M. methulotrophus is a Gram negative obligate methylotroph depending on the presence of reduced carbon compounds containing one or more carbon atoms, but containing no carbon-carbon bonds. This organism can synthesize all its cellular constituents from methanol, trimethylamine, dimethylamine, or methylamine. Conversion of methanol and the methylated amines to cell carbon involves the ultimate oxidation to formaldehyde and ammonia. While the methanol dehydrogenase is produced constitutively, the enzymes involved in the assimilation of the methylated amines are inducible. All three enzymes of the trimethylamine pathway are always induced regardless of the methylated amine substrate. Transposon mutagenesis was used to generate mutations in M.methylotrophus and an antibiotic selection procedure used to isolate mutants defective specifically in the trimethylamine pathway. Two mutants were characterised and subjected to further study. The mutant tmd 3 was unable to utilise trimethylamine, dimethylamine or methylamine as substrates and was shown to lack trimethylamine dehydrogenase by polyacrylamide gel electrophoresis. Enzyme studies confirmed the lack of the dehydrogenase within the mutant cells. The mutant mad 1, unable to use methylamine as a substrate, was shown via enzyme studies to contain trimethylamine dehydrogenase, and dimethylamine dehydrogenase, but to lack methylamine dehydrogenase activity. Molecular cloning of wild-type M. methylotrophus DNA in a broad host-range plasmid vector was used to isolate DNA fragments that could replace the mad 1 defect. An 11kb fragment was isolated that fully restored methylamine dehydrogenase activity to mad 1 cells. A 2.5kb fragment was subcloned and shown, by Southern blotting with 32P-labelled In5 DNA as a probe, to contain the site of integration of the In5 insertion, located to within a few hundred base pairs of the end. DNA sequencing, now in progress, has generated 600 base pairs of sequence from either end of the subcloned fragment, within which several regions of interest were noted.

No description available.

No description available.

Bacterial sepsis is a frequently occurring disease in the first weeks of life, posing a significant threat particularly to those born prematurely. The incidence of sepsis is determined by laboratory surveillance, only taking into account culture positive episodes of sepsis. However, in up to 80% of neonates treated for sepsis, blood cultures fail to grow an organism. Therefore a variety of molecular techniques have been trialled to overcome these diagnostic difficulties. To describe the current incidence and causal pathogens of neonatal sepsis in Europe in this thesis, systematic literature reviews on neonatal bacterial and fungal infections were conducted. The review highlighted in particular the discrepancy between incidences of culture positive and clinical sepsis. A further literature review assessed molecular diagnostic techniques that have been employed to determine pathogens of neonatal sepsis. Based on the results obtained from the systematic reviews, a series of molecular tests, including quantitative multiplex PCRs, a 16S rDNA broad range PCR and a Candida multiplex PCR were developed. These tests were applied to two sets of samples obtained from neonates with suspected and confirmed early and late onset sepsis in Europe, collected in two separate clinical trials. Results identified a large amount of bacteria (74% in EOS and 50% in LOS), however failed to detect all cultured pathogens. A large number of samples were positive for CoNS and Enterobacteriacae in both sample sets. In particular, in EOS, S. pneumoniae was shown to be more predominant than anticipated from the literature, and in LOS Enterococci were more prevalent. Of concern is a high number of polymicrobial infections detected by PCR. Universal definitions for clinical sepsis need to be established to enable surveillance and comparison across countries. Molecular diagnostics have the potential to become an important additional tool to describe the epidemiology of neonatal sepsis.

No description available.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

An outbreak of neonatal diarrhoea was investigated in a herd of known susceptibility to infection by K88- positive Escherichia coli. The probable cause of the majority of diarrhoea cases was identified as a K88- positive E. coli strain. Diarrhoea was confined to susceptible piglets born to resistant sows. Susceptible sows protected their susceptible offspring and resistant piglets were unaffected. Potential protective mechanisms of the colostrum of susceptible sows were compared to the same properties in colostrum of resistant sows. Colostrum from susceptible sows (a) inhibited the binding of K88 antigen to intestinal epithelial cell brush borders, and (b) opsonized E. coli strain G205 and promoted in-vitro killing by porcine neutrophils significantly better than colostrum from resistant sows. Fractionation of colostrum by gel filtration and ion-exchange chromatography permitted identification of the immunoglobulin classes most efficient in both these activities. Both IgM and IgA, but not IgG, inhibited binding of K88 antigen to brush borders and opsonic activity was predominantly mediated by IgM. The importance of opsonic activity depends on the ab.ility of neutrophils to phagocytose bacteria within the intestinal lumen. In intestinal ligated loop experiments emigration of neutrophils into the lumen and phagocytosed bacteria were demonstrated by light microscopy and scanning and transmission electron microscopy. Neutrophil emigration only occurred when piglets had received maternal antibody_ No equivalent neutrophil emigration was observed in susceptible .piglets deprived of colostrum. There was also little or no neutrophil response in colostrum-fed resistant piglets probably du~ to a lower concentration of bacterial antigens close to the epithelial cell surface .or lack of specific antibody. Experiments to determine the numbers of organisms killed by neutrophils in the intestinal lumen failed to demonstrate any reduction, attributable to phagocytosis, in viable K88-positive E. coli.

No description available.

No description available.

No description available.