a Departments of Infectious Diseases and b Microbiology, Institut Clínic d'Infeccions i Immunologia (ICII) and c Department of Internal Medicine, Hospital Clínic Universitari, Villarroel 170, 08036 Barcelona, Spain
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Abstract |
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Introduction |
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The main objectives of this study were: (i) to analyse the evolution of the susceptibility to fluoroquinolones of E. coli strains isolated from faecal flora during and after the administration of therapeutic doses of oral ciprofloxacin for 1 month; (ii) to investigate whether there is any relationship between the resistant strains isolated during treatment and susceptible strains in the gut before treatment.
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Materials and methods |
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The study involved 29 outpatients with acute prostatitis for whom the initial faecal sample was positive for E. coli and who attended the Infectious Disease Department of the Hospital Clínic (Barcelona, Spain), a 900 bed tertiary hospital. These patients were treated orally with ciprofloxacin 750 mg twice daily for 28 days. Compliance with prescribed treatment was monitored by means of a pill count of returned boxes of antibiotic and by interview. None of the patients had received antibiotics for at least 4 weeks before the study commenced.
Microbiological methods
Stool cultures were collected immediately before therapy commenced and every 2 weeks during and after treatment, until a predominant quinolone-susceptible E. coli strain was isolated. Fresh stools were collected in sterile containers and cultured immediately. One gram of faeces was homogenized in 9 mL of saline serum and plated by streaking 0.05 mL on to MacConkey agar. After inoculation, plates were incubated aerobically at 3537°C for 2 days. To recover the predominant E. coli strains, all morphologically different colonies suspected to be E. coli were subcultured in the same medium and identified by conventional methods. E. coli isolates were then stored in skimmed milk at -70°C, until antibiotic susceptibility tests and molecular studies were performed.
Antimicrobial susceptibility tests
Antibiotic susceptibility testing was performed by the Etest method (AB Biodisk, Solna, Sweden) following the manufacturer's instructions. E. coli ATCC 25922 was used as the quality control organism.
Epidemiological analysis
The epidemiological relationships among the E. coli strains isolated at different times from each patient were determined by repetitive extragenic palindromic PCR (REP-PCR). For REP-PCR, bacteria were grown on MacConkey agar overnight and one colony of each isolate was suspended in 25 µL of reaction mixture containing 20 mM TrisHCl pH 8.8, 100 mM KCl, 3.0 mM MgCl2, gelatin 0.1% (v/v) and 400 µM dNTPs. Primer was added at 1 µM, together with 2.5 U Taq polymerase. The reaction mixture was overlaid with mineral oil and DNA amplification was achieved with the following programme: 30 cycles of 94°C for 1 min, 40°C for 1 min and 65°C for 8 min, with a single final extension at 65°C for 16 min. Samples (10 µL) of each PCR end-product were analysed on agarose (1.5% w/v) gels. The primer used was 5'-GCGCCGICATGCGGCATT-3'.
Detection of mutations in the gyrA gene
To study the mechanisms of quinolone resistance in the strains isolated, PCR amplification and DNA sequencing of the quinolone resistance determining region (QRDR) of the gyrA gene were performed as described elsewhere.9 PCRs were carried out in a DNA Thermal Cycler 480 (Perkin-Elmer Cetus, Emeryville, CA, USA). Primers and free nucleotides were removed with a QiaQuick spin PCR purification kit (Qiagen, Inc., Chatsworth, CA, USA), according to the manufacturer's instructions; the sample was directly processed for DNA sequencing with Thermo Sequenase II Dye Terminator cycle sequencing kit (Amersham, Cleveland, OH, USA) and analysed in an automatic DNA sequencer (Applied Biosystems 377; Perkin-Elmer, Foster City, CA, USA). When the PCR product from the QRDR of the gyrA gene was analysed by digestion with HinfI, 20 µL of PCR mixture were incubated for 2.5 h with 10 U of restriction endonuclease. Digestion products were separated by electrophoresis in NuSieve (1.5% w/v) and agarose (1% w/v) gels.
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Results |
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The four patients whose strains were initially resistant to nalidixic acid and ciprofloxacin harboured the same strains during treatment (patients 2427 in the Table and the Figure
). In the two patients whose strains at the start of the study were resistant only to nalidixc acid, quinoloneresistant but genetically different strains were isolated during or just after treatment (patients 28 and 29 in the Table
).
Within 2 months of the end of quinolone treatment, the predominant E. coli strains in the faecal flora of all patients were susceptible to quinolones (nalidixic acid MIC < 4 mg/L, ciprofloxacin MIC < 0.02 mg/L) and genetically different from both the quinolone-susceptible strains isolated before treatment and from the quinolone-resistant strains isolated during treatment (Table and Figure
).
All the quinolone-resistant strains showed at least one mutation, specifically in the triplet encoding amino acid residue Ser-83 of GyrA.
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Discussion |
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Since our method only detected the dominant faecal E. coli strain in each patient, it can only be asserted with certainty that high-dose oral ciprofloxacin did not select resistant mutants from the susceptible strain detected initially. The origin of the resistant E. coli that emerged during treatment cannot be determined from the current data. Three scenarios are conceivable: (i) unmasking of ciprofloxacin-resistant organisms present in low numbers before therapy but undetected by the methodology; (ii) acquisition of an exogenous resistant strain during treatment; and (iii) selection in the host of highly resistant mutants from pre-existing intermediately susceptible organisms. In the particular setting, either of the first two possibilities is likely, considering that the use of quinolones in the community is widespread and that resistant strains selected in food animals exposed to quinolones may have been introduced into the food chain.24 We included in the study only patients who had not taken antibiotics during the month before taking the first sample, but we did not investigate the issue further. The presence of high-level quinolone-resistant E. coli strains in four of our patients at baseline could indicate previous exposure to quinolones. High rates of faecal carriage of quinolone-resistant E. coli strains in healthy people in our community and an extremely high prevalence of quinolone-resistant E. coli in the stools of farm animals in our geographical area have been reported.4 These data are consistent with the hypothesis that the resistant E. coli strains found in our patients were either already carried in low numbers before therapy or were acquired during ciprofloxacin administration. The possibility that high-level resistance was selected by ciprofloxacin from pre-existing intermediate-susceptible strains seems less likely, since in two patients, where the predominant strain was one with a single Ser-83 mutation before treatment, high-dose oral ciprofloxacin therapy simply suppressed its growth without selecting mutants with increased resistance.
The transient nature of the faecal carriage of high-level quinolone-resistant E. coli strains after ciprofloxacin therapy has been reported in neutropenic patients, but no information was given about the molecular epidemiology of the successive isolates.10 It seems probable that susceptible E. coli strains, in the absence of selective antibiotic pressure, are more fit than resistant ones, and replace them. With our methodology we could not discern whether the resistant strains disappeared completely, or remained at low density together with the quinolone-susceptible strains. Studies are in progress to clarify this issue.
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Acknowledgements |
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Notes |
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References |
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2 . Ena, J., Lopez-Perezagua, M. M., Martinez-Peinado, C., Cia-Barrio, M. A. & Ruiz-Lopez, I. (1998). Emergence of ciprofloxacin resistance in Escherichia coli isolates after widespread use of fluoroquinolones. Diagnostic Microbiology and Infectious Disease 30, 1037. [ISI][Medline]
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Tavío, M. M., Vila, J., Ruiz, J., Ruiz, J., Martín-Sánchez, A. M. & Jiménez de Anta, M. T. (1999). Mechanisms involved in the development of resistance to fluoroquinolones in Escherichia coli strains. Journal of Antimicrobial Chemotherapy 44, 73542.
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Perea, S., Hidalgo, M., Arcediano A., Ramos, M. J., Gomez, C., Hornedo, J. et al. (1999). Incidence and clinical impact of fluoroquinolone-resistant Escherichia coli in the faecal flora of cancer patients treated with high dose chemotherapy and ciprofloxacin prophylaxis. Journal of Antimicrobial Chemotherapy 44, 11720.
Received 20 November 2000; returned 20 March 2001; revised 15 May 2001; accepted 4 September 2001