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.

Klebsiella pneumoniae is a Gram negative, enteric bacterium that frequently causes disease in immunocompromised individuals. These types of infections are often associated with the presence of indwelling medical devices, which provide a site for the organism to attach and subsequently form a biofilm. A key component in K. pneumoniae biofilm formation in vitro is type 3 fimbriae. The two main components of this project have been to determine if type 3 fimbriae are an in vivo virulence factor using a mouse model of catheter associated urinary tract infection (CAUTI) and to examine the mechanism by which the production of type 3 fimbriae are regulated. Using a mouse model in which a silicone tube is implanted into the bladder of mice, mimicking the effects of catheterization, we have been able to show that type 3 fimbriae are required for colonization and persistence. Using different time points and conditions, we demonstrated that there are conditions when type 3 fimbriae alone are sufficient for colonization and other conditions where both type 1 and type 3 fimbriae have unique roles in colonization and persistence. Additionally, competition experiments showed that neither fimbrial mutant alone, or a double mutant in type 1 and type 3 fimbriae could compete with wildtype K. pneumoniae. In most animals, only wild-type bacteria were recovered by 24 hours post-inoculation. This work reinforced the role of type 1 fimbriae in pathogenesis and showed, for the first time, a role for type 3 fimbriae using an in vivo model. Our early work has indicated that type 3 fimbriae are regulated at least in part by the intracellular levels of the secondary messenger molecule cyclic di-GMP. Downstream from the type 3 fimbrial operon a gene encoding a phosphodiesterase is present; the product of this gene breaks down cyclic di-GMP. In the absence of this gene the levels of type 3 fimbrial expression are increased. Also adjacent to the mrk operon is a two-gene operon containing the determinants we have named mrkH and mrkI. mrkH encodes a PilZ domain containing protein, which we have shown binds cyclic di-GMP. Using a transcriptional fusion we have shown that the mrk gene promoter is activated modestly in the presence of MrkH, but when MrkH and MrkI are both present the activity is increased 100-fold. This has lead to the hypothesis that MrkH and MrkI interact, which we have been able to demonstrate using copurification procedures. This interaction appears to occur in a cyclic di-GMP dependent manner with the resulting protein complex binding to the mrk promoter region and activating the expression of type 3 fimbriae.