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

Thiopurine drugs are widely used in the maintenance of remission in pediatric inflammatory bowel disease (IBD) but, despite their proven efficacy, some patients do not respond to treatment. A contributing factor influencing the efficacy of drug therapies is the gut microbiota. Pediatric IBD is often accompanied by an altered microbiota with an increase of Enterobacteriaceae. We therefore investigated the impact of candidate bacterial strains, belonging to this family, on the efficacy and cytotoxicity and metabolism of thiopurines in vitro.Azathioprine (AZA), mercaptopurine (MP) and thioguanine (TG) were provided in vitro to bacteria for 4h at 37 degrees Celsius and the drug concentrations measured by UV spectrophotometry. Cytotoxic effects of thiopurines previously provided or not to bacteria were assessed by treatment of immortalized cell lines (NALM6 and JURKAT cells) for 72 h. Intracellular concentrations of thiopurine metabolites and incorporation of deoxythioguanosine (dTGUA) in DNA were determined after treatment of NALM6 and JURKAT with thiopurines provided to K. pneumoniae. Thiopurines were measured in K. pneumoniae lysates and bacterial conditioned media were used for metabolomic analyses. Shotgun metagenomic sequencing was performed on fecal samples of 8 CD pediatric patients.Incubation of thiopurines with K. pneumoniae, but not with E. coli and S. enterica reduced cytotoxic effects on NALM6 and JURKAT cells. Exposure to this bacterial strain decreased the concentration of AZA, MP and TG by 26, 35 and 36 percent respectively. Thiopurine metabolites were lower in NALM6 and JURKAT cells treated with drugs previously exposed to K. pneumoniae and the concentrations of metabolites clustered according to the exposure status (p = 0.041). Concentrations of dTGUA in DNA were lower in cells treated with drugs previously incubated with the bacterial strain. Internalization of the drugs could be demonstrated by detection of MP in the lysate of K. pneumoniae previously incubated with AZA or MP at 400 micromolar; whereas TG was measured in the lysate after bacterial incubation with this drug. Metabolomics revealed biotransformation of the drugs by K. pneumoniae, tentatively assigned to deconjugation, reduction, glycosylation, acetylation or conjugation with propionic acid. Incubation with thiopurine drugs led to changes in the secretion of endogenous bacterial metabolites. Furthermore, K. pneumoniae was associated to lower TGN concentrations in red blood cells of CD pediatric patients.We could show that K. pneumoniae decreases the cytotoxicity of thiopurines in vitro and elucidate the underlying mechanisms of interference. MP and TG are internalized in the bacteria, thus reducing the availability. We found evidence for biotransformation of the drugs as well as for drug induced modified bacterial metabolism. Furthermore, the pilot study on CD pediatric patients confirmed a role of K. pneumoniae in influencing the drug bioavailability in vivo.