Biliary stents inserted to relieve obstructive jaundice caused by biliary or pancreatic malignancies inevitably become occluded by microbial growth in the form of diverse microbial community biofilms. The scarce information available on these communities is based on cultivation methods, but such methods usually provide distorted overviews of community composition, so commonalities and differences in biliary stent communities are uncertain. We extracted DNA and RNA from the microbial communities of 11 biliary stents explanted from nine patients in hospitals from two different countries, amplified 16S rRNA and rDNA sequences, analysed the amplicons by the single-strand conformation polymorphism (SSCP) method, and sequenced and deduced phylogenetic assignments of the major amplicons representing the major biofilm community members. We used a Modified Robbins Device (MRD) to study de novo development of a stent biofilm from a patient stent microbial community. Single-strand conformation polymorphism fingerprinting revealed the same six abundant bacterial species, here designated Leitbakteria, namely Klebsiella pneumoniae, Enterococcus faecalis, Pseudomonas aeruginosa, Enterobacter aerogenes, and two unculturable bacteria distantly related to E. coli and Shigella sonnei, in all of the stent biofilm communities. In the experimental biliary stent system, a sequential colonization of the stent surface was observed, with P. aeruginosa being the pioneer colonizer, followed by K. pneumoniae and one of the unculturable Leitbakteria, followed by the remainder of the community. The overview of microbial biofilm communities of biliary stents gained by the use of culture-independent methods revealed new unculturable bacteria as major members of biliary stent biofilms, and the diversity of the abundant members of the stent biofilms is considerably lower than suggested from earlier studies based on cultivation methods, and that communities from different stents from different patients in different countries are remarkably similar and have similar major members, the stent Leitbakteria.

This study provides a comprehensive survey of the spatial and temporal bacterial composition of biliary stent biofilms. The bacterial diversity, distribution and dynamics of 59 biliary and 4 pancreatic stent communities from 40 patients being treated at two different hospitals, which implant stents either simultaneously or consecutively, were characterized by single-strand conformation polymorphism (SSCP) analysis. Fifty-one phylotypes belonging to 5 bacterial phyla and 24 bacterial families were detected across 63 stents. This is a much broader diversity than previously detected through culture-dependent methods, particularly in regard to the diversity of obligate anaerobes. Stent bacterial diversity was patient-dependent and more similar when stents were implanted simultaneously rather than consecutively. Stent bacterial community composition differed between hospitals specifically because of the difference in abundance of Bifidobacteria. Co-colonization of Veillonella sp., Streptococcus anginosus and organisms closely related to Fusobacterium nucleatum revealed a potentially important attachment and survival strategy that has yet to be reported in biliary stents. This work reveals a more complete survey of the identities of bacterial species that form biofilms in biliary stents, their co-colonization patterns and the natural variation in species composition between different patients, hospitals and locations along the stent. Consideration of the community composition from individual patients will allow tailoring of prophylactic antibiotic treatments and thus will make the management of stent biofilms more effective.