Urinary tract infection (UTI) is one of the most common forms of bacterial infection, resulting in around 175 million reported cases of uncomplicated UTI worldwide every year. More than 80% of UTI is caused by uropathogenic Escherichia coli (UPEC). Antibiotics are widely used for treating uncomplicated cases of UTIs but the recent emergence of various multidrug resistant strains has posed a serious challenge to conventional therapy. This project focuses on the characterization of the recently emerged uropathogenic clone of E. coli of serotype O25b:H4 and sequence type 131 (E. coli ST131).
Virulence profiling of ninety-one E. coli ST131 clinical strains from the United Kingdom and Australia detected the presence of numerous factors associated with uropathogenicity. The type 1 fimbrial adhesin, fimH (an important bladder colonization factor), was detected in 97.8% of strains. Other highly prevalent virulence factors included fyuA, iutA, traT, sat and iha. A draft genome sequence of EC958, a representative E. coli ST131 UTI isolate, was determined. The λ-Red mediated mutagenesis system was modified to enable genetic manipulation of the multidrug resistant EC958 strain.
Expression of type 1 fimbriae was found to be a critical determinant for bladder colonisation by E. coli ST131 in a mouse UTI model and in vitro biofilm formation. Despite the high prevalence of fimH, expression of type 1 fimbriae was not a conserved trait within this clonal lineage. This was attributed to the presence of an insertion element within fimB, which encodes an activator for fimbriae expression. The disruption of fimB resulted in slower off-to-on switching of type 1 fimbriae. PCR screening revealed that this insertion was present in 65% of all ST131 isolates in our collection. These strains had several conserved nucleotide changes within the promoter region of fimE (usually turns the fim switch off) and almost six times higher expression of fimE compared to strains with an intact fimB. In the absence of a functional FimB, the tyrosine recombinases FimE (the gene for which is located adjacent to fimB) and FimX (the gene for which is located at a different site on the chromosome) were found to be responsible for off-to-on fim switching. Analysis of randomly generated transposon mutants in fimE and fimX promoter-lacZ reporter strains led to the observation that the metabolic state of a cell, including intracellular GTP concentration and the level of the (p)ppGpp regulatory alarmone, was responsible for regulating type 1 fimbriae expression by altering the promoter activity of fimE and fimX. It was also observed that yubO, a plasmid-located conserved hypothetical gene, down-regulates type 1 fimbrial expression. These results shed light on the altered regulation of type 1 fimbriae expression in the clinically predominant sub-group of the E. coli ST131 clonal lineage.
EC958 was found to be resistant to killing by human serum, an important phenotype associated with survival in the bloodstream. A multiplexing transposon directed insertion-site sequencing method was designed to define the serum resistome (i.e. genes required for resistance to human serum) of EC958 and the essential genes required for in vitro growth. The serum resistome was made up of 56 genes, the majority of which encoded membrane proteins or factors involved in lipopolysaccharide (LPS) biosynthesis. Targeted mutagenesis confirmed a role in serum resistance for 82% of these genes. The murein lipoprotein Lpp, along with two lipid A-core biosynthesis enzymes WaaP and WaaG, were most strongly associated with serum resistance. The enterobacterial common antigen and colanic acid were also found to be important for mediating serum resistance in EC958. A novel function for two genes, hyxA and hyxR, in the regulation of O-antigen chain length was identified. In a separate part of the analysis, 315 genes were identified as essential for the in vitro growth of EC958, 73% of which were also essential for E. coli K-12.
To better study virulence mechanisms of UPEC, a novel application of the rpsL counter selection system was devised to construct capsule (kpsD) and O antigen (waaL) mutants and complemented derivatives of three non-ST131 UPEC strains: CFT073 (O6:K2:H1), RS218 (O18:K1:H7) and 1177 (O1:K1:H7). The O1, O6 and O18 antigens were found to be highly important for survival in human serum. The K2 antigen was moderately important, while K1 played an insignificant role in conferring serum resistance. In the presence of whole blood, both K1 and K2 antigens provided a survival advantage to the UPEC strains tested. The O6 antigen was found to be significantly more important than K2 for bladder colonization in a mouse UTI model. The mutagenesis strategy described can be applied to study other virulence factors with complex functions in vitro and in vivo.
Overall, this project provides novel insights into the genetic makeup and virulence of strains belonging to the globally disseminated multidrug resistant E. coli ST131 clone, with a particular focus on the fluoroquinolone resistant, FimH30 representative EC958 strain.