Download Slide 1

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® 29214 was isolated from a patient with a UTI. E. coli
ATCC® 700417 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) 4well 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 RMPI1640 as a control. Slides were placed in airtight jars and gassed with 5% CO2 for 15 seconds. Jars were
incubated at 37oC. 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.
A
400x
B
100x
C
Epithelial cell
200x
Epithelial cell
Attached
bacteria
Figure 1 E. coli attachment to human bladder epithelial cells. (A) After two hours, 29214 has begun to
stick to the tissue cells. (B) 700417 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).
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