Repeated Treatments with Chitosan in Combination with Antibiotics Completely Eradicate Uropathogenic Escherichia coli From Infected Mouse Urinary Bladders

Abstract

Uropathogenic Escherichia coli (UPEC), the primary causative agents of urinary tract infections, colonize and invade the epithelial cells of the bladder urothelium. Infection of immature urothelial cells can result in the formation of persistent intracellular reservoirs that are refractory to antibiotic treatments. Previously, we defined a novel therapeutic strategy that used the bladder cell exfoliant chitosan to deplete UPEC reservoirs. However, although a single treatment of chitosan followed by ciprofloxacin administration had a marked effect on reducing UPEC titers within the bladder, this treatment failed to prevent relapsing bacteriuria. We show here that repeated use of chitosan in conjunction with the antibiotic ciprofloxacin completely eradicates UPEC from the urinary tract and prevents the development of relapsing bouts of bacteriuria. In addition, microscopy revealed rapid restoration of bladder integrity following chitosan treatment, indicating that chitosan can be used to effectively combat recalcitrant bladder infections without causing lasting harm to the urothelium.

Uropathogenic Escherichia coli (UPEC) are the primary causative agents of acute and recurrent urinary tract infections (UTIs) [1]. UPEC are facultative intracellular bacteria that can invade, replicate, and persist within the epithelial cells of the urinary bladder [2]. The bladder urothelium is composed of terminally differentiated superficial cells called umbrella cells, which are in direct contact with the urine, and less differentiated intermediate and basal epithelial cells [3]. UPEC adhere to superficial cells and can subsequently invade and enter the host cytosol, where the bacteria are able to replicate to high levels, forming large biofilm-like intracellular bacterial communities (IBCs) [4, 5]. Ultimately, bacteria are released from IBCs and are expelled with the flow of urine or reinfect tissues within the urinary tract. The exfoliation of umbrella cells during the course of a UTI can also rid the host of IBCs, but this exposes underlying, less differentiated urothelial cells to infection and may facilitate the establishment of quiescent intracellular reservoirs [5–7]. The ability of UPEC to persist at subclinical levels within intracellular reservoirs, where they are nonsusceptible to most antibiotic treatments, likely contributes to the recurrence of UTIs following cessation of antibiotic treatments [8–11]. Although antibiotic treatments are very successful in treating acute cystitis, chronic and recurrent infections remain common despite clinical intervention [2].

The urothelium is a tight epithelium impermeable to urine and exogenous substances, such as antibiotics [8, 12, 13]. Under physiological conditions, the urothelium has an extremely slow turnover rate, replicating once every 10–12 months [14, 15]. However, after injury, the proliferation and differentiation of basal cells increase rapidly, leading to the fast renewal of intermediate and superficial cells and the reestablishment of the permeability barrier function [3, 12]. Several experimental methods for inducing urothelial injury have been developed as means to compromise and study the barrier function [16–21]. The intravesical application of the biopolymer chitosan serves as an easily controlled method to detach the superficial layer of urothelial cells [22]. Chitosan is a cationic polysaccharide composed of glucosamine and N-acetyl glucosamine, obtained by partial N-deacetylation of chitin. It is regarded as a biocompatible, biodegradable, and nontoxic polymer and has been used extensively as a vehicle for the delivery of vaccines in animals and humans [23–25]. It interacts with negatively charged macromolecules such as integrins on the cell membrane, causing the disruption of tight junctions and the exfoliation of epithelial cells [26–28]. Induced removal of urothelial cells by chitosan is followed by rapid regeneration of the urothelium [22, 29, 30]. Chitosan-induced exfoliation of superficial urothelial cells, in combination with antibiotic treatments, has been shown to reduce the burden of UPEC in a mouse UTI model but did not completely clear the pathogens from the urinary tract [29]. In this study, we describe an optimized chitosan treatment protocol in which repeated administration of chitosan in combination with ciprofloxacin allows for complete eradication of UPEC from the murine urinary bladder. Importantly, the approach, which relies on repeated chitosan administration with or without the antibiotic ciprofloxacin, does not seem to cause lasting damage to the urothelium.

METHODS

Animals

Experiments were performed on 10–14-week-old female C57BL/6JOLaHsd mice (Harlan, Udine, Italy). Mice were housed in open-barred cages with bedding (Lignocel ¾; Altromin, Rosenberg, Germany) under controlled conditions of temperature (22°C ± 1°C), humidity (55% ± 10%), and light (a cycle of 12 hours of light [from 7:00 am to 7:00 pm] followed by 12 hours of darkness) and had unlimited access to laboratory food and water. All procedures involving mice were approved by the National Ethical Committee and the Administration of the Republic of Slovenia for Food Safety, Veterinary, and Plant Protection (permit 34401–5/2011/5). Animal care and treatment were in accordance with Slovenian and international legislation and policy (directive 2010/63/EU) on the protection of animals used for scientific purposes.

Bacteria

The reference UPEC cystitis isolate UTI89 used in this study has been described previously [5]. Bacteria were inoculated from frozen stocks into Luria-Bertani broth and grown statically at 37°C for 48 hours. Bacterial cells were pelleted by centrifugation for 15 minutes at 3000 ×g and then resuspended in sterile phosphate-buffered saline (PBS) to a concentration of 10⁸ colony-forming units (CFU)/mL.

Reagents

Chitosan hydrochloride (86% deacetylated; Kraeber, Ellerbek, Germany) was prepared as a 0.5% (w/v) dispersion by mixing at room temperature overnight in phosphate buffer before adjusting the pH to 4.5. Chitosan solutions were used with or without addition of ciprofloxacin (800 µg/mL; Sigma-Aldrich, Steinheim, Germany).

Mouse Experiments

Mice were anesthetized with ketamine HCl (100 mg/kg intraperitoneally) and xylazine (10 mg/kg intraperitoneally), placed in the dorsal position, and inoculated with an 80-µL suspension of UTI89 (approximately 10⁷ CFU) in PBS via transurethral catheterization, using a sterile polyethylene catheter with a 0.28-mm inner diameter (Intramedic, Becton Dickinson, Sparks, Maryland). The bladder of each animal was emptied by gentle pressure on the abdomen before catheterization. Infusions were performed gradually and at a slow rate to avoid injury or vesicoureteral reflux. The catheter, sheathed over a 30-gauge needle connected to a 1-mL syringe, was retained in the urinary bladder for 60 minutes. Infected mice were left untreated or were treated with 0.5% chitosan or 0.5% chitosan plus ciprofloxacin, delivered in 80 µL by transurethral catheterization 1, 4, 7, and 11 days after inoculation with UTI89. At the indicated time points, mice were euthanized, and their bladders and kidneys were harvested aseptically, weighed, and homogenized in 1 mL of sterile tissue-culture-grade PBS containing 0.025% Triton X-100. Tissue homogenates were serially diluted and plated in duplicate on Luria-Bertani or cysteine-lactose-electrolyte–deficient agar plates. After an overnight incubation at 37°C, colonies were counted, and the number of CFU per gram of tissue was determined. Bacteria present in urine collected from mice were enumerated using Uricult Trio tests (Orion Diagnostica, Espoo, Finland). After overnight incubation at 37°C, the presence of bacteria was assessed as colonies on the plates.

Experimental Scheme

Mice were divided into 2 groups, uninfected mice (control group I; n = 13) and infected mice (n = 74). For short-term experiments, in which animals were euthanized 1 day or 14 days after infection, infected mice were subdivided into 2 control groups that received no exfoliant: control group II (n = 20), to verify bacterial cystitis 24 hours after infection, and control group III (n = 14), to verify bacterial cystitis simultaneously with verification of that in treated animals on day 14 after infection. Two therapeutic groups received 0.5% chitosan alone (n = 20) or 0.5% chitosan with ciprofloxacin (n = 20). Intravesical application of 80 µL of each exfoliant was performed 24 hours after initiation of the infection and then every third day, for a total of 4 times. Mice from both experimental groups were euthanized on the third day following the final intravesical application (day 14 after infection), and bladders and urine samples were collected. Samples from control groups I and II were collected on day 1 or day 14.

For long-term experiments, in which animals were euthanized 32 days after infection, successful induction of bacterial cystitis was tested 24 hours after infection (as in control group II), while 12 mice were divided into 2 groups: the therapeutic group (n = 6) received 0.5% chitosan with ciprofloxacin and control group II (n = 6) did not receive any exfoliant. Administration of treatments occurred as described above. Mice from control group II and the therapeutic group were euthanized 3 weeks after the final intravesical application (day 32 after infection).

Transmission Electron Microscopy

Excised bladders were cut into small pieces and fixed in a mixture of 4% paraformaldehyde and 2% glutaraldehyde in 0.2 M cacodylate buffer (pH 7.3) for 3 hours at 4ºC. Overnight rinsing in 0.33 M sucrose in 0.2 M cacodylate buffer was followed by postfixation with 1% OsO₄ for 1 hour. After dehydration in a series of ethanol concentrations, tissue samples were embedded in Epon (Serva Electrophoresis, Heidelberg, Germany). Epon semithin sections (1 µm) were stained with 1% Toluidine blue and 2% borate in distilled water for 20 seconds and observed with a Nikon Eclipse TE bright-field microscope (Amsterdam, Netherlands). Ultrathin sections were contrasted with uranyl acetate and lead citrate and examined with a Jeol 100 CX electron microscope (Tokio, Japan).

Scanning Electron Microscopy (SEM)

Fully filled and distended bladders were excised from animals, cut longitudinally into halves, and fixed for 3–4 hours at 4°C in a mixture of 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). The tissue samples were rinsed in 0.1 M cacodylate buffer and postfixed in 1% osmium tetroxide in the same buffer for 1 hour at 4°C. Specimens were dried at their respective critical points, sputter-coated with gold, and examined at 15 kV with a Jeol JSM 84 A scanning electron microscope (Tokio, Japan).

Immunofluorescence

Bladders were rinsed overnight in 30% sucrose, frozen, and cut into 5-µm-thick sections. After washing in PBS, sections were permeabilized in cold acetone for 5 minutes, blocked in 3% bovine serum albumin (BSA) in PBS, and incubated overnight at 4°C with mouse monoclonal antibodies against cytokeratin 20 (DAKO, Glostrup, Denmark) diluted 1:100 in 1% BSA in PBS. After washing in PBS, samples were incubated in the dark at 37°C for 1 hour with Alexa Fluor 555–labeled goat anti-rabbit secondary antibodies (Invitrogen, Molecular Probes, Eugene, Oregon) diluted 1:300 in 1% BSA in PBS. After prolonged washing in PBS, sections were incubated at 37°C for 1 hour with fluorescein isothiocyanate–labeled rabbit polyclonal antibodies against E. coli (dilution, 1:10; Abcam, Cambridge, United Kingdom). After additional washing in PBS, sections were mounted in the antibleaching mounting medium Vectashield, containing 4ʹ,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, California), to stain DNA. Negative controls, in which both primary antibodies were substituted with serum from nonimmunized animals, were evaluated to validate results. Samples were imaged using a Nikon Eclipse TE 300 fluorescence microscope.

Statistical Analysis

P values were determined by 2-tailed Mann-Whitney U tests, using Prism 5.01 software (GraphPad Software). The normality of data distribution was not assumed, so nonparametric tests were used for all mouse experiments. P values of <.05 were deemed significant.

RESULTS AND DISCUSSION

In previous work using a mouse UTI model system, we were able to greatly reduce the levels of detectable UPEC reservoirs within bladder tissue by treating UPEC-infected mice once with chitosan followed by 3–7 doses of the antibiotic sparfloxacin [29]. However, despite marked reduction of the bladder reservoir populations, we were unable to completely prevent relapsing (or recurrent) infections, as assessed by the occurrence of bacteriuria (≥10⁴ CFU/mL of urine) in mice over a 15-day period following cessation of the antibiotic treatments (data not shown) [29]. Nonetheless, these results were promising and prompted us to pursue optimization of a chitosan treatment protocol in which the UPEC reservoir populations could be more thoroughly eradicated.

To begin, adult female C57BL/6JOLaHsd mice were inoculated with the UPEC cystitis isolate UTI89 via transurethral catheterization. Mice were then left untreated or were given chitosan or chitosan with ciprofloxacin, via catheter, 1, 4, 7, and 14 day after inoculation with UTI89 (Figure 1). By 24 hours after inoculation, results of 100% of Uricult tests of urine specimens from infected mice were positive (Figure 2). Immunofluorescence microscopy confirmed the presence of intracellular UPEC populations within the urothelial cells, with bacteria seen dispersed within the cytoplasm of umbrella cells and more tightly associated within IBCs (Figures 3A and 3B). SEM of infected bladders revealed mostly immature urothelial cells that are exposed following desquamation of umbrella cells in response to infection (Figure 3C) [31]. The remaining umbrella cells exhibited ruffled, sponge-like apical surfaces that were often loaded with bacteria protruding from the interior of the cells (Figure 3D). By 14 days after inoculation, immunofluorescence microscopy and SEM indicated the persistence of UPEC within the different layers of the urothelium (Figure 4A), as well as on the urothelial surface (Figure 4B). Many of these bacteria were filamentous, as seen in previous studies [4, 5, 29]. At this day 14 time point, only 14% of the untreated mice had bacteriuria, based on results of Uricult tests, although bladders from all of these mice had high bacterial titers (Figure 2). By 32 days after inoculation, UPEC titers within the untreated mice had declined about 10-fold, and 17% of the animals were positive for bacteriuria. These results demonstrate that UPEC can persist at fairly steady levels for long periods within the bladders of C57BL/6JOLaHsd mice and are in line with previous findings indicating that the absence of bacteriuria does not necessarily reflect a lack of pathogens within the urothelium [8].

Figure 1.

Schematic overview of the experimental set up used in this study. Titer, bladder samples collection for bacterial titer analysis T, intravesical treatment with chitosan or chitosan plus ciprofloxacin.

Figure 2.

Repeated treatments with chitosan plus ciprofloxacin eliminate uropathogenic Escherichia coli reservoirs. Shown are bacterial titers in the bladders of mice that were infected with UTI89 and then left untreated or treated with 4 doses of chitosan alone or of chitosan plus ciprofloxacin. Bladders were recovered 1, 14, or 32 days after inoculation, as noted below the graph. Bars indicate median values. P values were determined by Mann-Whitney U tests and involve data for 4 mice on day 1 after infection and for 6 mice each on days 14 and 32 after infection. CFU, colony-forming units; Cipro, ciprofloxacin. ^aPositivity for bacteriuria was determined by Uricult assays.

Figure 3.

Uropathogenic Escherichia coli (UPEC) colonization of bladder urothelium. A and B, Immunofluorescent images of bladder sections from mice 1 day after inoculation with the UPEC isolate UTI89, showing bacteria (arrows), the well-established marker of terminally differentiated umbrella cells cytokeratin 20 (asterisks), and host nuclei (n). L, lumen of the urinary bladder. Dotted line represents lumenal surface of the urothelium. The arrow (B) indicates an intracellular bacterial community encircled also with dotted line. C, Scanning electron microscopy image showing that the majority of umbrella cells have exfoliated at the day 1 time point, revealing small, immature urothelial cells. Arrows indicate some of the few remaining umbrella cells. D, Higher magnification image of a remaining umbrella cell, which contains many bacteria protruding across the perforated apical plasma membrane. Scale bars denote 10 µm (A and B), 50 µm (C), and 1 µm (D).

Figure 4.

Persistence of uropathogenic Escherichia coli (UPEC) within the urothelium of untretaed mice. A, immunofluorescent image showing UPEC (arrowheads), cytokeratin 20 (asterisks), and host cell nuclei (n) in a bladder section from a mouse 14 days after inoculation with UTI89. B, Scanning electron microscopy image of the bladder surface 14 days after inoculation with UPEC. Image shows a bacterium that is visible on the umbrella cell (UC; arrow), as well as intermediate cells that have become exposed because of desquamation of UCs. Scale bars denote 10 µm. BC, basal cells; IC, intermediate cells; L, lumen of the urinary bladder.

The treatment of infected mice with 4 sequential doses of chitosan, with or without ciprofloxacin, reduced bladder bacterial titers below the limit of detection by 14 days after inoculation (Figure 2). Similar results were seen 32 days after inoculation, at which point no bacteria were recovered from the bladders of mice that had been treated with a combination of chitosan and ciprofloxacin 21 days earlier. As determined by Uricult tests, 10% of the mice given only the chitosan treatments had bacteriuria 14 days after inoculation with UTI89, whereas none of the animals receiving chitosan plus ciprofloxacin had bacteriuria at either the day 14 or day 32 time points. This is a substantial improvement over previous results obtained using a single dose of chitosan and antibiotics, which failed to completely eliminate UPEC reservoirs (data not shown) [29].

In total, these results indicate that repeated treatments with chitosan plus ciprofloxacin can effectively reduce and even eliminate UPEC bladder reservoirs, as well as the incidence of bacteriuria. By stimulating the necrotic death and exfoliation of umbrella cells, chitosan treatments trigger rapid regenerative and differentiation pathways within the urothelium that likely lead to the reactivation and resurgence of UPEC reservoirs [29]. Damage to the urothelium due to the administration of a single dose of chitosan is transient and does not cause notable or prolonged disruption of the urothelial barrier [22, 29, 30]. Using SEM, we found that the bladder urothelium could also rapidly recover from multiple rounds of exposure to chitosan (Figure 5). At the day 14 time point in our assays, the urothelial surface of infected bladders that had been treated with 4 doses of chitosan, with or without ciprofloxacin, was composed exclusively of maturing superficial urothelial cells of varying sizes, as previously observed with uninfected bladders that were treated with a single dose of chitosan [22]. In addition, no bacteria, host cell death, or evidence of ongoing urothelial desquamation were detected by SEM at this time point (Figure 5A and 5B).

Figure 5.

The urothelium recovers rapidly from multiple doses of chitosan. Scanning electron microscopy images show the urothelium at the day 14 time point, 3 days after administration of the last of 4 chitosan (A) or chitosan plus ciprofloxacin (B) treatments. Scale bars denote 10 µm.

In summary, the C57BL/6JOLaHsd mice used in this study were unable to resolve, on their own, bladder infections caused by UPEC. The spontaneous bouts of bacteriuria, coupled with persistent colonization of the bladder tissues, seen in this model may reflect conditions in patients with chronic, recurrent, or relapsing UTI. As a biodegradable cationic polymer with low toxicity, chitosan is a useful tool for the safe and controlled removal of urothelial cells. Previous studies indicate that chitosan stimulates urothelial cell desquamation and necrotic death by interacting with integrin receptors and causing the disassembly of tight junctions and the disruption of the host plasma membrane [12, 22, 32]. In addition, we have shown that urothelial regeneration after chitosan treatment is extremely fast, which limits any detrimental effects that chitosan has on the blood-urine barrier [22]. The potential therapeutic value of combinatorial treatments with chitosan and antibiotics such as ciprofloxacin requires further investigation, but the findings presented here provide a strong impetus for pursuing this approach as an option for individuals who have chronic UTIs that are unresolved by use of more classical antibiotic therapies.

Notes

Acknowledgments We thank Darja Plestenjak (Institute of Microbiology and Immunology, Faculty of Medicine, Ljubljana, Slovenia), for committed work in animal care, and Frank Rauh (FMC, Ewing, New Jersey), for providing chitosan.

Disclaimer. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health.

Financial support. This work was supported by the Slovenian Research Agency (program grant P3-0108), the National Institutes of Health (grant AI095647 to M. A. M.), the University of Utah Pathology Department (seed grant to M. A. M.), and the National Institute of Allergy and Infectious Diseases (training grant award T32AI055434 to M. G. B.

Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.