Oleksandr Ilchenko ^1,2 Elena Nikolaevskaya ³ Oksana Zinchenko ² Volodymyr Ivanytsia ² Cristina Prat Aymerich ⁴ Madeleine Ramstedt ⁵ Olena Rzhepishevska ^5*

• ¹ Department of Chemistry, Faculty of Science and Technology, Umeå University, Umeå, Västerbotten, Sweden
• ² Odessa I.I.Mechnikov National University, Odesa, Ukraine
• ³ Center for Socially Significant Diseases of Odesa Regional Council, Odesa, Ukraine
• ⁴ European Clinical Research Alliance on Infectious Diseases, Utrecht, Netherlands, Netherlands
• ⁵ Umeå University, Umeå, Västerbotten, Sweden

Introduction: tuberculosis (TB) is treated using a combination of four antibiotics based on the drug resistance of the infecting strain. Growing drug resistance of Mycobacterium tuberculosis (Mtb) requires novel antibiotics to secure effective regimens. Gallium (Ga) is being assessed as a repurposed drug against TB because it inhibits the growth and disrupts iron metabolism in Mtb. As Ga interaction with established antibiotics can be critical, we investigated how a combination of Ga with levofloxacin (Lfx) or linezolid (Lzd) affects growth and metabolome of a multidrug-resistant (MDR) Mtb clinical strain. Methods: Mtb was cultured using a BACTEC 960 system with 125 to 1000 μM Ga, and 250 to 500 μM Ga with 0.125 mg/L of Lfx or Lzd. For metabolome analysis, the antibacterials were used at the concentrations that inhibits the growth of bacteria but does not to kill them. Metabolites were extracted from Mtb cells and analysed using chromatography-mass spectrometry. Results: MDR Mtb strain responded to Ga in a dose-dependent manner. When compared to Ga alone, the increase in growth inhibition was statistically significant only for Ga/Lfx but not for Ga/Lzd. Moreover, Mtb exposure to Ga/Lfx or Ga/Lzd resulted in distinct metabolite profiles. Ga increased the levels of aconitate, fumarate and glucose in cells suggesting an inhibition of iron-dependent aconitase and fumarate hydratase and the inhibition of pentose phosphate pathway. Levels of glucose, succinic acid, citric acid and hexadecanoic acid followed a similar pattern in cells exposed to Ga and Ga/Lfx at 500 μM Ga but differed at Ga 250 μM. Discussion: In presence of Lfx, the Mtb metabolome features triggered by Ga are more pronounced than in presence of Lzd. Lfx affects nucleic acids and transcription, and, in this way, may potentiate Ga-dependent growth inhibition by preventing the expected redirection of metabolism used by bacteria to sidestep the iron-dependent enzymes.