Isolation and characterization of an Escherichia coli strain exhibiting partial tolerance to quinolones

Abstract

Quinolone antimicrobial agents rapidly kill bacteria by largely unknown mechanisms. To study this phenomenon, a strain of Escherichia coli inhibited but inefficiently killed by (i.e., partially tolerant to) norfloxacin was isolated and characterized. E. coli KL16 (norfloxacin MIC, 0.10 ,ug/ml; MBC, 0.20 ,Ig/ml) was mutagenized with nitrosoguanidine and cyclically exposed to 3 ,zg of norfloxacin per ml. After five cycles, a bacterial strain (DS1) which was killed 1,000-fold less than KL16 during 3 h of drug exposure was isolated. The MIC and MBC of norfloxacin for DS1 were 0.20 and 1.5 ,ug/ml, respectively. Over a range of norfloxacin concentrations, DS1 was killed 2 to 4 orders of magnitude less than KL16. DS1 grew more slowly than KL16 but after normalization for growth rate was killed four times less rapidly than KL16 at drug concentrations 10-fold higher than respective MICs. DS1 and KL16 cells ifiamented similarly upon exposure to norfloxacin. DS1 exhibited tolerance to other DNA gyrase A subunit antagonists (ofloxacin and ciprofloxacin) and DNA gyrase B subunit antagonists (novobiocin and coumermycin) but not to the aminoglycoside gentamicin, suggesting involvement of DNA gyrase. DS1 also appeared to be minimally tolerant to the P-lactam cefoxitin.DS1 exhibited increased susceptibility to the mutagen methyl methanesulfonate, implying a defect in DNA repair. This report describes the first use of quinolone enrichment for isolation of a bacterial strain partially tolerant to quinolones. The study of defects in such tolerant strains offers an approach to an increased understanding of the mechanisms of bacterial killing by quinolones.

Mutants of Escherichia coli K-12 exhibiting reduced killing by both quinolone and beta-lactam antimicrobial agents

“…Media were supplemented with 75 jig of diaminopimelic acid per ml for diaminopimelic acidrequiring strains. Determlinations of MICs and MBCs were performed as described previously (62), except for studies with mutants HM21 and HM23, for which initial inocula were 106 CFU (rather than 104 CFU) in 1 ml of broth. Time-killing studies and drug concentration-killing studies were done as described previously (62), except for drug concentration-killing studies with rifampin, for which bacteria were exposed to drug for 4 and 7 h as well as 3 h. All experiments were done two or more times.…”

“…To study the killing phenomenon, we isolated DS1, a mutant strain of E. coli KL16 which exhibits reduced killing by quinolones and antagonists of the DNA gyrase B subunit but not by the aminoglycoside gentamicin (62). DS1 unexpectedly exhibited reduced killing by the ,-lactam cefoxitin (62).…”

“…Exposure of E. coli to a quinolone results in an initial rapid decrease in cell viability followed by a slowing or cessation of killing (6,8,11,12,18,21,47,62). The refractory subpopulation is composed of persisters (3,24,37) which are neither resistant mutants nor mutants that are less effectively killed on growth and reexposure to drug (62). Additional characteristics of killing by quinolones include a paradoxical reduction in killing at high drug concentrations (9,12,47,59); reduced killing in the presence of chloramphenicol, rifampin, or other treatments that inhibit protein synthesis (suggesting a requirement for synthesis of a protein[s] for killing [8,9,11,12,47,48,51,59,65]); differences in the * Corresponding author.…”

“…In An important property of quinolones is their ability effectively to kill many bacterial species (11,12,47,60). Exposure of E. coli to a quinolone results in an initial rapid decrease in cell viability followed by a slowing or cessation of killing (6,8,11,12,18,21,47,62). The refractory subpopulation is composed of persisters (3, 24, 37) which are neither resistant mutants nor mutants that are less effectively killed on growth and reexposure to drug (62).…”

Single-cell protein induction dynamics reveals a period of vulnerability to antibiotics in persister bacteria

“…In previous work, we demonstrated that persistence originates in subpopulations of slowly or nongrowing bacteria that form part of the bacterial population already before the exposure to antibiotics (15). Two types of persistent bacteria were identified in WT and in the high-persistence (hip) strains of Escherichia coli (16,17): (i) Type I persisters, which enter at stationary phase (18), a dormant state that protects them from the lethal action of several antibiotics known to affect mainly actively growing cells (15,19); and (ii) Type II persisters, which do not require a starvation signal to enter the persister state and are continuously generated from the normally growing cells. In the present work, we focus on Type I persistence to antibiotic treatments such as those found in hipA7 (20), which has been observed in several WT bacterial strains and is today believed to be at the root of persistent infections (13,21).…”