Wnorowska et al. BMC Infectious Diseases (2019) 19:369 https://doi.org/10.1186/s12879-019-3994-3 RESEARCH ARTICLE Open Access Use of ceragenins as a potential treatment for urinary tract infections Urszula Wnorowska1, Ewelina Piktel1, Bonita Durnaś2, Krzysztof Fiedoruk3, Paul B. Savage4 and Robert Bucki1* Abstract Background: Urinary tract infections (UTIs) are one of the most common bacterial infections. High recurrence rates and the increasing antibiotic resistance among uropathogens constitute a large social and economic problem in current public health. We assumed that combination of treatment that includes the administration ceragenins (CSAs), will reinforce the effect of antimicrobial LL-37 peptide continuously produced by urinary tract epithelial cells. Such treatment might be an innovative approach to enhance innate antibacterial activity against multidrug- resistant E. coli. Methods: Antibacterial activity measured using killing assays. Biofilm formation was assessed using crystal violet staining. Viability of bacteria and bladder epithelial cells subjected to incubation with tested agents was determined using MTT assays. We investigated the effects of chosen molecules, both alone and in combinations against four clinical strains of E. coli, obtained from patients diagnosed with recurrent UTI. Results: We observed that the LL-37 peptide, whose concentration increases at sites of urinary infection, exerts increased bactericidal effect against E. coli when combined with ceragenins CSA-13 and CSA-131. Conclusion: We suggest that the employment of combination of natural peptide LL-37 with synthetic analogs might be a potential solution to treat urinary tract infections caused by drug-resistant bacteria. Keywords: Urinary tract infection, LL-37 peptide, Ceragenins, Bacterial drug resistance Background trimethoprim/sulphamethoxazole, may no longer be used Urinary tract infections (UTIs) are one of the most com- for empiric treatment due to high resistance rates [7]. mon bacterial infections worldwide. There are several risk Additionally, fluoroquinolones are characterized by factors associated with UTI development such as age, significant toxicity, manifested by disorders of the gastro- pregnancy, sexual intercourse, use of diaphragm and intestinal tract and inflammation of the connective tissue spermicide, delayed post-coital micturition, menopause, (permanent damage to tendons and cartilage) [8]. Signifi- catheterization, incontinence, antibiotic use and a previ- cant drug resistance, which considerably limits the useful- ous history of UTI [1]. The main etiological agent of UTIs ness in the therapy of UTIs, is also reported for is uropathogenic Escherichia coli (UPEC), which is re- doxycycline, a tetracycline antimicrobial characterized by sponsible for more than 80% of all urinary infections [2]. broad spectrum of antibacterial activity, favorable pharma- Importantly, most isolates of E. coli are multidrug resistant cokinetics and a good safety profile [9]. Furthermore, in [3]. High recurrence rates and progressively increasing re- the case of chronic and recurrent UTIs infection, the major sistance of microorganisms to conventional antibiotics, es- challenge is eradication of microbial biofilm, which consid- pecially among carbapenem-resistant Enterobacteriaceae, erably increases bacterial resistance to antibiotics [10]. makes UTIs a serious social and economic problem [4–6]. Among other factors explaining failure of antibiotic It should be noted that some conventional antibiotics used therapy of UTIs is ineffective eradication of intracellular conventionally to fight UTIs, such as fluoroquinolones or pathogens that might avoid antibiotic therapy and host immune response when after uptake are hiding as intra- * Correspondence: [email protected] cellular microorganism in cytosol of epithelial cells [11, 1Department of Microbiological and Nanobiomedical Engineering, Medical University of Bialystok, Mickiewicza 2c, 15-222, Bialystok, Poland 12]. It is important to note that a majority of currently Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wnorowska et al. BMC Infectious Diseases (2019) 19:369 Page 2 of 13 used antimicrobial agents, such as aminoglycosides and there is a small probability that the bacteria will develop β-lactams, is effective only against extracellular forms of resistance to this class of molecules [28]. Given these ob- pathogens due to the inability to penetrate and accumu- servations, ceragenins offer a possible effective method late in the host cells, while others (i.e., macrolides and for the prevention and treatment of bacterial UTIs. Im- fluoroquinolones), exhibit poor retention inside host portantly, results from animal-based studies indicate that cells [13]. Considering the obstacles in eradication of ceragenin CSA-13 undergoes urinary excretion, which intracellular pathogens and related with it, recurrence of strongly suggests the possibility of their application in intracellular bacteria-associated infections, it is sug- UTIs [29]. Nevertheless, it has not been yet determined gested that determination of the activity of antibiotics if combination therapy of LL-37 peptide with ceragenins against the intracellular form of the pathogen would can be used as potential treatment against urinary tract more accurate predict the clinical efficacy of agents in in infections. vivo settings. Since intracellular activity of an antibiotic Considering the results described above, we decided to is determined by a variety of factors, from which ability investigate the effects of combinations of LL-37 with to reach the eukaryotic cell membrane and its subcellu- selected ceragenins using an in vivo setting of urinary lar localization, are the most prominent, we hypothe- infection: cultures of E. coli in planktonic and biofilm sized that membrane-active agents, such as host form and E. coli-infected urinary bladder cells. In this antibacterial peptides and ceragenins, may demonstrate study, we determined the protective effect of LL-37 pep- efficiency in reaching the cytosol. tide and ceragenins in relation to epithelium cells of the Substantial efforts have been expended in development urinary tract, which can promote survival of uropatho- of endogenous antimicrobial peptides (AMPs) as new gens internalized into the cells and thereby provide the therapeutic options suitable in the treatment of potential to re-infect the host after antibiotic treatment. drug-resistant microbial infections. Well studied AMPs Considering successful LL-37/ceragenin-mediated eradi- include human cathelicidin LL-37, which plays an im- cation of clinical strains of E. coli observed during our portant role in the first line of mucosal immunity against study, we suggest that therapy with ceragenins will invading pathogens [14, 15]. In contrast to conventional reinforce endogenous LL-37 peptide activity and may be antibiotics, development of resistance against AMPs is an important option in the treatment of UTIs. unlikely due to natural origin of AMPs and their mech- anism of action. AMPs associate with bacterial mem- Methods branes, leading to perturbations and loss of permeability Bacterial strains used in the study barrier function and membrane polarization. Notably, Clinical strains of E. coli were obtained from patients di- AMPs, as components of innate host defense, are pro- agnosed with urinary tract infection at Independent Pub- duced by urothelium and renal intercalated cells, both lic Province Hospital of Jan Sniadecki in Bialystok. The constitutively and in response to microbial assault and susceptibilities of tested bacterial strains to conventional are reported to be less toxic than other antibiotics [16, antibiotics were established by the agar disc diffusion 17]. A recent study by Ibrahim et al. indicated that levels method on MHA agar according to the guidelines of the of LL-37 are substantially increased among patients with EUCAST (ang. The European Committee on Antimicro- UTI, both in the patient’s urine, and in the plasma of pa- bial Susceptibility Testing). Zones of inhibition were tients during the period of infection of the urinary tract measured to the nearest millimeter and recorded. Sus- [18]. In addition, apart from the broad spectrum of anti- ceptibility tests showed that all E. coli isolates were re- microbial activity against bacteria, viruses, fungi and par- sistant to ampicillin, and two of them were resistant to asites, AMPs have been found to be key modulators of sulfamethoxazolum/trimethoprimum (Table 1). immunity and inflammation, playing critical roles in in- nate immunity [19–22]. Ceragenins (CSAs), synthetic mimics of endogenous Materials AMPs, are recognized as a novel, effective antibiotic LL-37 peptide was purchased from Lipopharm.pl class due to a variety of attractive features, including (Zblewo, Poland), and the purity of LL-37 was > 98% (as broad spectrum of killing