Transcript Slide 1
Biofilm Formation and Adherence of Uropathogenic E. coli Steven Nus, Adam Ostendorf & Jeffrey Voreis Winter 2004 Introduction Materials & Methods Biofilms are a group of sessile bacteria attached to a surface. They secrete a slime matrix which surrounds and protects them. The films are formed by acylhomoserine lactone signals produced by individual bacterial cells. These chemicals 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) in an interesting strain of this species. UPEC is the leading cause of urinary tract infections (UTI) that are extremely common in women, accounting for over 8 million doctor visits annually. This species accounts for 70% to 95% of known infections. 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 to protect themselves from immune responses. Therefore, understanding these mechanisms and the conditions under which they are expressed may be the key to reducing the pathogenicity of this organism. 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. Ten-Minute Interval Growth: 150L of a bacteria/PBS suspension and 600L of 1/10 TSB +10% sucrose was added to each well of (6) 4-well slides. They were placed on a shaker at 50 rpm at 37o C. Every ten minutes, contents of a single well were extracted and rinsed twice with PBS. 300L of 3.7% formaldehyde was added to the well and incubated at 37o C for 10 minutes before three more PBS rinses. The process lasted 70 minutes until all wells were empty. Then 300L of DAPI was added to each well and the slides were incubated at 37o C for 15 minutes. The DAPI was removed from each well and rinsed twice with PBS. 48 hours Growth Experiment: 50L of the respective strain and PBS suspension and 200L of 1/10 TSB + 10% sucrose was added to each well in (9) 8-well slides. Slides were put on a shaker at 50 rpm at 37o C. Then slides were fixed and stained using DAPI as previously described. Biolfilm Accumulation Experiment: We filled 12 compartments of 24well plates with 1.5mL of 1/10 TSB + 10% sucrose and 50L of a bacteria and PBS suspension. We used 2 plates per strain for 6 total plates. They were placed on a shaker at 100 rpm at 37o C. At designated times, media from 1 well in each plate was removed, and the well was rinsed twice with PBS. After all periods, 1mL of 0.5% crystal violet was added and incubated for 15 minutes. We rinsed the wells 3 times with filtered Nanopure® H2O. Wells were allowed to dry, then filled with 2mL of 95% EtOH. The liquid was extracted and placed into cuvette, from which optical density was then measured at 590 nm. Bladder Cell Experiment: 10L 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 RPMI1640/bacteria mixture to a well on each slide. One well contained only RMPI-1640 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. Results & Discussion Stock E. coli A E. coli 29214 400x E. coli 700417 400x B 400x C 50 min Conclusions D 400x 400x E 400x F The uropathogenic strains of E. coli (UPEC) did not exhibit dramatically increased growth ability when compared to the stock laboratory strain, which was considered our control. E. coli 700417, a uropathogenic strain, appears to form biofilms and extracellular slime faster than the other strains studied. 70 min The UPEC did not display greater affinity for binding to human bladder epithelial cells when compared to the control. G 200x 200x H 200x I The UPEC displayed a higher pathogenicity towards the bladder cells than did the control. 120 min 200x Figure 1 The growth patterns of three strains of E. coli. In general, the stock strain and 29214 lagged behind the growth of 700417, as demonstrated by Fig 1(A, B, C). At 70 minutes, the three appeared to have similar population densities under microscopic inspection in Fig 1(D, E, F). The three strains filled the field after two hours, but the stock E. coli’s growth appeared to trail slightly behind the uropathogenic strains as shown in Fig(G, H, I). According to the two time courses examining growth, one of 70 minutes with 10 minute intervals and another spanning 48 hours, E. coli growth appears to be the same in all three strains after two or three hours. Growth held study from three hours to the end of the experiment at 48 hours. This is possibly due to limiting factors such as nutrient availability, waste accumulation, and spatial inhibition. We would like to thank Dr. Roy Ventullo, Dr. J. Keith McClung, Dr. Edward Westen, Julie Paladino, and the Wartburg College Biology Department for their guidance and support throughout this project. Figure 2 After 8 hours appeared like this picture of strain 700417. A 400x B 100x C Epithelial cell 200x Epithelial cell Attached bacteria 1 log Absorbance (arb. units) Figure 3 Biofilm production for three strains of E. coli. Biofilms were allowed to accumulate in well plates, were dried and stained with crystal violet. All strains developed increasingly massive biofilms over time. The stock strain and 29214 exhibited an accumulation of biofilm that increased over the period from 0 hours to 3 hours. After 3 hours, the mass of biofilm for both strains remained fairly constant. 700417 appeared to form a biofilm almost immediately. After their initial formation, the biofilms remained constant. We hypothesized all strains would form biofilms which would increase in size over time. We also hypothesized the stock strain would not produce as much material as the uropathogenic strains. There appears to be no appreciable difference in total biofilm formation by E. coli 29214 and the stock strain. The 700417 strain appears to be a fast colonizer and possibly carries a residual film of organic matter which would explain the initial appearance of significant mass. Acknowledgements Figure 4 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). 0.1 Over the course of a 48 hour trial, the three strains of E. coli displayed varying degrees of adherence and pathogenicity. After examining the fields on all slides, we could determine little to no difference in adherence to epithelial cells. The three strains attached to the tissue cells, but they also stuck to the glass slide despite rinsing multiple times with PBS while rocking. The adherence to the cells may have been due to the expression of type 1-pili. An extracellular layer of proteins and biofilms may have facilitated adhesion to the slide. Stock Strain E. coli 29214 E. coli 700417 0.01 0 2 4 6 8 10 12 14 Time (hrs.) 16 18 20 22 24 The tissue cells began degrading five hours after inoculation, yet those with the stock strain did not seem as severely damaged. The control cells appeared normal at the end of the trial, so we believe the uropathogenic strains exhibited additional virulence factors, or more robust metabolic activity, which harmed the epithelial cells to a greater degree.