Thesis (Ph.D.)--Virginia Commonwealth University, 2010. / Prepared for: Dept. of Clinical Laboratory Sciences. Title from title-page of electronic thesis. Bibliography: leaves 127-148.

A dissertation submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Medicine. / The emergence of strains of S. pneumoniae resistant to penicillin and to other antibiotics, and the spread of that resistance over the world, have become major concerns and increase the need for epidemiological surveillance. The following typing methods have been used to detect strain variability in pneumococci: Serotyping, antibiotic susceptibility profiles, multilocus enzyme electrophoresis (MLEE), penicillin-binding protein (PBP) profiles, pulse-field gel electrophoresis (pFGE), and ribotyping. Serotyping, antibiograms, and MLEE only detect phenotypic variability. PBP gene profiles, PFGE, and ribotyping detect genotypic differences but these techniques are labour intensive and time consuming. Random amplified polymorphic DNA (RAPD) is a new technique that bas proved useful for typing bacteria, fungi, and parasites, but has not been. studied using pneumococci. Unlike conventional polymerase chain reaction (peR), RAPD utilizes single, short primers, usually 10 oligonucleotides in length. As the primer is short and low astringency annealing temperatures are used, there will be many complimentary sites scattered randomly throughout a bacterium's genome, When such sites occur a few hundred base pairs away from each other and on opposite DNA strands, the enclosed region can be amplified by peR This results in numerous discrete target fragments which can be separated by agarose-gel electrophoresis and ethidium bromide staining. RAPD requires no sequence information and it scans the whole genome rather than relying on hypervariability within one specific gene. The aims of this study were: to determine strain variability using RAPD, to determine the reproducibility ofRAPD, and to demonstrate intercontinental spread of a multiresistant pneumococcal strain. The following strains were evaluated: a) 10 strains from a day-care centre (DCC), the index case being a 3 year old girl 'with otitis media. An aunt from Spain had recently been staying with the family. The other strains were isolated from class mates and siblings of the index case.; b) 18 clinical isolates from Seoul, Korea; and c) 11 epidemiologically unrelated isolates from South Africa, including the reference strain, R6. Two DNA extraction methods were used. The first involving lysis with sodmm-dodecyl-sulphaze and proteinase K. Proteins were removed with phenol-chloroform, and the DNA precipitated with ethanol. The second method involved incubating the cells at 95 0C for 15 microlitres, followed by centrifugation. 2 microlitres of the supernatant was then used for each PCR reaction, Three primers were evaluated. After 01uimisation of the RAPDreaction for pneumococci, the final peR mixtures per 50 ul was: primer (4 plY1), template (0.5 ng), nuc1eotides (300 pMeach), magnesium (4 mM~, and Taq polymerase (2 U). 35 cycles were used with an annealing temperature of 35'C. Both DNA extraction methods: gave reproducible results but were not comparable to each other. All 10 strains from the DCC gave the same banding pattern as the Spanish done for all 3 primers. 7 of the Korean strains gave the same banding pattern as the Spanish clone using the first two primers, however one strain showed an additional band using the third primer. Of the remaining 22 strains, 21 different banding patterns were obtained. This study has shown that RAPD is a simple and rapid technique that can distinguish strain variation among pneumococci. The reproducibility is excellent within the same laboratory. Finally using RAPD. this study identified a Spanish multiresistant 23F clone in South Africa and Korea. / Andrew Chakane 2018

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references. Also available online.

Qi Wei. / "November 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 158-185). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Thesis (M.S.)--University of Alabama at Birmingham, 2006. / Title from first page of PDF file (viewed Feb. 19, 2009). Includes bibliographical references (p. 35-44).

Lic.-avh. (sammanfattning) Stockholm : Karolinska institutet, 2009.

Objetivo: Analisar através de metodologia molecular a microbiota fecal de recém-nascidos (RN) saudáveis, em aleitamento materno exclusivo. Materiais e métodos: Amostras fecais de dez RN foram avaliadas no 2º, 7º e 30º dias de vida (DV), através de sequenciamento do 16S rDNA bacteriano. Real-time PCR para bifidobacterias foi empregado nas amostras de 30 dias. Resultados: A diversidade bacteriana fecal aumentou do 2º para o 30º DV. E. coli predominou no 2º e 7º DV, e Clostridium no 30º DV. Usando real-time PCR, bifidobacterias foram identificadas em todas as amostras de 30 dias. Conclusão: Enterobacterias predominaram na primeira semana de vida. Aos 30 DV observou-se uma maior diversidade bacteriana, com predomínio de Clostridium.. A técnica inicial não permitiu identificar bifidobacterias. / Purpose: To evaluate by molecular methodology the fecal microbiota of healthy newborns, exclusively breastfed. Materials and methods: Fecal samples from ten neonates were analyzed on 2nd, 7th and 30th day of life, using 16S rDNA sequencing and real-time PCR for bifidobacteria. Results: The fecal bacteria diversity increased from the second to the 30th day of life. E. coli was predominant in the fecal samples from the 2nd and 7th day of life, and Clostridium.in the samples of the 30th day. Using real-time PCR bifidobacteria were identified in all 30th day samples. Conclusion: Enterobacteria were predominant in the first week of life. On 30th day of life a greater bacterial diversity was observed with predominance of Clostridium. The initial technique didnt allow the identification of bifidobacteria.

Objetivo: Analisar através de metodologia molecular a microbiota fecal de recém-nascidos (RN) saudáveis, em aleitamento materno exclusivo. Materiais e métodos: Amostras fecais de dez RN foram avaliadas no 2º, 7º e 30º dias de vida (DV), através de sequenciamento do 16S rDNA bacteriano. Real-time PCR para bifidobacterias foi empregado nas amostras de 30 dias. Resultados: A diversidade bacteriana fecal aumentou do 2º para o 30º DV. E. coli predominou no 2º e 7º DV, e Clostridium no 30º DV. Usando real-time PCR, bifidobacterias foram identificadas em todas as amostras de 30 dias. Conclusão: Enterobacterias predominaram na primeira semana de vida. Aos 30 DV observou-se uma maior diversidade bacteriana, com predomínio de Clostridium.. A técnica inicial não permitiu identificar bifidobacterias. / Purpose: To evaluate by molecular methodology the fecal microbiota of healthy newborns, exclusively breastfed. Materials and methods: Fecal samples from ten neonates were analyzed on 2nd, 7th and 30th day of life, using 16S rDNA sequencing and real-time PCR for bifidobacteria. Results: The fecal bacteria diversity increased from the second to the 30th day of life. E. coli was predominant in the fecal samples from the 2nd and 7th day of life, and Clostridium.in the samples of the 30th day. Using real-time PCR bifidobacteria were identified in all 30th day samples. Conclusion: Enterobacteria were predominant in the first week of life. On 30th day of life a greater bacterial diversity was observed with predominance of Clostridium. The initial technique didnt allow the identification of bifidobacteria.

In under a century, melioidosis, the infection due to Burkholderia pseudomallei, has emerged from Whitmore’s series of glanders-like infections amongst the morphia addicts in Burma to a major cause of mortality in northeastern Thailand and northern Australia. Also endemic in other parts of south-east Asia, melioidosis may have varied presentations ranging from severe, overwhelming infection to chronic, low grade disease. Observational evidence had suggested that granulocyte colony stimulating factor (G-CSF), a naturally occurring substance produced by the body in response to infection, may have been useful in reducing the high mortality associated with the more severe forms of this infection. Other observations linked the occurrence of this disease to various environmental factors, such as contamination of drinking water and the annual rainfall. This thesis explores and attempts to quantify these associations. There are three parts to this thesis. In the first part, I reviewed the epidemiology and management of patients with melioidosis. The use of G-CSF and meropenem was associated with a fall in mortality, although other factors may have at least partially contributed to this effect. In the second part, I progressed towards a clinical trial of G-CSF. There was no other evidence supporting the use of G-CSF in severe sepsis and ethical issues precluded a trial in Darwin. There was not evidence from laboratory models of G-CSF action in melioidosis to support the use of G-CSF in patients, although there remained some doubt regarding the applicability of such models to human disease. I examined clinical methods to identify patients at high risk of death from melioidosis. A simple scoring system based on clinical and laboratory parameters was developed and externally validated. However, clinical definitions of severe sepsis appeared to be better predictors of mortality. A clinical trial based on clinical definitions was commenced in Thailand. In the final part, I explored the question of whether different strains or B. pseudomallei or different environmental conditions caused different patterns of infection. There was no evidence that strain types of this bacterium determine the pattern or severity of disease, but weather conditions appeared to influence the distribution of disease in northern Australia.

The possibility of identifying single nucleotide polymorphisms (SNPs) that would be useful for rapid bacterial typing was investigated. Neisseria meningitidis was the organism chosen for modelling the approach since informative SNPs could be found amongst the sequence data available for multi-locus sequence typing (MLST) at http://www.mlst.net. The hypothesis tested was that a small number of SNPs located within the seven gene fragments sequenced for MLST provide information equivalent to MLST. Preliminary investigations revealed that a small number of SNPs could be utilised to highly discriminate sequence types (STs) of clinical interest. Laboratory procedures demonstrated that SNP fingerprinting of N. meningitidis isolates is achievable. Further tests showed that laboratory identification of a defining SNP in the genome of isolates was to be a practical method of obtaining relevant typing information. Identification of the most discriminating SNPs amongst the ever-increasing amount of MLST sequence data summoned the need for computer-based assistance. Two methods of SNP selection devised by the author of this thesis were translated into computer-based algorithms by contributing team members. Software for two computer programs was produced. The algorithms facilitate the optimal selection of SNPs useful for (1) distinguishing specific STs and (2) differentiating non-specific STs. Current input information can be obtained from the MLST database and consequently the programs can be applied to any bacterial species for which MLST data have been entered. The two algorithms for the selection of SNPs were designed to serve contrasting purposes. The first of these was to determine the ST identity of isolates from an outbreak of disease. In this case, isolates would be tested for their membership to any of the STs known to be associated with disease. It was shown that one SNP per ST could distinguish each of four hyperinvasive STs of N. meningitidis from between 92.5% and 97.5% of all other STs. With two SNPs per ST, between 96.7% and 99.0% discrimination is achieved. The SNPs were selected from MLST loci with the assistance of the first algorithm which scores SNPs according to the number of base mismatches in a sequence alignment between an allele of an ST of interest and alleles belonging to all other STs at a specified locus. The second purpose was to determine whether or not isolates from different sources belong to the same ST, regardless of their actual ST identity. It was shown that with seven SNPs, four sample STs of N. meningitidis could, on average, be discriminated from 97.1% of all other STs. The SNPs were selected with the aid of the second algorithm which scores SNPs at MLST loci for the relative frequency of each nucleotide base in a sequence alignment as a measure of the extent of their polymorphism. A third algorithm for selecting SNPs has been discussed. By altering the method of scoring SNPs, it is possible to overcome the limitations inherent in the two algorithms that were utilised for finding SNPs. In addition, the third approach caters for finding SNPs that distinguish members of a complex from non-members.