Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Porphyromonas gingivalis à um patÃgeno extremamente associado com a etiologia da periodontite crÃnica e agressiva. O objetivo deste estudo foi avaliar atravÃs de reaÃÃo em cadeia da polimerase em tempo real (Real Time-PCR) a presenÃa de Porphyromonas gingivalis (Pg) e dos genÃtipos fimA II e IV em indivÃduos com periodontite agressiva generalizada (PAG). Quarenta indivÃduos com PAG (29,7  8,1 anos) foram analisados clinicamente - Ãndice de Placa (IP), Ãndice Gengival (IG), profundidade de sondagem (PS), nÃvel de inserÃÃo clÃnico (NIC) - e microbiologicamente, atravÃs de Real Time PCR, quanto à presenÃa de Pg e dos genÃtipos fimA II e IV. Amostras de biofilme subgengival foram colhidas do sÃtio proximal com maior PS e maior NIC. MÃdias de PS e NIC desses sÃtios foram respectivamente: 9,5  2,2 mm e 10,2  2,8 mm. P. gingivalis foi observado em 26 (65%) dos indivÃduos. O genÃtipo fimA II foi verificado em 16 (61,53%) enquanto o genÃtipo fimA IV em 7 (26,92%) dos que apresentaram P. gingivalis. Entretanto, nÃo foi observada diferenÃa estatÃstica entre os parÃmetros clÃnicos dos indivÃduos que apresentaram ou nÃo o microrganismo ou seus respectivos genÃtipos. TambÃm nÃo foi verificada associaÃÃo entre a presenÃa dos genÃtipos e idade ou gÃnero dos pacientes. Os dados sugerem uma associaÃÃo entre genÃtipos fimA II de Porphyromonas gingivalis quando da ocorrÃncia deste microrganismo em indivÃduos com periodontite agressiva generalizada. / Porphyromonas gingivalis is a pathogen strongly associated with the etiology of chronic and aggressive periodontitis. The purpose of this study was to evaluate by Real-Time polymerase chain reaction (Real Time-PCR) the presence of Porphyromonas gingivalis (Pg) and fimA genotypes type II and type IV in patients with generalized aggressive periodontitis (GAgP). Forty individuals with aggressive periodontitis (AgP) (29.7  8.1 years) were clinical analyzed through plaque index (PI), gingival index (GI), probing depth (PD), clinical attachment level (CAL) and microbiologically, by Real Time-PCR for the presence of Pg and fimA genotypes type II and type IV. Subgingival biofilm samples were collected from the interproximal periodontal sites (> PD and > CAL). The PD and CAL average of this sites were respectively: 9,5  2,2 mm e 10,2  2,8 mm. P. gingivalis was observed in 26 (65%) of individuals. FimA genotypes type II was detected in 16 (61,53%) while fimA genotypes type IV in 7 (26,92%) of those with P. gingivalis. However, no differences were observed between the clinical parameters of patients who presented or not the organism or its genotypes. There was also no association between the presence of genotypes and age or gender of patients. The data suggest an association between P. gingivalis fimA genotypes upon the occurrence of this microorganism in patients with generalized aggressive periodontitis.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 133-156).

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

E. coli enteropatogênica atípica (aEPEC) é um importante patótipo causador de diarréia. As cepas de aEPEC não produzem BFP, sugerindo que outras fimbriais estão envolvidas na adesão de aEPEC ao hospedeiro. ECP é encontrada na maioria das cepas patogênicas e não patogênicas de E. coli. O objetivo do estudo foi avaliar a expressão e produção de ECP em cepas de aEPEC. A presença do operon ecp foi avaliada por PCR e a expressão avaliada por PCR utilizando cDNA obtido a partir do cultivo bacteriano em LB, DMEM e DMEM pré-condicionado. A produção de ECP foi avaliada por imunofluorescência. Foi observado que a expressão, por todas as cepas, só ocorreu quando foram cultivadas em DMEM pré-condicionado. Houve expressão diferencial do operon com o cultivo em LB ou DMEM. A produção de ECP foi observada apenas puma cepa quando cultivada em LB. Já em DMEM, três cepas produziram ECP. Houve um aumento do número de cepas produtoras da fímbria com o cultivo em DMEM pre condicionado. Esses resultados sugerem que a sinalização celular pode interferir na expressão e produção da ECP. / Atypical enteropathogenic E. coli (aEPEC) is an pathotype that causes diarrhea. aEPEC does not produce BFP, suggesting that other pili must be involved in aEPEC adhesion to the host cell. ECP is found in most pathogenic and non-pathogenic E. coli. The objective of this study was to evaluate the expression and production of ECP in aEPEC. The presence of ecp operon was assessed by PCR and the expression evaluated by PCR using cDNA obtained from bacterial growth in LB, DMEM and preconditioned DMEM. ECP production was evaluated by immunofluorescence. It was observed that the operon expression by all strains only occurred when they were grown in preconditioned DMEM. There was differential expression of ecp operon when strains were grown in LB or DMEM. ECP production was observed only by one strain when grown in LB. Three strains producted ECP grown in DMEM, but there was a higher production of the pili when strains were grown in DMEM preconditionated. These results suggest that cellular signaling may interfere with the expression and production of ECP.

E. coli enteropatogênica atípica (aEPEC) é um importante patótipo causador de diarréia. As cepas de aEPEC não produzem BFP, sugerindo que outras fimbriais estão envolvidas na adesão de aEPEC ao hospedeiro. ECP é encontrada na maioria das cepas patogênicas e não patogênicas de E. coli. O objetivo do estudo foi avaliar a expressão e produção de ECP em cepas de aEPEC. A presença do operon ecp foi avaliada por PCR e a expressão avaliada por PCR utilizando cDNA obtido a partir do cultivo bacteriano em LB, DMEM e DMEM pré-condicionado. A produção de ECP foi avaliada por imunofluorescência. Foi observado que a expressão, por todas as cepas, só ocorreu quando foram cultivadas em DMEM pré-condicionado. Houve expressão diferencial do operon com o cultivo em LB ou DMEM. A produção de ECP foi observada apenas puma cepa quando cultivada em LB. Já em DMEM, três cepas produziram ECP. Houve um aumento do número de cepas produtoras da fímbria com o cultivo em DMEM pre condicionado. Esses resultados sugerem que a sinalização celular pode interferir na expressão e produção da ECP. / Atypical enteropathogenic E. coli (aEPEC) is an pathotype that causes diarrhea. aEPEC does not produce BFP, suggesting that other pili must be involved in aEPEC adhesion to the host cell. ECP is found in most pathogenic and non-pathogenic E. coli. The objective of this study was to evaluate the expression and production of ECP in aEPEC. The presence of ecp operon was assessed by PCR and the expression evaluated by PCR using cDNA obtained from bacterial growth in LB, DMEM and preconditioned DMEM. ECP production was evaluated by immunofluorescence. It was observed that the operon expression by all strains only occurred when they were grown in preconditioned DMEM. There was differential expression of ecp operon when strains were grown in LB or DMEM. ECP production was observed only by one strain when grown in LB. Three strains producted ECP grown in DMEM, but there was a higher production of the pili when strains were grown in DMEM preconditionated. These results suggest that cellular signaling may interfere with the expression and production of ECP.

DNA adenine methyltransferase (Dam) plays an important role in different bacterial functions. It has been shown that Dam is required for regulation of bacterial replication initiation and is required for proofreading newly synthesized DNA through methylation directed mismatch repair. Dam is also involved in the regulation of different genes and is required for virulence in several different bacterial genera though its degree of importance depends on the specific bacteria being studied. During this work, a Dam-negative strain (JSM1) was constructed in Klebsiella pneumoniae strain 43816 to ascertain its importance for K. pneumoniae viability and virulence. To test JSM1 for expression of fimbrial virulence factors, agglutinations were used to detect the presence of type three and type one fimbriae, respectively. No differences between 43816 and JSM1 were discernable. Similarly, JSM1 production of capsular material appeared to be unaltered. K. pneumoniae JSM1 virulence in a murine model was examined following intranasal or intraperitoneal inoculation, and it was determined that JSM1 is partially attenuated. Quantitative analysis of 43816 and JSM1 biofilm growth revealed only slight decreases in JSM1 biofilm mass and thickness, but live/dead staining of developed biofilms showed decreased JSM1 biofilm viability over time compared to 43816 biofilms. JSM1 was also examined for alterations in the frequency of spontaneous antibiotic resistance mutations and tested for increased susceptibility to various DNA damaging agents, and statistically significant differences were found for some of the spontaneous antibiotic resistance mutation frequencies tested. Fimbriae in K. pneumoniae are important virulence factors which facilitate respiratory and urinary tract infections in vivo. They also contribute to formation of biofilms which are believed to cause chronic infections and increased antibiotic resistance. Searches for homologous regions within the Klebsiella chromosome using the chaperone and usher components of E. coli type 1 fimbriae revealed five putative fimbrial gene clusters on the Klebsiella chromosome which had not been characterized. Mutations created within select gene clusters did not yield detectable deficiencies in biofilm formation or murine respiratory virulence. However, based on the multiplicity of fimbrial expression observed in Salmonella enterica serovar Typhimurium, combinational mutations may be required prior to detection of a discernable phenotype.

Catheter-associated urinary tract infection (CAUTI) is one of the most common nosocomial infections and is caused by a range of different uropathogens, particularly by uropathogenic Escherichia coli (UPEC). Amongst the different virulence factors, biofilm formation and bacterial aggregation, often mediated by cell surface structures such as fimbriae, are common traits among uropathogens that cause CAUTI. In this study, a collection of UPEC isolates were screened for virulence genes and phenotypes associated with urinary tract infections such as biofilm formation and mannose-sensitive haemagglutination. Two strains, E. coli MS2027 (which formed a strong biofilm) and E. coli M184 (which aggregated strongly) were analysed in detail to determine the molecular mechanisms associated with these phenotypes. Transposon mutagenesis of E. coli MS2027 identified type 3 fimbriae as the factor responsible for its strong biofilm growth. Further screening revealed the presence of type 3 fimbriae in uropathogenic Citrobacter freundii, Citrobacter koseri, Klebsiella oxytoca, Klebsiella pneumoniae and other E. coli. Phylogenetic analysis of the type 3 fimbrial (mrkABCD) genes from these strains revealed they clustered into five distinct clades (A-E) ranging from one to twenty-three members. The majority of the sequences grouped in clade A, which was represented by the mrk gene cluster from the genome sequenced K. pneumoniae strain MGH78578. We demonstrated that type 3 fimbriae are functionally expressed by different Gram negative nosocomial pathogens and present evidence to suggest that they contribute significantly to catheter colonisation. The type 3 fimbrial genes from E. coli MS2027 were found to be located on a conjugative plasmid. Sequencing and annotation revealed that this 42,644 bp plasmid, named pMAS2027, contains 58 putative genes. Bioinformatic analysis identified pMAS2027 as an incompatibility X (IncX1) plasmid. Plasmid pMAS2027 contained genes encoding two important virulence factors, type 3 fimbriae and a type IV secretion (T4S) system. The biofilm ability was solely based on the expression of type 3 fimbriae and not the T4S system. The T4S system, however, accounted for the conjugative ability of pMAS2027. Differential tagging with fluorescent reporter genes demonstrated conjugative transfer of pMAS2027 between cells during biofilm growth. Finaly, transposon mutagenesis of E. coli M184 revealed a number of putative genes potentially responsible for bacterial aggregation. Of these, genes involved in the synthesis of the enterobacterial common antigen (ECA) were shown to be associated with an aggregation phenotype.

Catheter-associated urinary tract infection (CAUTI) is one of the most common nosocomial infections and is caused by a range of different uropathogens, particularly by uropathogenic Escherichia coli (UPEC). Amongst the different virulence factors, biofilm formation and bacterial aggregation, often mediated by cell surface structures such as fimbriae, are common traits among uropathogens that cause CAUTI. In this study, a collection of UPEC isolates were screened for virulence genes and phenotypes associated with urinary tract infections such as biofilm formation and mannose-sensitive haemagglutination. Two strains, E. coli MS2027 (which formed a strong biofilm) and E. coli M184 (which aggregated strongly) were analysed in detail to determine the molecular mechanisms associated with these phenotypes. Transposon mutagenesis of E. coli MS2027 identified type 3 fimbriae as the factor responsible for its strong biofilm growth. Further screening revealed the presence of type 3 fimbriae in uropathogenic Citrobacter freundii, Citrobacter koseri, Klebsiella oxytoca, Klebsiella pneumoniae and other E. coli. Phylogenetic analysis of the type 3 fimbrial (mrkABCD) genes from these strains revealed they clustered into five distinct clades (A-E) ranging from one to twenty-three members. The majority of the sequences grouped in clade A, which was represented by the mrk gene cluster from the genome sequenced K. pneumoniae strain MGH78578. We demonstrated that type 3 fimbriae are functionally expressed by different Gram negative nosocomial pathogens and present evidence to suggest that they contribute significantly to catheter colonisation. The type 3 fimbrial genes from E. coli MS2027 were found to be located on a conjugative plasmid. Sequencing and annotation revealed that this 42,644 bp plasmid, named pMAS2027, contains 58 putative genes. Bioinformatic analysis identified pMAS2027 as an incompatibility X (IncX1) plasmid. Plasmid pMAS2027 contained genes encoding two important virulence factors, type 3 fimbriae and a type IV secretion (T4S) system. The biofilm ability was solely based on the expression of type 3 fimbriae and not the T4S system. The T4S system, however, accounted for the conjugative ability of pMAS2027. Differential tagging with fluorescent reporter genes demonstrated conjugative transfer of pMAS2027 between cells during biofilm growth. Finaly, transposon mutagenesis of E. coli M184 revealed a number of putative genes potentially responsible for bacterial aggregation. Of these, genes involved in the synthesis of the enterobacterial common antigen (ECA) were shown to be associated with an aggregation phenotype.

Catheter-associated urinary tract infection (CAUTI) is one of the most common nosocomial infections and is caused by a range of different uropathogens, particularly by uropathogenic Escherichia coli (UPEC). Amongst the different virulence factors, biofilm formation and bacterial aggregation, often mediated by cell surface structures such as fimbriae, are common traits among uropathogens that cause CAUTI. In this study, a collection of UPEC isolates were screened for virulence genes and phenotypes associated with urinary tract infections such as biofilm formation and mannose-sensitive haemagglutination. Two strains, E. coli MS2027 (which formed a strong biofilm) and E. coli M184 (which aggregated strongly) were analysed in detail to determine the molecular mechanisms associated with these phenotypes. Transposon mutagenesis of E. coli MS2027 identified type 3 fimbriae as the factor responsible for its strong biofilm growth. Further screening revealed the presence of type 3 fimbriae in uropathogenic Citrobacter freundii, Citrobacter koseri, Klebsiella oxytoca, Klebsiella pneumoniae and other E. coli. Phylogenetic analysis of the type 3 fimbrial (mrkABCD) genes from these strains revealed they clustered into five distinct clades (A-E) ranging from one to twenty-three members. The majority of the sequences grouped in clade A, which was represented by the mrk gene cluster from the genome sequenced K. pneumoniae strain MGH78578. We demonstrated that type 3 fimbriae are functionally expressed by different Gram negative nosocomial pathogens and present evidence to suggest that they contribute significantly to catheter colonisation. The type 3 fimbrial genes from E. coli MS2027 were found to be located on a conjugative plasmid. Sequencing and annotation revealed that this 42,644 bp plasmid, named pMAS2027, contains 58 putative genes. Bioinformatic analysis identified pMAS2027 as an incompatibility X (IncX1) plasmid. Plasmid pMAS2027 contained genes encoding two important virulence factors, type 3 fimbriae and a type IV secretion (T4S) system. The biofilm ability was solely based on the expression of type 3 fimbriae and not the T4S system. The T4S system, however, accounted for the conjugative ability of pMAS2027. Differential tagging with fluorescent reporter genes demonstrated conjugative transfer of pMAS2027 between cells during biofilm growth. Finaly, transposon mutagenesis of E. coli M184 revealed a number of putative genes potentially responsible for bacterial aggregation. Of these, genes involved in the synthesis of the enterobacterial common antigen (ECA) were shown to be associated with an aggregation phenotype.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 52-68).