Antimicrobial Susceptibility Testing of Antimicrobial Peptides Requires New and Standardized Testing Structures

Abstract

The need for optimized as well as standardized test systems of novel antimicrobial peptides (AMPs) was discussed by experts in the field at the International Meeting on Antimicrobial Peptides (IMAP) 2017 and the 2019 Gordon Research Conference (GRC) on Antimicrobial Peptides, and a survey related to this topic was circulated to participants to collate opinions. The survey included questions ranging from the relevance of susceptibility testing for understanding the mode of action of AMPs, to the importance of optimization and a degree of standardization of test methods and their clinical relevance. Based on the survey results, suggestions for future improvements in the research field are made.

INTRODUCTION

Antimicrobial peptides (AMPs) are a common endogenous defense mechanism against pathogens and occur in all classes of life.¹ AMPs are relatively small peptides (4–50 amino acid residues) that are generally positively charged and often contain an amphipathic conformation with antimicrobial properties, including cathelicidins, defensins, or protegrins, among others. Advances in synthetic biology techniques, chemical synthesis, and structural understanding have led to improvements in the antimicrobial spectrum and tissue compatibility of AMPs.^2,3 Because of their diversity and multiple mechanisms of action, these peptides offer an opportunity to overcome the global health crisis of antimicrobial resistance,⁴ although despite the promise shown by AMPs it has been difficult to translate this into clinical approval. AMPs are capable of broadly targeting pathogenic microbes, including bacteria, protozoa, fungi, and viruses.⁵ The most commonly described mechanisms of action of AMPs are displayed in Figure 1 and include membranolytic and non-membranotytic mechanisms to inhibit/kill pathogens. For example, when certain AMPs reach a threshold concentration and spontaneously insert themselves into pathogen membranes, they can lead to different models of pore and ion channel formation, eventually leading to cell lysis and death.⁶ Other specific or less common modes of action of AMPs include membrane thickening/thinning,⁷ septum formation,⁶ charged lipid clustering,⁸ nucleic acid targeting,⁶ anion carriers,⁹ electroporation,¹⁰ nonlytic membrane depolarization,¹¹ and nonbilayer intermediates.¹²

Figure 1.

Depiction of selected modes of action of AMPs. Binding to biological membranes changes the conformation of the AMP. The subsequent membranolytic and non-membranolytic mechanisms lead to cell death.

The development of resistance to AMPs does not seem to occur, if at all, at the rate of resistance to conventional antibiotics.^13,14 It is important to be able to accurately assess clinically effective doses and treatment regimens of AMPs, not least to prevent bacterial resistance by underdosing. In order to accurately determine and predict the antimicrobial activity of AMPs, reliable and reproducible susceptibility assays are urgently needed. Susceptibility testing is influenced by many factors as outlined in our recent review.¹⁵ The composition of media used is as important as the pH value, the ionic strength, and the presence of proteases and metal ions. In addition, temperature, oxygen content, and plasticware play a significant role on the outcome of the testing.⁹ Therefore, it is critical to take as many of these factors as possible into consideration when designing any protocol for susceptibility testing for AMPs which can be used as a basis for predicting the in vivo activity and clinical efficacy. The need for optimized test systems was discussed by experts in the field at the International Meeting on Antimicrobial Peptides (IMAP) 2017 and the 2019 Gordon Research Conference (GRC) on Antimicrobial Peptides, and the different observations made were subsequently collected in a survey, the results of which are reported here.

METHODS

The survey was developed in 2019 (see the Supporting Information). The questions covered a variety of topics related to AMPs, ranging from the relevance of susceptibility testing for understanding the mode of action of AMPs, to the importance of optimization and standardization of test methods and their physiological/clinical relevance. Questions were also asked about possible official recognition of antimicrobial susceptibility testing (AST) for AMPs by organizations including EUCAST (European Committee on Antimicrobial Susceptibility Testing) and CLSI (Clinical and Laboratory Standards Institute) as well as the most appropriate means to describe such methods in publications.

RESULTS

The survey link was opened by invited participants 56 times, and 43 responses were collected. From these, 37 completed responses were included in the analysis. A list of statements summarizing the degree of agreement on the closed questions is given in Table 1. The results display a broad consensus that standardized test methods are important and essential for AST of AMPs and that these test methods should reflect the physiological (i.e., in vivo) environment in which AMP will have to function as closely as possible. Due to the diverse structures and modes of action of AMPs, the majority of participants did not expect that a single method for testing all peptides can be devised. Interestingly, there was no consensus on the relevance of standard AST for understanding the biology and potential of AMPs as new therapeutic agents. The majority of responses (81.08%) indicated that currently there is no awareness of the need for new testing methods in the regulatory landscape. Most participants (75.68%) also reported that new susceptibility testing methods are insufficiently described in published peer-reviewed papers.

Table 1.

Results of a Survey among Participants of IMAP (International Meeting on Antimicrobial Peptides) 2017 and 2019 Gordon Research Conference on Antimicrobial Peptides on the Requirements for Antimicrobial Susceptibility Testing (AST) of Antimicrobial Peptides (AMPs)/Host Defense Peptides (HDPs)

The importance of standardized AST methods for AMPs, adapted to physiological conditions was again emphasized in the open questions of the survey (Table S1). Aspects of susceptibility testing method development and its dissemination were listed as being important. In addition to the method’s requirement to mimic the physiological environment, the importance of having a method that is simple, cheap, robust, reproducible, and automatable was noted. Otherwise, routine testing of large sample quantities (pathogens and/or AMPs) would not be feasible. Key factors for in vitro testing include pH, ion and bicarbonate concentration, and the presence of serum in the medium. Storage, dilution, structure, and mechanism of action should be considered on the peptide side as well as whether the biological matrix used (e.g., growth medium, blood, serum, etc.) has an inhibitory effect on the tested peptides. Stability, purity, and cytotoxicity must also be taken into account when working with peptides. Besides the peptides, the pathogens must also be considered. The inoculum density, the growth phase and conditions, as well as biofilms, nongrowing microorganisms, persister cells, small colony variants, and spores are important factors. In general, the site of infection, the polymicrobial nature of infections, the presence of immune cells, as well as the potential immune status of donors and possible synergies should be considered and also reflected by in vitro systems. Possible development of resistance to AMPs should also be assessed. Some participants thought that simulating a local environment by in vitro systems in which the peptide will later be applied (e.g., topically to the skin, intravenously, or nebulized into the lungs) is more important than a general standardized in vitro method for a peptide. It would be beneficial to test conventional antibiotics, as well as AMPs, in a physiological relevant new media or test system in order to obtain reference values. For AMP AST methods to be more comprehensive and the values derived more comparable between laboratories and existing AST methods, many survey respondents stated that they would like to see AST methods for AMP described better and in more detail in publications. It is recommended that publications with incomplete descriptions of methodologies should not be cited. As an alternative resource to share/access information, the establishment of a website for publication of test methods or special journals for methods would be welcomed. The inclusion of comprehensive and clear AST protocols for AMPs in the CLSI guidelines was also proposed.

CONCLUSION

The research and development of AMPs as new potential classes of antimicrobials is thriving and expanding on a global basis. Optimized and where possible, standardized susceptibility testing methods for these promising antimicrobial candidates are needed to more accurately predict their therapeutic potential as well as to enable direct comparisons among different AMPs. Due to the diversity of AMP structures and modes of action and the different physiological environments in which they will be applied as therapies, a range of defined test methods will be necessary. Close cooperation with relevant institutions including CLSI, EUCAST, USCAST, regulatory bodies, and International Organization for Standardization (ISO) will be important going forward for the recognition and adoption of these new methods.