Identification of bacteria associated with Dinoflagellates (Dinophyceae) Alexandrium spp. using tyramide signal amplification fluorescent in situ hybridization and confocal microscopy

In the marine environment, phytoplankton and bacterioplankton can be physically associated. Such association has recently been hypothesized to be involved in the toxicity of the dinoflagellate genus Alexandrium. However, the methods, which have been used so far to identify, localize, and quantify bacteria associated with phytoplankton, are either destructive, time consuming, or lack precision. In the present study we combined tyramide signal amplification–fluorescent in situ hybridization (TSA-FISH) with confocal microscopy to determine the physical association of dinoflagellate cells with bacteria. Dinoflagellate attached microflora was successfully identified with TSA-FISH, whereas FISH using monolabeled probes failed to detect bacteria, because of the dinoflagellate autofluorescence. Bacteria attached to entire dinoflagellates were further localized and distinguished from those attached to empty theca, by using calcofluor and DAPI, two fluorochromes that stain dinoflagellate theca and DNA, respectively. The contribution of specific bacterial taxa of attached microflora was assessed by double hybridization. Endocytoplasmic and endonuclear bacteria were successfully identified in the nonthecate dinoflagellate Gyrodinium instriatum. In contrast, intracellular bacteria were not observed in either toxic or nontoxic strains of Alexandrium spp. Finally, the method was successfully tested on natural phytoplankton assemblages, suggesting that this combination of techniques could prove a useful tool for the simultaneous identification, localization, and quantification of bacteria physically associated with dinoflagellates and more generally with phytoplankton.

Gender comparisons in non-acute cardiac symptom recognition and subsequent help-seeking decisions: a mixed methods study protocol

Introduction: Coronary heart disease (CHD) is one of the leading causes of death in both men and women worldwide. Despite the common misconception that CHD is a ‘man's disease’, it is now well accepted that women endure worse clinical outcomes than men following CHD-related events. A number of studies have explored whether or not gender differences exist in patients presenting with CHD, and specifically whether women delay seeking help for cardiac conditions. UK and overseas studies on help-seeking for emergency cardiac events are contradictory, yet suggest that women often delay help-seeking. In addition, no studies have looked at presumed cardiac symptoms outside an emergency situation. Given the lack of understanding in this area, an explorative qualitative study on the gender differences in help-seeking for a non-emergency cardiac events is needed. Methods and analysis: A purposive sample of 20–30 participants of different ethnic backgrounds and ages attending a rapid access chest pain clinic will be recruited to achieve saturation. Semistructured interviews focusing on help-seeking decision-making for apparent cardiac symptoms will be undertaken. Interview data will be analysed thematically using qualitative software (NVivo) to understand any similarities and differences between the way men and women construct help-seeking. Findings will also be used to inform the preliminary development of a cardiac help-seeking intentions questionnaire. Ethics and dissemination: Ethical approvals were sought and granted. Namely, the University of Westminster (sponsor) and St Georges NHS Trust REC, and the Trust Research and Development Office granted approval to host the study on the Queen Mary's Roehampton site. The study is low risk, with interviews being conducted on hospital premises during working hours. Investigators will disseminate findings via presentations and publications. Participants will receive a written summary of the key findings.

Presence and characterisation of ecotin in F. necrophorum

Fusobacterium necrophorum is a causative agent of Lemierre’s syndrome (LS) in humans. LS is characterised by thrombophlebitis of the jugular vein and bacteraemia. Disseminated intravascular coagulation is also a documented symptom. F. necrophorum is a Gram-negative, anaerobic bacterium known to possess virulence genes such as a haemolysin, filamentous haemagglutinin and leukotoxin, which target host blood components. Ecotin is a serine protease inhibitor that has not previously been characterised in F. necrophorum, but in E.coli has been shown to have a potent anticoagulant effect. Next generation and Sanger sequencing were used to confirm the presence of the ecotin gene in the genomes of a collection of F. necrophorum clinical and reference strains. When translated, it was found to be a highly conserved protein made up of159 amino acids. Enzyme/substrate inhibition assays demonstrated that F. necrophorum ecotin inhibits human plasma kallikrein and human neutrophil elastase in a dose-dependent manner. Data will also be presented on the anticoagulant effects of ecotin during activated partial thromboplastin time, thrombin time and prothrombin time tests on human donor blood. The mechanisms for how this organism reaches the bloodstream and the significance of this serine protease inhibitor during F. necrophorum infections remain to be elucidated

Comparison of virulence genes found in draft genomes of F. necrophorum

Fusobacterium necrophorum is a causative agent of persistent sore throat syndrome, tonsillar abscesses and Lemierre’s syndrome (LS) in humans. LS is characterised by thrombophlebitis of the jugular vein and bacteraemia. It is a Gram-negative, anaerobic bacterium which to date has no available reference genome. Draft genomes suggest it to be a single circular chromosome of approximately 2.2Mb. A reference strain of each of the two F. necrophorum subspecies and a clinical isolate from a LS patient were sequenced on a Roche 454 GS-FLX+. Sequence data was assembled using Roche GS Assembler and the resulting contigs annotated using xBASE, Pfam and BLAST. The annotation data was mined for gene products associated with virulence revealing a leukotoxin, haemolysin, filamentous haemagglutinnin, adhesin, hemin receptor, phage genes, CRISPR-associated proteins, ecotin and a putative type V secretion system. Data will be presented on comparative genomics of the three strains, with a focus on putative virulence genes. Tools such as Artemis Comparison Tool and ClustalO were used for sequence alignments and PhyML was used to generate phylogenetic trees. Conserved motifs associated with virulence were also located. Understanding variations at the genomic level may help to explain the increased virulence of some F. necrophorum strains.

In-situ development of self-defensive antibacterial biomaterials: phenol-g-keratin-EC based bio-composites with characteristics for biomedical applications

Recently, the development of highly inspired biomaterials with multi-functional characteristics has gained considerable attention, especially in biomedical, and other health-related areas of the modern world. It is well-known that the lack of antibacterial potential has significantly limited biomaterials for many challenging applications such as infection free wound healing and/or tissue engineering etc. In this perspective, herein, a series of novel bio-composites with natural phenols as functional entities and keratin-EC as a base material were synthesised by laccase-assisted grafting. Subsequently, the resulting composites were removed from their respective casting surfaces, critically evaluated for their antibacterial and biocompatibility features and information is also given on their soil burial degradation profile. In-situ synthesised phenol-g-keratin-EC bio-composites possess strong anti-bacterial activity against Gram-positive and Gram-negative bacterial strains i.e., B. subtilis NCTC 3610, P. aeruginosa NCTC 10662, E. coli NTCT 10418 and S. aureus NCTC 6571. More specifically, 10HBA-g-keratin-EC and 20T-g-keratin-EC composites were 100% resistant to colonisation against all of the aforementioned bacterial strains, whereas, 15CA-g-keratin-EC and 15GA-g-keratin-EC showed almost negligible colonisation up to a variable extent. Moreover, at various phenolic concentrations used, the newly synthesised composites remained cytocompatible with human keratinocyte-like HaCaT, as an obvious cell ingrowth tendency was observed and indicated by the neutral red dye uptake assay. From the degradation point of view, an increase in the degradation rate was recorded during their soil burial analyses. Our investigations could encourage greater utilisation of natural materials to develop bio-composites with novel and sophisticated characteristics for potential applications.

In Vitro Assessment of the Synergy between Polymyxin B (PMB) and Polymyxin B Nonapeptide (PMBN) and Antibiotics on Biofilms from Diabetic Foot Infections

Background: The increasing resistance of Gram-negative bacteria isolated from nosocomial infections and chronic wounds, such as diabetic foot ulcers has renewed research interests in the use of polymyxins in the treatment of multidrug resistant infections. The added resistance conferred by biofilm development in such infections and the absence of novel antibiotics presuppose that polymyxins are the likely drugs of choice in spite of their nephrotoxicity. The effects of PMB and PMBN have been previously assessed on planktonic bacteria isolated from various infections. Methods: This current study assessed the synergy between a PMB/PMBN and two antibiotics (ceftazidime and levofloxacin) in an attempt to develop a strategy for biofilm disruption using the Minimum Biofilm Eradication Concentration Physiology and Genetic assay (MBEC™ P & G, Innovotech Inc, Edmonton, Alberta, Canada) according to manufacturer’s instructions. Klebsiella pneumoniae (K. pneumoniae) and Proteus mirabilis (P. mirabilis) biofilms of initial broth suspensions of 108 colony forming units per mL, cultivated on the pegs of the MBEC device were challenged with 5120 µg/mL of both ceftazidime and levofloxacin in a ten-fold dilution assay and in the presence of 100 and 500 µg/mL PMB and PMBN. Results: From table of results (Table 1), it can be deduced that both ceftazidime and levofloxacin are very effective in inhibiting biofilm development (as shown by percentage inhibition (PI)) when augmented with PMB and PMBN. This is about 100-fold increase in efficacy when compared to the antibiotics used on their own. The percentage reduction (PR) in biofilm was also increased considerably when PMB and PMBN concentrations were increased to 500 µg/mL. PMB was more effective than its less antibacterial derivative PMBN. Levofloxacin was also found to be more effective than ceftazidime when combined with both PMB and PMBN due to its enhanced cell-membrane permeability and as an anti-DNA replication uncoupling agent. Conclusion: The above results indicate that the synergy between antibiotics and cell membrane permeabilising agents may provide alternate strategies towards biofilm eradication

Development of novel antibacterial active, HaCaT biocompatible and biodegradable CA-g-P(3HB)-EC biocomposites with caffeic acid as a functional entity

We have developed novel composites by grafting caffeic acid (CA) onto the P(3HB)-EC based material and laccase from Trametes versicolor was used for grafting purposes. The resulting composites were designated as CA-g-P(3HB)-EC i.e., P(3HB)-EC (control), 5CA-g-P(3HB)-EC, 10CA-g-P(3HB)-EC, 15CA-g-P(3HB)-EC and 20CA-g-P(3HB)-EC. An FT-IR (Fourier-transform infrared spectroscopy) was used to examine the functional and elemental groups of the control and laccase-assisted graft composites. Evidently, 15CA-g-P(3HB)-EC composite exhibited resilient antibacterial activity against Gram-positive and Gram-negative bacterial strains, respectively. Moreover, a significant level of biocompatibility and biodegradability of the CA-g-P(3HB)-EC composites was also achieved with the human keratinocytes-like HaCaT cells and soil burial evaluation, respectively. In conclusion, the newly developed novel composites with multi characteristics could well represent the new wave of biomaterials for medical applications, and more specifically have promising future in the infection free would dressings, burn and/or skin regeneration field due to their sophisticated characteristics.

Multivariate Moments Expansion Density: Application of the Dynamic Equicorrelation Model

In this study, we propose a new semi-nonparametric (SNP) density model for describing the density of portfolio returns. This distribution, which we refer to as the multivariate moments expansion (MME), admits any non-Gaussian (multivariate) distribution as its basis because it is specified directly in terms of the basis density’s moments. To obtain the expansion of the Gaussian density, the MME is a reformulation of the multivariate Gram-Charlier (MGC), but the MME is much simpler and tractable than the MGC when positive transformations are used to produce well-defined densities. As an empirical application, we extend the dynamic conditional equicorrelation (DECO) model to an SNP framework using the MME. The resulting model is parameterized in a feasible manner to admit two-stage consistent estimation and it represents the DECO as well as the salient non-Gaussian features of portfolio return distributions. The in- and out-of-sample performance of a MME-DECO model of a portfolio of 10 assets demonstrate that it can be a useful tool for risk management purposes.