Mechanisms and Consequences of Bacterial Resistance to Antimicrobial Peptides

Home , Bacteriocin, Serial passage

Drug Resistance Updates 26 (2016) 43–57

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Drug Resistance Updates

jo urnal homepage: www.elsevier.com/locate/drup

Invited review

Mechanisms and consequences of bacterial resistance to antimicrobial peptides

∗

D.I. Andersson , D. Hughes, J.Z. Kubicek-Sutherland

Uppsala University, Department of Medical Biochemistry and Microbiology, Box 582, SE-751 23 Uppsala, Sweden

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

Article history: Cationic antimicrobial peptides (AMPs) are an intrinsic part of the human innate immune system. Over

Received 14 March 2016

100 different human AMPs are known to exhibit broad-spectrum antibacterial activity. Because of the

Received in revised form 7 April 2016

increased frequency of resistance to conventional antibiotics there is an interest in developing AMPs as an

Accepted 11 April 2016

alternative antibacterial therapy. Several cationic peptides that are derivatives of AMPs from the human

innate immune system are currently in clinical development. There are also ongoing clinical studies

Keywords:

aimed at modulating the expression of AMPs to boost the human innate immune response. In this review

Antimicrobial peptides

we discuss the potential problems associated with these therapeutic approaches. There is considerable

Anti-bacterial drugs

Resistance experimental data describing mechanisms by which bacteria can develop resistance to AMPs. As for any

type of drug resistance, the rate by which AMP resistance would emerge and spread in a population

Innate immunity

Selection of bacteria in a natural setting will be determined by a complex interplay of several different factors,

Bacterial infections including the mutation supply rate, the ﬁtness of the resistant mutant at different AMP concentrations,

and the strength of the selective pressure. Several studies have already shown that AMP-resistant bac-

terial mutants display broad cross-resistance to a variety of AMPs with different structures and modes

of action. Therefore, routine clinical administration of AMPs to treat bacterial infections may select for

resistant bacterial pathogens capable of better evading the innate immune system. The ramiﬁcations of

therapeutic levels of exposure on the development of AMP resistance and bacterial pathogenesis are not

yet understood. This is something that needs to be carefully studied and monitored if AMPs are used in

clinical settings.

license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

1. Introduction to AMPs ...... 44

1.1. Deﬁnition of AMPs and early history ...... 44

2. Structures of AMPs ...... 44

3. AMP structure inﬂuences activity ...... 45

4. Biological roles of AMPs ...... 45

4.1. Expression of AMPs ...... 45

4.2. Antibacterial activity (direct) ...... 45

4.3. Immune modulation (may indirectly be antibacterial) ...... 45

5. Mode of action of AMPs ...... 45

5.1. Membrane interaction speciﬁcity ...... 45

5.2. Membrane and cell wall activity of AMPs ...... 46

5.3. AMPs activity may also involve intracellular targets ...... 46

6. Intrinsic resistance to AMPs ...... 46

6.1. Membrane modiﬁcations in Gram-negative bacteria ...... 46

6.2. Membrane modiﬁcations in Gram-positive bacteria ...... 47

6.3. Other intrinsic AMP resistance mechanisms ...... 47

6.4. Methods to identify intrinsic AMP resistance mechanisms ...... 48

∗

Corresponding author. Tel.: +46 18 4714175.

E-mail address: [email protected] (D.I. Andersson).

http://dx.doi.org/10.1016/j.drup.2016.04.002

44 D.I. Andersson et al. / Drug Resistance Updates 26 (2016) 43–57

7. Acquired resistance ...... 48

7.1. Methods to select for mutants with acquired AMP resistance ...... 48

7.2. Mechanisms of acquired resistance ...... 49

7.2.1. Naturally occurring acquired resistance ...... 49

7.2.2. Serial passage ...... 50

7.2.3. Direct plating ...... 50

7.3. What determines the risk of acquiring resistance to AMPs? ...... 51

7.3.1. Mutation supply rate ...... 51

7.3.2. Mutant ﬁtness ...... 51

7.3.3. Selection strength ...... 52

7.3.4. Compensatory evolution ...... 52

7.3.5. Epistatic interactions ...... 52

8. Therapeutic use of AMPs...... 52

9. Concluding remarks...... 54

Acknowledgement ...... 54

References ...... 54

1. Introduction to AMPs difference in the genetic origins of AMPs and NRPS-antibiotics has

consequences for the compositions of the ﬁnal products. Ribosoma-

1.1. Deﬁnition of AMPs and early history lly produced AMPs contain only the normal complement of amino

acids found in proteins, sometimes with post-translational modiﬁ-

Cationic antimicrobial peptides (AMPs) are relatively small pep- cations. NRPS-produced antibiotics, in contrast, are unconstrained

tides that, by deﬁnition, have a net positive charge, and exhibit by the limitations of ribosomal translation, and usually contain

some, often broad-spectrum, antimicrobial activity. This rather tau- a mixture of normal amino acids together with non-canonical

tological deﬁnition encompasses a great diversity of molecules, amino acids not found in any proteins (Walsh et al., 2013). In

at both the sequence and the structural level. An online antimi- addition, genes for some of the human ␣-defensins (HNP1 and

crobial peptide database, APD3 (Wang et al., 2016), accessible at HNP3) are present in multiple copies and are inherited unequally

http://aps.unmc.edu/AP/, lists over 2600 examples of AMPs from by different individuals, giving rise to individuals with two or

all kingdoms of life: bacteria, archaea, and eukaryotes (including three copies of either or both of these genes, potentially affect-

plants, animals, fungi and protists). AMPs can act directly to protect ing the levels of AMPs produced among individuals (Mars et al.,

animals against a wide variety of bacterial, viral, fungal and proto- 1995).

zoan infections and because of this are often referred to as host Human AMPs vary in length from 5 to 149 amino acids

defense peptides. In animals, both vertebrates and invertebrates, (Bangalore et al., 1990; Cash et al., 2006). The great majority are

AMPs also function as a universal and important part of the innate cationic peptides, but the net charge of different human AMPs

immune system, modulating activities that promote protection ranges from −3 to +20. AMPs have been identiﬁed in differ-

against infections by microorganisms. The APD3 database cur- ent human excretions, tissues and cell types, including saliva,

rently lists 112 different human host defense peptides of which tears, sweat and milk, on the skin and tongue, in bone mar-

100 have experimentally determined antibacterial activities. The row, plasma, kidneys, liver, heart, brain, eyes, intestine, sperm,

focus of this review is on the importance of human AMPs in pro- urinary tract, amniotic ﬂuid, and respiratory tract, and in many dif-

tecting against bacterial infections, on the mechanisms of intrinsic ferent cell types including epithelial/mucosal cells, macrophages,

and acquired bacterial resistance to AMPs, and on the opportuni- neutrophils, natural killer cells, monocytes, eosinophilic leuko-

ties and potential consequences associated with therapeutic use of cytes, Paneth cells, T-cells and B-cells (http://aps.unmc.edu/AP/

AMPs. ). Research into the functions of AMPs goes back at least to the

AMPs are not unique as peptide-based small molecules with time of Alexander Fleming who discovered lysozyme (Fleming and

antibacterial activity. Many of the antibiotics produced by microor- Allison, 1922). However, it is in the past few decades, especially

ganisms are peptide-based molecules. These are produced by after the discovery of cationic bacteriocidal peptides in polymor-

non-ribosomal peptide synthesis (NRPS), a process that involves phonuclear leukocytes (Zeya and Spitznagel, 1963), that interest

the expression of large arrays of genes encoding multiple enzymes and research has expanded explosively. During the 1990s impor-

that work in succession to catalyze the sequence of chemi- tant new classes of AMPs were discovered in humans, including

cal reactions required to synthetize the antibiotic (Baltz, 2006; the ␤-defensins (Bensch et al., 1995) and the cathelicidins (Gallo

Hughes, 2003). Peptide-based antibiotics include ␤-lactams such et al., 1997) and this stimulated research into understanding the

as penicillin, cyclic peptide antibiotics like the polymixins and bac- number and variety of human AMPs and, more recently, into their

itracin, glycopeptides like vancomycin (Yim et al., 2014), and the potential applications in antimicrobial therapy to control bacterial

lipopeptide daptomycin, one of the most recently introduced novel infections.

antibiotic classes (Robbel and Marahiel, 2010). The AMPs discussed

in this review differ from peptide-based antibiotics in the mech-

anism of their synthesis and also in their amino acid make-up. 2. Structures of AMPs

AMPs, including those made in human cells, are in contrast to

NRPS-antibiotics, produced by the normal process of ribosomal Defensins and cathelicidin were recognized early on as

translation on an mRNA template. The primary product is usu- important components of the antimicrobial mechanisms of

ally a pre-protein that is then processed to the ﬁnal length of polymorphonuclear leukocytes (PMNs), and were classiﬁed as

the active AMP. For example, the human cathelicidin AMP, LL-37, belonging to signiﬁcantly different structural families (Lehrer and

is produced as a precursor protein, human cationic antimicrobial Ganz, 2002; Zanetti et al., 1995). A classiﬁcation of AMPs into four

protein-18 kDa (hCAP18), and the mature LL-37 peptide is the result major families, based on their 3D structures, has been adopted by

of its processing by proteinase 3 (Gudmundsson et al., 1996). This the APD3 database (Wang et al., 2016).

D.I. Andersson et al. / Drug Resistance Updates 26 (2016) 43–57 45

(i) The alpha family is composed of AMPs with helical structures, bacteria, LPS, TNF␣, and IL-1 (Diamond et al., 1996; Fang et al., 2003;

for example, the human cathelicidin LL-37 (Agerberth et al., Singh et al., 1998).

1995).

(ii) The beta family is composed of AMPs with ␤-strands, for exam- 4.2. Antibacterial activity (direct)

ple, the human ␣-defensins (Selsted et al., 1985).

(iii) The alphabeta family comprises AMPs with both ␣-helical and Human cathelicidin and human defensins have the capacity

␤-strands structures in the same 3D fold, for example, the to kill a wide variety of Gram-positive and Gram-negative bacte-

human ␤-defensins (Harder et al., 1997). The sequences of both ria in vitro (Ganz et al., 1985; Garcia et al., 2001b; Harder et al.,

the ␣- and ␤-defensins include six cysteine residues that sta- 2001, 1997; Ouellette and Selsted, 1996; Zaiou et al., 2003). Like

bilize the structure by forming three intramolecular disulﬁde small molecule antibiotics, this antibacterial activity of AMPs is

bonds. ␤-Sheet AMPs are frequently cyclic molecules with the dose-dependent and bacteriocidal activities are usually observed at

␮

structure constrained by the intramolecular disulﬁde bridges. M concentrations. Evidence for their antibacterial efﬁcacy in vivo

The ␣- and ␤-defensins differ in the bonding pattern of their comes from studies using infections in animal models (Huang et al.,

cysteines, probably reﬂecting their different phylogenetic ori- 2002), and also from infection studies using transgenic or knock-

gins. In total, humans encode six different ␣-defensins, over out mice defective in the production of various AMPs (Moser et al.,

30 different ␤-defensins, but only one ␣-helical cathelicidin, 2002; Salzman et al., 2003; Wilson et al., 1999).

LL-37 (Bauer and Shafer, 2015; Wang, 2014).

(iv) The non-alphabeta family contains AMPs with neither ␣- 4.3. Immune modulation (may indirectly be antibacterial)

helical nor ␤-strands, for example, the antimicrobial peptide

indolicidin, isolated from cattle (Selsted et al., 1992). Because AMPs are produced by epithelial cells and by circulating immune

3D structures are known for less than 400 of the over 2600 cells including neutrophils and macrophages, and are among

identified AMPs, this classification system should be regarded the first immune effectors encountered by an invading microbe

as provisional and other classiﬁcation systems are concurrently (Jenssen et al., 2006). The human cathelicidin LL-37 modulates

in use, based on properties such as covalent bonding patterns, the innate immune response by acting as a chemoattractant for

hydrophobicity, net charge, or molecular targets (Wang, 2015). neutrophils, monocytes, and mast cells (Niyonsaba et al., 2002b;

Yang et al., 2000) and human ␤-defensins chemoattract leuko-

cytes (Garcia et al., 2001a; Niyonsaba et al., 2002a; Territo et al.,

␤

3. AMP structure inﬂuences activity 1989). -Defensins also attract dendritic cells that can act directly

by phagocytizing and killing pathogens (Liu, 2001) or by produc-

The antibacterial properties of AMPs are associated with two ing cytokines, chemokines and other AMPs that then participate in

interrelated features of the peptides: their net charge, which for innate antimicrobial activity (Duits et al., 2002). Thus cathelicidin,

the great majority of AMPs is positive, and their propensity to be defensins and chemokines overlap in their chemotactic activity and

amphipathic, meaning that they can fold into structures with both affects on innate immunity.

a hydrophobic and a hydrophilic surface. These features together

facilitate the interaction of AMPs with the negatively charged com- 5. Mode of action of AMPs

ponents of the bacterial envelope (e.g., lipopolysaccharides (LPS)

and techoic acids (TA)) on the surfaces of Gram-negative and Gram- 5.1. Membrane interaction speciﬁcity

positive bacteria, respectively, and with the negatively charged

phospholipids of the bacterial membrane (Morgera et al., 2008; Selective toxicity, killing bacterial pathogens without damaging

Wang, 2008). For example, AMPs like LL-37 are initially attracted host tissue, is a feature that is crucial for AMPs. However, while

to the bacterial surface by electrostatic interactions. LL-37 interacts it has been demonstrated that AMPs can permeabilize bacterial

with lipid bilayers such that the amphipathic helix is oriented par- membranes and cell walls, and that this ability correlates with their

allel to the surface of the bilayer (Henzler Wildman et al., 2003). antibacterial effects, the actual mode of bacterial killing by AMPs

It is widely believed that after the initial electrostatic interaction remains an area where there is still a poor level of understand-

with the bacterial surface that the amphipathic nature of AMPs ing. One clue to the potential speciﬁcity of killing by membrane

allows them to insert into the bacterial cell membrane, forming disruption is that there are signiﬁcant differences in the lipid com-

a pore that disrupts membrane integrity, leading to osmotic lysis position of bacterial and eukaryotic cell membranes (Sohlenkamp

of the bacterial cell. However, as discussed below, there is evidence and Geiger, 2016; Teixeira et al., 2012). Bacterial membranes are

that this view of the AMP antibacterial mode of action may be an rich in negatively charged phospholipids such as phosphatidyl-

oversimpliﬁcation. glycerol (PG), cardiolipin (CL) and phosphatidylserine (PS) that are

2+ 2+

stabilized by divalent cations such as Mg and Ca . In addition,

Gram-negative bacteria have an outer membrane containing LPS

4. Biological roles of AMPs that acts as a permeability barrier reducing access to the cyto-

plasmic membrane. In contrast to bacteria, human cell membranes

4.1. Expression of AMPs are rich in zwitterionic phospholipids with a neutral net charge,

such as phosphatidylethanolamine (PE), phosphatidylcholine (PC),

Human AMPs are expressed in a wide variety of cells of the and sphingomyelin (SM). The human AMP LL-37 can discriminate

immune system, including neutrophils, NK, and T cells, as well as in between different membranes based on their lipid constituents,

the epithelial cells lining tissues facing the external environment preferentially inserting into lipids containing PG, typical of bacte-

(Yang et al., 2004). The expression of these AMPs can be consti- rial membranes, but not into membranes containing PC or PE, as

tutive (e.g. ␣- and ␤-defensins in neutrophils, and ␣-defensins in found in human red blood cells (Neville et al., 2006). These impor-

paneth cells), inducible (e.g. ␣-defensins in CD8T cells, ␤-defensins tant differences between bacterial and eukaryotic membranes,

in epithelial cells) or both constitutive and inducible (e.g. catheli- where bacteria have anionic lipids exposed on the surface whereas

cidins and ␤-defensins in a variety of cells, including neutrophils, eukaryotic membranes sequester anionic lipids in the monolayer

epithelial cells, and macrophages). Expression of most of the ␤- facing the interior of the cell, may explain the selectivity of AMPs

defensins is also induced by proinﬂammatory stimuli, including for bacterial membranes. Human cell membranes also contain

46 D.I. Andersson et al. / Drug Resistance Updates 26 (2016) 43–57

signiﬁcant amounts of cholesterol that affect the ﬂuidity of phos- accumulate at the site of cell division and at the cell poles, suppor-

pholipids in the membrane, and increase the stability of the lipid ting the notion that at least part of its antimicrobial activity might

bilayers (Tytler et al., 1995). Accordingly, a number of distinguish- be exerted in the cytoplasm (Chileveru et al., 2015). Indolicidin,

ing features including the presence of cholesterol, the membrane a bovine cathelicidin with broad-spectrum bacteriocidal activity,

potential, and the asymmetric distribution of phospholipids in the has been shown not to lyse bacterial cells but to inhibit DNA syn-

membrane, may be important for reducing the binding of AMPs to thesis (Subbalakshmi and Sitaram, 1998) and shown in vitro to

human cells (Lai and Gallo, 2009). interact with duplex DNA (Ghosh et al., 2014). Analogs of buforin

antimicrobial peptides were also shown to exert their bacterioci-

5.2. Membrane and cell wall activity of AMPs dal activity on E. coli by binding to DNA and RNA after penetrating

the cell membrane (Hao et al., 2013). One possibility is that AMP

The bacteriocidal effect of AMPs is widely believed to be due targeting can be promiscuous and dependent on local concentra-

to the formation of pores in the bacterial cytoplasmic membrane, tion, either causing lethal membrane disruption or translocation

resulting in a loss of control over ion ﬂows across the membrane into the cytoplasm to interfere with cellular metabolism (Sugiarto

and cell death. There is considerable in vitro data showing that AMPs and Yu, 2007). One of the approaches to test the signiﬁcance of

can disrupt lipid bilayers, supporting this basic model. Exactly how intracellular interactions in the bacteriocidal effects of AMPs is to

AMPs form pores in bacterial membranes is less certain. The initial select for mutants with reduced susceptibility to AMPs and then

step is believed to involve electrostatic interactions between the genetically map the selected mutations. In the following sections

positively charged amino acids in AMPs and the negatively charged this approach is described, with the twin aims of determining the

LPS, or phospholipid head group of the membrane, resulting in probability of resistance developing in the case that AMPs are used

AMPs accumulating on the surface of the membrane (Epand et al., more widely in therapy, and to gain a better understanding of the

2015). After reaching some threshold concentration, AMPs may molecular targets of AMPs, whether at the cell wall, cell membrane,

self-assemble on the membrane and incorporate into it creating or intracellularly.

a pore in the process. Several models for AMP-directed membrane

pore formation have been suggested: (i) the barrel-stave model,

6. Intrinsic resistance to AMPs

proposes that peptides insert perpendicularly in the bilayer, asso-

ciating together to form a pore; (ii) the carpet mechanism, proposes

Bacteria encounter AMPs frequently in their natural environ-

that peptides adsorb parallel to the bilayer and, after reaching suf-

ments and have evolved mechanisms to resist their action. Intrinsic

ﬁcient density of coverage, produce a detergent-like effect that

resistance to AMPs can occur via passive or inducible mechanisms.

disintegrates the membrane; and (iii) the toroidal pore mechanism,

Passive AMP resistance occurs in some bacterial species includ-

proposes that peptides insert perpendicularly in the bilayer and

ing Proteus, Morganella, Providencia, Serratia, and Burkholderia as a

induce a local membrane curvature with the pore lined partly by

result of an inherently more positively charged lipid A that reduces

peptides and partly by phospholipid head groups (Melo et al., 2009).

AMP interaction (Manniello et al., 1978; Viljanen and Vaara, 1984).

Recent research suggests that the interactions between different

The induction of AMP-resistance in other bacteria is tightly reg-

AMPs and bacterial membranes may involve much more speciﬁc

ulated in response to environmental conditions and serves as a

interactions than suggested by these generic models of pore for-

mechanism for bacterial survival in various natural environments

mation. For example, phosphatidylethanolamine (PE), present at

in which they would experience the threat of AMPs. Inducible (also

a high concentration on the surface of bacterial membranes but

referred to as adaptive) resistance results in transient molecular

sequestered on the cytoplasmic leaﬂet of mammalian membranes,

modiﬁcations in both Gram-negative and Gram-positive bacteria

acts as a high afﬁnity lipid receptor for several AMPs (Phoenix et al.,

(summarized in Fig. 1). Reversibility offsets the energetic burden

2015). Current evidence suggests that many AMPs, including the

imposed by many of these modiﬁcations, which largely affect the

plant cyclotides, use speciﬁc receptors such as PE, in their inter-

composition of the bacterial membrane. The molecular basis of

actions with membranes (Stromstedt et al., 2016). Other speciﬁc

inducible AMP resistance has been reviewed extensively for both

components of bacterial membranes that are targeted by AMPs

Gram-negative and Gram-positive bacteria (Anaya-Lopez et al.,

include LPS, lipoteichoic acid (LTA) and the peptidoglycan precur-

2013; Gunn, 2001; Kraus and Peschel, 2006; Matamouros and

sor lipid II (Schmitt et al., 2015). Lipid II seems to be a favorite target.

Miller, 2015; Nawrocki et al., 2014; Nizet, 2006; Yeaman and Yount,

The antibiotic activity of the human ␤-defensin 3 (Sass et al., 2010)

2003). A common mechanism of AMP resistance in bacteria is the

and human ␣-defensin 1 (de Leeuw et al., 2010; Varney et al., 2013),

incorporation of positively charged molecules into their cell sur-

relies on their selective binding of lipid II to block bacterial cell

face in order to reduce the interaction and binding of cationic AMPs.

wall biosynthesis. Accordingly, the bacteriocidal activities of many

Inactivation of these mechanisms results in enhanced susceptibility

human AMPs may have little or nothing to do with generic effects

to AMPs, including components of the host innate immune system

on the integrity of bacterial cytoplasmic membranes and more to

and often in reduced virulence.

do with speciﬁc target interactions that happen to be localized to

the bacterial cell wall and/or membrane.

6.1. Membrane modiﬁcations in Gram-negative bacteria

5.3. AMPs activity may also involve intracellular targets

Intrinsic AMP resistance has been most extensively studied in

AMPs have also been shown to traverse the cell membrane Salmonella enterica serovar Typhimurium in which a variety of

and to block essential cellular processes without causing extensive LPS modiﬁcations are triggered by environmental stimuli includ-

membrane damage (Brogden, 2005; Patrzykat et al., 2002) raising ing nutrient starvation (McLeod and Spector, 1996), low pH, low

the question of whether some of their antibacterial activities are magnesium, and high iron (Gunn and Miller, 1996) as well as in

dependent on intracellular targeting. Thus, analogs of the human various host tissues (Kubicek-Sutherland et al., 2015). The PmrAB,

␤

-defensin 4 were shown to translocate across the outer and inner PhoPQ and Rcs regulatory systems mediate many of these modiﬁ-

membranes of Escherichia coli without causing cell lysis, suggesting cations in order to support bacterial survival in vivo. The addition

that the observed bacteriocidal activity might depend on cytoplas- of 4-aminoarabinose (Ara4N) to lipid A, which creates a less nega-

mic interactions (Sharma and Nagaraj, 2015). Human ␣-defensin 5 tively charged LPS and reduces binding afﬁnity of AMPs, is regulated

was also shown to translocate into the cytoplasm of E. coli and to by the pmrCAB and pmrHFIJKLM operons in S. Typhimurium (Gunn

D.I. Andersson et al. / Drug Resistance Updates 26 (2016) 43–57 47

Fig. 1. Summary of intrinsic antimicrobial peptide resistance mechanism in Gram-negative and Gram-positive bacteria. The main pathways resulting in transient high-level

AMP resistance in bacteria are membrane modiﬁcations, increased efﬂux and proteolytic degradation.

et al., 2000). Inactivation of pmrA or pmrB results in attenuated viru- morphology, adhesion, virulence and antimicrobial resistance

lence when administered orally but not intraperitoneally indicating (Brown et al., 2013). Although critical to cellular function, the

the importance of Ara4N modiﬁcation in overcoming the innate anionic properties of TAs make them a target for the binding of

immunity in the intestine. PmrA can also be regulated by the PhoPQ positively charged AMPs. Resistance to AMPs is often conferred in a

system via the protein PmrD, which prevents dephosphorylation of transient manner via TA modiﬁcations that reduce the overall neg-

PmrA resulting in continued activation (Roland et al., 1994). Other ative charge in the bacterial membrane. The GraRS (also known as

PhoPQ regulated genes that mediate AMP resistance and virulence aps) sensor/regulator system senses AMPs in the environment and

in S. Typhimurium include slyA, encoding a virulence regulatory induces AMP resistance in Staphylococcus aureus by activating the

protein; ugtL, encoding an inner membrane protein that forms d-alanylation of TA, the incorporation of lysylphosphatidylglycerol

monophosphorylated lipid A; pagP, encoding an outer membrane in the bacterial membrane, and activation of the vraFG AMP trans-

protein involved in palmitoylation of lipid A; and yqjA, an inner porter (Li et al., 2007). d-Alanine ester modiﬁcations are made via

membrane protein of unknown function (Guo et al., 1998; Shi et al., components of the dlt operon in response to low divalent cation

2+ 2+

2004a,b). The Rcs phosphorelay system regulates AMP resistance concentrations (Mg and Ca ) and the presence of cationic AMPs

in S. Typhimurium by activating expression of the uncharacter- (Koprivnjak et al., 2006). Inactivation of the dlt operon results in

ized periplasmic protein YdeI, which is required for AMP resistance increased susceptibility to AMPs in S. aureus (Peschel et al., 1999),

and oral virulence in mice (Erickson and Detweiler, 2006). Other Bacillus subtilis (Cao and Helmann, 2004), Group A streptococci

AMP resistance and virulence determinants in S. Typhimurium (GAS) (Kristian et al., 2005), Group B streptococci (GBS) (Poyart

include surA, tolB, and pgm. SurA and TolB are involved in mem- et al., 2003), Listeria monocytogenes (Abachin et al., 2002), and

brane stabilization while Pgm (phosphoglucomutase) is involved Lactobacillus reuteri (Walter et al., 2007). Similarly, the MprF pro-

in LPS biosynthesis (Paterson et al., 2009; Tamayo et al., 2002). tein (multiple peptide resistance factor) mediates the addition of

The PhoPQ system also plays a role in AMP resistance and vir- -lysine to membrane phospholipids thereby reducing their overall

ulence in Pseudomonas aeruginosa (Gooderham et al., 2009), and negative charge and conferring AMP resistance in S. aureus (Ernst

PmrAB regulated LPS modiﬁcations confer AMP resistance in Acine- et al., 2009), L. monocytogenes (Thedieck et al., 2006), and Bacillus

tobacter baumannii (Adams et al., 2009). In Vibrio cholerae, the anthracis (Samant et al., 2009). Inactivation of graR or mprF results

almEFG operon encodes the glycylation of LPS, which confers resis- in enhanced susceptibility to AMPs and attenuation of virulence in

tance to polymyxins (Henderson et al., 2014). In P. aeruginosa the mice (Kraus et al., 2008; Li et al., 2007; Yang et al., 2012).

arnBCADTEF operon mediates the addition of Ara4N to lipid A con-

ferring AMP resistance (McPhee et al., 2003). The anionic capsular

6.3. Other intrinsic AMP resistance mechanisms

polysaccharide of Klebsiella pneumoniae, P. aeruginosa, and Strep-

tococcus pneumoniae, has also been shown to confer resistance to

Besides membrane modiﬁcation, other effective methods for

polymyxin B and human ␣-defensin 1 by binding cationic AMPs

dampening the antimicrobial effects of AMPs are efﬂux and pro-

(Llobet et al., 2008).

teolytic degradation. In S. Typhimurium AMPs are exported by the

sap (sensitivity to antimicrobial peptides) efﬂux system and the

6.2. Membrane modiﬁcations in Gram-positive bacteria putative ABC transporter encoded by the yejABEF, both of which are

required for virulence in mice (Eswarappa et al., 2008; Groisman

In Gram-positive bacteria, AMP-resistance occurs following et al., 1992b). In S. aureus, the chromosomal vraFG gene encodes the

the incorporation of positively charged molecules into cell wall ABC transporter-dependent efﬂux pump, also regulated by GraRS

teichoic acids (TA), which play an essential role in cell division, (Li et al., 2007). Also, the plasmid-encoded qacA gene encodes a

48 D.I. Andersson et al. / Drug Resistance Updates 26 (2016) 43–57

Fig. 2. Methods used for isolation of AMP-resistant mutants. (A) Serial passage involves the growth of bacteria in liquid media containing progressively increasing concen-

trations of AMPs. (B) Selection on plates involves direct plating of bacteria on agar plates containing AMP concentrations above the MIC.

proton motive force-dependent efﬂux pump (Kupferwasser et al., unintended background mutations that are typically not accounted

1999). Efﬂux pumps that act on AMPs have also been identiﬁed for in large-scale screens.

in Neisseria gonorrhoeae (Shafer et al., 1998) and Yersinia spp. Once a gene or pathway has been identiﬁed, a subsequent

(Bengoechea and Skurnik, 2000). Proteases that degrade AMPs more targeted approach can provide direct information about the

include PgtE in S. Typhimurium (Guina et al., 2000) and OpmT in relationship between individual genes and AMP resistance. The

E. coli (Stumpe et al., 1998). inactivation of individual genes of interest has helped to identify

genes within a regulon that may be overlooked in a large-scale

approach. Narrowing the analysis down to several genes of interest

6.4. Methods to identify intrinsic AMP resistance mechanisms also supports the testing of resistance to a variety of AMPs, includ-

ing those that are more expensive. However, this targeted approach

To date the molecular basis of intrinsic AMP resistance has been requires prior elucidation of candidate genes and their regulatory

characterized largely using gene inactivation studies. These stud- pathways and still does not include essential genes. Lastly, the

ies have been conducted using both large-scale (genome-wide) largest problem with gene inactivation studies is the inability to

and targeted (single gene) approaches. Screening approaches to assess the molecular basis of acquired (stable) AMP resistance.

identify genes associated with AMP resistance typically involve

transposon or chemical mutagenesis of a wild type AMP-resistant

bacterial isolate to create a collection of mutants, which are then 7. Acquired resistance

screened phenotypically for resulting AMP hyper-susceptibility.

Such studies have identiﬁed genes conferring AMP resistance in 7.1. Methods to select for mutants with acquired AMP resistance

S. Typhimurium (Fields et al., 1989; Groisman et al., 1992b), E. coli

(Groisman et al., 1992a), Group A Streptococcus (GAS) (Nizet et al., Considering the continued high interest in AMPs as potential

2001); S. aureus (Peschel et al., 1999, 2001); and Neisseria menin- therapeutic drugs for bacterial infections surprisingly few stud-

gitidis (Tzeng et al., 2005). Transposon mutagenesis can also be ies have tried to assess the risk of resistance development and

performed on a bacterial strain with a mutant background in order explore the mechanisms for acquired resistance. Partly this might

to assess the contribution of particular genes in a resistance path- be explained by the long-held, and erroneous, belief that resistance

way. For instance, mutagenesis was performed in S. Typhimurium to AMPs is very difﬁcult to acquire and that it therefore is not a

on a constitutively active pmrA mutant allowing for the initial iden- big concern (Batoni et al., 2011; Ghosh and Haldar, 2015; Nizet,

tiﬁcation of non-regulatory genes conferring AMP resistance, pmrE 2006; Yeaman and Yount, 2003). However, as several recent stud-

(also known as pagA and ugd) and pmrF, both of which are involved ies have shown, resistance to AMPs can in fact evolve at high rates

in lipid A modiﬁcation (Gunn et al., 1998). (at least in vitro), generating mutants with, sometimes high, levels

These large-scale screens have provided tremendous insights of resistance.

into the molecular basis of intrinsic resistance, however there are Typically two different methods have been used to select

disadvantages to this methodology. Drawbacks from a large-scale mutants with acquired resistance to AMPs (Fig. 2). The ﬁrst,

knockout approach include the difﬁculty in observing small and most used, method involves serial passage of bacteria in

changes in AMP susceptibility that often require time-kill assays media containing AMPs at concentrations near the MIC, which are

to detect; AMP-resistance mechanisms involving multiple genes progressively increased during the course of the experiment as

are not directly assayed; and the involvement of essential genes resistant mutants are enriched. After different time points bacte-

can not be characterized. This method can also be limited by the ria are recovered and tested for resistance by broth micro-dilution

high cost of certain AMPs, which can make large-scale phenotypic (to obtain MICs of the AMPs) and/or time-kill assays (Lofton et al.,

screening very expensive and the approach has thus far been 2013). Generally the latter method is more sensitive in detecting

largely limited to less expensive peptides. Transposon mutagene- small differences in susceptibility (Lofton et al., 2013). The advan-

sis can also involve harsh conditions often resulting in additional tage with serial passage is that it is relatively easy to perform and

D.I. Andersson et al. / Drug Resistance Updates 26 (2016) 43–57 49

Table 1

Summary of known mechanisms of acquired resistance to antimicrobial peptides.

Organism Method for isolation AMP resistance Genes involved in Proposed mechanism Reference

AMP resistance

Staphylococcus aureus Clinical isolates LL-37, human hemB Inactivation results in small Glaser et al. (2014)

␤-defensin 2, human colony variant (SCV)

␤-defensin 3, phenotype with reduced AMP

lactoferricin B binding/uptake

Escherichia coli, Klebsiella Clinical isolates Colistin, polymyxin B mcr-1 Encodes a PEtN transferase Hu et al. (2016)

pneumoniae modiﬁes lipid A to reduce

anionic charge

Listeria monocytogenes Direct plating with Leucocin A mptACD Unknown Gravesen et al. (2002)

leucocin A

Acinetobacter baumannii Direct plating with Colistin, polymyxin B lpxA, lpxD, or lpxC Inactivation results in Moffatt et al. (2010)

colistin complete loss of LPS

production, reduced AMP

binding

Salmonella Typhimurium DES mutagenesis Polymyxin, CAP37, phoQ Constitutive activation in low Gunn and Miller (1996)

CAP57, protamine, Mg of phoP-regulated LPS

polylysine modiﬁcations reduce anionic

charge

Direct plating with Colistin, polymyxin B pmrA, pmrB Constitutive activation of Roland et al. (1993)

colistin pmrAB-regulated Ara4N and and Sun et al. (2009)

PEtN LPS modiﬁcation reduce

anionic charge

Direct plating with PR-39 sbmA Inactivation reduces AMP Pranting et al. (2008)

PR-39 uptake

Direct plating with Protamine, colistin, hemA, hemB, hemC, Inactivation results in small Pranting and

protamine lactoferricin, human hemL colony variant (SCV) Andersson (2010)

␣-defensin 1 phenotype with reduced AMP

binding/uptake

Serial passage with LL-37, CNY100HL, pmrB, phoP Constitutive activation of Lofton et al. (2013)

LL-37 or CNY100HL wheat germ histones various LPS modiﬁcations

reducing anionic charge

Ara4N, 4-aminoarabinose; PEtN, phosphoethanolamine.

that it requires low amounts of AMPs since growth volumes are 7.2. Mechanisms of acquired resistance

small. However, disadvantages are that it only selects for high ﬁt-

ness mutants (i.e. the mutant spectrum will be biased) and because The molecular basis of acquired resistance has been charac-

selection is stepwise the mutants often contain more than one terized in a handful of bacterial pathogens through isolation of

mutation, necessitating subsequent genetic reconstitution experi- naturally occurring resistant isolates, serial passage in the presence

ments to conﬁrm the role of each individual mutations. Likewise, of AMPs and direct plating with AMPs (summarized in Table 1).

because the selection is not lethal and growth often occurs dur-

ing several hundred generations, media adaptation mutations will

almost always be acquired (Lofton et al., 2013). These mutations 7.2.1. Naturally occurring acquired resistance

confer ﬁtness increases that are unrelated to the AMP-selection and Recently, for the ﬁrst time AMP resistance has been shown to

are identiﬁable by performing a parallel experiment in the absence be horizontally transferable in E. coli. A plasmid containing the

of AMP. mcr-1 gene was shown to mediate colistin resistance by encoding

An alternative approach, and which if possible is preferable, is a phosphoethanolamine modiﬁcation to lipid A (Liu et al., 2016).

to perform a direct selection on agar plates containing AMP lev- The mcr-1 containing plasmid was initially isolated in Chinese live-

els above the MIC. The advantages with this approach are that the stock animals, and since its initial characterization the mcr-1 gene

mutants generally only contain one mutation (alleviating the need has been identiﬁed retroactively in 3 of 1267 human fecal micro-

for genetic reconstruction, a big advantage in organisms that are biome samples taken from China prior to 2011 indicating animal

genetically intractable) and that also resistant mutants with severe to human gene transfer (Hu et al., 2016).

ﬁtness reductions are recoverable. Furthermore, if this experi- In S. aureus the small colony variant (SCV) phenotype, character-

ment is performed as a classical Luria–Delbruck ﬂuctuation test ized by slow growth rate, diminished transmembrane potential and

with many independent cultures (Pranting and Andersson, 2010; altered metabolism, has been associated with resistance to several

Pranting et al., 2008; Sun et al., 2009), this approach allows deter- AMPs including LL-37, human ␤-defensins 2 and 3, RNase 7 and

mination of mutation rates to resistance. The main problem with lactoferricin B (Glaser et al., 2014; Samuelsen et al., 2005b). The

the use of agar plate selections is that it requires high amounts of SCV phenotype can be induced by the intracellular environment at

−3 −7

AMPs and that many AMPs are less active/inactive in the complex a rate of 10 (spontaneous rate is less than 10 ) in S. aureus in

environment of an agar plate (Dhawan et al., 1997). Furthermore, order to evade the host immune response (Vesga et al., 1996). The

if resistance evolution requires the formation of several mutations lactoferricin B resistant phenotype of SCVs in S. aureus was shown to

the direct selection method is unlikely to yield results, making the be a result of reduced metabolic activity and not of reduced trans-

serial passage method preferable. membrane potential or uptake of the peptide (Samuelsen et al.,

For both of the above methods, it is possible to explore additional 2005b). Glaser et al. (2014) also showed that clinically derived S.

mutational pathways and resistance mechanisms by artiﬁcially aureus SCVs as well as a hemB auxotroph displayed reduced suscep-

increasing the mutation rate. For example, chemical mutagenesis or tibility to LL-37 and human defensins. S. aureus has also displayed

various types of mutator strains have been used to this end (Hoang constitutive resistance to platelet microbicidal proteins (PMPs) by

et al., 2012; Silverman et al., 2001). increasing membrane ﬂuidity via expression of unsaturated lipids

50 D.I. Andersson et al. / Drug Resistance Updates 26 (2016) 43–57

(Bayer et al., 2000) or reducing membrane potential (Yeaman et al., in vitro, however in a mouse model of sepsis this mutation resulted

1998). in minor attenuation. Disruption of waaY results in reduced hep-

tose phosphorylation of the LPS core, thereby diminishing the

7.2.2. Serial passage interaction between AMPs and the bacterial membrane. Although

Samuelsen et al. (2005a) described the rapid induction of S. these individual mutations each conferred only minor enhance-

aureus resistance to the AMP lactoferricin B (active form of bovine ments in AMP resistance, combining all three mutations in a single

lactoferrin) with over 30-fold increase in resistance following only mutant further increased AMP resistance. pmrB and waaY muta-

four serial passages in medium containing increasing concen- tions appeared together in two of four isolates following serial

trations of the peptide. High-level lactoferricin B resistance was passage with LL-37 or CNY100HL indicating a possible epistatic

maintained when passaged in medium without peptide. Follow- effect between these mutations both of which lead to a reduction

ing 30 passages in the absence of peptide, all strains displayed at in AMP interaction with LPS.

least a partially stable resistance phenotype with elevated MICs up

to 10-fold higher than wild type although the exact mechanism of 7.2.3. Direct plating

resistance was not deﬁned. Lactoferricin B resistant mutants also Roland et al. isolated spontaneous AMP resistant S.

displayed low-level cross-resistance to indolicidin and magainin. Typhimurium by directly plating on agar plates containing

These experiments suggest that in S. aureus the acquisition of low- various concentrations of polymyxin E (colistin). Characteriza-

level stable resistance can be achieved rapidly, within as little as tion of the resulting AMP-resistant mutants yielded a mutant

four passages in the presence of lactoferricin B, while high-level sta- containing a pmrA mutant allele (pmrA505) that displays con-

ble resistance requires the acquisition of multiple mutations over stitutive activation of PmrAB leading to increased Ara4N and

time. phosphoethanolamine covalent modiﬁcation of LPS. pmrA505

Perron et al. (2006) showed that stable AMP resistance can arise displays a 1000-fold increase in resistance to polymyxin B and a

in E. coli and Pseudomonas ﬂuorescens following continuous expo- 2- to 4-fold increase in resistance to neutrophil proteins (Roland

sure (600–700 generations) to gradually increasing concentrations et al., 1993). Colistin-resistant mutants in S. Typhimurium were

of the AMP pexiganan, a magainin analog currently in clinical devel- also obtained by direct plating on agar containing colistin at a

opment. Pexiganan resistance was observed with 32- to 512-fold concentration 10 or 50 times the MIC of wild type (Sun et al., 2009).

and 2- to 64-fold increase in resistance to the parental strains Spontaneous resistant mutants displayed 2- to 35-fold increased

for P. fluorescens and E. coli, respectively without an in vitro fit- colistin resistance with little to no fitness cost in a mouse model

ness cost. However, the exact mechanism of resistance was not of sepsis. 44 independent mutants were characterized and 27

determined. Similarly, it was shown that S. aureus could develop different missense mutations were identiﬁed in pmrA or pmrB, the

stable AMP resistance following serial passage in medium contain- two-component regulatory system. The colistin-resistant mutants

ing increasing concentrations of pexiganan (Habets and Brockhurst, displayed 2- to 15-fold increases in expression of pmrH indicating

2012). These mutants often displayed 10- to 50-fold increased a dysregulation of pmrAB-regulated genes. Stable colistin resis-

resistance to pexiganan and cross-resistance to human ␣-defensin tance following direct plating was also observed in A. baumannii

HNP1. The larger the level of pexiganan resistance, the higher the following the complete loss of LPS due to the inactivation of one

in vitro ﬁtness cost. Further serial passage in the absence of pexi- of three genes involved in lipid A biosynthesis (lpxA, lpxD, or lpxC)

ganan resulted in compensatory mutations ameliorating the ﬁtness (Moffatt et al., 2010).

cost while maintaining AMP resistance, but the molecular basis of Similarly, a PhoQ conditionally constitutive allele (phoQ24 or

AMP resistance was not deﬁned. This study suggests that the costs pho-24) displayed ∼10-fold increase in phoP-activated genes (pags)

associated with pexiganan resistance in S. aureus can be easily com- (Miller and Mekalanos, 1990). When grown in low levels of Mg ,

pensated resulting in the persistence of mutants capable of evading the phoQ24 allele confers polymyxin resistance as a result of

critical components of the human innate immune system. increased expression of PmrAB (Gunn and Miller, 1996). A simi-

Acquired AMP resistance in S. Typhimurium has been examined lar phenotype was observed following over-expression of dltABCD

by following continuous exposure (∼500 generations) to increas- in wild-type S. aureus, which increases d-alanylation of TA and

ing concentrations of LL-37 (a human cathelicidin), CNY100HL reduces cell surface negative charge (Peschel et al., 1999).

(derivative of human C3 complement peptide CNY21), or wheat Stable resistance to the pig cathelicidin PR-39 occurred in S.

germ histones (WGH) (Lofton et al., 2013). This resulted in the Typhimurium following direct plating on PR-39 containing agar

rapid isolation of stable resistant mutants that also displayed at a concentration 1.5 times the MIC of wild type (Pranting et al.,

2- to 4-fold increases in resistance to the AMPs not used for 2008). The resulting spontaneous PR-39 mutants were stable and

selection. AMP resistant isolates where shown to contain muta- displayed 2.5 to 3.5 fold increases in PR-39 resistance over wild

tions in two-component regulatory systems (pmrB or phoP) or in type. All of the AMP resistant mutants isolated contained mutations

LPS biosynthesis pathways (waaY, also referred to as rfaY). phoP in sbmA, a putative ABC transporter. The isolated mutants displayed

mutants containing a single amino acid substitution showed sim- similar phenotypes to that of a sbmA strain indicating that a loss

ilar levels of resistance to all three AMPs tested and had a ﬁtness of function genotype resulting in reduced PR-39 uptake into the cell

cost in vitro, however no signiﬁcant virulence defect was detected is responsible for the increased AMP resistance. The inactivation of

in a mouse model of sepsis (Lofton et al., 2015). The phoP mutant sbmA and the resulting PR-39 resistance did not confer a ﬁtness cost

also displayed a reduced O-antigen chain length, enhanced toler- in vitro or in a mouse model of sepsis.

ance of low pH and bile, and increased pagP expression regardless of S. Typhimurium mutants resistant to protamine, an arginine-

environmental Mg ion concentrations, indicating a constitutively rich AMP isolated from salmon sperm cells, were isolated by direct

active phenotype. Mutations in pmrB, single amino acid substitut- plating on agar containing protamine at a concentration of around

ions, occurred without an impact on bacterial ﬁtness in vitro or 3 times the wild type MIC (Pranting and Andersson, 2010). Spon-

in vivo in mice via intraperitoneal infection, yielding low-level taneous mutants were 2- to 20-fold more resistant to protamine

AMP resistance. Activation of the PmrAB two-component regula- than wild type, and displayed cross-resistance to other AMPs (col-

tory system supports the Ara4N modiﬁcation of lipid A, resulting istin, lactoferricin, human ␣-defensin HNP1). Resistance mutations

in reduced negative charge and diminished AMP binding. An inac- were identiﬁed as deletions, nonsense or missense in haem biosyn-

tivating frameshift mutation in waaY also resulted individually in thesis (hemA, hemB, hemC, hemL) with 14 different mutations in

low-level AMP resistance without an impact on bacterial ﬁtness four steps of haem biosynthesis were observed indicating that the

D.I. Andersson et al. / Drug Resistance Updates 26 (2016) 43–57 51

Table 2

Rates and ﬁtness effects of mutations causing resistance to antibiotics or AMPs.

a b b

Drug class Species Mutation rate Fitness MIC increase Mutation Reference

AMP

−6

PR-39 S. enterica 0.4 × 10 No change 4-fold sbmA Pranting et al. (2008)

LL-37 S. enterica ND No change/reduced 6-fold waaY, pmrB, phoP Lofton et al. (2013)

CNY-100HL S. enterica ND No change/reduced 2- to 3-fold waaY, pmrB, phoP Lofton et al. (2013)

WGH S. enterica ND No change/reduced >15-fold waaY, pmrB, phoP Lofton et al. (2013)

Pexiganan P. ﬂuorescens ND Weakly reduced 2- to 32-fold ND Perron et al. (2006)

Pexiganan E. coli ND ND 2- to -32-fold ND Perron et al. (2006)

−6

Colistin S. enterica 0.6 10 Weakly reduced 2- to 35-fold pmrAB Sun et al. (2009)

−7

Protamine S. enterica 2.3 × 10 Strongly reduced 2- to 20-fold hemACL/cydC Pranting and Andersson (2010)

Antibiotics

−9

Rifampicin E. coli, S. enterica 5 × 10 Reduced >200-fold rpoB Brandis et al. (2015)

−9

Ciproﬂoxacin E. coli <1 × 10 No change 16-fold gyrA Chin and Neu (1987)

Fusidic acid S. enterica ND Reduced 8- to 500-fold fusA Nagaev et al. (2001)

−8 −5

Mecillinam E. coli 8 10 to 2 × 10 No change/reduced 2- to 768-fold >38 genes Thulin et al. (2015)

−9

Mupirocin S. enterica 1 × 10 Reduced >64-fold ileS Paulander et al. (2007)

Per cell per generation.

As compared to susceptible parental strain.

Wheat germ histones.

lack of an end product in this pathway is responsible for protamine of the population size and rates of mutation (or HGT) to AMP resis-

resistance. Mutations in the hem genes often result in defects in tance. Mutation rates to AMP resistance have been measured in

−7 −6

electron transport and respiration, which results in slow growth, several cases and they are typically in the range of 10 to 10

reduced membrane potential and thus lowered afﬁnity for bind- per generation per cell (Pranting and Andersson, 2010; Pranting

ing and uptake of AMPs. All protamine-resistant mutants displayed et al., 2008; Sun et al., 2009), making them comparable to antibi-

−5 −10

large defects in bacterial ﬁtness with relative growth rates ranging otics (10 to 10 per generation per cell). Population sizes within

from 0.25 to 0.85 as compared to the wild type. The tremendous ﬁt- an infected human are generally poorly known but in a few cases

ness cost resulted in instability of the protamine resistance in some we have numbers for the total bacterial population size. For exam-

mutants, particularly hemC and hemA mutants, and compensatory ple, for urinary tract infections the total population sizes in a full

6 10

evolution by serial passage in the absence of protamine resulted bladder of 300 ml is in the range of 10 –10 per bladder and for

in the loss of protamine resistance. Three mutants displayed non- pulmonary tuberculosis infections the number of bacteria per ml

5 6

sense mutations in cydC, a cysteine/glutathione ABC transporter of sputum can be 10 –10 and even higher in the lungs (Hughes

thought to be involved in maintaining cellular redox balance. A and Andersson, 2015). Similarly, for meningitis and bronchitis the

cydC mutant would therefore display reduced membrane potential bacterial counts can be as high as 10 per ml, and overall these data

as well, resulting in AMP resistance. suggest that the total bacterial population sizes in an infected indi-

Spontaneous high-level bacteriocin resistance (over 2000-fold) vidual typically is so high that pre-existing AMP resistant mutants

occurred in L. monocytogenes following direct plating due to are expected to be present (i.e. population size x mutation rate >1)

increased expression of two putative ␤-glucoside-speciﬁc phos- when treatment is initiated.

photransferase systems leading to reduced interaction with AMPs

(Gravesen et al., 2002).

7.3.2. Mutant ﬁtness

7.3. What determines the risk of acquiring resistance to AMPs? The ﬁtness of a drug-resistant pathogen (in the absence and

presence of drug) is a key parameter in determining evolution-

As for any type of drug resistance, the rate by which AMP ary success—i.e. the probability that it will emerge, ﬁx, transmit

resistance would emerge and spread in a population of bacte- and remain—within a host population, as has been demonstrated

ria in a natural setting is determined by a complex interplay of in different studies for antibiotic resistant bacteria (Andersson and

several different factors, including the mutation supply rate; the Hughes, 2011). With regard to ﬁtness measurements of AMP resis-

ﬁtness of the resistant mutant at different AMP concentrations and tant mutants data is scarce and only a few studies have explored this

the strength of the selective pressure (AMP levels) (Hughes and in some detail (Lofton et al., 2013, 2015; Pranting and Andersson,

Andersson, 2015). Also, the occurrence of compensatory evolution 2010; Pranting et al., 2008). As is generally observed for antibi-

where ﬁtness costs might be reduced by additional mutations, and otic resistant mutants, AMP resistant variants show some type of

epistatic interactions involving resistance genes and drugs (Hughes reduction in ﬁtness, even though cases are known when with the

and Andersson, 2015) that may generate cross-resistance and/or assays used, the resistant mutants appeared as ﬁt as the congenic

collateral sensitivity, complicates the picture even further. In addi- susceptible strain (Pranting et al., 2008). For example, PR-39 resis-

tion, numerous other epidemiological factors (e.g. host population tant S. Typhimurium mutants showed no apparent reduction in

structure, density, immunity, etc.) will affect how a putative AMP ﬁtness as measured by growth rate, starvation survival and growth

resistant clone would spread in a population. Several of the above in mice. In other cases, S. Typhimurium mutants resistant to colistin,

parameters have been defined for antibiotic and antiviral resis- LL-37, CNY 100HL and wheat germ histones, fitness was signifi-

tances but with regard to AMP resistance few have been explored cantly reduced but the reductions were for some mutants relatively

in any detail (summarized in Table 2). minor, suggesting that these mutants might persist quite well in a

host population, even without a selective pressure (Lofton et al.,

7.3.1. Mutation supply rate 2013, 2015). Yet for protamine resistant S. Typhimurium mutants

The generation of genetic heterogeneity, in this case mutants the reductions were so severe that it is unlikely that they could

that are resistant/less susceptible to AMPs, in a bacterial popula- remain in the host population without a strong selection (Pranting

tion is largely determined by the mutation supply rate, the product and Andersson, 2010).

52 D.I. Andersson et al. / Drug Resistance Updates 26 (2016) 43–57

7.3.3. Selection strength phenomena for antibiotic resistance and they imply that collateral

Bacterial pathogens are, as a result of treatments, exposed to a sensitivity can potentially be applied to clinical situations to reduce

wide range of antibiotic concentrations, generating a variable range the rate of resistance evolution (Imamovic and Sommer, 2013;

of selective pressures. As shown by recent studies, not only concen- Munck et al., 2014). Thus, treatment would combine two drugs

trations well above MIC (lethal selection) can select for resistance where resistance evolution to one drug increases the susceptibility

but mutants may also emerge during non-lethal selection far below to the other. Again, for AMP resistant mutants the extent of

the MIC, where their rate of enrichment is determined by the ﬁtness collateral sensitivity for different AMPs and/or antibiotics has not

difference between susceptible and resistant cells (Gullberg et al., been extensively explored, even though a few cases have been

2011). Since only a few peptide antibiotics (e.g. colistin) have had reported (Berti et al., 2015; Garcia-Quintanilla et al., 2015).

any widespread clinical use, and as of yet no in vitro studies have

been performed to study selection at different AMP concentrations,

it is difﬁcult to assess the impact of selection strength on which 8. Therapeutic use of AMPs

mutant types emerge. It is notable that for AMPs, selection for resis-

tant mutants could occur at two levels: (i) due to therapeutic use Widespread antibiotic resistance has led to the urgent need

of AMPs (which might generate very strong selective pressures), for discovery of novel antimicrobials to treat bacterial infections.

and (ii) due to continuous selection provided by AMPs produced in The broad-spectrum bactericidal activity of AMPs makes them a

the human body and to which bacterial pathogens can be exposed. promising candidate for therapeutic use. There are only a handful

At present, we know that the pressure exerted by therapeutic of AMPs currently in clinical use, and of these, polymyxin B and

use is weak (simply because few AMPs are used clinically) but colistin (polymyxin E) are the most characterized in a clinical set-

the pressure exerted by human-speciﬁc AMPs is largely unknown. ting (Falagas and Kasiakou, 2005; Landman et al., 2008; Zavascki

However, one recent study showed that for a waaY mutant resis- et al., 2007). The polymyxins are produced by the bacterium

tant to several AMPs the dosage of LL-37 (0.1–1.5 mg/L) required to Bacillus polymyxa and were introduced into clinical practice in

select for and maintain the mutant in the population is similar to the 1950s. The polymyxins have been administered intravenously

the concentrations of AMPs found in, for example, secretions near to treat multi-drug resistant infections caused by Gram-negative

host epithelial cells (Lofton et al., 2013). This ﬁnding implies that pathogens, as an aerosol to treat bronchopulmonary infections,

naturally produced AMPs will exert signiﬁcant selective pressures. and intraventricularly to treat meningitis (Landman et al., 2008). In

However, the deleterious nature of this particular waaY mutation addition to its use for systemic treatment of complicated infections,

(reduced tolerance of serum, acidic conditions, and survival in a polymyxin B is used in topical formulations to treat and prevent

mouse model of sepsis) likely explains its absence in pathogenic skin, eye and ear infections. The high incidence of nephrotoxi-

isolates (Lofton et al., 2015). city and neurotoxicity associated with intravenous administration

has resulted in limited usage and study of these drugs over the

7.3.4. Compensatory evolution past 50 years (Falagas and Kasiakou, 2006). Regardless, the emer-

Most mutations that occur in any organism are deleterious but gence of multi-drug resistant pathogens has led to an increase in

compensatory evolution, where the costs are reduced or elimi- the therapeutic use of polymyxins despite limited pharmacologi-

nated by additional mutations, might increase the probability of cal and susceptibility studies. Conﬂicting clinical breakpoints have

maintenance. A number of experimental and clinical studies have emerged due to the sensitivity of polymyxins, like other AMPs,

shown that the ﬁtness cost of antibiotic resistance can be efﬁciently to environmental conditions, resulting in discrepancies between

and rapidly reduced by compensatory mutations and that compen- in vitro and in vivo efﬁcacy. These discrepancies have hampered

sated mutants can rapidly sweep a resistant population of bacteria the clinical development of AMPs to date (Falagas and Kasiakou,

(Andersson and Hughes, 2010). To what extent this process will 2005; Landman et al., 2008; Zavascki et al., 2007).

inﬂuence evolution will depend on the mutation rates of compen- Currently, there are many AMPs in clinical development for

sation, the ﬁtness of compensated mutants and the population size the treatment of various bacterial pathogens, but the major-

of the resistant mutant. One of the few studies of compensatory ity of these are intended for topical use only (Table 3). This

evolution involving AMPs was made using pexiganan and S. aureus is likely a direct result of the toxicity observed following sys-

but the actual mutations selected were not determined (Habets and temic administration of polymyxin B and colistin (Falagas and

Brockhurst, 2012). Kasiakou, 2006). The only AMP in clinical trials for intravenous

administration is human-derived Lactoferrin 1–11 (hLF1–11) to

7.3.5. Epistatic interactions treat life-threatening infections that occur in stem cell trans-

Recent data from bacteria show that epistasis between resis- plantation patients. Although a study has shown that hLF1–11 is

tance mutations is pervasive and that it might have an impact on well-tolerated in single and multiple doses as high as 5 mg follow-

resistance evolution (Hughes and Andersson, 2015). For example, ing intravenous administration (Velden et al., 2009), it is unclear

combinations of ﬁtness-reducing chromosomal resistance muta- whether this dose will be sufﬁcient to treat an infection since col-

tions and/or resistance plasmids can either generate no epistasis, istin has been administered up to a total of 720 mg per day in 3

positive epistasis (a double mutant has a higher ﬁtness than divided doses (Michalopoulos et al., 2005).

expected from the sum of costs of individual mutations), or neg- Aside from hLF1–11, there is an AMP-derivative from the human

ative epistasis (a double mutant has a lower ﬁtness than expected innate immune system in phase II clinical trials. OP-145, derived

from the sum of costs of individual mutations). All of these types of from the human cathelicidin LL-37, and formulated in eardrops,

epistasis have been observed in experimental studies. At present, is being tested to treat chronic bacterial ear infections (Malanovic

this type of epistasis has not been systematically studied for AMP et al., 2015). Other AMPs derived from naturally occurring animal

resistance mutations. immune components include Pexiganan, Iseganan and Omiganan.

Another type of epistasis involves the phenomenon where Pexiganan is an analog of magainin, an AMP originally isolated from

resistance to one drug alters susceptibility to other drugs. Thus, the skin of the African clawed frog, that has gone through clinical

resistance to one drug class might either increase resistance to trials twice to treat diabetic foot ulcers and is currently in phase

another (cross-resistance), or result in increased susceptibility III (Lamb and Wiseman, 1998; Lipsky et al., 2008). Another animal-

(Macvanin and Hughes, 2005), so-called collateral sensitivity. derived AMP is a synthetic protegrin originally isolated from pig

Recent large-scale studies have shown that both are common leucocytes termed Iseganan developed as a rinse for the potential

D.I. Andersson et al. / Drug Resistance Updates 26 (2016) 43–57 53

Table 3

Antimicrobial peptides in clinical development.

Peptide AMP source (host) Status Administration Indication Company

OP-145 LL-37 (human) Phase I/II Ear drops Chronic bacterial ear infection OctoPlus Inc.

hLF1–11 (Lactoferrin) Lactoferrin 1–11 (human) Not speciﬁed Intravenous Neutropenic stem cell AM-Pharma B.V.

transplantation patients

Pexiganan (MSI-78) Magainin (frog) Phase III Topical cream Diabetic foot infection Dipexium Pharmaceuticals, Inc.

Phase III Topical cream Diabetic foot ulcers MacroChem Corporation

Iseganan (IB-367) Protegrin-1 (porcine leukocytes) Phase III Mouth wash Prevention of National Cancer Institute (NCI) chemotherapy-induced

mucositis

Phase II/III Mouth wash Prevention of IntraBiotics Pharmaceuticals ventilator-associated

pneumonia

Omiganan (MBI 226, Indolicidin (bovine neutrophils) Phase III Topical cream Topical skin antisepsis, Mallinckrodt

CLS001) prevention of catheter

infections

Phase III Topical cream Rosacea Cutanea Life Sciences, Inc.

Phase II Topical cream Usual type vulvar Cutanea Life Sciences, Inc.

intraepithelial neoplasia (uVIN)

Phase II Topical cream Moderate to severe Cutanea Life Sciences, Inc.

inﬂammatory acne vulgaris

Phase II Topical cream Mild to moderate atopic Cutanea Life Sciences, Inc.

dermatitis

Lytixar (LTX-109) Synthetic antimicrobial Phase II Topical cream Uncomplicated Gram-positive Lytix Biopharma AS

peptidomimetic skin infections

Phase I/IIa Nasal Nasal carriers of Staphylococcus Lytix Biopharma AS

aureus

C16G2 Synthetic speciﬁcally targeted Phase II Mouth wash Prevent tooth decay caused by C3 Jian, Inc.

antimicrobial peptide Streptococcus mutans

treatment of mucositis, as an aerosol to treat respiratory infec- many ongoing clinical trials aimed at elucidating the utility of vita-

tions, and as a gel to treat pneumonia (Toney, 2002). Omiganan min D supplements in combating bacterial diseases ranging from

is a derivative of indolicidin, which was isolated from bovine neu- uncomplicated diarrheal diseases to life-threatening pulmonary

trophils and is in phase II/III clinical trials to treat skin infections. tuberculosis. Other compounds that have been shown to induce

Aside from naturally occurring AMPs, there are also several purely expression of host antimicrobial peptides include pimecrolimus

synthetic AMPs in clinical trials (Melo et al., 2006). The synthetic applied topically for the treatment of dermatitis (eczema) (Buchau

AMP LTX-109 is in phase II trials for the treatment of uncomplicated et al., 2008) and phenylbutyrate administered orally to treat various

Gram-positive skin infections as well as to prevent nasal carriage bacterial infections (Steinmann et al., 2009).

of S. aureus (Nilsson et al., 2015). C16G2 is another synthetic AMP There are also AMPs used in the clinic with indications other

in phase II trials indicated for the prevention of tooth decay caused than as antimicrobials. For example, protamine has been used

by Streptococcus mutans (Kaplan et al., 2011). clinically as a heparin antagonist to prevent blood clotting dur-

Aside from the direct administration of antimicrobial peptides, ing cardiac surgery, vascular surgery, and interventional radiology.

there are several ongoing clinical studies aimed at modulating Recently, protamine has been proposed as a drug to treat obesity

the expression of AMPs by the body to boost the innate immune due to its ability to inhibit lipase activity and decrease the absorp-

response (Table 4). Many recent ﬁndings have elucidated the effects tion of dietary fat (Duarte-Vazquez et al., 2009). Routine clinical

of dietary nutrients on modulating the innate immune response, use of protamine will inevitably result in bacterial exposure and

including the expression of AMPs, in order to assess the poten- subsequent development of resistance, regardless of whether its

tial of utilizing such dietary supplements as therapeutics. Since intended use is as an antimicrobial or not. Pranting and Andersson

many microbial infections are associated with inhibition of the (2010) have already shown in S. Typhimurium that protamine-

innate immune response (Diacovich and Gorvel, 2010), it is thought resistant mutants display cross-resistance to a variety of other

that enhancing the immune system will be an effective strategy AMPs (including components of the human innate immune sys-

to combat infection. Vitamin D3 has been shown to directly reg- tem) as well as to clinically prescribed antibiotics (gentamicin and

ulate expression of the human antimicrobial peptides LL-37 and streptomycin), which would make infections more persistent and

␤

-defensin 2 (Wang et al., 2004; Weber et al., 2005). There are difﬁcult to treat.

Table 4

Selected antimicrobial peptide modulating compounds in clinical development.

Intervention Administration Location of targeted AMPs Status Indication Study sponsor

Vitamin D3 Oral Skin and saliva Phase II Atopic dermatitis and National Institute of Allergy

psoriasis and Infectious Diseases (NIAID)

Vitamin D3 Oral Lung Not speciﬁed Active tuberculosis Atlanta VA Medical Center

Phenylbutyrate and Oral Lung Phase II Pulmonary tuberculosis International Centre for

vitamin D3 Diarrhoeal Disease Research,

Bangladesh

Lung, blood Phase II Pulmonary tuberculosis Karolinska Institutet

Blood Phase II HIV Karolinska Institutet

Sodium butyrate Enema Colon Phase II Shigellosis International Centre for

Diarrhoeal Disease Research,

Bangladesh

Pimecrolimus Topical Skin Phase IV Dermatitis National Jewish Health

54 D.I. Andersson et al. / Drug Resistance Updates 26 (2016) 43–57

mechanisms would impact ﬁtness and virulence of the mutants.

However, from the limited number of studies done we can also

conclude that (i) it is not difﬁcult for bacteria to acquire AMP resis-

tance by mutation, (ii) a number of mechanisms exist by which this

can occur, (iii) some of these mechanisms are not associated with

any extensive loss of ﬁtness, and (iv) often these resistance muta-

tions confer cross-resistance to human AMPs that are part of innate

immunity.

The latter is a point of special concern since it might imply that

therapeutic use of AMPs could select for mutants that become resis-

tant to key components of our own immune system. This potential

problem has been pointed out by several researchers and with more

data being available on acquired resistance mechanisms and cross-

resistance it seems clear that this is something that need to be

carefully studied and monitored when AMPs are increasingly used

in clinical settings.

Acknowledgement

Fig. 3. Potential consequence of therapeutic administration of antimicrobial pep- This work was supported by grants from the Swedish Research

tides. Routine clinical administration of AMPs to treat bacterial infections will select

Council to DIA and DH.

for resistant mutants more capable of evading the innate immune system and

causing persistent infections. Thus alternate antimicrobial therapies will be imple-

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