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

Ute Hofmann, Elke Schaeffeler, Gabriele Stocco, Marianna Lucafò, Giuliana Decorti, Matteo Bramuzzo, Mathias Haag, Matthias Schwab, Martina Franzin, Cristina Lagatolla, Sofia Sindici Forgiarini, Sylvia Karin Neef, Manola Comar, Barbara Bellich

Journal: British journal of pharmacology 2025;182(18):4281-4298

PMID: 40437943

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Abstract

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.

Address: Department of Advanced Translational Diagnostics, Institute for Maternal and Child Health, IRCCS 'Burlo Garofolo', Trieste, Italy.; Department of Life Sciences, University of Trieste, Trieste, Italy.; Department of Medical, Surgical and Health Sciences, University of Trieste, Trieste, Italy.; Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.; University of Tuebingen, Tuebingen, Germany.; Department of Medical, Surgical and Health Sciences, University of Trieste, Trieste, Italy.; Department of Advanced Translational Microbiology, Institute for Maternal and Child Health, IRCCS 'Burlo Garofolo', Trieste, Italy.; Pediatric Gastroenterology, Digestive Endoscopy and Clinical Nutrition Unit, Department of Pediatric, Institute for Maternal and Child Health IRCCS 'Burlo Garofolo', Trieste, Italy.; Department of Advanced Translational Diagnostics, Institute for Maternal and Child Health, IRCCS 'Burlo Garofolo', Trieste, Italy.; Department of Medical, Surgical and Health Sciences, University of Trieste, Trieste, Italy.; Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.; Departments of Clinical Pharmacology, and of Biochemistry and Pharmacy, University of Tuebingen, Tuebingen, Germany.

DOI: 10.1111/bph.70089

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MeSH Terms: Humans, Klebsiella pneumoniae, Biotransformation, Jurkat Cells, Inflammatory Bowel Diseases, Feces, Gastrointestinal Microbiome, Mercaptopurine, Cell Survival, Child