With increasing awareness of the urinary microbiome in refractory DO, the mechanism of intracellular localisation has received much interest. Intracellular localisation of bacteria within the urothelium reduces antibiotic efficacy and limits immune cell penetration, thus promoting survival of the uropathogen within the bladder. The aim of this study was to examine the capacity for common uropathogens to localise within the urothelium, and to determine what virulence factors are present in common uropathogens.

Three strains of uropathogenic Escherichia coli (E. coli), Enterococcus faecalis (E. faecalis) and Group B Steptococcus (GBS) were isolated by the clinical microbiology department from the urine of infected patients. Assays were performed to elucidate virulence mechanisms associated with intracellular localisation: yeast cell agglutination to test for FimH, an adhesin required for E. coli adherence to the urothelium [1]; a biofilm assay, as increased biofilm formation can enhance E. faecalis infection [2]; and haemolytic activity (using horse blood agar) as increasing toxicity is important for GBS penetration of cell membranes [3].

Intracellular localisation was confirmed by co-culturing each uropathogenic strain with RT4 urothelial cells for 2hours, followed by a further 2hour incubation with gentamycin to kill extracellular bacteria. The cells were stained using DAPI (nuclear material), Wheat-germ Agglutinin (urothelial cell membrane), and specific anti-bacterial antibodies (uropathogen membrane) to visualise the location of the uropathogen by confocal microscopy.

Two of the three E. coli strains tested positive for FimH in the yeast agglutination assay; all were deemed haemolytic but had either weak or no capacity to form biofilms (Table 1). Intracellular localisation was observed for E. coli strain 2 by confocal microscopy, but could not be confirmed with strain 1 as it was highly virulent and destroyed all urothelial cells in culture. E. coli strain 3 adhered to RT4 urothelial cells, but no intracellular localisation was observed. 

The three E. faecalis strains demonstrated no capacity for yeast cell agglutination (Table 1). However, all three were haemolytic, with a strong capacity to produce biofilms (Table 1). The E. faecalis strains were also observed to localise within the RT4 urothelial cells. 

All GBS strains were strongly haemolytic and produced strong biofilms (Table 1). Subsequent confocal imaging showed inconclusive results for intracellular localisation of GBS due to cross-reactivity of the bacterial membrane with the WGA antibody. Careful examination of the confocal images through orthogonal views suggests GBS is capable of intracellular localisation however this requires further confirmation with additional antibodies.

The results of the current study demonstrate that the uropathogens E. coli, E. faecalis and GBS, all possess virulence factors that have been associated with the capacity to localize within the urothelial cell. This may enhance their protection from antibiotics and the host immune system, potentiating their survival and thus their proliferation within the bladder [1]. 

Our results show that E. coli strain 3 did not agglutinate yeast cells and was unable to localise internally, corroborating the results of multiple studies that determined FimH is required for attachment and internalisation of E. coli e.g. [1]. All E. faecalis strains studied were localised within urothelial cells, and all were strong producers of biofilms. The rate of adherence, invasion and subsequent survival of E. faecalis within a cell has been shown to be dependent upon biofilm production [2]. 

The conclusions that can be drawn from studies with the GBS strains are limited by the cross reactivity between the GBS membrane and the antibody used to label the urothelial cell membrane. However, it appears that all three strains were capable of intracellular localisation and all three had a capacity for biofilm production as well as haemolytic activity. Therefore, GBS may enhance their localisation within the urothelium through both biofilm formation and their haemolytic activity.

Uropathogens possess a variety of virulence mechanisms, both unique to their species and/or common across species, which may allow for their localisation in the urothelium and their continued survival within the urinary tract.

Rosen DA, Pinkner JS, Walker JN, Elam JS, Jones JM & Hultgren SJ (2008). Molecular variations in Klebsiella pneumoniae and Escherichia coli FimH affect function and pathogenesis in the urinary tract. Infect Immun 76, 3346-3356.
Elhadidy M & Zahran E (2014). Biofilm mediates Enterococcus faecalis adhesion, invasion and survival into bovine mammary epithelial cells. Lett Appl Microbiol 58, 248-254.
Rajagopal L (2009). Understanding the regulation of group B Streptococcal virulence factors. Future Microbiol 4, 201-221.