Antimicrobial Susceptibility Testing of Antimicrobial Peptides To Better Predict Efficacy

REVIEW
published: 07 July 2020

doi: 10.3389/fcimb.2020.00326
Antimicrobial Susceptibility Testing

of Antimicrobial Peptides to Better
Predict Efficacy
Derry K. Mercer 1*, Marcelo D. T. Torres 2 , Searle S. Duay 3 , Emma Lovie 1 , Laura Simpson 1 ,
Maren von Köckritz-Blickwede 4 , Cesar de la Fuente-Nunez 2 , Deborah A. O’Neil 1 and
Alfredo M. Angeles-Boza 3
1
NovaBiotics Ltd, Aberdeen, United Kingdom, 2 Machine Biology Group, Departments of Psychiatry and Microbiology,
Institute for Biomedical Informatics, Institute for Translational Medicine and Therapeutics, Perelman School of Medicine, Penn
Institute for Computational Science, and Department of Bioengineering, University of Pennsylvania, Philadelphia, PA,
United States, 3 Department of Chemistry, Institute of Materials Science, University of Connecticut, Storrs, CT, United States,
4
Institute for Biochemistry, University of Veterinary Medicine, Hanover, Germany
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
Edited by:
Florie Desriac, almost 60 years ago for testing conventional antibiotics, are not necessarily fit for purpose
Université de Caen when it comes to determining the susceptibility of microorganisms to AMP. Without
Normandie, France
careful consideration of the parameters comprising AST there is a risk of failing to identify
Reviewed by:
Julien Verdon,
novel antimicrobials at a time when antimicrobial resistance (AMR) is leading the planet
University of Poitiers, France toward a post-antibiotic era. More physiologically/clinically relevant AST will allow better
Carlos Juan,
determination of the preclinical activity of drug candidates and allow the identification
Instituto de Investigación Sanitaria de
Palma (IdISPa), Spain of lead compounds. An important consideration is the efficacy of AMP in biological
*Correspondence: matrices replicating sites of infection, e.g., blood/plasma/serum, lung bronchiolar lavage
Derry K. Mercer fluid/sputum, urine, biofilms, etc., as this will likely be more predictive of clinical efficacy.
derry@novabiotics.co.uk
Additionally, specific AST for different target microorganisms may help to better predict
Specialty section: efficacy of AMP in specific infections. In this manuscript, we describe what we believe
This article was submitted to are the key considerations for AST of AMP and hope that this information can better
Clinical Microbiology,
a section of the journal
guide the preclinical development of AMP toward becoming a new generation of urgently
Frontiers in Cellular and Infection needed antimicrobials.
Microbiology
Keywords: antimicrobial peptide (AMP), antimicrobial susceptibility testing, antibiotic, antifungal, host defence
Received: 06 March 2020 peptide (HDP)
Accepted: 29 May 2020
Published: 07 July 2020
Citation: ANTIMICROBIAL RESISTANCE

Mercer DK, Torres MDT, Duay SS,
Lovie E, Simpson L, Antimicrobial resistance (AMR) is a global health crisis. The over-use and inappropriate
von Köckritz-Blickwede M, prescribing of antibiotics has set us on a path toward a post-antibiotic era where our existing
de la Fuente-Nunez C, O’Neil DA and
armory of antibiotics will no longer be effective. Increasing numbers of microorganisms are already
Angeles-Boza AM (2020) Antimicrobial
Susceptibility Testing of Antimicrobial
becoming widely resistant to existing antibiotic classes (O’Neill, 2016; Barlow, 2018; Chatterjee
Peptides to Better Predict Efficacy. et al., 2018; Van Puyvelde et al., 2018; Roope et al., 2019). The need for new antimicrobial agents is
Front. Cell. Infect. Microbiol. 10:326. more important now than it has ever been. So too is the need for better antimicrobial stewardship;
doi: 10.3389/fcimb.2020.00326 prudent and appropriate use of antimicrobials.
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 1 July 2020 | Volume 10 | Article 326
Mercer et al. Antimicrobial Susceptibility Testing of AMP
AMR is responsible for 700,000 deaths per annum globally to form pores or to enter cells (Jenssen et al., 2006; Fjell et al.,
(Blair et al., 2015; European Commission, 2017) but this is 2011; Aoki and Ueda, 2013). HDP and the AMP derived from
forecast to increase to 10 million deaths annually by 2050 these scaffolds are versatile molecules with a wide diversity
(killing more people than cancer and diabetes) if the measures of structural and physicochemical properties, and are able to
highlighted above are not urgently implemented successfully to target microorganisms through diverse mechanisms of action,
address drug resistant infections (O’Neill, 2016). This assessment although the most common mechanism of action is membrane
was based on scenarios for rising drug resistance and economic perturbation/lysis (Brogden, 2005; Hancock and Sahl, 2006; Le
growth to 2050 for six major pathogens/infectious diseases; et al., 2017; Pyne et al., 2017; Kumar et al., 2018; Aisenbrey et al.,
Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, 2019). For the purposes of this manuscript, we will refer to both
malaria, tuberculosis and HIV (O’Neill, 2014). According to the AMP and HDP as AMP.
Centers for Disease Control and Prevention (CDC), in the US Achieving precise control over AMP properties and
alone over 2.8 million infections per year are caused by antibiotic- understanding how peptides behave in different environments
resistant bacteria, causing more than 35,000 deaths in the US are still challenges in the field (Naafs, 2018). The understanding
per annum and US$55 billion in increased healthcare costs and of AMP features and details of their mechanism/s of action are
lost productivity (CDC, 2019). Strikingly, in 2009–2010 in the still not clear and have been the target of many studies (Porto
US almost 20% of pathogens reported from Hospital-acquired et al., 2018; Torres et al., 2018; Torres and de la Fuente-Nunez,
infections (HAI) were multidrug-resistant (Sievert et al., 2013). 2019; Yount et al., 2019). Some of the most promising approaches
Most of the antibiotics available today are broad-spectrum to describe the role of structural and physicochemical properties
molecules derived from agents that have been in use for more on AMP antimicrobial activity are those involving computer-
than 30 years and as well as “failing” through resistance, based strategies combined with high-throughput experiments
oftentimes have unintended side effects, such as toxicity (Lee et al., 2018; de la Fuente-Nunez, 2019; Torres and de la
toward beneficial commensal bacteria and mammalian cells, Fuente-Nunez, 2019). Recent advances in computational biology
and triggering inflammatory responses (Lepore et al., 2019). have allowed the development of new molecular descriptors,
Research efforts are ongoing to discover and develop new, which enable the discovery of potent AMP through exploitation
more effective and safe antimicrobial agents that can overcome of their vast sequence space (Awale et al., 2017; Lin et al., 2018).
bacterial resistance mechanisms, occasionally even presenting Genetic and pattern recognition algorithms are examples of
selective activity toward single bacterial species or specific strains successful tools that have been used for the generation of AMP
of bacteria (de la Fuente-Nunez et al., 2017b). antibiotics that display antimicrobial activity both in vitro and
even in animal models (Lipkin and Lazaridis, 2017; Cipcigan
et al., 2018; Pane et al., 2018; Pfeil et al., 2018; Porto et al.,
ANTIMICROBIAL PEPTIDES/HOST 2018; Rondon-Villarreal and Pinzon-Reyes, 2018). For example,
DEFENCE PEPTIDES Guavanin 2, an AMP generated by means of a genetic algorithm
through a descriptive function that considered amphipathic
Antimicrobial peptides (AMP) have potential as a new distribution, net charge and hydrophobicity, was bactericidal
therapeutic class of antimicrobials and are one of the most at low concentrations, causing the disruption of Pseudomonas
promising scaffolds being explored for the generation of much- aeruginosa membranes by hyperpolarization of the membrane
needed novel antibacterials and antifungals. The blueprint for and displaying anti-infective activity in a mouse model (Porto
many AMP as drugs are endogenous Host Defence Peptides et al., 2018).
(HDP); relatively small peptides (4–50 amino acid residues) In vivo studies in animals have demonstrated that AMP
that are generally positively charged and often contain an provide protection against microbial infection and that their
amphipathic conformation (Jiang et al., 2009; Mercer et al., 2019; absence results in an increased risk of infectious disease (Rivas-
Torres et al., 2019). In the context of this manuscript AMP refers Santiago et al., 2009). In some cases, protection against infection
to all peptides with antimicrobial properties, whereas HDP are is relatively generalised, i.e. effected by a number of AMP, such as
essential innate host defence effector molecules and are amongst the combination of drosomycin and metchnikowin and defence
the “first responders” in all eukaryotic and some prokaryotic against Candida albicans infection in Drosophila melanogaster
organisms to infectious challenge or an inflammation (Zasloff, (Imler and Bulet, 2005; Hanson et al., 2019), whereas in
2002; Hassan et al., 2012; Mansour et al., 2014; Kang et al., others interactions are very specific, e.g., diptericin and defence
2017). As well as having direct antimicrobial activity against against Providencia rettgeri infection in D. melanogaster (Hanson
bacteria, fungi and parasites, HDP can modulate the host et al., 2019). Clinical correlations between AMP production
immune response, hence being termed host defence peptides and protection against infection exist that extend to humans
(Hancock and Sahl, 2006; Mansour et al., 2014). HDP are (Hancock et al., 2016; Mangoni et al., 2016; de la Fuente-Nunez
often classified according to the structure they tend to adopt et al., 2017a; Coates et al., 2018), as patients with impaired
in hydrophilic/hydrophobic interfaces, such as the interface of epithelial AMP production (e.g., atopic dermatitis/eczema) are
microbial cell membranes and the extracellular environment, more susceptible to secondary infection, unlike those with
e. g., α-helix, β-sheet, etc. (Wang, 2015). Often, ∼50% of their increased AMP production (e.g., psoriasis) (Ong et al., 2002;
sequence comprises hydrophobic and aliphatic residues that Yamasaki and Gallo, 2008). Thus, it appears to be clear that AMP
facilitate interactions with and translocation across membranes function as antimicrobials in vivo.
Despite the promise of AMP as novel antimicrobials, a lack of to facilitate the determination of the likelihood of resistance
optimization and standardization of experimental conditions for development, (iii) to provide estimates of likely in vivo and
antimicrobial susceptibility testing (AST), including exposure to critically, clinical efficacy when testing compounds in biological
different pH, salt solutions, serum half-life, and media/biological matrices replicating sites of infection, e.g., blood/plasma/serum,
matrices used during AST (Mahlapuu et al., 2016; Torres lung bronchiolar lavage fluid/sputum, urine, biofilms, etc.
et al., 2019) has been a major block to confirming efficacy (Breteler et al., 2011; Macia et al., 2014; Bottger et al., 2017; Ersoy
potential from the outset in AMP drug development pathways. et al., 2017; Nizet, 2017; Savini et al., 2017; Starr and Wimley,
Standardization of experimental conditions for assessing the 2017; Haney et al., 2019).
antimicrobial properties of AMP, and the difficulties encountered
therein, are the subject of this manuscript. It is widely believed
that AMP represent a group of molecules with the potential for ANTIMICROBIAL SUSCEPTIBILITY
development into a new generation of antimicrobials and for TESTING METHODS FOR EXISTING
which “standard” AST protocols can significantly underestimate CLASSES OF ANTIMICROBIALS
the AMP efficacy as antimicrobial drug candidates. In this
era of increasing levels of AMR worldwide, drug development Most AST, and its interpretation, is conducted using
professionals cannot afford to ignore potential antimicrobial internationally recognised standards developed by bodies
drug candidates simply because they do not perform well using including the International Organization for Standardization
“standard” laboratory test methods. Should AMP be successfully (ISO), Clinical and Laboratory Standards Institute (CLSI), the
developed as therapeutics, due consideration needs to be given European Committee on Antimicrobial Susceptibility Testing
to manufacturing peptides on a large scale and safe and ethical (EUCAST), The United States Committee on Antimicrobial
disposal of manufacturing by-products and unused peptides. Susceptibility Testing (USCAST) and the US Food and Drug
More than 60 peptide-based drugs have been already approved Administration (FDA) Center for Drug Evaluation and Research
by the Food and Drug Administration (FDA) and more than (CDER) (Table 1) (Magiorakos et al., 2012; Kahlmeter, 2015;
400 are in pre/clinical development (Aoki and Ueda, 2013; da Humphries et al., 2019). Susceptibility Test Interpretive Criteria
Costa et al., 2015; Ageitos et al., 2016; Mahlapuu et al., 2016; Lee (STIC), also known as “breakpoints,” are used to determine
et al., 2019). Of these, at least 70 are AMP, with more than 25 the optimal dose of antimicrobials for treating infection
in clinical trials (Koo and Seo, 2019). The peptide therapeutics and are based on those published by the CLSI, EUCAST,
market was valued at >$23 Bn (US) in 2017 and is predicted USCAST and the FDA. In December 2017 the FDA launched
to be worth >$43 Bn (US) by 2024 (Zion Market Research, the Antimicrobial Susceptibility Test Interpretive Criteria
2018). Additionally, peptide-based antimicrobials have been website (https://www.fda.gov/drugs/development-resources/
successfully used in the clinic for a number of years, including fda-recognised-antimicrobial-susceptibility-test-interpretive-
the antibacterials colistin, vancomycin, daptomycin and the criteria) which includes STIC similar to those published by
antifungals of the echinocandin class (Hancock and Chapple, CLSI and EUCAST. Different documents describe breakpoints
1999; Mercer and O’Neil, 2013). The peptide components of for bacteria, yeasts, filamentous fungi (moulds) and other
the complex molecules are, in most cases, cyclic (head-to-tail microorganisms (Table 1). Despite many similarities and
cyclization) or restricted (side chain-to-side chain or side chain- agreements, there remains some lack of harmonisation between
to-end cyclization) or conjugated with other organic compounds, AST methods from different organisations (Pfaller et al., 2011,
such as carbohydrates or lipids. Cyclization and/or conjugation 2014; Chowdhary et al., 2015; Kahlmeter, 2015; Brown et al.,
confer AMP longer half-life and increased bioavailability, thus 2016; Sanguinetti and Posteraro, 2018; Simjee et al., 2018;
improving the probability of achieving a successful treatment Cusack et al., 2019). Interpretive categories most commonly
(Greber and Dawgul, 2017). assigned are susceptible (S), indicative of a high probability
of a successful outcome, and resistant (R), indicative of a low
probability of a successful outcome, although in less common
ANTIMICROBIAL SUSCEPTIBILITY cases other categories include; non-susceptible, intermediate,
TESTING susceptible-dose dependent and area of technical uncertainty
(See the documents in Table 1 for details about these interpretive
Antimicrobial susceptibility testing (AST) determines the categories). An alternative STIC is the Epidemiological Cutoff
concentration of an antimicrobial that inhibits microbial growth, Value (ECV CLSI, 2018f or ECOFF EUCAST, 2019c). The
for both microbicidal and microbiostatic agents (Brown et al., ECV/ECOFF is defined as the MIC that separates a population
2016; Sanguinetti and Posteraro, 2018; Humphries et al., 2019; into isolates with and those without acquired or mutational
van Belkum et al., 2019). The importance of accurate AST in at resistance based on their phenotypic MIC value. An ECV is not a
least guiding antibiotic use in the clinic cannot be underestimated “breakpoint” as there is no clinical outcome or clinical trial data.
(Doern et al., 1994; Kumar et al., 2009; Weiss et al., 2012; Holmes Thus, an ECV is not a predictor of clinical success, but allows for
et al., 2016). prediction of whether an isolate has possible resistance to a given
During the development of novel antimicrobials, AST is antimicrobial (Turnidge et al., 2006; Lockhart et al., 2017). For
vitally important; (i) to determine the preclinical activity of drug conventional antimicrobials with known resistance mechanisms,
candidates and allow the identification of lead compounds, (ii) it is easier to define an ECV/ECOFF than a breakpoint, but for
TABLE 1 | Internationally recognised standards for Antimicrobial Susceptibility Testing (AST) and Susceptibility Testing Interpretive Criteria (STIC)/Breakpoints.
Organisation Antimicrobial susceptibility testing document Interpretive criteria document References
Bacteria
CLSI Methods for Dilution Antimicrobial Susceptibility Tests for Performance Standards for Antimicrobial CLSI, 2011, 2015, 2018a,b,c,d,
Bacteria That Grow Aerobically. M07, ED11. Susceptibility Testing (M100 ED29) 2019
Methods for Antimicrobial Susceptibility Testing of Performance Standards for Susceptibility Testing of
Anaerobic Bacteria. M11, ED9. Mycobacteria, Nocardia spp., and Other Aerobic
Susceptibility Testing of Mycobacteria, Nocardia spp., and Actinomycetes. M62, ED1.
Other Aerobic Actinomycetes. M24, ED3.
Methods for Antimicrobial Susceptibility Testing for Human
Mycoplasmas. M43, ED1.
Methods for Antimicrobial Dilution and Disk Susceptibility
Testing of Infrequently Isolated or Fastidious Bacteria.
M45, ED3.
EUCAST Antimicrobial susceptibility testing: EUCAST disk diffusion The European Committee on Antimicrobial EUCAST, 2019a,b
method, Version 7.0. Susceptibility Testing. Breakpoint tables for
EUCAST uses ISO 20776-1 for other bacterial interpretation of MICs and zone diameters. Version
AST methods 9.0, 2019
FDA Antibacterial Susceptibility Test Interpretive Criteria, https://www.fda.gov/drugs/
2018 development-resources/
antibacterial-susceptibility-test-
interpretive-criteria
USCAST 2019 USCAST Interpretive tables http://www.uscast.org/
ISO Clinical laboratory testing and in vitro diagnostic test ISO, 2019
systems—Susceptibility testing of infectious agents and
evaluation of performance of antimicrobial susceptibility
test devices - Part 1: Reference method for testing the
in vitro activity of antimicrobial agents against rapidly
growing aerobic bacteria involved in infectious diseases.
ISO20776-1.
Yeasts
CLSI Reference Method for Broth Dilution Antifungal Performance Standards for Antifungal Susceptibility CLSI, 2017a,b, 2018e,f
Susceptibility Testing of Yeasts. M27, ED4. Testing of Yeasts, M60, S1.
Method for Antifungal Disk Diffusion Susceptibility Testing Epidemiological Cutoff Values for Antifungal
of Yeasts. M44, ED3. Susceptibility Testing, M59, ED2.
EUCAST Method for the determination of broth dilution minimum The European Committee on Antimicrobial EUCAST, 2017a, 2018
inhibitory concentrations of antifungal agents for yeasts. Susceptibility Testing: Breakpoint tables for
E.DEF 7.3.1. interpretation of MICs. Version 9.0, 2018
Filamentous
Fungi
CLSI Reference Method for Broth Dilution Antifungal Performance Standards for Antifungal Susceptibility CLSI, 2010, 2017c,d, 2018f
Susceptibility Testing of Filamentous Fungi. M38, ED3. Testing of Filamentous Fungi. M61, ED1.
Method for Antifungal Disk Diffusion Susceptibility Testing Epidemiological Cutoff Values for Antifungal
of Nondermatophyte Filamentous Fungi. M51, ED1. Susceptibility Testing, M59, ED2.
EUCAST Method for the determination of broth dilution minimum The European Committee on Antimicrobial EUCAST, 2017b, 2018
inhibitory concentrations of antifungal agents for conidia Susceptibility Testing: Breakpoint tables for
forming moulds. E.DEF 9.3.1. interpretation of MICs. Version 9.0, 2018
AMP, where resistance mechanisms are not necessarily known, is almost exclusively used (Pfaller and Diekema, 2012; Ostrosky-
or present, it is much more difficult (if not impossible) to define Zeichner and Andes, 2017). The time taken for conventional
ECV/ECOFF, let alone a breakpoint. If that is the case, then AST varies considerably, depending on the infectious agent, and
defining STIC for AMP will require an entirely new definition. normally is only performed after the pathogen has been cultured
The most commonly used manual AST methods are disk and identified at the species level (van Belkum et al., 2019). For
diffusion and broth microdilution, although many large hospital bacteria this can be as quick as 24–48 h, but for fungi, isolation
laboratories use automated systems, due to improvements in and identification can take days, if not weeks, rather than hours
convenience and flexibility, such as BD PhoenixTM , Beckman and AST may take 48 h or longer (CLSI, 2012, 2017a,b; EUCAST,
Coulter MicroScan, bioMerieux Vitek R
2, Accelerate Diagnostics 2017a,b). Any delay in appropriate antimicrobial therapy can
PhenoTest and Thermo Fisher SensititreTM (Syal et al., 2017). In lead to increased mortality for severe infections (Delaloye and
the case of antifungal susceptibility testing, broth microdilution Calandra, 2014; Liu et al., 2017). There is, therefore, an urgent
need for more rapid AST (Sanguinetti and Posteraro, 2017; Kim TABLE 2 | Factors influencing antimicrobial activity of AMP.
et al., 2018; Cansizoglu et al., 2019; Idelevich and Becker, 2019).
In vitro Ex vivo In vivo
The standards for AST available from ISO, EUCAST and
CLSI were first implemented almost 60 years ago (World Health pH and ionic strength Biological matrices Animal models of
Organization, 1961) and have remained largely unchanged (e.g., blood) infection
since then in a “one size fits all” approach and are used Temperature Mammalian cells Pharmacokinetics
largely unquestioningly by many users (Nizet, 2017). However, Medium type/composition Intracellular Pharmacodynamics
some antimicrobials (and microorganisms) do not work pathogens
in these standards and require modifications to the testing Nutrient concentrations Air:Liquid or Metabolic
procedures, either by the use of additives to standard media Solid:liquid interface interactions
to generate representative efficacy values or by the use of Buffer Infection models Polypharmacy
(drug- drug
alternative or modified media for fastidious microorganisms
interactions)
(e.g., Haemophilus Test Medium for Haemophilus influenzae
Bicarbonate Formulation and
and H. parainfluenzae and the addition of 2.5–5.0% (v/v) delivery
lysed horse blood to cation-adjusted Mueller-Hinton (CA- Metal ions Polymicrobial
MH) medium when testing streptococci) (CLSI, 2018a,b). infections
Interestingly, EUCAST developed a different medium for use Salt (NaCl)
with fastidious bacteria (including streptococci and Haemophilus Polysorbate-80
spp.; a modified version of MH agar, with the addition of Synergy/Antagonism with other
5% mechanically defibrinated horse blood and 20 mg/L β- antimicrobials
nicotinamide adenine dinucleotide (β-NAD) (Matuschek Inoculum size
et al., 2018). Although most efficacy end-points are 100% Growth Phase (e.g., biofilms,
growth inhibition, there may be a lesser burden for some persisters, spores, small colony
pathogen/antimicrobial combinations (e.g., ≥50% growth variants, and other phenotypic
variants)
inhibition for fluconazole, flucytosine and ketoconazole for
Charge effects
non-dermatophyte moulds CLSI, 2017c) or the determination
Solubility
of minimum effective concentrations (MEC), rather than MIC
Laboratory materials
(e.g., the MEC of echinocandins vs. filamentous fungi is defined
as “the lowest concentration of an antifungal agent that leads to Proteolysis
the growth of small, rounded, compact hyphal forms compared Biological macromolecules (e.g.,
protein, DNA)
with the hyphal growth seen in the control well” CLSI, 2017c).
Oxygen (hyper-, norm- and
Consideration of other assay parameters, such as those described hypoxia)
in Table 2, perhaps require attention when conducting these
Mono/Polymicrobial interactions
standard procedures or when they are updated.
When using the same AST method, e.g., broth microdilution, In the context of this manuscript, ex vivo refers to experiment parameters that are not in a
living host organism (out of the living), but are simulating in vivo conditions. In vivo refers
results can be influenced by factors such as medium age,
to experiments conducted in/on a living host organism.
presence of polysorbate 80 and ion content (Bradford et al.,
2005; Fernandez-Mazarrasa et al., 2009; Sader et al., 2012;
Sutherland and Nicolau, 2014) as can non-compliance with
AST standards (Mouton et al., 2018; Turner and Ashley, 2019). reporting of false negatives that occurs when 5% (w/v) sodium
Examples where modifications to existing AST methods have chloride is used (Huang et al., 1993; Brown, 2001). Additionally,
been successfully implemented include the lipopeptide antibiotic CLSI recommends that when testing for oxacillin resistance
daptomycin. Daptomycin requires physiological concentrations in staphylococci samples should be incubated at 33–35◦ C, as
of calcium (50 mg/L) in the medium for optimal efficacy or testing at temperatures above 35◦ C may not detect mecA-
otherwise MIC values can be up to 32-fold higher, clearly mediated resistance (CLSI, 2018a). Therefore, antimicrobial
affecting whether an isolate could be sensitive or resistant substance-specific changes can be made to “standard” AST
(Eliopoulos et al., 1986; Campeau et al., 2018) and therefore the methods, so there is no reason why this should not be
CA-MH broth or agar is supplemented with additional Ca2+ possible for AMP in pre-/clinical development. Additionally,
(CLSI, 2018a,b). The lipoglycopeptides antibiotics (including AMP-specific end points do not necessarily have to equate
oritavancin, dalbavancin, and teicoplanin) are subject to binding to 100% growth inhibition. Obviously, any and all deviations
to laboratory plasticware (Arhin et al., 2008; Ross et al., 2014) from “standard” protocols will require rigorous and detailed
and therefore CLSI recommends addition of 0.002% (v/v) justification and validation.
polysorbate 80 (Tween 80) to CA-MHB to prevent such binding AMP represent one such group of molecules for which
(CLSI, 2018a). When testing staphylococci for sensitivity to these “standard” protocols can significantly underestimate their
oxacillin (MRSA phenotype) it is recommended to supplement efficacy potential. In this era of increasing levels of AMR
media with 2% (w/v) sodium chloride as this enhances the worldwide, can drug developers really afford to ignore potential
expression of mecA-mediated oxacillin resistance and reduces the antimicrobial drug candidates simply because they do not
perform well using “standard” test methods? AST of AMP

is normally performed using the broth microdilution (or
macrodilution) procedure, as many AMP are positively charged
molecules (and are dependent on that positive charge for
activity Jiang et al., 2009; Mercer et al., 2019; Torres et al.,
2019). Positively charged AMP interact with negatively charged
components in agar and neutralising their activity, meaning that
disk diffusion AST methods significantly under-estimate activity
or mask it completely (Kunin and Edmondson, 1968; Lehrer
et al., 1991), as is the case for the peptide antibiotics daptomycin
(Humphries et al., 2013) and colistin (Albur et al., 2014; Poirel
et al., 2017; Matuschek et al., 2018).
If AMP are to realise their potential as a future generation of
anti-infective therapeutics, AST methods will require approval
from regulatory authorities and buy-in from organisations such
as EUCAST, CLSI, and ISO (Kahlmeter, 2015; CLSI, 2018g). To
do this, any AST method for AMP or modification to an AST
method, at least for EUCAST, must be calibrated to the ISO broth
microdilution technique, often as part of the formal accreditation
process (Kahlmeter, 2015; ISO, 2019). Therefore, any AST
method must be accurate, robust, reproducible, have clinical
utility and validity and, ultimately, be amenable to automation.
FIGURE 1 | Two different mechanisms of action proposed for
membrane-active AMPs. The lipid bilayer is represented by spheres, while the
AMPs are represented by green helical cartoons.
ANTIMICROBIAL SUSCEPTIBILITY
TESTING OF AMP
It is clear that current AST methodologies are not “fit-for-
purpose” for determining the activity of AMP in vitro. When this because currently used test methods are not fit for purpose?
considering a single antimicrobial and a single species of For example, Roversi et al. determined that for the cathelicidin
microorganism, inter-laboratory variation and biological peptide PMAP-23 to kill E. coli ATCC25922 required 106 -107
variation (i.e., variation between strains) can still cause a broad bound peptides per cell (1–10 µM; 2.7–27 mg/L) (Roversi et al.,
distribution of wild-type MIC values (Annis and Craig, 2005; 2014). This compares to a typical susceptible MIC distribution of
Hombach et al., 2016; Mouton et al., 2019). Therefore, efforts to ciprofloxacin vs. E. coli of 0.004–0.064 mg/L (0.012–0.193 µM)
introduce standardised AST methods for AMP may turn out to (R > 0.5 mg/L; ECOFF = 0.064 mg/L) (http://www.eucast.org)
be an extremely onerous and difficult task (Jepson et al., 2016). and which specifically targets the enzymes DNA gyrase and
Previous attempts to standardise AST for AMP have met with topoisomerase IV, thereby preventing DNA replication. PMAP-
limited success. The method devised by the Hancock lab (http:// 23 kills bacteria by the “carpet” model (Orioni et al., 2009), so
cmdr.ubc.ca/bobh/method/modified-mic-method-for-cationic- this is likely to be more peptide molecules than required for pore-
antimicrobial-peptides/), was an adaptation of the CLSI broth forming peptides, or those with more specific mechanisms of
microdilution procedure (CLSI, 2018a), but has not been widely action (Figure 1).
adopted. The main differences between the two methods were A lack of antimicrobial activity of AMP, or attenuation
careful choice of labware (polypropylene or Sigmacote-coated of activity, has been observed for AMP when assayed under
glass), diluent used (0.01% (v/v) acetic acid containing 0.2% conditions in which existing, conventional antibiotic classes
(w/v) bovine serum albumin) and determining an MIC50 are active. This has been ascribed to a variety of features,
(reduction in growth of ≥50%) in the Hancock method. A including inactivation by physiological concentrations of
comparison of the two methods revealed differences in MIC NaCl and divalent metal cations or serum/plasma. However,
in which the Hancock method generated MIC values that were manipulation of media conditions, including making them more
predominantly lower than the CLSI method (Giacometti et al., physiologically relevant, could potentially reverse the inhibitory
2000). In the two decades since the publication of this method, effects of compounds such as NaCl and proteins. For example,
interest in AMP has grown significantly, so consideration of AST addition of sodium bicarbonate was able to reverse the inhibitory
for AMP is perhaps overdue a review and update. effects of physiological NaCl concentration against a number of
Membrane-active AMP are typically associated with higher different AMP, including the human cathelicidin LL-37, human
MIC values than those of conventional antibiotics, although this β-defensin 2 (hBD-2) and Cryptidin-4, albeit not dermcidin
is not always the case. Is this simply because the membrane (Dorschner et al., 2006). Many of the factors for consideration
represents a very large target, especially for AMP that kill via the described here have also been investigated for their effects on the
carpet mechanism and even some pore-formers (Figure 1), or is activity of conventional antimicrobials with varying outcomes
(Hajdu et al., 2010; Erdogan-Yildirim et al., 2011; Ersoy et al., synergistic activity as observed with the cationic, membrane-
2017; Oesterreicher et al., 2019). active polymyxins. This is clearly an area that requires more
It has been suggested that the discovery and preclinical detailed investigation before any recommendation specific to
development of new antimicrobials should target pathogens AMP susceptibility testing can be made. In the method described
as they are found at sites of infection, rather than the by the Hancock laboratory (http://cmdr.ubc.ca/bobh/method/
potentially different phenotype demonstrated in microbiological modified-mic-method-for-cationic-antimicrobial-peptides/) for
growth media (Dorschner et al., 2006; Ersoy et al., 2017). One AST of AMP, they recommend the use of a diluent of 0.01%
reason for this is because standard AST does not take into acetic acid containing 0.2% bovine serum albumin (BSA) to
account the potential influence (positive or negative) of the reduce peptide binding to plastic surfaces (Wiegand et al., 2008),
host cell environment on microbial susceptibility and resistance although it is difficult to ascertain whether this recommendation
(Sutherland and Nicolau, 2014; Haney et al., 2019). In order to has been broadly adopted. Even when it comes to the choice of
create a standardised AST procedure suitable for AMP there are filter for filter-sterilisation of AMP-containing solutions, caution
a number of factors that will need to be considered (Table 2). may be required, as cationic AMP may bind to negatively charged
Whilst many of the factors described in Table 2 may have an membranes, such as cellulose acetate (Wiegand et al., 2008).
impact on the design of AST for AMP, or the MIC of individual The choice of laboratory plasticware is similarly problematic
AMP, combinations of changes to these factors must also be as there are a number of publications that indicate that cationic
considered (Oesterreicher et al., 2019). AMP bind to polypropylene, polystyrene and borosilicate glass,
the most commonly used materials used in the manufacture of
labware (Chico et al., 2003; Kristensen et al., 2015), although
Laboratory Materials the extent of binding may be similar regardless of the type of
A number of publications have demonstrated that the results material used (Sanchez-Gomez et al., 2008; Kristensen et al.,
of AST of many antimicrobials, not just AMP, can be affected 2015). This situation also applies to some antibiotics (see above)
by the choice of laboratory plasticware for use with the broth and even antifungals such as the echinocandins (Fothergill et al.,
microdilution procedure and even the choice of tubes used for 2016; Arendrup et al., 2019). In the study of Kristensen and
preparing reagents (Singhal et al., 2018; Kavanagh et al., 2019). co-workers, binding to the surface of the tubes (irrespective of
This also applies to AST of AMP (Otvos and Cudic, 2007; material) was saturable and relatively more peptide bound at
Wiegand et al., 2008; Kristensen et al., 2015). low concentrations, so a simple solution may be to keep peptide
Some peptide-based antibiotics and AMP need to be prepared concentrations as high as possible to minimise the percentage
in alternative solvents, or with additives included in the lost to protein binding. Irrespective of this, this would lead to
media/diluent to prevent binding to the surfaces of tubes an underestimation of AMP efficacy when trying to determine
and plates. The lipoglycopeptides, including oritavancin and MIC values at lower peptide concentrations. The use of low
telavancin, must be solubilised in dimethyl sulfoxide (DMSO) protein-binding plasticware demonstrated reduced binding of
or with the addition of a surfactant, 0.002% (v/v) polysorbate selected AMP when compared to polystyrene, polypropylene and
80 (Tween 80), added to the water to prevent binding (Arhin borosilicate glass (Kristensen et al., 2015), but they unfortunately
et al., 2008; Ross et al., 2014). When DMSO was used in place did not carry out AST in this study, so it is not currently possible
of water as solvent for AST of the echinocandins (lipopeptide to assess whether the use of low protein-binding labware would
antifungals) caspofungin and micafungin, MIC values were lower lead to a reduction in MIC for AMP without further studies and,
and MIC ranges were narrower (Alastruey-Izquierdo et al., additionally, this is unlikely to provide a low cost solution.
2012) as was the case when water was supplemented with 50% The possibility of using different diluents, additives, or
bovine serum albumin (Arendrup et al., 2011; Garcia-Effron changes to laboratory plasticware clearly warrants further
et al., 2011). In contrast, even though colistin (cyclic lipopeptide) investigation, but perhaps the best recommendations are to be as
binds to plastics (Karvanen et al., 2017), the CLSI and EUCAST consistent as possible with your choices of materials and diluents
recommend broth microdilution for AST of colistin, but without and keep AMP concentrations high where possible to minimise
added surfactant (Hindler and Humphries, 2013; CLSI-EUCAST, the relative amount of peptide loss by binding.
2016), as polysorbate 80 can act synergistically with polymyxins
and reduce MICs (Brown and Winsley, 1968; Ezadi et al., Media Composition
2019), presumably due to interactions with lipopolysaccharides Most AST using AMP is conducted using broth dilution
in the Gram negative outer membrane (Correa et al., 2017). methods described by EUCAST or CLSI, or close variants
Additionally, polysorbate has antibacterial properties of its own thereof. For bacteria, when conducting broth dilution AST,
(Brown et al., 1979; Figura et al., 2012). In the case of AMP, both organisations recommend the use of Mueller-Hinton broth
the potential use of polysorbate is more complex as the addition (MHB) containing 10–12.5 mg/L Mg2+ and 20–25 mg/L Ca2+
of polysorbate 20 improved antiviral activity of LL-37 and (CLSI, 2018a; EUCAST, 2019a), whereas for testing of fungi, both
magainin-2B amide, but reduced antibacterial activity (Ulaeto recommend RMPI-1640 medium (RPMI), but with different
et al., 2016), however the impact of polysorbate on AMP activity concentrations of glucose; 2.0 g/L for CLSI test methods and
has not been extensively studied. The effect of polysorbate 20.0 g/L for EUCAST test methods (CLSI, 2017a,c; EUCAST,
for the prevention of binding of AMP to laboratory plastics 2017a,b). The reasons for the almost universal adoption of MHB
would need to be carefully investigated in light of possible for antibacterial susceptibility testing are unclear, but MHB
serves as a poor simulation of normal human body fluids (Ersoy azithromycin also synergised with the AMP LL-37 against MDR
et al., 2017; Nizet, 2017). Antifungal susceptibility testing using P. aeruginosa and MDR Acinetobacter baumannii. When tested
RPMI-1640 liquid medium (CLSI, 2017a,c; EUCAST, 2017a,b) in vivo, azithromycin also resulted in clearance of A. baumannii
more closely simulates normal human body fluids, whereas CLSI and S. maltophilia, sensitising S. maltophilia to neutrophil killing
disk diffusion testing of fungi and yeasts again uses Mueller- (Lin et al., 2015; Kumaraswamy et al., 2016). A series of 20 AMP
Hinton agar (MHA) (CLSI, 2010, 2018e). The effect of media were isolated from a surface-displayed peptide library and tested
type on AST results has been examined in a number of studies for activity in MHB via broth microdilution, but only 2 of the
(Schwab et al., 1999; Sajjan et al., 2001; Dorschner et al., 2006; peptides, and cecropin P1, demonstrated an MIC, but when the
Kumaraswamy et al., 2016; Ersoy et al., 2017; Tucker et al., 2018). MBC was determined in 10 mM Tris, pH 7.4 + 25 mM NaCl,
The use of MHB for AST of cationic AMP may be an unsuitable all AMP were bactericidal (MBC ≤128 µM) against at least 1 of
medium due to the high content of anionic amino acids in 4 Gram negative bacteria (Tucker et al., 2018). In other cases,
hydrolysed casein. MHB contains 17.5 g/L acid hydrolysate the use of diluted nutrient media results in improved efficacy of
of casein, with only 3 g/L beef extract and 1.5 g/L starch, AMP in AST. The MIC of the lactoferricin derivative HLopt2
as well as additional Ca2+ (20–25 mg/L final concentration) against selected Candida spp. was >250 mg/L, but when tested
and Mg2+ (10–12.5 mg/L final concentration). Casein contains in BHI diluted 1:100 the minimum microbicidal concentrations
25% anionic amino acids and these can interfere with cationic (MMC) were 2–31 mg/L. Similarly, the MIC in CA-MH vs. P.
AMP activity and cause them to precipitate (Turner et al., aeruginosa and S. aureus was much higher (125 and 63 mg/L,
1998). Turner and co-workers compared the activity of LL- respectively) than the MMC when tested in 1:100-diluted BHI
37 and protegrin (PG-1) in conventional MHB and MHB that (4 and 2 mg/L, respectively) (Ptaszynska et al., 2019). In some
had first been passed through an anion exchange column to cases, the choice of media type can have detrimental effects on
deplete the MHB of anionic compounds and demonstrated that AMP activity. For example, the semi-synthetic AMP Lin-SB056-
MICs against E. coli ML-35p, P. aeruginosa MR3007, Bacillus 1 was bactericidal against 6 P. aeruginosa in 1% TSB (MBC
subtilis and S. aureus 930918-3 were 3 - >20-fold higher in = 1.56–3.12 mg/L), whereas activity was lost in 80% artificial
standard MHB compared to MHB passed through an anion sputum medium, closely resembling the CF lung, except in the
exchange column (Turner et al., 1998). The choice of media presence of additional EDTA (ethylenediaminetetraacetic acid),
can make a significant impact on AMP efficacy. Incubation for where bactericidal activity was restored (Maisetta et al., 2017).
2 h with 5 µM of the cationic AMP D4E1 resulted in 100% The use of a more physiologically representative cell culture
killing of ∼1 × 105 cfu of S. aureus when incubated in RPMI- medium (RPMI-1640) may be better suited for AST of AMP, and
1640 liquid medium buffered with 30 mM HEPES, whereas the is certainly more representative of conditions in vivo than CA-
use of MHB, CA-MHB, nutrient broth + 125 mM NaCl or MHB or other nutrient-rich microbiological growth media and
Tryptone soya broth (TSB) resulted in less killing (68.6, 22.2, that the presence of higher concentrations of anionic substances
56.1, and 5.3%, respectively). Similar trends were observed for adversely affects the efficacy of cationic AMP. In a caveat to this,
P. aeruginosa ATCC27853 and another cationic AMP (D2A21) it has recently been observed that the composition of a number
(Schwab et al., 1999). In the Schwab study, a range of different of cell culture media, including RPMI-1640, do not simulate
buffers were also tested (PBS, Tris-NaCl, citrate-phosphate, saline bodily fluids particularly closely (McKee and Komarova, 2017),
and phosphate), with a trend for less killing in the phosphate so perhaps further work is required to develop media that more
buffers. Interestingly, osmolarity did not have any effect on the closely simulates mammalian bodily fluids.
activity of D2A21 against a range of bacterial pathogens isolated
from patients with cystic fibrosis (CF) (Schwab et al., 1999). Solubility and Aggregation
When determining the efficacy of the histatin derivative P-113, Given that most AMP have a net positive charge and hydrophilic
Sajjan and co-workers observed little activity in CA-MHB, but regions, aqueous solubility at concentrations required for AST
when this was diluted 20-fold the MIC was 3.1 mg/L, indicating are not problematic, although some AMP aggregate and become
that one or more components of CA-MHB were inhibitory to insoluble at relatively high concentrations in certain media and
the antimicrobial activity of P-113 against P. aeruginosa. Use of in the presence of selected anions. This can result in a loss
an alternative medium (LM broth) that attained similar growth of activity, stability and/or increased cytotoxicity (Frokjaer and
and growth rates to CA-MHB, but with reduced concentrations Otzen, 2005; Ratanji et al., 2014; Haney et al., 2017). Hydrophobic
of monovalent and divalent salts, retained the efficacy of P-113 regions in peptides are known to self-associate and drive the
to a range of bacterial CF pathogens, but did not affect the formation of aggregates (Kim and Hecht, 2006), so modifications
efficacy of tobramycin, ceftazidime or imipenem, indicating the to AMP design may reduce/prevent aggregation (Haney et al.,
detrimental effect of monovalent and/or divalent salts on AMP 2017). For example, the AMP Temporin L forms aggregates
efficacy (Sajjan et al., 2001). in water and due to an extended hydrophobic region, but
The activity of azithromycin against different isolates of substitution of glutamine to lysine at position 3 significantly
Stenotrophomonas maltophilia was examined in cation-adjusted reduced aggregation in water and improved its antiendotoxin
MHB (CA-MHB) and RPMI-1640 liquid medium supplemented properties (Srivastava and Ghosh, 2013), whereas substitution
with 10% Luria-Bertani broth (LB) (Kumaraswamy et al., 2016). with arginine at the same position improved activity against
In this study MICs in MHB were 32–256 mg/L, whereas in P. aeruginosa (and is also likely to attenuate aggregation)
RPMI, MIC values were significantly lower (0.03–0.25 mg/L) and (Mangoni et al., 2011). For example, significant aggregation of the
immunomodulatory AMP IDR-1018 occurred in the presence of of their innate immune system functions (van der Does et al.,
phosphate, benzoate, nitrate, and citrate, but less aggregation was 2019) that are present in most biological matrices. For example,
observed in the presence of acetate, chloride, water and, perhaps LL-37 has direct interactions with ≥16 proteins/receptors, that
significantly, bicarbonate (see later). IDR-1018 also exhibited subsequently interact with >1000 secondary effector proteins
aggregation in 10% RPMI and 1% MEM tissue culture media and when used to stimulate monocytes >900 gene expression
and also co-precipitated with serum proteins in a concentration- changes were observed (Hancock et al., 2016). It is, therefore, not
dependent manner, in many cases adversely affected the desired unreasonable to assume that many AMP will interact with host
immunomodulatory properties of the peptide and increased cells and that the effects of these interactions may not necessarily
cytotoxicity (Hartlieb et al., 2017). Protegrin-4 (PG-4) also be desirable. This may not need to be assessed when considering
aggregates in the presence of phosphate (>2.0 mg/ml PG-4 in in vitro AST, but would need consideration, for example, at later
50 mM sodium phosphate buffer, pH 7.4), forming amyloid-like stages of the drug development process. Significant efforts have
fibrils and retained antimicrobial activity against B. subtilis (Gour been made to improve AMP activity in blood, plasma and/or
et al., 2019). LL-37 exists in equilibrium between monomers and serum (Hamamoto et al., 2002; Knappe et al., 2010; Nguyen
oligomers in solution at low concentrations and oligomerizes et al., 2010; Chu et al., 2013; Dong et al., 2018; Kumar et al.,
in the presence of zwitterionic membranes (Johansson et al., 2018). When considering AMP activity in blood, serum or plasma
1998; Oren et al., 1999). Dermaseptin S9 formed aggregates the source of the blood and the method of collection must
and amyloid-like fibrils and the peptide binds to membranes in be taken into account. Most blood samples are collected in
an aggregated state (Caillon et al., 2013). Human α-defensin 6 tubes (vacutainers) containing an anticoagulant to prevent blood
(HD6) is a 32-residue cysteine-rich peptide that lacks the broad- clotting and it is known that the presence of an anticoagulant
spectrum antimicrobial activity observed for other human α- can affect AMP activity. For example, EDTA and citrate are
defensins. Strikingly, HD6 oligomerises to form “nanonets,” due known to enhance the efficacy of some AMP as they can chelate
to the disposition of hydrophobic residues in the HD6 primary divalent cations which are inhibitory to the activity of a number
structure, that entrap microbes and prevent invasive pathogens of AMP (Wei and Bobek, 2005; Walkenhorst et al., 2014; Maisetta
such as Salmonella enterica serovar Typhimurium and Listeria et al., 2017; Umerska et al., 2018; Grassi et al., 2019) and EDTA
monocytogenes from entering host cells in the gastrointestinal and heparin can inactivate proteases found in blood, including
tract (Chu et al., 2012; Chairatana and Nolan, 2017). An metalloproteases (EDTA), thrombin and Factor Xa (heparin) that
in silico analysis of the aggregative potential of AMP and non- may prevent AMP hydrolysis (Bowen and Remaley, 2014; Bottger
antimicrobial peptides revealed that AMP demonstrate very low et al., 2017; Rawlings et al., 2018).
in vitro aggregation propensity, but high in vivo aggregation The stability of proline-rich AMP (apidaecin and oncocin
propensity. Non-antimicrobial peptides can be divided in two derivatives) was examined in murine blood, serum and plasma
main groups, presenting either high or low values for both in and, perhaps surprisingly, the general trend was for greatest
vivo and in vitro aggregation. These results suggest that most stability in whole blood, followed by plasma and least stable
AMP demonstrate minimal aggregation in aqueous solution, but in serum, albeit only over a 1 h incubation period, and that
promote aggregation in a more hydrophobic environment (i.e., substitution of L-arginine residues for D-arginine or ornithine
the bacteria cell membrane) (Torrent et al., 2011), something improved stability in blood, serum and plasma (Bottger et al.,
borne out in many experimental studies. Thus, when conducting 2017). In another study, pre-incubation of a panel of AMP
AST with AMP, consideration of the solute/s used can be with red blood cells (RBC) (1 × 109 RBC/ml) before exposure
important and, in most cases, it would be advantageous to use to the pathogen significantly increased the MIC of most AMP,
aqueous solutions where possible for formulation of drugs for with a similar inhibitory effect caused by serum, even though
human or animal use, to minimise aggregation and to carefully the affinity of AMP for bacteria was much greater than for
assess peptide solubility in the media used for AST. RBC. Thus, serum binding and binding to host cells for AMP
intended for systemic delivery requires consideration and can be
Biological Matrices adapted to AST testing in the presence of serum or host cells
When testing the efficacy of any antimicrobial, logic dictates that (Starr et al., 2016). However, when the AMP DNS-PMAP23 or
it would be practical to test efficacy in the biological matrix at esculentin-1a(1-21)NH2 was added directly to a mixture of RBC
the site of infection, e.g., blood, sputum, urine, etc., but this is and E. coli, no significant inhibition of antibacterial activity took
unlikely to be practical for routine screening. However, prior to place (Savini et al., 2017), indicating that experimental set-up,
the initiation of in vivo efficacy studies it would be sensible to and by extension the nature of the infection being potentially
determine the efficacy of AMP in relevant biological matrices. treated, is an important consideration. In another study, in the
Proteolytic degradation of AMP is often considered a major absence of host cells, WLBU2 (12.5 µM) retained activity in the
weakness limiting their potential therapeutic application, as is presence of 98% human serum, whereas LL-37 was not active
binding to biological matrices, including serum/plasma proteins at concentrations up to 100 µM (Deslouches et al., 2005) and
(Wang et al., 1998; Sivertsen et al., 2014), nucleic acids (Park therefore the effect of biological matrices on AMP activity may
et al., 1998; Hsu et al., 2005), ribosomes (Mardirossian et al., 2014) be peptide-specific.
other proteins (Tu et al., 2011) bacterial cell walls (Malanovic The efficacy of the histatin-derived AMP, P-113, was tested in
and Lohner, 2016) and lipopolysaccharide (Piers et al., 1994; Sun diluted sputum from CF patients and no activity vs. P. aeruginosa
and Shang, 2015). Many AMP can also bind to host cells as part was observed. When the stability of P-113 was determined in
sputum, half-lives of 2.8–58.5 min were determined, probably Despite the effect of pH on AMP activity (which in many
preventing activity in sputum. By switching the composition of cases has not been investigated), AST of AMP is predominantly
P-113 from all L-enantiomer amino acids to all D-enantiomer carried out at neutral pH and the effect of pH modulation
amino acids (P-113D), the activity against P. aeruginosa was is not considered. When considering the relevance of pH on
comparable to the L-enantiomer peptide, but P-113D was stable AST of AMP, the main consideration should be of the pH
in CF sputum for 7 d. When the efficacy of P-113D was tested in at the site of infection and during delivery to the site of
CF sputum, an additional 1 log kill of P. aeruginosa was attained infection. Many assume that this is close to neutral, as an often
in 1 h and efficacy was enhanced further by pre-treatment of the cited physiological pH is 7.4, although pH can range from
sputum with recombinant human deoxyribonuclease (rhDNase; 7.0 to 9.0 in blood (Kellum, 2000). However, physiological pH
Pulmozyme R
), a therapeutic used in some CF patients (Sajjan values cover a relatively broad range, including pH 5.0 in the
et al., 2001). Interestingly, the activity of an all L-isomer of the macrophage phagosome a site where intracellular pathogens
AMP temporin lost activity in faeces within 30 min, whereas the such as K. pneumoniae, Salmonella typhimurium, E. coli and
all D-isomer version retained activity for 30 min, but activity Mycobacterium tuberculosis can reside (Underhill and Ozinsky,
was lost after 24 h (Oh et al., 2000). Thus, testing of AMP in 2002) and between 4 and 7 on the skin surface, with a most
biological matrices can be factored into AST and the earlier this is frequently determined pH range of 4.0–5.9 (Lambers et al., 2006),
conducted will have a bearing on lead selection for AMP intended although the pH of chronic wounds can be alkaline (pH 7.15–
for specific infections. 8.9) (Gethin, 2007). Skin infection can cause an elevation in skin
pH, as can other conditions, such as diabetes mellitus, that can
pH and Ionic Strength lead to increased risk of infection. Additionally, wound healing
More than 30 AMP are known to have pH-dependent activity, is associated with less acidic pH which can influence microbial
including LL-37, histatins, psoriasin, and lactoferrin, with greater colonisation and infection (Rippke et al., 2018). In the urinary
activity predominantly observed at lower pH values, especially tract the pH of normal urine is slightly acidic (pH 6–7.5), but
for histidine-containing AMP such as clavanins (Lee et al., 1997; a range as wide as 4.0–8.0 is normal. During infection this can
Malik et al., 2016; Alvares et al., 2017). Changes in pH can affect rise to 9.0 (e.g., “urea-splitting” pathogens such as Proteus spp.,
ionic interactions between membranes and AMP by changing Klebsiella pneumoniae, or Ureaplasma urealyticum) and is a clear
the protonation states of functional groups on the membrane indicator of a UTI (Bono and Reygaert, 2019). Conventional AST
and/or AMP, as well as effects on the ionic strength of the is conducted at pH 7.2–7.4 for bacteria and pH 7 for fungi which
solution (Walkenhorst, 2016). Cationic AMP are normally more may be appropriate for many infections, but consideration of the
efficacious at neutral and lower pH due to the loss of net pH (and the buffer used to attain this) for testing should be taken
positive charge at alkaline pH (Malik et al., 2016). For example, into account when investigating target pathogens or infections at
the efficacy of LL-37 against C. albicans was greater at pH 4.5 sites with different pH.
(81% death) when compared to pH 5.5 (79% death) and pH
7.2 (40% death) (Lopez-Garcia et al., 2005). Localised pH can Oxygen (Hyper-, Norm-, and Hypoxia)
significantly impact the interaction of AMP with membranes and Antimicrobial susceptibility testing is normally conducted under
their subsequent ability to perturb the membrane (Malik et al., conditions of normoxia, yet in tissues the amount of oxygen
2016; Alvares et al., 2017). This can reflect their predominant range between <1 and 11% oxygen, whereas in vitro experiments
site of action, e.g., skin. The ionic strength of the buffer can also are normally performed in 19.95% oxygen, an artificially high
influence efficacy, with reduced efficacy often observed at higher concentration relative to tissue concentrations. For example, in
ionic strengths (or an over-estimation of activity at low ionic normal air the oxygen partial pressure is 160 mmHg, whereas
strengths) (Sanchez-Gomez et al., 2008; Walkenhorst et al., 2013). in alveoli, this is reduced to 110 mmHg, in the brain 23–48
Additionally, the choice of buffer can affect AMP efficacy, with mmHg and in the colon, only 3–4 mmHg (Carreau et al., 2011).
greater efficacy observed in MOPS compared to phosphate buffer Areas of hypoxia are features of sites of bacterial infection,
at similar pH and ionic strength (Walkenhorst, 2016). However, healing wounds and other diseased tissues (Murdoch et al., 2005)
there are a number of exceptions to this, that may be peptide- and hypoxia can induce the expression of hBD-2 (Nickel et al.,
dependent or organism dependent. Walkenhorst and co-workers 2012). Thus, physiological oxygen concentrations vary widely,
observed the expected trend of enhanced activity at lower pH yet are largely not considered in the context of antimicrobial
values for a family of peptides versus Gram negative bacteria and efficacy, unless specifically considering activity against anaerobes
C. albicans, but the opposite effect was observed against S. aureus, or microaerophiles. Given the membrane disruption mechanism
with enhanced efficacy at higher pH values and hypothesized that of action of many AMP, should oxygen be a factor affecting their
this was due to changes to teichoic acids in the S. aureus cell activity? The plectasin-derived AMP, NZ2114 and MP1102, were
wall, making the peptidoglycan layer less negatively charged at bactericidal by membrane lysis vs. Clostridium perfringens under
neutral and acidic pH (Walkenhorst et al., 2013). Interestingly, a anaerobic conditions (>3 log kill in <60 min) (Zheng et al.,
linear form of esculentin 2EM caused greater cell lysis at pH 8.0 2017), whereas LL-37 and hBD-3 (< 5 mg/L) were bactericidal
compared to pH 6.0 and this correlated with increased α-helicity against C. difficile under anaerobic conditions (Nuding et al.,
of the peptide (Malik et al., 2016), indicating that the effect of 2014). The activity of human defensins against anaerobic bacteria
pH on AMP activity cannot be readily predicted and needs to be revealed that human α-defensin 5 and hBD-1 were minimally
determined where necessary. active against a panel of 25 strict anaerobes, hBD-2 demonstrated
relatively weak activity against most strict anaerobes, whereas importance physiologically, it is not routinely considered when
hBD-3 was active against 18 of 25 strict anaerobes tested conducting AST or in the maintenance of pH during AST.
(Nuding et al., 2009). Interestingly, human α-defensin 6 (HD- Even though AST of fungi uses RPMI-1640 liquid medium, a
6), the second most abundant AMP produced by Paneth mammalian cell culture medium, this is buffered to pH 7.0
cells in the small intestine (Wehkamp et al., 2005), does not with MOPS (CLSI, 2017a,c), rather than sodium bicarbonate
demonstrate direct antimicrobial activity under standard aerobic as it would be when culturing mammalian cells and using a
conditions, but demonstrated direct killing of Bifidobacterium CO2 incubator (5% CO2 ). Sodium bicarbonate has antibacterial,
adolescentis, Lactobacillus acidophilus, and Bif. breve, Bif. longum antifungal and antibiofilm properties of its own, but only at
and Streptococcus salivarius subsp. thermophilus under reducing supra-physiological concentrations (≥50 mM) (Corral et al.,
conditions (mimicking anaerobiosis) (Schroeder et al., 2015). Oh 1988; Xie et al., 2010; Letscher-Bru et al., 2013; Dobay et al., 2018;
and co-workers tested 16 CAMEL peptides (cecropin-melittin Farha et al., 2018). Bicarbonate acts as a selective dissipater of the
hybrids) against a selection of anaerobes (Peptostreptococcus spp., trans-membrane pH gradient, a component of the proton motive
C. difficile, Bacteroides fragilis, Prevotella, spp., Fusobacterium force (along with the membrane potential) and can enhance
nucleatum and Propionibacterium spp.; n = 60) under anaerobic the activity of AMP, including LL-37, α-defensin, indolicidin,
conditions and all were active (MIC90 = 1–32 mg/L) (Oh et al., protegrin and bactenecin and selected antibiotics, including
2000). Piscidins and ixosin are AMP that contain the copper- and aminoglycosides, macrolides and selected fluoroquinolones. The
nickel-binding ATCUN motif. Bactericidal activity under aerobic activity of AMP was enhanced as both the AMP and bicarbonate
conditions is enhanced when these AMP bind copper, but under perturb bacterial membrane potential and in the case of
anaerobic conditions two piscidins (p1 and p3) and ixosin retain antibiotics, enhancement of activity was predominantly limited
activity, but this does not depend on the presence of copper ions to those whose uptake is driven by the membrane potential
(Libardo et al., 2016; Oludiran et al., 2019). Thus, it would appear (Farha et al., 2018). Interestingly, tobramycin (aminoglycoside)
that antimicrobial activity of AMP under anaerobic conditions is activity against isolates of P. aeruginosa was enhanced in
dependent on the AMP used and for the purposes of AMP vs. the presence of bicarbonate against planktonic cells, but the
anaerobes would need to be assessed on a case-by-case basis. combination promoted biofilm growth (Kaushik et al., 2016).
The relevance of bicarbonate addition during AST of selected
Proteolysis antibiotics against bacteria has been investigated. When CA-MH
Susceptibility to proteolysis is often viewed as one of the most broth was supplemented with physiological levels of bicarbonate,
significant limitations when trying to develop peptide drugs, this improved the predictive value of AST for treatment of
including AMP (Vlieghe et al., 2010; Lecaille et al., 2016; Starr in vivo infections for a number of antibiotic and pathogen
and Wimley, 2017). When conducting AST with AMP, protease combinations, potentially due to structural changes to bacteria
production by the pathogen of interest is an obvious potential and changes in gene expression (Ersoy et al., 2017). When
cause of reduction in/or loss of activity (Stumpe et al., 1998; analysing the effect of bicarbonate on the sensitivity of isolates
Schmidtchen et al., 2002; Nesuta et al., 2017; Rapala-Kozik et al., of MRSA to anti-staphylococcal β-lactams, two phenotypes
2018). Additionally, if testing were to be carried out in biological became apparent; those that became susceptible on bicarbonate
matrices other than growth media (e.g., blood, saliva etc.), then supplementation and those that were unaffected. In the isolates
proteolysis by relevant host proteases could adversely affect AMP that became susceptible, bicarbonate supplementation caused
activity (Knappe et al., 2010; Lecaille et al., 2016; Starr et al., reduced expression of mecA and sarA, which led to decreased
2016; Bottger et al., 2017; Starr and Wimley, 2017). To determine production of penicillin-binding protein 2a and correlated with
whether proteolysis could occur during AST, protease inhibitors sensitivity to β-lactams in a rabbit infective endocarditis model
could be included in the system (Shin et al., 2010). However, comparable to that of MSSA isolates. Additionally, bicarbonate
this is not necessarily as simple as it appears. A number of responsive isolates demonstrated lower survival when the β-
protease inhibitors, including EDTA and citrate, can enhance lactam was combined with LL-37 in vitro and this may have
AMP activity by chelation of metal ions, or by other unknown enhanced the efficacy seen in vivo (Ersoy et al., 2019).
mechanisms, and this activity would need to be determined prior Dorschner and colleagues observed inhibition of S. aureus
to their use in AST (Wei and Bobek, 2005; Walkenhorst et al., growth by LL-37 was greater in MEM, a cell culture medium,
2014; Maisetta et al., 2017; Umerska et al., 2018; Grassi et al., when compared with Tryptone Soy Broth (a nutrient-rich
2019). bacterial growth medium) containing the same concentrations
of NaCl and FBS (Dorschner et al., 2006). By analysis of
Bicarbonate individual components of MEM, they determined that it
Bicarbonate (NaHCO3 ) is relatively common in mammalian was the presence of physiological concentrations of sodium
tissues (NaHCO3 ; 24.90 ± 1.79 mM in human blood Wishart bicarbonate that enhanced membrane-permeabilising activity
et al., 2018) and the bicarbonate buffer system, sodium of the following AMP: LL-37, mCRAMP (murine cathelicidin-
bicarbonate in balance with carbonic acid, helps to maintain related antimicrobial peptide), PR-39 (a porcine cathelicidin),
the physiological pH, including blood, interstitial fluid and hBD-2 (human β-defensin 2), but not dermcidin (an anionic
the upper gastro-intestinal tract (Boron and Boulpaep, 2005). AMP from human skin and sweat) and not in a pH-dependent
However, bicarbonate warrants additional consideration beyond manner. The presence of bicarbonate; may also have ameliorated
its capacity for maintaining pH homeostasis. Despite its the inhibitory effect of the 150 mM NaCl in the medium used,
as NaCl concentrations of >50 mM can inhibit the activity of not when heated to 90◦ C (Ebbensgaard et al., 2015). However,
many AMP (Goldman et al., 1997; Travis et al., 2000). Culturing this is not the case for all AMP. The AMP epinecidin-1 was not
E. coli O29 in the presence of NaHCO3 affected the expression stable at elevated temperatures and demonstrated a 32 - >64-
of a number of virulence-related genes that could increase fold increase in MIC against S. aureus following incubation at
susceptibility to AMP (Dorschner et al., 2006). The addition of 60–100◦ C for 5 min (Huang et al., 2017). As is well-documented,
25 mM bicarbonate enhanced the activity of the AMP tritrpticin AMP have been isolated from almost all life-forms, including
against the protozoan Trichomonas vaginalis (Infante et al., arctic and Antarctic fish. Moronecidin (isolated from the hybrid
2011). Conversely, selected S. aureus small-colony variants (SCV) striped bass) and 2 derivatives were assessed for efficacy against
were less susceptible to LL-37 when incubated in the presence Psychrobacter spp. PAMC25501 at 5–15◦ C and E. coli DH5α at
of 50 mM NaHCO3 ; (∼2 x physiological concentration) (Zhang 15–37◦ C and no differences in MIC were observed at different
et al., 2018), although the effect of physiological bicarbonate temperatures (Shin et al., 2017). The activity of the piscidin-
concentrations was not examined. Small colony variants (SCV) like AMP, chionodracine (isolated from the icefish Chiondraco
are slow-growing sub-populations of bacteria with altered hamatus) was more active at 25 than 37◦ C against E. coli (MIC
metabolism and reduced antibiotic susceptibility which, in the 20 and 5 mg/L, respectively) and B. cereus (MIC 10 and 5 mg/L,
case of S. aureus, can cause persisting and recurrent infections respectively) (Buonocore et al., 2012), although this may reflect
(Baumert et al., 2002). S. aureus SCV are already known to be adaptation to the low temperature environment from which they
less susceptible to a number of AMP when compared to wild- were isolated.
type cells (Koo et al., 1996; Sadowska et al., 2002; Samuelsen
et al., 2005), although this effect was not observed when a Metal Ions
cationic antimicrobial polypeptide was tested against S. aureus Transition metal ions influence the activity of AMP in a variety
SCV (Mercer et al., 2017). It would therefore be relevant to of ways. In some cases, the activity of AMP are dependent on
examine the effects of media supplementation with physiological the presence of metal ions (Paulmann et al., 2012; Melino et al.,
concentrations of bicarbonate (25 mM; 2.1 g/L) when conducting 2014; Alexander et al., 2018; Jezowska-Bojczuk and Stokowa-
AST with AMP to generate results possibly more predictive Soltys, 2018; Agbale et al., 2019), whereas in others the presence
of efficacy in vivo. Such results must be viewed with caution, of metal ions can cause reduction, or even complete abrogation,
however, in light of the biofilm promoting effects in combination of activity (Friedrich et al., 1999; Deslouches et al., 2005). In most
with tobramycin (Kaushik et al., 2016). circumstances, the effect of metal ions must be considered on a
case-by-case basis as different ions will produce a distinct effect.
Temperature For example, LL-37 is inactive in the presence of ≥3 µM MgCl2 ,
The effect of temperature on the activity of AMP during whereas the de novo designed AMP, WLBU2, remains active at
AST has not been widely investigated, as most AMP are the same concentration. LL-37 is also less potent in the presence
intended for use against infectious diseases AST is predominantly of ≥1 µM CaCl2 , whereas the MIC of WLBU2 increases by ∼4-
conducted at body temperature (∼37◦ C) or at temperatures fold in the presence of ≥6 µM CaCl2 , (Deslouches et al., 2005).
recommended in CLSI or EUCAST guidelines (30–37◦ C). Activity of the cecropin-melittin hybrid peptide variants was ≥4-
Thus, it seems most relevant to conduct AST at physiological fold increased in the presence of 3–5 mM MgCl2 (representing
temperatures. At low temperatures membrane bilayers undergo the serum concentration of divalent cations), although one
a reversible change of state from a fluid (disordered, liquid variant is insensitive to the effects of 3 mM MgCl2 , but not to
crystalline) to a non-fluid (ordered, gel) array of the fatty acyl 5 mM MgCl2 (Friedrich et al., 1999).
chains (de Mendoza, 2014) and this increase in membrane Susceptibility testing of K. pneumoniae against tetracycline
rigidity can lead to reduced AMP efficacy/resistance (Cole and in tissue culture medium predicts resistance, whereas testing
Nizet, 2016; Joo et al., 2016). Interestingly, the P. aeruginosa in MHB and LPM pH 5.5 media predicts susceptibility. Mice
quorum-sensing molecule 2-n-heptyl-4-hydroxyquinoline N- infected with K. pneumoniae and treated with tetracycline
oxide (HQNO) increases membrane fluidity in S. aureus, so it survive infection mirroring the results obtained in pH 5.5
would be intriguing to determine whether this increases the media, conditions that resemble best the environment in which
sensitivity of S. aureus to AMP (Orazi et al., 2019). HQNO can tetracycline interacts with this pathogen since K. pneumoniae
also induce S. aureus to adopt the SCV phenotype (Hoffman et al., resides within macrophage phagosomes (Ersoy et al., 2017).
2006) and this may also affect AMP activity. The effect of high and This example highlights the importance of mimicking the
low temperatures on storage or preparation of AMP, as well as environment of phagosomes when dealing with intracellular
activity, has received consideration in a number of studies (Wei pathogens. Besides K. pneumoniae, other important pathogens
et al., 2007; Zhang et al., 2011; Ji et al., 2014; Lee et al., 2014; Jiao that thrive in an intracellular environment include Salmonella
et al., 2019). For example, many AMP demonstrate stability at spp. (de Jong et al., 2012; Helaine et al., 2014), Mycobacterium
temperatures below 100◦ C. For example, the thermal stability of tuberculosis (Pieters, 2008) and Legionella pneumophila (Escoll
8 AMP (Cap18, Cap11, Cap-11-1-18m2, Cecropin B, Cecropin et al., 2013). Streptococcus pyogenes is also suspected of sharing
P1, Indolicidin and Sub5) was assessed by heating them to 70 this lifestyle (Hertzen et al., 2012).
or 90◦ C for up to 30 min before conducting AST. All AMP were After phagosomes internalize their cargo, acidification of
stable following heating, with only Sub5 demonstrating an MIC the interior takes place in a rapid process. For example, the
increase (4 to 8 mg/L) after being heated to 70◦ C for 30 min, but phagocytic compartment of a bone marrow-derived macrophage
TABLE 3 | Concentration of copper and zinc ions within phagolysosomes of S. pyogenes mutants defective in Zn efflux had a lower survival
peritoneal macrophages during infection by three Mycobacterium spp. within the hosts.
Element Time M. smegmatis M. avium M. tuberculosis
There is also evidence for removal of Zn ions from
phagosomes during infections and excellent reviews on the
Cu 1h 9.9 ± 5.5 µM 28.3 ± 11.4 µM 426 ± 393 µM topic exist. In this work, we wanted to limit ourselves to those
24 h 24.8 ± 0.65 µM 17.3 ± 10.3 µM 24.7 ± 9.5 µM environments in which the concentration of Zn ions increased.
Zn 1h 70.5 ± 37.3 µM 134.6 ± 38.8 µM 37.8 ± 25.2 µM Besides the phagocytic environment, there are other sites
24 h 260 ± 117 µM 120.8 ± 31.1 µM 459 ± 271 µM of microbial infection in which copper and zinc ions are
found at concentrations in which they can affect the activity of
Concentrations were determined using hard x-ray microprobe with sub-optical resolution
(Wagner et al., 2005).
antimicrobial agents. For instance, during urinary tract infection
by the pathogens Proteus mirabilis and K. pneumoniae, copper
is found at micromolar concentration as a host response to
the infection (Hyre et al., 2017). Additional in vivo studies
phagosome following internalization of an immunoglobulin G- demonstrated that Cu-deficient mice are more susceptible to
coated particle reaches a pH of 5.0 or below within 10–12 min uropathogenic E. coli infection, indicating that copper release
of internalization of an immunoglobulin G (IgG)-coated particle into urine is an important innate defence mechanism. An
(Yates et al., 2005). The acidic, hydrolytically competent additional human fluid that contains metal ions at concentrations
environment of the phagolysosome in combination with other high enough to affect the antimicrobial activity of antibiotics
antimicrobial effectors typically lead to the death and digestion is sweat (Troy et al., 2007). Copper concentrations range from
of most non-pathogenic microbes. It is in this environment that 4.6 ± 0.4 µM to 20 ± 10 µM, whereas zinc concentrations
the intracellular pathogens mentioned above survive and thrive, can be as high as 630 µM. Copper levels in human saliva
and more importantly, it is where the interaction between an also have been reported to range from 1.6 µM to 7.5 µM
antimicrobial agent and the pathogen will take place. Besides (Dreizen et al., 1952; Borello, 1976). Human saliva also contains
the low pH, other antimicrobial effectors fill the phagocytic zinc ions with reports indicating a maximum concentration
compartment, including metal ions. of 6.7 mM (Sejdini et al., 2018). Clearly, copper and zinc
Metal ions such as copper and zinc have been observed ion interactions with antimicrobial agents is plausible outside
at high concentrations in the phagocytic milieu as a response phagocytic compartments.
to certain types of infections. Wagner et al. using a hard x- Antibiotics, with their richness of functional groups, are
ray microprobe with suboptical resolution reported that upon poised for metal ion coordination. The result of this interaction
infection by the human pathogens M. tuberculosis and M. avium can range from antagonism to synergism, although the former is
or with avirulent M. smegmatis the concentration of copper and the most common outcome. Back in 1946, Eisner et al. reported
zinc ions within phagolysosomes of peritoneal macrophages can the inactivation of penicillin by zinc salts (sulphate, acetate,
be as high as 426 ± 393 µM and 459 ± 271 µM, respectively chloride, and oxide) (Eisner and Porzecanski, 1946). Amoxicillin
(Table 3) (Wagner et al., 2005). Additional indirect evidence and ampicillin are also readily degraded by zinc ions (Navarro
for the presence of copper ions in the intracellular battlefield et al., 2003). Tetracycline and several of its derivatives avidly bind
against pathogens include the observation that in IFN-γ and LPS- copper and zinc ions to form 2:1 complexes (Doluisio and Martin,
activated macrophages the levels of the Ctr1 Cu importer are 1963; Brion et al., 1985). Indeed, the formation of tetracycline-
elevated (White et al., 2009). Moreover, the Cu pump ATP7A zinc complexes is suspected to affect the metabolism of the drug
is overexpressed and localized to the phagolysosome, suggesting and adversely impact its antibiotic activity (Doluisio and Martin,
accumulation of Cu within this compartment. Interestingly, 1963; Brion et al., 1985). Other antibiotics are known to bind
macrophage exposure to the Cu chelator tetrathiomolybdate metal ions (e.g., quinolones Seedher and Agarwal, 2010; Uivarosi,
(TTM) results in increased survival of S. typhimurium (Achard 2013 and aminoglycosides Lesniak et al., 2003). Interestingly,
et al., 2012). Besides the quantitative determination of Zn by many chelates of quinolones show equal or enhanced activity
Wagner and co-workers, there is additional evidence for a role compared to that of the parent antibiotic. The reason for
of host Zn in the direct overload killing of invading pathogens the superior activity of the quinolone-metal complexes is not
(Djoko et al., 2015). Using a Zn-responsive fluorescent probe, it clear. Overall, the impact of metal binding on the activity of
was observed that infection of human macrophages with either many antibiotics is undeniable and deserves attention during
E. coli or M. tuberculosis leads to an increase in the intracellular susceptibility assays.
levels of Zn (Botella et al., 2011). Consistent with the hypothesis The effect on the antimicrobial activity of AMP of the presence
that Zn directly kills phagocytosed pathogens, it was observed of Cu and Zn ions in the assay media is difficult to predict.
that bacterial mutants defective in Zn export (zntA and ctpC Typically, compounds that act as simple metal chelators will see
in E. coli and M. tuberculosis, respectively) showed decrease a decrease in their antimicrobial activity as the concentration of
survival within these human macrophages. Similar observations chelated ions increase. An example of an antimicrobial peptide
of increased Zn concentrations within phagocytic cells have that acts as a metal chelator is the tick peptide microplusin, which
been observed upon infection with Histoplasma capsulatum specifically chelates Cu2+ ions (Figure 2). The opposite effect,
(Subramanian Vignesh et al., 2013), and Streptococcus pyogenes that is the increase in activity in the presence of these metal
(Ong et al., 2015). As observed for E. coli and M. tuberculosis, ions, also takes place although it has received less attention. In
2014; Libardo and Angeles-Boza, 2014). The Zn2+ ions are

required for the formation of ion channels as they promote the
formation of a trimer of dimers. One final example that is worth
mentioning corresponds to the synthetic ∗ ARVA peptides, which
contain no histidines, that were selected from a combinatorial
library and shown to be synergistic with copper and zinc ions
(Walkenhorst et al., 2014; Walkenhorst, 2016). Thus, there is
mounting evidence that the activity of AMP is affected by the
presence of copper and zinc ions.
An important question that must be asked at this point is
whether the presence of other divalent cations would resemble
the effect of Cu and Zn ions? The short answer is no, as the effects
on the activity of AMP by Ca2+ and Mg2+ ions often differ from
those effects that originate from the presence of Cu and Zn ions.
For example, the presence of CaCl2 and MgCl2 does not affect
the activity of microplusin; however, similar concentrations of
CuCl2 have a negative impact in the activity of the AMP (Silva
et al., 2009). Similarly, the presence of Ca2+ ions decrease the
antimicrobial activity of clavanin A, an AMP found in phagocytic
cells, whereas Zn2+ ions increased its activity (Juliano et al.,
2017). In short, Ca2+ and Mg2+ ions do not have the same effect
as Cu and Zn ions in the antimicrobial activity of AMP.
Excluding the highest and lowest values of the metal
concentrations in the set of values shown in Table 3, the
average concentrations of Cu and Zn ions are 24 and 146 µM,
FIGURE 2 | Structure of microplusin solved by NMR solution spectroscopy respectively; therefore, we recommend using Cu and Zn
(PDB ID: 2KNJ). For illustration purposes the Cu2+ ion has been added, concentrations of 25 and 150 µM, respectively if the phagosomal
shown as brown sphere, to its putative binding site. The four labelled residues, environment is to be mimicked. [Zn2+ ] = 150 µM is well above
shown as red liquorice, are highly likely to bind Cu2+ ion due to their proximity
to the ion. The other residues, shown as blue liquorice, are also capable of
the concentration required to maximize the potency of clavanin
binding Cu ion, as suggested by the NMR experiment (Silva et al., 2009). A 16-fold at pH 5.5 (Juliano et al., 2017). Likewise, 150 µM is in
the upper range of the Zn concentrations that were synergistic
with ∗ ARVA peptides (Walkenhorst et al., 2014). Since Ersoy has
successfully used LPM (low magnesium medium) media at pH
the case of copper ions, several antimicrobial peptides have been 5.5 to mimic the phagolysosome compartment, it is suggested
recently reported to utilize this ion to enhance its bactericidal that using this media in combination with Cu and Zn ions.
activity (Libardo et al., 2014, 2015, 2016, 2017; Hayden et al., CuCl2 and ZnCl2 can be used as sources for these ions. If
2015; Alexander et al., 2017). One of the earliest examples after performing the assay in the presence of copper and zinc
of the AMP requirement for Zn2+ is that of bacitracin, a ions as suggested above one is suspicious of synergy between
mixture of chemically related dodecapeptides isolated from any of these metal ions and the AMP, the best assay to follow
Bacillus subtilis and Bacillus licheniformis. Bacitracin, a drug and is the one described by Walkenhorst et al. which allows the
food additive, has a multimodal mechanism of action (Storm determination of FIC values using a 96-well plate. CuCl2 and
and Strominger, 1973; Storm, 1974; Karala and Ruddock, 2010; ZnCl2 were used in liquid test medium (LTM) and the procedure
Dickerhof et al., 2011). There is consensus in that the main mode is clearly described in the paper of Walkenhorst and co-workers
of action involves the inhibition of bacterial cell-wall biosynthesis (Walkenhorst et al., 2014).
by targeting extracellular, membrane-associated pyrophosphate
groups. Remarkably, recognition of the pyrophosphate group of Salt (NaCl)
the lipid target is mediated by a zinc ion which organizes the N- The effect of NaCl on AMP activity needs to be taken into
terminal region of the peptide and neutralizes the large charge account depending on the site of infection as the concentration
of the ligand, thus allowing direct antibiotic–lipid interactions can vary significantly, from ∼140 mM in blood (Li et al., 2016),
(Storm and Strominger, 1973; Economou et al., 2013). Other 12–52 mM in saliva (Ferguson and Botchway, 1979) and 8–14
peptides with enhanced its antimicrobial activity in the presence mmol/mmol creatinine in urine (Sasaki et al., 1998), whereas
of Zn2+ are the anionic human AMP DCD-1 and its derivative in certain infectious disease states this can be much higher (up
DCD-1L. Both peptides are present in eccrine sweat and are to 180 mM in CF sputum Grandjean Lapierre et al., 2017 and
highly active at pH values that range from 5.5 to 7.4 and retain 300 mM in infected urine Withman et al., 2013). Distinct from
their activities against several microorganisms when the ionic other metal ions and salts, some AMP are sensitive to higher salt
strength of the medium is increased by adding up to 150 mM concentrations. For example, human β-defensin 1 retains activity
NaCl (Dennison et al., 2006; Song et al., 2013; Becucci et al., at low NaCl concentrations (≤50 mM), but this is rapidly lost
when the NaCl concentration increases above 75 mM (Goldman point the cell densities specified in CLSI, ISO and EUCAST
et al., 1997), which are physiologically relevant (Wishart et al., documents (Table 1) are not unreasonable starting points, but
2018). This sensitivity was to the Na+ ion, not the Cl− ion. consideration should also be given to the pathogen and site of
Similarly, the activity of LL-37 is reduced significantly in the infection, as in vivo information may be available to better guide
presence of 100 mM NaCl (Turner et al., 1998) and the activities the likely cell density during infection and this would clearly be
of defensins are similarly reduced (Bals et al., 1998; Garcia more relevant to take into consideration.
et al., 2001). The tryptophan-rich AMP, Pac-525, demonstrated
reduced activity in the presence of 100–300 mM NaCl, whereas
the Pac-525 derivative, D-Nal-Pac-525 (all D-amino acids and Growth Phase: Biofilms, Persister Cells
tryptophan residues replaced with D-β-naphthylalanine) was Spores and Small Colony Variants (SCV)
not sensitive to these NaCl concentrations (Wang et al., 2009). When conducting broth dilution AST using CLSI or EUCAST
The histatin derivative P-113 was also sensitive to high NaCl protocols, bacteria are normally prepared when actively growing
concentrations (as well as additional Ca2+ and Mg2+ in the (exponential phase) or in the stationary phase, depending on
media) (Rothstein et al., 2001). Conversely, clavanin retained whether the inoculum is prepared by the broth culture or
activity in 100 mM NaCl and Clavanin AK retained activity in colony suspension methods, respectively (CLSI, 2018a; ISO,
300 mM NaCl (Lee et al., 1997), the de novo designed peptide 2019), but the potential influence of growth phase on AST
SHAP1 retained activity in the presence of 200 mM NaCl (Kim results is not taken into consideration. Similarly, the growth
et al., 2014), the Streptococcus mutans-specific AMP IMB-2 phase is not factored in when preparing yeast inocula (CLSI,
retained ∼85% activity in the presence of 134 mM NaCl (Mai 2017a). Conversely, AST of filamentous fungi starts with an
et al., 2011) and the RR12 AMP retained activity in 300 mM inoculum prepared from a spore suspension (CLSI, 2017c) that
NaCl (Mohanram and Bhattacharjya, 2016). Therefore, when can take a number of hours to germinate into actively growing
considering AST of AMP, the concentration of NaCl at the site hyphae. In most cases, AST of conventional antibiotics and
of infection is an important factor and the effects of NaCl on antifungals requires actively metabolising/growing cells, as their
AMP activity will need to be assessed on a case-by-case basis. If mechanism of action relies on physiological processes, including
the tested AMP is salt-sensitive, there are a number of possible DNA/RNA replication (fluoroquinolones and flucytosine),
strategies available to reduce salt-sensitivity (Harwig et al., 1996; protein biosynthesis (tetracyclines and macrolides), cell wall
Friedrich et al., 1999; Tam et al., 2000; Park et al., 2004; Yu et al., biosynthesis (β-lactams and echinocandins) and membrane
2011; Chu et al., 2013). biosynthesis (polyenes and azoles) (Silver, 2011; Perfect, 2017).
Therefore, it is important that microorganisms are actively
Inoculum Size growing, or have the capacity to do so, when AST is performed.
The efficacy of an antimicrobial during AST will necessarily Membrane-active AMP, on the other hand, are active against
depend on the size of inoculum tested. The inoculum effect metabolically inactive as well as active cells; a major potential
is a well-documented laboratory phenomenon that can be therapeutic advantage, e.g., (Mercer et al., 2017). As such, it
described as a significant elevation in antibiotic MIC when is not critical that cells be metabolically active when AST is
the number of inoculated microorganisms is increased (Brook, conducted for these AMP, and it is only by following convention
1989; Smith and Kirby, 2018; Idelevich and Becker, 2019). than most AST of AMP is done on actively metabolising cells. At
Elevated MIC (reduced efficacy) values as a function of increased sites of infection, microorganisms are rarely found growing in
cell density has been observed for a limited number of AMP, the exponential phase on nutrient-rich media sources; therefore,
including LL-37 (Snoussi et al., 2018), ARVA (Starr et al., the importance of microbes existing in other growth phases
2016) and DNS-PMAP 23 (Savini et al., 2017). In cultures requires consideration and there is evidence that slower growing
with a low inoculum density, MICs of AMP and antibiotics pathogens are more virulent than faster growing pathogens
can reach a plateau value that is independent of cell density (Leggett et al., 2017). Additionally, a number of different
(Udekwu et al., 2009; Savini et al., 2017). parameters influencing growth phase can alter virulence gene
An additional consideration must be the presence of host expression in pathogens, including toxin and adhesin production
cells as these can effectively contribute to the inoculum effect. in E. coli (Crofts et al., 2018) and hyphal development in Candida
For example, conducting AST in the presence of host cells, spp. (Su et al., 2018).
e.g., red blood cells, can cause a loss of activity against the Given the heterogeneity of microbial populations within an
target pathogen due to binding of the AMP to these host cells, infection site, a range of cell morphologies and environmental
even if selectivity is lower than for the pathogen (Starr et al., conditions will exist and these will have an impact on
2016). However, bactericidal concentrations of AMP can be antimicrobial susceptibility. Given this heterogeneity, it is
achieved in the presence of host cells (Savini et al., 2017) and relevant to test antimicrobial efficacy, including that of AMP,
this should also be possible clinically. Thus, when conducting vs. microbes existing in different growth states (Radlinski
AST of AMP the size microbial inoculum (cfu/ml) must be and Conlon, 2018; Dewachter et al., 2019). Specialised
given due consideration and a balance reached between low cell slow or non-growing forms of microbial pathogens include
density resulting in efficacy that is unachievable in vivo and high biofilms (Desai et al., 2014; Reichhardt et al., 2016; Koo et al.,
cell density that results in under-estimation of activity and the 2017; Wolfmeier et al., 2018; Orazi and O’Toole, 2019), spores
potential loss of promising therapeutic candidates. As a starting (Setlow, 2014; Gil et al., 2017), persister cells (Fisher et al., 2017;
Wuyts et al., 2018; Balaban et al., 2019) and small colony variants (Libardo et al., 2017); (3) inhibition of the alarmone system
(SCV) (Proctor et al., 2006; Johns et al., 2015). In some cases, to avoid the bacterial stringent response, e.g., peptide 1018
there are even combinations of these cells, e.g., persister cells (de la Fuente-Nunez et al., 2014); (4) downregulation of genes
and/or SCV within biofilms (Mirani et al., 2015; Waters et al., responsible for biofilm formation, e.g., hBD-3 (Zhu et al.,
2016; Wuyts et al., 2018; Yan and Bassler, 2019). A common 2013); and (5) immunomodulatory properties may also confer
feature of microbial growth in these forms is normally reduced additional in vivo anti-biofilm properties. Interestingly, some
susceptibility to antibiotics and antifungals. The efficacy of AMP are able to prevent biofilm formation at concentrations
AMP has been tested against cells demonstrating differing significantly below their MIC, or have anti-biofilm properties,
growth modes. but without antibacterial activity (for examples see (Pletzer
At the simplest level, a microbial biofilm is a surface- and Hancock, 2016; Mercer et al., 2017; Haney et al., 2018a;
associated community of microorganisms surrounded by an Shahrour et al., 2019), indicating a separate mechanism of
extracellular polymeric matrix. It is estimated that more than action from membrane disruption/direct killing. The membrane
80% of microbial infections are caused by microbes growing disruption properties of AMP have been the main focus of this
as biofilms (Romling and Balsalobre, 2012), and therefore their manuscript and will remain so for the discussion of AST of AMP
determination of their susceptibility to antimicrobial agents, and biofilms.
including AMP, is of paramount importance when developing Examples of AMP that can kill biofilm microbes by
novel antimicrobial agents. An increasing understanding of membrane permeabilization include the eosinophil cationic
biofilm infections has led to the appreciation that many infections protein-derived AMP, RN3(5-17P22-36), that demonstrated
are polymicrobial in nature and may contain diverse species membrane-permeabilising activity (Sytox green membrane
of bacteria, fungi and viruses (Peters et al., 2012; Wolcott permeabilization) against established biofilms of P. aeruginosa,
et al., 2013; Mihai et al., 2015; Todd and Peters, 2019) and albeit at 2–8-fold higher concentrations than required for
a number of models of polymicrobial biofilms have recently the same activity vs. planktonic P. aeruginosa (Pulido et al.,
been developed (Gabrilska and Rumbaugh, 2015). Polymicrobial 2016). The esculentin-1a-derived AMP, Esc(1-21), also caused
biofilms are of relevance in many infections, including chronic permeabilization of the membranes of planktonic cells and
wound infections (Clinton and Carter, 2015), CF lung infections biofilms of P. aeruginosa PAO1 (Sytox green membrane
(Lopes et al., 2015), bacterial vaginosis (Jung et al., 2017) permeabilization and β-galactosidase release) (Luca et al., 2013),
and medical device-associated infections (Wi and Patel, 2018). and LL-37 and selected truncated versions (LL-31, LL7-37,
Microbes growing in biofilms can be up to 1000-fold more LL13-37, and LL7-31), albeit at relatively high concentrations
tolerant to antimicrobials than their planktonically growing (20–100 µM) (measured by propidium iodide (PI) uptake)
counterparts (Costerton et al., 1995; Hoiby et al., 2010), so against pre-grown P. aeruginosa PAO1 biofilms (Nagant et al.,
more effective therapeutic options are urgently required. A 2012). The lactoferricin-derived AMP, LF11-215, LF11-324 and
number of mechanisms are responsible for biofilm antibiotic- a lipopeptide derivative, DI-MB-LF11-324, caused membrane
tolerance including; (1) reduced diffusion of antibiotics through permeabilization of P. aeruginosa PAO1 in biofilms (PI uptake)
the biofilm matrix, (2) sequestration of antibiotics by the biofilm at 10 × MIC concentrations (Sanchez-Gomez et al., 2015). The
matrix; (3) presence of slow-growing and persister cells (see related AMP Seg6L and Seg6D both demonstrated antibiofilm
below) refractory to antibiotics targeting bacterial metabolism; properties with Seg6D predominantly causing cell lysis, whereas
and (4) increased exchange of antibiotic resistance genes on Seg6L degraded the biofilm by detaching live cells, rather than
mobile genetic elements by cells in close proximity (Stewart direct killing, demonstrating that relatively minor changes to
and Costerton, 2001; Hall and Mah, 2017; Orazi and O’Toole, AMP composition (substitution of 5 Seg6L amino acids for
2019). The potential role of AMP as therapeutics to treat biofilm D-isoform amino acids) can substantially affect antimicrobial
infections has received significantly more attention than activity properties (Segev-Zarko et al., 2015). Some AMP are also
vs. spores, persister cells or SCV and has been the subject membrane-active against fungal biofilms (van Dijck et al.,
of several review articles in the last few years (Batoni et al., 2018). Tyrocidines are cationic cyclodecapeptides with broad-
2016; de la Fuente-Nunez et al., 2016; Pletzer and Hancock, spectrum antimicrobial activity and the tyrocidines (TrcA, TrcB,
2016; Delattin et al., 2017; Grassi et al., 2017b; van Dijck and TrcC) were able to disrupt the membrane of Candida
et al., 2018; Von Borowski et al., 2018; Yasir et al., 2018; albicans growing as biofilms (PI uptake) at concentrations similar
Shahrour et al., 2019). A detailed analysis of the potential role to the planktonic MIC (∼12.5 µM), but significantly higher
of AMP in the treatment of biofilm infections is beyond the concentrations were required to eradicate biofilms (Troskie et al.,
scope of this manuscript, but some key points with respect to 2014). Similarly, the peptidomimetic mPE was able to disrupt the
AST will be addressed in the following paragraphs. Membrane membrane of Candida albicans growing as biofilms (PI uptake)
disruption is the most common mechanism of action of cationic at concentrations 6–12-fold higher (50–100 mg/L) than the MIC
AMP and remains important in some instances for anti-biofilm vs. planktonic cells (Hua et al., 2010). Studies of the activity
properties of AMP, but when assessing anti-biofilm properties, of AMP vs. polymicrobial biofilms are less frequently reported,
other attributes of AMP may be of equal or greater importance but the Herpes simplex-derived AMP gH625-GCGKKKK was
(Yasir et al., 2018), including; (1) blocking of/interference with able to prevent formation of mixed species biofilms consisting
bacterial cell signalling systems, e.g., LL-37 (Overhage et al., of Candida tropicalis and S. aureus or C. tropicalis and Serratia
2008); (2) degradation of the biofilm matrix, e.g., piscidin-3 marcescens as well as eradicating mixed biofilms containing
these microbes (de Alteriis et al., 2018) as did melittin against routine AST of biofilms (Macia et al., 2014). The most commonly
a polymicrobial biofilm consisting of selected dairy isolates used are the minimal biofilm inhibitory concentration (MBIC),
of bacteria (P. aeruginosa, Staphylococcus haemolyticus, K. defined as the lowest concentration of an antimicrobial at which
pneumoniae, and Aeromonas caviae) (Galdiero et al., 2019). there is no time-dependent increase in the mean number of
The de novo AMP ASP-1 was able to eradicate polymicrobial biofilm viable cells (Moskowitz et al., 2004), or the minimal
biofilms containing S, aureus, P. aeruginosa, A. baumannii, biofilm-eradication concentration (MBEC), defined as the lowest
and K. pneumoniae when formulated into a polyurethane-based concentration of antibiotic required to eradicate the biofilm (Ceri
dressing (Bayramov et al., 2018) and the Komodo dragon- et al., 1999). In the case of membrane-active AMP, evidence
derived peptide DRGN-1 demonstrated moderate eradication of membrane permeabilization must be provided, for example,
activity against mixed biofilms containing P. aeruginosa and S. the use of fluorescence microscopy (standard, confocal laser
aureus (Chung et al., 2017). Luo and co-workers tested a library scanning microscopy or scanning electron microscopy) using
of peptoids for antibiofilm activity and demonstrated that 3 membrane-impermeant fluorophores that allow demonstration
peptoids from the library were able to eradicate mixed species of membrane permeabilization, including PI (Boulos et al., 1999)
biofilms (formed for 8 h) containing C. albicans and S. aureus or or Sytox dyes (Roth et al., 1997) or reporter genes. Recently,
C. albicans and E. coli (Luo et al., 2017). Haney and co-workers proposed two quick, easy, reproducible
In general, concentrations of AMP required to permeabilise and inexpensive methods; one for assessing biofilm inhibition
membranes in biofilms are higher than for their planktonic and the other for assessing biofilm eradication, to determine
counterparts and this may reflect sequestration (for example, by the activity of AMP against biofilms (Haney et al., 2018b). Both
extracellular DNA or other extracellular polymeric substances), methods were based on the use of microtitre plates for detection
hydrolysis of AMP in the biofilm, increased microbial cell and utilised the crystal violet protocol for determining biofilm
density or may reflect changes to individual microorganisms biomass (O’Toole and Kolter, 1998; O’Toole, 2011) and in the
residing within the biofilm, as bacteria growing in biofilms eradication assay they assessed metabolic activity within the
can have altered membrane permeability (Orazi and O’Toole, biofilm using the tetrazolium chloride dye (triphenyl tetrazolium
2019), which may affect the activity of membrane-active chloride) (Brown et al., 2013; Sabaeifard et al., 2014). These
AMP. Membranes of S. aureus, Listeria monocytogenes, P. methods are similar to many reported in the literature and
aeruginosa, Salmonella Typhimurium growing as biofilms appear suitable for simplified analysis of biofilm prevention and
contain significantly higher proportions of saturated fatty eradication and should also be readily adapted for use in the
acids compared to cells growing planktonically. Increases in analysis of fungal biofilms, but do not provide an assessment
saturated fatty acids (in particular long-chain fatty acids are of the effects of AMP on the bacterial membrane, which would
concomitant to decreases in branched-chain fatty acid content, require use of some of the methods outlined above.
which can lead to increased membrane rigidity and stability The efficacy of AMP vs. bacterial and fungal spores has been
(Denich et al., 2003; Dubois-Brissonnet et al., 2016). the subject of a relatively limited number of studies. Bacterial
Approved standards exist for AST of both bacteria and endospores with relevance to human infectious diseases are
fungi (Table 1), but these apply only to microbes growing mainly produced by Clostridiodes spp. (Clostridium spp.) and
planktonically. Approved AST standards for microorganisms Bacillus spp. Bacterial endospores are metabolically dormant
growing as biofilms do not exist and a number of different and environmentally resistant and are capable of surviving
methods are used (Macia et al., 2014; Magana et al., 2018), making antibiotic exposure, extremes of temperature, desiccation and
direct comparisons of different studies difficult to undertake. As ionizing radiation (Higgins and Dworkin, 2012). The cathelicidin
described above, microorganisms growing as biofilms are more family AMP PG-1, BMAP28, and LL-37 demonstrated sporicidal
tolerant of antimicrobials than microbes growing planktonically, activity against B. anthracis spores in the low mg/L range,
so the results of conventional AST cannot be used to accurately whereas SMAP-29, CAP-18 were not effective and NA-CATH
predict the results of biofilm AST (Macia et al., 2014). As an added and mCRAMP demonstrated activity only at high concentrations
level of complexity, the level of biofilm antimicrobial tolerance (1,000 mg/L) (Blower et al., 2018). The AMP chrysophin-3 also
may also be influenced by the methods used to establish and demonstrated sporicidal activity vs. B. anthracis (Pinzon-Arango
monitor the biofilms. et al., 2013) and activity of PG-1 vs. spores of B. anthracis
At present it is not possible to specify an optimal testing had been previously reported (Lisanby et al., 2008). The AMP
procedure for assessing the activity of AMP against biofilms, TC19, TC84, BP2 and the lantibiotic nisin A were tested for
given their heterogenous nature and the complexity underlying efficacy against spores of Bacillus subtilis and were bactericidal
their development (Haney et al., 2018a; Magana et al., 2018). against germinated spores by perturbing the inner membrane,
When reporting activity of AMP vs. biofilms it is vital, initially, thus preventing outgrowth to vegetative cells, but were not able
to specify whether biofilm prevention or eradication is being to prevent germination (Omardien et al., 2018). Presumably, this
described. When describing the anti-biofilm properties of AMP is because the inner membrane of endospores is immobile and
appropriate methodological details must be provided, including becomes fluid only during spore germination (Setlow, 2006).
device used to grow the biofilm and the parameters used to Other than this, the only peptides with activity vs. spores are the
establish anti-biofilm activity. Most commonly, biofilms are lantibiotics nisin (Gut et al., 2008) and subtilin (Liu and Hansen,
grown in multi-well plates or the Calgary device, which are 1993), both of which were only active against germinated spores,
simpler and cheaper than flow-cell devices and better suited to as above. Assessing the efficacy of AMP vs. bacterial spores for
AST is relatively simple, by substituting a spore suspension for growth, atypical colony morphology, and an ability to persist
the normal inoculum, but in order to fully understand the precise in mammalian cells. SCV are characterized by down-regulation
effect of AMP on spores requires more specialised techniques, of genes for metabolism and virulence, while genes important
such as those described by Omardien et al. (2018). As spores are for adhesion, persistence and biofilm formation are often
not normally the cause of the disease, rather this is vegetatively up-regulated (Proctor et al., 2006; Johns et al., 2015; Kahl
growing cells, the importance of AMP activity against spores is of et al., 2016). SCV are frequently deficient in electron
more relevance to disease prevention, rather than cure. transport (menadione and haemin auxotrophs) or thiamine
Activity specifically against fungal spores (conidia) has biosynthesis (thymidine auxotrophs) and these phenotypes
received limited attention compared to activity against can be reversed by supplementation with menadione,
vegetatively growing fungi or yeast. Fungal spores are often haemin or thymidine, respectively (Proctor et al., 2006).
the infectious propagule that initiates disease, e.g., inhalation Additionally, SCV are less susceptible to various antibiotics
of Aspergillus spp. spores into the lung, but the disease is including aminoglycosides, such as tobramycin, trimethoprim-
caused once the spores germinate and the fungi begin vegetative sulfamethoxazole, fluorquinolones, fusidic acid, and even to
growth (Kosmidis and Denning, 2015). As with efficacy vs. antiseptics like triclosan (Kahl, 2014; Evans, 2015; Bui et al.,
bacterial endospores, sporicidal activity is of more relevance 2017). SCV are relatively commonly isolated from infections and
to disease prevention than cure. Nevertheless, activity against are also responsible for latent or recurrent infections, often once
vegetatively growing cells and spores is a desirable property antibiotic selection is removed, and are often present in chronic
for any antifungal AMP. The thaumatin-like protein osmotin infections (Proctor et al., 2006; Johns et al., 2015). In a number of
demonstrated antifungal activity against fungal hyphae of cases, intracellular SCV have been isolated from sites of infection,
predominantly plant-pathogenic fungi and caused lysis of including fibroblasts, osteoblasts, macrophages and endothelial
spores of Fusarium moniliforme, F. oxysporum, Trichoderma cells (Kahl et al., 2016) and intracellular presence can induce SCV
longibrachatum, and Verticillium dahlaie (Abad et al., 1996) formation (Vesga et al., 1996). SCV normally constitute a minor
as did the synthetic undecapeptides Pep3, BP15, BP20, BP33, sub-population of cells at the site of infection and therefore their
and BP76 vs. F. oxysporum conidia (Badosa et al., 2009). isolation in sufficient numbers for the purposes of AST can be
The antifungal peptide drosomycin, originally isolated from challenging (Johns et al., 2015; Kahl et al., 2016). SCV have been
Drosophila spp., inhibited fungal spore germination (Zhang identified in diverse range of bacterial genera and isolated from
and Zhu, 2009) as did bombinins H2 and H4 which were active clinical specimens, including S. aureus, S. epidermidis, S. capitis,
against spores of Phytophthora nicotianae (Matejuk et al., 2010). P. aeruginosa, Salmonella serovars, Burkholderia spp., Vibrio
Peptide KK14 and derivatives inhibited spore germination of cholerae, Shigella spp., Brucella melitensis, E. coli, Lactobacillus
F. culmorum, Penicillium expansum, and A. niger, albeit with acidophilus, Serratia marcescens, and Neisseria gonorrhoeae
reduced activity vs. germinated conidia (Thery et al., 2019) as (Proctor et al., 2006; Johns et al., 2015; Kahl et al., 2016). At
did the AMP O3 TR and derivatives (Thery et al., 2018). Rabbit present, there are no approved methods for AST of SCV, and
neutrophil cationic peptides, however, were not active against S. aureus SCV do not grow well in CA-MH broth, making it
ungerminated spores of Aspergillus fumigatus or Rhizopus difficult to generate meaningful results from broth-dilution
oryzae (Levitz et al., 1986). When carrying out broth dilution AST (Precit et al., 2016). Precit and co-workers developed
AST using filamentous fungi, the inoculum used is a spore a disk diffusion method suitable for AST of S. aureus SCV
(conidial) suspension, but no attempt is made to determine (Precit et al., 2016), but this method is unlikely to work well
whether the antifungal used prevents spore germination or is with AMP due to the know interactions of cationic AMP with
solely active against germinated spores/hyphae. The measure the negatively charged sulphate and sugar components of the
of inhibition is optical density or a visual (i.e., not using agaropectin in agar (Lehrer et al., 1991). Another difficulty
microscopy) determination of growth/no growth (CLSI, 2017c; when working with SCV is their reversion to a normal colony
EUCAST, 2017b), except in the case of echinocandins using phenotype (Proctor et al., 2006; Johns et al., 2015; Kahl et al.,
EUCAST methodology in which the Minimum Effective 2016). Other studies have described AST of SCV (see Table S1
Concentration (MEC) endpoint is determined (EUCAST, in Precit et al., 2016 for a complete list), including direct use of
2017b). Thus, it is clear that echinocandins do not prevent the CLSI broth dilution procedure (Gao et al., 2010; Singh et al.,
spore germination (i.e., not sporicidal), but this cannot be 2010) and modifications of this, including prolonged incubation
determined for other antifungals, including AMP, without times (von Eiff et al., 1997; Samuelsen et al., 2005; Mercer
additional analyses, such as microscopic analysis over time et al., 2017) and media changes (e.g., Brain Heart Infusion
(Abad et al., 1996). Alternatively, spores could be exposed to (BHI) Kahl et al., 1998; Yagci et al., 2013). Naturally, AST of
AMP for only limited time periods in which spore germination intracellular SCV adds a further level of complexity and the
could not have taken place (i.e., killing activity can have authors are not aware of any studies on the activity of AMP
affected only spores) followed by plating on non-selective against intracellular SCV. For experimental purposes, SCV of S.
media to determination sporicidal activity (Badosa et al., aureus (with the characteristics of clinical SCV) can be selected
2009). for in vitro by incubation in the presence of the P. aeruginosa
Small colony variants (SCV) constitute a slow-growing quorum sensing molecule 4-hydroxy-2-heptylquinoline-N-oxide
sub-population of bacteria that are characterized by impaired (HQNO) (Hoffman et al., 2006), although the authors are not
aware whether this works with other staphylococci or other wild-type S. aureus (Mercer et al., 2017). In another study,
bacterial genera. It was reported that SCV of E. coli could be S. aureus SCV were less susceptible to the AMP protamine,
generated following exposure to 2-methyl-1,4-napthoquinone but equally susceptible to magainin and HNP-1, whereas SCV
(Colwell, 1946) or copper ions (Weed and Longfellow, 1954) were more susceptible to dermaseptin than the wild-type S.
and that mutants in a number of bacterial genes can generate aureus. Interestingly, exposure to sub-MIC concentrations of
SCV (Santos and Hirshfield, 2016; Tikhomirova et al., 2018; protamine selected for SCV in vitro (Sadowska et al., 2002).
Al Ahmar et al., 2019; Vidovic et al., 2019). The importance of Zhang and co-workers reported that wild-type and SCV of S.
SCV in the context of infection means that AMP activity vs. aureus were equally susceptible to LL-37, but that SCV became
SCV could represent an important method for the treatment less susceptible in the presence of bicarbonate, adding a further
of chronic, recurrent and antibiotic resistant infections, but has level of complexity of AST of SCV (Zhang et al., 2018). The
received only limited attention in the context of AMP. Glaser above results appear to indicate that susceptibility of SCV
and co-workers demonstrated that SCV of S. aureus were less of S. aureus to AMP needs to be determined on a case-by-
susceptible to the skin-derived AMP RNase7, hBD-2, hBD-3, case basis and potentially that he method of AST adopted is
and LL-37 than wild-type S. aureus as was a hemB mutant when also important.
compared to the complemented mutant, with the exception Persister cells are dormant or slow-growing variants of normal
of LL-37. SCV were also less susceptible to the killing activity wild-type cells, forming a sub-population that are highly tolerant
of human stratum corneum (Glaser et al., 2014). Similarly, to antimicrobials (despite no genetic basis for resistance), but
SCV of P. aeruginosa were also less susceptible to LL-37 than that can revert back to wild-type growth and sensitivity. Persister
the wild-type (Pestrak et al., 2018). S. aureus SCV were less cells can be responsible for recalcitrance of infections and
susceptible to the AMP lactoferricin B and thrombin-induced relapse following treatment and the emergence of antibiotic
microbicidal protein (tPMP) when compared to the wild-type resistance (Fisher et al., 2017; Balaban et al., 2019). Most work
and in the case of lactoferricin B, this was irrespective of the to-date has been carried out on bacterial persister cells, however,
underlying auxotrophy (Koo et al., 1996; Samuelsen et al., 2005). fungal persister cells have been isolated from biofilms and
Conversely, an antimicrobial polypeptide (NP108) retained that share many of the characteristics of bacterial persisters,
comparable efficacy vs. S. aureus SCV when compared to including tolerance to high doses of antifungals (Bojsen et al.,
TABLE 4 | Clinically approved peptide antimicrobials.
Antimicrobial Drug name Company Class Source Mol Wt Application MOA Dosing References
Colistin Coly-Mycin Generic Lipopeptide Bacillus colistinus 1155.4 Antibacterial; Membrane IV, IM and Karaiskos et al.,
(Polymyxin E) Gram - disruption Inhalation 2017
Dalbavancina Dalvance Durata Lipoglycopeptide Semi-synthetic 1816.7 Antibacterial; Cell wall IV Bassetti et al.,
Therapeutics Gram + biosynthesis 2018
Daptomycin Cubicin Merck Lipopeptide Streptomyces roseosporus 1620.6 Antibacterial; Membrane IV Gonzalez-Ruiz
(Cubist) Gram + disruption et al., 2016
Gramicidin D NA Generic Linear peptides Bacillus brevis 1882.2 Antibacterial; Membrane Topical Burkhart et al.,
Gram + disruption 1999
Gramicidin S NA Generic Cyclic peptide Bacillus brevis 1141.4 Antibacterial; Membrane Topical Mogi and Kita,
Gram + and - disruption 2009
Oritavancinb Orbactiv Melinta Lipoglycopeptide Semi-synthetic 1793.1 Antibacterial; Cell wall IV Saravolatz and
Therapeutics Gram + biosynthesis Stein, 2015
Polymyxin B NA Generic Lipopeptide Bacillus polymyxa 1203.5 Antibacterial; Membrane IV and IM Rigatto et al., 2019
Gram - disruption
Teicoplanin Targocid NPS Pharma Glycopeptide Actinoplanes teichomyceticus 1879.6 Antibacterial; Cell wall IV and IM Campoli-Richards
Gram + biosynthesis et al., 1990
Telavancinb Vibativ Theravance Lipoglycopeptide Semi-synthetic 1755.7 Antibacterial; Cell wall IV Higgins et al.,
Gram + biosynthesis 2005
Vancomycin NA Generic Glycopeptide Streptomyces orientalis 1449.3 Antibacterial; Cell wall Oral and IV Alvarez et al., 2016
Gram + biosynthesis
Anidulafungin Eraxis Pfizer Echinocandin Semi-synthetic 1140.2 Antifungal Cell wall IV Mayr et al., 2011
(lipopeptide) biosynthesis
Caspofungin Cancidas Merck Echinocandin Semi-synthetic 1093.3 Antifungal Cell wall IV Song and
(lipopeptide) biosynthesis Stevens, 2016
Micafungin Mycamine Astellas Echinocandin Semi-synthetic 1270.3 Antifungal Cell wall IV Scott, 2012
(lipopeptide) biosynthesis
IV, Intravenous; IM, Intramuscular; NA, Not applicable; a Dalbavancin is a semi-synthetic derivative of Teicoplanin; b Oritavancin and Telavancin are semi-synthetic derivatives
of Vancomycin.
2017; Wuyts et al., 2018). Given the importance of persister Interactions of AMP With
cells in infection, it is important to develop antimicrobial Antibiotics/Antifungals
agents capable of eradicating persister cells and the efficacy A large number of publications have described in vitro synergy
of AMP against bacterial persister cells has been examined in of selected AMP with other AMP (Zerweck et al., 2017; Hanson
a limited number of studies. The dendrimeric AMP, 2D-24, et al., 2019), antibiotics (for example Cassone and Otvos, 2010;
was active against persister cells of P. aeruginosa PAO1 and Sakoulas et al., 2014; Soren et al., 2015; Pollini et al., 2017;
a mucoid mutant PDO300, as well as planktonic and biofilm Pizzolato-Cezar et al., 2019) or antifungals (Duggineni et al.,
cells (Bahar et al., 2015). Trp/Arg-containing AMP successfully 2007; Singh et al., 2017), although this is not always the case
killed planktonic and biofilm persister cells of E. coli (Chen (He et al., 2015) and is something that is no doubt under-
et al., 2011), and the aryl-alkyl-lysine NCK-10 against persister reported. Conjugates of AMP and antibiotics, organometallic
cells, planktonic cells and biofilms of S. aureus (Ghosh et al., compounds, gold nanoparticles or to create AMP polymers
2015) and the LL-37 derivative SAAP-148 (de Breij et al., 2018). to increase efficacy, to reduce toxicity and/or to improve
Additionally, the temporin analogue, TB_L1FK, the β-defensin formulation have become of increasing interest over recent years
derivative, C5, and the dendrimeric AMP, Den-SB056, were (for examples, see Reinhardt and Neundorf, 2016; Rajchakit and
equally effective against in vitro generated persister and wild- Sarojini, 2017; David et al., 2018; Sun et al., 2018) and will
type cells of P. aeruginosa and S. aureus (Grassi et al., 2017b). undoubtedly require additional consideration when it comes to
Persister cells of some bacterial species can be generated in the AST. In vitro synergy is often species- or even strain-specific
laboratory by antibiotic exposure (Dorr et al., 2009; Sulaiman and is often specific to certain antimicrobials, so there are
et al., 2018) or treatment with specific chemicals, including no general rules with respect to predicting synergy. Whilst
E. coli, P. aeruginosa, and S. aureus using the uncoupling in vitro synergy between antibiotics or antifungals demonstrates
agent carbonyl cyanide m-chlorophenyl hydrazone (CCCP) potential, there is limited evidence that synergy translates into
(Kwan et al., 2013; Grassi et al., 2017a) and E. coli using the clinic, beyond β-lactam/β-lactamase combinations such as
salicylate (Wang et al., 2017), facilitating AST of persisters piperacillin/tazobactam and ceftazidime/avibactam, so perhaps
with AMP. it might be set to temper this enthusiasm until detailed
TABLE 5 | AMP in clinical development (adapted from Koo and Seo, 2019).
AMP Source Targetc Phase Company Administrationd
EA-230 hCG derivative Sepsis and renal failure protection II Exponential biotherapies Iv
CZEN-002 α-MSH derivative Anti-fungal IIb Zengen Top
D2A21 Synthetic Burn wound infections III Demegen Top
XMP-629 BPI derivative Impetigo and acne rosacea IIIb Xoma Ltd. Top
Neuprex(rBPI21) BPR derivative Peadiatric meningococcemia IIIb Xoma Ltd. iv
Delmitide(RDP58) HLA class I derivative Inflammatory bowel disease IIa Genzyme Top
Ghrelin Endogenous HDP Chronic respiratory failure IIa University of Miyazaki; Papworth Hospital iv
hLF1-11 Lactoferricin derivative MRSA, K. pneumoniae, L. monocytogenes I/II AM-Pharma iv
C16G2 Synthetic Tooth decay by Streptococcus mutans II C3 Jian Inc. Mouthwash
SGX942(Dusquetide) Synthetic Oral mucositis III Soligenix Oral rinse
DPK-060 Kininogen derivative Acute external otitis II ProMore pharma Ear drops
PXL01 Lactoferrin analogue Postsurgical adhesions III ProMore pharma Top
PAC113 Histatin 5 analogue Oral candidiasis IIa Pacgen biopharmaceuticals Mouth rinse
POL7080 Protegrin analogue P. aeruginosa, K. pneumoniae III Polyphor Ltd. iv
LTX-109 (Lytixar) Synthetic G+, MRSA skin infection; impetigo IIa Lytix biopharma Top
OP-145 LL-37 derivative Chronic middle ear infection IIa Dr. Reddy’s research Ear drops
LL-37 Human cathelicidin Leg ulcer IIb ProMore pharma Top
Novexatin (NP213) Cyclic cationic peptide Fungal nail infection IIa NovaBiotics Ltd. Top
p2TA (AB103) Synthetic Necrotizing soft tissue infections III Atox Bio Ltd. iv
Iseganan (IB-367) Protegrin analogue Pneumonia, stomatitis IIIb IntraBiotics pharmaceuticals Top
Pexiganan (MSI-78) Magainin analogue Diabetic foot ulcers IIIb Dipexium pharmaceuticals Top
Omiganan (CLS001) Indolicidin derivative Rosacea III Cutanea life sciences Top
a Clinical
trial completed.
b Clinical
trial discontinued.
c Target microorgansim: G+ - Gram positive; MRSA – methicillin-resistant S. aureus.
d Route of administration: top - topical; iv – intravenous.
standardisation of testing methods and clinical trials have chemistries and new functional families. AMP are clearly a group
been carried out (Doern, 2014). When conducting AST with of molecules with significant potential as a new class of therapy
combinations of AMP with conventional antimicrobials care to address the urgent need for better and AMR-mitigating
needs to be taken analysing antimicrobials where solvent other antibacterial and antifungal therapies. Among other advantages
than water are used (e.g., DMSO), or where conventional media highlighted in this and other reviews, AMP offer great versatility
requires supplements, including blood, NaCl and polysorbate in terms of chemical functionality. Unfortunately, “standard”
(see above for the potential effects of these compounds on AST protocols can significantly underestimate the efficacy of
AMP), as they may impact upon AMP activity. Dimethyl AMP and improved, more predictive methods are required to
sulfoxide (DMSO) can function as a membrane permeabiliser identify leads and facilitate faster progress of AMP into pre-
and can depolarize membranes (Yu and Quinn, 1998) and /clinical development. These changes to “standard” protocols
clearly, this may enhance the apparent activity of membrane will require rigorous and detailed justification and a degree of
permeabilising AMP. validation. New AST of AMP will have to take into account that
many AMP depend on their positive charge for activity as well
AMP IN CLINICAL DEVELOPMENT as the many other factors we have enumerated in this review
(see Table 2). Despite the manifold factors that influence AMP
A limited number of antibiotics comprising a peptide element activity, we are confident that research toward developing more
are in clinical use (Table 4), but as yet, no AMP have been appropriate AST for AMP is headed in the right direction. New
approved as therapies although a number are in clinical trials protocols will, in the near future, accelerate the discovery process
(Table 5). Koo and Seo, in a comprehensive review of AMP drug of novel AMP therapeutics.
development, state that approximately half of AMP currently in
development are at the preclinical stage, while a third have moved AUTHOR CONTRIBUTIONS
forward to clinical trials, most of those are currently in phase II.
Approximately 15% of the AMP failed during one of these stages DM, SD, MT, EL, LS, MK-B, CF-N, DO’N, and AA-B contributed
(Koo and Seo, 2019). These numbers are much more promising to the writing of this manuscript. DM, MT, CF-N, DO’N, and
than the ones presented by Lau and Dunn for previous years, AA-B contributed to the editing of this manuscript. All authors
where the number of discontinued AMP trials was higher than contributed to the article and approved the submitted version.
50% (Lau and Dunn, 2018). To increase the clinical pipeline of
AMP drug candidates and more importantly, approval of these FUNDING
potentially AMR game-changing antimicrobials, better and more
appropriate AST (and in vivo models, although this not a topic This work was partially supported by the National Science
for this manuscript) is critical. Foundation (MCB1715494 to AA-B). Cesar de la Fuente-
Nunez holds a Presidential Professorship at the University of
CONCLUSIONS Pennsylvania, is a recipient of the Langer Prize by the AIChE
Foundation, and acknowledges funding from the Institute for
Antimicrobial resistance is a global health problem that will Diabetes, Obesity, and Metabolism and the Penn Mental Health
require the survey of a large number of molecules with a variety of AIDS Research Center of the University of Pennsylvania.
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published: 07 July 2020

Antimicrobial Susceptibility Testing

During the development of antimicrobial peptides (AMP) as potential therapeutics,

Citation: ANTIMICROBIAL RESISTANCE

Organisation Antimicrobial susceptibility testing document Interpretive criteria document References

perform well using “standard” test methods? AST of AMP

2014; Libardo and Angeles-Boza, 2014). The Zn2+ ions are

TABLE 4 | Clinically approved peptide antimicrobials.

AMP Source Targetc Phase Company Administrationd

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