Isolation of fluoroquinolone-resistant rectal Escherichia coli after treatment of acute uncomplicated cystitis

Kalpana Gupta*, Thomas M. Hooton and Walter E. Stamm

Department of Medicine/Division of Allergy and Infectious Diseases, 1959 NE Pacific St., Box 356523, University of Washington, Seattle, WA 98195, USA


* Corresponding author and present address. Department of Medicine/Section of Infectious Diseases, Yale University School of Medicine and VA CT HCS, 950 Campbell Avenue, 11-ACSLG, West Haven, CT 06516, USA. Tel: +1-203-932-5711, ext. 4224; Fax: +1-203-937-4926; E-mail: Kalpana.gupta{at}yale.edu

Received 21 September 2004; returned 6 November 2004; revised 14 April 2005; accepted 20 April 2005


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Objectives: Given increasing rates of co-trimoxazole resistance among uropathogens causing acute uncomplicated cystitis, fluoroquinolones, nitrofurantoin and fosfomycin are often considered as alternative empirical therapy. The choice between these drugs should depend in part on whether they are associated with the isolation of drug-resistant microbial flora. We conducted a randomized treatment trial to assess the effects of ciprofloxacin, nitrofurantoin and fosfomycin on the rectal microbial flora of women with acute uncomplicated cystitis, including isolation of fluoroquinolone-resistant strains.

Methods: Pre-menopausal women presenting with acute uncomplicated cystitis were randomized to treatment with 3 days of ciprofloxacin, 7 days of nitrofurantoin, or a single dose of fosfomycin. Women were followed for 1 month for evaluation of clinical and microbiological responses as well as for isolation of resistant rectal E. coli.

Results: Sixty-two women (25 ciprofloxacin, 17 nitrofurantoin, 20 fosfomycin) were enrolled and eligible for analysis. All three regimens were well tolerated and resulted in >90% clinical and bacteriological cure. The prevalence of rectal E. coli was markedly decreased by ciprofloxacin and fosfomycin, but not by nitrofurantoin. One woman treated with ciprofloxacin had emergence of two ciprofloxacin-resistant rectal E. coli strains within 10 days of completing therapy. No emergence of resistance was observed in the other two treatment groups.

Conclusions: This study demonstrates that fluoroquinolone-resistant E. coli remain infrequent in the rectal flora of women with uncomplicated cystitis in Seattle. However, a 3 day course of a fluoroquinolone for treatment of uncomplicated cystitis was followed by isolation of fluoroquinolone-resistant rectal E. coli in one patient.

Keywords: urinary tract infections , drug resistance , E. coli


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Given increasing rates of antimicrobial resistance to co-trimoxazole among uropathogens causing acute uncomplicated cystitis in women, fluoroquinolones are increasingly being chosen for empirical therapy of this common infection.1 However, widespread use of fluoroquinolones for the empiric treatment of uncomplicated cystitis has not been recommended due to concern that this will promote bacterial resistance.1 To date, no published studies have demonstrated that short course fluoroquinolone therapy for acute cystitis in women actually results in the selection of resistant Escherichia coli. We conducted a randomized treatment trial to assess the effects of ciprofloxacin, nitrofurantoin and fosfomycin on the microbial flora of women with acute cystitis, including isolation of fluoroquinolone-resistant strains.


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Study population

Women aged 18–45 years presenting to the university healthcare centre with symptoms of acute uncomplicated cystitis were offered the opportunity to participate. Subjects were also recruited through advertisements at the University of Washington campus. Additional inclusion criteria included being non-pregnant, in good general health, and having pyuria as measured by a haemocytometer counting chamber and a urine culture with ≥102 cfu/mL of a uropathogen. Women who were pregnant, lactating, not regularly taking contraceptives, or who had a history of chronic conditions such as diabetes, known anatomical abnormalities of the urinary tract, allergy to any of the three study drugs, or recent (<2 weeks) exposure to an oral or parenteral antimicrobial were not eligible for the study.

Study procedures

Study participants were randomized to treatment with ciprofloxacin 250 mg twice daily for 3 days, nitrofurantoin 100 mg twice daily for 7 days, or fosfomycin 3 g single dose, and were followed prospectively for 1 month. Urine and rectal cultures were performed at baseline and at 1–3, 10–14 and 28–30 days after therapy. Written informed consent was obtained from all participants, and study procedures were approved by the University of Washington Human Subjects Review Committee.

Laboratory methods

Culture and identification of urine specimens were performed using standard laboratory methods.2 The Kirby–Bauer disc method was used to determine antimicrobial susceptibilities of uropathogens and rectal isolates.2 Rectal swabs were collected using a portacult transport medium and transferred to the laboratory and inoculated within 24 h of collection. The swabs were plated directly onto 5% Sheep Blood agar and MacConkey agar and then streaked for isolation using a sterile loop. Flat lactose-fermenting colonies that were indole-positive were identified as E. coli. To test for selection of resistant E. coli in the rectal flora during therapy, each morphologically different colony type of E. coli on sheep's blood agar was isolated from each patient at each visit and stocked individually at –70°C in glycerol/trypticase soy broth. At the completion of the study, the E. coli strains were taken out of the freezer, reisolated, and tested for susceptibility using NCCLS guidelines to the study drug with which the patient was treated. Strain identity was assessed using XbaI digests in pulsed-field gel electrophoresis.3 Haemolysin production and papG genotypes were determined using previously described methodology.4,5

Statistical methods

The prevalence of rectal E. coli was calculated by dividing the number of women with any rectal E. coli isolated by the total number of women sampled for rectal E. coli at each visit. The difference in prevalence between treatment groups was tested using the {chi}2 test. A binomial 95% confidence interval was calculated for the point estimate of the percentage of women with resistant strains detected in the ciprofloxacin treatment arm.


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A total of 74 women with symptoms of acute uncomplicated cystitis volunteered to participate in the study. Of these, 11 did not have a positive urine culture and thus were not eligible to continue the study. One woman did not make any follow-up visits and was not considered eligible for analysis. Thus, a total of 62 women were enrolled, were randomized to treatment with nitrofurantoin (17 women), fosfomycin (20 women) or ciprofloxacin (25 women), and were eligible for analysis. The demographic characteristics of the women in each treatment arm were similar. The women were mostly young (mean age 23), white, unmarried, and college educated. The majority of women had a previous history of one or more urinary tract infections (UTIs).

Causative agents and response to therapy

The majority of UTIs were caused by E. coli alone (77%) or in combination with another uropathogen (enterococci, Staphylococcus saprophyticus, Klebsiella pneumoniae, Proteus mirabilis, Enterobacter sp.) (15%). The remaining UTIs were caused by enterococci, Klebsiella sp., Staphylococcus saprophyticus and Citrobacter sp. (1–2% each). Overall, clinical and bacterial cure was achieved in >90% of women in each treatment group.

Isolation of resistant E. coli

Rectal colonization with E. coli was present in 94% of women at the enrolment visit. There was a significant reduction in the prevalence of rectal E. coli after treatment with ciprofloxacin and fosfomycin, but not after treatment with nitrofurantoin (P < 0.001) (Figure 1). Two ciprofloxacin-resistant rectal E. coli strains were isolated from a single patient in the ciprofloxacin treatment group ~10 days after completing therapy (study day 14). The patient had a history of a UTI in the preceding 6–8 weeks, but no other UTIs in her lifetime and no antibiotic usage for at least 2 weeks prior to enrolment. She was sexually active with one male partner and reported no changes in sexual partners or birth control method during the study period. She did not require additional antibiotic therapy during the study period.



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Figure 1. Changes in prevalence of rectal E. coli after treatment of UTI. The percentage of women with rectal E. coli decreased markedly in women treated with ciprofloxacin and fosfomycin, but not nitrofurantoin (P < 0.001, {chi}2 test).

 
The ciprofloxacin-resistant strains were resistant to multiple drugs (Table 1). These strains were not genetically related to the strains isolated from the subject's rectal flora before therapy, and were replaced by different, ciprofloxacin-susceptible, strains by the 30 day visit. Neither strain exhibited ß-haemolysis on sheep blood agar or had papG alleles as assessed by PCR. The patient did not have a clinically apparent recurrence of UTI during the study period, nor did she have asymptomatic bacteriuria at follow-up visits.


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Table 1. Susceptibility of rectal E. coli in a woman treated with a 3 day course of ciprofloxacin for acute uncomplicated cystitis

 
No other subjects in the ciprofloxacin group had isolation of ciprofloxacin-resistant rectal E. coli. Thus, 1/25 or 4.0% (95% CI, 0.1%; 20.4%) of women in the ciprofloxacin group had isolation of fluoroquinolone-resistant rectal E. coli. All rectal E. coli strains isolated from the subjects in the nitrofurantoin and fosfomycin treatment groups were susceptible to the study drug with which the subject had been treated.


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In this study, we demonstrate the emergence of two closely-related ciprofloxacin-resistant E. coli strains in one patient 10 days after completing a 3 day regimen of ciprofloxacin for treatment of acute cystitis. To our knowledge, this is the first study that demonstrates emergence of fluoroquinolone-resistant E. coli after short course therapy of uncomplicated UTI. In studies done in the mid-1980s, Schaeffer and Sisney6 examined the effects of 10 days of norfloxacin treatment in 20 women with acute cystitis and found no selection of resistant E. coli in the faecal or vaginal flora during or up to 1 week after therapy. Similarly, Hooton et al.7 did not find any emergence of resistant coliforms in the rectal flora after 3 day and 7 day regimens of ofloxacin therapy for acute uncomplicated cystitis, although 19% of women treated with a 7 day regimen of co-trimoxazole had emergence of co-trimoxazole-resistant rectal coliforms. More recent studies involving single dose prophylaxis with ciprofloxacin in men undergoing urological procedures and a 28 day treatment regimen in men with prostatitis have shown an increase in ciprofloxacin-resistant intestinal E. coli after treatment.8,9 It is likely that fluoroquinolone resistance among faecal E. coli has increased, perhaps accounting for the ability to demonstrate selection of resistant strains in the latter two studies.

Although fluoroquinolone-resistant E. coli remain infrequent as a cause of uncomplicated UTI in the USA, they are being seen with increasing frequency in such patients in Spain, for example.1 Concomitantly, fluoroquinolone-resistant E. coli have been shown to be highly prevalent in food animals given fluoroquinolone-containing feeds in Spain and elsewhere.10 Given the widespread use of fluoroquinolones in both humans and animals, it is probable that fluoroquinolone-resistant E. coli are increasingly likely to be present in colonic flora. Under the selective pressure provided by even a short 3 day treatment regimen for acute cystitis, resistant strains may predominate at least transiently. To cause UTI, fluoroquinolone-resistant E. coli would need to persist in the rectal flora and eventually colonize the vaginal introitus. Strains possessing urovirulence determinants would be more likely to eventually cause UTI.

Importantly, the ciprofloxacin-resistant strains in this study were not highly uropathogenic as evidenced by lack of P-fimbriae and ß-haemolysis, and did not result in a symptomatic UTI during the follow-up period. They were also genetically different from the susceptible strains that were isolated before therapy and post-therapy. This suggests that exposure to ciprofloxacin either resulted in selection of resistant E. coli strains which were present in quantities too low to be identified prior to drug exposure or in the acquisition of resistant E. coli strains after drug exposure. Studies of patients with complicated UTI have also found that fluoroquinolone-resistant E. coli which are isolated during or shortly after fluoroquinolone exposure are genetically different from susceptible E. coli isolated prior to drug exposure.8,9 Of note, none of the 37 women in the other two treatment regimens in the study (a single dose of fosfomycin or 7 days of nitrofurantoin) demonstrated emergence of intestinal E. coli strains resistant to the treatment drug or to ciprofloxacin. Although we report this occurrence in only one case out of 25 women who were randomized to ciprofloxacin, the findings indicate that selection for fluoroquinolone resistance can occur even with short course therapy of acute cystitis. With increasing use of fluoroquinolones for outpatient UTI in women, this low rate of selection pressure could translate into significantly increased population-based rates of fluoroquinolone resistance and adversely impact treatment options.


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K. G. has served as a consultant for, received research support from, and/or speaking honoraria from Procter & Gamble, Inc., Ortho McNeil, and Bayer Pharmaceutical. T. M. H. has served as a consultant for, received research support from, and/or speaking honoraria from Procter & Gamble, Inc. Ortho McNeil, and Bayer Pharmaceutical. W. E. S. has served as a consultant for and received research support from Procter & Gamble, Inc., Ortho McNeil, and Bayer Pharmaceutical.


    Acknowledgements
 
We gratefully acknowledge the assistance of Pacita Roberts in providing statistical expertise, Kathy Agnew, Marsha Cox and Sheila Manuguid in performing the detailed microbiology, and Carol Winter, ARNP, and Natalie DeShaw, Research Study Coordinator, for patient care and database management. This work was supported in part by a grant from Proctor and Gamble, Inc. and by Public Health Service grants DK 53369 and DK 02660. Presented in part at the 43rd International Conference of Antimicrobial Agents and Chemotherapy in Chicago, October 2003.


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1. Gupta K. Emerging antibiotic resistance in urinary tract pathogens. Infect Dis Clin North Am 2002; 17: 243–60.[ISI]

2. Murray PR, Baron EJ, Jorgensen JH et al. Manual of Clinical Microbiology, 8th edn. Washington, DC, USA: ASM Press, 2003.

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9. Wagenlehner F, Stower-Hoffmann J, Schneider-Brachert W et al. Influence of a prophylactic single dose of ciprofloxacin on the level of resistance of Escherichia coli to fluoroquinolones in urology. Int J Antimicrob Agents 2000; 15: 207–11.[CrossRef][ISI][Medline]

10. Saenz Y, Zarazaga M, Brinas L et al. Antibiotic resistance in Escherichia coli isolates obtained from animals, foods and humans in Spain. Int J Antimicrob Agents 2001; 18: 353–8.[CrossRef][ISI][Medline]