Assessment of commercial chromogenic solid media for the detection of non-O157 Shiga toxin-producing Escherichia coli (STEC).

Detection of Shiga toxin-producing Escherichia coli (STEC) has evolved significantly since the introduction of sorbitol-MacConkey agar. This study compares four chromogenic media (CHROMagar™ STEC, Rainbow® O157 agar, CHROMagar™ O157, and Colorex® O157) in their identification of non-O157 STEC. When 161 non-O157 STEC were directly inoculated onto each medium, detection rates on CHROMagar™ STEC, Rainbow® O157 agar, CHROMagar™ O157 and Colorex® O157 were 90%, 70%, 3.7% and 6.8%, respectively. Tellurite minimal inhibitory concentrations (MICs) correlated with growth on CHROMagar™ STEC as 20 of 22 isolates with poor or no growth had MICs ≤1µg/mL. Stool spiking experiments revealed that CHROMagar™ STEC had the highest recovery of the six most common non-O157 STEC, ranging from 30% (in mucoid stool) to 98% (in watery stool). When using clinical stool samples, CHROMagar™ STEC had a sensitivity, specificity, positive predictive value, and negative predictive value of 84.6%, 87%, 13.9%, and 99.6%, respectively.

Molecular profiling of Escherichia coli O157:H7 and non-O157 strains isolated from humans and cattle in Alberta, Canada.

Virulence markers in Shiga toxin-producing Escherichia coli (STEC) and their association with diseases remain largely unknown. This study determines the importance of 44 genetic markers for STEC (O157 and non-O157) from human clinical cases and their correlation to disease outcome. STEC isolated from a cattle surveillance program were also included. The virulence genes tested were present in almost all O157:H7 isolates but highly variable in non-O157 STEC isolates. Patient age was a significant determinant of clinical outcome.

Off-pump coronary artery bypass surgery and acute kidney injury: a meta-analysis of randomized controlled trials.

BACKGROUND AND OBJECTIVES: Off-pump coronary artery bypass grafting (CABG) has been advocated to cause less inflammation, morbidity, and mortality than the more traditional on-pump technique. This meta-analysis compares these two surgical techniques with respect to causing acute kidney injury (AKI). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This study searched for randomized controlled trials in MEDLINE and abstracts from the proceedings of scientific meetings through February 2010. Included were trials comparing off-pump to on-pump CABG that reported the incidence of AKI, as defined by a mixture of criteria including biochemical parameter/urine output/dialysis requirement. Mortality was evaluated among the studies that reported kidney-related outcomes. For primary and subgroup analyses, fixed-effect meta-analyses of odds ratios (OR) were performed. RESULTS: In 22 identified trials (4819 patients), the weighted incidence of AKI in the on-pump CABG group was 4.0% (95% confidence interval [CI] 1.8%, 8.5%), dialysis requirement 2.4% (95% CI 1.6%, 3.7%), and mortality 2.6% (95% CI 1.6%, 4.0%). By meta-analysis, off-pump CABG was associated with a 40% lower odds of postoperative AKI (OR 0.60; 95% CI 0.43, 0.84; P = 0.003) and a nonsignificant 33% lower odds for dialysis requirement (OR 0.67; 95% CI 0.40, 1.12; P = 0.12). Within the selected trials, off-pump CABG was not associated with a significant decrease in mortality. CONCLUSIONS: Off-pump CABG may be associated with a lower incidence of postoperative AKI but may not affect dialysis requirement, a serious complication of cardiac surgery. However, the different definitions of AKI used in individual trials and methodological concerns preclude definitive conclusions.

Identification of separate structural features that affect rate and cation concentration dependence of self-cleavage by the Neurospora VS ribozyme.

The cleavage site of the Neurospora VS ribozyme is located in an internal loop in a hairpin called stem-loop I. Stem-loop I undergoes a cation-dependent structural change to adopt a conformation, termed shifted, that is required for activity. Using site-directed mutagenesis and kinetic analyses, we show here that the insertion of a single-stranded linker between stem-loop I and the rest of the ribozyme increases the observed self-cleavage rate constant by 2 orders of magnitude without affecting the Mg(2+) requirement of the reaction. A distinct set of mutations that favors the formation of the shifted conformation of stem-loop I decreases the Mg(2+) requirement by an order of magnitude with little or no effect on the observed cleavage rate under standard reaction conditions. Similar trends were seen in reactions that contained Li(+) instead of Mg(2+). Mutants with lower ionic requirements also exhibited increased thermostability, providing evidence that the shifted conformation of stem-loop I favors the formation of the active conformation of the RNA. In natural, multimeric VS RNA, where a given ribozyme core is flanked by one copy of stem-loop I immediately upstream and another copy 0.7 kb downstream, cleavage at the downstream site is strongly preferred, providing evidence that separation of stem-loop I from the ribozyme core reflects the naturally evolved organization of the RNA.

Membrane permeabilization, orientation, and antimicrobial mechanism of subtilosin A.

Subtilosin A is an antimicrobial peptide produced by the soil bacterium Bacillus subtilis that possesses bactericidal activity against a diverse range of bacteria, including Listeria monocytogenes. Recent structural studies have found that subtilosin A is posttranslationally modified in a unique way, placing it in a new class of bacteriocins. In this study, in order to understand the mechanism of membrane-disruption by subtilosin A, the interaction of the peptide with model phospholipid bilayers is characterized using fluorescence, solid-state NMR and differential scanning calorimetry (DSC) experiments. Our results in this study show that subtilosin A interacts with the lipid head group region of bilayer membranes in a concentration dependent manner. Fluorescence experiments reveal the interaction of subtilosin A with small unilamellar vesicles (SUVs) composed of POPC, POPG and E. coli total lipids, and that at least one edge of the molecule is buried in membrane bilayers. At high concentrations, it induces leakage from SUVs of POPC and POPE/POPG (7:3) mixture. (15)N solid-state NMR data suggests that the cyclic peptide is partially inserted into bilayers, which is in agreement with the fluorescence data. (31)P and (2)H NMR experiments and DSC data support the hypothesis that subtilosin A adopts a partially buried orientation in lipid bilayers, by showing that it induces a conformational change in the lipid headgroup and disordering in the hydrophobic region of bilayers. These results suggest that the lipid perturbation observed in this study may be one of the consequences of subtilosin A binding to lipid bilayers, which results in membrane permeabilization at high peptide concentrations.

Ab initio study of (13)C(alpha) chemical shift anisotropy tensors in peptides.

This study reports magnitudes and the orientation of the (13)C(alpha) chemical shift anisotropy (CSA) tensors of peptides obtained using quantum chemical calculations. The dependency of the CSA tensor parameters on the energy optimization of hydrogen atom positions and hydrogen bonding effects and the use of zwitterionic peptides in the calculations are examined. Our results indicate that the energy optimization of the hydrogen atom positions in crystal structures is necessary to obtain accurate CSA tensors. The inclusion of intermolecular effects such as hydrogen bonding in the calculations provided better agreement between the calculated and experimental values; however, the use of zwitterionic peptides in calculations, with or without the inclusion of hydrogen bonding, did not improve the results. In addition, our calculated values are in good agreement with tensor values obtained from solid-state NMR experiments on glycine-containing tripeptides. In the case of peptides containing an aromatic residue, calculations on an isolated peptide yielded more accurate isotropic shift values than the calculations on extended structures of the peptide. The calculations also suggested that the presence of an aromatic ring in the extended crystal peptide structure influences the magnitude of the delta(22) which the present level of ab initio calculations are unable to reproduce.

Exceptionally fast self-cleavage by a Neurospora Varkud satellite ribozyme.

Most of the small ribozymes, including those that have been investigated as potential therapeutic agents, appear to be rather poor catalysts. These RNAs use an internal phosphoester transfer mechanism to catalyze site-specific RNA cleavage with apparent cleavage rate constants typically <2 min(-1). We have identified variants of one of these, the Neurospora Varkud satellite ribozyme, that self-cleaves with experimentally measured apparent rate constants of up to 10 s(-1) (600 min(-1)), approximately 2 orders of magnitude faster than any previously characterized self-cleaving RNA. We describe structural features of the cleavage site loop and an adjacent helix that affect the apparent rate constants for cleavage and ligation and the equilibrium between them. These data show that the phosphoester transfer ribozymes can catalyze reactions with rate constants much larger than previously appreciated and in the range of those of protein enzymes that perform similar reactions.

How Does an Amide-N Chemical Shift Tensor Vary in Peptides?

This study addresses a void in the existing literature on the amide-(15)N chemical shift anisotropy (CSA) tensor of peptides: a systematic investigation of how the tensor varies in different peptides. Amide-(15)N CSA tensors for several dipeptides are obtained using quantum chemical calculations, as well as for a series of model Ala-X and X-Ala sequences in both α-helical and ß-sheet conformations (where X is one of the naturally occurring amino acids). The calculated values show a significant variation in both isolated and extended peptide structures. Hydrogen bonding at both the carbonyl group and the N-H bond of the peptide plane is shown to affect the principal values of the tensor. Calculations on model peptides indicate that the amide-(15)N CSA tensor is dependent on atoms located within a distance of five bonds. Consequently, the tensor of a given peptide residue is unaffected by residues other than those adjacent to it, which implies that the amide-(15)N CSA tensor should be considered in the context of tripeptide sequences. This further suggests that the amide-(15)N CSA tensor of the second residue of a given tripeptide sequence may be extrapolated to the same sequence in any other polypeptide or protein, given the same backbone conformation and intermolecular environment. These conclusions will facilitate future NMR structural studies of proteins.