A thesis submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg. in fulfilment of the requirements of Doctor of Philosophy / Haemophilus influenzae and Haemophilus parainfluenzae are obligate human parasites that form part of the flora of the mucous membranes. They are normally present in the mouth and upper respiratory tract of healthy individuals. H. influenzae is known as a major pathogen in children while H. parainfluenzae is an opportunistic pathogen which is also found in the urogenital tract. H. ducreyi, however; has only been isolated from genital ulcers in patients suffering from chancroid. The various species of the genus Haemophilus are characterised by their requirements for two growth factors, X factor or haemin and V factor or nicotinamide adenine dinucleotide (NAD). According to present taxonomic criteria and laboratory practice, isolates belonging to the genus Haemophilus that grow in the absence of NAD cannot be identified as H. influenzae or H. poraintluenzae. However, in 1989 four unusual clinical strains of H. parainfluenzae were isolated which were found to be V factor-independent. (Abbreviation abstract) / Andrew Chakane 2019

A research report submitted to the School of Public Health, Faculty of Health Sciences, University of the Witwatersrand, in partial fulfilment of the requirements of the degree of Masters in Science in Epidemiology and Biostatistics Johannesburg, April 2013 / Haemophilus influenzae type b disease (Hib) was a major cause of childhood disease but has nearly been eliminated in developed countries after the introduction of Hib conjugate vaccine. An increase in non-typeable H. influenzae (NTHi) disease was observed in some countries post-Hib vaccination which prompted concerns about possible serotype replacement. Hib disease in children <5 years of age decreased by a minimum estimate of 71% in South Africa after the Hib vaccine introduction in 1999. The epidemiology of Hib disease among persons >5 year olds and NTHi disease in all ages has not been described in South Africa before. The aim of this study is therefore to describe the trends and factors associated with invasive Hib and NTHi disease in South Africa, 2003-2009 among persons of all ages.

Thesis (M.S.)--University of Missouri, School of Mines and Metallurgy, 1921. / The entire thesis text is included in file. Typescript. Illustrated by author. Title from title screen of thesis/dissertation PDF file (viewed May 29, 2009) Includes bibliographical references (p. 25).

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 161-166). Also available on the Internet.

Thesis (M.S.P.H.)--University of Michigan, 1943.

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.

Thesis (M.S.P.H.)--University of Michigan, 1943.

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.

Made available in DSpace on 2012-05-07T15:02:16Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 000004.pdf: 872019 bytes, checksum: b8e894b7b58911018e4a590c0266cbe1 (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / A bactéria Haemophilus da espécie influenzae, pode causar no homem diversas infecções, invasivas ou não. O H. influenzae do tipo b (Hib), antes da introdução da vacina conjugada contra Hib, associava-se a infecções como epiglotite, artrite séptica, bacteriemia, pneumonia septicemia e meningite, principalmente em crianças. As outras espécies tipáveis (a,c,d,e,f) e não tipáveis estavam associadas a infecções do trato respiratório adquiridas na comunidade. Após a introdução dessa vacina, houve redução expressiva das doenças causadas pelo Hib em diversas partes do mundo, inclusive no Brasil. Atualmente, outros tipos da espécie H. influenzae (Hi) que não o Hib estão sendo isoladas não somente em crianças, causando infecções graves. O ressurgimento de casos por Hib, em crianças vacinadas, tem sido reportado como possível falha vacinal. Esses fatos preocupam a Saúde Pública no mundo, demonstrando que mudanças epidemiológicas podem estar ocorrendo devido à seleção de cepas após a introdução da vacina. Neste estudo, mudanças genéticas na região capsular do Hi foram encontradas em cepas isoladas nos períodos pré e pós-vacinal de três estados brasileiros. Através do estudo de susceptibilidade foram encontradas cepas produtoras de β-lactamase (BLA+) e não produtoras resistentes a ampicilina (BLNAR). A análise de redução da susceptibilidade às quinolonas foi avaliada utilizando-se o ácido nalidíxico como marcador, confrontando-se a resistência deste antibiótico com os marcadores moleculares das regiões determinantes de resistência a quinolonas (QRDRs) através do sequenciamento dos genes gyrA e parC que codificam a síntese das proteínas GyrA e ParC. Os resultados obtidos demonstraram que o ácido nalidíxico é o antibiótico mais indicado desse grupo para detectar a redução da sensibilidade. / The bacteria specie Haemophilus influenzae can cause several infections on men, either invasive or not. Before the introduction of conjugated vaccine against type b H. influenzae (Hib), it was associated to infections like epiglottitis, septic arthritis, bacteremia, pneumonia, septicemia and meningitis, occurring mostly in children. The other typeable species (a,c,d,e,f) and not typeable (NT) were associated to infections of respiratory trait acquired in the community. After introducing this vaccine, expressive reduction of diseases caused by Hib has been observed worldwide, including Brazil. Recently, different types of H. influenzae species other than Hib have been isolated in children and adults, causing severe infections. The reemergence of Hib cases in vaccinated children has been reported as a possible vaccine failure. These facts are a concern to public health departments around the world showing that epidemiologic changes may happen due to the selection of strains after vaccine introduction. In this study, genetic changes in the capsular region of Hi were found in isolated strains in the pre and post vaccinal periods in three Brazilian states. Through susceptibility studies, β-lactamase producer strains were found as well as non producer resistant to ampicilin (BLNAR). Analysis of susceptibility reduction to quilonones was evaluated by using nalidixic acid as a marker, and comparing the resistance to this antibiotic to molecular markers of the determining regions of resistance to quilonones (QRDRs) through sequencing of gyrA and parC genes that codify the synthesis of GyrA and ParC proteins. The results show that nalidixic acid is the most suitable antibiotic of this group able to detect sensibility reduction to quinolones. This study shows that there is a need for routine and research laboratories to evaluate and develop simple and quick methods to indicate possible capsule changes and emergence of resistant strains, to help epidemiological and sanitary surveillance.

Haemophilus influenzae and related species have a unique requirement for externally-provided NAD; therefore, several pyridine nucleotide-requiring enzymes become important for the survival of these pathogens. Haemophilus influenzae ATP:NMN adenylyltransferase was partially purified 15-fold with a 27% yield using dye affinity chromatography. Affinity chromatography was also used to purify NAD kinase from Haemophilus influenzae, 18-fold with a 32% yield. Substrate specificity studies of these enzymes demonstrated the enzymes to function with 3-acetylpyridine analogs of their respective substrates. A membrane-bound NMN glycohydrolase was demonstrated in Haemophilus influenzae. The enzyme functions with 3-acetylpyridine mononucleotide as a substrate, and is inhibited effectively by 3-aminopyridine mononucleotide. The possible involvement of this enzyme in the transport of NMN into the cytoplasm is discussed Growth inhibition studies demonstrated that 3-aminopyridine mononucleotide is a potent inhibitor of growth of the organism and could inhibit growth by inhibiting the transport of NMN. The previously reported inhibition of growth by the 3-aminopyridine adenine dinucleotide was attributed to the formation of the mononucleotide through the reaction catalyzed by the Haemophilus influenzae periplasmic nucleotide pyrophosphatase. A cytosolic lactate dehydrogenase, specific for D(-)-lactate was purified to electrophoretic homogeneity 2100-fold with a 14% yield. The purified enzyme was demonstrated to be a tetramer of M, = 135,000. It catalyzes essentially the reduction of pyruvate with very low activity observed for the oxidation of D(-)-lactate. An optimum pH of 7.2 was determined for the reduction of pyruvate with NADH as the coenzyme. Several NADH analogs, altered either in the pyridine or purine moiety, functioned as coenzymes. Coenzyme-competitive inhibition by adenosine derivatives demonstrated important interactions of the pyrophosphate region of the coenzyme in binding with the enzyme. Several structural analogs of NADH and pyruvate were evaluated as selective inhibitors of the enzyme. Chemical modification of the purified D-lactate dehydrogenase was effectively achieved by micromolar concentrations of several N-alkylmaleimides. Positive chain length effects in the inactivation by maleimides indicated the presence of a hydrophobic region close to the sulfhydryl groups being modified. The product of the reaction catalyzed by D-lactate dehydrogenase, D(-)-lactate, provides the substrate for a membrane-bound D-lactate oxidase. The D-lactate oxidase converts D(-)-lactate back to pyruvate and transfers electrons to the respiratory chain. No cytosolic L(+)-lactate dehydrogenase was found in Haemophilus influenzae; however, the organism possesses an L-lactate oxidase associated with the cell membrane. The L-lactate oxidase is also part of the respiratory chain, and utilizes exogenous L(+)-lactate to give pyruvate for the organism to use as a carbon source. Haemophilus influenzae and related species have a unique requirement for externally-provided NAD; therefore, several pyridine nucleotide-requiring enzymes become important for the survival of these pathogens. Haemophilus influenzae ATP:NMN adenylyltransferase was partially purified 15-fold with a 27% yield using dye affinity chromatography. Affinity chromatography was also used to purify NAD kinase from Haemophilus influenzae, 18-fold with a 32% yield. Substrate specificity studies of these enzymes demonstrated the enzymes to function with 3-acetylpyridine analogs of their respective substrates. A membrane-bound NMN glycohydrolase was demonstrated in Haemophilus influenzae. The enzyme functions with 3-acetylpyridine mononucleotide as a substrate, and is inhibited effectively by 3-aminopyridine mononucleotide. The possible involvement of this enzyme in the transport of NMN into the cytoplasm is discussed. Growth inhibition studies demonstrated that 3-aminopyridine mononucleotide is a potent inhibitor of growth of the organism and could inhibit growth by inhibiting the transport of NMN. The previously reported inhibition of growth by the 3-aminopyridine adenine dinucleotide was attributed to the formation of the mononucleotide through the reaction catalyzed by the Haemophilus influenzae periplasmic nucleotide pyrophosphatase. A cytosolic lactate dehydrogenase, specific for D(-)-lactate was purified to electrophoretic homogeneity 2100-fold with a 14% yield. The purified enzyme was demonstrated to be a tetramer of M, = 135,000. It catalyzes essentially the reduction of pyruvate with very low activity observed for the oxidation of D(-)-lactate. An optimum pH of 7.2 was determined for the reduction of pyruvate with NADH as the coenzyme. Several NADH analogs, altered either in the pyridine or purine moiety, functioned as coenzymes. Coenzyme-competitive inhibition by adenosine derivatives demonstrated important interactions of the pyrophosphate region of the coenzyme in binding with the enzyme. Several structural analogs of NADH and pyruvate were evaluated as selective inhibitors of the enzyme. Chemical modification of the purified D-lactate dehydrogenase was effectively achieved by micromolar concentrations of several N-alkylmaleimides. Positive chain length effects in the inactivation by maleimides indicated the presence of a hydrophobic region close to the sulfhydryl groups being modified. The product of the reaction catalyzed by D-lactate dehydrogenase, D(-)-lactate, provides the substrate for a membrane-bound D-lactate oxidase. The D-lactate oxidase converts D(-)-lactate back to pyruvate and transfers electrons to the respiratory chain. No cytosolic L(+)-lactate dehydrogenase was found in Haemophilus influenzae; however, the organism possesses an L-lactate oxidase associated with the cell membrane. The L-lactate oxidase is also part of the respiratory chain, and utilizes exogenous L(+)-lactate to give pyruvate for the organism to use as a carbon source. / Ph. D.