Antimicrobial Susceptibility Testing of Antimicrobial Peptides to Better Predict Efficacy

Abstract

During the development of antimicrobial peptides (AMP) as potential therapeutics, antimicrobial susceptibility testing (AST) stands as an essential part of the process in identification and optimisation of candidate AMP. Standard methods for AST, developed almost 60 years ago for testing conventional antibiotics, are not necessarily fit for purpose when it comes to determining the susceptibility of microorganisms to AMP. Without careful consideration of the parameters comprising AST there is a risk of failing to identify novel antimicrobials at a time when antimicrobial resistance (AMR) is leading the planet toward a post-antibiotic era. More physiologically/clinically relevant AST will allow better determination of the preclinical activity of drug candidates and allow the identification of lead compounds. An important consideration is the efficacy of AMP in biological matrices replicating sites of infection, e.g., blood/plasma/serum, lung bronchiolar lavage fluid/sputum, urine, biofilms, etc., as this will likely be more predictive of clinical efficacy. Additionally, specific AST for different target microorganisms may help to better predict efficacy of AMP in specific infections. In this manuscript, we describe what we believe are the key considerations for AST of AMP and hope that this information can better guide the preclinical development of AMP toward becoming a new generation of urgently needed antimicrobials.

Innate Inspiration: Antifungal Peptides and Other Immunotherapeutics From the Host Immune Response

“…Antimicrobial peptides are promising candidates as therapeutics for the treatment of fungal infection and are much needed in clinical practice due to the limited array of treatment options and increasing resistance to existing antifungals. Unfortunately, we are not seeing enough drug candidates making it through the drug development pipeline, as in vitro and in vivo testing approaches are not always appropriate and/or optimised for AMP (268). The same is true in part for clinical efficacy trials which must be appropriate for AMP (end points in particular).…”

“…We say "small" as the authors acknowledge that many clinically used antimicrobials do not obey the traditional definition of small, i.e., <500 Da, from Lipinski's rule of five (266), but are nevertheless generally smaller than most AMP. Evaluation of AMP as antimicrobial drug candidates begins with in vitro antimicrobial susceptibility testing in which a number of key parameters need to be taken into consideration, including media composition, growth phase, oxygen, temperature and other biological matrices (Table 2) (267,268). This also applies to in vitro cytotoxicity testing (269,270), formulation and delivery considerations (see Section Formulation and Delivery) and the choice of models for in vivo testing (271).…”

“…This also applies to in vitro cytotoxicity testing (269,270), formulation and delivery considerations (see Section Formulation and Delivery) and the choice of models for in vivo testing (271). It is probable that with adequate consideration given to the factors outlined above and also appropriately designed clinical trials there would be significantly more AMP in preclinical and clinical development and the importance of this is described in detail in a new review of the subject (268).…”

Synergies with and Resistance to Membrane-Active Peptides

“…Groups, such as the Clinical Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing yearly define “breakpoints” in microorganisms as the MIC values above which an organism may be called “resistant” to a given drug [ 28 , 29 ]. There are a myriad of challenges to consider when determining breakpoints for MAPs and seemingly no consensus on how to compare them [ 30 ]. The breakpoints are influenced by the exact MAP mechanism of action, as well as how susceptible they are to degradation.…”

Antimicrobial Activities of LL-37 Fragment Mutant-Poly (Lactic-Co-Glycolic) Acid Conjugate against Staphylococcus aureus, Escherichia coli, and Candida albicans

“…Antimicrobial peptides (AMPs), as host defense peptides, show potential as new therapeutic classes of antimicrobials [ 1 ]. These peptides exert biological activities by interacting with the plasma membrane to disrupt the membrane and lyse the cell, or are taken up by the target cell depending on the amino acid composition of the peptide [ 2 ].…”