The effects of nalidixic acid and four fluoroquinolones on DNA, RNA, and protein synthesis in the presence and absence of 20 mg of chloramphenicol per liter were examined by comparing the killing kinetics, MIC, morphological response, and maximum concentration to induce recA in Eschenichia coli. All agents demonstrated paradoxical killing kinetics, in that above an optimum concentration the rate of bactericidal action was slower. Filamentation of E. coli AB1157 was observed with all quinolones up to the optimum bactericidal concentration. Addition of chloramphenicol reduced the bactericidal activity, inhibited filamentation, and abolished recA induction, but it had no effect on DNA synthesis inhibition by any of the agents. Excellent correlation was obtained between the concentration required to inhibit DNA synthesis by 50%, the MIC, the maximum concentration to induce recA, and the optimum bactericidal concentration. Evidence from this study and previously published data suggest that the primary mechanism of action of quinolones is independent of the SOS response and does not require active protein synthesis; however, induction of recA and SOS responses is consequential and enhances cell death.The precise mechanisms of action of quinolone (including fluoroquinolone) antimicrobial agents have yet to be determined; however, one of the major interactions of quinolones is that with the enzyme DNA gyrase (topoisomerase II). It has been shown by two assays (one that measures the conversion of relaxed plasmid DNA to its native supercoiled form and the other that measures the production of linear DNA in a cleavage reaction) that quinolones inhibit the supercoiling activity of DNA gyrase (10,15,32,36). It has been postulated that nalidixic acid inhibits the resealing of DNA that occurs at the replication fork catalyzed by DNA gyrase, thereby preventing supercoiling (11). It is proposed that the complex of quinolone plus DNA gyrase is bound to the DNA, forming a replication fork barrier and allowing the accumulation of gapped or single-stranded DNA (25), which has been shown to accumulate in nalidixic acid-treated Escherichia coli (7). Quinolones induce the SOS response (DNA repair mechanism [4,14,22,23]), the inducing signal for which is thought to be gapped DNA (25). While there is a large body of evidence that DNA gyrase is the major target of quinolones, measurements of the concentration of a quinolone required to inhibit in vitro supercoiling activity often produce values that are 10-fold higher than the MIC (10,16,27). The poor correlation is unlikely to be due to a partially denatured DNA gyrase preparation (A. Maxwell, personal communication).The concentration of a quinolone needed to inhibit in vivo DNA synthesis by 50% (IC50) and quinolone MICs show an excellent correlation (2; J. M. Diver, L. J. V. Piddock, and R. Wise, Proc. 15th Int. Congr. Chemother., abstr. 985, 1987), and it has therefore been hypothesized that the initial reaction in the cascade of events causing quinolone-induced * Corresponding author...

The bactericidal effects of five quinolones (at the optimum bactericidal concentration for strain AB1157) on 15 strains of Escherichia coli with mutations in genes for the SOS response or cell division was studied by a viable-count method. The kill rate data were normalized for growth rate and compared to those for the wild type, AB1157. Similar MICs of enoxacin and fleroxacin were obtained for all mutants; however, different mutants had differing susceptibilities to ciprofloxacin, norfloxacin, and nalidixic acid. Killing kinetic studies showed that mutants with constitutive RecA expression (recA730 and spr-55 mutants) survived longer than AB1157 with all quinolones. Mutants deficient in SOS induction, e.g., recA430 and kx43 mutants, also survived longer, suggesting that induction of the SOS response by quinolones is harmful to wild-type cells.Recombination repair-deficient mutants (recB21, recC22, and recD1009 mutants) were killed more rapidly than AB1157, as were excision repair mutants, except with nalidixic acid. Mutants which were unable to filament (sfiAll and sfiB114 mutants) survived longer than AB1157 with all agents, but a mutant defective in the Lon protease was killed more quickly. It was concluded that (i) recombination and excision repair were involved in the repair of quinolone-damaged DNA and (ii) continuous induction (in response to exposure to quinolones) of the SOS response, and hence induction of the cell division inhibitor SfiA, causes cell filamentation and thereby contributes to the bactericidal activity of quinolones.The bactericidal mechanism of quinolones is unclear; however, it has been proposed that the initial event is the inhibition of DNA synthesis (5, 35) by interference with the nick-sealing activity of DNA gyrase (9,14,29 There is excellent correlation between the concentration of quinolones required to inhibit DNA synthesis in Escherichia coli by 50% and the MIC (5, 31). However, the inhibition of protein synthesis by chloramphenicol decreases the bactericidal activity of quinolones (36) and inhibits induction of RecA (32) but has no effect on the inhibition of DNA synthesis (7, 31). Therefore, it has been proposed that the primary event in the bactericidal action of quinolones is the inhibition of DNA synthesis due to the interaction with DNA gyrase; induction of the SOS response is consequential but also contributes to the bactericidal activity of these agents (31).Nalidixic acid and UV light have been shown to induce RecA, the SOS regulatory protein in E. coli (13,18) tion between the concentration of a quinolone causing maximum RecA induction, the optimum bactericidal concentration (OBC), and the MIC (30,31,37). At concentrations above the OBC and the maximum RecA-inducing concentration, quinolones inhibit protein synthesis, which is required for expression of SOS genes.The physiological consequences of SOS induction include the inhibition of cell division and enhanced DNA repair (38). Induction of the SOS response may enhance the survival of a damaged cell because of incr...

The NCTC type strains of Escherichia coli, Enterobacter cloacae, Serratia marcescens and Klebsiella pneumoniae were exposed to 3, 5 and 10 x MIC of nalidixic acid, enoxacin, ciprofloxacin, PD 117596 and PD 127391. From each strain a mutant with a high MIC of quinolones alone (gyrA) and a mutant with intermediate resistance to quinolones, some beta-lactams, chloramphenicol and tetracycline (multiply resistant, m-r) were selected on agar containing antibiotics. The gyrA mutants required a higher concentration of quinolone to inhibit DNA synthesis by 50% but quinolone uptake kinetics and outer membrane profile were the same as the wild type. The m-r mutants had similar DNA synthesis IC50 as the wild type, decreased quinolone uptake kinetics and had decreased expression of an OMP of approximately 40 kD. The gyrA and m-r mutants were then exposed to 3, 5 and 10 x MIC of the same quinolones and new mutants (F2) selected. The F2 mutants from the gyrA mutants displayed a further increase in quinolone MIC; the F2 mutants from the m-r mutants had several phenotypes: high quinolone MICs with cross resistance to other agents, high quinolone resistance alone, or intermediate quinolone resistance alone. Most F2 mutants had MICs above the recommended breakpoint concentrations for quinolones. The F2 mutants often had altered biochemical profiles (API 20E), however, only in the case of E. cloacae did this affect speciation with the strains being identified as Rhanella aquatalis.

The SOS response is induced in Escherichia coli by agents that damage DNA, such as quinolone antibiotics. It has been proposed that induction of the SOS response by these agents may have a role in the mechanism of quinolone action. SOS mutants derived from Escherichia coli AB1157 were investigated by susceptibility testing and killing kinetic studies at various quinolone concentrations to determine whether SOS response induction was protective or damaging to quinolone-treated bacteria. Susceptibility testing showed some differences between the SOS mutants, but killing kinetic studies demonstrated further differences, some of which could be explained with respect to the SOS phenotype. The effect of ciprofloxacin and nalidixic acid on the mutants cannot be explained with respect to the SOS phenotype, although the presence of a defective SOS response makes the bacteria less sensitive to the action of these agents. Evidence is provided that the induction of the SOS response may be protective to fleroxacin and enoxacin treated bacteria. These results suggest that quinolones may not have a common mechanism of action, as was first thought.