Use of ceragenins as a potential treatment for urinary tract infections

Clinical strains of E. coli were obtained from patients diagnosed with urinary tract infection at Independent Public Province Hospital of Jan Sniadecki in Bialystok. The susceptibilities of tested bacterial strains to conventional antibiotics were established by the agar disc diffusion method on MHA agar according to the guidelines of the EUCAST (ang. The European Committee on Antimicrobial Susceptibility Testing). Zones of inhibition were measured to the nearest millimeter and recorded. Susceptibility tests showed that all E. coli isolates were resistant to ampicillin, and two of them were resistant to sulfamethoxazolum/trimethoprimum (Table 1).

LL-37 peptide was purchased from Lipopharm.pl (Zblewo, Poland), and the purity of LL-37 was > 98% (as determined by high-performance liquid chromatography [HPLC]). The ceragenins, CSA-13 and CSA-131, were synthesized as described previously [30]. Conventional antibiotics (sulfamethoxazolum/trimethoprimum, ciprofloxacin, norfloxacin, gentamicin, fosfomycin, meropenem, amoxicillin/clavulanic acid, cefepime, cefotaxime, ampicillin and doxycyclinum) were purchased from OXOID (England) and Polfa Tarchomin (Poland).

Human bladder epithelial cell line T24 (ATCC® HTB­4™), purchased from American Type Culture Collection (ATCC), was cultured at 37 °C with 5% CO₂ in McCoy’s 5A medium (ATCC, catalog no. 30–2007) supplemented with 10% fetal bovine serum (FBS, ATCC, catalog no. 30–2020). All cell culture-based experiments, with the exception of MTT assays, were performed in serum-free conditions. For this purpose, FBS-containing growth medium was replaced with serum-free medium 4 h before the addition of tested agents at indicated concentrations.

Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of tested compounds were determined using bacteria at the logarithmic phase of growth. Antibacterial agents: LL-37, CSA-13, and CSA-131 and combination of LL-37 with doxycycline (DOX) and ceragenins were tested against four clinical strains of E. coli (~ 10⁵ CFU/mL). The MIC and MBC values were determined in Luria-Bertani broth (LB) using the microdilution method described by the reference Clinical and Laboratory Standards Institute (CLSI). The MIC values were determined versus bacterial concentrations of ~ 10⁵ CFU/mL, and the MBC was performed by plating each sample on LB agar.

The killing activity of LL-37, doxycycline, ceragenins CSA-13 and CSA-131 and combinations of LL-37 with doxycycline, CSA-13 and CSA-131 were determined against four E. coli isolates. Individual colonies of bacteria were diluted to 10⁵ CFU/ml with sterile phosphate-buffered saline (PBS, pH 7.0). The assays were run with different concentrations of LL-37, doxycycline, CSA-13 and CSA-131 (ranging from 2 μM to 50 μM) individually, and combinations of LL-37 with doxycycline, LL-37 with CSA-13 and LL-37 with CSA-131 (ranging from 2 μM to 50 μM; in 1:1 ratio). After 30 min of incubation at 37 °C, samples were diluted 10- to 1000-fold and 10 μl aliquots of each dilution were plated on agar and incubated overnight at 37 °C to determine the number of viable colonies. The colony-forming units (CFU/ml) of the individual samples were determined from the dilution factor. Presented data are an average from three individual experiments. To confirm the killing activity of tested agents, detection of metabolic activity in E. coli in suspensions upon treatment with different concentrations of tested compounds (2–50 μM) and their combinations (1:1 ratio) was assessed. After incubation of bacteria with tested compounds (at 37 °C for 1 h), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT reagent, Sigma Aldrich, USA) at final concentration of 0.5 mg/mL was added to each well, and the 96-well plates were incubated at 37 °C for 1–2 h in the dark. Then, 100 μl of dimethyl sulfoxide (DMSO) was added to each well and the plate was left for 1 h at room temperature to allow the color to develop. The optical density was measured at 550 nm using Varioskan LUX (Thermo Fisher Scientific, Waltham, MA, USA).

Formation of biofilm by UTI-associated clinical isolates of E. coli in the presence of different concentrations (2–50 μM) of tested compounds and their combinations (1:1 ratio) was assessed using crystal violet (CV) staining (0.1%) in 96-well polystyrene microtiter plates. After incubation of bacterial samples with indicated agents (37 °C for 48 h), the plates were washed with PBS to remove unattached bacteria and stained using 0.1% (w/v) crystal violet for 15 min at room temperature. Crystal violet was removed, solubilized in 95% ethanol and plates were scanned at 570 nm using Labsystem Varioscan Lux (Thermo Fisher Scientific, Waltham, MA, USA) to determine the optical density of the dye attached to stained biofilms.

The cytotoxic effect of tested compounds and their combinations (1:1 ratio) against bladder epithelial cells was evaluated using MTT assay. For this purpose, T24 (ATCC® HTB­4™) cells were seeded into each well of a 96-well flat-bottom microtiter plate (Sarstedt, Newton, NC, USA) to adhere overnight. After 24 h from seeding, cells were treated with LL-37, doxycycline, CSA-13 and CSA-131 (2, 4, 10, 20 and 50 μM) and combinations of LL-37 with doxycycline, LL-37 with CSA-13 and LL-37 with CSA-131 (ranging from 2 μM to 50 μM; in 1:1 ratio). After 1 h incubation of cells with indicated therapeutic agents (37 °C with 5% CO₂), the MTT salt working solution (100 μL/well at the final concentration of 0.5 mg/mL) was added to each well and incubated for another 2–4 h. Working medium was removed and DMSO was added (100 μL/well) to solubilize formazan crystals. Absorbance was measured at 550 nm using Varioskan LUX. The percentage of cell viability was calculated as (absorbance of treated cells/absorbance of untreated cells) × 100%.

To assess the activity of tested compounds against intracellular pathogens, T24 (ATCC® HTB­4™) cells were seeded at the density of 10⁵ cells/well in a 24-well flat-bottom microtiter plate (Sarstedt, Newton, NC, USA) and cultured at 37 °C with 5% CO₂ for 24 h to form a confluent monolayer. After this time, medium was removed and wells were washed twice with PBS. Clinical isolates of E. coli at log-phase (1 mL, 2 × 10⁸ CFU/mL) were then added to each well and incubated for 2 h at 37 °C. Cells were washed three times with PBS, 5 μg/mL of gentamycin was added to each well to eliminate extracellular E. coli and plates were left for further incubation for 2 h. Control wells were washed twice with PBS and T24 (ATCC® HTB­4™) cells were lysed with 0.1% Triton X-100 in PBS for 10 min at 37 °C and plated on agar plates overnight. Subsequently, different molar concentrations (2–50 μM) of LL-37, doxycycline, CSA-13 and CSA-131 and combinations of LL-37 with doxycycline/CSA-13/CSA-131 were added to the remaining wells for 2 h at 37 °C. After incubation, cells were washed three times with PBS and lysed with 0.1% Triton X-100 for 10 min at 37 °C. To determine the viability of intracellular E. coli upon treatment with tested agents, obtained samples were diluted 10- to 1000-fold and ten-microliter aliquots of each dilution were spotted on agar plates for ~ 18 h at 37 °C. The CFU (CFU/ml) of the individual samples of intracellular E. coli were determined from the dilution factor and were used to calculate the percentage of bacterial outgrowth.

The antibacterial activities of conventional antibiotics against clinical strains of E.coli obtained from patients diagnosed with UTI were assessed using a disc diffusion assay and are shown in Table 1.

We found that 50% of the E. coli isolates were resistant to sulfamethoxazolum / trimethoprimum, which is well-established broad spectrum antibiotic combination with potent bactericidal activity against clinically pathogens causing UTIs. In addition, 100% of E. coli strains were resistant to ampicillin. E.coli strains employed in this study were sensitive to other conventional antibiotics checked in our experimental settings such as meropenem.

To determine the bactericidal activities of LL-37, DOX and ceragenins against extracellular E.coli strains, we used a conventional bacterial killing assay (Fig. 1) and MIC/MBC measurements (Table 2).

Susceptibility data from our experiments demonstrate that ceragenins (CSA-13, CSA-131) have stronger bactericidal activities against E. coli than LL-37 or doxycycline against clinical strains of pathogen. Additionally, when compared to ceragenins alone, the combinations of LL-37 with ceragenins had significantly stronger activities against E. coli (Fig. 1a, b, c and d), with the most prominent effect observed for the combination of LL-37 with CSA-131. Importantly, at doses that exert bactericidal effects (i.e. 2–10 μM), low toxicity against mammalian cells was observed (Fig. 2). The MICs and MBCs of LL-37, DOX, ceragenins and combination of LL-37 with DOX and ceragenins against clinical isolates of E. coli are shown in Table 2. Overall, the in vitro activities of the combination of LL-37 with CSA-131 (average MIC for all tested strains ~ 2.5 μg/mL) was somewhat greater than CSA-131 (MIC ~ 4 μg/mL) and was much greater than that of LL-37 (MIC ~ 80 μg/mL) both used individually. Similar results were obtained with CSA-13. Doxycycline activity was not increased when combined with LL-37 peptide. To confirm these observations, we assessed the metabolic activity recorded in bacterial samples after 1 h of treatment with different antibacterial agents at varied concentrations. For this purpose, MTT assay, based on the conversion of MTT component to purple, non-soluble formazan precipitate by viable cells with active metabolism, was employed. As expected, the antibacterial activities of ceragenins, but not LL-37 or doxycycline resulted in significant decrease of E.coli cell viability (Fig. 3 a-d). The antibacterial activities of ceragenins in combination with LL-37 were strongly higher than with doxycycline. These observations show that combinations of ceragenins with LL-37 are suitable for developing effective treatments against E. coli UTIs.

Quantification of biofilm mass with the crystal violet assay (Fig. 4) showed that ceragenins alone exert significant activity against E. coli and effectively prevent biofilm formation. Moreover, LL-37 in combination with ceragenins had considerable efficacy in preventing biofilm formation by E. coli compared to LL-37 with DOX, which were unable to inhibit biofilm formation at tested concentrations. In some cases, more than 50% higher activity of ceragenins in combination with LL-37 at the same molar ratio was observed. Notably, DOX alone showed no antibiofilm properties against E. coli.

The results of cell viability assays after the incubation of the T24 cell line with antibacterial agents at different concentrations are displayed in Fig. 2. LL-37 peptide, doxycycline and ceragenins alone and in combination with LL-37, at low concentration (i.e. 1–10 μM), did not significantly affect the survival of T24 cells. Higher doses of these antimicrobial agents caused a dose-dependent cytotoxic response resulting in cells lysis. Nevertheless, strong bactericidal effects were observed at lower, non-cytotoxic doses of tested agents, which highlights their safety in the treatment of E. coli-caused UTIs.

To investigate whether combination therapies of LL-37 peptide with doxycycline/ceragenins are more effective than the antibacterial compounds alone in an in vitro model mimicking the site of infection, we evaluated the killing effectiveness of antimicrobial agents against intracellular pathogens, i.e. bacterial cells with the ability to escape antibiotic therapy and re-infect the host after the end of treatment. Tested agents were investigated at molar combinations ranging from 5 to 10 μM, since higher doses were toxic against tested cell line (Fig. 2). The combination of LL-37 with ceragenins (CSA-13 and CSA-131) was found to be more effective in eliminating intracellular E. coli than LL-37, CSAs or doxycycline alone. Significantly, we showed that combination of LL-37 with CSA-131 killed approximately 65 and 79% of the intracellular E. coli at 5 μM and 10 μM concentrations, respectively (Fig. 5). In contrast, at the same concentration antibacterial agents alone killed fewer intracellular organisms: LL-37 killed 8–12%, doxycycline killed 6–20%, CSA-13 killed 29–45% and CSA-131 killed 41–57%, depending on the tested bacterial strain. Importantly, the intracellular bacterial percent killing increased with higher concentration of combination of LL-37 with CSAs (Fig. 5 a-d). Consequently, 10 μM of LL-37 with CSA-131 completely eradicated the intracellular E. coli strain (Fig. 5c). The applied ceragenins had an increased bactericidal effect for E. coli in combination with the LL-37 peptide, the concentration of which increases at the site during UTIs. Importantly, these results suggest that this combination could prevent the recurrence of infections through elimination of intracellular bacteria.