Biochimica et Biophysica Acta 1858 (2016) 926–935

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Biochimica et Biophysica Acta

journal homepage: www.elsevier.com/locate/bbamem

Using adjuvants and environmental factors to modulate the activity of

antimicrobial peptides☆
William F. Walkenhorst
Loyola University New Orleans, Department of Chemistry and Biochemistry, 6363 St. Charles Avenue, New Orleans, LA 70118, United States

a r t i c l e i n f o a b s t r a c t

Article history: The increase in antibiotic resistant and multi-drug resistant bacterial infections has serious implications for the
Received 16 October 2015 future of health care. The difficulty in finding both new microbial targets and new drugs against existing targets
Received in revised form 22 December 2015 adds to the concern. The use of combination and adjuvant therapies are potential strategies to counter this threat.
Accepted 29 December 2015
Antimicrobial peptides (AMPs) are a promising class of antibiotics (ABs), particularly for topical and surface ap-
Available online 2 January 2016
plications. Efforts have been directed toward a number of strategies, including the use of conventional ABs com-
Keywords:
bined with AMPs, and the use of potentiating agents to increase the performance of AMPs. This review focuses on
Antimicrobial peptides combination strategies such as adjuvants and the manipulation of environmental variables to improve the effica-
Environmental factors cy of AMPs as potential therapeutic agents. This article is part of a Special Issue entitled: Antimicrobial peptides
Adjuvants edited by Karl Lohner and Kai Hilpert.
Antibiotic targets © 2015 Elsevier B.V. All rights reserved.
Resistance mechanisms
Synergy

1. Introduction drug classes, oxazolidinones such as linezolid (2000) and lipopeptides

The discovery and subsequent development of antibiotics have
arguably been among the most important medical advances in history
[1–3]. The precarious nature of existence without access to modern
antibiotics is illustrated by 2012 World Health Organization data from
sub-Saharan Africa, where more than 2 million deaths (N20% of total
deaths) were attributed to a variety of infectious diseases, excluding
HIV and malaria, which together accounted for an additional 2 million
deaths [4]. The introduction of sulfa-drugs (sulfonamides) and penicillin
(β-lactams) in the early decades of the 20th century led to the golden
age of antibiotics in the 1950s and 1960s during which time many dif-
ferent classes of antibiotics with a variety of different microbial targets
were developed [5,6].
Successful microbial targets (Fig. 1) have included a variety of enzy-
matic steps in the synthesis of peptidoglycan (PepG) of the bacterial cell
wall, different sites involved in ribosomal protein synthesis, enzymes of
folic acid metabolism, DNA synthesis (DNA gyrase), RNA synthesis (RNA
polymerase) and the microbial plasma membrane [2,3,7,8]. The pattern
of antibiotic development over the last 50 years, however, is a cause for
concern. Most of the new ABs introduced since 1962 have simply been
modifications of existing ABs resulting in 2nd and 3rd generation
drugs against the same targets [2,3,5,7,9]. Even the two most recent
☆ This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl
Lohner and Kai Hilpert.
E-mail address: walken@loyno.edu.
such as daptomycin (2003), which inhibit new bacterial targets, were
actually discovered nearly 20 years earlier [2]. Furthermore, there has
been a dearth of new broad spectrum ABs over the last 40 years, with
a particular need for new methods for treating Gram negative infections
as most of the ABs marketed since 2000 have improved activity against
only Gram positive organisms [6,7,9]. The development of new ABs has
also been severely hindered by an increasingly stringent regulatory en-
vironment including the possibly shortsighted requirement that new
drugs be as good or better (FDA noninferiority) as existing treatments
rather than simply effective, although recent changes such as the
REMS program and 21st Century Cures Act have attempted to address
some of these issues [2,7,10,11]. Given that treatments for cancer, mi-
crobial infections, and HIV, for example, all have problems with multi-
drug resistance, it seems unwise to limit the number of available drug
choices. Fewer options may exacerbate the difficulties in treating resis-
tant organisms and may also limit competition and lead to higher drug
prices. Another important consideration is that, by their very nature, AB
treatments for episodic infections suffer in competition with the more
lucrative development of drugs for chronic conditions such as diabetes,
arthritis, and heart disease [1,6,7,10,12].
In order to increase the number and diversity of antimicrobial
targets, a variety of new types of screens, including the use of modern
genomic methods have been employed, but with mixed success [6,10,
13–15]. Although a number of potential new targets have been identi-
fied such as peptide deformylase and enzymes of the Type II fatty acid
synthesis pathway, most new leads continue to be natural products

http://dx.doi.org/10.1016/j.bbamem.2015.12.034
0005-2736/© 2015 Elsevier B.V. All rights reserved.
 W.F. Walkenhorst / Biochimica et Biophysica Acta 1858 (2016) 926–935
Fig. 1. Targets of antibiotics. Ten different categories of antibiotic targets are depicted. The most effective antibiotics target cell wall synthesis, DNA gyrase, ribosomal protein synthesis (30S
and 50S subunits), and folic acid metabolism.
The figure is reproduced, with permission, from Ref. [158].
with the recent discovery of teixobactin the result of a new method de-
signed to allow testing of uncultivated soil bacteria to identify potential
new antibiotics [13,16–18].
The paucity of new antimicrobial targets is problematic due to the
increasingly formidable problem of microbial resistance to ABs. In re-
cent years, an increase in the incidence of antibiotic resistant and multi-
drug resistant infections has threatened to reverse many of the gains of
the antibiotic era. In 2013, the Centers for Disease Control (CDC) report-
ed that more than 2 million infections and 23,000 deaths are attributed
to antibiotic resistant organisms annually in the USA [7,19]. Microbial
resistance is not a new occurrence, however. Resistance has been
observed for nearly every antibiotic, often occurring fairly quickly after
introduction of the antibiotic. For example, resistance against penicillin
[8], sulfonamide, streptomycin [3], and more recently daptomycin [9]
were all observed shortly after their introduction and in the case of pen-
icillin, before its widespread therapeutic use [17]. In retrospect, this is
not surprising as many ABs are naturally occurring molecules produced
by soil microbes in an ancient competition between ABs and resistance
mechanisms that has been ongoing for billions of years [8,12,20,21].
The naturally occurring AMPs present an interesting case. AMPs
have evolved in species as diverse as bacteria to plants to humans and
yet remain effective against most microbes [22]. AMPs are part of the in-
nate immune system in both vertebrates and invertebrates and act
through a binding event in which the positively charged peptides inter-
act with the anionic plasma membranes of susceptible microorganisms
[23,24]. Although the synthesis of the cell wall components of peptido-
glycan has been a common target for many ABs, the microbial plasma
membrane and other cell surface targets such as the lipopolysaccharide
outer membrane (LPS) and lipid A components of Gram negative
organisms, and the teichoic acids (TA) of Gram positive organisms
have been underexploited targets for commercial ABs [13,25]. Although
AMPs primarily target the plasma membrane [24,26, see also ***this
issue], it has been observed [27], that pH dependent interactions with
other charged molecules of the microbial cell wall or lipopolysaccharide
layers [26,28,29] are also important. Following binding of the plasma
membrane, the amphipathic AMPs then interact with the nonpolar
membrane lipids [30] and cause cell death due to membrane disruption,
although other mechanisms have also been implicated [31].
927
AMPs have several important advantages in addition to their low
levels of natural resistance. AMPs sterilize rapidly at micromolar con-
centration, are able to target quiescent cells, and often have very
broad spectrum activity. Disadvantages of AMPs include all of those
common to peptide drugs: (a) they are subject to proteolysis in the di-
gestive tract, thus limiting oral administration, (b) peptide drugs tend to
be expensive, and (c) intravenous and subcutaneous injections are also
limited due to the possibility of both proteolysis and immunogenicity
[22]. A significant disadvantage, unique to AMPs, is that in addition to
targeting microbial membranes, AMPs also target red blood cell and
other host membranes, although typically at higher concentrations.
The resulting cytolysis is an impediment to the development of AMPs
as systemic drugs and efforts are underway by a number of groups to in-
crease the therapeutic index (cell selectivity) for this class of drugs
[32–36].
Though limited in common routes of delivery, AMPs are well suited
for topical and other surface applications [37–39]. In fact, most clinical
trials to date involve the treatment of diabetic ulcers and other skin in-
fections such as thrush, as well as infections related to cystic fibrosis
[40], while additional applications such as in treatments for eye infec-
tions and as additives to cosmetics are under investigation [22,39,41].
A potential advantage of topical applications highlighted in this review
is that one can adjust environmental factors such as pH, ionic strength,
and the concentration of specific ions, and make use of adjuvants to po-
tentiate the activity of the AMPs. Thus, a comprehensive understanding
of the effect of environmental factors and adjuvants is needed to further
the development and application of AMPs as successful drug therapies.
2. Resistance mechanisms
Multidrug resistant bacteria are widespread and are encountered in-
creasingly often in infections [2,3,5]. Bacteria have developed a number
of common methods of acquiring resistance (Fig. 2). Among these are:
(a) enzymatic modification of ABs to inactivate them, (b) mutation of
AB target to prevent binding of ABs, (c) overexpression of target mole-
cules, (d) use of an alternate pathway to bypass the action of the AB,
(e) efflux pumps in the plasma membrane to prevent buildup of ABs
within cell, (f) mechanisms to decrease permeability or entry of ABs,
928 W.F. Walkenhorst / Biochimica et Biophysica Acta 1858 (2016) 926–935
Fig. 2. Mechanisms of antibiotic resistance. The major mechanisms used by bacteria to resist antibiotics (blue spheres) are shown. These include target modification, alternate (bypass)
pathways, enzymatic modification of the drug, overexpression of the target molecule, sequestration by specific binding proteins (decoys) and mechanisms to prevent drug penetration
or increase efflux from the cell.
The figure is reproduced, with permission, from Ref. [42].
and (g) production of AB binding proteins which may include alternate
target molecules which act as decoys [42]. An additional mechanism in-
volves switching to persistent, non-metabolically active states such as a
biofilms [3,5,17,43–46]. Alterations of cell wall or membrane charge can
be classified as examples of (b) or (f), depending on the type of AB.
One of the advantages of AMPs is that only limited resistance is
found across a wide range of species of microbe. In addition, unlike
the situation for conventional ABs, resistance to AMPs is not readily in-
ducible for most organisms [47,48]. The lack of resistance to AMPs is
often attributed to: (a) the energetic cost associated with entirely
changing the properties of the cellular membrane, and (b) the lack of
a definite structured (protein) target susceptible to evolution of resis-
tance via genetic mutation [23,31,38].
A number of limited resistance mechanisms have been identified,
however, in both Gram negative and Gram positive strains of common
opportunistic bacteria such as Escherichia coli, Salmonella enterica, Pseu-
domonas aeruginosa, and Staphylococcus aureus. In Gram negative spe-
cies the lipopolysaccharide (LPS) and lipid A components are often
modified to incorporate positively charged amino groups such as
found in aminoarabinose and ethanolamine, or to modify chain length
[49–54]. The human pathogenic O157:H7 strain of E. coli, for example,
contains an LPS layer that has been modified by the addition of free
amino containing groups such as glucosamine and ethanolamine
[55–57].
Many Gram positive organisms contain copious amounts of anionic
wall teichoic acids and lipoteichoic acids (TAs) in their peptidoglycan
(PepG) layer which are similarly modified [58,59]. The TAs contain
ribitol phosphates which give the PepG layer of Gram positive organisms
a net negative charge. In a number of Gram positive species, such as
S. aureus, the ribitol alcohol groups are esterified to D-Ala residues
containing free amino groups [60,61]. Strains of S. aureus containing
reduced levels of D-Ala in their teichoic acids exhibit an increased sensi-
tivity to AMPs as well as a reduced incidence of sepsis [58,62–64]. In ad-
dition, S. aureus can also modify its plasma membrane by incorporation
of L-lysine [65,66]. Exceptionally AMP resistant bacteria are found, how-
ever, in several species of bacteria such as Burkholderia, Serratia, Proteus,
and Providencia [67–69]. The mechanism of resistance to AMPs is not yet
fully understood although members of Burkholderia are known to make
use of aminoarabinose as well as multiple efflux pumps of the resistance-
nodulation-division (RND) family [42,54,70,71].
3. Combination therapy as a strategy to combat resistance
The search for improved compounds and new formulations to com-
bat microbial infections is, out of necessity, a constant process so as to
stay one step ahead of the acquisition of resistance by pathogenic micro-
organisms [37]. The widespread occurrence of resistance to single drugs
has led to the use of combination therapy such as multi-drug cocktails
against a variety of maladies including tuberculosis, HIV, and cancer
[72–75]. Combination therapies often but not always [11,76] improve
clinical outcomes and may lower the chances of developing resistance
although the acquisition of multidrug resistance is an increasing prob-
lem [9,72,74,77–80]. A variety of strategies to increase antibiotic effec-
tiveness through the use of combination therapy have been proposed
and several have been available clinically for a number of years [5,7].
For example, the cotrimoxazole formulations (Septra® and Bactrim®)
have been sold commercially since 1969 to target different steps in
the pathway for synthesis of folic acid while drugs such as Augmentin®
which combines amoxicillin with a β-lactamase inhibitor such as
clavulanic acid have been available since 1981 [12]. The latter drug com-
bination inhibits one of the mechanisms of resistance described in the
previous section (enzymatic degradation of the AB) as opposed to the
former, which simply administers a second AB along with the first.
Other strategies are under development to counter additional mecha-
nisms of resistance such as combining efflux pump inhibitors, or cell
wall or membrane permeabilizing agents in conjunction with an antibi-
otic [7,46]. AMPs fit into this paradigm in several ways. Numerous stud-
ies have probed the ability of AMPs combined with conventional ABs to
 W.F. Walkenhorst / Biochimica et Biophysica Acta 1858 (2016) 926–935
enhance performance, perhaps via increased permeability of the plasma
membrane to a conventional AB, while combining efflux pump inhibi-
tors with AMPs has also been proposed [74]. Various types of combina-
tion therapy involving AMPs will be explored in more detail in the
sections that follow.
4. Effects of ionic strength and specific ions on AMP activity
Perhaps the simplest type of adjuvant therapy consists of simple
modification of the environmental factors in a formulation to facilitate
the action of the antibiotic. AMPs are particularly well suited to this
type of approach since they will most likely be used in topical or other
surface applications such as in ointments, lotions, and eye and mouth
washes that are amenable to variation of environmental conditions. It
is equally important to identify factors that impede AMP performance
as those that enhance activity.
The effects of salt concentration on AMP activity have been the sub-
ject of a number of studies [81,82]. It is well known that high monova-
lent salt concentrations (N 100 mM) interfere with AMP activity
Fig. 3. Ionic strength and specific ion data. In panel A, the minimum sterilizing
concentrations (MSC) of ARVA are plotted versus total ionic strength of KCl solutions in
10 mM phosphate buffer for E. coli (black) and S. aureus (open). In panel B, specific ion
effects are observed for the divalent ions Ca2+ and Mg2+ that differ between the Gram
negative E. coli and P. aeruginosa compared to the Gram positive S. aureus (see legend).
929
(Fig. 3A) due to the electrostatic nature of the initial binding step to mi-
crobial membranes and are likely to be clinically relevant [83–85]. For
example, cystic fibrosis patients often exhibit lung infections by
P. aeruginosa that have been linked to the higher salt concentrations
(≈ 120 mM) found in such patients. As a result, a number of studies
have focused on increasing the salt tolerance of AMPs. Many strategies
have been explored such as increasing cationicity [86], increasing pep-
tide stability [82,87,88], and testing AMPs isolated from marine organ-
isms. The β-defensins and magainins are generally less effective as the
NaCl concentration increases above 100 mM but salt tolerant AMPs
have been identified that are active at NaCl concentrations up to
300 mM [27,82,89] and even as high as 450 mM in AMPs from some ma-
rine organisms [90]. Most investigations of the effect of added salt have
been conducted in media or using radial diffusion assays in agar. Both
media and agar contain a variety of charged molecules including buffer-
ing agents and salts such as NaCl. While the presence of rich media dur-
ing the incubation step may better mimic physiological conditions, the
use of minimal media may be preferred to conduct initial salt and pH
studies as it provides a more controlled background in which to assess
the effect of various environmental factors on AMP activity. The use of
ionic strength (Fig. 3) rather than salt concentration is preferred as
ionic buffer species can often contribute substantially to the ionic
strength, particularly if polyvalent buffers such as phosphate are used
[91].
Divalent ions often interfere with AMP activity at concentrations
many times lower than predicted by the ionic strength equation and
as such fall in the category of specific ion effects (Fig. 3B). Divalent
Ca2+ and Mg2+ ions severely impact the activity of AMPs at low mM
concentrations. The activity of several mammalian AMPs, for example,
was found to be greatly reduced, particularly against Gram negative
organisms [38,82,92]. To bind the plasma membrane, AMPs must first
cross the lipopolysaccharide (LPS) outer membrane of Gram negative
bacteria. The peptides are thought to displace divalent Ca2 + and
Mg2 + ions upon binding LPS and initiate a self-promoted uptake
process [26,28,93–95].
Although the very strong adverse effect of Ca2+ and Mg2+ ions on
activity of AMPs against Gram negative organisms is well documented,
our lab has found similar, though less pronounced, effects for activity of
an AMP against the Gram positive S. aureus (Fig. 3B) which are still
many times larger than the effect expected for a simple ionic strength
effect occurring via charge screening. The differences between the
Gram positive and Gram negative organisms probably reflect the
known differences in Ca2+ and Mg2 + metal binding sites by the LPS
layer of Gram negative organisms compared to the outer PepG layer of
Gram positive organisms [96–99]. Specific ion effects that differ be-
tween E. coli and S. aureus have also been observed for (Na)2SO4, possi-
bly due to interactions between the sulfate oxoanion and the arginine
guanidino groups of AMPs or with charged molecules of the bacterial
cell walls [100–102]. In a similar fashion, the use of the cationic buffer
MOPS has been observed to allow killing by AMPs at lower concentra-
tions than the use of the oxoanionic phosphate buffer at similar pH
and ionic strength (unpublished data).
5. Effects of pH on AMP activity
Another environmental factor to consider is the influence of pH on
AMP activity, which has been studied for several peptides [81,83,89,
103]. Changes in pH can affect ionic interactions between the AMPs
and the plasma membrane directly by changing the protonation states
of functional groups on either the peptide or the bacterial surface, but
can potentially affect activity indirectly due to a change in the ionic
strength of the solution. Depending on the pKas of the functional groups
involved, a change in pH can change the net charge of the AMP or of the
target molecules on the bacterial cell wall or membranes and thus affect
peptide binding. According to the Henderson–Hasselbalch equation,
however, the relative concentrations of the various charged species of
930 W.F. Walkenhorst / Biochimica et Biophysica Acta 1858 (2016) 926–935
the buffer also change as the pH changes. The change in ionic strength
with pH can be large due to the z2 term in the ionic strength equation,
especially for polyvalent ions such as phosphate. Therefore, it is impor-
tant to take ionic strength into account in any pH study [27].
Studies on the effect of pH have commonly been conducted over
narrow pH ranges such as 5.5 to 7.5 [89,103]. The histidine-rich
clavanins, for example, exhibited a large increase in activity as the pH
was lowered from pH 7.4 to 5.5 [89]. The most likely explanation is
that the net positive charge on clavanin increases as the pH is dropped
to below histidine's pKa value near 6, thus increasing the affinity of
the AMP for the microbial membrane. More recently, several studies
have been conducted over a wider range of pH values and salt concen-
trations [81,83,104]. A comprehensive study of the effects of both pH
and ionic strength found some interesting and potentially clinically rel-
evant effects on the activity of the AMP ARVA (a linear 12 amino acid
synthetic peptide) against a panel of four microorganisms as described
below [27]. The minimum sterilizing concentration (MSC) against two
Gram negative species (E. coli, P. aeruginosa) was found to be linear
over the pH range from 4 to 9 with enhanced activity at low pH. The
yeast Candida albicans exhibited a similar behavior. The effect for the
Gram negative species was also found to be linear with peptide net
charge. At pH 4 ARVA has a net charge of + 4, while at pH 9 the net
charge is approximately + 3, resulting in a 4-fold to 6-fold increase in
the concentration of peptide required for sterilization. This is consistent
with other observations that increasing the number or clustering of pos-
itive charges affects the activity of AMPs [67,86,105]. Moieties of the
typical Gram negative LPS and PG layers as well as the Candida cell
wall appear to have few groups with pKas that titrate in the pH 4 to
pH 9 range [62,106,107].
The MSC of ARVA against the Gram positive S. aureus, on the other
hand, while also linear between pH 4 and pH 9, exhibited the opposite
slope and showed enhanced killing at high pH despite the smaller net
charge on ARVA at this pH, and remarkably, at MSC values comparable
to those for the Gram negative organisms at low pH. The pH behavior
for ARVA against S. aureus is attributed to changes in the net charge of
the PG layer due to deprotonation of the amino groups of the D-Ala res-
idues esterified to the TAs of the Gram positive organism described in
the section above on resistance mechanisms. Thus, the cell wall be-
comes much more anionic at high pH.
These results suggest that using environmental factors such as pH
may be an effective strategy to counter common resistance mechanisms
and increase the effectiveness of AMPs [27] and emphasize the impor-
tance of the initial binding step on the activity of AMPs. Investigating
the interactions of AMPs with the outer cell wall layers may be impor-
tant to improve the efficacy of AMPs, rather than focusing only on the
plasma membrane, as ionic interactions with a variety of charged mol-
ecules on microbial membranes and cell walls may play a critical role
in antimicrobial activity. Tailoring the pH in a species or strain depen-
dent fashion may be a strategy to improve AMP performance in topical
applications and may be applicable for pathogenic strains that alter their
cell surface charge.
6. Combination therapy: the search for synergy with AMPs
Combination therapies have several potential advantages so long as
there are no antagonistic interactions between the drugs that limit their
effectiveness [72,75]. If the two drugs show additivity or synergy, addi-
tional advantages may result apart from those related to preventing de-
velopment of resistance [78]. For example, lower doses of both drugs
can be used, which may allow cost savings as well as the possibility of
fewer side effects. If one compound is much more expensive than the
other, as is often the case with peptide drugs, the best combination of
therapeutic effect and cost may be obtained by adjusting the relative ra-
tios of the two compounds.
Various methods have been developed for characterizing additivity
and synergy [72–77,108,109]. The commonly used Loewe definitions
of synergy, additivity, indifference, and antagonism [110,111] are
based on the following relations:
FICA ¼ MSCCa =MSCA and FICB ¼ MSCCb =MSCB
where FICA is the fractional inhibitory concentration for compound A
defined as the ratio of the MSC for compound A when combined with
compound B (MSCCa) divided by the MSC of compound A alone
(MSCA) and similarly for FICB. The value of the overall FIC value used
to determine synergy or additivity is a constant designated FICC and is
calculated simply as FICC = FICA + FICB. Interactions are often described
using the following values of FICC: a value b0.5 indicating synergy, a
value between 0.5 and 2.0 additivity, a value between 2.0 and 4.0 indif-
ference, and a value N 4.0 antagonism [110,111].
A variety of compounds have been used in combination with AMPs
in an effort to find formulations effective against common infectious or-
ganisms. Among the stratagems employed have been the following:
(a) combination of AMPs with other AMPs, (b) combination of AMPs
with conventional ABs [74,112], (c) combination of AMPs with toxic cat-
ions, and (d) combination of AMPs with chelators or toxic anions. For
the purposes of this review, the focus will be mainly on the latter two
categories.
Data to test for synergy can be collected by several methods includ-
ing the checkerboard assay [113], agar diffusion methods, and time-kill
curves [described and compared in [77]]. The checkerboard assay is
most commonly carried out in 96-well plates in which compound 1 is
serially diluted across the rows of the plate while compound 2 is serially
diluted down the columns. Agar diffusion methods use a similar ap-
proach, except that the sizes of inhibition zones against the microbe of
interest are measured as the concentrations of the two compounds are
varied perpendicularly across the agar plate.
The checkerboard assay has been widely used, but has several weak-
nesses including reproducibility and ambiguity in interpreting the data
[74]. Recently, He et al. introduced a new method to detect and analyze
interactions between drugs in combination treatments [112]. Using a
direct parallel synergy (DPS) assay, they tested a panel of four conven-
tional ABs pairwise in combination with four different AMPs. In the
DPS assay, the individual MSC (or MIC) values are measured in separate
columns of a 96-well plate while the MSC values when two compounds
are combined are measured in a third column. The data from the com-
bined and individual columns are then combined in a ratio to determine
FIC values. They found no examples of synergy in any of the sixteen
combinations despite the widespread occurrence of synergy in the liter-
ature in similar studies using the checkerboard assay. The well
established synergistic combination of magainin II with β-lactam ABs,
however, did exhibit synergy in the DPS assay. Thus, use of the DPS
assay may limit the number of false positives in studies of synergy.
7. Combinations of metal ions with AMPs
Metals and toxic ions such as copper, silver, and gold have been used
in medical applications for thousands of years [114–116]. More recently,
they have been employed in new formulations to combat biofilms and
improve wound care [117–119], often in the form of nanoparticle and
surface technologies [120,121]. Gold complexes and gold nanoparticles
have been observed to have antimicrobial activity [122,123]. Similarly,
silver and silver nanoparticles have been widely employed as antimi-
crobial agents [124,125]. Antimicrobial activity by both gold and silver
nanoparticles is believed to be due mostly to release of metal ions, but
the size and chemical composition of the nanoparticles plays a role as
well [126]. The toxicity of metal ions such as gold, silver, and copper is
attributed both to the formation of reactive oxygen species (ROS) inside
the cytoplasm and to reactivity toward cellular thiol species in enzymes
and respiratory proteins [127,128]. Some recent work has suggested
that conventional ABs may also kill by inducing ROS and that strategies
that enhance ROS formation may be a means to find new ABs [129,130],
 W.F. Walkenhorst / Biochimica et Biophysica Acta 1858 (2016) 926–935 931

but other concurrent studies found no evidence for involvement for ROS
in the activity of conventional ABs [131,132]. The involvement of ROS in
antimicrobial activity of metals, however, is well established and the
combination of AMPs with metals or metal nanoparticles may show
promise as in a recent example of synergy observed between polymyxin
B and silver nanoparticles against Gram negative bacteria [133].
Zinc and copper are toxic to prokaryotic cells at submillimolar con-
centrations and to eukaryotic cells at higher concentrations [134,135].
The toxic effects of copper are usually attributed to Fenton type reac-
tions creating ROS related oxidative stress or to reaction with cellular
sulfhydryl species but more recent evidence suggests that disruption
of iron–sulfur proteins may also be involved [136,137]. Zinc is added
to a number of oral care products [138,139] and has bacteriostatic ef-
fects toward a variety of aerobic and anaerobic organisms. Zinc may
act by increasing the membrane permeability of protons to interfere
with F-ATPase activity as well as being an inhibitor of glycolytic
enzymes [138,140].
Synergy has been reported for the fish AMP piscidin 2 when
combined with copper [141] and for a family of human peptidoglycan
recognition proteins (PGRPs) combined with zinc [142]. It should be
noted that the latter do not target the plasma membrane as do most cat-
ionic AMPs. Most eukaryotic organisms are less sensitive to the effects of
toxic ions and can tolerate higher concentrations of such ions [116,134].
Since most microbes can readily develop resistance to heavy metals
[143] the use of combination therapy can act to limit such resistance.
In addition, copper and silver have been shown to induce some bacteria
to enter the viable but nonculturable state (VBNC) which AMPs are
uniquely suited to kill given that they target the plasma membrane
and do not require metabolically active cells [144,145]. At the same
time, additivity or synergy can allow the use of lower concentrations
of toxic ions, thus limiting potential side effects.
Recently, we employed the DPS assay described above to investigate
the effects of combining the AMP ARVA with CuCl2 and ZnCl2 [146]. An
advantage of this assay is that it is amenable to a large number of repe-
titions, and that alternate concentration ratios can be readily compared.
A useful means of displaying data that we often employ is depicted in
Fig. 4. FICA and FICB are not constants as they can vary as the ratios of
compounds A and B are varied but the FICC value obtained by adding
FICA and FICB is a constant. For this reason, FICC is often preferred.
When one compound is more expensive or more toxic than another,
however, the individual FICA and FICB values become more important.
By graphing the FICA value versus FICB (FICIon versus FICARVA here),
one can obtain the FICC value by simply adding the x- and
y-coordinates and can readily identify synergistic interactions as
those falling on or below the red lines and additive interactions as
those on or below the black lines. All three bacterial species tested,
both Gram positive and Gram negative, showed either synergistic
or strongly additive effects between the divalent ions Zn 2 +
(diamonds) or Cu2 + (filled circles) and ARVA (Fig. 4A–C). The con-
centration of ARVA required for sterilization was lowered 10 to 25

Fig. 4. Data from DPS assay. Values for FICIon are plotted versus FICARVA for each of the four toxic ions. CuCl2 data are plotted as circles, EDTA as triangles, NaF as squares, and ZnCl2 as
diamonds. Data with filled symbols are from one set of peptide:ion ratios while open symbols are data using altered ratios of peptide compared to the toxic ion [146]. Panels A–D
show data collected against E. coli (A), P. aeruginosa (B), S. aureus (C), and C. albicans (D). The FICC value is obtained by adding the x- and y-coordinates (FICIon + FICARVA). Data
exhibiting synergy will fall on or below the red lines while those with additivity fall on or below the black lines in each panel.
Adapted from Ref. [146].
932 W.F. Walkenhorst / Biochimica et Biophysica Acta 1858 (2016) 926–935
fold (FICARVA = 0.04 to 0.1) into the 100–250 nM range for E. coli,
P. aeruginosa, and S. aureus.
The values of FICA and FICB are a function of the concentration ratios
chosen for the two species. Alternate starting ratios of the two com-
pounds (ARVA:CuCl2) were tested against E. coli (open circles in
Fig. 4A). Increasing the concentration of ARVA while decreasing the con-
centration of CuCl2 in the initial well decreases the FICIon from about 0.5
down to 0.14 (for CuCl2) while increasing the FICARVA from 0.11 to 0.33.
The FICC however changes much less than either of the individual FIC
values, verifying the additive effect of the two compounds. While two
of the values for ARVA with CuCl2 fall above the red line (Fig. 4A) with
FICC ~ 0.6, the third value of FICC falls below the red line (FICC = 0.47)
indicating synergy. To decide which set of conditions is preferred, the
actual concentrations of the two species must also be considered. If
the cost of ARVA was the limiting factor, one might choose the strongly
additive condition (closed circle, Fig. 4A) but if the toxicity of CuCl2 was
important at the concentrations involved, one could choose the syner-
gistic data point (open circle), which actually requires a higher ARVA
concentration.
8. Combination of toxic and bacteriostatic anions with AMPs
EDTA and NaF are commonly used in the food industry [147], in oral
health applications [116,138,148,149], and in the medical technology
industry [119,150,151] to inhibit bacterial growth. Fluoride ion is toxic
only at relatively high concentrations in most organisms. A variety of ef-
fects are attributed to fluoride including enzyme toxicity, mimicry of
phosphate by metal-fluoride complexes, and strong inhibition of F-
ATPases due to increased proton permeability of membranes with HF
formation [116,138,152]. Some oral bacterial species, however, are
inhibited in the micromolar to low millimolar (~1–200 ppm) concen-
tration range [149,153]. Recently, Mai et al. [154] observed synergy be-
tween 10 ppm NaF and a AMP against the oral bacteria Streptococcus
mutans. EDTA and similar molecules function in metal chelation,
which is the most likely mechanism for their antimicrobial activity. By
starving growing bacteria of divalent metals such as Ca2+, Mg2+, and
micronutrients such as Zn2+, Cu2+, and Mn2+, EDTA can act in a bacte-
riostatic fashion to prevent bacterial growth. EDTA can also weaken cell
walls by removing bound Ca2+ and Mg2+ which crosslink and strength-
en bacterial cell walls [59,107,155]. In combination with amine buffers,
EDTA has long been known to have inhibitory effects on bacteria [156].
EDTA has also been used to limit biofilms [118], to prevent infections
and sterilize catheters [150,151], and in combinations with AMPs or
other antimicrobials [151,152,157]. Wei et al. [83] observed synergy be-
tween the AMP, MUC7, derived from saliva, and EDTA against a patho-
genic strain of C. albicans isolated from dentures.
In our recent work [146], both Gram negative species exhibited syn-
ergy with EDTA paired with ARVA while the Gram positive S. aureus
showed only simple additivity (filled triangles, Fig. 4A–C). To further
understand this result, one must consider the actual concentrations of
the two species involved. Classifications such as synergy and additivity
are useful, but do not tell the whole story. For example, the [EDTA]
needed to kill S. aureus in the presence of ARVA was ~600 μM compared
to about twice that for the two Gram negative organisms, while the
FICARVA values were nearly identical for all three bacteria. Variation of
the ARVA:EDTA ratio (open triangles, Fig. 4A, C), allowed killing as
low as 200–300 μM EDTA for all three bacteria with FICARVA values be-
tween 0.2 and 0.4, thus still allowing substantially less of the compara-
tively expensive ARVA to be used. Likewise, the simple additivity result
obtained with ARVA:EDTA for C. albicans (Fig. 4D) was actually as good
or better than the synergism observed for the two Gram negative spe-
cies. All three were able to sterilize at concentrations of ARVA of
~ 250 nM but ~ 1 mM EDTA was required for this effect for the two
Gram negative organisms while with Candida this result required only
274 μM EDTA.
A potential mechanism to explain synergy between EDTA or other
chelators with AMPs is that removal of divalent calcium and magnesium
ions may make the cell wall much more anionic, thus enhancing the
electrostatic attractions between cationic AMPs and the microbial cell
wall. NaF showed only simple additivity with ARVA for the three bacte-
rial species (filled squares) but had a FICARVA value below 0.4 for
S. aureus. In general, C. albicans showed larger reductions in FICARVA
than for FICIon, which is useful if the goal is to use less of the more expen-
sive AMP.
9. Conclusions
The proliferation of AB and multidrug resistance, particularly in nos-
ocomial infections, makes the development of new ABs and AB formula-
tions a necessity. Many of the future applications of AMPs will likely be
surface applications where the environment is amenable to manipula-
tion. The full range of techniques reviewed above is available for use
in improving AMP performance, including combinations of two or
more antimicrobial agents with additive or synergistic actions and
adjusting environment factors so as to maximize AMP activity. In addi-
tion, there exists the possibility for combining adjuvants and environ-
mental factors to account for species and strain dependent variation in
microbial cell envelopes. A better understanding of the effects of
environmental factors such as pH, ionic strength, specific ions, and sur-
face properties on AMP activity will facilitate the development of new
applications for AMPs and new formulations as potential drug therapies
[27,37,41]. The use of low concentrations of relatively inexpensive com-
pounds such as ZnCl2 or CuCl2, NaF, or Na2EDTA should help to lower
the costs for AMP drug formulations.
Transparency document
The Transparency document associated with this article can be
found, in the online version.
Acknowledgments
We thank the Wimley laboratory of Tulane University Medical
Center for providing the peptide used in some studies as well as for
sharing their newly developed synergy assay, and appreciate the techni-
cal assistance of Jing He and Charles Starr for peptide preparation, and
thank Justine Sundrud for assistance with data collection. This work
was partially funded by the Louisiana Board of Regents ENH-PKSFI
(LEQSF(2007–12)-ENH-PKSFI-PES-03) program to Dr. Frank Jordan,
Loyola University.
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