PII-203 - KLEBSIELLA PNEUMONIAE CONTRIBUTES TO REDUCE THIOPURINES CYTOTOXICITY IN VITRO: POSSIBLE IMPLICATIONS OF BACTERIAL METABOLISM

M. Franzin¹, C. Lagatolla², M. Haag³, S. Neef³, V. Iebba², M. Comar¹, E. Schaeffeler³, B. Bellich¹, M. Bramuzzo¹, G. Decorti², M. Lucafò², U. Hofmann³, M. Schwab³, G. Stocco²; ¹Institute for Maternal and Child Health IRCCS Burlo Garofolo, ²University of Trieste, ³Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology.

Background: Pediatric inflammatory bowel disease (pIBD) patients undergo thiopurine treatment, but some patients do not respond. Gut bacteria influence efficacy of therapies and the overgrowth of Enterobacteriaceae in gut microbiota of pIBD patients. Therefore, this study investigated if candidate Enterobacteriaceae, potentially harmful and abundant, modulate thiopurines effects and concentration.

Methods: Azathioprine (AZA), mercaptopurine (MP) and thioguanine (TG) (400 µM) were exposed in vitro to E. coli, S. enterica and K. pneumoniae for 4h at 37°C and drug concentration was measured by UV. Cytotoxicity of drugs exposed was assessed by a 72h-treatment on NALM6 and JURKAT cells by MTT assay. Concentrations of intracellular thiopurine metabolites and deoxythioguanosine (dTGUA) in DNA were evaluated after 72h-treament with drugs exposed or not to K. pneumoniae. Thiopurines were measured in K. pneumoniae lysates. Metabolomics was performed on K. pneumoniae conditioned media. Bacterial relative abundance was analyzed by shotgun metagenomic sequencing in fecal stools and thiopurine metabolites were quantified in erythrocytes of 8 pIBD patients.

Results: Exposure of thiopurines to K. pneumoniae, but not to E. coli and S. enterica, reduced the concentrations of AZA, MP and TG by 26, 35 and 36% respectively (t-test p < 0.05), with MP detected in K. pneumoniae bacteria incubated with AZA or MP (2.71 µM ±0.10 and 128.90 µM ±1.25) and TG after incubation with TG (142.85 µM ±1.35). NALM6 and JURKAT cells treated with drugs previously exposed to K. pneumoniae, showed significantly reduced cytotoxic effects, intracellular thiopurine metabolites and incorporation in DNA of dTGUA. Metabolomics putatively identified endogenous bacterial metabolites related to metabolism of thiopurines, as well as drug-derived molecules, through reactions of deconjugation, reduction, glycosylation, acetylation or conjugation with propionic acid. K. pneumoniae fecal abundance was associated with lower thiopurine metabolites concentrations in erythrocytes of pIBD patients (rho = -0.79, p= 0.019).

Conclusion: K. pneumoniae reduces the effects of thiopurines in vitro by internalising them. Metabolomics indicates drugs biotransformation and K. pneumoniae abundance may mediate clinical efficacy in pIBD.