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Background Proton pump inhibitor ( PPI) use is associated with an increased risk of Clostridium difficile infection ( CDI), though the mechanism is unclear. PPI induced alterations to the gut microbiome may facilitate the emergence of CDI, though the effects of PPIs on gut microbiota are not well characterised. [Correction added on 10 March 2016, after first online publication: microflora has been changed to microbiota throughout the article.] Aim To compare the faecal microbiomes of long-term PPI users to those with no history of PPI use. Methods We used a population-based database to identify individuals with ≥5 years of continuous PPI use along with non- PPI using controls. Stool samples were subjected to microbiological analysis, with hierarchical clustering at genus level, along with alpha and beta diversity measures comparing the two groups. Metadata was accounted for using quantile regression to eliminate potential confounding variables in taxonomic abundance comparisons. Results Sixty-one subjects (32 PPI, 29 controls) were analysed. While no significant differences in alpha diversity were found between the PPI users and controls, a moderate shift of the PPI users away from the non- PPI user cluster in the beta diversity was observed. After controlling for pertinent confounders, we discovered a decrease in Bacteroidetes and an increase in Firmicutes at the phylum level. We also performed species classifications and found Holdemania filiformis and Pseudoflavonifractor capillosus to be increased and decreased in the PPI cohort, respectively. Conclusions Long-term PPIs use has an effect on the gut microbiome. The alteration in the ratio of Firmicutes to Bacteroidetes may pre-dispose to the development of CDI. [ABSTRACT FROM AUTHOR]