School of Medicine and Health Sciences Poster Presentations

Title

Counting angels on the head of a pin—a new in vitro model for the study of biofilm formation on urinary catheters

Document Type

Poster

Abstract Category

Basic Biomedical Sciences

Keywords

UTI, urinary catheter, biofilm, in vitro model,

Publication Date

Spring 5-1-2019

Abstract

Catheter-associated urinary tract infections (CAUTIs) are one of the most common nosocomial infections, resulting in over 560,000 infections, 8,000 deaths and about one billion dollars in medical costs yearly in the US. CAUTIs involve biofilm formation on non-vital catheter surfaces which are not treatable with antibiotics alone. Despite several decades of research, a urinary catheter engineered 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 biofilm research, which have mostly relied upon 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. We propose a novel in vitro model, using urinary tract infection (UTI) patient urine samples to more closely mirror in vivo biofilm formation conditions. We have previously reported that biofilms grown up in our in vitro model are far more scant and heterologous than those customarily produced under standard laboratory conditions. This makes the traditional microscopy-based means for quantitating bacterial biofilms less accurate. To this end we have employed ddPCR-based 16S enumeration as a more accurate means for quantifying biofilms. Patients with symptoms and laboratory finding consistent with UTI were enrolled in the study, and 50-100 cc of urine was collected. Within one hour of collection, 5 mL aliquots of urine were pipetted into 3 wells of a sterile plate. Silicone tablets was placed into each of the 3 wells. Plates were then incubated at 36C with rocking at 80rpm for 96 hours. Silicone tablets were then removed, washed, stained with Syto-9 and imaged with an epifluorescence microscope. DNA was then extracted from the silicone tablets using a commercial DNA extraction kit (MP Bio), and ddPCR was used to amplify and quantify bacterial 16S. 46 subjects were enrolled; ddPCR-based 16Sresults largely mirrored image-based assessment of biofilm burden, however, in a far more quantifiable manner. This new technique will make it possible to screen potential anti-biofilm modalities in a model that much more closely reflects the conditions faced by uropathogens in the harsh environment of the urinary bladder. Using this approach, identifying antibiofilm strategies that can work is more likely. ddPCR-based quantification of biofilm 16S is a more precise tool for quantifying biofilms, making comparisons between treatments and controls more meaningful.

Open Access

1

Comments

Presented at Research Days 2019.

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Counting angels on the head of a pin—a new in vitro model for the study of biofilm formation on urinary catheters

Catheter-associated urinary tract infections (CAUTIs) are one of the most common nosocomial infections, resulting in over 560,000 infections, 8,000 deaths and about one billion dollars in medical costs yearly in the US. CAUTIs involve biofilm formation on non-vital catheter surfaces which are not treatable with antibiotics alone. Despite several decades of research, a urinary catheter engineered 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 biofilm research, which have mostly relied upon 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. We propose a novel in vitro model, using urinary tract infection (UTI) patient urine samples to more closely mirror in vivo biofilm formation conditions. We have previously reported that biofilms grown up in our in vitro model are far more scant and heterologous than those customarily produced under standard laboratory conditions. This makes the traditional microscopy-based means for quantitating bacterial biofilms less accurate. To this end we have employed ddPCR-based 16S enumeration as a more accurate means for quantifying biofilms. Patients with symptoms and laboratory finding consistent with UTI were enrolled in the study, and 50-100 cc of urine was collected. Within one hour of collection, 5 mL aliquots of urine were pipetted into 3 wells of a sterile plate. Silicone tablets was placed into each of the 3 wells. Plates were then incubated at 36C with rocking at 80rpm for 96 hours. Silicone tablets were then removed, washed, stained with Syto-9 and imaged with an epifluorescence microscope. DNA was then extracted from the silicone tablets using a commercial DNA extraction kit (MP Bio), and ddPCR was used to amplify and quantify bacterial 16S. 46 subjects were enrolled; ddPCR-based 16Sresults largely mirrored image-based assessment of biofilm burden, however, in a far more quantifiable manner. This new technique will make it possible to screen potential anti-biofilm modalities in a model that much more closely reflects the conditions faced by uropathogens in the harsh environment of the urinary bladder. Using this approach, identifying antibiofilm strategies that can work is more likely. ddPCR-based quantification of biofilm 16S is a more precise tool for quantifying biofilms, making comparisons between treatments and controls more meaningful.