Rapid selection of organisms with increasing resistance on subinhibitory concentrations of norfloxacin in agar

Tenney, James H.; Maack, R W; Chippendale, G. R.

doi:10.1128/aac.23.1.188

Cited by 83 publications

(56 citation statements)

References 5 publications

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“…In E. coli, overexpression of MarA causes decreased expression of the OmpF porin (6) as well as increased expression of the multidrug efflux pump AcrAB (23), thereby conferring resistance to a large number of antimicrobial agents (for a review, see reference 1). Studies with mutants selected in vitro have been confined to descriptions of phenotypic differences (MICs, outer membrane profiles, accumulation of FQs) or have focused on mutations in the quinolone resistance-determining regions (QRDRs) of gyrA and/or parC (10,25,27,29). In this in vitro study we sought to understand the role of the AcrAB efflux pump in the FQ resistance mediated by topoisomerase mutations acquired during selection on ofloxacin.…”

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Ineffectiveness of Topoisomerase Mutations in Mediating Clinically Significant Fluoroquinolone Resistance in Escherichia coli in the Absence of the AcrAB Efflux Pump

Oethinger

Kern

Jellen-Ritter

et al. 2000

Antimicrob Agents Chemother

191

121

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Fluoroquinolone-resistant mutants, selected from a wild-type Escherichia coli K-12 strain and its Mar mutant by exposure to increasing levels of ofloxacin on solid medium, were analyzed by Northern (RNA) blot analysis, sequencing, and radiolabelled ciprofloxacin accumulation studies. Mutations in the target gene gyrA (DNA gyrase), the regulatory gene marR, and additional, as yet unidentified genes (genes that probably affect efflux mediated by the multidrug efflux pump AcrAB) all contributed to fluoroquinolone resistance. Inactivation of the acrAB locus made all strains, including those with target gene mutations, hypersusceptible to fluoroquinolones and certain other unrelated drugs. These studies indicate that, in the absence of the AcrAB pump, gyrase mutations fail to produce clinically relevant levels of fluoroquinolone resistance.

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Ineffectiveness of Topoisomerase Mutations in Mediating Clinically Significant Fluoroquinolone Resistance in Escherichia coli in the Absence of the AcrAB Efflux Pump

Oethinger

Kern

Jellen-Ritter

et al. 2000

Antimicrob Agents Chemother

191

121

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“…However, the new agents could not inhibit the bacterial growth of strain GN14181 at high concentrations. Resistance to new fluoroquinolones, such as NFX (MIC, 100 ,ug/ml), at so high a level has not been attained even with serial passage of E. coli on increasing drug concentrations in the laboratory (11,22).…”

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Purification and properties of DNA gyrase from a fluoroquinolone-resistant strain of Escherichia coli

Sato¹,

Inoue²,

Fujii³

et al. 1986

Antimicrob Agents Chemother

114

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Subunit A and B proteins of DNA gyrase were separately purified from fluoroquinolone-resistant Escherichia coli GN14181 (MIC of ofloxacin, 100 gLg/ml) and susceptible strain KL-16. The supercoiling activities of reconstituted Ar+Br (r, resistant) and Ar+Bs (s, susceptible) were 250-fold more resistant to new fluoroquinolones thsn those of As+Bs and As+Br.DNA gyrase was discovered by Geilert et al. (8) as an activity in Escherichia coli that catalyzes the ATP-coupled negative supercoiling of DNA. In addition to supercoiling activity, the enzyme causes the relaxation of supercoiling DNA in the absence of ATP, and the binding of the enzyme to DNA leads to double-strand breakage at specific sites promoted by quinolones and sodium dodecyl sulfate (4,5,10,17,20). The enzyme activities result from breakage and rejoining of both strands of duplex DNA (3,5). The active gyrase holoenzyme is a tetramer composed of two subunits, A2 and B2 (10, 15). It is believed that subunit A protein is the target of quinolones because nalidixic acid-resistant gyrA mutants are also resistant to quinolones (11,12). New fluoroquinolones, such as ofloxacin, norfloxacin, ciprofloxacin, and enoxacin, have broad spectra against gramnegative and -positive bacteria (13,14,16,19), and they strongly inhibit the supercoiling activity of DNA gyrase from E. coli . However, there have been no reports on the inhibitory properties of DNA gyrase from clinical isolates.In this report, we present the purification and properties of subunit A and B proteins of DNA gyrase from a fluoroquinolone-resistant isolate, E. coli GN14181.E. coli GN14181 was isolated from an upper urinary tract infection and identified by the method ofBergey's Manual of Systematic Bacteriology (3). E. coli K-12 strains KL-16 (Hfr, thi, relA) and MH-5, its gyrAr derivative, were also used (2, 9).We received the following compounds as gifts from their manufacturers: ofloxacin (OFX), norfloxacin (NFX), ciprofloxacin (CPX), enoxacin (ENX), pipemidic acid, and nalidixic acid (NA). Novobiocin (NB) and coumermycin Al (COU) were purchased from Sigma Chemical Co. purified by using a heparin-Sepharose CL-6B (Pharmacia Fine Chemicals) column. Active fractions, which eluted around 0.3 M NaCl, were pooled after dialysis against TGED buffer. The B subunit was eluted with 5 M urea from an NB-Sepharose column.The assay of supercoiling activity was modified from previous reports (6, 7). One unit of enzyme activity was defined as the amount that brought 50% of relaxed pBR322 to the supercoiled position in agarose gel electrophoresis as described by Gellert et al. (8). The reaction mixture containing subunit A and B proteins (each, 0.1 ,ug of protein), drug solution, and pBR322 relaxed by topoisomerase I (Bethesda Research Laboratories, Inc.) was incubated at 37°C for 1 h, and the reaction was stopped by the addition of proteinase K (20 ,ug/ml). The mixtures were subjected to 0.8% agarose gel electrophoresis. The gel was stained with ethidium bromide (0.5 ,ug/ml) and photographed by using UV light. The ne...

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“…The frequency of single-step resistant variants was expressed as the ratio of the number of CFU grown in the presence of the antibiotic (at twice the MIC) to the number of CFU of the control grown without imipenem, as described previously (2). Along with these assays, the imipenem-sensitive S. algae strain was also subjected to a serial passage experiment with imipenem, as described by Tenney et al (18). In brief, overnight growth of the S. algae strain on Mueller-Hinton agar was swabbed onto Mueller-Hinton agar plates containing one-half the MIC of imipenem.…”

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Treatment Failure Due to Emergence of Resistance to Carbapenem during Therapy for Shewanella algae Bacteremia

Kim¹,

Kang²,

Lee³

et al. 2006

J Clin Microbiol

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We describe a case of bacteremia due to imipenem-susceptible Shewanella algae. Despite treatment with imipenem, the patient developed a spinal epidural abscess, from which imipenem-resistant S. algae was isolated. The development of resistance should be monitored when S. algae infection is treated with imipenem, even though the strain is initially susceptible to imipenem. CASE REPORTA 65-year-old man underwent distal pancreatectomy with cholecystectomy because of intraductal papillary mucinous tumor. On hospital day 12 (postoperative day 7), the patient complained of chills and fever. Abdominal sonography showed fluid collection in the abdomen, and percutaneous drainage was performed. Cultures of blood and percutaneous drainage fluid yielded Shewanella algae, which was susceptible to ceftazidime, cefepime, aztreonam, imipenem, and amikacin. It was resistant to piperacillin and gentamicin, as determined by disk diffusion testing. Based on this result, treatment with imipenem (500 mg intravenously [i.v.] every 6 h [q6h]) was instituted. Despite the treatment, the patient remained febrile. On hospital day 25, the patient became agitated and disoriented, and signs of meningismus were evident on his examination. His cerebrospinal fluid (CSF) contained 180 white blood cells/ mm 3 . The CSF protein concentration was 307 mg/dl (reference range, 15 to 45 mg/dl), and the CSF sugar concentration was 41 mg/dl (reference range, 40 to 80 mg/dl). The CSF culture was negative. Imipenem treatment was changed to meropenem treatment. On hospital day 32, he complained of pain in his neck, and weakness of his left arm and left leg developed suddenly. Magnetic resonance imaging (MRI) revealed a spinal epidural abscess extending from the fourth to the sixth cervical vertebrae. The patient underwent hemilaminectomy and discectomy of C4 and C5 with drainage of the epidural abscess, which yielded S. algae on culture.

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Rapid selection of organisms with increasing resistance on subinhibitory concentrations of norfloxacin in agar

Cited by 83 publications

References 5 publications

Ineffectiveness of Topoisomerase Mutations in Mediating Clinically Significant Fluoroquinolone Resistance in Escherichia coli in the Absence of the AcrAB Efflux Pump

Ineffectiveness of Topoisomerase Mutations in Mediating Clinically Significant Fluoroquinolone Resistance in Escherichia coli in the Absence of the AcrAB Efflux Pump

Purification and properties of DNA gyrase from a fluoroquinolone-resistant strain of Escherichia coli

Treatment Failure Due to Emergence of Resistance to Carbapenem during Therapy for Shewanella algae Bacteremia

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