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Using rpoS, tolC, ompF, and recA knockouts, we investigated their effect on the physiological response and lethality of cipro oxacin in E. coli growing at different rates on glucose, succinate or acetate. We have shown that, regardless of the strain, the degree of changes in respiration, membrane potential, NAD/NADH ratio, ATP and glutathione (GSH) strongly depends on the initial growth rate and the degree of its inhibition. The deletion of the regulator of the general stress response RpoS, although it in uenced the expression of antioxidant genes, did not signi cantly affect the tolerance to cipro oxacin at all growth rates. The mutant lacking TolC, which is a component of many E. coli e ux pumps, showed the same sensitivity to cipro oxacin as the parent. The absence of porin OmpF slowed down the entry of cipro oxacin into cells, prolonged growth and shifted the optimal bactericidal concentration towards higher values. De ciency of RecA, a regulator of the SOS response, dramatically altered the late phase of the SOS response (SOS-dependent cell death), preventing respiratory inhibition and a drop in membrane potential. The recA mutation inverted GSH uxes across the membrane and abolished cipro oxacininduced H 2 S production. At all growth rates, this mutation reduced logCFU ml -1 by about 3 orders of magnitude. All studied mutants showed an inverse linear relationship between logCFU ml -1 and the speci c growth rate, that is, the activity of antibiotic targets. Mutations shifted the plot of this dependence relative to the parental strain according to their signi cance for cipro oxacin tolerance.

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The ability of hydrogen sulfide (H2S) to protect bacteria from bactericidal antibiotics has previously been described. The main source of H2S is the desulfurization of cysteine, which is either synthesized by cells from sulfate or transported from the medium, depending on its composition. Applying electrochemical sensors and a complex of biochemical and microbiological methods, changes in growth, respiration, membrane potential, SOS response, H2S production and bacterial survival under the action of bactericidal ciprofloxacin and bacteriostatic chloramphenicol in commonly used media were studied. Chloramphenicol caused a sharp inhibition of metabolism in all studied media. The physiological response of bacteria to ciprofloxacin strongly depended on its dose. In rich LB medium, cells retained metabolic activity at higher concentrations of ciprofloxacin than in minimal M9 medium. This decreased number of surviving cells (CFU) by 2–3 orders of magnitude in LB compared to M9 medium, and shifted optimal bactericidal concentration (OBC) from 0.3 µg/mL in M9 to 3 µg/mL in LB. Both drugs induced transient production of H2S in M9 medium. In media containing cystine, H2S was produced independently of antibiotics. Thus, medium composition significantly modifies physiological response of E. coli to bactericidal antibiotic, which should be taken into account when interpreting data and developing drugs.