Preclinical testing of a broad-spectrum antimicrobial endotracheal tube coated with an innate immune synthetic mimic.

BACKGROUND: Endotracheal tubes provide an abiotic surface on which bacteria and fungi form biofilms, and the release of endotoxins and planktonic organisms can cause damaging inflammation and infections. OBJECTIVES: Ceragenins are small molecule mimics of antimicrobial peptides with broad-spectrum antibacterial and antifungal activity, and a ceragenin may be used to provide antimicrobial protection to the abiotic surface of an endotracheal tube. METHODS: A hydrogel film, containing CSA-131, was generated on endotracheal tubes. Elution of CSA-131 was quantified in drip-flow and static systems, antifungal and antibacterial activity was measured with repeated inoculation in growth media, biofilm formation was observed through electron microscopy, safety was determined by intubation of pigs with coated and uncoated endotracheal tubes. RESULTS: Optimized coatings containing CSA-131 provided controlled elution of CSA-131, with concentrations released of less than 1 µg/mL. The eluting ceragenin prevented fungal and bacterial colonization of coated endotracheal tubes for extended periods, while uncoated tubes were colonized by bacteria and fungi. Coated tubes were well tolerated in intubated pigs. CONCLUSIONS: Thin films containing CSA-131 provide protection against microbial colonization of endotracheal tubes. This protection prevents fungal and bacterial biofilm formation on the tubes and reduces endotoxin associated with tubes. This coating is well suited for decreasing the adverse effects of intubation associated with infection and inflammation.

Ceragenins are active against drug-resistant Candida auris clinical isolates in planktonic and biofilm forms.

Background: Candida auris has emerged as a serious threat to human health. Of particular concern are the resistance profiles of many clinical isolates, with some being resistant to multiple classes of antifungals. Objectives: Measure susceptibilities of C. auris isolates, in planktonic and biofilm forms, to ceragenins (CSAs). Determine the effectiveness of selected ceragenins in gel and cream formulations in eradicating fungal infections in tissue explants. Materials and methods: A collection of 100 C. auris isolates available at CDC was screened for susceptibility to a lead ceragenin. A smaller collection was used to characterize antifungal activities of other ceragenins against organisms in planktonic and biofilm forms. Effects of ceragenins on fungal cells and biofilms were observed via microscopy. An ex vivo model of mucosal fungal infection was used to evaluate formulated forms of lead ceragenins. Results: Lead ceragenins displayed activities comparable to those of known antifungal agents against C. auris isolates with MICs of 0.5-8 mg/L and minimum fungicidal concentrations (MFCs) of 2-64 mg/L. No cross-resistance with other antifungals was observed. Fungal cell morphology was altered in response to ceragenin treatment. Ceragenins exhibited activity against sessile organisms in biofilms. Gel and cream formulations including 2% CSA-44 or CSA-131 resulted in reductions of over 4 logs against established fungal infections in ex vivo mucosal tissues. Conclusions: Ceragenins demonstrated activity against C. auris, suggesting that these compounds warrant further study to determine whether they can be used for topical applications to skin and mucosal tissues for treatment of infections with C. auris and other fungi.

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.

Proteomic Analysis of Resistance of Gram-Negative Bacteria to Chlorhexidine and Impacts on Susceptibility to Colistin, Antimicrobial Peptides, and Ceragenins.

Use of chlorhexidine in clinical settings has led to concerns that repeated exposure of bacteria to sub-lethal doses of chlorhexidine might result in chlorhexidine resistance and cross resistance with other cationic antimicrobials including colistin, endogenous antimicrobial peptides (AMPs) and their mimics, ceragenins. We have previously shown that colistin-resistant Gram-negative bacteria remain susceptible to AMPs and ceragenins. Here, we investigated the potential for cross resistance between chlorhexidine, colistin, AMPs and ceragenins by serial exposure of standard strains of Gram-negative bacteria to chlorhexidine to generate resistant populations of organisms. Furthermore, we performed a proteomics study on the chlorhexidine-resistant strains and compared them to the wild-type strains to find the pathways by which bacteria develop resistance to chlorhexidine. Serial exposure of Gram-negative bacteria to chlorhexidine resulted in four- to eight-fold increases in minimum inhibitory concentrations (MICs). Chlorhexidine-resistant organisms showed decreased susceptibility to colistin (8- to 32-fold increases in MICs) despite not being exposed to colistin. In contrast, chlorhexidine-resistant organisms had the same MICs as the original strains when tested with representative AMPs (LL-37 and magainin I) and ceragenins (CSA-44 and CSA-131). These results imply that there may be a connection between the emergence of highly colistin-resistant Gram-negative pathogens and the prevalence of chlorhexidine usage. Yet, use of chlorhexidine may not impact innate immune defenses (e.g., AMPs) and their mimics (e.g., ceragenins). Here, we also show that chlorhexidine resistance is associated with upregulation of proteins involved in the assembly of LPS for outer membrane biogenesis and virulence factors in Pseudomonas aeruginosa. Additionally, resistance to chlorhexidine resulted in elevated expression levels of proteins associated with chaperones, efflux pumps, flagella and cell metabolism. This study provides a comprehensive overview of the evolutionary proteomic changes in P. aeruginosa following exposure to chlorhexidine and colistin. These results have important clinical implications considering the continuous application of chlorhexidine in hospitals that could influence the emergence of colistin-resistant strains.

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.