Effect of fluoroquinolone treatment on growth of and toxin production by epidemic and nonepidemic clostridium difficile strains in the cecal contents of mice

Abstract

Several recent outbreaks of Clostridium difficile-associated disease (CDAD) have been attributed to the emergence of an epidemic strain with increased resistance to fluoroquinolone antibiotics. Some clinical studies have suggested that fluoroquinolones with enhanced antianaerobic activity (i.e., gatifloxacin and moxifloxacin) may have a greater propensity to induce CDAD than ciprofloxacin and levofloxacin do. We examined the effects of subcutaneous fluoroquinolone treatment on in vitro growth of and toxin production by epidemic and nonepidemic C. difficile isolates in cecal contents of mice and evaluated the potential for these agents to inhibit fluoroquinolone-susceptible isolates during treatment. When C. difficile isolates were inoculated into cecal contents collected 2 days after the final antibiotic dose, gatifloxacin and moxifloxacin promoted significantly more growth and toxin production than ciprofloxacin and levofloxacin did. During treatment, gatifloxacin and moxifloxacin inhibited growth of fluoroquinolone-susceptible but not fluoroquinolone-resistant isolates. Ciprofloxacin and levofloxacin promoted growth of C. difficile when administered at higher doses (i.e., 20 times the human dose in mg/kg of body weight), and levofloxacin inhibited growth of fluoroquinolone-susceptible, but not fluoroquinolone-resistant, C. difficile isolates when administered in combination with ceftriaxone. Thus, fluoroquinolones with enhanced antianaerobic activity (i.e., gatifloxacin and moxifloxacin) promoted C. difficile growth to a greater extent than did ciprofloxacin and levofloxacin in this model. However, our findings suggest that fluoroquinolones may exert selective pressure favoring the emergence of epidemic fluoroquinolone-resistant C. difficile strains by inhibiting fluoroquinolone-susceptible but not fluoroquinolone-resistant isolates during treatment and that agents such as levofloxacin or ciprofloxacin can exert such selective pressure when administered in combination with antibiotics that disrupt the anaerobic microflora.

FIG. 1.

Effect of antibiotic treatment on growth of (A) and toxin production by (B) Clostridium difficile in the cecal contents of mice. Strains 3 and 4 were epidemic BI strains. Mice received daily subcutaneous antibiotic treatment for 5 days. Two days after the final antibiotic dose, cecal contents were collected and inoculated with 10⁴ CFU/ml of the C. difficile test strains. Samples were incubated anaerobically for 48 h, and then serial dilutions were plated onto selective medium for quantification of C. difficile and assayed for toxin production. Mean toxin titers are expressed as the reciprocal of the highest serial 10-fold dilution that gave positive results. For ciprofloxacin and levofloxacin, 12× and 20× refer to 12 and 20 times the initial dosage, respectively. Error bars represent standard errors.

FIG. 2.

Effect of antibiotic treatment on growth of Clostridium difficile in the cecal contents of mice during treatment. Mice received daily subcutaneous antibiotic treatment for 5 days. Two hours after the final antibiotic dose, cecal contents were collected and inoculated with 10⁴ CFU/ml of the C. difficile test strains. Samples were incubated anaerobically for 48 h, and then serial dilutions were plated onto selective medium for quantification of C. difficile. Error bars represent standard errors. FQ, fluoroquinolone.

FIG. 3.

Effect of clindamycin treatment on growth of clindamycin-resistant (strain 4) and clindamycin-susceptible (strains 5 and 6) Clostridium difficile strains in the cecal contents of mice during or after treatment. Mice received daily subcutaneous clindamycin treatment for 5 days. Two hours (during treatment) or 2 days (after treatment) after the final antibiotic dose, cecal contents were collected and inoculated with 10⁴ CFU/ml of the C. difficile test strains. Samples were incubated anaerobically for 48 h, and then serial dilutions were plated onto selective medium for quantification of C. difficile. Error bars represent standard errors.