Introduction. Materials and methods

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1 Journal of Antimicrobial Chemotherapy (2002) 49, JAC Molecular epidemiology and evolution of resistance to quinolones in Escherichia coli after prolonged administration of ciprofloxacin in patients with prostatitis Juan P. Horcajada a *, Jordi Vila b, Antonio Moreno-Martínez a, Joaquim Ruiz b, Jose Antonio Martínez a, Miquel Sánchez c, Eladio Soriano a and Josep Mensa a Departments of a Infectious Diseases and b Microbiology, Institut Clínic d Infeccions i Immunologia (ICII) and c Department of Internal Medicine, Hospital Clínic Universitari, Villarroel 170, Barcelona, Spain The emergence and evolution of quinolone-resistant Escherichia coli in faeces of patients with prostatitis treated with high-dose oral ciprofloxacin for 1 month were studied. In 11 of 23 patients, from whom only quinolone-susceptible E. coli was isolated before treatment, quinolone-resistant strains, genetically distinct from the quinolone-susceptible ones, predominated during and just after therapy. Two months after treatment, these were completely displaced by quinolone-susceptible E. coli, genetically distinct from the E. coli isolated before and during therapy. Hence, during ciprofloxacin therapy, half of the patients were transiently colonized with new, quinolone-resistant strains of E. coli. Introduction The wide use of fluoroquinolones in both human and animal medicine has led to a significant increase in the level of resistance to these antimicrobial agents in clinical isolates of Escherichia coli from several geographical areas. 1 4 Patients receiving fluoroquinolones for long periods, either to prevent recurrent urinary tract infections or for selective intestinal decontamination, may be colonized by quinolone-resistant Enterobacteriaceae in the intestinal tract. 5 7 However, neither the origin of these resistant strains nor the evolution of intestinal colonization after the selective pressure exerted by the quinolones is removed has been studied. The resistant strains could be newly acquired exogenous resistant strains or resistant mutants arising from strains already in the intestinal tract. The selection of quinolone-resistant mutants correlates with ratios of the selective concentration of quinolone and MIC 8 mg/l. The use of fluoroquinolones with little intrinsic activity, the prescription of relatively low doses, such as in the course of prophylaxis of urinary tract infections, and the partial inactivation of fluoroquinolones in stools, are conditions that determine concentration/mic ratio and, therefore, favour the selection of resistant mutants. In contrast, selection of resistant mutants is likely to be less frequent if the patient is prescribed ciprofloxacin at therapeutic doses. 8 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. Materials and methods Patients 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. *Corresponding author. Tel: ; Fax: ; jhorcaja@clinic.ub.es 2002 The British Society for Antimicrobial Chemotherapy 55

2 J. P. Horcajada et al. 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 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 was used as the quality control organism. 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 L 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. Results Before ciprofloxacin administration, the predominant E. coli strains in the faecal flora were susceptible to quinolones in 23 (79%) of 29 patients studied (patients 1 23 in Table). In the remaining six patients (21%), the initial strains were resistant to nalidixic acid (patients in the Table and the Figure) and four of these were also resistant to ciprofloxacin (patients 24 27). For 12 (52%) of the 23 patients with quinolone-susceptible E. coli strains at the start of treatment, no growth of E. coli was observed from stool cultures made during treatment, whereas for each of the remaining 11 patients (48%) a quinolone-resistant strain was isolated during treatment 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 L of reaction mixture containing 20 mm Tris HCl ph 8.8, 100 mm KCl, 3.0 mm MgCl 2, gelatin 0.1% (v/v) and 400 M dntps. Primer was added at 1 M, 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 L) of each PCR end-product were analysed on agarose (1.5% w/v) gels. The primer used was 5 -GCGCCGICATGCGGC- ATT-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 Figure. REP-PCR patterns. Patient 24: lane 1, quinoloneresistant E. coli before treatment; lane 2, quinolone-resistant E. coli with the same REP-PCR pattern during treatment; lane 3, quinolone-susceptible E. coli with different REP-PCR pattern after treatment. Patient 13: lane 1, quinolone-susceptible E. coli before treatment; lane 2, quinolone-resistant E. coli with different REP-PCR pattern during treatment; lane 3, quinolone-susceptible E. coli with different REP-PCR pattern after treatment. Patient 1: lane 1, quinolone-susceptible E. coli before treatment; lane 2, quinolone-susceptible E. coli with different REP-PCR pattern after treatment. Lane M, DNA molecular weight markers (Gibco-BRL). 56

3 Epidemiology of E. coli quinolone resistance Table. Evolution of susceptibility to quinolones and molecular epidemiology of the E. coli isolates Before treatment During treatment a 1st month after treatment 2nd month after treatment Patient no. MIC NAL MIC CIP MIC NAL MIC CIP clonal identity b MIC NAL MIC CIP clonal identity b MIC NAL MIC CIP clonal identity b NBG NBG NBG no NBG NBG NBG no NBG NBG NBG NBG NBG NBG no NBG NBG NBG NBG NBG NBG no NBG NBG NBG NBG NBG NBG no NBG NBG NBG NBG NBG NBG no NBG NBG NBG NBG NBG NBG no NBG NBG NBG NBG NBG NBG no NBG NBG NBG NBG NBG NBG no NBG NBG NBG NBG NBG NBG no NBG NBG NBG NBG NBG NBG no NBG NBG NBG NBG NBG NBG no >256 >32 no no >256 >32 no no >256 >32 no >256 3 no no >256 >32 no >256 >32 no no NBG NBG NBG >256 >32 no no NBG NBG NBG >256 >32 no no NBG NBG NBG > no no NBG NBG NBG >256 >32 no no NBG NBG NBG >256 >32 no no NBG NBG NBG > no no >256 8 no NBG NBG NBG no 24 >256 >32 >256 >32 yes no 25 >256 >32 >256 >32 yes no 26 >256 >32 NBG NBG NBG >256 >32 yes no 27 >256 >32 >256 >32 yes >256 >32 yes no 28 > >256 >32 no NBG NBG NBG no 29 > NBG NBG NBG > no no MIC NAL, MIC of nalidixic acid (mg/l). MIC CIP, MIC of ciprofloxacin (mg/l). NBG, no bacterial growth. a Ciprofloxacin 750 mg bd po for 28 days. b Clonal identity with the strain isolated immediately before. 57

4 J. P. Horcajada et al. or from the first stool culture performed 15 days after treatment finished (patients in the Table and the Figure). The MICs of nalidixic acid for these 11 strains were 256 mg/l and the MICs of ciprofloxacin were 32 mg/l in eight (72%) of the 11 strains. All the quinolone-resistant strains were genetically different from the quinolonesusceptible strains recovered before starting quinolone treatment. The four patients whose strains were initially resistant to nalidixic acid and ciprofloxacin harboured the same strains during treatment (patients 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. Discussion The study illustrates the dynamics of the predominant E. coli faecal flora in 29 males with prostatitis given highdose oral ciprofloxacin therapy. Before treatment the predominant E. coli strains were susceptible to nalidixic acid and ciprofloxacin in 23 (79%) and 26 (90%) patients, respectively. During or shortly after therapy of these patients, the dominant quinolone-susceptible E. coli strains were no longer detected; in eight cases they were substituted by genetically unrelated strains resistant to ciprofloxacin; in three patients new strains, resistant to nalidixic acid but susceptible to ciprofloxacin, emerged. In the remaining four patients where the dominant pretherapy E. coli strains were resistant to ciprofloxacin, the same organisms persisted during therapy and for a short time thereafter. Finally, after ciprofloxacin therapy ceased, all patients acquired new quinolone-susceptible E. coli that were genetically unrelated to previous isolates, as the dominant strains. 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. 2 4 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. Acknowledgements The competent assistance of Victoria Lasheras is gratefully acknowledged. This work was supported, in part, by grant SAF 97/0091 from Plan Nacional I D, Spain and Beca Agustí Pumarola from the Societat Catalana de Malalties Infeccioses. References 1. Cometta, A., Calandra, T., Bille, J. & Glauser, P. (1994). Escherichia coli resistant to fluoroquinolones in patients with cancer and neutropenia. New England Journal of Medicine 330, 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 fluoro- 58

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