Characterization of biofilms in biliary stents and potential factors involved in occlusion

ARTICLE HIGHLIGHTS

Research background

Since its introduction in 1979, biliary plastic stents have been a landmark achievement in the field of endoscopic retrograde cholangiopancreatography for the relief of obstructed biliary system by a non-surgical approach. The limiting factors for these plastic stents are their diameter and the tendency to get occluded. The maximum diameter of plastic stent that can be placed is 11.5 Fr requiring a duodenoscope accessory channel diameter of 4.2 mm. This limitation of the maximum diameter leads to the tendency for them to get occluded due to the formation of biofilm causing recurrent obstruction and need for repeat procedures subsequently leading to increased medical costs and poor quality of life. The cardinal step in the process of stent occlusion is bacterial colonization. Various studies including scanning electron microscopic observations have shown that the clogging material found in biliary stents consists of bacterial biofilm, biliary sludge and duodenal refluxate of dietary fibers. Biofilm is formed by microbes embedded in an exopolysaccharide matrix which also engulfs “foreign bodies” of various sizes. Its ultrastructure reveals voids and channels required for nutrient diffusion and molecular signaling.

Research motivation

Despite multiple studies elucidating the various organisms and the formation of biofilms, various factors involved in the formation of these biofilms are not well studied. The proposed mechanism of biofilm formation initiates with the process of priming of the stent surface with various proteins followed by microbial adherence and subsequently formation of an exopolysaccharide matrix to embed the microbial colonies and other “foreign bodies” to give rise to the final mature biofilm. However, proper characterization of biofilm formation in plastic stents has to be adequately elucidated before steps for its prevention can be made successful. Components of the biofilm such as protein and polysaccharides developing in biliary stents have never been quantified in previous studies.

Research objectives

The main objectives of this study were to elucidate the various bacteria implicated in biofilm formation in biliary plastic stents, to quantify the principal constituents (namely proteins and polysaccharide) of biofilm mass and the possible predisposing factors in relation to biofilm formation in the stents. This prospective study evaluated the extracellular polymeric substance such as protein and polysaccharide in the biofilms as well as microbes occluding the biliary stents in patients who had retrieval of biliary stents (7 Fr and 10 Fr) and analyzed predisposing factors involved in the process of occlusion of the stents. Our results showed that the presence of cholangitis at the time of stent insertion and smaller diameter of stents were found to have higher protein concentration, whereas male gender and age above 60 years had higher polysaccharide concentration, predisposing to higher propensity of biofilm formation. Longer (≥ 6 mo) indwelling time of stents was associated with higher biofilm formation and protein concentration, elucidating the time-dependent process of biofilm formation. Our data suggest that plastic stents should be replaced between 3-6 mo. Further studies can be done to explore the origin of the bacteria grown in biofilms. Strategies to prevent biofilm formation can also be planned and investigated.

Research methods

This was a prospective study conducted at a tertiary care hospital in Northern India (Postgraduate Institute of Medical Education and Research, Chandigarh, India) from April 2011 to March 2014. All consecutive patients who required an elective or emergency biliary stent exchange/removal were enrolled and clinical details of each patient were noted. The stents were retrieved through video duodenoscope and transferred into sterile containers for processing. For molecular identification of bacterial species, the encrusted material enclosed within the stent was cultured aerobically and anaerobically and the microbial DNA was extracted. PCR was standardized using the universal 16S rRNA gene-specific primers for determining the DNA sequence for commonly known bacteria. Molecular identification of unknown bacteria involved in biofilm formation was done using the Density Gradient Gel Electrophoresis. The amplified PCR products were sequenced commercially using bands which were different from commonly known bands. Data obtained after sequencing were compared with the National Center of Biotechnology Information GenBank data base, using standard nucleotide blast search tools. The major molecules in the biofilms such as protein and polysaccharide were estimated in the biofilm mass by modified Lowry’s method and anthrone method respectively. The outcome measures were quantification of biofilm protein, polysaccharides and the organisms and their relation with gender, age, etiology of biliary diseases, stent indwelling time, stent size and the presence of cholangitis. Statistical analysis for this study was performed using SPSS version 20.0 using χ² test and Fisher’s exact test to investigate the relationship between various parameters.

Research results

Higher protein concentration in the biofilm was noted in patients with cholangitis as compared to those without cholangitis. Cholangitis and protein concentration increased the likelihood of biofilm formation in these patients explaining higher stent occlusion rates in infected bile. Male gender and age above 60 years had higher polysaccharide concentration, predisposing to higher propensity of biofilm formation. Smaller diameter stents depicted higher protein concentration predisposing to early biofilm formation thereby indicating the use of larger diameter stents. 10 Fr stents had lower concentration of protein deposition in the biofilm compared to 7 Fr stents and hence explains the longer patency rates. PCR and sequencing helped to detect several commonly known and unknown microorganisms in most of the stents. Time dependent process of biofilm formation was demonstrated by greater quantity of biofilm mass deposition on increasing length of stent indwelling time. Longer indwelling time of stents has a greater likelihood of accumulating higher biofilm formation and patients should be followed-up between 3-6 mo to avoid complications.

Research conclusions

Presence of cholangitis at the time of stent insertion and smaller diameter of stents were found to have higher protein concentration, whereas male gender and age above 60 years had higher polysaccharide concentration, predisposing to higher propensity of biofilm formation. Longer indwelling time of stents was associated with higher biofilm formation and protein concentration elucidating the time-dependent process of biofilm formation. Our study suggests that plastic stents should be replaced between 3-6 mo. Plastic stents retrieved from patients with biliary tract disease showed polymicrobial organisms with higher protein content among patients with cholangitis and those with smaller diameter stents. Longer indwelling duration had more biofilm formation. Presence of cholangitis at the time of stent insertion and smaller diameter of stents were found to have higher protein concentration, whereas male gender and age above 60 years had higher polysaccharide concentration, predisposing to higher tendency of biofilm formation. Longer indwelling stent duration was associated with higher biofilm formation and protein concentration, revealing the time-dependent progression of biofilm formation. Longer indwelling time of stents, smaller diameter stents, male gender and age above 60 years are associated with more biofilm formation. Data on comorbidities such as diabetes in patients should be checked for predisposition, if any, of biofilm formation. Culture results of patients with cholangitis at the time of stent insertion will help to correlate with organisms grown in the biofilms. Protein concentration in the biofilm was significantly higher in patients with cholangitis, lower in the 10 Fr group than the 7 Fr group, and significantly higher in stents of ≥ 6 mo of indwelling time. Polysaccharide concentration in biofilms of stents of male gender as well as in patients with age > 60 years was significant. The most common bacteria identified by PCR alone and/or sequencing were Pseudomonas (n = 38), Citrobacter (n = 23), Klebsiella (n = 22), Staphylococcus (n = 20), Serratia (n = 16), Escherichia coli (n = 14), Streptococcus (n = 13), Enterococcus (n = 13), Aeromonas (n = 12), Proteus (n = 10) and Enterobacter (n = 9). Longer indwelling time of stents and smaller diameter stents are associated with more biofilm formation. Larger diameter (10 Fr) stents should be preferred for the relief of obstructed biliary system by a non-surgical approach in patients with benign or malignant biliary disease. Stents should not be kept in-situ for more than 3-6 mo.

Research perspectives

Our study suggests that 10 Fr stents should be preferred over the 7 Fr stents and stents should be replaced between 3-6 mo. Attempts to prevent biofilm formation should be investigated. Ultrastructural characterization of biofilms in occluded stents should also be done.