School of Medicine and Health Sciences Poster Presentations

Title

Novel in-vitro model for assessment of biofilm formation by uropathogens

Document Type

Poster

Abstract Category

Immunology/Infectious Diseases

Keywords

CAUTI, Anti-biofilm, Catheters, Fluorescence Microscopy, Uropathogens

Publication Date

Spring 5-1-2019

Abstract

Catheter-associated urinary tract infections (CAUTI's) are one of the most common nosocomial infections, resulting in over 560,000 infections, 8,000 deaths, and upwards of $1.7 billion in added medical costs each year in the US. Despite several decades of research, a urinary catheter designed to inhibit biofilm formation continues to elude clinical adoption. One reason for this poor track record relates to the in-vitro models employed for urinary catheter research, which have mostly relied upon nutrient-rich defined media, and laboratory bacterial strains. These in-vitro models poorly mimic in-vivo conditions under which CAUTIs develop, and lead to failed therapeutic candidates in the clinical domain. To address this problem, we have devised a more clinically relevant in-vitro model for assessing biofilm inhibition on non-vital surfaces. A total of 46 subjects met the clinical criteria for urinary tract infection (UTI) and were enrolled from an urban emergency department. 100 mL of UTI urine was collected and transported to the laboratory. 1 cm2 flat silicone surface that was either uncoated or coated with one of two enzymes previously shown to inhibit biofilm formation were individually incubated in 5 ml of fresh uti urine for 4 days at 37°C with rocking. Subsequently, each silicone surface was then removed, stained with a fluorescent nuclear stain and imaged with an epifluorescence microscope. Biofilm images were evaluated with Image J software. Samples were stored and subsequently DNA was extracted for additional biofilm assessment using universal 16S primers in conjunction with ddPCR. Urine culture results were extracted from patient medical records for use in data analysis. Of enrolled subjects, 37 had culture results indicating uropathogens and were included in data analysis. Silicone surfaces coated with amylase, (active amylase has previously shown antibiofilm activity), demonstrated a significant increase in biofilm coverage whether all uropathogens were evaluated, or just those urine samples that grew out E. coli. Silicone surfaces coated with acylase, an AI-1 inhibitor, showed a similar trend which, however, did not reach statistical significance. We have shown that biofilm formation on silicone surfaces by clinical uropathogens in a clinically relevant medium (UTI urine) can be assessed via image analysis. Further we have shown that engineered enzymatic surface coatings previously shown to inhibit biofilm formation by representative strains of biofilm-forming bacteria did not inhibit biofilms on silicone surfaces in our model. Work to assess biofilms with greater sensitivity using ddPCR is currently ongoing.

Open Access

1

Comments

Presented at Research Days 2019.

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Novel in-vitro model for assessment of biofilm formation by uropathogens

Catheter-associated urinary tract infections (CAUTI's) are one of the most common nosocomial infections, resulting in over 560,000 infections, 8,000 deaths, and upwards of $1.7 billion in added medical costs each year in the US. Despite several decades of research, a urinary catheter designed to inhibit biofilm formation continues to elude clinical adoption. One reason for this poor track record relates to the in-vitro models employed for urinary catheter research, which have mostly relied upon nutrient-rich defined media, and laboratory bacterial strains. These in-vitro models poorly mimic in-vivo conditions under which CAUTIs develop, and lead to failed therapeutic candidates in the clinical domain. To address this problem, we have devised a more clinically relevant in-vitro model for assessing biofilm inhibition on non-vital surfaces. A total of 46 subjects met the clinical criteria for urinary tract infection (UTI) and were enrolled from an urban emergency department. 100 mL of UTI urine was collected and transported to the laboratory. 1 cm2 flat silicone surface that was either uncoated or coated with one of two enzymes previously shown to inhibit biofilm formation were individually incubated in 5 ml of fresh uti urine for 4 days at 37°C with rocking. Subsequently, each silicone surface was then removed, stained with a fluorescent nuclear stain and imaged with an epifluorescence microscope. Biofilm images were evaluated with Image J software. Samples were stored and subsequently DNA was extracted for additional biofilm assessment using universal 16S primers in conjunction with ddPCR. Urine culture results were extracted from patient medical records for use in data analysis. Of enrolled subjects, 37 had culture results indicating uropathogens and were included in data analysis. Silicone surfaces coated with amylase, (active amylase has previously shown antibiofilm activity), demonstrated a significant increase in biofilm coverage whether all uropathogens were evaluated, or just those urine samples that grew out E. coli. Silicone surfaces coated with acylase, an AI-1 inhibitor, showed a similar trend which, however, did not reach statistical significance. We have shown that biofilm formation on silicone surfaces by clinical uropathogens in a clinically relevant medium (UTI urine) can be assessed via image analysis. Further we have shown that engineered enzymatic surface coatings previously shown to inhibit biofilm formation by representative strains of biofilm-forming bacteria did not inhibit biofilms on silicone surfaces in our model. Work to assess biofilms with greater sensitivity using ddPCR is currently ongoing.