Effect of anaerobic growth on quinolone lethality with Escherichia coli

Abstract

Quinolone activity against Escherichia coli was examined during aerobic growth, aerobic treatment with chloramphenicol, and anaerobic growth. Nalidixic acid, norfloxacin, ciprofloxacin, and PD161144 were lethal for cultures growing aerobically, and the bacteriostatic activity of each quinolone was unaffected by anaerobic growth. However, lethal activity was distinct for each quinolone with cells treated aerobically with chloramphenicol or grown anaerobically. Nalidixic acid failed to kill cells under both conditions; norfloxacin killed cells when they were grown anaerobically but not when they were treated with chloramphenicol; ciprofloxacin killed cells under both conditions but required higher concentrations than those required with cells grown aerobically; and PD161144, a C-8-methoxy fluoroquinolone, was equally lethal under all conditions. Following pretreatment with nalidixic acid, a shift to anaerobic conditions or the addition of chloramphenicol rapidly blocked further cell death. Formation of quinolone-gyrase-DNA complexes, observed as a sodium dodecyl sulfate (SDS)-dependent drop in cell lysate viscosity, occurred during aerobic and anaerobic growth and in the presence and in the absence of chloramphenicol. However, lethal chromosome fragmentation, detected as a drop in viscosity in the absence of SDS, occurred with nalidixic acid treatment only under aerobic conditions in the absence of chloramphenicol. With PD161144, chromosome fragmentation was detected when the cells were grown aerobically and anaerobically and in the presence and in the absence of chloramphenicol. Thus, all quinolones tested appear to form reversible bacteriostatic complexes containing broken DNA during aerobic growth, during anaerobic growth, and when protein synthesis is blocked; however, the ability to fragment chromosomes and to rapidly kill cells under these conditions depends on quinolone structure.

FIG. 1.

Effect of anaerobic conditions on growth of E. coli. Wild-type strain DM4100 was grown under aerobic conditions (filled circles), and at the time indicated by the arrow, part of the culture was shifted to anaerobic conditions (open circles) by passing an anaerobic gas mixture (85% N₂, 10% H₂, and 5% CO₂) through the culture. Culture turbidity was measured with a Klett-Summerson colorimeter.

FIG. 2.

Effect of anaerobic conditions on quinolone lethality. Exponentially growing E. coli (strain DM4100) was treated with nalidixic acid (A), norfloxacin (B), ciprofloxacin (C), or PD161144 (D) for 2 h under aerobic conditions (filled circles), anaerobic conditions (open circles) initiated 20 min before drug addition, or aerobic conditions that included treatment with 20 μg/ml chloramphenicol (filled triangles) added 10 min before the quinolone. For panel C, cells growing anaerobically were treated with chloramphenicol for 10 min, followed by treatment with ciprofloxacin (open triangles). At the end of the incubations, the cells were diluted aerobically, applied to drug-free agar, and incubated to determine the fraction of CFU relative to the number of CFU at the time of quinolone addition. Replicate experiments with each compound produced similar results.

FIG. 3.

Concordance of chloramphenicol and anaerobic effects on quinolone-mediated lethality. Exponentially growing cells (strain DM4100) were treated with 50 μg/ml nalidixic acid (10 times the MIC; filled circles), and after 90 min (arrow) a portion of the culture was shifted to anaerobic conditions (open circles) or was treated with chloramphenicol at 20 μg/ml (filled triangles). At the indicated times, aliquots were processed as described in the legend to Fig. 2. Replicate experiments produced similar results.

FIG. 4.

Effects of gyrA and parC resistance mutations on lethal action of norfloxacin and PD161144. Exponentially growing E. coli mutants were treated with the indicated concentrations of norfloxacin (A to C) or PD161144 (D and E) for 2 h under aerobic conditions (filled circles), anaerobic conditions (open circles), or aerobic conditions following treatment for 10 min with 20 μg/ml chloramphenicol (filled triangles). Following incubation, aliquots were processed as described in the legend to Fig. 2. (A and D) mutant with parC-mediated resistance (strain KD1373); (B and E) mutant with gyrA-mediated resistance (strain KD2750); (C and F) mutant with gyrA- and parC-mediated resistance (strain KD2329). The dotted lines in panels A, B, D, and E represent the results for wild-type cells treated with the quinolone (data from Fig. 2). Replicate experiments with each drug and each strain produced similar results.

FIG. 5.

Quinolone-mediated chromosome fragmentation detected by lysate viscosity. Wild-type E. coli (strain DM4100) was grown exponentially under aerobic or anaerobic conditions and then treated for 2 h with 50 μg/ml nalidixic acid (Nal; 10 times the MIC) (A) or with 0.8 μg/ml PD161144 (10 times the MIC) (B). Cells were also treated with no drug (squares). Cells were gently lysed under aerobic or anerobic conditions, and lysate viscosity was measured as described in Materials and Methods. Open symbols, cells grown and lysed under anaerobic conditions in the absence (circles) or the presence of SDS (triangles); filled symbols, cells grown and lysed under aerobic conditions in the absence (circles) or the presence of SDS (triangles). (C) Effect of 20 μg/ml chloramphenicol (Cm) alone (squares) and in combination with either nalidixic acid (circles) or PD161144 (triangles). SDS was added to (filled symbols) or omitted from (open symbols) the cell lysates. Replicate experiments produced similar results.

FIG. 6.

Schematic representation of quinolone action. (a) Binding of gyrase to DNA; (b) formation of quinolone-gyrase-DNA complexes; (c) lethal chromosome fragmentation that requires ongoing protein synthesis under aerobic conditions; (d) Lethal chromosome fragmentation that requires ongoing protein synthesis but not aerobic conditions; (e) lethal chromosome fragmentation that does not require ongoing protein synthesis or aerobic conditions; (f) DNA breakage detected after treatment of cell lysate with an ionic detergent, such as SDS. (Inset) The use of lethal pathways by each quinolone is indicated; question marks indicate a lack of evidence for particular pathways (see Discussion).