Precise and rapid assessment of Escherichia coli adherence to vaginal epithelial cells by flow cytometry.
BACKGROUND
In the pathogenesis of Escherichia coli urinary tract infections (UTIs) in women, infecting bacteria adhere to vaginal and periurethral epithelial cells prior to ascending to the bladder and causing infection. Complex interactions among specific bacterial adhesins and various host factors appear to influence adherence of E. coli to mucosal surfaces such as the urogenital epithelium. To conduct population-based studies assessing host epithelial cell determinants that influence bacterial attachment, a method of measuring bacterial adherence utilizing clinically derived epithelial cell samples is needed.


METHODS
We developed and standardized an efficient, accurate, high-throughput method for analyzing the adherence of uropathogenic E. coli to clinical samples containing a large number of exfoliated vaginal epithelial cells (VEC). Three wild-type E. coli strains isolated from women with UTI (IA2 expressing pap-encoded, class II fimbriae only; F24 expressing pap-encoded, class II and type 1 fimbriae; and F20, without pap-encoded or type I fimbriae) were transformed with gfpmut3, encoding green fluorescent protein, incubated with VECs, and analyzed by flow cytometry.


RESULTS
Enumeration of the binding of each E. coli strain to 10,000 VECs showed reproducible, highly significant strain-dependent differences in adherence to VECs. Differential analysis of the relative contributions of type 1 pili and P fimbrial-mediated binding to the adherence phenotype was performed. It demonstrated that IA2 binding was dependent entirely on P fimbriae, whereas F24 binding was dependent on both P and type 1 fimbriae.


CONCLUSIONS
This method has great potential for use in high-throughput analyses of clinically derived epithelial cell samples and will be valuable in population-based investigations of host-parasite interactions in UTI utilizing VECs collected from specific patient groups.
Stapleton
Ann E
AE
Department of Medicine, Division of Allergy and Infectious Diseases, University of Washington, Seattle, Washington 98195, USA. stapl@u.washington.edu
Fennell
Cynthia L
CL
Coder
David M
DM
Wobbe
Cheryl L
CL
Roberts
Pacita L
PL
Stamm
Walter E
WE
D001422:Bacterial Adhesion / Q000502:physiology*; D004847:Epithelial Cells / Q000382:microbiology* / Q000473:pathology; D004926:Escherichia coli / Q000145:classification / Q000472:pathogenicity / Q000502:physiology*; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D015203:Reproducibility of Results; D014552:Urinary Tract Infections / Q000382:microbiology* / Q000473:pathology; D014621:Vagina / Q000382:microbiology* / Q000473:pathology