Klebsiella pneumoniae contributes to altered cytotoxicity of thiopurines in vitro: Possible implications of biotransformation and bacterial metabolism

Background and purpose: Thiopurines are used in paediatric inflammatory bowel disease (IBD), but some patients do not respond. Because the gut microbiota influences drug efficacy and IBD-patient microbiota presents increased bacterial abundance, we investigated the impact of candidate Enterobacteriaceae on drug cytotoxicity, metabolism and efficacy. Experimental approach: Thiopurines were exposed in vitro to bacteria for 4 h at 37°C and drug concentrations measured by UV spectrophotometry. Cytotoxic effects and drug metabolite concentrations on NALM6 and JURKAT cells were determined after treatment with thiopurines exposed or not to bacteria. Drugs were measured in Klebsiella pneumoniae lysates and bacterial conditioned media were used for metabolomic analyses. Shotgun metagenomic sequencing was performed on eight IBD-patient faecal stools. Key results: Incubation of thiopurines with K. pneumoniae, but not Escherichia coli and Salmonella enterica, reduced thiopurine concentrations and cytotoxicity on NALM6 and JURKAT cells. Thiopurine metabolites were lower in cells treated with drugs previously exposed to K. pneumoniae. Internalisation of drugs was demonstrated by their detection in lysates after bacterial incubation. Untargeted metabolomics revealed biotransformation of thiopurines by K. pneumoniae, as reactions of deconjugation, reduction, glycosylation, acetylation or conjugation with propionic acid. Incubation with thiopurines led to changes in the secretion of endogenous bacterial metabolites. K. pneumoniae faecal abundance was associated with lower thiopurine metabolite concentrations in erythrocytes of paediatric IBD-patients. Conclusions and Implications: K. pneumoniae decreases the cytotoxicity of thiopurines through internalisation of MP and TG. We revealed potential bacterial drug biotransformation, as well as negative correlations between bacterial abundance and drug metabolites.