Synergistic Effect of SOS Response and GATC Methylome Suppression on Antibiotic Stress Survival in Escherichia coli

Abstract

The suppression of the SOS response has been shown to enhance the in vitro activity of quinolones. Furthermore, Dam-dependent base methylation has an impact on susceptibility to other antimicrobials affecting DNA synthesis. Here, we investigated the interplay between these two processes, alone and in combination, in terms of antimicrobial activity. A genetic strategy was used employing single- and double-gene mutants for the SOS response (recA gene) and the Dam methylation system (dam gene) in isogenic models of Escherichia coli both susceptible and resistant to quinolones. Regarding the bacteriostatic activity of quinolones, a synergistic sensitization effect was observed when the Dam methylation system and the recA gene were suppressed. In terms of growth, after 24 h in the presence of quinolones, the Δdam ΔrecA double mutant showed no growth or delayed growth compared to the control strain. In bactericidal terms, spot tests showed that the Δdam ΔrecA double mutant was more sensitive than the ΔrecA single mutant (about 10- to 10²-fold) and the wild type (about 10³- to 10⁴-fold) in both susceptible and resistant genetic backgrounds. Differences between the wild type and the Δdam ΔrecA double mutant were confirmed by time-kill assays. The suppression of both systems, in a strain with chromosomal mechanisms of quinolone resistance, prevents the evolution of resistance. This genetic and microbiological approach demonstrated the enhanced sensitization of E. coli to quinolones by dual targeting of the recA (SOS response) and Dam methylation system genes, even in a resistant strain model.

Keywords: Dam methylation system; antibiotic stress; quinolones; recA gene; resistance reversion.

FIG 1

Impact of recA gene inactivation and Dam methylation system suppression on cell growth in the presence of sublethal concentrations of quinolone. (A) OD values for E. coli BW25113 and all genetic combinations at ciprofloxacin concentrations of 0.002 μg/mL over 24 h. (C) OD values for E. coli BW15 and all genetic combinations at ciprofloxacin concentrations of 0.25 μg/mL over 24 h. (B and D) OD values with no antibiotics over 24 h. Data are presented as the means from at least three independent measurements. Standard deviations are indicated by error bars.

FIG 2

Survival of E. coli BW25113 mutants (A) and E. coli BW15 mutants (B) treated with ciprofloxacin as determined by spot tests. Liquid cultures grown overnight were serially diluted and plated onto LB agar with and without ciprofloxacin concentrations of 0.001 μg/mL (BW25113 group) and 0.375 μg/mL (BW15 group). The results shown are representative of data from at least three independent experiments.

FIG 3

The combination of recA gene inactivation and Dam methylation system suppression enhances the synergistic bactericidal activity of quinolones. Viable bacterial counts of E. coli BW25113, E. coli BW25113 Δdam, E. coli BW25113 ΔrecA, and E. coli BW25113 Δdam ΔrecA (A) and viable bacterial counts of E. coli BW15, E. coli BW15 Δdam, E. coli BW15 ΔrecA, and E. coli BW15 Δdam ΔrecA (B) were determined in time-kill assays at ciprofloxacin (CIP) concentrations of 8× MIC for each strain during 24 h. Data are presented as the means from at least three independent measurements. Error bars represent standard deviations.

FIG 4

Curves showing the culture growth of E. coli BW25113 and all genetic combinations (A) and E. coli BW15 and all genetic combinations (B) with increasing concentrations of antibiotics. Shown are representations of the numbers of viable populations over time. Sixteen populations of each strain were propagated under increasing concentrations of ciprofloxacin, starting with 6.25 × 10⁻⁵ μg/mL for the E. coli BW25113 group and 1.25 × 10⁻² μg/mL of the antibiotic on day 1 and doubling the antibiotic concentration each day. The dashed vertical line represents the wild-type and E. coli Δdam ΔrecA (BW25113 and BW15) MICs of ciprofloxacin.

FIG 5

Diagram illustrating synergistic quinolone sensitization by targeting the SOS response and the Dam methylation system.