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1.
Anaerobe ; 71: 102422, 2021 Oct.
Article in English | MEDLINE | ID: mdl-34343672

ABSTRACT

Clostridioides difficile R20291 is the most studied PCR-Ribotype 027 isolate. The two predominant lineages of this hypervirulent strain, however, exhibit substantive phenotypic differences and possess genomes that differ by a small number of nucleotide changes. It is important that the source of R20291 is taken into account in research outcomes.


Subject(s)
Clostridioides/genetics , Clostridium Infections/microbiology , Polymorphism, Single Nucleotide , Clostridioides/classification , Clostridioides/isolation & purification , Clostridioides difficile/classification , Clostridioides difficile/genetics , Clostridioides difficile/isolation & purification , Genome, Bacterial , Humans , Phylogeny
2.
PLoS One ; 8(2): e56051, 2013.
Article in English | MEDLINE | ID: mdl-23405251

ABSTRACT

Sophisticated genetic tools to modify essential biological processes at the molecular level are pivotal in elucidating the molecular pathogenesis of Clostridium difficile, a major cause of healthcare associated disease. Here we have developed an efficient procedure for making precise alterations to the C. difficile genome by pyrE-based allelic exchange. The robustness and reliability of the method was demonstrated through the creation of in-frame deletions in three genes (spo0A, cwp84, and mtlD) in the non-epidemic strain 630Δerm and two genes (spo0A and cwp84) in the epidemic PCR Ribotype 027 strain, R20291. The system is reliant on the initial creation of a pyrE deletion mutant, using Allele Coupled Exchange (ACE), that is auxotrophic for uracil and resistant to fluoroorotic acid (FOA). This enables the subsequent modification of target genes by allelic exchange using a heterologous pyrE allele from Clostridium sporogenes as a counter-/negative-selection marker in the presence of FOA. Following modification of the target gene, the strain created is rapidly returned to uracil prototrophy using ACE, allowing mutant phenotypes to be characterised in a PyrE proficient background. Crucially, wild-type copies of the inactivated gene may be introduced into the genome using ACE concomitant with correction of the pyrE allele. This allows complementation studies to be undertaken at an appropriate gene dosage, as opposed to the use of multicopy autonomous plasmids. The rapidity of the 'correction' method (5-7 days) makes pyrE(-) strains attractive hosts for mutagenesis studies.


Subject(s)
Bacterial Proteins/genetics , Clostridioides difficile/genetics , Genetic Vectors/genetics , Genome, Bacterial/genetics , Alleles , Clostridioides difficile/growth & development , DNA, Bacterial/genetics , Genetic Complementation Test , Phenotype , Sequence Deletion
3.
Biomaterials ; 31(10): 2806-15, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20045554

ABSTRACT

Poly(3-hydroxybutyrate) (P(3HB)) foams exhibiting highly interconnected porosity (85% porosity) were prepared using a unique combination of solvent casting and particulate leaching techniques by employing commercially available sugar cubes as porogen. Bioactive glass (BG) particles of 45S5 Bioglass grade were introduced in the scaffold microstructure, both in micrometer ((m-BG), <5 microm) and nanometer ((n-BG), 30 nm) sizes. The in vitro bioactivity of the P(3HB)/BG foams was confirmed within 10 days of immersion in simulated body fluid and the foams showed high level of protein adsorption. The foams interconnected porous microstructure proved to be suitable for MG-63 osteoblast cell attachment and proliferation. The foams implanted in rats as subcutaneous implants resulted in a non-toxic and foreign body response after one week of implantation. In addition to showing bioactivity and biocompatibility, the P(3HB)/BG composite foams also exhibited bactericidal properties, which was tested on the growth of Staphylococcus aureus. An attempt was made at developing multifunctional scaffolds by incorporating, in addition to BG, selected concentrations of Vitamin E or/and carbon nanotubes. P(3HB) scaffolds with multifunctionalities (viz. bactericidal, bioactive, electrically conductive, antioxidative behaviour) were thus produced, which paves the way for next generation of advanced scaffolds for bone tissue engineering.


Subject(s)
Hydroxybutyrates/pharmacology , Polyesters/pharmacology , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Adsorption/drug effects , Animals , Bacterial Adhesion/drug effects , Biocompatible Materials/pharmacology , Cell Line , Cell Proliferation/drug effects , Ceramics/pharmacology , Colony Count, Microbial , Culture Media , Humans , Hydrogen-Ion Concentration/drug effects , Implants, Experimental , Microbial Sensitivity Tests , Microscopy, Electron, Scanning , Nanotubes, Carbon , Porosity/drug effects , Rats , Rats, Sprague-Dawley , Staphylococcus aureus/cytology , Staphylococcus aureus/drug effects , Staphylococcus aureus/growth & development , Staphylococcus aureus/ultrastructure , Thermogravimetry , Vitamin E/pharmacology
4.
Biomacromolecules ; 10(4): 691-9, 2009 Apr 13.
Article in English | MEDLINE | ID: mdl-19296657

ABSTRACT

P(3HB), is one of the most well studied polyhydroxyalkanoates. It is biodegradable, biocompatible, exhibits thermoplastic properties and can be produced from renewable carbon sources. The commercial exploitation of P(3HB) has been mainly held back by its high production costs. Hence, a lot of research is required to optimize P(3HB) fermentation conditions. In this study we have focused on the effects of impeller speed and pH on P(3HB) production in Bacillus cereus SPV. Four different impeller speeds, 50, 125, 250, and 500 rpm, were used. The highest amount of P(3HB) accumulation was achieved using 125 rpm impeller speed (34% dcw) and this was attributed to optimal cell growth rate. Also, pH-stat fermentations were carried out at pH 3.0, 6.8, and 10. This study confirmed that lack of P(3HB) degradation during unbuffered Bacillus fermentations is due to the low pH conditions. This observation is crucial for the industrial exploitation of the genus Bacillus for the production of P(3HB).


Subject(s)
Bacillus cereus/metabolism , Biomechanical Phenomena , Fermentation , Hydroxybutyrates/metabolism , Polyesters/metabolism , Bacillus cereus/genetics , Culture Media , Hydrogen-Ion Concentration , Hydroxybutyrates/chemistry , Magnetic Resonance Spectroscopy , Polyesters/chemistry
5.
J R Soc Interface ; 6(33): 401-9, 2009 Apr 06.
Article in English | MEDLINE | ID: mdl-18765336

ABSTRACT

This study investigated the possibility of incorporating alpha-tocopherol (vitamin E) into poly(3hydroxybutyrate) (P(3HB))/Bioglass composites, which are being developed for bone tissue engineering matrices. P(3HB) films with 20 wt% Bioglass and 10 wt% vitamin E were prepared using the solvent casting technique. Addition of vitamin E significantly improved the hydrophilicity of the composites along with increasing the total protein adsorption. The presence of protein adsorbed on the composite surface was further confirmed using X-ray photoelectron spectroscopy analysis. Preliminary cell culture studies using MG-63 human osteoblasts showed that the addition of vitamin E in the P(3HB)/20 wt% Bioglass films significantly increased cell proliferation. The results achieved in this study confirmed the possibility of incorporating vitamin E as a suitable additive in P(3HB)/Bioglass composites to engineer the surface of the composites by promoting higher protein adsorption and increasing the hydrophilicity.


Subject(s)
Antioxidants/chemistry , Ceramics/chemistry , Hydroxybutyrates/chemistry , Polyesters/chemistry , Vitamin E/chemistry , Adsorption , Animals , Antioxidants/pharmacology , Blood Proteins/chemistry , Cattle , Cell Adhesion/drug effects , Cell Growth Processes/drug effects , Cell Line, Tumor , Ceramics/pharmacology , Humans , Hydroxybutyrates/pharmacology , Microscopy, Electron, Scanning , Osteoblasts/cytology , Osteoblasts/drug effects , Polyesters/pharmacology , Surface Properties , Tissue Engineering/methods , Vitamin E/pharmacology
6.
Biomaterials ; 29(12): 1750-61, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18255139

ABSTRACT

This study compares the effects of introducing micro (m-BG) and nanoscale (n-BG) bioactive glass particles on the various properties (thermal, mechanical and microstructural) of poly(3hydroxybutyrate) (P(3HB))/bioactive glass composite systems. P(3HB)/bioactive glass composite films with three different concentrations of m-BG and n-BG (10, 20 and 30 wt%, respectively) were prepared by a solvent casting technique. The addition of n-BG particles had a significant stiffening effect on the composites, modulus when compared with m-BG. However, there were no significant differences in the thermal properties of the composites due to the addition of n-BG and m-BG particles. The systematic addition of n-BG particles induced a nanostructured topography on the surface of the composites, which was not visible by SEM in m-BG composites. This surface effect induced by n-BG particles considerably improved the total protein adsorption on the n-BG composites compared to the unfilled polymer and the m-BG composites. A short term in vitro degradation (30 days) study in simulated body fluid (SBF) showed a high level of bioactivity as well as higher water absorption for the P(3HB)/n-BG composites. Furthermore, a cell proliferation study using MG-63 cells demonstrated the good biocompatibility of both types of P(3HB)/bioactive glass composite systems. The results of this investigation confirm that the addition of nanosized bioactive glass particles had a more significant effect on the mechanical and structural properties of a composite system in comparison with microparticles, as well as enhancing protein adsorption, two desirable effects for the application of the composites in tissue engineering.


Subject(s)
Glass/chemistry , Hydroxybutyrates/chemistry , Hydroxybutyrates/pharmacology , Nanostructures/administration & dosage , Nanostructures/chemistry , Osteoblasts/drug effects , Polyesters/chemistry , Polyesters/pharmacology , Body Fluids/chemistry , Cell Line , Ceramics , Compomers/chemistry , Compomers/pharmacology , Humans , Manufactured Materials , Materials Testing , Microspheres , Nanostructures/ultrastructure , Osteoblasts/cytology , Particle Size , Surface Properties
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