Leishmania respiration profiling as a One Health functional marker associated with antimony resistance in human and canine leishmaniases

Rapid functional tools for assessing antimony susceptibility in Leishmania are needed to strengthen resistance surveillance across human and veterinary settings. We developed a real-time oxygen-consumption assay using the Resipher analyzer and evaluated normalized oxygen-consumption rate (OCR) as a functional correlate of antimony (Sb) resistance in Leishmania infantum and Leishmania donovani . A density of 1 × 10 7 parasites/mL and a 48-h readout provided the most robust analytical window, and rotenone markedly reduced OCR, demonstrating assay responsiveness to respiratory perturbation. Antimony-resistant laboratory lines showed lower OCR/proliferation ratios than the wild-type strain. This phenotype was reproduced in ten canine L. infantum field isolates and ten human L. donovani clinical isolates. OCR/proliferation ratio was inversely correlated with promastigote ( L. infantum : r = −0.8930, p = 0.0008; L. donovani : r = −0.8470, p = 0.0019) and intracellular-amastigote Sb EC 50 values ( L. infantum : r = −0.8787, p = 0.0008; L. donovani : r = −0.7964, p = 0.0058). Under isolate-specific Sb EC 90 exposure, resistant isolates accumulated less ROS, which was positively associated with OCR/proliferation ratio ( L. infantum : r = 0.6927, p = 0.0390; L. donovani : r = 0.7560, p = 0.0114). Low-OCR isolates were also less sensitive to rotenone and antimycin A, indicating broader bioenergetic remodeling. Normalized OCR is a functional correlate of Sb susceptibility and a complementary phenotypic screening approach for One Health surveillance. Future studies incorporating larger and more genetically diverse isolate collections, prospective susceptibility testing, and inter-laboratory standardization will enable the definition of robust analytical thresholds and facilitate implementation in resistance-surveillance programs.