Antifungal susceptibilities, in vitro production of virulence factors and activities of ceragenins against Candida spp. isolated from vulvovaginal candidiasis.

Vulvovaginal candidiasis (VVC) is the second most common cause of vaginitis after bacterial vaginosis, affecting millions of women worldwide every year. Candida albicans is the most frequent agent of VVC followed by other species of Candida such as C. glabrata and C. parapsilosis. Out of a total of 100 clinical isolates of Candida spp. obtained from patients diagnosed with VVC, 84 were identified as C. albicans, while the remaining isolates were identified as non--albicans Candida strains. Phospholipases and proteinases were produced by a majority of the C. albicans strains and esterases and hemolysins a minority of these strains. Among the non-C. albicans strains, only a few of the strains produced these proteins. Nearly all of the isolates formed biofilms. Our results showed that the butoconazole, clotrimazole, and fluconazole were active against C. albicans and less so against the non-albicans Candida strains. The MIC90 of amphotericin B and nystatins were 2 and 4 µg/ml, respectively, against either C. albicans or non-albicans Candida spp. Representative ceragenins (CSA-13, CSA-131, and CSA-138), developed as mimics of endogenous antimicrobial peptides, were active against fluconazole-resistant strains, both alone and in combination with fluconazole. These results suggest the potential use of ceragenins in treating VVC, including infections caused by fluconazole-resistant isolates.

Antibacterial and Antifungal Activities of Poloxamer Micelles Containing Ceragenin CSA-131 on Ciliated Tissues.

Ceragenins were designed as non-peptide mimics of endogenous antimicrobial peptides, and they display broad-spectrum antibacterial and antifungal activities, including the ability to eradicate established biofilms. These features of ceragenins make them attractive potential therapeutics for persistent infections in the lung, including those associated with cystic fibrosis. A characteristic of an optimal therapeutic for use in the lungs and trachea is the exertion of potent antimicrobial activities without damaging the cilia that play a critical role in these tissues. In previous work, potent antimicrobial activities of ceragenin CSA-131 have been reported; however, we found in ex vivo studies that this ceragenin, at concentrations necessary to eradicate established biofilms, also causes loss of cilia function. By formulating CSA-131 in poloxamer micelles, cilia damage was eliminated and antimicrobial activity was unaffected. The ability of CSA-131, formulated with a poloxamer, to reduce the populations of fungal pathogens in tracheal and lung tissue was also observed in ex vivo studies. These findings suggest that CSA-131, formulated in micelles, may act as a potential therapeutic for polymicrobial and biofilm-related infections in the lung and trachea.

Susceptibility of Colistin-Resistant, Gram-Negative Bacteria to Antimicrobial Peptides and Ceragenins.

The susceptibility of colistin-resistant clinical isolates of Klebsiella pneumoniae to ceragenins and antimicrobial peptides (AMPs) suggests that there is little to no cross-resistance between colistin and ceragenins/AMPs and that lipid A modifications are found in bacteria with modest changes in susceptibility to ceragenins and with high levels of resistance to colistin. These results suggest that there are differences in the resistance mechanisms to colistin and ceragenins/AMPs.

Antimicrobial Growth Promoters Altered the Function but Not the Structure of Enteric Bacterial Communities in Broiler Chicks ± Microbiota Transplantation.

Non-antibiotic alternatives to antimicrobial growth promoters (AGPs) are required, and understanding the mode of action of AGPs may facilitate the development of effective alternatives. The temporal impact of the conventional antibiotic AGP, virginiamycin, and an AGP alternative, ceragenin (CSA-44), on the structure and function of the broiler chicken cecal microbiota was determined using next-generation sequencing and 1H-nuclear magnetic resonance spectroscopy (NMR)-based metabolomics. To elucidate the impact of enteric bacterial diversity, oral transplantation (±) of cecal digesta into 1-day-old chicks was conducted. Microbiota transplantation resulted in the establishment of a highly diverse cecal microbiota in recipient chicks that did not change between day 10 and day 15 post-hatch. Neither virginiamycin nor CSA-44 influenced feed consumption, weight gain, or feed conversion ratio, and did not affect the structure of the cecal microbiota in chicks possessing a low or high diversity enteric microbiota. However, metabolomic analysis of the cecal contents showed that the metabolome of cecal digesta was affected in birds administered virginiamycin and CSA-44 as a function of bacterial community diversity. As revealed by metabolomics, glycolysis-related metabolites and amino acid synthesis pathways were impacted by virginiamycin and CSA-44. Thus, the administration of AGPs did not influence bacterial community structure but did alter the function of enteric bacterial communities. Hence, alterations to the functioning of the enteric microbiota in chickens may be the mechanism by which AGPs impart beneficial health benefits, and this possibility should be examined in future research.

Antimicrobial Activity of Ceragenins against Vancomycin-Susceptible and -Resistant Enterococcus spp.

Ceragenins (CSAs) are a new class of antimicrobial agents designed to mimic the activities of endogenous antimicrobial peptides. In this study, the antibacterial activities of various ceragenins (CSA-13, CSA-44, CSA-90, CSA-131, CSA-138, CSA-142, and CSA-192), linezolid, and daptomycin were assessed against 50 non-repeated Enterococcus spp. (17 of them vancomycin-resistant Enterococcus-VRE) isolated from various clinical specimens. Among the ceragenins evaluated, the MIC50 and MIC90 values of CSA-44 and CSA-192 were the lowest (2 and 4 µg/mL, respectively), and further studies were continued with these two ceragenins. Potential interactions between CSA-44 or CSA-192 and linezolid were tested and synergistic interactions were seen with the CSA-192-linezolid combination against three Enterococcus spp., one of them VRE. The effects of CSA-44 and CSA-192 on the MIC values of vancomycin were also investigated, and the largest MIC change was seen in the vancomycin-CSA-192 combination. The in vivo effects of CSA-44 and CSA-192 were evaluated in a Caenorhabditis elegans model system. Compared to no treatment, increased survival was observed with C. elegans when treated with ceragenins. In conclusion, CSA-44 and CSA-192 appear to be good candidates (alone or in combination) for the treatment of enterococcal infections, including those from VRE.

Ceragenin CSA-44 as a Means to Control the Formation of the Biofilm on the Surface of Tooth and Composite Fillings.

Recurrent oral infections, as manifested by endodontic and periodontal disease, are often caused by Enterococcus faecalis (E. faecalis) and Candida albicans (C. albicans). Here, we assessed the anti-biofilm activity of ceragenin CSA-44 against these microbes growing as a biofilm in the presence of saliva on the surface of human teeth and dental composite (composite filling) subjected to mechanical stresses. Methods: Biofilm mass analysis was performed using crystal violet (CV) staining. The morphology, viscoelastic properties of the biofilm after CSA-44 treatment, and changes in the surface of the composite in response to biofilm presence were determined by AFM microscopy. Results: CSA-44 prevented biofilm formation and reduced the mass of biofilm formed by tested microorganisms on teeth and dental composite. Conclusion: The ability of CSA-44 to prevent the formation and to reduce the presence of established biofilm on tooth and composite filling suggests that it can serve as an agent in the development of new methods of combating oral pathogens and reduce the severity of oral infections.

New ß-Lactam Antibiotics and Ceragenins - A Study to Assess Their Potential in Treatment of Infections Caused by Multidrug-Resistant Strains of Pseudomonas aeruginosa.

BACKGROUND: The increasing number of infections caused by antibiotic resistant strains of Pseudomonas aeruginosa posed a very serious challenge for clinical practice. This standing is driving scientists to develop new antibiotics against these microorganisms. METHODS: In this study, we measured the MIC/MBC values and estimated the ability of tested molecules to prevent bacterial biofilm formation to explore the effectiveness of ß-lactam antibiotics ceftolozane/tazobactam, ceftazidime/avibactam, meropenem/vaborbactam, and ceragenins CSA-13, CSA-44, and CSA-131 against 150 clinical isolates of Pseudomonas aeruginosa that were divided into five groups, based on their antibiotic resistance profiles to beta-lactams. Selected strains of microorganisms from each group were also subjected to prolonged incubations (20 passages) with ceragenins to probe the development of resistance towards those molecules. Cytotoxicity of tested ceragenins was evaluated using human red blood cell (RBCs) hemolysis and microscopy observations of human lung epithelial A549 cells after ceragenin treatment. Poloxamer 407 (pluronic F-127) at concentrations ranging from 0.5% to 5% was tested as a potential drug delivery substrate to reduce ceragenin toxicity. RESULTS: Collected data proved that ceragenins at low concentrations are highly active against clinical strains of Pseudomonas aeruginosa regardless of their resistance mechanisms to conventional antibiotics. Ceragenins also show low potential for resistance development, high antibiofilm activity, and controlled toxicity when used together with poloxamer 407. CONCLUSION: This data strongly supports the need for further study directed to develop this group of molecules as new antibiotics to fighting infections caused by antibiotic resistant strains of Pseudomonas aeruginosa.

The application of ceragenins to orthopedic surgery and medicine.

Osteomyelitis and infections associated with orthopedic implants represent a significant burden of disease worldwide. Ceragenins (CSAs) are a relatively new class of small-molecule antimicrobials that target a broad range of Gram-positive and Gram-negative bacteria as well as fungi, viruses, and parasites. This review sets the context of the need for new antimicrobial strategies by cataloging the common pathogens associated with orthopedic infection and highlighting the increasing challenges of managing antibiotic-resistant bacterial strains. It then comparatively describes the antimicrobial properties of CSAs with a focus on the CSA-13 family. More recently developed members of this family such as CSA-90 and CSA-131 may have a particular advantage in an orthopedic setting as they possess secondary pro-osteogenic properties. In this context, we consider several new preclinical studies that demonstrate the utility of CSAs in orthopedic models. Emerging evidence suggests that CSAs are effective against antibiotic-resistant Staphylococcus aureus strains and can prevent the formation of biofilms. There remains considerable scope for developing CSA-based treatments, either as coatings for orthopedic implants or as local or systemic antibiotics to prevent bone infection.

Antibacterial and Antibiofilm Activities of Ceragenins against Pseudomonas aeruginosa Clinical Isolates.

OBJECTIVES: Pseudomonas aeruginosa can cause life-threatening infections that are difficult to treat due to its high resistance to antibiotics and its ability to form antibiotic tolerant biofilms. Ceragenins, designed to mimic the activities of antimicrobial peptides, represent a promising new group of antibacterial agents that display potent anti-P. aeruginosa activity. The aim of this study was to evaluate the antibacterial and antibiofilm activities of ceragenins in comparison to colistin and ciprofloxacin against P. aeruginosa strains. MATERIALS AND METHODS: Biofilm formation and determination of minimum inhibitory concentration (MIC) values of ceragenins (CSA-13, CSA-44, CSA-131, and CSA-138), ciprofloxacin, and colistin were evaluated against 25 P. aeruginosa isolates. Four good biofilm-producing strains were chosen for biofilm studies, and sessile MICs and inhibition of molecule adhesion and biofilm formation were evaluated. RESULTS: The MIC50 (µg/mL) values of CSA-13, CSA-44, CSA-131, CSA-138, ciprofloxacin, and colistin were 8, 8, 8, 16, 1, and 2, respectively. The sessile MICs for molecules were greater than planktonic MICs. CSA-13, CSA-44, and CSA-131 were more efficient after 4 h incubation while CSA-138, ciprofloxacin and colistin were more efficient after 1 h incubation. The most efficient agent for inhibition of adhesion was colistin (up to 45%). CSA-131, CSA-138, and colistin were the most efficient agents for inhibition of biofilm formation (up to 90%). CONCLUSION: Our study highlights the potential of CSA-131 and CSA-138 as potential alternative agents to conventional antibiotics for the eradication of biofilms of P. aeruginosa.

Effects of ceragenins and conventional antimicrobials on Candida albicans and Staphylococcus aureus mono and multispecies biofilms.

It is known that synergy between Candida albicans and Staphylococcus aureus results in enhanced biofilm formation and increased resistance to antimicrobials. Ceragenins (CSAs) are derivatives of cholic acid designed to mimic the antimicrobial activities of endogenous antimicrobial peptides. In this study, various CSAs were tested on C. albicans and methicillin-susceptible S. aureus or methicillin-resistant S. aureus mono or multispecies biofilms at 2 different concentrations (16 and 64 µg/mL) and compared with conventional antimicrobials. CSA-8 was active agent both with mono and multispecies biofilms (P < 0.05). Among antifungals, amphotericin B and, among antibacterials, ciprofloxacin and gentamicin were active agents against all studied microorganisms. This study suggests that CSAs, especially CSA-8, have useful antibiofilm effects against monomicrobial or fungal-bacterial multispecies biofilms.

Ceragenin CSA-13 as free molecules and attached to magnetic nanoparticle surfaces induce caspase-dependent apoptosis in human breast cancer cells via disruption of cell oxidative balance.

Natural antimicrobial peptides and ceragenins, as non-peptide amphipathic mimics, have been proposed as anti-cancer agents. To date, it has been confirmed that cathelicidin LL-37 and ceragenin CSA-13, both in free form and immobilized on the surface of magnetic nanoparticles (MNP@LL-37, MNP@CSA-13) induce apoptosis in colon cancer cells. Nevertheless, the question remains whether ceragenins, as synthetic analogs of LL-37 peptide and mimicking a number of its properties, act as antineoplastic agents in breast cancer cells, where LL-37 peptide stimulates oncogenesis. Considering potential anticancer activity, we determined whether CSA-13 and MNP@CSA-13 might be effective against breast cancer cells. Our study provides evidence that both CSA-13 and MNP@CSA-13 decreased viability and inhibit proliferation of MCF-7 and MDA-MB-231 cells despite the protumorigenic properties of LL-37 peptide. Flow cytometry-based analyses revealed that ceragenin treatment results in increases in dead and PI-negative/low-viability cells, which was associated with glutathione (GSH) depletion and increased reactive oxygen species (ROS) generation followed by mitochondrial membrane depolarization, caspase activation, and DNA fragmentation. These findings demonstrate that both CSA-13 and MNP@CSA-13 cause disruption of the oxidative balance of cancer cells. This novel mechanism of ceragenin-mediated eradication of cancer cells suggest that these agents may be developed as a possible treatment of breast cancer.

Susceptibility of Multidrug-Resistant Bacteria, Isolated from Water and Plants in Nigeria, to Ceragenins.

The continuous emergence of multidrug resistant pathogens is a major global health concern. Although antimicrobial peptides (AMPs) have shown promise as a possible means of combatting multidrug resistant strains without readily engendering resistance, costs of production and targeting by proteases limit their utility. Ceragenins are non-peptide AMP mimics that overcome these shortcomings while retaining broad-spectrum antimicrobial activity. To further characterize the antibacterial activities of ceragenins, their activities against a collection of environmental isolates of bacteria were determined. These isolates were isolated in Nigeria from plants and water. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of selected ceragenins and currently available antimicrobials against these isolates were measured to determine resistance patterns. Using scanning electron microscopy (SEM), we examined the morphological changes in bacterial membranes following treatment with ceragenins. Finally, we investigated the effectiveness of ceragenins in inhibiting biofilm formation and destroying established biofilms. We found that, despite high resistance to many currently available antimicrobials, including colistin, environmental isolates in planktonic and biofilm forms remain susceptible to ceragenins. Additionally, SEM and confocal images of ceragenin-treated cells confirmed the effective antibacterial and antibiofilm activity of ceragenins.

Magnetic nanoparticles enhance the anticancer activity of cathelicidin LL-37 peptide against colon cancer cells.

The pleiotropic activity of human cathelicidin LL-37 peptide includes an ability to suppress development of colon cancer cells. We hypothesized that the anticancer activity of LL-37 would improve when attached to the surface of magnetic nanoparticles (MNPs). Using colon cancer culture (DLD-1 cells and HT-29 cells), we evaluated the effects of MNPs, LL-37 peptide, its synthetic analog ceragenin CSA-13, and two novel nanosystems, ie, MNP@LL-37 and MNP@CSA-13, on cancer cell viability and apoptosis. Treatment of cancer cells with the LL-37 peptide linked to MNPs (MNP@LL-37) caused a greater decrease in cell viability and a higher rate of apoptosis compared with treatment using free LL-37 peptide. Additionally, we observed a strong ability of ceragenin CSA-13 and MNP@CSA-13 to induce apoptosis of DLD-1 cells. We found that both nanosystems were successfully internalized by HT-29 cells, and cathelicidin LL-37 and ceragenin CSA-13 might play a key role as novel homing molecules. These results indicate that the previously described anticancer activity of LL-37 peptide against colon cancer cells might be significantly improved using a theranostic approach.

Maghemite, silver, ceragenin conjugate particles for selective binding and contrast of bacteria.

New synthesis techniques are providing increasing control over many inorganic nanoparticle characteristics, facilitating the creation of new multifunctional theranostics. This report proposes the synthesis and testing of a combination nanoparticle comprised of a maghemite core for enhanced T2 MRI contrast diagnostics, a colloidal silver shell acting as an antimicrobial and therapeutic vehicle, and a ceragenin (CSA-124) surfactant providing microbial adhesion. A polyacrylic acid functionalized maghemite nanoparticle is synthesized by a high temperature organic phase reduction followed by thiol functionalization and gold cluster seeding. A silver shell is formed through AgNO3 reduction, and an oriented monolayer of the thiolated ceragenin, is bound through a self-assembly process. The process and products are characterized throughout synthesis through TEM, DLS, FT-IR, UV-Vis, ICP-OES, HPLC-ESI-TOF-MS, DC magnetization and susceptibility, X-ray diffraction, and in vitro MRI. Synthesized Diagnostic Antimicrobial Nanoparticles (DANs) were found to have a spherical morphology with a diameter of 32.47±1.83 nm, hydrodynamic diameter of 53.05±1.20 nm, maximum magnetic moment of 12 emu/g NP (54 emu/g Fe) with little variation due to temperature, and are predominantly paramagnetic. In vitro MRI studies show that DANs contrast well at concentrations as low as 9 ppm, and successfully adhere to Staphylococcus aureus. DAN MIC was determined to be approximately 12 ppm and 24 ppm against S. aureus and Escherichia coli respectively.