a Microbiology, Department of Clinical Sciences, School of Medicine, University of Las Palmas de Gran Canaria, Dr Pasteur, 35080 Las Palmas de Gran Canaria; b Department of Microbiology, Hospital Clinic, School of Medicine, University of Barcelona, Villarroel, 170, 08036 Barcelona, Spain; c Department of Science and Biomedical Technology, Universitá degli Studi dellAquila, Via Vetoio, Loc. Coppito, 67100 LAquila, Italy
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Abstract |
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Introduction |
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Materials and methods |
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Spontaneous resistant mutants were selected from two C. freundii clinical isolates (DM1 and DM2) with increasing fluoroquinolone concentrations (2x and 4x the MIC) on drug-containing MacConkey agar (Difco, Detroit, MI, USA). They are named after their parent isolate, followed by the initial letter of the selective agent used in the first, second and third selective steps: i.e. C for ciprofloxacin (Bayer, Barcelona, Spain), L for lomefloxacin (Searle, Madrid, Spain), T for temafloxacin (Abbott, Madrid, Spain), S for sparfloxacin (RhônePoulenc Rorer, Madrid, Spain) and O for ofloxacin (Roussel, Madrid, Spain). Only mutants in which norfloxacin MIC was 2 mg/L were studied. Escherichia coli JF699 (OmpA deficient isolate), E. coli JF703 (OmpF deficient isolate) and E. coli KL16 (donated by Dr Berlyn, E. coli Genetic Stock Center, New Haven, CT, USA), and E. coli ATCC-25922, Klebsiella pneumoniae ATCC-10031 and C. freundii NCTC-9750 type isolate (tp) (donated by Dr Uruburu, CECT, Valencia, Spain) were used as controls.
Antibiotics and susceptibility tests
Tetracycline, nalidixic acid and norfloxacin were from Sigma (Madrid, Spain). MICs were determined by an agar dilution method according to NCCLS guidelines.7
Amplification and DNA sequencing of the quinolone resistance-determining region of the gyrA, gyrB and parC genes
They were performed using the previously described methods and oligonucleotide primers.4,5 The CATCGCCGCGAACGATTCGG primer was also used for parC gene amplification.
Preparation and analysis of outer membrane proteins (OMP) and lipopolysaccharide (LPS)
They were prepared and separated by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDSPAGE) using methods described previously.810
Measurements of norfloxacin accumulation
Norfloxacin accumulation was measured by the method previously described,5 simultaneously in each parent isolate and the quinolone-resistant mutants derived from it, with and without the presence of 50 and 100 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP). The total bacterial suspension of each isolate (prepared at an optical density of 1.5 at 520 nm wavelength) was divided into three parts and 50 or 100 µM CCCP were added to two parts 10 min before norfloxacin 10 mg/L. Norfloxacin concentration in each cellular extract was measured at least six times by bioassay using K. pneumoniae ATCC-10031. The accepted standard deviation for all the norfloxacin uptake results was always 5% with respect to each mean value of the three measurements that were taken at 5, 10, 15 and 20 min, with and without CCCP.
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Results and discussion |
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In the present study, the electrophoretic profiles of OMP or LPS were the same in the ten mutants and their parent isolates. The DM1 and DM2 isolates, and the ten quinolone-resistant mutants expressed a new band with an apparent molecular weight of 46 kDa that was not expressed in the fully sensitive NCTC-9750 (tp) (Figure). Furthermore, the ten mutants and parent isolates did not express a band with an electrophoretic mobility in ureaSDSpolyacrylamide gels similar to E. coli OmpC, although NCTC-9750 did express it (Figure
). Both parent isolates and the ten mutants showed a LPS smooth phenotype, as did C. freundii NCTC-9750 (Figure
). Therefore, it seems that permeability was not involved in the development of resistance to quinolones in the ten mutants studied with respect to their parent isolates.
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These results suggest that the development of resistance to fluoroquinolones in nine selected mutants of C. freundii resulted from a combination of enhanced efflux and a single substitution in the GyrA subunit. However, only enhanced active efflux plus the pre-existent single mutation in the gyrB gene can explain the fluoroquinolone MICs in the TL isolate.
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Notes |
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References |
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Weigel, L. M., Steward, C. D. & Tenover, F. C. (1998). gyrA mutations associated with fluoroquinolone resistance in eight species of Enterobacteriaceae. Antimicrobial Agents and Chemotherapy 42, 26617.
4 . Vila, J., Ruiz, J., Goñi, P. & Jiménez de Anta, M. T. (1996). Detection of mutations in parC in quinolone-resistant clinical isolates of Escherichia coli. Antimicrobial Agents and Chemotherapy 40, 4913.[Abstract]
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7 . National Committee for Clinical Laboratory Standards. (1996). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyThird Edition: Approved Standard M7-A3. NCCLS, Villanova, PA.
8 . Sawai, T., Hiruma, R., Kawana, N., Kaneko, M., Taniyasu, F. & Inami, A. (1982). Outer membrane permeation of ß-lactam antibiotics in Escherichia coli, Proteus mirabilis, and Enterobacter cloacae. Antimicrobial Agents and Chemotherapy 22, 58592.[ISI][Medline]
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Received 15 June 1999; returned 19 August 1999; revised 17 September 1999; accepted 22 October 1999