Evaluation of Immunocompetent Urinary Tract Infected Balb/C Mouse Model For the Study of Antibiotic Resistance Development Using Escherichia Coli CFT073 Infection

Abstract

Urinary tract infections (UTI) are common worldwide and are becoming increasingly difficult to treat because of the development of antibiotic resistance. Immunocompetent murine models of human UTI have been used to study pathogenesis and treatment but not for investigating resistance development after treatment with antibiotics. In this study, intravesical inoculation of uropathogenic Escherichia coli CFT073 in immunocompetent Balb/c mice was used as a model of human UTI. The value of the model in investigating antibiotic exposure on in vivo emergence of antibiotic resistance was examined. Experimentally infected mice were treated with 20 or 200 mg/kg ampicillin, 5 or 50 mg/kg ciprofloxacin, or 100 or 1000 mg/kg of fosfomycin. Ampicillin and ciprofloxacin were given twice daily at 8 h intervals, and fosfomycin was given once daily. Antibiotic treatment began 24 h after bacterial inoculation and ended after 72 h following the initial treatment. Although minimum inhibitory concentrations (MIC) for the experimental strain of E. coli were exceeded at peak concentrations in tissues and consistently in urine, low levels of bacteria persisted in tissues in all experiments. E. coli from bladder tissue, kidney, and urine grew on plates containing 1× MIC of antibiotic, but none grew at 3× MIC. This model is not suitable for studying emergent resistance but might serve to examine bacterial persistence.

Keywords: CFT073 UPEC strain; Mouse UTI model; ampicillin; antibiotic resistance; ciprofloxacin; fosfomycin.

Figure 1

Natural history of Escherichia coli CFT073 strain urinary tract infections (UTI) in a Balb/c mouse strain. Mice were inoculated into urinary bladder with 10⁸ colony forming units (CFU) of E. coli by transurethral catheterization on day 0. Following infection, mice were sacrificed at day 1, 3, 7, 8, and 10 post-infection and the bacterial counts (number of CFU) from urinary bladder (per gm) (A), kidneys (per gm) (B), and urine (per mL) (C) are shown at different post-infection days. Value zero was plotted as the limit of detection. Solid horizontal line represents the median bacterial count for the groups. The data shown was pooled from three separate experiments done at different times; n = 18 for day 1 and 2, n = 13 for day 7, n = 7 for day 8, and n = 17 for day 10. For urine samples, the number of animals varies based on the presence of urine in the urinary bladder at the time of necropsy.

Figure 1

Natural history of Escherichia coli CFT073 strain urinary tract infections (UTI) in a Balb/c mouse strain. Mice were inoculated into urinary bladder with 10⁸ colony forming units (CFU) of E. coli by transurethral catheterization on day 0. Following infection, mice were sacrificed at day 1, 3, 7, 8, and 10 post-infection and the bacterial counts (number of CFU) from urinary bladder (per gm) (A), kidneys (per gm) (B), and urine (per mL) (C) are shown at different post-infection days. Value zero was plotted as the limit of detection. Solid horizontal line represents the median bacterial count for the groups. The data shown was pooled from three separate experiments done at different times; n = 18 for day 1 and 2, n = 13 for day 7, n = 7 for day 8, and n = 17 for day 10. For urine samples, the number of animals varies based on the presence of urine in the urinary bladder at the time of necropsy.

Figure 2

Single dose pharmacokinetics of ampicillin, ciprofloxacin, and fosfomycin antibiotics in female Balb/c mice. Groups of six mice were administered orally with 200 mg/kg of ampicillin, 50 mg/kg of ciprofloxacin and 500 mg/kg of fosfomycin. Plasma were sampled at 0, 15, and 30 min, 1, 2, 4, 8, 12, and 24 h time points. Urinary bladder, kidneys and urine were sampled at 0, 30 min, 2, 8, and 24 h time points during necropsy. Each symbol represents mean and standard deviation from six animals.

Figure 2

Single dose pharmacokinetics of ampicillin, ciprofloxacin, and fosfomycin antibiotics in female Balb/c mice. Groups of six mice were administered orally with 200 mg/kg of ampicillin, 50 mg/kg of ciprofloxacin and 500 mg/kg of fosfomycin. Plasma were sampled at 0, 15, and 30 min, 1, 2, 4, 8, 12, and 24 h time points. Urinary bladder, kidneys and urine were sampled at 0, 30 min, 2, 8, and 24 h time points during necropsy. Each symbol represents mean and standard deviation from six animals.

Figure 3

Representative photomicrographs of saline control (A,B), 1 DPI (C,D), and 10 DPI (E,F). Control urinary bladders exhibit a normal urothelium with minimal mucosal lymphocytes and edema. Intracellular bacterial communities (black arrow) admixed with neutrophilic infiltrates, marked urothelial hyperplasia, and mucosal edema with lymphocytic inflammation are observed at 1 DPI. At 10 DPI, urothelial hyperplasia is minimal and variable lymphohistiocytic mucosal infiltrates with rare neutrophils are still apparent. A, C, E @ 4× and B, D, F @ 60×. H&E stain. DPI = days post infection.

Figure 3

Representative photomicrographs of saline control (A,B), 1 DPI (C,D), and 10 DPI (E,F). Control urinary bladders exhibit a normal urothelium with minimal mucosal lymphocytes and edema. Intracellular bacterial communities (black arrow) admixed with neutrophilic infiltrates, marked urothelial hyperplasia, and mucosal edema with lymphocytic inflammation are observed at 1 DPI. At 10 DPI, urothelial hyperplasia is minimal and variable lymphohistiocytic mucosal infiltrates with rare neutrophils are still apparent. A, C, E @ 4× and B, D, F @ 60×. H&E stain. DPI = days post infection.

Figure 4

The long term study with repeated courses of fosfomycin (500 mg/kg) showing the infection, treatment and sample collection schedules.