400x100x200x ABC Epithelial cell Attached bacteria Figure 1 E. coli attachment to human bladder epithelial cells. (A) After two hours, has begun to stick to the tissue cells. (B) floods the field as the bladder cells have deteriorated 18 hours after inoculation. (C) After 29 hours, stock E. coli congregate and attach on a bladder cell; this strain does not appear as pathogenic because the tissue cells seem healthier at 29 hours when compared to those in Fig 1(B). Biofilm Formation and Adherence of Uropathogenic E. coli Jeffrey Voreis, Steven Nus & Adam Ostendorf Winter 2004, BI 412 Introduction Biofilms are a group of sessile bacteria attached to a surface. They secrete a slime matrix which surrounds and protects them. Chemical signals accumulate and trigger gene expression which may initialize biofilm production. Growing these films is difficult in the laboratory because they form very slowly. Sessile bacteria secrete antigens and stimulate antibody production. Biofilms facilitate survival of bacteria because they protect against antimicrobial attacks due to the thick layer of slime and its slow diffusion rate. Furthermore, limited nutrition leads to a metabolic rate reduction that also protects the bacteria encased in a biofilm by reducing their susceptibility to metabolic attacks. By using a biofilm for protection, bacteria may infect surrounding tissues while avoiding attack. Uropathenogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTI). UTIs are extremely common in women, accounting for over 8 million doctor visits annually. During a UTI, E. coli coats the surface of bladder epithelial cells. Type 1-pili expressed by UPEC are required for stable attachment and pathogenicity. When type 1-pili are present on the bacteria, it is able to invade the urothelium by interacting with uroplakins on the tissue cell. E. coli has the ability to adhere to epithelial cells and secrete a biofilm for protection. Therefore, understanding these mechanisms and the conditions under which they are expressed may be the key to reducing the pathogenicity of this organism. We hypothesize that the UPEC strains will adhere quicker to the tissue cells and cause more degradation than the non-uropathogenic strain. Materials & Methods Note about the strains used: Escherichia coli ATCC® was isolated from a patient with a UTI. E. coli ATCC® came from a patient with recurrent UTIs. The third strain was our control, a nonpathogenic E. coli strain from Dr. Ventullo’s microbiology laboratory. The two ATCC strains are uropathogenic strains of E. coli. Bladder Cell Experiment: 10  L of bacteria was added to 0.8ml of RPMI-1640 in a sterile centrifuge tube. (8) 4- well slides containing human bladder epithelial cells were washed with 0.6mL PBS and rocked for 30 seconds. Then we added 0.8mL of RPMI-1640/bacteria mixture to a well on each slide. One well contained only RMPI as a control. Slides were placed in airtight jars and gassed with 5% CO 2 for 15 seconds. Jars were incubated at 37 o C. At designated times, media was removed from each well on 1 slide and the well was rinsed once with PBS. Wells were then rinsed twice and rocked with PBS. Then slides were fixed and stained using DAPI as previously described. The wells were removed from each slide. SloFade was added and a coverslip with fingernail polish sealed the slide. The fixing and staining procedure was repeated for each time period. Conclusions  No significant differences of adherence between different strains  UPEC strains appeared to degrade the tissue cells at a higher rate than the control strain  UPEC strains exhibited additional virulence factors or more robust metabolic activity which may account for the increased degradation of the tissue cells