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.When DNA topoisomerases bind to bacterial DNA, they form complexes that are trapped by the quinolone antibacterials (for a review, see reference 6). These ternary drug-enzyme-DNA complexes block DNA replication (5, 27, 29, 32), RNA synthesis (22, 33), and cell growth (2, 12). A key feature of the complexes is that the trapped DNA moiety is broken, with its ends constrained through covalent linkage to the GyrA protein (24, 25). As a result, quinolone concentrations sufficient to block replication do not relax chromosomal DNA supercoiling (29). When the quinolone is removed, the breaks are readily resealed (11, 31) and the inhibitory effects of the compounds are reversed (13,21,27). Irreversible events that lead to rapid cell death occur at higher quinolone concentrations (1, 20). We have proposed that rapid cell death arises from chromosome fragmentation that occurs when doublestrand DNA breaks are released from the protein-mediated constraint present in ternary complexes. At rapidly lethal quinolone concentrations, supercoils cannot be maintained (1) and fragmented DNA is obtained under conditions that allow resealing at bacteriost...

Antibiotics induce redox-related physiological alterations as part of their lethality

“…6C). These results extend observations by others that anaerobic culture and treatment inhibits killing by quinolones (97).…”

Quinolone-Mediated Bacterial Death

“…1, steps d and e). Such a factor is likely to be short-lived, since the lethal action of nalidixic acid is rapidly blocked by chloramphenicol or by a shift to anaerobiosis (10,51). Although mutants have been obtained that block nalidixic acid lethality without affecting bacteriostatic action (80; X. Zhao, unpublished observation), the genes involved have proven difficult to identify.…”

“…Additional correlations are revealed by two environmental perturbations: anaerobic growth and inhibition of protein synthesis by chloramphenicol. For first-generation quinolones, both perturbations block lethal action and chromosome fragmentation (51,53). However, neither prevents cleaved complex formation, as shown by the presence of DNA breakage when cell lysates are treated with SDS.…”

Hydroxyl Radicals Are Involved in Cell Killing by the Bacterial Topoisomerase I Cleavage Complex

“…The events that follow the stabilization of the covalent topoisomerase complex, leading to bacterial cell death, remain to be elucidated. Previous studies on the effect of DNA replication and protein synthesis on quinolone-mediated cell death suggest that there may be multiple pathways involved, depending on the individual quinolone structure and growth conditions (8,20,27,44). More recent studies suggest that at least part of the bactericidal action of quinolones can be attributed to oxidative damage from reactive oxygen species (10,12,18).…”

“…This study demonstrates that besides the similarity in processing of the covalent protein-DNA complex, type IA and type IIA topoisomerase cleavage complexes also share a common bactericidal pathway. Based on studies of the effects of DNA replication and protein synthesis inhibition, it was proposed that multiple pathways of cell killing are involved in quinolone action, and the individual quinolone structure may be a factor for certain pathways (8,20,27,44). Hydroxyl radical formation has recently been implicated in the cell death caused by bactericidal antibiotics, including quinolones (10).…”