Clostridium botulinum Group II Isolate Phylogenomic Profiling Using Whole-Genome Sequence Data.

Clostridium botulinum group II isolates (n = 163) from different geographic regions, outbreaks, and neurotoxin types and subtypes were characterized in silico using whole-genome sequence data. Two clusters representing a variety of botulinum neurotoxin (BoNT) types and subtypes were identified by multilocus sequence typing (MLST) and core single nucleotide polymorphism (SNP) analysis. While one cluster included BoNT/B4/F6/E9 and nontoxigenic members, the other comprised a wide variety of different BoNT/E subtype isolates and a nontoxigenic strain. In silico MLST and core SNP methods were consistent in terms of clade-level isolate classification; however, core SNP analysis showed higher resolution capability. Furthermore, core SNP analysis correctly distinguished isolates by outbreak and location. This study illustrated the utility of next-generation sequence-based typing approaches for isolate characterization and source attribution and identified discrete SNP loci and MLST alleles for isolate comparison.

Common distal elements orchestrate CIITA isoform-specific expression in multiple cell types.

Major histocompatibility class II (MHC-II) expression is critical for immune responses and is controlled by the MHC-II transactivator CIITA. CIITA is primarily regulated at the transcriptional level and is expressed from three main promoters with myeloid, lymphoid and interferon (IFN)-γ-treated non-hematopoietic cells using promoters pI, pIII and pIV, respectively. Recent studies in non-hematopoietic cells suggest that a series of distal regulatory elements may be involved in regulating CIITA transcription. To identify distal elements in B cells, a DNase I hypersensitivity screen was performed, revealing a series of potential novel regulatory elements. These elements were analyzed computationally and biochemically. Several regions displayed active histone modifications and/or enhanced expression of a reporter gene. Four of the elements interacted with pIII in B cells. These same four regions were also found to interact with pI in splenic dendritic cells (spDC). Intriguingly, examination of the above interactions in pI-knockout-derived spDC showed a switch to the next available promoter, pIII. Extensive DNA methylation was found at the pI region in B cells, suggesting that this promoter is not accessible in B cells. Thus, CIITA expression is likely mediated in hematopoietic cells by common elements with promoter accessibility having a part in promoter choice.

Two novel toxin variants revealed by whole-genome sequencing of 175 Clostridium botulinum type E strains.

We sequenced 175 Clostridium botulinum type E strains isolated from food, clinical, and environmental sources from northern Canada and analyzed their botulinum neurotoxin (bont) coding sequences (CDSs). In addition to bont/E1 and bont/E3 variant types, neurotoxin sequence analysis identified two novel BoNT type E variants termed E10 and E11. Strains producing type E10 were found along the eastern coastlines of Hudson Bay and the shores of Ungava Bay, while strains producing type E11 were only found in the Koksoak River region of Nunavik. Strains producing BoNT/E3 were widespread throughout northern Canada, with the exception of the coast of eastern Hudson Bay.

Late Cretaceous ammonoids show that drivers of diversification are regionally heterogeneous.

Palaeontologists have long sought to explain the diversification of individual clades to whole biotas at global scales. Advances in our understanding of the spatial distribution of the fossil record through geological time, however, has demonstrated that global trends in biodiversity were a mosaic of regionally heterogeneous diversification processes. Drivers of diversification must presumably have also displayed regional variation to produce the spatial disparities observed in past taxonomic richness. Here, we analyse the fossil record of ammonoids, pelagic shelled cephalopods, through the Late Cretaceous, characterised by some palaeontologists as an interval of biotic decline prior to their total extinction at the Cretaceous-Paleogene boundary. We regionally subdivide this record to eliminate the impacts of spatial sampling biases and infer regional origination and extinction rates corrected for temporal sampling biases using Bayesian methods. We then model these rates using biotic and abiotic drivers commonly inferred to influence diversification. Ammonoid diversification dynamics and responses to this common set of diversity drivers were regionally heterogeneous, do not support ecological decline, and demonstrate that their global diversification signal is influenced by spatial disparities in sampling effort. These results call into question the feasibility of seeking drivers of diversity at global scales in the fossil record.

Identification of Reticulitermes Subterranean Termites (Blattodea: Rhinotermitidae) in the Eastern United States Using Inter-Simple Sequence Repeats.

In the eastern United States, there are nine species of subterranean termites in three genera: Reticulitermes (six species), Coptotermes (two species), and Prorhinotermes (one species). These species serve as important ecological players by decomposing cellulose material, and some are important structural pests. Many of these species are difficult to discriminate morphologically and require examining the reproductive or soldier castes, which can be difficult to collect. While some genetic tools have been developed for species identification, they are often expensive and time-consuming. To help facilitate identification, we developed a more cost-effective and rapid genetic method to identify Reticulitermes species by screening 10 PCR primers that amplified inter-simple sequence repeats (ISSRs) in other termite species. From these, one primer was amplified in all five focal Reticulitermes species and contained conserved, species-specific fragments. We further screened this identification method on samples of each species covering a diversity of mitochondrial DNA haplotypes and localities. This identification method utilizing ISSRs can be used to quickly identify five species of Reticulitermes subterranean termites in the eastern United States in a matter of hours, providing a useful technique for pest management as well as future ecological research.

Physical and genetic characterization of an outer-membrane protein (OmpM1) containing an N-terminal S-layer-like homology domain from the phylogenetically Gram-positive gut anaerobe Mitsuokella multacida.

Thin sectioning and freeze-fracture-etch of the ovine ruminal isolate Mitsuokella multacida strain 46/5(2) revealed a Gram-negative envelope ultra-structure consisting of a peptidoglycan wall overlaid by an outer membrane. Sodium-dodecyl-sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of whole cells, cell envelopes and Triton X-100 extracted envelopes in combination with thin-section and N-terminal sequence analyses demonstrated that the outer membrane contained two major proteins (45 and 43 kDa) sharing identical N-termini (A-A-N-P-F-S-D-V-P-A-D-H-W-A-Y-D). A gene encoding a protein with a predicted N-terminus identical to those of the 43 and 45 kDa outer-membrane proteins was cloned. The 1290 bp open reading frame encoded a 430 amino acid polypeptide with a predicted molecular mass of 47,492 Da. Cleavage of a predicted 23 amino acid leader sequence would yield a protein with a molecular mass of 45,232 Da. Mass spectroscopic analysis confirmed that the cloned gene (ompM1) encoded the 45 kDa outer-membrane protein. The N-terminus of the mature OmpM1 protein (residues 24-70) shared homology with surface-layer homology (SLH) domains found in a wide variety of regularly structured surface-layers (S-layers). However, the outer-membrane locale, resistance to denaturation by SDS and high temperatures and the finding that the C-terminal residue was a phenylalanine suggested that ompM1 encoded a porin. Threading analysis in combination with the identification of membrane spanning domains indicated that the C-terminal region of OmpM1 (residues 250-430) likely forms a 16-strand beta-barrel and appears to be related to the unusual N-terminal SLH-domain-containing beta-barrel-porins previously described in the cyanobacterium Synechococcus PCC6301.

Effect of staphylococcal alpha-hemolysin upon anion transport in the rabbit erythrocyte.

Equilibrium exchange of SO4(2-) was measured prior to and during hemolysis in rabbit erythrocytes exposed to staphylococcal alpha-hemolysin. The anion-transport protein of the rabbit erythrocyte has also been identified. Equilibrium exchange of SO4(2-) was measured by both efflux and influx of 35SO4(2-). The rate of influx of SO4(2-) in rabbit erythrocytes exposed to alpha-hemolysin was twice that of the untreated cells. The rate of SO4(2-) efflux was unchanged by alpha-hemolysin. Inhibition of anion exchange with 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) did not inhibit hemolysis, therefore, the increased influx of SO4(2-) may occur through a DIDS-insensitive pathway.

Roles of structural domains in the morphology and surface anchoring of the tetragonal paracrystalline array of Aeromonas hydrophila. Biochemical characterization of the major structural domain.

The tetragonally arranged S-layer of Aeromonas hydrophila contains two morphological domains. The mature S-layer protein of A. hydrophila has a subunit molecular weight of 52,000, and has been reported to contain two structural domains. Here a mutant has been isolated which produces an S-layer of subunit molecular weight 38,650 as determined by sedimentation analysis. This truncated S-protein was exported via the periplasm to the cell surface, but could not self-assemble into a tetragonal array or be anchored to the cell surface. Instead the truncated protein formed cup-like structures which were purified and characterized biochemically. Automated Edman degradation showed that the truncated protein comprised the amino-terminal structural domain of the S-protein. This domain had an increased hydrophobic amino acid content relative to the wild-type protein, and contained approximately 42% beta-sheet, 10% alpha-helix, and 19% beta-turn. Differences in alpha-helix and beta-turn contents between the wild-type and truncated proteins were observed when the effects of pH and SDS were examined, indicating that the carboxy terminus influences the effects of environmental change on the conformation of the S-protein. This lesser carboxy-terminal array also appears to be required for both correct array morphology, and array anchoring, while the greater amino-terminal domain appears to comprise the major morphological core of the surface array.

Cloth-based hybridization array system for the detection of Clostridium botulinum type A, B, E, and F neurotoxin genes.

A simple cloth-based hybridization array system was developed for the characterization of Clostridium botulinum isolates based on the botulinum neurotoxin serotype. Bacterial isolates were subjected to a multiplex PCR incorporating digoxigenin-dUTP and primers targeting the four botulinum neurotoxin gene serotypes (A, B, E, and F) predominantly involved in human illness, followed by hybridization of the amplicons with an array of toxin gene-specific oligonucleotide probes immobilized on polyester cloth and subsequent immunoenzymatic assay of the bound digoxigenin label. This system provided sensitive and specific detection of the different botulinum neurotoxin gene markers in a variety of C. botulinum strains, exhibiting the expected patterns of reactivity with a panel of target and nontarget organisms.

Evolutionary Patterns among Living and Fossil Kogiid Sperm Whales: Evidence from the Neogene of Central America.

Kogiids are known by two living species, the pygmy and dwarf sperm whale (Kogia breviceps and K. sima). Both are relatively rare, and as their names suggest, they are closely related to the sperm whale, all being characterized by the presence of a spermaceti organ. However, this organ is much reduced in kogiids and may have become functionally different. Here we describe a fossil kogiid from the late Miocene of Panama and we explore the evolutionary history of the group with special attention to this evolutionary reduction. The fossil consists of cranial material from the late Tortonian (~7.5 Ma) Piña facies of the Chagres Formation in Panama. Detailed comparison with other fossil and extant kogiids and the results of a phylogenetic analysis place the Panamanian kogiid, herein named Nanokogia isthmia gen. et sp. nov., as a taxon most closely related to Praekogia cedrosensis from the Messinian (~6 Ma) of Baja California and to Kogia spp. Furthermore our results show that reduction of the spermaceti organ has occurred iteratively in kogiids, once in Thalassocetus antwerpiensis in the early-middle Miocene, and more recently in Kogia spp. Additionally, we estimate the divergence between extant species of Kogia at around the late Pliocene, later than previously predicted by molecular estimates. Finally, comparison of Nanokogia with the coeval Scaphokogia cochlearis from Peru shows that these two species display a greater morphological disparity between them than that observed between the extant members of the group. We hypothesize that this reflects differences in feeding ecologies of the two species, with Nanokogia being more similar to extant Kogia. Nanokogia shows that kogiids have been part of the Neotropical marine mammal communities at least since the late Miocene, and gives us insight into the evolutionary history and origins of one of the rarest groups of living whales.

Thin aggregative fimbriae enhance Salmonella enteritidis biofilm formation.

Salmonella enteritidis enteropathogens produce a variety of potentially adherent fimbrial types including SEF14, SEF17, SEF18 and SEF21 (type I). In a simplified, pure culture, biofilm generating system the virulent isolate, S. enteritidis 3b, readily adhered to Teflon (polytetrafluoroethylene) and stainless steel forming thick cell aggregates. The inability of an isogenic SEF17-deficient mutant to form thick biofilms suggested a role for SEF17 in stabilizing cell-cell interactions during biofilm formation. Epifluorescent detection of SEF17 in biofilms confirmed the association of these fimbriae with aggregated cells but not with adherent mutants unable to produce SEF17. The reduced adherence observed with an isogenic SEF14/SEF21-deficient strain implicated the involvement of additional cell surface adherence factors, possibly including SEF21 (type I) fimbriae in the adherence of S. enteritidis to stainless steel or Teflon. The role of SEF17 fimbriae in biofilm formation and the contributions of SEF17 to the persistence of Salmonellae on surfaces and in food are discussed.

Antibacterial effect of protamine in combination with EDTA and refrigeration.

The antimicrobial effect of protamine (clupeine) on a range of gram-positive and gram-negative foodborne pathogens and spoilage bacteria, was evaluated using an agar dilution assay and a broth dilution assay with Alamar Blue as growth indicator. Protamine was tested alone at concentrations from 0 to 10,000 microg/ml, and in combination with EDTA (0.9 mM). Assays were performed at 5 degrees C, 10 degrees C, 18 degrees C and 30 degrees C to test the effect of temperature. Minimum inhibitory concentration (MIC) values ranged from 10 microg/ml for Brochothrix thermosphacta to no inhibition at 10,000 microg/ml for bacteria such as Aeromonas hydrophila, proteolytic strains of Clostridium botulinum, Hafnia alvei and Morganella morganii. The minimum bactericidal concentrations (MBCs) were generally higher than MICs. In combination with EDTA, MICs of protamine decreased for gram-negative test strains, whereas EDTA alone inhibited gram-positive strains. The effect of assay incubation temperature was variable and not clear for most strains. Concentrations of 100-750 microg/ml protamine inhibited the five non-proteolytic C. botulinum strains, while none of the eight proteolytic strains was inhibited, indicating the possible role of proteolytic enzymes in protecting cells from protamine. Clearing zones, indicative of proteolytic activity, were observed in the opaque TSB-agarose around colonies of some but not all protamine-resistant bacteria, suggesting that this is not the only resistance mechanism. Addition of 5% (w/v) gelatin to study the effect of an increased protein concentration in the agar dilution assay showed that electrostatic interactions between protamine and the protein decreased the antimicrobial efficacy of the peptide.

Growth and toxin production by Clostridium botulinum on inoculated fresh-cut packaged vegetables.

To determine the safety of fresh-cut vegetables packaged in modified atmosphere, challenge studies using both nonproteolytic and proteolytic strains of Clostridium botulinum were performed with a variety of fresh-cut packaged salads and vegetables stored at different temperatures. When vegetables were inoculated with spores of C. botulinum and incubated in low-O2 atmospheres, spore germination and growth and toxin production were observed. Botulinum toxin was produced by proteolytic types A and B on onion, butternut squash, rutabaga, salad, and stir-fry vegetables. Nonproteolytic C. botulinum produced toxin on butternut squash and salad. Nonproteolytic C. botulinum was capable of producing neurotoxin at temperatures as low as 5 degree C whereas proteolytic strains produced neurotoxin at 15 degrees C and higher. Although most samples were visibly spoiled before detection of botulinum toxin, samples of butternut squash and onion remained acceptable after detection of toxin. The strict maintenance of low temperatures (< 5 degrees C) is recommended in order to control the potential growth of C. botulinum on fresh-cut vegetables packaged in a modified atmosphere.

Survival and recovery of Escherichia coli O157:H7 in inoculated bottled water.

A methodology used to isolate Escherichia coli O157:H7 from water and survival of this pathogen in inoculated water is described. The methodology used in the isolation of E. coli O157:H7 included the use of selective plating on Sorbitol MacConkey agar (supplemented with potassium tellurite [2.5 mg/liter], cefixime [0.05 mg/liter], and cefsulodin [10 mg/liter], and modified hemorrhagic colitis agar (also supplemented with potassium tellurite [2.5 mg/liter]) and cefsulodin [10 mg/liter]). There were no significant differences (P < 0.05) between the recoveries of E. coli O157:H7 on these two selective media. Direct plating on these selective agars was used to determine the length of time that E. coli O157:H7 was able to grow, remain viable, and be resistant to the selective agents. E. coli O157:H7 survived in inoculated water for up to > 300 days, depending on the type of water. Observation by scanning electron microscopy indicated that E. coli O157:H7 cells attached to, and multiplied on, the container walls.

Growth and toxin production by Clostridium botulinum in English-style crumpets packaged under modified atmospheres.

To determine the safety of a high moisture bakery product, packaged under modified atmospheres, challenge studies were done on English-style crumpets (water activity [a(w)] 0.990, pH 6.5) inoculated postbaking with Clostridium botulinum types A and proteolytic B spores (5 X 10(2) spores/g). Products were packaged either in air, in air with an Ageless FX200 oxygen absorbent, or in a CO2/N2 (60:40) gas mixture, stored at ambient temperature (25 degrees C), and monitored for toxicity daily. All inoculated crumpets were toxic within 4 to 6 days and were organoleptically acceptable at the time of toxigenesis. Counts of C. botulinum increased to approximately 10(5) CFU/g at the time of toxicity. To determine the effect of baking on product safety, subsequent challenge studies were done on crumpets inoculated with 5 x 10(2) spores/g (baked weight basis) prior to baking. All crumpets were toxic after only 6 days, irrespective of packaging conditions, and toxigenesis again preceded spoilage. Temperature profile studies showed that the maximum internal temperature reached during baking was 97 degrees C, and the total baking process was equivalent to 0.03 min at 121 degrees C. The actual time to toxin production in both studies (4 to 6 days) correlated well with the predicted time (3.4 days) using the U.S. Department of Agriculture Pathogen Modeling Program (version 5.1) for proteolytic strains of C. botulinum. These studies confirm that high moisture bakery products, if contaminated with C. botulinum spores either pre- or postbaking, could pose a public health hazard, if packaged in air (in a high gas barrier package where O2 was depleted and CO2 was generated during storage) or under modified atmosphere packaging conditions and stored at ambient temperature.

Effect of pH and CO2 on growth and toxin production by Clostridium botulinum in English-style crumpets packaged under modified atmospheres.

The effect of pH and CO2 on both growth of and toxin production by Clostridium botulinum in English-style crumpets, packaged under modified atmospheres was investigated using a 2 x 2 factorial experiment. English-style crumpets (water activity, 0.990; pH 6.5 and 8.3) were inoculated with C. botulinum spores types A and proteolytic B (500 spores/g), packaged in either 60% CO2 (balance N2) or 100% CO2, stored at ambient temperature (25 degrees C), and monitored daily for toxicity. Toxin was detected after 4 days in crumpets packaged in 60% CO2, irrespective of initial product pH. Toxin production was delayed 1.5 to 3 days in crumpets packaged under 100% CO2. Analysis of variance indicated a significant interaction effect of pH and %CO2 on time of earliest toxin detection. Delay of toxin production was greatest for high pH (8.3) crumpets. All products were organoleptically acceptable at the time of toxigenesis, and therefore, high moisture-high pH bakery products, if contaminated with spores of C. botulinum, could become hazardous if packaged in atmospheres containing CO2.

Evaluation of a "heads-up" display for cardiopulmonary bypass.

Multiple variables must be analyzed during cardiopulmonary bypass in order to judge the adequacy of perfusion. Variables when viewed singly can be confusing and lead to inaccurate representation of the physiological status of the patient. Communication between the perfusionist and members of the surgical team requires accuracy and complete presentation of pertinent data. Toward this goal of improving the assimilation and processing of information during cardiopulmonary bypass, a multivariable computer-aided "Heads-up Display" (HUD) was developed. Modern jet pilots use heads-up display for rapid assimilation of information when making judgments about the performance of their aircraft and weapons systems. Heads-up display is an electronically generated display that is superimposed upon a pilot's forward field of view. An analogy between a jet pilot and a perfusionist can be made. A geometric form, a hexagon, is used as part of the heads-up display for cardiopulmonary bypass (CPB-HUD). The polygon represents a performance evaluation graph. Each of the six "spokes of a wheel" represents a physiological parameter. The represented variables are: cardiac index, peripheral vascular resistance, hematocrit, dynamic operating blood level, venous saturation, and mean arterial pressure. The perfusionist inputs target values. Target values are then compared to actual values and expressed as a percentage. If all targeted values are achieved, the graphical representation is a hexagon. The surgical team rapidly recognizes abnormal patterns that are outside individual target values. They include, but are not limited to, patterns of: vasoconstriction, vasodilatation, hypovolemia, decreased oxygen carrying capacity, and several others. The CPB-HUD has proved to be of value for planning, real time evaluation, retrospective analysis of cardiopulmonary bypass benchmark data, and as an aid in the teaching of new personnel concerned with cardiopulmonary bypass.

Management of anticoagulation following central nervous system hemorrhage in patients with high thromboembolic risk.

SUMMARY BACKGROUND: Patients who present with central nervous system (CNS) hemorrhage while on anticoagulation (AC) for thromboembolic (TE) risk factors are a challenge to manage. OBJECTIVE: We sought to inform decisions surrounding the timing and intensity of AC resumption by performing a systematic review. METHODS: Three reviewers screened publications from Medline and EMBASE and extracted data. Hemorrhagic and TE adverse events that occurred subsequent to the index hemorrhage were recorded, as was their timing relative to presentation and covariates that might influence their occurrence. RESULTS: Data were extracted from 63 publications detailing 492 patients; 7.7% of patients experienced hemorrhagic complications and 6.1% experienced TE complications. Hemorrhagic complications were more common within 72 h of presentation while TE complications were more common thereafter. Patients restarted on AC after 72 h were significantly more likely to have a TE complication (P = 0.006) and those restarted before 72 h were more likely to hemorrhage (P = 0.0727). Factors associated with re-hemorrhage included younger age, traumatic cause, subdural hematomas and failure to reverse AC. TE complications were more common in younger patients and those with spinal hemorrhage, multiple hemorrhages, and non-traumatic causes of the index hemorrhage. Re-initiation of AC at a lower intensity also significantly increased the risk of TE complications. INTERPRETATION: Our results suggest that it may be prudent to re-initiate AC earlier than previously thought, with the timing and intensity modified based on predictors of TE and hemorrhagic complications. These findings must be explored in a prospective study because of limitations inherent to the analyzed studies.