Identification of novel clostridium perfringens type E strains that carry an iota toxin plasmid with a functional enterotoxin gene

Clostridium perfringens enterotoxin (CPE) is a major virulence factor for human gastrointestinal diseases, such as food poisoning and antibiotic associated diarrhea. The CPE-encoding gene (cpe) can be chromosomal or plasmid-borne. Recent development of conventional PCR cpe-genotyping assays makes it possible to identify cpe location (chromosomal or plasmid) in type A isolates. Initial studies for developing cpe genotyping assays indicated that all cpe-positive strains isolated from sickened patients were typable by cpe-genotypes, but surveys of C. perfringens environmental strains or strains from feces of healthy people suggested that this assay might not be useful for some cpe-carrying type A isolates. In the current study, a pulsed-field gel electrophoresis Southern blot assay showed that four cpe-genotype untypable isolates carried their cpe gene on a plasmid of ~65 kb. Complete sequence analysis of the ~65 kb variant cpe-carrying plasmid revealed no intact IS elements and a disrupted cytosine methyltransferase (dcm) gene. More importantly, this plasmid contains a conjugative transfer region, a variant cpe gene and variant iota toxin genes. The toxin genes encoded by this plasmid are expressed based upon the results of RT-PCR assays. The ~65 kb plasmid is closely related to the pCPF4969 cpe plasmid of type A isolates. MLST analyses indicated these isolates belong to a unique cluster of C. perfringens. Overall, these isolates carrying a variant functional cpe gene and iota toxin genes represent unique type E strains. © 2011 Miyamoto et al

Sialidases Affect the Host Cell Adherence and Epsilon Toxin-Induced Cytotoxicity of Clostridium perfringens Type D Strain CN3718

Clostridium perfringens type B or D isolates, which cause enterotoxemias or enteritis in livestock, produce epsilon toxin (ETX). ETX is exceptionally potent, earning it a listing as a CDC class B select toxin. Most C. perfringens strains also express up to three different sialidases, although the possible contributions of those enzymes to type B or D pathogenesis remain unclear. Type D isolate CN3718 was found to carry two genes (nanI and nanJ) encoding secreted sialidases and one gene (nanH) encoding a cytoplasmic sialidase. Construction in CN3718 of single nanI, nanJ and nanH null mutants, as well as a nanI/nanJ double null mutant and a triple sialidase null mutant, identified NanI as the major secreted sialidase of this strain. Pretreating MDCK cells with NanI sialidase, or with culture supernatants of BMC206 (an isogenic CN3718 etx null mutant that still produces sialidases) enhanced the subsequent binding and cytotoxic effects of purified ETX. Complementation of BMC207 (an etx/nanH/nanI/nanJ null mutant) showed this effect is mainly attributable to NanI production. Contact between BMC206 and certain mammalian cells (e.g., enterocyte-like Caco-2 cells) resulted in more rapid sialidase production and this effect involved increased transcription of BMC206 nanI gene. BMC206 was shown to adhere to some (e.g. Caco-2 cells), but not all mammalian cells, and this effect was dependent upon sialidase, particularly NanI, expression. Finally, the sialidase activity of NanI (but not NanJ or NanH) could be enhanced by trypsin. Collectively these in vitro findings suggest that, during type D disease originating in the intestines, trypsin may activate NanI, which (in turn) could contribute to intestinal colonization by C. perfringens type D isolates and also increase ETX action

Organization of the cpe Locus in CPE-Positive Clostridium perfringens Type C and D Isolates

Clostridium perfringens enterotoxin (encoded by the cpe gene) contributes to several important human, and possibly veterinary, enteric diseases. The current study investigated whether cpe locus organization in type C or D isolates resembles one of the three (one chromosomal and two plasmid-borne) cpe loci commonly found amongst type A isolates. Multiplex PCR assays capable of detecting sequences in those type A cpe loci failed to amplify products from cpe-positive type C and D isolates, indicating these isolates possess different cpe locus arrangements. Therefore, restriction fragments containing the cpe gene were cloned and sequenced from two type C isolates and one type D isolate. The obtained cpe locus sequences were then used to construct an overlapping PCR assay to assess cpe locus diversity amongst other cpe-positive type C and D isolates. All seven surveyed cpe-positive type C isolates had a plasmid-borne cpe locus partially resembling the cpe locus of type A isolates carrying a chromosomal cpe gene. In contrast, all eight type D isolates shared the same plasmid-borne cpe locus, which differed substantially from the cpe locus present in other C. perfringens by containing two copies of an ORF with 67% identity to a transposase gene (COG4644) found in Tn1546, but not previously associated with the cpe gene. These results identify greater diversity amongst cpe locus organization than previously appreciated, providing new insights into cpe locus evolution. Finally, evidence for cpe gene mobilization was found for both type C and D isolates, which could explain their cpe plasmid diversity

Measurement of the branching fractions for B- --> D(*)+ pi- l- nu-bar and B0bar --> D(*)0 pi+ l- nu-bar

We report on a measurement of the branching fractions for B- --> D(*)+ pi- l- nu-bar and B0bar --> D(*)0 pi+ l- nu-bar with 275 million BBbar events collected at the Upsilon(4S) resonance with the Belle detector at KEKB. Events are tagged by fully reconstructing one of the B mesons in hadronic modes. We obtain Br(B- --> D+ pi- l- nu-bar) = (0.54 +/- 0.07(stat) +/- 0.07(syst) +/- 0.06(BR)) x 10^-2, Br(B- --> D*+ pi- l- nu-bar) = (0.67 +/- 0.11(stat) +/- 0.09(syst) +/- 0.03(BR)) x 10^-2, Br(B0bar --> D0 pi+ l- nu-bar) = (0.33 +/- 0.06(stat) +/- 0.06(syst) +/- 0.03(BR)) x 10^-2, Br(B0bar --> D*0 pi+ l- nu-bar) = (0.65 +/- 0.12(stat) +/- 0.08(syst) +/- 0.05(BR)) x 10^-2, where the third error comes from the error on Bbar --> D(*) l- nu-bar decays. Contributions from B0bar --> D*+ l- nu-bar decays are excluded in the measurement of B0bar --> D0 pi+ l- nu-bar.Comment: 6 pages, 10 figures, submitted to Physical Review D (Rapid Communication), the Lepton-Photon 2005 Conference (Uppsala, Sweden) and the HEP2005 Europhysics Conference (Lisboa, Portugal

Measurement of the wrong-sign decays D0 -> K+ pi- pi0 and D0 -> K+ pi- pi+ pi-, and search for CP violation

Using 281 fb^-1 of data from the Belle experiment recorded at or near the Upsilon(4S) resonance, we have measured the rates of the ``wrong-sign'' decays D0 -> K+ pi- pi0 and D0 -> K+ pi- pi+ pi- relative to those of the Cabibbo-favored decays D0 -> K- pi+ pi0 and D0 -> K- pi+ pi+ pi-. These wrong-sign decays proceed via a doubly Cabibbo-suppressed amplitude or via D0-D0bar mixing; the latter has not yet been observed. We obtain R_WS(Kpipi0)=[0.229 +/-0.017(stat.) +0.013-0.009(sys.)]% and R_WS(K3pi)=[0.320 +/-0.019(stat.) +0.018-0.013(sys.)]%. The CP asymmetries are measured to be -0.006 +/- 0.053 and -0.018 +/- 0.044 for the K+ pi- pi0 and K+ pi- pi+ pi- final states, respectively.Comment: 10 pages, 3 figures, submitted to PRL, Lepton-Photon 2005 Conference in Uppsala, Sweden and HEP2005 Europhysics Conference in Lisboa, Portuga

Measurement of D0 -> pilnu (Klnu) Form Factors and Absolute Branching Fractions

Using a 282 1/fb data sample collected by the Belle experiment at the KEKB e+e- collider, we study D0 decays to K-l+nu and pi-l+nu final states. The D0 flavor and momentum are tagged through a full reconstruction of the recoiling charm meson and additional mesons from fragmentation. The reconstruction method provides very good resolution in neutrino momentum and in q^2 = (p_l+p_nu)^2. Normalizing to the total number of D0 tags, we measure the absolute branching fractions to be B(D0 -> Klnu) =(3.45 +- 0.07stat +- 0.20syst)% and B(D0 -> pilnu) = (0.255 +- 0.019stat +- 0.016syst)% and the semi-leptonic form factors (within the modified pole model) f+^K(0) = 0.695 +- 0.007stat +- 0.022syst and f+^pi(0) = 0.624 +- 0.020stat +- 0.030syst.Comment: 9 pages, 2 figures, submitted to Phys. Rev. Let

Measurement of phi_3 with Dalitz plot analysis of B+ -> D(*)K(*)+ decay

We present a measurement of the unitarity triangle angle phi_3 using a Dalitz plot analysis of the K0_S pi+ pi- decay of the neutral D meson from the B+- -> D(*)K(*)+- process. The method employs the interference between D0 and D0bar to extract the angle phi_3, strong phase Delta and the ratio r of suppressed and allowed amplitudes. We apply this method to a 357 fb-1 data sample collected by the Belle experiment. The analysis uses three modes: B+ -> DK+, B+ -> D*K+ with D* -> Dpi0, and B+ -> DK*+ with K*+ -> K0_S pi+, as well as the corresponding charge-conjugate modes. From a combined maximum likelihood fit to the three modes, we obtain phi_3=53+15-18(stat)+-3(syst)+-9(model) degrees. The corresponding two standard deviation interval is 8<phi_3<111 degrees.Comment: 18 pages, 6 figures, 7 tables. To be submitted to Phys. Rev.

Formulation and in vitro evaluation of fast dissolving tablets of metoprolol tartrate

The demand for fast dissolving tablets has been growing during the last decade, especially for elderly and children who have swallowing difficulties. In the present work, fast dissolving tablets of metoprolol tartrate, were prepared using sodium starch glycolate, sodium croscarmellose and crospovidone as superdisintegrants, by the direct compression method. The tablets prepared were evaluated for various parameters including weight variation, hardness, friability, in vitro dispersion time, drug-polymer interaction, drug content water absorption ratio, wetting time, in vitro drug release, FTIR and DSC studies. The tablets prepared by the direct compression method had a weight variation in the range of 145 mg to 152 mg, which is below ± 7.5%, a hardness of 3.6 kg/cm² to 4.5 kg/cm², percentage friability of 0.46% to 0.73%, in vitro dispersion time of 18 s to 125 s, drug content uniformity of between 98.12% and 100.03%, a water absorption ratio of 67% to 87%, wetting time of 32 sec. to 64 sec., and an in vitro drug release of 53.92% - 98.82% within 15 min. The IR spectral analysis and DSC study showed no drug interaction with formulation additives of the tablet, and the formulations indicated no significant changes in hardness, friability, drug content or in vitro drug release. Fast dissolving tablets of metoprolol tartrate have enhanced dissolution and will lead to improved bioavailability and more effective therapy

Measurements of CP Violation in B0→D∗−π+B^0 \to D^{*-}\pi^+ and B0→D−π+B^0 \to D^- \pi^+ Decays

We report measurements of time dependent decay rates for $B^0 \to D^{(*)-}\pi^+$ decays and extraction of CP violation parameters that depend on $\phi_3$. Using fully reconstructed $D^{(*)}\pi$ events and partially reconstructed $D^{*}\pi$ events from a data sample that contains 386 million $B\bar{B}$ pairs that was collected near the $\Upsilon(4S)$ resonance, with the Belle detector at the KEKB asymmetric energy $e^+ e^-$ collider, we obtain the CP violation parameters $S^+ (D^{(*)}\pi)$ and $S^- (D^{(*)}\pi)$. We obtain $S^+ (D^* \pi) = 0.049 \pm 0.020(\mathrm{stat}) \pm 0.011(\mathrm{sys})$, $S^- (D^* \pi) = 0.031 \pm 0.019(\mathrm{stat}) \pm 0.011(\mathrm{sys})$, and $S^+ (D \pi) = 0.031 \pm 0.030(\mathrm{stat}) \pm 0.012(\mathrm{sys})$, $S^- (D \pi) = 0.068 \pm 0.029(\mathrm{stat}) \pm 0.012(\mathrm{sys})$. These results are an indication of CP violation in $B^0 \to D^{*-}\pi^+$ and $B^0 \to D^- \pi^+$ decays at the $2.5 \sigma$ and $2.2 \sigma$ levels, respectively. If we use the values of $R_{D^{(*)}\pi}$ that are derived using assumptions of factorization and SU(3) symmetry, the branching fraction measurements for the $D_s^{(*)} \pi$ modes, and lattice QCD calculations, we can restrict the allowed region of $|\sin (2\phi_1 + \phi_3)|$ to be above 0.44 and 0.52 at 68% confidence level from the $D^* \pi$ and $D \pi$ modes, respectively.Comment: 14 pages, 12 figures, submitted to Physical Review