Thesis (M.Sc.) -- University of the North, 1982 / Refer to the document

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A library of 18 coliform bacteria strains was obtained from different sampling points in the drinking water distribution system in Lexington, KY, over a three month period in 2006. To investigate the cause of the coliform occurrence we conducted a microbial source tracking study using phenotypic (API 20E, Biolog, and Vitek) and genotypic (pulsed field gel electrophoresis (PFGE) and ribotyping) analyses to determine the degree of genetic variation among isolates. Characterization of isolates by PFGE and ribotyping showed that coliform events in the distribution system were related and a regrowth problem may exist due to biofilm formation. The ability of a persistent Enterobacter cloacae strain to adhere and form biofilm was found to depend on environmental conditions such as temperature, pipe material, soiled surface, chlorine and nutrient levels with higher temperatures and nutrient levels promoting adherence. Considerable variation in adherence and biofilm formation was observed among representative Enterobacter isolates.

Refrigeration has been found to cause cell elongation in mesophilic enteric organisms like commensal Escherichia coli and E. coli O157:H7. As elongated cells may divide into multiple daughter cells, they may result in underestimation of pathogen numbers in foods when plate counts are used. When E. coli cultures were incubated at 6°C for ≤10 days, cells grew by elongation, did not divide, and lost viability (LIVE/DEAD vitality stain) at similar rates. Substantial fractions of cells in cultures elongating at 6°C were inactivated by an abrupt shift to 37°C. Direct microscopic observation of cells transferred to 37ºC after 5 days at 6°C showed that few or no cells of normal size (≤4µm) divided, while elongated cells (>4 µm) formed multiple daughter cells. Thus the threat from mesophilic pathogens with a low infective dose may be underestimated in refrigerated foods. It was also found that E. coli O157:H7 cultures containing elongated cells prepared at 6 or 15 °C have greater potential to attach to food contact surfaces than those grown at higher temperatures. Interestingly, at 6°C cell elongation was inhibited by ≥ 100 mg/l cinnamaldehyde and ≥ 200 mg/l cinnamaldehyde was lethal. In contrast, at 37°C 200 mg/l cinnamaldehyde initially delayed multiplication of E. coli cells by causing cell elongation, but from 2 to 4 h, growth resumed and cells reverted to normal length. To understand this transient behaviour, genome-wide transcriptional analysis of E. coli O157:H7 was performed at 2 and 4 h exposure to cinnamaldehyde in conjunction with reverse phase-high performance liquid chromatography analysis for cinnamaldehyde and other cinnamic compounds. At 2 h exposure, cinnamaldehyde induced expression of many oxidative stress-related genes, reduced expression of genes involved in DNA replication, synthesis of protein, O-antigen and fimbriae. At 4 h many repressive effects of cinnamaldehyde on E. coli O157:H7 gene expression were reversed. Data indicated that by 4 h, E. coli O157:H7 was able to convert cinnamaldehyde into the less toxic cinnamic alcohol using alcohol dehydrogenase or aldehyde reductase enzymes (YqhD and DkgA). The results also showed that the antimicrobial activity of cinnamaldehyde was likely attributable to its carbonyl aldehyde group.

Refrigeration has been found to cause cell elongation in mesophilic enteric organisms like commensal Escherichia coli and E. coli O157:H7. As elongated cells may divide into multiple daughter cells, they may result in underestimation of pathogen numbers in foods when plate counts are used. When E. coli cultures were incubated at 6°C for ≤10 days, cells grew by elongation, did not divide, and lost viability (LIVE/DEAD vitality stain) at similar rates. Substantial fractions of cells in cultures elongating at 6°C were inactivated by an abrupt shift to 37°C. Direct microscopic observation of cells transferred to 37ºC after 5 days at 6°C showed that few or no cells of normal size (≤4µm) divided, while elongated cells (>4 µm) formed multiple daughter cells. Thus the threat from mesophilic pathogens with a low infective dose may be underestimated in refrigerated foods. It was also found that E. coli O157:H7 cultures containing elongated cells prepared at 6 or 15 °C have greater potential to attach to food contact surfaces than those grown at higher temperatures. Interestingly, at 6°C cell elongation was inhibited by ≥ 100 mg/l cinnamaldehyde and ≥ 200 mg/l cinnamaldehyde was lethal. In contrast, at 37°C 200 mg/l cinnamaldehyde initially delayed multiplication of E. coli cells by causing cell elongation, but from 2 to 4 h, growth resumed and cells reverted to normal length. To understand this transient behaviour, genome-wide transcriptional analysis of E. coli O157:H7 was performed at 2 and 4 h exposure to cinnamaldehyde in conjunction with reverse phase-high performance liquid chromatography analysis for cinnamaldehyde and other cinnamic compounds. At 2 h exposure, cinnamaldehyde induced expression of many oxidative stress-related genes, reduced expression of genes involved in DNA replication, synthesis of protein, O-antigen and fimbriae. At 4 h many repressive effects of cinnamaldehyde on E. coli O157:H7 gene expression were reversed. Data indicated that by 4 h, E. coli O157:H7 was able to convert cinnamaldehyde into the less toxic cinnamic alcohol using alcohol dehydrogenase or aldehyde reductase enzymes (YqhD and DkgA). The results also showed that the antimicrobial activity of cinnamaldehyde was likely attributable to its carbonyl aldehyde group.

Nesta tese estudamos os repertórios das famílias multigênicas var (domínio DBLa), rif e stevor, importantes fatores de virulência, de Plasmodium falciparum em isolados brasileiros. Mostramos que estas famílias multigênicas podem ser conservadas entre diferentes isolados no Brasil, sugerindo que o repertório de genes variantes pode ser limitado em algumas localidades. Identificamos menos polimorfismos nos genes stevor que os genes var ou rif. Constatamos que genes var podem ser conservados ao longo do tempo em isolados brasileiros. Identificamos também os domínios DBLa e DBLb de genes var associados a citoaderência a CHOICAM1 em isolados de Rondônia. Mediante ensaios de ELISA, mostramos que domínios DBLbICAM foram reconhecidos de modo heterogêneo. Por citometria de fluxo, demonstramos que o parasita aderente em ICAM1 foi reconhecido majoritariamente pelo plasma de pacientes com infecção sintomática e menos de infecção assintomática. Além disso, identificamos por PCR em tempo real os genes var transcritos no clone 3D7 aderente em diferentes linhagens de células CHO. / Herein, we studied the repertoire of multigenic families of var (DBLa domain), rif and stevor from brazilian isolates of Plasmodium falciparum. We showed that these multigenic families could be conserved among different isolates from Brazil, suggesting that the repertoire of variant genes can be limited in certain areas. We identified less polymorphism in stevor genes than in var or rif genes. We also demonstrated that var genes can be conserved over time in Brazilian isolates and identified the DBLa and DBLb domains from var genes associated with cytoadherence to CHOICAM1 in Rondonian isolates. ELISA assays showed that these DBLbICAM domains were recognized in a heterogeneous fashion. By flow cytometry, we demonstrated that ICAM1-adherent parasites were stronger recognized by plasma from symptomatic infections than from non-symptomatic infections. Finally, we profiled the var gene transcripts in 3D7 parasites adherent to different CHO cells by real time PCR.

Nesta tese estudamos os repertórios das famílias multigênicas var (domínio DBLa), rif e stevor, importantes fatores de virulência, de Plasmodium falciparum em isolados brasileiros. Mostramos que estas famílias multigênicas podem ser conservadas entre diferentes isolados no Brasil, sugerindo que o repertório de genes variantes pode ser limitado em algumas localidades. Identificamos menos polimorfismos nos genes stevor que os genes var ou rif. Constatamos que genes var podem ser conservados ao longo do tempo em isolados brasileiros. Identificamos também os domínios DBLa e DBLb de genes var associados a citoaderência a CHOICAM1 em isolados de Rondônia. Mediante ensaios de ELISA, mostramos que domínios DBLbICAM foram reconhecidos de modo heterogêneo. Por citometria de fluxo, demonstramos que o parasita aderente em ICAM1 foi reconhecido majoritariamente pelo plasma de pacientes com infecção sintomática e menos de infecção assintomática. Além disso, identificamos por PCR em tempo real os genes var transcritos no clone 3D7 aderente em diferentes linhagens de células CHO. / Herein, we studied the repertoire of multigenic families of var (DBLa domain), rif and stevor from brazilian isolates of Plasmodium falciparum. We showed that these multigenic families could be conserved among different isolates from Brazil, suggesting that the repertoire of variant genes can be limited in certain areas. We identified less polymorphism in stevor genes than in var or rif genes. We also demonstrated that var genes can be conserved over time in Brazilian isolates and identified the DBLa and DBLb domains from var genes associated with cytoadherence to CHOICAM1 in Rondonian isolates. ELISA assays showed that these DBLbICAM domains were recognized in a heterogeneous fashion. By flow cytometry, we demonstrated that ICAM1-adherent parasites were stronger recognized by plasma from symptomatic infections than from non-symptomatic infections. Finally, we profiled the var gene transcripts in 3D7 parasites adherent to different CHO cells by real time PCR.