Combinatorial small-molecule therapy prevents uropathogenic Escherichia coli catheter-associated urinary tract infections in mice

Abstract

Catheter-associated urinary tract infections (CAUTIs) constitute the majority of nosocomial urinary tract infections (UTIs) and pose significant clinical challenges. These infections are polymicrobial in nature and are often associated with multidrug-resistant pathogens, including uropathogenic Escherichia coli (UPEC). Urinary catheterization elicits major histological and immunological alterations in the bladder that can favor microbial colonization and dissemination in the urinary tract. We report that these biological perturbations impact UPEC pathogenesis and that bacterial reservoirs established during a previous UPEC infection, in which bacteriuria had resolved, can serve as a nidus for subsequent urinary catheter colonization. Mannosides, small molecule inhibitors of the type 1 pilus adhesin, FimH, provided significant protection against UPEC CAUTI by preventing bacterial invasion and shifting the UPEC niche primarily to the extracellular milieu and on the foreign body. By doing so, mannosides potentiated the action of trimethoprim-sulfamethoxazole in the prevention and treatment of CAUTI. In this study, we provide novel insights into UPEC pathogenesis in the context of urinary catheterization, and demonstrate the efficacy of novel therapies that target critical mechanisms for this infection. Thus, we establish a proof-of-principle for the development of mannosides to prevent and eventually treat these infections in the face of rising antibiotic-resistant uropathogens.

Fig 1

Uropathogenic E. coli produces IBCs in the superficial umbrella cells of implanted bladders. (A) Representative images of splayed bladders of female C57BL/6Ncr mice infected with UTI89 at 6 hpi in the absence (nonimplanted) or presence (implanted) of implants after LacZ staining. Each black arrow indicates a purple speck, indicative of an IBC. (B) Quantification of IBC formation following LacZ staining at 6 hpi. Each symbol represents IBC number from a single mouse from two independent experiments with n = 5/group. The P value was obtained using the Mann-Whitney U test. (C) Representative CLSM images of whole bladders from nonimplanted and implanted animals infected with UTI89 ectopically expressing GFP (green), stained with DNA dye SYTO83 (red), and an Alexa Fluor 633 conjugate of WGA (blue) reveal the presence of IBC within umbrella cells. Scale bar, 20 μm.

Fig 2

UPEC reservoir reactivation can lead to urinary implant and bladder colonization. Graphs represent bacterial titers in log scale recovered from implants and homogenized bladders of nonimplanted or implanted for 3 days (A) or 5 days (B) animals that were nonbacteriuric and previously infected for 14 dpi with UTI89HK::GFP. Horizontal dashed lines represent the limit of detection for viable bacteria. Each symbol represents a mouse from two independent experiments with n = 10 to 20/group/experiment. The horizontal bars represent the median of each data set. The P value was determined by using the Mann-Whitney U test.

Fig 3

Deletion of fimH reduces biofilm formation and attenuates UPEC virulence. Graphs represent crystal violet-based quantification (A) and CFU enumeration in a logarithmic scale (log scale) (B) of 24-h-old UTI89 and UTI89ΔfimH (ΔfimH) biofilms under human urine flow on silicone tubing at 37°C, indicating that the ΔfimH strain is defective in biofilm formation under these conditions. The bars represent means of three independent experiments, and error bars indicate the standard errors of the mean. The P values were determined using the Mann-Whitney U test. (C) Graph representing bacterial titers in log scale recovered from implants and homogenized bladders of nonimplanted (open symbols) and implanted (closed symbols) infected with either UTI89 (squares) or UTI89ΔfimH (circles) for 24 h. Horizontal dashed lines represent the limit of detection for viable bacteria. Each symbol represents a mouse from at least two independent experiments with n = 5/group. The horizontal bars represent the median of each data set. *, P < 0.05; ***, P < 0.0005 (as determined by the Mann-Whitney U test).

Fig 4

Mannoside treatment prevents IBC formation and UPEC virulence when used in combination with TMP-SMZ. (A) Graph representing IBC enumeration from LacZ staining of splayed bladders of female C57BL/6Ncr mice treated i.p. with mannoside or saline prior to transurethral implantation and inoculation with UTI89 at 6 hpi. Each symbol represents the IBC number from a single mouse from two independent experiments with n = 5/group. The P value was determined using the Mann-Whitney U test. (B) Graph representing bacterial titers in log scale recovered at 6 hpi from implants and homogenized bladders of animals treated with saline (○), mannoside (□),TMP-SMZ (●), and TMP-SMZ plus mannoside (■) prior to urinary implantation and inoculation with UTI89. Horizontal dashed lines represent the limit of detection for viable bacteria. Each symbol represents a mouse from at least two independent experiments with n = 5/group. The horizontal bars represent the median of each data set. *, P < 0.05; **, P < 0.0005; ***, P < 0.0005. ns, P > 0.05. Significance was evaluated by using the Mann-Whitney U test.