression of miR-19a in 100 pairs of bladder cancer (C) and adjacent non-neoplastic tissues (N). (C) Normalized expression of miR-19a in 100 pairs of bladder cancer and adjacent normal tissues. (D) The correlation of miR-19a expression with tumor grades of bladder cancer tissues. miR-19a is up-regulated in bladder cancer tissues compared with the corresponding adjacent non- neoplastic tissues To further analyze the expression of miR-19a in patients with bladder cancer, we measured the levels of miR-19a in 100 pairs of bladder cancer tissues (C) and the adjacent non-neoplastic tissues (N).

tularensis subsp. holarctica). Figure 7 Cytospin preparation of infected U 937 cell culture followed by specific detection of the facultative pathogen F. tularensis subsp. novicida (MOI 10:1, 24 h). (A: phase contrast microscopy;

B: FISH, probe EUB338-6-FAM; C: FISH, probe Bwnov168-Cy3). An automated blood culture system (BACTEC, BD, P505-15 manufacturer Heidelberg, Germany) was used to grow bacterial cells from each representative strain initially used for 23S rRNA gene sequencing. The culture bottles were spiked with 5 ml of human blood and the bacteria grown on HCA medium. Depending on the subspecies and the initial inoculum size, growth in aerobic blood culture bottles occurred between two to eleven days of incubation. Bacterial cells from each subspecies were strongly labeled with their corresponding probes as well as the EUB338 probe used for positive control (Table 3). Table 3 Identification of different F. philomiragia and F. tularensis subspp. in positive blood culture using FISH.   Bwall1448 (35% FA) Bwphi1448 + Bwall1448c (50%FA) Bwhol1151 + Bwhol1151c (35%FA) Bwnov168 + Bwnov168c (35%FA) Bwtume168II + Bwtume168c (20%FA) Bwmed1379 + Bwmed1379c (20%FA) F. tul. subsp. holarctica + – + – - – F. tul. subsp. mediasiatica + – - – + + F. tul. subsp. novicida + – - + – - F. philomiragia + + – - – - Blood culture bottles

were inoculated with 5 ml venous blood spiked with 102CFU of each GF120918 cell line different strain. +: positive hybridization -: negative reaction, no fluorescence In mixed samples containing bacterial cells from different strains

(e.g. type A as well as type B) both populations could be easily separated by whole cell hybridization with distinctly labeled probes (Fig. 8). By this approach, for instance, one type A bacterial cell can be detected and unequivocally identified in 1.000 type B cells. Figure 8 Mixed sample of bacterial cells from F. tularensis tularensis (ATCC 6223) and F. tularensis subsp. holarctica LVS (ratio 100:1). Contamination lower than 1% could be identified using appropriate probe sets. (A: FISH staining with probe EUB338-6-FAM for staining of all bacteria in liquid samples. B: Specific many staining of F. tularensis subsp. holarctica). Discussion Tularemia is a rare but dangerous zoonosis, which is endemic in almost all countries of the Northern Hemisphere. In some areas like Central and Southern Europe as well as Turkey, tularemia is an emerging or re-emerging disease representing a significant threat for public health [33–35]. Its causative agent, F. tularensis, is regarded as a potential biological warfare or bioterrorism agent of the highest category. For these reasons clinical and public health laboratories are urged to provide rapid and reliable diagnostic tools for the sensitive detection and identification of F.