Study of the contribution of active defense mechanisms to ciprofloxacin tolerance in Escherichia coli growing at different rates
Using rpoS, tolC, ompF, and recA knockouts,
we investigated their effect on the physiological
response and lethality of ciprofloxacin 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 influenced the expression of antioxidant
genes, did not significantly affect the tolerance to
ciprofloxacin at all growth rates. The mutant lacking
TolC, which is a component of many E. coli efflux
pumps, showed the same sensitivity to ciprofloxacin as
the parent. The absence of porin OmpF slowed down
the entry of ciprofloxacin into cells, prolonged growth
and shifted the optimal bactericidal concentration
towards higher values. Deficiency 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 fluxes across the membrane and abolished
ciprofloxacin-induced H2S production. All studied
mutants showed an inverse linear relationship between
logCFU ml-1 and the specific growth rate. Mutations
shifted the plot of this dependence relative to the
parental strain according to their significance for
ciprofloxacin tolerance. The crucial role of the SOS
system is confirmed by dramatic shift down of this plot
in the recA mutant.
