Characterization of Bacterial Biofilm Composition in Occluded Plastic Biliary Stents

Abstract

Purpose: Plastic biliary stents are an effective treatment for biliary obstruction. Despite being resolutive and accessible, they are known to have a low patency rate, estimated at 3 to 6 months. This can be attributed to the formation of bacterial biofilm, which leads to the luminal obstruction of the stent. The aim of this study is to identify the bacterial composition of biofilms from obstructed plastic biliary stents removed through ERCP.

Methods: Obstructed plastic biliary stents were retrieved from patients undergoing ERCP. The stents were fragmented into three segments of 2.0 cm each: proximal, medial, and distal. Gram staining was performed on each fragment, followed by assessment using optical microscopy. Subsequently, 4 µm cross-sections were made of each fragment, with subsequent analysis by confocal microscopy. The material from the inside of the stents was also placed in culture medium and colony-forming units were counted.

Results: Optical microscopy and analysis by confocal microscopy showed a seemingly higher number of bacterial colonies in the distal portion of the stents compared to the proximal and medial regions. A greater presence of bacteria in the distal segments of the stents was confirmed, with growth reaching up to 10¹⁴, while growth in the proximal and medial segments was only observed up to 10⁹ and 10⁸, respectively. Biochemical identification using Gram staining identified both Gram-positive and Gram-negative species: Enterococcus faecium; Aeromonas hydrophila/caviae; Escherichia coli; Enterobacter cloacae; Citrobacter freundii; Klebsiella oxytoca; Proteus vulgaris; Proteus mirabilis; Pantoea sp; Morganella morganii.

Conclusion: The composition of the biofilm in biliary stents confirmed to be polymicrobial. The distal portion of the stents is likely the most frequent site of obstruction. New strategies, such as the development and improvement of plastic stents, should be considered to slow this growth and enhance permeability.

Keywords: ERCP; biliary system; biofilm.

Figure 1

Left: Endoscopic view of an occluded biliary stent. Right: Endoscopic removal of a prosthesis occluded by ERCP, with the aid of a polypectomy snare.

Figure 2

Schematic representation of the occluded, fragmented segments of the stents for analysis.

Figure 3

CFU/mL count of the stent in the proximal third.

Figure 4

CFU/mL count of the stent in the medial third.

Figure 5

CFU/mL count of the stent in the distal third.

Figure 6

Quantification of CFU/mL in the proximal, medial, and distal portions of the stents at different dilutions.

Figure 7

Gram staining images of samples containing biofilm from different regions of the biliary catheter: (A) proximal region, (B) medial region, and (C) distal region. Optical microscopy, 1000x magnification.

Figure 8

Images of cross-sections from the medial region of a biliary catheter containing bacterial biofilm stained with propidium iodide. Confocal laser microscopy. Magnification 620x (A and B).

Figure 9

Images of cross-sections from the distal region of a biliary catheter containing bacterial biofilm stained with propidium iodide. Confocal laser microscopy. Magnification 620x (A–C).

Figure 10

Three-dimensional images of cross-sections from the distal region of a biliary catheter containing bacterial biofilm stained with propidium iodide. Confocal laser microscopy. Magnification 620x (A and B).