Mechanisms of quinolone resistance in a clinical isolate of Escherichia coli highly resistant to fluoroquinolones but susceptible to nalidixic acid

Abstract

Two associated resistance mechanisms were found in a nalidixic acid-susceptible (4 ,ug/ml) but fluoroquinolone-resistant (8 to 16 ,ug/ml) strain of Escherichia coli Q2 selected under norfloxacin therapy. As compared with the susceptible E. coli Ql isolated before treatment, changes in outer membrane proteins and lipopolysaccharides in Q2 were associated with a 1.5-to 3-fold decrease in the uptake of fluoroquinolones but not nalidixic acid. A 50% inhibition of DNA synthesis in toluene-permeabilized cells of the resistant strain E. coli Q2 required up to 500-fold increased quantities of fluoroquinolones, whereas such inhibition was obtained in both E. coli Ql and Q2 with similar amounts of nalidixic acid. Selection from E. coli Ql on norfloxacin of one-step resistant mutants resembling E. coli Q2 was unsuccessful. From these results we infer that a decrease in outer membrane permeability, associated with a peculiar alteration of the DNA gyrase, was responsible for the unusual quinolone resistance phenotype of E. coli Q2.Quinolones are synthetic antibiotics which can be grouped into the older compounds nalidixic acid, pipemidic acid, piromidic acid, and flumequine and the newer, fluorinated quinolones such as norfloxacin pefloxacin, ofloxacin, and ciprofloxacin. The latter have comparatively enlarged spectra of activity as well as better intrinsic activities. Many studies have been carried out with quinolone-resistant gramnegative bacilli. Two main mechanisms of resistance which may occur either alone or combined have been found (15): (i) modification of the DNA gyrase in either subunit A (8, 10, 15, 20) or B (16, 21), which can give rise to moderate or high levels of resistance, and (ii) decreased uptake of quinolones, which correlates with a decrease in the quantity of porins (5, 10, 13-15). Another not totally elucidated mechanism involving the marA gene may be responsible for low levels of resistance (7).It was the purpose of this study to describe the mechanism(s) which could explain the particular profiles of resistance to fluroroquinolones and susceptibility to nalidixic acid observed in a clinical isolate of Escherichia coli. Hinton agar by using a Steers-type replicator device with ca. 104 CFU per spot and were read after 18 h of incubation at 37°C. MICs of novobiocin were determined as the quantities of antibiotic necessary to totally inhibit the growth of 100 CFU per plate after 18 h of incubation at 37°C (21). Selection of resistant mutants. Mutants resistant to norfloxacin were selected in vitro on plates containing a gradient of the antibiotic from 0 to 8 pug/ml.Extraction of OM proteins and LPS. Cell membranes were prepared from 200 ml of exponential-phase cultures at an optical density at 650 nm of 0.4 as previously described (19). Outer membrane (OM) proteins were obtained as follows: 100 jig of total-membrane protein was incubated for 45 min at 25°C in 70 RI of sodium phosphate buffer (50 mM, pH 7.0) containing 0.3% of N-laurylsarcosine N197 (ICN Biochemicals, Cleveland, Ohio) and centrifuged at ...

Laboratory Detection of Haemophilus influenzae with Decreased Susceptibility to Nalidixic Acid, Ciprofloxacin, Levofloxacin, and Moxifloxacin Due to gyrA and parC Mutations

“…This resistance phenotype was not detected in Spanish isolates, and isolates with this phenotype may represent a different quinolone-resistant subpopulation. Strains with similar resistance phenotypes have been reported among other bacteria, such as Staphylococcus aureus (26) and Escherichia coli (21); but this is a new phenomenon in H. influenzae. Novel mechanisms of resistance have been proposed for isolates of this phenotype, such as multidrug efflux (26) and decreased outer membrane permeability (21).…”

“…Other alternative b These proposed diameters were based on the distribution of the population in Fig. 2. resistance mechanisms, such as an alteration in outer membrane permeability, may operate in these strains (21). The nalidixic acid-resistant (MICs, Ͼ32 g/ml), ciprofloxacin-susceptible phenotype has been described to be strongly prevalent among clinical E. coli isolates in Spain (27).…”

“…Nalidixic acid has received special attention for use in a screening test for determination of the susceptibilities of gram-negative bacteria to quinolones (6,9,18) and the detection of strains with unique phenotypes of quinolone resistance (21,26,27).…”

New Quinolone Resistance Phenomenon in Salmonella enterica : Nalidixic Acid-Susceptible Isolates with Reduced Fluoroquinolone Susceptibility

“…A population with such a resistance pattern has not been previously described. Anecdotal reports have been published on other enterobacteria showing similar resistance characteristics, but such strains have been solitary, as well as rare (12).…”

Development and Validation of a Diagnostic DNA Microarray To Detect Quinolone-Resistant Escherichia coli among Clinical Isolates

“…In addition, quinolone-resistant E. coli could be a potential threat to neutropenic patients with leukemia who receive a quinolone as prophylaxis (36). The molecular background of quinolone resistance is missense mutations (single-nucleotide exchanges) in the target enzyme genes and, less importantly, the reduction of quinolone accumulation inside the cells (2,10,16,22,27). In gram-negative organisms, such as E. coli, the primary target of fluoroquinolones is the DNA gyrase (3, 11).…”