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.
| Identifer | oai:union.ndltd.org:ADTP/286206 |
| Creators | Yanwen Cheryl-lynn Ong |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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