A preliminary study of bacterial genetic determinants in human gallbladder. A focus on bile resistance

[Background/Aims] The human gastrointestinal microbiota and its relationship with different physiological states has been characterized in detail in the last years. However, the microbiota of bile and gallbladder has been scarcely studied. Also, the functions of the autochthonous biliary microorganisms and the characteristics that allow the survival in this environment have not been investigated. In this context, we have constructed a metagenomic library from human bile of liver donors, in order to analyse the activities responsible for bacterial survival in presence of bile. [Methods] For the generation of the library we used total DNA isolated from human bile. Fragments with a size of approximately 20-30kb were cloned in the vector pCC1FOS™ (Epicentre®) and transformed into competent Escherichia coli. The pool of E. coli clones was tested in different concentrations of bile salts in order to detect highly resistant clones. [Results] Growth experiments allowed us to select five clones with a bile resistance phenotype. The selected clones were able to grow in a bile concentration at least 10 times higher than the E. coli strain containing the empty vector. The five fosmids were sequenced using Illumina technology and the bioinformatic analysis of the sequences showed the presence of a variety of potential genes that could play a role in bile resistance, coding for putative proteins involved in oxidative stress response, as well as DNA repair proteins, transmembrane pumps and lipopolysaccharide biosynthesis enzymes. Interestingly, the majority of DNA sequences displayed a low homology with genomic sequences of known microorganisms, suggesting that the native biliary microorganisms harbouring the DNA inserts could be new microbial taxons. [Conclusions] Five DNA fragments from biliary microorganisms, able to confer bile resistance in E. coli, were identified in our study. Further work is needed in order to unravel the specific DNA sequences responsible for the observed