Brian Hilton and Dr. Paul Savage, Department of Chemistry and Biochemistry
Antimicrobial resistance has increasingly become a global concern. The World Health Organization classified antimicrobial resistance as a “serious threat [that] is no longer a prediction for the future, it is happening right now in every region of the world and has the potential to affect anyone, of any age, in any country”. Dr. Keiji Fukuda, WHO’s Assistant Director-General for Health Security, explained that “A post-antibiotic era—in which common infections and minor injuries can kill—far from being an apocalyptic fantasy, is instead a very real possibility for the 21st century”. Considerable effort is being expended to develop new chemotherapeutics to treat microbial infections and avoid entering a post antibiotic era in which our arsenal of antibiotics are no longer effective at controlling microbial growth.
Antimicrobial peptides have received a significant amount of attention due to their broad range antimicrobial activity. However, there are two major obstacles that have made their application difficult. First, the compounds are peptides so they are therefore susceptible to proteases. The general protease activity in the body results in relatively short half lives for these compounds. Second, the nature of the compounds as peptide-based therapeutics results in their relative high cost of production. These drawbacks, in part, have resulted in difficulty developing them as mainstream therapeutic agents. This is part of the reason why Dr. Paul Savage in the BYU Department of Chemistry and Biochemistry invented non-peptide compounds to mimic antimicrobial peptide activity. These compounds are called ceragenins or cationic steroid antimicrobials (CSAs).
Cationic steroid antimicrobials mimic the antimicrobial peptide structure and also have been shown to mimic their antimicrobial mechanism of activity. Ceragenins, like AMPs, are facially amphipathic cationic compounds; however, ceragenins are cholic acid based derivatives and are therefore not susceptible to proteases. Ceragenins circumvent the two drawbacks of AMP clinical application; that is, they are steroid based so they are both cheaper to produce and not degraded by endogenous proteases. Because of their structure, CSAs are able to electrostatically interact with the bacterial membrane to depolarize the cells sufficiently to cause cell death. It has been shown that ceragenins are able to cross the outer membranes of gram-negative bacteria and cause ion flux through the inner membrane causing membrane depolarization, which correlates with their antibacterial activity. Studies also suggests that only minor defects in the membrane, caused by CSAs, result in bacterial death. Similar ion fluxes have been observed in cells treated with AMPs thus indicating that ceragenins are indeed mimicking their activity. Various studies have shown that the similarity in mechanism between CSAs and AMPs also results in CSA sharing the broad spectrum antimicrobial activity of AMPs.
Recently, there has been ever increasing concern with respect to new strains of colistin resistant gram-negative bacteria. Colistin is an effective “last resort” drug used in emergency situations against most types of gram-negative bacteria—especially those that are multidrug resistant. In other words, when all other drugs prove unable to eradicate a bacterial infection, colistin is used as a “last resort” to eliminate the infection. Our research focused on the gram-negative, colistin-resistant bacteria, Klebsiella pneumoniae. Klebsiella pneumoniae, a gram-negative bacterium, is especially pertinent to the global discussion over colistin-resistance because of its ability to transfer the plasmid-mediated MCR-1 gene—the gene that grants colistin resistance—via horizontal gene transfer to other species of bacteria and thereby spreading bacterial resistance at an alarming rate. In other words, K. pneumoniae, beyond being especially pathogenic, is also a potentially extra-dangerous carrier and distributor of colistin resistance.
For our research group, the question arose: Do colistin-resistant bacteria show susceptibility to ceragenins? This question is especially interesting because colistin, AMPs, and CSAs all share similar structures. Colistin, like AMPs, is peptide based and a cationic amphiphile. Since ceragenin structure is modeled after AMPs, it is likely that colistin, antimicrobial peptides, and CSAs function via similar mechanisms which begs the question of whether they share similar antimicrobial activity. In reality, two questions were the foundation of our inquiry: (1) Are colistin-resistant bacteria susceptible to AMPs and CSAs? (2) Does generation of resistance to colistin occur at the same rate as potential generation of resistance to AMPs and ceragenins? (3) Since the primary mechanism for bacterial resistance to AMPs and ceragenins is through modification of the lipid A portion of lipopolysaccharide (LPS), how important are these modifications in resistance of gram-negative bacteria to colistin, AMPs and ceragenins?
In order to explore these three questions, our research group carried out several different assays and analyses. These include: minimum inhibitory concentration (MIC) measurements, minimum bactericidal concentration (MBC) assessment, time-kill assay interpretation, serial passaging tests. To carry out these tests, six clinical isolates of colistin-resistant Klebsiella pneumoniae were contrasted with wild type K. pneumoniae. In order to gauge the effectiveness of CSAs against K. pneumoniae, the pathogen’s susceptibility to representative ceragenins was compared with that of colistin and three AMPs, specifically: LL-37, cecropin A and magainin 1. The results are therefore interpreted as comparisons between the AMPs, colistin, and CSAs potency toward the different strains.
First, minimum inhibitory concentrations of ceragenins, AMPs, and colistin were measured and compared. Notably, CSA-44 and CSA-131 were the most effective ceragenins at inhibiting K. pneumoniae growth for both the wild type strain and the clinical isolates; There was little or no variation in MIC value between the resistant and wild type strains. MIC values of colistin against the resistant strains was 8-100 times higher than the MIC values of colistin against the wild type. The MIC values of AMPs varied from one AMP to another. All in all, the MIC results demonstrated that colistin-resistance is not correlated with CSA-resistance, at least for K. pneumoniae.
Next, we conducted serial passaging to compare the rate at which representative gram-negative bacteria generate resistance to CSA-131 and colistin. We serially exposed K. pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii with colistin and CSA-131 for respectively 10 and 30 twenty-four hour periods. MIC values were monitored throughout each passaging cycle. Although MICs for colistin began at 1-2 ug/mL for all three strains, they rose to ≥ 350 μg/mL after 10 cycles. On the other hand, MICs for CSA-131 began at 1-2 ug/mL and rose to just 2-8 ug/mL after 30 passages. Surprisingly, representative gram-negative bacteria did not become remarkably more resistant to CSA-131 after 30 serial passage cycles. This shows that potential generation of resistance to CSA is much slower than generation of resistance to colistin for these gram-negative bacteria.
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. These results suggest that there are differences in resistance mechanisms to colistin and ceragenins/AMPs.