Departament de Microbiologia, IDIBAPS, Hospital Clínic, Facultat de Medicina, Universitat de Barcelona, Villarroel 170, 08036-Barcelona, Spain
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Abstract |
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Introduction |
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Mutations of amino acid codons 426 and 447 of the gyrB gene of E. coli are also responsible for the acquisition of quinolone resistance,10,19,20 although their relative importance differs in clinical and in-vitro E. coli quinolone-resistant strains. While quinolone-resistant strains show a similar in-vitro frequency of mutations in gyrA and gyrB genes,10 a clear predominance of gyrA gene mutations over those of gyrB gene has been shown in quinolone-resistant clinical isolates.12,17
There is a considerable sequence similarity between the genes that encode the A and B subunits of the DNA gyrase and those that encode them for the topoisomerase IV. Recently, it has been shown that topoisomerase IV is a quinolone target in E. coli3,21 and that changes at residues Ser-80 and Glu-84 of ParC (A subunit) may contribute to decreasing fluoroquinolone susceptibility.6,22,23,24Conversely, the role of ParE, (B subunit of topoisomerase IV) in the acquisition of quinolone resistance in clinical isolates of E. coli seems to be irrelevant,6,25 although mutations in in-vitro quinolone-resistant strains of E. coli have been described by Breines et al.26 in an analogous position to gyrB gene mutations responsible for quinolone resistance development.
Changes in quinolone accumulation by increased efflux or decreased uptake have also been linked to quinolone resistance acquisition. While the former is usually linked to some kind of pump which actively expels the drug, the latter has been associated with changes in the outer membrane proteins.
Alterations in the gyrA gene of a quinolone-resistant strain of C. freundii were shown for the first time indirectly with an experiment in which the supercoiling activity of the mutant's DNA gyrase was resistant to quinolone compounds.5 In 1988, Aoyama et al.27 studied norfloxacin uptake and membrane profiles in a clinically resistant strain and recently, mutations in gyrA and parC have been described in quinolone-resistant clinical strains.11 However, a comprehensive study of all possible resistance mechanisms in clinical isolates of C. freundii has not yet been reported.
In this work, we have determined the mechanisms of quinolone resistance in clinical isolates of C. freundii by means of sequencing the QRDR of the gyrA, gyrB and parC genes and determining quinolone uptake and outer membrane protein profiles.
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Materials and methods |
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A total of 12 clinical isolates of C. freundii were recovered from different biological samples, from either in-patients or out-patients, submitted to the Clinical Microbiology Laboratory at the Hospital Clinic of Barcelona, Spain.
Antimicrobial susceptibility testing
Susceptibility testing was performed by an agar dilution method in accordance with the guidelines established by the National Committee for Clinical Laboratory Standards.28 Approximately 104 cfu/spot of each isolate was inoculated with a multipoint replicator on to freshly prepared medium containing serial dilutions of ciprofloxacin (Bayer, Leverkusen, Germany), nalidixic acid (Prodesfarma, Barcelona, Spain) or chloramphenicol (Sigma, St Louis, MO, USA)
REP (repetitive extragenic palindromic) PCR
REPPCR was carried out using the primer 5' GCG CCG ICA TGC GGC ATT 3' under the following conditions: 30 cycles of 1 min at 94°C, 1 min at 40°C, 1 min at 65°C and a final extension at 65°C for 16 min. The reaction was prepared using 5 µL of boiled bacterial suspension, 1 µL of 5 µM primer and a PCR bead (Pharmacia Biotech, Uppsala, Sweden) in a final volume of 25 µL. Five microlitres of the amplification product was separated in a 12.5% precast polyacrylamide gel using a GenePhor apparatus (Pharmacia Biotech) and silver-stained using the Pharmacia Biotech DNA silver staining kit.
Ciprofloxacin uptake
Ciprofloxacin uptake was determined as previously described by Asuquo & Piddock.29
Amplification and DNA sequencing of quinolone resistance determining region (QRDR) in gyrA and parC genes
The PCR amplification of the QRDR gyrA gene was carried out using the primers and following the conditions previously described by Vila et al.16 The parC QRDR was amplified using the sense primer described for E. coli23 and an antisense consensus primer designed by comparing several published parC gene sequences. The sequence of the antisense primer was 5'-CAT CGC CGC GAA CGA TTC GG-3'. The PCR conditions were the same as for gyrA amplification.16 To amplify the gyrB fragment, primers and conditions used were as described previously.16 The PCR reactions were performed using a DNA thermal cycler 480 (Perkin-Elmer Cetus, Emeryville, CA, USA). Amplified DNA products were resolved by electrophoresis in agarose gels (2%, w/v) containing 0.5 mg/L of ethidium bromide. PCR products were recovered directly from the agarose gel and purified. DNA sequencing was performed with the TaqDyeDeoxyTerminator Cycle Sequencing kit (Applied Biosystems) and analysed in an automatic DNA sequencer (Applied Biosystems 377).
The EMBL accession numbers for the partial sequences are: gyrA, AF064797; parC, AF064798 and gyrB, AF071877.
Outer membrane protein profile analysis
Outer membrane proteins were prepared with N-lauroylsarcosine as previously described.30 Proteins were separated by electrophoresis using 10% urea-SDS-polyacrylamide gel and silver stained using the Pharmacia-Biotech protein silver staining kit.
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Results |
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Discussion |
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Even though the topoisomerase-mediated resistance (specifically mutations in gyrA and parC genes) can explain in general terms the MICs of the strains under study, differences within group B (resistant to both quinolones tested) need further explanation. C. freundii outer membrane proteins have been described previously.27 As in E. coli there are three major proteins, although their electrophoretic mobilities are different. A decrease in the expression of an outer membrane porin has been associated with a decrease in quinolone uptake.27 In our study, no major differences were observed in the expression of the isolates' OmpC, OmpF and OmpA profiles.
Differences did appear, however, in the amount of ciprofloxacin accumulated. Strains 1.44 and 1.38 belong to the same REPPCR type, have the same gyrase and topoisomerase mutations but differ in their ciprofloxacin MIC. Such a difference could be attributed to strain 1.38 having a more important active efflux than 1.44, which is suggested by the former's increased ciprofloxacin uptake in the presence of CCCP. This suggestion is supported by the fact that the chloramphenicol MIC for strain 1.38 was higher than that for strain 1.44. This is probably due to the fact that the active efflux system pumping ciprofloxacin out of the cell also takes chloramphenicol.
In summary, in C. freundii, as in E. coli, high levels of quinolone resistance are due to mutations in gyrA and parC genes, with the former having the primary priority point mutation. In addition, an overexpressed active efflux pump responsible for increasing already high levels of resistance to ciprofloxacin was also observed in resistant strains.
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Acknowledgments |
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Notes |
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References |
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Received 11 October 1998; returned 14 December 1998; revised 18 June 1999; accepted 27 July 1999