Studies on the regulation of solvent production and endospore formation in Clostridium Acetobutylicum P262

The aim of this study was to characterise the relationships between solventogenesis and endospore formation in Clostridium acetobutylicum strain P262. Growth and endospore formation was monitored in a number of complex and minimal media and as a result of these studies a new defined sporulation medium was developed. The defined system produced high levels of solvents and supported 60 80% sporulation in C. acetobutylicum P262. Endospore formation occurred near-synchronously, enabling this system to be used in correlative physiological and morphological studies. Five other Type Culture Clostridium strains grew and sporulated less well in the C. acetobutylicum minimal medium (CAMM). These variations emphasise the importance of strain differences amongst the Clostridia. Two well defined physiological phases, the acidogenic phase and the solventogenic phase, which characterise the industrial ABE fermentation process were observed in CAMM

Secondary metabolites produced by Staphylococcus species and their antimicrobial activities against ESKAPE group of bacteria

Abstract: Background: ESKAPE-related infections pose a serious challenge to the health sector as they are known to be resistant to the current antibiotics. This led to the investigation of secondary metabolites from Staphylococcus species [(S. saprophyticus (ATCC 35552), S. epidermidis (ATCC 51625) and S. aureus (isolated from milk)] as a possible solution to alleviate ESKAPE-related conditions. Methods: Secondary metabolites were screened by Gas chromatography coupled with mass spectrophotometry (GC/MS/MS), then purified using silica gel and thin layer chromatography plates. The normal distribution for prominent secondary was achieved by using the Kruskal Wallis Test. Compounds were then identified using nuclear magnetic resonance and such were tested for antimicrobial properties against ESKAPE bacteria by autobiography and microtiter plates respectively. The cytotoxicity studies were carried out against Vero cells using 3-(4,5-dimethyl-2-thiazole)-2,5 diphenyl-2- H bromide tetrazolium (MTT) assay. The Spearman`s correlation analysis method was used to evaluate if values were statistically significant. Results: Different classes of compounds were identified, among others were hetrocyclic compounds, amino acid and alcohols. The data analysis for prominent compounds revealed the percentage peak area average was not normally distributed. To achieve normal distribution for prominent secondary, Kruskal Wallis Test was carried to reach the value that was statistically significant (p= 0.396). The identified compounds include: polycyclic aromatic hydrocarbon (Fluoranthene) and (heterocyclic compound) 3-methyl-2-phenyl- 1H-pyrrole from S. aureus. Two compounds from S. epidermidis were: amide (oleamide) and fatty acid (ester methyl palmitate), and four from S...D.Tech. (Biomedical Technology

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Prophylaxis of disease caused by bacterial pathogens of man

This thesis reports research undertaken which will lead to improved pretreatments and therapies for disease caused by Clostridium perfringens, Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei. C. perfringens is thought to be the most widely distributed bacterial pathogen and is the most important Clostridial species associated with enteric disease in domesticated animals. During warfare C. perfringens has been a significant causes of mortality. Between 1 and 10% of wounded personnel developed gas gangrene during the 1st and 2nd world wars. The ability of the bacterium to cause a range of diseases is due largely to the differential production of toxins. The first reported cloning and nucleotide sequencing of three of the four major toxins (α, β and ε-toxins) is documented in this thesis. The regulation of expression of α-toxin in C. perfringens has been investigated and methods for the expression of recombinant proteins in E. coli have been devised This information has been used to develop improved PCR-based diagnostic tests, and to investigate structure-function relationships. A high resolution crystal structure of a-toxin (phospholipase C) is reported. Using molecular and biophysical techniques, the functions of the two domains of the protein have been determined. Residues that play roles in the interaction of the toxin with host cell membranes have been identified using site-directed mutagenesis. This work has also provided a major insight into the structures and functions of related phospholipases C (the zincmetallophospholipases C) from other bacterial pathogens. This pioneering work with α-toxin is recognised by invitations to write reviews and book chapters on this subject and on bacterial phospholipases C. C. perfringens β-toxin has been shown to be related to pore forming toxins such as Staphylococcus aureus α-toxin. This finding suggests, for the first time, the mode of action of β-toxin. The interaction of C. perfringens ε-toxin with host cells has been investigated and progress made in identifying the cell-surface receptor for the toxin. Genetically engineered toxoids have been devised which induce high-level protection against α and ε-toxins. These vaccines are currently being developed by industry for veterinary use. Similar approaches have been used to devise a recombinant vaccine against Clostridium botulinum toxin F. The wider applications of toxins as therapeutics have also been investigated, and a novel cancer drug delivery system based on targeted lysis of drug-containing liposomes by α-toxin has been devised and patented. F. tularensis is the etiological agent of tularemia, a disease of man that is found in most countries in the Northern hemisphere and most frequently in Scandinavia, N. America, Japan and N. Russia. In this thesis the efficacy of antibiotics for the prevention and treatment of experimental tularemia is documented. Two surface antigens (lipopolysaccharide and FopA) have been evaluated as sub-unit vaccines. Of these, lipopolysaccharide shows potential as a protective antigen. However, because of the paucity of information available on this bacterium, a wider approach to vaccine development, involving the determination of the genome sequence of a fully virulent strain of F. tularensis has been undertaken. A preliminary analysis of the genome sequence is reported here, which has allowed the identification of targets for the development of a rationally attenuated mutant for use as a live vaccine. Y. pestis is generally recognised to have caused three major pandemics of disease, and credible estimates indicate that together these resulted in 200 million deaths. WHO figures indicate that there is a continuing public health problem from plague, especially in Africa, Asia and South America. In this thesis existing vaccines and antibiotics have been evaluated for the prevention and treatment of plague and found to have limitations. A number of approaches to the development of an improved vaccine have been investigated including rationally attenuated strains of the bacterium and isolated surface antigens. A sub-unit vaccine against plague has been devised based on recombinant forms of the F1- and V-antigens. This vaccine provides high level protection against both bubonic and pneumonic plague. This recombinant sub-unit vaccine has been patented and is currently in phase I clinical trials in man. This vaccine has been formulated for single oral or intranasal delivery, using microencapsulated or Salmonella-based delivery systems. Methods for enhancing the stability and efficacy of these vaccines have been investigated. Reviews on plague and plague vaccines have been written, confirming the status of the author as a world leader in this field. The work to devise an improved vaccine has also provided insight into the molecular basis of pathogencity of Y. pestis. A phoP / phoQ regulatory system has been discovered in the bacterium, which plays a key role in survival of the bacterium within macrophages. The V-antigen has been shown to be surface located to play a key role in the translocation of effector proteins into host cells. The biogenesis of the F1-capsular antigen has been investigated at a genetic and biophysical level. In order to underpin future work with this pathogen, the genome sequence is currently being determined. This work has already provided major new insights into the evolution of this pathogen. B. pseudomallei (formerly Pseudomonas pseudomallei) is found primarily in S. E. Asia, N. Australia and other tropical areas of the world. Melioidosis has recently appeared in temperate zones, including mainland France and the UK possible as a consequence of increased international travel. Acute disease can be treated with antibiotics but the bacterium can persist in the host and subsequent disease episodes can occur. In this thesis ciprofloxacin and doxycyline have been are evaluated and shown to have significant limitations for the treatment of melioidodis. In the longer term there is a requirement for an effective vaccine against melioidosis, and work is reported here to devise the genetic tools which will be necessary for the genetic manipulation of the bacterium, with a view towards the identification of virulence determinants