Elena G. Salina ^1* Billy A. Martini ¹ Vladimir V. Sorokin ² Andrey L. Mulyukin ²

• ¹ Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia
• ² Winogradsky Institute of Microbiology, Research Center of Biotechnology of the Russian Aca, Moscow, Russia

Regarding the current need in predictive persisting models for Mycobacterium abscessus, we adopted the classical assay for studying drug-tolerant bacterial persisters, focusing on the behavior of a small mycobacterial antibiotic-insensitive subpopulation during prolonged exposure to moxifloxacin. Our study showed a widely ranging response of M. abscessus depending on the antibiotic concentration, the growth stage of mycobacterial cultures, and the availability of potassium ions in the medium. Middle logarithmic cultures, which were initially grown in the balanced or K + -free medium, contained small sup-populations to be competent for prolonged and stable survival in the presence of moxifloxacin. The response of mid-log cultures to the antibiotic attack involved killing with further re-growth upon exposure to 1 -2 MBCs of moxifloxacin or profound elimination of antibiotic insensitive subpopulation to < 10 2 CFU/mL in the presence of moxifloxacin at 16 MBCs. A stage when the number of viable survived cells was constant occurred for stationary-phase cultures grown in the complete medium and exposed to high moxifloxacin dose. On the contrary, antibiotic insensitive subpopulations in stationary-phase M. abscessus cultures that faced potassium deficiency underwent gradual killing during exposure to moxifloxacin in a wide concentration range (1 -16 MBCs). Electron microscopy studies demonstrated that singular cells were rapidly destructed after relatively short-term exposure to moxifloxacin, unlike cells in aggregates or clumps which disappeared afterwards and provided an explanation for the delayed biocidal effect. A small subpopulation that survived under harsh moxifloxacin pressure was remarkably heterogenous in cell morphology and fine structure and was composed of ovoid forms and cell-wall deficient cells with reduced size. The obtained results predict a possibility of distinct effects from the same antibiotic dose on M. abscessus cells depending on their physiological state and abundance in an infected cell or tissue. Taken together, our study may contribute to the development of strategies for combatting recalcitrant survivor subpopulations.